Substrate specificity of bacterial DD-peptidases (penicillin-binding proteins).

PubMed

Pratt, R F

2008-07-01

The DD-peptidase enzymes (penicillin-binding proteins) catalyze the final transpeptidation reaction of bacterial cell wall (peptidoglycan) biosynthesis. Although there is now much structural information available about these enzymes, studies of their activity as enzymes lag. It is now established that representatives of two low-molecular-mass classes of DD-peptidases recognize elements of peptidoglycan structure and rapidly react with substrates and inhibitors incorporating these elements. No members of other DD-peptidase classes, including the high-molecular-mass enzymes, essential for bacterial growth, appear to interact strongly with any particular elements of peptidoglycan structure. Rational design of inhibitors for these enzymes is therefore challenging.

Adamantoylated biologically active small peptides and glycopeptides structurally related to the bacterial peptidoglycan.

PubMed

Frkanec, Ruža; Vranešić, Branka; Tomić, Srdjanka

2013-01-01

A large number of novel synthetic compounds representing smaller parts of original peptidoglycan molecules have been synthesized and found to possess versatile biological activity, particularly immunomodulating properties. A series of compounds containing the adamantyl residues coupled to peptides and glycopeptides characteristic for bacterial peptidoglycan was described. The new adamantylpeptides and adamantylglycopeptides were prepared starting from N-protected racemic adamantylglycine and dipeptide L-Ala-D-isoglutamine. The adamantyl glycopeptides were obtained by coupling the adamantyltripeptides with alpha-D-mannose moiety through spacer molecule of fixed chirality. Since the starting material was D,L-(adamantyl-glycine) the condensation products with the dipeptide were mixtures of diastereoisomers. The obtained diastereoisomers were separated, characterized, and tested for immunostimulating activity. An HPLC method for purity testing was developed and adapted for the particular compounds.

Peptidoglycan microarray as a novel tool to explore protein-ligand recognition.

PubMed

Wang, Ning; Hirata, Akiyoshi; Nokihara, Kiyoshi; Fukase, Koichi; Fujimoto, Yukari

2016-11-04

Peptidoglycan is a giant bag-shaped molecule essential for bacterial cell shape and resistance to osmotic stresses. The activity of a large number of bacterial surface proteins involved in cell growth and division requires binding to this macromolecule. Recognition of peptidoglycan by immune effectors is also crucial for the establishment of the immune response against pathogens. The availability of pure and chemically defined peptidoglycan fragments is a major technical bottleneck that has precluded systematic studies of the mechanisms underpinning protein-mediated peptidoglycan recognition. Here, we report a microarray strategy suitable to carry out comprehensive studies to characterize proteins-peptidoglycan interactions. We describe a method to introduce a functional group on peptidoglycan fragments allowing their stable immobilization on amorphous carbon chip plates to minimize nonspecific binding. Such peptidoglycan microarrays were used with a model peptidoglycan binding protein-the human peptidoglycan recognition protein-S (hPGRP-S). We propose that this strategy could be implemented to carry out high-throughput analyses to study peptidoglycan binding proteins. © 2016 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 422-429, 2016. © 2016 Wiley Periodicals, Inc.

Molecular cloning and characterization of a short peptidoglycan recognition protein from silkworm Bombyx mori.

PubMed

Yang, P-J; Zhan, M-Y; Ye, C; Yu, X-Q; Rao, X-J

2017-12-01

Peptidoglycan is the major bacterial component recognized by the insect immune system. Peptidoglycan recognition proteins (PGRPs) are a family of pattern-recognition receptors that recognize peptidoglycans and modulate innate immune responses. Some PGRPs retain N-acetylmuramoyl-L-alanine amidase (Enzyme Commission number: 3.5.1.28) activity to hydrolyse bacterial peptidoglycans. Others have lost the enzymatic activity and work only as immune receptors. They are all important modulators for innate immunity. Here, we report the cloning and functional analysis of PGRP-S4, a short-form PGRP from the domesticated silkworm, Bombyx mori. The PGRP-S4 gene encodes a protein of 199 amino acids with a signal peptide and a PGRP domain. PGRP-S4 was expressed in the fat body, haemocytes and midgut. Its expression level was significantly induced by bacterial challenges in the midgut. The recombinant PGRP-S4 bound bacteria and different peptidoglycans. In addition, it inhibited bacterial growth and hydrolysed an Escherichia coli peptidoglycan in the presence of Zn 2+ . Scanning electron microscopy showed that PGRP-S4 disrupted the bacterial cell surface. PGRP-S4 further increased prophenoloxidase activation caused by peptidoglycans. Taken together, our data suggest that B. mori PGRP-S4 has multiple functions in immunity. © 2017 The Royal Entomological Society.

Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics

USDA-ARS?s Scientific Manuscript database

Virion-associated peptidoglycan hydrolases (VAPGH) are phage-encoded lytic enzymes that locally degrade the peptidoglycan (PG) of the bacterial cell wall during infection. Their action usually generates a small hole through which the phage tail crosses the cell envelope to inject the phage genetic m...

Another Brick in the Wall: a Rhamnan Polysaccharide Trapped inside Peptidoglycan of Lactococcus lactis.

PubMed

Sadovskaya, Irina; Vinogradov, Evgeny; Courtin, Pascal; Armalyte, Julija; Meyrand, Mickael; Giaouris, Efstathios; Palussière, Simon; Furlan, Sylviane; Péchoux, Christine; Ainsworth, Stuart; Mahony, Jennifer; van Sinderen, Douwe; Kulakauskas, Saulius; Guérardel, Yann; Chapot-Chartier, Marie-Pierre

2017-09-12

Polysaccharides are ubiquitous components of the Gram-positive bacterial cell wall. In Lactococcus lactis , a polysaccharide pellicle (PSP) forms a layer at the cell surface. The PSP structure varies among lactococcal strains; in L. lactis MG1363, the PSP is composed of repeating hexasaccharide phosphate units. Here, we report the presence of an additional neutral polysaccharide in L. lactis MG1363 that is a rhamnan composed of α-l-Rha trisaccharide repeating units. This rhamnan is still present in mutants devoid of the PSP, indicating that its synthesis can occur independently of PSP synthesis. High-resolution magic-angle spinning nuclear magnetic resonance (HR-MAS NMR) analysis of whole bacterial cells identified a PSP at the surface of wild-type cells. In contrast, rhamnan was detected only at the surface of PSP-negative mutant cells, indicating that rhamnan is located underneath the surface-exposed PSP and is trapped inside peptidoglycan. The genetic determinants of rhamnan biosynthesis appear to be within the same genetic locus that encodes the PSP biosynthetic machinery, except the gene tagO encoding the initiating glycosyltransferase. We present a model of rhamnan biosynthesis based on an ABC transporter-dependent pathway. Conditional mutants producing reduced amounts of rhamnan exhibit strong morphological defects and impaired division, indicating that rhamnan is essential for normal growth and division. Finally, a mutation leading to reduced expression of lcpA , encoding a protein of the LytR-CpsA-Psr (LCP) family, was shown to severely affect cell wall structure. In lcpA mutant cells, in contrast to wild-type cells, rhamnan was detected by HR-MAS NMR, suggesting that LcpA participates in the attachment of rhamnan to peptidoglycan. IMPORTANCE In the cell wall of Gram-positive bacteria, the peptidoglycan sacculus is considered the major structural component, maintaining cell shape and integrity. It is decorated with other glycopolymers, including

Different walls for rods and balls: the diversity of peptidoglycan

PubMed Central

Turner, Robert D; Vollmer, Waldemar; Foster, Simon J

2014-01-01

Peptidoglycan performs the essential role of resisting turgor in the cell walls of most bacteria. It determines cell shape, and its biosynthesis is the target for many important antibiotics. The fundamental chemical building blocks of peptidoglycan are conserved: repeating disaccharides cross-linked by peptides. However, these blocks come in many varieties and can be assembled in different ways. So beyond the fundamental similarity, prodigious chemical, organizational and architectural diversity is revealed. Here, we track the evolution of our current understanding of peptidoglycan and underpinning technical and methodological developments. The origin and function of chemical diversity is discussed with respect to some well-studied example species. We then explore how this chemistry is manifested in elegant and complex peptidoglycan organization and how this is interpreted in different and sometimes controversial architectural models. We contend that emerging technology brings about the possibility of achieving a complete understanding of peptidoglycan chemistry, through architecture, to the way in which diverse species and populations of cells meet the challenges of maintaining viability and growth within their environmental niches, by exploiting the bioengineering versatility of peptidoglycan. PMID:24405365

Bacterial Diterpene Synthases: New Opportunities for Mechanistic Enzymology and Engineered Biosynthesis

PubMed Central

Smanski, Michael J.; Peterson, Ryan M.; Huang, Sheng-Xiong; Shen, Ben

2012-01-01

Diterpenoid biosynthesis has been extensively studied in plants and fungi, yet cloning and engineering diterpenoid pathways in these organisms remain challenging. Bacteria are emerging as prolific producers of diterpenoid natural products, and bacterial diterpene synthases are poised to make significant contributions to our understanding of terpenoid biosynthesis. Here we will first survey diterpenoid natural products of bacterial origin and briefly review their biosynthesis with emphasis on diterpene synthases (DTSs) that channel geranylgeranyl diphosphate to various diterpenoid scaffolds. We will then highlight differences of DTSs of bacterial and higher organism origins and discuss the challenges in discovering novel bacterial DTSs. We will conclude by discussing new opportunities for DTS mechanistic enzymology and applications of bacterial DTS in biocatalysis and metabolic pathway engineering. PMID:22445175

Tunicamycins: translocase-I inhibitors that target bacterial cell wall and mammalian N-glycoproteins. The potential for selective inhibitors

USDA-ARS?s Scientific Manuscript database

Tunicamycins are a heterologous family of nucleoside antibiotics that target the biosynthesis of bacterial peptidoglycan and eukaryotic N-glycoproteins. The mechanism of action is known, with the tunicamycin-Mg2+ complex established as a transition state analog for hexosamine-1-phosphate: prenol pho...

Phosphorylated hydroxyethylamines as novel inhibitors of the bacterial cell wall biosynthesis enzymes MurC to MurF.

PubMed

Sova, Matej; Kovac, Andreja; Turk, Samo; Hrast, Martina; Blanot, Didier; Gobec, Stanislav

2009-12-01

Enzymes involved in the biosynthesis of bacterial peptidoglycan represent important targets for development of new antibacterial drugs. Among them, Mur ligases (MurC to MurF) catalyze the formation of the final cytoplasmic precursor UDP-N-acetylmuramyl-pentapeptide from UDP-N-acetylmuramic acid. We present the design, synthesis and biological evaluation of a series of phosphorylated hydroxyethylamines as new type of small-molecule inhibitors of Mur ligases. We show that the phosphate group attached to the hydroxyl moiety of the hydroxyethylamine core is essential for good inhibitory activity. The IC(50) values of these inhibitors were in the micromolar range, which makes them a promising starting point for the development of multiple inhibitors of Mur ligases as potential antibacterial agents. In addition, 1-(4-methoxyphenylsulfonamido)-3-morpholinopropan-2-yl dihydrogen phosphate 7a was discovered as one of the best inhibitors of MurE described so far.

Different walls for rods and balls: the diversity of peptidoglycan.

PubMed

Turner, Robert D; Vollmer, Waldemar; Foster, Simon J

2014-03-01

Peptidoglycan performs the essential role of resisting turgor in the cell walls of most bacteria. It determines cell shape, and its biosynthesis is the target for many important antibiotics. The fundamental chemical building blocks of peptidoglycan are conserved: repeating disaccharides cross-linked by peptides. However, these blocks come in many varieties and can be assembled in different ways. So beyond the fundamental similarity, prodigious chemical, organizational and architectural diversity is revealed. Here, we track the evolution of our current understanding of peptidoglycan and underpinning technical and methodological developments. The origin and function of chemical diversity is discussed with respect to some well-studied example species. We then explore how this chemistry is manifested in elegant and complex peptidoglycan organization and how this is interpreted in different and sometimes controversial architectural models. We contend that emerging technology brings about the possibility of achieving a complete understanding of peptidoglycan chemistry, through architecture, to the way in which diverse species and populations of cells meet the challenges of maintaining viability and growth within their environmental niches, by exploiting the bioengineering versatility of peptidoglycan. © 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.

Peptidoglycan turnover and recycling in Gram-positive bacteria.

PubMed

Reith, Jan; Mayer, Christoph

2011-10-01

Bacterial cells are protected by an exoskeleton, the stabilizing and shape-maintaining cell wall, consisting of the complex macromolecule peptidoglycan. In view of its function, it could be assumed that the cell wall is a static structure. In truth, however, it is steadily broken down by peptidoglycan-cleaving enzymes during cell growth. In this process, named cell wall turnover, in one generation up to half of the preexisting peptidoglycan of a bacterial cell is released from the wall. This would result in a massive loss of cell material, if turnover products were not be taken up and recovered. Indeed, in the Gram-negative model organism Escherichia coli, peptidoglycan recovery has been recognized as a complex pathway, named cell wall recycling. It involves about a dozen dedicated recycling enzymes that convey cell wall turnover products to peptidoglycan synthesis or energy pathways. Whether Gram-positive bacteria also recover their cell wall is currently questioned. Given the much larger portion of peptidoglycan in the cell wall of Gram-positive bacteria, however, recovery of the wall material would provide an even greater benefit in these organisms compared to Gram-negatives. Consistently, in many Gram-positives, orthologs of recycling enzymes were identified, indicating that the cell wall may also be recycled in these organisms. This mini-review provides a compilation of information about cell wall turnover and recycling in Gram-positive bacteria during cell growth and division, including recent findings relating to muropeptide recovery in Bacillus subtilis and Clostridium acetobutylicum from our group. Furthermore, the impact of cell wall turnover and recycling on biotechnological processes is discussed.

An Unusual Mutation Results in the Replacement of Diaminopimelate with Lanthionine in the Peptidoglycan of a Mutant Strain of Mycobacterium smegmatis†

PubMed Central

Consaul, Sandra A.; Wright, Lori F.; Mahapatra, Sebabrata; Crick, Dean C.; Pavelka, Martin S.

2005-01-01

Mycobacterial peptidoglycan contains l-alanyl-d-iso-glutaminyl-meso-diaminopimelyl-d-alanyl-d-alanine peptides, with the exception of the peptidoglycan of Mycobacterium leprae, in which glycine replaces the l-alanyl residue. The third-position amino acid of the peptides is where peptidoglycan cross-linking occurs, either between the meso-diaminopimelate (DAP) moiety of one peptide and the penultimate d-alanine of another peptide or between two DAP residues. We previously described a collection of spontaneous mutants of DAP-auxotrophic strains of Mycobacterium smegmatis that can grow in the absence of DAP. The mutants are grouped into seven classes, depending on how well they grow without DAP and whether they are sensitive to DAP, temperature, or detergent. Furthermore, the mutants are hypersusceptible to β-lactam antibiotics when grown in the absence of DAP, suggesting that these mutants assemble an abnormal peptidoglycan. In this study, we show that one of these mutants, M. smegmatis strain PM440, utilizes lanthionine, an unusual bacterial metabolite, in place of DAP. We also demonstrate that the abilities of PM440 to grow without DAP and use lanthionine for peptidoglycan biosynthesis result from an unusual mutation in the putative ribosome binding site of the cbs gene, encoding cystathionine β-synthase, an enzyme that is a part of the cysteine biosynthetic pathway. PMID:15716431

Diversity of Innate Immune Recognition Mechanism for Bacterial Polymeric meso-Diaminopimelic Acid-type Peptidoglycan in Insects

PubMed Central

Yu, Yang; Park, Ji-Won; Kwon, Hyun-Mi; Hwang, Hyun-Ok; Jang, In-Hwan; Masuda, Akiko; Kurokawa, Kenji; Nakayama, Hiroshi; Lee, Won-Jae; Dohmae, Naoshi; Zhang, Jinghai; Lee, Bok Luel

2010-01-01

In Drosophila, the synthesis of antimicrobial peptides in response to microbial infections is under the control of the Toll and immune deficiency (Imd) signaling pathway. The Toll signaling pathway responds mainly to the lysine-type peptidoglycan of Gram-positive bacteria and fungal β-1,3-glucan, whereas the Imd pathway responds to the meso-diaminopimelic acid (DAP)-type peptidoglycan of Gram-negative bacteria and certain Gram-positive bacilli. Recently we determined the activation mechanism of a Toll signaling pathway biochemically using a large beetle, Tenebrio molitor. However, DAP-type peptidoglycan recognition mechanism and its signaling pathway are still unclear in the fly and beetle. Here, we show that polymeric DAP-type peptidoglycan, but not its monomeric form, formed a complex with Tenebrio peptidoglycan recognition protein-SA, and this complex activated the three-step proteolytic cascade to produce processed Spätzle, a Toll receptor ligand, and induced Drosophila defensin-like antimicrobial peptide in Tenebrio larvae similarly to polymeric lysine-type peptidoglycan. Monomeric DAP-type peptidoglycan induced Drosophila diptericin-like antimicrobial peptide in Tenebrio hemocytes. In addition, both polymeric and monomeric DAP-type peptidoglycans induced expression of Tenebrio peptidoglycan recognition protein-SC2, which is DAP-type peptidoglycan-selective N-acetylmuramyl-l-alanine amidase that functions as a DAP-type peptidoglycan scavenger, appearing to function as a negative regulator of the DAP-type peptidoglycan signaling by cleaving DAP-type peptidoglycan in Tenebrio larvae. Taken together, these results demonstrate that molecular recognition mechanism for polymeric DAP-type peptidoglycan is different between Tenebrio larvae and Drosophila adults, providing biochemical evidences of biological diversity of innate immune responses in insects. PMID:20702416

Cell wall peptidoglycan architecture in Bacillus subtilis

PubMed Central

Hayhurst, Emma J.; Kailas, Lekshmi; Hobbs, Jamie K.; Foster, Simon J.

2008-01-01

The bacterial cell wall is essential for viability and shape determination. Cell wall structural dynamics allowing growth and division, while maintaining integrity is a basic problem governing the life of bacteria. The polymer peptidoglycan is the main structural component for most bacteria and is made up of glycan strands that are cross-linked by peptide side chains. Despite study and speculation over many years, peptidoglycan architecture has remained largely elusive. Here, we show that the model rod-shaped bacterium Bacillus subtilis has glycan strands up to 5 μm, longer than the cell itself and 50 times longer than previously proposed. Atomic force microscopy revealed the glycan strands to be part of a peptidoglycan architecture allowing cell growth and division. The inner surface of the cell wall has a regular macrostructure with ≈50 nm-wide peptidoglycan cables [average 53 ± 12 nm (n = 91)] running basically across the short axis of the cell. Cross striations with an average periodicity of 25 ± 9 nm (n = 96) along each cable are also present. The fundamental cabling architecture is also maintained during septal development as part of cell division. We propose a coiled-coil model for peptidoglycan architecture encompassing our data and recent evidence concerning the biosynthetic machinery for this essential polymer. PMID:18784364

Nisin, alone and combined with peptidoglycan-modulating antibiotics: activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant enterococci.

PubMed

Brumfitt, W; Salton, M R J; Hamilton-Miller, J M T

2002-11-01

We have sought ways to circumvent resistance, by combining nisin with other antibiotics known to target bacterial cell wall biosynthesis. Twenty strains each of methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococci (VRE) were tested in vitro by standardized methods against nisin alone and combined with bacitracin, ramoplanin and chloramphenicol. Ramoplanin was the most potent compound, and bacitracin had the least activity. Two-way synergy was observed with nisin and ramoplanin. However, chloramphenicol was clearly antagonistic to the activity of nisin. Observations of synergy between nisin and ramoplanin against MRSA and VRE offer a promising approach to the concept of combining nisin with inhibitors of cell wall peptidoglycan. Further investigations are needed in order to develop this approach as a clinical possibility.

Bacterial cellulose biosynthesis: diversity of operons, subunits, products, and functions.

PubMed

Römling, Ute; Galperin, Michael Y

2015-09-01

Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits - which differ among various taxa - affect the enzymatic activity and product yield in vivo by modulating (i) the expression of the biosynthesis apparatus, (ii) the export of the nascent β-D-glucan polymer to the cell surface, and (iii) the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of resulting biofilms, which is particularly important for the interactions of bacteria with higher organisms - leading to rhizosphere colonization and modulating the virulence of cellulose-producing bacterial pathogens inside and outside of host cells. We review the organization of four principal types of cellulose synthase operon found in various bacterial genomes, identify additional bcs genes that encode components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms and in the choice between acute infection and persistence in the host. Copyright © 2015 Elsevier Ltd. All rights reserved.

Bacterial cellulose biosynthesis: diversity of operons, subunits, products and functions

PubMed Central

Römling, Ute; Galperin, Michael Y.

2015-01-01

Summary Recent studies of bacterial cellulose biosynthesis, including structural characterization of a functional cellulose synthase complex, provided the first mechanistic insight into this fascinating process. In most studied bacteria, just two subunits, BcsA and BcsB, are necessary and sufficient for the formation of the polysaccharide chain in vitro. Other subunits – which differ among various taxa – affect the enzymatic activity and product yield in vivo by modulating expression of biosynthesis apparatus, export of the nascent β-D-glucan polymer to the cell surface, and the organization of cellulose fibers into a higher-order structure. These auxiliary subunits play key roles in determining the quantity and structure of the resulting biofilm, which is particularly important for interactions of bacteria with higher organisms that lead to rhizosphere colonization and modulate virulence of cellulose-producing bacterial pathogens inside and outside of host cells. Here we review the organization of four principal types of cellulose synthase operons found in various bacterial genomes, identify additional bcs genes that encode likely components of the cellulose biosynthesis and secretion machinery, and propose a unified nomenclature for these genes and subunits. We also discuss the role of cellulose as a key component of biofilms formed by a variety of free-living and pathogenic bacteria and, for the latter, in the choice between acute infection and persistence in the host. PMID:26077867

Crystal structure of the glycosidase family 73 peptidoglycan hydrolase FlgJ

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hashimoto, Wataru; Ochiai, Akihito; Momma, Keiko

Glycoside hydrolase (GH) categorized into family 73 plays an important role in degrading bacterial cell wall peptidoglycan. The flagellar protein FlgJ contains N- and C-terminal domains responsible for flagellar rod assembly and peptidoglycan hydrolysis, respectively. A member of family GH-73, the C-terminal domain (SPH1045-C) of FlgJ from Sphingomonas sp. strain A1 was expressed in Escherichia coli, purified, and characterized. SPH1045-C exhibited bacterial cell lytic activity most efficiently at pH 6.0 and 37 deg. C. The X-ray crystallographic structure of SPH1045-C was determined at 1.74 A resolution by single-wavelength anomalous diffraction. The enzyme consists of two lobes, {alpha} and {beta}. Amore » deep cleft located between the two lobes can accommodate polymer molecules, suggesting that the active site is located in the cleft. Although SPH1045-C shows a structural homology with family GH-22 and GH-23 lysozymes, the arrangement of the nucleophile/base residue in the active site is specific to each peptidoglycan hydrolase.« less

Peptidoglycan sensing by octopaminergic neurons modulates Drosophila oviposition

PubMed Central

Kurz, C Leopold; Charroux, Bernard; Chaduli, Delphine; Viallat-Lieutaud, Annelise; Royet, Julien

2017-01-01

As infectious diseases pose a threat to host integrity, eukaryotes have evolved mechanisms to eliminate pathogens. In addition to develop strategies reducing infection, animals can engage in behaviors that lower the impact of the infection. The molecular mechanisms by which microbes impact host behavior are not well understood. We demonstrate that bacterial infection of Drosophila females reduces oviposition and that peptidoglycan, the component that activates Drosophila antibacterial response, is also the elicitor of this behavioral change. We show that peptidoglycan regulates egg-laying rate by activating NF-κB signaling pathway in octopaminergic neurons and that, a dedicated peptidoglycan degrading enzyme acts in these neurons to buffer this behavioral response. This study shows that a unique ligand and signaling cascade are used in immune cells to mount an immune response and in neurons to control fly behavior following infection. This may represent a case of behavioral immunity. DOI: http://dx.doi.org/10.7554/eLife.21937.001 PMID:28264763

Substrate specificity of low-molecular mass bacterial DD-peptidases.

PubMed

Nemmara, Venkatesh V; Dzhekieva, Liudmila; Sarkar, Kumar Subarno; Adediran, S A; Duez, Colette; Nicholas, Robert A; Pratt, R F

2011-11-22

The bacterial DD-peptidases or penicillin-binding proteins (PBPs) catalyze the formation and regulation of cross-links in peptidoglycan biosynthesis. They are classified into two groups, the high-molecular mass (HMM) and low-molecular mass (LMM) enzymes. The latter group, which is subdivided into classes A-C (LMMA, -B, and -C, respectively), is believed to catalyze DD-carboxypeptidase and endopeptidase reactions in vivo. To date, the specificity of their reactions with particular elements of peptidoglycan structure has not, in general, been defined. This paper describes the steady-state kinetics of hydrolysis of a series of specific peptidoglycan-mimetic peptides, representing various elements of stem peptide structure, catalyzed by a range of LMM PBPs (the LMMA enzymes, Escherichia coli PBP5, Neisseria gonorrhoeae PBP4, and Streptococcus pneumoniae PBP3, and the LMMC enzymes, the Actinomadura R39 dd-peptidase, Bacillus subtilis PBP4a, and N. gonorrhoeae PBP3). The R39 enzyme (LMMC), like the previously studied Streptomyces R61 DD-peptidase (LMMB), specifically and rapidly hydrolyzes stem peptide fragments with a free N-terminus. In accord with this result, the crystal structures of the R61 and R39 enzymes display a binding site specific to the stem peptide N-terminus. These are water-soluble enzymes, however, with no known specific function in vivo. On the other hand, soluble versions of the remaining enzymes of those noted above, all of which are likely to be membrane-bound and/or associated in vivo and have been assigned particular roles in cell wall biosynthesis and maintenance, show little or no specificity for peptides containing elements of peptidoglycan structure. Peptidoglycan-mimetic boronate transition-state analogues do inhibit these enzymes but display notable specificity only for the LMMC enzymes, where, unlike peptide substrates, they may be able to effectively induce a specific active site structure. The manner in which LMMA (and HMM) DD

Bacterial Cell Mechanics.

PubMed

Auer, George K; Weibel, Douglas B

2017-07-25

Cellular mechanical properties play an integral role in bacterial survival and adaptation. Historically, the bacterial cell wall and, in particular, the layer of polymeric material called the peptidoglycan were the elements to which cell mechanics could be primarily attributed. Disrupting the biochemical machinery that assembles the peptidoglycan (e.g., using the β-lactam family of antibiotics) alters the structure of this material, leads to mechanical defects, and results in cell lysis. Decades after the discovery of peptidoglycan-synthesizing enzymes, the mechanisms that underlie their positioning and regulation are still not entirely understood. In addition, recent evidence suggests a diverse group of other biochemical elements influence bacterial cell mechanics, may be regulated by new cellular mechanisms, and may be triggered in different environmental contexts to enable cell adaptation and survival. This review summarizes the contributions that different biomolecular components of the cell wall (e.g., lipopolysaccharides, wall and lipoteichoic acids, lipid bilayers, peptidoglycan, and proteins) make to Gram-negative and Gram-positive bacterial cell mechanics. We discuss the contribution of individual proteins and macromolecular complexes in cell mechanics and the tools that make it possible to quantitatively decipher the biochemical machinery that contributes to bacterial cell mechanics. Advances in this area may provide insight into new biology and influence the development of antibacterial chemotherapies.

Biosynthesis of the tunicamycin antibiotics proceeds via unique exo-glycal intermediates

NASA Astrophysics Data System (ADS)

Wyszynski, Filip J.; Lee, Seung Seo; Yabe, Tomoaki; Wang, Hua; Gomez-Escribano, Juan Pablo; Bibb, Mervyn J.; Lee, Soo Jae; Davies, Gideon J.; Davis, Benjamin G.

2012-07-01

The tunicamycins are archetypal nucleoside antibiotics targeting bacterial peptidoglycan biosynthesis and eukaryotic protein N-glycosylation. Understanding the biosynthesis of their unusual carbon framework may lead to variants with improved selectivity. Here, we demonstrate in vitro recapitulation of key sugar-manipulating enzymes from this pathway. TunA is found to exhibit unusual regioselectivity in the reduction of a key α,β-unsaturated ketone. The product of this reaction is shown to be the preferred substrate for TunF—an epimerase that converts the glucose derivative to a galactose. In Streptomyces strains in which another gene (tunB) is deleted, the biosynthesis is shown to stall at this exo-glycal product. These investigations confirm the combined TunA/F activity and delineate the ordering of events in the metabolic pathway. This is the first time these surprising exo-glycal intermediates have been seen in biology. They suggest that construction of the aminodialdose core of tunicamycin exploits their enol ether motif in a mode of C-C bond formation not previously observed in nature, to create an 11-carbon chain.

A novel mechanism of programmed cell death in bacteria by toxin-antitoxin systems corrupts peptidoglycan synthesis.

PubMed

Mutschler, Hannes; Gebhardt, Maike; Shoeman, Robert L; Meinhart, Anton

2011-03-01

Most genomes of bacteria contain toxin-antitoxin (TA) systems. These gene systems encode a toxic protein and its cognate antitoxin. Upon antitoxin degradation, the toxin induces cell stasis or death. TA systems have been linked with numerous functions, including growth modulation, genome maintenance, and stress response. Members of the epsilon/zeta TA family are found throughout the genomes of pathogenic bacteria and were shown not only to stabilize resistance plasmids but also to promote virulence. The broad distribution of epsilon/zeta systems implies that zeta toxins utilize a ubiquitous bacteriotoxic mechanism. However, whereas all other TA families known to date poison macromolecules involved in translation or replication, the target of zeta toxins remained inscrutable. We used in vivo techniques such as microscropy and permeability assays to show that pneumococcal zeta toxin PezT impairs cell wall synthesis and triggers autolysis in Escherichia coli. Subsequently, we demonstrated in vitro that zeta toxins in general phosphorylate the ubiquitous peptidoglycan precursor uridine diphosphate-N-acetylglucosamine (UNAG) and that this activity is counteracted by binding of antitoxin. After identification of the product we verified the kinase activity in vivo by analyzing metabolite extracts of cells poisoned by PezT using high pressure liquid chromatograpy (HPLC). We further show that phosphorylated UNAG inhibitis MurA, the enzyme catalyzing the initial step in bacterial peptidoglycan biosynthesis. Additionally, we provide what is to our knowledge the first crystal structure of a zeta toxin bound to its substrate. We show that zeta toxins are novel kinases that poison bacteria through global inhibition of peptidoglycan synthesis. This provides a fundamental understanding of how epsilon/zeta TA systems stabilize mobile genetic elements. Additionally, our results imply a mechanism that connects activity of zeta toxin PezT to virulence of pneumococcal infections

Sex-dependent alterations in motor and anxiety-like behavior of aged bacterial peptidoglycan sensing molecule 2 knockout mice.

PubMed

Arentsen, Tim; Khalid, Roksana; Qian, Yu; Diaz Heijtz, Rochellys

2018-01-01

Peptidoglycan recognition proteins (PGRPs) are key sensing-molecules of the innate immune system that specifically detect bacterial peptidoglycan (PGN) and its derivates. PGRPs have recently emerged as potential key regulators of normal brain development and behavior. To test the hypothesis that PGRPs play a role in motor control and anxiety-like behavior in later life, we used 15-month old male and female peptidoglycan recognition protein 2 (Pglyrp2) knockout (KO) mice. Pglyrp2 is an N-acetylmuramyl-l-alanine amidase that hydrolyzes PGN between the sugar backbone and the peptide chain (which is unique among the mammalian PGRPs). Using a battery of behavioral tests, we demonstrate that Pglyrp2 KO male mice display decreased levels of anxiety-like behavior compared with wild type (WT) males. In contrast, Pglyrp2 KO female mice show reduced rearing activity and increased anxiety-like behavior compared to WT females. In the accelerated rotarod test, however, Pglyrp2 KO female mice performed better compared to WT females (i.e., they had longer latency to fall off the rotarod). Further, Pglyrp2 KO male mice exhibited decreased expression levels of synaptophysin, gephyrin, and brain-derived neurotrophic factor in the frontal cortex, but not in the amygdala. Pglyrp2 KO female mice exhibited increased expression levels of spinophilin and alpha-synuclein in the frontal cortex, while exhibiting decreased expression levels of synaptophysin, gephyrin and spinophilin in the amygdala. Our findings suggest a novel role for Pglyrp2asa key regulator of motor and anxiety-like behavior in late life. Copyright © 2017. Published by Elsevier Inc.

Virtual screening for potential inhibitors of bacterial MurC and MurD ligases.

PubMed

Tomašić, Tihomir; Kovač, Andreja; Klebe, Gerhard; Blanot, Didier; Gobec, Stanislav; Kikelj, Danijel; Mašič, Lucija Peterlin

2012-03-01

Mur ligases are bacterial enzymes involved in the cytoplasmic steps of peptidoglycan biosynthesis and are viable targets for antibacterial drug discovery. We have performed virtual screening for potential ATP-competitive inhibitors targeting MurC and MurD ligases, using a protocol of consecutive hierarchical filters. Selected compounds were evaluated for inhibition of MurC and MurD ligases, and weak inhibitors possessing dual inhibitory activity have been identified. These compounds represent new scaffolds for further optimisation towards multiple Mur ligase inhibitors with improved inhibitory potency.

Inhibitors targeting on cell wall biosynthesis pathway of MRSA.

PubMed

Hao, Haihong; Cheng, Guyue; Dai, Menghong; Wu, Qinghua; Yuan, Zonghui

2012-11-01

Methicillin resistant Staphylococcus aureus (MRSA), widely known as a type of new superbug, has aroused world-wide concern. Cell wall biosynthesis pathway is an old but good target for the development of antibacterial agents. Peptidoglycan and wall teichoic acids (WTAs) biosynthesis are two main processes of the cell wall biosynthesis pathway (CWBP). Other than penicillin-binding proteins (PBPs), some key factors (Mur enzymes, lipid I or II precursor, etc.) in CWBP are becoming attractive molecule targets for the discovery of anti-MRSA compounds. A number of new compounds, with higher affinity for PBPs or with inhibitory activity on such molecule targets in CWBP of MRSA, have been in the pipeline recently. This review concludes recent research achievements and provides a complete picture of CWBP of MRSA, including the peptidoglycan and wall teichoic acids synthesis pathway. The potential inhibitors targeting on CWBP are subsequently presented to improve development of novel therapeutic strategies for MRSA.

Mur Ligase Inhibitors as Anti-bacterials: A Comprehensive Review.

PubMed

Sangshetti, Jaiprakash N; Joshi, Suyog S; Patil, Rajendra H; Moloney, Mark G; Shinde, Devanand B

2017-01-01

Exploring a new target for antibacterial drug discovery has gained much attention because of the emergence of Multidrug Resistance (MDR) strains of bacteria. To overcome this problem the development of novel antibacterial was considered as highest priority task and was one of the biggest challenge since multiple factors were involved. The bacterial peptidoglycan biosynthetic pathway has been well documented in the last few years and has been found to be imperative source for the development of novel antibacterial agents with high target specificity as they are essential for bacterial survival and have no homologs in humans. We have therefore reviewed the process of peptidoglycan biosynthesis which involves various steps like formation of UDP-Nacetylglucosamine (GlcNAc), UDP-N-acetylmuramic acid (MurNAc) and lipid intermediates (Lipid I and Lipid II) which are controlled by various enzymes like GlmS, GlmM, GlmU enzyme, followed by Mur Ligases (MurAMurF) and finally by MraY and MurG respectively. These four amide ligases MurC-MurF can be used as the source for the development of novel multi-target antibacterial agents as they shared and conserved amino acid regions, catalytic mechanisms and structural features. This review begins with the need for novel antibacterial agents and challenges in their development even after the development of bacterial genomic studies. An overview of the peptidoglycan monomer formation, as a source of disparity in this process is presented, followed by detailed discussion of structural and functional aspects of all Mur enzymes and different chemical classes of their inhibitors along with their SAR studies and inhibitory potential. This review finally emphasizes on different patents and novel Mur inhibitors in the development phase. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Detection of bacterial infection by a technetium-99m-labeled peptidoglycan aptamer.

PubMed

Ferreira, Iêda Mendes; de Sousa Lacerda, Camila Maria; Dos Santos, Sara Roberta; de Barros, André Luís Branco; Fernandes, Simone Odília; Cardoso, Valbert Nascimento; de Andrade, Antero Silva Ribeiro

2017-09-01

Nuclear medicine clinicians are still waiting for the optimal scintigraphic imaging agents capable of distinguishing between infection and inflammation, and between fungal and bacterial infections. Aptamers have several properties that make them suitable for molecular imaging. In the present study, a peptidoglycan aptamer (Antibac1) was labeled with 99m Tc and evaluated by biodistribution studies and scintigraphic imaging in infection-bearing mice. Labeling with 99m Tc was performed by the direct method and the complex stability was evaluated in saline, plasma and in the molar excess of cysteine. The biodistribution and scintigraphic imaging studies with the 99m Tc-Antibac1 were carried out in two different experimental infection models: Bacterial-infected mice (S. aureus) and fungal-infected mice (C. albicans). A 99m Tc radiolabeled library, consisting of oligonucleotides with random sequences, was used as a control for both models. Radiolabeling yields were superior to 90% and 99m Tc-Antibac1 was highly stable in presence of saline, plasma, and cysteine up to 6h. Scintigraphic images of S. aureus infected mice at 1.5 and 3.0h after 99m Tc-Antibac1 injection showed target to non-target ratios of 4.7±0.9 and 4.6±0.1, respectively. These values were statistically higher than those achieved for the 99m Tc-library at the same time frames (1.6±0.4 and 1.7±0.4, respectively). Noteworthy, 99m Tc-Antibac1 and 99m Tc-library showed similar low target to non-target ratios in the fungal-infected model: 2.0±0.3 and 2.0±0.6for 99m Tc-Antibac1 and 2.1±0.3 and 1.9 ± 0.6 for 99m Tc-library, at the same times. These findings suggest that the 99m Tc-Antibac1 is a feasible imaging probe to identify a bacterial infection focus. In addition, this radiolabeled aptamer seems to be suitable in distinguishing between bacterial and fungal infection. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Peptidoglycan Hydrolases of Escherichia coli

PubMed Central

van Heijenoort, Jean

2011-01-01

Summary: The review summarizes the abundant information on the 35 identified peptidoglycan (PG) hydrolases of Escherichia coli classified into 12 distinct families, including mainly glycosidases, peptidases, and amidases. An attempt is also made to critically assess their functions in PG maturation, turnover, elongation, septation, and recycling as well as in cell autolysis. There is at least one hydrolytic activity for each bond linking PG components, and most hydrolase genes were identified. Few hydrolases appear to be individually essential. The crystal structures and reaction mechanisms of certain hydrolases having defined functions were investigated. However, our knowledge of the biochemical properties of most hydrolases still remains fragmentary, and that of their cellular functions remains elusive. Owing to redundancy, PG hydrolases far outnumber the enzymes of PG biosynthesis. The presence of the two sets of enzymes acting on the PG bonds raises the question of their functional correlations. It is difficult to understand why E. coli keeps such a large set of PG hydrolases. The subtle differences in substrate specificities between the isoenzymes of each family certainly reflect a variety of as-yet-unidentified physiological functions. Their study will be a far more difficult challenge than that of the steps of the PG biosynthesis pathway. PMID:22126997

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)

DOE PAGES

Sychantha, David; Jones, Carys S.; Little, Dustin J.; ...

2017-10-27

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain.more » We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.« less

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sychantha, David; Jones, Carys S.; Little, Dustin J.

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain.more » We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens.« less

In vitro characterization of the antivirulence target of Gram-positive pathogens, peptidoglycan O-acetyltransferase A (OatA)

PubMed Central

Sychantha, David; Jones, Carys S.; Little, Dustin J.; Howell, P. Lynne

2017-01-01

The O-acetylation of the essential cell wall polymer peptidoglycan occurs in most Gram-positive bacterial pathogens, including species of Staphylococcus, Streptococcus and Enterococcus. This modification to peptidoglycan protects these pathogens from the lytic action of the lysozymes of innate immunity systems and, as such, is recognized as a virulence factor. The key enzyme involved, peptidoglycan O-acetyltransferase A (OatA) represents a particular challenge to biochemical study since it is a membrane associated protein whose substrate is the insoluble peptidoglycan cell wall polymer. OatA is predicted to be bimodular, being comprised of an N-terminal integral membrane domain linked to a C-terminal extracytoplasmic domain. We present herein the first biochemical and kinetic characterization of the C-terminal catalytic domain of OatA from two important human pathogens, Staphylococcus aureus and Streptococcus pneumoniae. Using both pseudosubstrates and novel biosynthetically-prepared peptidoglycan polymers, we characterized distinct substrate specificities for the two enzymes. In addition, the high resolution crystal structure of the C-terminal domain reveals an SGNH/GDSL-like hydrolase fold with a catalytic triad of amino acids but with a non-canonical oxyanion hole structure. Site-specific replacements confirmed the identity of the catalytic and oxyanion hole residues. A model is presented for the O-acetylation of peptidoglycan whereby the translocation of acetyl groups from a cytoplasmic source across the cytoplasmic membrane is catalyzed by the N-terminal domain of OatA for their transfer to peptidoglycan by its C-terminal domain. This study on the structure-function relationship of OatA provides a molecular and mechanistic understanding of this bacterial resistance mechanism opening the prospect for novel chemotherapeutic exploration to enhance innate immunity protection against Gram-positive pathogens. PMID:29077761

Structure of Bordetella pertussis peptidoglycan

DOE Office of Scientific and Technical Information (OSTI.GOV)

Folkening, W.J.; Nogami, W.; Martin, S.A.

1987-09-01

Bordetella pertussis Tohama phases I and III were grown to the late-exponential phase in liquid medium containing (/sup 3/H)diaminopimelic acid and treated by a hot (96/sup 0/C) sodium dodecyl sulfate extraction procedure. Washed sodium dodecyl sulfate-insoluble residue from phases I and III consisted of complexes containing protein (ca. 40%) and peptidoglycan (60/sup 6/). Subsequent treatment with proteinase K yielded purified peptidoglycan which contained N-acetylglucosamine, N-acetylmuramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 and <2% protein. Radiochemical analyses indicated that /sup 3/H added in diaminopimelic acid was present in peptidoglycan-protein complexes and purified peptidoglycan as diaminopimelicmore » acid exclusively and that pertussis peptidoglycan was not O acetylated, consistent with it being degraded completely by hen egg white lysozyme. Muramidase-derived disaccharide peptide monomers and peptide-cross-linked dimers and higher oligomers were isolated by molecular-sieve chromatography; from the distribution of these peptidoglycan fragments, the extent of peptide cross-linking of both phase I and III peptidoglycan was calculated to be ca. 48%. Unambiguous determination of the structure of muramidase-derived pepidoglycan fragments by fast atom bombardment-mass spectrometry and tandem mass spectrometry indicated that the pertussis peptidoglycan monomer fraction was surprisingly homogeneous, consisting of >95% N-acetylglucosaminyl-N-acetylmuramyl-alanyl-glutamyl-diaminopimelyl-alanine.« less

Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division

PubMed Central

Packiam, Mathanraj; Hsu, Yen-Pang; Tekkam, Srinivas; Hall, Edward; Rittichier, Jonathan T.; VanNieuwenhze, Michael; Brun, Yves V.; Maurelli, Anthony T.

2016-01-01

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe’s developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host. PMID:27144308

Pathogenic Chlamydia Lack a Classical Sacculus but Synthesize a Narrow, Mid-cell Peptidoglycan Ring, Regulated by MreB, for Cell Division.

PubMed

Liechti, George; Kuru, Erkin; Packiam, Mathanraj; Hsu, Yen-Pang; Tekkam, Srinivas; Hall, Edward; Rittichier, Jonathan T; VanNieuwenhze, Michael; Brun, Yves V; Maurelli, Anthony T

2016-05-01

The peptidoglycan (PG) cell wall is a peptide cross-linked glycan polymer essential for bacterial division and maintenance of cell shape and hydrostatic pressure. Bacteria in the Chlamydiales were long thought to lack PG until recent advances in PG labeling technologies revealed the presence of this critical cell wall component in Chlamydia trachomatis. In this study, we utilize bio-orthogonal D-amino acid dipeptide probes combined with super-resolution microscopy to demonstrate that four pathogenic Chlamydiae species each possess a ≤ 140 nm wide PG ring limited to the division plane during the replicative phase of their developmental cycles. Assembly of this PG ring is rapid, processive, and linked to the bacterial actin-like protein, MreB. Both MreB polymerization and PG biosynthesis occur only in the intracellular form of pathogenic Chlamydia and are required for cell enlargement, division, and transition between the microbe's developmental forms. Our kinetic, molecular, and biochemical analyses suggest that the development of this limited, transient, PG ring structure is the result of pathoadaptation by Chlamydia to an intracellular niche within its vertebrate host.

Toll-like receptor 2-mediated peptidoglycan uptake by immature intestinal epithelial cells from apical side and exosome-associated transcellular transcytosis

PubMed Central

Bu, Heng-Fu; Wang, Xiao; Tang, Yi; Koti, Viola; Tan, Xiao-Di

2015-01-01

Peptidoglycan is a potent immune adjuvant derived from bacterial cell walls. Previous investigations suggest that intestinal epithelium may absorb peptidoglycan from the lumen. Nonetheless, how peptidoglycan is taken up and crosses intestinal epithelium remains largely unclear. Here, we first characterized peptidoglycan transport in vitro using IEC-18 and HT29-CL19A cells, which represent less mature epithelial cells in intestinal crypts. With fluorescent microscopy, we visualized internalization of dual-labeled peptidoglycan by enterocytes. Engulfed peptidoglycan was found to form a complex with peptidoglycan recognition protein-3, which may facilitate delivering peptidoglycan in vivo. Utilizing electronic microscopy, we revealed that uptake of apical peptidoglycan across intestinal epithelial monolayers was involved in phagocytosis, multivesicular body formation, and exosome secretion. We also studied transport of peptidoglycan using the transwell system. Our data indicated that apically loaded peptidoglycan was exocytosed to the basolateral compartment with exosomes by HT29-CL19A cells. The peptidoglycan-contained basolateral exosome extracts induced macrophage activation. Through gavaging mice with labeled peptidoglycan, we found that luminal peptidoglycan was taken up by columnar epithelial cells in crypts of the small intestine. Furthermore, we showed that pre-confluent immature but not post-confluent mature C2BBe1 cells engulfed peptidoglycan via a toll-like receptor 2-dependent manner. Together, our findings suggest that (1) crypt-based immature intestinal epithelial cells play an important role in transport of luminal peptidoglycan over the intestinal epithelium; and (2) luminal peptidoglycan is transcytosed across intestinal epithelia via a toll-like receptor 2-meciated phagocytosis-multivesicular body-exosome pathway. The absorbed peptidoglycan and its derivatives may facilitate maintenance of intestinal immune homeostasis. PMID:20020500

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis.

PubMed

Rueff, Anne-Stéphanie; Chastanet, Arnaud; Domínguez-Escobar, Julia; Yao, Zhizhong; Yates, James; Prejean, Maria-Victoria; Delumeau, Olivier; Noirot, Philippe; Wedlich-Söldner, Roland; Filipe, Sergio R; Carballido-López, Rut

2014-01-01

MreB proteins play a major role during morphogenesis of rod-shaped bacteria by organizing biosynthesis of the peptidoglycan cell wall. However, the mechanisms underlying this process are not well understood. In Bacillus subtilis, membrane-associated MreB polymers have been shown to be associated to elongation-specific complexes containing transmembrane morphogenetic factors and extracellular cell wall assembly proteins. We have now found that an early intracellular step of cell wall synthesis is also associated to MreB. We show that the previously uncharacterized protein YkuR (renamed DapI) is required for synthesis of meso-diaminopimelate (m-DAP), an essential constituent of the peptidoglycan precursor, and that it physically interacts with MreB. Highly inclined laminated optical sheet microscopy revealed that YkuR forms uniformly distributed foci that exhibit fast motion in the cytoplasm, and are not detected in cells lacking MreB. We propose a model in which soluble MreB organizes intracellular steps of peptidoglycan synthesis in the cytoplasm to feed the membrane-associated cell wall synthesizing machineries. © 2013 John Wiley & Sons Ltd.

Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth.

PubMed

Desai, Janish; Wang, Yang; Wang, Ke; Malwal, Satish R; Oldfield, Eric

2016-10-06

We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron-withdrawing aryl-alkyl side chains which inhibited the growth of Gram-negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ∼1-4 μg mL -1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially "rescued" by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (∼2-6 μg mL -1 ) against Gram-positive but not Gram-negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ∼1-2 μg mL -1 . © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Chemometric Analysis of Bacterial Peptidoglycan Reveals Atypical Modifications That Empower the Cell Wall against Predatory Enzymes and Fly Innate Immunity.

PubMed

Espaillat, Akbar; Forsmo, Oskar; El Biari, Khouzaima; Björk, Rafael; Lemaitre, Bruno; Trygg, Johan; Cañada, Francisco Javier; de Pedro, Miguel A; Cava, Felipe

2016-07-27

Peptidoglycan is a fundamental structure for most bacteria. It contributes to the cell morphology and provides cell wall integrity against environmental insults. While several studies have reported a significant degree of variability in the chemical composition and organization of peptidoglycan in the domain Bacteria, the real diversity of this polymer is far from fully explored. This work exploits rapid ultraperformance liquid chromatography and multivariate data analysis to uncover peptidoglycan chemical diversity in the Class Alphaproteobacteria, a group of Gram negative bacteria that are highly heterogeneous in terms of metabolism, morphology and life-styles. Indeed, chemometric analyses revealed novel peptidoglycan structures conserved in Acetobacteria: amidation at the α-(l)-carboxyl of meso-diaminopimelic acid and the presence of muropeptides cross-linked by (1-3) l-Ala-d-(meso)-diaminopimelate cross-links. Both structures are growth-controlled modifications that influence sensitivity to Type VI secretion system peptidoglycan endopeptidases and recognition by the Drosophila innate immune system, suggesting relevant roles in the environmental adaptability of these bacteria. Collectively our findings demonstrate the discriminative power of chemometric tools on large cell wall-chromatographic data sets to discover novel peptidoglycan structural properties in bacteria.

Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology.

PubMed

Lenz, Robert W; Marchessault, Robert H

2005-01-01

The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate), PHB, as a granular component in bacterial cells proceeded without any of the controversies which marked the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to personalities and locations involved with the events covering a discovery timespan of 75 years.

PG-Metrics: A chemometric-based approach for classifying bacterial peptidoglycan data sets and uncovering their subjacent chemical variability

PubMed Central

Kumar, Keshav; Espaillat, Akbar; Cava, Felipe

2017-01-01

Bacteria cells are protected from osmotic and environmental stresses by an exoskeleton-like polymeric structure called peptidoglycan (PG) or murein sacculus. This structure is fundamental for bacteria’s viability and thus, the mechanisms underlying cell wall assembly and how it is modulated serve as targets for many of our most successful antibiotics. Therefore, it is now more important than ever to understand the genetics and structural chemistry of the bacterial cell walls in order to find new and effective methods of blocking it for the treatment of disease. In the last decades, liquid chromatography and mass spectrometry have been demonstrated to provide the required resolution and sensitivity to characterize the fine chemical structure of PG. However, the large volume of data sets that can be produced by these instruments today are difficult to handle without a proper data analysis workflow. Here, we present PG-metrics, a chemometric based pipeline that allows fast and easy classification of bacteria according to their muropeptide chromatographic profiles and identification of the subjacent PG chemical variability between e.g. bacterial species, growth conditions and, mutant libraries. The pipeline is successfully validated here using PG samples from different bacterial species and mutants in cell wall proteins. The obtained results clearly demonstrated that PG-metrics pipeline is a valuable bioanalytical tool that can lead us to cell wall classification and biomarker discovery. PMID:29040278

Accumulation of Peptidoglycan O-Acetylation Leads to Altered Cell Wall Biochemistry and Negatively Impacts Pathogenesis Factors of Campylobacter jejuni.

PubMed

Ha, Reuben; Frirdich, Emilisa; Sychantha, David; Biboy, Jacob; Taveirne, Michael E; Johnson, Jeremiah G; DiRita, Victor J; Vollmer, Waldemar; Clarke, Anthony J; Gaynor, Erin C

2016-10-21

Campylobacter jejuni is a leading cause of bacterial gastroenteritis in the developed world. Despite its prevalence, its mechanisms of pathogenesis are poorly understood. Peptidoglycan (PG) is important for helical shape, colonization, and host-pathogen interactions in C. jejuni Therefore, changes in PG greatly impact the physiology of this organism. O-acetylation of peptidoglycan (OAP) is a bacterial phenomenon proposed to be important for proper cell growth, characterized by acetylation of the C6 hydroxyl group of N-acetylmuramic acid in the PG glycan backbone. The OAP gene cluster consists of a PG O-acetyltransferase A (patA) for translocation of acetate into the periplasm, a PG O-acetyltransferase B (patB) for O-acetylation, and an O-acetylpeptidoglycan esterase (ape1) for de-O-acetylation. In this study, reduced OAP in ΔpatA and ΔpatB had minimal impact on C. jejuni growth and fitness under the conditions tested. However, accumulation of OAP in Δape1 resulted in marked differences in PG biochemistry, including O-acetylation, anhydromuropeptide levels, and changes not expected to result directly from Ape1 activity. This suggests that OAP may be a form of substrate level regulation in PG biosynthesis. Ape1 acetylesterase activity was confirmed in vitro using p-nitrophenyl acetate and O-acetylated PG as substrates. In addition, Δape1 exhibited defects in pathogenesis-associated phenotypes, including cell shape, motility, biofilm formation, cell surface hydrophobicity, and sodium deoxycholate sensitivity. Δape1 was also impaired for chick colonization and adhesion, invasion, intracellular survival, and induction of IL-8 production in INT407 cells in vitro The importance of Ape1 in C. jejuni biology makes it a good candidate as an antimicrobial target. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives as multiple inhibitors of bacterial Mur ligases (MurC-MurF).

PubMed

Perdih, Andrej; Hrast, Martina; Barreteau, Hélène; Gobec, Stanislav; Wolber, Gerhard; Solmajer, Tom

2014-08-01

Enzymes catalyzing the biosynthesis of bacterial peptidoglycan represent traditionally a collection of highly selective targets for novel antibacterial drug design. Four members of the bacterial Mur ligase family-MurC, MurD, MurE and MurF-are involved in the intracellular steps of peptidoglycan biosynthesis, catalyzing the synthesis of the peptide moiety of the Park's nucleotide. In our previous virtual screening campaign, a chemical class of benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole derivatives exhibiting dual MurD/MurE inhibition properties was discovered. In the present study we further investigated this class of compounds by performing inhibition assays on all four Mur ligases (MurC-MurF). Furthermore, molecular dynamics (MD) simulation studies of one of the initially discovered compound 1 were performed to explore its geometry as well as its energetic behavior based on the Linear Interaction Energy (LIE) method. Further in silico virtual screening (VS) experiments based on the parent active compound 1 were conducted to optimize the discovered series. Selected hits were assayed against all Escherichia coli MurC-MurF enzymes in biochemical inhibition assays and molecules 10-14 containing benzene-1,3-dicarboxylic acid 2,5-dimethylpyrrole coupled with five member-ring rhodanine moiety were found to be multiple inhibitors of the whole MurC-MurF cascade of bacterial enzymes in the micromolar range. Steady-state kinetics studies suggested this class to act as competitive inhibitors of the MurD enzyme towards d-Glu. These compounds represent novel valuable starting point in the development of novel antibacterial agents. Copyright © 2014 Elsevier Ltd. All rights reserved.

Encyclopedia of bacterial gene circuits whose presence or absence correlate with pathogenicity--a large-scale system analysis of decoded bacterial genomes.

PubMed

Shestov, Maksim; Ontañón, Santiago; Tozeren, Aydin

2015-10-13

Bacterial infections comprise a global health challenge as the incidences of antibiotic resistance increase. Pathogenic potential of bacteria has been shown to be context dependent, varying in response to environment and even within the strains of the same genus. We used the KEGG repository and extensive literature searches to identify among the 2527 bacterial genomes in the literature those implicated as pathogenic to the host, including those which show pathogenicity in a context dependent manner. Using data on the gene contents of these genomes, we identified sets of genes highly abundant in pathogenic but relatively absent in commensal strains and vice versa. In addition, we carried out genome comparison within a genus for the seventeen largest genera in our genome collection. We projected the resultant lists of ortholog genes onto KEGG bacterial pathways to identify clusters and circuits, which can be linked to either pathogenicity or synergy. Gene circuits relatively abundant in nonpathogenic bacteria often mediated biosynthesis of antibiotics. Other synergy-linked circuits reduced drug-induced toxicity. Pathogen-abundant gene circuits included modules in one-carbon folate, two-component system, type-3 secretion system, and peptidoglycan biosynthesis. Antibiotics-resistant bacterial strains possessed genes modulating phagocytosis, vesicle trafficking, cytoskeletal reorganization, and regulation of the inflammatory response. Our study also identified bacterial genera containing a circuit, elements of which were previously linked to Alzheimer's disease. Present study produces for the first time, a signature, in the form of a robust list of gene circuitry whose presence or absence could potentially define the pathogenicity of a microbiome. Extensive literature search substantiated a bulk majority of the commensal and pathogenic circuitry in our predicted list. Scanning microbiome libraries for these circuitry motifs will provide further insights into the complex

Pyrazolopyrimidines establish MurC as a vulnerable target in Pseudomonas aeruginosa and Escherichia coli.

PubMed

Hameed P, Shahul; Manjrekar, Praveena; Chinnapattu, Murugan; Humnabadkar, Vaishali; Shanbhag, Gajanan; Kedari, Chaitanyakumar; Mudugal, Naina Vinay; Ambady, Anisha; de Jonge, Boudewijn L M; Sadler, Claire; Paul, Beena; Sriram, Shubha; Kaur, Parvinder; Guptha, Supreeth; Raichurkar, Anandkumar; Fleming, Paul; Eyermann, Charles J; McKinney, David C; Sambandamurthy, Vasan K; Panda, Manoranjan; Ravishankar, Sudha

2014-10-17

The bacterial peptidoglycan biosynthesis pathway provides multiple targets for antibacterials, as proven by the clinical success of β-lactam and glycopeptide classes of antibiotics. The Mur ligases play an essential role in the biosynthesis of the peptidoglycan building block, N-acetyl-muramic acid-pentapeptide. MurC, the first of four Mur ligases, ligates l-alanine to UDP-N-acetylmuramic acid, initiating the synthesis of pentapeptide precursor. Therefore, inhibiting the MurC enzyme should result in bacterial cell death. Herein, we report a novel class of pyrazolopyrimidines with subnanomolar potency against both Escherichia coli and Pseudomonas aeruginosa MurC enzymes, which demonstrates a concomitant bactericidal activity against efflux-deficient strains. Radio-labeled precursor incorporation showed these compounds selectively inhibited peptidoglycan biosynthesis, and genetic studies confirmed the target of pyrazolopyrimidines to be MurC. In the presence of permeability enhancers such as colistin, pyrazolopyrimidines exhibited low micromolar MIC against the wild-type bacteria, thereby, indicating permeability and efflux as major challenges for this chemical series. Our studies provide biochemical and genetic evidence to support the essentiality of MurC and serve to validate the attractiveness of target for antibacterial discovery.

Enzymatic Reductive Dehalogenation Controls the Biosynthesis of Marine Bacterial Pyrroles.

PubMed

El Gamal, Abrahim; Agarwal, Vinayak; Rahman, Imran; Moore, Bradley S

2016-10-12

Enzymes capable of performing dehalogenating reactions have attracted tremendous contemporary attention due to their potential application in the bioremediation of anthropogenic polyhalogenated persistent organic pollutants. Nature, in particular the marine environment, is also a prolific source of polyhalogenated organic natural products. The study of the biosynthesis of these natural products has furnished a diverse array of halogenation biocatalysts, but thus far no examples of dehalogenating enzymes have been reported from a secondary metabolic pathway. Here we show that the penultimate step in the biosynthesis of the highly brominated marine bacterial product pentabromopseudilin is catalyzed by an unusual debrominase Bmp8 that utilizes a redox thiol mechanism to remove the C-2 bromine atom of 2,3,4,5-tetrabromopyrrole to facilitate oxidative coupling to 2,4-dibromophenol. To the best of our knowledge, Bmp8 is first example of a dehalogenating enzyme from the established genetic and biochemical context of a natural product biosynthetic pathway.

The Biosynthesis of Capuramycin-type Antibiotics

PubMed Central

Cai, Wenlong; Goswami, Anwesha; Yang, Zhaoyong; Liu, Xiaodong; Green, Keith D.; Barnard-Britson, Sandra; Baba, Satoshi; Funabashi, Masanori; Nonaka, Koichi; Sunkara, Manjula; Morris, Andrew J.; Spork, Anatol P.; Ducho, Christian; Garneau-Tsodikova, Sylvie; Thorson, Jon S.; Van Lanen, Steven G.

2015-01-01

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5′-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5′-aldehyde transaldolase were uncovered, suggesting that C–C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5′-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures. PMID:25855790

RAV transcription factors are essential for disease resistance against cassava bacterial blight via activation of melatonin biosynthesis genes.

PubMed

Wei, Yunxie; Chang, Yanli; Zeng, Hongqiu; Liu, Guoyin; He, Chaozu; Shi, Haitao

2018-01-01

With 1 AP2 domain and 1 B3 domain, 7 MeRAVs in apetala2/ethylene response factor (AP2/ERF) gene family have been identified in cassava. However, the in vivo roles of these remain unknown. Gene expression assays showed that the transcripts of MeRAVs were commonly regulated after Xanthomonas axonopodis pv manihotis (Xam) and MeRAVs were specifically located in plant cell nuclei. Through virus-induced gene silencing (VIGS) in cassava, we found that MeRAV1 and MeRAV2 are essential for plant disease resistance against cassava bacterial blight, as shown by the bacterial propagation of Xam in plant leaves. Through VIGS in cassava leaves and overexpression in cassava leave protoplasts, we found that MeRAV1 and MeRAV2 positively regulated melatonin biosynthesis genes and the endogenous melatonin level. Further investigation showed that MeRAV1 and MeRAV2 are direct transcriptional activators of 3 melatonin biosynthesis genes in cassava, as evidenced by chromatin immunoprecipitation-PCR in cassava leaf protoplasts and electrophoretic mobility shift assay. Moreover, cassava melatonin biosynthesis genes also positively regulated plant disease resistance. Taken together, this study identified MeRAV1 and MeRAV2 as common and upstream transcription factors of melatonin synthesis genes in cassava and revealed a model of MeRAV1 and MeRAV2-melatonin biosynthesis genes-melatonin level in plant disease resistance against cassava bacterial blight. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Bacterial SPOR domains are recruited to septal peptidoglycan by binding to glycan strands that lack stem peptides

PubMed Central

Yahashiri, Atsushi; Jorgenson, Matthew A.; Weiss, David S.

2015-01-01

Bacterial SPOR domains bind peptidoglycan (PG) and are thought to target proteins to the cell division site by binding to “denuded” glycan strands that lack stem peptides, but uncertainties remain, in part because septal-specific binding has yet to be studied in a purified system. Here we show that fusions of GFP to SPOR domains from the Escherichia coli cell-division proteins DamX, DedD, FtsN, and RlpA all localize to septal regions of purified PG sacculi obtained from E. coli and Bacillus subtilis. Treatment of sacculi with an amidase that removes stem peptides enhanced SPOR domain binding, whereas treatment with a lytic transglycosylase that removes denuded glycans reduced SPOR domain binding. These findings demonstrate unequivocally that SPOR domains localize by binding to septal PG, that the physiologically relevant binding site is indeed a denuded glycan, and that denuded glycans are enriched in septal PG rather than distributed uniformly around the sacculus. Accumulation of denuded glycans in the septal PG of both E. coli and B. subtilis, organisms separated by 1 billion years of evolution, suggests that sequential removal of stem peptides followed by degradation of the glycan backbone is an ancient feature of PG turnover during bacterial cell division. Linking SPOR domain localization to the abundance of a structure (denuded glycans) present only transiently during biogenesis of septal PG provides a mechanism for coordinating the function of SPOR domain proteins with the progress of cell division. PMID:26305949

Roles of tRNA in cell wall biosynthesis

PubMed Central

Dare, Kiley; Ibba, Michael

2013-01-01

Recent research into various aspects of bacterial metabolism such as cell wall and antibiotic synthesis, degradation pathways, cellular stress, and amino acid biosynthesis has elucidated roles of aminoacyl-transfer ribonucleic acid (aa-tRNA) outside of translation. Although the two enzyme families responsible for cell wall modifications, aminoacyl-phosphatidylglycerol synthases (aaPGSs) and Fem, were discovered some time ago, they have recently become of intense interest for their roles in the antimicrobial resistance of pathogenic microorganisms. The addition of positively charged amino acids to phosphatidylglycerol (PG) by aaPGSs neutralizes the lipid bilayer making the bacteria less susceptible to positively charged antimicrobial agents. Fem transferases utilize aa-tRNA to form peptide bridges that link strands of peptidoglycan. These bridges vary among the bacterial species in which they are present and play a role in resistance to antibiotics that target the cell wall. Additionally, the formation of truncated peptides results in shorter peptide bridges and loss of branched linkages which makes bacteria more susceptible to antimicrobials. A greater understanding of the structure and substrate specificity of this diverse enzymatic family is necessary to aid current efforts in designing potential bactericidal agents. These two enzyme families are linked only by the substrate with which they modify the cell wall, aa-tRNA; their structure, cell wall modification processes and the physiological changes they impart on the bacterium differ greatly. PMID:22262511

Principal coordinate analysis assisted chromatographic analysis of bacterial cell wall collection: A robust classification approach.

PubMed

Kumar, Keshav; Cava, Felipe

2018-04-10

In the present work, Principal coordinate analysis (PCoA) is introduced to develop a robust model to classify the chromatographic data sets of peptidoglycan sample. PcoA captures the heterogeneity present in the data sets by using the dissimilarity matrix as input. Thus, in principle, it can even capture the subtle differences in the bacterial peptidoglycan composition and can provide a more robust and fast approach for classifying the bacterial collection and identifying the novel cell wall targets for further biological and clinical studies. The utility of the proposed approach is successfully demonstrated by analysing the two different kind of bacterial collections. The first set comprised of peptidoglycan sample belonging to different subclasses of Alphaproteobacteria. Whereas, the second set that is relatively more intricate for the chemometric analysis consist of different wild type Vibrio Cholerae and its mutants having subtle differences in their peptidoglycan composition. The present work clearly proposes a useful approach that can classify the chromatographic data sets of chromatographic peptidoglycan samples having subtle differences. Furthermore, present work clearly suggest that PCoA can be a method of choice in any data analysis workflow. Copyright © 2018 Elsevier Inc. All rights reserved.

An endogenous nanomineral chaperones luminal antigen and peptidoglycan to intestinal immune cells

NASA Astrophysics Data System (ADS)

Powell, Jonathan J.; Thomas-McKay, Emma; Thoree, Vinay; Robertson, Jack; Hewitt, Rachel E.; Skepper, Jeremy N.; Brown, Andy; Hernandez-Garrido, Juan Carlos; Midgley, Paul A.; Gomez-Morilla, Inmaculada; Grime, Geoffrey W.; Kirkby, Karen J.; Mabbott, Neil A.; Donaldson, David S.; Williams, Ifor R.; Rios, Daniel; Girardin, Stephen E.; Haas, Carolin T.; Bruggraber, Sylvaine F. A.; Laman, Jon D.; Tanriver, Yakup; Lombardi, Giovanna; Lechler, Robert; Thompson, Richard P. H.; Pele, Laetitia C.

2015-05-01

In humans and other mammals it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer's patches, small areas of the intestine concentrated with particle-scavenging immune cells. In wild-type mice, intestinal immune cells containing these naturally formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1’, whereas in NOD1/2 double knockout mice, which cannot recognize peptidoglycan, programmed death-ligand 1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and show how this helps to shape intestinal immune homeostasis.

Constructing the wonders of the bacterial world: biosynthesis of complex enzymes.

PubMed

Sargent, Frank

2007-03-01

The prokaryotic cytoplasmic membrane not only maintains cell integrity and forms a barrier between the cell and its outside environment, but is also the location for essential biochemical processes. Microbial model systems provide excellent bases for the study of fundamental problems in membrane biology including signal transduction, chemotaxis, solute transport and, as will be the topic of this review, energy metabolism. Bacterial respiration requires a diverse array of complex, multi-subunit, cofactor-containing redox enzymes, many of which are embedded within, or located on the extracellular side of, the membrane. The biosynthesis of these enzymes therefore requires carefully controlled expression, assembly, targeting and transport processes. Here, focusing on the molybdenum-containing respiratory enzymes central to anaerobic respiration in Escherichia coli, recent descriptions of a chaperone-mediated 'proofreading' system involved in coordinating assembly and export of complex extracellular enzymes will be discussed. The paradigm proofreading chaperones are members of a large group of proteins known as the TorD family, and recent research in this area highlights common principles that underpin biosynthesis of both exported and non-exported respiratory enzymes.

Phenyl thiazolyl urea and carbamate derivatives as new inhibitors of bacterial cell-wall biosynthesis.

PubMed

Francisco, Gerardo D; Li, Zhong; Albright, J Donald; Eudy, Nancy H; Katz, Alan H; Petersen, Peter J; Labthavikul, Pornpen; Singh, Guy; Yang, Youjun; Rasmussen, Beth A; Lin, Yang-I; Mansour, Tarek S

2004-01-05

Over 50 phenyl thiazolyl urea and carbamate derivatives were synthesized for evaluation as new inhibitors of bacterial cell-wall biosynthesis. Many of them demonstrated good activity against MurA and MurB and gram-positive bacteria including MRSA, VRE and PRSP. 3,4-Difluorophenyl 5-cyanothiazolylurea (3p) with clog P of 2.64 demonstrated antibacterial activity against both gram-positive and gram-negative bacteria.

Characterisation of ATP-dependent Mur ligases involved in the biogenesis of cell wall peptidoglycan in Mycobacterium tuberculosis.

PubMed

Munshi, Tulika; Gupta, Antima; Evangelopoulos, Dimitrios; Guzman, Juan David; Gibbons, Simon; Keep, Nicholas H; Bhakta, Sanjib

2013-01-01

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery.

Characterisation of ATP-Dependent Mur Ligases Involved in the Biogenesis of Cell Wall Peptidoglycan in Mycobacterium tuberculosis

PubMed Central

Munshi, Tulika; Gupta, Antima; Evangelopoulos, Dimitrios; Guzman, Juan David; Gibbons, Simon; Keep, Nicholas H.; Bhakta, Sanjib

2013-01-01

ATP-dependent Mur ligases (Mur synthetases) play essential roles in the biosynthesis of cell wall peptidoglycan (PG) as they catalyze the ligation of key amino acid residues to the stem peptide at the expense of ATP hydrolysis, thus representing potential targets for antibacterial drug discovery. In this study we characterized the division/cell wall (dcw) operon and identified a promoter driving the co-transcription of mur synthetases along with key cell division genes such as ftsQ and ftsW. Furthermore, we have extended our previous investigations of MurE to MurC, MurD and MurF synthetases from Mycobacterium tuberculosis. Functional analyses of the pure recombinant enzymes revealed that the presence of divalent cations is an absolute requirement for their activities. We also observed that higher concentrations of ATP and UDP-sugar substrates were inhibitory for the activities of all Mur synthetases suggesting stringent control of the cytoplasmic steps of the peptidoglycan biosynthetic pathway. In line with the previous findings on the regulation of mycobacterial MurD and corynebacterial MurC synthetases via phosphorylation, we found that all of the Mur synthetases interacted with the Ser/Thr protein kinases, PknA and PknB. In addition, we critically analyzed the interaction network of all of the Mur synthetases with proteins involved in cell division and cell wall PG biosynthesis to re-evaluate the importance of these key enzymes as novel therapeutic targets in anti-tubercular drug discovery. PMID:23555903

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria

PubMed Central

Mistou, Michel-Yves; Sutcliffe, Iain C.; van Sorge, Nina M.

2016-01-01

The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. PMID:26975195

Bacterial glycobiology: rhamnose-containing cell wall polysaccharides in Gram-positive bacteria.

PubMed

Mistou, Michel-Yves; Sutcliffe, Iain C; van Sorge, Nina M

2016-07-01

The composition of the Gram-positive cell wall is typically described as containing peptidoglycan, proteins and essential secondary cell wall structures called teichoic acids, which comprise approximately half of the cell wall mass. The cell walls of many species within the genera Streptococcus, Enterococcus and Lactococcus contain large amounts of the sugar rhamnose, which is incorporated in cell wall-anchored polysaccharides (CWP) that possibly function as homologues of well-studied wall teichoic acids (WTA). The presence and chemical structure of many rhamnose-containing cell wall polysaccharides (RhaCWP) has sometimes been known for decades. In contrast to WTA, insight into the biosynthesis and functional role of RhaCWP has been lacking. Recent studies in human streptococcal and enterococcal pathogens have highlighted critical roles for these complex polysaccharides in bacterial cell wall architecture and pathogenesis. In this review, we provide an overview of the RhaCWP with regards to their biosynthesis, genetics and biological function in species most relevant to human health. We also briefly discuss how increased knowledge in this field can provide interesting leads for new therapeutic compounds and improve biotechnological applications. © FEMS 2016.

Transpeptidase activity of penicillin-binding protein SpoVD in peptidoglycan synthesis conditionally depends on the disulfide reductase StoA.

PubMed

Bukowska-Faniband, Ewa; Hederstedt, Lars

2017-07-01

Endospore cortex peptidoglycan synthesis is not required for bacterial growth but essential for endospore heat resistance. It therefore constitutes an amenable system for research on peptidoglycan biogenesis. The Bacillus subtilis sporulation-specific class B penicillin-binding protein (PBP) SpoVD and many homologous PBPs contain two conserved cysteine residues of unknown function in the transpeptidase domain - one as residue x in the SxN catalytic site motif and the other in a flexible loop near the catalytic site. A disulfide bond between these residues blocks the function of SpoVD in cortex synthesis. With a combination of experiments with purified proteins and B. subtilis mutant cells, it was shown that in active SpoVD the two cysteine residues most probably interact by hydrogen bonding and that this is important for peptidoglycan synthesis in vivo. It was furthermore demonstrated that the sporulation-specific thiol-disulfide oxidoreductase StoA reduces SpoVD and that requirement of StoA for cortex synthesis can be suppressed by two completely different types of structural alterations in SpoVD. It is concluded that StoA plays a critical role mainly during maturation of SpoVD in the forespore outer membrane. The findings advance our understanding of essential PBPs and redox control of extra-cytoplasmic protein disulfides in bacterial cells. © 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.

Antibacterial Targets in Fatty Acid Biosynthesis

PubMed Central

Wright, H. Tonie; Reynolds, Kevin A.

2008-01-01

Summary The fatty acid biosynthesis pathway is an attractive but still largely unexploited target for development of new anti-bacterial agents. The extended use of the anti-tuberculosis drug isoniazid and the antiseptic triclosan, which are inhibitors of fatty acid biosynthesis, validates this pathway as a target for anti-bacterial development. Differences in subcellular organization of the bacterial and eukaryotic multi-enzyme fatty acid synthase systems offer the prospect of inhibitors with host vs. target specificity. Platensimycin, platencin, and phomallenic acids, newly discovered natural product inhibitors of the condensation steps in fatty acid biosynthesis, represent new classes of compounds with antibiotic potential. An almost complete catalogue of crystal structures for the enzymes of the type II fatty acid biosynthesis pathway can now be exploited in the rational design of new inhibitors, as well as the recently published crystal structures of type I FAS complexes. PMID:17707686

Homology modeling and docking analyses of M. leprae Mur ligases reveals the common binding residues for structure based drug designing to eradicate leprosy.

