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Sample records for iglabcd virulence operon

  1. Genetic Characterization of a Streptococcus mutans LraI Family Operon and Role in Virulence

    PubMed Central

    Kitten, Todd; Munro, Cindy L.; Michalek, Suzanne M.; Macrina, Francis L.

    2000-01-01

    Proteins belonging to the LraI (for “lipoprotein receptor antigen”) family function as adhesins in several streptococci, as a virulence factor for endocarditis in at least one of these species, and potentially as metal transporters in many bacteria. We have identified and characterized the chromosomal locus containing the LraI family gene (designated sloC) from Streptococcus mutans, an agent of dental caries and endocarditis in humans. Northern blot analysis indicated that sloC is cotranscribed with three other genes. As with other LraI operons, the sloA and sloB genes apparently encode components of an ATP-binding cassette transport system. The product of the fourth gene, sloR, has homology to the metal-dependent regulator from Corynebacterium diphtheriae, DtxR. A potential binding site for SloR was identified upstream from the sloABCR operon and was conserved upstream from LraI operons in several other streptococci. Potential SloR homologs were identified in the unfinished genomic sequences from two of these, S. pneumoniae and S. pyogenes. Mutagenesis of sloC in S. mutans resulted in apparent loss of expression of the entire operon as assessed by Northern blot analysis. The sloC mutant was indistinguishable from its wild-type parent in a gnotobiotic rat model of caries but was significantly less virulent in a rat model of endocarditis. Virulence for endocarditis was restored by correction of the sloC mutation but not by provision of the sloC gene in trans, suggesting that virulence requires the expression of other genes in the sloC operon. PMID:10899841

  2. Contribution of the nos-pdt Operon to Virulence Phenotypes in Methicillin-Sensitive Staphylococcus aureus

    PubMed Central

    Almand, Erin A.; Rivera, Frances E.; Shaw, Lindsey N.; Richardson, Anthony R.; Rice, Kelly C.

    2014-01-01

    Nitric oxide (NO) is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS) enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT) enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS) were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and potential

  3. Contribution of the nos-pdt operon to virulence phenotypes in methicillin-sensitive Staphylococcus aureus.

    PubMed

    Sapp, April M; Mogen, Austin B; Almand, Erin A; Rivera, Frances E; Shaw, Lindsey N; Richardson, Anthony R; Rice, Kelly C

    2014-01-01

    Nitric oxide (NO) is emerging as an important regulator of bacterial stress resistance, biofilm development, and virulence. One potential source of endogenous NO production in the pathogen Staphylococcus aureus is its NO-synthase (saNOS) enzyme, encoded by the nos gene. Although a role for saNOS in oxidative stress resistance, antibiotic resistance, and virulence has been recently-described, insights into the regulation of nos expression and saNOS enzyme activity remain elusive. To this end, transcriptional analysis of the nos gene in S. aureus strain UAMS-1 was performed, which revealed that nos expression increases during low-oxygen growth and is growth-phase dependent. Furthermore, nos is co-transcribed with a downstream gene, designated pdt, which encodes a prephenate dehydratase (PDT) enzyme involved in phenylalanine biosynthesis. Deletion of pdt significantly impaired the ability of UAMS-1 to grow in chemically-defined media lacking phenylalanine, confirming the function of this enzyme. Bioinformatics analysis revealed that the operon organization of nos-pdt appears to be unique to the staphylococci. As described for other S. aureus nos mutants, inactivation of nos in UAMS-1 conferred sensitivity to oxidative stress, while deletion of pdt did not affect this phenotype. The nos mutant also displayed reduced virulence in a murine sepsis infection model, and increased carotenoid pigmentation when cultured on agar plates, both previously-undescribed nos mutant phenotypes. Utilizing the fluorescent stain 4-Amino-5-Methylamino-2',7'-Difluorofluorescein (DAF-FM) diacetate, decreased levels of intracellular NO/reactive nitrogen species (RNS) were detected in the nos mutant on agar plates. These results reinforce the important role of saNOS in S. aureus physiology and virulence, and have identified an in vitro growth condition under which saNOS activity appears to be upregulated. However, the significance of the operon organization of nos-pdt and potential

  4. Virulence dependent and independent regulation of the Bordetella pertussis cya operon.

    PubMed Central

    Laoide, B M; Ullmann, A

    1990-01-01

    The Bordetella pertussis adenylate cyclase (cya) operon is composed of four open reading frames, cyaA, B, D and E (Glaser et al., 1988, EMBO J., 7, 3997-4004). The cyaA gene encodes a virulence factor, cyclolysin, a bifunctional protein exhibiting both adenylate cyclase and haemolytic activities while the cyaB, D and E gene products are necessary for cyclolysin transport. We show that the cyaA gene is activated by a promoter located 115 bp upstream from the translational start codon and that transcription is only activated in virulent strains. Termination of transcription occurs 3' to the cyaA structural gene, however there appears to be some read-through into the downstream genes, resulting in full length cyaABDE transcripts. We also identify a second start site of transcription 30 bp upstream from the cyaB gene, in the intergenic cyaA--cyaB region. Transcription is activated from this site in both Vir+ and Vir- strains. Thus, the expression of the virulence associated cyclolysin is positively controlled via a trans-acting protein encoded by the bvg locus while the transport genes show a lower level of constitutive expression which is independent of virulence control. Images Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:1691098

  5. Characterization of a Mycobacterium avium subsp. avium Operon Associated with Virulence and Drug Detoxification

    PubMed Central

    Viale, Mariana Noelia; Imperiale, Belén; Gioffre, Andrea Karina; Colombatti Olivieri, María Alejandra; Moyano, Roberto Damián; Morcillo, Nora; Santangelo, María de la Paz; Davis, William; Romano, María Isabel

    2014-01-01

    The lprG-p55 operon of Mycobacterium tuberculosis and Mycobacterium bovis is involved in the transport of toxic compounds. P55 is an efflux pump that provides resistance to several drugs, while LprG is a lipoprotein that modulates the host's immune response against mycobacteria. The knockout mutation of this operon severely reduces the replication of both mycobacterial species during infection in mice and increases susceptibility to toxic compounds. In order to gain insight into the function of LprG in the Mycobacterium avium complex, in this study, we assayed the effect of the deletion of lprG gene in the D4ER strain of Mycobacterium avium subsp. avium. The replacement of lprG gene with a hygromycin cassette caused a polar effect on the expression of p55. Also, a twofold decrease in ethidium bromide susceptibility was observed and the resistance to the antibiotics rifampicin, amikacin, linezolid, and rifabutin was impaired in the mutant strain. In addition, the mutation decreased the virulence of the bacteria in macrophages in vitro and in a mice model in vivo. These findings clearly indicate that functional LprG and P55 are necessary for the correct transport of toxic compounds and for the survival of MAA in vitro and in vivo. PMID:24967408

  6. The dlt Operon of Bacillus cereus Is Required for Resistance to Cationic Antimicrobial Peptides and for Virulence in Insects▿

    PubMed Central

    Abi Khattar, Z.; Rejasse, A.; Destoumieux-Garzón, D.; Escoubas, J. M.; Sanchis, V.; Lereclus, D.; Givaudan, A.; Kallassy, M.; Nielsen-Leroux, C.; Gaudriault, S.

    2009-01-01

    The dlt operon encodes proteins that alanylate teichoic acids, the major components of cell walls of gram-positive bacteria. This generates a net positive charge on bacterial cell walls, repulsing positively charged molecules and conferring resistance to animal and human cationic antimicrobial peptides (AMPs) in gram-positive pathogenic bacteria. AMPs damage the bacterial membrane and are the most effective components of the humoral immune response against bacteria. We investigated the role of the dlt operon in insect virulence by inactivating this operon in Bacillus cereus, which is both an opportunistic human pathogen and an insect pathogen. The ΔdltBc mutant displayed several morphological alterations but grew at a rate similar to that for the wild-type strain. This mutant was less resistant to protamine and several bacterial cationic AMPs, such as nisin, polymyxin B, and colistin, in vitro. It was also less resistant to molecules from the insect humoral immune system, lysozyme, and cationic AMP cecropin B from Spodoptera frugiperda. ΔdltBc was as pathogenic as the wild-type strain in oral infections of Galleria mellonella but much less virulent when injected into the hemocoels of G. mellonella and Spodoptera littoralis. We detected the dlt operon in three gram-negative genera: Erwinia (Erwinia carotovora), Bordetella (Bordetella pertussis, Bordetella parapertussis, and Bordetella bronchiseptica), and Photorhabdus (the entomopathogenic bacterium Photorhabdus luminescens TT01, the dlt operon of which did not restore cationic AMP resistance in ΔdltBc). We suggest that the dlt operon protects B. cereus against insect humoral immune mediators, including hemolymph cationic AMPs, and may be critical for the establishment of lethal septicemia in insects and in nosocomial infections in humans. PMID:19767427

  7. Identification of the Staphylococcus aureus vfrAB Operon, a Novel Virulence Factor Regulatory Locus

    PubMed Central

    Daly, Seth M.; Hall, Pamela R.; Bayles, Kenneth W.

    2014-01-01

    During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not been characterized previously. Interestingly, a ΔvfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrB mutant did not restore hemolytic activity in the ΔvfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary for S. aureus pathogenesis. PMID:24549328

  8. Identification of the Staphylococcus aureus vfrAB operon, a novel virulence factor regulatory locus.

    PubMed

    Bose, Jeffrey L; Daly, Seth M; Hall, Pamela R; Bayles, Kenneth W

    2014-05-01

    During a screen of the Nebraska Transposon Mutant Library, we identified 71 mutations in the Staphylococcus aureus genome that altered hemolysis on blood agar medium. Although many of these mutations disrupted genes known to affect the production of alpha-hemolysin, two of them were associated with an apparent operon, designated vfrAB, that had not been characterized previously. Interestingly, a ΔvfrB mutant exhibited only minor effects on the transcription of the hla gene, encoding alpha-hemolysin, when grown in broth, as well as on RNAIII, a posttranscriptional regulatory RNA important for alpha-hemolysin translation, suggesting that VfrB may function at the posttranscriptional level. Indeed, a ΔvfrB mutant had increased aur and sspAB protease expression under these conditions. However, disruption of the known secreted proteases in the ΔvfrB mutant did not restore hemolytic activity in the ΔvfrB mutant on blood agar. Further analysis revealed that, in contrast to the minor effects of VfrB on hla transcription when strains were cultured in liquid media, the level of hla transcription was decreased 50-fold in the absence of VfrB on solid media. These results demonstrate that while VfrB represses protease expression when strains are grown in broth, hla regulation is highly responsive to factors associated with growth on solid media. Intriguingly, the ΔvfrB mutant displayed increased pathogenesis in a model of S. aureus dermonecrosis, further highlighting the complexity of VfrB-dependent virulence regulation. The results of this study describe a phenotype associated with a class of highly conserved yet uncharacterized proteins found in Gram-positive bacteria, and they shed new light on the regulation of virulence factors necessary for S. aureus pathogenesis. PMID:24549328

  9. Crosstalk between virulence loci: regulation of Salmonella enterica pathogenicity island 1 (SPI-1) by products of the std fimbrial operon.

    PubMed

    López-Garrido, Javier; Casadesús, Josep

    2012-01-01

    Invasion of intestinal epithelial cells is a critical step in Salmonella infection and requires the expression of genes located in Salmonella pathogenicity island 1 (SPI-1). A key factor for SPI-1 expression is DNA adenine (Dam) methylation, which activates synthesis of the SPI-1 transcriptional activator HilD. Dam-dependent regulation of hilD is postranscriptional (and therefore indirect), indicating the involvement of unknown cell functions under Dam methylation control. A genetic screen has identified the std fimbrial operon as the missing link between Dam methylation and SPI-1. We show that all genes in the std operon are part of a single transcriptional unit, and describe three previously uncharacterized ORFs (renamed stdD, stdE, and stdF). We present evidence that two such loci (stdE and stdF) are involved in Dam-dependent control of Salmonella SPI-1: in a Dam(-) background, deletion of stdE or stdF suppresses SPI-1 repression; in a Dam(+) background, constitutive expression of StdE and/or StdF represses SPI-1. Repression of SPI-1 by products of std operon explains the invasion defect of Salmonella Dam(-) mutants, which constitutively express the std operon. Dam-dependent repression of std in the ileum may be required to permit invasion, as indicated by two observations: constitutive expression of StdE and StdF reduces invasion of epithelial cells in vitro (1,000 fold) and attenuates Salmonella virulence in the mouse model (>60 fold). In turn, crosstalk between std and SPI-1 may play a role in intestinal infections by preventing expression of SPI-1 in the caecum, an intestinal compartment in which the std operon is known to be expressed.

  10. Assessment of Mycobacterium bovis Deleted in p27-p55 Virulence Operon as Candidate Vaccine against Tuberculosis in Animal Models

    PubMed Central

    Bianco, María V.; Clark, Simon; Blanco, Federico C.; Garbaccio, Sergio; García, Elizabeth; Cataldi, Angel A.; Bigi, Fabiana

    2014-01-01

    A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response. PMID:24588000

  11. Assessment of Mycobacterium bovis deleted in p27-p55 virulence operon as candidate vaccine against tuberculosis in animal models.

    PubMed

    Bianco, María V; Clark, Simon; Blanco, Federico C; Garbaccio, Sergio; García, Elizabeth; Cataldi, Angel A; Williams, Ann; Bigi, Fabiana

    2014-01-01

    A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.

  12. [Insertional Inactivation of Virulence Operon in Population of Persistent Bordetella pertussis Bacteria].

    PubMed

    Karataev, G I; Sinyashina, L N; Medkova, A Yu; Semin, E G; Shevtsova, Z V; Matua, A Z; Kondzariya, I G; Amichba, A A; Kubrava, D T; Mikvabia, Z Ya

    2016-04-01

    Avirulent B. pertussis bacteria containing IS elements in the bvgAS operon were detected during the study of whooping cough patients and bacilli carriers. The present work is devoted to the study of the accumulation dynamics and the mechanisms of generation of persistent forms of the B. pertussis bacteria in lower monkeys as the most adequate model for extrapolation ofthe experiment results to humans. By means of the real-time PCR method, it was established that the B. pertussis bacteria lived more than three months in the upper respiratory tract after a single intranasal monkey infection; the period was reduced to 14-28 days during repeated infection. An increase in the portion of B. pertussis Bvg mutants in the population to tens of percent from the total number of registered bacteria was registered. The experimental confirmation ofthe development and accumulation of avirulent B. pertussis Bvg mutants during the development of the infectious process was obtained. Further study of the composition of the B. pertussis persistent bacteria population at different stages of the disease will make it possible to formulate new approaches to the whooping cough diagnostics and prevention and creation of fundamentally new drugs.

  13. [Insertional Inactivation of Virulence Operon in Population of Persistent Bordetella pertussis Bacteria].

    PubMed

    Karataev, G I; Sinyashina, L N; Medkova, A Yu; Semin, E G; Shevtsova, Z V; Matua, A Z; Kondzariya, I G; Amichba, A A; Kubrava, D T; Mikvabia, Z Ya

    2016-04-01

    Avirulent B. pertussis bacteria containing IS elements in the bvgAS operon were detected during the study of whooping cough patients and bacilli carriers. The present work is devoted to the study of the accumulation dynamics and the mechanisms of generation of persistent forms of the B. pertussis bacteria in lower monkeys as the most adequate model for extrapolation ofthe experiment results to humans. By means of the real-time PCR method, it was established that the B. pertussis bacteria lived more than three months in the upper respiratory tract after a single intranasal monkey infection; the period was reduced to 14-28 days during repeated infection. An increase in the portion of B. pertussis Bvg mutants in the population to tens of percent from the total number of registered bacteria was registered. The experimental confirmation ofthe development and accumulation of avirulent B. pertussis Bvg mutants during the development of the infectious process was obtained. Further study of the composition of the B. pertussis persistent bacteria population at different stages of the disease will make it possible to formulate new approaches to the whooping cough diagnostics and prevention and creation of fundamentally new drugs. PMID:27529975

  14. The yrpAB operon of Yersinia ruckeri encoding two putative U32 peptidases is involved in virulence and induced under microaerobic conditions.

    PubMed

    Navais, Roberto; Méndez, Jessica; Pérez-Pascual, David; Cascales, Desirée; Guijarro, José A

    2014-07-01

    In an attempt to dissect the virulence mechanisms of Yersinia ruckeri two adjacent genes, yrpA and yrpB, encoding putative peptidases belonging to the U32 family, were analyzed. Similar genes, with the same genetic organization were identified in genomic analysis of human-pathogenic yersiniae. RT-PCR studies indicated that these genes form an operon in Y. ruckeri. Transcriptional studies using an yrpB::lacZY fusion showed high levels of expression of these genes in the presence of peptone in the culture medium, as well as under oxygen-limited conditions. These two factors had a synergic effect on gene induction when both were present simultaneously during bacterial incubation, which indicates the important role that environmental conditions in the fish gut can play in the regulation of specific genes. LD 50 experiments using an yrpA insertional mutant strain demonstrated the participation of this gene in the virulence of Y. ruckeri.

  15. A Homologue of an Operon Required for DNA Transfer in Agrobacterium Is Required in Brucella abortus for Virulence and Intracellular Multiplication

    PubMed Central

    Sieira, Rodrigo; Comerci, Diego J.; Sánchez, Daniel O.; Ugalde, Rodolfo A.

    2000-01-01

    As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment. PMID:10940027

  16. The sloABCR Operon of Streptococcus mutans Encodes an Mn and Fe Transport System Required for Endocarditis Virulence and Its Mn-Dependent Repressor

    PubMed Central

    Paik, Sehmi; Brown, Arunsri; Munro, Cindy L.; Cornelissen, Cynthia Nau; Kitten, Todd

    2003-01-01

    Streptococcus mutans belongs to the viridans group of oral streptococci, which is the leading cause of endocarditis in humans. The LraI family of lipoproteins in viridans group streptococci and other bacteria have been shown to function as virulence factors, adhesins, or ABC-type metal transporters. We previously reported the identification of the S. mutans LraI operon, sloABCR, which encodes components of a putative metal uptake system composed of SloA, an ATP-binding protein, SloB, an integral membrane protein, and SloC, a solute-binding lipoprotein, as well as a metal-dependent regulator, SloR. We report here the functional analysis of this operon. By Western blotting, addition of Mn to the growth medium repressed SloC expression in a wild-type strain but not in a sloR mutant. Other metals tested had little effect. Cells were also tested for aerobic growth in media stripped of metals then reconstituted with Mg and either Mn or Fe. Fe at 10 μM supported growth of the wild-type strain but not of a sloA or sloC mutant. Mn at 0.1 μM supported growth of the wild-type strain and sloR mutant but not of sloA or sloC mutants. The combined results suggest that the SloABC proteins transport both metals, although the SloR protein represses this system only in response to Mn. These conclusions are supported by 55Fe uptake studies with Mn as a competitor. Finally, a sloA mutant demonstrated loss of virulence in a rat model of endocarditis, suggesting that metal transport is required for endocarditis pathogenesis. PMID:14526007

  17. Bicarbonate-mediated transcriptional activation of divergent operons by the virulence regulatory protein, RegA, from Citrobacter rodentium.

    PubMed

    Yang, Ji; Hart, Emily; Tauschek, Marija; Price, G Dean; Hartland, Elizabeth L; Strugnell, Richard A; Robins-Browne, Roy M

    2008-04-01

    Regulation of virulence gene expression plays a central role in the pathogenesis of enteric bacteria as they encounter diverse environmental conditions in the gastrointestinal tract of their hosts. In this study, we investigated environmental regulation of two putative virulence determinants adcA and kfc by RegA, an AraC/XylS-like regulator, from Citrobacter rodentium, and identified bicarbonate as the environmental signal which induced transcription of adcA and kfc through RegA. Primer extension experiments showed that adcA and kfc were divergently transcribed from sigma(70) promoters. In vivo and in vitro experiments demonstrated that bicarbonate facilitated and stabilized the binding of RegA to an operator located between the two promoters. The interaction of RegA with its DNA target resulted in the formation of a nucleosome-like structure, which evidently displaced the histone-like proteins, H-NS and StpA, from the adcA and kfc promoter regions, leading to transcriptional derepression. In addition, our results indicated that RegA also behaved as a Class I activator by directly stimulating transcription initiation by RNA polymerase. This is the first report to describe the molecular mechanism by which an environmental chemical stimulates transcription of virulence-associated genes of an enteric pathogen through an AraC/XlyS-like activator.

  18. The Agrobacterium tumefaciens virulence gene chvE is part of a putative ABC-type sugar transport operon.

    PubMed

    Kemner, J M; Liang, X; Nester, E W

    1997-04-01

    The Agrobacterium tumefaciens virulence determinant ChvE is a periplasmic binding protein which participates in chemotaxis and virulence gene induction in response to monosaccharides which occur in the plant wound environment. The region downstream of the A. tumefaciens chvE gene was cloned and sequenced for nucleotide and expression analysis. Three open reading frames transcribed in the same direction as chvE were revealed. The first two, together with chvE, encode putative proteins of a periplasmic binding protein-dependent sugar uptake system, or ABC-type (ATP binding cassette) transporter. The third open reading frame encodes a protein of unknown function. The deduced transporter gene products are related on the amino acid level to bacterial sugar transporters and probably function in glucose and galactose uptake. We have named these genes gguA, -B, and -C, for glucose galactose uptake. Mutations in gguA, gguB, or gguC do not affect virulence of A. tumefaciens on Kalanchoe diagremontiana; growth on 1 mM galactose, glucose, xylose, ribose, arabinose, fucose, or sucrose; or chemotaxis toward glucose, galactose, xylose, or arabinose. PMID:9079938

  19. Comparative Bioinformatics and Experimental Analysis of the Intergenic Regulatory Regions of Bacillus cereus hbl and nhe Enterotoxin Operons and the Impact of CodY on Virulence Heterogeneity

    PubMed Central

    Böhm, Maria-Elisabeth; Krey, Viktoria M.; Jeßberger, Nadja; Frenzel, Elrike; Scherer, Siegfried

    2016-01-01

    Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5′ intergenic regions (5′ IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5′ IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5′ untranslated regions (5′ UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5′ UTR in B. cereus INRA C3 showed that the entire 331 bp 5′ UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5′ UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5′ IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5′ UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. Plc

  20. Comparative Bioinformatics and Experimental Analysis of the Intergenic Regulatory Regions of Bacillus cereus hbl and nhe Enterotoxin Operons and the Impact of CodY on Virulence Heterogeneity.

    PubMed

    Böhm, Maria-Elisabeth; Krey, Viktoria M; Jeßberger, Nadja; Frenzel, Elrike; Scherer, Siegfried

    2016-01-01

    Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5' intergenic regions (5' IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5' IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5' untranslated regions (5' UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5' UTR in B. cereus INRA C3 showed that the entire 331 bp 5' UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5' UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5' IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5' UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. PlcR binding sites are

  1. Comparative Bioinformatics and Experimental Analysis of the Intergenic Regulatory Regions of Bacillus cereus hbl and nhe Enterotoxin Operons and the Impact of CodY on Virulence Heterogeneity.

    PubMed

    Böhm, Maria-Elisabeth; Krey, Viktoria M; Jeßberger, Nadja; Frenzel, Elrike; Scherer, Siegfried

    2016-01-01

    Bacillus cereus is a food contaminant with greatly varying enteropathogenic potential. Almost all known strains harbor the genes for at least one of the three enterotoxins Nhe, Hbl, and CytK. While some strains show no cytotoxicity, others have caused outbreaks, in rare cases even with lethal outcome. The reason for these differences in cytotoxicity is unknown. To gain insight into the origin of enterotoxin expression heterogeneity in different strains, the architecture and role of 5' intergenic regions (5' IGRs) upstream of the nhe and hbl operons was investigated. In silico comparison of 142 strains of all seven phylogenetic groups of B. cereus sensu lato proved the presence of long 5' IGRs upstream of the nheABC and hblCDAB operons, which harbor recognition sites for several transcriptional regulators, including the virulence regulator PlcR, redox regulators ResD and Fnr, the nutrient-sensitive regulator CodY as well as the master regulator for biofilm formation SinR. By determining transcription start sites, unusually long 5' untranslated regions (5' UTRs) upstream of the nhe and hbl start codons were identified, which are not present upstream of cytK-1 and cytK-2. Promoter fusions lacking various parts of the nhe and hbl 5' UTR in B. cereus INRA C3 showed that the entire 331 bp 5' UTR of nhe is necessary for full promoter activity, while the presence of the complete 606 bp hbl 5' UTR lowers promoter activity. Repression was caused by a 268 bp sequence directly upstream of the hbl transcription start. Luciferase activity of reporter strains containing nhe and hbl 5' IGR lux fusions provided evidence that toxin gene transcription is upregulated by the depletion of free amino acids. Electrophoretic mobility shift assays showed that the branched-chain amino acid sensing regulator CodY binds to both nhe and hbl 5' UTR downstream of the promoter, potentially acting as a nutrient-responsive roadblock repressor of toxin gene transcription. PlcR binding sites are

  2. Group A streptococcal growth phase-associated virulence factor regulation by a novel operon (Fas) with homologies to two-component-type regulators requires a small RNA molecule.

    PubMed

    Kreikemeyer, B; Boyle, M D; Buttaro, B A; Heinemann, M; Podbielski, A

    2001-01-01

    A novel growth phase-associated two-component-type regulator, Fas (fibronectin/fibrinogen binding/haemolytic activity/streptokinase regulator), of Streptococcus pyogenes was identified in the M1 genome sequence, based on homologies to the histidine protein kinase (HPK) and response regulator (RR) part of the Staphylococcus aureus Agr and Streptococcus pneumoniae Com quorum-sensing systems. The fas operon, present in all 12 tested M serotypes, was transcribed as polycystronic message (fasBCA) and contained genes encoding two potential HPKs (FasB and FasC) and one RR (FasA). Downstream of fasBCA, we identified a small 300 nucleotide monocistronic transcript, designated fasX, that did not appear to encode true peptide sequences. Measurements of luciferase promoter fusions revealed a growth phase-associated transcription of fasBCA and fasX, with peak activities during the late exponential phase. Insertional mutagenesis disrupting fasBCA and fasA led to a phenotype similar to agr-null mutations in S. aureus, with prolonged expression of extracellular matrix protein-binding adhesins and reduced expression of secreted virulence factors such as streptokinase and streptolysin S. In addition, fasX transcription was dependent on the RR FasA; however, deletion mutagenesis of fasX resulted in a similar phenotype to that of the fasBCA or fasA mutants. Complementation of the fasX deletion mutant, with the fasX gene expressed in trans from a plasmid, restored the wild-type fasBCA regulation pattern. This strongly suggested that fasX, a putative non-translated RNA, is the main effector molecule of the fas regulon. However, using spent culture supernatants from wild-type and fas mutant strains, we were not able to show an influence on the logarithmic growth phase expression of fas and dependent genes. Thus, despite structural and functional similarities between fas and agr, to date the fas operon appears not to be involved in group A streptococcal (GAS) quorum-sensing regulation

  3. Operons in eukaryotes.

    PubMed

    Blumenthal, Thomas

    2004-11-01

    It was thought that polycistronic transcription is a characteristic of bacteria and archaea, where many of the genes are clustered in operons composed of two to more than ten genes. By contrast, the genes of eukaryotes are generally considered to be monocistronic, each with its own promoter at the 5' end and a transcription terminator at the 3' end; however, it has recently become clear that not all eukaryotic genes are transcribed monocistronically. Numerous instances of polycistronic transcription in eukaryotes, from protists to chordates, have been reported. These can be divided into two broad types. Dicistronic transcription units specify a messenger RNA (mRNA) encoding two separate genes that is transported to the cytoplasm and translated in that form. Presumably, internal ribosome entry sites (IRES), or some form of translational re-initiation following the stop codon, are responsible for allowing translation of the downstream gene. In the other type, the initial transcript is processed by 3' end cleavage and trans-splicing to create monocistronic mRNAs that are transported to the cytoplasm and translated. Like bacterial operons, eukaryotic operons often result in co-expression of functionally related proteins.

  4. Analysis of expression profile of mce operon genes (mce1, mce2, mce3 operon) in different Mycobacterium tuberculosis isolates at different growth phases

    PubMed Central

    Singh, Pratibha; Katoch, V.M.; Mohanty, K.K.; Chauhan, Devendra Singh

    2016-01-01

    Background & objectives: Mycobacterium tuberculosis (M. tuberculosis) has four homologous mammalian cell entry (mce) operons (mce1-4) that encode exported proteins and have a possible role in the virulence mechanism of this pathogen. The expression of mce operon is considered to be complex and not completely understood. Although expression of mce operon at different in vitro growth phases has been studied earlier, its expression in different M. tuberculosis isolates under different growth phases is not yet studied. The present preliminary study was conducted on a limited number of isolates to know the trend of expression pattern of mce operon genes in different M. tuberculosis isolates under different growth stages. Methods: In this study, we monitored the transcriptional profile of selected mce operon genes (mce1A, mce1D, mce2A, mce2D, mce3A, mce3C) in different M. tuberculosis isolates (MDR1, MDR2, and sensitive isolate) at early exponential and stationary phases using real-time quantitative PCR. Results: The expression ratio of all selected mce operon genes in all M. tuberculosis isolates was reduced at the initial phase and increased substantially at a later phase of growth. Higher expression of mce1 operon genes was found in all M. tuberculosis isolates as compared to other mce operon genes (mce2 and mce3 operons) at stationary growth phase. Interpretation & conclusions: The higher expression of mce operon genes at stationary phase (as compared to early exponential phase) suggested growth phase dependent expression of mce operon genes. This indicated that the mce operon genes might have a role in M. tuberculosis survival and adaptation on the onset of adverse condition like stationary phase. Identification of differentially expressed genes will add to our understanding of the bacilli involved in adaptation to different growth conditions. PMID:27377506

  5. Modeling operon dynamics: the tryptophan and lactose operons as paradigms.

    PubMed

    Mackey, Michael C; Santillán, Moisés; Yildirim, Necmettin

    2004-03-01

    Understanding the regulation of gene control networks and their ensuing dynamics will be a critical component in the understanding of the mountain of genomic data being currently collected. This paper reviews recent mathematical modeling work on the tryptophan and lactose operons which are, respectively, the classical paradigms for repressible and inducible operons. PMID:15127892

  6. The mbo Operon Is Specific and Essential for Biosynthesis of Mangotoxin in Pseudomonas syringae

    PubMed Central

    Carrión, Víctor J.; Arrebola, Eva; Cazorla, Francisco M.; Murillo, Jesús; de Vicente, Antonio

    2012-01-01

    Mangotoxin is an antimetabolite toxin produced by certain Pseudomonas syringae pv. syringae strains. This toxin is an oligopeptide that inhibits ornithine N-acetyl transferase, a key enzyme in the biosynthesis of ornithine and arginine. Previous studies have reported the involvement of the putative nonribosomal peptide synthetase MgoA in virulence and mangotoxin production. In this study, we analyse a new chromosomal region of P. syringae pv. syringae UMAF0158, which contains six coding sequences arranged as an operon (mbo operon). The mbo operon was detected in only mangotoxin-producing strains, and it was shown to be essential for the biosynthesis of this toxin. Mutants in each of the six ORFs of the mbo operon were partially or completely impaired in the production of the toxin. In addition, Pseudomonas spp. mangotoxin non-producer strains transformed with the mbo operon gained the ability to produce mangotoxin, indicating that this operon contains all the genetic information necessary for mangotoxin biosynthesis. The generation of a single transcript for the mbo operon was confirmed and supported by the allocation of a unique promoter and Rho-independent terminator. The phylogenetic analysis of the P. syringae strains harbouring the mbo operon revealed that these strains clustered together. PMID:22615797

  7. Operon prediction in Pyrococcus furiosus

    PubMed Central

    Tran, Thao T.; Dam, Phuongan; Su, Zhengchang; Poole, Farris L.; Adams, Michael W. W.; Zhou, G. Tong; Xu, Ying

    2007-01-01

    Identification of operons in the hyperthermophilic archaeon Pyrococcus furiosus represents an important step to understanding the regulatory mechanisms that enable the organism to adapt and thrive in extreme environments. We have predicted operons in P.furiosus by combining the results from three existing algorithms using a neural network (NN). These algorithms use intergenic distances, phylogenetic profiles, functional categories and gene-order conservation in their operon prediction. Our method takes as inputs the confidence scores of the three programs, and outputs a prediction of whether adjacent genes on the same strand belong to the same operon. In addition, we have applied Gene Ontology (GO) and KEGG pathway information to improve the accuracy of our algorithm. The parameters of this NN predictor are trained on a subset of all experimentally verified operon gene pairs of Bacillus subtilis. It subsequently achieved 86.5% prediction accuracy when applied to a subset of gene pairs for Escherichia coli, which is substantially better than any of the three prediction programs. Using this new algorithm, we predicted 470 operons in the P.furiosus genome. Of these, 349 were validated using DNA microarray data. PMID:17148478

  8. The Life-cycle of Operons

    SciTech Connect

    Price, Morgan N.; Arkin, Adam P.; Alm, Eric J.

    2005-11-18

    Operons are a major feature of all prokaryotic genomes, but how and why operon structures vary is not well understood. To elucidate the life-cycle of operons, we compared gene order between Escherichia coli K12 and its relatives and identified the recently formed and destroyed operons in E. coli. This allowed us to determine how operons form, how they become closely spaced, and how they die. Our findings suggest that operon evolution is driven by selection on gene expression patterns. First, both operon creation and operon destruction lead to large changes in gene expression patterns. For example, the removal of lysA and ruvA from ancestral operons that contained essential genes allowed their expression to respond to lysine levels and DNA damage, respectively. Second, some operons have undergone accelerated evolution, with multiple new genes being added during a brief period. Third, although most operons are closely spaced because of a neutral bias towards deletion and because of selection against large overlaps, highly expressed operons tend to be widely spaced because of regulatory fine-tuning by intervening sequences. Although operon evolution seems to be adaptive, it need not be optimal: new operons often comprise functionally unrelated genes that were already in proximity before the operon formed.

  9. The htpAB operon of Legionella pneumophila cannot be deleted in the presence of the groE chaperonin operon of Escherichia coli.