PubMed

Shanmugam, Anusuya; Natarajan, Jeyakumar

2012-06-01

Multi drug resistance capacity for Mycobacterium leprae (MDR-Mle) demands the profound need for developing new anti-leprosy drugs. Since most of the drugs target a single enzyme, mutation in the active site renders the antibiotic ineffective. However, structural and mechanistic information on essential bacterial enzymes in a pathway could lead to the development of antibiotics that targets multiple enzymes. Peptidoglycan is an important component of the cell wall of M. leprae. The biosynthesis of bacterial peptidoglycan represents important targets for the development of new antibacterial drugs. Biosynthesis of peptidoglycan is a multi-step process that involves four key Mur ligase enzymes: MurC (EC:6.3.2.8), MurD (EC:6.3.2.9), MurE (EC:6.3.2.13) and MurF (EC:6.3.2.10). Hence in our work, we modeled the three-dimensional structure of the above Mur ligases using homology modeling method and analyzed its common binding features. The residues playing an important role in the catalytic activity of each of the Mur enzymes were predicted by docking these Mur ligases with their substrates and ATP. The conserved sequence motifs significant for ATP binding were predicted as the probable residues for structure based drug designing. Overall, the study was successful in listing significant and common binding residues of Mur enzymes in peptidoglycan pathway for multi targeted therapy.

A peptidoglycan recognition protein from Sciaenops ocellatus is a zinc amidase and a bactericide with a substrate range limited to Gram-positive bacteria.

PubMed

Li, Mo-Fei; Zhang, Min; Wang, Chun-Lin; Sun, Li

2012-02-01

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRPs are highly conserved in invertebrates and vertebrates including fish. However, the biological function of teleost PGRP remains largely uninvestigated. In this study, we identified a PGRP homologue, SoPGLYRP-2, from red drum (Sciaenops ocellatus) and analyzed its activity and potential function. The deduced amino acid sequence of SoPGLYRP-2 is composed of 482 residues and shares 46-94% overall identities with known fish PGRPs. SoPGLYRP-2 contains at the C-terminus a single zinc amidase domain with conserved residues that form the catalytic site. Quantitative RT-PCR analysis detected SoPGLYRP-2 expression in multiple tissues, with the highest expression occurring in liver and the lowest expression occurring in brain. Experimental bacterial infection upregulated SoPGLYRP-2 expression in kidney, spleen, and liver in time-dependent manners. To examine the biological activity of SoPGLYRP-2, purified recombinant proteins representing the intact SoPGLYRP-2 (rSoPGLYRP-2) and the amidase domain (rSoPGLYRP-AD) were prepared from Escherichia coli. Subsequent analysis showed that rSoPGLYRP-2 and rSoPGLYRP-AD (i) exhibited comparable Zn(2+)-dependent peptidoglycan-lytic activity and were able to recognize and bind to live bacterial cells, (ii) possessed bactericidal effect against Gram-positive bacteria and slight bacteriostatic effect against Gram-negative bacteria, (iii) were able to block bacterial infection into host cells. These results indicate that SoPGLYRP-2 is a zinc-dependent amidase and a bactericide that targets preferentially at Gram-positive bacteria, and that SoPGLYRP-2 is likely to play a role in host innate immune defense during bacterial infection. Copyright © 2011 Elsevier Ltd. All rights reserved.

Peptidoglycan precursor from Fusobacterium nucleatum contains lanthionine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fredriksen, A.; Vasstrand, E.N.; Jensen, H.B.

1991-01-01

Fusobacterium nucleatum was grown in the presence of ({sup 14}C)UDP. By means of sequential precipitation and chromatographic separation of the cytoplasmic content, a peptidoglycan ({sup 14}C)UDP pentapeptide containing lanthionine was isolated. This finding indicates that lanthionine is synthesized and incorporated as such during the assembly of the peptidoglycan.

An Endogenous Nanomineral Chaperones Luminal Antigen and Peptidoglycan to Intestinal Immune Cells

PubMed Central

Powell, Jonathan J; Thomas-McKay, Emma; Thoree, Vinay; Robertson, Jack; Hewitt, Rachel E; Skepper, Jeremy N; Brown, Andy; Hernandez-Garrido, Juan Carlos; Midgley, Paul A; Gomez-Morilla, Inmaculada; Grime, Geoffrey W; Kirkby, Karen J; Mabbott, Neil A; Donaldson, David S; Williams, Ifor R; Rios, Daniel; Girardin, Stephen E; Haas, Carolin T; Bruggraber, Sylvaine FA; Laman, Jon D; Tanriver, Yakup; Lombardi, Giovanna; Lechler, Robert; Thompson, Richard P H; Pele, Laetitia C

2015-01-01

In humans and other mammals, it is known that calcium and phosphate ions are secreted from the distal small intestine into the lumen. However, why this secretion occurs is unclear. Here, we show that the process leads to the formation of amorphous magnesium-substituted calcium phosphate nanoparticles that trap soluble macromolecules, such as bacterial peptidoglycan and orally-fed protein antigens, in the lumen and transport them to immune cells of the intestinal tissue. The macromolecule-containing nanoparticles utilize epithelial M cells to enter Peyer’s patches - small areas of the intestine concentrated with particle-scavenging immune cells. In wild type mice, intestinal immune cells containing these naturally-formed nanoparticles expressed the immune tolerance-associated molecule ‘programmed death-ligand 1 (PD-L1)’, whereas in NOD1/2 double knock-out mice, which cannot recognize peptidoglycan, PD-L1 was undetected. Our results explain a role for constitutively formed calcium phosphate nanoparticles in the gut lumen and how this helps to shape intestinal immune homeostasis. PMID:25751305

Structure of Pneumococcal Peptidoglycan Hydrolase LytB Reveals Insights into the Bacterial Cell Wall Remodeling and Pathogenesis*

PubMed Central

Bai, Xiao-Hui; Chen, Hui-Jie; Jiang, Yong-Liang; Wen, Zhensong; Huang, Yubin; Cheng, Wang; Li, Qiong; Qi, Lei; Zhang, Jing-Ren; Chen, Yuxing; Zhou, Cong-Zhao

2014-01-01

Streptococcus pneumoniae causes a series of devastating infections in humans. Previous studies have shown that the endo-β-N-acetylglucosaminidase LytB is critical for pneumococcal cell division and nasal colonization, but the biochemical mechanism of LytB action remains unknown. Here we report the 1.65 Å crystal structure of the catalytic domain (residues Lys-375–Asp-658) of LytB (termed LytBCAT), excluding the choline binding domain. LytBCAT consists of three structurally independent modules: SH3b, WW, and GH73. These modules form a “T-shaped” pocket that accommodates a putative tetrasaccharide-pentapeptide substrate of peptidoglycan. Structural comparison and simulation revealed that the GH73 module of LytB harbors the active site, including the catalytic residue Glu-564. In vitro assays of hydrolytic activity indicated that LytB prefers the peptidoglycan from the lytB-deficient pneumococci, suggesting the existence of a specific substrate of LytB in the immature peptidoglycan. Combined with in vitro cell-dispersing and in vivo cell separation assays, we demonstrated that all three modules are necessary for the optimal activity of LytB. Further functional analysis showed that the full catalytic activity of LytB is required for pneumococcal adhesion to and invasion into human lung epithelial cells. Structure-based alignment indicated that the unique modular organization of LytB is highly conserved in its orthologs from Streptococcus mitis group and Gemella species. These findings provided structural insights into the pneumococcal cell wall remodeling and novel hints for the rational design of therapeutic agents against pneumococcal growth and thereby the related diseases. PMID:25002590

Structures of the Peptidoglycan N-Acetylglucosamine Deacetylase Bc1974 and Its Complexes with Zinc Metalloenzyme Inhibitors.

PubMed

Giastas, Petros; Andreou, Athena; Papakyriakou, Athanasios; Koutsioulis, Dimitris; Balomenou, Stavroula; Tzartos, Socrates J; Bouriotis, Vassilis; Eliopoulos, Elias E

2018-02-06

The cell wall peptidoglycan is recognized as a primary target of the innate immune system, and usually its disintegration results in bacterial lysis. Bacillus cereus, a close relative of the highly virulent Bacillus anthracis, contains 10 polysaccharide deacetylases. Among these, the peptidoglycan N-acetylglucosamine deacetylase Bc1974 is the highest homologue to the Bacillus anthracis Ba1977 that is required for full virulence and is involved in resistance to the host's lysozyme. These metalloenzymes belong to the carbohydrate esterase family 4 (CE4) and are attractive targets for the development of new anti-infective agents. Herein we report the first X-ray crystal structures of the NodB domain of Bc1974, the conserved catalytic core of CE4s, in the unliganded form and in complex with four known metalloenzyme inhibitors and two amino acid hydroxamates that target the active site metal. These structures revealed the presence of two conformational states of a catalytic loop known as motif-4 (MT4), which were not observed previously for peptidoglycan deacetylases, but were recently shown in the structure of a Vibrio clolerae chitin deacetylase. By employing molecular docking of a substrate model, we describe a catalytic mechanism that probably involves initial binding of the substrate in a receptive, more open state of MT4 and optimal catalytic activity in the closed state of MT4, consistent with the previous observations. The ligand-bound structures presented here, in addition to the five Bc1974 inhibitors identified, provide a valuable basis for the design of antibacterial agents that target the peptidoglycan deacetylase Ba1977.

Human SAP is a novel peptidoglycan recognition protein that induces complement- independent phagocytosis of Staphylococcus aureus

PubMed Central

An, Jang-Hyun; Kurokawa, Kenji; Jung, Dong-Jun; Kim, Min-Jung; Kim, Chan-Hee; Fujimoto, Yukari; Fukase, Koichi; Coggeshall, K. Mark; Lee, Bok Luel

2014-01-01

The human pathogen Staphylococcus aureus is responsible for many community-acquired and hospital-associated infections and is associated with high mortality. Concern over the emergence of multidrug-resistant strains has renewed interest in the elucidation of host mechanisms that defend against S. aureus infection. We recently demonstrated that human serum mannose-binding lectin (MBL) binds to S. aureus wall teichoic acid (WTA), a cell wall glycopolymer, a discovery that prompted further screening to identify additional serum proteins that recognize S. aureus cell wall components. In this report, we incubated human serum with 10 different S. aureus mutants and determined that serum amyloid P component (SAP) bound specifically to a WTA-deficient S. aureus ΔtagO mutant, but not to tagO-complemented, WTA-expressing cells. Biochemical characterization revealed that SAP recognizes bacterial peptidoglycan as a ligand and that WTA inhibits this interaction. Although SAP binding to peptidoglycan was not observed to induce complement activation, SAP-bound ΔtagO cells were phagocytosed by human polymorphonuclear leukocytes in an Fcγ receptor-dependent manner. These results indicate that SAP functions as a host defense factor, similar to other peptidoglycan recognition proteins and nucleotide-binding oligomerization domain (NOD)-like receptors. PMID:23966633

Crystallographic and Molecular Dynamics Analysis of Loop Motions Unmasking the Peptidoglycan-Binding Site in Stator Protein MotB of Flagellar Motor

PubMed Central

Nahar, Musammat F.; Buckle, Ashley M.; Roujeinikova, Anna

2011-01-01

Background The C-terminal domain of MotB (MotB-C) shows high sequence similarity to outer membrane protein A and related peptidoglycan (PG)-binding proteins. It is believed to anchor the power-generating MotA/MotB stator unit of the bacterial flagellar motor to the peptidoglycan layer of the cell wall. We previously reported the first crystal structure of this domain and made a puzzling observation that all conserved residues that are thought to be essential for PG recognition are buried and inaccessible in the crystal structure. In this study, we tested a hypothesis that peptidoglycan binding is preceded by, or accompanied by, some structural reorganization that exposes the key conserved residues. Methodology/Principal Findings We determined the structure of a new crystalline form (Form B) of Helicobacter pylori MotB-C. Comparisons with the existing Form A revealed conformational variations in the petal-like loops around the carbohydrate binding site near one end of the β-sheet. These variations are thought to reflect natural flexibility at this site required for insertion into the peptidoglycan mesh. In order to understand the nature of this flexibility we have performed molecular dynamics simulations of the MotB-C dimer. The results are consistent with the crystallographic data and provide evidence that the three loops move in a concerted fashion, exposing conserved MotB residues that have previously been implicated in binding of the peptide moiety of peptidoglycan. Conclusion/Significance Our structural analysis provides a new insight into the mechanism by which MotB inserts into the peptidoglycan mesh, thus anchoring the power-generating complex to the cell wall. PMID:21533052

Robust peptidoglycan growth by dynamic and variable multi-protein complexes.

PubMed

Pazos, Manuel; Peters, Katharina; Vollmer, Waldemar

2017-04-01

In Gram-negative bacteria such as Escherichia coli the peptidoglycan sacculus resides in the periplasm, a compartment that experiences changes in pH value, osmolality, ion strength and other parameters depending on the cell's environment. Hence, the cell needs robust peptidoglycan growth mechanisms to grow and divide under different conditions. Here we propose a model according to which the cell achieves robust peptidoglycan growth by employing dynamic multi-protein complexes, which assemble with variable composition from freely diffusing sets of peptidoglycan synthases, hydrolases and their regulators, whereby the composition of the active complexes depends on the cell cycle state - cell elongation or division - and the periplasmic growth conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

Biosynthesis of Bacterial Cellulose/Carboxylic Multi-Walled Carbon Nanotubes for Enzymatic Biofuel Cell Application

PubMed Central

Lv, Pengfei; Feng, Quan; Wang, Qingqing; Li, Guohui; Li, Dawei; Wei, Qufu

2016-01-01

Novel nanocomposites comprised of bacterial cellulose (BC) with carboxylic multi-walled carbon nanotubes (c-MWCNTs) incorporated into the BC matrix were prepared through a simple method of biosynthesis. The biocathode and bioanode for the enzyme biological fuel cell (EBFC) were prepared using BC/c-MWCNTs composite injected by laccase (Lac) and glucose oxidase (GOD) with the aid of glutaraldehyde (GA) crosslinking. Biosynthesis of BC/c-MWCNTs composite was characterized by digital photos, scanning electron microscope (SEM), and Fourier Transform Infrared (FTIR). The experimental results indicated the successful incorporation of c-MWCNTs into the BC. The electrochemical and biofuel performance were evaluated by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The power density and current density of EBFCs were recorded at 32.98 µW/cm3 and 0.29 mA/cm3, respectively. Additionally, the EBFCs also showed acceptable stability. Preliminary tests on double cells indicated that renewable BC have great potential in the application field of EBFCs. PMID:28773310

The Capsular Polysaccharide of Staphylococcus aureus Is Attached to Peptidoglycan by the LytR-CpsA-Psr (LCP) Family of Enzymes*

PubMed Central

Chan, Yvonne Gar-Yun; Kim, Hwan Keun; Schneewind, Olaf; Missiakas, Dominique

2014-01-01

Envelope biogenesis in bacteria involves synthesis of intermediates that are tethered to the lipid carrier undecaprenol-phosphate. LytR-CpsA-Psr (LCP) enzymes have been proposed to catalyze the transfer of undecaprenol-linked intermediates onto the C6-hydroxyl of MurNAc in peptidoglycan, thereby promoting attachment of wall teichoic acid (WTA) in bacilli and staphylococci and capsular polysaccharides (CPS) in streptococci. S. aureus encodes three lcp enzymes, and a variant lacking all three genes (Δlcp) releases WTA from the bacterial envelope and displays a growth defect. Here, we report that the type 5 capsular polysaccharide (CP5) of Staphylococcus aureus Newman is covalently attached to the glycan strands of peptidoglycan. Cell wall attachment of CP5 is abrogated in the Δlcp variant, a defect that is best complemented via expression of lcpC in trans. CP5 synthesis and peptidoglycan attachment are not impaired in the tagO mutant, suggesting that CP5 synthesis does not involve the GlcNAc-ManNAc linkage unit of WTA and may instead utilize another Wzy-type ligase to assemble undecaprenyl-phosphate intermediates. Thus, LCP enzymes of S. aureus are promiscuous enzymes that attach secondary cell wall polymers with discrete linkage units to peptidoglycan. PMID:24753256

Super-resolution microscopy reveals cell wall dynamics and peptidoglycan architecture in ovococcal bacteria.

PubMed

Wheeler, Richard; Mesnage, Stéphane; Boneca, Ivo G; Hobbs, Jamie K; Foster, Simon J

2011-12-01

Cell morphology and viability in Eubacteria is dictated by the architecture of peptidoglycan, the major and essential structural component of the cell wall. Although the biochemical composition of peptidoglycan is well understood, how the peptidoglycan architecture can accommodate the dynamics of growth and division while maintaining cell shape remains largely unknown. Here, we elucidate the peptidoglycan architecture and dynamics of bacteria with ovoid cell shape (ovococci), which includes a number of important pathogens, by combining biochemical analyses with atomic force and super-resolution microscopies. Atomic force microscopy analysis showed preferential orientation of the peptidoglycan network parallel to the short axis of the cell, with distinct architectural features associated with septal and peripheral wall synthesis. Super-resolution three-dimensional structured illumination fluorescence microscopy was applied for the first time in bacteria to unravel the dynamics of peptidoglycan assembly in ovococci. The ovococci have a unique peptidoglycan architecture and growth mode not observed in other model organisms. © 2011 Blackwell Publishing Ltd.

Escherichia coli peptidoglycan structure and mechanics as predicted by atomic-scale simulations.

PubMed

Gumbart, James C; Beeby, Morgan; Jensen, Grant J; Roux, Benoît

2014-02-01

Bacteria face the challenging requirement to maintain their shape and avoid rupture due to the high internal turgor pressure, but simultaneously permit the import and export of nutrients, chemical signals, and virulence factors. The bacterial cell wall, a mesh-like structure composed of cross-linked strands of peptidoglycan, fulfills both needs by being semi-rigid, yet sufficiently porous to allow diffusion through it. How the mechanical properties of the cell wall are determined by the molecular features and the spatial arrangement of the relatively thin strands in the larger cellular-scale structure is not known. To examine this issue, we have developed and simulated atomic-scale models of Escherichia coli cell walls in a disordered circumferential arrangement. The cell-wall models are found to possess an anisotropic elasticity, as known experimentally, arising from the orthogonal orientation of the glycan strands and of the peptide cross-links. Other features such as thickness, pore size, and disorder are also found to generally agree with experiments, further supporting the disordered circumferential model of peptidoglycan. The validated constructs illustrate how mesoscopic structure and behavior emerge naturally from the underlying atomic-scale properties and, furthermore, demonstrate the ability of all-atom simulations to reproduce a range of macroscopic observables for extended polymer meshes.

Phylogenetic mapping of bacterial morphology

NASA Technical Reports Server (NTRS)

Siefert, J. L.; Fox, G. E.

1998-01-01

The availability of a meaningful molecular phylogeny for bacteria provides a context for examining the historical significance of various developments in bacterial evolution. Herein, the classical morphological descriptions of selected members of the domain Bacteria are mapped upon the genealogical ancestry deduced from comparison of small-subunit rRNA sequences. For the species examined in this study, a distinct pattern emerges which indicates that the coccus shape has arisen and accumulated independently multiple times in separate lineages and typically survived as a persistent end-state morphology. At least two other morphologies persist but have evolved only once. This study demonstrates that although bacterial morphology is not useful in defining bacterial phylogeny, it is remarkably consistent with that phylogeny once it is known. An examination of the experimental evidence available for morphogenesis as well as microbial fossil evidence corroborates these findings. It is proposed that the accumulation of persistent morphologies is a result of the biophysical properties of peptidoglycan and their genetic control, and that an evolved body-plan strategy based on peptidoglycan may have been a fate-sealing step in the evolution of Bacteria. More generally, this study illustrates that significant evolutionary insights can be obtained by examining biological and biochemical data in the context of a reliable phylogenetic structure.

Targeting the permeability barrier and peptidoglycan recycling pathways to disarm Pseudomonas aeruginosa against the innate immune system

PubMed Central

Moya, Bartolomé; Munar-Bestard, Marta; Zamorano, Laura; Cabot, Gabriel; Blázquez, Jesús; Ayala, Juan A.; Oliver, Antonio

2017-01-01

Antimicrobial resistance is a continuously increasing threat that severely compromises our antibiotic arsenal and causes thousands of deaths due to hospital-acquired infections by pathogens such as Pseudomonas aeruginosa, situation further aggravated by the limited development of new antibiotics. Thus, alternative strategies such as those targeting bacterial resistance mechanisms, virulence or potentiating the activity of our immune system resources are urgently needed. We have recently shown that mutations simultaneously causing the peptidoglycan recycling blockage and the β-lactamase AmpC overexpression impair the virulence of P.aeruginosa. These findings suggested that peptidoglycan metabolism might be a good target not only for fighting antibiotic resistance, but also for the attenuation of virulence and/or potentiation of our innate immune weapons. Here we analyzed the activity of the innate immune elements peptidoglycan recognition proteins (PGRPs) and lysozyme against P. aeruginosa. We show that while lysozyme and PGRPs have a very modest basal effect over P. aeruginosa, their bactericidal activity is dramatically increased in the presence of subinhibitory concentrations of the permeabilizing agent colistin. We also show that the P. aeruginosa lysozyme inhibitors seem to play a very residual protective role even in permeabilizing conditions. In contrast, we demonstrate that, once the permeability barrier is overpassed, the activity of lysozyme and PGRPs is dramatically enhanced when inhibiting key peptidoglycan recycling components (such as the 3 AmpDs, AmpG or NagZ), indicating a decisive protective role for cell-wall recycling and that direct peptidoglycan-binding supports, at least partially, the activity of these enzymes. Finally, we show that recycling blockade when occurring simultaneously with AmpC overexpression determines a further decrease in the resistance against PGRP2 and lysozyme, linked to quantitative changes in the cell-wall. Thus, our

Biosynthesis of Sulfur-Containing tRNA Modifications: A Comparison of Bacterial, Archaeal, and Eukaryotic Pathways

PubMed Central

Čavužić, Mirela; Liu, Yuchen

2017-01-01

Post-translational tRNA modifications have very broad diversity and are present in all domains of life. They are important for proper tRNA functions. In this review, we emphasize the recent advances on the biosynthesis of sulfur-containing tRNA nucleosides including the 2-thiouridine (s2U) derivatives, 4-thiouridine (s4U), 2-thiocytidine (s2C), and 2-methylthioadenosine (ms2A). Their biosynthetic pathways have two major types depending on the requirement of iron–sulfur (Fe–S) clusters. In all cases, the first step in bacteria and eukaryotes is to activate the sulfur atom of free l-cysteine by cysteine desulfurases, generating a persulfide (R-S-SH) group. In some archaea, a cysteine desulfurase is missing. The following steps of the bacterial s2U and s4U formation are Fe–S cluster independent, and the activated sulfur is transferred by persulfide-carrier proteins. By contrast, the biosynthesis of bacterial s2C and ms2A require Fe–S cluster dependent enzymes. A recent study shows that the archaeal s4U synthetase (ThiI) and the eukaryotic cytosolic 2-thiouridine synthetase (Ncs6) are Fe–S enzymes; this expands the role of Fe–S enzymes in tRNA thiolation to the Archaea and Eukarya domains. The detailed reaction mechanisms of Fe–S cluster depend s2U and s4U formation await further investigations. PMID:28287455

Interactions between late acting proteins required for peptidoglycan synthesis during sporulation

PubMed Central

Fay, Allison; Meyer, Pablo; Dworkin, Jonathan

2010-01-01

The requirement of peptidoglycan synthesis for growth complicates the analysis of interactions between proteins involved in this pathway. In particular, the later steps that involve membrane-linked substrates have proven largely recalcitrant to in vivo analysis. Here we have taken advantage of the peptidoglycan synthesis that occurs during sporulation in Bacillus subtilis to examine the interactions between SpoVE, a non-essential, sporulation-specific homolog of the well-conserved and essential SEDS proteins, and SpoVD, a non-essential class B penicillin binding protein (PBP). We found that localization of SpoVD is dependent on SpoVE and that SpoVD protects SpoVE from in vivo proteolysis. Co-immunoprecipitations and Fluorescence Resonance Energy Transfer experiments indicated that SpoVE and SpoVD interact and co-affinity purification in E. coli demonstrated that this interaction is direct. Finally, we generated a functional protein consisting of a SpoVE-SpoVD fusion and found that a loss-of-function point mutation in either part of the fusion resulted in a loss of function of the entire fusion that was not complemented by a wild type protein. Thus, SpoVE has a direct and functional interaction with SpoVD and this conclusion will facilitate understanding the essential function SpoVE and related SEDS proteins such as FtsW and RodA play in bacterial growth and division. PMID:20417640

Biosynthesis of Bacterial Cell Walls.

DTIC Science & Technology

amino acid accumulation level in Lactobacillus plantarum and Streptococcus faecalis despite a normal initial transport rate. In the course of these...biosynthesis of a dipeptide, D-alanylcysteine; A demonstration that a pantothenic acid deficiency renders L. plantarum unusually sensitive to lysozyme digestion...A quantitative description of the lipid constituents of S. faecalis and L. plantarum ; An investigation of the biochemical basis of a marked lytic

Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC).

PubMed

Pazos, Manuel; Otten, Christian; Vollmer, Waldemar

2018-03-20

Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by 'attacking' enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius . The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II.

[Alteration of cholera toxin biosynthesis in Vibrio cholerae 01 as a result of temperate phage 139 integration into bacterial chromosome].

PubMed

Eroshenko, G A; Smirnova, N I

2002-01-01

Infection of V. cholerae 01 (classical and eltor biovars) cells with the temperate cholera phage 139 derived from V. cholerae serogroup 0139 followed by integration of the phage genome into the bacterial chromosome significantly increased the production of cholera toxin, the main virulence factor. The level of toxin biosynthesis in the lysogenic V. cholerae classical strain increased 3-fold and that in V. eltor thirty times in comparison with the parental strains. Increased production of cholera toxin was not associated with an increase in the number of copies of genes involved in its biosynthesis but seemed to be due to changes in toxinogenesis regulation.

A new metabolic cell wall labeling method reveals peptidoglycan in Chlamydia trachomatis

PubMed Central

Liechti, G.; Kuru, E.; Hall, E.; Kalinda, A.; Brun, Y. V.; VanNieuwenhze, M.; Maurelli, A. T.

2014-01-01

Peptidoglycan (PG), an essential structure in the cell walls of the vast majority of bacteria, is critical for division and maintaining cell shape and hydrostatic pressure1. Bacteria comprising the Chlamydiales were thought to be one of the few exceptions. Chlamydia encodes genes for PG biosynthesis2–7 and exhibits susceptibility to "anti-PG" antibiotics8,9, yet attempts to detect PG in any chlamydial species have proven unsuccessful (the ‘chlamydial anomaly’10). We employed a novel approach to metabolically label chlamydial PG using D-amino acid dipeptide probes and click chemistry. Replicating Chlamydia trachomatis was labeled with the probes throughout its biphasic, developmental life cycle, and differential probe incorporation experiments conducted in the presence of ampicillin is consistent with the presence of chlamydial PG modifying enzymes. These findings culminate 50 years of speculation and debate concerning the chlamydial anomaly and are the strongest evidence to date that chlamydial species possess functional PG. PMID:24336210

The Role of Bacterial Protein Tyrosine Phosphatases in the Regulation of the Biosynthesis of Secreted Polysaccharides

PubMed Central

Morona, Renato

2014-01-01

Abstract Significance: Tyrosine phosphorylation and associated protein tyrosine phosphatases are gaining prominence as critical mechanisms in the regulation of fundamental processes in a wide variety of bacteria. In particular, these phosphatases have been associated with the control of the biosynthesis of capsular polysaccharides and extracellular polysaccharides, critically important virulence factors for bacteria. Recent Advances: Deletion and overexpression of the phosphatases result in altered polysaccharide biosynthesis in a range of bacteria. The recent structures of associated auto-phosphorylating tyrosine kinases have suggested that the phosphatases may be critical for the cycling of the kinases between monomers and higher order oligomers. Critical Issues: Additional substrates of the phosphatases apart from cognate kinases are currently being identified. These are likely to be critical to our understanding of the mechanism by which polysaccharide biosynthesis is regulated. Future Directions: Ultimately, these protein tyrosine phosphatases are an attractive target for the development of novel antimicrobials. This is particularly the case for the polymerase and histidinol phosphatase family, which is predominantly found in bacteria. Furthermore, the determination of bacterial tyrosine phosphoproteomes will likely help to uncover the fundamental roles, mechanism, and critical importance of these phosphatases in a wide range of bacteria. Antioxid. Redox Signal. 20, 2274–2289. PMID:24295407

Synthesis of avibactam derivatives and activity on β-lactamases and peptidoglycan biosynthesis enzymes of mycobacteria.

PubMed

Edoo, Zainab; Iannazzo, Laura; Compain, Fabrice; Li de la Sierra Gallay, Inès; van Tilbeurgh, Herman; Fonvielle, Matthieu; Bouchet, Flavie; Le Run, Eva; Mainardi, Jean-Luc; Arthur, Michel; Ethève-Quelquejeu, Mélanie; Hugonnet, Jean-Emmanuel

2018-03-30

There is a renewed interest for β-lactams for treating infections due to Mycobacterium tuberculosis and M. abscessus since their β-lactamases are inhibited by classical (clavulanate) or new generation (avibactam) inhibitors, respectively. Here, we report access to an azido derivative of the diazabicyclooctane (DBO) scaffold of avibactam for functionalization by the Huisgen-Sharpless cycloaddition reaction. The amoxicillin-DBO combinations were active indicating that the triazole ring is compatible with drug penetration (minimal inhibitory concentration of 16 µg/ml for both species). Mechanistically, β-lactamase inhibition was not sufficient to account for the potentiation of amoxicillin by DBOs. Thus, we investigated the latter compounds as inhibitors of L,D-transpeptidases (LDTs), which are the main peptidoglycan polymerases in mycobacteria. The DBOs acted as slow-binding inhibitors of LDTs by S-carbamoylation indicating that optimization of DBOs for LDT inhibition is an attractive strategy to obtain drugs selectively active on mycobacteria. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Functional characterization of a short peptidoglycan recognition protein, PGRP5 in grass carp Ctenopharyngodon idella.

PubMed

Li, Jun Hua; Chang, Ming Xian; Xue, Na Na; Nie, P

2013-08-01

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from insects to mammals, recognize bacterial peptidoglycan (PGN) and function in antibacterial innate immunity. In this study, a short-form PGRP, designated as gcPGRP5 was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP5 is composed of 180 residues with a conserved PGRP domain at the C-terminus. The gcPGRP5 gene consists of four exons and three introns, spacing approximately 2.3 kb in genomic sequence. Phylogenetic analysis demonstrated that the gcPGRP5 is clustered with other PGRP-S identified in teleost fish. The gcPGRP5 is constitutively expressed in all organs/tissues examined, and its expression was significantly induced in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and PGN. Fluorescence analysis showed that gcPGRP5 is distributed in cytoplasm of CIK cells, and cell lysates from CIK cells transfected with pTurbo-gcPGRP5-GFP and ptGFP1-gcPGRP5 plasmids display the binding activity and peptidoglycan-lytic amidase activity toward Lys-PGN from Staphylococcus aureus and Dap-PGN from Bacillus subtilis. Furthermore, heat-shock protein70 (Hsp70), and MyD88, an adaptor molecule in Toll-like receptor pathway, had an increased expression in CIK cells overexpressed with gcPGRP5. It is thus indicated that gcPGRP5 exhibits amidase activity, and also possesses roles in anti-stress, and in Toll-like receptor signaling pathway. Copyright © 2013 Elsevier Ltd. All rights reserved.

Cell wall elongation mode in Gram-negative bacteria is determined by peptidoglycan architecture.

PubMed

Turner, Robert D; Hurd, Alexander F; Cadby, Ashley; Hobbs, Jamie K; Foster, Simon J

2013-01-01

Cellular integrity and morphology of most bacteria is maintained by cell wall peptidoglycan, the target of antibiotics essential in modern healthcare. It consists of glycan strands, cross-linked by peptides, whose arrangement determines cell shape, prevents lysis due to turgor pressure and yet remains dynamic to allow insertion of new material, and hence growth. The cellular architecture and insertion pattern of peptidoglycan have remained elusive. Here we determine the peptidoglycan architecture and dynamics during growth in rod-shaped Gram-negative bacteria. Peptidoglycan is made up of circumferentially oriented bands of material interspersed with a more porous network. Super-resolution fluorescence microscopy reveals an unexpected discontinuous, patchy synthesis pattern. We present a consolidated model of growth via architecture-regulated insertion, where we propose only the more porous regions of the peptidoglycan network that are permissive for synthesis.

Invariant amino acids in the Mur peptide synthetases of bacterial peptidoglycan synthesis and their modification by site-directed mutagenesis in the UDP-MurNAc:L-alanine ligase from Escherichia coli.

PubMed

Bouhss, A; Mengin-Lecreulx, D; Blanot, D; van Heijenoort, J; Parquet, C

1997-09-30

The comparison of the amino acid sequences of 20 cytoplasmic peptidoglycan synthetases (MurC, MurD, MurE, MurF, and Mpl) from various bacterial organisms has allowed us to detect common invariants: seven amino acids and the ATP-binding consensus sequence GXXGKT/S all at the same position in the alignment. The Mur synthetases thus appeared as a well-defined class of closely functionally related proteins. The conservation of a constant backbone length between certain invariants suggested common structural motifs. Among the other enzymes catalyzing a peptide bond formation driven by ATP hydrolysis to ADP and Pi, only folylpoly-gamma-l-glutamate synthetases presented the same common conserved amino acid residues, except for the most N-terminal invariant D50. Site-directed mutageneses were carried out to replace the K130, E174, H199, N293, N296, R327, and D351 residues by alanine in the MurC protein from Escherichia coli taken as model. For this purpose, plasmid pAM1005 was used as template, MurC being highly overproduced in this genetic setting. Analysis of the Vmax values of the mutated proteins suggested that residues K130, E174, and D351 are essential for the catalytic process whereas residues H199, N293, N296, and R327 were not. Mutations K130A, H199A, N293A, N296A, and R327A led to important variations of the Km values for one or more substrates, thereby indicating that these residues are involved in the structure of the active site and suggesting that the binding order of the substrates could be ATP, UDP-MurNAc, and alanine. The various mutated murC plasmids were tested for their effects on the growth, cell morphology, and peptidoglycan cell content of a murC thermosensitive strain at 42 degrees C. The observed effects (complementation, altered morphology, and reduced peptidoglycan content) paralleled more or less the decreased values of the MurC activity of each mutant.

Staphylococcus aureus Peptidoglycan Stem Packing by Rotational-Echo Double Resonance NMR Spectroscopy

PubMed Central

Kim, Sung Joon; Singh, Manmilan; Preobrazhenskaya, Maria; Schaefer, Jacob

2013-01-01

Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with D-[1-13C]alanine and L-[15N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between 13C and 15N of labeled amino acids incorporated in the peptidoglycan, and from those labels to 19F of a glycopeptide drug specifically bound to the peptidoglycan. The observed 13C-15N average distance of 4.1 to 4.4 Å between D- and L-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus. PMID:23617832

Bacterial Cell Wall Precursor Phosphatase Assays Using Thin-layer Chromatography (TLC) and High Pressure Liquid Chromatography (HPLC)

PubMed Central

Pazos, Manuel; Otten, Christian; Vollmer, Waldemar

2018-01-01

Peptidoglycan encases the bacterial cytoplasmic membrane to protect the cell from lysis due to the turgor. The final steps of peptidoglycan synthesis require a membrane-anchored substrate called lipid II, in which the peptidoglycan subunit is linked to the carrier lipid undecaprenol via a pyrophosphate moiety. Lipid II is the target of glycopeptide antibiotics and several antimicrobial peptides, and is degraded by ‘attacking’ enzymes involved in bacterial competition to induce lysis. Here we describe two protocols using thin-layer chromatography (TLC) and high pressure liquid chromatography (HPLC), respectively, to assay the digestion of lipid II by phosphatases such as Colicin M or the LXG toxin protein TelC from Streptococcus intermedius. The TLC method can also monitor the digestion of undecaprenyl (pyro)phosphate, whereas the HPLC method allows to separate the di-, mono- or unphosphorylated disaccharide pentapeptide products of lipid II. PMID:29651453

Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales

PubMed Central

Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole; Dubost, Lionel; Marie, Arul; Shikura, Noriyasu; Mainardi, Jean-Luc; Rice, Louis B.

2014-01-01

Synthesis of peptidoglycan precursors ending in d-lactate (d-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by l,d-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in d-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of d-Lac into cytoplasmic precursors. This was due to a d,d-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of d-Lac for d-Ala and Gly. The contribution of l,d-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-d,d-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal d-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by d,d-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of l,d-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-d,d-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that l,d-transpeptidases merely act as a tolerance mechanism in this bacterium. PMID:24395229

Peptidoglycan cross-linking in glycopeptide-resistant Actinomycetales.

PubMed

Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole; Dubost, Lionel; Marie, Arul; Shikura, Noriyasu; Mainardi, Jean-Luc; Rice, Louis B; Arthur, Michel

2014-01-01

Synthesis of peptidoglycan precursors ending in D-lactate (D-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by L,D-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in D-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of D-Lac into cytoplasmic precursors. This was due to a D,D-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of D-Lac for D-Ala and Gly. The contribution of L,D-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-D,D-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal D-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by D,D-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of L,D-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-D,D-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that L,D-transpeptidases merely act as a tolerance mechanism in this bacterium.

Circumferential gap propagation in an anisotropic elastic bacterial sacculus

NASA Astrophysics Data System (ADS)

Taneja, Swadhin; Levitan, Benjamin A.; Rutenberg, Andrew D.

2014-01-01

We have modeled stress concentration around small gaps in anisotropic elastic sheets, corresponding to the peptidoglycan sacculus of bacterial cells, under loading corresponding to the effects of turgor pressure in rod-shaped bacteria. We find that under normal conditions the stress concentration is insufficient to mechanically rupture bacteria, even for gaps up to a micron in length. We then explored the effects of stress-dependent smart autolysins, as hypothesized by A. L. Koch [Adv. Microb. Physiol. 24, 301 (1983), 10.1016/S0065-2911(08)60388-4; Res. Microbiol. 141, 529 (1990), 10.1016/0923-2508(90)90017-K]. We show that the measured anisotropic elasticity of the peptidoglycan (PG) sacculus can lead to stable circumferential propagation of small gaps in the sacculus. This is consistent with the recent observation of circumferential propagation of PG-associated MreB patches in rod-shaped bacteria. We also find a bistable regime of both circumferential and axial gap propagation, which agrees with behavior reported in cytoskeletal mutants of B. subtilis. We conclude that the elastic anisotropies of a bacterial sacculus, as characterized experimentally, may be relevant for maintaining rod-shaped bacterial growth.

Entrapment of peptidoglycans and adamantyltripeptides into liposomes: an HPLC assay for determination of encapsulation efficiency.

PubMed

Frkanec, Ruza; Travas, Dijana; Krstanović, Marina; Spoljar, Beata Halassy; Ljevaković, Durdica; Vranesić, Branka; Frkanec, Leo; Tomasić, Jelka

2003-11-01

The encapsulation of different immunomodulating peptides, the peptidoglycan monomer, its semisynthetic derivatives (Adamant-1-yl)-acetyl-peptidoglycan monomer and Boc-Tyr-peptidoglycan monomer, respectively, and of two diastereoisomers of adamantyltripeptides into the large negatively charged multilamellar liposomes was investigated. The reproducible quantitative method using HPLC was established for the determination of the entrapped compounds. It was shown that the tested compounds could be efficiently incorporated into liposomes using either the film or modified film method. The results confirmed that the peptidoglycans with lipophilic substituents and particularly the adamantyltripeptides were incorporated into liposomes with higher efficiency than the peptidoglycan monomer using either of the described methods. Liposome preparations were stable at 4 degrees C up to seven days as shown by minimal leaking of the entrapped material.

Peptidoglycan architecture can specify division planes in Staphylococcus aureus.

PubMed

Turner, Robert D; Ratcliffe, Emma C; Wheeler, Richard; Golestanian, Ramin; Hobbs, Jamie K; Foster, Simon J

2010-06-15

Division in Staphylococci occurs equatorially and on specific sequentially orthogonal planes in three dimensions, resulting, after incomplete cell separation, in the 'bunch of grapes' cluster organization that defines the genus. The shape of Staphylococci is principally maintained by peptidoglycan. In this study, we use Atomic Force Microscopy (AFM) and fluorescence microscopy with vancomycin labelling to examine purified peptidoglycan architecture and its dynamics in Staphylococcus aureus and correlate these with the cell cycle. At the presumptive septum, cells were found to form a large belt of peptidoglycan in the division plane before the centripetal formation of the septal disc; this often had a 'piecrust' texture. After division, the structures remain as orthogonal ribs, encoding the location of past division planes in the cell wall. We propose that this epigenetic information is used to enable S. aureus to divide in sequentially orthogonal planes, explaining how a spherical organism can maintain division plane localization with fidelity over many generations.

The Disruptive Effect of Lysozyme on the Bacterial Cell Wall Explored by an "In-Silico" Structural Outlook

ERIC Educational Resources Information Center

Primo, Emiliano D.; Otero, Lisandro H.; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter

2018-01-01

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall…

Structure-Function Analysis of Staphylococcus aureus Amidase Reveals the Determinants of Peptidoglycan Recognition and Cleavage*

PubMed Central

Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

2014-01-01

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen. PMID:24599952

Structure-function analysis of Staphylococcus aureus amidase reveals the determinants of peptidoglycan recognition and cleavage.

PubMed

Büttner, Felix Michael; Zoll, Sebastian; Nega, Mulugeta; Götz, Friedrich; Stehle, Thilo

2014-04-18

The bifunctional major autolysin AtlA of Staphylococcus aureus cleaves the bacterium's peptidoglycan network (PGN) at two distinct sites during cell division. Deletion of the enzyme results in large cell clusters with disordered division patterns, indicating that AtlA could be a promising target for the development of new antibiotics. One of the two functions of AtlA is performed by the N-acetylmuramyl-l-alanine amidase AmiA, which cleaves the bond between the carbohydrate and the peptide moieties of PGN. To establish the structural requirements of PGN recognition and the enzymatic mechanism of cleavage, we solved the crystal structure of the catalytic domain of AmiA (AmiA-cat) in complex with a peptidoglycan-derived ligand at 1.55 Å resolution. The peptide stem is clearly visible in the structure, forming extensive contacts with protein residues by docking into an elongated groove. Less well defined electron density and the analysis of surface features indicate likely positions of the carbohydrate backbone and the pentaglycine bridge. Substrate specificity analysis supports the importance of the pentaglycine bridge for fitting into the binding cleft of AmiA-cat. PGN of S. aureus with l-lysine tethered with d-alanine via a pentaglycine bridge is completely hydrolyzed, whereas PGN of Bacillus subtilis with meso-diaminopimelic acid directly tethered with d-alanine is not hydrolyzed. An active site mutant, H370A, of AmiA-cat was completely inactive, providing further support for the proposed catalytic mechanism of AmiA. The structure reported here is not only the first of any bacterial amidase in which both the PGN component and the water molecule that carries out the nucleophilic attack on the carbonyl carbon of the scissile bond are present; it is also the first peptidoglycan amidase complex structure of an important human pathogen.

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate

PubMed Central

Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K.; Liu, Peter; Pantua, Homer; Abbas, Alexander R.; Nickerson, Nicholas N.; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min

2017-01-01

ABSTRACT Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli. Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro, the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro. The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo. Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for “group 2” capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. PMID:28536290

Genes Sufficient for Synthesizing Peptidoglycan are Retained in Gymnosperm Genomes, and MurE from Larix gmelinii can Rescue the Albino Phenotype of Arabidopsis MurE Mutation.

PubMed

Lin, Xiaofei; Li, Ningning; Kudo, Hiromi; Zhang, Zhe; Li, Jinyu; Wang, Li; Zhang, Wenbo; Takechi, Katsuaki; Takano, Hiroyoshi

2017-03-01

The endosymbiotic theory states that plastids are derived from a single cyanobacterial ancestor that possessed a cell wall. Peptidoglycan (PG), the main component of the bacteria cell wall, gradually degraded during plastid evolution. PG-synthesizing Mur genes have been found to be retained in the genomes of basal streptophyte plants, although many of them have been lost from the genomes of angiosperms. The enzyme encoded by bacterial MurE genes catalyzes the formation of the UDP-N-acetylmuramic acid (UDP-MurNAc) tripeptide in bacterial PG biosynthesis. Knockout of the MurE gene in the moss Physcomitrella patens resulted in defects of chloroplast division, whereas T-DNA-tagged mutants of Arabidopsis thaliana for MurE revealed inhibition of chloroplast development but not of plastid division, suggesting that AtMurE is functionally divergent from the bacterial and moss MurE proteins. Here, we could identify 10 homologs of bacterial Mur genes, including MurE, in the recently sequenced genomes of Picea abies and Pinus taeda, suggesting the retention of the plastid PG system in gymnosperms. To investigate the function of gymnosperm MurE, we isolated an ortholog of MurE from the larch, Larix gmelinii (LgMurE) and confirmed its presence as a single copy per genome, as well as its abundant expression in the leaves of larch seedlings. Analysis with a fusion protein combining green fluorescent protein and LgMurE suggested that it localizes in chloroplasts. Cross-species complementation assay with MurE mutants of A. thaliana and P. patens showed that the expression of LgMurE cDNA completely rescued the albefaction defects in A. thaliana but did not rescue the macrochloroplast phenotype in P. patens. The evolution of plastid PG and the mechanism behind the functional divergence of MurE genes are discussed in the context of information about plant genomes at different evolutionary stages. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of

Genome wide transcriptional profiling of Herbaspirillum seropedicae SmR1 grown in the presence of naringenin.

PubMed

Tadra-Sfeir, Michelle Z; Faoro, Helisson; Camilios-Neto, Doumit; Brusamarello-Santos, Liziane; Balsanelli, Eduardo; Weiss, Vinicius; Baura, Valter A; Wassem, Roseli; Cruz, Leonardo M; De Oliveira Pedrosa, Fábio; Souza, Emanuel M; Monteiro, Rose A

2015-01-01

Herbaspirillum seropedicae is a diazotrophic bacterium which associates endophytically with economically important gramineae. Flavonoids such as naringenin have been shown to have an effect on the interaction between H. seropedicae and its host plants. We used a high-throughput sequencing based method (RNA-Seq) to access the influence of naringenin on the whole transcriptome profile of H. seropedicae. Three hundred and four genes were downregulated and seventy seven were upregulated by naringenin. Data analysis revealed that genes related to bacterial flagella biosynthesis, chemotaxis and biosynthesis of peptidoglycan were repressed by naringenin. Moreover, genes involved in aromatic metabolism and multidrug transport efllux were actived.

Bacterial RNA isolation.

PubMed

Ares, Manuel

2012-09-01

In this bacterial RNA isolation protocol, an "RNA-protective" treatment is followed by lysozyme digestion of the peptidoglycan component of the cell wall. EDTA promotes the loss of the outer membrane of Gram-negative bacteria and allows the lysozyme better access to the peptidoglycan. Cells begin to lyse during digestion in hypotonic lysozyme buffer and lysis is completed by sodium dodecyl sulfate (SDS) and hot phenol:chloroform:isoamyl alcohol (PCA) extraction. SDS and hot phenol disrupt membranes, denature protein (including RNase), and strip proteins from RNA. The separation of the organic phase from the aqueous phase is achieved using Phase Lock Gel, an inert material with a density intermediate between the organic and aqueous samples. The sample is split into three phases: from bottom to top, these are phenol and chloroform (organic phase), the inert gel with the interface material, and the aqueous phase with the RNA. The gel acts as a physical barrier between the sample and the organic phase plus interface. Following organic extraction, the RNA is concentrated by ethanol precipitation.

Peptidoglycan Association of Murein Lipoprotein Is Required for KpsD-Dependent Group 2 Capsular Polysaccharide Expression and Serum Resistance in a Uropathogenic Escherichia coli Isolate.

PubMed

Diao, Jingyu; Bouwman, Catrien; Yan, Donghong; Kang, Jing; Katakam, Anand K; Liu, Peter; Pantua, Homer; Abbas, Alexander R; Nickerson, Nicholas N; Austin, Cary; Reichelt, Mike; Sandoval, Wendy; Xu, Min; Whitfield, Chris; Kapadia, Sharookh B

2017-05-23

Murein lipoprotein (Lpp) and peptidoglycan-associated lipoprotein (Pal) are major outer membrane lipoproteins in Escherichia coli Their roles in cell-envelope integrity have been documented in E. coli laboratory strains, and while Lpp has been linked to serum resistance in vitro , the underlying mechanism has not been established. Here, lpp and pal mutants of uropathogenic E. coli strain CFT073 showed reduced survival in a mouse bacteremia model, but only the lpp mutant was sensitive to serum killing in vitro The peptidoglycan-bound Lpp form was specifically required for preventing complement-mediated bacterial lysis in vitro and complement-mediated clearance in vivo Compared to the wild-type strain, the lpp mutant had impaired K2 capsular polysaccharide production and was unable to respond to exposure to serum by elevating capsular polysaccharide amounts. These properties correlated with altered cellular distribution of KpsD, the predicted outer membrane translocon for "group 2" capsular polysaccharides. We identified a novel Lpp-dependent association between functional KpsD and peptidoglycan, highlighting important interplay between cell envelope components required for resistance to complement-mediated lysis in uropathogenic E. coli isolates. IMPORTANCE Uropathogenic E. coli (UPEC) isolates represent a significant cause of nosocomial urinary tract and bloodstream infections. Many UPEC isolates are resistant to serum killing. Here, we show that a major cell-envelope lipoprotein (murein lipoprotein) is required for serum resistance in vitro and for complement-mediated bacterial clearance in vivo This is mediated, in part, through a novel mechanism by which murein lipoprotein affects the proper assembly of a key component of the machinery involved in production of "group 2" capsules. The absence of murein lipoprotein results in impaired production of the capsule layer, a known participant in complement resistance. These results demonstrate an important role for

Modification of Helicobacter pylori peptidoglycan enhances NOD1 activation and promotes cancer of the stomach

DOE PAGES

Suarez, Giovanni; Romero-Gallo, Judith; Piazuelo, M. Blanca; ...

2015-03-02

Helicobacter pylori is the strongest known risk factor for gastric carcinogenesis. One cancer-linked locus is the cag pathogenicity island, which translocates components of peptidoglycan (PGN) into host cells. NOD1 is an intracellular immune receptor that senses PGN from Gram-negative bacteria and responds by inducing autophagy and activating NF-κB, leading to inflammation-mediated bacterial clearance; however chronic pathogens can evade NOD1-mediated clearance by altering PGN structure. We previously demonstrated that the H. pylori cag+ strain 7.13 rapidly induces gastric cancer in Mongolian gerbils. Using 2D-DIGE and mass spectrometry, we identified a novel mutation within the gene encoding the peptidoglycan deacetylase PgdA; therefore,more » we sought to define the role of H. pylori PgdA in NOD1-dependent activation of NF-κB, inflammation, and cancer. Co-culture of H. pylori strain 7.13 or its pgdA$-$ isogenic mutant with AGS gastric epithelial cells or HEK293 epithelial cells expressing a NF-κB reporter revealed that pgdA inactivation significantly decreased NOD1-dependent NF-κB activation and autophagy. Infection of Mongolian gerbils with an H. pylori pgdA$-$ mutant strain led to significantly decreased levels of inflammation and malignant lesions in the stomach; however, pre-activation of NOD1 prior to bacterial challenge reciprocally suppressed inflammation and cancer in response to wild-type H. pylori. Expression of NOD1 differs in human gastric cancer specimens compared to non-cancer samples harvested from the same patients. In conclusion, these results indicate that PGN deacetylation plays an important role in modulating host inflammatory responses to H. pylori, allowing the bacteria to persist and induce carcinogenic consequences in the gastric niche.« less

Glycopeptide antibiotic biosynthesis.

PubMed

Yim, Grace; Thaker, Maulik N; Koteva, Kalinka; Wright, Gerard

2014-01-01

Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new antibiotics into the market and the emergence of glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective antibiotic treatment difficult. We have now learned a great deal about how bacteria produce antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important antibiotics.

High-throughput, Highly Sensitive Analyses of Bacterial Morphogenesis Using Ultra Performance Liquid Chromatography*

PubMed Central

Desmarais, Samantha M.; Tropini, Carolina; Miguel, Amanda; Cava, Felipe; Monds, Russell D.; de Pedro, Miguel A.; Huang, Kerwyn Casey

2015-01-01

The bacterial cell wall is a network of glycan strands cross-linked by short peptides (peptidoglycan); it is responsible for the mechanical integrity of the cell and shape determination. Liquid chromatography can be used to measure the abundance of the muropeptide subunits composing the cell wall. Characteristics such as the degree of cross-linking and average glycan strand length are known to vary across species. However, a systematic comparison among strains of a given species has yet to be undertaken, making it difficult to assess the origins of variability in peptidoglycan composition. We present a protocol for muropeptide analysis using ultra performance liquid chromatography (UPLC) and demonstrate that UPLC achieves resolution comparable with that of HPLC while requiring orders of magnitude less injection volume and a fraction of the elution time. We also developed a software platform to automate the identification and quantification of chromatographic peaks, which we demonstrate has improved accuracy relative to other software. This combined experimental and computational methodology revealed that peptidoglycan composition was approximately maintained across strains from three Gram-negative species despite taxonomical and morphological differences. Peptidoglycan composition and density were maintained after we systematically altered cell size in Escherichia coli using the antibiotic A22, indicating that cell shape is largely decoupled from the biochemistry of peptidoglycan synthesis. High-throughput, sensitive UPLC combined with our automated software for chromatographic analysis will accelerate the discovery of peptidoglycan composition and the molecular mechanisms of cell wall structure determination. PMID:26468288

Renew or die: The molecular mechanisms of peptidoglycan recycling and antibiotic resistance in Gram-negative pathogens.

PubMed

Domínguez-Gil, Teresa; Molina, Rafael; Alcorlo, Martín; Hermoso, Juan A

2016-09-01

Antimicrobial resistance is one of the most serious health threats. Cell-wall remodeling processes are tightly regulated to warrant bacterial survival and in some cases are directly linked to antibiotic resistance. Remodeling produces cell-wall fragments that are recycled but can also act as messengers for bacterial communication, as effector molecules in immune response and as signaling molecules triggering antibiotic resistance. This review is intended to provide state-of-the-art information about the molecular mechanisms governing this process and gather structural information of the different macromolecular machineries involved in peptidoglycan recycling in Gram-negative bacteria. The growing body of literature on the 3D structures of the corresponding macromolecules reveals an extraordinary complexity. Considering the increasing incidence and widespread emergence of Gram-negative multidrug-resistant pathogens in clinics, structural information on the main actors of the recycling process paves the way for designing novel antibiotics disrupting cellular communication in the recycling-resistance pathway. Copyright © 2016. Published by Elsevier Ltd.

Synthesis and biological evaluation of N-acylhydrazones as inhibitors of MurC and MurD ligases.

PubMed

Sink, Roman; Kovac, Andreja; Tomasić, Tihomir; Rupnik, Veronika; Boniface, Audrey; Bostock, Julieanne; Chopra, Ian; Blanot, Didier; Masic, Lucija Peterlin; Gobec, Stanislav; Zega, Anamarija

2008-09-01

The Mur ligases have an essential role in the intracellular biosynthesis of bacterial peptidoglycan, and they represent attractive targets for the design of novel antibacterials. A series of compounds with an N-acylhydrazone scaffold were synthesized and screened for inhibition of the MurC and MurD enzymes from Escherichia coli. Compounds with micromolar inhibitory activities against both MurC and MurD were identified, and some of them also showed antibacterial activity.

Analysis of five complete genome sequences for members of the class Peribacteria in the recently recognized Peregrinibacteria bacterial phylum

DOE PAGES

Anantharaman, Karthik; Brown, Christopher T.; Burstein, David; ...

2016-01-28

Five closely related populations of bacteria from the Candidate Phylum (CP) Peregrinibacteria, part of the bacterial Candidate Phyla Radiation (CPR), were sampled from filtered groundwater obtained from an aquifer adjacent to the Colorado River near the town of Rifle, CO, USA. Here, we present the first complete genome sequences for organisms from this phylum. These bacteria have small genomes and, unlike most organisms from other lineages in the CPR, have the capacity for nucleotide synthesis. They invest significantly in biosynthesis of cell wall and cell envelope components, including peptidoglycan, isoprenoids via the mevalonate pathway, and a variety of amino sugarsmore » including perosamine and rhamnose. The genomes encode an intriguing set of large extracellular proteins, some of which are very cysteine-rich and may function in attachment, possibly to other cells. Strain variation in these proteins is an important source of genotypic variety. Overall, the cell envelope features, combined with the lack of biosynthesis capacities for many required cofactors, fatty acids, and most amino acids point to a symbiotic lifestyle. Furthermore, phylogenetic analyses indicate that these bacteria likely represent a new class within the Peregrinibacteria phylum, although they ultimately may be recognized as members of a separate phylum. In conclusion, we propose the provisional taxonomic assignment as ‘ Candidatus Peribacter riflensis’, Genus Peribacter, Family Peribacteraceae, Order Peribacterales, Class Peribacteria in the phylum Peregrinibacteria.« less

Neither Lys- and DAP-type peptidoglycans stimulate mouse or human innate immune cells via Toll-like receptor 2

PubMed Central

Langer, Marybeth; Girton, Alanson W.; Popescu, Narcis I.; Burgett, Tarea; Metcalf, Jordan P.

2018-01-01

Peptidoglycan (PGN), a major component of bacterial cell walls, is a pathogen-associated molecular pattern (PAMP) that causes innate immune cells to produce inflammatory cytokines that escalate the host response during infection. In order to better understand the role of PGN in infection, we wanted to gain insight into the cellular receptor for PGN. Although the receptor was initially identified as Toll-like receptor 2 (TLR2), this receptor has remained controversial and other PGN receptors have been reported. We produced PGN from live cultures of Bacillus anthracis and Staphylococcus aureus and tested samples of PGN isolated during the purification process to determine at what point TLR2 activity was removed, if at all. Our results indicate that although live B. anthracis and S. aureus express abundant TLR2 ligands, highly-purified PGN from either bacterial source is not recognized by TLR2. PMID:29474374

Genome wide transcriptional profiling of Herbaspirillum seropedicae SmR1 grown in the presence of naringenin

PubMed Central

Tadra-Sfeir, Michelle Z.; Faoro, Helisson; Camilios-Neto, Doumit; Brusamarello-Santos, Liziane; Balsanelli, Eduardo; Weiss, Vinicius; Baura, Valter A.; Wassem, Roseli; Cruz, Leonardo M.; De Oliveira Pedrosa, Fábio; Souza, Emanuel M.; Monteiro, Rose A.

2015-01-01

Herbaspirillum seropedicae is a diazotrophic bacterium which associates endophytically with economically important gramineae. Flavonoids such as naringenin have been shown to have an effect on the interaction between H. seropedicae and its host plants. We used a high-throughput sequencing based method (RNA-Seq) to access the influence of naringenin on the whole transcriptome profile of H. seropedicae. Three hundred and four genes were downregulated and seventy seven were upregulated by naringenin. Data analysis revealed that genes related to bacterial flagella biosynthesis, chemotaxis and biosynthesis of peptidoglycan were repressed by naringenin. Moreover, genes involved in aromatic metabolism and multidrug transport efllux were actived. PMID:26052319

Peptidoglycan inhibits progesterone and androstenedione production in bovine ovarian theca cells.

PubMed

Magata, F; Horiuchi, M; Miyamoto, A; Shimizu, T

2014-08-01

Uterine bacterial infection perturbs uterine and ovarian functions in postpartum dairy cows. Peptidoglycan (PGN) produced by gram-positive bacteria has been shown to disrupt the ovarian function in ewes. The aim of this study was to determine the effect of PGN on steroid production in bovine theca cells at different stages of follicular development. Bovine theca cells isolated from pre- and post-selection ovarian follicles (<8.5mm and >8.5mm in diameter, respectively) were cultured in vitro and challenged with PGN. Steroid production was evaluated by measuring progesterone (P4) and androstenedione (A4) concentration in culture media after 48 h or 96 h of culture. Bovine theca cells expressed PGN receptors including Toll-like receptor 2 and nucleotide-binding oligomerization domain 1 and 2. Treatment with PGN (1, 10, or 50 μg/ml) led to a decrease in P4 and A4 production by theca cells in both pre- and post-selection follicles. The mRNA expression of steroidogenic enzymes were decreased by PGN treatment. Moreover, A4 production was further suppressed when theca cells of post-selection follicles were simultaneously treated by PGN and lipopolysaccharide (0.1, 1, or 10 μg/ml). These findings indicate that bacterial toxins may act locally on ovarian steroidogenic cells and compromise follicular development in postpartum dairy cows. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Adjuvant activity of peptidoglycan monomer and its metabolic products.

PubMed

Halassy, Beata; Krstanović, Marina; Frkanec, Ruza; Tomasić, Jelka

2003-02-14

Peptidoglycan monomer (PGM) is a natural compound of bacterial origin. It is a non-toxic, non-pyrogenic, water-soluble immunostimulator potentiating humoral immune response to ovalbumin (OVA) in mice. It is fast degraded and its metabolic products-the pentapeptide (PP) and the disaccharide (DS)-are excreted from the mammalian organism upon parenteral administration. The present study investigates: (a). whether PGM could influence the long-living memory generation; (b). whether metabolic products retain adjuvant properties of the parent compound and contribute to its adjuvanticity. We report now that mice immunised twice with OVA+PGM had significantly higher anti-OVA IgG levels upon challenge with antigen alone 6 months later in comparison to control group immunised with OVA only. PP and DS were prepared enzymatically in vitro as apyrogenic and chemically pure compounds. When mice were immunised with OVA plus PP and DS, respectively, the level of anti-OVA IgGs in sera was not higher than in mice immunised with OVA alone, while PGM raised the level of specific antibodies. Results implicate that the adjuvant active molecule, capable of enhancing long-living memory generation, is PGM itself, and none of its metabolic products.

The Absence of a Mature Cell Wall Sacculus in Stable Listeria monocytogenes L-Form Cells Is Independent of Peptidoglycan Synthesis.

PubMed

Studer, Patrick; Borisova, Marina; Schneider, Alexander; Ayala, Juan A; Mayer, Christoph; Schuppler, Markus; Loessner, Martin J; Briers, Yves

2016-01-01

L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan.

Chemistry of Peptidoglycan in Mycobacterium tuberculosis Life Cycle: An off-the-wall Balance of Synthesis and Degradation.

PubMed

Squeglia, Flavia; Ruggiero, Alessia; Berisio, Rita

2018-02-21

The cell wall envelope of mycobacteria is structurally distinct from that of both Gram-positive and Gram-negative bacteria. In Mycobacterium tuberculosis, this cell wall has unique structural features and plays a crucial role in drug resistance and macrophage survival under stress conditions. Peptidoglycan is the major constituent of this cell wall, with an important structural role, giving structural strength, and counteracting the osmotic pressure of the cytoplasm. Synthesis of this complex polymer takes place in three stages that occur at three different locations in the cell, from the cytoplasm to the external side of the cell membrane, where polymerization occurs. A fine balance of peptidoglycan synthesis and degradation is responsible for a plethora of molecular mechanisms which are key to the pathogenicity of M. tuberculosis. Enlargement of mycobacterial cells can occur through the synthesis of new peptidoglycan, autolysis of old peptidoglycan, or a combination of both processes. Here, we discuss the chemical aspects of peptidoglycan synthesis and degradation, in relation to metabolic stages of M. tuberculosis. Going from inside the mycobacterial cytoplasm to outside its membrane, we describe the assembly line of peptidoglycan synthesis and polymerization, and continue with its depolymerization events and their consequences on mycobacterial life and resuscitation from dormancy. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Terahertz vibrational signature of bacterial spores arising from nanostructure decorated endospore surface.

PubMed

Datta, Debopam; Stroscio, Michael A; Dutta, Mitra; Zhang, Weidong; Brown, Elliott R

2018-05-03

This theoretical effort is the first to explore the possible hypothesis that terahertz optical activity of Bacillus spores arises from normal vibrational modes of spore coat subcomponents in the terahertz frequency range. Bacterial strains like Bacillus and Clostridium form spores with a hardened coating made of peptidoglycan to protect its genetic material in harsh conditions. In recent years, electron microscopy and atomic force microscopy has revealed that bacterial spore surfaces are decorated with nanocylinders and honeycomb nanostructures. In this article, a simple elastic continuum model is used to describe the vibration of these nanocylinders mainly in Bacillus subtilis, which also leads to the conclusion that the terahertz signature of these spores arises from the vibration of these nanostructures. Three vibrating modes: radial/longitudinal, torsional and flexural, have been identified and discussed for the nanocylinders. The effect of bound water, which shifts the vibration frequency, is also discussed. The peptidoglycan molecule consists of polar and charged amino acids; hence, the sporal surface local vibrations interact strongly with the terahertz radiation. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The structural biology of phenazine biosynthesis

PubMed Central

Blankenfeldt, Wulf; Parsons, James F.

2014-01-01

The phenazines are a class of over 150 nitrogen-containing aromatic compounds of bacterial and archeal origin. Their redox properties not only explain their activity as broad-specificity antibiotics and virulence factors but also enable them to function as respiratory pigments, thus extending their importance to the primary metabolism of phenazine-producing species. Despite their discovery in the mid-19th century, the molecular mechanisms behind their biosynthesis have only been unraveled in the last decade. Here, we review the contribution of structural biology that has led to our current understanding of phenazine biosynthesis. PMID:25215885

Growth mechanics of bacterial cell wall and morphology of bacteria

NASA Astrophysics Data System (ADS)

Jiang, Hongyuan; Sun, Sean

2010-03-01

The peptidoglycan cell wall of bacteria is responsible for maintaining the cell shape and integrity. During the bacterial life cycle, the growth of the cell wall is affected by mechanical stress and osmotic pressure internal to the cell. We develop a theory to describe cell shape changes under the influence of mechanical forces. We find that the theory predicts a steady state size and shape for bacterial cells ranging from cocci to spirillum. Moreover, the theory suggest a mechanism by which bacterial cytoskeletal proteins such as MreB and crescentin can maintain the shape of the cell. The theory can also explain the several recent experiments on growing bacteria in micro-environments.

Primary structure of the wall peptidoglycan of leprosy-derived corynebacteria.

PubMed Central

Janczura, E; Leyh-Bouille, M; Cocito, C; Ghuysen, J M

1981-01-01

The cell walls isolated from axenically grown leprosy-derived corynebacteria were submitted to various chemical and enzymatic degradations. The glycan strands of the wall peptidoglycan are essentially composed of N-acetylglycosaminyl-N-acetylmuramic acid disaccharide units. Small amounts of N-acetylglycosaminyl-N-glycolylmuramic acid (less than 10%) were also detected. The muramic acid residues of adjacent glycan strands are substituted by amidated tetrapeptide units which, in turn, are cross-linked through direct linkages extending between the C-terminal D-alanine residue of one tetrapeptide and the mesodiaminopimelic acid residue of another tetrapeptide. Such a structure is very similar to that of the wall peptidoglycan found in the taxonomically related microorganisms of the Corynebacterium, Mycobacterium, and Nocardia groups. PMID:7462160

Role of N-acetylglucosaminidase and N-acetylmuramidase activities in Enterococcus faecalis peptidoglycan metabolism.

PubMed

Mesnage, Stéphane; Chau, Françoise; Dubost, Lionel; Arthur, Michel

2008-07-11

Identification of the full complement of peptidoglycan hydrolases detected by zymogram in Enterococcus faecalis extracts led to the characterization of two novel hydrolases that we named AtlB and AtlC. Both enzymes have a similar modular organization comprising a central catalytic domain fused to two LysM peptidoglycan-binding modules. AtlB and AtlC displayed N-acetylmuramidase activity, as demonstrated by tandem mass spectrometry analyses of peptidoglycan fragments generated by the purified enzymes. The genes encoding AtlB and AtlC were deleted either alone or in combination with the gene encoding AtlA, a previously described N-acetylglucosaminidase. No autolytic activity was detected in the triple mutant indicating that AtlA, AtlB, and AtlC account for the major hydrolytic activities in E. faecalis. Analysis of cell size distribution by flow cytometry showed that deletion of atlA resulted in the formation of long chains. Thus, AtlA digests the septum and is required for cell separation after cell division. We found that AtlB could act as a surrogate for AtlA, although the enzyme was less efficient at septum digestion. Deletion of atlC had no impact on cell morphology. Labeling of the peptidoglycan with N-[14C]acetylglucosamine revealed an unusually slow turnover as compared with model organisms, almost completely dependent upon the combined activities of AtlA and AtlB. In contrast to atlA, the atlB and atlC genes are located in putative prophages. Because AtlB and AtlC were produced in the absence of cell lysis or production of phage progeny, these enzymes may have been hijacked by E. faecalis to contribute to peptidoglycan metabolism.

The Biosynthesis of Capuramycin-type Antibiotics: IDENTIFICATION OF THE A-102395 BIOSYNTHETIC GENE CLUSTER, MECHANISM OF SELF-RESISTANCE, AND FORMATION OF URIDINE-5'-CARBOXAMIDE.

PubMed

Cai, Wenlong; Goswami, Anwesha; Yang, Zhaoyong; Liu, Xiaodong; Green, Keith D; Barnard-Britson, Sandra; Baba, Satoshi; Funabashi, Masanori; Nonaka, Koichi; Sunkara, Manjula; Morris, Andrew J; Spork, Anatol P; Ducho, Christian; Garneau-Tsodikova, Sylvie; Thorson, Jon S; Van Lanen, Steven G

2015-05-29

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Bacterial α2-macroglobulins: colonization factors acquired by horizontal gene transfer from the metazoan genome?

PubMed Central

Budd, Aidan; Blandin, Stephanie; Levashina, Elena A; Gibson, Toby J

2004-01-01

Background Invasive bacteria are known to have captured and adapted eukaryotic host genes. They also readily acquire colonizing genes from other bacteria by horizontal gene transfer. Closely related species such as Helicobacter pylori and Helicobacter hepaticus, which exploit different host tissues, share almost none of their colonization genes. The protease inhibitor α2-macroglobulin provides a major metazoan defense against invasive bacteria, trapping attacking proteases required by parasites for successful invasion. Results Database searches with metazoan α2-macroglobulin sequences revealed homologous sequences in bacterial proteomes. The bacterial α2-macroglobulin phylogenetic distribution is patchy and violates the vertical descent model. Bacterial α2-macroglobulin genes are found in diverse clades, including purple bacteria (proteobacteria), fusobacteria, spirochetes, bacteroidetes, deinococcids, cyanobacteria, planctomycetes and thermotogae. Most bacterial species with bacterial α2-macroglobulin genes exploit higher eukaryotes (multicellular plants and animals) as hosts. Both pathogenically invasive and saprophytically colonizing species possess bacterial α2-macroglobulins, indicating that bacterial α2-macroglobulin is a colonization rather than a virulence factor. Conclusions Metazoan α2-macroglobulins inhibit proteases of pathogens. The bacterial homologs may function in reverse to block host antimicrobial defenses. α2-macroglobulin was probably acquired one or more times from metazoan hosts and has then spread widely through other colonizing bacterial species by more than 10 independent horizontal gene transfers. yfhM-like bacterial α2-macroglobulin genes are often found tightly linked with pbpC, encoding an atypical peptidoglycan transglycosylase, PBP1C, that does not function in vegetative peptidoglycan synthesis. We suggest that YfhM and PBP1C are coupled together as a periplasmic defense and repair system. Bacterial α2-macroglobulins might

Peptidoglycan-associated lipoprotein (Pal) of Gram-negative bacteria: function, structure, role in pathogenesis and potential application in immunoprophylaxis.

PubMed

Godlewska, Renata; Wiśniewska, Katarzyna; Pietras, Zbigniew; Jagusztyn-Krynicka, Elzbieta Katarzyna

2009-09-01

The protein Pal (peptidoglycan-associated lipoprotein) is anchored in the outer membrane (OM) of Gram-negative bacteria and interacts with Tol proteins. Tol-Pal proteins form two complexes: the first is composed of three inner membrane Tol proteins (TolA, TolQ and TolR); the second consists of the TolB and Pal proteins linked to the cell's OM. These complexes interact with one another forming a multiprotein membrane-spanning system. It has recently been demonstrated that Pal is essential for bacterial survival and pathogenesis, although its role in virulence has not been clearly defined. This review summarizes the available data concerning the structure and function of Pal and its role in pathogenesis.

AtlA functions as a peptidoglycan lytic transglycosylase in the Neisseria gonorrhoeae type IV secretion system.

PubMed

Kohler, Petra L; Hamilton, Holly L; Cloud-Hansen, Karen; Dillard, Joseph P

2007-08-01

Type IV secretion systems require peptidoglycan lytic transglycosylases for efficient secretion, but the function of these enzymes is not clear. The type IV secretion system gene cluster of Neisseria gonorrhoeae encodes two peptidoglycan transglycosylase homologues. One, LtgX, is similar to peptidoglycan transglycosylases from other type IV secretion systems. The other, AtlA, is similar to endolysins from bacteriophages and is not similar to any described type IV secretion component. We characterized the enzymatic function of AtlA in order to examine its role in the type IV secretion system. Purified AtlA was found to degrade macromolecular peptidoglycan and to produce 1,6-anhydro peptidoglycan monomers, characteristic of lytic transglycosylase activity. We found that AtlA can functionally replace the lambda endolysin to lyse Escherichia coli. In contrast, a sensitive measure of lysis demonstrated that AtlA does not lyse gonococci expressing it or gonococci cocultured with an AtlA-expressing strain. The gonococcal type IV secretion system secretes DNA during growth. A deletion of ltgX or a substitution in the putative active site of AtlA severely decreased DNA secretion. These results indicate that AtlA and LtgX are actively involved in type IV secretion and that AtlA is not involved in lysis of gonococci to release DNA. This is the first demonstration that a type IV secretion peptidoglycanase has lytic transglycosylase activity. These data show that AtlA plays a role in type IV secretion of DNA that requires peptidoglycan breakdown without cell lysis.

Characterization of Mutants Deficient in the l,d-Carboxypeptidase (DacB) and WalRK (VicRK) Regulon, Involved in Peptidoglycan Maturation of Streptococcus pneumoniae Serotype 2 Strain D39▿†

PubMed Central

Barendt, Skye M.; Sham, Lok-To; Winkler, Malcolm E.

2011-01-01

Peptidoglycan (PG) hydrolases play critical roles in the remodeling of bacterial cell walls during division. PG hydrolases have been studied extensively in several bacillus species, such as Escherichia coli and Bacillus subtilis, but remain relatively uncharacterized in ovococcus species, such as Streptococcus pneumoniae (pneumococcus). In this work, we identified genes that encode proteins with putative PG hydrolytic domains in the genome of S. pneumoniae strain D39. Knockout mutations in these genes were constructed, and the resulting mutants were characterized in comparison with the parent strain for growth, cell morphology, PG peptide incorporation, and in some cases, PG peptide composition. In addition, we characterized deletion mutations in nonessential genes of unknown function in the WalRKSpn two-component system regulon, which also contains the essential pcsB cell division gene. Several mutants did not show overt phenotypes, which is perhaps indicative of redundancy. In contrast, two new mutants showed distinct defects in PG biosynthesis. One mutation was in a gene designated dacB (spd_0549), which we showed encodes an l,d-carboxypeptidase involved in PG maturation. Notably, dacB mutants, similar to dacA (d,d-carboxypeptidase) mutants, exhibited defects in cell shape and septation, consistent with the idea that the availability of PG peptide precursors is important for proper PG biosynthesis. Epistasis analysis indicated that DacA functions before DacB in d-Ala removal, and immunofluorescence microscopy showed that DacA and DacB are located over the entire surface of pneumococcal cells. The other mutation was in WalRKSpn regulon gene spd_0703, which encodes a putative membrane protein that may function as a type of conserved streptococcal shape, elongation, division, and sporulation (SEDS) protein. PMID:21378199

Bacterial genome mining of enzymatic tools for alkyne biosynthesis

PubMed Central

Zhu, Xuejun; Su, Michael; Manickam, Kadhirvel; Zhang, Wenjun

2015-01-01

The alkyne is an important functionality widely used in material science, pharmaceutical science, and chemical biology, but the importance of this functionality is contrasted by the very limited number of enzymes known to be involved in alkyne biosynthesis. We recently reported the first known carrier protein-dependent pathway for terminal alkyne formation, and in silico analysis suggested that this mechanism could be widespread in bacteria. In this paper, we screened additional homologous gene cassettes presumed to be involved in alkyne biosynthesis using both in vitro biochemical study and an E. coli-polyketide synthase (PKS) reporting system for in vivo analysis. We discovered and characterized a new terminal alkyne biosynthetic pathway comprised of TtuA, B, and C from Teredinibacter turnerae T7901. While the acyl-CoA ligase homolog (TtuA) demonstrated promiscuity in the activation and loading of medium-chain fatty acids onto the carrier protein (TtuC), the desaturase homolog (TtuB) showed stringent substrate specificity towards C10 fatty acyl moieties. In addition, TtuB was demonstrated to be a bifunctional desaturase/acetylenase that efficiently catalyzed two sequential O2-dependent dehydrogenation reactions. A novel terminal-alkyne bearing polyketide was further produced upon co-expression of ttuABC and a PKS gene in E. coli. The discovery and characterization of TtuA, B, and C provides us with a new bifunctional desaturase/acetylenase for mechanistic and structural study and expands the scarce enzyme inventory for the biosynthesis of the alkyne functionality, which has important applications in synthetic and chemical biology. PMID:26441143

Peptidoglycan recognition protein genes and their roles in the innate immune pathways of the red flour beetle, Tribolium castaneum.

PubMed

Koyama, Hiroaki; Kato, Daiki; Minakuchi, Chieka; Tanaka, Toshiharu; Yokoi, Kakeru; Miura, Ken

2015-11-01

We have previously demonstrated that the functional Toll and IMD innate immune pathways indeed exist in the model beetle, Tribolium castaneum while the beetle's pathways have broader specificity in terms of microbial activation than that of Drosophila. To elucidate the molecular basis of this broad microbial activation, we here focused on potential upstream sensors of the T. castaneum innate immune pathways, peptidoglycan recognition proteins (PGRPs). Our phenotype analyses utilizing RNA interference-based comprehensive gene knockdown followed by bacterial challenge suggested: PGRP-LA functions as a pivotal sensor of the IMD pathway for both Gram-negative and Gram-positive bacteria; PGRP-LC acts as an IMD pathway-associated sensor mainly for Gram-negative bacteria; PGRP-LE also has some roles in Gram-negative bacterial recognition of the IMD pathway. On the other hand, we did not obtain clear phenotype changes by gene knockdown of short-type PGRP genes, probably because of highly inducible nature of these genes. Our results may collectively account for the promiscuous bacterial activation of the T. castaneum innate immune pathways at least in part. Copyright © 2015 Elsevier Inc. All rights reserved.

Involvement of Pacific oyster CgPGRP-S1S in bacterial recognition, agglutination and granulocyte degranulation.

PubMed

Iizuka, Masao; Nagasaki, Toshihiro; Takahashi, Keisuke G; Osada, Makoto; Itoh, Naoki

2014-03-01

Peptidoglycan recognition protein (PGRP) recognizes invading bacteria through their peptidoglycans (PGN), a component of the bacterial cell wall. Insect PGRPs contribute to effective immune systems as inducers of other host defense responses, while this function has not been reported from PGRP of bivalves. In this study, recombinant CgPGRP-S1S (rCgPGRP-S1S), produced in the mantle and the gill, was synthesized and used to elucidate the immunological function of CgPGRP-S1S. rCgPGRP-S1S bound specifically to DAP-type PGN and to Escherichia coli cells, but not to other DAP-type PGN-containing bacterial species, Vibrio anguillarum, or Bacillus subtilis. Antibacterial activity was not detected, but E. coli cells were agglutinated. Moreover, in addition to these direct interactions with bacterial cells, rCgPGRP-S1S induced secretion of granular contents by hemocyte degranulation. Taken together, these results suggest for the first time that a PGRP of bivalves is, just as in insects, involved in host defense, not only by direct interaction with bacteria, but also by triggering other defense pathways. Copyright © 2013 Elsevier Ltd. All rights reserved.

Physics of Bacterial Morphogenesis

PubMed Central

Sun, Sean X.; Jiang, Hongyuan

2011-01-01

Summary: Bacterial cells utilize three-dimensional (3D) protein assemblies to perform important cellular functions such as growth, division, chemoreception, and motility. These assemblies are composed of mechanoproteins that can mechanically deform and exert force. Sometimes, small-nucleotide hydrolysis is coupled to mechanical deformations. In this review, we describe the general principle for an understanding of the coupling of mechanics with chemistry in mechanochemical systems. We apply this principle to understand bacterial cell shape and morphogenesis and how mechanical forces can influence peptidoglycan cell wall growth. We review a model that can potentially reconcile the growth dynamics of the cell wall with the role of cytoskeletal proteins such as MreB and crescentin. We also review the application of mechanochemical principles to understand the assembly and constriction of the FtsZ ring. A number of potential mechanisms are proposed, and important questions are discussed. PMID:22126993

From cells to muropeptide structures in 24 h: Peptidoglycan mapping by UPLC-MS

PubMed Central

Kühner, Daniel; Stahl, Mark; Demircioglu, Dogan D.; Bertsche, Ute

2014-01-01

Peptidoglycan (PGN) is ubiquitous in nearly all bacterial species. The PGN sacculus protects the cells against their own internal turgor making PGN one of the most important targets for antibacterial treatment. Within the last sixty years PGN composition has been intensively studied by various methods. The breakthrough was the application of HPLC technology on the analysis of muropeptides. However, preparation of pure PGN relied on a very time consuming method of about one week. We established a purification protocol for both Gram-positive and Gram-negative bacteria which can be completely performed in plastic reaction tubes yielding pure muropeptides within 24 hours. The muropeptides can be analyzed by UPLC-MS, allowing their immediate determination. This new rapid method provides the feasibility to screen PGN composition even in high throughput, making it a highly useful tool for basic research as well as for the pharmaceutical industry. PMID:25510564

The UbiK protein is an accessory factor necessary for bacterial ubiquinone (UQ) biosynthesis and forms a complex with the UQ biogenesis factor UbiJ.

PubMed

Loiseau, Laurent; Fyfe, Cameron; Aussel, Laurent; Hajj Chehade, Mahmoud; Hernández, Sara B; Faivre, Bruno; Hamdane, Djemel; Mellot-Draznieks, Caroline; Rascalou, Bérengère; Pelosi, Ludovic; Velours, Christophe; Cornu, David; Lombard, Murielle; Casadesús, Josep; Pierrel, Fabien; Fontecave, Marc; Barras, Frédéric

2017-07-14

Ubiquinone (UQ), also referred to as coenzyme Q, is a widespread lipophilic molecule in both prokaryotes and eukaryotes in which it primarily acts as an electron carrier. Eleven proteins are known to participate in UQ biosynthesis in Escherichia coli , and we recently demonstrated that UQ biosynthesis requires additional, nonenzymatic factors, some of which are still unknown. Here, we report on the identification of a bacterial gene, yqiC , which is required for efficient UQ biosynthesis, and which we have renamed ubiK Using several methods, we demonstrated that the UbiK protein forms a complex with the C-terminal part of UbiJ, another UQ biogenesis factor we previously identified. We found that both proteins are likely to contribute to global UQ biosynthesis rather than to a specific biosynthetic step, because both ubiK and ubiJ mutants accumulated octaprenylphenol, an early intermediate of the UQ biosynthetic pathway. Interestingly, we found that both proteins are dispensable for UQ biosynthesis under anaerobiosis, even though they were expressed in the absence of oxygen. We also provide evidence that the UbiK-UbiJ complex interacts with palmitoleic acid, a major lipid in E. coli Last, in Salmonella enterica , ubiK was required for proliferation in macrophages and virulence in mice. We conclude that although the role of the UbiK-UbiJ complex remains unknown, our results support the hypothesis that UbiK is an accessory factor of Ubi enzymes and facilitates UQ biosynthesis by acting as an assembly factor, a targeting factor, or both. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Understanding Mircrobial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2016-10-01

limitation, we have developed an expanded metabolic labeling approach that chemically tags lipopolysaccharide, capsular polysaccharide , and peptidoglycan...click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide , inflammatory...bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide , inflammatory bowel disease, microbiome, microbiota

Biosynthesis of highly porous bacterial cellulose nanofibers

NASA Astrophysics Data System (ADS)

Hosseini, Hadi; Kokabi, Mehrdad; Mousavi, Seyyed Mohammad

2018-01-01

Bacterial cellulose nanofibers (BCNFs) as a sustainable and biodegradable polymer has drawn tremendous research attention in tissue engineering, bacterial sensors and drug delivery due to its extraordinary properties such as high purity, high crystallinity, high water absorption capacity and excellent mechanical strength in the wet state. This awesome properties, is attributed to BCNFs structure, therefore its characterization is important. In this work, the bacterial strain, Gluconacetobacter xylinus (PTCC 1734, obtained from Iranian Research Organization for Science and Technology (IROST)), was used to produce BCNFs hydrogel using bacterial fermentation under static condition at 29 °C for 10 days in the incubator. Then, the biosynthesized BCNFs wet gel, were dried at ambient temperature and pressure and characterized using Brunauer-Emmett-Teller (BET) and Field emission scanning electron microscopy (FE-SEM) analysis. FESEM image displayed highly interconnected and porous structure composed of web-like continuous, nanofibers with an average diameter of 48.5±2.1 nm. BET result analysis depicted BCNFs dried at ambient conditions had IV isotherm type, according to the IUPAC classification, indicating that BCNFs dried at ambient condition is essentially mesoporous. On the other hand, BET results depicted, mesoporous structure is around 85%. In addition, Specific surface area (SBET) obtained 81.45 m2/g. These results are in accordance with the FESEM observation.

The Peptidoglycan-Binding Protein SjcF1 Influences Septal Junction Function and Channel Formation in the Filamentous Cyanobacterium Anabaena.

PubMed

Rudolf, Mareike; Tetik, Nalan; Ramos-León, Félix; Flinner, Nadine; Ngo, Giang; Stevanovic, Mara; Burnat, Mireia; Pernil, Rafael; Flores, Enrique; Schleiff, Enrico

2015-06-30

Filamentous, heterocyst-forming cyanobacteria exchange nutrients and regulators between cells for diazotrophic growth. Two alternative modes of exchange have been discussed involving transport either through the periplasm or through septal junctions linking adjacent cells. Septal junctions and channels in the septal peptidoglycan are likely filled with septal junction complexes. While possible proteinaceous factors involved in septal junction formation, SepJ (FraG), FraC, and FraD, have been identified, little is known about peptidoglycan channel formation and septal junction complex anchoring to the peptidoglycan. We describe a factor, SjcF1, involved in regulation of septal junction channel formation in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. SjcF1 interacts with the peptidoglycan layer through two peptidoglycan-binding domains and is localized throughout the cell periphery but at higher levels in the intercellular septa. A strain with an insertion in sjcF1 was not affected in peptidoglycan synthesis but showed an altered morphology of the septal peptidoglycan channels, which were significantly wider in the mutant than in the wild type. The mutant was impaired in intercellular exchange of a fluorescent probe to a similar extent as a sepJ deletion mutant. SjcF1 additionally bears an SH3 domain for protein-protein interactions. SH3 binding domains were identified in SepJ and FraC, and evidence for interaction of SjcF1 with both SepJ and FraC was obtained. SjcF1 represents a novel protein involved in structuring the peptidoglycan layer, which links peptidoglycan channel formation to septal junction complex function in multicellular cyanobacteria. Nonetheless, based on its subcellular distribution, this might not be the only function of SjcF1. Cell-cell communication is central not only for eukaryotic but also for multicellular prokaryotic systems. Principles of intercellular communication are well established for eukaryotes, but the

A novel type of peptidoglycan-binding domain highly specific for amidated D-Asp cross-bridge, identified in Lactobacillus casei bacteriophage endolysins.

PubMed

Regulski, Krzysztof; Courtin, Pascal; Kulakauskas, Saulius; Chapot-Chartier, Marie-Pierre

2013-07-12

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-L-alanine amidase, whereas Lc-Lys-2 is a γ-D-glutamyl-L-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with D-Ala(4)→D-Asx-L-Lys(3) in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting D-Ala(4)→L-Ala-(L-Ala/L-Ser)-L-Lys(3); moreover, they do not lyse the L. lactis mutant containing only the nonamidated D-Asp cross-bridge, i.e. D-Ala(4)→D-Asp-L-Lys(3). In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 L-Lys(3)-D-Asn-L-Lys(3) bridges replacing the wild-type 4→3 D-Ala(4)-D-Asn-L-Lys(3) bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly D-Asn but not PG with only the nonamidated D-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the D-Asn interpeptide bridge of PG.

Peptidoglycan from Fermentation By-Product Triggers Defense Responses in Grapevine

PubMed Central

Chen, Yang; Takeda, Taito; Aoki, Yoshinao; Fujita, Keiko; Suzuki, Shunji; Igarashi, Daisuke

2014-01-01

Plants are constantly under attack from a variety of microorganisms, and rely on a series of complex detection and response systems to protect themselves from infection. Here, we found that a by-product of glutamate fermentation triggered defense responses in grapevine, increasing the expression of defense response genes in cultured cells, foliar chitinase activity, and resistance to infection by downy mildew in leaf explants. To identify the molecule that triggered this innate immunity, we fractionated and purified candidates extracted from Corynebacterium glutamicum, a bacterium used in the production of amino acids by fermentation. Using hydrolysis by lysozyme, a silkworm larva plasma detection system, and gel filtration analysis, we identified peptidoglycan as inducing the defense responses. Peptidoglycans of Escherichia coli, Bacillus subtilis, and Staphylococcus aureus also generated similar defensive responses. PMID:25427192

Host-induced bacterial cell wall decomposition mediates pattern-triggered immunity in Arabidopsis

PubMed Central

Liu, Xiaokun; Grabherr, Heini M; Willmann, Roland; Kolb, Dagmar; Brunner, Frédéric; Bertsche, Ute; Kühner, Daniel; Franz-Wachtel, Mirita; Amin, Bushra; Felix, Georg; Ongena, Marc; Nürnberger, Thorsten; Gust, Andrea A

2014-01-01

Peptidoglycans (PGNs) are immunogenic bacterial surface patterns that trigger immune activation in metazoans and plants. It is generally unknown how complex bacterial structures such as PGNs are perceived by plant pattern recognition receptors (PRRs) and whether host hydrolytic activities facilitate decomposition of bacterial matrices and generation of soluble PRR ligands. Here we show that Arabidopsis thaliana, upon bacterial infection or exposure to microbial patterns, produces a metazoan lysozyme-like hydrolase (lysozyme 1, LYS1). LYS1 activity releases soluble PGN fragments from insoluble bacterial cell walls and cleavage products are able to trigger responses typically associated with plant immunity. Importantly, LYS1 mutant genotypes exhibit super-susceptibility to bacterial infections similar to that observed on PGN receptor mutants. We propose that plants employ hydrolytic activities for the decomposition of complex bacterial structures, and that soluble pattern generation might aid PRR-mediated immune activation in cell layers adjacent to infection sites. DOI: http://dx.doi.org/10.7554/eLife.01990.001 PMID:24957336

UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis

PubMed Central

White, Mark D.; Payne, Karl A.P.; Fisher, Karl; Marshall, Stephen A.; Parker, David; Rattray, Nicholas J.W.; Trivedi, Drupad K.; Goodacre, Royston; Rigby, Stephen E.J.; Scrutton, Nigel S.; Hay, Sam; Leys, David

2016-01-01

Ubiquinone, or coenzyme Q, is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains1. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor2. Despite structural and biochemical characterization of UbiX as an FMN-binding protein, no decarboxylase activity has been detected3–4. We report here that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD5. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases6–7, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyl transferase mechanism of UbiX resembles that of the terpene synthases8. The active site environment is dominated by π-systems, which assist phosphate-C1’ bond breakage following FMN reduction, leading to formation of the N5-C1’ bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3’-C6 bond. The study establishes the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoire. PMID:26083743

Salicylic acid biosynthesis is enhanced and contributes to increased biotrophic pathogen resistance in Arabidopsis hybrids

PubMed Central

Yang, Li; Li, Bosheng; Zheng, Xiao-yu; Li, Jigang; Yang, Mei; Dong, Xinnian; He, Guangming; An, Chengcai; Deng, Xing Wang

2015-01-01

Heterosis, the phenotypic superiority of a hybrid over its parents, has been demonstrated for many traits in Arabidopsis thaliana, but its effect on defence remains largely unexplored. Here, we show that hybrids between some A. thaliana accessions show increased resistance to the biotrophic bacterial pathogen Pseudomonas syringae pv. tomato (Pst) DC3000. Comparisons of transcriptomes between these hybrids and their parents after inoculation reveal that several key salicylic acid (SA) biosynthesis genes are significantly upregulated in hybrids. Moreover, SA levels are higher in hybrids than in either parent. Increased resistance to Pst DC3000 is significantly compromised in hybrids of pad4 mutants in which the SA biosynthesis pathway is blocked. Finally, increased histone H3 acetylation of key SA biosynthesis genes correlates with their upregulation in infected hybrids. Our data demonstrate that enhanced activation of SA biosynthesis in A. thaliana hybrids may contribute to their increased resistance to a biotrophic bacterial pathogen. PMID:26065719

Molecular mapping of the cell wall polysaccharides of the human pathogen Streptococcus agalactiae

NASA Astrophysics Data System (ADS)

Beaussart, Audrey; Péchoux, Christine; Trieu-Cuot, Patrick; Hols, Pascal; Mistou, Michel-Yves; Dufrêne, Yves F.

2014-11-01

The surface of many bacterial pathogens is covered with polysaccharides that play important roles in mediating pathogen-host interactions. In Streptococcus agalactiae, the capsular polysaccharide (CPS) is recognized as a major virulence factor while the group B carbohydrate (GBC) is crucial for peptidoglycan biosynthesis and cell division. Despite the important roles of CPS and GBC, there is little information available on the molecular organization of these glycopolymers on the cell surface. Here, we use atomic force microscopy (AFM) and transmission electron microscopy (TEM) to analyze the nanoscale distribution of CPS and GBC in wild-type (WT) and mutant strains of S. agalactiae. TEM analyses reveal that in WT bacteria, peptidoglycan is covered with a very thin (few nm) layer of GBC (the ``pellicle'') overlaid by a 15-45 nm thick layer of CPS (the ``capsule''). AFM-based single-molecule mapping with specific antibody probes shows that CPS is exposed on WT cells, while it is hardly detected on mutant cells impaired in CPS production (ΔcpsE mutant). By contrast, both TEM and AFM show that CPS is over-expressed in mutant cells altered in GBC expression (ΔgbcO mutant), indicating that the production of the two surface glycopolymers is coordinated in WT cells. In addition, AFM topographic imaging and molecular mapping with specific lectin probes demonstrate that removal of CPS (ΔcpsE), but not of GBC (ΔgbcO), leads to the exposure of peptidoglycan, organized into 25 nm wide bands running parallel to the septum. These results indicate that CPS forms a homogeneous barrier protecting the underlying peptidoglycan from environmental exposure, while the presence of GBC does not prevent peptidoglycan detection. This work shows that single-molecule AFM, combined with high-resolution TEM, represents a powerful platform for analysing the molecular arrangement of the cell wall polymers of bacterial pathogens.

Positioning cell wall synthetic complexes by the bacterial morphogenetic proteins MreB and MreD.

PubMed

White, Courtney L; Kitich, Aleksandar; Gober, James W

2010-05-01

In Caulobacter crescentus, intact cables of the actin homologue, MreB, are required for the proper spatial positioning of MurG which catalyses the final step in peptidoglycan precursor synthesis. Similarly, in the periplasm, MreC controls the spatial orientation of the penicillin binding proteins and a lytic transglycosylase. We have now found that MreB cables are required for the organization of several other cytosolic murein biosynthetic enzymes such as MraY, MurB, MurC, MurE and MurF. We also show these proteins adopt a subcellular pattern of localization comparable to MurG, suggesting the existence of cytoskeletal-dependent interactions. Through extensive two-hybrid analyses, we have now generated a comprehensive interaction map of components of the bacterial morphogenetic complex. In the cytosol, this complex contains both murein biosynthetic enzymes and morphogenetic proteins, including RodA, RodZ and MreD. We show that the integral membrane protein, MreD, is essential for lateral peptidoglycan synthesis, interacts with the precursor synthesizing enzymes MurG and MraY, and additionally, determines MreB localization. Our results suggest that the interdependent localization of MreB and MreD functions to spatially organize a complex of peptidoglycan precursor synthesis proteins, which is required for propagation of a uniform cell shape and catalytically efficient peptidoglycan synthesis.

Genetic basis for the resistance of Staphylococcus aureus to peptidoglycan hydrolase by comparative transcriptome and whole genome sequence analysis

USDA-ARS?s Scientific Manuscript database

Background: Lysostaphin is a glycyl-glycine bacteriocin peptidoglycan hydrolase secreted by Staphylococcus simulans for degrading the peptidoglycan moieties in Staphylococcus aureus cell walls which result in cell lysis. There are known mechanisms of resistance to lysostaphin, e.g. serine in place...

Rhizocola hellebori gen. nov., sp. nov., an actinomycete of the family Micromonosporaceae containing 3,4-dihydroxydiaminopimelic acid in the cell-wall peptidoglycan.

PubMed

Matsumoto, Atsuko; Kawaguchi, Yoko; Nakashima, Takuji; Iwatsuki, Masato; Ōmura, Satoshi; Takahashi, Yōko

2014-08-01

An actinomycete strain, K12-0602(T), was isolated from the root of a Helleborus orientalis plant in Japan. The 16S rRNA gene sequence of strain K12-0602(T) showed that it had a close relationship with members of the family Micromonosporaceae and the 16S rRNA gene sequence similarity values between strain K12-0602(T) and type strains of type species of 27 genera belonging to the family Micromonosporaceae were below 96.2%. MK-9 (H4) and MK-9 (H6) were detected as major menaquinones, and galactose, xylose, mannose and ribose were present in the whole-cell hydrolysate. The acyl type of the peptidoglycan was glycolyl. Major fatty acids were iso-C(15 : 0), iso-C(16 : 0), C(17 : 1)ω9c and anteiso-C(17 : 0). Phosphatidylethanolamine was detected as the phospholipid corresponding to phospholipid type II. The G+C content of the genomic DNA was 67 mol%. Analyses of the cell-wall peptidoglycan by TLC and LC/MS showed that it was composed of alanine, glycine, hydroxylglutamic acid and an unknown amino acid, which was subsequently determined to be 3,4-dihydroxydiaminopimelic acid using instrumental analyses, including NMR and mass spectrometry. On the basis of the phylogenetic analysis and chemotaxonomic characteristics, strain K12-0602(T) represents a novel species of a new genus in the family Micromonosporaceae, for which the name Rhizocola hellebori gen. nov., sp. nov. is proposed. The type strain of the type species is K12-0602(T) ( = NBRC 109834(T) = DSM 45988(T)). This is the first report, to our knowledge, of 3,4-dihydroxydiaminopimelic acid being found as a diamino acid in bacterial cell-wall peptidoglycan. © 2014 IUMS.

Inhibitor design strategy based on an enzyme structural flexibility: a case of bacterial MurD ligase.

PubMed

Perdih, Andrej; Hrast, Martina; Barreteau, Hélène; Gobec, Stanislav; Wolber, Gerhard; Solmajer, Tom

2014-05-27

Increasing bacterial resistance to available antibiotics stimulated the discovery of novel efficacious antibacterial agents. The biosynthesis of the bacterial peptidoglycan, where the MurD enzyme is involved in the intracellular phase of the UDP-MurNAc-pentapeptide formation, represents a collection of highly selective targets for novel antibacterial drug design. In our previous computational studies, the C-terminal domain motion of the MurD ligase was investigated using Targeted Molecular Dynamic (TMD) simulation and the Off-Path Simulation (OPS) technique. In this study, we present a drug design strategy using multiple protein structures for the identification of novel MurD ligase inhibitors. Our main focus was the ATP-binding site of the MurD enzyme. In the first stage, three MurD protein conformations were selected based on the obtained OPS/TMD data as the initial criterion. Subsequently, a two-stage virtual screening approach was utilized combining derived structure-based pharmacophores with molecular docking calculations. Selected compounds were then assayed in the established enzyme binding assays, and compound 3 from the aminothiazole class was discovered to act as a dual MurC/MurD inhibitor in the micomolar range. A steady-state kinetic study was performed on the MurD enzyme to provide further information about the mechanistic aspects of its inhibition. In the final stage, all used conformations of the MurD enzyme with compound 3 were simulated in classical molecular dynamics (MD) simulations providing atomistic insights of the experimental results. Overall, the study depicts several challenges that need to be addressed when trying to hit a flexible moving target such as the presently studied bacterial MurD enzyme and show the possibilities of how computational tools can be proficiently used at all stages of the drug discovery process.

Biosynthesis of Gold Nanoparticles Using Pseudomonas Aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Abd El-Aziz, M.; Badr, Y.; Mahmoud, M. A.

2007-02-14

Pseudomonas aeruginosa were used for extracellular biosynthesis of gold nanoparticles (Au NPs). Consequently, Au NPs were formed due to reduction of gold ion by bacterial cell supernatant of P. aeruginos ATCC 90271, P. aeruginos (2) and P. aeruginos (1). The UV-Vis. and fluorescence spectra of the bacterial as well as chemical prepared Au NPs were recorded. Transmission electron microscopy (TEM) micrograph showed the formation of well-dispersed gold nanoparticles in the range of 15-30 nm. The process of reduction being extracellular and may lead to the development of an easy bioprocess for synthesis of Au NPs.

Bacterial Cell Growth Inhibitors Targeting Undecaprenyl Diphosphate Synthase and Undecaprenyl Diphosphate Phosphatase.

PubMed

Wang, Yang; Desai, Janish; Zhang, Yonghui; Malwal, Satish R; Shin, Christopher J; Feng, Xinxin; Sun, Hong; Liu, Guizhi; Guo, Rey-Ting; Oldfield, Eric

2016-10-19

We synthesized a series of benzoic acids and phenylphosphonic acids and investigated their effects on the growth of Staphylococcus aureus and Bacillus subtilis. One of the most active compounds, 5-fluoro-2-(3-(octyloxy)benzamido)benzoic acid (7, ED 50 ∼0.15 μg mL -1 ) acted synergistically with seven antibiotics known to target bacterial cell-wall biosynthesis (a fractional inhibitory concentration index (FICI) of ∼0.35, on average) but had indifferent effects in combinations with six non-cell-wall biosynthesis inhibitors (average FICI∼1.45). The most active compounds were found to inhibit two enzymes involved in isoprenoid/bacterial cell-wall biosynthesis: undecaprenyl diphosphate synthase (UPPS) and undecaprenyl diphosphate phosphatase (UPPP), but not farnesyl diphosphate synthase, and there were good correlations between bacterial cell growth inhibition, UPPS inhibition, and UPPP inhibition. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock.

PubMed

Van Amersfoort, Edwin S; Van Berkel, Theo J C; Kuiper, Johan

2003-07-01

Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.

Molecular cloning and functional characterization of peptidoglycan recognition protein 6 in grass carp Ctenopharyngodon idella.

PubMed

Li, Jun Hua; Yu, Zhang Long; Xue, Na Na; Zou, Peng Fei; Hu, Jing Yu; Nie, P; Chang, Ming Xian

2014-02-01

Peptidoglycan recognition proteins (PGRPs) are pattern recognition molecules of innate immunity. In this study, a long-form PGRP, designated as gcPGRP6, was identified from grass carp Ctenopharyngodon idella. The deduced amino acid sequence of gcPGRP6 is composed of 464 residues with a conserved PGRP domain at the C-terminus. The gcPGRP6 gene consists of four exons and three introns, spacing approximately 2.7 kb of genomic sequence. Phylogenetic analysis demonstrated that gcPGRP6 is clustered closely with zebrafish PGLYRP6, and formed a long-type PGRP subfamily together with PGLYRP2 members identified in teleosts and mammals. Real-time PCR and Western blotting analyses revealed that gcPGRP6 is constitutively expressed in organs/tissues examined, and its expression was significantly induced in liver and intestine of grass carp in response to PGN stimulation and in CIK cells treated with lipoteichoic acid (LTA), polyinosinic polycytidylic acid (Poly I:C) and peptidoglycan (PGN). Immunofluorescence microscopy and Western blotting analyses revealed that gcPGRP6 is effectively secreted to the exterior of CIK cells. The over-expression of gcPGRP6 in CIK cells leads to the activation of NF-κB and the inhibition of intracellular bacterial growth. Moreover, cell lysates from CIK cells transfected with pTurbo-gcPGRP6-GFP plasmid display the binding activity towards Lys-type PGN from Staphylococcus aureus and DAP-type PGN from Bacillus subtilis. Furthermore, proinflammatory cytokine IL-2 and intracellular PGN receptor NOD2 had a significantly increased expression in CIK cells overexpressed with gcPGRP6. It is demonstrated that the PGRP6 in grass carp has a role in binding PGN, in inhibiting the growth of intracellular bacteria, and in activating NF-κB, as well as in regulating innate immune genes. Copyright © 2013 Elsevier Ltd. All rights reserved.

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics.

PubMed

Price, Morgan N; Zane, Grant M; Kuehl, Jennifer V; Melnyk, Ryan A; Wall, Judy D; Deutschbauer, Adam M; Arkin, Adam P

2018-01-01

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics

DOE Office of Scientific and Technical Information (OSTI.GOV)

Price, Morgan N.; Zane, Grant M.; Kuehl, Jennifer V.

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. Here, we studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fillmore » 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.« less

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics

DOE PAGES

Price, Morgan N.; Zane, Grant M.; Kuehl, Jennifer V.; ...

2018-01-11

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. Here, we studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fillmore » 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes.« less

Filling gaps in bacterial amino acid biosynthesis pathways with high-throughput genetics

PubMed Central

Kuehl, Jennifer V.; Melnyk, Ryan A.; Deutschbauer, Adam M.; Arkin, Adam P.

2018-01-01

For many bacteria with sequenced genomes, we do not understand how they synthesize some amino acids. This makes it challenging to reconstruct their metabolism, and has led to speculation that bacteria might be cross-feeding amino acids. We studied heterotrophic bacteria from 10 different genera that grow without added amino acids even though an automated tool predicts that the bacteria have gaps in their amino acid synthesis pathways. Across these bacteria, there were 11 gaps in their amino acid biosynthesis pathways that we could not fill using current knowledge. Using genome-wide mutant fitness data, we identified novel enzymes that fill 9 of the 11 gaps and hence explain the biosynthesis of methionine, threonine, serine, or histidine by bacteria from six genera. We also found that the sulfate-reducing bacterium Desulfovibrio vulgaris synthesizes homocysteine (which is a precursor to methionine) by using DUF39, NIL/ferredoxin, and COG2122 proteins, and that homoserine is not an intermediate in this pathway. Our results suggest that most free-living bacteria can likely make all 20 amino acids and illustrate how high-throughput genetics can uncover previously-unknown amino acid biosynthesis genes. PMID:29324779

A homogeneous, high-throughput-compatible, fluorescence intensity-based assay for UDP-N-acetylenolpyruvylglucosamine reductase (MurB) with nanomolar product detection.

PubMed

Shapiro, Adam B; Livchak, Stephania; Gao, Ning; Whiteaker, James; Thresher, Jason; Jahić, Haris; Huang, Jian; Gu, Rong-Fang

2012-03-01

A novel assay for the NADPH-dependent bacterial enzyme UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is described that has nanomolar sensitivity for product formation and is suitable for high-throughput applications. MurB catalyzes an essential cytoplasmic step in the synthesis of peptidoglycan for the bacterial cell wall, reduction of UDP-N-acetylenolpyruvylglucosamine to UDP-N-acetylmuramic acid (UNAM). Interruption of this biosynthetic pathway leads to cell death, making MurB an attractive target for antibacterial drug discovery. In the new assay, the UNAM product of the MurB reaction is ligated to L-alanine by the next enzyme in the peptidoglycan biosynthesis pathway, MurC, resulting in hydrolysis of adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The ADP is detected with nanomolar sensitivity by converting it to oligomeric RNA with polynucleotide phosphorylase and detecting the oligomeric RNA with a fluorescent dye. The product sensitivity of the new assay is 1000-fold greater than that of the standard assay that follows the absorbance decrease resulting from the conversion of NADPH to NADP(+). This sensitivity allows inhibitor screening to be performed at the low substrate concentrations needed to make the assay sensitive to competitive inhibition of MurB.

De novo transcriptome assembly and analysis of differential gene expression following peptidoglycan (PGN) challenge in Antheraea pernyi.

PubMed

Liu, Yu; Xin, Zhao-Zhe; Zhang, Dai-Zhen; Zhu, Xiao-Yu; Wang, Ying; Chen, Li; Tang, Bo-Ping; Zhou, Chun-Lin; Chai, Xin-Yue; Tian, Ji-Wu; Liu, Qiu-Ning

2018-06-01

Antheraea pernyi is not only an important economic insect, it is increasingly employed as a model organism due to a variety of advantages, including ease of rearing and experimental manipulation compared with other Lepidoptera. Peptidoglycan (PGN) is a major component of the bacterial cell wall, and interactions between PGN and A. pernyi cause a series of physiological changes in the insect. In the present study, we constructed cDNA libraries from a A. pernyi PGN-infected group and a control group stimulated with phosphate-buffered saline (PBS). The transcriptome was de novo assembled using the Trinity platform, and 1698 differentially expressed genes (DEGs) were identified, comprising 894 up-regulated and 804 down-regulated genes. To further investigate immune-related DEGs, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed. GO analysis identified major immune-related GO terms and KEGG enrichment indicated gene responses to three pathways related to the insect immune system. Several homologous genes related to the immune response of the A. pernyi fat body post-PGN infection were identified and categorised. Taken together, the results provide insight into the complex molecular mechanisms of the responses to bacterial infection at the transcriptional level. Copyright © 2018 Elsevier B.V. All rights reserved.

Proteome analysis of Arabidopsis seedlings exposed to bacterial volatiles.

PubMed

Kwon, Young Sang; Ryu, Choong-Min; Lee, Soohyun; Park, Hyo Bee; Han, Ki Soo; Lee, Jung Han; Lee, Kyunghee; Chung, Woo Sik; Jeong, Mi-Jeong; Kim, Hee Kyu; Bae, Dong-Won

2010-11-01

Plant root-associated bacteria (rhizobacteria) elicit plant basal immunity referred to as induced systemic resistance (ISR) against multiple pathogens. Among multi-bacterial determinants involving such ISR, the induction of ISR and promotion of growth by bacterial volatile compounds was previously reported. To exploit global de novo expression of plant proteins by bacterial volatiles, proteomic analysis was performed after exposure of Arabidopsis plants to the rhizobacterium Bacillus subtilis GB03. Ethylene biosynthesis enzymes were significantly up-regulated. Analysis by quantitative reverse transcriptase polymerase chain reaction confirmed that ethylene biosynthesis-related genes SAM-2, ACS4, ACS12, and ACO2 as well as ethylene response genes, ERF1, GST2, and CHIB were up-regulated by the exposure to bacterial volatiles. More interestingly, the emission of bacterial volatiles significantly up-regulated both key defense mechanisms mediated by jasmonic acid and salicylic acid signaling pathways. In addition, high accumulation of antioxidant proteins also provided evidence of decreased sensitivity to reactive oxygen species during the elicitation of ISR by bacterial volatiles. The present results suggest that the proteomic analysis of plant defense responses in bacterial volatile-mediated ISR can reveal the mechanisms of plant basal defenses orchestrated by endogenous ethylene production pathways and the generation of reactive oxygen species.

A Methyl 4-Oxo-4-phenylbut-2-enoate with in Vivo Activity against MRSA that Inhibits MenB in the Bacterial Menaquinone Biosynthesis Pathway

PubMed Central

Matarlo, Joe S.; Lu, Yang; Daryaee, Fereidoon; Daryaee, Taraneh; Ruzsicska, Bela; Walker, Stephen G.; Tonge, Peter J.

2016-01-01

4-Oxo-4-phenyl-but-2-enoates inhibit MenB, the 1,4-dihydroxyl-2-naphthoyl-CoA synthase in the bacterial menaquinone (MK) biosynthesis pathway, through the formation of an adduct with coenzyme A (CoA). Here, we show that the corresponding methyl butenoates have MIC values as low as 0.35–0.75 µg/mL against drug sensitive and resistant strains of Staphylococcus aureus. Mode of action studies on the most potent compound, methyl 4-(4-chlorophenyl)-4-oxobut-2-enoate (1), reveal that 1 is converted into the corresponding CoA adduct in S. aureus cells, and that this adduct binds to the S. aureus MenB (saMenB) with a Kd value of 2 µM. The antibacterial spectrum of 1 is limited to bacteria that utilize MK for respiration, and the activity of 1 can be complemented with exogenous MK or menadione. Finally, treatment of methicillin-resistant S. aureus (MRSA) with 1 results in the small colony variant phenotype and thus 1 phenocopies knockout of the menB gene. Taken together the data indicate that the antibacterial activity of 1 results from a specific effect on MK biosynthesis. We also evaluated the in vivo efficacy of 1 using two mouse models of MRSA infection. Notably, compound 1 increased survival in a systemic infection model and resulted in a dose-dependent decrease in bacterial load in a thigh infection model, validating MenB as a target for the development of new anti-MRSA candidates. PMID:27294200

Long-Range Activation of Systemic Immunity through Peptidoglycan Diffusion in Drosophila

PubMed Central

Gendrin, Mathilde; Welchman, David P.; Poidevin, Mickael; Hervé, Mireille; Lemaitre, Bruno

2009-01-01

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient RelishE20 flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that RelishE20 flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila. PMID:20019799

Long-range activation of systemic immunity through peptidoglycan diffusion in Drosophila.

PubMed

Gendrin, Mathilde; Welchman, David P; Poidevin, Mickael; Hervé, Mireille; Lemaitre, Bruno

2009-12-01

The systemic immune response of Drosophila is known to be induced both by septic injury and by oral infection with certain bacteria, and is characterized by the secretion of antimicrobial peptides (AMPs) into the haemolymph. To investigate other possible routes of bacterial infection, we deposited Erwinia carotovora (Ecc15) on various sites of the cuticle and monitored the immune response via expression of the AMP gene Diptericin. A strong response was observed to deposition on the genital plate of males (up to 20% of a septic injury response), but not females. We show that the principal response to genital infection is systemic, but that some AMPs, particularly Defensin, are induced locally in the genital tract. At late time points we detected bacteria in the haemolymph of immune deficient Relish(E20) flies, indicating that the genital plate can be a route of entry for pathogens, and that the immune response protects flies against the progression of genital infection. The protective role of the immune response is further illustrated by our observation that Relish(E20) flies exhibit significant lethality in response to genital Ecc15 infections. We next show that a systemic immune response can be induced by deposition of the bacterial elicitor peptidoglycan (PGN), or its terminal monomer tracheal cytotoxin (TCT), on the genital plate. This immune response is downregulated by PGRP-LB and Pirk, known regulators of the Imd pathway, and can be suppressed by the overexpression of PGRP-LB in the haemolymph compartment. Finally, we provide strong evidence that TCT can activate a systemic response by crossing epithelia, by showing that radiolabelled TCT deposited on the genital plate can subsequently be detected in the haemolymph. Genital infection is thus an intriguing new model for studying the systemic immune response to local epithelial infections and a potential route of entry for naturally occurring pathogens of Drosophila.

Helicobacter pylori modulates host cell responses by CagT4SS-dependent translocation of an intermediate metabolite of LPS inner core heptose biosynthesis

PubMed Central

Faber, Eugenia; Bats, Simon H.; Murillo, Tatiana; Speidel, Yvonne; Coombs, Nina

2017-01-01

Highly virulent Helicobacter pylori cause proinflammatory signaling inducing the transcriptional activation and secretion of cytokines such as IL-8 in epithelial cells. Responsible in part for this signaling is the cag pathogenicity island (cagPAI) that codetermines the risk for pathological sequelae of an H. pylori infection such as gastric cancer. The Cag type IV secretion system (CagT4SS), encoded on the cagPAI, can translocate various molecules into cells, the effector protein CagA, peptidoglycan metabolites and DNA. Although these transported molecules are known to contribute to cellular responses to some extent, a major part of the cagPAI-induced signaling leading to IL-8 secretion remains unexplained. We report here that biosynthesis of heptose-1,7-bisphosphate (HBP), an important intermediate metabolite of LPS inner heptose core, contributes in a major way to the H. pylori cagPAI-dependent induction of proinflammatory signaling and IL-8 secretion in human epithelial cells. Mutants defective in the genes required for synthesis of HBP exhibited a more than 95% reduction of IL-8 induction and impaired CagT4SS-dependent cellular signaling. The loss of HBP biosynthesis did not abolish the ability to translocate CagA. The human cellular adaptor TIFA, which was described before to mediate HBP-dependent activity in other Gram-negative bacteria, was crucial in the cagPAI- and HBP pathway-induced responses by H. pylori in different cell types. The active metabolite was present in H. pylori lysates but not enriched in bacterial supernatants. These novel results advance our mechanistic understanding of H. pylori cagPAI-dependent signaling mediated by intracellular pattern recognition receptors. They will also allow to better dissect immunomodulatory activities by H. pylori and to improve the possibilities of intervention in cagPAI- and inflammation-driven cancerogenesis. PMID:28715499

Exogenous Indole Regulates Lipopeptide Biosynthesis in Antarctic Bacillus amyloliquefaciens Pc3.

PubMed

Ding, Lianshuai; Zhang, Song; Guo, Wenbin; Chen, Xinhua

2018-05-28

Bacillus amyloliquefaciens Pc3 was isolated from Antarctic seawater with antifungal activity. In order to investigate the metabolic regulation mechanism in the biosynthesis of lipopeptides in B. amyloliquefaciens Pc3, GC/MS-based metabolomics was used when exogenous indole was added. The intracellular metabolite profiles showed decreased asparagine, aspartic acid, glutamine, glutamic acid, threonine, valine, isoleucine, hexadecanoic acid, and octadecanoic acid in the indole-treated groups, which were involved in the biosynthesis of lipopeptides. B. amyloliquefaciens Pc3 exhibited a growth promotion, bacterial total protein increase, and lipopeptide biosynthesis inhibition upon the addition of indole. Besides this, real-time PCR analysis further revealed that the transcription of lipopeptide biosynthesis genes ituD, fenA , and srfA-A were downregulated by indole with 22.4-, 21.98-, and 26.0-fold, respectively. It therefore was speculated that as the metabolic flux of most of the amino acids and fatty acids were transferred to the synthesis of proteins and biomass, lipopeptide biosynthesis was weakened owing to the lack of precursor amino acids and fatty acids.

Lipopolysaccharide and cAMP modify placental calcitriol biosynthesis reducing antimicrobial peptides gene expression.

PubMed

Olmos-Ortiz, Andrea; García-Quiroz, Janice; Avila, Euclides; Caldiño-Soto, Felipe; Halhali, Ali; Larrea, Fernando; Díaz, Lorenza

2018-06-01

Calcitriol, the hormonal form of vitamin D 3 (VD), stimulates placental antimicrobial peptides expression; nonetheless, the regulation of calcitriol biosynthesis in the presence of bacterial products and its consequence on placental innate immunity have scarcely been addressed. We investigated how some bacterial products modify placental VD metabolism and its ability to induce antimicrobial peptides gene expression. Cultured human trophoblasts biosynthesized calcitriol only in the presence of its precursor calcidiol, a process that was inhibited by cyclic-AMP but stimulated by lipopolysaccharide (LPS). Intracrine calcitriol upregulated cathelicidin, S100A9, and β-defensins (HBDs) gene expression, while LPS further stimulated HBD2 and S100A9. Unexpectedly, LPS significantly repressed cathelicidin basal mRNA levels and drastically diminished calcidiol ability to induce it. Meanwhile, cyclic-AMP, which is used by many microbes to avoid host defenses, suppressed calcitriol biosynthesis, resulting in significant inhibition of most VD-dependent microbicidal peptides gene expression. While LPS stimulated calcitriol biosynthesis, cyclic-AMP inhibited it. LPS downregulated cathelicidin mRNA expression, whereas cyclic-AMP antagonized VD-dependent-upregulation of most antimicrobial peptides. These findings reveal LPS and cyclic-AMP involvement in dampening placental innate immunity, highlighting the importance of cyclic-AMP in the context of placental infection and suggesting its participation to facilitate bacterial survival. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Relationship between Glycolysis and Exopolysaccharide Biosynthesis in Lactococcus lactis

PubMed Central

Ramos, Ana; Boels, Ingeborg C.; de Vos, Willem M.; Santos, Helena

2001-01-01

The relationships between glucose metabolism and exopolysaccharide (EPS) production in a Lactococcus lactis strain containing the EPS gene cluster (Eps+) and in nonproducer strain MG5267 (Eps−) were characterized. The concentrations of relevant phosphorylated intermediates in EPS and cell wall biosynthetic pathways or glycolysis were determined by 31P nuclear magnetic resonance. The concentrations of two EPS precursors, UDP-glucose and UDP-galactose, were significantly lower in the Eps+ strain than in the Eps− strain. The precursors of the peptidoglycan pathway, UDP-N-acetylglucosamine and UDP-N-acetylmuramoyl-pentapeptide, were the major UDP-sugar derivatives detected in the two strains examined, but the concentration of the latter was greater in the Eps+ strain, indicating that there is competition between EPS synthesis and cell growth. An intermediate in biosynthesis of histidine and nucleotides, 5-phosphorylribose 1-pyrophosphate, accumulated at concentrations in the millimolar range, showing that the pentose phosphate pathway was operating. Fructose 1,6-bisphosphate and glucose 6-phosphate were the prominent glycolytic intermediates during exponential growth of both strains, whereas in the stationary phase the main metabolites were 3-phosphoglyceric acid, 2-phosphoglyceric acid, and phosphoenolpyruvate. The activities of relevant enzymes, such as phosphoglucose isomerase, α-phosphoglucomutase, and UDP-glucose pyrophosphorylase, were identical in the two strains. 13C enrichment on the sugar moieties of pure EPS showed that glucose 6-phosphate is the key metabolite at the branch point between glycolysis and EPS biosynthesis and ruled out involvement of the triose phosphate pool. This study provided clues for ways to enhance EPS production by genetic manipulation. PMID:11133425

Nanoconjugated vancomycin: new opportunities for the development of anti-VRSA agents

NASA Astrophysics Data System (ADS)

Prasad Chakraborty, Subhankari; Sahu, Sumanta Kumar; Mahapatra, Santanu Kar; Santra, Susmita; Bal, Manjusri; Roy, Somenath; Pramanik, Panchanan

2010-03-01

More than 90% of Staphylococcus strains are resistant to penicillin. In 1961 S. aureus developed resistance to methicillin (MRSA), invalidating almost all antibiotics, including the most potent β-lactams. Vancomycin, a glycopeptide antibiotic, was used for the treatment of MRSA in 1980. Vancomycin inhibits the bio-synthesis of peptidoglycan and the assembly of NAM-NAG-polypeptide into the growing peptidoglycan chain. Vancomycin resistant S. aureus (VRSA) first appeared in the USA in 2002. Folic acid tagged chitosan nanoparticles are used as Trojan horses to deliver vancomycin into bacterial cells. These nanoparticles are biocompatible and biodegradable semisynthetic polymers. These nanosized vehicles enhance the transport of vancomycin across epithelial surfaces and show its efficient drug action, which has been understood from studies of the minimum inhibitory concentration and minimum bactericidal concentration of nanoparticles of a chitosan derivative loaded with vancomycin. Tolerance values distinctly show that vancomycin loaded into nanoconjugate is very effective and has a strong bactericidal effect on VRSA.

Staphylococcus aureus Tissue Infection During Sepsis Is Supported by Differential Use of Bacterial or Host-Derived Lipoic Acid

PubMed Central

Alonzo, Francis

2016-01-01

To thrive in diverse environments, bacteria must shift their metabolic output in response to nutrient bioavailability. In many bacterial species, such changes in metabolic flux depend upon lipoic acid, a cofactor required for the activity of enzyme complexes involved in glycolysis, the citric acid cycle, glycine catabolism, and branched chain fatty acid biosynthesis. The requirement of lipoic acid for metabolic enzyme activity necessitates that bacteria synthesize the cofactor and/or scavenge it from environmental sources. Although use of lipoic acid is a conserved phenomenon, the mechanisms behind its biosynthesis and salvage can differ considerably between bacterial species. Furthermore, low levels of circulating free lipoic acid in mammals underscore the importance of lipoic acid acquisition for pathogenic microbes during infection. In this study, we used a genetic approach to characterize the mechanisms of lipoic acid biosynthesis and salvage in the bacterial pathogen Staphylococcus aureus and evaluated the requirements for both pathways during murine sepsis. We determined that S. aureus lipoic acid biosynthesis and salvage genes exist in an arrangement that directly links redox stress response and acetate biosynthesis genes. In addition, we found that lipoic acid salvage is dictated by two ligases that facilitate growth and lipoylation in distinct environmental conditions in vitro, but that are fully compensatory for survival in vivo. Upon infection of mice, we found that de novo biosynthesis or salvage promotes S. aureus survival in a manner that depends upon the infectious site. In addition, when both lipoic acid biosynthesis and salvage are blocked S. aureus is rendered avirulent, implying an inability to induce lipoic acid-independent metabolic programs to promote survival. Together, our results define the major pathways of lipoic acid biosynthesis and salvage in S. aureus and support the notion that bacterial nutrient acquisition schemes are instrumental

Staphylococcus aureus Tissue Infection During Sepsis Is Supported by Differential Use of Bacterial or Host-Derived Lipoic Acid.

PubMed

Zorzoli, Azul; Grayczyk, James P; Alonzo, Francis

2016-10-01

To thrive in diverse environments, bacteria must shift their metabolic output in response to nutrient bioavailability. In many bacterial species, such changes in metabolic flux depend upon lipoic acid, a cofactor required for the activity of enzyme complexes involved in glycolysis, the citric acid cycle, glycine catabolism, and branched chain fatty acid biosynthesis. The requirement of lipoic acid for metabolic enzyme activity necessitates that bacteria synthesize the cofactor and/or scavenge it from environmental sources. Although use of lipoic acid is a conserved phenomenon, the mechanisms behind its biosynthesis and salvage can differ considerably between bacterial species. Furthermore, low levels of circulating free lipoic acid in mammals underscore the importance of lipoic acid acquisition for pathogenic microbes during infection. In this study, we used a genetic approach to characterize the mechanisms of lipoic acid biosynthesis and salvage in the bacterial pathogen Staphylococcus aureus and evaluated the requirements for both pathways during murine sepsis. We determined that S. aureus lipoic acid biosynthesis and salvage genes exist in an arrangement that directly links redox stress response and acetate biosynthesis genes. In addition, we found that lipoic acid salvage is dictated by two ligases that facilitate growth and lipoylation in distinct environmental conditions in vitro, but that are fully compensatory for survival in vivo. Upon infection of mice, we found that de novo biosynthesis or salvage promotes S. aureus survival in a manner that depends upon the infectious site. In addition, when both lipoic acid biosynthesis and salvage are blocked S. aureus is rendered avirulent, implying an inability to induce lipoic acid-independent metabolic programs to promote survival. Together, our results define the major pathways of lipoic acid biosynthesis and salvage in S. aureus and support the notion that bacterial nutrient acquisition schemes are instrumental

Peptidoglycan Recycling in Gram-Positive Bacteria Is Crucial for Survival in Stationary Phase

PubMed Central

Borisova, Marina; Gaupp, Rosmarie; Duckworth, Amanda; Schneider, Alexander; Dalügge, Désirée; Mühleck, Maraike; Deubel, Denise; Unsleber, Sandra; Yu, Wenqi; Muth, Günther; Bischoff, Markus; Götz, Friedrich

2016-01-01

ABSTRACT Peptidoglycan recycling is a metabolic process by which Gram-negative bacteria reutilize up to half of their cell wall within one generation during vegetative growth. Whether peptidoglycan recycling also occurs in Gram-positive bacteria has so far remained unclear. We show here that three Gram-positive model organisms, Staphylococcus aureus, Bacillus subtilis, and Streptomyces coelicolor, all recycle the sugar N-acetylmuramic acid (MurNAc) of their peptidoglycan during growth in rich medium. They possess MurNAc-6-phosphate (MurNAc-6P) etherase (MurQ in E. coli) enzymes, which are responsible for the intracellular conversion of MurNAc-6P to N-acetylglucosamine-6-phosphate and d-lactate. By applying mass spectrometry, we observed accumulation of MurNAc-6P in MurNAc-6P etherase deletion mutants but not in either the isogenic parental strains or complemented strains, suggesting that MurQ orthologs are required for the recycling of cell wall-derived MurNAc in these bacteria. Quantification of MurNAc-6P in ΔmurQ cells of S. aureus and B. subtilis revealed small amounts during exponential growth phase (0.19 nmol and 0.03 nmol, respectively, per ml of cells at an optical density at 600 nm [OD600] of 1) but large amounts during transition (0.56 nmol and 0.52 nmol) and stationary (0.53 nmol and 1.36 nmol) phases. The addition of MurNAc to ΔmurQ cultures greatly increased the levels of intracellular MurNAc-6P in all growth phases. The ΔmurQ mutants of S. aureus and B. subtilis showed no growth deficiency in rich medium compared to the growth of the respective parental strains, but intriguingly, they had a severe survival disadvantage in late stationary phase. Thus, although peptidoglycan recycling is apparently not essential for the growth of Gram-positive bacteria, it provides a benefit for long-term survival. PMID:27729505

Sequence and transcriptional analysis of the genes responsible for curdlan biosynthesis in Agrobacterium sp. ATCC 31749 under simulated dissolved oxygen gradients conditions.

PubMed

Zhang, Hong-Tao; Zhan, Xiao-Bei; Zheng, Zhi-Yong; Wu, Jian-Rong; Yu, Xiao-Bin; Jiang, Yun; Lin, Chi-Chung

2011-07-01

Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

Lyme disease and relapsing fever Borrelia elongate through zones of peptidoglycan synthesis that mark division sites of daughter cells.

PubMed

Jutras, Brandon Lyon; Scott, Molly; Parry, Bradley; Biboy, Jacob; Gray, Joe; Vollmer, Waldemar; Jacobs-Wagner, Christine

2016-08-16

Agents that cause Lyme disease, relapsing fever, leptospirosis, and syphilis belong to the phylum Spirochaetae-a unique lineage of bacteria most known for their long, spiral morphology. Despite the relevance to human health, little is known about the most fundamental aspects of spirochete growth. Here, using quantitative microscopy to track peptidoglycan cell-wall synthesis, we found that the Lyme disease spirochete Borrelia burgdorferi displays a complex pattern of growth. B. burgdorferi elongates from discrete zones that are both spatially and temporally regulated. In addition, some peptidoglycan incorporation occurs along the cell body, with the notable exception of a large region at the poles. Newborn cells inherit a highly active zone of peptidoglycan synthesis at midcell that contributes to elongation for most of the cell cycle. Concomitant with the initiation of nucleoid separation and cell constriction, second and third zones of elongation are established at the 1/4 and 3/4 cellular positions, marking future sites of division for the subsequent generation. Positioning of elongation zones along the cell is robust to cell length variations and is relatively precise over long distances (>30 µm), suggesting that cells ‟sense" relative, as opposed to absolute, cell length to establish zones of peptidoglycan synthesis. The transition from one to three zones of peptidoglycan growth during the cell cycle is also observed in relapsing fever Borrelia. However, this mode of growth does not extend to representative species from other spirochetal genera, suggesting that this distinctive growth mode represents an evolutionary divide in the spirochete phylum.

PGRP-SD, an Extracellular Pattern-Recognition Receptor, Enhances Peptidoglycan-Mediated Activation of the Drosophila Imd Pathway.

PubMed

Iatsenko, Igor; Kondo, Shu; Mengin-Lecreulx, Dominique; Lemaitre, Bruno

2016-11-15

Activation of the innate immune response in Metazoans is initiated through the recognition of microbes by host pattern-recognition receptors. In Drosophila, diaminopimelic acid (DAP)-containing peptidoglycan from Gram-negative bacteria is detected by the transmembrane receptor PGRP-LC and by the intracellular receptor PGRP-LE. Here, we show that PGRP-SD acted upstream of PGRP-LC as an extracellular receptor to enhance peptidoglycan-mediated activation of Imd signaling. Consistent with this, PGRP-SD mutants exhibited impaired activation of the Imd pathway and increased susceptibility to DAP-type bacteria. PGRP-SD enhanced the localization of peptidoglycans to the cell surface and hence promoted signaling. Moreover, PGRP-SD antagonized the action of PGRP-LB, an extracellular negative regulator, to fine-tune the intensity of the immune response. These data reveal that Drosophila PGRP-SD functions as an extracellular receptor similar to mammalian CD14 and demonstrate that, comparable to lipopolysaccharide sensing in mammals, Drosophila relies on both intra- and extracellular receptors for the detection of bacteria. Copyright © 2016 Elsevier Inc. All rights reserved.

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly

PubMed Central

van Teeffelen, Sven; Wang, Siyuan; Furchtgott, Leon; Huang, Kerwyn Casey; Wingreen, Ned S.; Shaevitz, Joshua W.; Gitai, Zemer

2011-01-01

Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yet-unidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis. PMID:21903929

The bacterial actin MreB rotates, and rotation depends on cell-wall assembly.

PubMed

van Teeffelen, Sven; Wang, Siyuan; Furchtgott, Leon; Huang, Kerwyn Casey; Wingreen, Ned S; Shaevitz, Joshua W; Gitai, Zemer

2011-09-20

Bacterial cells possess multiple cytoskeletal proteins involved in a wide range of cellular processes. These cytoskeletal proteins are dynamic, but the driving forces and cellular functions of these dynamics remain poorly understood. Eukaryotic cytoskeletal dynamics are often driven by motor proteins, but in bacteria no motors that drive cytoskeletal motion have been identified to date. Here, we quantitatively study the dynamics of the Escherichia coli actin homolog MreB, which is essential for the maintenance of rod-like cell shape in bacteria. We find that MreB rotates around the long axis of the cell in a persistent manner. Whereas previous studies have suggested that MreB dynamics are driven by its own polymerization, we show that MreB rotation does not depend on its own polymerization but rather requires the assembly of the peptidoglycan cell wall. The cell-wall synthesis machinery thus either constitutes a novel type of extracellular motor that exerts force on cytoplasmic MreB, or is indirectly required for an as-yet-unidentified motor. Biophysical simulations suggest that one function of MreB rotation is to ensure a uniform distribution of new peptidoglycan insertion sites, a necessary condition to maintain rod shape during growth. These findings both broaden the view of cytoskeletal motors and deepen our understanding of the physical basis of bacterial morphogenesis.

Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis

PubMed Central

Tran, Anh T.; Watson, Emma E.; Pujari, Venugopal; Conroy, Trent; Dowman, Luke J.; Giltrap, Andrew M.; Pang, Angel; Wong, Weng Ruh; Linington, Roger G.; Mahapatra, Sebabrata; Saunders, Jessica; Charman, Susan A.; West, Nicholas P.; Bugg, Timothy D. H.; Tod, Julie; Dowson, Christopher G.; Roper, David I.; Crick, Dean C.; Britton, Warwick J.; Payne, Richard J.

2017-01-01

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis. PMID:28248311

Sansanmycin natural product analogues as potent and selective anti-mycobacterials that inhibit lipid I biosynthesis

NASA Astrophysics Data System (ADS)

Tran, Anh T.; Watson, Emma E.; Pujari, Venugopal; Conroy, Trent; Dowman, Luke J.; Giltrap, Andrew M.; Pang, Angel; Wong, Weng Ruh; Linington, Roger G.; Mahapatra, Sebabrata; Saunders, Jessica; Charman, Susan A.; West, Nicholas P.; Bugg, Timothy D. H.; Tod, Julie; Dowson, Christopher G.; Roper, David I.; Crick, Dean C.; Britton, Warwick J.; Payne, Richard J.

2017-03-01

Tuberculosis (TB) is responsible for enormous global morbidity and mortality, and current treatment regimens rely on the use of drugs that have been in use for more than 40 years. Owing to widespread resistance to these therapies, new drugs are desperately needed to control the TB disease burden. Herein, we describe the rapid synthesis of analogues of the sansanmycin uridylpeptide natural products that represent promising new TB drug leads. The compounds exhibit potent and selective inhibition of Mycobacterium tuberculosis, the etiological agent of TB, both in vitro and intracellularly. The natural product analogues are nanomolar inhibitors of Mtb phospho-MurNAc-pentapeptide translocase, the enzyme responsible for the synthesis of lipid I in mycobacteria. This work lays the foundation for the development of uridylpeptide natural product analogues as new TB drug candidates that operate through the inhibition of peptidoglycan biosynthesis.

Lysin Motif–Containing Proteins LYP4 and LYP6 Play Dual Roles in Peptidoglycan and Chitin Perception in Rice Innate Immunity[W][OA

PubMed Central

Liu, Bing; Li, Jian-Feng; Ao, Ying; Qu, Jinwang; Li, Zhangqun; Su, Jianbin; Zhang, Yang; Liu, Jun; Feng, Dongru; Qi, Kangbiao; He, Yanming; Wang, Jinfa; Wang, Hong-Bin

2012-01-01

Plant innate immunity relies on successful detection of microbe-associated molecular patterns (MAMPs) of invading microbes via pattern recognition receptors (PRRs) at the plant cell surface. Here, we report two homologous rice (Oryza sativa) lysin motif–containing proteins, LYP4 and LYP6, as dual functional PRRs sensing bacterial peptidoglycan (PGN) and fungal chitin. Live cell imaging and microsomal fractionation consistently revealed the plasma membrane localization of these proteins in rice cells. Transcription of these two genes could be induced rapidly upon exposure to bacterial pathogens or diverse MAMPs. Both proteins selectively bound PGN and chitin but not lipopolysaccharide (LPS) in vitro. Accordingly, silencing of either LYP specifically impaired PGN- or chitin- but not LPS-induced defense responses in rice, including reactive oxygen species generation, defense gene activation, and callose deposition, leading to compromised resistance against bacterial pathogen Xanthomonas oryzae and fungal pathogen Magnaporthe oryzae. Interestingly, pretreatment with excess PGN dramatically attenuated the alkalinization response of rice cells to chitin but not to flagellin; vice versa, pretreatment with chitin attenuated the response to PGN, suggesting that PGN and chitin engage overlapping perception components in rice. Collectively, our data support the notion that LYP4 and LYP6 are promiscuous PRRs for PGN and chitin in rice innate immunity. PMID:22872757

Impact of peptidoglycan O-acetylation on autolytic activities of the Enterococcus faecalis N-acetylglucosaminidase AtlA and N-acetylmuramidase AtlB.

PubMed

Emirian, Aurélie; Fromentin, Sophie; Eckert, Catherine; Chau, Françoise; Dubost, Lionel; Delepierre, Muriel; Gutmann, Laurent; Arthur, Michel; Mesnage, Stéphane

2009-09-17

Autolysins are potentially lethal enzymes that partially hydrolyze peptidoglycan for incorporation of new precursors and septum cleavage after cell division. Here, we explored the impact of peptidoglycan O-acetylation on the enzymatic activities of Enterococcus faecalis major autolysins, the N-acetylglucosaminidase AtlA and the N-acetylmuramidase AtlB. We constructed isogenic strains with various O-acetylation levels and used them as substrates to assay E. faecalis autolysin activities. Peptidoglycan O-acetylation had a marginal inhibitory impact on the activities of these enzymes. In contrast, removal of cell wall glycopolymers increased the AtlB activity (37-fold), suggesting that these polymers negatively control the activity of this enzyme.

PhzA/B catalyzes the formation of the tricycle in phenazine biosynthesis.

USDA-ARS?s Scientific Manuscript database

Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but man...

Purine biosynthesis is the bottleneck in trimethoprim-treated Bacillus subtilis.

PubMed

Stepanek, Jennifer Janina; Schäkermann, Sina; Wenzel, Michaela; Prochnow, Pascal; Bandow, Julia Elisabeth

2016-10-01

Trimethoprim is a folate biosynthesis inhibitor. Tetrahydrofolates are essential for the transfer of C 1 units in several biochemical pathways including purine, thymine, methionine, and glycine biosynthesis. This study addressed the effects of folate biosynthesis inhibition on bacterial physiology. Two complementary proteomic approaches were employed to analyze the response of Bacillus subtilis to trimethoprim. Acute changes in protein synthesis rates were monitored by radioactive pulse labeling of newly synthesized proteins and subsequent 2DE analysis. Changes in protein levels were detected using gel-free quantitative MS. Proteins involved in purine and histidine biosynthesis, the σ B -dependent general stress response, and sporulation were upregulated. Most prominently, the PurR-regulon required for de novo purine biosynthesis was derepressed indicating purine depletion. The general stress response was activated energy dependently and in a subpopulation of treated cultures an early onset of sporulation was observed, most likely triggered by low guanosine triphosphate levels. Supplementation of adenosine triphosphate, adenosine, and guanosine to the medium substantially decreased antibacterial activity, showing that purine depletion becomes the bottleneck in trimethoprim-treated B. subtilis. The frequently prescribed antibiotic trimethoprim causes purine depletion in B. subtilis, which can be complemented by supplementing purines to the medium. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Structure-Function Analysis of MurJ Reveals a Solvent-Exposed Cavity Containing Residues Essential for Peptidoglycan Biogenesis in Escherichia coli

PubMed Central

Butler, Emily K.; Davis, Rebecca M.; Bari, Vase; Nicholson, Paul A.

2013-01-01

Gram-negative bacteria such as Escherichia coli build a peptidoglycan (PG) cell wall in their periplasm using the precursor known as lipid II. Lipid II is a large amphipathic molecule composed of undecaprenyl diphosphate and a disaccharide-pentapeptide that PG-synthesizing enzymes use to build the PG sacculus. During PG biosynthesis, lipid II is synthesized at the cytoplasmic face of the inner membrane and then flipped across the membrane. This translocation of lipid II must be assisted by flippases thought to shield the disaccharide-pentapeptide as it crosses the hydrophobic core of the membrane. The inner membrane protein MurJ is essential for PG biogenesis and homologous to known and putative flippases of the MOP (multidrug/oligo-saccharidyl-lipid/polysaccharide) exporter superfamily, which includes flippases that translocate undecaprenyl diphosphate-linked oligosaccharides across the cytoplasmic membranes of bacteria. Consequently, MurJ has been proposed to function as the lipid II flippase in E. coli. Here, we present a three-dimensional structural model of MurJ generated by the I-TASSER server that suggests that MurJ contains a solvent-exposed cavity within the plane of the membrane. Using in vivo topological studies, we demonstrate that MurJ has 14 transmembrane domains and validate features of the MurJ structural model, including the presence of a solvent-exposed cavity within its transmembrane region. Furthermore, we present functional studies demonstrating that specific charged residues localized in the central cavity are essential for function. Together, our studies support the structural homology of MurJ to MOP exporter proteins, suggesting that MurJ might function as an essential transporter in PG biosynthesis. PMID:23935042

The SPOR Domain, a Widely Conserved Peptidoglycan Binding Domain That Targets Proteins to the Site of Cell Division.

PubMed

Yahashiri, Atsushi; Jorgenson, Matthew A; Weiss, David S

2017-07-15

Sporulation-related repeat (SPOR) domains are small peptidoglycan (PG) binding domains found in thousands of bacterial proteins. The name "SPOR domain" stems from the fact that several early examples came from proteins involved in sporulation, but SPOR domain proteins are quite diverse and contribute to a variety of processes that involve remodeling of the PG sacculus, especially with respect to cell division. SPOR domains target proteins to the division site by binding to regions of PG devoid of stem peptides ("denuded" glycans), which in turn are enriched in septal PG by the intense, localized activity of cell wall amidases involved in daughter cell separation. This targeting mechanism sets SPOR domain proteins apart from most other septal ring proteins, which localize via protein-protein interactions. In addition to SPOR domains, bacteria contain several other PG-binding domains that can exploit features of the cell wall to target proteins to specific subcellular sites. Copyright © 2017 American Society for Microbiology.

Control of Lung Inflammation by Microbiome and Peptidoglycan Recognition Protein

DTIC Science & Technology

2017-07-01

AWARD NUMBER: W81XWH-16-1-0230 TITLE: Control of Lung Inflammation by Microbiome and Peptidoglycan Recognition Protein PRINCIPAL INVESTIGATOR...the Army position, policy or decision unless so designated by other documentation. REPORT DOCUMENTATION PAGE Form Approved OMB No. 0704-0188...for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO

Life in the "old bag" yet: structure of peptidoglycan L,D-carboxypeptidases.

PubMed

Cadby, Ian T; Lovering, Andrew L

2014-07-08

In this issue of Structure, Hoyland and colleagues describe the structure of a peptidoglycan L,D-carboxypeptidase in both substrate-bound and apoenzyme forms. These studies reveal the basis for enzyme specificity and assist greatly in a field where form and function overlap. Copyright © 2014 Elsevier Ltd. All rights reserved.

Combined scanning transmission X-ray and electron microscopy for the characterization of bacterial endospores.

PubMed

Jamroskovic, Jan; Shao, Paul P; Suvorova, Elena; Barak, Imrich; Bernier-Latmani, Rizlan

2014-09-01

Endospores (also referred to as bacterial spores) are bacterial structures formed by several bacterial species of the phylum Firmicutes. Spores form as a response to environmental stress. These structures exhibit remarkable resistance to harsh environmental conditions such as exposure to heat, desiccation, and chemical oxidants. The spores include several layers of protein and peptidoglycan that surround a core harboring DNA as well as high concentrations of calcium and dipicolinic acid (DPA). A combination of scanning transmission X-ray microscopy, scanning transmission electron microscopy, and energy dispersive spectroscopy was used for the direct quantitative characterization of bacterial spores. The concentration and localization of DPA, Ca(2+) , and other elements were determined and compared for the core and cortex of spores from two distinct genera: Bacillus subtilis and Desulfotomaculum reducens. This micro-spectroscopic approach is uniquely suited for the direct study of individual bacterial spores, while classical molecular and biochemical methods access only bulk characteristics. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

PubMed Central

Verbree, Carolin T.; Dätwyler, Steven M.; Meile, Susanne; Eichenseher, Fritz; Donovan, David M.; Loessner, Martin J.

2017-01-01

ABSTRACT Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have screened a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk, using a microtiter plate-based protocol. Nine suitable PGH constructs were identified by this approach and further compared in time-kill assays for their efficacy against S. aureus in heat-treated milk. The three most active enzymes (lysostaphin, Ami2638A, and CHAPK_CWT-LST) reduced S. aureus in milk to undetectable numbers within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity in most combinations, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The most effective PGH combination completely eradicated S. aureus from milk, with no more bacteria being detected within 24 h after addition of the enzymes, corresponding to a reduction of >9 log units compared to the control. Efficacy was also retained at different inoculum levels (3 versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas both Ami2638A and lysostaphin retained their activity, reducing bacterial numbers by >3.5 log units within 3 h. IMPORTANCE Staphylococci and S. aureus in particular are a major cause of bovine mastitis, an inflammation of the mammary gland in cows associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary

Rate and topography of peptidoglycan synthesis during cell division in Escherichia coli: Concept of a leading edge

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wientjes, F.B.; Nanninga, N.

1989-06-01

The rate at which the peptidoglycan of Escherichia coli is synthesized during the division cycle was studied with two methods. One method involved synchronization of E. coli MC4100 lysA cultures by centrifugal elutriation and subsequent pulse-labeling of the synchronously growing cultures with (meso-{sup 3}H)diaminopimelic acid (({sup 3}H)Dap). The second method was autoradiography of cells pulse-labeled with ({sup 3}H)Dap. It was found that the peptidoglycan is synthesized at a more or less exponentially increasing rate during the division cycle with a slight acceleration in this rate as the cells start to constrict. Apparently, polar cap formation requires synthesis of extra surfacemore » components, presumably to accommodate for a change in the surface-to-volume ratio. Furthermore, it was found that the pool size of Dap was constant during the division cycle. Close analysis of the topography of ({sup 3}H)Dap incorporation at the constriction site revealed that constriction proceeded by synthesis of peptidoglycan at the leading edge of the invaginating cell envelope. During constriction, no reallocation of incorporation occurred, i.e., the incorporation at the leading edge remained high throughout the process of constriction. Impairment of penicillin-binding protein 3 by mutation or by the specific {beta}-lactam antibiotic furazlocillin did not affect ({sup 3}H)Dap incorporation during initiation of constriction. However, the incorporation at the constriction site was inhibited in later stages of the constriction process. It is concluded that during division at least two peptidoglycan-synthesizing systems are operating sequentially.« less

Paleoproterozoic sterol biosynthesis and the rise of oxygen

NASA Astrophysics Data System (ADS)

Gold, David A.; Caron, Abigail; Fournier, Gregory P.; Summons, Roger E.

2017-03-01

Natural products preserved in the geological record can function as ‘molecular fossils’, providing insight into organisms and physiologies that existed in the deep past. One important group of molecular fossils is the steroidal hydrocarbons (steranes), which are the diagenetic remains of sterol lipids. Complex sterols with modified side chains are unique to eukaryotes, although simpler sterols can also be synthesized by a few bacteria. Sterol biosynthesis is an oxygen-intensive process; thus, the presence of complex steranes in ancient rocks not only signals the presence of eukaryotes, but also aerobic metabolic processes. In 1999, steranes were reported in 2.7 billion year (Gyr)-old rocks from the Pilbara Craton in Australia, suggesting a long delay between photosynthetic oxygen production and its accumulation in the atmosphere (also known as the Great Oxidation Event) 2.45-2.32 Gyr ago. However, the recent reappraisal and rejection of these steranes as contaminants pushes the oldest reported steranes forward to around 1.64 Gyr ago (ref. 6). Here we use a molecular clock approach to improve constraints on the evolution of sterol biosynthesis. We infer that stem eukaryotes shared functionally modern sterol biosynthesis genes with bacteria via horizontal gene transfer. Comparing multiple molecular clock analyses, we find that the maximum marginal probability for the divergence time of bacterial and eukaryal sterol biosynthesis genes is around 2.31 Gyr ago, concurrent with the most recent geochemical evidence for the Great Oxidation Event. Our results therefore indicate that simple sterol biosynthesis existed well before the diversification of living eukaryotes, substantially predating the oldest detected sterane biomarkers (approximately 1.64 Gyr ago), and furthermore, that the evolutionary history of sterol biosynthesis is tied to the first widespread availability of molecular oxygen in the ocean-atmosphere system.

Paleoproterozoic sterol biosynthesis and the rise of oxygen.