    PubMed

    Nasrallah, Gheyath K; Gagnon, Elizabeth; Orton, Dennis J; Garduño, Rafael A

    2011-11-01

    HtpB, the chaperonin of the intracellular bacterial pathogen Legionella pneumophila , displays several virulence-related functions in vitro. To confirm HtpB's role in vivo, host infections with an htpB deletion mutant would be required. However, we previously reported that the htpAB operon (encoding co-chaperonin and chaperonin) is essential. We attempted here to delete htpAB in a L. pneumophila strain carrying the groE operon (encoding the Escherichia coli co-chaperonin and chaperonin). The groE operon was inserted into the chromosome of L. pneumophila Lp02, and then allelic replacement of htpAB with a gentamicin resistance cassette was attempted. Although numerous potential postallelic replacement transformants showed a correct selection phenotype, we still detected htpAB by PCR and full-size HtpB by immunoblot. Southern blot and PCR analysis indicated that the gentamicin resistance cassette had apparently integrated in a duplicated htpAB region. However, we showed by Southern blot that strain Lp02, and the Lp02 derivative carrying the groE operon, have only one copy of htpAB. These results confirmed that the htpAB operon cannot be deleted, not even in the presence of the groE operon, and suggested that attempts to delete htpAB under strong phenotypic selection result in aberrant genetic recombinations that could involve duplication of the htpAB locus. PMID:22029459

  10. Investigating Evolutionary Dynamics of RHA1 Operons

    PubMed Central

    Chen, Yong; Geng, Dandan; Ehrhardt, Kristina; Zhang, Shaoqiang

    2016-01-01

    Grouping genes as operons is an important genomic feature of prokaryotic organisms. The comprehensive understanding of the operon organizations would be helpful to decipher transcriptional mechanisms, cellular pathways, and the evolutionary landscape of prokaryotic genomes. Although thousands of prokaryotes have been sequenced, genome-wide investigation of the evolutionary dynamics (division and recombination) of operons among these genomes remains unexplored. Here, we systematically analyzed the operon dynamics of Rhodococcus jostii RHA1 (RHA1), an oleaginous bacterium with high potential applications in biofuel, by comparing 340 prokaryotic genomes that were carefully selected from different genera. Interestingly, 99% of RHA1 operons were observed to exhibit evolutionary events of division and recombination among the 340 compared genomes. An operon that encodes all enzymes related to histidine biosynthesis in RHA1 (His-operon) was found to be segmented into smaller gene groups (sub-operons) in diverse genomes. These sub-operons were further reorganized with different functional genes as novel operons that are related to different biochemical processes. Comparatively, the operons involved in the functional categories of lipid transport and metabolism are relatively conserved among the 340 compared genomes. At the pathway level, RHA1 operons found to be significantly conserved were involved in ribosome synthesis, oxidative phosphorylation, and fatty acid synthesis. These analyses provide evolutionary insights of operon organization and the dynamic associations of various biochemical pathways in different prokaryotes. PMID:27398020

  11. The Life-cycle of Operons

    SciTech Connect

    Price, Morgan N.; Arkin, Adam P.; Alm, Eric J.

    2007-03-15

    Operons are a major feature of all prokaryotic genomes, buthow and why operon structures vary is not well understood. To elucidatethe life-cycle of operons, we compared gene order between Escherichiacoli K12 and its relatives and identified the recently formed anddestroyed operons in E. coli. This allowed us to determine how operonsform, how they become closely spaced, and how they die. Our findingssuggest that operon evolution may be driven by selection on geneexpression patterns. First, both operon creation and operon destructionlead to large changes in gene expression patterns. For example, theremoval of lysA and ruvA from ancestral operons that contained essentialgenes allowed their expression to respond to lysine levels and DNAdamage, respectively. Second, some operons have undergone acceleratedevolution, with multiple new genes being added during a brief period.Third, although genes within operons are usually closely spaced becauseof a neutral bias toward deletion and because of selection against largeoverlaps, genes in highly expressed operons tend to be widely spacedbecause of regulatory fine-tuning by intervening sequences. Althoughoperon evolution may be adaptive, it need not be optimal: new operonsoften comprise functionally unrelated genes that were already inproximity before the operon formed.

  12. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  13. Detecting uber-operons in prokaryotic genomes.

    PubMed

    Che, Dongsheng; Li, Guojun; Mao, Fenglou; Wu, Hongwei; Xu, Ying

    2006-01-01

    We present a study on computational identification of uber-operons in a prokaryotic genome, each of which represents a group of operons that are evolutionarily or functionally associated through operons in other (reference) genomes. Uber-operons represent a rich set of footprints of operon evolution, whose full utilization could lead to new and more powerful tools for elucidation of biological pathways and networks than what operons have provided, and a better understanding of prokaryotic genome structures and evolution. Our prediction algorithm predicts uber-operons through identifying groups of functionally or transcriptionally related operons, whose gene sets are conserved across the target and multiple reference genomes. Using this algorithm, we have predicted uber-operons for each of a group of 91 genomes, using the other 90 genomes as references. In particular, we predicted 158 uber-operons in Escherichia coli K12 covering 1830 genes, and found that many of the uber-operons correspond to parts of known regulons or biological pathways or are involved in highly related biological processes based on their Gene Ontology (GO) assignments. For some of the predicted uber-operons that are not parts of known regulons or pathways, our analyses indicate that their genes are highly likely to work together in the same biological processes, suggesting the possibility of new regulons and pathways. We believe that our uber-operon prediction provides a highly useful capability and a rich information source for elucidation of complex biological processes, such as pathways in microbes. All the prediction results are available at our Uber-Operon Database: http://csbl.bmb.uga.edu/uber, the first of its kind.

  14. Loss of the lac operon contributes to Salmonella invasion of epithelial cells through derepression of flagellar synthesis.

    PubMed

    Jiang, Lingyan; Ni, Zhiwei; Wang, Lei; Feng, Lu; Liu, Bin

    2015-03-01

    Salmonella, a genus that is closely related to Escherichia coli, includes many pathogens of humans and other animals. A notable feature that distinguishes Salmonella from E. coli is lactose negativity, because the lac operon is lost in most Salmonella genomes. Here, we expressed the lac operon in Salmonella enterica serovar Typhimurium and compared the virulence of the Lac(+) strain to that of the wild-type strain in a murine model, invasion assays, and macrophage replication assays. We showed that the Lac(+) strain is attenuated in vivo and the attenuation of virulence is caused by its defect in epithelial cell invasion. However, the invasion-defective phenotype is unrelated to lactose utilization. Through sequencing and the comparison of the transcriptome profile between the Lac(+) and wild-type strains during invasion, we found that most flagellar genes were markedly downregulated in the Lac(+) strain, while other genes associated with invasion, such as the majority of genes encoded in Salmonella pathogenicity island 1, were not differentially expressed. Moreover, we discovered that lacA is the major repressor of flagellar gene expression in the lac operon. In conclusion, these data demonstrate that the lac operon decreases Salmonella invasion of epithelial cells through repression of flagellar biosynthesis. As the ability to invade epithelial cells is a critical virulence determinant of Salmonella, our results provide important evidence that the loss of the lac operon contributes to the evolution of Salmonella pathogenicity. PMID:25362512

  15. Loss of the lac operon contributes to Salmonella invasion of epithelial cells through derepression of flagellar synthesis.

    PubMed

    Jiang, Lingyan; Ni, Zhiwei; Wang, Lei; Feng, Lu; Liu, Bin

    2015-03-01

    Salmonella, a genus that is closely related to Escherichia coli, includes many pathogens of humans and other animals. A notable feature that distinguishes Salmonella from E. coli is lactose negativity, because the lac operon is lost in most Salmonella genomes. Here, we expressed the lac operon in Salmonella enterica serovar Typhimurium and compared the virulence of the Lac(+) strain to that of the wild-type strain in a murine model, invasion assays, and macrophage replication assays. We showed that the Lac(+) strain is attenuated in vivo and the attenuation of virulence is caused by its defect in epithelial cell invasion. However, the invasion-defective phenotype is unrelated to lactose utilization. Through sequencing and the comparison of the transcriptome profile between the Lac(+) and wild-type strains during invasion, we found that most flagellar genes were markedly downregulated in the Lac(+) strain, while other genes associated with invasion, such as the majority of genes encoded in Salmonella pathogenicity island 1, were not differentially expressed. Moreover, we discovered that lacA is the major repressor of flagellar gene expression in the lac operon. In conclusion, these data demonstrate that the lac operon decreases Salmonella invasion of epithelial cells through repression of flagellar biosynthesis. As the ability to invade epithelial cells is a critical virulence determinant of Salmonella, our results provide important evidence that the loss of the lac operon contributes to the evolution of Salmonella pathogenicity.

  16. The cyanase operon and cyanate metabolism.

    PubMed

    Anderson, P M; Sung, Y C; Fuchs, J A

    1990-12-01

    Cyanase is an inducible enzyme in E. coli that catalyzes bicarbonate-dependent decomposition of cyanate. It is encoded as part of an operon we have named the cyn operon, which includes three genes in the following order: cynT (cyanate permease), cynS (cyanase), and cynX (protein of unknown function). The direction of transcription is opposite to that of the lac operon, and the 3'-end of the cyn operon overlaps the 3'-end of the lac operon by 98 nucleotides. The gene cynR (regulatory protein) is located upstream from the cyn operon, and its transcription is opposite that of the cyn operon. The genes of the cyn operon and the cynR gene have been cloned, sequenced and over-expressed. Cyanate at concentrations of about 1 mM is toxic to strains of E. coli lacking the cyanase gene, but strains in which the inducible gene for cyanase is present can grow on cyanate as the sole source of nitrogen at concentrations as high as 20 mM. The presence of cyanase itself is not sufficient to overcome cyanate toxicity--the permease must also be present. Strains lacking the cyanase gene, but having a functional permease gene, are extremely sensitive to cyanate. Uptake of cyanate involves the product of the permease gene in an energy-dependent process. It appears that the cyn operon has evolved to function in detoxification/decomposition of cyanate arising from both intra- and extracellular sources.

  17. Problem-solving test: Tryptophan operon mutants.

    PubMed

    Szeberényi, József

    2010-09-01

    Terms to be familiar with before you start to solve the test: tryptophan, operon, operator, repressor, inducer, corepressor, promoter, RNA polymerase, chromosome-polysome complex, regulatory gene, cis-acting element, trans-acting element, plasmid, transformation. PMID:21567855

  18. Virulence Determination

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This chapter reviews the in vitro and in vivo assays that are available for determination of pathogenic potential of Listeria monocytogenes bacteria, highlighting the value of using multiplex PCR for rapid and accurate assessment of listerial virulence....

  19. Detection of pap, sfa, afa, foc, and fim Adhesin-Encoding Operons in Uropathogenic Escherichia coli Isolates Collected From Patients With Urinary Tract Infection

    PubMed Central

    Rahdar, Masoud; Rashki, Ahmad; Miri, Hamid Reza; Rashki Ghalehnoo, Mehdi

    2015-01-01

    Background: Uropathogenic Escherichia coli (UPEC) with its virulence factors is the most prevalent cause of urinary tract infection (UTI). Objectives; This study aimed to determine the occurrence of fim, pap, sfa, and afa genes among 100 UPEC isolates collected from patients diagnosed with UTI. Materials and Methods A total of 100 UPEC isolates were obtained from urine samples of patients with UTI. The prevalence of 5 virulence genes encoding type 1 fimbriae (fimH), pili associated with pyelonephritis (pap), S and F1C fimbriae (sfa and foc) and afimbrial adhesins (afa) were determined through PCR method. We also investigated the phylogenetic background of all isolates. In addition, the distribution of adhesin-encoding operons between the phylogroups was assessed. Results: The prevalence of genes encoding for fimbrial adhesive systems was 95% for fim, 57% for pap, 16% for foc, and 81% for sfa. The operons encoding for afa afimbrial adhesins were identified in 12% of isolates. The various combinations of detected genes were designated as virulence patterns. The fim gene, which occurred in strains from all phylogenetic groups (A, B1, B2, and D) was evaluated and no significant differences were found among these groups. Conversely, significant differences were observed in relation to pap, afa, foc, and sfa operons. Conclusions: These results indicate that the PCR method is a powerful genotypic assay for the detection of adhesin-encoding operons. Thus, this assay can be recommended for clinical use to detect virulent urinary E. coli strains, as well as epidemiological studies. PMID:26464770

  20. Gene inactivation of a chemotaxis operon in the pathogen Leptospira interrogans.

    PubMed

    Lambert, Ambroise; Wong Ng, Jérôme; Picardeau, Mathieu

    2015-01-01

    Chemotaxis may have an important role in the infection process of pathogenic Leptospira spp.; however, little is known about the regulation of flagellar-based motility in these atypical bacteria. We generated a library of random transposon mutants of the pathogen L. interrogans, which included a mutant with insertion in the first gene of an operon containing the chemotaxis genes cheA, cheW, cheD, cheB, cheY and mcp. The disrupted gene encodes a putative histidine kinase (HK). The HK mutant was motile and virulent, but swarm plate and capillary assays suggested that chemotaxis was reduced in this mutant. Further analysis of bacterial trajectories by videomicroscopy showed that the ability of this mutant to reverse was significantly impaired in comparison to wild-type strain. Our data therefore show that this operon is required for full chemotaxis of Leptospira spp.

  1. Agrobacterium tumefaciens virE operon encodes a single-stranded DNA-binding protein.

    PubMed

    Das, A

    1988-05-01

    The virulence (vir) genes of Agrobacterium tumefaciens Ti plasmid are essential for transformation of plant cells. Overproduction of a virE-encoded gene product in Escherichia coli was achieved by construction of an operon fusion with the E. coli tryptophan (trp) operon. The virE2 gene product in E. coli partitioned into the insoluble membrane fraction. The protein was solubilized by treatment with 4 M urea at 0 degree C. DNA-protein binding experiments showed that a strong single-stranded (ss) DNA-binding activity was present in protein fractions containing the virE2 gene product. The binding was highly specific with little or no binding observed with either double-stranded DNA or ssRNA. No significant binding to Ti plasmid DNA sequences was observed. Protein blotting studies indicated that the ssDNA-binding activity was associated with the 68-kDa virE2 polypeptide. PMID:2452439

  2. Evaluation of the Role of the opgGH Operon in Yersinia pseudotuberculosis and Its Deletion during the Emergence of Yersinia pestis

    PubMed Central

    Quintard, Kévin; Dewitte, Amélie; Reboul, Angéline; Madec, Edwige; Bontemps-Gallo, Sébastien; Dondeyne, Jacqueline; Marceau, Michaël; Simonet, Michel

    2015-01-01

    The opgGH operon encodes glucosyltransferases that synthesize osmoregulated periplasmic glucans (OPGs) from UDP-glucose, using acyl carrier protein (ACP) as a cofactor. OPGs are required for motility, biofilm formation, and virulence in various bacteria. OpgH also sequesters FtsZ in order to regulate cell size according to nutrient availability. Yersinia pestis (the agent of flea-borne plague) lost the opgGH operon during its emergence from the enteropathogen Yersinia pseudotuberculosis. When expressed in OPG-negative strains of Escherichia coli and Dickeya dadantii, opgGH from Y. pseudotuberculosis restored OPGs synthesis, motility, and virulence. However, Y. pseudotuberculosis did not produce OPGs (i) under various growth conditions or (ii) when overexpressing its opgGH operon, its galUF operon (governing UDP-glucose), or the opgGH operon or Acp from E. coli. A ΔopgGH Y. pseudotuberculosis strain showed normal motility, biofilm formation, resistance to polymyxin and macrophages, and virulence but was smaller. Consistently, Y. pestis was smaller than Y. pseudotuberculosis when cultured at ≥37°C, except when the plague bacillus expressed opgGH. Y. pestis expressing opgGH grew normally in serum and within macrophages and was fully virulent in mice, suggesting that small cell size was not advantageous in the mammalian host. Lastly, Y. pestis expressing opgGH was able to infect Xenopsylla cheopis fleas normally. Our results suggest an evolutionary scenario whereby an ancestral Yersinia strain lost a factor required for OPG biosynthesis but kept opgGH (to regulate cell size). The opgGH operon was presumably then lost because OpgH-dependent cell size control became unnecessary. PMID:26150539

  3. RbsR Activates Capsule but Represses the rbsUDK Operon in Staphylococcus aureus

    PubMed Central

    Lei, Mei G.

    2015-01-01

    ABSTRACT Staphylococcus aureus capsule is an important virulence factor that is regulated by a large number of regulators. Capsule genes are expressed from a major promoter upstream of the cap operon. A 10-bp inverted repeat (IR) located 13 bp upstream of the −35 region of the promoter was previously shown to affect capsule gene transcription. However, little is known about transcriptional activation of the cap promoter. To search for potential proteins which directly interact with the cap promoter region (Pcap), we directly analyzed the proteins interacting with the Pcap DNA fragment from shifted gel bands identified by electrophoretic mobility shift assay. One of these regulators, RbsR, was further characterized and found to positively regulate cap gene expression by specifically binding to the cap promoter region. Footprinting analyses showed that RbsR protected a DNA region encompassing the 10-bp IR. Our results further showed that rbsR was directly controlled by SigB and that RbsR was a repressor of the rbsUDK operon, involved in ribose uptake and phosphorylation. The repression of rbsUDK by RbsR could be derepressed by d-ribose. However, d-ribose did not affect RbsR activation of capsule. IMPORTANCE Staphylococcus aureus is an important human pathogen which produces a large number of virulence factors. We have been using capsule as a model virulence factor to study virulence regulation. Although many capsule regulators have been identified, the mechanism of regulation of most of these regulators is unknown. We show here that RbsR activates capsule by direct promoter binding and that SigB is required for the expression of rbsR. These results define a new pathway wherein SigB activates capsule through RbsR. Our results further demonstrate that RbsR inhibits the rbs operon involved in ribose utilization, thereby providing an example of coregulation of metabolism and virulence in S. aureus. Thus, this study further advances our understanding of staphylococcal

  4. Teaching the Big Ideas of Biology with Operon Models

    ERIC Educational Resources Information Center

    Cooper, Robert A.

    2015-01-01

    This paper presents an activity that engages students in model-based reasoning, requiring them to predict the behavior of the trp and lac operons under different environmental conditions. Students are presented six scenarios for the "trp" operon and five for the "lac" operon. In most of the scenarios, specific mutations have…

  5. Origin of bistability in the lac Operon.

    PubMed

    Santillán, M; Mackey, M C; Zeron, E S

    2007-06-01

    Multistability is an emergent dynamic property that has been invoked to explain multiple coexisting biological states. In this work, we investigate the origin of bistability in the lac operon. To do this, we develop a mathematical model for the regulatory pathway in this system and compare the model predictions with other experimental results in which a nonmetabolizable inducer was employed. We investigate the effect of lactose metabolism using this model, and show that it greatly modifies the bistable region in the external lactose (Le) versus external glucose (Ge) parameter space. The model also predicts that lactose metabolism can cause bistability to disappear for very low Ge. We have also carried out stochastic numerical simulations of the model for several values of Ge and Le. Our results indicate that bistability can help guarantee that Escherichia coli consumes glucose and lactose in the most efficient possible way. Namely, the lac operon is induced only when there is almost no glucose in the growing medium, but if Le is high, the operon induction level increases abruptly when the levels of glucose in the environment decrease to very low values. We demonstrate that this behavior could not be obtained without bistability if the stability of the induced and uninduced states is to be preserved. Finally, we point out that the present methods and results may be useful to study the emergence of multistability in biological systems other than the lac operon. PMID:17351004

  6. Indole and 7‐hydroxyindole diminish Pseudomonas aeruginosa virulence

    PubMed Central

    Lee, Jintae; Attila, Can; Cirillo, Suat L. G.; Cirillo, Jeffrey D.; Wood, Thomas K.

    2009-01-01

    Summary Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7‐hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)‐regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI‐opmD multidrug efflux pump and genes involved in the synthesis of QS‐regulated virulence factors including pyocyanin (phz operon), 2‐heptyl‐3‐hydroxy‐4(1H)‐quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole‐related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa. PMID:21261883

  7. Indole and 7-hydroxyindole diminish Pseudomonas aeruginosa virulence.

    PubMed

    Lee, Jintae; Attila, Can; Cirillo, Suat L G; Cirillo, Jeffrey D; Wood, Thomas K

    2009-01-01

    Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7-hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)-regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI-opmD multidrug efflux pump and genes involved in the synthesis of QS-regulated virulence factors including pyocyanin (phz operon), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole-related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa. PMID:21261883

  8. Comparative functional analysis of the lac operons in Streptococcus pyogenes.

    PubMed

    Loughman, Jennifer A; Caparon, Michael G

    2007-04-01

    Having no known environmental reservoir, Streptococcus pyogenes, a bacterium responsible for a wider variety of human diseases than any other bacterial species, must rely on its host for metabolic substrates. Although a streptococcal aldolase, LacD.1, has been adapted to virulence gene regulation, both LacD.1 and a paralogous protein, LacD.2, are predicted to function in the tagatose 6-phosphate pathway for lactose and galactose utilization. In order to gain insight into the mechanism of the LacD.1 regulatory pathway and the role of genome context in the emergence of LacD.1's novel regulatory functions, we compared the function and regulation of the Lac.1 and Lac.2 loci. The Lac.1 operon is not inducible, and regulation by LacD.1 is independent of a functional tagatose 6-phosphate pathway and enhanced by the conserved truncation of upstream Lac.1 genes. In contrast, Lac.2 expression is sensitive to environmental carbohydrates, and LacD.2, not LacD.1, contributes to growth on galactose. Thus, we conclude that the Lac.1 locus has been specialized to participate in regulation, leaving efficient utilization of carbohydrate sources to the Lac.2 locus. The adaptation of LacD for transcription regulation may be an underappreciated strategy among prokaryotes, as homologues of this multifaceted enzyme are present in a broad range of species. PMID:17371500

  9. 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.

  10. 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. PMID:26077867

  11. 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

  12. Distribution of horizontally transferred heavy metal resistance operons in recent outbreak bacteria.

    PubMed

    Reva, Oleg; Bezuidt, Oliver

    2012-03-01

    Mankind is confronted by the outbreaks of highly virulent and multi-drug resistant pathogens. The outbreak strains often belong to well-known diseases associated species such as Salmonella, Klebsiella and Mycobacterium, but even normally commensal and environmental microorganisms may suddenly acquire properties of virulent bacteria and cause nosocomial infections. The acquired virulence is often associated with lateral exchange of pathogenicity genomic islands containing drug and heavy metal resistance determinants. Metal ions are used by the immune system of macro-organisms against bactericidal agents. The ability to control heavy metal homeostasis is a factor that allows the survival of pathogenic microorganisms in macrophages. In this paper, we investigate the origin of heavy metal resistance operons in the recent outbreak strains and the possible routes which may lead to acquisitions of these genes by potentially new pathogens. We hypothesize that new outbreak microorganisms appear intermittently on an intersection of the non-specialized, genetically naïve strains of potential pathogens and virulence factor comprising vectors (plasmid and/or phages) newly generated in the environmental microflora. Global contamination of the environment and climate change may also have an effect toward the acceleration and appearance of new pathogens.

  13. gltBDF operon of Escherichia coli.

    PubMed Central

    Castaño, I; Bastarrachea, F; Covarrubias, A A

    1988-01-01

    A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega. Images PMID:2448295

  14. Study of the in vivo role of Mce2R, the transcriptional regulator of mce2 operon in Mycobacterium tuberculosis

    PubMed Central

    2013-01-01

    Background Tuberculosis is one of the leading causes of mortality throughout the world. Mycobacterium tuberculosis, the agent of human tuberculosis, has developed strategies involving proteins and other compounds called virulence factors to subvert human host defences and damage and invade the human host. Among these virulence-related proteins are the Mce proteins, which are encoded in the mce1, mce2, mce3 and mce4 operons of M. tuberculosis. The expression of the mce2 operon is negatively regulated by the Mce2R transcriptional repressor. Here we evaluated the role of Mce2R during the infection of M. tuberculosis in mice and macrophages and defined the genes whose expression is in vitro regulated by this transcriptional repressor. Results We used a specialized transduction method for generating a mce2R mutant of M. tuberculosis H37Rv. Although we found equivalent replication of the MtΔmce2R mutant and the wild type strains in mouse lungs, overexpression of Mce2R in the complemented strain (MtΔmce2RComp) significantly impaired its replication. During in vitro infection of macrophages, we observed a significantly increased association of the late endosomal marker LAMP-2 to MtΔmce2RComp-containing phagosomes as compared to MtΔmce2R and the wild type strains. Whole transcriptional analysis showed that Mce2R regulates mainly the expression of the mce2 operon, in the in vitro conditions studied. Conclusions The findings of the current study indicate that Mce2R weakly represses the in vivo expression of the mce2 operon in the studied conditions and argue for a role of the proteins encoded in Mce2R regulon in the arrest of phagosome maturation induced by M. tuberculosis. PMID:24007602

  15. Novel Twin Streptolysin S-Like Peptides Encoded in the sag Operon Homologue of Beta-Hemolytic Streptococcus anginosus

    PubMed Central

    Tabata, Atsushi; Nakano, Kota; Ohkura, Kazuto; Tomoyasu, Toshifumi; Kikuchi, Ken; Whiley, Robert A.

    2013-01-01

    Streptococcus anginosus is a member of the anginosus group streptococci, which form part of the normal human oral flora. In contrast to the pyogenic group streptococci, our knowledge of the virulence factors of the anginosus group streptococci, including S. anginosus, is not sufficient to allow a clear understanding of the basis of their pathogenicity. Generally, hemolysins are thought to be important virulence factors in streptococcal infections. In the present study, a sag operon homologue was shown to be responsible for beta-hemolysis in S. anginosus strains by random gene knockout. Interestingly, contrary to pyogenic group streptococci, beta-hemolytic S. anginosus was shown to have two tandem sagA homologues, encoding streptolysin S (SLS)-like peptides, in the sag operon homologue. Gene deletion and complementation experiments revealed that both genes were functional, and these SLS-like peptides were essential for beta-hemolysis in beta-hemolytic S. anginosus. Furthermore, the amino acid sequence of these SLS-like peptides differed from that of the typical SLS of S. pyogenes, especially in their propeptide domain, and an amino acid residue indicated to be important for the cytolytic activity of SLS in S. pyogenes was deleted in both S. anginosus homologues. These data suggest that SLS-like peptides encoded by two sagA homologues in beta-hemolytic S. anginosus may be potential virulence factors with a different structure essential for hemolytic activity and/or the maturation process compared to the typical SLS present in pyogenic group streptococci. PMID:23292771

  16. Virulence Attributes of Low-Virulence Organisms

    PubMed Central

    1994-01-01

    The vast majority of infections involving female pelvic structures arise from organisms that are members of the normal flora. In addition, exogenous organisms that invade through the lower genital tract must interact with organisms that are part of the host's flora. In contrast to the concept that the normal flora is entirely innocuous, recent research has begun to identify what appear to be virulence attributes among these ordinarily low-virulence organisms. Most of our understanding of virulence has been derived from highly virulent organisms, of which Neisseria gonorrhoeae provides an example of relevance to the female genital tract. A review of the virulence factors of the gonococcus is presented to serve as an example of the variety of virulence properties associated with pathogenic bacteria. Molecular biology has begun to clarify one of the important paradigms of pathogenic bacteriology—that bacteria change their expression of virulence properties in response to their location within a host or to the stage of infection. Thus, infection involves not only the possession of virulence factors, but also the carefully controlled use of those factors. Virulence is often controlled by the coordinate expression of many virulence-associated genes in response to one environmental signal. With regard to low- virulence organisms present in the female lower genital tract, we are beginning to identify some of their virulence attributes. Examples from the work of our laboratory include the hemolysin of Gardnerella vaginalis and an immunosuppressive mycotoxin produced by Candida albicans. Demonstrating the coordinate expression (or other control mechanisms) of virulence factors in these sometimes innocuous and sometimes inimical organisms represents the next frontier in the study of normal vaginal microbiology. PMID:18475373

  17. HosA, a MarR Family Transcriptional Regulator, Represses Nonoxidative Hydroxyarylic Acid Decarboxylase Operon and Is Modulated by 4-Hydroxybenzoic Acid.

    PubMed

    Roy, Ajit; Ranjan, Akash

    2016-02-23

    Members of the Multiple antibiotic resistance Regulator (MarR) family of DNA binding proteins regulate transcription of a wide array of genes required for virulence and pathogenicity of bacteria. The present study reports the molecular characterization of HosA (Homologue of SlyA), a MarR protein, with respect to its target gene, DNA recognition motif, and nature of its ligand. Through a comparative genomics approach, we demonstrate that hosA is in synteny with nonoxidative hydroxyarylic acid decarboxylase (HAD) operon and is present exclusively within the mutS-rpoS polymorphic region in nine different genera of Enterobacteriaceae family. Using molecular biology and biochemical approach, we demonstrate that HosA binds to a palindromic sequence downstream to the transcription start site of divergently transcribed nonoxidative HAD operon and represses its expression. Furthermore, in silico analysis showed that the recognition motif for HosA is highly conserved in the upstream region of divergently transcribed operon in different genera of Enterobacteriaceae family. A systematic chemical search for the physiological ligand revealed that 4-hydroxybenzoic acid (4-HBA) interacts with HosA and derepresses HosA mediated repression of the nonoxidative HAD operon. Based on our study, we propose a model for molecular mechanism underlying the regulation of nonoxidative HAD operon by HosA in Enterobacteriaceae family. PMID:26818787

  18. Genetic analysis of the virE operon of the Agrobacterium Ti plasmid pTiA6.

    PubMed

    McBride, K E; Knauf, V C

    1988-04-01

    The virE operon of the Agrobacterium tumefaciens Ti plasmid pTiA6 encodes at least one trans-acting protein involved in the expression of virulence. Two open reading frames designated virE1 and virE2 code for polypeptides of 7 and 60 kilodaltons (kDa), respectively, that can be visualized after expression in Escherichia coli minicells. To determine which virE sequences are required for virulence, a strain deleted for the entire locus [strain KE1(pTiA6 delta E)] was constructed and tested for the ability to be complemented by subclones with and without site-directed mutations in the virE operon. One subclone containing only virE1 and virE2 as well as upstream promoter sequences was sufficient to restore full virulence on the host plant Kalanchoe daigremontiana. However, some other virulence locus representing a host range determinant appeared to be deleted from strain KE1(pTiA6 delta E), since virE1 and virE2 were not sufficient to fully restore virulence on wounded tomato plants. virE operon constructs with specific lesions in either virE1 or virE2 were impaired for complementation of pTiA6 delta E. Several mutations specific for the promoter-proximal virE1 locus appeared to have a polar effect on expression of the virE2-encoded 60-kDa protein. However, virE2::lacZ fusion constructs suggest that this effect is not at the level of transcription or translation. Collectively, these data indicate that both the 7- and the 60-kDa polypeptides are virulence determinants for the Ti plasmid pTiA6 and suggest that the 60-kDa protein may be less stable in the absence of the 7-kDa protein. PMID:2832362

  19. Comparative Genomics Analysis of Streptococcus tigurinus Strains Identifies Genetic Elements Specifically and Uniquely Present in Highly Virulent Strains.

    PubMed

    Diene, Seydina M; François, Patrice; Zbinden, Andrea; Entenza, José Manuel; Resch, Grégory

    2016-01-01

    Streptococcus tigurinus is responsible for severe invasive infections such as infective endocarditis, spondylodiscitis and meningitis. As described, S. tigurinus isolates AZ_3aT and AZ_14 were highly virulent (HV phenotype) in an experimental model of infective endocarditis and showed enhanced adherence and invasion of human endothelial cells when compared to low virulent S. tigurinus isolate AZ_8 (LV phenotype). Here, we sought whether genetic determinants could explain the higher virulence of AZ_3aT and AZ_14 isolates. Several genetic determinants specific to the HV strains were identified through extensive comparative genomics amongst which some were thought to be highly relevant for the observed HV phenotype. These included i) an iron uptake and metabolism operon, ii) an ascorbate assimilation operon, iii) a newly acquired PI-2-like pilus islets described for the first time in S. tigurinus, iv) a hyaluronate metabolism operon, v) an Entner-Doudoroff pathway of carbohydrates metabolism, and vi) an alternate pathways for indole biosynthesis. We believe that the identified genomic features could largely explain the phenotype of high infectivity of the two HV S. tigurinus strains. Indeed, these features include determinants that could be involved at different stages of the disease such as survival of S. tigurinus in blood (iron uptake and ascorbate metabolism operons), initial attachment of bacterial pathogen to the damaged cardiac tissue and/or vegetation that formed on site (PI-2-like pilus islets), tissue invasion (hyaluronate operon and Entner-Doudoroff pathway) and regulation of pathogenicity (indole biosynthesis pathway). PMID:27505001

  20. Comparative Genomics Analysis of Streptococcus tigurinus Strains Identifies Genetic Elements Specifically and Uniquely Present in Highly Virulent Strains

    PubMed Central

    Diene, Seydina M.; François, Patrice; Zbinden, Andrea; Entenza, José Manuel

    2016-01-01

    Streptococcus tigurinus is responsible for severe invasive infections such as infective endocarditis, spondylodiscitis and meningitis. As described, S. tigurinus isolates AZ_3aT and AZ_14 were highly virulent (HV phenotype) in an experimental model of infective endocarditis and showed enhanced adherence and invasion of human endothelial cells when compared to low virulent S. tigurinus isolate AZ_8 (LV phenotype). Here, we sought whether genetic determinants could explain the higher virulence of AZ_3aT and AZ_14 isolates. Several genetic determinants specific to the HV strains were identified through extensive comparative genomics amongst which some were thought to be highly relevant for the observed HV phenotype. These included i) an iron uptake and metabolism operon, ii) an ascorbate assimilation operon, iii) a newly acquired PI-2-like pilus islets described for the first time in S. tigurinus, iv) a hyaluronate metabolism operon, v) an Entner-Doudoroff pathway of carbohydrates metabolism, and vi) an alternate pathways for indole biosynthesis. We believe that the identified genomic features could largely explain the phenotype of high infectivity of the two HV S. tigurinus strains. Indeed, these features include determinants that could be involved at different stages of the disease such as survival of S. tigurinus in blood (iron uptake and ascorbate metabolism operons), initial attachment of bacterial pathogen to the damaged cardiac tissue and/or vegetation that formed on site (PI-2-like pilus islets), tissue invasion (hyaluronate operon and Entner-Doudoroff pathway) and regulation of pathogenicity (indole biosynthesis pathway). PMID:27505001

  1. The pap Operon of Avian Pathogenic Escherichia coli Strain O1:K1 Is Located on a Novel Pathogenicity Island

    PubMed Central

    Kariyawasam, Subhashinie; Johnson, Timothy J.; Nolan, Lisa K.