PubMed

Gold, David A; Caron, Abigail; Fournier, Gregory P; Summons, Roger E

2017-03-16

Natural products preserved in the geological record can function as 'molecular fossils', providing insight into organisms and physiologies that existed in the deep past. One important group of molecular fossils is the steroidal hydrocarbons (steranes), which are the diagenetic remains of sterol lipids. Complex sterols with modified side chains are unique to eukaryotes, although simpler sterols can also be synthesized by a few bacteria. Sterol biosynthesis is an oxygen-intensive process; thus, the presence of complex steranes in ancient rocks not only signals the presence of eukaryotes, but also aerobic metabolic processes. In 1999, steranes were reported in 2.7 billion year (Gyr)-old rocks from the Pilbara Craton in Australia, suggesting a long delay between photosynthetic oxygen production and its accumulation in the atmosphere (also known as the Great Oxidation Event) 2.45-2.32 Gyr ago. However, the recent reappraisal and rejection of these steranes as contaminants pushes the oldest reported steranes forward to around 1.64 Gyr ago (ref. 6). Here we use a molecular clock approach to improve constraints on the evolution of sterol biosynthesis. We infer that stem eukaryotes shared functionally modern sterol biosynthesis genes with bacteria via horizontal gene transfer. Comparing multiple molecular clock analyses, we find that the maximum marginal probability for the divergence time of bacterial and eukaryal sterol biosynthesis genes is around 2.31 Gyr ago, concurrent with the most recent geochemical evidence for the Great Oxidation Event. Our results therefore indicate that simple sterol biosynthesis existed well before the diversification of living eukaryotes, substantially predating the oldest detected sterane biomarkers (approximately 1.64 Gyr ago), and furthermore, that the evolutionary history of sterol biosynthesis is tied to the first widespread availability of molecular oxygen in the ocean-atmosphere system.

Microbial Biosynthesis of Silver Nanoparticles in Different Culture Media.

PubMed

Luo, Ke; Jung, Samuel; Park, Kyu-Hwan; Kim, Young-Rok

2018-01-31

Microbial biosynthesis of metal nanoparticles has been extensively studied for the applications in biomedical sciences and engineering. However, the mechanism for their synthesis through microorganism is not completely understood. In this study, several culture media were investigated for their roles in the microbial biosynthesis of silver nanoparticles (AgNPs). The size and morphology of the synthesized AgNPs were analyzed by UV-vis spectroscopy, Fourier-transform-infrared (FT-IR), transmission electron microscopy (TEM), and dynamic light scattering (DLS). The results demonstrated that nutrient broth (NB) and Mueller-Hinton broth (MHB) among tested media effectively reduced silver ions to form AgNPs with different particle size and shape. Although the involved microorganism enhanced the reduction of silver ions, the size and shape of the particles were shown to mainly depend on the culture media. Our findings suggest that the growth media of bacterial culture play an important role in the synthesis of metallic nanoparticles with regard to their size and shape. We believe our findings would provide useful information for further exploration of microbial biosynthesis of AgNPs and their biomedical applications.

Downregulation of the Musca domestica peptidoglycan recognition protein SC (PGRP-SC) leads to overexpression of antimicrobial peptides and tardy pupation.

PubMed

Gao, Yifu; Tang, Ting; Gu, Jihai; Sun, Lingling; Gao, Xiaobin; Ma, Xianyong; Wang, Xiaochun; Liu, Fengsong; Wang, Jianhui

2015-10-01

PGRP (peptidoglycan recognition protein) is a conserved protein family that recognizes the peptidoglycan in bacterial cell wall and causes the activation of various innate immune responses. Previous studies have reported that PGRP-SCs in Drosophila dampen the activation of Immune Deficiency (Imd) pathway to microbial infection, and participate in the lifespan extension of the insects. To facilitate understanding the function of PGRP-SCs from an evolutionary angle, we identified and functionally characterized the PGRP-SC gene in the housefly Musca domestica, a species that has adapted to a septic environment much harsher than the natural habitat of Drosophila. The gene designated as MdPGRP-SC was found most abundantly expressed in the 3rd instar larvae, and is expressed at this developmental stage predominantly in the gut. MdPGRP-SC was virtually unchanged in whole larvae after a septic injury at the second larval instar, while two antimicrobial peptides (AMPs), diptericin and attacin, were upregulated in the first 24h but not later. Through dsRNA microinjection, MdPGRP-SC was knocked down by RNA interference (RNAi), and caused the significant increased expression of diptericin and attacin. The pupation of MdPGRP-SC-depleted larvae was severely suppressed compared to controls. Opposite to the expression trend of MdPGRP-SC, a spontaneous active expression of diptericin and attacin was found in pre-pupae but not in third instar larvae. Taken together, our study reveals that downregulation of MdPGRP-SC leads to the overexpression of the AMPs, and is involved in the larvae-to-pupa transition of housefly. Copyright © 2015 Elsevier Ltd. All rights reserved.

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization.

PubMed

Perdih, Andrej; Hrast, Martina; Pureber, Kaja; Barreteau, Hélène; Grdadolnik, Simona Golič; Kocjan, Darko; Gobec, Stanislav; Solmajer, Tom; Wolber, Gerhard

2015-06-01

Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/D-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/D-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8-11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

Furan-based benzene mono- and dicarboxylic acid derivatives as multiple inhibitors of the bacterial Mur ligases (MurC-MurF): experimental and computational characterization

NASA Astrophysics Data System (ADS)

Perdih, Andrej; Hrast, Martina; Pureber, Kaja; Barreteau, Hélène; Grdadolnik, Simona Golič; Kocjan, Darko; Gobec, Stanislav; Solmajer, Tom; Wolber, Gerhard

2015-06-01

Bacterial resistance to the available antibiotic agents underlines an urgent need for the discovery of novel antibacterial agents. Members of the bacterial Mur ligase family MurC-MurF involved in the intracellular stages of the bacterial peptidoglycan biosynthesis have recently emerged as a collection of attractive targets for novel antibacterial drug design. In this study, we have first extended the knowledge of the class of furan-based benzene-1,3-dicarboxylic acid derivatives by first showing a multiple MurC-MurF ligase inhibition for representatives of the extended series of this class. Steady-state kinetics studies on the MurD enzyme were performed for compound 1, suggesting a competitive inhibition with respect to ATP. To the best of our knowledge, compound 1 represents the first ATP-competitive MurD inhibitor reported to date with concurrent multiple inhibition of all four Mur ligases (MurC-MurF). Subsequent molecular dynamic (MD) simulations coupled with interaction energy calculations were performed for two alternative in silico models of compound 1 in the UMA/ d-Glu- and ATP-binding sites of MurD, identifying binding in the ATP-binding site as energetically more favorable in comparison to the UMA/ d-Glu-binding site, which was in agreement with steady-state kinetic data. In the final stage, based on the obtained MD data novel furan-based benzene monocarboxylic acid derivatives 8- 11, exhibiting multiple Mur ligase (MurC-MurF) inhibition with predominantly superior ligase inhibition over the original series, were discovered and for compound 10 it was shown to possess promising antibacterial activity against S. aureus. These compounds represent novel leads that could by further optimization pave the way to novel antibacterial agents.

Cross-Linked Peptidoglycan Mediates Lysostaphin Binding to the Cell Wall Envelope of Staphylococcus aureus†

PubMed Central

Gründling, Angelika; Schneewind, Olaf

2006-01-01

Staphylococcus simulans bv. staphylolyticus secretes lysostaphin, a bacteriocin that cleaves pentaglycine cross bridges in the cell wall of Staphylococcus aureus. The C-terminal cell wall-targeting domain (CWT) of lysostaphin is required for selective binding of this bacteriocin to S. aureus cells; however, the molecular target for this was unknown. We used purified green fluorescent protein fused to CWT (GFP-CWT) to reveal species-specific association of the reporter with staphylococci. GFP-CWT bound S. aureus cells as well as purified peptidoglycan sacculi. The addition of cross-linked murein, disaccharides linked to interconnected wall peptides, blocked GFP-CWT binding to staphylococci, whereas murein monomers or lysostaphin-solubilized cell wall fragments did not. S. aureus strain Newman variants lacking the capacity for synthesizing polysaccharide capsule (capFO), poly-N-acetylglucosamine (icaAC), lipoprotein (lgt), cell wall-anchored proteins (srtA), or the glycolipid anchor of lipoteichoic acid (ypfP) bound GFP-CWT similar to wild-type staphylococci. A tagO mutant strain, defective in the synthesis of polyribitol wall teichoic acid attached to the cell wall envelope, displayed increased GFP-CWT binding. In contrast, a femAB mutation, reducing both the amount and the length of peptidoglycan cross-linking (monoglycine cross bridges), showed a dramatic reduction in GFP-CWT binding. Thus, the CWT domain of lysostaphin directs the bacteriocin to cross-linked peptidoglycan, which also serves as the substrate for its glycyl-glycine endopeptidase domain. PMID:16547033

Cross-linked peptidoglycan mediates lysostaphin binding to the cell wall envelope of Staphylococcus aureus.

PubMed

Gründling, Angelika; Schneewind, Olaf

2006-04-01

Staphylococcus simulans bv. staphylolyticus secretes lysostaphin, a bacteriocin that cleaves pentaglycine cross bridges in the cell wall of Staphylococcus aureus. The C-terminal cell wall-targeting domain (CWT) of lysostaphin is required for selective binding of this bacteriocin to S. aureus cells; however, the molecular target for this was unknown. We used purified green fluorescent protein fused to CWT (GFP-CWT) to reveal species-specific association of the reporter with staphylococci. GFP-CWT bound S. aureus cells as well as purified peptidoglycan sacculi. The addition of cross-linked murein, disaccharides linked to interconnected wall peptides, blocked GFP-CWT binding to staphylococci, whereas murein monomers or lysostaphin-solubilized cell wall fragments did not. S. aureus strain Newman variants lacking the capacity for synthesizing polysaccharide capsule (capFO), poly-N-acetylglucosamine (icaAC), lipoprotein (lgt), cell wall-anchored proteins (srtA), or the glycolipid anchor of lipoteichoic acid (ypfP) bound GFP-CWT similar to wild-type staphylococci. A tagO mutant strain, defective in the synthesis of polyribitol wall teichoic acid attached to the cell wall envelope, displayed increased GFP-CWT binding. In contrast, a femAB mutation, reducing both the amount and the length of peptidoglycan cross-linking (monoglycine cross bridges), showed a dramatic reduction in GFP-CWT binding. Thus, the CWT domain of lysostaphin directs the bacteriocin to cross-linked peptidoglycan, which also serves as the substrate for its glycyl-glycine endopeptidase domain.

Peptidoglycan recognition proteins in Drosophila immunity.

PubMed

Kurata, Shoichiro

2014-01-01

Innate immunity is the front line of self-defense against infectious non-self in vertebrates and invertebrates. The innate immune system is mediated by germ-line encoding pattern recognition molecules (pathogen sensors) that recognize conserved molecular patterns present in the pathogens but absent in the host. Peptidoglycans (PGN) are essential cell wall components of almost all bacteria, except mycoplasma lacking a cell wall, which provides the host immune system an advantage for detecting invading bacteria. Several families of pattern recognition molecules that detect PGN and PGN-derived compounds have been indentified, and the role of PGRP family members in host defense is relatively well-characterized in Drosophila. This review focuses on the role of PGRP family members in the recognition of invading bacteria and the activation and modulation of immune responses in Drosophila. Copyright © 2013 Elsevier Ltd. All rights reserved.

Evolutionarily distinct bacteriophage endolysins featuring conserved peptidoglycan cleavage sites protect mice from MRSA infection

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus is a Gram-positive pathogen relevant for both human and animal health. With multi-drug resistant S. aureus strains becoming increasingly prevalent, alternative therapeutics are urgently needed. Bacteriophage endolysins (peptidoglycan hydrolases, PGH) are capable of killing Gra...

Killing of Staphylococci by θ-Defensins Involves Membrane Impairment and Activation of Autolytic Enzymes

PubMed Central

Wilmes, Miriam; Stockem, Marina; Bierbaum, Gabriele; Schlag, Martin; Götz, Friedrich; Tran, Dat Q.; Schaal, Justin B.; Ouellette, André J.; Selsted, Michael E.; Sahl, Hans-Georg

2014-01-01

θ-Defensins are cyclic antimicrobial peptides expressed in leukocytes of Old world monkeys. To get insight into their antibacterial mode of action, we studied the activity of RTDs (rhesus macaque θ-defensins) against staphylococci. We found that in contrast to other defensins, RTDs do not interfere with peptidoglycan biosynthesis, but rather induce bacterial lysis in staphylococci by interaction with the bacterial membrane and/or release of cell wall lytic enzymes. Potassium efflux experiments and membrane potential measurements revealed that the membrane impairment by RTDs strongly depends on the energization of the membrane. In addition, RTD treatment caused the release of Atl-derived cell wall lytic enzymes probably by interaction with membrane-bound lipoteichoic acid. Thus, the premature and uncontrolled activity of these enzymes contributes strongly to the overall killing by θ-defensins. Interestingly, a similar mode of action has been described for Pep5, an antimicrobial peptide of bacterial origin. PMID:25632351

A Molecular Description of Cellulose Biosynthesis

PubMed Central

McNamara, Joshua T.; Morgan, Jacob L.W.; Zimmer, Jochen

2016-01-01

Cellulose is the most abundant biopolymer on Earth, and certain organisms from bacteria to plants and animals synthesize cellulose as an extracellular polymer for various biological functions. Humans have used cellulose for millennia as a material and an energy source, and the advent of a lignocellulosic fuel industry will elevate it to the primary carbon source for the burgeoning renewable energy sector. Despite the biological and societal importance of cellulose, the molecular mechanism by which it is synthesized is now only beginning to emerge. On the basis of recent advances in structural and molecular biology on bacterial cellulose synthases, we review emerging concepts of how the enzymes polymerize glucose molecules, how the nascent polymer is transported across the plasma membrane, and how bacterial cellulose biosynthesis is regulated during biofilm formation. Additionally, we review evolutionary commonalities and differences between cellulose synthases that modulate the nature of the cellulose product formed. PMID:26034894

Structural studies on molecular interactions between camel peptidoglycan recognition protein, CPGRP-S, and peptidoglycan moieties N-acetylglucosamine and N-acetylmuramic acid.

PubMed

Sharma, Pradeep; Yamini, Shavait; Dube, Divya; Singh, Amar; Sinha, Mau; Dey, Sharmistha; Mitra, Dipendra K; Kaur, Punit; Sharma, Sujata; Singh, Tej P

2012-06-22

Peptidoglycan (PGN) consists of repeating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), which are cross-linked by short peptides. It is well known that PGN forms a major cell wall component of bacteria making it an important ligand for the recognition by peptidoglycan recognition proteins (PGRPs) of the host. The binding studies showed that PGN, GlcNAc, and MurNAc bind to camel PGRP-S (CPGRP-S) with affinities corresponding to dissociation constants of 1.3 × 10(-9), 2.6 × 10(-7), and 1.8 × 10(-7) M, respectively. The crystal structure determinations of the complexes of CPGRP-S with GlcNAc and MurNAc showed that the structures consist of four crystallographically independent molecules, A, B, C, and D, in the asymmetric unit that exists as A-B and C-D units of two neighboring linear polymers. The structure determinations showed that compounds GlcNAc and MurNAc bound to CPGRP-S at the same subsite in molecule C. Both GlcNAc and MurNAc form several hydrogen bonds and extensive hydrophobic interactions with protein atoms, indicating the specific nature of their bindings. Flow cytometric studies showed that PGN enhanced the secretions of TNF-α and IL-6 from human peripheral blood mononuclear cells. The introduction of CPGRP-S to the PGN-challenged cultured peripheral blood mononuclear cells reduced the expressions of proinflammatory cytokines, TNF-α and IL-6. This showed that CPGRP-S inhibited PGN-induced production of proinflammatory cytokines and down-regulated macrophage-mediated inflammation, indicating its potential applications as an antibacterial agent.

Biosynthesis of coral settlement cue tetrabromopyrrole in marine bacteria by a uniquely adapted brominase–thioesterase enzyme pair

PubMed Central

El Gamal, Abrahim; Agarwal, Vinayak; Diethelm, Stefan; Rahman, Imran; Schorn, Michelle A.; Sneed, Jennifer M.; Louie, Gordon V.; Whalen, Kristen E.; Mincer, Tracy J.; Noel, Joseph P.; Paul, Valerie J.; Moore, Bradley S.

2016-01-01

Halogenated pyrroles (halopyrroles) are common chemical moieties found in bioactive bacterial natural products. The halopyrrole moieties of mono- and dihalopyrrole-containing compounds arise from a conserved mechanism in which a proline-derived pyrrolyl group bound to a carrier protein is first halogenated and then elaborated by peptidic or polyketide extensions. This paradigm is broken during the marine pseudoalteromonad bacterial biosynthesis of the coral larval settlement cue tetrabromopyrrole (1), which arises from the substitution of the proline-derived carboxylate by a bromine atom. To understand the molecular basis for decarboxylative bromination in the biosynthesis of 1, we sequenced two Pseudoalteromonas genomes and identified a conserved four-gene locus encoding the enzymes involved in its complete biosynthesis. Through total in vitro reconstitution of the biosynthesis of 1 using purified enzymes and biochemical interrogation of individual biochemical steps, we show that all four bromine atoms in 1 are installed by the action of a single flavin-dependent halogenase: Bmp2. Tetrabromination of the pyrrole induces a thioesterase-mediated offloading reaction from the carrier protein and activates the biosynthetic intermediate for decarboxylation. Insights into the tetrabrominating activity of Bmp2 were obtained from the high-resolution crystal structure of the halogenase contrasted against structurally homologous halogenase Mpy16 that forms only a dihalogenated pyrrole in marinopyrrole biosynthesis. Structure-guided mutagenesis of the proposed substrate-binding pocket of Bmp2 led to a reduction in the degree of halogenation catalyzed. Our study provides a biogenetic basis for the biosynthesis of 1 and sets a firm foundation for querying the biosynthetic potential for the production of 1 in marine (meta)genomes. PMID:27001835

Purification, crystallization and preliminary X-ray analysis of Escherichia coli UDP-N-acetylmuramoyl:L-alanine ligase (MurC).

PubMed

Deva, Taru; Pryor, KellyAnn D; Leiting, Barbara; Baker, Edward N; Smith, Clyde A

2003-08-01

UDP-N-acetylmuramoyl:L-alanine ligase (MurC) is involved in the pathway leading from UDP-N-glucosamine to the UDP-N-acetylmuramoyl:pentapeptide unit, which is the building block for the peptidoglycan layer found in all bacterial cell walls. The pathways leading to the biosynthesis of the peptidoglycan layer are important targets for the development of novel antibiotics, since animal cells do not contain these pathways. MurC is the first of four similar ATP-dependent amide-bond ligases which share primary and tertiary structural similarities. The crystal structures of three of these have been determined by X-ray crystallography, giving insights into the binding of the carbohydrate substrate and the ATP. Diffraction-quality crystals of the enzyme MurC have been obtained in both native and selenomethionine forms and X-ray diffraction data have been collected at the Se edge at a synchrotron source. The crystals are orthorhombic, with unit-cell parameters a = 73.9, b = 93.6, c = 176.8 A, and diffraction has been observed to 2.6 A resolution.

Peptidoglycan and lipoteichoic acid, components of the streptococcal cell wall, have marked and differential effects on adhesion molecule expression and the production of reactive oxygen species in human whole blood leukocytes.

PubMed

Saetre, T; Kähler, H; Foster, S J; Lyberg, T

2000-07-01

To elucidate the pathophysiology of infections with Streptococcus pyogenes we applied flow cytometric techniques to study dose-response and time-related effects of the streptococcal cell-wall-derived components lipoteichoic acid (LTA 0.005 to 50 microg/ml) and peptidoglycan (10 and 100 microg/ml) on the expression of leukocyte adhesion molecules, the CD14 receptor, and the production of leukocyte reactive oxygen species (ROS). LTA (50 microg/ml, 1-2 h) markedly increased the expression of CD11b (approximately 5-fold), CD11c (approximately 2-fold) and CD11a. Concomitantly, CD62L was downregulated (60%). Peptidoglycan alone or in combination with LTA had little effect on adhesion molecules, except for an amplification of the downregulation of CD62L to 90%. Monocyte CD14 expression was doubled by LTA. Leukocyte ROS production was 10-fold and 5-fold increased by peptidoglycan in granulocytes and monocytes, respectively. LTA alone had no effect, while the combination of peptidoglycan with LTA doubled the increase in ROS caused by peptidoglycan. LTA and peptidoglycan had marked and differential effects: LTA caused mainly adhesion molecule modulation, whereas peptidoglycan mainly increased ROS production. These changes are important in inflammatory cell activation and recruitment, intracellular microbial killing and adverse tissue injury.

Bacterial Exopolysaccharide mediated heavy metal removal: A Review on biosynthesis, mechanism and remediation strategies.

PubMed

Gupta, Pratima; Diwan, Batul

2017-03-01

Heavy metal contamination has been recognized as a major public health risk, particularly in developing countries and their toxicological manifestations are well known. Conventional remediation strategies are either expensive or they generate toxic by-products, which adversely affect the environment. Therefore, necessity for an environmentally safe strategy motivates interest towards biological techniques. One of such most profoundly driven approach in recent times is biosorption through microbial biomass and their products. Extracellular polymeric substances are such complex blend of high molecular weight microbial (prokaryotic and eukaryotic) biopolymers. They are mainly composed of proteins, polysaccharides, uronic acids, humic substances, lipids etc. One of its essential constituent is the exopolysaccharide (EPS) released out of self defense against harsh conditions of starvation, pH and temperature, hence it displays exemplary physiological, rheological and physio-chemical properties. Its net anionic makeup allows the biopolymer to effectively sequester positively charged heavy metal ions. The polysaccharide has been expounded deeply in this article with reference to its biosynthesis and emphasizes heavy metal sorption abilities of polymer in terms of mechanism of action and remediation. It reports current investigation and strategic advancements in dealing bacterial cells and their EPS in diverse forms - mixed culture EPS, single cell EPS, live, dead or immobilized EPS. A significant scrutiny is also involved highlighting the existing challenges that still lie in the path of commercialization. The article enlightens the potential of EPS to bring about bio-detoxification of heavy metal contaminated terrestrial and aquatic systems in highly sustainable, economic and eco-friendly manner.

Pyrimidine Biosynthesis in Lactobacillus leichmannii

PubMed Central

Hutson, Judith Y.; Downing, Mancourt

1968-01-01

Tracer studies of pyrimidine biosynthesis in Lactobacillus leichmannii (ATCC 7830) indicated that, while aspartate is utilized in the usual manner, the guanido carbon of arginine, rather than carbon dioxide, is utilized as a pyrimidine precursor. The guanido carbon of arginine also contributes, to some extent, to the carbon dioxide pool utilized for purine biosynthesis. The enzyme of the first reaction leading from arginine to pyrimidines, arginine deiminase, was investigated in crude bacterial extracts. It was inhibited by thymidylic acid and purine ribonucleotides, and to a lesser extent by purine deoxynucleotides and deoxycytidylic acid. Under the assay conditions employed, a number of nucleotides had no effect on the enzyme activity of the aspartate transcarbamylase of L. leichmannii. Growth of the cells in media containing uracil, compared to growth in media without uracil, resulted in a four- to fivefold decrease in the concentrations of aspartate transcar-bamylase and dihydroorotase and a twofold increase in the concentration of arginine deiminase, as estimated from specific enzyme activity in crude extracts of the cells. A small increase in specific enzyme activity of ornithine transcarbamylase and carbamate kinase was also observed in extracts obtained from cells grown on uracil. No appreciable change in concentration of any of the five enzymes studied was detected when the cells were grown in media containing thymidine or guanylic acid. A hypothetical scheme which suggests a relationship between the control of purine and pyrimidine biosynthesis in this bacterium and which is consistent with the experimental results obtained is presented. PMID:5686000

Pneumocandin biosynthesis: involvement of a trans-selective proline hydroxylase.

PubMed

Houwaart, Stefanie; Youssar, Loubna; Hüttel, Wolfgang

2014-11-03

Echinocandins are cyclic nonribosomal hexapeptides based mostly on nonproteinogenic amino acids and displaying strong antifungal activity. Despite previous studies on their biosynthesis by fungi, the origin of three amino acids, trans-4- and trans-3-hydroxyproline, as well as trans-3-hydroxy-4-methylproline, is still unknown. Here we describe the identification, overexpression, and characterization of GloF, the first eukaryotic α-ketoglutarate/Fe(II) -dependent proline hydroxylase from the pneumocandin biosynthesis cluster of the fungus Glarea lozoyensis ATCC 74030. In in vitro transformations with L-proline, GloF generates trans-4- and trans-3-hydroxyproline simultaneously in a ratio of 8:1; the latter reaction was previously unknown for proline hydroxylase catalysis. trans-4-Methyl-L-proline is converted into the corresponding trans-3-hydroxyproline. All three hydroxyprolines required for the biosynthesis of the echinocandins pneumocandins A0 and B0 in G. lozoyensis are thus provided by GloF. Sequence analyses revealed that GloF is not related to bacterial proline hydroxylases, and none of the putative proteins with high sequence similarity in the databases has been characterized so far. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Comparative genomics study for the identification of drug and vaccine targets in Staphylococcus aureus: MurA ligase enzyme as a proposed candidate.

PubMed

Ghosh, Soma; Prava, Jyoti; Samal, Himanshu Bhusan; Suar, Mrutyunjay; Mahapatra, Rajani Kanta

2014-06-01

Now-a-days increasing emergence of antibiotic-resistant pathogenic microorganisms is one of the biggest challenges for management of disease. In the present study comparative genomics, metabolic pathways analysis and additional parameters were defined for the identification of 94 non-homologous essential proteins in Staphylococcus aureus genome. Further study prioritized 19 proteins as vaccine candidates where as druggability study reports 34 proteins suitable as drug targets. Enzymes from peptidoglycan biosynthesis, folate biosynthesis were identified as candidates for drug development. Furthermore, bacterial secretory proteins and few hypothetical proteins identified in our analysis fulfill the criteria of vaccine candidates. As a case study, we built a homology model of one of the potential drug target, MurA ligase, using MODELLER (9v12) software. The model has been further selected for in silico docking study with inhibitors from the DrugBank database. Results from this study could facilitate selection of proteins for entry into drug design and vaccine production pipelines. Copyright © 2014 Elsevier B.V. All rights reserved.

Engineering membrane and cell-wall programs for tolerance to toxic chemicals: Beyond solo genes.

PubMed

Sandoval, Nicholas R; Papoutsakis, Eleftherios T

2016-10-01

Metabolite toxicity in microbes, particularly at the membrane, remains a bottleneck in the production of fuels and chemicals. Under chemical stress, native adaptation mechanisms combat hyper-fluidization by modifying the phospholipids in the membrane. Recent work in fluxomics reveals the mechanism of how membrane damage negatively affects energy metabolism while lipidomic and transcriptomic analyses show that strains evolved to be tolerant maintain membrane fluidity under stress through a variety of mechanisms such as incorporation of cyclopropanated fatty acids, trans-unsaturated fatty acids, and upregulation of cell wall biosynthesis genes. Engineered strains with modifications made in the biosynthesis of fatty acids, peptidoglycan, and lipopolysaccharide have shown increased tolerance to exogenous stress as well as increased production of desired metabolites of industrial importance. We review recent advances in elucidation of mechanisms or toxicity and tolerance as well as efforts to engineer the bacterial membrane and cell wall. Copyright © 2016 Elsevier Ltd. All rights reserved.

Compound-specific nitrogen isotope analysis of D-alanine, L-alanine, and valine: application of diastereomer separation to delta15N and microbial peptidoglycan studies.

PubMed

Takano, Yoshinori; Chikaraishi, Yoshito; Ogawa, Nanako O; Kitazato, Hiroshi; Ohkouchi, Naohiko

2009-01-01

We have developed an analytical method to determine the compound-specific nitrogen isotope compositions of individual amino acid enantiomers using gas chromatography/combustion/isotope ratio mass spectrometry. A novel derivatization of amino acid diastereomers by optically active (R)-(-)-2-butanol or (S)-(+)-2-butanol offers two advantages for nitrogen isotope analysis. First, chromatographic chiral separation can be achieved without the use of chiral stationary-phase columns. Second, the elution order of these compounds on the chromatogram can be switched by a designated esterification reaction. We applied the method to the compound-specific nitrogen isotope analysis of D- and L-alanine in a peptidoglycan derived from the cell walls of cultured bacteria (Firmicutes and Actinobacteria; Enterococcus faecalis, Staphylococcus aureus, Staphylococcus staphylolyticus, Lactobacillus acidophilus, Bacillus subtilis, Micrococcus luteus, and Streptomyces sp.), natural whole bacterial cells (Bacillus subtilis var. natto), (pseudo)-peptidoglycan from archaea (Methanobacterium sp.), and cell wall from eukaryota (Saccharomyces cerevisiae). We observed statistically significant differences in nitrogen isotopic compositions; e.g., delta15N ( per thousand vs air) in Staphylococcus staphylolyticus for d-alanine (19.2 +/- 0.5 per thousand, n = 4) and L-alanine (21.3 +/- 0.8 per thousand, n = 4) and in Bacillus subtilis for D-alanine (6.2 +/- 0.2 per thousand, n = 3) and L-alanine (8.2 +/- 0.4 per thousand, n = 3). These results suggest that enzymatic reaction pathways, including the alanine racemase reaction, produce a nitrogen isotopic difference in amino acid enantiomers, resulting in 15N-depleted D-alanine. This method is expected to facilitate compound-specific nitrogen isotope studies of amino acid stereoisomers.

The Role of Biotin in Bacterial Physiology and Virulence: a Novel Antibiotic Target for Mycobacterium tuberculosis.

PubMed

Salaemae, Wanisa; Booker, Grant W; Polyak, Steven W

2016-04-01

Biotin is an essential cofactor for enzymes present in key metabolic pathways such as fatty acid biosynthesis, replenishment of the tricarboxylic acid cycle, and amino acid metabolism. Biotin is synthesized de novo in microorganisms, plants, and fungi, but this metabolic activity is absent in mammals, making biotin biosynthesis an attractive target for antibiotic discovery. In particular, biotin biosynthesis plays important metabolic roles as the sole source of biotin in all stages of the Mycobacterium tuberculosis life cycle due to the lack of a transporter for scavenging exogenous biotin. Biotin is intimately associated with lipid synthesis where the products form key components of the mycobacterial cell membrane that are critical for bacterial survival and pathogenesis. In this review we discuss the central role of biotin in bacterial physiology and highlight studies that demonstrate the importance of its biosynthesis for virulence. The structural biology of the known biotin synthetic enzymes is described alongside studies using structure-guided design, phenotypic screening, and fragment-based approaches to drug discovery as routes to new antituberculosis agents.

Widespread occurrence of secondary lipid biosynthesis potential in microbial lineages.

PubMed

Shulse, Christine N; Allen, Eric E

2011-01-01

Bacterial production of long-chain omega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), is constrained to a narrow subset of marine γ-proteobacteria. The genes responsible for de novo bacterial PUFA biosynthesis, designated pfaEABCD, encode large, multi-domain protein complexes akin to type I iterative fatty acid and polyketide synthases, herein referred to as "Pfa synthases". In addition to the archetypal Pfa synthase gene products from marine bacteria, we have identified homologous type I FAS/PKS gene clusters in diverse microbial lineages spanning 45 genera representing 10 phyla, presumed to be involved in long-chain fatty acid biosynthesis. In total, 20 distinct types of gene clusters were identified. Collectively, we propose the designation of "secondary lipids" to describe these biosynthetic pathways and products, a proposition consistent with the "secondary metabolite" vernacular. Phylogenomic analysis reveals a high degree of functional conservation within distinct biosynthetic pathways. Incongruence between secondary lipid synthase functional clades and taxonomic group membership combined with the lack of orthologous gene clusters in closely related strains suggests horizontal gene transfer has contributed to the dissemination of specialized lipid biosynthetic activities across disparate microbial lineages.

Phosphorylation of the Streptococcus pneumoniae cell wall biosynthesis enzyme MurC by a eukaryotic-like Ser/Thr kinase.

PubMed

Falk, Shaun P; Weisblum, Bernard

2013-03-01

Streptococcus pneumoniae contains a single Ser/Thr kinase-phosphatase pair known as StkP-PhpP. Here, we report the interaction of StkP-PhpP with S. pneumoniae UDP-N-acetylmuramoyl:L-alanine ligase, MurC, an enzyme that synthesizes an essential intermediate of the cell wall peptidoglycan pathway. Combinatorial phage display using StkP as target selected the peptide sequence YEVCGSDTVGC as an interacting partner and subsequently confirmed by ELISA. The phage peptide sequence YEVCGSDTVGC aligns closely with the MurC motif spanning S. pneumoniae amino acid coordinates 31-37. We show that MurC is phosphorylated by StkP and that phosphoMurC is dephosphorylated by PhpP. These data suggest a link between StkP-PhpP with the coordinated regulation of cell wall biosynthesis via MurC. © 2012 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.

Desleucyl-Oritavancin with a Damaged d-Ala-d-Ala Binding Site Inhibits the Transpeptidation Step of Cell-Wall Biosynthesis in Whole Cells of Staphylococcus aureus.

PubMed

Kim, Sung Joon; Singh, Manmilan; Sharif, Shasad; Schaefer, Jacob

2017-03-14

We have used solid-state nuclear magnetic resonance to characterize the exact nature of the dual mode of action of oritavancin in preventing cell-wall assembly in Staphylococcus aureus. Measurements performed on whole cells labeled selectively in vivo have established that des-N-methylleucyl-N-4-(4-fluorophenyl)benzyl-chloroeremomycin, an Edman degradation product of [ 19 F]oritavancin, which has a damaged d-Ala-d-Ala binding aglycon, is a potent inhibitor of the transpeptidase activity of cell-wall biosynthesis. The desleucyl drug binds to partially cross-linked peptidoglycan by a cleft formed between the drug aglycon and its biphenyl hydrophobic side chain. This type of binding site is present in other oritavancin-like glycopeptides, which suggests that for these drugs a similar transpeptidase inhibition occurs.

Biosynthesis and function of simple amides in Xenorhabdus doucetiae.

PubMed

Bode, Edna; He, Yue; Vo, Tien Duy; Schultz, Roland; Kaiser, Marcel; Bode, Helge B

2017-11-01

Xenorhabdus doucetiae, the bacterial symbiont of the entomopathogenic nematode Steinernema diaprepesi produces several different fatty acid amides. Their biosynthesis has been studied using a combination of analysis of gene deletions and promoter exchanges in X. doucetiae and heterologous expression of candidate genes in E. coli. While a decarboxylase is required for the formation of all observed phenylethylamides and tryptamides, the acyltransferase XrdE encoded in the xenorhabdin biosynthesis gene cluster is responsible for the formation of short chain acyl amides. Additionally, new, long-chain and cytotoxic acyl amides were identified in X. doucetiae infected insects and when X. doucetiae was grown in Galleria Instant Broth (GIB). When the bioactivity of selected amides was tested, a quorum sensing modulating activity was observed for the short chain acyl amides against the two different quorum sensing systems from Chromobacterium and Janthinobacterium. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Engineering fatty acid biosynthesis in microalgae for sustainable biodiesel.

PubMed

Blatti, Jillian L; Michaud, Jennifer; Burkart, Michael D

2013-06-01

Microalgae are a promising feedstock for biodiesel and other liquid fuels due to their fast growth rate, high lipid yields, and ability to grow in a broad range of environments. However, many microalgae achieve maximal lipid yields only under stress conditions hindering growth and providing compositions not ideal for biofuel applications. Metabolic engineering of algal fatty acid biosynthesis promises to create strains capable of economically producing fungible and sustainable biofuels. The algal fatty acid biosynthetic pathway has been deduced by homology to bacterial and plant systems, and much of our understanding is gleaned from basic studies in these systems. However, successful engineering of lipid metabolism in algae will necessitate a thorough characterization of the algal fatty acid synthase (FAS) including protein-protein interactions and regulation. This review describes recent efforts to engineer fatty acid biosynthesis toward optimizing microalgae as a biodiesel feedstock. Copyright © 2013 Elsevier Ltd. All rights reserved.

The cell shape proteins MreB and MreC control cell morphogenesis by positioning cell wall synthetic complexes.

PubMed

Divakaruni, Arun V; Baida, Cyril; White, Courtney L; Gober, James W

2007-10-01

MreB, the bacterial actin homologue, is thought to function in spatially co-ordinating cell morphogenesis in conjunction with MreC, a protein that wraps around the outside of the cell within the periplasmic space. In Caulobacter crescentus, MreC physically associates with penicillin-binding proteins (PBPs) which catalyse the insertion of intracellularly synthesized precursors into the peptidoglycan cell wall. Here we show that MreC is required for the spatial organization of components of the peptidoglycan-synthesizing holoenzyme in the periplasm and MreB directs the localization of a peptidoglycan precursor synthesis protein in the cytosol. Additionally, fluorescent vancomycin (Van-FL) labelling revealed that the bacterial cytoskeletal proteins MreB and FtsZ, as well as MreC and RodA, were required for peptidoglycan synthetic activity. MreB and FtsZ were found to be required for morphogenesis of the polar stalk. FtsZ was required for a cell cycle-regulated burst of peptidoglycan synthesis early in the cell cycle resulting in the synthesis of cross-band structures, whereas MreB was required for lengthening of the stalk. Thus, the bacterial cytoskeleton and cell shape-determining proteins such as MreC, function in concert to orchestrate the localization of cell wall synthetic complexes resulting in spatially co-ordinated and efficient peptidoglycan synthetic activity.

Exploration of the binding modes of buffalo PGRP1 receptor complexed with meso-diaminopimelic acid and lysine-type peptidoglycans by molecular dynamics simulation and free energy calculation.

PubMed

Sahoo, Bikash Ranjan; Dubey, Praveen Kumar; Goyal, Shubham; Bhoi, Gopal Krushna; Lenka, Santosh Kumar; Maharana, Jitendra; Pradhan, Sukanta Kumar; Kataria, Ranjit Singh

2014-09-05

The peptidoglycan recognition proteins (PGRPs) are the key components of innate-immunity, and are highly specific for the recognition of bacterial peptidoglycans (PGN). Among different mammalian PGRPs, the PGRP1 binds to murein PGN of Gram-positive bacteria (lysine-type) and also have bactericidal activity towards Gram-negative bacteria (diaminopimelic acid or Dap-type). Buffaloes are the major sources of milk and meat in Asian sub-continents and are highly exposed to bacterial infections. The PGRP activates the innate-immune signaling, but their studies has been confined to limited species due to lack of structural and functional information. So, to understand the structural constituents, 3D model of buffalo PGRP1 (bfPGRP1) was constructed and conformational and dynamics properties of bfPGRP1 was studied. The bfPGRP1 model highly resembled human and camel PGRP structure, and shared a highly flexible N-terminus and centrally placed L-shaped cleft. Docking simulation of muramyl-tripeptide, tetrapeptide, pentapeptide-Dap-(MTP-Dap, MTrP-Dap and MPP-Dap) and lysine-type (MTP-Lys, MTrP-Lys and MPP-Lys) in AutoDock 4.2 and ArgusLab 4.0.1 anticipated β1, α2, α4, β4, and loops connecting β1-α2, α2-β2, β3-β4 and α4-α5 as the key interacting domains. The bfPGRP1-ligand complex molecular dynamics simulation followed by free binding energy (BE) computation conceded BE values of -18.30, -35.53, -41.80, -25.03, -24.62 and -22.30 kJ mol(-1) for MTP-Dap, MTrP-Dap, MPP-Dap, MTP-Lys, MTrP-Lys and MPP-Lys, respectively. The groove-surface and key binding residues involved in PGN-Dap and Lys-type interaction intended by the molecular docking, and were also accompanied by significant BE values directed their importance in pharmacogenomics, and warrants further in vivo studies for drug targeting and immune signaling pathways exploration. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Reversed-phase high-performance liquid chromatographic method for the determination of peptidoglycan monomers and structurally related peptides and adamantyltripeptides.

PubMed

Krstanović, Marina; Frkanec, Ruza; Vranesić, Branka; Ljevaković, Durdica; Sporec, Vesna; Tomasić, Jelka

2002-06-25

The reversed-phase HPLC method using UV detection was developed for the determination of (a) immunostimulating peptidoglycan monomers represented by the basic structure GlcNAc-MurNAc-L-Ala-D-isoGln-meso-DAP(omegaNH(2))-D-Ala-D-Ala (PGM) and two more lipophilic derivatives, Boc-Tyr-PGM and (Ada-1-yl)-CH(2)-CO-PGM, (b) two diastereomeric immunostimulating adamantyltripeptides L- and D-(adamant-2-yl)-Gly-L-Ala-D-isoGln and (c) peptides obtained by the enzyme hydrolyses of peptidoglycans and related peptides. The enzymes used, N-acetylmuramyl-L-alanine amidase and an L,D-aminopeptidase are present in mammalian sera and are involved in the metabolism of peptidoglycans and related peptides. Appropriate solvent systems were chosen with regard to structure and lipophilicity of each compound. As well, different gradient systems within the same solvent system had to be applied in order to achieve satisfactory separation and retention time. HPLC separation was developed with the aim to use this method for the study of the stability of the tested compounds, the purity during preparation and isolation and for following the enzyme hydrolyses.

Bacterial actin MreB assembles in complex with cell shape protein RodZ.

PubMed

van den Ent, Fusinita; Johnson, Christopher M; Persons, Logan; de Boer, Piet; Löwe, Jan

2010-03-17

Bacterial actin homologue MreB is required for cell shape maintenance in most non-spherical bacteria, where it assembles into helical structures just underneath the cytoplasmic membrane. Proper assembly of the actin cytoskeleton requires RodZ, a conserved, bitopic membrane protein that colocalises to MreB and is essential for cell shape determination. Here, we present the first crystal structure of bacterial actin engaged with a natural partner and provide a clear functional significance of the interaction. We show that the cytoplasmic helix-turn-helix motif of Thermotoga maritima RodZ directly interacts with monomeric as well as filamentous MreB and present the crystal structure of the complex. In vitro and in vivo analyses of mutant T. maritima and Escherichia coli RodZ validate the structure and reveal the importance of the MreB-RodZ interaction in the ability of cells to propagate as rods. Furthermore, the results elucidate how the bacterial actin cytoskeleton might be anchored to the membrane to help constrain peptidoglycan synthesis in the periplasm.

The RNA Chaperone Hfq Regulates Antibiotic Biosynthesis in the Rhizobacterium Pseudomonas aeruginosa M18

PubMed Central

Wang, Guohao; Li, Sainan; Huang, Jiaofang; Wei, Xue; Li, Yaqian

2012-01-01

The rhizosphere microbe Pseudomonas aeruginosa M18 shows strong antifungal activities, mainly due to the biosynthesis of antibiotics like pyoluteorin (Plt) and phenazine-1-carboxylic acid (PCA). The ubiquitous RNA chaperone Hfq regulates bacterial virulence and stress tolerance through global posttranscriptional regulation. Here, we explored the molecular mechanism by which Hfq controls antibiotic biosynthesis in P. aeruginosa M18. The robust downregulation of Plt biosynthesis by Hfq was mediated exclusively by the posttranscriptional downregulation of the plt transcriptional activator PltR. Hfq posttranscriptionally repressed phzM expression and consequently reduced the conversion of PCA to pyocyanin. However, Hfq positively controlled the phz2 operon and PCA biosynthesis through both QscR-mediated transcriptional regulation at the promoter and an unknown regulation at the operator. Also, Hfq was shown to directly bind at the mRNA 5′ untranslated leaders of pltR, qscR, and phzM. These three negatively regulated target genes of Hfq shared a similar secondary structure with a short single-stranded AU-rich spacer (a potential Hfq-binding motif) linking two stem-loops. Taken together, these results indicate that Hfq, potentially in collaboration with unknown small noncoding RNAs (sRNAs), tightly controls antibiotic biosynthesis through both direct posttranscriptional inhibition and indirect transcriptional regulation. PMID:22427627

Use of antibody to membrane adenosine triphosphatase in the study of bacterial relatioships.

PubMed

Whiteside, T L; De Siervo, A J; Salton, M R

1971-03-01

An antiserum to Ca(2+)-activated adenosine triphosphatase from membranes of Micrococcus lysodeikticus cross-reacted in agar gels with membrane adenosine triphosphatases from other pigmented micrococci and related species. Species of Micrococcus and Sarcina showed different levels of inhibition of adenosine triphosphatase activities in heterologous reactions with antiserum. Inter- and intraspecific relationships based on the inhibition reaction were compared with an independent parameter, namely the quantitative and qualitative composition of the bacterial membrane phospholipids and fatty acids. The guanine plus cytosine contents in the deoxyribonucleic acid of the species studied correlated well with the serological cross-reactivity of adenosine triphosphatases from their membranes. The types of cross-bridges found in the peptidoglycans of these cocci were also compared with the other properties. The results suggest that an antiserum specific for a major membrane protein may be a reliable and most useful adjunct in studying bacterial serotaxonomy.

Genomic and Transcriptomic Analyses of Indole-3-Acetic Acid Biosynthesis in Diatoms

NASA Astrophysics Data System (ADS)

Lim, R.; Armbrust, V.

2016-02-01

Indole-3-acetic acid (IAA) is a major plant growth hormone and a common mediator of plant-bacterial interactions. Recently, IAA has also been found to play a role in interactions between diatoms and bacteria, with IAA production by an associated Sulfitobacter leading to increased growth rates in the marine diatom Pseudo-nitzschia multiseries. It is unclear, however, if diatoms themselves are able to synthesize IAA and whether this capability is widespread throughout Bacillariophyta. Four major tryptophan-dependent IAA biosynthesis pathways have been identified in plants and bacteria, each denoted by the first intermediate downstream of tryptophan: the indole-3-pyruvate (IPyA), tryptamine (TAM), indole-3-acetaldoxime (IAOx) and indole-3-acetamide (IAM) pathways. To investigate the possibility of IAA biosynthesis in diatoms, we first analyzed publicly available genomes of raphid pennates P. multiseries, Phaeodactylum tricornutum, Fragilariopsis cylindrus and centric Thalassiosira pseudonana for potential homologs to plant and bacterial IAA biosynthesis genes. The P. multiseries, F. cylindrus and P. tricornutum genomes encode downstream enzymes for bacterial TAM and IAM and plant IPyA pathways. The more evolutionarily ancient T. pseudonana encodes one TAM enzyme in its genome. To investigate the potential distribution of these pathways more broadly, we surveyed the transcriptomes of 11 diatom species that include representatives from all four Bacillariophyta classes. Datasets used were sequenced as part of the Marine Microbial Eukaryote Transcriptome Sequencing Project (MMETSP) and obtained from cultures maintained axenically. Transcripts associated with the TAM pathway were most frequently detected, with potential homologs to required enzymes identified in 10 of the 11 species examined. Transcripts homologous to rate-limiting IPyA enzymes were detected in six species. Only two centric and araphid pennate species expressed transcripts associated with enzymes in the

Cloning, Expression and Characterization of UDP-N-Acetylglucosamine Enolpyruvyl Transferase (MurA) from Wolbachia Endosymbiont of Human Lymphatic Filarial Parasite Brugia malayi

PubMed Central

Shahab, Mohd; Verma, Meenakshi; Pathak, Manisha; Mitra, Kalyan; Misra-Bhattacharya, Shailja

2014-01-01

Wolbachia, an endosymbiont of filarial nematode, is considered a promising target for treatment of lymphatic filariasis. Although functional characterization of the Wolbachia peptidoglycan assembly has not been fully explored, the Wolbachia genome provides evidence for coding all of the genes involved in lipid II biosynthesis, a part of peptidoglycan biosynthesis pathway. UDP-N-acetylglucosamine enolpyruvyl transferase (MurA) is one of the lipid II biosynthesis pathway enzymes and it has inevitably been recognized as an antibiotic target. In view of the vital role of MurA in bacterial viability and survival, MurA ortholog from Wolbachia endosymbiont of Brugia malayi (wBm-MurA) was cloned, expressed and purified for further molecular characterization. The enzyme kinetics and inhibition studies were undertaken using fosfomycin. wBm-MurA was found to be expressed in all the major life stages of B. malayi and was immunolocalized in Wolbachia within the microfilariae and female adults by the confocal microscopy. Sequence analysis suggests that the amino acids crucial for enzymatic activity are conserved. The purified wBm-MurA was shown to possess the EPSP synthase (3-phosphoshikimate 1-carboxyvinyltransferase) like activity at a broad pH range with optimal activity at pH 7.5 and 37°C temperature. The apparent affinity constant (K m) for the substrate UDP-N-acetylglucosamine was found to be 0.03149 mM and for phosphoenolpyruvate 0.009198 mM. The relative enzymatic activity was inhibited ∼2 fold in presence of fosfomycin. Superimposition of the wBm-MurA homology model with the structural model of Haemophilus influenzae (Hi-MurA) suggests binding of fosfomycin at the same active site. The findings suggest wBm-MurA to be a putative antifilarial drug target for screening of novel compounds. PMID:24941309

Chemotherapy of Bacterial Plasmids

DTIC Science & Technology

1979-01-29

of drug resistance is interrupted 1960 Fukasawa. Watanabe by blender treatment of mixed cultures The term " R-factor" is introduced for the 1960 M ...bacterial . -.co DNA biosynthesis (rev. in [53]). At a concentration oI I!319- M tl:2 -,- O Oil of 6.25x 106 ,l1which did not inhibit the growth A of...sensitized 96 15;! .,\\litsuhdshi. S . I~ohe. S.. Inoue. M :bi, _"). 131 (1976, those bacteria to inhibitions by ampicillin and cepha- -. ~ J.. et al .J

Functional Analysis of the Cytoskeleton Protein MreB from Chlamydophila pneumoniae

PubMed Central

Gaballah, Ahmed; Kloeckner, Anna; Otten, Christian; Sahl, Hans-Georg; Henrichfreise, Beate

2011-01-01

In rod-shaped bacteria, the bacterial actin ortholog MreB is considered to organize the incorporation of cell wall precursors into the side-wall, whereas the tubulin homologue FtsZ is known to tether incorporation of cell wall building blocks at the developing septum. For intracellular bacteria, there is no need to compensate osmotic pressure by means of a cell wall, and peptidoglycan has not been reliably detected in Chlamydiaceae. Surprisingly, a nearly complete pathway for the biosynthesis of the cell wall building block lipid II has been found in the genomes of Chlamydiaceae. In a previous study, we discussed the hypothesis that conservation of lipid II biosynthesis in cell wall-lacking bacteria may reflect the intimate molecular linkage of cell wall biosynthesis and cell division and thus an essential role of the precursor in cell division. Here, we investigate why spherical-shaped chlamydiae harbor MreB which is almost exclusively found in elongated bacteria (i.e. rods, vibrios, spirilla) whereas they lack the otherwise essential division protein FtsZ. We demonstrate that chlamydial MreB polymerizes in vitro and that polymerization is not inhibited by the blocking agent A22. As observed for MreB from Bacillus subtilis, chlamydial MreB does not require ATP for polymerization but is capable of ATP hydrolysis in phosphate release assays. Co-pelleting and bacterial two-hybrid experiments indicate that MreB from Chlamydophila (Chlamydia) pneumoniae interacts with MurF, MraY and MurG, three key components in lipid II biosynthesis. In addition, MreB polymerization is improved in the presence of MurF. Our findings suggest that MreB is involved in tethering biosynthesis of lipid II and as such may be necessary for maintaining a functional divisome machinery in Chlamydiaceae. PMID:22022378

Functional analysis of the cytoskeleton protein MreB from Chlamydophila pneumoniae.

PubMed

Gaballah, Ahmed; Kloeckner, Anna; Otten, Christian; Sahl, Hans-Georg; Henrichfreise, Beate

2011-01-01

In rod-shaped bacteria, the bacterial actin ortholog MreB is considered to organize the incorporation of cell wall precursors into the side-wall, whereas the tubulin homologue FtsZ is known to tether incorporation of cell wall building blocks at the developing septum. For intracellular bacteria, there is no need to compensate osmotic pressure by means of a cell wall, and peptidoglycan has not been reliably detected in Chlamydiaceae. Surprisingly, a nearly complete pathway for the biosynthesis of the cell wall building block lipid II has been found in the genomes of Chlamydiaceae. In a previous study, we discussed the hypothesis that conservation of lipid II biosynthesis in cell wall-lacking bacteria may reflect the intimate molecular linkage of cell wall biosynthesis and cell division and thus an essential role of the precursor in cell division. Here, we investigate why spherical-shaped chlamydiae harbor MreB which is almost exclusively found in elongated bacteria (i.e. rods, vibrios, spirilla) whereas they lack the otherwise essential division protein FtsZ. We demonstrate that chlamydial MreB polymerizes in vitro and that polymerization is not inhibited by the blocking agent A22. As observed for MreB from Bacillus subtilis, chlamydial MreB does not require ATP for polymerization but is capable of ATP hydrolysis in phosphate release assays. Co-pelleting and bacterial two-hybrid experiments indicate that MreB from Chlamydophila (Chlamydia) pneumoniae interacts with MurF, MraY and MurG, three key components in lipid II biosynthesis. In addition, MreB polymerization is improved in the presence of MurF. Our findings suggest that MreB is involved in tethering biosynthesis of lipid II and as such may be necessary for maintaining a functional divisome machinery in Chlamydiaceae.

A Crystal Structure of a Dimer of the Antibiotic Ramoplanin Illustrates Membrane Positioning and a Potential Lipid II Docking Interface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hamburger, J.; Hoertz, A; Lee, A

2009-01-01

The glycodepsipeptide antibiotic ramoplanin A2 is in late stage clinical development for the treatment of infections from Gram-positive pathogens, especially those that are resistant to first line antibiotics such as vancomycin. Ramoplanin A2 achieves its antibacterial effects by interfering with production of the bacterial cell wall; it indirectly inhibits the transglycosylases responsible for peptidoglycan biosynthesis by sequestering their Lipid II substrate. Lipid II recognition and sequestration occur at the interface between the extracellular environment and the bacterial membrane. Therefore, we determined the structure of ramoplanin A2 in an amphipathic environment, using detergents as membrane mimetics, to provide the most physiologicallymore » relevant structural context for mechanistic and pharmacological studies. We report here the X-ray crystal structure of ramoplanin A2 at a resolution of 1.4 {angstrom}. This structure reveals that ramoplanin A2 forms an intimate and highly amphipathic dimer and illustrates the potential means by which it interacts with bacterial target membranes. The structure also suggests a mechanism by which ramoplanin A2 recognizes its Lipid II ligand.« less

Structure and Function of the Bi-Directional Bacterial Flagellar Motor

PubMed Central

Morimoto, Yusuke V.; Minamino, Tohru

2014-01-01

The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor–stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor. PMID:24970213

A directed-overflow and damage-control N -glycosidase in riboflavin biosynthesis

DOE PAGES

Frelin, Océane; Huang, Lili; Hasnain, Ghulam; ...

2015-02-15

Plants and bacteria synthesize the essential human micronutrient riboflavin (vitamin B2) via the same multistep pathway. The early intermediates of this pathway are notoriously reactive, and may be overproduced in vivo because riboflavin biosynthesis enzymes lack feedback controls. Here we demonstrate disposal of riboflavin intermediates by COG3236 (DUF1768), a protein of previously unknown function that is fused to two different riboflavin pathway enzymes in plants and bacteria (RIBR and RibA, respectively). We present cheminformatic, biochemical, genetic, and genomic evidence to show that: (i) plant and bacterial COG3236 proteins cleave the N-glycosidic bond of the first two intermediates of riboflavin biosynthesis,more » yielding relatively innocuous products; (ii) certain COG3236 proteins are in a multienzyme riboflavin biosynthesis complex that gives them privileged access to riboflavin intermediates; and (iii) COG3236 action in Arabidopsis thaliana and Escherichia coli helps maintain flavin levels. We find COG3236 proteins thus illustrate two emerging principles in chemical biology: directed overflow metabolism, in which excess flux is diverted out of a pathway, and the pre-emption of damage from reactive metabolites.« less

Use of Antibody to Membrane Adenosine Triphosphatase in the Study of Bacterial Relationships1

PubMed Central

Whiteside, Theresa L.; De Siervo, August J.; Salton, Milton R. J.

1971-01-01

An antiserum to Ca2+-activated adenosine triphosphatase from membranes of Micrococcus lysodeikticus cross-reacted in agar gels with membrane adenosine triphosphatases from other pigmented micrococci and related species. Species of Micrococcus and Sarcina showed different levels of inhibition of adenosine triphosphatase activities in heterologous reactions with antiserum. Inter- and intraspecific relationships based on the inhibition reaction were compared with an independent parameter, namely the quantitative and qualitative composition of the bacterial membrane phospholipids and fatty acids. The guanine plus cytosine contents in the deoxyribonucleic acid of the species studied correlated well with the serological cross-reactivity of adenosine triphosphatases from their membranes. The types of cross-bridges found in the peptidoglycans of these cocci were also compared with the other properties. The results suggest that an antiserum specific for a major membrane protein may be a reliable and most useful adjunct in studying bacterial serotaxonomy. Images PMID:4323299

MurD enzymes: some recent developments.

PubMed

Šink, Roman; Barreteau, Hélène; Patin, Delphine; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Blanot, Didier

2013-12-01

The synthesis of the peptide stem of bacterial peptidoglycan involves four enzymes, the Mur ligases (MurC, D, E and F). Among them, MurD is responsible for the ATP-dependent addition of d-glutamic acid to UDP-MurNAc-l-Ala, a reaction which involves acyl-phosphate and tetrahedral intermediates. Like most enzymes of peptidoglycan biosynthesis, MurD constitutes an attractive target for the design and synthesis of new antibacterial agents. Escherichia coli MurD has been the first Mur ligase for which the tridimensional (3D) structure was solved. Thereafter, several co-crystal structures with different ligands or inhibitors were released. In the present review, we will deal with work performed on substrate specificity, reaction mechanism and 3D structure of E. coli MurD. Then, a part of the review will be devoted to recent work on MurD orthologs from species other than E. coli and to cellular organization of Mur ligases and in vivo regulation of the MurD activity. Finally, we will review the different classes of MurD inhibitors that have been designed and assayed to date with the hope of obtaining new antibacterial compounds.

Peptidoglycan-Sensing Receptors Trigger the Formation of Functional Amyloids of the Adaptor Protein Imd to Initiate Drosophila NF-κB Signaling.

PubMed

Kleino, Anni; Ramia, Nancy F; Bozkurt, Gunes; Shen, Yanfang; Nailwal, Himani; Huang, Jing; Napetschnig, Johanna; Gangloff, Monique; Chan, Francis Ka-Ming; Wu, Hao; Li, Jixi; Silverman, Neal

2017-10-17

In the Drosophila immune response, bacterial derived diaminopimelic acid-type peptidoglycan binds the receptors PGRP-LC and PGRP-LE, which through interaction with the adaptor protein Imd leads to activation of the NF-κB homolog Relish and robust antimicrobial peptide gene expression. PGRP-LC, PGRP-LE, and Imd each contain a motif with some resemblance to the RIP Homotypic Interaction Motif (RHIM), a domain found in mammalian RIPK proteins forming functional amyloids during necroptosis. Here we found that despite sequence divergence, these Drosophila cryptic RHIMs formed amyloid fibrils in vitro and in cells. Amyloid formation was required for signaling downstream of Imd, and in contrast to the mammalian RHIMs, was not associated with cell death. Furthermore, amyloid formation constituted a regulatable step and could be inhibited by Pirk, an endogenous feedback regulator of this pathway. Thus, diverse sequence motifs are capable of forming amyloidal signaling platforms, and the formation of these platforms may present a regulatory point in multiple biological processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Unraveling novel broad-spectrum antibacterial targets in food and waterborne pathogens using comparative genomics and protein interaction network analysis.

PubMed

Jadhav, Ankush; Shanmugham, Buvaneswari; Rajendiran, Anjana; Pan, Archana

2014-10-01

Food and waterborne diseases are a growing concern in terms of human morbidity and mortality worldwide, even in the 21st century, emphasizing the need for new therapeutic interventions for these diseases. The current study aims at prioritizing broad-spectrum antibacterial targets, present in multiple food and waterborne bacterial pathogens, through a comparative genomics strategy coupled with a protein interaction network analysis. The pathways unique and common to all the pathogens under study (viz., methane metabolism, d-alanine metabolism, peptidoglycan biosynthesis, bacterial secretion system, two-component system, C5-branched dibasic acid metabolism), identified by comparative metabolic pathway analysis, were considered for the analysis. The proteins/enzymes involved in these pathways were prioritized following host non-homology analysis, essentiality analysis, gut flora non-homology analysis and protein interaction network analysis. The analyses revealed a set of promising broad-spectrum antibacterial targets, present in multiple food and waterborne pathogens, which are essential for bacterial survival, non-homologous to host and gut flora, and functionally important in the metabolic network. The identified broad-spectrum candidates, namely, integral membrane protein/virulence factor (MviN), preprotein translocase subunits SecB and SecG, carbon storage regulator (CsrA), and nitrogen regulatory protein P-II 1 (GlnB), contributed by the peptidoglycan pathway, bacterial secretion systems and two-component systems, were also found to be present in a wide range of other disease-causing bacteria. Cytoplasmic proteins SecG, CsrA and GlnB were considered as drug targets, while membrane proteins MviN and SecB were classified as vaccine targets. The identified broad-spectrum targets can aid in the design and development of antibacterial agents not only against food and waterborne pathogens but also against other pathogens. Copyright © 2014 Elsevier B.V. All rights

Bacterial cell-wall recycling

PubMed Central

Johnson, Jarrod W.; Fisher, Jed F.; Mobashery, Shahriar

2012-01-01

Many Gram-negative and Gram-positive bacteria recycle a significant proportion of the peptidoglycan components of their cell walls during their growth and septation. In many—and quite possibly all—bacteria, the peptidoglycan fragments are recovered and recycled. While cell-wall recycling is beneficial for the recovery of resources, it also serves as a mechanism to detect cell-wall–targeting antibiotics and to regulate resistance mechanisms. In several Gram-negative pathogens, anhydro-MurNAc-peptide cell-wall fragments regulate AmpC β-lactamase induction. In some Gram-positive organisms, short peptides derived from the cell wall regulate the induction of both β-lactamase and β-lactam-resistant penicillin-binding proteins. The involvement of peptidoglycan recycling with resistance regulation suggests that inhibitors of the enzymes involved in the recycling might synergize with cell-wall-targeted antibiotics. Indeed, such inhibitors improve the potency of β-lactams in vitro against inducible AmpC β-lactamase-producing bacteria. We describe the key steps of cell-wall remodeling and recycling, the regulation of resistance mechanisms by cell-wall recycling, and recent advances toward the discovery of cell-wall recycling inhibitors. PMID:23163477

Morphology, Growth, and Size Limit of Bacterial Cells

NASA Astrophysics Data System (ADS)

Jiang, Hongyuan; Sun, Sean X.

2010-07-01

Bacterial cells utilize a living peptidoglycan network (PG) to separate the cell interior from the surroundings. The shape of the cell is controlled by PG synthesis and cytoskeletal proteins that form bundles and filaments underneath the cell wall. The PG layer also resists turgor pressure and protects the cell from osmotic shock. We argue that mechanical influences alter the chemical equilibrium of the reversible PG assembly and determine the cell shape and cell size. Using a mechanochemical approach, we show that the cell shape can be regarded as a steady state of a growing network under the influence of turgor pressure and mechanical stress. Using simple elastic models, we predict the size of common spherical and rodlike bacteria. The influence of cytoskeletal bundles such as crescentin and MreB are discussed within the context of our model.

Dislocation-mediated growth of bacterial cell walls

PubMed Central

Amir, Ariel; Nelson, David R.

2012-01-01

Recent experiments have illuminated a remarkable growth mechanism of rod-shaped bacteria: proteins associated with cell wall extension move at constant velocity in circles oriented approximately along the cell circumference [Garner EC, et al., (2011) Science 333:222–225], [Domínguez-Escobar J, et al. (2011) Science 333:225–228], [van Teeffelen S, et al. (2011) PNAS 108:15822–15827]. We view these as dislocations in the partially ordered peptidoglycan structure, activated by glycan strand extension machinery, and study theoretically the dynamics of these interacting defects on the surface of a cylinder. Generation and motion of these interacting defects lead to surprising effects arising from the cylindrical geometry, with important implications for growth. We also discuss how long range elastic interactions and turgor pressure affect the dynamics of the fraction of actively moving dislocations in the bacterial cell wall. PMID:22660931

Anaerobic biosynthesis of the lower ligand of vitamin B12

PubMed Central

Hazra, Amrita B.; Han, Andrew W.; Mehta, Angad P.; Mok, Kenny C.; Osadchiy, Vadim; Begley, Tadhg P.; Taga, Michiko E.

2015-01-01

Vitamin B12 (cobalamin) is required by humans and other organisms for diverse metabolic processes, although only a subset of prokaryotes is capable of synthesizing B12 and other cobamide cofactors. The complete aerobic and anaerobic pathways for the de novo biosynthesis of B12 are known, with the exception of the steps leading to the anaerobic biosynthesis of the lower ligand, 5,6-dimethylbenzimidazole (DMB). Here, we report the identification and characterization of the complete pathway for anaerobic DMB biosynthesis. This pathway, identified in the obligate anaerobic bacterium Eubacterium limosum, is composed of five previously uncharacterized genes, bzaABCDE, that together direct DMB production when expressed in anaerobically cultured Escherichia coli. Expression of different combinations of the bza genes revealed that 5-hydroxybenzimidazole, 5-methoxybenzimidazole, and 5-methoxy-6-methylbenzimidazole, all of which are lower ligands of cobamides produced by other organisms, are intermediates in the pathway. The bza gene content of several bacterial and archaeal genomes is consistent with experimentally determined structures of the benzimidazoles produced by these organisms, indicating that these genes can be used to predict cobamide structure. The identification of the bza genes thus represents the last remaining unknown component of the biosynthetic pathway for not only B12 itself, but also for three other cobamide lower ligands whose biosynthesis was previously unknown. Given the importance of cobamides in environmental, industrial, and human-associated microbial metabolism, the ability to predict cobamide structure may lead to an improved ability to understand and manipulate microbial metabolism. PMID:26246619

Enhanced staphylolytic activity of the Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 HydH5 virion associated peptidoglycan hydrolase: fusions, deletions and synergy with LysH5

USDA-ARS?s Scientific Manuscript database

Virion-associated peptidoglycan hydrolases have a potential as antimicrobial agents due to their ability to lyse Gram positive bacteria on contact. In this work, our aim was to improve the lytic activity of HydH5, a virion associated peptidoglycan hydrolase from the Staphylococcus aureus bacteriopha...

Structure and function of the bi-directional bacterial flagellar motor.

PubMed

Morimoto, Yusuke V; Minamino, Tohru

2014-02-18

The bacterial flagellum is a locomotive organelle that propels the bacterial cell body in liquid environments. The flagellum is a supramolecular complex composed of about 30 different proteins and consists of at least three parts: a rotary motor, a universal joint, and a helical filament. The flagellar motor of Escherichia coli and Salmonella enterica is powered by an inward-directed electrochemical potential difference of protons across the cytoplasmic membrane. The flagellar motor consists of a rotor made of FliF, FliG, FliM and FliN and a dozen stators consisting of MotA and MotB. FliG, FliM and FliN also act as a molecular switch, enabling the motor to spin in both counterclockwise and clockwise directions. Each stator is anchored to the peptidoglycan layer through the C-terminal periplasmic domain of MotB and acts as a proton channel to couple the proton flow through the channel with torque generation. Highly conserved charged residues at the rotor-stator interface are required not only for torque generation but also for stator assembly around the rotor. In this review, we will summarize our current understanding of the structure and function of the proton-driven bacterial flagellar motor.

Harnessing cell-to-cell variations to probe bacterial structure and biophysics

NASA Astrophysics Data System (ADS)

Cass, Julie A.

Advances in microscopy and biotechnology have given us novel insights into cellular biology and physics. While bacteria were long considered to be relatively unstructured, the development of fluorescence microscopy techniques, and spatially and temporally resolved high-throughput quantitative studies, have uncovered that the bacterial cell is highly organized, and its structure rigorously maintained. In this thesis I will describe our gateTool software, designed to harness cell-to-cell variations to probe bacterial structure, and discuss two exciting aspects of structure that we have employed gateTool to investigate: (i) chromosome organization and the cellular mechanisms for controlling DNA dynamics, and (ii) the study of cell wall synthesis, and how the genes in the synthesis pathway impact cellular shape. In the first project, we develop a spatial and temporal mapping of cell-cycle-dependent chromosomal organization, and use this quantitative map to discover that chromosomal loci segregate from midcell with universal dynamics. In the second project, I describe preliminary time- lapse and snapshot imaging analysis suggesting phentoypical coherence across peptidoglycan synthesis pathways.

Corrected and Republished from: Identification of Peptidoglycan Hydrolase Constructs with Synergistic Staphylolytic Activity in Cow's Milk

PubMed Central

Verbree, Carolin T.; Dätwyler, Steven M.; Meile, Susanne; Eichenseher, Fritz; Donovan, David M.; Loessner, Martin J.

2017-01-01

ABSTRACT Peptidoglycan hydrolases (PGHs) have been suggested as novel therapeutics for the treatment of bovine mastitis. However, activity in the presence of cow's milk is an important requirement for drugs administered into the bovine udder. We have used a microtiter plate-based protocol to screen a library of >170 recombinant PGHs, including engineered bacteriophage endolysins, for enzymes with activity against Staphylococcus aureus in milk. Eight suitable PGH constructs were identified by this approach, and their efficacies against S. aureus in heat-treated milk were compared by time-kill assays. The two most active enzymes (lysostaphin and CHAPK_CWT-LST) reduced S. aureus numbers in milk to undetectable levels within minutes at nanomolar concentrations. Due to their different peptidoglycan cleavage sites, these PGH constructs revealed synergistic activity, as demonstrated by checkerboard assays, spot assays, and time-kill experiments. Furthermore, they proved active against a selection of staphylococcal mastitis isolates from different geographical regions when applied individually or in synergistic combination. The PGH combination completely eradicated S. aureus from milk: no more bacteria were detected within 24 h after the addition of the enzymes, corresponding to a reduction of >9 log units from the level in the control. Efficacy was also retained at different inoculum levels (3 log versus 6 log CFU/ml) and when S. aureus was grown in milk as opposed to broth prior to the experiments. In raw cow's milk, CHAPK_CWT-LST showed reduced efficacy, whereas lysostaphin retained its activity, reducing bacterial numbers by >3.5 log units within 3 h. IMPORTANCE Staphylococci, and S. aureus in particular, are a major cause of bovine mastitis, an inflammation of the mammary gland in cows that is associated with high costs and risks for consumers of milk products. S. aureus-induced mastitis, commonly treated by intramammary infusion of antibiotics, is characterized by

Modeling central metabolism and energy biosynthesis across microbial life.

PubMed

Edirisinghe, Janaka N; Weisenhorn, Pamela; Conrad, Neal; Xia, Fangfang; Overbeek, Ross; Stevens, Rick L; Henry, Christopher S

2016-08-08

Automatically generated bacterial metabolic models, and even some curated models, lack accuracy in predicting energy yields due to poor representation of key pathways in energy biosynthesis and the electron transport chain (ETC). Further compounding the problem, complex interlinking pathways in genome-scale metabolic models, and the need for extensive gapfilling to support complex biomass reactions, often results in predicting unrealistic yields or unrealistic physiological flux profiles. To overcome this challenge, we developed methods and tools ( http://coremodels.mcs.anl.gov ) to build high quality core metabolic models (CMM) representing accurate energy biosynthesis based on a well studied, phylogenetically diverse set of model organisms. We compare these models to explore the variability of core pathways across all microbial life, and by analyzing the ability of our core models to synthesize ATP and essential biomass precursors, we evaluate the extent to which the core metabolic pathways and functional ETCs are known for all microbes. 6,600 (80 %) of our models were found to have some type of aerobic ETC, whereas 5,100 (62 %) have an anaerobic ETC, and 1,279 (15 %) do not have any ETC. Using our manually curated ETC and energy biosynthesis pathways with no gapfilling at all, we predict accurate ATP yields for nearly 5586 (70 %) of the models under aerobic and anaerobic growth conditions. This study revealed gaps in our knowledge of the central pathways that result in 2,495 (30 %) CMMs being unable to produce ATP under any of the tested conditions. We then established a methodology for the systematic identification and correction of inconsistent annotations using core metabolic models coupled with phylogenetic analysis. We predict accurate energy yields based on our improved annotations in energy biosynthesis pathways and the implementation of diverse ETC reactions across the microbial tree of life. We highlighted missing annotations that were essential to

Regulation of transcription by eukaryotic-like serine-threonine kinases and phosphatases in Gram-positive bacterial pathogens

PubMed Central

Wright, David P; Ulijasz, Andrew T

2014-01-01

Bacterial eukaryotic-like serine threonine kinases (eSTKs) and serine threonine phosphatases (eSTPs) have emerged as important signaling elements that are indispensable for pathogenesis. Differing considerably from their histidine kinase counterparts, few eSTK genes are encoded within the average bacterial genome, and their targets are pleiotropic in nature instead of exclusive. The growing list of important eSTK/P substrates includes proteins involved in translation, cell division, peptidoglycan synthesis, antibiotic tolerance, resistance to innate immunity and control of virulence factors. Recently it has come to light that eSTK/Ps also directly modulate transcriptional machinery in many microbial pathogens. This novel form of regulation is now emerging as an additional means by which bacteria can alter their transcriptomes in response to host-specific environmental stimuli. Here we focus on the ability of eSTKs and eSTPs in Gram-positive bacterial pathogens to directly modulate transcription, the known mechanistic outcomes of these modifications, and their roles as an added layer of complexity in controlling targeted RNA synthesis to enhance virulence potential. PMID:25603430

Gene Transfers Shaped the Evolution of De Novo NAD+ Biosynthesis in Eukaryotes

PubMed Central

Ternes, Chad M.; Schönknecht, Gerald

2014-01-01

NAD+ is an essential molecule for life, present in each living cell. It can function as an electron carrier or cofactor in redox biochemistry and energetics, and serves as substrate to generate the secondary messenger cyclic ADP ribose and nicotinic acid adenine dinucleotide phosphate. Although de novo NAD+ biosynthesis is essential, different metabolic pathways exist in different eukaryotic clades. The kynurenine pathway starting with tryptophan was most likely present in the last common ancestor of all eukaryotes, and is active in fungi and animals. The aspartate pathway, detected in most photosynthetic eukaryotes, was probably acquired from the cyanobacterial endosymbiont that gave rise to chloroplasts. An evolutionary analysis of enzymes catalyzing de novo NAD+ biosynthesis resulted in evolutionary trees incongruent with established organismal phylogeny, indicating numerous gene transfers. Endosymbiotic gene transfers probably introduced the aspartate pathway into eukaryotes and may have distributed it among different photosynthetic clades. In addition, several horizontal gene transfers substituted eukaryotic genes with bacterial orthologs. Although horizontal gene transfer is accepted as a key mechanism in prokaryotic evolution, it is supposed to be rare in eukaryotic evolution. The essential metabolic pathway of de novo NAD+ biosynthesis in eukaryotes was shaped by numerous gene transfers. PMID:25169983

Bacterial Actins.

PubMed

Izoré, Thierry; van den Ent, Fusinita

2017-01-01

A diverse set of protein polymers, structurally related to actin filaments contributes to the organization of bacterial cells as cytomotive or cytoskeletal filaments. This chapter describes actin homologs encoded by bacterial chromosomes. MamK filaments, unique to magnetotactic bacteria, help establishing magnetic biological compasses by interacting with magnetosomes. Magnetosomes are intracellular membrane invaginations containing biomineralized crystals of iron oxide that are positioned by MamK along the long-axis of the cell. FtsA is widespread across bacteria and it is one of the earliest components of the divisome to arrive at midcell, where it anchors the cell division machinery to the membrane. FtsA binds directly to FtsZ filaments and to the membrane through its C-terminus. FtsA shows altered domain architecture when compared to the canonical actin fold. FtsA's subdomain 1C replaces subdomain 1B of other members of the actin family and is located on the opposite side of the molecule. Nevertheless, when FtsA assembles into protofilaments, the protofilament structure is preserved, as subdomain 1C replaces subdomain IB of the following subunit in a canonical actin filament. MreB has an essential role in shape-maintenance of most rod-shaped bacteria. Unusually, MreB filaments assemble from two protofilaments in a flat and antiparallel arrangement. This non-polar architecture implies that both MreB filament ends are structurally identical. MreB filaments bind directly to membranes where they interact with both cytosolic and membrane proteins, thereby forming a key component of the elongasome. MreB filaments in cells are short and dynamic, moving around the long axis of rod-shaped cells, sensing curvature of the membrane and being implicated in peptidoglycan synthesis.