    2006-01-01

    We have identified a 56-kb pathogenicity island (PAI) in avian pathogenic Escherichia coli strain O1:K1 (APEC-O1). This PAI, termed PAI IAPEC-O1, is integrated adjacent to the 3′ end of the pheV tRNA gene. It carries putative virulence genes of APEC (pap operon), other E. coli genes (tia and ireA), and a 1.5-kb region unique to APEC-O1. The kps gene cluster required for the biosynthesis of polysialic acid capsule was mapped to a location immediately downstream of this PAI. PMID:16369033

  2. Transcriptional Activation of the tad Type IVb Pilus Operon by PypB in Yersinia enterocolitica▿

    PubMed Central

    Schilling, Jennifer; Wagner, Karin; Seekircher, Stephanie; Greune, Lilo; Humberg, Verena; Schmidt, M. Alexander; Heusipp, Gerhard

    2010-01-01

    Type IV pili are virulence factors in various bacteria and mediate, among other functions, the colonization of diverse surfaces. Various subclasses of type IV pili have been identified, but information on pilus expression, biogenesis, and the associated phenotypes is sparse for the genus Yersinia. We recently described the identification of PypB as a transcriptional regulator in Yersinia enterocolitica. Here we show that the pypB gene is associated with the tad locus, a genomic island that is widespread among bacterial and archaeal species. The genetic linkage of pypB with the tad locus is conserved throughout the yersiniae but is not found among other bacteria carrying the tad locus. We show that the genes of the tad locus form an operon in Y. enterocolitica that is controlled by PypB and that pypB is part of this operon. The tad genes encode functions necessary for the biogenesis of the Flp subfamily of type IVb pili initially described for Aggregatibacter actinomycetemcomitans to mediate a tight-adherence phenotype. In Y. enterocolitica, the Flp pilin protein shows some peculiarities in its amino acid sequence that imply similarities as well as differences compared to typical motifs found in the Flp subtype of type IVb pili. Flp is expressed and processed after PypB overproduction, resulting in microcolony formation but not in increased adherence to biotic or abiotic surfaces. Our data describe the transcriptional regulation of the tad type IVb pilus operon by PypB in Y. enterocolitica but fail to show most previously described phenotypes associated with this type of pilus in other bacteria. PMID:20472801

  3. Transient repression of the lac operon.

    PubMed

    Tyler, B; Loomis, W F; Magasanik, B

    1967-12-01

    Severe transient repression of constitutive or induced beta-galactosidase synthesis occurs upon the addition of glucose to cells of Escherichia coli growing on glycerol, succinic acid, or lactic acid. Only mutants particularily well adapted to growth on glucose exhibit this phenomenon when transferred to a glucose-containing medium. No change in ribonucleic acid (RNA) metabolism was observed during transient repression. We could show that transient repression is pleiotropic, affecting all products of the lac operon. It occurs in a mutant insensitive to catabolite repression. It is established much more rapidly than catabolite repression, and is elicited by glucose analogues that are phosphorylated but not further catabolized by the cell. Thus, transient repression is not a consequence of the exclusion of inducer from the cell, does not require catabolism of the added compound, and does not involve a gross change in RNA metabolism. We conclude that transient repression is distinct from catabolite repression. PMID:4864411

  4. The fas operon of Rhodococcus fascians encodes new genes required for efficient fasciation of host plants.

    PubMed Central

    Crespi, M; Vereecke, D; Temmerman, W; Van Montagu, M; Desomer, J

    1994-01-01

    Three virulence loci (fas, att, and hyp) of Rhodococcus fascians D188 have been identified on a 200-kb conjugative linear plasmid (pFiD188). The fas locus was delimited to a 6.5-kb DNA fragment by insertion mutagenesis, single homologous disruptive recombination, and in trans complementation of different avirulent insertion mutants. The locus is arranged as a large operon containing six open reading frames whose expression is specifically induced during the interaction with host plants. One predicted protein is homologous to P-450 cytochromes from actinomycetes. The putative ferredoxin component is of a novel type containing additional domains homologous to transketolases from chemoautotrophic, photosynthetic, and methylotrophic microorganisms. Genetic analysis revealed that fas encodes, in addition to the previously identified ipt, at least two new genes that are involved in fasciation development, one of which is only required on older tobacco plants. PMID:8169198

  5. High accuracy operon prediction method based on STRING database scores.

    PubMed

    Taboada, Blanca; Verde, Cristina; Merino, Enrique

    2010-07-01

    We present a simple and highly accurate computational method for operon prediction, based on intergenic distances and functional relationships between the protein products of contiguous genes, as defined by STRING database (Jensen,L.J., Kuhn,M., Stark,M., Chaffron,S., Creevey,C., Muller,J., Doerks,T., Julien,P., Roth,A., Simonovic,M. et al. (2009) STRING 8-a global view on proteins and their functional interactions in 630 organisms. Nucleic Acids Res., 37, D412-D416). These two parameters were used to train a neural network on a subset of experimentally characterized Escherichia coli and Bacillus subtilis operons. Our predictive model was successfully tested on the set of experimentally defined operons in E. coli and B. subtilis, with accuracies of 94.6 and 93.3%, respectively. As far as we know, these are the highest accuracies ever obtained for predicting bacterial operons. Furthermore, in order to evaluate the predictable accuracy of our model when using an organism's data set for the training procedure, and a different organism's data set for testing, we repeated the E. coli operon prediction analysis using a neural network trained with B. subtilis data, and a B. subtilis analysis using a neural network trained with E. coli data. Even for these cases, the accuracies reached with our method were outstandingly high, 91.5 and 93%, respectively. These results show the potential use of our method for accurately predicting the operons of any other organism. Our operon predictions for fully-sequenced genomes are available at http://operons.ibt.unam.mx/OperonPredictor/. PMID:20385580

  6. cAMP Regulation of the lactose operon.

    PubMed

    Szeberenyi, Jozsef

    2004-05-01

    Terms to be familiar with before you start to solve the test: lactose operon, adenylate cyclase, cAMP, catabolite activator protein (CAP), expression plasmid, lac operator, lac repressor, lactose, glucose, promoter, cis- and trans-acting factors. PMID:21706723

  7. Evolution and Biophysics of the Escherichia coli lac Operon

    NASA Astrophysics Data System (ADS)

    Ray, J. Christian; Igoshin, Oleg; Quan, Selwyn; Monds, Russell; Cooper, Tim; Balázsi, Gábor

    2011-03-01

    To understand, predict, and control the evolution of living organisms, we consider biophysical effects and molecular network architectures. The lactose utilization system of E. coli is among the most well-studied molecular networks in biology, making it an ideal candidate for such studies. Simulations show how the genetic architecture of the wild-type operon attenuates large metabolic intermediate fluctuations that are predicted to occur in an equivalent system with the component genes on separate operons. Quantification of gene expression in the lac operon evolved in growth conditions containing constant lactose, alternating with glucose, or constant glucose, shows characteristic gene expression patterns depending on conditions. We are simulating these conditions to show context-dependent biophysical sources and costs of different lac operon architectures.

  8. Virulence regulation in Citrobacter rodentium: the art of timing.

    PubMed

    Yang, Ji; Tauschek, Marija; Hart, Emily; Hartland, Elizabeth L; Robins-Browne, Roy M

    2010-05-01

    The mouse enteric pathogen Citrobacter rodentium, like its human counterpart, enteropathogenic Escherichia coli, causes attaching and effacing lesions in the intestinal epithelium of its host. This phenotype requires virulence factors encoded by the locus for enterocyte effacement (LEE) pathogenicity island. For timely expression of these virulence determinants at the site of infection and for efficient delivery of some virulence factors into epithelial cells, C. rodentium utilizes a positive regulatory loop involving the LEE-encoded regulatory proteins Ler, GrlA and GrlR to control LEE expression. Several transcription factors not encoded by LEE, some of which respond to specific environmental signals, also participate in this regulatory loop. Recently, we identified a non-LEE encoded, AraC-like regulatory protein, RegA, which plays a key role in the ability of C. rodentium to colonize the intestine. RegA functions by activating the transcription of a number of horizontally acquired operons encoding virulence-associated factors, such as autotransporters, fimbriae, a dispersin-like protein and its transporter. In addition, RegA represses transcription of a number of housekeeping genes. Importantly, RegA requires a gut-specific environmental signal, bicarbonate, to exert its effects on gene expression. In our proposed model, when C. rodentium senses bicarbonate ions in the gastrointestinal tract, RegA directs the bacterium to reduce the production of proteins involved in normal cellular functions, while enhancing the production of factors required for colonization and virulence.

  9. Expression of the Oligopeptide Permease Operon of Moraxella catarrhalis Is Regulated by Temperature and Nutrient Availability

    PubMed Central

    Jones, Megan M.

    2015-01-01

    Moraxella catarrhalis causes otitis media in children and exacerbations of chronic obstructive pulmonary disease in adults. Together, these two conditions contribute to enormous morbidity and mortality worldwide. The oligopeptide permease (opp) ABC transport system is a nutritional virulence factor important for the utilization of peptides. The substrate binding protein OppA, which binds peptides for uptake, is a potential vaccine antigen, but little was known about the regulation of gene expression. The five opp genes oppB, oppC, oppD, oppF, and oppA are in the same open reading frame. Sequence analysis predicted two promoters, one located upstream of oppB and one within the intergenic region between oppF and oppA. We have characterized the gene cluster as an operon with two functional promoters and show that cold shock at 26°C for ≤0.5 h and the presence of a peptide substrate increase gene transcript levels. Additionally, the putative promoter upstream of oppA contributes to the transcription of oppA but is not influenced by the same environmental cues as the promoter upstream of oppB. We conclude that temperature and nutrient availability contribute to the regulation of the Opp system, which is an important nutritional virulence factor in M. catarrhalis. PMID:26099587

  10. yadBC of Yersinia pestis, a New Virulence Determinant for Bubonic Plague▿ †

    PubMed Central

    Forman, Stanislav; Wulff, Christine R.; Myers-Morales, Tanya; Cowan, Clarissa; Perry, Robert D.; Straley, Susan C.

    2008-01-01

    In all Yersinia pestis strains examined, the adhesin/invasin yadA gene is a pseudogene, yet Y. pestis is invasive for epithelial cells. To identify potential surface proteins that are structurally and functionally similar to YadA, we searched the Y. pestis genome for open reading frames with homology to yadA and found three: the bicistronic operon yadBC (YPO1387 and YPO1388 of Y. pestis CO92; y2786 and y2785 of Y. pestis KIM5), which encodes two putative surface proteins, and YPO0902, which lacks a signal sequence and likely is nonfunctional. In this study we characterized yadBC regulation and tested the importance of this operon for Y. pestis adherence, invasion, and virulence. We found that loss of yadBC caused a modest loss of invasiveness for epithelioid cells and a large decrease in virulence for bubonic plague but not for pneumonic plague in mice. PMID:18025093

  11. [Evolutionary engineering in Salmonella: emergence of hybrid virulence-resistance plasmids in non-typhoid serotypes].

    PubMed

    Mendoza, María Del Carmen; Herrero, Ana; Rodicio, María Rosario

    2009-01-01

    An example of evolutive engineering in bacterial pathogens is the emergence of hybrid virulence-resistance (VR) plasmids in Salmonella enterica, resulting from an association between antimicrobial resistance determinants and specific virulence plasmids of the S. typhimurium and S. choleraesuis serotypes. VR plasmids all possess the spv (Salmonella plasmid virulence) operon, which is involved in systemic infection; however, they differ in the presence of other virulence determinants and in the resistance gene profile. VR plasmids of S. typhimurium have been found in Europe, and show resistance regions with different levels of complexity that can include class 1 integrons and various transposons. VR plasmids of S. choleraesuis, detected in strains isolated in Taiwan, only confer resistance to ampicillin and sulfonamides. Both serotypes are zoonotic and the presence of hybrid VR plasmids may confer an adaptive advantage under certain conditions, resulting in bacterial strains that are more difficult to treat and have a higher epidemic potential.

  12. Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains.

    PubMed Central

    Steck, T R; Kado, C I

    1990-01-01

    Certain virulence region operons of the Agrobacterium tumefaciens Ti plasmid promoted conjugative Ti plasmid transfer. Mutations in the vir region of pTiC58 inhibited conjugative plasmid transfer between A. tumefaciens strains. Mutations in virA, virG, 5' virB, and virE had the greatest effect on plasmid transfer, and mutations in virC had no effect. Transfer inhibition in vir mutants occurred in the presence or absence of acetosyringone. PMID:2318813

  13. The protein burden of lac operon products.

    PubMed

    Koch, A L

    1983-01-01

    A new approach to measuring the slowing of growth due to the manufacture of proteins not needed by a bacterium is presented. An entire single colony of Escherichia coli was used to start a chemostat culture that was then given a selective pressure by the addition of phenylgalactoside (phi-gal). This enriched the population for constitutive mutants that produced beta-galactosidase without induction and could split phi-gal, consume the galactose, and grow faster. When the phi-gal was removed, the constitutives grew slower than the parental strain and were gradually lost. This procedure allows competition experiments to be carried out with minimum effects due to genetic drift. Experiments with both strains having wild-type and mutant permease genes were conducted. With the former the selective disadvantage was initially much greater than expected from the simplest hypothesis that extra unused proteins would slow growth in proportion to their fraction of the total protein synthesis. This phase was followed by a second phase where the selective disadvantage was smaller than predicted by this simple hypothesis. With a very slowly reverting permease negative strain the selective disadvantage, and therefore the protein burden, was found to be much smaller and not statistically different from zero. Thus, while one would expect under carbon and energy limitation in the chemostat the protein burden to be larger than under unlimited conditions, it is so small that even the refined technique used here could not measure it accurately. It is certainly less than the fraction of 'waste' protein synthesis; but it could be between zero and the fraction of the cells' energy and carbon budget spent on manufacture of the proteins of the lac operon. PMID:6361271

  14. Contribution of YopB to virulence of Yersinia enterocolitica.

    PubMed

    Hartland, E L; Bordun, A M; Robins-Browne, R M

    1996-06-01

    The 70-kb virulence plasmid, pYV, of Yersinia enterocolitica encodes a number of secreted proteins (Yops) which are essential for virulence. YopD, the 33-kDa product of the lcrGVHyopBD operon, appears to be involved in delivering YopE and YopH (the Yersinia protein tyrosine phosphatase) into target cells. These proteins then act in concert to cause cytotoxicity in host cells. Previously, we reported that bacteria carrying transposon insertions in yopD are not cytotoxic for macrophages, show impaired tyrosine phosphatase activity in host cells, and are avirulent for mice (E. L. Hartland, S. P. Green, W. A. Phillips, and R. M. Robins-Browne, Infect. Immun. 62:4445-4453, 1994). trans complementation of yopD mutants of Y. enterocolitica with the yopD gene restores all these properties. In this study, we show that polar mutations in proximal genes of the lcrGVHyopBD operon also abrogated bacterial virulence and the capacity to induce cytotoxicity in mouse bone marrow-derived macrophages and HEp-2 epithelial cells. Moreover, trans complementation of a yopBD mutant with the yopD gene alone was not sufficient to restore the ability of the bacteria to cause cytotoxicity. Further work showed that YopB was required for cytotoxicity, dephosphorylation of host proteins, and virulence for mice. These findings indicate that YopB and YopD may serve a related function in Y. enterocolitica and that they may act together to deliver intracellularly acting Yops to their respective targets in host cells.

  15. Contribution of YopB to virulence of Yersinia enterocolitica.

    PubMed Central

    Hartland, E L; Bordun, A M; Robins-Browne, R M

    1996-01-01

    The 70-kb virulence plasmid, pYV, of Yersinia enterocolitica encodes a number of secreted proteins (Yops) which are essential for virulence. YopD, the 33-kDa product of the lcrGVHyopBD operon, appears to be involved in delivering YopE and YopH (the Yersinia protein tyrosine phosphatase) into target cells. These proteins then act in concert to cause cytotoxicity in host cells. Previously, we reported that bacteria carrying transposon insertions in yopD are not cytotoxic for macrophages, show impaired tyrosine phosphatase activity in host cells, and are avirulent for mice (E. L. Hartland, S. P. Green, W. A. Phillips, and R. M. Robins-Browne, Infect. Immun. 62:4445-4453, 1994). trans complementation of yopD mutants of Y. enterocolitica with the yopD gene restores all these properties. In this study, we show that polar mutations in proximal genes of the lcrGVHyopBD operon also abrogated bacterial virulence and the capacity to induce cytotoxicity in mouse bone marrow-derived macrophages and HEp-2 epithelial cells. Moreover, trans complementation of a yopBD mutant with the yopD gene alone was not sufficient to restore the ability of the bacteria to cause cytotoxicity. Further work showed that YopB was required for cytotoxicity, dephosphorylation of host proteins, and virulence for mice. These findings indicate that YopB and YopD may serve a related function in Y. enterocolitica and that they may act together to deliver intracellularly acting Yops to their respective targets in host cells. PMID:8675342

  16. Boolean models can explain bistability in the lac operon.

    PubMed

    Veliz-Cuba, Alan; Stigler, Brandilyn

    2011-06-01

    The lac operon in Escherichia coli has been studied extensively and is one of the earliest gene systems found to undergo both positive and negative control. The lac operon is known to exhibit bistability, in the sense that the operon is either induced or uninduced. Many dynamical models have been proposed to capture this phenomenon. While most are based on complex mathematical formulations, it has been suggested that for other gene systems network topology is sufficient to produce the desired dynamical behavior. We present a Boolean network as a discrete model for the lac operon. Our model includes the two main glucose control mechanisms of catabolite repression and inducer exclusion. We show that this Boolean model is capable of predicting the ON and OFF steady states and bistability. Further, we present a reduced model which shows that lac mRNA and lactose form the core of the lac operon, and that this reduced model exhibits the same dynamics. This work suggests that the key to model qualitative dynamics of gene systems is the topology of the network and Boolean models are well suited for this purpose. PMID:21563979

  17. Cost-benefit tradeoffs in engineered lac operons.

    PubMed

    Eames, Matt; Kortemme, Tanja

    2012-05-18

    Cells must balance the cost and benefit of protein expression to optimize organismal fitness. The lac operon of the bacterium Escherichia coli has been a model for quantifying the physiological impact of costly protein production and for elucidating the resulting regulatory mechanisms. We report quantitative fitness measurements in 27 redesigned operons that suggested that protein production is not the primary origin of fitness costs. Instead, we discovered that the lac permease activity, which relates linearly to cost, is the major physiological burden to the cell. These findings explain control points in the lac operon that minimize the cost of lac permease activity, not protein expression. Characterizing similar relationships in other systems will be important to map the impact of cost/benefit tradeoffs on cell physiology and regulation. PMID:22605776

  18. Genome Data from DOOR: a Database for prOkaryotic OpeRons

    DOE Data Explorer

    DOOR (Database of prOkaryotic OpeRons) is an operon database developed by Computational Systems Biology Lab (CSBL) at University of Georgia. Although the operons in the database are based on prediction, there are some unique features. These are: • A algorithm is consistently best at all aspects including sensitivity and specificity for both true positives and true negatives, and the overall accuracy reaches 90 percent. The prediction algorithm is based on this paper: P. Dam, V. Olman, K. Harris, Z. Su, Y. Xu., Operon prediction using both genome-specific and general genomic information, Nucleic Acids Res., 35(1):288-98, 2007 • DOOR provides one of the largest data sets of operon information available to the public. DOOR provides operons for 675 prokaryotic genomes. Although most of operons in DOOR are not verified by experiments, the creators are also trying to provide some limited literature information, which is extracted from ODB. They emphasize that if the users are looking for strictly experimentally verified operons, they should look into DBTBS and RegulonDB first. • Operons which include RNA genes, which are rarely seen in other operon databases especially for predicted operon databases • Defined the similarity scores between operons, which is based on weighted maximum matching between operons. Similar operon groups can be used to predict accurate orthologous genes,and their upstream regions can be used to find the consensus binding motifs. • Integration of two motif finding programs in the database: MEME and CUBIC. DOOR provides an Organism View for browsing, a gene search tool, an operon search tool, and the operon prediction interface.[Text taken and edited from http://csbl1.bmb.uga.edu/OperonDB/tutorial.php

  19. Operon Formation is Driven by Co-Regulation and Not by Horizontal Gene Transfer

    SciTech Connect

    Price, Morgan N.; Huang, Katherine H.; Arkin, Adam P.; Alm, Eric J.

    2005-04-12

    Although operons are often subject to horizontal gene transfer (HGT), non-HGT genes are particularly likely to be in operons. To resolve this apparent discrepancy and to determine whether HGT is involved in operon formation, we examined the evolutionary history of the genes and operons in Escherichia coli K12. We show that genes that have homologs in distantly related bacteria but not in close relatives of E. coli (indicating HGTi) form new operons at about the same rates as native genes. Furthermore, genes in new operons are no more likely than other genes to have phylogenetic trees that are inconsistent with the species tree. In contrast, essential genes and ubiquitous genes without paralogs (genes believed to undergo HGT rarely) often form new operons. We conclude that HGT is not associated with operon formation, but instead promotes the prevalence of pre-existing operons. To explain operon formation, we propose that new operons reduce the amount of regulatory information required to specify optimal expression patterns. Consistent with this hypothesis, operons have greater amounts of conserved regulatory sequences than do individually transcribed genes.

  20. Characterization of the Cobalamin and Fep Operons in Methylobium petrolphilum PM1

    SciTech Connect

    Ewing, J

    2005-09-06

    The bacterium Methylobium petroleophilum PM1 is economically important due to its ability to degrade methyl tert-butyl ether (MTBE), a fuel additive. Because PM1 is a representative of all MTBE degraders, it is important to understand the transport pathways critical for the organism to survive in its particular environment. In this study, the cobalamin pathway and select iron transport genes will be characterized to help further understand all metabolic pathways in PM1. PM1 contains a total of four cobalamin operons. A single operon is located on the chromosome. Located on the megaplasmid are two tandem repeats of cob operons and a very close representative of the cob operon located on the chromosome. The fep operon, an iron transport mechanism, lies within the multiple copies of the cob operon. The cob operon and the fep operon appear to be unrelated except for a shared need for the T-on-B-dependent energy transduction complex to assist the operons in moving large molecules across the outer membrane of the cell. A genomic study of the cob and the fep operons with that of phylogenetically related organisms helped to confirm the identity of the cob and fep operons and to represent the pathways. More study of the pathways should be done to find the relationship that positions the two seemingly unrelated cob and fep genes together in what appears to be one operon.

  1. Development of a Lac Operon Concept Inventory (LOCI)

    PubMed Central

    Stefanski, Katherine M.; Gardner, Grant E.; Seipelt-Thiemann, Rebecca L.

    2016-01-01

    Concept inventories (CIs) are valuable tools for educators that assess student achievement and identify misconceptions held by students. Results of student responses can be used to adjust or develop new instructional methods for a given topic. The regulation of gene expression in both prokaryotes and eukaryotes is an important concept in genetics and one that is particularly challenging for undergraduate students. As part of a larger study examining instructional methods related to gene regulation, the authors developed a 12-item CI assessing student knowledge of the lac operon. Using an established protocol, the authors wrote open-ended questions and conducted in-class testing with undergraduate microbiology and genetics students to discover common errors made by students about the lac operon and to determine aspects of item validity. Using these results, we constructed a 12-item multiple-choice lac operon CI called the Lac Operon Concept Inventory (LOCI), The LOCI was reviewed by two experts in the field for content validity. The LOCI underwent item analysis and was assessed for reliability with a sample of undergraduate genetics students (n = 115). The data obtained were found to be valid and reliable (coefficient alpha = 0.994) with adequate discriminatory power and item difficulty. PMID:27252300

  2. Development of a Lac Operon Concept Inventory (LOCI).

    PubMed

    Stefanski, Katherine M; Gardner, Grant E; Seipelt-Thiemann, Rebecca L

    2016-01-01

    Concept inventories (CIs) are valuable tools for educators that assess student achievement and identify misconceptions held by students. Results of student responses can be used to adjust or develop new instructional methods for a given topic. The regulation of gene expression in both prokaryotes and eukaryotes is an important concept in genetics and one that is particularly challenging for undergraduate students. As part of a larger study examining instructional methods related to gene regulation, the authors developed a 12-item CI assessing student knowledge of the lac operon. Using an established protocol, the authors wrote open-ended questions and conducted in-class testing with undergraduate microbiology and genetics students to discover common errors made by students about the lac operon and to determine aspects of item validity. Using these results, we constructed a 12-item multiple-choice lac operon CI called the Lac Operon Concept Inventory (LOCI), The LOCI was reviewed by two experts in the field for content validity. The LOCI underwent item analysis and was assessed for reliability with a sample of undergraduate genetics students (n = 115). The data obtained were found to be valid and reliable (coefficient alpha = 0.994) with adequate discriminatory power and item difficulty. PMID:27252300

  3. Characterization of the virE operon of the Agrobacterium Ti plasmid pTiA6.

    PubMed

    Winans, S C; Allenza, P; Stachel, S E; McBride, K E; Nester, E W

    1987-01-26

    The Agrobacterium tumefaciens Ti plasmid contains at least six transcriptional units (designated vir loci) which are essential for efficient crown gall tumorigenesis. Mutations in one of these loci, virE, result in a sharply attenuated virulence phenotype. In the present communication, we have analyzed the virE operon at the molecular level. This locus contains open reading frames coding for two hydrophilic proteins having molecular weights of approximately 7,000 daltons and 60,500 daltons. Using a maxicell strain of E. coli, we have visualized two proteins encoded by virE which correspond in size to these open reading frames. Analysis of codon usage of virE and seven other vir loci indicates that, in contrast to E. coli, all possible codons for a given amino acid are utilized at approximately the same frequency. PMID:3547330

  4. Identification and characterization of an operon of Helicobacter pylori that is involved in motility and stress adaptation.

    PubMed Central

    Beier, D; Spohn, G; Rappuoli, R; Scarlato, V

    1997-01-01

    We identified a novel stress-responsive operon (sro) of Helicobacter pylori that contains seven genes which are likely to be involved in cellular functions as diverse as chemotaxis, heat shock response, ion transport, and posttranslational protein modification. The products of three of these genes show amino acid homologies to known proteins, such as the flagellar motor switch protein CheY, a class of heat shock proteins, and the ribosomal protein L11 methyltransferase, and to a phosphatidyltransferase. In addition to containing an open reading frame of unknown function, the product of which is predicted to be membrane associated, the sro locus contains three open reading frames that have previously been described as constituting two separate loci, the ftsH gene and the copAP operon of H. pylori. Knockout mutants showed that CheY is essential for bacterial motility and that CopA, but not CopP, relieves copper toxicity. Transcriptional analyses indicated that this locus is regulated by a single promoter and that a positive effect on transcription is exerted by the addition of copper to the medium and by temperature upshift from 37 to 45 degrees C. The possible role of this locus in H. pylori virulence is discussed. PMID:9244252

  5. LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

    PubMed Central

    Sycz, Gabriela; Carrica, Mariela Carmen; Tseng, Tong-Seung; Bogomolni, Roberto A.; Briggs, Winslow R.; Goldbaum, Fernando A.; Paris, Gastón

    2015-01-01

    Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms. PMID:25993430

  6. LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus.

    PubMed

    Sycz, Gabriela; Carrica, Mariela Carmen; Tseng, Tong-Seung; Bogomolni, Roberto A; Briggs, Winslow R; Goldbaum, Fernando A; Paris, Gastón

    2015-01-01

    Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms.

  7. Transcription of the Streptococcus pyogenes hyaluronic acid capsule biosynthesis operon is regulated by previously unknown upstream elements.

    PubMed

    Falaleeva, Marina; Zurek, Oliwia W; Watkins, Robert L; Reed, Robert W; Ali, Hadeel; Sumby, Paul; Voyich, Jovanka M; Korotkova, Natalia

    2014-12-01

    The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence.

  8. Transcription of the Streptococcus pyogenes Hyaluronic Acid Capsule Biosynthesis Operon Is Regulated by Previously Unknown Upstream Elements

    PubMed Central

    Falaleeva, Marina; Zurek, Oliwia W.; Watkins, Robert L.; Reed, Robert W.; Ali, Hadeel; Sumby, Paul; Voyich, Jovanka M.

    2014-01-01

    The important human pathogen Streptococcus pyogenes (group A Streptococcus [GAS]) produces a hyaluronic acid (HA) capsule that plays critical roles in immune evasion. Previous studies showed that the hasABC operon encoding the capsule biosynthesis enzymes is under the control of a single promoter, P1, which is negatively regulated by the two-component regulatory system CovR/S. In this work, we characterize the sequence upstream of P1 and identify a novel regulatory region controlling transcription of the capsule biosynthesis operon in the M1 serotype strain MGAS2221. This region consists of a promoter, P2, which initiates transcription of a novel small RNA, HasS, an intrinsic transcriptional terminator that inefficiently terminates HasS, permitting read-through transcription of hasABC, and a putative promoter which lies upstream of P2. Electrophoretic mobility shift assays, quantitative reverse transcription-PCR, and transcriptional reporter data identified CovR as a negative regulator of P2. We found that the P1 and P2 promoters are completely repressed by CovR, and capsule expression is regulated by the putative promoter upstream of P2. Deletion of hasS or of the terminator eliminates CovR-binding sequences, relieving repression and increasing read-through, hasA transcription, and capsule production. Sequence analysis of 44 GAS genomes revealed a high level of polymorphism in the HasS sequence region. Most of the HasS variations were located in the terminator sequences, suggesting that this region is under strong selective pressure. We discovered that the terminator deletion mutant is highly resistant to neutrophil-mediated killing and is significantly more virulent in a mouse model of GAS invasive disease than the wild-type strain. Together, these results are consistent with the naturally occurring mutations in this region modulating GAS virulence. PMID:25287924

  9. Requirement of norD for Brucella suis Virulence in a Murine Model of In Vitro and In Vivo Infection

    PubMed Central

    Loisel-Meyer, Séverine; Jiménez de Bagüés, Maria Pilar; Bassères, Eugénie; Dornand, Jacques; Köhler, Stephan; Liautard, Jean-Pierre; Jubier-Maurin, Véronique

    2006-01-01

    A mutant of Brucella suis bearing a Tn5 insertion in norD, the last gene of the operon norEFCBQD, encoding nitric oxide reductase, was unable to survive under anaerobic denitrifying conditions. The norD strain exhibited attenuated multiplication within nitric oxide-producing murine macrophages and rapid elimination in mice, hence demonstrating that norD is essential for Brucella virulence. PMID:16495577

  10. A mutation in tdcA attenuates the virulence of Salmonella enterica serovar Typhimurium.

    PubMed

    Lim, Sangyong; Kim, Minjeong; Choi, Jeongjoon; Ryu, Sangryeol

    2010-05-01

    The Salmonella tdc operon encodes enzymes belonging to a metabolic pathway that degrades L-serine and L-threonine. The upregulation of the tdc operon and increased virulence of Salmonella grown under oxygen-limiting conditions prompted us to investigate the role of the tdc operon in the pathogenesis of Salmonella Typhimurium. A Salmonella strain carrying a null mutation in tdcA, which encodes the transcriptional activator of the tdc operon, was impaired in mice infected intraperitoneally with the bacterium. In addition, the Salmonella tdcA mutant showed reduced replication compared with the parental strain in cultured animal cells, although their growth rates were similar in various culture media. To understand the function of TdcA in pathogenesis, we performed two-dimensional gel electrophoresis and found that flagellar and PhoP-regulated proteins were affected by the tdcA mutation. The results of beta-galactosidase assays and FACS analysis showed that, among the four PhoP-dependent genes tested, the expression of ssaG, which is located in Salmonella pathogenicity island 2 (SPI2), was reduced in the tdcA mutant, especially in the intracellular environment of macrophages. Taken together, our data suggest that tdcA plays an important role in the pathogenesis of Salmonella.

  11. Regulation of the bgl operon of Escherichia coli by transcriptional antitermination.

    PubMed Central

    Schnetz, K; Rak, B

    1988-01-01

    The bgl operon of Escherichia coli encodes all functions necessary for the regulated uptake and utilization of aryl beta-glucosides. The operon is unusual, however, in that it is cryptic in wild-type strains, requiring activation by mutational events. The vast majority of these mutations are due to transposition of insertion elements into the promoter region of the operon. In this report we show that integration of IS5 into the vicinity of the bgl promoter (P0) enhances its activity by greater than 60-fold thereby activating the operon. In the activated state the operon is subject to induction by substrate. Recent studies have shown that induction of the bgl operon by substrate involves antitermination within the leader of the operon. We now show that substrate-dependent regulation involves specific termination/antitermination of transcription at two signal structures flanking the first gene of the operon, bglG. Antitermination is mediated by the product of gene bglG. In the absence of substrate this antitermination is prevented by the action of the product of gene bglF (the second gene of the operon), which encodes the beta-glucoside-specific transport protein (enzymeIIBgl of the phosphoenolpyruvate-dependent phosphotransferase system, PTS) resulting in repression of the operon. The bgl promoter (P0) is not subject to substrate-dependent regulation. The bgl operon has two additional promoters (P1 and P2) located within the terminators, which could also participate in regulation. Images PMID:2846278

  12. Cryptosporidium Pathogenicity and Virulence

    PubMed Central

    Bouzid, Maha; Chalmers, Rachel M.; Tyler, Kevin M.

    2013-01-01

    Cryptosporidium is a protozoan parasite of medical and veterinary importance that causes gastroenteritis in a variety of vertebrate hosts. Several studies have reported different degrees of pathogenicity and virulence among Cryptosporidium species and isolates of the same species as well as evidence of variation in host susceptibility to infection. The identification and validation of Cryptosporidium virulence factors have been hindered by the renowned difficulties pertaining to the in vitro culture and genetic manipulation of this parasite. Nevertheless, substantial progress has been made in identifying putative virulence factors for Cryptosporidium. This progress has been accelerated since the publication of the Cryptosporidium parvum and C. hominis genomes, with the characterization of over 25 putative virulence factors identified by using a variety of immunological and molecular techniques and which are proposed to be involved in aspects of host-pathogen interactions from adhesion and locomotion to invasion and proliferation. Progress has also been made in the contribution of host factors that are associated with variations in both the severity and risk of infection. Here we provide a review comprised of the current state of knowledge on Cryptosporidium infectivity, pathogenesis, and transmissibility in light of our contemporary understanding of microbial virulence. PMID:23297262

  13. Parasitoid wasp virulence

    PubMed Central

    Mortimer, Nathan T

    2013-01-01

    In nature, larvae of the fruit fly Drosophila melanogaster are commonly infected by parasitoid wasps. Following infection, flies mount an immune response termed cellular encapsulation in which fly immune cells form a multilayered capsule that covers and kills the wasp egg. Parasitoids have thus evolved virulence factors to suppress cellular encapsulation. To uncover the molecular mechanisms underlying the antiwasp response, we and others have begun identifying and functionally characterizing these virulence factors. Our recent work on the Drosophila parasitoid Ganaspis sp.1 has demonstrated that a virulence factor encoding a SERCA-type calcium pump plays an important role in Ganaspis sp.1 virulence. This venom SERCA antagonizes fly immune cell calcium signaling and thereby prevents the activation of the encapsulation response. In this way, the study of wasp virulence factors has revealed a novel aspect of fly immunity, namely a role for calcium signaling in fly immune cell activation, which is conserved with human immunity, again illustrating the marked conservation between fly and mammalian immune responses. Our findings demonstrate that the cellular encapsulation response can serve as a model of immune cell function and can also provide valuable insight into basic cell biological processes. PMID:24088661

  14. Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae.

    PubMed

    Manzoor, Irfan; Shafeeq, Sulman; Afzal, Muhammad; Kuipers, Oscar P

    2015-01-01

    In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5'-ATTTGAACATTATTCAAGT-3') in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon.

  15. Modelling, property verification and behavioural equivalence of lactose operon regulation.

    PubMed

    Pinto, Marcelo Cezar; Foss, Luciana; Mombach, José Carlos Merino; Ribeiro, Leila

    2007-02-01

    Understanding biochemical pathways is one of the biggest challenges in the field of molecular biology nowadays. Computer science can contribute in this area by providing formalisms and tools to simulate and analyse pathways. One formalism that is suited for modelling concurrent systems is Milner's Calculus of Communicating Systems (CCS). This paper shows the viability of using CCS to model and reason about biochemical networks. As a case study, we describe the regulation of lactose operon. After describing this operon formally using CCS, we validate our model by automatically checking some known properties for lactose regulation. Moreover, since biological systems tend to be very complex, we propose to use multiple descriptions of the same system at different levels of abstraction. The compatibility of these multiple views can be assured via mathematical proofs of observational equivalence. PMID:16620804

  16. Selection and Neutrality in Lactose Operons of Escherichia Coli

    PubMed Central

    Dean, A. M.

    1989-01-01

    The kinetics of the permeases and β-galactosidases of six lactose operons which had been transduced into a common genetic background from natural isolates of Escherichia coli were investigated. The fitnesses conferred by the operons were determined using chemostat competition experiments in which lactose was the sole growth-limiting factor. The cell wall is demonstrated to impose a resistance to the diffusion of galactosides at low substrate concentrations. A steady state model of the flux of lactose through the metabolic pathway (diffusion, uptake and hydrolysis) is shown to be proportional to fitness. This metabolic model is used to explain why an approximately twofold range in activity among the permease alleles confers a 13% range in fitness, whereas a similar range in activity among alleles of the β-galactosidase confers a 0.5% range in fitness. This metabolic model implies that selection need not be maximized when a resource is scarce. PMID:2513251

  17. Gene organization and structure of the Streptomyces lividans gal operon.

    PubMed Central

    Adams, C W; Fornwald, J A; Schmidt, F J; Rosenberg, M; Brawner, M E

    1988-01-01

    We present the gene organization and DNA sequence of the Streptomyces lividans galactose utilization genes. Complementation of Escherichia coli galE, galT, or galK mutants and DNA sequence analysis were used to demonstrate that the galactose utilization genes are organized within an operon with the gene order galT, galE, and galK. Comparison of the inferred protein sequences for the S. lividans gal gene products to the corresponding E. coli and Saccharomyces carlbergensis sequences identified regions of structural homology within each of the galactose utilization enzymes. Finally, we discuss a potential relationship between the gene organization of the operon and the functional roles of the gal enzymes in cellular metabolism. Images PMID:3335481

  18. Evolution of bacterial trp operons and their regulation.

    PubMed

    Merino, Enrique; Jensen, Roy A; Yanofsky, Charles

    2008-04-01

    Survival and replication of most bacteria require the ability to synthesize the amino acid L-tryptophan whenever it is not available from the environment. In this article we describe the genes, operons, proteins, and reactions involved in tryptophan biosynthesis in bacteria, and the mechanisms they use in regulating tryptophan formation. We show that although the reactions of tryptophan biosynthesis are essentially identical, gene organization varies among species--from whole-pathway operons to completely dispersed genes. We also show that the regulatory mechanisms used for these genes vary greatly. We address the question--what are some potential advantages of the gene organization and regulation variation associated with this conserved, important pathway? PMID:18374625

  19. The Salmonella typhimurium mar locus: molecular and genetic analyses and assessment of its role in virulence.

    PubMed Central

    Sulavik, M C; Dazer, M; Miller, P F

    1997-01-01

    The marRAB operon is a regulatory locus that controls multiple drug resistance in Escherichia coli. marA encodes a positive regulator of the antibiotic resistance response, acting by altering the expression of unlinked genes. marR encodes a repressor of marRAB transcription and controls the production of MarA in response to environmental signals. A molecular and genetic study of the homologous operon in Salmonella typhimurium was undertaken, and the role of marA in virulence in a murine model was assessed. Expression of E. coli marA (marAEC) present on a multicopy plasmid in S. typhimurium resulted in a multiple antibiotic resistance (Mar) phenotype, suggesting that a similar regulon exists in this organism. A genomic plasmid library containing S. typhimurium chromosomal sequences was introduced into an E. coli strain that was deleted for the mar locus and contained a single-copy marR'-'lacZ translational fusion. Plasmid clones that contained both S. typhimurium marR (marRSt) and marA (marASt) genes were identified as those that were capable of repressing expression of the fusion and which resulted in a Mar phenotype. The predicted amino acid sequences of MarRSt, MarASt, and MarBSt were 91, 86, and 42% identical, respectively, to the same genes from E. coli, while the operator/promoter region of the operon was 86% identical to the same 98-nucleotide-upstream region in E. coli. The marRAB transcriptional start sites for both organisms were determined by primer extension, and a marRABSt transcript of approximately 1.1 kb was identified by Northern blot analysis. Its accumulation was shown to be inducible by sodium salicylate. Open reading frames flanking the marRAB operon were also conserved. An S. typhimurium marA disruption strain was constructed by an allelic exchange method and compared to the wild-type strain for virulence in a murine BALB/c infection model. No effect on virulence was noted. The endogenous S. typhimurium plasmid that is associated with virulence

  20. ArgR is an essential local transcriptional regulator of the arcABC operon in Streptococcus suis and is crucial for biological fitness in an acidic environment.

    PubMed

    Fulde, Marcus; Willenborg, Joerg; de Greeff, Astrid; Benga, Laurentiu; Smith, Hilde E; Valentin-Weigand, Peter; Goethe, Ralph

    2011-02-01

    Streptococcus suis is one of the most important pathogens in pigs and can also cause severe infections in humans. Despite its clinical relevance, very little is known about the factors that contribute to its virulence. Recently, we identified a new putative virulence factor in S. suis, the arginine deiminase system (ADS), an arginine catabolic enzyme system encoded by the arcABC operon, which enables S. suis to survive in an acidic environment. In this study, we focused on ArgR, an ADS-associated regulator belonging to the ArgR/AhrC arginine repressor family. Using an argR knockout strain we were able to show that ArgR is essential for arcABC operon expression and necessary for the biological fitness of S. suis. By cDNA expression microarray analyses and quantitative real-time RT-PCR we found that the arcABC operon is the only gene cluster regulated by ArgR, which is in contrast to the situation in many other bacteria. Reporter gene analysis with gfp under the control of the arcABC promoter demonstrated that ArgR is able to activate the arcABC promoter. Electrophoretic mobility shift assays with fragments of the arcABC promoter and recombinant ArgR, and chromatin immunoprecipitation with antibodies directed against ArgR, revealed that ArgR interacts with the arcABC promoter in vitro and in vivo by binding to a region from -147 to -72 bp upstream of the transcriptional start point. Overall, our results show that in S. suis, ArgR is an essential, system-specific transcriptional regulator of the ADS that interacts directly with the arcABC promoter in vivo.

  1. Promoter of the Mycoplasma pneumoniae rRNA operon.

    PubMed Central

    Hyman, H C; Gafny, R; Glaser, G; Razin, S

    1988-01-01

    RNA transcripts starting from the 5' end of the single Mycoplasma pneumoniae rRNA operon were analyzed by several methods. By primer extension analysis a start site was found 62 nucleotides upstream from the start site of the 16S rRNA. This site was preceded by a putative Pribnow box; however, a defined -35 recognition region was absent. The cloned rRNA operon was transcribed in vitro by using purified RNA polymerase of Escherichia coli. A single start site could be demonstrated within a few nucleotides of the start site found by primer extension analysis of M. pneumoniae transcripts. When fragments from the cloned operon were used as hybridization probes, S1 nuclease mapping yielded a single transcript extending approximately 193 nucleotides upstream from the 16S rRNA start site. The region surrounding this endpoint did not resemble any known promoter sequence. Dot blot hybridization of M. pneumoniae RNA to three oligonucleotides consisting of nucleotides -5 to -21, -38 to -54, and -112 to -132 (from the start of the 16S rRNA gene) indicated that most rRNA transcripts were processed at the stem site preceding the 16S rRNA gene. The majority of the longer precursor transcripts, extending beyond this point, did not extend further upstream to an oligonucleotide consisting of nucleotides -112 to -132. It was concluded that transcription of the rRNA operon of M. pneumoniae is initiated by a single promoter. The nucleotide sequence of the region is presented. Images PMID:2838465

  2. Elucidation of operon structures across closely related bacterial genomes.

    PubMed

    Zhou, Chuan; Ma, Qin; Li, Guojun

    2014-01-01

    About half of the protein-coding genes in prokaryotic genomes are organized into operons to facilitate co-regulation during transcription. With the evolution of genomes, operon structures are undergoing changes which could coordinate diverse gene expression patterns in response to various stimuli during the life cycle of a bacterial cell. Here we developed a graph-based model to elucidate the diversity of operon structures across a set of closely related bacterial genomes. In the constructed graph, each node represents one orthologous gene group (OGG) and a pair of nodes will be connected if any two genes, from the corresponding two OGGs respectively, are located in the same operon as immediate neighbors in any of the considered genomes. Through identifying the connected components in the above graph, we found that genes in a connected component are likely to be functionally related and these identified components tend to form treelike topology, such as paths and stars, corresponding to different biological mechanisms in transcriptional regulation as follows. Specifically, (i) a path-structure component integrates genes encoding a protein complex, such as ribosome; and (ii) a star-structure component not only groups related genes together, but also reflects the key functional roles of the central node of this component, such as the ABC transporter with a transporter permease and substrate-binding proteins surrounding it. Most interestingly, the genes from organisms with highly diverse living environments, i.e., biomass degraders and animal pathogens of clostridia in our study, can be clearly classified into different topological groups on some connected components.

  3. Structural characterization of the Salmonella typhimurium LT2 umu operon.

    PubMed Central

    Thomas, S M; Crowne, H M; Pidsley, S C; Sedgwick, S G

    1990-01-01

    The umuDC operon of Escherichia coli encodes functions required for mutagenesis induced by radiation and a wide variety of chemicals. The closely related organism Salmonella typhimurium is markedly less mutable than E. coli, but a umu homolog has recently been identified and cloned from the LT2 subline. In this study the nucleotide sequence and structure of the S. typhimurium LT2 umu operon have been determined and its gene products have been identified so that the molecular basis of umu activity might be understood more fully. S. typhimurium LT2 umu consists of a smaller 417-base-pair (bp) umuD gene ending 2 bp upstream of a larger 1,266-bp umuC gene. The only apparent structural difference between the two operons is the lack of gene overlap. An SOS box identical to that found in E. coli is present in the promoter region upstream of umuD. The calculated molecular masses of the umuD and umuC gene products were 15.3 and 47.8 kilodaltons, respectively, which agree with figures determined by transpositional disruption and maxicell analysis. The S. typhimurium and E. coli umuD sequences were 68% homologous and encoded products with 71% amino acid identity; the umuC sequences were 71% homologous and encoded products with 83% amino acid identity. Furthermore, the potential UmuD cleavage site and associated catalytic sites could be identified. Thus the very different mutagenic responses of S. typhimurium LT2 and E. coli cannot be accounted for by gross differences in operon structure or gene products. Rather, the ability of the cloned S. typhimurium umuD gene to give stronger complementation of E. coli umuD77 mutants in the absence of a functional umuC gene suggests that Salmonella UmuC protein normally constrains UmuD protein activity. Images PMID:2203737

  4. Elucidation of operon structures across closely related bacterial genomes.

    PubMed

    Zhou, Chuan; Ma, Qin; Li, Guojun

    2014-01-01

    About half of the protein-coding genes in prokaryotic genomes are organized into operons to facilitate co-regulation during transcription. With the evolution of genomes, operon structures are undergoing changes which could coordinate diverse gene expression patterns in response to various stimuli during the life cycle of a bacterial cell. Here we developed a graph-based model to elucidate the diversity of operon structures across a set of closely related bacterial genomes. In the constructed graph, each node represents one orthologous gene group (OGG) and a pair of nodes will be connected if any two genes, from the corresponding two OGGs respectively, are located in the same operon as immediate neighbors in any of the considered genomes. Through identifying the connected components in the above graph, we found that genes in a connected component are likely to be functionally related and these identified components tend to form treelike topology, such as paths and stars, corresponding to different biological mechanisms in transcriptional regulation as follows. Specifically, (i) a path-structure component integrates genes encoding a protein complex, such as ribosome; and (ii) a star-structure component not only groups related genes together, but also reflects the key functional roles of the central node of this component, such as the ABC transporter with a transporter permease and substrate-binding proteins surrounding it. Most interestingly, the genes from organisms with highly diverse living environments, i.e., biomass degraders and animal pathogens of clostridia in our study, can be clearly classified into different topological groups on some connected components. PMID:24959722

  5. Salmonella enterica Typhimurium fljBA operon stability: implications regarding the origin of Salmonella enterica I 4,[5],12:i:.

    PubMed

    Tomiyama, M P O; Werle, C H; Milanez, G P; Nóbrega, D B; Pereira, J P; Calarga, A P; Flores, F; Brocchi, M

    2015-01-01

    Salmonella enterica subsp enterica serovar 4,5,12:i:- has been responsible for many recent Salmonella outbreaks worldwide. Several studies indicate that this serovar originated from S. enterica subsp enterica serovar Typhimurium, by the loss of the flagellar phase II gene (fljB) and adjacent sequences. However, at least two different clones of S. enterica 4,5,12:i:- exist that differs in the molecular events responsible for fljB deletion. The aim of this study was to test the stability of the fljBA operon responsible for the flagellar phase variation under different growth conditions in order to verify if its deletion is a frequent event that could explain the origin and dissemination of this serovar. In fact, coding sequences for transposons are present near this operon and in some strains, such as S. enterica Typhimurium LT2, the Fels-2 prophage gene is inserted near this operon. The presence of mobile DNA could confer instability to this region. In order to examine this, the cat (chloramphenicol acetyltransferase) gene was inserted adjacent to the fljBA operon so that deletions involving this genomic region could be identified. After growing S. enterica chloramphenicol-resistant strains under different conditions, more than 104 colonies were tested for the loss of chloramphenicol resistance. However, none of the colonies were sensitive to chloramphenicol. These data suggest that the origin of S. enterica serovar 4,5,12:i:- from Typhimurium by fljBA deletion is not a frequent event. The origin and dissemination of 4,5,12:i:- raise several questions about the role of flagellar phase variation in virulence. PMID:26782556

  6. Salmonella enterica Typhimurium fljBA operon stability: implications regarding the origin of Salmonella enterica I 4,[5],12:i:.

    PubMed

    Tomiyama, M P O; Werle, C H; Milanez, G P; Nóbrega, D B; Pereira, J P; Calarga, A P; Flores, F; Brocchi, M

    2015-12-29

    Salmonella enterica subsp enterica serovar 4,5,12:i:- has been responsible for many recent Salmonella outbreaks worldwide. Several studies indicate that this serovar originated from S. enterica subsp enterica serovar Typhimurium, by the loss of the flagellar phase II gene (fljB) and adjacent sequences. However, at least two different clones of S. enterica 4,5,12:i:- exist that differs in the molecular events responsible for fljB deletion. The aim of this study was to test the stability of the fljBA operon responsible for the flagellar phase variation under different growth conditions in order to verify if its deletion is a frequent event that could explain the origin and dissemination of this serovar. In fact, coding sequences for transposons are present near this operon and in some strains, such as S. enterica Typhimurium LT2, the Fels-2 prophage gene is inserted near this operon. The presence of mobile DNA could confer instability to this region. In order to examine this, the cat (chloramphenicol acetyltransferase) gene was inserted adjacent to the fljBA operon so that deletions involving this genomic region could be identified. After growing S. enterica chloramphenicol-resistant strains under different conditions, more than 104 colonies were tested for the loss of chloramphenicol resistance. However, none of the colonies were sensitive to chloramphenicol. These data suggest that the origin of S. enterica serovar 4,5,12:i:- from Typhimurium by fljBA deletion is not a frequent event. The origin and dissemination of 4,5,12:i:- raise several questions about the role of flagellar phase variation in virulence.

  7. Identification of the Bacillus subtilis pur operon repressor.

    PubMed

    Weng, M; Nagy, P L; Zalkin, H

    1995-08-01

    Transcription of the Bacillus subtilis pur operon is repressed in response to a signal of excess adenine. We have purified the repressor protein and have identified, cloned, and overexpressed the purR regulatory gene that controls transcription initiation of the operon. B. subtilis purR encodes a 62-kDa homodimer that binds to the pur operon control region. The PurR binding site which overlaps the promoter encompasses approximately 110 bp. The protein-DNA interaction is inhibited by 5-phosphoribosyl 1-pyrophosphate. A mutation that deletes the repressor binding site or one that disrupts purR abolishes binding activity in vitro and repression of transcription in vivo in response to the excess adenine signal. These results lead to a model in which an excess-adenine signal is transmitted to PurR via the 5-phosphoribosyl 1-pyrophosphate pool. In addition, purR is autoregulated. There is no structural or mechanistic similarity between the B. subtilis and Escherichia coli purine repressors.

  8. Regulation of Tryptophan Operon Expression in the Archaeon Methanothermobacter thermautotrophicus

    PubMed Central

    Xie, Yunwei; Reeve, John N.

    2005-01-01

    Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between trpY and trpE, inhibiting trpY transcription in the absence of tryptophan and both trpY and trpEGCFBAD transcription in the presence of tryptophan. TrpY apparently inhibits trpY transcription by blocking RNA polymerase access to the site of trpY transcription initiation and represses trpEGCFBAD transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other Euryarchaea and that there is only one tryptophan codon in the entire trpEGCFBAD operon (trpB encodes W175), expression of the trp operon may also be regulated in vivo by the supply of charged tRNATrp available to translate the second codon of the trpY mRNA. PMID:16159776

  9. Regulation of tryptophan operon expression in the archaeon Methanothermobacter thermautotrophicus.

    PubMed

    Xie, Yunwei; Reeve, John N

    2005-09-01

    Conserved trp genes encode enzymes that catalyze tryptophan biosynthesis in all three biological domains, and studies of their expression in Bacteria and eukaryotes have revealed a variety of different regulatory mechanisms. The results reported here provide the first detailed description of an archaeal trp gene regulatory system. We have established that the trpEGCFBAD operon in Methanothermobacter thermautotrophicus is transcribed divergently from a gene (designated trpY) that encodes a tryptophan-sensitive transcription regulator. TrpY binds to TRP box sequences (consensus, TGTACA) located in the overlapping promoter regions between trpY and trpE, inhibiting trpY transcription in the absence of tryptophan and both trpY and trpEGCFBAD transcription in the presence of tryptophan. TrpY apparently inhibits trpY transcription by blocking RNA polymerase access to the site of trpY transcription initiation and represses trpEGCFBAD transcription by preventing TATA box binding protein (TBP) binding to the TATA box sequence. Given that residue 2 (W2) is the only tryptophan in TrpY and in TrpY homologues in other Euryarchaea and that there is only one tryptophan codon in the entire trpEGCFBAD operon (trpB encodes W175), expression of the trp operon may also be regulated in vivo by the supply of charged tRNA(Trp) available to translate the second codon of the trpY mRNA. PMID:16159776

  10. rrndb: the Ribosomal RNA Operon Copy Number Database.

    PubMed

    Klappenbach, J A; Saxman, P R; Cole, J R; Schmidt, T M

    2001-01-01

    The Ribosomal RNA Operon Copy Number Database (rrndb) is an Internet-accessible database containing annotated information on rRNA operon copy number among prokaryotes. Gene redundancy is uncommon in prokaryotic genomes, yet the rRNA genes can vary from one to as many as 15 copies. Despite the widespread use of 16S rRNA gene sequences for identification of prokaryotes, information on the number and sequence of individual rRNA genes in a genome is not readily accessible. In an attempt to understand the evolutionary implications of rRNA operon redundancy, we have created a phylogenetically arranged report on rRNA gene copy number for a diverse collection of prokaryotic microorganisms. Each entry (organism) in the rrndb contains detailed information linked directly to external websites including the Ribosomal Database Project, GenBank, PubMed and several culture collections. Data contained in the rrndb will be valuable to researchers investigating microbial ecology and evolution using 16S rRNA gene sequences. The rrndb web site is directly accessible on the WWW at http://rrndb.cme. msu.edu.

  11. Growth and sporulation defects in Bacillus subtilis mutants with a single rrn operon can be suppressed by amplification of the rrn operon.

    PubMed

    Yano, Koichi; Masuda, Kenta; Akanuma, Genki; Wada, Tetsuya; Matsumoto, Takashi; Shiwa, Yuh; Ishige, Taichiro; Yoshikawa, Hirofumi; Niki, Hironori; Inaoka, Takashi; Kawamura, Fujio

    2016-01-01

    The genome of Bacillus subtilis strain 168 encodes ten rRNA (rrn) operons. We previously reported that strains with only a single rrn operon had a decreased growth and sporulation frequency. We report here the isolation and characterization of suppressor mutants from seven strains that each have a single rrn operon (rrnO, A, J, I, E, D or B). The suppressor mutants for strain RIK656 with a single rrnO operon had a higher frequency of larger colonies. These suppressor mutants had not only increased growth rates, but also increased sporulation frequencies and ribosome levels compared to the parental mutant strain RIK656. Quantitative PCR analyses showed that all these suppressor mutants had an increased number of copies of the rrnO operon. Suppressor mutants were also isolated from the six other strains with single rrn operons (rrnA, J, I, E, D or B). Next generation and capillary sequencing showed that all of the suppressor mutants had tandem repeats of the chromosomal locus containing the remaining rrn operon (amplicon). These amplicons varied in size from approximately 9 to 179 kb. The amplifications were likely to be initiated by illegitimate recombination between non- or micro-homologous sequences, followed by unequal crossing-over during DNA replication. These results are consistent with our previous report that rrn operon copy number has a major role in cellular processes such as cell growth and sporulation.

  12. Molecular epidemiology of adhesin and hemolysin virulence factors among uropathogenic Escherichia coli.

    PubMed

    Arthur, M; Johnson, C E; Rubin, R H; Arbeit, R D; Campanelli, C; Kim, C; Steinbach, S; Agarwal, M; Wilkinson, R; Goldstein, R

    1989-02-01

    The pap, prs, pil, and hly operons of the pyelonephritic Escherichia coli isolate J96 code for the expression of P, F, and type 1 adhesins and the production of hemolysin, respectively; the afaI operon of the pyelonephritic E. coli KS52 encodes an X adhesin. Using different segments of these operons as probes, colony hybridizations were performed on 97 E. coli urinary tract and 40 fecal clinical isolates to determine (i) the presence in the infecting bacteria of nucleotide sequences related to virulence operons, and (ii) the phenotypic properties associated with such sequences. Coexpression of P and F adhesins encoded by pap-related sequences was detected more frequently among isolates from patients with pyelonephritis (32 of 49, 65%) than among those with cystitis (11 of 48, 23%; P less than 0.0001) or from fecal specimens (6 of 40, 15%; P less than 0.0001). Therefore, the expression of both adhesins appears to be critical in the colonization of the upper urinary tract. In contrast, afaI-related sequences were detected significantly more frequently among isolates from patients with cystitis, suggesting that this class of X adhesin may have a role in lower urinary tract infections. Urinary tract isolates differed from fecal isolates by a low incidence of type 1 adhesin expression among pil probe-positive isolates. hly-related sequences were only detected in pap probe-positive isolates. The frequency of hemolysin production among pap probe-positive isolates was not associated with a particular pattern of infection. The distribution of these virulence factors was similar in the presence or absence of reflux, indicating that structural abnormalities of the urinary tract did not facilitate colonization by adhesin-negative isolates.

  13. Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers.

    PubMed

    Grosso-Becerra, María Victoria; Croda-García, Gerardo; Merino, Enrique; Servín-González, Luis; Mojica-Espinosa, Raúl; Soberón-Chávez, Gloria

    2014-10-28

    In a number of bacterial pathogens, the production of virulence factors is induced at 37 °C; this effect is often regulated by mRNA structures formed in the 5' untranslated region (UTR) that block translation initiation of genes at environmental temperatures. At 37 °C, the RNA structures become unstable and ribosomes gain access to their binding sites in the mRNAs. Pseudomonas aeruginosa is an important opportunistic pathogen and the expression of many of its virulence-associated traits is regulated by the quorum-sensing (QS) response, but the effect of temperature on virulence-factor expression is not well-understood. The aim of this work is the characterization of the molecular mechanism involved in thermoregulation of QS-dependent virulence-factor production. We demonstrate that traits that are dependent on the QS transcriptional regulator RhlR have a higher expression at 37 °C, correlating with a higher RhlR concentration as measured by Western blot. We also determined, using gene fusions and point mutations, that RhlR thermoregulation is a posttranscriptional effect dependent on an RNA thermometer of the ROSE (Repression Of heat-Shock gene Expression) family. This RNA element regulates the expression of the rhlAB operon, involved in rhamnolipid production, and of the downstream rhlR gene. We also identified a second functional thermometer in the 5' UTR of the lasI gene. We confirmed that these RNA thermometers are the main mechanism of thermoregulation of QS-dependent gene expression in P. aeruginosa using quantitative real-time PCR. This is the first description, to our knowledge, of a ROSE element regulating the expression of virulence traits and of an RNA thermometer controlling multiple genes in an operon through a polar effect.

  14. Regulation of Pseudomonas aeruginosa virulence factors by two novel RNA thermometers

    PubMed Central

    Grosso-Becerra, María Victoria; Croda-García, Gerardo; Merino, Enrique; Servín-González, Luis; Mojica-Espinosa, Raúl; Soberón-Chávez, Gloria

    2014-01-01

    In a number of bacterial pathogens, the production of virulence factors is induced at 37 °C; this effect is often regulated by mRNA structures formed in the 5′ untranslated region (UTR) that block translation initiation of genes at environmental temperatures. At 37 °C, the RNA structures become unstable and ribosomes gain access to their binding sites in the mRNAs. Pseudomonas aeruginosa is an important opportunistic pathogen and the expression of many of its virulence-associated traits is regulated by the quorum-sensing (QS) response, but the effect of temperature on virulence-factor expression is not well-understood. The aim of this work is the characterization of the molecular mechanism involved in thermoregulation of QS-dependent virulence-factor production. We demonstrate that traits that are dependent on the QS transcriptional regulator RhlR have a higher expression at 37 °C, correlating with a higher RhlR concentration as measured by Western blot. We also determined, using gene fusions and point mutations, that RhlR thermoregulation is a posttranscriptional effect dependent on an RNA thermometer of the ROSE (Repression Of heat-Shock gene Expression) family. This RNA element regulates the expression of the rhlAB operon, involved in rhamnolipid production, and of the downstream rhlR gene. We also identified a second functional thermometer in the 5′ UTR of the lasI gene. We confirmed that these RNA thermometers are the main mechanism of thermoregulation of QS-dependent gene expression in P. aeruginosa using quantitative real-time PCR. This is the first description, to our knowledge, of a ROSE element regulating the expression of virulence traits and of an RNA thermometer controlling multiple genes in an operon through a polar effect. PMID:25313031

  15. New insights into regulation of the tryptophan biosynthetic operon in Gram-positive bacteria.

    PubMed

    Gutierrez-Preciado, A; Jensen, R A; Yanofsky, C; Merino, E

    2005-08-01

    The tryptophan operon of Bacillus subtilis serves as an excellent model for investigating transcription regulation in Gram-positive bacteria. In this article, we extend this knowledge by analyzing the predicted regulatory regions in the trp operons of other fully sequenced Gram-positive bacteria. Interestingly, it appears that in eight of the organisms examined, transcription of the trp operon appears to be regulated by tandem T-box elements. These regulatory elements have recently been described in the trp operons of two bacterial species. Single T-box elements are commonly found in Gram-positive bacteria in operons encoding aminoacyl tRNA synthetases and proteins performing other functions. Different regulatory mechanisms appear to be associated with variations of trp gene organization within the trp operon. PMID:15953653

  16. The ars operon of Escherichia coli confers arsenical and antimonial resistance.

    PubMed Central

    Carlin, A; Shi, W; Dey, S; Rosen, B P

    1995-01-01

    The chromosomally encoded arsenical resistance (ars) operon subcloned into a multicopy plasmid was found to confer a moderate level of resistance to arsenite and antimonite in Escherichia coli. When the operon was deleted from the chromosome, the cells exhibited hypersensitivity to arsenite, antimonite, and arsenate. Expression of the ars genes was inducible by arsenite. By Southern hybridization, the operon was found in all strains of E. coli examined but not in Salmonella typhimurium, Pseudomonas aeruginosa, or Bacillus subtilis. PMID:7860609

  17. Comparison of tryptophan biosynthetic operon regulation in different Gram-positive bacterial species.

    PubMed

    Gutiérrez-Preciado, Ana; Yanofsky, Charles; Merino, Enrique

    2007-09-01

    The tryptophan biosynthetic operon has been widely used as a model system for studying transcription regulation. In Bacillus subtilis, the trp operon is primarily regulated by a tryptophan-activated RNA-binding protein, TRAP. Here we show that in many other Gram-positive species the trp operon is regulated differently, by tRNA(Trp) sensing by the RNA-based T-box mechanism, with T-boxes arranged in tandem. Our analyses reveal an apparent relationship between trp operon organization and the specific regulatory mechanism(s) used. PMID:17555843

  18. Agrobacterium virulence gene induction.

    PubMed

    Gelvin, Stanton B

    2006-01-01

    The ability of Agrobacterium to transform plants and other organisms is under highly regulated genetic control. Two Virulence (Vir) proteins, VirA and VirG, function as a two-component regulatory system to sense particular phenolic compounds synthesized by wounded plant tissues. Induction by these phenolic compounds, in the presence of certain neutral or acid sugars, results in activation of other vir genes, leading to the processing of T-DNA from the Ti-plasmid and transfer of T-DNA to recipient host cells. Many plant, and most nonplant, species do not provide sufficient quantities of the correct phenolic compounds to permit efficient Agrobacterium-mediated genetic transformation to occur. In order to transform these species, phenolic inducing compounds must be added to agrobacteria before and/or during cocultivation of recipient cells with the bacteria. This chapter discusses conditions for efficient induction of Agrobacterium virulence genes by phenolic compounds. PMID:16988335

  19. Characterization of the mannitol catabolic operon of Corynebacterium glutamicum.

    PubMed

    Peng, Xue; Okai, Naoko; Vertès, Alain A; Inatomi, Ken-Ichi; Inui, Masayuki; Yukawa, Hideaki

    2011-09-01

    Corynebacterium glutamicum encodes a mannitol catabolic operon, which comprises three genes: the DeoR-type repressor coding gene mtlR (sucR), an MFS transporter gene (mtlT), and a mannitol 2-dehydrogenase gene (mtlD). The mtlR gene is located upstream of the mtlTD genes in the opposite orientation. In spite of this, wild-type C. glutamicum lacks the ability to utilize mannitol. This wild-type phenotype results from the genetic regulation of the genes coding for mannitol transport and catalytic proteins mediated by the autoregulated MtlR protein since mtlR mutants grow on mannitol as the sole carbon source. MtlR binds to sites near the mtlR (two sites) and mtlTD promoters (one site downstream of the promoter), with the consensus sequence 5'-TCTAACA-3' being required for its binding. The newly discovered operon comprises the three basic functional elements required for mannitol utilization: regulation, transport, and metabolism to fructose, further processed to the common intermediate of glycolysis fructose-6-phosphate. When relieved from MtlR repression, C. glutamicum, which lacks a functional fructokinase, excretes the fructose derived from mannitol and imports it by the fructose-specific PTS. In order to use mannitol from seaweed biomass hydrolysates as a carbon source for the production of useful commodity chemicals and materials, an overexpression system using the tac promoter was developed. For congruence with the operon, we propose to rename sucR as the mtlR gene. PMID:21655984

  20. RegR virulence regulon of rabbit-specific enteropathogenic Escherichia coli strain E22.

    PubMed

    Srikhanta, Yogitha N; Hocking, Dianna M; Praszkier, Judyta; Wakefield, Matthew J; Robins-Browne, Roy M; Yang, Ji; Tauschek, Marija

    2013-04-01

    AraC-like regulators play a key role in the expression of virulence factors in enteric pathogens, such as enteropathogenic Escherichia coli (EPEC), enterotoxigenic E. coli, enteroaggregative E. coli, and Citrobacter rodentium. Bioinformatic analysis of the genome of rabbit-specific EPEC (REPEC) strain E22 (O103:H2) revealed the presence of a gene encoding an AraC-like regulatory protein, RegR, which shares 71% identity to the global virulence regulator, RegA, of C. rodentium. Microarray analysis demonstrated that RegR exerts 25- to 400-fold activation on transcription of several genes encoding putative virulence-associated factors, including a fimbrial operon (SEF14), a serine protease, and an autotransporter adhesin. These observations were confirmed by proteomic analysis of secreted and heat-extracted surface-associated proteins. The mechanism of RegR-mediated activation was investigated by using its most highly upregulated gene target, sefA. Transcriptional analyses and electrophoretic mobility shift assays showed that RegR activates the expression of sefA by binding to a region upstream of the sefA promoter, thereby relieving gene silencing by the global regulatory protein H-NS. Moreover, RegR was found to contribute significantly to virulence in a rabbit infection experiment. Taken together, our findings indicate that RegR controls the expression of a series of accessory adhesins that significantly enhance the virulence of REPEC strain E22. PMID:23340312

  1. Dynamic behavior in mathematical models of the tryptophan operon

    NASA Astrophysics Data System (ADS)

    Santillán, Moisés; Mackey, Michael C.

    2001-03-01

    This paper surveys the general theory of operon regulation as first formulated by Goodwin and Griffith, and then goes on to consider in detail models of regulation of tryptophan production by Bliss, Sinha, and Santillán and Mackey, and the interrelationships between them. We further give a linear stability analysis of the Santillán and Mackey model for wild type E. coli as well as three different mutant strains that have been previously studied in the literature. This stability analysis indicates that the tryptophan production systems should be stable, which is in accord with our numerical results.