Purification, crystallization and preliminary X-ray diffraction analysis of GatD, a glutamine amidotransferase-like protein from Staphylococcus aureus peptidoglycan

PubMed Central

Vieira, Diana; Figueiredo, Teresa A.; Verma, Anil; Sobral, Rita G.; Ludovice, Ana M.; de Lencastre, Hermínia; Trincao, Jose

2014-01-01

Amidation of peptidoglycan is an essential feature in Staphylococcus aureus that is necessary for resistance to β-lactams and lysozyme. GatD, a 27 kDa type I glutamine amidotransferase-like protein, together with MurT ligase, catalyses the amidation reaction of the glutamic acid residues of the peptidoglycan of S. aureus. The native and the selenomethionine-derivative proteins were crystallized using the sitting-drop vapour-diffusion method with polyethylene glycol, sodium acetate and calcium acetate. The crystals obtained diffracted beyond 1.85 and 2.25 Å, respectively, and belonged to space group P212121. X-ray diffraction data sets were collected at Diamond Light Source (on beamlines I02 and I04) and were used to obtain initial phases. PMID:24817726

Phenotypic and genotypic characterization of peptidoglycan hydrolases of Lactobacillus sakei

PubMed Central

Najjari, Afef; Amairi, Houda; Chaillou, Stéphane; Mora, Diego; Boudabous, Abdellatif; Zagorec, Monique; Ouzari, Hadda

2015-01-01

Lactobacillus sakei, a lactic acid bacterium naturally found in fresh meat and sea products, is considered to be one of the most important bacterial species involved in meat fermentation and bio-preservation. Several enzymes of Lb. sakei species contributing to microbial safeguarding and organoleptic properties of fermented-meat were studied. However, the specific autolytic mechanisms and associated enzymes involved in Lb. sakei are not well understood. The autolytic phenotype of 22 Lb. sakei strains isolated from Tunisian meat and seafood products was evaluated under starvation conditions, at pH 6.5 and 8.5, and in the presence of different carbon sources. A higher autolytic rate was observed when cells were grown in the presence of glucose and incubated at pH 6.5. Almost all strains showed high resistance to mutanolysin, indicating a minor role of muramidases in Lb. sakei cell lysis. Using Micrococcus lysodeikticus cells as a substrate in activity gels zymogram, peptidoglycan hydrolase (PGH) patterns for all strains was characterized by two lytic bands of ∼80 (B1) and ∼70 kDa (B2), except for strain BMG.167 which harbored two activity signals at a lower MW. Lytic activity was retained in high salt and in acid/basic conditions and was active toward cells of Lb. sakei, Listeria monocytogenes, Listeria ivanovii and Listeria innocua. Analysis of five putative PGH genes found in the Lb. sakei 23 K model strain genome, indicated that one gene, lsa1437, could encode a PGH (N-acetylmuramoyl-L-alanine amidase) containing B1 and B2 as isoforms. According to this hypothesis, strain BMG.167 showed an allelic version of lsa1437 gene deleted of one of the five LysM domains, leading to a reduction in the MW of lytic bands and the high autolytic rate of this strain. Characterization of autolytic phenotype of Lb. sakei should expand the knowledge of their role in fermentation processes where they represent the dominant species. PMID:26843981

The disruptive effect of lysozyme on the bacterial cell wall explored by an in-silico structural outlook.

PubMed

Primo, Emiliano D; Otero, Lisandro H; Ruiz, Francisco; Klinke, Sebastián; Giordano, Walter

2018-01-01

The bacterial cell wall, a structural unit of peptidoglycan polymer comprised of glycan strands consisting of a repeating disaccharide motif [N-acetylglucosamine (NAG) and N-acetylmuramylpentapeptide (NAM pentapeptide)], encases bacteria and provides structural integrity and protection. Lysozymes are enzymes that break down the bacterial cell wall and disrupt the bacterial life cycle by cleaving the linkage between the NAG and NAM carbohydrates. Lab exercises focused on the effects of lysozyme on the bacterial cell wall are frequently incorporated in biochemistry classes designed for undergraduate students in diverse fields as biology, microbiology, chemistry, agronomy, medicine, and veterinary medicine. Such exercises typically do not include structural data. We describe here a sequence of computer tasks designed to illustrate and reinforce both physiological and structural concepts involved in lysozyme effects on the bacterial cell-wall structure. This lab class usually lasts 3.5 hours. First, the instructor presents introductory concepts of the bacterial cell wall and the effect of lysozyme on its structure. Then, students are taught to use computer modeling to visualize the three-dimensional structure of a lysozyme in complex with bacterial cell-wall fragments. Finally, the lysozyme inhibitory effect on a bacterial culture is optionally proposed as a simple microbiological assay. The computer lab exercises described here give students a realistic understanding of the disruptive effect of lysozymes on the bacterial cell wall, a crucial component in bacterial survival. © 2017 by The International Union of Biochemistry and Molecular Biology, 46(1):83-90, 2018. © 2017 The International Union of Biochemistry and Molecular Biology.

Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1).

PubMed

Demidenko, Aleksandr; Akberdin, Ilya R; Allemann, Marco; Allen, Eric E; Kalyuzhnaya, Marina G

2016-01-01

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1) . Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE , was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE -knockout mutants and farE -overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE -strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.

Fatty acid biosynthesis pathways in Methylomicrobium buryatense 5G(B1)

DOE PAGES

Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; ...

2017-01-10

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fattymore » acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of FA transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for FA-biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the FA profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. As a result, the gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.« less

Fatty acid biosynthesis pathways in Methylomicrobium buryatense 5G(B1)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fattymore » acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of FA transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for FA-biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the FA profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. As a result, the gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph.« less

Fatty Acid Biosynthesis Pathways in Methylomicrobium buryatense 5G(B1)

PubMed Central

Demidenko, Aleksandr; Akberdin, Ilya R.; Allemann, Marco; Allen, Eric E.; Kalyuzhnaya, Marina G.

2017-01-01

Methane utilization by methanotrophic bacteria is an attractive application for biotechnological conversion of natural or biogas into high-added-value products. Haloalcaliphilic methanotrophic bacteria belonging to the genus Methylomicrobium are among the most promising strains for methane-based biotechnology, providing easy and inexpensive cultivation, rapid growth, and the availability of established genetic tools. A number of methane bioconversions using these microbial cultures have been discussed, including the derivation of biodiesel, alkanes, and OMEGA-3 supplements. These compounds are derived from bacterial fatty acid pools. Here, we investigate fatty acid biosynthesis in Methylomicrobium buryatense 5G(B1). Most of the genes homologous to typical Type II fatty acid biosynthesis pathways could be annotated by bioinformatics analyses, with the exception of fatty acid transport and regulatory elements. Different approaches for improving fatty acid accumulation were investigated. These studies indicated that both fatty acid degradation and acetyl- and malonyl-CoA levels are bottlenecks for higher level fatty acid production. The best strain generated in this study synthesizes 111 ± 2 mg/gDCW of extractable fatty acids, which is ~20% more than the original strain. A candidate gene for fatty acid biosynthesis regulation, farE, was identified and studied. Its deletion resulted in drastic changes to the fatty acid profile, leading to an increased pool of C18-fatty acid methyl ester. The FarE-regulon was further investigated by RNA-seq analysis of gene expression in farE-knockout mutants and farE-overexpressing strains. These gene profiles highlighted a novel set of enzymes and regulators involved in fatty acid biosynthesis. The gene expression and fatty acid profiles of the different farE-strains support the hypothesis that metabolic fluxes upstream of fatty acid biosynthesis restrict fatty acid production in the methanotroph. PMID:28119683

Potential of the virion-associated peptidoglycan hydrolase HydH5 and its derivative fusion proteins in milk biopreservation

USDA-ARS?s Scientific Manuscript database

Bacteriophage lytic enzymes have recently attracted considerable interest as novel antimicrobials against Gram-positive bacteria. In this work, antimicrobial activity in milk of HydH5 [(a virion-associated peptidoglycan hydrolase (VAPGH) encoded by the Staphylococcus aureus bacteriophage vB_SauS-ph...

Bacterial Fimbriae and Their Peptides Activate Human Gingival Epithelial Cells through Toll-Like Receptor 2

PubMed Central

Asai, Yasuyuki; Ohyama, Yoshinori; Gen, Keika; Ogawa, Tomohiko

2001-01-01

Gingival epithelial cells are a central component of the barrier between oral microflora and internal tissues. Host responses to periodontopathic bacteria and surface components containing fimbriae are thought to be important in the development and progression of periodontal diseases. To elucidate this mechanism, we established immortalized human gingival epithelial cells (HGEC) that were transfected with human papillomavirus. HGEC predominantly expressed Toll-like receptor (TLR) 2, but not TLR4 or CD14. They also induced interleukin-8 (IL-8) production when stimulated with Porphyromonas gingivalis fimbriae and Staphylococcus aureus peptidoglycan, but not Escherichia coli-type synthetic lipid A. Furthermore, an active synthetic peptide composed of residues 69 to 73 (ALTTE) of the fimbrial subunit protein, derived from P. gingivalis and similar to a common component of cell wall peptidoglycans in parasitic bacteria, N-acetylmuramyl-l-alanyl-d-isoglutamine (MDP), significantly induced IL-8 production and NF-κB activation in HGEC, and these cytokine-producing activities were augmented by a complex of soluble CD14 and lipopolysaccharide-binding protein (LBP). IL-8 production in HGEC stimulated with these bacterial components was clearly inhibited by mouse monoclonal antibody to human TLR2. These findings suggest that P. gingivalis fimbrial protein and its active peptide are capable of activating HGEC through TLR2. PMID:11705912

Crystal Structures of the SpoIID Lytic Transglycosylases Essential for Bacterial Sporulation.

PubMed

Nocadello, Salvatore; Minasov, George; Shuvalova, Ludmilla S; Dubrovska, Ievgeniia; Sabini, Elisabetta; Anderson, Wayne F

2016-07-15

Bacterial spores are the most resistant form of life known on Earth and represent a serious problem for (i) bioterrorism attack, (ii) horizontal transmission of microbial pathogens in the community, and (iii) persistence in patients and in a nosocomial environment. Stage II sporulation protein D (SpoIID) is a lytic transglycosylase (LT) essential for sporulation. The LT superfamily is a potential drug target because it is active in essential bacterial processes involving the peptidoglycan, which is unique to bacteria. However, the absence of structural information for the sporulation-specific LT enzymes has hindered mechanistic understanding of SpoIID. Here, we report the first crystal structures with and without ligands of the SpoIID family from two community relevant spore-forming pathogens, Bacillus anthracis and Clostridium difficile. The structures allow us to visualize the overall architecture, characterize the substrate recognition model, identify critical residues, and provide the structural basis for catalysis by this new family of enzymes. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release

PubMed Central

Greene, Neil G.; Narciso, Ana R.; Filipe, Sergio R.; Camilli, Andrew

2015-01-01

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens. PMID:26114646

Peptidoglycan Branched Stem Peptides Contribute to Streptococcus pneumoniae Virulence by Inhibiting Pneumolysin Release.

PubMed

Greene, Neil G; Narciso, Ana R; Filipe, Sergio R; Camilli, Andrew

2015-06-01

Streptococcus pneumoniae (the pneumococcus) colonizes the human nasopharynx and is a significant pathogen worldwide. Pneumolysin (Ply) is a multi-functional, extracellular virulence factor produced by this organism that is critical for pathogenesis. Despite the absence of any apparent secretion or cell surface attachment motifs, Ply localizes to the cell envelope of actively growing cells. We sought to characterize the consequences of this surface localization. Through functional assays with whole cells and subcellular fractions, we determined that Ply activity and its release into the extracellular environment are inhibited by peptidoglycan (PG) structure. The ability of PG to inhibit Ply release was dependent on the stem peptide composition of this macromolecule, which was manipulated by mutation of the murMN operon that encodes proteins responsible for branched stem peptide synthesis. Additionally, removal of choline-binding proteins from the cell surface significantly reduced Ply release to levels observed in a mutant with a high proportion of branched stem peptides suggesting a link between this structural feature and surface-associated choline-binding proteins involved in PG metabolism. Of clinical relevance, we also demonstrate that a hyperactive, mosaic murMN allele associated with penicillin resistance causes decreased Ply release with concomitant increases in the amount of branched stem peptides. Finally, using a murMN deletion mutant, we observed that increased Ply release is detrimental to virulence during a murine model of pneumonia. Taken together, our results reveal a novel role for branched stem peptides in pneumococcal pathogenesis and demonstrate the importance of controlled Ply release during infection. These results highlight the importance of PG composition in pathogenesis and may have broad implications for the diverse PG structures observed in other bacterial pathogens.

Fluorescence detection-based functional assay for high-throughput screening for MraY.

PubMed

Stachyra, Thérèse; Dini, Christophe; Ferrari, Paul; Bouhss, Ahmed; van Heijenoort, Jean; Mengin-Lecreulx, Dominique; Blanot, Didier; Biton, Jacques; Le Beller, Dominique

2004-03-01

We have developed a novel assay specific to MraY, which catalyzes the first membrane step in the biosynthesis of bacterial cell wall peptidoglycan. This was accomplished by using UDP-MurNAc-N(epsilon)-dansylpentapeptide, a fluorescent derivative of the MraY nucleotide substrate, and a partially purified preparation of MraY solubilized from membranes of an Escherichia coli overproducing strain. Two versions of the assay were developed, one consisting of the high-pressure liquid chromatography separation of the substrate and product (dansylated lipid I) and the other, without separation and adapted to the high-throughput format, taking advantage of the different fluorescence properties of the nucleotide and lipid I in the reaction medium. The latter assay was validated with a set of natural and synthetic MraY inhibitors.

The biology of Mur ligases as an antibacterial target.

PubMed

Kouidmi, Imène; Levesque, Roger C; Paradis-Bleau, Catherine

2014-10-01

With antibiotic resistance mechanisms increasing in diversity and spreading among bacterial pathogens, the development of new classes of antibacterial agents against judiciously chosen targets is a high-priority task. The biochemical pathway for peptidoglycan biosynthesis is one of the best sources of antibacterial targets. Within this pathway are the Mur ligases, described in this review as highly suitable targets for the development of new classes of antibacterial agents. The amide ligases MurC, MurD, MurE and MurF function with the same catalytic mechanism and share conserved amino acid regions and structural features that can conceivably be exploited for the design of inhibitors that simultaneously target more than one enzyme. This would provide multi-target antibacterial weapons with minimized likelihood of target-mediated resistance development. © 2014 John Wiley & Sons Ltd.

Design and Synthesis of New Peptidomimetics as Potential Inhibitors of MurE.

PubMed

Zivec, Matej; Turk, Samo; Blanot, Didier; Gobec, Stanislav

2011-03-01

With the continuing emergence and spread of multidrug-resistant bacteria, there is an urgent need for the development of new antimicrobial agents. One possible source of new antibacterial targets is the biosynthesis of the bacterial cell-wall peptidoglycan. The assembly of the peptide stem is carried out by four essential enzymes, known as the Mur ligases (MurC, D, E and F). We have designed and synthesised a focused library of compounds as potential inhibitors of UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:L-lysine ligase (MurE) from Staphylococcus aureus. This was achieved using two approaches: (i) synthesis of transition-state analogues based on the methyleneamino core; and (ii) synthesis of MurE reaction product analogues. Two methyleneamino-based compounds are identified as initial hits for inhibitors of MurE.

Biosynthesis of titanium dioxide nanoparticles using Bacillus amyloliquefaciens culture and enhancement of its photocatalytic activity for the degradation of a sulfonated textile dye Reactive Red 31.

PubMed

Khan, Razia; Fulekar, M H

2016-08-01

The present study aims at exploiting Bacillus amyloliquefaciens for the biosynthesis of titanium dioxide nanoparticles and also investigates role of bacterial enzymes in the biosynthesis of titanium dioxide nanoparticles. Bacterial synthesized as well as metal doped titanium dioxide nanoparticles were characterized by X-ray diffractometer (XRD), Fourier transform infrared spectroscopy (FTIR), Transmission electron microscopy (TEM), Energy dispersive X-ray spectroscopy (EDAX). Amylase activity (43.37IU) in culture supernatant evinced a potential involvement of extracellular enzyme in TiO2 nanoparticle biosynthesis. Crystallite size of bio-synthesized nanoparticles was found to be in the range of 15.23-87.6nm. FTIR spectroscopy and native-PAGE (Polyacrylamide Gel Electrophoresis) clearly indicated involvement of alpha amylase in biosynthesis of TiO2 nanoparticles and in their stabilization. TEM micrographs of the synthesized titanium dioxide nanoparticles revealed the formation of spherical nanoparticles with a size range of 22.11-97.28nm. Photocatalytic degradation of Reactive Red 31 (RR31) dye was carried out using bio-synthesized TiO2 nanoparticles under UV radiation. Photocatalytic activity of synthesized nanoparticles was enhanced by Ag, La, Zn and Pt doping. Platinum doped TiO2 showed highest potential (90.98%) in RR31 degradation as compared to undoped (75.83%). Copyright © 2016 Elsevier Inc. All rights reserved.

Rapid Inhibition Profiling in Bacillus subtilis to Identify the Mechanism of Action of New Antimicrobials.

PubMed

Lamsa, Anne; Lopez-Garrido, Javier; Quach, Diana; Riley, Eammon P; Pogliano, Joe; Pogliano, Kit

2016-08-19

Increasing antimicrobial resistance has become a major public health crisis. New antimicrobials with novel mechanisms of action (MOA) are desperately needed. We previously developed a method, bacterial cytological profiling (BCP), which utilizes fluorescence microscopy to rapidly identify the MOA of antimicrobial compounds. BCP is based upon our discovery that cells treated with antibiotics affecting different metabolic pathways generate different cytological signatures, providing quantitative information that can be used to determine a compound's MOA. Here, we describe a system, rapid inhibition profiling (RIP), for creating cytological profiles of new antibiotic targets for which there are currently no chemical inhibitors. RIP consists of the fast, inducible degradation of a target protein followed by BCP. We demonstrate that degrading essential proteins in the major metabolic pathways for DNA replication, transcription, fatty acid biosynthesis, and peptidoglycan biogenesis in Bacillus subtilis rapidly produces cytological profiles closely matching that of antimicrobials targeting the same pathways. Additionally, RIP and antibiotics targeting different steps in fatty acid biosynthesis can be differentiated from each other. We utilize RIP and BCP to show that the antibacterial MOA of four nonsteroidal anti-inflammatory antibiotics differs from that proposed based on in vitro data. RIP is a versatile method that will extend our knowledge of phenotypes associated with inactivating essential bacterial enzymes and thereby allow for screening for molecules that inhibit novel essential targets.

Genes involved in long-chain alkene biosynthesis in Micrococcus luteus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Beller, Harry R.; Goh, Ee-Been; Keasling, Jay D.

2010-01-07

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which four decades ago was reported to biosynthesize iso- and anteiso branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty-acid overproducing E. coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3more » (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-CoA produced the same C27 monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or -ACP) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (?-ketoacyl-ACP synthase III), which catalyzes decarboxylative Claisen

Genes involved in long-chain alkene biosynthesis in Micrococcus luteus.

PubMed

Beller, Harry R; Goh, Ee-Been; Keasling, Jay D

2010-02-01

Aliphatic hydrocarbons are highly appealing targets for advanced cellulosic biofuels, as they are already predominant components of petroleum-based gasoline and diesel fuels. We have studied alkene biosynthesis in Micrococcus luteus ATCC 4698, a close relative of Sarcina lutea (now Kocuria rhizophila), which 4 decades ago was reported to biosynthesize iso- and anteiso-branched, long-chain alkenes. The underlying biochemistry and genetics of alkene biosynthesis were not elucidated in those studies. We show here that heterologous expression of a three-gene cluster from M. luteus (Mlut_13230-13250) in a fatty acid-overproducing Escherichia coli strain resulted in production of long-chain alkenes, predominantly 27:3 and 29:3 (no. carbon atoms: no. C=C bonds). Heterologous expression of Mlut_13230 (oleA) alone produced no long-chain alkenes but unsaturated aliphatic monoketones, predominantly 27:2, and in vitro studies with the purified Mlut_13230 protein and tetradecanoyl-coenzyme A (CoA) produced the same C(27) monoketone. Gas chromatography-time of flight mass spectrometry confirmed the elemental composition of all detected long-chain alkenes and monoketones (putative intermediates of alkene biosynthesis). Negative controls demonstrated that the M. luteus genes were responsible for production of these metabolites. Studies with wild-type M. luteus showed that the transcript copy number of Mlut_13230-13250 and the concentrations of 29:1 alkene isomers (the dominant alkenes produced by this strain) generally corresponded with bacterial population over time. We propose a metabolic pathway for alkene biosynthesis starting with acyl-CoA (or-ACP [acyl carrier protein]) thioesters and involving decarboxylative Claisen condensation as a key step, which we believe is catalyzed by OleA. Such activity is consistent with our data and with the homology (including the conserved Cys-His-Asn catalytic triad) of Mlut_13230 (OleA) to FabH (beta-ketoacyl-ACP synthase III), which

Genetic characterization of enzymes involved in the priming steps of oxytetracycline biosynthesis in Streptomyces rimosus.

PubMed

Wang, Peng; Gao, Xue; Chooi, Yit-Heng; Deng, Zixin; Tang, Yi

2011-08-01

Tetracyclines are clinically important aromatic polyketides whose biosynthesis is catalysed by bacterial type II polyketide synthases (PKSs). Tetracyclines are biosynthesized starting with an amide-containing malonamate starter unit and the resulting C-2 carboxyamide is critical for the antibiotic activities. In this work, we genetically verified that an amidotransferase, OxyD, and a thiolase, OxyP, are involved in the biosynthesis and incorporation of the starter unit. First, two mutations, R248T and D268N, were found to be present in OxyD* encoded in Streptomyces rimosus ATCC 13224, a strain that produces the acetate-primed 2-acetyl-2-decarboxyamido-oxytetracycline (ADOTC) instead of the malonamate-primed oxytetracycline (OTC). Homology modelling suggested that in particular D268N may inactivate OxyD. Complementation of S. rimosus ATCC 13224 with wild-type OxyD restored OTC biosynthesis, thereby confirming the essential role of OxyD in the synthesis of the amide starter unit. Second, using a series of knockout and complementation approaches, we demonstrated that OxyP is most likely involved in maintaining fidelity of the amide-priming process via hydrolysis of the competing acetate priming starter units. While the inactivation of OxyP does not eliminate OTC biosynthesis, the ratio of acetate-primed ADOTC to malonamate-primed OTC is significantly increased. This suggests that OxyP plays an ancillary role in OTC biosynthesis and is important for minimizing the levels of ADOTC, a shunt product that has much weaker antibiotic activities than OTC.

Evolution of the Kdo2-lipid A Biosynthesis in Bacteria

DOE Office of Scientific and Technical Information (OSTI.GOV)

S Opiyo; R Pardy; H Moriyama

BACKGROUND: Lipid A is the highly immunoreactive endotoxic center of lipopolysaccharide (LPS). It anchors the LPS into the outer membrane of most Gram-negative bacteria. Lipid A can be recognized by animal cells, triggers defense-related responses, and causes Gram-negative sepsis. The biosynthesis of Kdo2-lipid A, the LPS substructure, involves with nine enzymatic steps. RESULTS: In order to elucidate the evolutionary pathway of Kdo2-lipid A biosynthesis, we examined the distribution of genes encoding the nine enzymes across bacteria. We found that not all Gram-negative bacteria have all nine enzymes. Some Gram-negative bacteria have no genes encoding these enzymes and others have genesmore » only for the first four enzymes (LpxA, LpxC, LpxD, and LpxB). Among the nine enzymes, five appeared to have arisen from three independent gene duplication events. Two of such events happened within the Proteobacteria lineage, followed by functional specialization of the duplicated genes and pathway optimization in these bacteria. CONCLUSIONS: The nine-enzyme pathway, which was established based on the studies mainly in Escherichia coli K12, appears to be the most derived and optimized form. It is found only in E. coli and related Proteobacteria. Simpler and probably less efficient pathways are found in other bacterial groups, with Kdo2-lipid A variants as the likely end products. The Kdo2-lipid A biosynthetic pathway exemplifies extremely plastic evolution of bacterial genomes, especially those of Proteobacteria, and how these mainly pathogenic bacteria have adapted to their environment.« less

Immersion Refractometry of Isolated Bacterial Cell Walls

PubMed Central

Marquis, Robert E.

1973-01-01

Immersion-refractometric and light-scattering measurements were adapted to determinations of average refractive indices and physical compactness of isolated bacterial cell walls. The structures were immersed in solutions containing various concentrations of polymer molecules that cannot penetrate into wall pores, and then an estimate was made of the polymer concentration or the refractive index of the polymer solution in which light scattering was reduced to zero. Because each wall preparation was heterogeneous, the refractive index of the medium for zero light scattering had to be estimated by extrapolation. Refractive indices for walls suspended in bovine serum albumin solutions ranged from 1.348 for walls of the rod form of Arthrobacter crystallopoietes to 1.382 for walls of the teichoic acid deficient, 52A5 strain of Staphylococcus aureus. These indices were used to calculate approximate values for solids content per milliliter, and the calculated values agreed closely with those estimated from a knowledge of dextran-impermeable volumes per gram, dry weight, of the walls. When large molecules such as dextrans or serum albumin were used for immersion refractometry, the refractive indices obtained were for entire walls, including both wall polymers and wall water. When smaller molecules that can penetrate wall pores to various extents were used with Micrococcus lysodeikticus walls, the average, apparent refractive index of the structures increased as the molecular size of probing molecules was decreased. It was possible to obtain an estimate of 1.45 to 1.46 for the refractive index of wall polymers, predominantly peptidoglycans in this case, by extrapolating the curve for refractive index versus molecular radius to a value of 0.2 nm, the approximate radius of a water molecule. This relatively low value for polymer refractive index was interpreted as evidence in favor of the amorphous, elastic model of peptidoglycan structure and against the crystalline, rigid

Adenosine diphosphate sugar pyrophosphatase prevents glycogen biosynthesis in Escherichia coli

PubMed Central

Moreno-Bruna, Beatriz; Baroja-Fernández, Edurne; Muñoz, Francisco José; Bastarrica-Berasategui, Ainara; Zandueta-Criado, Aitor; Rodríguez-López, Milagros; Lasa, Iñigo; Akazawa, Takashi; Pozueta-Romero, Javier

2001-01-01

An adenosine diphosphate sugar pyrophosphatase (ASPPase, EC 3.6.1.21) has been characterized by using Escherichia coli. This enzyme, whose activities in the cell are inversely correlated with the intracellular glycogen content and the glucose concentration in the culture medium, hydrolyzes ADP-glucose, the precursor molecule of glycogen biosynthesis. ASPPase was purified to apparent homogeneity (over 3,000-fold), and sequence analyses revealed that it is a member of the ubiquitously distributed group of nucleotide pyrophosphatases designated as “nudix” hydrolases. Insertional mutagenesis experiments leading to the inactivation of the ASPPase encoding gene, aspP, produced cells with marginally low enzymatic activities and higher glycogen content than wild-type bacteria. aspP was cloned into an expression vector and introduced into E. coli. Transformed cells were shown to contain a dramatically reduced amount of glycogen, as compared with the untransformed bacteria. No pleiotropic changes in the bacterial growth occurred in both the aspP-overexpressing and aspP-deficient strains. The overall results pinpoint the reaction catalyzed by ASPPase as a potential step of regulating glycogen biosynthesis in E. coli. PMID:11416161

Tyramine and phenylethylamine biosynthesis by food bacteria.

PubMed

Marcobal, Angela; De las Rivas, Blanca; Landete, José María; Tabera, Laura; Muñoz, Rosario

2012-01-01

Tyramine poisoning is caused by the ingestion of food containing high levels of tyramine, a biogenic amine. Any foods containing free tyrosine are subject to tyramine formation if poor sanitation and low quality foods are used or if the food is subject to temperature abuse or extended storage time. Tyramine is generated by decarboxylation of the tyrosine through tyrosine decarboxylase (TDC) enzymes derived from the bacteria present in the food. Bacterial TDC have been only unequivocally identified and characterized in Gram-positive bacteria, especially in lactic acid bacteria. Pyridoxal phosphate (PLP)-dependent TDC encoding genes (tyrDC) appeared flanked by a similar genetic organization in several species of lactic acid bacteria, suggesting a common origin by a single mobile genetic element. Bacterial TDC are also able to decarboxylate phenylalanine to produce phenylethylamine (PEA), another biogenic amine. The molecular knowledge of the genes involved in tyramine production has led to the development of molecular methods for the detection of bacteria able to produce tyramine and PEA. These rapid and simple methods could be used for the analysis of the ability to form tyramine by bacteria in order to evaluate the potential risk of tyramine biosynthesis in food products.

The cell wall hydrolase Pmp23 is important for assembly and stability of the division ring in Streptococcus pneumoniae.

PubMed

Jacq, Maxime; Arthaud, Christopher; Manuse, Sylvie; Mercy, Chryslène; Bellard, Laure; Peters, Katharina; Gallet, Benoit; Galindo, Jennifer; Doan, Thierry; Vollmer, Waldemar; Brun, Yves V; VanNieuwenhze, Michael S; Di Guilmi, Anne Marie; Vernet, Thierry; Grangeasse, Christophe; Morlot, Cecile

2018-05-15

Bacterial division is intimately linked to synthesis and remodeling of the peptidoglycan, a cage-like polymer that surrounds the bacterial cell, providing shape and mechanical resistance. The bacterial division machinery, which is scaffolded by the cytoskeleton protein FtsZ, includes proteins with enzymatic, structural or regulatory functions. These proteins establish a complex network of transient functional and/or physical interactions which preserve cell shape and cell integrity. Cell wall hydrolases required for peptidoglycan remodeling are major contributors to this mechanism. Consistent with this, their deletion or depletion often results in morphological and/or division defects. However, the exact function of most of them remains elusive. In this work, we show that the putative lysozyme activity of the cell wall hydrolase Pmp23 is important for proper morphology and cell division in the opportunistic human pathogen Streptococcus pneumoniae. Our data indicate that active Pmp23 is required for proper localization of the Z-ring and the FtsZ-positioning protein MapZ. In addition, Pmp23 localizes to the division site and interacts directly with the essential peptidoglycan synthase PBP2x. Altogether, our data reveal a new regulatory function for peptidoglycan hydrolases.

Modeling central metabolism and energy biosynthesis across microbial life

DOE PAGES

Edirisinghe, Janaka N.; Weisenhorn, Pamela; Conrad, Neal; ...

2016-08-08

Here, automatically generated bacterial metabolic models, and even some curated models, lack accuracy in predicting energy yields due to poor representation of key pathways in energy biosynthesis and the electron transport chain (ETC). Further compounding the problem, complex interlinking pathways in genome-scale metabolic models, and the need for extensive gapfilling to support complex biomass reactions, often results in predicting unrealistic yields or unrealistic physiological flux profiles. As a result, to overcome this challenge, we developed methods and tools to build high quality core metabolic models (CMM) representing accurate energy biosynthesis based on a well studied, phylogenetically diverse set of modelmore » organisms. We compare these models to explore the variability of core pathways across all microbial life, and by analyzing the ability of our core models to synthesize ATP and essential biomass precursors, we evaluate the extent to which the core metabolic pathways and functional ETCs are known for all microbes. 6,600 (80 %) of our models were found to have some type of aerobic ETC, whereas 5,100 (62 %) have an anaerobic ETC, and 1,279 (15 %) do not have any ETC. Using our manually curated ETC and energy biosynthesis pathways with no gapfilling at all, we predict accurate ATP yields for nearly 5586 (70 %) of the models under aerobic and anaerobic growth conditions. This study revealed gaps in our knowledge of the central pathways that result in 2,495 (30 %) CMMs being unable to produce ATP under any of the tested conditions. We then established a methodology for the systematic identification and correction of inconsistent annotations using core metabolic models coupled with phylogenetic analysis. In conclusion, we predict accurate energy yields based on our improved annotations in energy biosynthesis pathways and the implementation of diverse ETC reactions across the microbial tree of life. We highlighted missing annotations that were essential

Modeling central metabolism and energy biosynthesis across microbial life

DOE Office of Scientific and Technical Information (OSTI.GOV)

Edirisinghe, Janaka N.; Weisenhorn, Pamela; Conrad, Neal

Here, automatically generated bacterial metabolic models, and even some curated models, lack accuracy in predicting energy yields due to poor representation of key pathways in energy biosynthesis and the electron transport chain (ETC). Further compounding the problem, complex interlinking pathways in genome-scale metabolic models, and the need for extensive gapfilling to support complex biomass reactions, often results in predicting unrealistic yields or unrealistic physiological flux profiles. As a result, to overcome this challenge, we developed methods and tools to build high quality core metabolic models (CMM) representing accurate energy biosynthesis based on a well studied, phylogenetically diverse set of modelmore » organisms. We compare these models to explore the variability of core pathways across all microbial life, and by analyzing the ability of our core models to synthesize ATP and essential biomass precursors, we evaluate the extent to which the core metabolic pathways and functional ETCs are known for all microbes. 6,600 (80 %) of our models were found to have some type of aerobic ETC, whereas 5,100 (62 %) have an anaerobic ETC, and 1,279 (15 %) do not have any ETC. Using our manually curated ETC and energy biosynthesis pathways with no gapfilling at all, we predict accurate ATP yields for nearly 5586 (70 %) of the models under aerobic and anaerobic growth conditions. This study revealed gaps in our knowledge of the central pathways that result in 2,495 (30 %) CMMs being unable to produce ATP under any of the tested conditions. We then established a methodology for the systematic identification and correction of inconsistent annotations using core metabolic models coupled with phylogenetic analysis. In conclusion, we predict accurate energy yields based on our improved annotations in energy biosynthesis pathways and the implementation of diverse ETC reactions across the microbial tree of life. We highlighted missing annotations that were essential

Molecular modeling, simulation and virtual screening of MurD ligase protein from Salmonella typhimurium LT2.

PubMed

Samal, Himanshu Bhusan; Das, Jugal Kishore; Mahapatra, Rajani Kanta; Suar, Mrutyunjay

2015-01-01

The Mur enzymes of the peptidoglycan biosynthesis pathway constitute ideal targets for the design of new classes of antimicrobial inhibitors in Gram-negative bacteria. We built a homology model of MurD of Salmonella typhimurium LT2 using MODELLER (9v12) software. 'The homology model was subjected to energy minimization by molecular dynamics (MD) simulation study with GROMACS software for a simulation time of 20 ns in water environment. The model was subjected for virtual screening study from the Zinc Database using Dockblaster software. Inhibition assay for the best inhibitor, 3-(amino methyl)-n-(4-methoxyphenyl) aniline, by flow cytometric analysis revealed the effective inhibition of peptidoglycan biosynthesis. Results from this study provide new insights for the molecular understanding and development of new antibacterial drugs against the pathogen. Copyright © 2015 Elsevier Inc. All rights reserved.

Identification of novel inhibitors of Pseudomonas aeruginosa MurC enzyme derived from phage-displayed peptide libraries.

PubMed

El Zoeiby, Ahmed; Sanschagrin, François; Darveau, André; Brisson, Jean-Robert; Levesque, Roger C

2003-03-01

The machinery of peptidoglycan biosynthesis is an ideal site at which to look for novel antimicrobial targets. Phage display was used to develop novel peptide inhibitors for MurC, an essential enzyme involved in the early steps of biosynthesis of peptidoglycan monomer. We cloned and overexpressed the murA, -B and -C genes from Pseudomonas aeruginosa in the pET expression vector, adding a His-tag to their C termini. The three proteins were overproduced in Escherichia coli and purified to homogeneity in milligram quantities. MurA and -B were combinatorially used to synthesize the MurC substrate UDP-N-acetylmuramate, the identity of which was confirmed by mass spectrometry and nuclear magnetic resonance analysis. Two phage-display libraries were screened against MurC in order to identify peptide ligands to the enzyme. Three rounds of biopanning were carried out, successively increasing elution specificity from round 1 to 3. The third round was accomplished with both non-specific elution and competitive elution with each of the three MurC substrates, UDP-N-acetylmuramic acid (UNAM), ATP and L-alanine. The DNA of 10 phage, selected randomly from each group, was extracted and sequenced, and consensus peptide sequences were elucidated. Peptides were synthesized and tested for inhibition of the MurC-catalysed reaction, and two peptides were shown to be inhibitors of MurC activity with IC(50)s of 1.5 and 0.9 mM, respectively. The powerful selection technique of phage display allowed us to identify two peptide inhibitors of the essential bacterial enzyme MurC. The peptide sequences represent the basis for the synthesis of inhibitory peptidomimetic molecules.

On the mechanism of peptidoglycan binding and cleavage by the endo-specific lytic transglycosylase MltE from Escherichia coli.

PubMed

Fibriansah, Guntur; Gliubich, Francesca I; Thunnissen, Andy-Mark W H

2012-11-13

The lytic transglycosylase MltE from Escherichia coli is a periplasmic, outer membrane-attached enzyme that cleaves the β-1,4-glycosidic bonds between N-acetylmuramic acid and N-acetylglucosamine residues in the cell wall peptidoglycan, producing 1,6-anhydromuropeptides. Here we report three crystal structures of MltE: in a substrate-free state, in a binary complex with chitopentaose, and in a ternary complex with the glycopeptide inhibitor bulgecin A and the murodipeptide N-acetylglucosaminyl-N-acetylmuramyl-l-Ala-d-Glu. The substrate-bound structures allowed a detailed analysis of the saccharide-binding interactions in six subsites of the peptidoglycan-binding groove (subsites -4 to +2) and, combined with site-directed mutagenesis analysis, confirmed the role of Glu64 as catalytic acid/base. The structures permitted the precise modeling of a short glycan strand of eight saccharide residues, providing evidence for two additional subsites (+3 and +4) and revealing the productive conformational state of the substrate at subsites -1 and +1, where the glycosidic bond is cleaved. Full accessibility of the peptidoglycan-binding groove and preferential binding of an N-acetylmuramic acid residue in a (4)C(1) chair conformation at subsite +2 explain why MltE shows only endo- and no exo-specific activity toward glycan strands. The results further indicate that catalysis of glycosidic bond cleavage by MltE proceeds via distortion toward a sofa-like conformation of the N-acetylmuramic acid sugar ring at subsite -1 and by anchimeric assistance of the sugar's N-acetyl group, as shown previously for the lytic transglycosylases Slt70 and MltB.

Digestion of peptidoglycan near the cross-link is necessary for the growth of Bacillus subtilis.

PubMed

Hashimoto, Masayuki; Matsushima, Hiroaki; Suparthana, I Putu; Ogasawara, Hiroshi; Yamamoto, Hiroki; Teng, ChingHao; Sekiguchi, Junichi

2018-03-01

Bacterial cells are covered with peptidoglycan (PG) layer(s), serving as the cellular exoskeleton. The PG sacculus changes its shape during cell growth, and thus both the synthesis and disassembly of PG are important for cell proliferation. In Bacillus subtilis, four dl-endopeptidases (DLEPases; LytE, LytF, CwlO and CwlS) are involved in the maintenance of cell morphology. The lytE cwlO double mutant exhibits synthetic lethality and defective cell elongation, while the lytE lytF cwlS triple mutant exhibits defective cell separation, albeit with septum formation. LytE is involved in both cell separation and elongation. We propose that DLEPases have varied roles in cell separation and elongation. To determine these roles, the catalytic domain of LytE was substituted with another catalytic domain that digests the other bonds in PG. By using the chimeric enzymes, we assessed the suppression of the synthetic lethality by the cell elongation defect and the disruption of chain morphology by the cell separation defect. All the constructed chimeric enzymes suppressed the cell separation defect, restoring the chain morphology. Digestion at any position of PG broke the linkage between two daughter cells, releasing them from each other. However, only d,d-endopeptidases suppressed the lack of DLEPase in the lytE cwlO double mutant. This indicated that the release of tension on the expanding PG sacculus is not the sole essential function of DLEPases. Considering that the structure of the digested PG is important for cell elongation, the digested product might be reused in the growth process in some way.

Stable Analogues of OSB-AMP: Potent Inhibitors of MenE, the o-Succinylbenzoate-CoA Synthetase from Bacterial Menaquinone Biosynthesis

PubMed Central

Lu, Xuequan; Zhou, Rong; Sharma, Indrajeet; Li, Xiaokai; Kumar, Gyanendra; Swaminathan, Subramanyam

2012-01-01

MenE, the o-succinylbenzoate (OSB)-CoA synthetase from bacterial menaquinone biosynthesis, is a promising new antibacterial target. Sulfonyladenosine analogues of the cognate reaction intermediate, OSB-AMP, have been developed as inhibitors of the MenE enzymes from Mycobacterium tuberculosis (mtMenE), Staphylococcus aureus (saMenE) and Escherichia coli (ecMenE). Both a free carboxylate and ketone moiety on the OSB side chain are required for potent inhibitory activity. OSB-AMS (4) is a competitive inhibitor of mtMenE with respect to ATP (Ki = 5.4 ± 0.1 nM) and a non-competitive inhibitor with respect to OSB (Ki = 11.2 ± 0.9 nM). These data are consistent with a bi uni uni bi ping-pong kinetic mechanism for these enzymes. In addition, OSB-AMS inhibits saMenE with Kiapp of 22 ± 8 nM and ecMenE with KiOSB=128±5nM. Putative active site residues, Arg-222, which may interact with the OSB aromatic carboxylate, and Ser-302, which may bind the OSB ketone oxygen, have been identified through computational docking of OSB-AMP with the unliganded crystal structure of saMenE. A pH-dependent interconversion of the free keto acid and lactol forms of the inhibitors is also described, along with implications for inhibitor design. PMID:22109989

Otitis media induced by peptidoglycan-polysaccharide (PGPS) in TLR2-deficient (Tlr2(-/-)) mice for developing drug therapy.

PubMed

Zhang, Xiaolin; Zheng, Tihua; Sang, Lu; Apisa, Luke; Zhao, Hongchun; Fu, Fenghua; Wang, Qingzhu; Wang, Yanfei; Zheng, Qingyin

2015-10-01

Toll like receptor 2 (TLR2) signaling can regulate the pathogenesis of otitis media (OM). However, the precise role of TLR2 signaling in OM has not been clarified due to the lack of an optimal animal model. Peptidoglycan-polysaccharide (PGPS) of the bacterial cell wall can induce inflammation by activating the TLR2 signaling. This study aimed at examining the pathogenic characteristics of OM induced by PGPS in Tlr2(-/-) mice, and the potential therapeutic effect of sodium aescinate (SA) in this model. Wild-type (WT) and Tlr2(-/-) mice were inoculated with streptococcal PGPS into their middle ears (MEs) and treated intravenously with vehicle or SA daily beginning at 3days prior to PGPS for 6 consecutive days. The pathologic changes of individual mice were evaluated longitudinally. In comparison with WT mice, Tlr2(-/-) mice were susceptible to PGPS-induced OM. Tlr2(-/-) mice displayed greater hearing loss, tympanic membrane damage, ME mucosal thickening, longer inflammation state, cilia and goblet cell loss. SA-treatment decreased neutrophil infiltration, modulated TLR2-related gene expression and improved ciliary organization. PGPS induced a relatively stable OM in Tlr2(-/-) mice, providing a new model for OM research. Treatment with SA mitigated the pathogenic damage in the ME and may be valuable for intervention of OM. Copyright © 2015 Elsevier B.V. All rights reserved.

Establishing a Role for Bacterial Cellulose in Environmental Interactions: Lessons Learned from Diverse Biofilm-Producing Proteobacteria

PubMed Central

Augimeri, Richard V.; Varley, Andrew J.; Strap, Janice L.

2015-01-01

Bacterial cellulose (BC) serves as a molecular glue to facilitate intra- and inter-domain interactions in nature. Biosynthesis of BC-containing biofilms occurs in a variety of Proteobacteria that inhabit diverse ecological niches. The enzymatic and regulatory systems responsible for the polymerization, exportation, and regulation of BC are equally as diverse. Though the magnitude and environmental consequences of BC production are species-specific, the common role of BC-containing biofilms is to establish close contact with a preferred host to facilitate efficient host–bacteria interactions. Universally, BC aids in attachment, adherence, and subsequent colonization of a substrate. Bi-directional interactions influence host physiology, bacterial physiology, and regulation of BC biosynthesis, primarily through modulation of intracellular bis-(3′→5′)-cyclic diguanylate (c-di-GMP) levels. Depending on the circumstance, BC producers exhibit a pathogenic or symbiotic relationship with plant, animal, or fungal hosts. Rhizobiaceae species colonize plant roots, Pseudomonadaceae inhabit the phyllosphere, Acetobacteriaceae associate with sugar-loving insects and inhabit the carposphere, Enterobacteriaceae use fresh produce as vehicles to infect animal hosts, and Vibrionaceae, particularly Aliivibrio fischeri, colonize the light organ of squid. This review will highlight the diversity of the biosynthesis and regulation of BC in nature by discussing various examples of Proteobacteria that use BC-containing biofilms to facilitate host–bacteria interactions. Through discussion of current data we will establish new directions for the elucidation of BC biosynthesis, its regulation and its ecophysiological roles. PMID:26635751

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE PAGES

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.; ...

2015-09-15

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes

Insights into substrate specificity of NlpC/P60 cell wall hydrolases containing bacterial SH3 domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. In addition, these enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structuremore » consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.Peptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling enzymes

The Redundancy of Peptidoglycan Carboxypeptidases Ensures Robust Cell Shape Maintenance in Escherichia coli

PubMed Central

Peters, Katharina; Kannan, Suresh; Rao, Vincenzo A.; Biboy, Jacob; Vollmer, Daniela; Erickson, Stephen W.; Lewis, Richard J.

2016-01-01

ABSTRACT Peptidoglycan (PG) is an essential structural component of the bacterial cell wall and maintains the integrity and shape of the cell by forming a continuous layer around the cytoplasmic membrane. The thin PG layer of Escherichia coli resides in the periplasm, a unique compartment whose composition and pH can vary depending on the local environment of the cell. Hence, the growth of the PG layer must be sufficiently robust to allow cell growth and division under different conditions. We have analyzed the PG composition of 28 mutants lacking multiple PG enzymes (penicillin-binding proteins [PBPs]) after growth in acidic or near-neutral-pH media. Statistical analysis of the muropeptide profiles identified dd-carboxypeptidases (DD-CPases) that were more active in cells grown at acidic pH. In particular, the absence of the DD-CPase PBP6b caused a significant increase in the pentapeptide content of PG as well as morphological defects when the cells were grown at acidic pH. Other DD-CPases (PBP4, PBP4b, PBP5, PBP6a, PBP7, and AmpH) and the PG synthase PBP1B made a smaller or null contribution to the pentapeptide-trimming activity at acidic pH. We solved the crystal structure of PBP6b and also demonstrated that the enzyme is more stable and has a lower Km at acidic pH, explaining why PBP6b is more active at low pH. Hence, PBP6b is a specialized DD-CPase that contributes to cell shape maintenance at low pH, and E. coli appears to utilize redundant DD-CPases for normal growth under different conditions. PMID:27329754

Role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins in AmpC expression, β-lactam resistance, and peptidoglycan structure.

PubMed

Ropy, Alaa; Cabot, Gabriel; Sánchez-Diener, Irina; Aguilera, Cristian; Moya, Bartolome; Ayala, Juan A; Oliver, Antonio

2015-07-01

This study aimed to characterize the role of Pseudomonas aeruginosa low-molecular-mass penicillin-binding proteins (LMM PBPs), namely, PBP4 (DacB), PBP5 (DacC), and PBP7 (PbpG), in peptidoglycan composition, β-lactam resistance, and ampC regulation. For this purpose, we constructed all single and multiple mutants of dacB, dacC, pbpG, and ampC from the wild-type P. aeruginosa PAO1 strain. Peptidoglycan composition was determined by high-performance liquid chromatography (HPLC), ampC expression by reverse transcription-PCR (RT-PCR), PBP patterns by a Bocillin FL-binding test, and antimicrobial susceptibility by MIC testing for a panel of β-lactams. Microscopy and growth rate analyses revealed no apparent major morphological changes for any of the mutants compared to the wild-type PAO1 strain. Of the single mutants, only dacC mutation led to significantly increased pentapeptide levels, showing that PBP5 is the major dd-carboxypeptidase in P. aeruginosa. Moreover, our results indicate that PBP4 and PBP7 play a significant role as dd-carboxypeptidase only if PBP5 is absent, and their dd-endopeptidase activity is also inferred. As expected, the inactivation of PBP4 led to a significant increase in ampC expression (around 50-fold), but, remarkably, the sequential inactivation of the three LMM PBPs produced a much greater increase (1,000-fold), which correlated with peptidoglycan pentapeptide levels. Finally, the β-lactam susceptibility profiles of the LMM PBP mutants correlated well with the ampC expression data. However, the inactivation of ampC in these mutants also evidenced a role of LMM PBPs, especially PBP5, in intrinsic β-lactam resistance. In summary, in addition to assessing the effect of P. aeruginosa LMM PBPs on peptidoglycan structure for the first time, we obtained results that represent a step forward in understanding the impact of these PBPs on β-lactam resistance, apparently driven by the interplay between their roles in AmpC induction, �

Engineering Pseudomonas for phenazine biosynthesis, regulation, and biotechnological applications: a review.

PubMed

Bilal, Muhammad; Guo, Shuqi; Iqbal, Hafiz M N; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

2017-10-03

Pseudomonas strains are increasingly attracting considerable attention as a valuable bacterial host both for basic and applied research. It has been considered as a promising candidate to produce a variety of bioactive secondary metabolites, particularly phenazines. Apart from the biotechnological perspective, these aromatic compounds have the notable potential to inhibit plant-pathogenic fungi and thus are useful in controlling plant diseases. Nevertheless, phenazines production is quite low by the wild-type strains that necessitated its yield improvement for large-scale agricultural applications. Metabolic engineering approaches with the advent of plentiful information provided by systems-level genomic and transcriptomic analyses enabled the development of new biological agents functioning as potential cell factories for producing the desired level of value-added bioproducts. This study presents an up-to-date overview of recombinant Pseudomonas strains as the preferred choice of host organisms for the biosynthesis of natural phenazines. The biosynthetic pathway and regulatory mechanism involved in the phenazine biosynthesis are comprehensively discussed. Finally, a summary of biological functionalities and biotechnological applications of the phenazines is also provided.

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway*

PubMed Central

Henrich, Erik; Ma, Yi; Engels, Ina; Münch, Daniela; Otten, Christian; Schneider, Tanja; Henrichfreise, Beate; Sahl, Hans-Georg; Dötsch, Volker; Bernhard, Frank

2016-01-01

Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cell-free expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were co-translationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY·nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin. PMID:26620564

Biosynthesis of polybrominated aromatic organic compounds by marine bacteria

PubMed Central

Agarwal, Vinayak; El Gamal, Abrahim A.; Yamanaka, Kazuya; Poth, Dennis; Kersten, Roland D.; Schorn, Michelle; Allen, Eric E.; Moore, Bradley S.

2014-01-01

Polybrominated diphenyl ethers (PBDEs) and polybrominated bipyrroles are natural products that bioaccumulate in the marine food chain. PBDEs have attracted widespread attention due to their persistence in the environment and potential toxicity to humans. However, the natural origins of PBDE biosynthesis are not known. Here we report marine bacteria as producers of PBDEs and establish a genetic and molecular foundation for their production that unifies paradigms for the elaboration of bromophenols and bromopyrroles abundant in marine biota. We provide biochemical evidence of marine brominase enzymes revealing decarboxylative-halogenation enzymology previously unknown among halogenating enzymes. Biosynthetic motifs discovered in our study were used to mine sequence databases to discover unrealized marine bacterial producers of organobromine compounds. PMID:24974229

Cloning and analysis of peptidoglycan recognition protein-LC and immune deficiency from the diamondback moth, Plutella xylostella.

PubMed

Zhan, Ming-Yue; Yang, Pei-Jin; Rao, Xiang-Jun

2018-02-01

Peptidoglycan (PGN) exists in both Gram-negative and Gram-positive bacteria as a component of the cell wall. PGN is an important target to be recognized by the innate immune system of animals. PGN recognition proteins (PGRP) are responsible for recognizing PGNs. In Drosophila melanogaster, PGRP-LC and IMD (immune deficiency) are critical for activating the Imd pathway. Here, we report the cloning and analysis of PGRP-LC and IMD (PxPGRP-LC and PxIMD) from diamondback moth, Plutella xylostella (L.), the insect pest of cruciferous vegetables. PxPGRP-LC gene consists of six exons encoding a polypeptide of 308 amino acid residues with a transmembrane region and a PGRP domain. PxIMD cDNA encodes a polypeptide of 251 amino acid residues with a death domain. Sequence comparisons indicate that they are characteristic of Drosophila PGRP-LC and IMD homologs. PxPGRP-LC and PxIMD were expressed in various tissues and developmental stages. Their mRNA levels were affected by bacterial challenges. The PGRP domain of PxPGRP-LC lacks key residues for the amidase activity, but it can recognize two types of PGNs. Overexpression of full-length and deletion mutants in Drosophila S2 cells induced expression of some antimicrobial peptide genes. These results indicate that PxPGRP-LC and PxIMD may be involved in the immune signaling of P. xylostella. This study provides a foundation for further studies of the immune system of P. xylostella. © 2017 Wiley Periodicals, Inc.

Cellulose biosynthesis by the beta-proteobacterium, Chromobacterium violaceum.

PubMed

Recouvreux, Derce O S; Carminatti, Claudimir A; Pitlovanciv, Ana K; Rambo, Carlos R; Porto, Luismar M; Antônio, Regina V

2008-11-01

The Chromobacterium violaceum ATCC 12472 genome was sequenced by The Brazilian National Genome Project Consortium. Previous annotation reported the presence of cellulose biosynthesis genes in that genome. Analysis of these genes showed that, as observed in other bacteria, they are organized in two operons. In the present work, experimental evidences of the presence of cellulose in the extracellular matrix of the biofilm produced by C. violaceum in static cultures are shown. Biofilm samples were enzymatically digested by cellulase, releasing glucose units, suggesting the presence of cellulose as an extracellular matrix component. Fluorescence microscopy observations showed that C. violaceum produces a cellulase-sensitive extracellular matrix composed of fibers able to bind calcofluor. C. violaceum grows on medium containing Congo red, forming brown-red colonies. Together, these results suggest that cellulase-susceptible matrix material is cellulose. Scanning electronic microscopy analysis showed that the extracellular matrix exhibited a network of microfibrils, typical of bacterial cellulose. Although cellulose production is widely distributed between several bacterial species, including at least the groups of Gram-negative proteobacteria alpha and gamma, we give for the first time experimental evidence for cellulose production in beta-proteobacteria.

Bacterial Adaptation to Antibiotics through Regulatory RNAs.

PubMed

Felden, Brice; Cattoir, Vincent

2018-05-01

The extensive use of antibiotics has resulted in a situation where multidrug-resistant pathogens have become a severe menace to human health worldwide. A deeper understanding of the principles used by pathogens to adapt to, respond to, and resist antibiotics would pave the road to the discovery of drugs with novel mechanisms. For bacteria, antibiotics represent clinically relevant stresses that induce protective responses. The recent implication of regulatory RNAs (small RNAs [sRNAs]) in antibiotic response and resistance in several bacterial pathogens suggests that they should be considered innovative drug targets. This minireview discusses sRNA-mediated mechanisms exploited by bacterial pathogens to fight against antibiotics. A critical discussion of the newest findings in the field is provided, with emphasis on the implication of sRNAs in major mechanisms leading to antibiotic resistance, including drug uptake, active drug efflux, drug target modifications, biofilms, cell walls, and lipopolysaccharide (LPS) biosynthesis. Of interest is the lack of knowledge about sRNAs implicated in Gram-positive compared to Gram-negative bacterial resistance. Copyright © 2018 American Society for Microbiology.

Spatial Patterning of Newly-Inserted Material during Bacterial Cell Growth

NASA Astrophysics Data System (ADS)

Ursell, Tristan

2012-02-01

In the life cycle of a bacterium, rudimentary microscopy demonstrates that cell growth and elongation are essential characteristics of cellular reproduction. The peptidoglycan cell wall is the main load-bearing structure that determines both cell shape and overall size. However, simple imaging of cellular growth gives no indication of the spatial patterning nor mechanism by which material is being incorporated into the pre-existing cell wall. We employ a combination of high-resolution pulse-chase fluorescence microscopy, 3D computational microscopy, and detailed mechanistic simulations to explore how spatial patterning results in uniform growth and maintenance of cell shape. We show that growth is happening in discrete bursts randomly distributed over the cell surface, with a well-defined mean size and average rate. We further use these techniques to explore the effects of division and cell wall disrupting antibiotics, like cephalexin and A22, respectively, on the patterning of cell wall growth in E. coli. Finally, we explore the spatial correlation between presence of the bacterial actin-like cytoskeletal protein, MreB, and local cell wall growth. Together these techniques form a powerful method for exploring the detailed dynamics and involvement of antibiotics and cell wall-associated proteins in bacterial cell growth.[4pt] In collaboration with Kerwyn Huang, Stanford University.

Activity Augmentation of Amphioxus Peptidoglycan Recognition Protein BbtPGRP3 via Fusion with a Chitin Binding Domain

PubMed Central

Wang, Wen-Jie; Cheng, Wang; Luo, Ming; Yan, Qingyu; Yu, Hong-Mei; Li, Qiong; Cao, Dong-Dong; Huang, Shengfeng; Xu, Anlong; Mariuzza, Roy A.; Chen, Yuxing; Zhou, Cong-Zhao

2015-01-01

Peptidoglycan recognition proteins (PGRPs), which have been identified in most animals, are pattern recognition molecules that involve antimicrobial defense. Resulting from extraordinary expansion of innate immune genes, the amphioxus encodes many PGRPs of diverse functions. For instance, three isoforms of PGRP encoded by Branchiostoma belcheri tsingtauense, termed BbtPGRP1~3, are fused with a chitin binding domain (CBD) at the N-terminus. Here we report the 2.7 Å crystal structure of BbtPGRP3, revealing an overall structure of an N-terminal hevein-like CBD followed by a catalytic PGRP domain. Activity assays combined with site-directed mutagenesis indicated that the individual PGRP domain exhibits amidase activity towards both DAP-type and Lys-type peptidoglycans (PGNs), the former of which is favored. The N-terminal CBD not only has the chitin-binding activity, but also enables BbtPGRP3 to gain a five-fold increase of amidase activity towards the Lys-type PGNs, leading to a significantly broadened substrate spectrum. Together, we propose that modular evolution via domain shuffling combined with gene horizontal transfer makes BbtPGRP1~3 novel PGRPs of augmented catalytic activity and broad recognition spectrum. PMID:26479246

Biochemical characterisation of the chlamydial MurF ligase, and possible sequence of the chlamydial peptidoglycan pentapeptide stem.

PubMed

Patin, Delphine; Bostock, Julieanne; Chopra, Ian; Mengin-Lecreulx, Dominique; Blanot, Didier

2012-06-01

Chlamydiaceae are obligate intracellular bacteria that do not synthesise detectable peptidoglycan although they possess an almost complete arsenal of genes encoding peptidoglycan biosynthetic activities. In this paper, the murF gene from Chlamydia trachomatis was shown to be capable of complementing a conditional Escherichia coli mutant impaired in UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase activity. Recombinant MurF from C. trachomatis was overproduced and purified from E. coli. It exhibited ATP-dependent UDP-MurNAc-X-γ-D-Glu-meso-A(2)pm:D-Ala-D-Ala ligase activity in vitro. No significant difference of kinetic parameters was seen when X was L-Ala, L-Ser or Gly. The L-Lys-containing UDP-MurNAc-tripeptide was a poorer substrate as compared to the meso-A(2)pm-containing one. Based on the respective substrate specificities of the chlamydial MurC, MurE, MurF and Ddl enzymes, a sequence L-Ala/L-Ser/Gly-γ-D-Glu-meso-A(2)pm-D-Ala-D-Ala is expected for the chlamydial pentapeptide stem, with Gly at position 1 being less likely.

Enterococcus faecalis Constitutes an Unusual Bacterial Model in Lysozyme Resistance▿

PubMed Central

Hébert, Laurent; Courtin, Pascal; Torelli, Riccardo; Sanguinetti, Maurizio; Chapot-Chartier, Marie-Pierre; Auffray, Yanick; Benachour, Abdellah

2007-01-01

Lysozyme is an important and widespread compound of the host constitutive defense system, and it is assumed that Enterococcus faecalis is one of the few bacteria that are almost completely lysozyme resistant. On the basis of the sequence analysis of the whole genome of E. faecalis V583 strain, we identified two genes that are potentially involved in lysozyme resistance, EF_0783 and EF_1843. Protein products of these two genes share significant homology with Staphylococcus aureus peptidoglycan O-acetyltransferase (OatA) and Streptococcus pneumoniae N-acetylglucosamine deacetylase (PgdA), respectively. In order to determine whether EF_0783 and EF_1843 are involved in lysozyme resistance, we constructed their corresponding mutants and a double mutant. The ΔEF_0783 mutant and ΔEF_0783 ΔEF_1843 double mutant were shown to be more sensitive to lysozyme than the parental E. faecalis JH2-2 strain and ΔEF_1843 mutant were. However, compared to other bacteria, such as Listeria monocytogenes or S. pneumoniae, the tolerance of ΔEF_0783 and ΔEF_0783 ΔEF_1843 mutants towards lysozyme remains very high. Peptidoglycan structure analysis showed that EF_0783 modifies the peptidoglycan by O acetylation of N-acetyl muramic acid, while the EF_1843 deletion has no obvious effect on peptidoglycan structure under the same conditions. Moreover, the EF_0783 and EF_1843 deletions seem to significantly affect the ability of E. faecalis to survive within murine macrophages. In all, while EF_0783 is currently involved in the lysozyme resistance of E. faecalis, peptidoglycan O acetylation and de-N-acetylation are not the main mechanisms conferring high levels of lysozyme resistance to E. faecalis. PMID:17785473

The C-terminal periplasmic domain of MotB is responsible for load-dependent control of the number of stators of the bacterial flagellar motor.

PubMed

Castillo, David J; Nakamura, Shuichi; Morimoto, Yusuke V; Che, Yong-Suk; Kami-Ike, Nobunori; Kudo, Seishi; Minamino, Tohru; Namba, Keiichi

2013-01-01

The bacterial flagellar motor is made of a rotor and stators. In Salmonella it is thought that about a dozen MotA/B complexes are anchored to the peptidoglycan layer around the motor through the C-terminal peptidoglycan-binding domain of MotB to become active stators as well as proton channels. MotB consists of 309 residues, forming a single transmembrane helix (30-50), a stalk (51-100) and a C-terminal peptidoglycan-binding domain (101-309). Although the stalk is dispensable for torque generation by the motor, it is required for efficient motor performance. Residues 51 to 72 prevent premature proton leakage through the proton channel prior to stator assembly into the motor. However, the role of residues 72-100 remains unknown. Here, we analyzed the torque-speed relationship of the MotB(Δ72-100) motor. At a low speed near stall, this mutant motor produced torque at the wild-type level. Unlike the wild-type motor, however, torque dropped off drastically by slight decrease in external load and then showed a slow exponential decay over a wide range of load by its further reduction. Since it is known that the stator is a mechano-sensor and that the number of active stators changes in a load-dependent manner, we interpreted this unusual torque-speed relationship as anomaly in load-dependent control of the number of active stators. The results suggest that residues 72-100 of MotB is required for proper load-dependent control of the number of active stators around the rotor.

Phosphorylation of the Peptidoglycan Synthase PonA1 Governs the Rate of Polar Elongation in Mycobacteria

PubMed Central

Kieser, Karen J.; Baer, Christina E.; Barczak, Amy K.; Meniche, Xavier; Chao, Michael C.; Rego, E. Hesper; Sassetti, Christopher M.; Fortune, Sarah M.; Rubin, Eric J.

2015-01-01

Cell growth and division are required for the progression of bacterial infections. Most rod-shaped bacteria grow by inserting new cell wall along their mid-section. However, mycobacteria, including the human pathogen Mycobacterium tuberculosis, produce new cell wall material at their poles. How mycobacteria control this different mode of growth is incompletely understood. Here we find that PonA1, a penicillin binding protein (PBP) capable of transglycosylation and transpeptidation of cell wall peptidoglycan (PG), is a major governor of polar growth in mycobacteria. PonA1 is required for growth of Mycobacterium smegmatis and is critical for M. tuberculosis during infection. In both cases, PonA1’s catalytic activities are both required for normal cell length, though loss of transglycosylase activity has a more pronounced effect than transpeptidation. Mutations that alter the amount or the activity of PonA1 result in abnormal formation of cell poles and changes in cell length. Moreover, altered PonA1 activity results in dramatic differences in antibiotic susceptibility, suggesting that a balance between the two enzymatic activities of PonA1 is critical for survival. We also find that phosphorylation of a cytoplasmic region of PonA1 is required for normal activity. Mutations in a critical phosphorylated residue affect transglycosylase activity and result in abnormal rates of cell elongation. Together, our data indicate that PonA1 is a central determinant of polar growth in mycobacteria, and its governance of cell elongation is required for robust cell fitness during both host-induced and antibiotic stress. PMID:26114871

The Sweet Tooth of Bacteria: Common Themes in Bacterial Glycoconjugates

PubMed Central

Tytgat, Hanne L. P.

2014-01-01

SUMMARY Humans have been increasingly recognized as being superorganisms, living in close contact with a microbiota on all their mucosal surfaces. However, most studies on the human microbiota have focused on gaining comprehensive insights into the composition of the microbiota under different health conditions (e.g., enterotypes), while there is also a need for detailed knowledge of the different molecules that mediate interactions with the host. Glycoconjugates are an interesting class of molecules for detailed studies, as they form a strain-specific barcode on the surface of bacteria, mediating specific interactions with the host. Strikingly, most glycoconjugates are synthesized by similar biosynthesis mechanisms. Bacteria can produce their major glycoconjugates by using a sequential or an en bloc mechanism, with both mechanistic options coexisting in many species for different macromolecules. In this review, these common themes are conceptualized and illustrated for all major classes of known bacterial glycoconjugates, with a special focus on the rather recently emergent field of glycosylated proteins. We describe the biosynthesis and importance of glycoconjugates in both pathogenic and beneficial bacteria and in both Gram-positive and -negative organisms. The focus lies on microorganisms important for human physiology. In addition, the potential for a better knowledge of bacterial glycoconjugates in the emerging field of glycoengineering and other perspectives is discussed. PMID:25184559

Phytoplankton-bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge.

PubMed

Bertrand, Erin M; McCrow, John P; Moustafa, Ahmed; Zheng, Hong; McQuaid, Jeffrey B; Delmont, Tom O; Post, Anton F; Sipler, Rachel E; Spackeen, Jenna L; Xu, Kai; Bronk, Deborah A; Hutchins, David A; Allen, Andrew E

2015-08-11

Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton-bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton-bacterial interactions in both positive and negative feedback loops.

Phytoplankton–bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge

PubMed Central

Bertrand, Erin M.; McCrow, John P.; Moustafa, Ahmed; Zheng, Hong; McQuaid, Jeffrey B.; Delmont, Tom O.; Post, Anton F.; Sipler, Rachel E.; Spackeen, Jenna L.; Xu, Kai; Bronk, Deborah A.; Hutchins, David A.; Allen, Andrew E.

2015-01-01

Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton–bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton–bacterial interactions in both positive and negative feedback loops. PMID:26221022

Chemiluminescence enzyme immunoassay using ProteinA-bacterial magnetite complex

NASA Astrophysics Data System (ADS)

Matsunaga, Tadashi; Sato, Rika; Kamiya, Shinji; Tanaka, Tsuyosi; Takeyama, Haruko

1999-04-01

Bacterial magnetic particles (BMPs) which have ProteinA expressed on their surface were constructed using magA which is a key gene in BMP biosynthesis in the magnetic bacterium Magnetospirillum sp. AMB-1. Homogenous chemiluminescence enzyme immunoassay using antibody bound ProteinA-BMP complexes was developed for detection of human IgG. A good correlation between the luminescence yield and the concentration of human IgG was obtained in the range of 1-10 3 ng/ml.

Metabolic plasticity for isoprenoid biosynthesis in bacteria.

PubMed

Pérez-Gil, Jordi; Rodríguez-Concepción, Manuel

2013-05-15

Isoprenoids are a large family of compounds synthesized by all free-living organisms. In most bacteria, the common precursors of all isoprenoids are produced by the MEP (methylerythritol 4-phosphate) pathway. The MEP pathway is absent from archaea, fungi and animals (including humans), which synthesize their isoprenoid precursors using the completely unrelated MVA (mevalonate) pathway. Because the MEP pathway is essential in most bacterial pathogens (as well as in the malaria parasites), it has been proposed as a promising new target for the development of novel anti-infective agents. However, bacteria show a remarkable plasticity for isoprenoid biosynthesis that should be taken into account when targeting this metabolic pathway for the development of new antibiotics. For example, a few bacteria use the MVA pathway instead of the MEP pathway, whereas others possess the two full pathways, and some parasitic strains lack both the MVA and the MEP pathways (probably because they obtain their isoprenoids from host cells). Moreover, alternative enzymes and metabolic intermediates to those of the canonical MVA or MEP pathways exist in some organisms. Recent work has also shown that resistance to a block of the first steps of the MEP pathway can easily be developed because several enzymes unrelated to isoprenoid biosynthesis can produce pathway intermediates upon spontaneous mutations. In the present review, we discuss the major advances in our knowledge of the biochemical toolbox exploited by bacteria to synthesize the universal precursors for their essential isoprenoids.

Molecular characteristics of hemoglobins in blood clam and their immune responses to bacterial infection.

PubMed

Xu, Bin; Zhang, Yanan; Jing, Zhao; Fan, Tingjun

2017-06-01

Bivalve hemoglobins have antibacterial activities, while the underlying mechanisms remain poorly understood. In our study, three full-length cDNAs of hemoglobins from blood clam skHbs were obtained, encoding putative polypeptides of 147, 150, and 152 amino acids, respectively. Predicted advanced protein structures showed that the skHbs had amphipathic antibacterial structures, displayed the typical structural characteristics of proteins with globin-like fold containing numerous alpha-helixes, and forming a homodimeric skHbI and a heterotetrameric skHbII complex. After injected with alive and heat-killed Gram-positive bacteria Bacillus subtilis, the mRNA levels of skHbI and skHbII were both significantly upregulated through increasing the expression of peptidoglycan recognition protein-like (PGRP-like) protein and Toll-like receptor (TLR-like) protein induced by peptidoglycan on the surface of the bacteria, but there were no obvious differences in their protein levels. Besides, reactive oxygen species (ROS) was detected to participate in the resistance to B. subtilis. These implied that skHbs could involve in the innate immune responses to Gram-positive bacterial infection directly with their amphipathic structures and indirectly by increasing ROS production through PGRP triggering Toll pathway. In conclusion, our findings reveal the structural characteristics of skHbs and their mechanism against Gram-positive bacteria thereby providing the molecular evidence for fundamental innate antibacterial activities by invoking respiratory proteins. Copyright © 2017 Elsevier B.V. All rights reserved.

Stable analogues of OSB-AMP: potent inhibitors of MenE, the o-succinylbenzoate-CoA synthetase from bacterial menaquinone biosynthesis.

PubMed

Lu, Xuequan; Zhou, Rong; Sharma, Indrajeet; Li, Xiaokai; Kumar, Gyanendra; Swaminathan, Subramanyam; Tonge, Peter J; Tan, Derek S

2012-01-02

MenE, the o-succinylbenzoate (OSB)-CoA synthetase from bacterial menaquinone biosynthesis, is a promising new antibacterial target. Sulfonyladenosine analogues of the cognate reaction intermediate, OSB-AMP, have been developed as inhibitors of the MenE enzymes from Mycobacterium tuberculosis (mtMenE), Staphylococcus aureus (saMenE) and Escherichia coli (ecMenE). Both a free carboxylate and a ketone moiety on the OSB side chain are required for potent inhibitory activity. OSB-AMS (4) is a competitive inhibitor of mtMenE with respect to ATP (K(i) =5.4±0.1 nM) and a noncompetitive inhibitor with respect to OSB (K(i) =11.2±0.9 nM). These data are consistent with a Bi Uni Uni Bi Ping-Pong kinetic mechanism for these enzymes. In addition, OSB-AMS inhibits saMenE with K(i)(app) =22±8 nM and ecMenE with K(i)(OSB) =128±5 nM. Putative active-site residues, Arg222, which may interact with the OSB aromatic carboxylate, and Ser302, which may bind the OSB ketone oxygen, have been identified through computational docking of OSB-AMP with the unliganded crystal structure of saMenE. A pH-dependent interconversion of the free keto acid and lactol forms of the inhibitors is also described, along with implications for inhibitor design. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity

PubMed Central

Anderson, Mark T.; Mitchell, Lindsay A.

2017-01-01

biosynthesis results in decreased phlA and flagellar gene transcription, which can be restored by supplying bacteria with exogenous cysteine. These results identify a previously unrecognized role for CysE and cysteine in the secretion of S. marcescens phospholipase and in bacterial motility. PMID:28559296

Fabrication of microtemplates for the control of bacterial immobilization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Miyahara, Yasuhiro; Mitamura, Koji; Saito, Nagahiro

2009-09-15

The authors described a region-selective immobilization methods of bacteria by using superhydrophobic/superhydrophilic and superhydrophobic/poly(ethylene glycol) (PEG) micropatterns for culture scaffold templates. In the case of superhydrophobic/superhydrophilic micropatterns, the superhydrophobic surface was prepared first by microwave-plasma enhanced chemical vapor deposition (MPECVD) from trimethylmethoxysilane. Then the superhydrophilic regions were fabricated by irradiating the superhydrophobic surface with vuv light through a stencil mask. In the case of the superhydrophobic/PEG micropatterned surfaces, PEG surfaces were fabricated first by chemical reaction of ester groups of p-nitrophenyl PEG with NH{sub 2} group of NH{sub 2}-terminated self assembled monolayer from n-6-hexyl-3-aminopropyltrimethoxysilane. The superhydrophobic regions were fabricated bymore » MPECVD thorough a stencil mask. In this study four bacteria were selected from viewpoint of peptidoglycan cell wall (E. coli versus B. subtilis), extracellular polysaccharide (E.coli versus P. stutzeri, P. aeruginosa), and growth rate (P. stutzeri versus P. aeruginosa). The former micropattern brought discrete adhesions of E. coli and B. subtilis specifically on the hydrophobic regions, Furthermore, using the superhydrophobic/PEG micropattern, adhesion of bacteria expanded for E. coli, B. subtilis, P. stutzeri, and P. aeruginosa. They observed a high bacterial adhesion onto superhydrophobic surfaces and the inhibitive effect of bacterial adhesion on PEG surfaces.« less

Biosynthesis of human myeloperoxidase.

PubMed

Nauseef, William M

2018-03-15

Members of Chordata peroxidase subfamily [1] expressed in mammals, including myeloperoxidase (MPO), eosinophil peroxidase (EPO), lactoperoxidase (LPO), and thyroid peroxidase (TPO), express conserved motifs around the heme prosthetic group essential for their activity, a calcium-binding site, and at least two covalent bonds linking the heme group to the protein backbone. Although most studies of the biosynthesis of these peroxidases have focused on MPO, many of the features described occur during biosynthesis of other members of the protein subfamily. Whereas MPO biosynthesis includes events typical for proteins generated in the secretory pathway, the importance and consequences of heme insertion are events uniquely associated with peroxidases. This Review summarizes decades of work elucidating specific steps in the biosynthetic pathway of human MPO. Discussion includes cotranslational glycosylation and subsequent modifications of the N-linked carbohydrate sidechains, contributions by molecular chaperones in the endoplasmic reticulum, cleavage of the propeptide from proMPO, and proteolytic processing of protomers and dimerization to yield mature MPO. Parallels between the biosynthesis of MPO and TPO as well as the impact of inherited mutations in the MPO gene on normal biosynthesis will be summarized. Lastly, specific gaps in our knowledge revealed by this review of our current understanding will be highlighted. Copyright © 2018 Elsevier Inc. All rights reserved.