  2. Regulation of the L-arabinose operon of Escherichia coli.

    PubMed

    Schleif, R

    2000-12-01

    Over forty years of research on the L-arabinose operon of Escherichia coli have provided insights into the mechanism of positive regulation of gene activity. This research also discovered DNA looping and the mechanism by which the regulatory protein changes its DNA-binding properties in response to the presence of arabinose. As is frequently seen in focused research on biological subjects, the initial studies were primarily genetic. Subsequently, the genetic approaches were augmented by physiological and then biochemical studies. Now biophysical studies are being conducted at the atomic level, but genetics still has a crucial role in the study of this system.

  3. Unprecedented high-resolution view of bacterial operon architecture revealed by RNA sequencing.

    PubMed

    Conway, Tyrrell; Creecy, James P; Maddox, Scott M; Grissom, Joe E; Conkle, Trevor L; Shadid, Tyler M; Teramoto, Jun; San Miguel, Phillip; Shimada, Tomohiro; Ishihama, Akira; Mori, Hirotada; Wanner, Barry L

    2014-01-01

    We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3' transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5' ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. Importance: We precisely mapped the 5' and 3' ends of RNA transcripts across the E. coli K-12 genome by using a single-nucleotide analytical approach. Our resulting high-resolution transcriptome maps show that ca. one-third of E. coli operons are

  4. Unity in organisation and regulation of catabolic operons in Lactobacillus plantarum, Lactococcus lactis and Listeria monocytogenes.

    PubMed

    Andersson, Ulrika; Molenaar, Douwe; Rådström, Peter; de Vos, Willem M

    2005-04-01

    Global regulatory circuits together with more specific local regulators play a notable role when cells are adapting to environmental changes. Lactococcus lactis is a lactic acid bacterium abundant in nature fermenting most mono- and disaccharides. Comparative genomics analysis of the operons encoding the proteins and enzymes crucial for catabolism of lactose, maltose and threhalose revealed an obvious unity in operon organisation . The local regulator of each operon was located in a divergent transcriptional direction to the rest of the operon including the transport protein-encoding genes. Furthermore, in all three operons a catabolite responsive element (CRE) site was detected inbetween the gene encoding the local regulator and one of the genes encoding a sugar transport protein. It is evident that regardless of type of transport system and catabolic enzymes acting upon lactose, maltose and trehalose, respectively, Lc. lactis shows unity in both operon organisation and regulation of these catabolic operons. This knowledge was further extended to other catabolic operons in Lc. lactis and the two related bacteria Lactobacillus plantarum and Listeria monocytogenes. Thirty-nine catabolic operons responsible for degradation of sugars and sugar alcohols in Lc. lactis, Lb. plantarum and L. monocytogenes were investigated and the majority of those possessed the same organisation as the lactose, maltose and trehalose operons of Lc. lactis. Though, the frequency of CRE sites and their location varied among the bacteria. Both Lc. lactis and Lb. plantarum showed CRE sites in direct proximity to genes coding for proteins responsible for sugar uptake. However, in L. monocytogenes CRE sites were not frequently found and not in the vicinity of genes encoding transport proteins, suggesting a more local mode of regulation of the catabolic operons found and/or the use of inducer control in this bacterium. PMID:15900965

  5. Unprecedented High-Resolution View of Bacterial Operon Architecture Revealed by RNA Sequencing

    PubMed Central

    Creecy, James P.; Maddox, Scott M.; Grissom, Joe E.; Conkle, Trevor L.; Shadid, Tyler M.; Teramoto, Jun; San Miguel, Phillip; Shimada, Tomohiro; Ishihama, Akira; Mori, Hirotada

    2014-01-01

    ABSTRACT We analyzed the transcriptome of Escherichia coli K-12 by strand-specific RNA sequencing at single-nucleotide resolution during steady-state (logarithmic-phase) growth and upon entry into stationary phase in glucose minimal medium. To generate high-resolution transcriptome maps, we developed an organizational schema which showed that in practice only three features are required to define operon architecture: the promoter, terminator, and deep RNA sequence read coverage. We precisely annotated 2,122 promoters and 1,774 terminators, defining 1,510 operons with an average of 1.98 genes per operon. Our analyses revealed an unprecedented view of E. coli operon architecture. A large proportion (36%) of operons are complex with internal promoters or terminators that generate multiple transcription units. For 43% of operons, we observed differential expression of polycistronic genes, despite being in the same operons, indicating that E. coli operon architecture allows fine-tuning of gene expression. We found that 276 of 370 convergent operons terminate inefficiently, generating complementary 3′ transcript ends which overlap on average by 286 nucleotides, and 136 of 388 divergent operons have promoters arranged such that their 5′ ends overlap on average by 168 nucleotides. We found 89 antisense transcripts of 397-nucleotide average length, 7 unannotated transcripts within intergenic regions, and 18 sense transcripts that completely overlap operons on the opposite strand. Of 519 overlapping transcripts, 75% correspond to sequences that are highly conserved in E. coli (>50 genomes). Our data extend recent studies showing unexpected transcriptome complexity in several bacteria and suggest that antisense RNA regulation is widespread. PMID:25006232

  6. The copYAZ Operon Functions in Copper Efflux, Biofilm Formation, Genetic Transformation, and Stress Tolerance in Streptococcus mutans

    PubMed Central

    Singh, Kamna; Senadheera, Dilani B.; Lévesque, Céline M.

    2015-01-01

    ABSTRACT In bacteria, copper homeostasis is closely monitored to ensure proper cellular functions while avoiding cell damage. Most Gram-positive bacteria utilize the copYABZ operon for copper homeostasis, where copA and copB encode copper-transporting P-type ATPases, whereas copY and copZ regulate the expression of the cop operon. Streptococcus mutans is a biofilm-forming oral pathogen that harbors a putative copper-transporting copYAZ operon. Here, we characterized the role of copYAZ operon in the physiology of S. mutans and delineated the mechanisms of copper-induced toxicity in this bacterium. We observed that copper induced toxicity in S. mutans cells by generating oxidative stress and disrupting their membrane potential. Deletion of the copYAZ operon in S. mutans strain UA159 resulted in reduced cell viability under copper, acid, and oxidative stress relative to the viability of the wild type under these conditions. Furthermore, the ability of S. mutans to form biofilms and develop genetic competence was impaired under copper stress. Briefly, copper stress significantly reduced cell adherence and total biofilm biomass, concomitantly repressing the transcription of the gtfB, gtfC, gtfD, gbpB, and gbpC genes, whose products have roles in maintaining the structural and/or functional integrity of the S. mutans biofilm. Furthermore, supplementation with copper or loss of copYAZ resulted in significant reductions in transformability and in the transcription of competence-associated genes. Copper transport assays revealed that the ΔcopYAZ strain accrued significantly large amounts of intracellular copper compared with the amount of copper accumulation in the wild-type strain, thereby demonstrating a role for CopYAZ in the copper efflux of S. mutans. The complementation of the CopYAZ system restored copper expulsion, membrane potential, and stress tolerance in the copYAZ-null mutant. Taking these results collectively, we have established the function of the S. mutans

  7. Transcription antitermination regulation of the Pseudomonas aeruginosa amidase operon.

    PubMed Central

    Wilson, S A; Wachira, S J; Norman, R A; Pearl, L H; Drew, R E

    1996-01-01

    In vivo titration experiments have demonstrated a direct interaction between the Pseudomonas aeruginosa transcription antiterminator, AmiR, and the mRNA leader sequence of the amidase operon. A region of 39 nucleotides has been identified which is sufficient to partially titrate out the AmiR available for antitermination. Site-directed mutagenesis has shown that the leader open reading frame has no role in the antitermination reaction, and has identified two critical elements at the 5' and 3' ends of the proposed AmiR binding site which are independently essential for antitermination. A T7 promoter/RNA polymerase-driven system shows AmiR-mediated antitermination, demonstrating a lack of promoter/polymerase specificity. Using the operon negative regulator, AmiC, immobilized on a solid support and gel filtration chromatography, an AmiC-AmiR complex has been identified and isolated. Complex stability and molecular weight assayed by gel filtration alter depending on the type of amide bound to AmiC. AmiC-AmiR-anti-inducer is a stable dimer-dimer complex and the addition of the inducer, acetamide, causes a conformational change which alters the complex stability and either this new configuration or dissociated AmiR interacts with the leader mRNA to cause antitermination. Images PMID:8918468

  8. Transcriptional Regulation of the Streptococcus salivarius 57.I Urease Operon

    PubMed Central

    Chen, Yi-Ywan M.; Weaver, Cheryl A.; Mendelsohn, David R.; Burne, Robert A.

    1998-01-01

    The Streptococcus salivarius 57.I ure cluster was organized as an operon, beginning with ureI, followed by ureABC (structural genes) and ureEFGD (accessory genes). Northern analyses revealed transcripts encompassing structural genes and transcripts containing the entire operon. A ς70-like promoter could be mapped 5′ to ureI (PureI) by primer extension analysis. The intensity of the signal increased when cells were grown at an acidic pH and was further enhanced by excess carbohydrate. To determine the function(s) of two inverted repeats located 5′ to PureI, transcriptional fusions of the full-length promoter region (PureI), or a deletion derivative (PureIΔ100), and a promoterless chloramphenicol acetyltransferase (CAT) gene were constructed and integrated into the chromosome to generate strains PureICAT and PureIΔ100CAT, respectively. CAT specific activities of PureICAT were repressed at pH 7.0 and induced at pH 5.5 and by excess carbohydrate. In PureIΔ100CAT, CAT activity was 60-fold higher than in PureICAT at pH 7.0 and pH induction was nearly eliminated, indicating that expression was negatively regulated. Thus, it was concluded that PureI was the predominant, regulated promoter and that regulation was governed by a mechanism differing markedly from other known mechanisms for bacterial urease expression. PMID:9791132

  9. The cox protein of bacteriophage P2 inhibits the formation of the repressor protein and autoregulates the early operon.

    PubMed Central

    Saha, S; Haggård-Ljungquist, E; Nordström, K

    1987-01-01

    The cox gene is the first gene of the early operon of bacteriophage P2. The early promoter Pe and the repressor promoter Pc are located close to each other and in such a way that their transcripts have opposite polarity and show an overlap of about 30 nucleotides. The expression of the early operon and of the C gene was studied in vivo by using fusions to a promoterless cat (chloramphenicol acetyl transferase) gene. The results show that the Cox protein negatively autoregulates the early operon and inhibits the formation of the repressor C. By measuring the efficiency of plating of a series of P2 virulent deletion mutants on bacteria carrying a cloned cox gene, the site of action of the Cox protein was mapped within the Pe-Pc region. The stimulatory effect of the C protein on expression of the Pc promoter was found to be due to inhibition of transcription from Pc; this was demonstrated by mutating Pe which showed that loss of transcription from Pe stimulated transcription from Pc. Hence, this is a case of regulation of gene expression by convergent transcription. By cloning the region C-Pe-Pc-cox such that the cat and kan genes are expressed from Pc and Pe, respectively, it was shown that only one of the resistances (Cm and Km) was expressed. This mimics the choice between lysogeny and lytic growth of the phage. The 'lysogenic' state was very stable whereas the 'lytic' state flipped to the 'lysogenic' at a somewhat higher frequency. The presence of a cloned cox gene drastically stimulated the formation of free phage from a P2-lysogen and dramatically reduced the frequency of lysogenization after P2 infection. We conclude that the pleiotropic effects of the cox (control of excision) gene, namely effects on lysogenization, formation of free phage and site-specific P2 recombination, can be explained by the effect of the Cox protein on the activity of the promoters Pc and Pe. Images Fig. 2. PMID:2826134

  10. Staphylococcus aureus CodY Negatively Regulates Virulence Gene Expression▿

    PubMed Central

    Majerczyk, Charlotte D.; Sadykov, Marat R.; Luong, Thanh T.; Lee, Chia; Somerville, Greg A.; Sonenshein, Abraham L.

    2008-01-01

    CodY is a global regulatory protein that was first discovered in Bacillus subtilis, where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G+C gram-positive bacteria, including Staphylococcus aureus. The introduction of a codY-null mutation into two S. aureus clinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin (hla), the accessory gene regulator (agr) (RNAII and RNAIII/hld), and the operon responsible for the production of PIA (icaADBC). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors of S. aureus. PMID:18156263

  11. In silico evolved lac operons exhibit bistability for artificial inducers, but not for lactose.

    PubMed

    van Hoek, M J A; Hogeweg, P

    2006-10-15

    Bistability in the lac operon of Escherichia coli has been widely studied, both experimentally and theoretically. Experimentally, bistability has been observed when E. coli is induced by an artificial, nonmetabolizable, inducer. However, if the lac operon is induced with lactose, the natural inducer, bistability has not been demonstrated. We derive an analytical expression that can predict the occurrence of bistability both for artificial inducers and lactose. We find very different conditions for bistability in the two cases. Indeed, for artificial inducers bistability is predicted, but for lactose the condition for bistability is much more difficult to satisfy. Moreover, we demonstrate that in silico evolution of the lac operon generates an operon that avoids bistability with respect to lactose, but does exhibit bistability with respect to artificial inducers. The activity of this evolved operon strikingly resembles the experimentally observed activity of the operon. Thus our computational experiments suggest that the wild-type lac operon, which regulates lactose metabolism, is not a bistable switch. Nevertheless, for engineering purposes, this operon can be used as a bistable switch with artificial inducers. PMID:16877514

  12. Functional Operons in Secondary Metabolic Gene Clusters in Glarea lozoyensis (Fungi, Ascomycota, Leotiomycetes)

    PubMed Central

    Yue, Qun; Chen, Li; Li, Yan; Bills, Gerald F.; Zhang, Xinyu; Xiang, Meichun; Li, Shaojie; Che, Yongsheng; Niu, Xuemei

    2015-01-01

    ABSTRACT Operons are multigene transcriptional units which occur mostly in prokaryotes but rarely in eukaryotes. Protein-coding operons have not been reported in the Fungi even though they represent a very diverse kingdom of organisms. Here, we report a functional operon involved in the secondary metabolism of the fungus Glarea lozoyensis belonging to Leotiomycetes (Ascomycota). Two contiguous genes, glpks3 and glnrps7, encoding polyketide synthase and nonribosomal peptide synthetase, respectively, are cotranscribed into one dicistronic mRNA under the control of the same promoter, and the mRNA is then translated into two individual proteins, GLPKS3 and GLNRPS7. Heterologous expression in Aspergillus nidulans shows that the GLPKS3-GLNRPS7 enzyme complex catalyzes the biosynthesis of a novel pyrrolidinedione-containing compound, xenolozoyenone (compound 1), which indicates the operon is functional. Although it is structurally similar to prokaryotic operons, the glpks3-glnrps7 operon locus has a monophylogenic origin from fungi rather than having been horizontally transferred from prokaryotes. Moreover, two additional operons, glpks28-glnrps8 and glpks29-glnrps9, were verified at the transcriptional level in the same fungus. This is the first report of protein-coding operons in a member of the Fungi. PMID:26081635

  13. Thermodynamic modeling of variations in the rate of RNA chain elongation of E. coli rrn operons.

    PubMed

    Fange, David; Mellenius, Harriet; Dennis, Patrick P; Ehrenberg, Måns

    2014-01-01

    Previous electron-microscopic imaging has shown high RNA polymerase occupation densities in the 16S and 23S encoding regions and low occupation densities in the noncoding leader, spacer, and trailer regions of the rRNA (rrn) operons in E. coli. This indicates slower transcript elongation within the coding regions and faster elongation within the noncoding regions of the operon. Inactivation of four of the seven rrn operons increases the transcript initiation frequency at the promoters of the three intact operons and reduces the time for RNA polymerase to traverse the operon. We have used the DNA sequence-dependent standard free energy variation of the transcription complex to model the experimentally observed changes in the elongation rate along the rrnB operon. We also model the stimulation of the average transcription rate over the whole operon by increasing rate of transcript initiation. Monte Carlo simulations, taking into account initiation of transcription, translocation, and backward and forward tracking of RNA polymerase, partially reproduce the observed transcript elongation rate variations along the rrn operon and fully account for the increased average rate in response to increased frequency of transcript initiation.

  14. The pseudogenes of Mycobacterium leprae reveal the functional relevance of gene order within operons

    PubMed Central

    Muro, Enrique M.; Mah, Nancy; Moreno-Hagelsieb, Gabriel; Andrade-Navarro, Miguel A.

    2011-01-01

    Almost 50 years following the discovery of the prokaryotic operon, the functional relevance of gene order within operons remains unclear. In this work, we take advantage of the eroded genome of Mycobacterium leprae to add evidence supporting the notion that functionally less important genes have a tendency to be located at the end of its operons. M. leprae’s genome includes 1133 pseudogenes and 1614 protein-coding genes and can be compared with the close genome of M. tuberculosis. Assuming M. leprae’s pseudogenes to represent dispensable genes, we have studied the position of these pseudogenes in the operons of M. leprae and of their orthologs in M. tuberculosis. We observed that both tend to be located in the 3′ (downstream) half of the operon (P-values of 0.03 and 0.18, respectively). Analysis of pseudogenes in all available prokaryotic genomes confirms this trend (P-value of 7.1 × 10−7). In a complementary analysis, we found a significant tendency for essential genes to be located at the 5′ (upstream) half of the operon (P-value of 0.006). Our work provides an indication that, in prokarya, functionally less important genes have a tendency to be located at the end of operons, while more relevant genes tend to be located toward operon starts. PMID:21051341

  15. The pseudogenes of Mycobacterium leprae reveal the functional relevance of gene order within operons.

    PubMed

    Muro, Enrique M; Mah, Nancy; Moreno-Hagelsieb, Gabriel; Andrade-Navarro, Miguel A

    2011-03-01

    Almost 50 years following the discovery of the prokaryotic operon, the functional relevance of gene order within operons remains unclear. In this work, we take advantage of the eroded genome of Mycobacterium leprae to add evidence supporting the notion that functionally less important genes have a tendency to be located at the end of its operons. M. leprae's genome includes 1133 pseudogenes and 1614 protein-coding genes and can be compared with the close genome of M. tuberculosis. Assuming M. leprae's pseudogenes to represent dispensable genes, we have studied the position of these pseudogenes in the operons of M. leprae and of their orthologs in M. tuberculosis. We observed that both tend to be located in the 3' (downstream) half of the operon (P-values of 0.03 and 0.18, respectively). Analysis of pseudogenes in all available prokaryotic genomes confirms this trend (P-value of 7.1 × 10(-7)). In a complementary analysis, we found a significant tendency for essential genes to be located at the 5' (upstream) half of the operon (P-value of 0.006). Our work provides an indication that, in prokarya, functionally less important genes have a tendency to be located at the end of operons, while more relevant genes tend to be located toward operon starts.

  16. Mutations in the control of virulence sensor gene from Streptococcus pyogenes after infection in mice lead to clonal bacterial variants with altered gene regulatory activity and virulence.

    PubMed

    Mayfield, Jeffrey A; Liang, Zhong; Agrahari, Garima; Lee, Shaun W; Donahue, Deborah L; Ploplis, Victoria A; Castellino, Francis J

    2014-01-01

    The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS- was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection.

  17. Mutations in the Control of Virulence Sensor Gene from Streptococcus pyogenes after Infection in Mice Lead to Clonal Bacterial Variants with Altered Gene Regulatory Activity and Virulence

    PubMed Central

    Mayfield, Jeffrey A.; Liang, Zhong; Agrahari, Garima; Lee, Shaun W.; Donahue, Deborah L.; Ploplis, Victoria A.; Castellino, Francis J.

    2014-01-01

    The cluster of virulence sensor (CovS)/responder (CovR) two-component operon (CovRS) regulates ∼15% of the genes of the Group A Streptococcal pyogenes (GAS) genome. Bacterial clones containing inactivating mutations in the covS gene have been isolated from patients with virulent invasive diseases. We report herein an assessment of the nature and types of covS mutations that can occur in both virulent and nonvirulent GAS strains, and assess whether a nonvirulent GAS can attain enhanced virulence through this mechanism. A group of mice were infected with a globally-disseminated clonal M1T1 GAS (isolate 5448), containing wild-type (WT) CovRS (5448/CovR+S+), or less virulent engineered GAS strains, AP53/CovR+S+ and Manfredo M5/CovR+S+. SpeB negative GAS clones from wound sites and/or from bacteria disseminated to the spleen were isolated and the covS gene was subjected to DNA sequence analysis. Numerous examples of inactivating mutations were found in CovS in all regions of the gene. The mutations found included frame-shift insertions and deletions, and in-frame small and large deletions in the gene. Many of the mutations found resulted in early translation termination of CovS. Thus, the covS gene is a genomic mutagenic target that gives GAS enhanced virulence. In cases wherein CovS− was discovered, these clonal variants exhibited high lethality, further suggesting that randomly mutated covS genes occur during the course of infection, and lead to the development of a more invasive infection. PMID:24968349

  18. Identification of a chromosomal gene controlling temperature-regulated expression of Shigella virulence.

    PubMed

    Maurelli, A T; Sansonetti, P J

    1988-04-01

    Genes required for the full expression of Shigella virulence are on both the chromosome and a large virulence-associated plasmid. Expression of one or more virulence (vir) genes is temperature-regulated, wild-type strains being virulent (invasive) when grown at 37 degrees C but phenotypically avirulent (noninvasive) at 30 degrees C. A vir::lac operon fusion located on the virulence plasmid, which brings the lac genes under control of a temperature-regulated vir gene promoter, was used to select regulatory mutants constitutive for the Lac+ phenotype at the nonpermissive temperature. A transposon Tn10-induced mutant that was Lac+ at 30 degrees C and 37 degrees C was isolated, and the Tn10 insertion was transduced into a wild-type strain. The transductants all simultaneously became deregulated for virulence and invaded HeLa cells equally well at 30 degrees C and 37 degrees C. Other virulence-associated phenotypes were also deregulated and expressed at 30 degrees C. Southern hybridization with a probe for Tn10 determined the insertion to be on the chromosome. Fine mapping by transduction with phage P1L4 positioned the mutation between the galU and trp genes. A cosmid cloned fragment of Shigella chromosomal DNA containing the region around galU was used in complementation studies and showed that the closely linked regulatory gene was able to complement, in trans, the Tn10-induced mutation. We propose that this mutation defines a regulatory gene, virR, and that insertion of Tn10 into this gene inactivates a repressor that normally blocks expression of vir genes at 30 degrees C. PMID:3282241

  19. Role of glucose and CcpA in capsule expression and virulence of Streptococcus suis.

    PubMed

    Willenborg, J; Fulde, M; de Greeff, A; Rohde, M; Smith, H E; Valentin-Weigand, P; Goethe, R

    2011-06-01

    Streptococcus suis is one of the most important pathogens in pigs and is also an emerging zoonotic agent. After crossing the epithelial barrier, S. suis causes bacteraemia, resulting in meningitis, endocarditis and bronchopneumonia. Since the host environment seems to be an important regulatory component for virulence, we related expression of virulence determinants of S. suis to glucose availability during growth and to the sugar metabolism regulator catabolite control protein A (CcpA). We found that expression of the virulence-associated genes arcB, representing arcABC operon expression, cps2A, representing capsular locus expression, as well as sly, ofs, sao and epf, differed significantly between exponential and early stationary growth of a highly virulent serotype 2 strain. Deletion of ccpA altered the expression of the surface-associated virulence factors arcB, sao and eno, as well as the two currently proven virulence factors in pigs, ofs and cps2A, in early exponential growth. Global expression analysis using a cDNA expression array revealed 259 differentially expressed genes in early exponential growth, of which 141 were more highly expressed in the CcpA mutant strain 10ΔccpA and 118 were expressed to a lower extent. Interestingly, among the latter genes, 18 could be related to capsule and cell wall synthesis. Correspondingly, electron microscopy characterization of strain 10ΔccpA revealed a markedly reduced thickness of the capsule. This phenotype correlated with enhanced binding to porcine plasma proteins and a reduced resistance to killing by porcine neutrophils. Taken together, our data demonstrate that CcpA has a significant effect on the capsule synthesis and virulence properties of S. suis.

  20. [Mutation analysis of the purine operon leader region in Bacillus subtilis].

    PubMed

    Lobanov, K V; korol'kova, N V; Eremina, S Iu; Érrais Lopes, L; Mironov, A S

    2011-07-01

    Expression of Bacillus subtilis purine (purE) operon is a subject of double negative control involving repressor protein PurR and a transcription terminator located in the operon leader region. We have performed site-directed mutagenesis of the specific motives, which are involved in formation of alternative hairpin structures, one of which produces transcription termination at the leader region ofpurEoperon. In vivo and in vitro analyses of the generated mutants have shown that purine bases, guanine and hypoxantine, serve as effector metabolites capable of increasing stability of terminating hairpin within the leader mRNA. Therefore, the leader RNA of purE operon serves as a sensor towards these metabolites and a riboswitch that provides premature termination of the operon transcription. The synergistic effect of the PurR repressor protein and a transcription terminator located at the leader region on the expression of purE operon was also revealed.

  1. Metabolic diversification--independent assembly of operon-like gene clusters in different plants.

    PubMed

    Field, Ben; Osbourn, Anne E

    2008-04-25

    Operons are clusters of unrelated genes with related functions that are a feature of prokaryotic genomes. Here, we report on an operon-like gene cluster in the plant Arabidopsis thaliana that is required for triterpene synthesis (the thalianol pathway). The clustered genes are coexpressed, as in bacterial operons. However, despite the resemblance to a bacterial operon, this gene cluster has been assembled from plant genes by gene duplication, neofunctionalization, and genome reorganization, rather than by horizontal gene transfer from bacteria. Furthermore, recent assembly of operon-like gene clusters for triterpene synthesis has occurred independently in divergent plant lineages (Arabidopsis and oat). Thus, selection pressure may act during the formation of certain plant metabolic pathways to drive gene clustering.

  2. Statistical Estimate of the Total Number of Operons Specific for Bacillus subtilis Sporulation

    PubMed Central

    Hranueli, D.; Piggot, P. J.; Mandelstam, J.

    1974-01-01

    As an alternative to exhaustive mapping, an attempt has been made to obtain a rough estimate of the total number of sporulation operons by statistical treatment. Sixteen sporulation mutants taken at random were characterized biochemically and morphologically. The mutations they contained were mapped to determine whether they fell into any one of 23 known operons. From the proportion that do so (10/16), it is calculated that the most probable number of sporulation operons is 37 (68% confidence limits of 31 and 46). If allowance is made for the fact that two of the operons apparently contain mutagenic “hot spots” and the calculation is amended accordingly, the most probable numbers of operons becomes 42 (limits 33 and 59). PMID:4212352

  3. Metabolic diversification--independent assembly of operon-like gene clusters in different plants.

    PubMed

    Field, Ben; Osbourn, Anne E

    2008-04-25

    Operons are clusters of unrelated genes with related functions that are a feature of prokaryotic genomes. Here, we report on an operon-like gene cluster in the plant Arabidopsis thaliana that is required for triterpene synthesis (the thalianol pathway). The clustered genes are coexpressed, as in bacterial operons. However, despite the resemblance to a bacterial operon, this gene cluster has been assembled from plant genes by gene duplication, neofunctionalization, and genome reorganization, rather than by horizontal gene transfer from bacteria. Furthermore, recent assembly of operon-like gene clusters for triterpene synthesis has occurred independently in divergent plant lineages (Arabidopsis and oat). Thus, selection pressure may act during the formation of certain plant metabolic pathways to drive gene clustering. PMID:18356490

  4. The PecT repressor coregulates synthesis of exopolysaccharides and virulence factors in Erwinia chrysanthemi.

    PubMed

    Condemine, G; Castillo, A; Passeri, F; Enard, C

    1999-01-01

    Erwinia chrysanthemi 3937 synthesizes an exopolysaccharide (EPS) composed of rhamnose, galactose, and galacturonic acid. Fourteen transcriptional fusions in genes required for EPS synthesis, named eps, were obtained by Tn5-B21 mutagenesis. Eleven of them are clustered on the chromosome and are repressed by PecT, a regulator of pectate lyase synthesis. In addition, expression of these fusions is repressed by the catabolite regulatory protein, CRP, and induced in low osmolarity medium. The three other mutations are located in genes that are not regulated by pecT. A 13-kb DNA fragment containing pecT-regulated eps genes has been cloned. All the genes identified on this fragment are transcribed in the same orientation and could form a large operon. The promoter region of this operon has been sequenced. It contains a JUMP-start sequence, a sequence required for the expression of polysaccharide-associated operons. E. chrysanthemi 3937 produces a systemic soft rot on its host Saintpaulia ionantha. An eps mutant was less efficient than the wild-type strain in initiating a maceration symptom, suggesting that production of EPS is required for the full expression of the E. chrysanthemi virulence. PMID:9885192

  5. [New method of construction of artificial translational-coupled operons in bacterial chromosome].

    PubMed

    Gulevich, A Iu; Skorokhodova, A Iu; Ermishev, V Iu; Krylov, A A; Minaeva, N I; Polonskaia, Z M; Zimenkov, D V; Biriukova, I V; Mashko, S V

    2009-01-01

    The new method of translational-coupled operons construction in bacterial chromosome has been developed on the basis of recombineering approach. It includes construction in vitro of the artificial operon with efficiently translated proximal cistron followed by its insertion E. coli chromosome, modification of the operon due to Red-driven insertion of the special "Junction" with excisable selective marker in the intercistronic region of the initial operon and excising the marker. The structure of this Junction has been designed and tested in the present investigation. It consists of: 1) E. coli rplC-rplD intercistronic region for placing the TAA-codon of the proximal operon's gene in the SD-sequence (TAAGGAG) of rplD; 2) Cm(R)-gene flanked by lambdaattL/R-sites in such a fashion that after lambdaInt/Xis-driven excision of the marker the residual lambdaattB-site would not contain the termination codons in frame with ATG of rplD; 3) E. coli trpE-trpD intercistronic region for location of ATG of trpD at the position of initiation codon of the distal gene of original operon. The general design of desired construction provides the conversion of the original two-cistronic operon into three-cistronic operon with translational-coupled genes, where the coupling of the artificial ORF (rplD'-lambdaattB-'trpE) with the proximal gene is occurred due to rplC-rplD intercistronic region and the coupling of this ORF with the distal gene--due to trpE-trpD. The experimental implementation of the described strategy was showed by construction of artificial operon P(tac-aroG4-serA5, where expression optimization of the distal serA5 gene was achieved via construction of three-cistronic operon with translational-coupled genes. PMID:19548541

  6. Virulence of enterococci.

    PubMed Central

    Jett, B D; Huycke, M M; Gilmore, M S

    1994-01-01

    Enterococci are commensal organisms well suited to survival in intestinal and vaginal tracts and the oral cavity. However, as for most bacteria described as causing human disease, enterococci also possess properties that can be ascribed roles in pathogenesis. The natural ability of enterococci to readily acquire, accumulate, and share extrachromosomal elements encoding virulence traits or antibiotic resistance genes lends advantages to their survival under unusual environmental stresses and in part explains their increasing importance as nosocomial pathogens. This review discusses the current understanding of enterococcal virulence relating to (i) adherence to host tissues, (ii) invasion and abscess formation, (iii) factors potentially relevant to modulation of host inflammatory responses, and (iv) potentially toxic secreted products. Aggregation substance, surface carbohydrates, or fibronectin-binding moieties may facilitate adherence to host tissues. Enterococcus faecalis appears to have the capacity to translocate across intact intestinal mucosa in models of antibiotic-induced superinfection. Extracellular toxins such as cytolysin can induce tissue damage as shown in an endophthalmitis model, increase mortality in combination with aggregation substance in an endocarditis model, and cause systemic toxicity in a murine peritonitis model. Finally, lipoteichoic acid, superoxide production, or pheromones and corresponding peptide inhibitors each may modulate local inflammatory reactions. Images PMID:7834601

  7. Long-Chain Fatty Acid Sensor, PsrA, Modulates the Expression of rpoS and the Type III Secretion exsCEBA Operon in Pseudomonas aeruginosa

    SciTech Connect

    Kang, Y.; Lunin, V. V.; Skarina, T.; Savchenko, A.; Schurr, M. J.; Hoang, T. T.

    2009-01-01

    The Pseudomonas aeruginosa PsrA autorepressor has dual roles as a repressor of the fadBA5{beta}-oxidation operon and an activator of the stationary-phase sigma factor rpoS and exsCEBA operon of the type III secretion system (TTSS). Previously, we demonstrated that the repression of the fadBA5 operon by PsrA is relieved by long-chain fatty acids (LCFAs). However, the signal affecting the activation of rpoS and exsC via PsrA is unknown. In this study, microarray and gene fusion data suggested that LCFA (e.g. oleate) affected the expression of rpoS and exsC. DNA binding studies confirmed that PsrA binds to the rpoS and exsC promoter regions. This binding was inhibited by LCFA, indicating that LCFA directly affects the activation of these two genes through PsrA. LCFA decreased rpoS and exsC expression, resulting in increased N-(butyryl)-l-homoserine-lactone quorum sensing signal and decreased ExoS/T production respectively. Based on the crystal structure of PsrA, site-directed mutagenesis of amino acid residues, within the hydrophobic channel thought to accommodate LCFA, created two LCFA-non-responsive PsrA mutants. The binding and activation of rpoS and exsC by these PsrA mutants was no longer inhibited by LCFA. These data support a mechanistic model where LCFAs influence PsrA regulation to control LCFA metabolism and some virulence genes in P. aeruginosa.

  8. Polarity effects in the lactose operon of Escherichia coli.

    PubMed

    Li, Yong; Altman, Sidney

    2004-05-21

    An intergenic RNA segment between lacY and lacA of the lactose operon in Escherichia coli is cleaved by RNase P, an endoribonuclease. The cleavage of the intergenic RNA was ten times less efficient than cleavage of a tRNA precursor in vitro. Fragments of the RNase P cleavage product are detectable in vivo in the wild-type strain but not in a mutant strain at the restrictive temperature. The cleavage product that contains lacA in the wild-type strain was quickly degraded. When this intergenic segment was cloned upstream of a reporter gene, the expression of the reporter gene was also inhibited substantially in wild-type E.coli, but not in a temperature sensitive mutant strain in RNase P at the restrictive temperature. These results support data regarding the natural polarity between lacZ versus lacA, the downstream gene. PMID:15123418

  9. The rise of operon-like gene clusters in plants.

    PubMed

    Boycheva, Svetlana; Daviet, Laurent; Wolfender, Jean-Luc; Fitzpatrick, Teresa B

    2014-07-01

    Gene clusters are common features of prokaryotic genomes also present in eukaryotes. Most clustered genes known are involved in the biosynthesis of secondary metabolites. Although horizontal gene transfer is a primary source of prokaryotic gene cluster (operon) formation and has been reported to occur in eukaryotes, the predominant source of cluster formation in eukaryotes appears to arise de novo or through gene duplication followed by neo- and sub-functionalization or translocation. Here we aim to provide an overview of the current knowledge and open questions related to plant gene cluster functioning, assembly, and regulation. We also present potential research approaches and point out the benefits of a better understanding of gene clusters in plants for both fundamental and applied plant science.

  10. Regulation of cobalamin biosynthetic operons in Salmonella typhimurium.

    PubMed Central

    Escalante-Semerena, J C; Roth, J R

    1987-01-01

    Transcription of cobalamin (cob) biosynthetic genes in Salmonella typhimurium is repressed by cobalamin and by molecular oxygen. These genes seem to be subject to catabolite repression, and they are maximally expressed under conditions of anaerobic respiration of glycerol-fumarate. A 215-fold increase in the expression of cob genes occurs when S. typhimurium shifts from aerobic growth on glucose to anaerobic respiration of glycerol-fumarate under strictly anoxic growth conditions. Exogenous cyclic AMP substantially stimulates the transcription of cob-lac fusions during aerobic growth. However, cyclic AMP is not absolutely required for the expression of the pathway, nor does it mediate the aerobic control. Cobalamin biosynthesis is not seen under aerobic growth conditions, even when transcription is stimulated by the addition of cyclic AMP. Hence, additional control mechanisms triggered by the presence of molecular oxygen must operate independently from transcription effects on the cob operons. PMID:3032913

  11. Evolution of mal ABC transporter operons in the Thermococcales and Thermotogales

    PubMed Central

    2008-01-01

    Background The mal genes that encode maltose transporters have undergone extensive lateral transfer among ancestors of the archaea Thermococcus litoralis and Pyrococcus furiosus. Bacterial hyperthermophiles of the order Thermotogales live among these archaea and so may have shared in these transfers. The genome sequence of Thermotoga maritima bears evidence of extensive acquisition of archaeal genes, so its ancestors clearly had the capacity to do so. We examined deep phylogenetic relationships among the mal genes of these hyperthermophiles and their close relatives to look for evidence of shared ancestry. Results We demonstrate that the two maltose ATP binding cassette (ABC) transporter operons now found in Tc. litoralis and P. furiosus (termed mal and mdx genes, respectively) are not closely related to one another. The Tc. litoralis and P. furiosus mal genes are most closely related to bacterial mal genes while their respective mdx genes are archaeal. The genes of the two mal operons in Tt. maritima are not related to genes in either of these archaeal operons. They are highly similar to one another and belong to a phylogenetic lineage that includes mal genes from the enteric bacteria. A unique domain of the enteric MalF membrane spanning proteins found also in these Thermotogales MalF homologs supports their relatively close relationship with these enteric proteins. Analyses of genome sequence data from other Thermotogales species, Fervidobacterium nodosum, Thermosipho melanesiensis, Thermotoga petrophila, Thermotoga lettingae, and Thermotoga neapolitana, revealed a third apparent mal operon, absent from the published genome sequence of Tt. maritima strain MSB8. This third operon, mal3, is more closely related to the Thermococcales' bacteria-derived mal genes than are mal1 and mal2. F. nodosum, Ts. melanesiensis, and Tt. lettingae have only one of the mal1-mal2 paralogs. The mal2 operon from an unknown species of Thermotoga appears to have been horizontally

  12. Characterization of the cyn operon in Escherichia coli K12.

    PubMed

    Sung, Y C; Fuchs, J A

    1988-10-15

    Escherichia coli can overcome the toxicity of environmental cyanate by hydrolysis of cyanate to ammonia and bicarbonate. This reaction is catalyzed by the enzyme cyanase, encoded by the cynS gene. The nucleotide sequence of cynS has been reported (Sung, Y.-c., Anderson, P. M., and Fuchs, J. A. (1987) J. Bacteriol. 169, 5224-5230). The nucleotide sequence of the complete cyn operon has now been determined. The cyn operon is approximately 2600 base pairs and includes cynT, cynS, and cynX, which encode cyanate permease, cyanase, and a protein of unknown function, respectively. Two cyanate-inducible transcripts of 1500 and 2500 nucleotides, respectively, were detected by Northern blot analysis. S1 nuclease mapping experiments indicated that two different cyn mRNAs have a common 5'-end and two different 3'-ends. One 3'-end was located within the coding region of cynX, whereas the other 3'-end includes the entire DNA sequence of cynX. The longer transcript contained 98 nucleotides complementary to lac mRNA produced by the predominant lac transcription termination sequence. Termination vectors were used to show that both 3'-ends were generated by sequences that caused transcriptional termination in vivo. Expression vectors were used to demonstrate that a protein corresponding to the expected size was synthesized from the DNA fragment containing the open reading frame designated cynX. The predicted amino acid sequence of cynX indicates that it is a very hydrophobic protein. The level of cynX expression was significantly less than that of cynT or cynS expression.

  13. The core promoter of the capsule operon of Streptococcus pneumoniae is necessary for colonization and invasive disease.

    PubMed

    Shainheit, Mara G; Mulé, Matthew; Camilli, Andrew

    2014-02-01

    Streptococcus pneumoniae is a commensal of the human nasopharynx but can cause invasive diseases, including otitis media, pneumonia, sepsis, and meningitis. The capsular polysaccharide (capsule) is a critical virulence factor required for both asymptomatic colonization and invasive disease, yet the expression level is different in each anatomical site. During colonization, reduced levels of capsule promote binding to the host epithelium and biofilm formation, while during systemic infection, increased capsule is required to evade opsonophagocytosis. How this regulation of capsule expression occurs is incompletely understood. To investigate the contribution of transcriptional regulation on capsule level in the serotype 4 strain TIGR4, we constructed two mutants harboring a constitutive promoter that was either comparably weaker (Pcat) or stronger (PtRNAGlu) than the wild-type (WT) capsule promoter, Pcps. Mild reductions in cpsA and cpsE transcript levels in the Pcat promoter mutant resulted in a 2-fold reduction in total amounts of capsule and in avirulence in murine models of lung and blood infection. Additionally, the PtRNAGlu mutant revealed that, despite expressing enhanced levels of cpsA and cpsE and possessing levels of capsule comparable to those of WT TIGR4, it was still significantly attenuated in all tested in vivo niches. Further analysis using chimeric promoter mutants revealed that the WT -10 and -35 boxes are required for optimal nasopharyngeal colonization and virulence. These data support the hypothesis that dynamic transcriptional regulation of the capsule operon is required and that the core promoter region plays a central role in fine-tuning levels of capsule to promote colonization and invasive disease.

  14. Postgenomic analysis of bacterial pathogens repertoire reveals genome reduction rather than virulence factors.

    PubMed

    Merhej, Vicky; Georgiades, Kalliopi; Raoult, Didier

    2013-07-01

    In the pregenomic era, the acquisition of pathogenicity islands via horizontal transfer was proposed as a major mechanism in pathogen evolution. Much effort has been expended to look for the contiguous blocks of virulence genes that are present in pathogenic bacteria, but absent in closely related species that are nonpathogenic. However, some of these virulence factors were found in nonpathogenic bacteria. Moreover, and contrary to expectation, pathogenic bacteria were found to lack genes (antivirulence genes) that are characteristic of nonpathogenic bacteria. The availability of complete genome sequences has led to a new era of pathogen research. Comparisons of genomes have shown that the most pathogenic bacteria have reduced genomes, with less ribosomal RNA and unorganized operons; they lack transcriptional regulators but have more genes that encode protein toxins, toxin-antitoxin (TA) modules, and proteins for DNA replication and repair, when compared with less pathogenic close relatives. These findings questioned the paradigm of virulence by gene acquisition and put forward the notion of genomic repertoire of virulence.

  15. N-acetylgalatosamine-Mediated Regulation of the aga Operon by AgaR in Streptococcus pneumoniae

    PubMed Central

    Afzal, Muhammad; Shafeeq, Sulman; Ahmed, Hifza; Kuipers, Oscar P.

    2016-01-01

    Here, we analyze the transcriptomic response of Streptococcus pneumoniae D39 to N-acetylgalactosamine (NAGa). Transcriptome comparison of S. pneumoniae D39 grown in NAGaM17 (0.5% NAGa + M17) to that grown in GM17 (0.5% Glucose + M17) revealed the elevated expression of various carbon metabolic genes/operons, including a PTS operon (denoted here as the aga operon), which is putatively involved in NAGa transport and utilization, in the presence of NAGa. We further studied the role of a GntR-family transcriptional regulator (denoted here as AgaR) in the regulation of aga operon. Our transcriptome and RT-PCR data suggest the role of AgaR as a transcriptional repressor of the aga operon. We predicted a 20-bp operator site of AagR (5′-ATAATTAATATAACAACAAA-3′) in the promoter region of the aga operon (PbgaC), which was further verified by mutating the AgaR operator site in the respective promoter. The role of CcpA in the additional regulation of the aga operon was elucidated by further transcriptome analyses and confirmed by quantitative RT-PCR.

  16. Impact of the deletion of the six mce operons in Mycobacterium smegmatis

    PubMed Central

    Klepp, Laura I.; Forrellad, Marina A.; Osella, Ana V.; Blanco, Federico C.; Stella, Emma J.; Bianco, María Verónica; Santangelo, María de la Paz; Sassetti, Cristopher; Jackson, Mary; Cataldi, Angel A.; Bigi, Fabiana; Morbidoni, Héctor R.

    2012-01-01

    The Mycobacterium smegmatis genome contains six operons designated mce (mammalian cell entry). These operons, which encode membrane and exported proteins, are highly conserved in pathogenic and non-pathogenic mycobacteria. Although the function of the Mce protein family has not yet been established in Mycobacterium smegmatis, the requirement of the mce4 operon for cholesterol utilization and uptake by Mycobacterium tuberculosis has recently been demonstrated. In this study, we report the construction of an M. smegmatis knock-out mutant deficient in the expression of all six mce operons. The consequences of these mutations were studied by analyzing physiological parameters and phenotypic traits. Differences in colony morphology, biofilm formation and aggregation in liquid cultures were observed, indicating that mce operons of M. smegmatis are implicated in the maintenance of the surface properties of the cell. Importantly, the mutant strain showed reduced cholesterol uptake when compared to the parental strain. Further cholesterol uptake studies using single mce mutant strains showed that the mutation of operon mce4 was reponsible for the cholesterol uptake failure detected in the sextuple mce mutant. This finding demonstrates that mce4 operon is involved in cholesterol transport in M. smegmatis. PMID:22353253

  17. Regulation of gene expression: cryptic β-glucoside (bgl) operon of Escherichia coli as a paradigm.

    PubMed

    Harwani, Dharmesh

    2014-01-01

    Bacteria have evolved various mechanisms to extract utilizable substrates from available resources and consequently acquire fitness advantage over competitors. One of the strategies is the exploitation of cryptic cellular functions encoded by genetic systems that are silent under laboratory conditions, such as the bgl (β-glucoside) operon of E. coli. The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to regulate its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. This is consistent with the observations that the activated operon confers a Growth Advantage in Stationary Phase (GASP) phenotype to Bgl(+) cells and exerts its regulation on at least twelve downstream target genes.

  18. Regulation of gene expression: cryptic β-glucoside (bgl) operon of Escherichia coli as a paradigm.

    PubMed

    Harwani, Dharmesh

    2014-01-01

    Bacteria have evolved various mechanisms to extract utilizable substrates from available resources and consequently acquire fitness advantage over competitors. One of the strategies is the exploitation of cryptic cellular functions encoded by genetic systems that are silent under laboratory conditions, such as the bgl (β-glucoside) operon of E. coli. The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to regulate its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. This is consistent with the observations that the activated operon confers a Growth Advantage in Stationary Phase (GASP) phenotype to Bgl(+) cells and exerts its regulation on at least twelve downstream target genes. PMID:25763016

  19. N-acetylgalatosamine-Mediated Regulation of the aga Operon by AgaR in Streptococcus pneumoniae

    PubMed Central

    Afzal, Muhammad; Shafeeq, Sulman; Ahmed, Hifza; Kuipers, Oscar P.

    2016-01-01

    Here, we analyze the transcriptomic response of Streptococcus pneumoniae D39 to N-acetylgalactosamine (NAGa). Transcriptome comparison of S. pneumoniae D39 grown in NAGaM17 (0.5% NAGa + M17) to that grown in GM17 (0.5% Glucose + M17) revealed the elevated expression of various carbon metabolic genes/operons, including a PTS operon (denoted here as the aga operon), which is putatively involved in NAGa transport and utilization, in the presence of NAGa. We further studied the role of a GntR-family transcriptional regulator (denoted here as AgaR) in the regulation of aga operon. Our transcriptome and RT-PCR data suggest the role of AgaR as a transcriptional repressor of the aga operon. We predicted a 20-bp operator site of AagR (5′-ATAATTAATATAACAACAAA-3′) in the promoter region of the aga operon (PbgaC), which was further verified by mutating the AgaR operator site in the respective promoter. The role of CcpA in the additional regulation of the aga operon was elucidated by further transcriptome analyses and confirmed by quantitative RT-PCR. PMID:27672623

  20. Overlapping mRNA transcripts of photosynthesis gene operons in Rhodobacter capsulatus.

    PubMed Central

    Wellington, C L; Beatty, J T

    1991-01-01

    The crtEF, bchCA, and puf operons of the facultative phototrophic bacterium Rhodobacter capsulatus encode gene products that are necessary for the formation of various components of the photosynthetic apparatus. The crtEF operon encodes two enzymes involved in the biosynthesis of carotenoids, the bchCA operon codes for two enzymes of the bacteriochlorophyll biosynthetic pathway, and the puf operon encodes four pigment-binding polypeptides as well as two polypeptides with less well understood functions. These three operons are adjacent to one another on the chromosome and are transcribed in the same direction. We present the results of RNA blotting and S1 nuclease protection end-mapping experiments which provide direct evidence that the mRNA transcripts of these three operons overlap. Therefore, it is likely that the crtEF, bchCA, and puf operons can be expressed as a single transcriptional unit, although RNA polymerase may initiate transcription at any of several promoters. Images PMID:1995590

  1. The Chlamydia trachomatis hyp operon is homologous to the groE stress response operon of Escherichia coli.

    PubMed

    Morrison, R P; Su, H; Lyng, K; Yuan, Y

    1990-08-01

    The Chlamydia trachomatis serovar A hyp operon was cloned, sequenced, and expressed in Escherichia coli. Two cotranscribed open reading frames, hypA and hypB, encoded polypeptides of 17 and 57 kilodaltons, respectively. The deduced amino acid sequences of serovar A HypA and HypB proteins were (respectively) 85 and 94% identical with HypA and HypB proteins of Chlamydia psittaci GPIC, and HypB was greater than 50% identical to 60-kilodalton stress response proteins from other procaryotes and eucaryotes. The sequence should be useful in defining the antigenic structure of the Chlamydia trachomatis HypB protein, a necessary step toward understanding the relationship between the immune response to this protein and the pathogenesis of human chlamydial diseases.

  2. Ancient origin of the tryptophan operon and the dynamics of evolutionary change.

    PubMed

    Xie, Gary; Keyhani, Nemat O; Bonner, Carol A; Jensen, Roy A

    2003-09-01

    The seven conserved enzymatic domains required for tryptophan (Trp) biosynthesis are encoded in seven genetic regions that are organized differently (whole-pathway operons, multiple partial-pathway operons, and dispersed genes) in prokaryotes. A comparative bioinformatics evaluation of the conservation and organization of the genes of Trp biosynthesis in prokaryotic operons should serve as an excellent model for assessing the feasibility of predicting the evolutionary histories of genes and operons associated with other biochemical pathways. These comparisons should provide a better understanding of possible explanations for differences in operon organization in different organisms at a genomics level. These analyses may also permit identification of some of the prevailing forces that dictated specific gene rearrangements during the course of evolution. Operons concerned with Trp biosynthesis in prokaryotes have been in a dynamic state of flux. Analysis of closely related organisms among the Bacteria at various phylogenetic nodes reveals many examples of operon scission, gene dispersal, gene fusion, gene scrambling, and gene loss from which the direction of evolutionary events can be deduced. Two milestone evolutionary events have been mapped to the 16S rRNA tree of Bacteria, one splitting the operon in two, and the other rejoining it by gene fusion. The Archaea, though less resolved due to a lesser genome representation, appear to exhibit more gene scrambling than the Bacteria. The trp operon appears to have been an ancient innovation; it was already present in the common ancestor of Bacteria and Archaea. Although the operon has been subjected, even in recent times, to dynamic changes in gene rearrangement, the ancestral gene order can be deduced with confidence. The evolutionary history of the genes of the pathway is discernible in rough outline as a vertical line of descent, with events of lateral gene transfer or paralogy enriching the analysis as interesting

  3. Virulence Potential of Fusogenic Orthoreoviruses

    PubMed Central

    Cheng, Peter K.C.; Lai, Mary Y.Y.; Leung, Peter C.K.; Wong, Kitty K.Y.; Lee, W.Y.; Lim, Wilina W.L.

    2012-01-01

    Several severe respiratory virus infections that have emerged during the past decade originated in animals, including bats. In Indonesia, exposure to bats has been associated with increased risk of acquiring orthoreovirus infection. Although orthoreovirus infections are mild and self-limiting, we explored their potential for evolution into a more virulent form. We used conventional virus culture, electron microscopy, and molecular sequencing to isolate and identify orthoreoviruses from 3 patients in whom respiratory tract infection developed after travel to Indonesia. Virus characterization by plaque-reduction neutralization testing showed antigenic similarity, but sequencing of the small segment genes suggested virus reassortment, which could lead to increased virulence. Bats as a reservoir might contribute to virus evolution and genetic diversity, giving orthoreoviruses the potential to become more virulent. Evolution of this virus should be closely monitored so that prevention and control measures can be taken should it become more virulent. PMID:22608100

  4. Quantitative approaches to the study of bistability in the lac operon of Escherichia coli.

    PubMed

    Santillán, Moisés; Mackey, Michael C

    2008-08-01

    In this paper, the history and importance of the lac operon in the development of molecular and systems biology are briefly reviewed. We start by presenting a description of the regulatory mechanisms in this operon, taking into account the most recent discoveries. Then we offer a survey of the history of the lac operon, including the discovery of its main elements and the subsequent influence on the development of molecular and systems biology. Next the bistable behaviour of the operon is discussed, both with respect to its discovery and its molecular origin. A review of the literature in which this bistable phenomenon has been studied from a mathematical modelling viewpoint is then given. We conclude with some brief remarks. PMID:18426771

  5. Quantitative approaches to the study of bistability in the lac operon of Escherichia coli.

    PubMed

    Santillán, Moisés; Mackey, Michael C

    2008-08-01

    In this paper, the history and importance of the lac operon in the development of molecular and systems biology are briefly reviewed. We start by presenting a description of the regulatory mechanisms in this operon, taking into account the most recent discoveries. Then we offer a survey of the history of the lac operon, including the discovery of its main elements and the subsequent influence on the development of molecular and systems biology. Next the bistable behaviour of the operon is discussed, both with respect to its discovery and its molecular origin. A review of the literature in which this bistable phenomenon has been studied from a mathematical modelling viewpoint is then given. We conclude with some brief remarks.

  6. Cycling expression and cooperative operator interaction in the trp operon of Escherichia coli.

    PubMed

    Hernández-Valdez, Areli; Santillán, Moisés; Zeron, Eduardo S

    2010-04-01

    Oscillatory behaviour in the tryptophan operon of an Escherichia coli mutant strain lacking the enzyme-inhibition regulatory mechanism has been observed by Bliss et al. but not confirmed by others. This behaviour could be important from the standpoint of synthetic biology, whose goals include the engineering of intracellular genetic oscillators. This work is devoted to investigating, from a mathematical modelling point of view, the possibility that the trp operon of the E. coli inhibition-free strain expresses cyclically. For that we extend a previously introduced model for the regulatory pathway of the tryptophan operon in Escherichia coli to account for the observed multiplicity and cooperativity of repressor binding sites. Thereafter we investigate the model dynamics using deterministic numeric solutions, stochastic simulations, and analytic studies. Our results suggest that a quasi-periodic behaviour could be observed in the trp operon expression level of single bacteria. PMID:20004672

  7. Transcript Cleavage, Attenuation, and an Internal Promoter in the Rhodobacter capsulatus puc Operon

    PubMed Central

    LeBlanc, H.; Lang, A. S.; Beatty, J. T.

    1999-01-01

    The stoichiometry of the structural proteins of the photosynthetic apparatus in purple photosynthetic bacteria is achieved primarily by complex regulation of the levels of mRNA encoding the different proteins, which has been studied in the greatest detail in the puf operon. Here we investigated the transcriptional and posttranscriptional regulation of the puc operon, which encodes the peripheral light harvesting complex LHII. We show that, analogous to the puf operon, a primary transcript encoding five puc genes is rapidly processed to generate more stable RNA subspecies. Contrary to previous hypotheses, translational coupling and regulation of puc transcription by puc gene products were found not to occur. A putative RNA stem-loop structure appears to attenuate transcription initiated at the puc operon major promoter. We also found that a minor pucD-internal promoter contributes to the levels of a message that encodes the LHII 14-kDa γ (PucE) protein. PMID:10438767

  8. [Proteolytic control of expression of Vibrio fischeri lux-operon genes in Escherichia coli cells].

    PubMed

    Mel'kina, O E; Manukhov, I V; Zavil'gel'skiĭ, G B

    2010-08-01

    The key elements of the regulatory system activating expression of the lux-operon genes in the sea bacteria Vibrio fischeri are the LuxR protein (an activator oftranscription) and N-(3-oxohexanoyl) L-homoserine lactone (an autoinducer, AI). It is shown that the ATP-dependent proteases ClpXP and Lon take part in the negative control of expression of the lux-operon genes and that AI protects the LuxR protein from proteolysis.

  9. Analysis of the puc Operon Promoter from Rhodobacter capsulatus

    PubMed Central

    Nickens, David G.; Bauer, Carl E.

    1998-01-01

    Expression of the Rhodobacter capsulatus puc operon, which codes for structural polypeptides of the light-harvesting-II peripheral antenna complex, is highly regulated in response to alterations in oxygen tension and light intensity. To obtain an understanding of the puc promoter region we report the high-resolution 5′ mapping of the puc mRNA transcriptional start site and DNA sequence analysis of the puc upstream regulatory sequence (pucURS). A ς70-type promoter sequence was identified (pucP1) which has a high degree of sequence similarity with carotenoid and bacteriochlorophyll biosynthesis promoters. Inspection of the DNA sequence also indicated the presence of two CrtJ and four integration host factor (IHF) binding sites. Transcriptional fusions of the pucURS fused to lacZ also confirmed that puc promoter activity is regulated by the transcriptional regulators IHF, CrtJ, and RegA. Gel retardation analysis using cell extracts indicates that mutations in IHF and RegA disrupt protein binding to DNA fragments containing the pucURS. PMID:9696778

  10. Anaerobically expressed Escherichia coli genes identified by operon fusion techniques.

    PubMed Central

    Choe, M; Reznikoff, W S

    1991-01-01

    Genes that are expressed under anaerobic conditions were identified by operon fusion techniques with a hybrid bacteriophage of lambda and Mu, lambda placMu53, which creates transcriptional fusions to lacZY. Cells were screened for anaerobic expression on XG medium. Nine strains were selected, and the insertion point of the hybrid phage in each strain was mapped on the Escherichia coli chromosome linkage map. The anaerobic and aerobic expression levels of these genes were measured by beta-galactosidase assays in different medium conditions and in the presence of three regulatory mutations (fnr, narL, and rpoN). The anaerobically expressed genes (aeg) located at minute 99 (aeg-99) and 75 (aeg-75) appeared to be partially regulated by fnr, and aeg-93 is tightly regulated by fnr. aeg-60 requires a functional rpoN gene for its anaerobic expression. aeg-46.5 is repressed by narL. aeg-65A and aeg-65C are partially controlled by fnr but only in media containing nitrate or fumarate. aeg-47.5 and aeg-48.5 were found to be anaerobically induced only in rich media. The effects of a narL mutation on aeg-46.5 expression were observed in all medium conditions regardless of the presence or absence of nitrate. This suggests that narL has a regulatory function in the absence of exogenously added nitrate. PMID:1917846

  11. Exploiting Bacterial Operons To Illuminate Human Iron-Sulfur Proteins.

    PubMed

    Andreini, Claudia; Banci, Lucia; Rosato, Antonio

    2016-04-01

    Organisms from all kingdoms of life use iron-sulfur proteins (FeS-Ps) in a multitude of functional processes. We applied a bioinformatics approach to investigate the human portfolio of FeS-Ps. Sixty-one percent of human FeS-Ps bind Fe4S4 clusters, whereas 39% bind Fe2S2 clusters. However, this relative ratio varies significantly depending on the specific cellular compartment. We compared the portfolio of human FeS-Ps to 12 other eukaryotes and to about 700 prokaryotes. The comparative analysis of the organization of the prokaryotic homologues of human FeS-Ps within operons allowed us to reconstruct the human functional networks involving the conserved FeS-Ps common to prokaryotes and eukaryotes. These functional networks have been maintained during evolution and thus presumably represent fundamental cellular processes. The respiratory chain and the ISC machinery for FeS-P biogenesis are the two conserved processes that involve the majority of human FeS-Ps. Purine metabolism is another process including several FeS-Ps, in which BOLA proteins possibly have a regulatory role. The analysis of the co-occurrence of human FeS-Ps with other proteins highlighted numerous links between the iron-sulfur cluster machinery and the response mechanisms to cell damage, from repair to apoptosis. This relationship probably relates to the production of reactive oxygen species within the biogenesis and degradation of FeS-Ps.

  12. A Novel Method for Accurate Operon Predictions in All SequencedProkaryotes

    SciTech Connect

    Price, Morgan N.; Huang, Katherine H.; Alm, Eric J.; Arkin, Adam P.

    2004-12-01

    We combine comparative genomic measures and the distance separating adjacent genes to predict operons in 124 completely sequenced prokaryotic genomes. Our method automatically tailors itself to each genome using sequence information alone, and thus can be applied to any prokaryote. For Escherichia coli K12 and Bacillus subtilis, our method is 85 and 83% accurate, respectively, which is similar to the accuracy of methods that use the same features but are trained on experimentally characterized transcripts. In Halobacterium NRC-1 and in Helicobacterpylori, our method correctly infers that genes in operons are separated by shorter distances than they are in E.coli, and its predictions using distance alone are more accurate than distance-only predictions trained on a database of E.coli transcripts. We use microarray data from sixphylogenetically diverse prokaryotes to show that combining intergenic distance with comparative genomic measures further improves accuracy and that our method is broadly effective. Finally, we survey operon structure across 124 genomes, and find several surprises: H.pylori has many operons, contrary to previous reports; Bacillus anthracis has an unusual number of pseudogenes within conserved operons; and Synechocystis PCC6803 has many operons even though it has unusually wide spacings between conserved adjacent genes.

  13. DOOR 2.0: presenting operons and their functions through dynamic and integrated views

    PubMed Central

    Mao, Xizeng; Ma, Qin; Zhou, Chuan; Chen, Xin; Zhang, Hanyuan; Yang, Jincai; Mao, Fenglou; Lai, Wei; Xu, Ying

    2014-01-01

    We have recently developed a new version of the DOOR operon database, DOOR 2.0, which is available online at http://csbl.bmb.uga.edu/DOOR/ and will be updated on a regular basis. DOOR 2.0 contains genome-scale operons for 2072 prokaryotes with complete genomes, three times the number of genomes covered in the previous version published in 2009. DOOR 2.0 has a number of new features, compared with its previous version, including (i) more than 250 000 transcription units, experimentally validated or computationally predicted based on RNA-seq data, providing a dynamic functional view of the underlying operons; (ii) an integrated operon-centric data resource that provides not only operons for each covered genome but also their functional and regulatory information such as their cis-regulatory binding sites for transcription initiation and termination, gene expression levels estimated based on RNA-seq data and conservation information across multiple genomes; (iii) a high-performance web service for online operon prediction on user-provided genomic sequences; (iv) an intuitive genome browser to support visualization of user-selected data; and (v) a keyword-based Google-like search engine for finding the needed information intuitively and rapidly in this database. PMID:24214966

  14. Regulatory regions of two transport operons under nitrogen control: nucleotide sequences.

    PubMed Central

    Higgins, C F; Ames, G F

    1982-01-01

    We have determined the nucleotide sequences of the regulatory regions from two amino acid transport operons from Salmonella typhimurium: dhuA, which regulates the histidine transport operon, and argTr, which regulates argT, the gene encoding the lysine-arginine-ornithine-binding protein, LAO. The promoter for the histidine transport operon has been identified from the sequence change in the promoter-up mutation dhuA1. Neither regulatory region has any of the features typical of the regulatory regions of the amino acid biosynthetic operons, indicating that regulation of at least these transport genes does not involve a transcription attenuation mechanism. We have identified three interesting features, present in both of these sequences, which may be of importance in the regulation of these and other operons: a "stem-loop-foot" structure, a region of specific homology, and a mirror symmetry. The region of mirror symmetry may be a protein recognition site important is regulating expression of these and other operons in response to nitrogen availability. Mirror symmetry as a structure for DNA-protein interaction sites has not been proposed previously. PMID:7041112

  15. Solving a discrete model of the lac operon using Z3

    NASA Astrophysics Data System (ADS)

    Gutierrez, Natalia A.

    2014-05-01

    A discrete model for the Lcac Operon is solved using the SMT-solver Z3. Traditionally the Lac Operon is formulated in a continuous math model. This model is a system of ordinary differential equations. Here, it was considerated as a discrete model, based on a Boolean red. The biological problem of Lac Operon is enunciated as a problem of Boolean satisfiability, and it is solved using an STM-solver named Z3. Z3 is a powerful solver that allows understanding the basic dynamic of the Lac Operon in an easier and more efficient way. The multi-stability of the Lac Operon can be easily computed with Z3. The code that solves the Boolean red can be written in Python language or SMT-Lib language. Both languages were used in local version of the program as online version of Z3. For future investigations it is proposed to solve the Boolean red of Lac Operon using others SMT-solvers as cvc4, alt-ergo, mathsat and yices.

  16. Organization and expression of photosynthesis genes and operons in anoxygenic photosynthetic proteobacteria.

    PubMed

    Liotenberg, Sylviane; Steunou, Anne-Soisig; Picaud, Martine; Reiss-Husson, Françoise; Astier, Chantal; Ouchane, Soufian

    2008-09-01

    Genes belonging to the same metabolic route are usually organized in operons in microbial genomes. For instance, most genes involved in photosynthesis were found clustered and organized in operons in photosynthetic Alpha- and Betaproteobacteria. The discovery of Gammaproteobacteria with a conserved photosynthetic gene cluster revives the questions on the role and the maintenance of such organization in proteobacteria. In this paper, we report the analysis of the structure and expression of the 14 kb cluster (crtEF-bchCXYZ-pufBALMC-crtADC) in the photosynthetic betaproteobacterium Rubrivivax gelatinosus, with the purpose of understanding the reasons and the biological constraints that might have led to the clustering of photosynthesis genes. The genetic analyses are substantiated by reverse transcription-PCR data which reveal the presence of a transcript encompassing the 14 genes and provide evidence of a polycistronic 'super-operon' organization starting at crtE and ending 14 kb downstream at the crtC gene. Furthermore, genetic analyses suggest that one of the selection pressures that may have driven and maintained the photosynthesis operons/super-operons in proteobacteria could very likely be the coexpression and regulation of the clustered genes/operon.

  17. Virulence of the Pseudomonas fluorescens clinical strain MFN1032 towards Dictyostelium discoideum and macrophages in relation with type III secretion system

    PubMed Central

    2012-01-01

    Background Pseudomonas fluorescens biovar I MFN1032 is a clinical isolate able to grow at 37°C. This strain displays secretion-mediated hemolytic activity involving phospholipase C and cyclolipopeptides, and a cell-associated hemolytic activity distinct from the secreted hemolytic activity. Cell-associated hemolysis is independent of biosurfactant production and remains in a gacA mutant. Disruption of the hrpU-like operon (the basal part of type III secretion system from rhizospheric strains) suppresses this activity. We hypothesized that this phenotype could reflect evolution of an ancestral mechanism involved in the survival of this species in its natural niche. In this study, we evaluated the hrpU-like operon’s contribution to other virulence mechanisms using a panel of Pseudomonas strains from various sources. Results We found that MFN1032 inhibited the growth of the amoebae Dictyostelium discoideum and that this inhibition involved the hrpU-like operon and was absent in a gacA mutant. MFN1032 was capable of causing macrophage lysis, if the hrpU-like operon was intact, and this cytotoxicity remained in a gacA mutant. Cell-associated hemolytic activity and macrophage necrosis were found in other P. fluorescens clinical isolates, but not in biocontrol P. fluorescens strains harbouring hrpU-like operon. The growth of Dictyostelium discoideum was inhibited to a different extent by P. fluorescens strains without correlation between this inhibition and hrpU-like operon sequences. Conclusions In P. fluorescens MFN1032, the basal part of type III secretion system plays a role in D. discoideum growth inhibition and macrophage necrosis. The inhibition of D. discoideum growth is dependent on the GacS/GacA system, while cell-associated hemolytic activity and macrophage lysis are not. Virulence against eukaryotic cells based on the hrpU-like operon may be more than just a stochastic evolution of a conserved system dedicated to survival in competition with natural

  18. FIS-dependent trans activation of stable RNA operons of Escherichia coli under various growth conditions.

    PubMed Central

    Nilsson, L; Verbeek, H; Vijgenboom, E; van Drunen, C; Vanet, A; Bosch, L

    1992-01-01

    In Escherichia coli transcription of the tRNA operon thrU (tufB) and the rRNA operon rrnB is trans-activated by the protein FIS. This protein, which stimulates the inversion of various viral DNA segments, binds specifically to a cis-acting sequence (designated UAS) upstream of the promoter of thrU (tufB) and the P1 promoter of the rrnB operon. There are indications that this type of regulation is representative for the regulation of more stable RNA operons. In the present investigation we have studied UAS-dependent transcription activation of the thrU (tufB) operon in the presence and absence of FIS during a normal bacterial growth cycle and after a nutritional shift-up. In early log phase the expression of the operon rises steeply in wild-type cells, whereafter it declines. Concomitantly, a peak of the cellular FIS concentration is observed. Cells in the stationary phase are depleted of FIS. The rather abrupt increase of transcription activation depends on the nutritional quality of the medium. It is not seen in minimal medium. After a shift from minimal to rich medium, a peak of transcription activation and of FIS concentration is measured. This peak gets higher as the medium gets more strongly enriched. We conclude that a correlation between changes of the UAS-dependent activation of the thrU (tufB) operon and changes of the cellular FIS concentration under a variety of experimental conditions exists. This correlation strongly suggests that the production of FIS responds to environmental signals, thereby trans-activating the operon. Cells unable to produce FIS (fis cells) also show an increase of operon transcription in the early log phase and after a nutritional shift-up, albeit less pronounced than that wild-type cells. Presumably it is controlled by the ribosome feedback regulatory system. cis activation of the operon by the upstream activator sequence is apparent in the absence of FIS. This activation is constant throughout the entire growth cycle and is

  19. Interference competition and parasite virulence.

    PubMed Central

    Massey, Ruth C.; Buckling, Angus; ffrench-Constant, Richard

    2004-01-01

    Within-host competition between parasites, a consequence of infection by multiple strains, is predicted to favour rapid host exploitation and greater damage to hosts (virulence). However, the inclusion of biological variables can drastically change this relationship. For example, if competing parasite strains produce toxins that kill each other (interference competition), their growth rates and virulence may be reduced relative to single-strain infections. Bacteriocins are antimicrobial toxins produced by bacteria that target closely related strains and species, and to which the producing strain is immune. We investigated competition between bacteriocin-producing, insect-killing bacteria (Photorhabdus and Xenorhabdus) and how this competition affected virulence in caterpillars. Where one strain could kill the other, and not vice versa, the non-killing strain was competitively excluded, and insect mortality was the same as that of the killing strain alone. However, when caterpillars were multiply infected by strains that could kill each other, we did not observe competitive exclusion and their virulence was less than single-strain infections. The ubiquity and diversity of bacteriocins among pathogenic bacteria suggest mixed infections will be, on average, less virulent than single infections. PMID:15255095

  20. Vibrio campbellii hmgA-mediated pyomelanization impairs quorum sensing, virulence, and cellular fitness

    PubMed Central

    Wang, Zheng; Lin, Baochuan; Mostaghim, Anahita; Rubin, Robert A.; Glaser, Evan R.; Mittraparp-arthorn, Pimonsri; Thompson, Janelle R.; Vuddhakul, Varaporn; Vora, Gary J.