How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function

PubMed Central

Otero, Lisandro H.; Rojas-Altuve, Alzoray; Llarrull, Leticia I.; Carrasco-López, Cesar; Kumarasiri, Malika; Lastochkin, Elena; Fishovitz, Jennifer; Dawley, Matthew; Hesek, Dusan; Lee, Mijoon; Johnson, Jarrod W.; Fisher, Jed F.; Chang, Mayland; Mobashery, Shahriar; Hermoso, Juan A.

2013-01-01

The expression of penicillin binding protein 2a (PBP2a) is the basis for the broad clinical resistance to the β-lactam antibiotics by methicillin-resistant Staphylococcus aureus (MRSA). The high-molecular mass penicillin binding proteins of bacteria catalyze in separate domains the transglycosylase and transpeptidase activities required for the biosynthesis of the peptidoglycan polymer that comprises the bacterial cell wall. In bacteria susceptible to β-lactam antibiotics, the transpeptidase activity of their penicillin binding proteins (PBPs) is lost as a result of irreversible acylation of an active site serine by the β-lactam antibiotics. In contrast, the PBP2a of MRSA is resistant to β-lactam acylation and successfully catalyzes the dd-transpeptidation reaction necessary to complete the cell wall. The inability to contain MRSA infection with β-lactam antibiotics is a continuing public health concern. We report herein the identification of an allosteric binding domain—a remarkable 60 Å distant from the dd-transpeptidase active site—discovered by crystallographic analysis of a soluble construct of PBP2a. When this allosteric site is occupied, a multiresidue conformational change culminates in the opening of the active site to permit substrate entry. This same crystallographic analysis also reveals the identity of three allosteric ligands: muramic acid (a saccharide component of the peptidoglycan), the cell wall peptidoglycan, and ceftaroline, a recently approved anti-MRSA β-lactam antibiotic. The ability of an anti-MRSA β-lactam antibiotic to stimulate allosteric opening of the active site, thus predisposing PBP2a to inactivation by a second β-lactam molecule, opens an unprecedented realm for β-lactam antibiotic structure-based design. PMID:24085846

Bacterial cell motility of Burkholderia gut symbiont is required to colonize the insect gut.

PubMed

Lee, Jun Beom; Byeon, Jin Hee; Jang, Ho Am; Kim, Jiyeun Kate; Yoo, Jin Wook; Kikuchi, Yoshitomo; Lee, Bok Luel

2015-09-14

We generated a Burkholderia mutant, which is deficient of an N-acetylmuramyl-l-alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile ΔamiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, ΔamiC mutant cells were unable to establish symbiotic association. But, ΔamiC mutant complemented with amiC gene restored in vivo symbiotic association. ΔamiC mutant cultured in minimal medium restored their motility with single-celled morphology. When ΔamiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Peptidoglycan Recognition Protein S2 From Silkworm Integument: Characterization, Microbe-Induced Expression, and Involvement in the Immune-Deficiency Pathway

PubMed Central

Yang, Jie; Wang, Xiaonan; Tang, Shunming; Shen, Zhongyuan; Wu, Jinmei

2015-01-01

Peptidoglycan recognition protein (PGRP) binds specifically to peptidoglycan and plays an important role as a pattern recognition receptor in the innate immunity of insects. The cDNA of a short-type PGRP, an open reading frame of 588 bp encoding a polypeptide of 196 amino acids, was cloned from Bombyx mori. A phylogenetic tree was constructed, and the results showed that BmPGRP-S2 was most similar to Drosophila melanogaster PGRP (DmPGRP-SA). The induced expression profile of BmPGRP-S2 in healthy Escherichia coli- and Bacillus subtilis-challenged B. mori was measured using semiquantitative reverse transcriptase polymerase chain reaction analysis. The expression of BmPGRP-S2 was upregulated at 24 h by E. coli and Ba. subtilis challenge. In addition, in the integument of B. mori, RNAi knockdown of BmPGRP-S2 caused an obvious reduction in the transcription expression of the transcription factor Relish and in antibacterial effector genes Attacin, Gloverin, and Moricin. The results indicated that BmPGRP-S2 participates in the signal transduction pathway of B. mori. PMID:25797797

Bacterial translocation and plasma cytokines during transcatheter and open-heart aortic valve implantation.

PubMed

Adrie, Christophe; Parlato, Marianna; Salmi, Lynda; Adib-Conquy, Minou; Bical, Olivier; Deleuze, Philippe; Fitting, Catherine; Cavaillon, Jean Marc; Monchi, Mehran

2015-01-01

To determine whether the good safety profile of transarterial aortic valve implantation (TAVI) is related to lower levels of systemic bacterial translocation and systemic inflammation compared with open-heart surgery. Transcatheter aortic valve implantation via the transfemoral approach is increasingly used in very high-risk patients with aortic stenosis. The outcomes seem similar to those after open-heart aortic valve replacement (OHAVR). Each of 26 consecutive high-risk patients (EuroSCORE >20% for risk of operative death) who underwent TAVI (cases) was matched to the first low-risk patient treated next in our department using elective OHAVR without coronary artery bypass (control subjects). We collected severity, outcome, and echocardiography indicators before and after surgery; complications; proinflammatory cytokine levels; and markers for microbial translocation. Despite greater illness severity, the TAVI patients had significantly lower vasopressor agent requirements, lower delirium rates, shorter hospital stays, and better hemodynamic findings compared with OHAVR patients. Vascular complications were more common after TAVI than after OHAVR (12, with seven requiring interventional therapy vs. 0, P = 0.006). Patients who underwent TAVI had lower blood transfusion requirements. Two TAVI patients died: one from iliac artery injury and the other from intracardiac prosthesis migration. Patients who underwent TAVI had lower plasma levels of endotoxin and bacterial peptidoglycan, as well as lower proinflammatory cytokine levels, suggesting less gastrointestinal bacterial translocation compared with OHAVR. Compared with OHAVR, TAVI was associated with decreases in bacterial translocation and inflammation. These differences may explain the lower delirium rate and better hemodynamic stability observed, despite the greater disease severity in TAVI patients.

The genetic and molecular basis for sunscreen biosynthesis in cyanobacteria

PubMed Central

Balskus, Emily P.; Walsh, Christopher T.

2011-01-01

UV-A and UV-B radiation are harmful to living systems, causing damage to biological macromolecules. An important strategy for dealing with UV exposure is the biosynthesis of small molecule sunscreens. Among such metabolites, the mycosporine and mycosporine-like amino acids (MAAs) are remarkable for their wide phylogenetic distribution and their unique chemical structures. Here we report the identification of a MAA biosynthetic gene cluster in a cyanobacterium and the discovery of analogous pathways in other sequenced organisms. We have expressed the cluster in a heterologous bacterial host and characterized all four biosynthetic enzymes in vitro. In addition to clarifying the origin of the MAAs, these efforts have revealed two unprecedented enzymatic strategies for imine formation. PMID:20813918

OmpA: A Flexible Clamp for Bacterial Cell Wall Attachment.

PubMed

Samsudin, Firdaus; Ortiz-Suarez, Maite L; Piggot, Thomas J; Bond, Peter J; Khalid, Syma

2016-12-06

The envelope of Gram-negative bacteria is highly complex, containing separate outer and inner membranes and an intervening periplasmic space encompassing a peptidoglycan (PGN) cell wall. The PGN scaffold is anchored non-covalently to the outer membrane via globular OmpA-like domains of various proteins. We report atomically detailed simulations of PGN bound to OmpA in three different states, including the isolated C-terminal domain (CTD), the full-length monomer, or the complete full-length dimeric form. Comparative analysis of dynamics of OmpA CTD from different bacteria helped to identify a conserved PGN-binding mode. The dynamics of full-length OmpA, embedded within a realistic representation of the outer membrane containing full-rough (Ra) lipopolysaccharide, phospholipids, and cardiolipin, suggested how the protein may provide flexible mechanical support to the cell wall. An accurate model of the heterogeneous bacterial cell envelope should facilitate future efforts to develop antibacterial agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Protein biosynthesis in mitochondria.

PubMed

Kuzmenko, A V; Levitskii, S A; Vinogradova, E N; Atkinson, G C; Hauryliuk, V; Zenkin, N; Kamenski, P A

2013-08-01

Translation, that is biosynthesis of polypeptides in accordance with information encoded in the genome, is one of the most important processes in the living cell, and it has been in the spotlight of international research for many years. The mechanisms of protein biosynthesis in bacteria and in the eukaryotic cytoplasm are now understood in great detail. However, significantly less is known about translation in eukaryotic mitochondria, which is characterized by a number of unusual features. In this review, we summarize current knowledge about mitochondrial translation in different organisms while paying special attention to the aspects of this process that differ from cytoplasmic protein biosynthesis.

Serine biosynthesis and transport defects.

PubMed

El-Hattab, Ayman W

2016-07-01

l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

Revised Model of Calcium and Magnesium Binding to the Bacterial Cell Wall

PubMed Central

Thomas, Kieth J.; Rice, Charles V.

2014-01-01

Metals bind to the bacterial cell wall yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane (lipoteichoic acid) or covalently bound to the cell wall (wall teichoic acid). The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, contradicting previous reports that calcium binding is 100% dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger and previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, meaning that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 µmol/mg and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of there being a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent, suggesting a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations. PMID:25315444

Genetically modified luminescent bacteria Ralostonia solanacerum, Pseudomonas syringae, Pseudomonas savastanoi, and wild type bacterium Vibrio fischeri in biosynthesis of gold nanoparticles from gold chloride trihydrate.

PubMed

Attaran, Neda; Eshghi, Hossein; Rahimizadeh, Mohammad; Mashreghi, Mansour; Bakavoli, Mehdi

2014-08-04

The effect of different genetically engineered bacteria, Pseudomonas syringae, Pseudomonas savastanoi, and Ralostonia solanacerum and also a natural marine bacterial species, Vibrio fischeri NRRL B-11177, is studied in producing gold nanoparticles. This is the first report about the biosynthesis of gold nanoparticles by natural and genetically engineered luminescent bacteria. These microorganisms reduced gold ions and produced fairly monodisperse nanoparticles. TEM analysis indicated that spherical nano gold particles in the different diameters and shapes were obtained at pH values of 6.64. In this biosynthesis protocol, the gold nanoparticles with desired shape and size can be prepared.

Transcriptional Response of Selenopolypeptide Genes and Selenocysteine Biosynthesis Machinery Genes in Escherichia coli during Selenite Reduction.

PubMed

Tetteh, Antonia Y; Sun, Katherine H; Hung, Chiu-Yueh; Kittur, Farooqahmed S; Ibeanu, Gordon C; Williams, Daniel; Xie, Jiahua

2014-01-01

Bacteria can reduce toxic selenite into less toxic, elemental selenium (Se(0)), but the mechanism on how bacterial cells reduce selenite at molecular level is still not clear. We used Escherichia coli strain K12, a common bacterial strain, as a model to study its growth response to sodium selenite (Na2SeO3) treatment and then used quantitative real-time PCR (qRT-PCR) to quantify transcript levels of three E. coli selenopolypeptide genes and a set of machinery genes for selenocysteine (SeCys) biosynthesis and incorporation into polypeptides, whose involvements in the selenite reduction are largely unknown. We determined that 5 mM Na2SeO3 treatment inhibited growth by ∼ 50% while 0.001 to 0.01 mM treatments stimulated cell growth by ∼ 30%. Under 50% inhibitory or 30% stimulatory Na2SeO3 concentration, selenopolypeptide genes (fdnG, fdoG, and fdhF) whose products require SeCys but not SeCys biosynthesis machinery genes were found to be induced ≥2-fold. In addition, one sulfur (S) metabolic gene iscS and two previously reported selenite-responsive genes sodA and gutS were also induced ≥2-fold under 50% inhibitory concentration. Our findings provide insight about the detoxification of selenite in E. coli via induction of these genes involved in the selenite reduction process.

Heterologous Expression of Bacterial Epoxyalkane:Coenzyme M Transferase and Inducible Coenzyme M Biosynthesis in Xanthobacter Strain Py2 and Rhodococcus rhodochrous B276

PubMed Central

Krum, Jonathan G.; Ensign, Scott A.

2000-01-01

Coenzyme M (CoM) (2-mercaptoethanesulfonic acid) biosynthesis is shown to be coordinately regulated with the expression of the enzymes of alkene and epoxide metabolism in the propylene-oxidizing bacteria Xanthobacter strain Py2 and Rhodococcus rhodochrous strain B276. These results provide the first evidence for the involvement of CoM in propylene metabolism by R. rhodochrous and demonstrate for the first time the inducible nature of eubacterial CoM biosynthesis. PMID:10762269

Complete characterization of the seventeen step moenomycin biosynthetic pathway

PubMed Central

Ostash, Bohdan; Doud, Emma; Lin, Cecilie; Ostash, Iryna; Perlstein, Deborah; Fuse, Shinichiro; Wolpert, Manuel; Kahne, Daniel; Walker, Suzanne

2009-01-01

The moenomycins are phosphoglycolipid antibiotics produced by Streptomyces ghanaensis and related organisms. The phosphoglycolipids are the only known active site inhibitors of the peptidoglycan glycosyltransferases, an important family of enzymes involved in the biosynthesis of the bacterial cell wall. Although these natural products have exceptionally potent antibiotic activity, pharmacokinetic limitations have precluded their clinical use. We previously identified the moenomycin biosynthetic gene cluster in order to facilitate biosynthetic approaches to new derivatives. Here we report a comprehensive set of genetic and enzymatic experiments that establish functions for the seventeen moenomycin biosynthetic genes involved in the synthesis moenomycin and variants. These studies reveal the order of assembly of the full molecular scaffold and define a subset of seven genes involved in the synthesis of bioactive analogs. This work will enable both in vitro and fermentation-based reconstitution of phosphoglycolipid scaffolds so that chemoenzymatic approaches to novel analogs can be explored. PMID:19640006

Regulation of cell wall biosynthesis.

PubMed

Zhong, Ruiqin; Ye, Zheng-Hua

2007-12-01

Plant cell walls differ in their amount and composition among various cell types and even in different microdomains of the wall of a given cell. Plants must have evolved regulatory mechanisms controlling biosynthesis, targeted secretion, and assembly of wall components to achieve the heterogeneity in cell walls. A number of factors, including hormones, the cytoskeleton, glycosylphosphatidylinositol-anchored proteins, phosphoinositides, and sugar nucleotide supply, have been implicated in the regulation of cell wall biosynthesis or deposition. In the past two years, there have been important discoveries in transcriptional regulation of secondary wall biosynthesis. Several transcription factors in the NAC and MYB families have been shown to be the key switches for activation of secondary wall biosynthesis. These studies suggest a transcriptional network comprised of a hierarchy of transcription factors is involved in regulating secondary wall biosynthesis. Further investigation and integration of the regulatory players participating in the making of cell walls will certainly lead to our understanding of how wall amounts and composition are controlled in a given cell type. This may eventually allow custom design of plant cell walls on the basis of our needs.

Essential role of Bordetella NadC in a quinolinate salvage pathway for NAD biosynthesis.

PubMed

Brickman, Timothy J; Suhadolc, Ryan J; McKelvey, Pamela J; Armstrong, Sandra K

2017-02-01

Nicotinamide adenine dinucleotide (NAD) is produced via de novo biosynthesis pathways and by salvage or recycling routes. The classical Bordetella bacterial species are known to be auxotrophic for nicotinamide or nicotinic acid. This study confirmed that Bordetella bronchiseptica, Bordetella pertussis and Bordetella parapertussis have the recycling/salvage pathway genes pncA and pncB, for use of nicotinamide or nicotinic acid, respectively, for NAD synthesis. Although these Bordetellae lack the nadA and nadB genes needed for de novo NAD biosynthesis, remarkably, they have one de novo pathway gene, nadC, encoding quinolinate phosphoribosyltransferase. Genomic analyses of taxonomically related Bordetella and Achromobacter species also indicated the presence of an 'orphan' nadC and the absence of nadA and nadB. When supplied as the sole NAD precursor, quinolinate promoted B. bronchiseptica growth, and the ability to use it required nadC. Co-expression of Bordetella nadC with the nadB and nadA genes of Paraburkholderia phytofirmans allowed B. bronchiseptica to grow in the absence of supplied pyridines, indicative of de novo NAD synthesis and functional confirmation of Bordetella NadC activity. Expression of nadC in B. bronchiseptica was influenced by nicotinic acid and by a NadQ family transcriptional repressor, indicating that these organisms prioritize their use of pyridines for NAD biosynthesis. © 2016 John Wiley & Sons Ltd.

Prebiotic oligosaccharides reduce proinflammatory cytokines in intestinal Caco-2 cells via activation of PPARγ and peptidoglycan recognition protein 3.

PubMed

Zenhom, Marwa; Hyder, Ayman; de Vrese, Michael; Heller, Knut J; Roeder, Thomas; Schrezenmeir, Jürgen

2011-05-01

Prebiotic oligosaccharides modulate the intestinal microbiota and beneficially affect the human body by reducing intestinal inflammation. This immunomodulatory effect was assumed to be bacterial in origin. However, some observations suggest that oligosaccharides may exert an antiinflammatory effect per se. We hypothesized that oligosaccharides affect the intestinal immunity via activation of peptidoglycan recognition protein 3 (PGlyRP3), which reduces the expression of proinflammatory cytokines. Caco-2 cells were treated with the oligosaccharides, α3-sialyllactose, or fructooligosaccharides (Raftilose p95), and the effects of these treatments on PGlyRP3 and PPARγ expression, the release and expression of some proinflammatory cytokines, and NF-κB translocation were tested. Both oligosaccharides had antiinflammatory activity; they significantly reduced IL-12 secretion in Caco-2 cells and gene expression of IL-12p35, IL-8, and TNFα. They also reduced the gene expression and nuclear translocation of NF-κB. Both oligosaccharides dose and time dependently induced the production of PGlyRP3, the silencing of which by transfection of Caco-2 cells with specific small interfering RNA targeting PGlyRP3 abolished the antiinflammatory role of both oligosaccharides. Incubation of Caco-2 cells with both oligosaccharides induced PPARγ. Antagonizing PPARγ by culturing the cells with GW9662 for 24 h inhibited the oligosaccharide-induced PGlyRP3 production and the antiinflammatory effect of the oligosaccharides. We conclude that oligosaccharides may exert an antiinflammatory effect by inducing the nuclear receptor PPARγ, which regulates the antiinflammatory PGlyRP3.

Targeting bacterial central metabolism for drug development.

PubMed

Murima, Paul; McKinney, John D; Pethe, Kevin

2014-11-20

Current antibiotics, derived mainly from natural sources, inhibit a narrow spectrum of cellular processes, namely DNA replication, protein synthesis, and cell wall biosynthesis. With the worldwide explosion of drug resistance, there is renewed interest in the investigation of alternate essential cellular processes, including bacterial central metabolic pathways, as a drug target space for the next generation of antibiotics. However, the validation of targets in central metabolism is more complex, as essentiality of such targets can be conditional and/or contextual. Bearing in mind our enhanced understanding of prokaryotic central metabolism, a key question arises: can central metabolism be bacteria's Achilles' heel and a therapeutic target for the development of new classes of antibiotics? In this review, we draw lessons from oncology and attempt to address some of the open questions related to feasibility of targeting bacterial central metabolism as a strategy for developing new antibacterial drugs. Copyright © 2014 Elsevier Ltd. All rights reserved.

Lytic activity of the virion-associated peptidoglycan hydrolase HydH5 of staphylococcus aureus bacteriophage vB_SauS-phiIPLA88

USDA-ARS?s Scientific Manuscript database

Staphylococcus aureus bacteriophage vB_SauS-phiIPLA88 (phiIPLA88) contains a virion-associated muralytic enzyme (HydH5) encoded by orf58, which is located in the morphogenetic module. Comparative bioinformatic analysis revealed that HydH5 significantly resembled other peptidoglycan hydrolases encode...

Cell-wall preparation containing poly-γ-D-glutamate covalently linked to peptidoglycan, a straightforward extractable molecule, protects mice against experimental anthrax infection.

PubMed

Candela, Thomas; Dumetz, Fabien; Tosi-Couture, Evelyne; Mock, Michèle; Goossens, Pierre L; Fouet, Agnès

2012-12-17

Bacillus anthracis is the causative agent of anthrax that is characterized by septicemia and toxemia. Many vaccine strategies were described to counteract anthrax infection. In contrast with veterinary live vaccines, currently human vaccines are acellular with the protective antigen, a toxin component, as the main constituent. However, in animal models this vaccine is less efficient than the live vaccine. In this study, we analyzed the protection afforded by a single extractable surface element. The poly-γ-D-glutamate capsule is covalently linked to the peptidoglycan. A preparation of peptidoglycan-linked poly-γ-D-glutamate (GluPG) was tested for its immunogenicity and its protective effect. GluPG injection, in mice, elicited the production of specific antibodies directed against poly-glutamate and partially protected the animals against lethal challenges with a non-toxinogenic strain. When combined to protective antigen, GluPG immunization conferred full protection against cutaneous anthrax induced with a fully virulent strain. Copyright © 2012 Elsevier Ltd. All rights reserved.

Mutagenesis of NosM Leader Peptide Reveals Important Elements in Nosiheptide Biosynthesis

PubMed Central

Jin, Liang; Wu, Xuri; Xue, Yanjiu; Jin, Yue; Wang, Shuzhen

2016-01-01

ABSTRACT Nosiheptide, a typical member of the ribosomally synthesized and posttranslationally modified peptides (RiPPs), exhibits potent activity against multidrug-resistant Gram-positive bacterial pathogens. The precursor peptide of nosiheptide (NosM) is comprised of a leader peptide with 37 amino acids and a core peptide containing 13 amino acids. To pinpoint elements in the leader peptide that are essential for nosiheptide biosynthesis, a collection of mutants with unique sequence features, including N- and C-terminal motifs, peptide length, and specific sites in the leader peptide, was generated by mutagenesis in vivo. The effects of various mutants on nosiheptide biosynthesis were evaluated. In addition to the necessity of a conserved motif LEIS box, native length and the N-terminal 12 amino acid residues were indispensable, and single-site substitutions of these 12 amino acid residues resulted in changes ranging from a greater-than-5-fold decrease to a 2-fold increase of nosiheptide production, depending on the sites and substituted residues. Moreover, although the C-terminal motif is not conservative, significant effects of this portion on nosiheptide production were also evident. Taken together, the present results further highlight the importance of the leader peptide in nosiheptide biosynthesis, and provide new insights into the diversity and specificity of leader peptides in the biosynthesis of various RiPPs. IMPORTANCE As a representative thiopeptide, nosiheptide exhibits excellent antibacterial activity. Although the biosynthetic gene cluster and several modification steps have been revealed, the presence and roles of the leader peptide within the precursor peptide of the nosiheptide gene cluster remain elusive. Thus, identification of specific elements in the leader peptide can significantly facilitate the genetic manipulation of the gene cluster for increasing nosiheptide production or generating diverse analogues. Given the complexity of the

Gram's Stain Does Not Cross the Bacterial Cytoplasmic Membrane.

PubMed

Wilhelm, Michael J; Sheffield, Joel B; Sharifian Gh, Mohammad; Wu, Yajing; Spahr, Christian; Gonella, Grazia; Xu, Bolei; Dai, Hai-Lung

2015-07-17

For well over a century, Hans Christian Gram's famous staining protocol has been the standard go-to diagnostic for characterizing unknown bacteria. Despite continuous and ubiquitous use, we now demonstrate that the current understanding of the molecular mechanism for this differential stain is largely incorrect. Using the fully complementary time-resolved methods: second-harmonic light-scattering and bright-field transmission microscopy, we present a real-time and membrane specific quantitative characterization of the bacterial uptake of crystal-violet (CV), the dye used in Gram's protocol. Our observations contradict the currently accepted mechanism which depicts that, for both Gram-negative and Gram-positive bacteria, CV readily traverses the peptidoglycan mesh (PM) and cytoplasmic membrane (CM) before equilibrating within the cytosol. We find that not only is CV unable to traverse the CM but, on the time-scale of the Gram-stain procedure, CV is kinetically trapped within the PM. Our results indicate that CV, rather than dyes which rapidly traverse the PM, is uniquely suited as the Gram stain.

Different polyamine pathways from bacteria have replaced eukaryotic spermidine biosynthesis in ciliates Tetrahymena thermophila and Paramecium tetaurelia.

PubMed

Li, Bin; Kim, Sok Ho; Zhang, Yang; Hanfrey, Colin C; Elliott, Katherine A; Ealick, Steven E; Michael, Anthony J

2015-09-01

The polyamine spermidine is absolutely required for growth and cell proliferation in eukaryotes, due to its role in post-translational modification of essential translation elongation factor eIF5A, mediated by deoxyhypusine synthase. We have found that free-living ciliates Tetrahymena and Paramecium lost the eukaryotic genes encoding spermidine biosynthesis: S-adenosylmethionine decarboxylase (AdoMetDC) and spermidine synthase (SpdSyn). In Tetrahymena, they were replaced by a gene encoding a fusion protein of bacterial AdoMetDC and SpdSyn, present as three copies. In Paramecium, a bacterial homospermidine synthase replaced the eukaryotic genes. Individual AdoMetDC-SpdSyn fusion protein paralogues from Tetrahymena exhibit undetectable AdoMetDC activity; however, when two paralogous fusion proteins are mixed, AdoMetDC activity is restored and spermidine is synthesized. Structural modelling indicates a functional active site is reconstituted by sharing critical residues from two defective protomers across the heteromer interface. Paramecium was found to accumulate homospermidine, suggesting it replaces spermidine for growth. To test this concept, a budding yeast spermidine auxotrophic strain was found to grow almost normally with homospermidine instead of spermidine. Biosynthesis of spermidine analogue aminopropylcadaverine, but not exogenously provided norspermidine, correlated with some growth. Finally, we found that diverse single-celled eukaryotic parasites and multicellular metazoan Schistosoma worms have lost the spermidine biosynthetic pathway but retain deoxyhypusine synthase. © 2015 John Wiley & Sons Ltd.

Pyridine-type alkaloid composition affects bacterial community composition of floral nectar

PubMed Central

Aizenberg-Gershtein, Yana; Izhaki, Ido; Santhanam, Rakesh; Kumar, Pavan; Baldwin, Ian T.; Halpern, Malka

2015-01-01

Pyridine-type alkaloids are most common in Nicotiana species. To study the effect of alkaloid composition on bacterial community composition in floral nectar, we compared the nicotine-rich wild type (WT) N. attenuata, the nicotine biosynthesis-silenced N. attenuata that was rich in anatabine and the anabasine-rich WT N. glauca plants. We found that the composition of these secondary metabolites in the floral nectar drastically affected the bacterial community richness, diversity and composition. Significant differences were found between the bacterial community compositions in the nectar of the three plants with a much greater species richness and diversity in the nectar from the transgenic plant. The highest community composition similarity index was detected between the two wild type plants. The different microbiome composition and diversity, caused by the different pyridine-type alkaloid composition, could modify the nutritional content of the nectar and consequently, may contribute to the change in the nectar consumption and visitation. These may indirectly have an effect on plant fitness. PMID:26122961

Pyridine-type alkaloid composition affects bacterial community composition of floral nectar.

PubMed

Aizenberg-Gershtein, Yana; Izhaki, Ido; Santhanam, Rakesh; Kumar, Pavan; Baldwin, Ian T; Halpern, Malka

2015-06-30

Pyridine-type alkaloids are most common in Nicotiana species. To study the effect of alkaloid composition on bacterial community composition in floral nectar, we compared the nicotine-rich wild type (WT) N. attenuata, the nicotine biosynthesis-silenced N. attenuata that was rich in anatabine and the anabasine-rich WT N. glauca plants. We found that the composition of these secondary metabolites in the floral nectar drastically affected the bacterial community richness, diversity and composition. Significant differences were found between the bacterial community compositions in the nectar of the three plants with a much greater species richness and diversity in the nectar from the transgenic plant. The highest community composition similarity index was detected between the two wild type plants. The different microbiome composition and diversity, caused by the different pyridine-type alkaloid composition, could modify the nutritional content of the nectar and consequently, may contribute to the change in the nectar consumption and visitation. These may indirectly have an effect on plant fitness.

Bacterial proteinases as targets for the development of second-generation antibiotics.

PubMed

Travis, J; Potempa, J

2000-03-07

The emergence of bacterial pathogen resistance to common antibiotics strongly supports the necessity to develop alternative mechanisms for combating drug-resistant forms of these infective organisms. Currently, few pharmaceutical companies have attempted to investigate the possibility of interrupting metabolic pathways other than those that are known to be involved in cell wall biosynthesis. In this review, we describe multiple, novel roles for bacterial proteinases during infection using, as a specific example, the enzymes from the organism Porphyromonas gingivalis, a periodontopathogen, which is known to be involved in the development and progression of periodontal disease. In this manner, we are able to justify the concept of developing synthetic inhibitors against members of this class of enzymes as potential second-generation antibiotics. Such compounds could not only prove valuable in retarding the growth and proliferation of bacterial pathogens but also lead to the use of this class of inhibitors against invasion by other infective organisms.

Specificity determinants for lysine incorporation in Staphylococcus aureus peptidoglycan as revealed by the structure of a MurE enzyme ternary complex.

PubMed

Ruane, Karen M; Lloyd, Adrian J; Fülöp, Vilmos; Dowson, Christopher G; Barreteau, Hélène; Boniface, Audrey; Dementin, Sébastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Dessen, Andréa; Roper, David I

2013-11-15

Formation of the peptidoglycan stem pentapeptide requires the insertion of both L and D amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-L-alanyl-D-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-L-Ala-γ-D-Glu-L-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between L-lysine and D,L-diaminopimelic acid, the predominant amino acid that replaces L-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of L-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for L-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic L-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly.

Specificity Determinants for Lysine Incorporation in Staphylococcus aureus Peptidoglycan as Revealed by the Structure of a MurE Enzyme Ternary Complex*

PubMed Central

Ruane, Karen M.; Lloyd, Adrian J.; Fülöp, Vilmos; Dowson, Christopher G.; Barreteau, Hélène; Boniface, Audrey; Dementin, Sébastien; Blanot, Didier; Mengin-Lecreulx, Dominique; Gobec, Stanislav; Dessen, Andréa; Roper, David I.

2013-01-01

Formation of the peptidoglycan stem pentapeptide requires the insertion of both l and d amino acids by the ATP-dependent ligase enzymes MurC, -D, -E, and -F. The stereochemical control of the third position amino acid in the pentapeptide is crucial to maintain the fidelity of later biosynthetic steps contributing to cell morphology, antibiotic resistance, and pathogenesis. Here we determined the x-ray crystal structure of Staphylococcus aureus MurE UDP-N-acetylmuramoyl-l-alanyl-d-glutamate:meso-2,6-diaminopimelate ligase (MurE) (E.C. 6.3.2.7) at 1.8 Å resolution in the presence of ADP and the reaction product, UDP-MurNAc-l-Ala-γ-d-Glu-l-Lys. This structure provides for the first time a molecular understanding of how this Gram-positive enzyme discriminates between l-lysine and d,l-diaminopimelic acid, the predominant amino acid that replaces l-lysine in Gram-negative peptidoglycan. Despite the presence of a consensus sequence previously implicated in the selection of the third position residue in the stem pentapeptide in S. aureus MurE, the structure shows that only part of this sequence is involved in the selection of l-lysine. Instead, other parts of the protein contribute substrate-selecting residues, resulting in a lysine-binding pocket based on charge characteristics. Despite the absolute specificity for l-lysine, S. aureus MurE binds this substrate relatively poorly. In vivo analysis and metabolomic data reveal that this is compensated for by high cytoplasmic l-lysine concentrations. Therefore, both metabolic and structural constraints maintain the structural integrity of the staphylococcal peptidoglycan. This study provides a novel focus for S. aureus-directed antimicrobials based on dual targeting of essential amino acid biogenesis and its linkage to cell wall assembly. PMID:24064214

Cysteine Biosynthesis Controls Serratia marcescens Phospholipase Activity.

PubMed

Anderson, Mark T; Mitchell, Lindsay A; Mobley, Harry L T

2017-08-15

biosynthesis results in decreased phlA and flagellar gene transcription, which can be restored by supplying bacteria with exogenous cysteine. These results identify a previously unrecognized role for CysE and cysteine in the secretion of S. marcescens phospholipase and in bacterial motility. Copyright © 2017 American Society for Microbiology.

Properties and applications of undecylprodigiosin and other bacterial prodigiosins.

PubMed

Stankovic, Nada; Senerovic, Lidija; Ilic-Tomic, Tatjana; Vasiljevic, Branka; Nikodinovic-Runic, Jasmina

2014-05-01

The growing demand to fulfill the needs of present-day medicine in terms of novel effective molecules has lead to reexamining some of the old and known bacterial secondary metabolites. Bacterial prodigiosins (prodiginines) have a long history of being re markable multipurpose compounds, best examined for their anticancer and antimalarial activities. Production of prodigiosin in the most common producer strain Serratia marcescens has been described in great detail. However, few reports have discussed the ecophysiological roles of these molecules in the producing strains, as well as their antibiotic and UV-protective properties. This review describes recent advances in the production process, biosynthesis, properties, and applications of bacterial prodigiosins. Special emphasis is put on undecylprodigiosin which has generally been a less studied member of the prodigiosin family. In addition, it has been suggested that proteins involved in undecylprodigiosin synthesis, RedG and RedH, could be a useful addition to the biocatalytic toolbox being able to mediate regio- and stereoselective oxidative cyclization. Judging by the number of recent references (216 for the 2007-2013 period), it has become clear that undecylprodigiosin and other bacterial prodigiosins still hold surprises in terms of valuable properties and applicative potential to medical and other industrial fields and that they still deserve continuing research curiosity.

Expression of a Peptidoglycan Hydrolase from Lytic Bacteriophages Atu_ph02 and Atu_ph03 Triggers Lysis of Agrobacterium tumefaciens

PubMed Central

Attai, Hedieh; Rimbey, Jeanette; Smith, George P.

2017-01-01

ABSTRACT To provide food security, innovative approaches to preventing plant disease are currently being explored. Here, we demonstrate that lytic bacteriophages and phage lysis proteins are effective at triggering lysis of the phytopathogen Agrobacterium tumefaciens. Phages Atu_ph02 and Atu_ph03 were isolated from wastewater and induced lysis of C58-derived strains of A. tumefaciens. The coinoculation of A. tumefaciens with phages on potato discs limited tumor formation. The genomes of Atu_ph02 and Atu_ph03 are nearly identical and are ∼42% identical to those of T7 supercluster phages. In silico attempts to find a canonical lysis cassette were unsuccessful; however, we found a putative phage peptidoglycan hydrolase (PPH), which contains a C-terminal transmembrane domain. Remarkably, the endogenous expression of pph in the absence of additional phage genes causes a block in cell division and subsequent lysis of A. tumefaciens cells. When the presumed active site of the N-acetylmuramidase domain carries an inactivating mutation, PPH expression causes extensive cell branching due to a block in cell division but does not trigger rapid cell lysis. In contrast, the mutation of positively charged residues at the extreme C terminus of PPH causes more rapid cell lysis. Together, these results suggest that PPH causes a block in cell division and triggers cell lysis through two distinct activities. Finally, the potent killing activity of this single lysis protein can be modulated, suggesting that it could be engineered to be an effective enzybiotic. IMPORTANCE The characterization of bacteriophages such as Atu_ph02 and Atu_ph03, which infect plant pathogens such as Agrobacterium tumefaciens, may be the basis of new biocontrol strategies. First, cocktails of diverse bacteriophages could be used as a preventative measure to limit plant diseases caused by bacteria; a bacterial pathogen is unlikely to simultaneously develop resistances to multiple bacteriophage species. The

Identification of an inhibitor of the MurC enzyme, which catalyzes an essential step in the peptidoglycan precursor synthesis pathway.

PubMed

Zawadzke, Laura E; Norcia, Michael; Desbonnet, Charlene R; Wang, Hong; Freeman-Cook, Kevin; Dougherty, Thomas J

2008-02-01

The pathway for synthesis of the peptidoglycan precursor UDP-N-acetylmuramyl pentapeptide is essential in Gram-positive and Gram-negative bacteria. This pathway has been exploited in the recent past to identify potential new antibiotics as inhibitors of one or more of the Mur enzymes. In the present study, a high-throughput screen was employed to identify potential inhibitors of the Escherichia coli MurC (UDP-N-acetylmuramic acid:L-alanine ligase), the first of four paralogous amino acid-adding enzymes. Inhibition of ATP consumed during the MurC reaction, using an adaptation of a kinase assay format, identified a number of potential inhibitory chemotypes. After nonspecific inhibition testing and chemical attractiveness were assessed, C-1 emerged as a compound for further characterization. The inhibition of MurC by this compound was confirmed in both a kinetic-coupled enzyme assay and a direct nuclear magnetic resonance product detection assay. C-1 was found to be a low micromolar inhibitor of the E. coli MurC reaction, with preferential inhibition by one of two enantiomeric forms. Experiments indicated that it was a competitive inhibitor of ATP binding to the MurC enzyme. Further work with MurC enzymes from several bacterial sources revealed that while the compound was equally effective at inhibiting MurC from genera (Proteus mirabilis and Klebsiella pneumoniae) closely related to E. coli, MurC enzymes from more distant Gram-negative species such as Haemophilus influenzae, Acinetobacter baylyi, and Pseudomonas aeruginosa were not inhibited.

Comparative analyses imply that the enigmatic sigma factor 54 is a central controller of the bacterial exterior

PubMed Central

2011-01-01

Background Sigma-54 is a central regulator in many pathogenic bacteria and has been linked to a multitude of cellular processes like nitrogen assimilation and important functional traits such as motility, virulence, and biofilm formation. Until now it has remained obscure whether these phenomena and the control by Sigma-54 share an underlying theme. Results We have uncovered the commonality by performing a range of comparative genome analyses. A) The presence of Sigma-54 and its associated activators was determined for all sequenced prokaryotes. We observed a phylum-dependent distribution that is suggestive of an evolutionary relationship between Sigma-54 and lipopolysaccharide and flagellar biosynthesis. B) All Sigma-54 activators were identified and annotated. The relation with phosphotransfer-mediated signaling (TCS and PTS) and the transport and assimilation of carboxylates and nitrogen containing metabolites was substantiated. C) The function annotations, that were represented within the genomic context of all genes encoding Sigma-54, its activators and its promoters, were analyzed for intra-phylum representation and inter-phylum conservation. Promoters were localized using a straightforward scoring strategy that was formulated to identify similar motifs. We found clear highly-represented and conserved genetic associations with genes that concern the transport and biosynthesis of the metabolic intermediates of exopolysaccharides, flagella, lipids, lipopolysaccharides, lipoproteins and peptidoglycan. Conclusion Our analyses directly implicate Sigma-54 as a central player in the control over the processes that involve the physical interaction of an organism with its environment like in the colonization of a host (virulence) or the formation of biofilm. PMID:21806785

Bacterial cell wall composition and the influence of antibiotics by cell-wall and whole-cell NMR

PubMed Central

Romaniuk, Joseph A. H.; Cegelski, Lynette

2015-01-01

The ability to characterize bacterial cell-wall composition and structure is crucial to understanding the function of the bacterial cell wall, determining drug modes of action and developing new-generation therapeutics. Solid-state NMR has emerged as a powerful tool to quantify chemical composition and to map cell-wall architecture in bacteria and plants, even in the context of unperturbed intact whole cells. In this review, we discuss solid-state NMR approaches to define peptidoglycan composition and to characterize the modes of action of old and new antibiotics, focusing on examples in Staphylococcus aureus. We provide perspectives regarding the selected NMR strategies as we describe the exciting and still-developing cell-wall and whole-cell NMR toolkit. We also discuss specific discoveries regarding the modes of action of vancomycin analogues, including oritavancin, and briefly address the reconsideration of the killing action of β-lactam antibiotics. In such chemical genetics approaches, there is still much to be learned from perturbations enacted by cell-wall assembly inhibitors, and solid-state NMR approaches are poised to address questions of cell-wall composition and assembly in S. aureus and other organisms. PMID:26370936

Studies on the genetic control of murine humoral response to immunization with a peptidoglycan-containing fraction extracted from Brucella melitensis.

PubMed

Cannat, A; Feingold, N; Caffin, J C; Serre, A

1979-01-01

A peptidoglycan containing fraction (fraction "5") extracted from Brucella melitensis has been injected in low infra-vaccinating doses into inbred mice. The genetic control of the resulting anti-Brucella humoral response has been studied in the C57BL/6 "good responder" X DBA2 "low responder" model. The results observed in F1, F2 and reciprocal backcrosses show that the "good responder" character, although transmitted as a dominant trait, is under polygenic control and independent of H2 haplotype, Ig allotype, sexual chromosoms or the "d" coat color gene. On the other hand, the phenotypic expression of at least one of the genes involved is sex-limited and influenced by hormonal environmental factors. Moreover the expression in females of one of these sex-dependent genes is associated with the "b" coat color gene. These results are discussed in terms of their possible relevance in spontaneous or vaccinal resistance to experimental brucellosis, of the relative role of the peptidoglycan and lipoprotein moieties in fraction "5" and of the possible importance of sex-dependent and chromosome 4-linked genetic factors for B-cell functions.

Multiparametric AFM reveals turgor-responsive net-like peptidoglycan architecture in live streptococci

NASA Astrophysics Data System (ADS)

Saar Dover, Ron; Bitler, Arkady; Shimoni, Eyal; Trieu-Cuot, Patrick; Shai, Yechiel

2015-05-01

Cell-wall peptidoglycan (PG) of Gram-positive bacteria is a strong and elastic multi-layer designed to resist turgor pressure and determine the cell shape and growth. Despite its crucial role, its architecture remains largely unknown. Here using high-resolution multiparametric atomic force microscopy (AFM), we studied how the structure and elasticity of PG change when subjected to increasing turgor pressure in live Group B Streptococcus. We show a new net-like arrangement of PG, which stretches and stiffens following osmotic challenge. The same structure also exists in isogenic mutants lacking surface appendages. Cell aging does not alter the elasticity of the cell wall, yet destroys the net architecture and exposes single segmented strands with the same circumferential orientation as predicted for intact glycans. Together, we show a new functional PG architecture in live Gram-positive bacteria.

Discovery of new inhibitors of the bacterial peptidoglycan biosynthesis enzymes MurD and MurF by structure-based virtual screening.

PubMed

Turk, Samo; Kovac, Andreja; Boniface, Audrey; Bostock, Julieanne M; Chopra, Ian; Blanot, Didier; Gobec, Stanislav

2009-03-01

The ATP-dependent Mur ligases (MurC, MurD, MurE and MurF) successively add L-Ala, D-Glu, meso-A(2)pm or L-Lys, and D-Ala-D-Ala to the nucleotide precursor UDP-MurNAc, and they represent promising targets for antibacterial drug discovery. We have used the molecular docking programme eHiTS for the virtual screening of 1990 compounds from the National Cancer Institute 'Diversity Set' on MurD and MurF. The 50 top-scoring compounds from screening on each enzyme were selected for experimental biochemical evaluation. Our approach of virtual screening and subsequent in vitro biochemical evaluation of the best ranked compounds has provided four novel MurD inhibitors (best IC(50)=10 microM) and one novel MurF inhibitor (IC(50)=63 microM).

Pseudomonas syringae Phytotoxins: Mode of Action, Regulation, and Biosynthesis by Peptide and Polyketide Synthetases

PubMed Central

Bender, Carol L.; Alarcón-Chaidez, Francisco; Gross, Dennis C.

1999-01-01

Coronatine, syringomycin, syringopeptin, tabtoxin, and phaseolotoxin are the most intensively studied phytotoxins of Pseudomonas syringae, and each contributes significantly to bacterial virulence in plants. Coronatine functions partly as a mimic of methyl jasmonate, a hormone synthesized by plants undergoing biological stress. Syringomycin and syringopeptin form pores in plasma membranes, a process that leads to electrolyte leakage. Tabtoxin and phaseolotoxin are strongly antimicrobial and function by inhibiting glutamine synthetase and ornithine carbamoyltransferase, respectively. Genetic analysis has revealed the mechanisms responsible for toxin biosynthesis. Coronatine biosynthesis requires the cooperation of polyketide and peptide synthetases for the assembly of the coronafacic and coronamic acid moieties, respectively. Tabtoxin is derived from the lysine biosynthetic pathway, whereas syringomycin, syringopeptin, and phaseolotoxin biosynthesis requires peptide synthetases. Activation of phytotoxin synthesis is controlled by diverse environmental factors including plant signal molecules and temperature. Genes involved in the regulation of phytotoxin synthesis have been located within the coronatine and syringomycin gene clusters; however, additional regulatory genes are required for the synthesis of these and other phytotoxins. Global regulatory genes such as gacS modulate phytotoxin production in certain pathovars, indicating the complexity of the regulatory circuits controlling phytotoxin synthesis. The coronatine and syringomycin gene clusters have been intensively characterized and show potential for constructing modified polyketides and peptides. Genetic reprogramming of peptide and polyketide synthetases has been successful, and portions of the coronatine and syringomycin gene clusters could be valuable resources in developing new antimicrobial agents. PMID:10357851

Characterization of Bacterial Communities Associated with the Tyrian Purple Producing Gland in a Marine Gastropod

PubMed Central

Ngangbam, Ajit Kumar; Baten, Abdul; Waters, Daniel L. E.; Whalan, Steve; Benkendorff, Kirsten

2015-01-01

Dicathais orbita is a marine mollusc recognised for the production of anticancer compounds that are precursors to Tyrian purple. This study aimed to assess the diversity and identity of bacteria associated with the Tyrian purple producing hypobranchial gland, in comparison with foot tissue, using a high-throughput sequencing approach. Taxonomic and phylogenetic analysis of variable region V1-V3 of 16S rRNA bacterial gene amplicons in QIIME and MEGAN were carried out. This analysis revealed a highly diverse bacterial assemblage associated with the hypobranchial gland and foot tissues of D. orbita. The dominant bacterial phylum in the 16S rRNA bacterial profiling data set was Proteobacteria followed by Bacteroidetes, Tenericutes and Spirochaetes. In comparison to the foot, the hypobranchial gland had significantly lower bacterial diversity and a different community composition, based on taxonomic assignment at the genus level. A higher abundance of indole producing Vibrio spp. and the presence of bacteria with brominating capabilities in the hypobranchial gland suggest bacteria have a potential role in biosynthesis of Tyrian purple in D. orbita. PMID:26488885

LipidII: Just Another Brick in the Wall?

PubMed Central

Scheffers, Dirk-Jan; Tol, Menno B.

2015-01-01

Nearly all bacteria contain a peptidoglycan cell wall. The peptidoglycan precursor molecule is LipidII, containing the basic peptidoglycan building block attached to a lipid. Although the suitability of LipidII as an antibacterial target has long been recognized, progress on elucidating the role(s) of LipidII in bacterial cell biology has been slow. The focus of this review is on exciting new developments, both with respect to antibacterials targeting LipidII as well as the emerging role of LipidII in organizing the membrane and cell wall synthesis. It appears that on both sides of the membrane, LipidII plays crucial roles in organizing cytoskeletal proteins and peptidoglycan synthesis machineries. Finally, the recent discovery of no less than three different categories of LipidII flippases will be discussed. PMID:26679002

Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yamada, Mototsugu, E-mail: mototsugu-yamada@meiji.co.jp; Watanabe, Takashi; Baba, Nobuyoshi

The selenomethionyl-substituted transpeptidase domain of penicillin-binding protein (PBP) 2B from S. pneumoniae was isolated from a limited proteolysis digest of the soluble form of recombinant PBP 2B and then crystallized. MAD data were collected to 2.4 Å resolution. Penicillin-binding protein (PBP) 2B from Streptococcus pneumoniae catalyzes the cross-linking of peptidoglycan precursors that occurs during bacterial cell-wall biosynthesis. A selenomethionyl (SeMet) substituted PBP 2B transpeptidase domain was isolated from a limited proteolysis digest of a soluble form of recombinant PBP 2B and then crystallized. The crystals belonged to space group P4{sub 3}2{sub 1}2, with unit-cell parameters a = b = 86.39,more » c = 143.27 Å. Diffraction data were collected to 2.4 Å resolution using the BL32B2 beamline at SPring-8. The asymmetric unit contains one protein molecule and 63.7% solvent.« less

Engineering microorganisms for improving polyhydroxyalkanoate biosynthesis.

PubMed

Chen, Guo-Qiang; Jiang, Xiao-Ran

2017-11-20

Biosynthesis of polyhydroxyalkanoates (PHA) has been studied since the 1920s. The biosynthesis pathways have been well understood and various attempts have been made to improve the PHA biosynthesis efficiency. Recent progresses have been focused on systematic improvements on PHA biosynthesis including changing growth pattern for rapid proliferation, engineering to enlarge cell sizes for more PHA accumulation space, reprogramming the PHA synthesis pathways using optimized RBS and promoter, redirecting metabolic flux to PHA synthesis using CRISPR/Cas9 tools, and very importantly, the employment of non-traditional host such as halophiles for reduced complexity on PHA production. All of the efforts should lead to ultrahigh PHA accumulation, controllable PHA compositions and molecular weights, open and continuous PHA production with gravity separation processes, resulting in competitive PHA production cost. Copyright © 2017 Elsevier Ltd. All rights reserved.

Discovery of novel bacterial RNA polymerase inhibitors: pharmacophore-based virtual screening and hit optimization.

PubMed

Hinsberger, Stefan; Hüsecken, Kristina; Groh, Matthias; Negri, Matthias; Haupenthal, Jörg; Hartmann, Rolf W

2013-11-14

The bacterial RNA polymerase (RNAP) is a validated target for broad spectrum antibiotics. However, the efficiency of drugs is reduced by resistance. To discover novel RNAP inhibitors, a pharmacophore based on the alignment of described inhibitors was used for virtual screening. In an optimization process of hit compounds, novel derivatives with improved in vitro potency were discovered. Investigations concerning the molecular mechanism of RNAP inhibition reveal that they prevent the protein-protein interaction (PPI) between σ(70) and the RNAP core enzyme. Besides of reducing RNA formation, the inhibitors were shown to interfere with bacterial lipid biosynthesis. The compounds were active against Gram-positive pathogens and revealed significantly lower resistance frequencies compared to clinically used rifampicin.

Regulation of Oil Biosynthesis in Algae

DTIC Science & Technology

2008-06-25

for future engineering purposes 3. Biochemical analysis of diacylglycerol acyltransferases ( DGATs ). These are key enzymes of oil biosynthesis...catalyzing the assembly of triacylglycerol in many organisms. 5 Genes predicted to encode DGATs and their role in triacylglycerol biosynthesis were identified

Observing cellulose biosynthesis and membrane translocation in crystallo

PubMed Central

Morgan, Jacob L.W.; McNamara, Joshua T.; Fischer, Michael; Rich, Jamie; Chen, Hong-Ming; Withers, Stephen G.; Zimmer, Jochen

2016-01-01

Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. In crystallo enzymology with the catalytically-active bacterial cellulose synthase BcsA-B complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a “finger helix” that contacts the polymer's terminal glucose. Cooperating with BcsA's gating loop, the finger helix moves ‘up’ and ‘down’ in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA’s transmembrane channel. This mechanism is validated experimentally by tethering BcsA's finger helix, which inhibits polymer translocation but not elongation. PMID:26958837

Glucose induces delocalization of a flagellar biosynthesis protein from the flagellated pole.

PubMed

Park, Soyoung; Park, Young-Ha; Lee, Chang-Ro; Kim, Yeon-Ran; Seok, Yeong-Jae

2016-09-01

To survive in a continuously changing environment, bacteria sense concentration gradients of attractants or repellents, and purposefully migrate until a more favourable habitat is encountered. While glucose is known as the most effective attractant, the flagellar biosynthesis and hence chemotactic motility has been known to be repressed by glucose in some bacteria. To date, the only known regulatory mechanism of the repression of flagellar synthesis by glucose is via downregulation of the cAMP level, as shown in a few members of the family Enterobacteriaceae. Here we show that, in Vibrio vulnificus, the glucose-mediated inhibition of flagellar motility operates by a completely different mechanism. In the presence of glucose, EIIA(Glc) is dephosphorylated and inhibits the polar localization of FapA (flagellar assembly protein A) by sequestering it from the flagellated pole. A loss or delocalization of FapA results in a complete failure of the flagellar biosynthesis and motility. However, when glucose is depleted, EIIA(Glc) is phosphorylated and releases FapA such that free FapA can be localized back to the pole and trigger flagellation. Together, these data provide new insight into a bacterial strategy to reach and stay in the glucose-rich area. © 2016 John Wiley & Sons Ltd.

Imaging mycobacterial growth and division with a fluorogenic probe.

PubMed

Hodges, Heather L; Brown, Robert A; Crooks, John A; Weibel, Douglas B; Kiessling, Laura L

2018-05-15

Control and manipulation of bacterial populations requires an understanding of the factors that govern growth, division, and antibiotic action. Fluorescent and chemically reactive small molecule probes of cell envelope components can visualize these processes and advance our knowledge of cell envelope biosynthesis (e.g., peptidoglycan production). Still, fundamental gaps remain in our understanding of the spatial and temporal dynamics of cell envelope assembly. Previously described reporters require steps that limit their use to static imaging. Probes that can be used for real-time imaging would advance our understanding of cell envelope construction. To this end, we synthesized a fluorogenic probe that enables continuous live cell imaging in mycobacteria and related genera. This probe reports on the mycolyltransferases that assemble the mycolic acid membrane. This peptidoglycan-anchored bilayer-like assembly functions to protect these cells from antibiotics and host defenses. Our probe, quencher-trehalose-fluorophore (QTF), is an analog of the natural mycolyltransferase substrate. Mycolyltransferases process QTF by diverting their normal transesterification activity to hydrolysis, a process that unleashes fluorescence. QTF enables high contrast continuous imaging and the visualization of mycolyltransferase activity in cells. QTF revealed that mycolyltransferase activity is augmented before cell division and localized to the septa and cell poles, especially at the old pole. This observed localization suggests that mycolyltransferases are components of extracellular cell envelope assemblies, in analogy to the intracellular divisomes and polar elongation complexes. We anticipate QTF can be exploited to detect and monitor mycobacteria in physiologically relevant environments.

Thiamin (Vitamin B1) Biosynthesis and Regulation: A Rich Source of Antimicrobial Drug Targets?

PubMed Central

Du, Qinglin; Wang, Honghai; Xie, Jianping

2011-01-01

Drug resistance of pathogens has necessitated the identification of novel targets for antibiotics. Thiamin (vitamin B1) is an essential cofactor for all organisms in its active form thiamin diphosphate (ThDP). Therefore, its metabolic pathways might be one largely untapped source of antibiotics targets. This review describes bacterial thiamin biosynthetic, salvage, and transport pathways. Essential thiamin synthetic enzymes such as Dxs and ThiE are proposed as promising drug targets. The regulation mechanism of thiamin biosynthesis by ThDP riboswitch is also discussed. As drug targets of existing antimicrobial compound pyrithiamin, the ThDP riboswitch might serves as alternative targets for more antibiotics. PMID:21234302

Biosynthesis of Gold and Silver Nanoparticles Using Extracts of Callus Cultures of Pumpkin (Cucurbita maxima).

PubMed

Iyer, R Indira; Panda, Tapobrata

2018-08-01

The potential of callus cultures and field-grown organs of pumpkin (Cucurbita maxima) for the biosynthesis of nanoparticles of the noble metals gold and silver has been investigated. Biosynthesis of AuNPs (gold nanoparticles) and AgNPs (silver nanoparticles) was obtained with flowers of C. maxima but not with pulp and seeds. With callus cultures established in MS-based medium the biogenesis of both AuNPs and AgNPs could be obtained. At 65 °C the biogenesis of AuNPs and AgNPs by callus extracts was enhanced. The AuNPs and AgNPs have been characterized by UV-visible spectroscopy, TEM, DLS and XRD. Well-dispersed nanoparticles, which exhibited a remarkable diversity in size and shape, could be visualized by TEM. Gold nanoparticles were found to be of various shapes, viz., rods, triangles, star-shaped particles, spheres, hexagons, bipyramids, discoid particles, nanotrapezoids, prisms, cuboids. Silver nanoparticles were also of diverse shapes, viz., discoid, spherical, elliptical, triangle-like, belt-like, rod-shaped forms and cuboids. EDX analysis indicated that the AuNPs and AgNPs had a high degree of purity. The surface charges of the generated AuNPs and AgNPs were highly negative as indicated by zeta potential measurements. The AuNPs and AgNPs exhibited remarkable stability in solution for more than four months. FTIR studies indicated that biomolecules in the callus extracts were associated with the biosynthesis and stabilisation of the nanoparticles. The synthesized AgNPs could catalyse degradation of methylene blue and exhibited anti-bacterial activity against E. coli DH5α. There is no earlier report of the biosynthesis of nanoparticles by this plant species. Callus cultures of Cucurbita maxima are effective alternative resources of biomass for synthesis of nanoparticles.

Pipecolic acid enhances resistance to bacterial infection and primes salicylic acid and nicotine accumulation in tobacco

PubMed Central

Vogel-Adghough, Drissia; Stahl, Elia; Návarová, Hana; Zeier, Jürgen

2013-01-01

Distinct amino acid metabolic pathways constitute integral parts of the plant immune system. We have recently identified pipecolic acid (Pip), a lysine-derived non-protein amino acid, as a critical regulator of systemic acquired resistance (SAR) and basal immunity to bacterial infection in Arabidopsis thaliana. In Arabidopsis, Pip acts as an endogenous mediator of defense amplification and priming. For instance, Pip conditions plants for effective biosynthesis of the phenolic defense signal salicylic acid (SA), accumulation of the phytoalexin camalexin, and expression of defense-related genes. Here, we show that tobacco plants respond to leaf infection by the compatible bacterial pathogen Pseudomonas syringae pv tabaci (Pstb) with a significant accumulation of several amino acids, including Lys, branched-chain, aromatic, and amide group amino acids. Moreover, Pstb strongly triggers, alongside the biosynthesis of SA and increases in the defensive alkaloid nicotine, the production of the Lys catabolites Pip and α-aminoadipic acid. Exogenous application of Pip to tobacco plants provides significant protection to infection by adapted Pstb or by non-adapted, hypersensitive cell death-inducing P. syringae pv maculicola. Pip thereby primes tobacco for rapid and strong accumulation of SA and nicotine following bacterial infection. Thus, our study indicates that the role of Pip as an amplifier of immune responses is conserved between members of the rosid and asterid groups of eudicot plants and suggests a broad practical applicability for Pip as a natural enhancer of plant disease resistance. PMID:24025239

Contribution of the Pmra Promoter to Expression of Genes in the Escherichia coli mra Cluster of Cell Envelope Biosynthesis and Cell Division Genes

PubMed Central

Mengin-Lecreulx, Dominique; Ayala, Juan; Bouhss, Ahmed; van Heijenoort, Jean; Parquet, Claudine; Hara, Hiroshi

1998-01-01

Recently, a promoter for the essential gene ftsI, which encodes penicillin-binding protein 3 of Escherichia coli, was precisely localized 1.9 kb upstream from this gene, at the beginning of the mra cluster of cell division and cell envelope biosynthesis genes (H. Hara, S. Yasuda, K. Horiuchi, and J. T. Park, J. Bacteriol. 179:5802–5811, 1997). Disruption of this promoter (Pmra) on the chromosome and its replacement by the lac promoter (Pmra::Plac) led to isopropyl-β-d-thiogalactopyranoside (IPTG)-dependent cells that lysed in the absence of inducer, a defect which was complemented only when the whole region from Pmra to ftsW, the fifth gene downstream from ftsI, was provided in trans on a plasmid. In the present work, the levels of various proteins involved in peptidoglycan synthesis and cell division were precisely determined in cells in which Pmra::Plac promoter expression was repressed or fully induced. It was confirmed that the Pmra promoter is required for expression of the first nine genes of the mra cluster: mraZ (orfC), mraW (orfB), ftsL (mraR), ftsI, murE, murF, mraY, murD, and ftsW. Interestingly, three- to sixfold-decreased levels of MurG and MurC enzymes were observed in uninduced Pmra::Plac cells. This was correlated with an accumulation of the nucleotide precursors UDP–N-acetylglucosamine and UDP–N-acetylmuramic acid, substrates of these enzymes, and with a depletion of the pool of UDP–N-acetylmuramyl pentapeptide, resulting in decreased cell wall peptidoglycan synthesis. Moreover, the expression of ftsZ, the penultimate gene from this cluster, was significantly reduced when Pmra expression was repressed. It was concluded that the transcription of the genes located downstream from ftsW in the mra cluster, from murG to ftsZ, is also mainly (but not exclusively) dependent on the Pmra promoter. PMID:9721276

Targeting the Bacterial Cytoskeleton of the Burkholderia cepacia Complex for Antimicrobial Development: A Cautionary Tale.

PubMed

Carnell, Sonya C; Perry, John D; Borthwick, Lee; Vollmer, Daniela; Biboy, Jacob; Facchini, Marcella; Bragonzi, Alessandra; Silipo, Alba; Vergunst, Annette C; Vollmer, Waldemar; Khan, Anjam C M; De Soyza, Anthony

2018-05-30

Burkholderia cepacia complex (BCC) bacteria are a group of opportunistic pathogens that cause severe lung infections in cystic fibrosis (CF). Treatment of BCC infections is difficult, due to the inherent and acquired multidrug resistance of BCC. There is a pressing need to find new bacterial targets for antimicrobials. Here, we demonstrate that the novel compound Q22, which is related to the bacterial cytoskeleton destabilising compound A22, can reduce the growth rate and inhibit growth of BCC bacteria. We further analysed the phenotypic effects of Q22 treatment on BCC virulence traits, to assess its feasibility as an antimicrobial. BCC bacteria were grown in the presence of Q22 with a broad phenotypic analysis, including resistance to H₂O₂-induced oxidative stress, changes in the inflammatory potential of cell surface components, and in-vivo drug toxicity studies. The influence of the Q22 treatment on inflammatory potential was measured by monitoring the cytokine responses of BCC whole cell lysates, purified lipopolysaccharide, and purified peptidoglycan extracted from bacterial cultures grown in the presence or absence of Q22 in differentiated THP-1 cells. BCC bacteria grown in the presence of Q22 displayed varying levels of resistance to H₂O₂-induced oxidative stress, with some strains showing increased resistance after treatment. There was strain-to-strain variation in the pro-inflammatory ability of bacterial lysates to elicit TNFα and IL-1β from human myeloid cells. Despite minimal toxicity previously shown in vitro with primary CF cell lines, in-vivo studies demonstrated Q22 toxicity in both zebrafish and mouse infection models. In summary, destabilisation of the bacterial cytoskeleton in BCC, using compounds such as Q22, led to increased virulence-related traits in vitro. These changes appear to vary depending on strain and BCC species. Future development of antimicrobials targeting the BCC bacterial cytoskeleton may be hampered if such effects

Phytosterols Play a Key Role in Plant Innate Immunity against Bacterial Pathogens by Regulating Nutrient Efflux into the Apoplast1[C][W][OA

PubMed Central

Wang, Keri; Senthil-Kumar, Muthappa; Ryu, Choong-Min; Kang, Li; Mysore, Kirankumar S.

2012-01-01

Bacterial pathogens colonize a host plant by growing between the cells by utilizing the nutrients present in apoplastic space. While successful pathogens manipulate the plant cell membrane to retrieve more nutrients from the cell, the counteracting plant defense mechanism against nonhost pathogens to restrict the nutrient efflux into the apoplast is not clear. To identify the genes involved in nonhost resistance against bacterial pathogens, we developed a virus-induced gene-silencing-based fast-forward genetics screen in Nicotiana benthamiana. Silencing of N. benthamiana SQUALENE SYNTHASE, a key gene in phytosterol biosynthesis, not only compromised nonhost resistance to few pathovars of Pseudomonas syringae and Xanthomonas campestris, but also enhanced the growth of the host pathogen P. syringae pv tabaci by increasing nutrient efflux into the apoplast. An Arabidopsis (Arabidopsis thaliana) sterol methyltransferase mutant (sterol methyltransferase2) involved in sterol biosynthesis also compromised plant innate immunity against bacterial pathogens. The Arabidopsis cytochrome P450 CYP710A1, which encodes C22-sterol desaturase that converts β-sitosterol to stigmasterol, was dramatically induced upon inoculation with nonhost pathogens. An Arabidopsis Atcyp710A1 null mutant compromised both nonhost and basal resistance while overexpressors of AtCYP710A1 enhanced resistance to host pathogens. Our data implicate the involvement of sterols in plant innate immunity against bacterial infections by regulating nutrient efflux into the apoplast. PMID:22298683

The mecillinam resistome reveals a role for peptidoglycan endopeptidases in stimulating cell wall synthesis in Escherichia coli.

PubMed

Lai, Ghee Chuan; Cho, Hongbaek; Bernhardt, Thomas G

2017-07-01

Bacterial cells are typically surrounded by an net-like macromolecule called the cell wall constructed from the heteropolymer peptidoglycan (PG). Biogenesis of this matrix is the target of penicillin and related beta-lactams. These drugs inhibit the transpeptidase activity of PG synthases called penicillin-binding proteins (PBPs), preventing the crosslinking of nascent wall material into the existing network. The beta-lactam mecillinam specifically targets the PBP2 enzyme in the cell elongation machinery of Escherichia coli. Low-throughput selections for mecillinam resistance have historically been useful in defining mechanisms involved in cell wall biogenesis and the killing activity of beta-lactam antibiotics. Here, we used transposon-sequencing (Tn-Seq) as a high-throughput method to identify nearly all mecillinam resistance loci in the E. coli genome, providing a comprehensive resource for uncovering new mechanisms underlying PG assembly and drug resistance. Induction of the stringent response or the Rcs envelope stress response has been previously implicated in mecillinam resistance. We therefore also performed the Tn-Seq analysis in mutants defective for these responses in addition to wild-type cells. Thus, the utility of the dataset was greatly enhanced by determining the stress response dependence of each resistance locus in the resistome. Reasoning that stress response-independent resistance loci are those most likely to identify direct modulators of cell wall biogenesis, we focused our downstream analysis on this subset of the resistome. Characterization of one of these alleles led to the surprising discovery that the overproduction of endopeptidase enzymes that cleave crosslinks in the cell wall promotes mecillinam resistance by stimulating PG synthesis by a subset of PBPs. Our analysis of this activation mechanism suggests that, contrary to the prevailing view in the field, PG synthases and PG cleaving enzymes need not function in multi-enzyme complexes

Intracellular Biosynthesis of Fluorescent CdSe Quantum Dots in Bacillus subtilis: A Strategy to Construct Signaling Bacterial Probes for Visually Detecting Interaction Between Bacillus subtilis and Staphylococcus aureus.

PubMed

Yan, Zheng-Yu; Ai, Xiao-Xia; Su, Yi-Long; Liu, Xin-Ying; Shan, Xiao-Hui; Wu, Sheng-Mei

2016-02-01

In this work, fluorescent Bacillus subtilis (B. subtilis) cells were developed as probes for imaging applications and to explore behaviorial interaction between B. subtilis and Staphylococcus aureus (S. aureus). A novel biological strategy of coupling intracellular biochemical reactions for controllable biosynthesis of CdSe quantum dots by living B. subtilis cells was demonstrated, through which highly luminant and photostable fluorescent B. subtilis cells were achieved with good uniformity. With the help of the obtained fluorescent B. subtilis cells probes, S. aureus cells responded to co-cultured B. subtilis and to aggregate. The degree of aggregation was calculated and nonlinearly fitted to a polynomial model. Systematic investigations of their interactions implied that B. subtilis cells inhibit the growth of neighboring S. aureus cells, and this inhibition was affected by both the growth stage and the amount of surrounding B. subtilis cells. Compared to traditional methods of studying bacterial interaction between two species, such as solid culture medium colony observation and imaging mass spectrometry detection, the procedures were more simple, vivid, and photostable due to the efficient fluorescence intralabeling with less influence on the cells' surface, which might provide a new paradigm for future visualization of microbial behavior.

Assessment of the bacterial impact on the post-mortem formation of zinc protoporphyrin IX in pork meat.

PubMed

Ghadiri Khozroughi, Amin; Kroh, Lothar W; Schlüter, Oliver; Rawel, Harshadrai

2018-08-01

The post-mortem accumulation of the heme biosynthesis metabolite zinc protoporphyrin IX (ZnPP) in porcine muscle is associated with both a meat-inherent and a bacterial enzymatic reaction during meat storage. To estimate the bacterial impact on ZnPP formation, meat and meat-like media were investigated by HPLC-FLD (and MALDI-TOF-MS) after inoculation with a representative microorganism (P. fluorescens). Results indicate the principal ability of meat-inherent bacteria to form ZnPP in meat extracts and meat-like media, but not on the meat muscle. Thus it was concluded that the ZnPP formation in meat is due to a meat-inherent enzymatic reaction induced by porcine ferrochelatase (FECH), while the bacterial (FECH) induced reaction seems to be not significant. Copyright © 2018. Published by Elsevier Ltd.

Nosiheptide Biosynthesis Featuring a Unique Indole Side Ring Formation on the Characteristic Thiopeptide Framework

PubMed Central

Yu, Yi; Duan, Lian; Zhang, Qi; Liao, Rijing; Ding, Ying; Pan, Haixue; Wendt-Pienkowski, Evelyn; Tang, Gongli; Shen, Ben; Liu, Wen

2009-01-01

Nosiheptide (NOS), belonging to the e series of thiopeptide antibiotics that exhibit potent activity against various bacterial pathogens, bears a unique indole side ring system and regiospecific hydroxyl groups on the characteristic macrocyclic core. Here, cloning, sequencing and characterization of the nos gene cluster from Streptomyces actuosus ATCC 25421 as a model for this series of thiopeptides has unveiled new insights into their biosynthesis. Bioinformatics-based sequence analysis and in vivo investigation into the gene functions show that NOS biosynthesis shares a common strategy with recently characterized b or c series thiopeptides for forming the characteristic macrocyclic core, which features a ribosomally synthesized precursor peptide with conserved posttranslational modifications. However, it apparently proceeds via a different route for tailoring the thiopeptide framework, allowing the final product to exhibit the distinct structural characteristics of e series thiopeptides, such as the indole side ring system. Chemical complementation supports the notion that the S-adenosylmethionine (AdoMet)-dependent protein NosL may play a central role in converting Trp to the key 3-methylindole moiety by an unusual carbon side chain rearrangement, most likely via a radical-initiated mechanism. Characterization of the indole side ring-opened analog of NOS from the nosN mutant strain is consistent with the proposed methyltransferase activity of its encoded protein, shedding light into the timing of the individual steps for indole side ring biosynthesis. These results also suggest the feasibility of engineering novel thiopeptides for drug discovery by manipulating the NOS biosynthetic machinery. PMID:19678698

Molecular modeling of Gram-positive bacteria peptidoglycan layer, selected glycopeptide antibiotics and vancomycin derivatives modified with sugar moieties.

PubMed

Ślusarz, Rafał; Szulc, Monika; Madaj, Janusz

2014-05-07

Proper understanding of the mechanisms of binding to Gram-positive bacteria cell wall layers-especially to the peptidoglycan (PG) layer, seems to be crucial for proper development of new drug candidates which are effective against these bacteria. In this work we have constructed two different models of the Gram-positive bacteria PG layer: the layered and the scaffold models. PG conformational changes during geometry optimization, models relaxation, and molecular dynamics were described and discussed. We have found that the border surface of both PG layer models differs from the surface located away from the edge of models and the chains formed by disaccharide units prefer helix-like conformation. This curling of PG chains significantly affects the shape of antibiotic-accessible surface and the process is thus crucial for new drug development. Glycopeptide antibiotics effective against Gram-positive bacteria, such as vancomycin and its semisynthetic derivatives-oritavancin and telavancin, bind to d-alanyl-d-alanine stem termini on the peptidoglycan precursors of the cell wall. This binding inhibits cross-linking between the peptides and subsequently prevents cell wall synthesis. In this study some of the aspects of conformational freedom of vancomycin and restrictions from the modifications of vancomycin structure introduced into oritavancin and telavancin and five other vancomycin derivatives (with addition of 2-acetamido-2-deoxy-β-d-galactopyranosylamine, 2-acetamido-2-deoxy-β-d-glucopyranosylamine, 1-amine-1-deoxy-d-glucitol, 2-amino-2-deoxy-d-galactitol, or 2-amino-2-deoxy-d-glucitol to the C-terminal amino acid group in the vancomycin) are presented and discussed. The resulting molecular dynamics trajectories, root mean square deviation changes of aglycon and saccharide moieties as well as a comparative study of possible interactions with cyclic and chain forms of modified groups have been carried out, measured, and analyzed. Energetically advantageous

Biochemical Principles and Functional Aspects of Pipecolic Acid Biosynthesis in Plant Immunity1[OPEN

PubMed Central

Kim, Denis; Schreiber, Stefan; Zeier, Tatyana; Schuck, Stefan; Reichel-Deland, Vanessa

2017-01-01

The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis (Arabidopsis thaliana), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/μ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae. Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction. PMID:28330936

The Zinc Finger Transcription Factor SlZFP2 Negatively Regulates Abscisic Acid Biosynthesis and Fruit Ripening in Tomato1

PubMed Central

Weng, Lin; Zhao, Fangfang; Li, Rong; Xu, Changjie; Chen, Kunsong

2015-01-01

Abscisic acid (ABA) regulates plant development and adaptation to environmental conditions. Although the ABA biosynthesis pathway in plants has been thoroughly elucidated, how ABA biosynthetic genes are regulated at the molecular level during plant development is less well understood. Here, we show that the tomato (Solanum lycopersicum) zinc finger transcription factor SlZFP2 is involved in the regulation of ABA biosynthesis during fruit development. Overexpression of SlZFP2 resulted in multiple phenotypic changes, including more branches, early flowering, delayed fruit ripening, lighter seeds, and faster seed germination, whereas down-regulation of its expression caused problematic fruit set, accelerated ripening, and inhibited seed germination. SlZFP2 represses ABA biosynthesis during fruit development through direct suppression of the ABA biosynthetic genes NOTABILIS, SITIENS, and FLACCA and the aldehyde oxidase SlAO1. We also show that SlZFP2 regulates fruit ripening through transcriptional suppression of the ripening regulator COLORLESS NON-RIPENING. Using bacterial one-hybrid screening and a selected amplification and binding assay, we identified the (A/T)(G/C)TT motif as the core binding sequence of SlZFP2. Furthermore, by RNA sequencing profiling, we found that 193 genes containing the SlZFP2-binding motifs in their promoters were differentially expressed in 2 d post anthesis fruits between the SlZFP2 RNA interference line and its nontransgenic sibling. We propose that SlZFP2 functions as a repressor to fine-tune ABA biosynthesis during fruit development and provides a potentially valuable tool for dissecting the role of ABA in fruit ripening. PMID:25637453

Peptidoglycan and Teichoic Acid Levels and Alterations in Staphylococcus aureus by Cell-Wall and Whole-Cell Nuclear Magnetic Resonance.