    2013-01-01

    Melanization due to the inactivation of the homogentisate-1,2-dioxygenase gene (hmgA) has been demonstrated to increase stress resistance, persistence, and virulence in some bacterial species but such pigmented mutants have not been observed in pathogenic members of the Vibrio Harveyi clade. In this study, we used Vibrio campbellii ATCC BAA-1116 as model organism to understand how melanization affected cellular phenotype, metabolism, and virulence. An in-frame deletion of the hmgA gene resulted in the overproduction of a pigment in cell culture supernatants and cellular membranes that was identified as pyomelanin. Unlike previous demonstrations in Vibrio cholerae, Burkholderia cepacia, and Pseudomonas aeruginosa, the pigmented V. campbellii mutant did not show increased UV resistance and was found to be ~2.7 times less virulent than the wild type strain in Penaeus monodon shrimp virulence assays. However, the extracted pyomelanin pigment did confer a higher resistance to oxidative stress when incubated with wild type cells. Microarray-based transcriptomic analyses revealed that the hmgA gene deletion and subsequent pyomelanin production negatively effected the expression of 129 genes primarily involved in energy production, amino acid, and lipid metabolism, and protein translation and turnover. This transcriptional response was mediated in part by an impairment of the quorum sensing regulon as transcripts of the quorum sensing high cell density master regulator LuxR and other operonic members of this regulon were significantly less abundant in the hmgA mutant. Taken together, the results suggest that the pyomelanization of V. campbellii sufficiently impairs the metabolic activities of this organism and renders it less fit and virulent than its isogenic wild type strain. PMID:24376440

  1. Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression.

    PubMed

    Kasper, Stephen H; Bonocora, Richard P; Wade, Joseph T; Musah, Rabi Ann; Cady, Nathaniel C

    2016-04-15

    The opportunistic pathogen Pseudomonas aeruginosa utilizes multiple quorum sensing (QS) pathways to coordinate an arsenal of virulence factors. We previously identified several cysteine-based compounds inspired by natural products from the plant Petiveria alliacea which are capable of antagonizing multiple QS circuits as well as reducing P. aeruginosa biofilm formation. To understand the global effects of such compounds on virulence factor production and elucidate their mechanism of action, RNA-seq transcriptomic analysis was performed on P. aeruginosa PAO1 exposed to S-phenyl-l-cysteine sulfoxide, the most potent inhibitor from the prior study. Exposure to this inhibitor down-regulated expression of several QS-regulated virulence operons (e.g., phenazine biosynthesis, type VI secretion systems). Interestingly, many genes that were differentially regulated pertain to the related metabolic pathways that yield precursors of pyochelin, tricarboxylic acid cycle intermediates, phenazines, and Pseudomonas quinolone signal (PQS). Activation of the MexT-regulon was also indicated, including the multidrug efflux pump encoded by mexEF-oprN, which has previously been shown to inhibit QS and pathogenicity. Deeper investigation of the metabolites involved in these systems revealed that S-phenyl-l-cysteine sulfoxide has structural similarity to kynurenine, a precursor of anthranilate, which is critical for P. aeruginosa virulence. By supplementing exogenous anthranilate, the QS-inhibitory effect was reversed. Finally, it was shown that S-phenyl-l-cysteine sulfoxide competitively inhibits P. aeruginosa kynureninase (KynU) activity in vitro and reduces PQS production in vivo. The kynurenine pathway has been implicated in P. aeruginosa QS and virulence factor expression; however, this is the first study to show that targeted inhibition of KynU affects P. aeruginosa gene expression and QS, suggesting a potential antivirulence strategy.

  2. Chemical Inhibition of Kynureninase Reduces Pseudomonas aeruginosa Quorum Sensing and Virulence Factor Expression.

    PubMed

    Kasper, Stephen H; Bonocora, Richard P; Wade, Joseph T; Musah, Rabi Ann; Cady, Nathaniel C

    2016-04-15

    The opportunistic pathogen Pseudomonas aeruginosa utilizes multiple quorum sensing (QS) pathways to coordinate an arsenal of virulence factors. We previously identified several cysteine-based compounds inspired by natural products from the plant Petiveria alliacea which are capable of antagonizing multiple QS circuits as well as reducing P. aeruginosa biofilm formation. To understand the global effects of such compounds on virulence factor production and elucidate their mechanism of action, RNA-seq transcriptomic analysis was performed on P. aeruginosa PAO1 exposed to S-phenyl-l-cysteine sulfoxide, the most potent inhibitor from the prior study. Exposure to this inhibitor down-regulated expression of several QS-regulated virulence operons (e.g., phenazine biosynthesis, type VI secretion systems). Interestingly, many genes that were differentially regulated pertain to the related metabolic pathways that yield precursors of pyochelin, tricarboxylic acid cycle intermediates, phenazines, and Pseudomonas quinolone signal (PQS). Activation of the MexT-regulon was also indicated, including the multidrug efflux pump encoded by mexEF-oprN, which has previously been shown to inhibit QS and pathogenicity. Deeper investigation of the metabolites involved in these systems revealed that S-phenyl-l-cysteine sulfoxide has structural similarity to kynurenine, a precursor of anthranilate, which is critical for P. aeruginosa virulence. By supplementing exogenous anthranilate, the QS-inhibitory effect was reversed. Finally, it was shown that S-phenyl-l-cysteine sulfoxide competitively inhibits P. aeruginosa kynureninase (KynU) activity in vitro and reduces PQS production in vivo. The kynurenine pathway has been implicated in P. aeruginosa QS and virulence factor expression; however, this is the first study to show that targeted inhibition of KynU affects P. aeruginosa gene expression and QS, suggesting a potential antivirulence strategy. PMID:26785289

  3. Antipolarity in the ilv Operon of Escherichia coli K-12

    PubMed Central

    Wechsler, James A.; Adelberg, E. A.

    1969-01-01

    The genes governing three of the enzymes of the isoleucine-valine biosynthetic pathway form the operon: operator-ilvA-ilvD-ilvE. The enzymes are: ilvA, l-threonine deaminase; ilvD, dihydroxy acid dehydrase; and ilvE, transaminase B. A nonsense mutation in the ilvD gene (D-ochre) and a nonsense mutation in the ilvE gene (E-amber) affect the properties of the proximal gene product, l-threonine deaminase (TD), in addition to inactivating the enzymes produced by the genes in which the mutations have occurred. The D-ochre mutation causes TD to move in diffusion and gel filtration experiments as though it were 30% smaller than the wild-type enzyme. The E-amber mutation causes TD to move in similar experiments as though it were much larger than the wild-type enzyme. Both mutations completely abolish the sensitivity of TD to l-isoleucine, the normal feedback inhibitor of the wild-type enzyme. The effects of the nonsense mutations on TD can be reversed in three ways: by genetic reversion of the D-ochre mutation; by treatment of the altered enzymes with 3.0 m urea; and by forming a heterozygous diploid, containing the wild-type allele as well as the mutant allele of ilvD or ilvE. The results suggest that the subunits of TD undergo abnormal aggregation in the presence of the partial polypeptides produced by the mutant alleles of ilvD or ilvE; multi-enzyme aggregates in extracts of wild type, however, could not be detected. PMID:4892370

  4. Kinetic approaches to lactose operon induction and bimodality.

    PubMed

    Michel, Denis

    2013-05-21

    The quasi-equilibrium approximation is acceptable when molecular interactions are fast enough compared to circuit dynamics, but is no longer allowed when cellular activities are governed by rare events. A typical example is the lactose operon (lac), one of the most famous paradigms of transcription regulation, for which several theories still coexist to describe its behaviors. The lac system is generally analyzed by using equilibrium constants, contradicting single-event hypotheses long suggested by Novick and Weiner (1957). Enzyme induction as an all-or-none phenomenon. Proc. Natl. Acad. Sci. USA 43, 553-566) and recently refined in the study of (Choi et al., 2008. A stochastic single-molecule event triggers phenotype switching of a bacterial cell. Science 322, 442-446). In the present report, a lac repressor (LacI)-mediated DNA immunoprecipitation experiment reveals that the natural LacI-lac DNA complex built in vivo is extremely tight and long-lived compared to the time scale of lac expression dynamics, which could functionally disconnect the abortive expression bursts and forbid using the standard modes of lac bistability. As alternatives, purely kinetic mechanisms are examined for their capacity to restrict induction through: (i) widely scattered derepression related to the arrival time variance of a predominantly backward asymmetric random walk and (ii) an induction threshold arising in a single window of derepression without recourse to nonlinear multimeric binding and Hill functions. Considering the complete disengagement of the lac repressor from the lac promoter as the probabilistic consequence of a transient stepwise mechanism, is sufficient to explain the sigmoidal lac responses as functions of time and of inducer concentration. This sigmoidal shape can be misleadingly interpreted as a phenomenon of equilibrium cooperativity classically used to explain bistability, but which has been reported to be weak in this system. PMID:23454080

  5. Ribosomal multi-operon diversity: an original perspective on the genus Aeromonas.

    PubMed

    Roger, Frédéric; Lamy, Brigitte; Jumas-Bilak, Estelle; Kodjo, Angeli; Marchandin, Hélène

    2012-01-01

    16S rRNA gene (rrs) is considered of low taxonomic interest in the genus Aeromonas. Here, 195 Aeromonas strains belonging to populations structured by multilocus phylogeny were studied using an original approach that considered Ribosomal Multi-Operon Diversity. This approach associated pulsed-field gel electrophoresis (PFGE) to assess rrn operon number and distribution across the chromosome and PCR-temporal temperature gel electrophoresis (TTGE) to assess rrs V3 region heterogeneity. Aeromonads harbored 8 to 11 rrn operons, 10 operons being observed in more than 92% of the strains. Intraspecific variability was low or nul except for A. salmonicida and A. aquariorum suggesting that large chromosomic rearrangements might occur in these two species while being extremely rarely encountered in the evolution of other taxa. rrn operon number at 8 as well as PFGE patterns were shown valuable for taxonomic purpose allowing resolution of species complexes. PCR-TTGE revealed a high rate of strains (41.5%) displaying intragenomic rrs heterogeneity. Strains isolated from human samples more frequently displayed intragenomic heterogeneity than strains recovered from non-human and environmental specimens. Intraspecific variability ranged from 0 to 76.5% of the strains. The observation of species-specific TTGE bands, the recovery of identical V3 regions in different species and the variability of intragenomic heterogeneity (1-13 divergent nucleotides) supported the occurrence of mutations and horizontal transfer in aeromonad rrs evolution. Altogether, the presence of a high number of rrn operon, the high proportion of strains harboring divergent rrs V3 region and the previously demonstrated high level of genetic diversity argued in favor of highly adaptative capabilities of aeromonads. Outstanding features observed for A. caviae supported the ongoing process of adaptation to a specialized niche represented by the gut, previously hypothesized. 16S rRNA gene is an informative marker

  6. [Regulation in the expression of alpha-galactosidase gene in raf operon in Escherichia coli].

    PubMed

    Su, T Z; Qi, S; Yun, W H; Xiu, L

    1989-06-01

    The alpha-galactosidase, coded for by the first structural gene rafA in the plasmid determined raf operon was an inducible enzyme. In contrast to lac or mel operon, raf operon has more strict structural specificity for inducers. The enzyme can be induced by melibiose and raffinose, or weakly by D-galactose, but not by structurally related sugars such as lactose, PNPG etc.. The alpha-galactosidase forming capacity as function of growth curve reached a single peak at the end of the logarithmic phase of the growth. The structure and regulation of raf operon is similar to those of lac operon. The repressormor-mediated negative control plays a major role in the regulation of raf operon, and cAMP-CAP mediated positive control is also involved in the regulation. When 0.4% glucose was added into the medium with other carbon sources, the expression of the enzyme was repressed by 2-3 fold. Transient catabolite repression has been observed neither in inducible nor constitutive alpha-galactosidase expression. Based on alpha-galactosidase assay, in mutant strains CA8306(cya) and CA8445 (cya, crp) the expression level of raf operon was only 9% and 2.5% of that in wild type strain respectively. The glucose effect or the repression in cya mutant can be abolished by 1-5 mmol cAMP. The constitutive alpha-galactosidase expression in cya and cry double mutant (CA8445) remains repressible by glucose, but irreversible by cAMP, suggesting cAMP-CAP complex is not the exclusive mediator of the catablite repression.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2551100

  7. Ribosomal Multi-Operon Diversity: An Original Perspective on the Genus Aeromonas

    PubMed Central

    Roger, Frédéric; Lamy, Brigitte; Jumas-Bilak, Estelle; Kodjo, Angeli; F., Carmagnol; E., Chachaty; C., Alba-Sauviat; C., Auvray; D., Barraud; Z., Benseddik; A., Bertrou; F., Bessis; H., Biessy; V., Blanc; Y., Boucaud-Maitre; P., Brunet; A., Michel; B., Cancet; J., Carrere; A., Cecille; G., Chambreuil; P., Chantelat; H., Chardon; C., Charrel; H., De Montclos; J.W., Decousser; J. M., Delarbre; A., Gravet; D., Deligne; C., Denoix; J., Deregnaucourt; F., Desroys du Roure; S., Dubourdieu; Z., El Harrif; C., Eloy; A., Evers; C., Febvre; D., Fevre; S., Gabriel; M. J., Galanti; E., Garnotel; M., Gavignet; F., Geffroy; G., Grise; I., Gros; I., Hermes; J., Heurte; E., Heusse; D., Jan; E., Jaouen; S., Laluque; R., Lamarca; Laurens, E.; A., Le Coustumier; E., Lecaillon; C., Lemble; M., Leneveu; S., Leotard; M. N., Letouzey; C., Malbrunot; O., Menouni; M., Morel; C., Olive; B., Pangon; J. G., Paul; J. M., Perez; P., Pouedras; D., Pressac; R., Sanchez; Y., Scat; A., Secher; J., Semon; D., Simeon; C., Simonin; J. P., Thellier; B., Tourand; A., Vachée; C., Varache; J., Vaucel; A. C., Vautrin; A., Verhaeghe; M., Villemain; L., Villeneuve; Marchandin, Hélène

    2012-01-01

    16S rRNA gene (rrs) is considered of low taxonomic interest in the genus Aeromonas. Here, 195 Aeromonas strains belonging to populations structured by multilocus phylogeny were studied using an original approach that considered Ribosomal Multi-Operon Diversity. This approach associated pulsed-field gel electrophoresis (PFGE) to assess rrn operon number and distribution across the chromosome and PCR-temporal temperature gel electrophoresis (TTGE) to assess rrs V3 region heterogeneity. Aeromonads harbored 8 to 11 rrn operons, 10 operons being observed in more than 92% of the strains. Intraspecific variability was low or nul except for A. salmonicida and A. aquariorum suggesting that large chromosomic rearrangements might occur in these two species while being extremely rarely encountered in the evolution of other taxa. rrn operon number at 8 as well as PFGE patterns were shown valuable for taxonomic purpose allowing resolution of species complexes. PCR-TTGE revealed a high rate of strains (41.5%) displaying intragenomic rrs heterogeneity. Strains isolated from human samples more frequently displayed intragenomic heterogeneity than strains recovered from non-human and environmental specimens. Intraspecific variability ranged from 0 to 76.5% of the strains. The observation of species-specific TTGE bands, the recovery of identical V3 regions in different species and the variability of intragenomic heterogeneity (1–13 divergent nucleotides) supported the occurrence of mutations and horizontal transfer in aeromonad rrs evolution. Altogether, the presence of a high number of rrn operon, the high proportion of strains harboring divergent rrs V3 region and the previously demonstrated high level of genetic diversity argued in favor of highly adaptative capabilities of aeromonads. Outstanding features observed for A. caviae supported the ongoing process of adaptation to a specialized niche represented by the gut, previously hypothesized. 16S rRNA gene is an informative

  8. Regulation of Tetralin Biodegradation and Identification of Genes Essential for Expression of thn Operons

    PubMed Central

    Martínez-Pérez, O.; Moreno-Ruiz, E.; Floriano, B.; Santero, E.

    2004-01-01

    The tetralin biodegradation genes of Sphingomonas macrogolitabida strain TFA are clustered in two closely linked and divergent operons. To analyze expression of both operons under different growth conditions, transcriptional and translational gene fusions of the first genes of each operon to lacZ have been constructed in plasmids unable to replicate in Sphingomonas and integrated by recombination into the genome of strain TFA. Expression analysis indicated that the transcription of both genes is induced in similar ways by the presence of tetralin. Gene expression in both operons is also subjected to overimposed catabolic repression. Two additional genes named thnR and thnY have been identified downstream of thnCA3A4 genes. ThnR is similar to LysR-type regulators, and mutational analysis indicated that ThnR is strictly required for expression of the thn operons. Unlike other LysR-type regulators, ThnR does not repress its own synthesis. In fact, ThnR activates its own expression, since thnR is cotranscribed with the thnCA3A4 genes. ThnY is similar to the ferredoxin reductase components of dioxygenase systems and shows the fer2 domain, binding a Cys4[2Fe-2S] iron sulfur center, and the FAD-binding domain, common to those reductases. However, it lacks the NAD-binding domain. Intriguingly, ThnY has a regulatory role, since it is also strictly required for expression of the thn operons. Given the similarity of ThnY to reductases and the possibility of its being present in the two redox states, it is tempting to speculate that ThnY is a regulatory component connecting expression of the thn operons to the physiological status of the cell. PMID:15342579

  9. Capsular switching as a strategy to increase pneumococcal virulence in experimental otitis media model.

    PubMed

    Sabharwal, Vishakha; Stevenson, Abbie; Figueira, Marisol; Orthopoulos, George; Trzciński, Krzysztof; Pelton, Stephen I

    2014-04-01

    We hypothesized that capsular switch event, in which pneumococcus acquires a new capsule operon by horizontal gene transfer, may result in emergence of strains with increased virulence in acute otitis media. Using serotype 6A strain from a patient with invasive pneumococcal disease and clonally distant serotype 6C strain isolated from asymptomatic carrier we created 6A:6C (6A background with 6C capsule) capsular transformants and applied whole genome macro-restriction analysis to assess conservation of the 6A chassis. Next, we assessed complement (C3) and antibodies deposition on surface of pneumococcal cells and tested capsule recipient, capsule donor and two 6A:6C transformants for virulence in chinchilla experimental otitis media model. Both 6A:6C(1 or 2) transformants bound less C3 compared to 6C capsule-donor strain but more compared to serotype 6A capsule-recipient strain. Pneumococci were present in significantly higher proportion of ears among animals challenged with either of two 6A:6C(1 or 2) transformants compared to chinchillas infected with 6C capsule-donor strain [p < 0.001] whereas a significantly decreased proportion of ears were infected with 6A:6C(1 or 2) transformants as compared to 6A capsule-recipient strain. Our observations though limited to two serotypes demonstrate that capsular switch events can result in Streptococcus pneumoniae strains of enhanced virulence for respiratory tract infection.

  10. Genetic and Biochemical Characterization of Glycerol Uptake in Mycoplasma mycoides subsp. mycoides SC: Its Impact on H2O2 Production and Virulence

    PubMed Central

    Vilei, Edy M.; Frey, Joachim

    2001-01-01

    Highly virulent strains of Mycoplasma mycoides subsp. mycoides SC belonging to the African cluster contain an operon with the genes gtsA, gtsB, and gtsC, encoding membrane ATP binding cassette transporter proteins GtsA, GtsB, and GtsC, which are involved in glycerol transport. Strain Afadé from the African cluster incorporated [U-14C]glycerol with a time-dependent increase. The less virulent strain L2 of the European cluster, which lacks gtsB and gtsC, failed to incorporate glycerol. Antibodies against GtsB noncompetitively inhibited glycerol uptake. l-α-Glycerophosphate was not transported by M. mycoides subsp. mycoides SC. It is postulated to be synthesized by phosphorylation of glycerol during transport and subsequently metabolized further to dihydroxyacetone phosphate accompanied by release of H2O2. Peroxide production in glycerol-containing growth medium was high for the African strain Afadé but very low for the European strain L2. Virtually no H2O2 was produced by both strains without glycerol. Hence, the efficient glycerol uptake system found in the virulent strain of the African cluster leads to a strong release of peroxide, a potential virulence factor which is lacking in the less virulent European strains. M. mycoides subsp. mycoides SC might have adopted, as a strategy for virulence, a highly efficient uptake system for glycerol which allows the production of an active metabolic intermediate that damages host cells. PMID:11139200

  11. Campylobacter virulence and survival factors.

    PubMed

    Bolton, Declan J

    2015-06-01

    Despite over 30 years of research, campylobacteriosis is the most prevalent foodborne bacterial infection in many countries including in the European Union and the United States of America. However, relatively little is known about the virulence factors in Campylobacter or how an apparently fragile organism can survive in the food chain, often with enhanced pathogenicity. This review collates information on the virulence and survival determinants including motility, chemotaxis, adhesion, invasion, multidrug resistance, bile resistance and stress response factors. It discusses their function in transition through the food processing environment and human infection. In doing so it provides a fundamental understanding of Campylobacter, critical for improved diagnosis, surveillance and control.

  12. Differences in virulence of Naegleria fowleri.

    PubMed

    De Jonckheere, J

    1979-10-01

    All pathogenic Naegleria fowleri isolated from the environment were highly virulent to mice when instilled intranasally. Axenic cultivation gradually decreased virulence of highly virulent strains. This decrease was most pronounced in environmental isolates and of minor importance in N. fowleri isolated from human cerebrospinal fluid. The low virulent strains obtained by continuous axenic cultivation appeared after clonation to consist of individuals with different virulence. Virulence could be enhanced in low virulent strains by brain passage and passages in Vero cell cultures, but could not be induced by these methods in nonvirulent strains isolated from the environment. Different mice strains showed different sensitivities to infection with pathogenic Naegleria. In addition, older mice were less sensitive than younger animals to low virulent strains. PMID:392414

  13. Differences in virulence of Naegleria fowleri.

    PubMed

    De Jonckheere, J

    1979-10-01

    All pathogenic Naegleria fowleri isolated from the environment were highly virulent to mice when instilled intranasally. Axenic cultivation gradually decreased virulence of highly virulent strains. This decrease was most pronounced in environmental isolates and of minor importance in N. fowleri isolated from human cerebrospinal fluid. The low virulent strains obtained by continuous axenic cultivation appeared after clonation to consist of individuals with different virulence. Virulence could be enhanced in low virulent strains by brain passage and passages in Vero cell cultures, but could not be induced by these methods in nonvirulent strains isolated from the environment. Different mice strains showed different sensitivities to infection with pathogenic Naegleria. In addition, older mice were less sensitive than younger animals to low virulent strains.

  14. Mutations upregulating the flhDC operon of Escherichia coli K-12.

    PubMed

    Lee, Changhan; Park, Chankyu

    2013-02-01

    Bacterial motility is governed by the flhDC master operon that is under the control of factors like OmpR, LrhA, HdfR, and H-NS. Previously, derivatives of the wild-type MG1655 strain of E. coli K-12 with enhanced motility were found to contain insertion sequences (ISs) in the regulatory region of the flhDC operon. Here, we report that not only integrations of IS insertion sequences into the regulatory region of the flhDC operon, but also a missense mutation in the lrhA gene enhances motility by relieving transcriptional repression of the flhDC operon. Two novel IS insertions were found upstream of flhDC. So far, the relationships between the trans- acting factors and the cis-acting regulatory sequences associated with the flhDC operon have not been clearly established. In this study, it was found that effects of the cis- and trans-acting mutations were acting in parallel, suggesting their apparently independent regulation of flagellar expression.

  15. Frequency of pap and pil operons in Escherichia coli strains associated with urinary infections.

    PubMed

    Perugini, M R; Vidotto, M C

    1996-03-01

    Strains of E. coli isolated from patients with urinary tract infection were examined for P and type 1 adhesin production by colony hybridization with pap and pil operons. The P pili probe detected 45 (46.4%) of the total of 97 strains studied and the type 1 pili probe detected 83 (85.6%). The pap operon was detected in 39 (53.4%) of 73 strains isolated from urine of patients with urinary disease and in 6 (25.0%) of 24 strains isolated from feces of healthy individuals employed as controls (P = 0.029), and the pil operon was detected in 67 (91.8%) of the urinary strains and in 16 (66.6%) of the fecal strains (P = 0.007). Our data did not show significant differences in frequency of P pili among isolates from pyelonephritis (78.5%), cystitis (45.8%) and asymptomatic bacteriuria (54.5%). Type 1 pili were not associated with the different types of infection; the frequency of these pili was 100% in pyelonephritis and in asymptomatic bacteriuria, and 87.5% in cystitis. The incidence of pap operon in strains isolated from pyelonephritis and from asymptomatic bacteriuria was higher in 11- to 40-year old women. These data show a high frequency of pap and pil operons among uropathogenic strains of E. coli, which seems to be an important factor in the development of urinary infection.

  16. Artificial citrate operon and Vitreoscilla hemoglobin gene enhanced mineral phosphate solubilizing ability of Enterobacter hormaechei DHRSS.

    PubMed

    Yadav, Kavita; Kumar, Chanchal; Archana, G; Kumar, G Naresh

    2014-10-01

    Mineral phosphate solubilization by bacteria is mediated through secretion of organic acids, among which citrate is one of the most effective. To overproduce citrate in bacterial systems, an artificial citrate operon comprising of genes encoding NADH-insensitive citrate synthase of E. coli and Salmonella typhimurium sodium-dependent citrate transporter was constructed. In order to improve its mineral phosphate solubilizing (MPS) ability, the citrate operon was incorporated into E. hormaechei DHRSS. The artificial citrate operon transformant secreted 7.2 mM citric acid whereas in the native strain, it was undetectable. The transformant released 0.82 mM phosphate in flask studies in buffered medium containing rock phosphate as sole P source. In fermenter studies, similar phenotype was observed under aerobic conditions. However, under microaerobic conditions, no citrate was detected and P release was not observed. Therefore, an artificial citrate gene cluster containing Vitreoscilla hemoglobin (vgb) gene under its native promoter, along with artificial citrate operon under constitutive tac promoter, was constructed and transformed into E. hormaechei DHRSS. This transformant secreted 9 mM citric acid under microaerobic conditions and released 1.0 mM P. Thus, incorporation of citrate operon along with vgb gene improves MPS ability of E. hormaechei DHRSS under buffered, microaerobic conditions mimicking rhizospheric environment.

  17. Promoter- and attenuator-related metabolic regulation of the Salmonella typhimurium histidine operon.

    PubMed Central

    Winkler, M E; Roth, D J; Hartman, P E

    1978-01-01

    Expression of the histidine (his) operon in Salmonella typhimurium was found to be positively correlated with the intracellular level of guanosine tetraphosphate (ppGpp). Limitation for amino acids other than histidine elicited a histidine-independent metabolic regulation of the operon. In bacteria grown at decreased growth rates, his operon expression was metabolically regulated up to a point, after which further decreases in growth rate no longer resulted in further enhancement of operon expression. Studies using strains carrying various regulatory and deletion mutations indicated that metabolic regulation is achieved predominantly by increased RNA chain initiations at the primary (P1) and internal (P2) promoters. Metabolic regulation ordinarly did not involve changes in RNA chain terminations at the attenuator site of the his operon. A model is proposed that involves ppGpp-induced changes in RNA polymerase initiation specificity at particular promoters. A second, special form of metabolic regulation may operate which also is histidine independent, but does involve relief of attenuation. PMID:342509

  18. [CpcHID operon as a new tool for classification and identification of Arthrospira platensis strains].

    PubMed

    Yang, Ling-yong; Wang, Zhi-ping; Cao, Xue-cheng; Chen, Xiao-yan; Xu, Bu-jin; Li, Xue-bin; Huang, Hui

    2006-12-01

    Arthrospira is a photoautotrophic filamentous cyanobacterium belonging to the family Oscillatoriaceae, phylum Cyanophyta. Morphological criteria alone were inadequate for classification of Arthrospira . To develop new molecular markers, in this study, the cpcHID operon, 16S rRNA and 16S-23S rRNA internally transcribed spacer (ITS) of seven Arthrospira platensis strains, Sp-10, Sp-2, Sp-9, Sp-1, Sp-1ll, Sp-3 and Sp-5, were cloned and sequenced. And the results of bioinformatics and molecular phylogenetics analyses with BioEdit 7.0, Clustal X 1.81 and Phylip 3.65 were as follows: (1) The sequences of cpcHID operon, 16S rRNA and ITS from the seven strains were highly homologous to the each corresponding gene based on multiple pair-wise comparison. (2) The mean absolute deviation of the G + C content, the ratio of different sites and the genetic distance coefficient based on the sequences of cpcHID operon in the seven strains were generally greater than that based on 16S rRNA and ITS region. (3) The phylogenetic dendrogram based on the sequences of cpcHID operon was almost same with that based on the sequences of 16S rRNA and ITS region. Therefore, it revealed that cpcHID operon could be applied as a new molecular marker to classification and identification of cyanobacterium, and more appropriate for species or strains determination due to its abundant information. PMID:17302170

  19. Evaluation of Virulence Gene Expression Patterns in Acinetobacter baumannii Using Quantitative Real-Time Polymerase Chain Reaction Array.

    PubMed

    Lannan, Ford M; O'conor, Daniel K; Broderick, Joseph C; Tate, Jamison F; Scoggin, Jacob T; Moran, Nicholas A; Husson, Christopher M; Hegeman, Erik M; Ogrydziak, Cole E; Singh, Sneha A; Vafides, Andrew G; Brinkley, Carl C; Goodin, Jeremy L

    2016-09-01

    According to the Centers for Disease Control's recently devised National Strategy for Combating Antibiotic-Resistant Bacteria, Acinetobacter baumannii is a "serious" threat level pathogen. A. baumannii's notoriety stems from the fact that a large number of modern strains are multidrug resistant and persist in the hospital setting, thus causing numerous deaths per year. It is imperative that research focus on a more fundamental understanding of the factors responsible for the success of A. baumannii. Toward this end, our group investigated virulence gene expression patterns in a recently characterized wound isolate, AB5075, using quantitative real-time polymerase chain reaction array. Notably, several genes showed statistically significant upregulation at 37°C compared to 25°C; MviM, Wbbj, CarO, and certain genes of the Bas, Bar, and Csu operons. Additionally, we found that in vitro biofilm formation by Csu transposon insertion mutant strains is attenuated. These findings validate previous reports that suggest a link between the Csu operon and biofilm formation. More importantly, our results demonstrate a successful method for evaluating the significance of previously identified virulence factors in a modern and clinically relevant strain of A. baumannii, thereby providing a path toward a more fundamental understanding of the pathogenicity of A. baumannii. PMID:27612361

  20. The ABC transporter YejABEF is required for resistance to antimicrobial peptides and the virulence of Brucella melitensis.

    PubMed

    Wang, Zhen; Bie, Pengfei; Cheng, Jie; Lu, Lin; Cui, Buyun; Wu, Qingmin

    2016-01-01

    The ability to resist the killing effects of host antimicrobial peptides (AMPs) plays a vital role in the virulence of pathogens. The Brucella melitensis NI genome has a gene cluster that encodes ABC transport. In this study, we constructed yejA1, yejA2, yejB, yejE, yejF, and whole yej operon deletion mutants, none of which exhibited discernible growth defect in TSB or minimal medium. Unlike their parental strain, the mutants showed a significantly increased sensitivity to acidic stress. The NIΔyejE and NIΔyejABEF mutants were also more sensitive than B. melitensis NI to polymyxin B, and the expression of yej operon genes was induced by polymyxin B. Moreover, cell and mouse infection assays indicated that NIΔyejE and NIΔyejABEF have restricted invasion and replication abilities inside macrophages and are rapidly cleared from the spleens of infected mice. These findings indicate that the ABC transporter YejABEF is required for the virulence of Brucella, suggesting that resistance to host antimicrobials is a key mechanism for Brucella to persistently survive in vivo. This study provided insights that led us to further investigate the potential correlation of AMP resistance with the mechanisms of immune escape and persistent infection by pathogens. PMID:27550726

  1. The ABC transporter YejABEF is required for resistance to antimicrobial peptides and the virulence of Brucella melitensis

    PubMed Central

    Wang, Zhen; Bie, Pengfei; Cheng, Jie; Lu, Lin; Cui, Buyun; Wu, Qingmin

    2016-01-01

    The ability to resist the killing effects of host antimicrobial peptides (AMPs) plays a vital role in the virulence of pathogens. The Brucella melitensis NI genome has a gene cluster that encodes ABC transport. In this study, we constructed yejA1, yejA2, yejB, yejE, yejF, and whole yej operon deletion mutants, none of which exhibited discernible growth defect in TSB or minimal medium. Unlike their parental strain, the mutants showed a significantly increased sensitivity to acidic stress. The NIΔyejE and NIΔyejABEF mutants were also more sensitive than B. melitensis NI to polymyxin B, and the expression of yej operon genes was induced by polymyxin B. Moreover, cell and mouse infection assays indicated that NIΔyejE and NIΔyejABEF have restricted invasion and replication abilities inside macrophages and are rapidly cleared from the spleens of infected mice. These findings indicate that the ABC transporter YejABEF is required for the virulence of Brucella, suggesting that resistance to host antimicrobials is a key mechanism for Brucella to persistently survive in vivo. This study provided insights that led us to further investigate the potential correlation of AMP resistance with the mechanisms of immune escape and persistent infection by pathogens. PMID:27550726

  2. Evaluation of Virulence Gene Expression Patterns in Acinetobacter baumannii Using Quantitative Real-Time Polymerase Chain Reaction Array.