PubMed

Romaniuk, Joseph A H; Cegelski, Lynette

2018-06-11

Gram-positive bacteria surround themselves with a multilayered macromolecular cell wall that is essential to cell survival and serves as a major target for antibiotics. The cell wall of Staphylococcus aureus is composed of two major structural components, peptidoglycan (PG) and wall teichoic acid (WTA), together creating a heterogeneous and insoluble matrix that poses a challenge to quantitative compositional analysis. Here, we present 13 C cross polarization magic angle spinning solid-state nuclear magnetic resonance (NMR) spectra of intact cell walls, purified PG, and purified WTA. The spectra reveal the clear molecular differences in the two polymers and enable quantification of PG and WTA in isolated cell walls, an attractive alternative to estimating teichoic acid content from a phosphate analysis of completely pyrolyzed cell walls. Furthermore, we discovered that unique PG and WTA spectral signatures could be identified in whole-cell NMR spectra and used to compare PG and WTA levels among intact bacterial cell samples. The distinguishing whole-cell 13 C NMR contributions associated with PG include the GlcNAc-MurNAc sugar carbons and glycyl α-carbons. WTA contributes carbons from the phosphoribitol backbone. Distinguishing 15 N spectral signatures include glycyl amide nitrogens in PG and the esterified d-alanyl amine nitrogens in WTA. 13 C NMR analysis was performed with samples at natural abundance and included 10 whole-cell sample comparisons. Changes consistent with altered PG and WTA content were detected in whole-cell spectra of bacteria harvested at different growth times and in cells treated with tunicamycin. This use of whole-cell NMR provides quantitative parameters of composition in the context of whole-cell activity.

Neutral Lipid Biosynthesis in Engineered Escherichia coli: Jojoba Oil-Like Wax Esters and Fatty Acid Butyl Esters

PubMed Central

Kalscheuer, Rainer; Stöveken, Tim; Luftmann, Heinrich; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

2006-01-01

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms. PMID:16461689

Neutral lipid biosynthesis in engineered Escherichia coli: jojoba oil-like wax esters and fatty acid butyl esters.

PubMed

Kalscheuer, Rainer; Stöveken, Tim; Luftmann, Heinrich; Malkus, Ursula; Reichelt, Rudolf; Steinbüchel, Alexander

2006-02-01

Wax esters are esters of long-chain fatty acids and long-chain fatty alcohols which are of considerable commercial importance and are produced on a scale of 3 million tons per year. The oil from the jojoba plant (Simmondsia chinensis) is the main biological source of wax esters. Although it has a multitude of potential applications, the use of jojoba oil is restricted, due to its high price. In this study, we describe the establishment of heterologous wax ester biosynthesis in a recombinant Escherichia coli strain by coexpression of a fatty alcohol-producing bifunctional acyl-coenzyme A reductase from the jojoba plant and a bacterial wax ester synthase from Acinetobacter baylyi strain ADP1, catalyzing the esterification of fatty alcohols and coenzyme A thioesters of fatty acids. In the presence of oleate, jojoba oil-like wax esters such as palmityl oleate, palmityl palmitoleate, and oleyl oleate were produced, amounting to up to ca. 1% of the cellular dry weight. In addition to wax esters, fatty acid butyl esters were unexpectedly observed in the presence of oleate. The latter could be attributed to solvent residues of 1-butanol present in the medium component, Bacto tryptone. Neutral lipids produced in recombinant E. coli were accumulated as intracytoplasmic inclusions, demonstrating that the formation and structural integrity of bacterial lipid bodies do not require specific structural proteins. This is the first report on substantial biosynthesis and accumulation of neutral lipids in E. coli, which might open new perspectives for the biotechnological production of cheap jojoba oil equivalents from inexpensive resources employing recombinant microorganisms.

Silkworm larvae plasma (SLP) assay for detection of bacteria: False positives secondary to inflammation in vivo.

PubMed

Ma, Michelle; Rice, Tyler A; Percopo, Caroline M; Rosenberg, Helene F

2017-01-01

The silkworm larvae plasma (SLP) assay has been developed as a means to detect bacterial peptidoglycan as a surrogate for live bacteria. Here, we present results that indicate that generation of melanin by this assay is not fully reliable as a surrogate marker for bacterial count. Published by Elsevier B.V.

(-)-Menthol biosynthesis and molecular genetics

NASA Astrophysics Data System (ADS)

Croteau, Rodney B.; Davis, Edward M.; Ringer, Kerry L.; Wildung, Mark R.

2005-12-01

(-)-Menthol is the most familiar of the monoterpenes as both a pure natural product and as the principal and characteristic constituent of the essential oil of peppermint ( Mentha x piperita). In this paper, we review the biosynthesis and molecular genetics of (-)-menthol production in peppermint. In Mentha species, essential oil biosynthesis and storage is restricted to the peltate glandular trichomes (oil glands) on the aerial surfaces of the plant. A mechanical method for the isolation of metabolically functional oil glands, has provided a system for precursor feeding studies to elucidate pathway steps, as well as a highly enriched source of the relevant biosynthetic enzymes and of their corresponding transcripts with which cDNA libraries have been constructed to permit cloning and characterization of key structural genes. The biosynthesis of (-)-menthol from primary metabolism requires eight enzymatic steps, and involves the formation and subsequent cyclization of the universal monoterpene precursor geranyl diphosphate to the parent olefin (-)-(4 S)-limonene as the first committed reaction of the sequence. Following hydroxylation at C3, a series of four redox transformations and an isomerization occur in a general “allylic oxidation-conjugate reduction” scheme that installs three chiral centers on the substituted cyclohexanoid ring to yield (-)-(1 R, 3 R, 4 S)-menthol. The properties of each enzyme and gene of menthol biosynthesis are described, as are their probable evolutionary origins in primary metabolism. The organization of menthol biosynthesis is complex in involving four subcellular compartments, and regulation of the pathway appears to reside largely at the level of gene expression. Genetic engineering to up-regulate a flux-limiting step and down-regulate a side route reaction has led to improvement in the composition and yield of peppermint oil.

Building and Breaking the Cell Wall in Four Acts: A Kinesthetic and Tactile Role-Playing Exercise for Teaching Beta-Lactam Antibiotic Mechanism of Action and Resistance †

PubMed Central

Popovich, John; Stephens, Michelle; Celaya, Holly; Suwarno, Serena; Barclay, Shizuka; Yee, Emily; Dean, David A.; Farris, Megan; Haydel, Shelley E.

2018-01-01

“Building and breaking the cell wall” is designed to review the bacterial cell envelope, previously learned in lower-division biology classes, while introducing new topics such as antibiotics and bacterial antibiotic resistance mechanisms. We developed a kinesthetic and tactile modeling activity where students act as cellular components and construct the cell wall. In the first two acts, students model a portion of the gram-positive bacterial cell envelope and then demonstrate in detail how the peptidoglycan is formed. Act III involves student demonstration of the addition of β-lactam antibiotics to the environment and how they inhibit the formation of peptidoglycan, thereby preventing bacterial replication. Using Staphylococcus aureus as a model for gram-positive bacteria, students finish the activity (Act IV) by acting out how S. aureus often becomes resistant to β-lactam antibiotics. A high level of student engagement was observed, and the activity received positive feedback. In an assessment administered prior to and two months after the activity, significant improvements in scores were observed (p < 0.0001), demonstrating increased understanding and retention. This activity allows students to (i) visualize, role play, and kinesthetically “build” the cell envelope and form the peptidoglycan layer, (ii) understand the mechanism of action for β-lactam antibiotics, as well as how gene acquisition and protein changes result in resistance, and (iii) work cooperatively and actively to promote long-term retention of the subject material. PMID:29904519

Prebiotic Oligosaccharides: Special Focus on Fructooligosaccharides, Its Biosynthesis and Bioactivity.

PubMed

Singh, Sudhir P; Jadaun, Jyoti Singh; Narnoliya, Lokesh K; Pandey, Ashok

2017-10-01

The bacterial groups in the gut ecosystem play key role in the maintenance of host's metabolic and structural functionality. The gut microbiota enhances digestion processing, helps in digestion of complex substances, synthesizes beneficial bioactive compounds, enhances bioavailability of minerals, impedes growth of pathogenic microbes, and prevents various diseases. It is, therefore, desirable to have an adequate intake of prebiotic biomolecules, which promote favorable modulation of intestinal microflora. Prebiotics are non-digestible and chemically stable structures that significantly enhance growth and functionality of gut microflora. The non-digestible carbohydrate, mainly oligosaccharides, covers a major part of total available prebiotics as dietary additives. The review describes the types of prebiotic low molecular weight carbohydrates, i.e., oligosaccharides, their structure, biosynthesis, functionality, and applications, with a special focus given to fructooligosaccharides (FOSs). The review provides an update on enzymes executing hydrolytic and fructosyltransferase activities producing prebiotic FOS biomolecules, and future perspectives.

Cytoplasmic peptidoglycan intermediate levels in Staphylococcus aureus.

PubMed

Vemula, Harika; Ayon, Navid J; Gutheil, William G

2016-02-01

Intracellular cytoplasmic peptidoglycan (PG) intermediate levels were determined in Staphylococcus aureus during log-phase growth in enriched media. Levels of UDP-linked intermediates were quantitatively determined using ion pairing LC-MS/MS in negative mode, and amine intermediates were quantitatively determined stereospecifically as their Marfey's reagent derivatives in positive mode. Levels of UDP-linked intermediates in S. aureus varied from 1.4 μM for UDP-GlcNAc-Enolpyruvyate to 1200 μM for UDP-MurNAc. Levels of amine intermediates (L-Ala, D-Ala, D-Ala-D-Ala, L-Glu, D-Glu, and L-Lys) varied over a range of from 860 μM for D-Ala-D-Ala to 30-260 mM for the others. Total PG was determined from the D-Glu content of isolated PG, and used to estimate the rate of PG synthesis (in terms of cytoplasmic metabolite flux) as 690 μM/min. The total UDP-linked intermediates pool (2490 μM) is therefore sufficient to sustain growth for 3.6 min. Comparison of UDP-linked metabolite levels with published pathway enzyme characteristics demonstrates that enzymes on the UDP-branch range from >80% saturation for MurA, Z, and C, to <5% saturation for MurB. Metabolite levels were compared with literature values for Escherichia coli, with the major difference in UDP-intermediates being the level of UDP-MurNAc, which was high in S. aureus (1200 μM) and low in E. coli (45 μM). Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

The cell wall components peptidoglycan and lipoteichoic acid from Staphylococcus aureus act in synergy to cause shock and multiple organ failure.

PubMed Central

De Kimpe, S J; Kengatharan, M; Thiemermann, C; Vane, J R

1995-01-01

Although the incidence of Gram-positive sepsis has risen strongly, it is unclear how Gram-positive organisms (without endotoxin) initiate septic shock. We investigated whether two cell wall components from Staphylococcus aureus, peptidoglycan (PepG) and lipoteichoic acid (LTA), can induce the inflammatory response and multiple organ dysfunction syndrome (MODS) associated with septic shock caused by Gram-positive organisms. In cultured macrophages, LTA (10 micrograms/ml), but not PepG (100 micrograms/ml), induces the release of nitric oxide measured as nitrite. PepG, however, caused a 4-fold increase in the production of nitrite elicited by LTA. Furthermore, PepG antibodies inhibited the release of nitrite elicited by killed S. aureus. Administration of both PepG (10 mg/kg; i.v.) and LTA (3 mg/kg; i.v.) in anesthetized rats resulted in the release of tumor necrosis factor alpha and interferon gamma and MODS, as indicated by a decrease in arterial oxygen pressure (lung) and an increase in plasma concentrations of bilirubin and alanine aminotransferase (liver), creatinine and urea (kidney), lipase (pancreas), and creatine kinase (heart or skeletal muscle). There was also the expression of inducible nitric oxide synthase in these organs, circulatory failure, and 50% mortality. These effects were not observed after administration of PepG or LTA alone. Even a high dose of LTA (10 mg/kg) causes only circulatory failure but no MODS. Thus, our results demonstrate that the two bacterial wall components, PepG and LTA, work together to cause systemic inflammation and multiple systems failure associated with Gram-positive organisms. Images Fig. 2 PMID:7479784

Molecular cloning and functional characterization of a short peptidoglycan recognition protein (HcPGRPS1) from the freshwater mussel, Hyriopsis cumingi.

PubMed

Yang, Ziyan; Li, Junhua; Li, Ying; Wu, Hongjuan; Wang, Xiaoyan

2013-12-01

Peptidoglycan recognition proteins (PGRPs), which are evolutionarily conserved from invertebrates to vertebrates, function as pattern-recognition and effector molecules in innate immunity. In the present study, a short-form PGRP, designated as HcPGRPS1 was identified from freshwater mussel Hyriopsis cumingi. The deduced amino acid sequence of HcPGRPS1 is composed of 235 residues which contains a conserved PGRP domain at the C-terminus. Sequence analysis showed that HcPGRPS1 shared high identities with other known PGRPs. The mRNA of HcPGRPS1 is constitutively expressed in a wide range of all tested tissues, with highest expression level in hepatopancreas, and its expression in tissues (gonad, nephridium, gill and foot) was up-regulated significantly after LPS or PGN stimulation (P<0.05). The recombinant protein of HcPGRPS1 exhibited binding activity and peptidoglycan-lytic amidase activity toward Lys-PGN from Staphylococcus aureus and DAP-PGN from Bacillus subtilis. Furthermore, recombinant HcPGRPS1 displayed strong antibacterial activity to both Gram-negative bacteria Escherichia coli, Aeromonas hydrophila, Aeromonas sobria and Gram-positive bacteria S. aureus in the presence of Zn(2+). These results suggested that HcPGRPS1 plays a multifunctional role in the defense and protection mechanisms of mussel innate immunity against infections. Copyright © 2013 Elsevier Ltd. All rights reserved.

Antitubercular nucleosides that inhibit siderophore biosynthesis: SAR of the glycosyl domain.

PubMed

Somu, Ravindranadh V; Wilson, Daniel J; Bennett, Eric M; Boshoff, Helena I; Celia, Laura; Beck, Brian J; Barry, Clifton E; Aldrich, Courtney C

2006-12-28

Tuberculosis is the leading cause of infectious disease mortality in the world by a bacterial pathogen. We previously demonstrated that a bisubstrate inhibitor of the adenylation enzyme MbtA, which is responsible for the second step of mycobactin biosynthesis, exhibited potent antitubercular activity. Here we systematically investigate the structure-activity relationships of the bisubstrate inhibitor glycosyl domain resulting in the identification of a carbocyclic analogue that possesses a KIapp value of 2.3 nM and MIC99 values of 1.56 microM against M. tuberculosis H37Rv. The SAR data suggest the intriguing possibility that the bisubstrate inhibitors utilize a transporter for entry across the mycobacterial cell envelope. Additionally, we report improved conditions for the expression of MbtA and biochemical analysis, demonstrating that MbtA follows a random sequential enzyme mechanism for the adenylation half-reaction.

Zinc disrupts central carbon metabolism and capsule biosynthesis in Streptococcus pyogenes.

PubMed

Ong, Cheryl-lynn Y; Walker, Mark J; McEwan, Alastair G

2015-06-01

Neutrophils release free zinc to eliminate the phagocytosed bacterial pathogen Streptococcus pyogenes (Group A Streptococcus; GAS). In this study, we investigated the mechanisms underpinning zinc toxicity towards this human pathogen, responsible for diseases ranging from pharyngitis and impetigo, to severe invasive infections. Using the globally-disseminated M1T1 GAS strain, we demonstrate that zinc stress impairs glucose metabolism through the inhibition of the glycolytic enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. In the presence of zinc, a metabolic shift to the tagatose-6-phosphate pathway allows conversion of D-galactose to dihydroxyacetone phosphate and glyceraldehyde phosphate, partially bypassing impaired glycolytic enzymes to generate pyruvate. Additionally, zinc inhibition of phosphoglucomutase results in decreased capsule biosynthesis. These data indicate that zinc exerts it toxicity via mechanisms that inhibit both GAS central carbon metabolism and virulence pathways.

Zinc disrupts central carbon metabolism and capsule biosynthesis in Streptococcus pyogenes

PubMed Central

Ong, Cheryl-lynn Y.; Walker, Mark J.; McEwan, Alastair G.

2015-01-01

Neutrophils release free zinc to eliminate the phagocytosed bacterial pathogen Streptococcus pyogenes (Group A Streptococcus; GAS). In this study, we investigated the mechanisms underpinning zinc toxicity towards this human pathogen, responsible for diseases ranging from pharyngitis and impetigo, to severe invasive infections. Using the globally-disseminated M1T1 GAS strain, we demonstrate that zinc stress impairs glucose metabolism through the inhibition of the glycolytic enzymes phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase. In the presence of zinc, a metabolic shift to the tagatose-6-phosphate pathway allows conversion of D-galactose to dihydroxyacetone phosphate and glyceraldehyde phosphate, partially bypassing impaired glycolytic enzymes to generate pyruvate. Additionally, zinc inhibition of phosphoglucomutase results in decreased capsule biosynthesis. These data indicate that zinc exerts it toxicity via mechanisms that inhibit both GAS central carbon metabolism and virulence pathways. PMID:26028191

Antitubercular Nucleosides that Inhibit Siderophore Biosynthesis: SAR of the Glycosyl Domain

PubMed Central

Somu, Ravindranadh V.; Wilson, Daniel; Bennett, Eric M.; Boshoff, Helena; Celia, Laura; Beck, Brian; Barry, Clifton E.; Aldrich, Courtney C.

2008-01-01

Tuberculosis (TB) is the leading cause of infectious disease mortality in the world by a bacterial pathogen. We previously demonstrated that a bisubstrate inhibitor of the adenylation enzyme MbtA, which is responsible for the second step of mycobactin biosynthesis, exhibited potent antitubercular activity. Here we systematically investigate the structure activity relationships of the bisubstrate inhibitor glycosyl domain resulting in the identification of a carbocyclic analogue that possesses a KIapp value of 2.3 nM and MIC99 values of 1.56 μM against M. tuberculosis H37Rv. The SAR data suggest the intriguing possibility that the bisubstrate inhibitors utilize a transporter for entry across the mycobacterial cell-envelope. Additionally, we report improved conditions for the expression of MbtA and biochemical analysis demonstrating that MbtA follows a random sequential enzyme mechanism for the adenylation half-reaction. PMID:17181146

Regulation of Oil Biosynthesis in Algae

DTIC Science & Technology

2011-03-14

transportation fuels can potentially be addressed by exploring oil (triacylglycerol) biosynthesis in microalgae . Many microalgae , including Chlamydomonas...biosynthesis in microalgae have not been studied at the molecular level. Chlamydomonas is being used as a microalgal model to identify genes and regulatory...of this phenomenon will shed light on the physiological significance of oil production in microalgae . A first paper describing this interesting

Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora.

PubMed

Mäe, A; Montesano, M; Koiv, V; Palva, E T

2001-09-01

Bacterial pheromones, mainly different homoserine lactones, are central to a number of bacterial signaling processes, including those involved in plant pathogenicity. We previously demonstrated that N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in the soft-rot phytopathogen Erwinia carotovora. In this pathogen, OHL controls the coordinate activation of genes encoding the main virulence determinants, extracellular plant cell wall degrading enzymes (PCWDEs), in a cell density-dependent manner. We suggest that E. carotovora employ quorum sensing to avoid the premature production of PCWDEs and subsequent activation of plant defense responses. To test whether modulating this sensory system would affect the outcome of a plant-pathogen interaction, we generated transgenic tobacco, producing OHL. This was accomplished by ectopic expression in tobacco of the E. carotovora gene expI, which is responsible for OHL biosynthesis. We show that expI-positive transgenic tobacco lines produced the active pheromone and partially complemented the avirulent phenotype of expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by exogenous addition of OHL to wild-type plants, which also resulted in increased resistance to E. carotovora.

De novo Biosynthesis of "Non-Natural" Thaxtomin Phytotoxins.

PubMed

Winn, Michael; Francis, Daniel; Micklefield, Jason

2018-03-30

Thaxtomins are diketopiperazine phytotoxins produced by Streptomyces scabies and other actinobacterial plant pathogens that inhibit cellulose biosynthesis in plants. Due to their potent bioactivity and novel mode of action there has been considerable interest in developing thaxtomins as herbicides for crop protection. To address the need for more stable derivatives, we have developed a new approach for structural diversification of thaxtomins. Genes encoding the thaxtomin NRPS from S. scabies, along with genes encoding a promiscuous tryptophan synthase (TrpS) from Salmonella typhimurium, were assembled in a heterologous host Streptomyces albus. Upon feeding indole derivatives to the engineered S. albus strain, tryptophan intermediates with alternative substituents are biosynthesized and incorporated by the NRPS to deliver a series of thaxtomins with different functionalities in place of the nitro group. The approach described herein, demonstrates how genes from different pathways and different bacterial origins can be combined in a heterologous host to create a de novo biosynthetic pathway to "non-natural" product target compounds. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Molecular adaptations of Herbaspirillum seropedicae during colonization of the maize rhizosphere.

PubMed

Balsanelli, Eduardo; Tadra-Sfeir, Michelle Z; Faoro, Helisson; Pankievicz, Vânia Cs; de Baura, Valter A; Pedrosa, Fábio O; de Souza, Emanuel M; Dixon, Ray; Monteiro, Rose A

2016-09-01

Molecular mechanisms of plant recognition and colonization by diazotrophic bacteria are barely understood. Herbaspirillum seropedicae is a Betaproteobacterium capable of colonizing epiphytically and endophytically commercial grasses, to promote plant growth. In this study, we utilized RNA-seq to compare the transcriptional profiles of planktonic and maize root-attached H. seropedicae SmR1 recovered 1 and 3 days after inoculation. The results indicated that nitrogen metabolism was strongly activated in the rhizosphere and polyhydroxybutyrate storage was mobilized in order to assist the survival of H. seropedicae during the early stages of colonization. Epiphytic cells showed altered transcription levels of several genes associated with polysaccharide biosynthesis, peptidoglycan turnover and outer membrane protein biosynthesis, suggesting reorganization of cell wall envelope components. Specific methyl-accepting chemotaxis proteins and two-component systems were differentially expressed between populations over time, suggesting deployment of an extensive bacterial sensory system for adaptation to the plant environment. An insertion mutation inactivating a methyl-accepting chemosensor induced in planktonic bacteria, decreased chemotaxis towards the plant and attachment to roots. In summary, analysis of mutant strains combined with transcript profiling revealed several molecular adaptations that enable H. seropedicae to sense the plant environment, attach to the root surface and survive during the early stages of maize colonization. © 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.

The neomuran origin of archaebacteria, the negibacterial root of the universal tree and bacterial megaclassification.

PubMed

Cavalier-Smith, T

2002-01-01

Prokaryotes constitute a single kingdom, Bacteria, here divided into two new subkingdoms: Negibacteria, with a cell envelope of two distinct genetic membranes, and Unibacteria, comprising the new phyla Archaebacteria and Posibacteria, with only one. Other new bacterial taxa are established in a revised higher-level classification that recognizes only eight phyla and 29 classes. Morphological, palaeontological and molecular data are integrated into a unified picture of large-scale bacterial cell evolution despite occasional lateral gene transfers. Archaebacteria and eukaryotes comprise the clade neomura, with many common characters, notably obligately co-translational secretion of N-linked glycoproteins, signal recognition particle with 7S RNA and translation-arrest domain, protein-spliced tRNA introns, eight-subunit chaperonin, prefoldin, core histones, small nucleolar ribonucleoproteins (snoRNPs), exosomes and similar replication, repair, transcription and translation machinery. Eubacteria (posibacteria and negibacteria) are paraphyletic, neomura having arisen from Posibacteria within the new subphylum Actinobacteria (possibly from the new class Arabobacteria, from which eukaryotic cholesterol biosynthesis probably came). Replacement of eubacterial peptidoglycan by glycoproteins and adaptation to thermophily are the keys to neomuran origins. All 19 common neomuran character suites probably arose essentially simultaneously during the radical modification of an actinobacterium. At least 11 were arguably adaptations to thermophily. Most unique archaebacterial characters (prenyl ether lipids; flagellar shaft of glycoprotein, not flagellin; DNA-binding protein lob; specially modified tRNA; absence of Hsp90) were subsequent secondary adaptations to hyperthermophily and/or hyperacidity. The insertional origin of protein-spliced tRNA introns and an insertion in proton-pumping ATPase also support the origin of neomura from eubacteria. Molecular co-evolution between

Biosynthesis of coenzyme Q in eukaryotes.

PubMed

Kawamukai, Makoto

2016-01-01

Coenzyme Q (CoQ) is a component of the electron transport chain that participates in aerobic cellular respiration to produce ATP. In addition, CoQ acts as an electron acceptor in several enzymatic reactions involving oxidation-reduction. Biosynthesis of CoQ has been investigated mainly in Escherichia coli and Saccharomyces cerevisiae, and the findings have been extended to various higher organisms, including plants and humans. Analyses in yeast have contributed greatly to current understanding of human diseases related to CoQ biosynthesis. To date, human genetic disorders related to mutations in eight COQ biosynthetic genes have been reported. In addition, the crystal structures of a number of proteins involved in CoQ synthesis have been solved, including those of IspB, UbiA, UbiD, UbiX, UbiI, Alr8543 (Coq4 homolog), Coq5, ADCK3, and COQ9. Over the last decade, knowledge of CoQ biosynthesis has accumulated, and striking advances in related human genetic disorders and the crystal structure of proteins required for CoQ synthesis have been made. This review focuses on the biosynthesis of CoQ in eukaryotes, with some comparisons to the process in prokaryotes.

Analysis of the Impact of Rosuvastatin on Bacterial Mevalonate Production Using a UPLC-Mass Spectrometry Approach.

PubMed

Nolan, J A; Kinsella, M; Hill, C; Joyce, S A; Gahan, C G M

2016-07-01

Statins are widely prescribed cholesterol-lowering medications and act through inhibition of the human enzyme 3-methylglutaryl coenzyme A reductase (HMG-R) which produces mevalonate (MVAL), a key substrate for cholesterol biosynthesis. Some important microbial species also express an isoform of HMG-R; however, the nature of the interaction between statins and bacteria is currently unclear and studies would benefit from protocols to quantify MVAL in complex microbial environments. The objective of this study was to develop a protocol for the analytical quantification of MVAL in bacterial systems and to utilise this approach to analyse the effects of Rosuvastatin (RSV) on bacterial MVAL formation. To determine the effective concentration range of RSV, we examined the dose-dependent inhibition of growth in the HMG-R(+) bacterial pathogens Listeria monocytogenes, Staphylococcus aureus and Enterococcus faecium at various concentrations of pure RSV. Growth inhibition generally correlated with a reduction in bacterial MVAL levels, particularly in culture supernatants at high RSV concentrations, as determined using our ultra-performance liquid chromatography mass spectrometry protocol. This work therefore outlines a refined protocol for the analysis of MVAL in microbial cultures and provides evidence for statin-mediated inhibition of bacterial HMG-R. Furthermore, we show that MVAL is readily transported and secreted from bacterial cells into the growth media.

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

PubMed Central

Andersen, Kasper R.; Kilstrup, Mogens; Martinussen, Jan; Switzer, Robert L.; Willemoës, Martin

2016-01-01

SUMMARY Phosphoribosyl diphosphate (PRPP) is an important intermediate in cellular metabolism. PRPP is synthesized by PRPP synthase, as follows: ribose 5-phosphate + ATP → PRPP + AMP. PRPP is ubiquitously found in living organisms and is used in substitution reactions with the formation of glycosidic bonds. PRPP is utilized in the biosynthesis of purine and pyrimidine nucleotides, the amino acids histidine and tryptophan, the cofactors NAD and tetrahydromethanopterin, arabinosyl monophosphodecaprenol, and certain aminoglycoside antibiotics. The participation of PRPP in each of these metabolic pathways is reviewed. Central to the metabolism of PRPP is PRPP synthase, which has been studied from all kingdoms of life by classical mechanistic procedures. The results of these analyses are unified with recent progress in molecular enzymology and the elucidation of the three-dimensional structures of PRPP synthases from eubacteria, archaea, and humans. The structures and mechanisms of catalysis of the five diphosphoryltransferases are compared, as are those of selected enzymes of diphosphoryl transfer, phosphoryl transfer, and nucleotidyl transfer reactions. PRPP is used as a substrate by a large number phosphoribosyltransferases. The protein structures and reaction mechanisms of these phosphoribosyltransferases vary and demonstrate the versatility of PRPP as an intermediate in cellular physiology. PRPP synthases appear to have originated from a phosphoribosyltransferase during evolution, as demonstrated by phylogenetic analysis. PRPP, furthermore, is an effector molecule of purine and pyrimidine nucleotide biosynthesis, either by binding to PurR or PyrR regulatory proteins or as an allosteric activator of carbamoylphosphate synthetase. Genetic analyses have disclosed a number of mutants altered in the PRPP synthase-specifying genes in humans as well as bacterial species. PMID:28031352

Ant Trail Pheromone Biosynthesis Is Triggered by a Neuropeptide Hormone

PubMed Central

Choi, Man-Yeon; Vander Meer, Robert K.

2012-01-01

Our understanding of insect chemical communication including pheromone identification, synthesis, and their role in behavior has advanced tremendously over the last half-century. However, endocrine regulation of pheromone biosynthesis has progressed slowly due to the complexity of direct and/or indirect hormonal activation of the biosynthetic cascades resulting in insect pheromones. Over 20 years ago, a neurohormone, pheromone biosynthesis activating neuropeptide (PBAN) was identified that stimulated sex pheromone biosynthesis in a lepidopteran moth. Since then, the physiological role, target site, and signal transduction of PBAN has become well understood for sex pheromone biosynthesis in moths. Despite that PBAN-like peptides (∼200) have been identified from various insect Orders, their role in pheromone regulation had not expanded to the other insect groups except for Lepidoptera. Here, we report that trail pheromone biosynthesis in the Dufour's gland (DG) of the fire ant, Solenopsis invicta, is regulated by PBAN. RNAi knock down of PBAN gene (in subesophageal ganglia) or PBAN receptor gene (in DG) expression inhibited trail pheromone biosynthesis. Reduced trail pheromone was documented analytically and through a behavioral bioassay. Extension of PBAN's role in pheromone biosynthesis to a new target insect, mode of action, and behavioral function will renew research efforts on the involvement of PBAN in pheromone biosynthesis in Insecta. PMID:23226278

Exploiting sulphur-carrier proteins from primary metabolism for 2-thiosugar biosynthesis

PubMed Central

Sasaki, Eita; Zhang, Xuan; Sun, He G.; Lu, Mei-Yeh Jade; Liu, Tsung-lin; Ou, Albert; Li, Jeng-yi; Chen, Yu-hsiang; Ealick, Steven E.; Liu, Hung-wen

2014-01-01

Sulphur is an essential element for life and exists ubiquitously in living systems1,2. Yet, how the sulphur atom is incorporated in many sulphur-containing secondary metabolites remains poorly understood. For C-S bond formation in primary metabolites, the major ionic sulphur sources are the protein-persulphide and protein-thiocarboxylate3,4. In each case, the persulphide and thiocarboxylate group on these sulphur-carrier (donor) proteins are post-translationally generated through the action of a specific activating enzyme. In all bacterial cases reported thus far, the genes encoding the enzyme that catalyzes the actual C-S bond formation reaction and its cognate sulphur-carrier protein co-exist in the same gene cluster5. To study 2-thiosugar production in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action appear similar to those of ThiG, the enzyme catalyzing thiazole formation in thiamin biosynthesis6,7. However, no sulphur-carrier protein gene could be located in the BE-7585A cluster. Subsequent genome sequencing revealed the presence of a few sulphur-carrier proteins likely involved in the biosynthesis of primary metabolites, but surprisingly only a single activating enzyme gene in the entire genome of A. orientalis. Further experiments showed that this activating enzyme is capable of adenylating each of these sulphur-carrier proteins, and likely also catalyzing the subsequent thiolation taking advantage of its rhodanese activity. A proper combination of these sulphur delivery systems is effective for BexX-catalyzed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. These studies represent the first complete characterization of a thiosugar formation in nature and also demonstrate the receptor promiscuity of the sulphur-delivery system in A. orientalis. Our

Pederin-type pathways of uncultivated bacterial symbionts: analysis of o-methyltransferases and generation of a biosynthetic hybrid.

PubMed

Zimmermann, Katrin; Engeser, Marianne; Blunt, John W; Munro, Murray H G; Piel, Jörn

2009-03-04

The complex polyketide pederin is a potent antitumor agent isolated from Paederus spp. rove beetles. We have previously isolated a set of genes from a bacterial endosymbiont that are good candidates for pederin biosynthesis. To biochemically study this pathway, we expressed three methyltransferases from the putative pederin pathway and used the partially unmethylated analogue mycalamide A from the marine sponge Mycale hentscheli as test substrate. Analysis by high-resolution MS/MS and NMR revealed that PedO regiospecifically methylates the marine compound to generate the nonnatural hybrid compound 18-O-methylmycalamide A with increased cytotoxicity. To our knowledge, this is the first biochemical evidence that invertebrates can obtain defensive complex polyketides from bacterial symbionts.

In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum

PubMed Central

Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

2017-01-01

ABSTRACT For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI. Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA. These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum. IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis, which use an individual nonaggregating type II fatty

Jasmonate-induced biosynthesis of andrographolide in Andrographis paniculata.

PubMed

Sharma, Shiv Narayan; Jha, Zenu; Sinha, Rakesh Kumar; Geda, Arvind Kumar

2015-02-01

Andrographolide is a prominent secondary metabolite found in Andrographis paniculata that exhibits enormous pharmacological effects. In spite of immense value, the normal biosynthesis of andrographolide results in low amount of the metabolite. To induce the biosynthesis of andrographolide, we attempted elicitor-induced activation of andrographolide biosynthesis in cell cultures of A. paniculata. This was carried out by using methyl jasmonate (MeJA) as an elicitor. Among the various concentrations of MeJA tested at different time periods, 5 µM MeJA yielded 5.25 times more andrographolide content after 24 h of treatment. The accumulation of andrographolide was correlated with the expression level of known regulatory genes (hmgs, hmgr, dxs, dxr, isph and ggps) of mevalonic acid (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways. These results established the involvement of MeJA in andrographolide biosynthesis by inducing the transcription of its biosynthetic pathways genes. The coordination of isph, ggps and hmgs expression highly influenced the andrographolide biosynthesis. © 2014 Scandinavian Plant Physiology Society.

Triterpenoid biosynthesis in Euphorbia lathyris latex

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hawkins, D.R.

1987-11-01

The structures of triterpenols, not previously been known, from Euphorbia lathyris latex are reported. A method for quantifying very small amounts of these compounds was developed. Concerning the biochemistry of the latex, no exogenous cofactors were required for the biosynthesis and the addition of compounds such as NADPAH and ATP do not stimulate the biosynthesis. The addition of DTE or a similar anti-oxidant was found to help reduce the oxidation of the latex, thus increasing the length of time that the latex remains active. The requirement of a divalent cation and the preference for Mn in the pellet was observed.more » The effect of several inhibitors on the biosynthesis of the triterpenoids was examined. Mevinolin was found to inhibit the biosynthesis of the triterpenoids from acetate, but not mevalonate. A dixon plot of the inhibition of acetate incorporation showed an I/sub 50/ concentration of 3.2 ..mu..M. Fenpropimorph was found to have little or no effect on the biosynthesis. Tridemorph was found to inhibit the biosynthesis of all of the triterpenoids with an I/sub 50/ of 4 ..mu..M. It was also observed that the cyclopropyl containing triterpenols, cycloartenol and 24-methylenecycloartenol were inhibited much more strongly than those containing an 8-9 double bond, lanosterol and 24-methylenelanosterol. The evidence indicates, but does not definetely prove, that lanosterol and 24-methylenelanosterol are not made from cycloartenol and 24-methylenecycloartenol via a ring-opening enzyme such as cycloeucalenol-obtusifoliol isomerase. The possibilty that cycloartenol is made via lanosterol was investigated by synthesizing 4-R-4-/sup 3/H-mevalonic acid and incubating latex with a mixture of this and /sup 14/C-mevalonic acid. From the /sup 3/H//sup 14/C ratio it was shown that cycloartenol and 24-methylenecycloartenol are not made via an intermediate containing as 8-9 double bond. 88 refs., 15 figs., 30 tabs.« less

Pro-inflammatory adjuvant properties of pigment-grade titanium dioxide particles are augmented by a genotype that potentiates interleukin 1β processing.

PubMed

Riedle, Sebastian; Pele, Laetitia C; Otter, Don E; Hewitt, Rachel E; Singh, Harjinder; Roy, Nicole C; Powell, Jonathan J

2017-12-08

Pigment-grade titanium dioxide (TiO 2 ) particles are an additive to some foods (E171 on ingredients lists), toothpastes, and pharma-/nutraceuticals and are absorbed, to some extent, in the human intestinal tract. TiO 2 can act as a modest adjuvant in the secretion of the pro-inflammatory cytokine interleukin 1β (IL-1β) when triggered by common intestinal bacterial fragments, such as lipopolysaccharide (LPS) and/or peptidoglycan. Given the variance in human genotypes, which includes variance in genes related to IL-1β secretion, we investigated whether TiO 2 particles might, in fact, be more potent pro-inflammatory adjuvants in cells that are genetically susceptible to IL-1β-related inflammation. We studied bone marrow-derived macrophages from mice with a mutation in the nucleotide-binding oligomerisation domain-containing 2 gene (Nod2 m/m ), which exhibit heightened secretion of IL-1β in response to the peptidoglycan fragment muramyl dipeptide (MDP). To ensure relevance to human exposure, TiO 2 was food-grade anatase (119 ± 45 nm mean diameter ± standard deviation). We used a short 'pulse and chase' format: pulsing with LPS and chasing with TiO 2 +/- MDP or peptidoglycan. IL-1β secretion was not stimulated in LPS-pulsed bone marrow-derived macrophages, or by chasing with MDP, and only very modestly so by chasing with peptidoglycan. In all cases, however, IL-1β secretion was augmented by chasing with TiO 2 in a dose-dependent fashion (5-100 μg/mL). When co-administered with MDP or peptidoglycan, IL-1β secretion was further enhanced for the Nod2 m/m genotype. Tumour necrosis factor α was triggered by LPS priming, and more so for the Nod2 m/m genotype. This was enhanced by chasing with TiO 2 , MDP, or peptidoglycan, but there was no additive effect between the bacterial fragments and TiO 2 . Here, the doses of TiO 2 that augmented bacterial fragment-induced IL-1β secretion were relatively high. In vivo, however, selected intestinal cells appear

Biochemistry of Mitochondrial Coenzyme Q Biosynthesis.

PubMed

Stefely, Jonathan A; Pagliarini, David J

2017-10-01

Coenzyme Q (CoQ, ubiquinone) is a redox-active lipid produced across all domains of life that functions in electron transport and oxidative phosphorylation and whose deficiency causes human diseases. Yet, CoQ biosynthesis has not been fully defined in any organism. Several proteins with unclear molecular functions facilitate CoQ biosynthesis through unknown means, and multiple steps in the pathway are catalyzed by currently unidentified enzymes. Here we highlight recent progress toward filling these knowledge gaps through both traditional biochemistry and cutting-edge 'omics' approaches. To help fill the remaining gaps, we present questions framed by the recently discovered CoQ biosynthetic complex and by putative biophysical barriers. Mapping CoQ biosynthesis, metabolism, and transport pathways has great potential to enhance treatment of numerous human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Genome-wide analysis of bacterial determinants of plant growth promotion and induced systemic resistance by Pseudomonas fluorescens.

PubMed

Cheng, Xu; Etalo, Desalegn W; van de Mortel, Judith E; Dekkers, Ester; Nguyen, Linh; Medema, Marnix H; Raaijmakers, Jos M

2017-11-01

Pseudomonas fluorescens strain SS101 (Pf.SS101) promotes growth of Arabidopsis thaliana, enhances greening and lateral root formation, and induces systemic resistance (ISR) against the bacterial pathogen Pseudomonas syringae pv. tomato (Pst). Here, targeted and untargeted approaches were adopted to identify bacterial determinants and underlying mechanisms involved in plant growth promotion and ISR by Pf.SS101. Based on targeted analyses, no evidence was found for volatiles, lipopeptides and siderophores in plant growth promotion by Pf.SS101. Untargeted, genome-wide analyses of 7488 random transposon mutants of Pf.SS101 led to the identification of 21 mutants defective in both plant growth promotion and ISR. Many of these mutants, however, were auxotrophic and impaired in root colonization. Genetic analysis of three mutants followed by site-directed mutagenesis, genetic complementation and plant bioassays revealed the involvement of the phosphogluconate dehydratase gene edd, the response regulator gene colR and the adenylsulfate reductase gene cysH in both plant growth promotion and ISR. Subsequent comparative plant transcriptomics analyses strongly suggest that modulation of sulfur assimilation, auxin biosynthesis and transport, steroid biosynthesis and carbohydrate metabolism in Arabidopsis are key mechanisms linked to growth promotion and ISR by Pf.SS101. © 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.

Inhibition of Staphyloxanthin Virulence Factor Biosynthesis in Staphylococcus aureus: In Vitro, in Vivo, and Crystallographic Results†

PubMed Central

Song, Yongcheng; Liu, Chia-I; Lin, Fu-Yang; No, Joo Hwan; Hensler, Mary; Liu, Yi-Liang; Jeng, Wen-Yih; Low, Jennifer; Liu, George Y.; Nizet, Victor; Wang, Andrew H.-J.; Oldfield, Eric

2009-01-01

The gold color of Staphylococcus aureus is derived from the carotenoid staphyloxanthin, a virulence factor for the organism. Here, we report the synthesis and activity of a broad variety of staphyloxanthin biosynthesis inhibitors that inhibit the first committed step in its biosynthesis, condensation of two farnesyl diphosphate (FPP) molecules to dehydrosqualene, catalyzed by the enzyme dehydrosqualene synthase (CrtM). The most active compounds are phosphonoacetamides that have low nanomolar Ki values for CrtM inhibition and are active in whole bacterial cells and in mice, where they inhibit S. aureus disease progression. We also report the X-ray crystallographic structure of the most active compound, N-3-(3-phenoxyphenyl)propylphosphonoacetamide (IC50 = 8 nM, in cells), bound to CrtM. The structure exhibits a complex network of hydrogen bonds between the polar headgroup and the protein, while the 3-phenoxyphenyl side chain is located in a hydrophobic pocket previously reported to bind farnesyl thiodiphosphate (FsPP), as well as biphenyl phosphonosulfonate inhibitors. Given the good enzymatic, whole cell, and in vivo pharmacologic activities, these results should help guide the further development of novel antivirulence factor-based therapies for S. aureus infections. PMID:19456099

Understanding Microbial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2017-10-01

pathogens and commensals. However, the technology available to track these molecules in host cells and tissues remains primitive. To address this...live, luminal bacteria into specific host intestinal immune cells and their subsequent degradation in host phagocytes. Notably, this approach also...click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide, inflammatory

Discovery of chlamydial peptidoglycan reveals bacteria with murein sacculi but without FtsZ

NASA Astrophysics Data System (ADS)

Pilhofer, Martin; Aistleitner, Karin; Biboy, Jacob; Gray, Joe; Kuru, Erkin; Hall, Edward; Brun, Yves V.; Vannieuwenhze, Michael S.; Vollmer, Waldemar; Horn, Matthias; Jensen, Grant J.

2013-12-01

Chlamydiae are important pathogens and symbionts with unique cell biological features. They lack the cell-division protein FtsZ, and the existence of peptidoglycan (PG) in their cell wall has been highly controversial. FtsZ and PG together function in orchestrating cell division and maintaining cell shape in almost all other bacteria. Using electron cryotomography, mass spectrometry and fluorescent labelling dyes, here we show that some environmental chlamydiae have cell wall sacculi consisting of a novel PG type. Treatment with fosfomycin (a PG synthesis inhibitor) leads to lower infection rates and aberrant cell shapes, suggesting that PG synthesis is crucial for the chlamydial life cycle. Our findings demonstrate for the first time the presence of PG in a member of the Chlamydiae. They also present a unique example of a bacterium with a PG sacculus but without FtsZ, challenging the current hypothesis that it is the absence of a cell wall that renders FtsZ non-essential.

Cloning and sequencing of Staphylococcus aureus murC, a gene essential for cell wall biosynthesis.

PubMed

Lowe, A M; Deresiewicz, R L

1999-01-01

Staphylococcus aureus is a major human pathogen that is increasingly resistant to clinically useful antimicrobial agents. While screening for S. aureus genes expressed during mammalian infection, we isolated murC. This gene encodes UDP-N-acetylmuramoyl-L-alanine synthetase, an enzyme essential for cell wall biosynthesis in a number of bacteria. S. aureus MurC has a predicted mass 49,182 Da and complements the temperature-sensitive murC mutation of E. coli ST222. Sequence data on the DNA flanking staphylococcal murC suggests that the local gene organization there parallels that found in B. subtilis, but differs from that found in gram-negative bacterial pathogens. MurC proteins represent promising targets for broad spectrum antimicrobial drug development.

Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: A molecular overview.

PubMed

Wang, Da-Zhi; Zhang, Shu-Fei; Zhang, Yong; Lin, Lin

2016-03-01

Paralytic shellfish toxins (PSTs) are a group of water soluble neurotoxic alkaloids produced by two different kingdoms of life, prokaryotic cyanobacteria and eukaryotic dinoflagellates. Owing to the wide distribution of these organisms, these toxic secondary metabolites account for paralytic shellfish poisonings around the world. On the other hand, their specific binding to voltage-gated sodium channels makes these toxins potentially useful in pharmacological and toxicological applications. Much effort has been devoted to the biosynthetic mechanism of PSTs, and gene clusters encoding 26 proteins involved in PST biosynthesis have been unveiled in several cyanobacterial species. Functional analysis of toxin genes indicates that PST biosynthesis in cyanobacteria is a complex process including biosynthesis, regulation, modification and export. However, less is known about the toxin biosynthesis in dinoflagellates owing to our poor understanding of the massive genome and unique chromosomal characteristics [1]. So far, few genes involved in PST biosynthesis have been identified from dinoflagellates. Moreover, the proteins involved in PST production are far from being totally explored. Thus, the origin and evolution of PST biosynthesis in these two kingdoms are still controversial. In this review, we summarize the recent progress on the characterization of genes and proteins involved in PST biosynthesis in cyanobacteria and dinoflagellates, and discuss the standing evolutionary hypotheses concerning the origin of toxin biosynthesis as well as future perspectives in PST biosynthesis. Paralytic shellfish toxins (PSTs) are a group of potent neurotoxins which specifically block voltage-gated sodium channels in excitable cells and result in paralytic shellfish poisonings (PSPs) around the world. Two different kingdoms of life, cyanobacteria and dinoflagellates are able to produce PSTs. However, in contrast with cyanobacteria, our understanding of PST biosynthesis in

Solanesol Biosynthesis in Plants.

PubMed

Yan, Ning; Liu, Yanhua; Zhang, Hongbo; Du, Yongmei; Liu, Xinmin; Zhang, Zhongfeng

2017-03-23

Solanesol is a non-cyclic terpene alcohol composed of nine isoprene units that mainly accumulates in solanaceous plants. Solanesol plays an important role in the interactions between plants and environmental factors such as pathogen infections and moderate-to-high temperatures. Additionally, it is a key intermediate for the pharmaceutical synthesis of ubiquinone-based drugs such as coenzyme Q10 and vitamin K2, and anti-cancer agent synergizers such as N-solanesyl-N,N'-bis(3,4-dimethoxybenzyl) ethylenediamine (SDB). In plants, solanesol is formed by the 2- C -methyl-d-erythritol 4-phosphate (MEP) pathway within plastids. Solanesol's biosynthetic pathway involves the generation of C5 precursors, followed by the generation of direct precursors, and then the biosynthesis and modification of terpenoids; the first two stages of this pathway are well understood. Based on the current understanding of solanesol biosynthesis, we here review the key enzymes involved, including 1-deoxy-d-xylulose 5-phosphate synthase (DXS), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR), isopentenyl diphosphate isomerase (IPI), geranyl geranyl diphosphate synthase (GGPPS), and solanesyl diphosphate synthase (SPS), as well as their biological functions. Notably, studies on microbial heterologous expression and overexpression of key enzymatic genes in tobacco solanesol biosynthesis are of significant importance for medical uses of tobacco.

A Deficiency in Arabinogalactan Biosynthesis Affects Corynebacterium glutamicum Mycolate Outer Membrane Stability▿

PubMed Central

Bou Raad, Roland; Méniche, Xavier; de Sousa-d'Auria, Celia; Chami, Mohamed; Salmeron, Christophe; Tropis, Marielle; Labarre, Cecile; Daffé, Mamadou; Houssin, Christine; Bayan, Nicolas

2010-01-01

Corynebacterineae is a specific suborder of Gram-positive bacteria that includes Mycobacterium tuberculosis and Corynebacterium glutamicum. The ultrastructure of the cell envelope is very atypical. It is composed of a heteropolymer of peptidoglycan and arabinogalactan (AG) covalently associated to an outer membrane. Five arabinosyltransferases are involved in the biosynthesis of AG in C. glutamicum. AftB catalyzes the transfer of Araf (arabinofuranosyl) onto the arabinan domain of the arabinogalactan to form terminal β(1 → 2)-linked Araf residues. Here we show that ΔaftB cells lack half of the arabinogalactan mycoloylation sites but are still able to assemble an outer membrane. In addition, we show that a ΔaftB mutant grown on a rich medium has a perturbed cell envelope and sheds a significant amount of membrane fragments in the external culture medium. These fragments contain mono- and dimycolate of trehalose and PorA/H, the major porin of C. glutamicum, but lack conventional phospholipids that typify the plasma membrane, suggesting that they are derived from the atypical mycolate outer membrane of the cell envelope. This is the first report of outer membrane destabilization in the Corynebacterineae, and it suggests that a strong interaction between the mycolate outer membrane and the underlying polymer is essential for cell envelope integrity. The presence of outer membrane-derived fragments (OMFs) in the external medium of the ΔaftB mutant is also a very promising tool for outer membrane characterization. Indeed, fingerprint analysis of major OMF-associated proteins has already led to the identification of 3 associated mycoloyltransferases and an unknown protein with a C-terminal hydrophobic anchoring domain reminiscent of that found for the S-layer protein PS2 of C. glutamicum. PMID:20363942

Biosynthesis of gold nanoparticles by the extreme bacterium Deinococcus radiodurans and an evaluation of their antibacterial properties.

PubMed

Li, Jiulong; Li, Qinghao; Ma, Xiaoqiong; Tian, Bing; Li, Tao; Yu, Jiangliu; Dai, Shang; Weng, Yulan; Hua, Yuejin

Deinococcus radiodurans is an extreme bacterium known for its high resistance to stresses including radiation and oxidants. The ability of D. radiodurans to reduce Au(III) and biosynthesize gold nanoparticles (AuNPs) was investigated in aqueous solution by ultraviolet and visible (UV/Vis) absorption spectroscopy, electron microscopy, X-ray diffraction (XRD), dynamic light scattering (DLS), Fourier transform infrared spectroscopy (FTIR) and X-ray photoelectron spectroscopy (XPS). D. radiodurans efficiently synthesized AuNPs from 1 mM Au(III) solution in 8 h. The AuNPs were of spherical, triangular and irregular shapes with an average size of 43.75 nm and a polydispersity index of 0.23 as measured by DLS. AuNPs were distributed in the cell envelope, across the cytosol and in the extracellular space. XRD analysis confirmed the crystallite nature of the AuNPs from the cell supernatant. Data from the FTIR and XPS showed that upon binding to proteins or compounds through interactions with carboxyl, amine, phospho and hydroxyl groups, Au(III) may be reduced to Au(I), and further reduced to Au(0) with the capping groups to stabilize the AuNPs. Biosynthesis of AuNPs was optimized with respect to the initial concentration of gold salt, bacterial growth period, solution pH and temperature. The purified AuNPs exhibited significant antibacterial activity against both Gram-negative ( Escherichia coli ) and Gram-positive ( Staphylococcus aureus ) bacteria by damaging their cytoplasmic membrane. Therefore, the extreme bacterium D. radiodurans can be used as a novel bacterial candidate for efficient biosynthesis of AuNPs, which exhibited potential in biomedical application as an antibacterial agent.

Identification and cloning of a type III polyketide synthase required for diffusible pigment biosynthesis in Saccharopolyspora erythraea.

PubMed

Cortés, Jesús; Velasco, Javier; Foster, Graham; Blackaby, Andrew P; Rudd, Brian A M; Wilkinson, Barrie

2002-06-01

The soluble, diffusible red-brown pigment produced by a Saccharopolyspora erythraea "red variant" has been shown to contain glycosylated and polymerized derivatives of 2,5,7-trihydroxy-1,4-naphthoquinone (flaviolin). Flaviolin is a spontaneous oxidation product of 1,3,6,8-tetrahydroxynaphthalene (THN), which is biosynthesized in bacteria by a chalcone synthase-like (CS-like) type III polyketide synthase (PKS). A fragment of the gene responsible for THN biosynthesis in S. erythraea E_8-7 was amplified by polymerase chain reaction (PCR) using degenerate primers based on conserved regions of known plant CS and bacterial CS-like genes. From the isolated fragment, a suicide vector was prepared, which was subsequently used to disrupt the red-brown pigment-producing (rpp) locus in S. erythraea, generating a mutant that displayed an albino phenotype. Chromosomal DNA from the albino mutant was subsequently used in a vector-recapture protocol to isolate a plasmid that contained an insert spanning the entire rpp locus. Sequencing of the insert revealed that the disrupted open reading frame (ORF) encodes a CS-like protein displaying 69% sequence identity to the rppA gene of Streptomyces griseus. The S. griseus rppA gene encodes RppA, the first characterized bacterial CS-like protein, which is sufficient in vitro for the synthesis of THN from malonyl-CoA. The rppA disruption mutant and rppA sequence provided a means by which to address the mechanism of diffusible pigment biosynthesis, as well as to investigate any link between this and the modulation of erythromycin A titre, which has been observed for S. erythraea variants.

Influence of oxidative and nitrosative stress on accumulation of diphosphate intermediates of the non-mevalonate pathway of isoprenoid biosynthesis in corynebacteria and mycobacteria.

PubMed

Artsatbanov, V Yu; Vostroknutova, G N; Shleeva, M O; Goncharenko, A V; Zinin, A I; Ostrovsky, D N; Kapreliants, A S

2012-04-01

Artificial generation of oxygen superoxide radicals in actively growing cultures of Mycobacterium tuberculosis, Myc. smegmatis, and Corynebacterium ammoniagenes is followed by accumulation in the bacterial cells of substantial amounts of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate (MEcDP) - an intermediate of the non-mevalonate pathway of isoprenoid biosynthesis (MEP) - most possibly due to the interaction of the oxygen radicals with the 4Fe-4S group in the active center and inhibition of the enzyme (E)-4-oxy-3-methylbut-2-enyl diphosphate synthase (IspG). Cadmium ions known to inhibit IspG enzyme in chloroplasts (Rivasseau, C., Seemann, M., Boisson, A. M., Streb, P., Gout, E., Douce, R., Rohmer, M., and Bligny, R. (2009) Plant Cell Environ., 32, 82-92), when added to culture of Myc. smegmatis, substantially increase accumulation of MEcDP induced by oxidative stress with no accumulation of other organic phosphate intermediates in the cell. Corynebacterium ammoniagenes'', well-known for its ability to synthesize large amounts of MEcDP, was also shown to accumulate this unique cyclodiphosphate in actively growing culture when NO at low concentration is artificially generated in the medium. A possible role of the MEP-pathway of isoprenoid biosynthesis and a role of its central intermediate MEcDP in bacterial response to nitrosative and oxidative stress is discussed.

Bacteriophage endolysins as novel antimicrobials

PubMed Central

Schmelcher, Mathias; Donovan, David M; Loessner, Martin J

2013-01-01

Endolysins are enzymes used by bacteriophages at the end of their replication cycle to degrade the peptidoglycan of the bacterial host from within, resulting in cell lysis and release of progeny virions. Due to the absence of an outer membrane in the Gram-positive bacterial cell wall, endolysins can access the peptidoglycan and destroy these organisms when applied externally, making them interesting antimicrobial candidates, particularly in light of increasing bacterial drug resistance. This article reviews the modular structure of these enzymes, in which cell wall binding and catalytic functions are separated, as well as their mechanism of action, lytic activity and potential as antimicrobials. It particularly focuses on molecular engineering as a means of optimizing endolysins for specific applications, highlights new developments that may render these proteins active against Gram-negative and intracellular pathogens and summarizes the most recent applications of endolysins in the fields of medicine, food safety, agriculture and biotechnology. PMID:23030422

The ng_ζ1 toxin of the gonococcal epsilon/zeta toxin/antitoxin system drains precursors for cell wall synthesis.

PubMed

Rocker, Andrea; Peschke, Madeleine; Kittilä, Tiia; Sakson, Roman; Brieke, Clara; Meinhart, Anton

2018-04-27

Bacterial toxin-antitoxin complexes are emerging as key players modulating bacterial physiology as activation of toxins induces stasis or programmed cell death by interference with vital cellular processes. Zeta toxins, which are prevalent in many bacterial genomes, were shown to interfere with cell wall formation by perturbing peptidoglycan synthesis in Gram-positive bacteria. Here, we characterize the epsilon/zeta toxin-antitoxin (TA) homologue from the Gram-negative pathogen Neisseria gonorrhoeae termed ng_ɛ1 / ng_ζ1. Contrary to previously studied streptococcal epsilon/zeta TA systems, ng_ɛ1 has an epsilon-unrelated fold and ng_ζ1 displays broader substrate specificity and phosphorylates multiple UDP-activated sugars that are precursors of peptidoglycan and lipopolysaccharide synthesis. Moreover, the phosphorylation site is different from the streptococcal zeta toxins, resulting in a different interference with cell wall synthesis. This difference most likely reflects adaptation to the individual cell wall composition of Gram-negative and Gram-positive organisms but also the distinct involvement of cell wall components in virulence.

In Vivo Roles of Fatty Acid Biosynthesis Enzymes in Biosynthesis of Biotin and α-Lipoic Acid in Corynebacterium glutamicum.

PubMed

Ikeda, Masato; Nagashima, Takashi; Nakamura, Eri; Kato, Ryosuke; Ohshita, Masakazu; Hayashi, Mikiro; Takeno, Seiki

2017-10-01

For fatty acid biosynthesis, Corynebacterium glutamicum uses two type I fatty acid synthases (FAS-I), FasA and FasB, in addition to acetyl-coenzyme A (CoA) carboxylase (ACC) consisting of AccBC, AccD1, and AccE. The in vivo roles of the enzymes in supplying precursors for biotin and α-lipoic acid remain unclear. Here, we report genetic evidence demonstrating that the biosynthesis of these cofactors is linked to fatty acid biosynthesis through the FAS-I pathway. For this study, we used wild-type C. glutamicum and its derived biotin vitamer producer BFI-5, which was engineered to express Escherichia coli bioBF and Bacillus subtilis bioI Disruption of either fasA or fasB in strain BFI-5 led to decreased production of biotin vitamers, whereas its amplification contributed to increased production, with a larger impact of fasA in both cases. Double disruptions of fasA and fasB resulted in no biotin vitamer production. The acc genes showed a positive effect on production when amplified simultaneously. Augmented fatty acid biosynthesis was also reflected in pimelic acid production when carbon flow was blocked at the BioF reaction. These results indicate that carbon flow down the FAS-I pathway is destined for channeling into the biotin biosynthesis pathway, and that FasA in particular has a significant impact on precursor supply. In contrast, fasB disruption resulted in auxotrophy for lipoic acid or its precursor octanoic acid in both wild-type and BFI-5 strains. The phenotypes were fully complemented by plasmid-mediated expression of fasB but not fasA These results reveal that FasB plays a specific physiological role in lipoic acid biosynthesis in C. glutamicum IMPORTANCE For the de novo biosynthesis of fatty acids, C. glutamicum exceptionally uses a eukaryotic multifunctional type I fatty acid synthase (FAS-I) system comprising FasA and FasB, in contrast to most bacteria, such as E. coli and B. subtilis , which use an individual nonaggregating type II fatty acid synthase

Understanding Microbial Sensing in Inflammatory Bowel Disease Using Click Chemistry

DTIC Science & Technology

2017-10-01

both pathogens and commensals. However, the technology available to track these molecules in host cells and tissues remains primitive. To address this...from live, luminal bacteria into specific host intestinal immune cells and their subsequent degradation in host phagocytes. Notably, this approach...Bioorthogonal click-chemistry, bacterial cell wall, bacterial outer membrane, peptidoglycan, lipopolysaccharide, endotoxin, capsular polysaccharide

Coupling between d-3-phosphoglycerate dehydrogenase and d-2-hydroxyglutarate dehydrogenase drives bacterial l-serine synthesis

PubMed Central

Zhang, Wen; Zhang, Manman; Gao, Chao; Zhang, Yipeng; Ge, Yongsheng; Guo, Shiting; Guo, Xiaoting; Zhou, Zikang; Liu, Qiuyuan; Zhang, Yingxin; Ma, Cuiqing; Tao, Fei; Xu, Ping

2017-01-01

l-Serine biosynthesis, a crucial metabolic process in most domains of life, is initiated by d-3-phosphoglycerate (d-3-PG) dehydrogenation, a thermodynamically unfavorable reaction catalyzed by d-3-PG dehydrogenase (SerA). d-2-Hydroxyglutarate (d-2-HG) is traditionally viewed as an abnormal metabolite associated with cancer and neurometabolic disorders. Here, we reveal that bacterial anabolism and catabolism of d-2-HG are involved in l-serine biosynthesis in Pseudomonas stutzeri A1501 and Pseudomonas aeruginosa PAO1. SerA catalyzes the stereospecific reduction of 2-ketoglutarate (2-KG) to d-2-HG, responsible for the major production of d-2-HG in vivo. SerA combines the energetically favorable reaction of d-2-HG production to overcome the thermodynamic barrier of d-3-PG dehydrogenation. We identified a bacterial d-2-HG dehydrogenase (D2HGDH), a flavin adenine dinucleotide (FAD)-dependent enzyme, that converts d-2-HG back to 2-KG. Electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETFQO) are also essential in d-2-HG metabolism through their capacity to transfer electrons from D2HGDH. Furthermore, while the mutant with D2HGDH deletion displayed decreased growth, the defect was rescued by adding l-serine, suggesting that the D2HGDH is functionally tied to l-serine synthesis. Substantial flux flows through d-2-HG, being produced by SerA and removed by D2HGDH, ETF, and ETFQO, maintaining d-2-HG homeostasis. Overall, our results uncover that d-2-HG–mediated coupling between SerA and D2HGDH drives bacterial l-serine synthesis. PMID:28827360

The role of magnetic iron oxide nanoparticles in the bacterially induced calcium carbonate precipitation.

PubMed

Seifan, Mostafa; Ebrahiminezhad, Alireza; Ghasemi, Younes; Samani, Ali Khajeh; Berenjian, Aydin

2018-04-01

Recently, magnetic iron oxide nanoparticles (IONs) have been used to control and modify the characteristics of concrete and mortar. Concrete is one of the most used materials in the world; however, it is susceptible to cracking. Over recent years, a sustainable biotechnological approach has emerged as an alternative approach to conventional techniques to heal the concrete cracks by the incorporation of bacterial cells and nutrients into the concrete matrix. Once cracking occurs, CaCO 3 is induced and the crack is healed. Considering the positive effects of IONs on the concrete properties, the effect of these nanoparticles on bacterial growth and CaCO 3 biosynthesis needs to be evaluated for their possible application in bio self-healing concrete. In the present work, IONs were successfully synthesized and characterized using various techniques. The presence of IONs showed a significant effect on both bacterial growth and CaCO 3 precipitation. The highest bacterial growth was observed in the presence of 150 μg/mL IONs. The highest concentration of induced CaCO 3 (34.54 g/L) was achieved when the bacterial cells were immobilized with 300 μg/mL of IONs. This study provides new data and supports the possibility of using IONs as a new tool in designing the next generation of bio self-healing concrete.

RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea

DOE PAGES

Mukai, Takahito; Crnković, Ana; Umehara, Takuya; ...

2017-05-09

ABSTRACT The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNA Cysis first acylated with phosphoserine (Sep) byO-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNA Cysby Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN,Euryarchaeota, TACK, and Asgard) andmore » two groups of bacteria (“CandidatusParcubacteria” andChloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNA Cysspecies with cysteinein vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS-SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea andChloroflexibacteria. We further discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.« less

RNA-Dependent Cysteine Biosynthesis in Bacteria and Archaea

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mukai, Takahito; Crnković, Ana; Umehara, Takuya

ABSTRACT The diversity of the genetic code systems used by microbes on earth is yet to be elucidated. It is known that certain methanogenic archaea employ an alternative system for cysteine (Cys) biosynthesis and encoding; tRNA Cysis first acylated with phosphoserine (Sep) byO-phosphoseryl-tRNA synthetase (SepRS) and then converted to Cys-tRNA Cysby Sep-tRNA:Cys-tRNA synthase (SepCysS). In this study, we searched all genomic and metagenomic protein sequence data in the Integrated Microbial Genomes (IMG) system and at the NCBI to reveal new clades of SepRS and SepCysS proteins belonging to diverse archaea in the four major groups (DPANN,Euryarchaeota, TACK, and Asgard) andmore » two groups of bacteria (“CandidatusParcubacteria” andChloroflexi). Bacterial SepRS and SepCysS charged bacterial tRNA Cysspecies with cysteinein vitro. Homologs of SepCysE, a scaffold protein facilitating SepRS-SepCysS complex assembly in Euryarchaeota class I methanogens, are found in a few groups of TACK and Asgard archaea, whereas the C-terminally truncated homologs exist fused or genetically coupled with diverse SepCysS species. Investigation of the selenocysteine (Sec)- and pyrrolysine (Pyl)-utilizing traits in SepRS-utilizing archaea and bacteria revealed that the archaea carrying full-length SepCysE employ Sec and that SepRS is often found in Pyl-utilizing archaea andChloroflexibacteria. We further discuss possible contributions of the SepRS-SepCysS system for sulfur assimilation, methanogenesis, and other metabolic processes requiring large amounts of iron-sulfur enzymes or Pyl-containing enzymes.« less

Racemization of alanine by the alanine racemases from Salmonella typhimurium and Bacillus stearothermophilus: energetic reaction profiles

DOE Office of Scientific and Technical Information (OSTI.GOV)

Faraci, W.S.; Walsh, C.T.

1988-05-03

Alanine racemases are bacterial pyridoxal 5'-phosphate (PLP) dependent enzymes providing D-alanine as an essential building block for biosynthesis of the peptidoglycan layer of the cell wall. Two isozymic alanine racemases, encoded by the dadB gene and the alr gene, from the Gram-negative mesophilic Salmonella typhimurium and one from the Gram-positive thermophilic Bacillus stearothermophilus have been examined for the racemization mechanism. Substrate deuterium isotope effects and solvent deuterium isotope effects have been measured in both L ..-->.. D and D..-->.. L directions for all three enzymes to assess the degree to which abstraction of the ..cap alpha..-proton or protonation of substratemore » PLP carbanion is limiting in catalysis. Additionally, experiments measuring internal return of ..cap alpha..-/sup 3/H from substrate to product and solvent exchange/substrate conversion experiments in /sup 3/H/sub 2/O have been used with each enzyme to examine the partitioning of substrate PLP carbanion intermediates and to obtain the relative heights of kinetically significant energy barriers in alanine racemase catalysis.« less

Isolation of temperature-sensitive mutations in murC of Staphylococcus aureus.

PubMed

Ishibashi, Mihoko; Kurokawa, Kenji; Nishida, Satoshi; Ueno, Kohji; Matsuo, Miki; Sekimizu, Kazuhisa

2007-09-01

Enzymes in the bacterial peptidoglycan biosynthesis pathway are important targets for novel antibiotics. Of 750 temperature-sensitive (TS) mutants of Gram-positive Staphylococcus aureus, six were complemented by the murC gene, which encodes the UDP-N-acetylmuramic acid:l-alanine ligase. Each mutation resulted in a single amino acid substitution and, in all cases, the TS phenotype was suppressed by high osmotic stress. In mutant strains with the G222E substitution, a decrease in the viable cell number immediately after shift to the restrictive temperature was observed. These results suggest that S. aureus MurC protein is essential for cell growth. The MurC H343Y mutation is located in the putative alanine recognition pocket. Consistent with this, allele-specific suppression was observed of the H343Y mutation by multiple copies of the aapA gene, which encodes an alanine transporter. The results suggest an in vivo role for the H343 residue of S. aureus MurC protein in high-affinity binding to L-alanine.

Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor

PubMed Central

2013-01-01

Background Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. Results We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production. Conclusions The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single

Plasmid-mediated resistance to protein biosynthesis inhibitors in staphylococci.

PubMed

Schwarz, Stefan; Fessler, Andrea T; Hauschild, Tomasz; Kehrenberg, Corinna; Kadlec, Kristina

2011-12-01

Protein biosynthesis inhibitors (PBIs) represent powerful antimicrobial agents for the control of bacterial infections. In staphylococci, numerous resistance genes are known to be involved in resistance to PBIs, most of which mediate resistance to a specific class/subclass of PBIs, though a few genes do confer a multidrug resistance phenotype-up to five classes/subclasses of PBIs. Plasmids play a key role in the dissemination of PBI resistance among staphylococci, as they primarily carry plasmid-borne PBI resistance genes; however, plasmids also can be vectors for transposon-borne PBI resistance genes. Small plasmids that carry single PBI resistance genes are widespread among staphylococci of human and animal origin. Various mechanisms exist by which they can recombine, form cointegrates, or integrate into chromosomal DNA or larger plasmids. We provide an overview of the current knowledge of plasmid-mediated PBI resistance in staphylococci, with particular reference to the currently known PBI resistance genes, their association with mobile genetic elements, and the recombination/integration processes that control their mobility. © 2011 New York Academy of Sciences.

Effect of liposomal formulations and immunostimulating peptidoglycan monomer (PGM) on the immune reaction to ovalbumin in mice.

PubMed

Habjanec, Lidija; Frkanec, Ruza; Halassy, Beata; Tomasić, Jelka

2006-01-01

The adjuvant activity of liposomes and immunostimulating peptidoglycan monomer (PGM) in different formulations has been studied in mice model using ovalbumin (OVA) as an antigen. PGM is a natural compound of bacterial origin with well-defined chemical structure: GlcNAc-MurNAc-L-Ala-D-isoGln-mesoDpm(epsilonNH2)-D-Ala-D-Ala. It is a non-toxic, non-pyrogenic, and water-soluble immunostimulator. The aim of this study was to investigate the influence of different liposomal formulations of OVA, with or without PGM, on the production of total IgG, as well as of IgG1 and IgG2a subclasses of OVA-specific antibodies (as indicators of Th2 and Th1 type of immune response, respectively). CBA mice were immunized s.c. with OVA mixed with liposomes, OVA with PGM mixed with liposomes, OVA encapsulated into liposomes and OVA with PGM encapsulated into liposomes. Control groups were OVA in saline, OVA with PGM in saline, and OVA in CFA/IFA adjuvant formulation. The entrapment efficacy of OVA was monitored by HPLC method. The adjuvant activity of the mixture of OVA and empty liposomes, the mixture of OVA, PGM, and liposomes and PGM encapsulated with OVA into liposomes on production of total anti-OVA IgG was demonstrated. The mixture of PGM and liposomes exhibited additive immunostimulating effect on the production of antigen-specific IgGs. The analysis of IgG subclasses revealed that encapsulation of OVA into liposomes favors the stimulation of IgG2a antibodies, indicating the switch toward the Th1 type of immune response. When encapsulated into liposomes or mixed with liposomes, PGM induced a switch from Th1 to Th2 type of immune response. It could be concluded that appropriate formulations of antigen, PGM, and liposomes differently affect the humoral immune response and direct the switch in the type of immune response (Th1/Th2).

Genetic Dissection of Tropodithietic Acid Biosynthesis by Marine Roseobacters▿ ‡

PubMed Central

Geng, Haifeng; Bruhn, Jesper Bartholin; Nielsen, Kristian F.; Gram, Lone; Belas, Robert

2008-01-01

The symbiotic association between the roseobacter Silicibacter sp. strain TM1040 and the dinoflagellate Pfiesteria piscicida involves bacterial chemotaxis to dinoflagellate-produced dimethylsulfoniopropionate (DMSP), DMSP demethylation, and ultimately a biofilm on the surface of the host. Biofilm formation is coincident with the production of an antibiotic and a yellow-brown pigment. In this report, we demonstrate that the antibiotic is a sulfur-containing compound, tropodithietic acid (TDA). Using random transposon insertion mutagenesis, 12 genes were identified as critical for TDA biosynthesis by the bacteria, and mutation in any one of these results in a loss of antibiotic activity (Tda−) and pigment production. Unexpectedly, six of the genes, referred to as tdaA-F, could not be found on the annotated TM1040 genome and were instead located on a previously unidentified plasmid (ca. 130 kb; pSTM3) that exhibited a low frequency of spontaneous loss. Homologs of tdaA and tdaB from Silicibacter sp. strain TM1040 were identified by mutagenesis in another TDA-producing roseobacter, Phaeobacter sp. strain 27-4, which also possesses two large plasmids (ca. 60 and ca. 70 kb, respectively), and tda genes were found by DNA-DNA hybridization in 88% of a diverse collection of nine roseobacters with known antibiotic activity. These data suggest that roseobacters may use a common pathway for TDA biosynthesis that involves plasmid-encoded proteins. Using metagenomic library databases and a bioinformatics approach, differences in the biogeographical distribution between the critical TDA synthesis genes were observed. The implications of these results to roseobacter survival and the interaction between TM1040 and its dinoflagellate host are discussed. PMID:18192410

Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Uehling, J.; Gryganskyi, A.; Hameed, K.

Endosymbiosis of bacteria by eukaryotes is a defining feature of cellular evolution. In addition to well-known bacterial origins for mitochondria and chloroplasts, multiple origins of bacterial endosymbiosis are known within the cells of diverse animals, plants and fungi. Early-diverging lineages of terrestrial fungi harbor endosymbiotic bacteria belonging to the Burkholderiaceae. Furthermore, we sequenced the metagenome of the soil-inhabiting fungus Mortierella elongata and assembled the complete circular chromosome of its endosymbiont, Mycoavidus cysteinexigens, which we place within a lineage of endofungal symbionts that are sister clade to Burkholderia. The genome of M. elongata strain AG77 features a core set of primarymore » metabolic pathways for degradation of simple carbohydrates and lipid biosynthesis, while the M. cysteinexigens (AG77) genome is reduced in size and function. Experiments using antibiotics to cure the endobacterium from the host demonstrate that the fungal host metabolism is highly modulated by presence/ absence of M. cysteinexigens. In independent comparative phylogenomic analyses of fungal and bacterial genomes we find that they are consistent with an ancient origin for M. elongata M. cysteinexigens symbiosis, most likely over 350 million years ago and concomitant with the terrestrialization of Earth and diversification of land fungi and plants.« less

Comparative genomics of Mortierella elongata and its bacterial endosymbiont Mycoavidus cysteinexigens

DOE PAGES

Uehling, J.; Gryganskyi, A.; Hameed, K.; ...

2017-01-11

Endosymbiosis of bacteria by eukaryotes is a defining feature of cellular evolution. In addition to well-known bacterial origins for mitochondria and chloroplasts, multiple origins of bacterial endosymbiosis are known within the cells of diverse animals, plants and fungi. Early-diverging lineages of terrestrial fungi harbor endosymbiotic bacteria belonging to the Burkholderiaceae. Furthermore, we sequenced the metagenome of the soil-inhabiting fungus Mortierella elongata and assembled the complete circular chromosome of its endosymbiont, Mycoavidus cysteinexigens, which we place within a lineage of endofungal symbionts that are sister clade to Burkholderia. The genome of M. elongata strain AG77 features a core set of primarymore » metabolic pathways for degradation of simple carbohydrates and lipid biosynthesis, while the M. cysteinexigens (AG77) genome is reduced in size and function. Experiments using antibiotics to cure the endobacterium from the host demonstrate that the fungal host metabolism is highly modulated by presence/ absence of M. cysteinexigens. In independent comparative phylogenomic analyses of fungal and bacterial genomes we find that they are consistent with an ancient origin for M. elongata M. cysteinexigens symbiosis, most likely over 350 million years ago and concomitant with the terrestrialization of Earth and diversification of land fungi and plants.« less

Inexpensive, renewable substrates for the fermentative biosynthesis of bacterial poly(hydroxyalkanoate)s with controlled monomeric compositions

USDA-ARS?s Scientific Manuscript database

Poly(hydroxyalkanoate)s (PHA’s) are well-known bacterial polyesters produced by many bacteria under nutrient-deficient conditions. While many PHA’s demonstrate comparable properties to the more popular petrochemical polymers, PHA applications are inhibited by high production costs. In an effort to r...

Identification of new genes in a cell envelope-cell division gene cluster of Escherichia coli: cell envelope gene murG.

PubMed Central

Salmond, G P; Lutkenhaus, J F; Donachie, W D

1980-01-01

We report the identification, cloning, and mapping of a new cell envelope gene, murG. This lies in a group of five genes of similar phenotype (in the order murE murF murG murC ddl) all concerned with peptidoglycan biosynthesis. This group is in a larger cluster of at least 10 genes, all of which are involved in some way with cell envelope growth. Images PMID:6998962

Biosynthesis and function of chondroitin sulfate.

PubMed

Mikami, Tadahisa; Kitagawa, Hiroshi

2013-10-01

Chondroitin sulfate proteoglycans (CSPGs) are principal pericellular and extracellular components that form regulatory milieu involving numerous biological and pathophysiological phenomena. Diverse functions of CSPGs can be mainly attributed to structural variability of their polysaccharide moieties, chondroitin sulfate glycosaminoglycans (CS-GAG). Comprehensive understanding of the regulatory mechanisms for CS biosynthesis and its catabolic processes is required in order to understand those functions. Here, we focus on recent advances in the study of enzymatic regulatory pathways for CS biosynthesis including successive modification/degradation, distinct CS functions, and disease phenotypes that have been revealed by perturbation of the respective enzymes in vitro and in vivo. Fine-tuned machineries for CS production/degradation are crucial for the functional expression of CS chains in developmental and pathophysiological processes. Control of enzymes responsible for CS biosynthesis/catabolism is a potential target for therapeutic intervention for the CS-associated disorders. Copyright © 2013 Elsevier B.V. All rights reserved.

Roles of the bacterial cell wall and capsule in induction of tumor necrosis factor alpha by type III group B streptococci.

PubMed Central

Vallejo, J G; Baker, C J; Edwards, M S

1996-01-01

Group B streptococci (GBS) are the major cause of sepsis and fatal shock in neonates in the United States. The precise role of tumor necrosis factor alpha (TNF-alpha) in the development of human GBS sepsis has not been defined; however, whole GBS have been shown to induce the production of this inflammatory cytokine. We sought to determine which bacterial cell wall components of GBS are responsible for triggering TNF-alpha production. Human cord blood monocytes were stimulated with encapsulated (COH1) or unencapsulated (COH1-13) whole type III GBS or with purified bacterial components, including type III capsular polysaccharide (III-PS), group B polysaccharide (GB-PS), lipoteichoic acid (LTA), or peptidoglycan (PG). Lipopolysaccharide from Escherichia coli served as a control. Supernatants were harvested at specific timed intervals, and TNF-alpha levels were measured by enzyme-linked immunosorbent assay. Monocytes exposed to COH1 and COH1-13 induced similar amounts of TNF-alpha. III-PS, GB-PS, LTA, and PG each induced TNF-alpha in a time- and concentration-dependent manner. However, TNF-alpha release was significantly greater after stimulation by the GB-PS or PG than after stimulation by III-PS or LTA (P < 0.05). Our findings indicate that GB-PS and PG are the bacterial cell wall components primarily evoking TNF-alpha release. These, alone or in concert with other factors, may be responsible for septic shock accompanying GBS sepsis. PMID:8945544

New 5-benzylidenethiazolidin-4-one inhibitors of bacterial MurD ligase: design, synthesis, crystal structures, and biological evaluation.

PubMed

Zidar, Nace; Tomašić, Tihomir; Šink, Roman; Kovač, Andreja; Patin, Delphine; Blanot, Didier; Contreras-Martel, Carlos; Dessen, Andréa; Premru, Manica Müller; Zega, Anamarija; Gobec, Stanislav; Mašič, Lucija Peterlin; Kikelj, Danijel

2011-11-01

Mur ligases (MurC-MurF), a group of bacterial enzymes that catalyze four consecutive steps in the formation of cytoplasmic peptidoglycan precursor, are becoming increasingly adopted as targets in antibacterial drug design. Based on the crystal structure of MurD cocrystallized with thiazolidine-2,4-dione inhibitor I, we have designed, synthesized, and evaluated a series of improved glutamic acid containing 5-benzylidenerhodanine and 5-benzylidenethiazolidine-2,4-dione inhibitors of MurD with IC(50) values up to 28 μM. Inhibitor 37, with an IC(50) of 34 μM, displays a weak antibacterial activity against S. aureus ATCC 29213 and E. faecalis ATCC 29212 with minimal inhibitory concentrations of 128 μg/mL. High-resolution crystal structures of MurD in complex with two new inhibitors (compounds 23 and 51) reveal details of their binding modes within the active site and provide valuable information for further structure-based optimization. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

Wall teichoic acids prevent antibody binding to epitopes within the cell wall of Staphylococcus aureus.

PubMed

Gautam, Samir; Kim, Taehan; Lester, Evan; Deep, Deeksha; Spiegel, David A

2016-01-15

Staphylococcus aureus is a Gram-positive bacterial pathogen that produces a range of infections including cellulitis, pneumonia, and septicemia. The principle mechanism in antistaphylococcal host defense is opsonization with antibodies and complement proteins, followed by phagocytic clearance. Here we use a previously developed technique for installing chemical epitopes in the peptidoglycan cell wall to show that surface glycopolymers known as wall teichoic acids conceal cell wall epitopes, preventing their recognition and opsonization by antibodies. Thus, our results reveal a previously unrecognized immunoevasive role for wall teichoic acids in S. aureus: repulsion of peptidoglycan-targeted antibodies.

A novel biological recovery approach for PHA employing selective digestion of bacterial biomass in animals.

PubMed

Ong, Su Yean; Zainab-L, Idris; Pyary, Somarajan; Sudesh, Kumar

2018-03-01

Polyhydroxyalkanoate (PHA) is a family of microbial polyesters that is completely biodegradable and possesses the mechanical and thermal properties of some commonly used petrochemical-based plastics. Therefore, PHA is attractive as a biodegradable thermoplastic. It has always been a challenge to commercialize PHA due to the high cost involved in the biosynthesis of PHA via bacterial fermentation and the subsequent purification of the synthesized PHA from bacterial cells. Innovative enterprise by researchers from various disciplines over several decades successfully reduced the cost of PHA production through the efficient use of cheap and renewable feedstock, precisely controlled fermentation process, and customized bacterial strains. Despite the fact that PHA yields have been improved tremendously, the recovery and purification processes of PHA from bacterial cells remain exhaustive and require large amounts of water and high energy input besides some chemicals. In addition, the residual cell biomass ends up as waste that needs to be treated. We have found that some animals can readily feed on the dried bacterial cells that contain PHA granules. The digestive system of the animals is able to assimilate the bacterial cells but not the PHA granules which are excreted in the form of fecal pellets, thus resulting in partial recovery and purification of PHA. In this mini-review, we will discuss this new concept of biological recovery, the selection of the animal model for biological recovery, and the properties and possible applications of the biologically recovered PHA.

Flavonoids: biosynthesis, biological functions, and biotechnological applications

PubMed Central

Falcone Ferreyra, María L.; Rius, Sebastián P.; Casati, Paula

2012-01-01

Flavonoids are widely distributed secondary metabolites with different metabolic functions in plants. The elucidation of the biosynthetic pathways, as well as their regulation by MYB, basic helix-loop-helix (bHLH), and WD40-type transcription factors, has allowed metabolic engineering of plants through the manipulation of the different final products with valuable applications. The present review describes the regulation of flavonoid biosynthesis, as well as the biological functions of flavonoids in plants, such as in defense against UV-B radiation and pathogen infection, nodulation, and pollen fertility. In addition, we discuss different strategies and achievements through the genetic engineering of flavonoid biosynthesis with implication in the industry and the combinatorial biosynthesis in microorganisms by the reconstruction of the pathway to obtain high amounts of specific compounds. PMID:23060891

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth.

PubMed

Turapov, Obolbek; Loraine, Jessica; Jenkins, Christopher H; Barthe, Philippe; McFeely, Daniel; Forti, Francesca; Ghisotti, Daniela; Hesek, Dusan; Lee, Mijoon; Bottrill, Andrew R; Vollmer, Waldemar; Mobashery, Shahriar; Cohen-Gonsaud, Martin; Mukamolova, Galina V

2015-07-01

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.

The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth

PubMed Central

Turapov, Obolbek; Loraine, Jessica; Jenkins, Christopher H.; Barthe, Philippe; McFeely, Daniel; Forti, Francesca; Ghisotti, Daniela; Hesek, Dusan; Lee, Mijoon; Bottrill, Andrew R.; Vollmer, Waldemar; Mobashery, Shahriar; Cohen-Gonsaud, Martin; Mukamolova, Galina V.

2015-01-01

PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture. PMID:26136255

Sulfate as a pivotal factor in regulation of Serratia sp. strain S2B pigment biosynthesis.

PubMed

Rastegari, Banafsheh; Karbalaei-Heidari, Hamid Reza

2016-10-01

In the present work, we investigated the prodiginine family as secondary metabolite members. Bacterial strain S2B, with the ability to produce red pigment, was isolated from the Sarcheshmeh copper mine in Iran. 16S rDNA gene sequencing revealed that the strain was placed in the Serratia genus. Pigment production was optimized using low-cost culture medium and the effects of various physicochemical factors were studied via statistical approaches. Purification of the produced pigment by silica gel column chromatography showed a strong red pigment fraction and a weaker orange band. Mass spectrometry, FT-IR spectroscopy and (1)H NMR analysis revealed that the red pigment was prodigiosin and the orange band was a prodigiosin-like analog, with molecular weights of 323 and 317 Da, respectively. Genotoxicity and cytotoxicity studies confirmed their membership in the prodiginine family. Analysis of the production pattern of the pigments in the presence of different concentrations of ammonium salts revealed the role of sulfate as an important factor in regulation of the pigment biosynthesis pathway. Overall, the data showed that regulation of the pigment biosynthesis pathway in Serratia sp. strain S2B was affected by inorganic micronutrients, particularly the sulfate ions. Copyright © 2016 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Qingping; Mengin-Lecreulx, Dominique; Liu, Xueqian W.

ABSTRACT Bacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A 2pm (A 2pm is diaminopimelic acid) cysteine amidase (ordl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminall-Ala. Their crystal structures revealed a highly conserved structure consistingmore » of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation. IMPORTANCEPeptidoglycan is a meshlike polymer that envelops the bacterial plasma membrane and bestows structural integrity. Cell wall lysins and recycling enzymes are part of a set of lytic enzymes that target covalent bonds connecting the amino acid and amino sugar building blocks of the PG network. These hydrolases are involved in processes such as cell growth and division, autolysis, invasion, and PG turnover and recycling. To avoid cleavage of unintended substrates, these enzymes have very selective substrate specificities. Our biochemical and structural analysis of three modular NlpC/P60 hydrolases, one lysin, and two recycling

Engineered Biosynthesis of a Novel Amidated Polyketide, Using the Malonamyl-Specific Initiation Module from the Oxytetracycline Polyketide Synthase

PubMed Central

Zhang, Wenjun; Ames, Brian D.; Tsai, Shiou-Chuan; Tang, Yi

2006-01-01

Tetracyclines are aromatic polyketides biosynthesized by bacterial type II polyketide synthases (PKSs). Understanding the biochemistry of tetracycline PKSs is an important step toward the rational and combinatorial manipulation of tetracycline biosynthesis. To this end, we have sequenced the gene cluster of oxytetracycline (oxy and otc genes) PKS genes from Streptomyces rimosus. Sequence analysis revealed a total of 21 genes between the otrA and otrB resistance genes. We hypothesized that an amidotransferase, OxyD, synthesizes the malonamate starter unit that is a universal building block for tetracycline compounds. In vivo reconstitution using strain CH999 revealed that the minimal PKS and OxyD are necessary and sufficient for the biosynthesis of amidated polyketides. A novel alkaloid (WJ35, or compound 2) was synthesized as the major product when the oxy-encoded minimal PKS, the C-9 ketoreductase (OxyJ), and OxyD were coexpressed in CH999. WJ35 is an isoquinolone compound derived from an amidated decaketide backbone and cyclized with novel regioselectivity. The expression of OxyD with a heterologous minimal PKS did not afford similarly amidated polyketides, suggesting that the oxy-encoded minimal PKS possesses novel starter unit specificity. PMID:16597959

High Ambient Temperature Represses Anthocyanin Biosynthesis through Degradation of HY5

PubMed Central

Kim, Sara; Hwang, Geonhee; Lee, Seulgi; Zhu, Jia-Ying; Paik, Inyup; Nguyen, Thom Thi; Kim, Jungmook; Oh, Eunkyoo

2017-01-01

Anthocyanins are flavonoid compounds that protect plant tissues from many environmental stresses including high light irradiance, freezing temperatures, and pathogen infection. Regulation of anthocyanin biosynthesis is intimately associated with environmental changes to enhance plant survival under stressful environmental conditions. Various factors, such as UV, visible light, cold, osmotic stress, and pathogen infection, can induce anthocyanin biosynthesis. In contrast, high temperatures are known to reduce anthocyanin accumulation in many plant species, even drastically in the skin of fruits such as grape berries and apples. However, the mechanisms by which high temperatures regulate anthocyanin biosynthesis in Arabidopsis thaliana remain largely unknown. Here, we show that high ambient temperatures repress anthocyanin biosynthesis through the E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1) and the positive regulator of anthocyanin biosynthesis ELONGATED HYPOCOTYL5 (HY5). We show that an increase in ambient temperature decreases expression of genes required in both the early and late steps of the anthocyanin biosynthesis pathway in Arabidopsis seedlings. As a result, seedlings grown at a high temperature (28°C) accumulate less anthocyanin pigment than those grown at a low temperature (17°C). We further show that high temperature induces the degradation of the HY5 protein in a COP1 activity-dependent manner. In agreement with this finding, anthocyanin biosynthesis and accumulation do not respond to ambient temperature changes in cop1 and hy5 mutant plants. The degradation of HY5 derepresses the expression of MYBL2, which partially mediates the high temperature repression of anthocyanin biosynthesis. Overall, our study demonstrates that high ambient temperatures repress anthocyanin biosynthesis through a COP1-HY5 signaling module. PMID:29104579

Discovery and Biochemical Characterization of the UDP-Xylose Biosynthesis Pathway in Sphaerobacter thermophilus.

PubMed

Gu, Bin; Laborda, Pedro; Wei, Shuang; Duan, Xu-Chu; Song, Hui-Bo; Liu, Li; Voglmeir, Josef

2016-01-01

The biosynthesis of UDP-xylose requires the stepwise oxidation/ decarboxylation of UDP-glucose, which is catalyzed by the enzymes UDPglucuronic acid dehydrogenase (UGD) and UDP-xylose synthase (UXS). UDPxylose biosynthesis is ubiquitous in animals and plants. However, only a few UGD and UXS isoforms of bacterial origin have thus far been biochemically characterized. Sphaerobacter thermophilus DSM 20745 is a bacterium isolated from heated sewage sludge, and therefore can be a valuable source of thermostable enzymes of biotechnological interest. However, no biochemical characterizations of any S. thermophilus enzymes have yet been reported. Herein, we describe the cloning and characterization of putative UGD (StUGD) and UXS (StUXS) isoforms from this organism. HPLC- and plate reader-based activity tests of the recombinantly expressed StUGD and StUXS showed that they are indeed active enzymes. Both StUGD and StUXS showed a temperature optimum of 70°C, and a reasonable thermal stability up to 60°C. No metal ions were required for enzymatic activities. StUGD had a higher pH optimum than StUXS. The simple purification procedures and the thermotolerance of StUGD and StUXS make them valuable biocatalysts for the synthesis of UDP-glucuronic acid and UDP-xylose at elevated temperatures. The biosynthetic potential of StUGD was further exemplified in a coupled enzymatic reaction with an UDP-glucuronosyltransferase, allowing the glucuronylation of the natural model substrate bilirubin.

Small-molecule inhibitors suppress the expression of both type III secretion and amylovoran biosynthesis genes in Erwinia amylovora.

PubMed

Yang, Fan; Korban, Schuyler S; Pusey, P Lawrence; Elofsson, Michael; Sundin, George W; Zhao, Youfu

2014-01-01

The type III secretion system (T3SS) and exopolysaccharide (EPS) amylovoran are two essential pathogenicity factors in Erwinia amylovora, the causal agent of the serious bacterial disease fire blight. In this study, small molecules that inhibit T3SS gene expression in E. amylovora under hrp (hypersensitive response and pathogenicity)-inducing conditions were identified and characterized using green fluorescent protein (GFP) as a reporter. These compounds belong to salicylidene acylhydrazides and also inhibit amylovoran production. Microarray analysis of E. amylovora treated with compounds 3 and 9 identified a total of 588 significantly differentially expressed genes. Among them, 95 and 78 genes were activated and suppressed by both compounds, respectively, when compared with the dimethylsulphoxide (DMSO) control. The expression of the majority of T3SS genes in E. amylovora, including hrpL and the avrRpt2 effector gene, was suppressed by both compounds. Compound 3 also suppressed the expression of amylovoran precursor and biosynthesis genes. However, both compounds induced significantly the expression of glycogen biosynthesis genes and siderophore biosynthesis, regulatory and transport genes. Furthermore, many membrane, lipoprotein and exported protein-encoding genes were also activated by both compounds. Similar expression patterns were observed for compounds 1, 2 and 4. Using crab apple flower as a model, compound 3 was capable of reducing disease development in pistils. These results suggest a common inhibition mechanism shared by salicylidene acylhydrazides and indicate that small-molecule inhibitors that disable T3SS function could be explored to control fire blight disease. © 2013 BSPP AND JOHN WILEY & SONS LTD.

Cloning, Sequencing, and Functional Analysis of an Iterative Type I Polyketide Synthase Gene Cluster for Biosynthesis of the Antitumor Chlorinated Polyenone Neocarzilin in “Streptomyces carzinostaticus”

PubMed Central

Otsuka, Miyuki; Ichinose, Koji; Fujii, Isao; Ebizuka, Yutaka

2004-01-01

Neocarzilins (NCZs) are antitumor chlorinated polyenones produced by “Streptomyces carzinostaticus” var. F-41. The gene cluster responsible for the biosynthesis of NCZs was cloned and characterized. DNA sequence analysis of a 33-kb region revealed a cluster of 14 open reading frames (ORFs), three of which (ORF4, ORF5, and ORF6) encode type I polyketide synthase (PKS), which consists of four modules. Unusual features of the modular organization is the lack of an obvious acyltransferase domain on modules 2 and 4 and the presence of longer interdomain regions more than 200 amino acids in length on each module. Involvement of the PKS genes in NCZ biosynthesis was demonstrated by heterologous expression of the cluster in Streptomyces coelicolor CH999, which produced the apparent NCZ biosynthetic intermediates dechloroneocarzillin A and dechloroneocarzilin B. Disruption of ORF5 resulted in a failure of NCZ production, providing further evidence that the cluster is essential for NCZ biosynthesis. Mechanistic consideration of NCZ formation indicates the iterative use of at least one module of the PKS, which subsequently releases its product by decarboxylation to generate an NCZ skeleton, possibly catalyzed by a type II thioesterase encoded by ORF7. This is a novel type I PKS system of bacterial origin for the biosynthesis of a reduced polyketide chain. Additionally, the protein encoded by ORF3, located upstream of the PKS genes, closely resembles the FADH2-dependent halogenases involved in the formation of halometabolites. The ORF3 protein could be responsible for the halogenation of NCZs, presenting a unique example of a halogenase involved in the biosynthesis of an aliphatic halometabolite. PMID:15328113

Cellulose biosynthesis: current views and evolving concepts.

PubMed

Saxena, Inder M; Brown, R Malcolm

2005-07-01

To outline the current state of knowledge and discuss the evolution of various viewpoints put forth to explain the mechanism of cellulose biosynthesis. * Understanding the mechanism of cellulose biosynthesis is one of the major challenges in plant biology. The simplicity in the chemical structure of cellulose belies the complexities that are associated with the synthesis and assembly of this polysaccharide. Assembly of cellulose microfibrils in most organisms is visualized as a multi-step process involving a number of proteins with the key protein being the cellulose synthase catalytic sub-unit. Although genes encoding this protein have been identified in almost all cellulose synthesizing organisms, it has been a challenge in general, and more specifically in vascular plants, to demonstrate cellulose synthase activity in vitro. The assembly of glucan chains into cellulose microfibrils of specific dimensions, viewed as a spontaneous process, necessitates the assembly of synthesizing sites unique to most groups of organisms. The steps of polymerization (requiring the specific arrangement and activity of the cellulose synthase catalytic sub-units) and crystallization (directed self-assembly of glucan chains) are certainly interlinked in the formation of cellulose microfibrils. Mutants affected in cellulose biosynthesis have been identified in vascular plants. Studies on these mutants and herbicide-treated plants suggest an interesting link between the steps of polymerization and crystallization during cellulose biosynthesis. * With the identification of a large number of genes encoding cellulose synthases and cellulose synthase-like proteins in vascular plants and the supposed role of a number of other proteins in cellulose biosynthesis, a complete understanding of this process will necessitate a wider variety of research tools and approaches than was thought to be required a few years back.

Biosynthesis of fibronectin by rabbit aorta.

PubMed

Takasaki, I; Chobanian, A V; Brecher, P

1991-09-15

The in vitro interactions between vascular cells and fibronectin have been shown to influence phenotypic expression of both cultured endothelial and smooth muscle cells. To more effectively assess the potential functional role of fibronectin in vivo in modulating vascular phenotypes, we have established methodology for studying fibronectin biosynthesis in the rabbit aorta using aortic rings that are morphologically and functionally intact and metabolically active. Aortic rings were incubated with 35S-labeled methionine in a supplemented physiological salt solution. The tissue was fractionated, and quantitative immunoprecipitation was performed using a polyclonal antibody directed against human plasma fibronectin. Newly synthesized fibronectin was most abundant in the fraction solubilized using 4% sodium dodecyl sulfate and in the incubation medium. In all fractions studied, fibronectin was present predominantly as a dimer with no detectable aggregates of fibronectin. Pulse-chase experiments showed that a substantial amount of newly synthesized fibronectin was found in the 4% sodium dodecyl sulfate extract after only 1 h, suggesting that fibronectin was rapidly incorporated into the extracellular matrix. The more soluble forms of newly synthesized fibronectin appeared to be the precursors for secreted fibronectin, and no precursor-product relationship between soluble and insoluble fibronectin was found. Dissection of aortic rings following incubation with labeled methionine showed that newly synthesized fibronectin was uniformally distributed in both intima-media and media-adventitia segments. Endothelial cell denudation caused only a 20% decrease of fibronectin biosynthesis concomitant with similar changes in total protein biosynthesis, consistent with the medial smooth muscle cell as the major source of newly synthesized fibronectin. Biosynthesis of fibronectin was increased following a 24-h preincubation of the aortic rings, and concomitant increases in steady

Peroxidase enzymes regulate collagen extracellular matrix biosynthesis.

PubMed

DeNichilo, Mark O; Panagopoulos, Vasilios; Rayner, Timothy E; Borowicz, Romana A; Greenwood, John E; Evdokiou, Andreas

2015-05-01

Myeloperoxidase and eosinophil peroxidase are heme-containing enzymes often physically associated with fibrotic tissue and cancer in various organs, without any direct involvement in promoting fibroblast recruitment and extracellular matrix (ECM) biosynthesis at these sites. We report herein novel findings that show peroxidase enzymes possess a well-conserved profibrogenic capacity to stimulate the migration of fibroblastic cells and promote their ability to secrete collagenous proteins to generate a functional ECM both in vitro and in vivo. Mechanistic studies conducted using cultured fibroblasts show that these cells are capable of rapidly binding and internalizing both myeloperoxidase and eosinophil peroxidase. Peroxidase enzymes stimulate collagen biosynthesis at a post-translational level in a prolyl 4-hydroxylase-dependent manner that does not require ascorbic acid. This response was blocked by the irreversible myeloperoxidase inhibitor 4-amino-benzoic acid hydrazide, indicating peroxidase catalytic activity is essential for collagen biosynthesis. These results suggest that peroxidase enzymes, such as myeloperoxidase and eosinophil peroxidase, may play a fundamental role in regulating the recruitment of fibroblast and the biosynthesis of collagen ECM at sites of normal tissue repair and fibrosis, with enormous implications for many disease states where infiltrating inflammatory cells deposit peroxidases. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Biosynthesis, structural architecture and biotechnological potential of bacterial tannase: a molecular advancement.

PubMed

Jana, Arijit; Halder, Suman Kumar; Banerjee, Amrita; Paul, Tanmay; Pati, Bikash Ranjan; Mondal, Keshab Chandra; Das Mohapatra, Pradeep Kumar

2014-04-01

Tannin-rich materials are abundantly generated as wastes from several agroindustrial activities. Therefore, tannase is an interesting hydrolase, for bioconversion of tannin-rich materials into value added products by catalyzing the hydrolysis of ester and depside bonds and unlocked a new prospect in different industrial sectors like food, beverages, pharmaceuticals, etc. Microorganisms, particularly bacteria are one of the major sources of tannase. In the last decade, cloning and heterologous expression of novel tannase genes and structural study has gained momentum. In this article, we have emphasized critically on bacterial tannase that have gained worldwide research interest for their diverse properties. The present paper delineate the developments that have taken place in understanding the role of tannase action, microbial sources, various cultivation aspects, downstream processing, salient biochemical properties, structure and active sites, immobilization, efforts in cloning and overexpression and with special emphasis on recent molecular and biotechnological achievements. Copyright © 2014 Elsevier Ltd. All rights reserved.

Biosynthesis of enediyne antitumor antibiotics.

PubMed

Van Lanen, Steven G; Shen, Ben

2008-01-01

The enediyne polyketides are secondary metabolites isolated from a variety of Actinomycetes. All members share very potent anticancer and antibiotic activity, and prospects for the clinical application of the enediynes has been validated with the recent marketing of two enediyne derivatives as anticancer agents. The biosynthesis of these compounds is of interest because of the numerous structural features that are unique to the enediyne family. The gene cluster for five enediynes has now been cloned and sequenced, providing the foundation to understand natures' means to biosynthesize such complex, exotic molecules. Presented here is a review of the current progress in delineating the biosynthesis of the enediynes with an emphasis on the model enediyne, C-1027.

Co-opting sulphur-carrier proteins from primary metabolic pathways for 2-thiosugar biosynthesis.

PubMed

Sasaki, Eita; Zhang, Xuan; Sun, He G; Lu, Mei-yeh Jade; Liu, Tsung-lin; Ou, Albert; Li, Jeng-yi; Chen, Yu-hsiang; Ealick, Steven E; Liu, Hung-wen

2014-06-19

Sulphur is an essential element for life and is ubiquitous in living systems. Yet how the sulphur atom is incorporated into many sulphur-containing secondary metabolites is poorly understood. For bond formation between carbon and sulphur in primary metabolites, the major ionic sulphur sources are the persulphide and thiocarboxylate groups on sulphur-carrier (donor) proteins. Each group is post-translationally generated through the action of a specific activating enzyme. In all reported bacterial cases, the gene encoding the enzyme that catalyses the carbon-sulphur bond formation reaction and that encoding the cognate sulphur-carrier protein exist in the same gene cluster. To study the production of the 2-thiosugar moiety in BE-7585A, an antibiotic from Amycolatopsis orientalis, we identified a putative 2-thioglucose synthase, BexX, whose protein sequence and mode of action seem similar to those of ThiG, the enzyme that catalyses thiazole formation in thiamine biosynthesis. However, no gene encoding a sulphur-carrier protein could be located in the BE-7585A cluster. Subsequent genome sequencing uncovered a few genes encoding sulphur-carrier proteins that are probably involved in the biosynthesis of primary metabolites but only one activating enzyme gene in the A. orientalis genome. Further experiments showed that this activating enzyme can adenylate each of these sulphur-carrier proteins and probably also catalyses the subsequent thiolation, through its rhodanese domain. A proper combination of these sulphur-delivery systems is effective for BexX-catalysed 2-thioglucose production. The ability of BexX to selectively distinguish sulphur-carrier proteins is given a structural basis using X-ray crystallography. This study is, to our knowledge, the first complete characterization of thiosugar formation in nature and also demonstrates the receptor promiscuity of the A. orientalis sulphur-delivery system. Our results also show that co-opting the sulphur-delivery machinery

Systematic bacterialization of yeast genes identifies a near-universally swappable pathway

PubMed Central

Kachroo, Aashiq H; Laurent, Jon M; Akhmetov, Azat; Szilagyi-Jones, Madelyn; McWhite, Claire D; Zhao, Alice; Marcotte, Edward M

2017-01-01

Eukaryotes and prokaryotes last shared a common ancestor ~2 billion years ago, and while many present-day genes in these lineages predate this divergence, the extent to which these genes still perform their ancestral functions is largely unknown. To test principles governing retention of ancient function, we asked if prokaryotic genes could replace their essential eukaryotic orthologs. We systematically replaced essential genes in yeast by their 1:1 orthologs from Escherichia coli. After accounting for mitochondrial localization and alternative start codons, 31 out of 51 bacterial genes tested (61%) could complement a lethal growth defect and replace their yeast orthologs with minimal effects on growth rate. Replaceability was determined on a pathway-by-pathway basis; codon usage, abundance, and sequence similarity contributed predictive power. The heme biosynthesis pathway was particularly amenable to inter-kingdom exchange, with each yeast enzyme replaceable by its bacterial, human, or plant ortholog, suggesting it as a near-universally swappable pathway. DOI: http://dx.doi.org/10.7554/eLife.25093.001 PMID:28661399

The intrinsic cephalosporin resistome of Listeria monocytogenes in the context of stress response, gene regulation, pathogenesis and therapeutics.

PubMed

Krawczyk-Balska, A; Markiewicz, Z

2016-02-01

Intrinsic resistance to antibiotics is a serious therapeutic problem in the case of many bacterial species. The Gram-positive human pathogen Listeria monocytogenes is intrinsically resistant to broad spectrum cephalosporin antibiotics, which are commonly used in therapy of bacterial infections. Besides three penicillin-binding proteins the intrinsic cephalosporin resistome of L. monocytogenes includes multidrug resistance transporter transporters, proteins involved in peptidoglycan biosynthesis and modification, cell envelope proteins with structural or general detoxification function, cytoplasmic proteins with unknown function and regulatory proteins. Analysis of the regulation of the expression of genes involved in the intrinsic resistance of L. monocytogenes to cephalosporins highlights the high complexity of control of the intrinsic resistance phenotype. The regulation of the transcription of the intrinsic resistome determinants involves the activity of eight regulators, namely LisR, CesR, LiaR, VirR, σ(B) , σ(H) , σ(L) and PrfA, of which the most prominent role play LisR, CesR and σ(B) . Furthermore, the vast majority of the intrinsic resistome determinants contribute to the tolerance of different stress conditions and virulence. A study indicates that O-acetyltransferase OatA is the most promising candidate for co-drug development since an agent targeting OatA should sensitize L. monocytogenes to certain antibiotics, therefore improving the efficacy of listeriosis treatment as well as food preservation measures. © 2015 The Society for Applied Microbiology.

The role of beta-ketoacyl-acyl carrier protein synthase III in the condensation steps of fatty acid biosynthesis in sunflower.

PubMed

González-Mellado, Damián; von Wettstein-Knowles, Penny; Garcés, Rafael; Martínez-Force, Enrique

2010-05-01

The beta-ketoacyl-acyl carrier protein synthase III (KAS III; EC 2.3.1.180) is a condensing enzyme catalyzing the initial step of fatty acid biosynthesis using acetyl-CoA as primer. To determine the mechanisms involved in the biosynthesis of fatty acids in sunflower (Helianthus annuus L.) developing seeds, a cDNA coding for HaKAS III (EF514400) was isolated, cloned and sequenced. Its protein sequence is as much as 72% identical to other KAS III-like ones such as those from Perilla frutescens, Jatropha curcas, Ricinus communis or Cuphea hookeriana. Phylogenetic study of the HaKAS III homologous proteins infers its origin from cyanobacterial ancestors. A genomic DNA gel blot analysis revealed that HaKAS III is a single copy gene. Expression levels of this gene, examined by Q-PCR, revealed higher levels in developing seeds storing oil than in leaves, stems, roots or seedling cotyledons. Heterologous expression of HaKAS III in Escherichia coli altered their fatty acid content and composition implying an interaction of HaKAS III with the bacterial FAS complex. Testing purified HaKAS III recombinant protein by adding to a reconstituted E. coli FAS system lacking condensation activity revealed a novel substrate specificity. In contrast to all hitherto characterized plant KAS IIIs, the activities of which are limited to the first cycles of intraplastidial fatty acid biosynthesis yielding C6 chains, HaKAS III participates in at least four cycles resulting in C10 chains.

Drosophila immunity: analysis of PGRP-SB1 expression, enzymatic activity and function.

PubMed

Zaidman-Rémy, Anna; Poidevin, Mickael; Hervé, Mireille; Welchman, David P; Paredes, Juan C; Fahlander, Carina; Steiner, Hakan; Mengin-Lecreulx, Dominique; Lemaitre, Bruno

2011-02-18

Peptidoglycan is an essential and specific component of the bacterial cell wall and therefore is an ideal recognition signature for the immune system. Peptidoglycan recognition proteins (PGRPs) are conserved from insects to mammals and able to bind PGN (non-catalytic PGRPs) and, in some cases, to efficiently degrade it (catalytic PGRPs). In Drosophila, several non-catalytic PGRPs function as selective peptidoglycan receptors upstream of the Toll and Imd pathways, the two major signalling cascades regulating the systemic production of antimicrobial peptides. Recognition PGRPs specifically activate the Toll pathway in response to Lys-type peptidoglycan found in most Gram-positive bacteria and the Imd pathway in response to DAP-type peptidoglycan encountered in Gram-positive bacilli-type bacteria and in Gram-negative bacteria. Catalytic PGRPs on the other hand can potentially reduce the level of immune activation by scavenging peptidoglycan. In accordance with this, PGRP-LB and PGRP-SC1A/B/2 have been shown to act as negative regulators of the Imd pathway. In this study, we report a biochemical and genetic analysis of PGRP-SB1, a catalytic PGRP. Our data show that PGRP-SB1 is abundantly secreted into the hemolymph following Imd pathway activation in the fat body, and exhibits an enzymatic activity towards DAP-type polymeric peptidoglycan. We have generated a PGRP-SB1/2 null mutant by homologous recombination, but its thorough phenotypic analysis did not reveal any immune function, suggesting a subtle role or redundancy of PGRP-SB1/2 with other molecules. Possible immune functions of PGRP-SB1 are discussed.

Bacterial natural product biosynthetic domain composition in soil correlates with changes in latitude on a continent-wide scale.

PubMed

Lemetre, Christophe; Maniko, Jeffrey; Charlop-Powers, Zachary; Sparrow, Ben; Lowe, Andrew J; Brady, Sean F

2017-10-31

Although bacterial bioactive metabolites have been one of the most prolific sources of lead structures for the development of small-molecule therapeutics, very little is known about the environmental factors associated with changes in secondary metabolism across natural environments. Large-scale sequencing of environmental microbiomes has the potential to shed light on the richness of bacterial biosynthetic diversity hidden in the environment, how it varies from one environment to the next, and what environmental factors correlate with changes in biosynthetic diversity. In this study, the sequencing of PCR amplicons generated using primers targeting either ketosynthase domains from polyketide biosynthesis or adenylation domains from nonribosomal peptide biosynthesis was used to assess biosynthetic domain composition and richness in soils collected across the Australian continent. Using environmental variables collected at each soil site, we looked for environmental factors that correlated with either high overall domain richness or changes in the domain composition. Among the environmental variables we measured, changes in biosynthetic domain composition correlate most closely with changes in latitude and to a lesser extent changes in pH. Although it is unclear at this time the exact mix of factors that may drive the relationship between biosynthetic domain composition and latitude, from a practical perspective the identification of a latitudinal basis for differences in soil metagenome biosynthetic domain compositions should help guide future natural product discovery efforts. Published under the PNAS license.

Nitrate, nitrite and nitric oxide reductases: from the last universal common ancestor to modern bacterial pathogens

PubMed Central

Vázquez-Torres, Andrés; Bäumler, Andreas

2016-01-01

The electrochemical gradient that ensues from the enzymatic activity of cytochromes such as nitrate reductase, nitric oxide reductase, and quinol oxidase contributes to the bioenergetics of the bacterial cell. Reduction of nitrogen oxides by bacterial pathogens can, however, be uncoupled from proton translocation and biosynthesis of ATP or NH4+, but still linked to quinol and NADH oxidation. Ancestral nitric oxide reductases, as well as cytochrome coxidases and quinol bo oxidases evolved from the former, are capable of binding and detoxifying nitric oxide to nitrous oxide. The NO-metabolizing activity associated with these cytochromes can be a sizable source of antinitrosative defense in bacteria during their associations with host cells. Nitrosylation of terminal cytochromes arrests respiration, reprograms bacterial metabolism, stimulates antioxidant defenses and alters antibiotic cytotoxicity. Collectively, the bioenergetics and regulation of redox homeostasis that accompanies the utilization of nitrogen oxides and detoxification of nitric oxide by cytochromes of the electron transport chain increases fitness of many Gram-positive and –negative pathogens during their associations with invertebrate and vertebrate hosts. PMID:26426528

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP.

PubMed

Bickford, Justin S; Nick, Harry S

2013-12-01

Isoprenoid lipid carriers are essential in protein glycosylation and bacterial cell envelope biosynthesis. The enzymes involved in their metabolism (synthases, kinases and phosphatases) are therefore critical to cell viability. In this review, we focus on two broad groups of isoprenoid pyrophosphate phosphatases. One group, containing phosphatidic acid phosphatase motifs, includes the eukaryotic dolichyl pyrophosphate phosphatases and proposed recycling bacterial undecaprenol pyrophosphate phosphatases, PgpB, YbjB and YeiU/LpxT. The second group comprises the bacterial undecaprenol pyrophosphate phosphatase, BacA/UppP, responsible for initial formation of undecaprenyl phosphate, which we predict contains a tyrosine phosphate phosphatase motif resembling that of the tumour suppressor, phosphatase and tensin homologue (PTEN). Based on protein sequence alignments across species and 2D structure predictions, we propose catalytic and lipid recognition motifs unique to BacA/UppP enzymes. The verification of our proposed active-site residues would provide new strategies for the development of substrate-specific inhibitors which mimic both the lipid and pyrophosphate moieties, leading to the development of novel antimicrobial agents.

Amelioration in secretion of hyperthermostable and Ca2+ -independent alpha-amylase of Geobacillus thermoleovorans by some polyamines and their biosynthesis inhibitor methylglyoxal-bis-guanylhydrazone.

PubMed

Uma Maheswar Rao, J L; Satyanarayana, T

2004-01-01

Effect of polyamines and their biosynthesis inhibitors on the production of hyperthermostable and Ca2+ -independent alpha-amylase by Geobacillus thermoleovorans MTCC 4220. The alpha-amylase was produced in starch-yeast extract-tryptone (SYT) broth with different polyamines (PA) and polyamine biosynthesis inhibitors, methylglyoxal-bis-guanylhydrazone (MGBG) and cyclohexylammonium sulphate (CHA) at 70 degrees C. The bacterial pellets were obtained after growing G. thermoleovorans at different temperatures, and used in determining total PA. The cell-free culture filtrates were used in alpha-amylase assays. During growth, total polyamines in biomass increased till 2 h, and thereafter, decreased gradually. The total polyamine content was very high in the biomass cultivated at 55 degrees C when compared with that of higher temperatures. Enzyme titre enhanced up to 70 degrees C, and thereafter declined. Extracellular enzyme and protein levels declined in the presence of exogenously added PA. The intracellular enzyme titres, however, were higher in putrescine (put) and spermidine (spd) than in spermine (spm). Polyamine biosynthesis inhibitor, MGBG enhanced secretion of alpha-amylase in a laboratory fermentor as well as shake flasks, although CHA did not affect it. The intracellular accumulation of put in the presence of MGBG appeared to enhance synthesis and secretion of alpha-amylase. Extracellular enzyme and protein levels were low in the presence of exogenously added PA, but their intracellular levels, however, were higher in put and spd than in spm. A substantial increase in the synthesis and secretion of alpha-amylase was attained in G. thermoleovorans in the presence of polyamine biosynthesis inhibitor MGBG.

Chirality and protein biosynthesis.

PubMed

Banik, Sindrila Dutta; Nandi, Nilashis

2013-01-01

Chirality is present at all levels of structural hierarchy of protein and plays a significant role in protein biosynthesis. The macromolecules involved in protein biosynthesis such as aminoacyl tRNA synthetase and ribosome have chiral subunits. Despite the omnipresence of chirality in the biosynthetic pathway, its origin, role in current pathway, and importance is far from understood. In this review we first present an introduction to biochirality and its relevance to protein biosynthesis. Major propositions about the prebiotic origin of biomolecules are presented with particular reference to proteins and nucleic acids. The problem of the origin of homochirality is unresolved at present. The chiral discrimination by enzymes involved in protein synthesis is essential for keeping the life process going. However, questions remained pertaining to the mechanism of chiral discrimination and concomitant retention of biochirality. We discuss the experimental evidence which shows that it is virtually impossible to incorporate D-amino acids in protein structures in present biosynthetic pathways via any of the two major steps of protein synthesis, namely aminoacylation and peptide bond formation reactions. Molecular level explanations of the stringent chiral specificity in each step are extended based on computational analysis. A detailed account of the current state of understanding of the mechanism of chiral discrimination during aminoacylation in the active site of aminoacyl tRNA synthetase and peptide bond formation in ribosomal peptidyl transferase center is presented. Finally, it is pointed out that the understanding of the mechanism of retention of enantiopurity has implications in developing novel enzyme mimetic systems and biocatalysts and might be useful in chiral drug design.

The expanding universe of alkaloid biosynthesis.

PubMed

De Luca, V; Laflamme, P

2001-06-01

Characterization of many of the major gene families responsible for the generation of central intermediates and for their decoration, together with the development of large genomics and proteomics databases, has revolutionized our capability to identify exotic and interesting natural-product pathways. Over the next few years, these tools will facilitate dramatic advances in our knowledge of the biosynthesis of alkaloids, which will far surpass that which we have learned in the past 50 years. These tools will also be exploited for the rapid characterization of regulatory genes, which control the development of specialized cell factories for alkaloid biosynthesis.

Cellulose Biosynthesis: Current Views and Evolving Concepts

PubMed Central

SAXENA, INDER M.; BROWN, R. MALCOLM

2005-01-01

• Aims To outline the current state of knowledge and discuss the evolution of various viewpoints put forth to explain the mechanism of cellulose biosynthesis. • Scope Understanding the mechanism of cellulose biosynthesis is one of the major challenges in plant biology. The simplicity in the chemical structure of cellulose belies the complexities that are associated with the synthesis and assembly of this polysaccharide. Assembly of cellulose microfibrils in most organisms is visualized as a multi-step process involving a number of proteins with the key protein being the cellulose synthase catalytic sub-unit. Although genes encoding this protein have been identified in almost all cellulose synthesizing organisms, it has been a challenge in general, and more specifically in vascular plants, to demonstrate cellulose synthase activity in vitro. The assembly of glucan chains into cellulose microfibrils of specific dimensions, viewed as a spontaneous process, necessitates the assembly of synthesizing sites unique to most groups of organisms. The steps of polymerization (requiring the specific arrangement and activity of the cellulose synthase catalytic sub-units) and crystallization (directed self-assembly of glucan chains) are certainly interlinked in the formation of cellulose microfibrils. Mutants affected in cellulose biosynthesis have been identified in vascular plants. Studies on these mutants and herbicide-treated plants suggest an interesting link between the steps of polymerization and crystallization during cellulose biosynthesis. • Conclusions With the identification of a large number of genes encoding cellulose synthases and cellulose synthase-like proteins in vascular plants and the supposed role of a number of other proteins in cellulose biosynthesis, a complete understanding of this process will necessitate a wider variety of research tools and approaches than was thought to be required a few years back. PMID:15894551

Roles of lignin biosynthesis and regulatory genes in plant development

PubMed Central

Yoon, Jinmi; Choi, Heebak

2015-01-01

Abstract Lignin is an important factor affecting agricultural traits, biofuel production, and the pulping industry. Most lignin biosynthesis genes and their regulatory genes are expressed mainly in the vascular bundles of stems and leaves, preferentially in tissues undergoing lignification. Other genes are poorly expressed during normal stages of development, but are strongly induced by abiotic or biotic stresses. Some are expressed in non‐lignifying tissues such as the shoot apical meristem. Alterations in lignin levels affect plant development. Suppression of lignin biosynthesis genes causes abnormal phenotypes such as collapsed xylem, bending stems, and growth retardation. The loss of expression by genes that function early in the lignin biosynthesis pathway results in more severe developmental phenotypes when compared with plants that have mutations in later genes. Defective lignin deposition is also associated with phenotypes of seed shattering or brittle culm. MYB and NAC transcriptional factors function as switches, and some homeobox proteins negatively control lignin biosynthesis genes. Ectopic deposition caused by overexpression of lignin biosynthesis genes or master switch genes induces curly leaf formation and dwarfism. PMID:26297385

Biosynthesis and molecular genetics of polyketides in marine dinoflagellates.

PubMed

Kellmann, Ralf; Stüken, Anke; Orr, Russell J S; Svendsen, Helene M; Jakobsen, Kjetill S

2010-03-31

Marine dinoflagellates are the single most important group of algae that produce toxins, which have a global impact on human activities. The toxins are chemically diverse, and include macrolides, cyclic polyethers, spirolides and purine alkaloids. Whereas there is a multitude of studies describing the pharmacology of these toxins, there is limited or no knowledge regarding the biochemistry and molecular genetics involved in their biosynthesis. Recently, however, exciting advances have been made. Expressed sequence tag sequencing studies have revealed important insights into the transcriptomes of dinoflagellates, whereas other studies have implicated polyketide synthase genes in the biosynthesis of cyclic polyether toxins, and the molecular genetic basis for the biosynthesis of paralytic shellfish toxins has been elucidated in cyanobacteria. This review summarises the recent progress that has been made regarding the unusual genomes of dinoflagellates, the biosynthesis and molecular genetics of dinoflagellate toxins. In addition, the evolution of these metabolic pathways will be discussed, and an outlook for future research and possible applications is provided.

Biosynthesis and Molecular Genetics of Polyketides in Marine Dinoflagellates

PubMed Central

Kellmann, Ralf; Stüken, Anke; Orr, Russell J. S.; Svendsen, Helene M.; Jakobsen, Kjetill S.

2010-01-01

Marine dinoflagellates are the single most important group of algae that produce toxins, which have a global impact on human activities. The toxins are chemically diverse, and include macrolides, cyclic polyethers, spirolides and purine alkaloids. Whereas there is a multitude of studies describing the pharmacology of these toxins, there is limited or no knowledge regarding the biochemistry and molecular genetics involved in their biosynthesis. Recently, however, exciting advances have been made. Expressed sequence tag sequencing studies have revealed important insights into the transcriptomes of dinoflagellates, whereas other studies have implicated polyketide synthase genes in the biosynthesis of cyclic polyether toxins, and the molecular genetic basis for the biosynthesis of paralytic shellfish toxins has been elucidated in cyanobacteria. This review summarises the recent progress that has been made regarding the unusual genomes of dinoflagellates, the biosynthesis and molecular genetics of dinoflagellate toxins. In addition, the evolution of these metabolic pathways will be discussed, and an outlook for future research and possible applications is provided. PMID:20479965

Preservation of protein globules and peptidoglycan in the mineralized cell wall of nitrate-reducing, iron(II)-oxidizing bacteria: a cryo-electron microscopy study.

PubMed

Miot, J; Maclellan, K; Benzerara, K; Boisset, N

2011-11-01

Iron-oxidizing bacteria are important actors of the geochemical cycle of iron in modern environments and may have played a key role all over Earth's history. However, in order to better assess that role on the modern and the past Earth, there is a need for better understanding the mechanisms of bacterial iron oxidation and for defining potential biosignatures to be looked for in the geologic record. In this study, we investigated experimentally and at the nanometre scale the mineralization of iron-oxidizing bacteria with a combination of synchrotron-based scanning transmission X-ray microscopy (STXM), scanning transmission electron microscopy (STEM) and cryo-transmission electron microscopy (cryo-TEM). We show that the use of cryo-TEM instead of conventional microscopy provides detailed information of the successive iron biomineralization stages in anaerobic nitrate-reducing iron-oxidizing bacteria. These results suggest the existence of preferential Fe-binding and Fe-oxidizing sites on the outer face of the plasma membrane leading to the nucleation and growth of Fe minerals within the periplasm of these cells that eventually become completely encrusted. In contrast, the septa of dividing cells remain nonmineralized. In addition, the use of cryo-TEM offers a detailed view of the exceptional preservation of protein globules and the peptidoglycan within the Fe-mineralized cell walls of these bacteria. These organic molecules and ultrastructural details might be protected from further degradation by entrapment in the mineral matrix down to the nanometre scale. This is discussed in the light of previous studies on the properties of Fe-organic interactions and more generally on the fossilization of mineral-organic assemblies. © 2011 Blackwell Publishing Ltd.

Peptide Epimerization Machineries Found in Microorganisms.

PubMed

Ogasawara, Yasushi; Dairi, Tohru

2018-01-01

D-Amino acid residues have been identified in peptides from a variety of eukaryotes and prokaryotes. In microorganisms, UDP- N -acetylmuramic acid pentapeptide (UDP-MurNAc-L-Ala-D-Glu-meso-diaminopimelate-D-Ala-D-Ala), a unit of peptidoglycan, is a representative. During its biosynthesis, D-Ala and D-Glu are generally supplied by racemases from the corresponding isomers. However, we recently identified a unique unidirectional L-Glu epimerase catalyzing the epimerization of the terminal L-Glu of UDP-MurNAc-L-Ala-L-Glu. Several such enzymes, introducing D-amino acid resides into peptides via epimerization, have been reported to date. This includes a L-Ala-D/L-Glu epimerase, which is possibly used during peptidoglycan degradation. In bacterial primary metabolisms, to the best of our knowledge, these two machineries are the only examples of peptide epimerization. However, a variety of peptides containing D-amino acid residues have been isolated from microorganisms as secondary metabolites. Their biosynthetic mechanisms have been studied and three different peptide epimerization machineries have been reported. The first is non-ribosomal peptide synthetase (NRPS). Excellent studies with dissected modules of gramicidin synthetase and tyrocidine synthetase revealed the reactions of the epimerization domains embedded in the enzymes. The obtained information is still utilized to predict epimerization domains in uncharacterized NRPSs. The second includes the biosynthetic enzymes of lantibiotics, which are ribosome-dependently supplied peptide antibiotics containing polycyclic thioether amino acids (lanthionines). A mechanism for the formation of the D-Ala moiety in lanthionine by two enzymes, dehydratases catalyzing the conversion of L-Ser into dehydroalanine and enzymes catalyzing nucleophilic attack of the thiol of cysteine into dehydroalanine, was clarified. Similarly, the formation of a D-Ala residue by reduction of the dehydroalanine residue was also reported. The last

In vitro biosynthesis of a universal t6A tRNA modification in Archaea and Eukarya

PubMed Central

Perrochia, Ludovic; Crozat, Estelle; Hecker, Arnaud; Zhang, Wenhua; Bareille, Joseph; Collinet, Bruno; van Tilbeurgh, Herman; Forterre, Patrick

2013-01-01

N6-threonylcarbamoyladenosine (t6A) is a modified nucleotide found in all transfer RNAs (tRNAs) decoding codons starting with adenosine. Its role is to facilitate codon–anticodon pairing and to prevent frameshifting during protein synthesis. Genetic studies demonstrated that two universal proteins, Kae1/YgjD and Sua5/YrdC, are necessary for t6A synthesis in Saccharomyces cerevisiae and Escherichia coli. In Archaea and Eukarya, Kae1 is part of a conserved protein complex named kinase, endopeptidase and other proteins of small size (KEOPS), together with three proteins that have no bacterial homologues. Here, we reconstituted for the first time an in vitro system for t6A modification in Archaea and Eukarya, using purified KEOPS and Sua5. We demonstrated binding of tRNAs to archaeal KEOPS and detected two distinct adenosine triphosphate (ATP)-dependent steps occurring in the course of the synthesis. Our data, together with recent reconstitution of an in vitro bacterial system, indicated that t6A cannot be catalysed by Sua5/YrdC and Kae1/YgjD alone but requires accessory proteins that are not universal. Remarkably, we observed interdomain complementation when bacterial, archaeal and eukaryotic proteins were combined in vitro, suggesting a conserved catalytic mechanism for the biosynthesis of t6A in nature. These findings shed light on the reaction mechanism of t6A synthesis and evolution of molecular systems that promote translation fidelity in present-day cells. PMID:23258706

YCZ-18 Is a New Brassinosteroid Biosynthesis Inhibitor

PubMed Central

Oh, Keimei; Matsumoto, Tadashi; Yamagami, Ayumi; Ogawa, Atushi; Yamada, Kazuhiro; Suzuki, Ryuichiro; Sawada, Takayuki; Fujioka, Shozo; Yoshizawa, Yuko; Nakano, Takeshi

2015-01-01

Plant hormone brassinosteroids (BRs) are a group of polyhydroxylated steroids that play critical roles in regulating broad aspects of plant growth and development. The structural diversity of BRs is generated by the action of several groups of P450s. Brassinazole is a specific inhibitor of C-22 hydroxylase (CYP90B1) in BR biosynthesis, and the application use of brassinazole has emerged as an effective way of complementing BR-deficient mutants to elucidate the functions of BRs. In this article, we report a new triazole-type BR biosynthesis inhibitor, YCZ-18. Quantitative analysis the endogenous levels of BRs in Arabidopsis indicated that YCZ-18 significantly decreased the BR contents in plant tissues. Assessment of the binding affinity of YCZ-18to purified recombinant CYP90D1 indicated that YCZ-18 induced a typical type II binding spectrum with a Kd value of approximately 0.79 μM. Analysis of the mechanisms underlying the dwarf phenotype associated with YCZ-18 treatment of Arabidopsis indicated that the chemically induced dwarf phenotype was caused by a failure of cell elongation. Moreover, dissecting the effect of YCZ-18 on the induction or down regulation of genes responsive to BRs indicated that YCZ-18 regulated the expression of genes responsible for BRs deficiency in Arabidopsis. These findings indicate that YCZ-18 is a potent BR biosynthesis inhibitor and has a new target site, C23-hydroxylation in BR biosynthesis. Application of YCZ-18 will be a good starting point for further elucidation of the detailed mechanism of BR biosynthesis and its regulation. PMID:25793645

Two tomato GDP-D-mannose epimerase isoforms involved in ascorbate biosynthesis play specific roles in cell wall biosynthesis and development.

PubMed

Mounet-Gilbert, Louise; Dumont, Marie; Ferrand, Carine; Bournonville, Céline; Monier, Antoine; Jorly, Joana; Lemaire-Chamley, Martine; Mori, Kentaro; Atienza, Isabelle; Hernould, Michel; Stevens, Rebecca; Lehner, Arnaud; Mollet, Jean Claude; Rothan, Christophe; Lerouge, Patrice; Baldet, Pierre

2016-08-01

GDP-D-mannose epimerase (GME, EC 5.1.3.18) converts GDP-D-mannose to GDP-L-galactose, and is considered to be a central enzyme connecting the major ascorbate biosynthesis pathway to primary cell wall metabolism in higher plants. Our previous work demonstrated that GME is crucial for both ascorbate and cell wall biosynthesis in tomato. The aim of the present study was to investigate the respective role in ascorbate and cell wall biosynthesis of the two SlGME genes present in tomato by targeting each of them through an RNAi-silencing approach. Taken individually SlGME1 and SlGME2 allowed normal ascorbate accumulation in the leaf and fruits, thus suggesting the same function regarding ascorbate. However, SlGME1 and SlGME2 were shown to play distinct roles in cell wall biosynthesis, depending on the tissue considered. The RNAi-SlGME1 plants harbored small and poorly seeded fruits resulting from alterations of pollen development and of pollination process. In contrast, the RNAi-SlGME2 plants exhibited vegetative growth delay while fruits remained unaffected. Analysis of SlGME1- and SlGME2-silenced seeds and seedlings further showed that the dimerization state of pectin rhamnogalacturonan-II (RG-II) was altered only in the RNAi-SlGME2 lines. Taken together with the preferential expression of each SlGME gene in different tomato tissues, these results suggest sub-functionalization of SlGME1 and SlGME2 and their specialization for cell wall biosynthesis in specific tomato tissues. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Hemosomegenesis and hemoglobin biosynthesis in vertebrates.

PubMed

Brunner Júnior, A; de Rizzo, E; Morena, D D; Cianciarullo, A M; Jared, C; Morena, P

1992-08-01

1. Ultrastructural observations on maturing rabbit embryo erythroid cells led to the finding of hemoglobinized organelles distinguishable from mitochondria due to their highly dense matrix, two or three longitudinally arranged double lamellae, and smaller diameters. Intraorganellar 50-60 A particles identical to those contained in the hemoglobinized cytoplasm were found. 2. Their hemoglobin (Hb) content was demonstrated by electrophoresis of the concentrated supernatant from the isolated, washed, and osmotically lysed organellar fraction. We have proposed that these organelles are the sites for heme integration into the globin (G) polypeptide chains and subunits assembly. The term hemosome has been suggested for such entities. 3. This hypothesis has been sustained by several analytical and experimental works based on the postulation that hemosomes should be found at higher frequencies where the Hb biosynthesis rate is more intensive, or where the induction of this biosynthesis is always dependent on the formation of hemosomes. 4. Maturing erythroid cells of the circulating embryo blood contain hemosomes in higher frequency than in liver erythroid cells, coinciding with the higher Hb biosynthesis rate in peripheral blood than in the liver. In bleeding anemia, the decay of Hb concentration parallels the reduction of the mean number of hemosomes per reticulocyte, in comparison with normal reticulocytes. 5. In HeLa cells and epithelial cultured cells induced to synthesize Hb, it was shown that this biosynthesis is ever concomitant with the formation of hemosomes and depends on the presence of erythropoietin, as occurs in erythroid cells. 6. Studies on hemosomegenesis and Hb biosynthesis experimentally effected in epithelial cultured cells, allowed the interpretation of the sequence of events leading to hemosome formation in maturing erythroid cells. Simultaneously with iron uptake, mitochondria differentiate to lamellated bodies and, successively, expansions rise for

PLANT VOLATILES. Biosynthesis of monoterpene scent compounds in roses.

PubMed

Magnard, Jean-Louis; Roccia, Aymeric; Caissard, Jean-Claude; Vergne, Philippe; Sun, Pulu; Hecquet, Romain; Dubois, Annick; Hibrand-Saint Oyant, Laurence; Jullien, Frédéric; Nicolè, Florence; Raymond, Olivier; Huguet, Stéphanie; Baltenweck, Raymonde; Meyer, Sophie; Claudel, Patricia; Jeauffre, Julien; Rohmer, Michel; Foucher, Fabrice; Hugueney, Philippe; Bendahmane, Mohammed; Baudino, Sylvie

2015-07-03

The scent of roses (Rosa x hybrida) is composed of hundreds of volatile molecules. Monoterpenes represent up to 70% percent of the scent content in some cultivars, such as the Papa Meilland rose. Monoterpene biosynthesis in plants relies on plastid-localized terpene synthases. Combining transcriptomic and genetic approaches, we show that the Nudix hydrolase RhNUDX1, localized in the cytoplasm, is part of a pathway for the biosynthesis of free monoterpene alcohols that contribute to fragrance in roses. The RhNUDX1 protein shows geranyl diphosphate diphosphohydrolase activity in vitro and supports geraniol biosynthesis in planta. Copyright © 2015, American Association for the Advancement of Science.

Novel drug targets in cell wall biosynthesis exploited by gene disruption in Pseudomonas aeruginosa.

PubMed

Elamin, Ayssar A; Steinicke, Susanne; Oehlmann, Wulf; Braun, Yvonne; Wanas, Hanaa; Shuralev, Eduard A; Huck, Carmen; Maringer, Marko; Rohde, Manfred; Singh, Mahavir

2017-01-01

For clinicians, Pseudomonas aeruginosa is a nightmare pathogen that is one of the top three causes of opportunistic human infections. Therapy of P. aeruginosa infections is complicated due to its natural high intrinsic resistance to antibiotics. Active efflux and decreased uptake of drugs due to cell wall/membrane permeability appear to be important issues in the acquired antibiotic tolerance mechanisms. Bacterial cell wall biosynthesis enzymes have been shown to be essential for pathogenicity of Gram-negative bacteria. However, the role of these targets in virulence has not been identified in P. aeruginosa. Here, we report knockout (k.o) mutants of six cell wall biosynthesis targets (murA, PA4450; murD, PA4414; murF, PA4416; ppiB, PA1793; rmlA, PA5163; waaA, PA4988) in P. aeruginosa PAO1, and characterized these in order to find out whether these genes and their products contribute to pathogenicity and virulence of P. aeruginosa. Except waaA k.o, deletion of cell wall biosynthesis targets significantly reduced growth rate in minimal medium compared to the parent strain. The k.o mutants showed exciting changes in cell morphology and colonial architectures. Remarkably, ΔmurF cells became grossly enlarged. Moreover, the mutants were also attenuated in vivo in a mouse infection model except ΔmurF and ΔwaaA and proved to be more sensitive to macrophage-mediated killing than the wild-type strain. Interestingly, the deletion of the murA gene resulted in loss of virulence activity in mice, and the virulence was restored in a plant model by unknown mechanism. This study demonstrates that cell wall targets contribute significantly to intracellular survival, in vivo growth, and pathogenesis of P. aeruginosa. In conclusion, these findings establish a link between cell wall targets and virulence of P. aeruginosa and thus may lead to development of novel drugs for the treatment of P. aeruginosa infection.

A Biotin Biosynthesis Gene Restricted to Helicobacter

PubMed Central

Bi, Hongkai; Zhu, Lei; Jia, Jia; Cronan, John E.

2016-01-01

In most bacteria the last step in synthesis of the pimelate moiety of biotin is cleavage of the ester bond of pimeloyl-acyl carrier protein (ACP) methyl ester. The paradigm cleavage enzyme is Escherichia coli BioH which together with the BioC methyltransferase allows synthesis of the pimelate moiety by a modified fatty acid biosynthetic pathway. Analyses of the extant bacterial genomes showed that bioH is absent from many bioC-containing bacteria and is replaced by other genes. Helicobacter pylori lacks a gene encoding a homologue of the known pimeloyl-ACP methyl ester cleavage enzymes suggesting that it encodes a novel enzyme that cleaves this intermediate. We isolated the H. pylori gene encoding this enzyme, bioV, by complementation of an E. coli bioH deletion strain. Purified BioV cleaved the physiological substrate, pimeloyl-ACP methyl ester to pimeloyl-ACP by use of a catalytic triad, each member of which was essential for activity. The role of BioV in biotin biosynthesis was demonstrated using a reconstituted in vitro desthiobiotin synthesis system. BioV homologues seem the sole pimeloyl-ACP methyl ester esterase present in the Helicobacter species and their occurrence only in H. pylori and close relatives provide a target for development of drugs to specifically treat Helicobacter infections. PMID:26868423

Coordination of peptidoglycan synthesis and outer membrane constriction during Escherichia coli cell division

PubMed Central

Gray, Andrew N; Egan, Alexander JF; van't Veer, Inge L; Verheul, Jolanda; Colavin, Alexandre; Koumoutsi, Alexandra; Biboy, Jacob; Altelaar, A F Maarten; Damen, Mirjam J; Huang, Kerwyn Casey; Simorre, Jean-Pierre; Breukink, Eefjan; den Blaauwen, Tanneke; Typas, Athanasios; Gross, Carol A; Vollmer, Waldemar

2015-01-01

To maintain cellular structure and integrity during division, Gram-negative bacteria must carefully coordinate constriction of a tripartite cell envelope of inner membrane, peptidoglycan (PG), and outer membrane (OM). It has remained enigmatic how this is accomplished. Here, we show that envelope machines facilitating septal PG synthesis (PBP1B-LpoB complex) and OM constriction (Tol system) are physically and functionally coordinated via YbgF, renamed CpoB (Coordinator of PG synthesis and OM constriction, associated with PBP1B). CpoB localizes to the septum concurrent with PBP1B-LpoB and Tol at the onset of constriction, interacts with both complexes, and regulates PBP1B activity in response to Tol energy state. This coordination links PG synthesis with OM invagination and imparts a unique mode of bifunctional PG synthase regulation by selectively modulating PBP1B cross-linking activity. Coordination of the PBP1B and Tol machines by CpoB contributes to effective PBP1B function in vivo and maintenance of cell envelope integrity during division. DOI: http://dx.doi.org/10.7554/eLife.07118.001 PMID:25951518

Alcohol, Intestinal Bacterial Growth, Intestinal Permeability to Endotoxin, and Medical Consequences

PubMed Central

Purohit, Vishnudutt; Bode, J. Christian; Bode, Christiane; Brenner, David A.; Choudhry, Mashkoor A.; Hamilton, Frank; Kang, Y. James; Keshavarzian, Ali; Rao, Radhakrishna; Sartor, R. Balfour; Swanson, Christine; Turner, Jerrold R.

2008-01-01

This report is a summary of the symposium on Alcohol, Intestinal Bacterial Growth, Intestinal Permeability to Endotoxin, and Medical Consequences, organized by National Institute on Alcohol Abuse and Alcoholism, Office of Dietary Supplements, and National Institute of Diabetes and Digestive and Kidney Diseases of National Institutes of Health in Rockville, Maryland, October 11, 2006. Alcohol exposure can promote the growth of Gram negative bacteria in the intestine which may result in accumulation of endotoxin. In addition, alcohol metabolism by Gram negative bacteria and intestinal epithelial cells can result in accumulation of acetaldehyde, which in turn can increase intestinal permeability to endotoxin by increasing tyrosine phosphorylation of tight junction and adherens junction proteins. Alcohol-induced generation of nitric oxide may also contribute to increased permeability to endotoxin by reacting with tubulin, which may cause damage to microtubule cytoskeleton and subsequent disruption of intestinal barrier function. Increased intestinal permeability can lead to increased transfer of endotoxin from the intestine to the liver and general circulation where endotoxin may trigger inflammatory changes in the liver and other organs. Alcohol may also increase intestinal permeability to peptidoglycan which can initiate inflammatory response in liver and other organs. In addition, acute alcohol exposure may potentiate the effect of burn injury on intestinal bacterial growth and permeability. Decreasing the number of Gram negative bacteria in the intestine can result in decreased production of endotoxin as well as acetaldehyde which is expected to decrease intestinal permeability to endotoxin. In addition, intestinal permeability may be preserved by administering epidermal growth factor, L-glutamine, oats supplementation, or zinc thereby preventing the transfer of endotoxin to the general circulation. Thus reducing the number of intestinal Gram negative bacteria and

Nucleoside antibiotics: biosynthesis, regulation, and biotechnology.

PubMed

Niu, Guoqing; Tan, Huarong

2015-02-01

The alarming rise in antibiotic-resistant pathogens has coincided with a decline in the supply of new antibiotics. It is therefore of great importance to find and create new antibiotics. Nucleoside antibiotics are a large family of natural products with diverse biological functions. Their biosynthesis is a complex process through multistep enzymatic reactions and is subject to hierarchical regulation. Genetic and biochemical studies of the biosynthetic machinery have provided the basis for pathway engineering and combinatorial biosynthesis to create new or hybrid nucleoside antibiotics. Dissection of regulatory mechanisms is leading to strategies to increase the titer of bioactive nucleoside antibiotics. Copyright © 2014. Published by Elsevier Ltd.

Functional Analysis of PDX2 from Arabidopsis, a Glutaminase Involved in Vitamin B6 Biosynthesis1[W][OA

PubMed Central

Tambasco-Studart, Marina; Tews, Ivo; Amrhein, Nikolaus; Fitzpatrick, Teresa B.

2007-01-01

Vitamin B6 is an essential metabolite in all organisms, being required as a cofactor for a wide variety of biochemical reactions. De novo biosynthesis of the vitamin occurs in microorganisms and plants, but animals must obtain it from their diet. Two distinct and mutually exclusive de novo pathways have been identified to date, namely deoxyxylulose 5-phosphate dependent, which is restricted to a subset of eubacteria, and deoxyxylulose 5-phosphate independent, present in archaea, fungi, plants, protista, and most eubacteria. In these organisms, pyridoxal 5′-phosphate (PLP) formation is catalyzed by a single glutamine amidotransferase (PLP synthase) composed of a glutaminase domain, PDX2, and a synthase domain, PDX1. Despite plants being an important source of vitamin B6, very little is known about its biosynthesis. Here, we provide information for Arabidopsis thaliana. The functionality of PDX2 is demonstrated, using both in vitro and in vivo analyses. The expression pattern of PDX2 is assessed at both the RNA and protein level, providing insight into the spatial and temporal pattern of vitamin B6 biosynthesis. We then provide a detailed biochemical analysis of the plant PLP synthase complex. While the active sites of PDX1 and PDX2 are remote from each other, coordination of catalysis is much more pronounced with the plant proteins than its bacterial counterpart, Bacillus subtilis. Based on a model of the PDX1/PDX2 complex, mutation of a single residue uncouples enzyme coordination and in turn provides tangible evidence for the existence of the recently proposed ammonia tunnel through the core of PDX1. PMID:17468224

Diverse Mechanisms of Sulfur Decoration in Bacterial tRNA and Their Cellular Functions

PubMed Central

Zheng, Chenkang; Black, Katherine A.; Dos Santos, Patricia C.

2017-01-01

Sulfur-containing transfer ribonucleic acids (tRNAs) are ubiquitous biomolecules found in all organisms that possess a variety of functions. For decades, their roles in processes such as translation, structural stability, and cellular protection have been elucidated and appreciated. These thionucleosides are found in all types of bacteria; however, their biosynthetic pathways are distinct among different groups of bacteria. Considering that many of the thio-tRNA biosynthetic enzymes are absent in Gram-positive bacteria, recent studies have addressed how sulfur trafficking is regulated in these prokaryotic species. Interestingly, a novel proposal has been given for interplay among thionucleosides and the biosynthesis of other thiocofactors, through participation of shared-enzyme intermediates, the functions of which are impacted by the availability of substrate as well as metabolic demand of thiocofactors. This review describes the occurrence of thio-modifications in bacterial tRNA and current methods for detection of these modifications that have enabled studies on the biosynthesis and functions of S-containing tRNA across bacteria. It provides insight into potential modes of regulation and potential evolutionary events responsible for divergence in sulfur metabolism among prokaryotes. PMID:28327539

Bacillus megaterium sporal peptidoglycan synthesis studied by high-resolution autoradiography.

PubMed

Frehel, C; Ryter, A

1980-11-01

Cells of a Dap- Lys- mutant strain of Bacillus megaterium were pulse labeled with [3H]diaminopimelic acid at different times of growth and sporulation. They were processed for radioactivity measurements and high-resolution autoradiography either just after the pulse or after a chase in a nonradioactive medium until refractile forespores started to appear at time (t)4,5. In the pulse-labeled cells, autoradiographs and radioactivity measurements showed that the radioactivity incorporated during a pulse decreased abruptly after t0 and stayed at a low level until t5, although the forespore wall and cortex were formed between t4 and t5. In the pulse-chased bacteria, the acid-insoluble radioactivity, as well as the number of silver grains on autoradiographs, increased during the chase in cells labeled at t1 to t2, whereas it decreased in those labeled before t0. Furthermore, analysis of silver grain distribution showed that, in stage IV bacteria, grains were distributed at the outside of the forespore, mostly on the sporangium cell wall, when pulse-labeling occurred before or at t0; they were located along the cortex and in the forespore cytoplasm when labeling was made at t1 or t2. These facts show that [3H]diaminopimelic acid necessary for spore envelope synthesis was incorporated before their morphological appearance. Free or small diaminopimelic acid precursors entered the sporangium between t1 and t2. The appearance of silver grains in the forespore cytoplasm suggests that the forespore is implicated in sporal peptidoglycan synthesis.

Investigating the Biosynthesis of Membrane-spanning Lipids Using Model Strains of Acidobacteria

NASA Astrophysics Data System (ADS)

Bradley, A. S.; Chubiz, L. M.

2016-12-01

Glycerol dialkyl glycerol tetraethers (GDGTs), deriving from the membrane-spanning lipids of microbes, are detected in a wide range of environments including marine and lacustrine waters, sediments, and in terrestrial soils. In sediments and soils, ratios of various GDGT structures form the basis of the TEX86 proxy based on isoprenoidal GDGTs derived from archaea, and the MBT/CBT proxy based on bacterial-derived branched GDGTs (brGDGTs), which is influenced by both temperature and pH. While the relationships of the proxy values to environmental variables have been empirically calibrated, much uncertainty remains in understanding genetic and physiological factors that affect the production of these lipid structures by microbes. In this study we compare two model bacterial strains - Edaphobacter aggregans WGB-1 , which has been previously demonstrated to produce brGDGTs (Damsté et al 2011) and Edaphobacter modestus JBG-1 (a non-brGDGT producer) to gain traction into understanding brGDGT production. We have sequenced each genome, facilitating comparisons that can be used to computationally generate hypotheses for genes involved in brGDGT biosynthesis. We will also report the results of initial experiments conducted to understand how the lipid profiles of each strain vary as a function of growth phase. Through a combination of genetic approaches and physiolotical experiments, we aim to bring new understanding to brGDGTs and how proxies derived from these lipids relate to environmental variables. Damsté et al. 2011 AEM 77: 4147

Valorization of lubricant-based wastewater for bacterial neutral lipids production: Growth-linked biosynthesis.

PubMed

Da Silva, Pedro D M P; Lima, Filipa; Alves, Maria Madalena; Bijmans, Martijn F M; Pereira, Maria Alcina

2016-09-15

Lipids produced by microorganisms are currently of great interest as raw material for either biofuels or oleochemicals production. Significant biosynthesis of neutral lipids, such as triacylglycerol (TAG) and wax esters (WE) are thought to be limited to a few strains. Hydrocarbonoclastic bacteria (HCB), key players in bioremediation of hydrocarbon contaminated ecosystems, are among this group of strains. Hydrocarbon rich wastewaters have been overlooked concerning their potential as raw material for microbial lipids production. In this study, lubricant-based wastewater was fed, as sole carbon source, to two HCB representative wild strains: Alcanivorax borkumensis SK2, and Rhodococcus opacus PD630. Neutral lipid production was observed with both strains cultivated under uncontrolled conditions of pH and dissolved oxygen. A. borkumensis SK2 was further investigated in a pH- and OD-controlled fermenter. Different phases were assessed separately in terms of lipids production and alkanes removal. The maximum TAG production rate occurred during stationary phase (4 mg-TAG/L h). The maximum production rate of WE-like compounds was 15 mg/L h, and was observed during exponential growth phase. Hydrocarbons removal was 97% of the gas chromatography (GC) resolved straight-chain alkanes. The maximum removal rate was observed during exponential growth phase (6 mg-alkanes/L h). This investigation proposes a novel approach for the management of lubricant waste oil, aiming at its conversion into valuable lipids. The feasibility of the concept is demonstrated under low salt (0.3%) and saline (3.3%) conditions, and presents clues for its technological development, since growth associated oil production opens the possibility for establishing continuous fermentation processes. Copyright © 2016 Elsevier Ltd. All rights reserved.