    PubMed

    Lannan, Ford M; O'conor, Daniel K; Broderick, Joseph C; Tate, Jamison F; Scoggin, Jacob T; Moran, Nicholas A; Husson, Christopher M; Hegeman, Erik M; Ogrydziak, Cole E; Singh, Sneha A; Vafides, Andrew G; Brinkley, Carl C; Goodin, Jeremy L

    2016-09-01

    According to the Centers for Disease Control's recently devised National Strategy for Combating Antibiotic-Resistant Bacteria, Acinetobacter baumannii is a "serious" threat level pathogen. A. baumannii's notoriety stems from the fact that a large number of modern strains are multidrug resistant and persist in the hospital setting, thus causing numerous deaths per year. It is imperative that research focus on a more fundamental understanding of the factors responsible for the success of A. baumannii. Toward this end, our group investigated virulence gene expression patterns in a recently characterized wound isolate, AB5075, using quantitative real-time polymerase chain reaction array. Notably, several genes showed statistically significant upregulation at 37°C compared to 25°C; MviM, Wbbj, CarO, and certain genes of the Bas, Bar, and Csu operons. Additionally, we found that in vitro biofilm formation by Csu transposon insertion mutant strains is attenuated. These findings validate previous reports that suggest a link between the Csu operon and biofilm formation. More importantly, our results demonstrate a successful method for evaluating the significance of previously identified virulence factors in a modern and clinically relevant strain of A. baumannii, thereby providing a path toward a more fundamental understanding of the pathogenicity of A. baumannii.

  3. Insights into Entamoeba histolytica virulence modulation.

    PubMed

    Padilla-Vaca, F; Anaya-Velázquez, F

    2010-08-01

    Entamoeba histolytica is able to invade human tissues by means of several molecules and biological properties related to the virulence. Pathogenic amebas use three major virulence factors, Gal/GalNAc lectin, amebapore and proteases, for lyse, phagocytose, kill and destroy a variety of cells and tissues in the host. Responses of the parasite to host components such as mucins and bacterial flora influence the behavior of pathogenic amebas altering their expression of virulence factors. The relative virulence of different strains of E. histolytica has been shown to vary as a consequence of changes in conditions of in vitro cultivation which implies substantial changes in basic metabolic aspects and factors directly and indirectly related to amebic virulence. Comparison of E. histolytica strains with different virulence phenotypes and under different conditions of growth will help to identify new virulence factor candidates and define the interplay between virulence factors and invasive phenotype. Virulence attenuate mutants of E. histolytica are useful also to uncover novel virulence determinants. The comparison of biological properties and virulence factors between E. histolytica and E. dispar, a non-pathogenic species, has been a useful approach to investigate the key factors involved in the experimental presentation of amebiasis and its complex regulation. The molecular mechanisms that regulate these variations in virulence are not yet known. Their elucidation will help us to better understand the gene expression plasticity that enables the effective adaptation of the ameba to changes in growth culture conditions and host factors.

  4. Molecular architecture of the regulatory Locus sae of Staphylococcus aureus and its impact on expression of virulence factors.

    PubMed

    Steinhuber, Andrea; Goerke, Christiane; Bayer, Manfred G; Döring, Gerd; Wolz, Christiane

    2003-11-01

    We characterized the sae operon, a global regulator for virulence gene expression in Staphylococcus aureus. A Tn917 sae mutant was obtained by screening a Tn917 library of the agr mutant ISP479Mu for clones with altered hemolytic activity. Sequence analysis of the sae operon revealed two additional open reading frames (ORFs) (ORF3 and ORF4) upstream of the two-component regulatory genes saeR and saeS. Four overlapping sae-specific transcripts (T1 to T4) were detected by Northern blot analysis, and the transcriptional initiation points were mapped by primer extension analysis. The T1, T2, and T3 mRNAs are probably terminated at the same stem-loop sequence downstream of saeS. The T1 message (3.1 kb) initiates upstream of ORF4, T2 (2.4 kb) initiates upstream of ORF3, and T3 (2.0 kb) initiates in front of saeR. T4 (0.7 kb) represents a monocistronic mRNA encompassing ORF4 only. sae-specific transcripts were detectable in all of the 40 different clinical S. aureus isolates investigated. Transcript levels were at maximum during the post-exponential growth phase. The sae mutant showed a significantly reduced rate of invasion of human endothelial cells, consistent with diminished transcription and expression of fnbA. The expression of type 5 capsular polysaccharide is activated in the sae mutant of strain Newman, as shown by immunofluorescence and promoter-reporter fusion experiments. In summary, the sae operon constitutes a four-component regulator system which acts on virulence gene expression in S. aureus.

  5. The fruRBA Operon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood

    PubMed Central

    Valdes, Kayla M.; Sundar, Ganesh S.; Vega, Luis A.; Belew, Ashton T.; Islam, Emrul; Binet, Rachel; El-Sayed, Najib M.

    2016-01-01

    Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogen Streptococcus pyogenes (the group A Streptococcus [GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where the fru locus (fruRBA) was the most induced. Reverse transcriptase PCR showed that fruRBA formed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entire fru operon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, and N-acetyl-d-galactosamine. The inactivation of sloR, a fruA homolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ΔfruR and ΔfruB mutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ΔfruA mutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment. PMID:26787724

  6. LeuO enhances butyrate-induced virulence expression through a positive regulatory loop in enterohaemorrhagic Escherichia coli.

    PubMed

    Takao, Miyuki; Yen, Hilo; Tobe, Toru

    2014-09-01

    Enterohaemorrhagic Escherichia coli (EHEC) causes bloody diarrhoea and other severe symptoms such as haemorrhagic uraemic syndrome. The expression of virulence genes on the locus for enterocyte effacement (LEE) and associated genes is regulated by a variety of factors, including transcriptional regulators and environmental signals. Butyrate, one of the major short-chain fatty acids present in the intestine, enhances expression of LEE genes and flagella biosynthesis genes in EHEC O157:H7, resulting in increased bacterial adherence and motility. Here, we show that expression of the leuO gene, which encodes a LysR-type transcriptional regulator, is enhanced by butyrate via Lrp, which is also necessary for butyrate-induced responses of LEE genes. LeuO expression induces prolonged activation of the promoter of LEE1 operon, including the ler gene, as well as virulence mechanisms such as microcolony formation. Activation of the LEE1 promoter by LeuO depends on another regulator, called Pch. The response of the leuO promoter to butyrate requires two virulence regulators, Pch and Ler, in addition to Lrp. Pch, Ler and Lrp bind the upstream region of the leuO promoter. Thus, leuO is involved in butyrate-enhanced expression of LEE genes through a positive feedback mechanism, but its expression and action on the LEE1 promoter are dependent on the virulence regulators Pch and Ler.

  7. The polyamine N-acetyltransferase-like enzyme PmvE plays a role in the virulence of Enterococcus faecalis.

    PubMed

    Martini, Cecilia; Michaux, Charlotte; Bugli, Francesca; Arcovito, Alessandro; Iavarone, Federica; Cacaci, Margherita; Paroni Sterbini, Francesco; Hartke, Axel; Sauvageot, Nicolas; Sanguinetti, Maurizio; Posteraro, Brunella; Giard, Jean-Christophe

    2015-01-01

    We previously showed that the mutant strain of Enterococcus faecalis lacking the transcriptional regulator SlyA is more virulent than the parental strain. We hypothesized that this phenotype was due to overexpression of the second gene of the slyA operon, ef_3001, renamed pmvE (for polyamine metabolism and virulence of E. faecalis). PmvE shares strong homologies with N(1)-spermidine/spermine acetyltransferase enzymes involved in the metabolism of polyamines. In this study, we used an E. faecalis strain carrying the recombinant plasmid pMSP3535-pmvE (V19/p3535-pmvE), which allows the induction of pmvE by addition of nisin. Thereby, we showed that the overexpression of PmvE increased the virulence of E. faecalis in the Galleria mellonella infection model, as well as the persistence within peritoneal macrophages. We were also able to show a direct interaction between the His-tagged recombinant PmvE (rPmvE) protein and putrescine by the surface plasmon resonance (SPR) technique on a Biacore instrument. Moreover, biochemical assays showed that PmvE possesses an N-acetyltransferase activity toward polyamine substrates. Our results suggest that PmvE contributes to the virulence of E. faecalis, likely through its involvement in the polyamine metabolism. PMID:25385793

  8. Cloning and characterization of the dnaKJ operon in Acetobacter aceti.

    PubMed

    Okamoto-Kainuma, Akiko; Yan, Wang; Fukaya, Masahiro; Tukamoto, Yoshinori; Ishikawa, Morio; Koizumi, Yukimichi

    2004-01-01

    The dnaKJ operon of Acetobacter aceti was cloned and sequenced. The profile of the gene configuration was similar to that of other alpha-proteobacteria. In the DnaK and DnaJ proteins of A. aceti, the characteristic domains/motifs reported in other organisms were well conserved. This operon was transcribed in response to a temperature shift and exposure to ethanol/acetic acid. The overexpression of this operon in A. aceti resulted in improved growth compared to the control strain at high temperature or in the presence of ethanol, suggesting a correlation to resistance against stressors present during fermentation, although the overexpression did not increase the resistance to acetic acid.

  9. The diversity of membrane transporters encoded in bacterial arsenic-resistance operons

    PubMed Central

    Wu, Shiyang; Lilley, Ross McCausland; Zhang, Ren

    2015-01-01

    Transporter-facilitated arsenite extrusion is the major pathway of arsenic resistance within bacteria. So far only two types of membrane-bound transporter proteins, ArsB and ArsY (ACR3), have been well studied, although the arsenic transporters in bacteria display considerable diversity. Utilizing accumulated genome sequence data, we searched arsenic resistance (ars) operons in about 2,500 bacterial strains and located over 700 membrane-bound transporters which are encoded in these operons. Sequence analysis revealed at least five distinct transporter families, with ArsY being the most dominant, followed by ArsB, ArsP (a recently reported permease family), Major Facilitator protein Superfamily (MFS) and Major Intrinsic Protein (MIP). In addition, other types of transporters encoded in the ars operons were found, but in much lower frequencies. The diversity and evolutionary relationships of these transporters with regard to arsenic resistance will be discussed. PMID:26020003

  10. Cloning and analysis of the rnc-era-recO operon from Pseudomonas aeruginosa.

    PubMed

    Powell, B; Peters, H K; Nakamura, Y; Court, D

    1999-08-01

    The rnc operon from Pseudomonas aeruginosa has been cloned and characterized. The three genes comprising this operon, rnc, era, and recO, are arranged similarly to those in some other gram-negative bacteria. Multicopy plasmids carrying the rnc operon of P. aeruginosa functionally complement mutations of the rnc, era, and recO genes in Escherichia coli. In particular, the P. aeruginosa era homolog rescues the conditional lethality of era mutants in E. coli, and the presumptive protein has 60% identity with the Era of E. coli. We discuss these data and evidence suggesting that a GTPase previously purified from P. aeruginosa and designated Pra is not an Era homolog. PMID:10438789

  11. Functional analysis of the Escherichia coli K-12 cyn operon transcriptional regulation.

    PubMed Central

    Lamblin, A F; Fuchs, J A

    1994-01-01

    The cynTSX operon enables Escherichia coli K-12 to degrade and use cyanate as a sole nitrogen source. The promoter of this operon is positively regulated by cyanate and the CynR protein. CynR, a member of the LysR family of regulatory proteins, binds specifically to a 136-bp DNA fragment containing both the cynR and the cynTSX promoters. In this study, we report the results of DNase I digestion studies showing that CynR protects a 60-bp region on the cynR coding strand and a 56-bp sequence on the cynTSX coding strand. CynR binding was not affected by cyanate or its structural homolog azide, a gratuitous inducer of the operon. However, CynR-induced bending of two different DNA fragments was detected. The amount of bending was decreased by cyanate. Images PMID:7961413

  12. Functional analysis of the Escherichia coli K-12 cyn operon transcriptional regulation.

    PubMed

    Lamblin, A F; Fuchs, J A

    1994-11-01

    The cynTSX operon enables Escherichia coli K-12 to degrade and use cyanate as a sole nitrogen source. The promoter of this operon is positively regulated by cyanate and the CynR protein. CynR, a member of the LysR family of regulatory proteins, binds specifically to a 136-bp DNA fragment containing both the cynR and the cynTSX promoters. In this study, we report the results of DNase I digestion studies showing that CynR protects a 60-bp region on the cynR coding strand and a 56-bp sequence on the cynTSX coding strand. CynR binding was not affected by cyanate or its structural homolog azide, a gratuitous inducer of the operon. However, CynR-induced bending of two different DNA fragments was detected. The amount of bending was decreased by cyanate.

  13. The dlt operon in the biosynthesis of D-alanyl-lipoteichoic acid in Lactobacillus casei.

    PubMed

    Neuhaus, F C; Heaton, M P; Debabov, D V; Zhang, Q

    1996-01-01

    The D-alanine incorporation system allows Lactobacillus casei to modulate the chemical properties of lipoteichoic acid (LTA) and hence control its proposed functions, i.e., regulation of autolysin action, metal ion binding, and the electromechanical properties of the cell wall. The system requires the D-alanine-D-alanyl carrier protein ligase (Dcl) and the D-alanyl carrier protein (Dcp). Our results indicate that the genes for these proteins are encoded in the dlt operon and that this operon contains at least 2 other genes, dltB and dltD. The aim of this paper is to describe the genetic organization of the operon, the role of the D-alanyl carrier protein, and the function of the putative protein encoded by dltB in the intramembranal translocation of the activated D-alanine. PMID:9158726

  14. ZnO nanoparticles inhibit Pseudomonas aeruginosa biofilm formation and virulence factor production.

    PubMed

    Lee, Jin-Hyung; Kim, Yong-Guy; Cho, Moo Hwan; Lee, Jintae

    2014-12-01

    The opportunistic pathogen Pseudomonas aeruginosa produces a variety of virulence factors, and biofilms of this bacterium are much more resistant to antibiotics than planktonic cells. Thirty-six metal ions have been investigated to identify antivirulence and antibiofilm metal ions. Zinc ions and ZnO nanoparticles were found to markedly inhibit biofilm formation and the production of pyocyanin, Pseudomonas quinolone signal (PQS), pyochelin, and hemolytic activity of P. aeruginosa without affecting the growth of planktonic cells. Transcriptome analyses showed that ZnO nanoparticles induce the zinc cation efflux pump czc operon and several important transcriptional regulators (porin gene opdT and type III repressor ptrA), but repress the pyocyanin-related phz operon, which explains observed phenotypic changes. A mutant study showed that the effects of ZnO nanoparticles on the control of pyocyanin production and biofilm formation require the czc regulator CzcR. In addition, ZnO nanoparticles markedly increased the cellular hydrophilicity of P. aeruginosa cells. Our results support that ZnO nanoparticles are potential antivirulence materials against recalcitrant P. aeruginosa infections and possibly other important pathogens. PMID:24958247

  15. Expression and regulation of the ery operon of Brucella melitensis in human trophoblast cells

    PubMed Central

    Zhang, Hui; Dou, Xiaoxia; Li, Zhiqiang; Zhang, Yu; Zhang, Jing; Guo, Fei; Wang, Yuanzhi; Wang, Zhen; Li, Tiansen; Gu, Xinli; Chen, Chuangfu

    2016-01-01

    Brucellosis is primarily a disease of domestic animals in which the bacteria localizes to fetal tissues such as embryonic trophoblast cells and fluids containing erythritol, which stimulates Brucella spp. growth. The utilization of erythritol is a characteristic of the genus Brucella. The ery operon contains four genes (eryA, eryB, eryC and eryD) for the utilization of erythritol, and plays a major role in the survival and multiplication of Brucella spp. The objective of the present study was to conduct a preliminary characterization of differential genes expression of the ery operon at several time points after Brucella infected embryonic trophoblast cells (HPT-8 cells). The result showed that the ery operon expression was higher in HPT-8 cells compared with the medium. The relative expression of eryA, eryB and eryC peaked at 2 h post-infection in HPT-8 cells, and eryD expression peaked at 3 h post-infection. The expression of eryA, eryB and eryC may be inhibited by increased eryD expression. However, the expression of the ery operon was stable in the presence of erythritol in cells. 2308Δery and 027Δery mutants of the ery operon were successfully constructed by homologous recombination, which were attenuated in RAW 264.7 murine macrophages. The characterization of the ery operon genes and their expression profiles in response to Brucella infection further contributes to our understanding of the molecular mechanisms of infection and the pathogenesis of brucellosis. PMID:27698777

  16. Fine-Tuned Transcriptional Regulation of Malate Operons in Enterococcus faecalis

    PubMed Central

    Mortera, Pablo; Espariz, Martín; Suárez, Cristian; Repizo, Guillermo; Deutscher, Josef; Alarcón, Sergio; Blancato, Víctor

    2012-01-01

    In Enterococcus faecalis, the mae locus is constituted by two putative divergent operons, maePE and maeKR. The first operon encodes a putative H+/malate symporter (MaeP) and a malic enzyme (MaeE) previously shown to be essential for malate utilization in this bacterium. The maeKR operon encodes two putative proteins with significant similarity to two-component systems involved in sensing malate and activating its assimilation in bacteria. Our transcriptional and genetic assays showed that maePE and maeKR are induced in response to malate by the response regulator MaeR. In addition, we observed that both operons were partially repressed in the presence of glucose. Accordingly, the cometabolism of this sugar and malate was detected. The binding of the complex formed by CcpA and its corepressor P-Ser-HPr to a cre site located in the mae region was demonstrated in vitro and explains the carbon catabolite repression (CCR) observed for the maePE operon. However, our results also provide evidence for a CcpA-independent CCR mechanism regulating the expression of both operons. Finally, a biomass increment of 40 or 75% was observed compared to the biomass of cells grown only on glucose or malate, respectively. Cells cometabolizing both carbon sources exhibit a higher rate of glucose consumption and a lower rate of malate utilization. The growth improvement achieved by E. faecalis during glucose-malate cometabolism might explain why this microorganism employs different regulatory systems to tightly control the assimilation of both carbon sources. PMID:22247139

  17. Expression and regulation of the ery operon of Brucella melitensis in human trophoblast cells

    PubMed Central

    Zhang, Hui; Dou, Xiaoxia; Li, Zhiqiang; Zhang, Yu; Zhang, Jing; Guo, Fei; Wang, Yuanzhi; Wang, Zhen; Li, Tiansen; Gu, Xinli; Chen, Chuangfu

    2016-01-01

    Brucellosis is primarily a disease of domestic animals in which the bacteria localizes to fetal tissues such as embryonic trophoblast cells and fluids containing erythritol, which stimulates Brucella spp. growth. The utilization of erythritol is a characteristic of the genus Brucella. The ery operon contains four genes (eryA, eryB, eryC and eryD) for the utilization of erythritol, and plays a major role in the survival and multiplication of Brucella spp. The objective of the present study was to conduct a preliminary characterization of differential genes expression of the ery operon at several time points after Brucella infected embryonic trophoblast cells (HPT-8 cells). The result showed that the ery operon expression was higher in HPT-8 cells compared with the medium. The relative expression of eryA, eryB and eryC peaked at 2 h post-infection in HPT-8 cells, and eryD expression peaked at 3 h post-infection. The expression of eryA, eryB and eryC may be inhibited by increased eryD expression. However, the expression of the ery operon was stable in the presence of erythritol in cells. 2308Δery and 027Δery mutants of the ery operon were successfully constructed by homologous recombination, which were attenuated in RAW 264.7 murine macrophages. The characterization of the ery operon genes and their expression profiles in response to Brucella infection further contributes to our understanding of the molecular mechanisms of infection and the pathogenesis of brucellosis.

  18. Organization and regulation of the ilvGEDA operon in Salmonella typhimurium LT2.

    PubMed Central

    Berg, C M; Shaw, K J

    1981-01-01

    A total of 102 isoleucine- and isoleucine-valine-requiring (ilv) mutants induced by insertion of the transposable element Tn10 have been classified to cistron by growth requirement, cross-feeding behavior, and enzyme assays. The mutations are in a polycistronic operon transcribed in the order ilvGEDA and in a monocistronic operon ilvC. Analysis of distal gene expression in these polar insertion mutants revealed the existence of two constitutive interval promoters, one preceding ilvE and the other preceding ilvD. Images PMID:7007356

  19. CodY Deletion Enhances In Vivo Virulence of Community-Associated Methicillin-Resistant Staphylococcus aureus Clone USA300

    PubMed Central

    Boyle-Vavra, Susan; Roux, Agnès; Ebine, Kazumi; Sonenshein, Abraham L.; Daum, Robert S.

    2012-01-01

    The Staphylococcus aureus global regulator CodY responds to nutrient availability by controlling the expression of target genes. In vitro, CodY represses the transcription of virulence genes, but it is not known if CodY also represses virulence in vivo. The dominant community-associated methicillin-resistant S. aureus (CA-MRSA) clone, USA300, is hypervirulent and has increased transcription of global regulators and virulence genes; these features are reminiscent of a strain defective in CodY. Sequence analysis revealed, however, that the codY genes of USA300 and other sequenced S. aureus isolates are not significantly different from the codY genes in strains known to have active CodY. codY was expressed in USA300, as well as in other pulsotypes assessed. Deletion of codY from a USA300 clinical isolate resulted in modestly increased expression of the global regulators agr and saeRS, as well as the gene encoding the toxin alpha-hemolysin (hla). A substantial increase (>30-fold) in expression of the lukF-PV gene, encoding part of the Panton-Valentine leukocidin (PVL), was observed in the codY mutant. All of these expression differences were reversed by complementation with a functional codY gene. Moreover, purified CodY protein bound upstream of the lukSF-PV operon, indicating that CodY directly represses expression of lukSF-PV. Deletion of codY increased the virulence of USA300 in necrotizing pneumonia and skin infection. Interestingly, deletion of lukSF-PV from the codY mutant did not attenuate virulence, indicating that the hypervirulence of the codY mutant was not explained by overexpression of PVL. These results demonstrate that CodY is active in USA300 and that CodY-mediated repression restrains the virulence of USA300. PMID:22526672

  20. Identification and transcriptional analysis of the Escherichia coli htrE operon which is homologous to pap and related pilin operons.

    PubMed Central

    Raina, S; Missiakas, D; Baird, L; Kumar, S; Georgopoulos, C

    1993-01-01

    We have characterized a new Escherichia coli operon consisting of two genes, ecpD and htrE. The ecpD gene encodes a 27-kDa protein which is 40% identical at the amino acid level to the pilin chaperone PapD family of proteins. Immediately downstream of the ecpD gene is the htrE gene. The htrE gene encodes a polypeptide of 95 kDa which is processed to a 92-kDa mature species. The HtrE protein is 38% identical to the type II pilin porin protein PapC. The ecpD htrE operon is located at 3.3 min on the genetic map, corresponding to the region from kbp 153 to 157 of the E. coli physical map. The htrE gene was identified on the basis of a Tn5 insertion mutation which resulted in a temperature-sensitive growth phenotype above 43.5 degrees C. The transcription of this operon is induced with a temperature shift from 22 to 37 or 42 degrees C but not to higher temperatures, e.g., 50 degrees C. Consistent with this result, the temperature-induced transcription was shown to be independent of the rpoH gene product (sigma 32). The transcription of this operon was further shown to require functional integration host factor protein, since himA or himD mutant bacteria possessed lower levels of ecpD htrE transcripts. Among the three transcriptional start sites discovered, one, defined by the P2 promoter, was found to be under the positive regulation of the katF (rpoS) gene, which encodes a putative sigma factor required for the transcription of many growth phase-regulated genes. Images PMID:8102362

  1. Positions of Trp codons in the leader peptide-coding region of the at operon influence anti-trap synthesis and trp operon expression in Bacillus licheniformis.

    PubMed

    Levitin, Anastasia; Yanofsky, Charles

    2010-03-01

    Tryptophan, phenylalanine, tyrosine, and several other metabolites are all synthesized from a common precursor, chorismic acid. Since tryptophan is a product of an energetically expensive biosynthetic pathway, bacteria have developed sensing mechanisms to downregulate synthesis of the enzymes of tryptophan formation when synthesis of the amino acid is not needed. In Bacillus subtilis and some other Gram-positive bacteria, trp operon expression is regulated by two proteins, TRAP (the tryptophan-activated RNA binding protein) and AT (the anti-TRAP protein). TRAP is activated by bound tryptophan, and AT synthesis is increased upon accumulation of uncharged tRNA(Trp). Tryptophan-activated TRAP binds to trp operon leader RNA, generating a terminator structure that promotes transcription termination. AT binds to tryptophan-activated TRAP, inhibiting its RNA binding ability. In B. subtilis, AT synthesis is upregulated both transcriptionally and translationally in response to the accumulation of uncharged tRNA(Trp). In this paper, we focus on explaining the differences in organization and regulatory functions of the at operon's leader peptide-coding region, rtpLP, of B. subtilis and Bacillus licheniformis. Our objective was to correlate the greater growth sensitivity of B. licheniformis to tryptophan starvation with the spacing of the three Trp codons in its at operon leader peptide-coding region. Our findings suggest that the Trp codon location in rtpLP of B. licheniformis is designed to allow a mild charged-tRNA(Trp) deficiency to expose the Shine-Dalgarno sequence and start codon for the AT protein, leading to increased AT synthesis. PMID:20061467

  2. Tn9 and IS1 inserts in a ribosomal ribonucleic acid operon of Escherichia coli are incompletely polar.

    PubMed Central

    Brewster, J M; Morgan, E A

    1981-01-01

    Transcription is known to be coupled to translation in many or all bacterial operons which code for proteins. In these operons, nonsense codons which prevent normal translation often result in premature termination of transcription (polarity). However, efficient transcription of ribosomal ribonucleic acid operons (rrn operons) occurs, although rrn transcripts are not translated. It therefore seemed possible that insertion sequences and transposable elements which are polar in protein-coding operons might not be polar in rrn operons. Previously, it has been shown (E. A. Morgan, Cell 21:257-265, 1980) that Tn10 is incompletely polar in the rrnX operon. Here we show that the transposon Tn9 and the insertion sequence IS1 also incompletely polar in rrnX. In normal cells expression of sequences distal to the insertions can be detected by genetic methods. In ultraviolet-irradiated cells expression of distal sequences is about 80% of that observed in uninterrupted rrnX operons. These observations provide evidence that ribonucleic acid polymerase molecules beginning at rrnX promoters can read through Tn9 and IS1 and that, at least in ultraviolet-irradiated cells, read-through is very efficient. Images PMID:6171559

  3. Thermodynamic Modeling of Variations in the Rate of RNA Chain Elongation of E. coli rrn Operons

    PubMed Central

    Fange, David; Mellenius, Harriet; Dennis, Patrick P.; Ehrenberg, Måns

    2014-01-01

    Previous electron-microscopic imaging has shown high RNA polymerase occupation densities in the 16S and 23S encoding regions and low occupation densities in the noncoding leader, spacer, and trailer regions of the rRNA (rrn) operons in E. coli. This indicates slower transcript elongation within the coding regions and faster elongation within the noncoding regions of the operon. Inactivation of four of the seven rrn operons increases the transcript initiation frequency at the promoters of the three intact operons and reduces the time for RNA polymerase to traverse the operon. We have used the DNA sequence-dependent standard free energy variation of the transcription complex to model the experimentally observed changes in the elongation rate along the rrnB operon. We also model the stimulation of the average transcription rate over the whole operon by increasing rate of transcript initiation. Monte Carlo simulations, taking into account initiation of transcription, translocation, and backward and forward tracking of RNA polymerase, partially reproduce the observed transcript elongation rate variations along the rrn operon and fully account for the increased average rate in response to increased frequency of transcript initiation. PMID:24411237

  4. The dnaKJ operon of Agrobacterium tumefaciens: transcriptional analysis and evidence for a new heat shock promoter.

    PubMed

    Segal, G; Ron, E Z

    1995-10-01

    The dnaKJ operon of Agrobacterium tumefaciens was cloned and sequenced and was found to be highly homologous to previously analyzed dnaKJ operons. Transcription of this operon in A. tumefaciens was stimulated by heat shock as well as by exposure to ethanol and hydrogen peroxide. There were two transcripts representing the dnaKJ operon: one containing the dnaK and dnaJ genes and the second containing only the dnaK gene. Primer extension analysis indicated that transcription started from the same site in heat-shocked cells and in untreated cells. The upstream regulatory region of the dnaKJ operon of A. tumefaciens does not contain the highly conserved inverted repeat sequence previously found in the groESL operon of this bacterium, as well as in many other groE and dnaK operons. Sequence analysis of the promoter region of several groESL and dnaK operons from alpha-purple proteobacteria indicates the existence of a putative promoter sequence different from the known consensus promoter sequences recognized by the Escherichia coli vegetative or heat shock sigma factor. This promoter may constitute the heat shock promoter of these alpha-purple proteobacteria.

  5. Virulence Effects and Signaling Partners Modulated by Brucella melitensis Light-sensing Histidine Kinase

    NASA Astrophysics Data System (ADS)

    Gourley, Christopher R.

    The facultative intracellular pathogen Brucella melitensis utilizes diverse virulence factors. A Brucella light sensing histidine kinase can influence in vitro virulence of the bacteria during intracellular infection. First, we demonstrated that the B. melitensis light sensing kinase (BM-LOV-HK) affects virulence in an IRF-1-/- mouse model of infection. Infection with a Δ BM-LOV-HK strain resulted in less bacterial colonization of IRF-1-/- spleens and extended survivorship compared to mice infected with wild type B. melitensis 16M. Second, using PCR arrays, we observed less expression of innate and adaptive immune system activation markers in ΔBM-LOV-HK infected mouse spleens than wild type B. melitensis 16M infected mouse spleens 6 days after infection. Third, we demonstrated by microarray analysis of B. melitensis that deletion of BM-LOV-HK alters bacterial gene expression. Downregulation of genes involved in control of the general stress response system included the alternative sigma factor RpoE1 and its anti-anti sigma factor PhyR. Conversely, genes involved in flagella production, quorum sensing, and the type IV secretion system (VirB operon) were upregulated in the Δ BM-LOV-HK strain compared to the wild type B. melitensis 16M. Analysis of genes differentially regulated in Δ BM-LOV-HK versus the wild type strain indicated an overlap of 110 genes with data from previous quorum sensing regulator studies of Δ vjbR and/ΔblxR(babR) strains. Also, several predicted RpoE1 binding sites located upstream of genes were differentially regulated in the ΔBM-LOV-HK strain. Our results suggest BM-LOV-HK is important for in vivo Brucella virulence, and reveals that BM-LOV-HK directly or indirect regulates members of the Brucella quorum sensing, type IV secretion, and general stress systems.

  6. Salmonella-secreted Virulence Factors

    SciTech Connect

    Heffron, Fred; Niemann, George; Yoon, Hyunjin; Kidwai, Afshan S.; Brown, Roslyn N.; McDermott, Jason E.; Smith, Richard D.; Adkins, Joshua N.

    2011-05-01

    In this short review we discuss secreted virulence factors of Salmonella, which directly affect Salmonella interaction with its host. Salmonella secretes protein to subvert host defenses but also, as discussed, to reduce virulence thereby permitting the bacteria to persist longer and more successfully disperse. The type III secretion system (TTSS) is the best known and well studied of the mechanisms that enable secretion from the bacterial cytoplasm to the host cell cytoplasm. Other secretion systems include outer membrane vesicles, which are present in all Gram-negative bacteria examined to date, two-partner secretion, and type VI secretion will also be addressed. Excellent reviews of Salmonella secreted effectors have focused on themes such as actin rearrangements, vesicular trafficking, ubiquitination, and the activities of the virulence factors themselves. This short review is based on S. Typhimurium infection of mice because it is a model of typhoid like disease in humans. We have organized effectors in terms of events that happen during the infection cycle and how secreted effectors may be involved.

  7. The Long-Chain Fatty Acid Sensor, PsrA, Modulates The Expression of rpoS and the Type III Secretion exsCEBA-Operon in Pseudomonas aeruginosa

    PubMed Central

    Kang, Yun; Lunin, Vladimir V.; Skarina, Tatiana; Savchenko, Alexei; Schurr, Michael J.; Hoang, Tung T.

    2009-01-01

    Summary The Pseudomonas aeruginosa PsrA autorepressor has dual roles as a repressor of the fadBA5 β-oxidation-operon and an activator of the stationary-phase sigma factor rpoS and exsCEBA-operon of the type III secretion system (TTSS). Previously, we demonstrated that the repression of the fadBA5-operon by PsrA is relieved by long-chain fatty acids (LCFA). However, the signal affecting the activation of rpoS and exsC via PsrA is unknown. In this study, microarray and gene-fusion data suggested that LCFA (e.g. oleate) affected the expression of rpoS and exsC. DNA binding studies confirmed that PsrA binds to the rpoS and exsC promoter regions. This binding was inhibited by LCFA, indicating that LCFA directly affects the activation of these two genes through PsrA. LCFA decreased rpoS and exsC expression, resulting in increased N-(butyryl)-l-homoserine-lactone quorum-sensing signal and decreased ExoS/T production, respectively. Based on the crystal structure of PsrA, site-directed mutagenesis of amino acid residues, within the hydrophobic channel thought to accommodate LCFA, created two LCFA-nonresponsive PsrA mutants. The binding and activation of rpoS and exsC by these PsrA mutants was no longer inhibited by LCFA. These data support a mechanistic model where LCFA influence PsrA regulation to control LCFA metabolism and some virulence genes in P. aeruginosa. PMID:19508282

  8. Determining the bistability parameter ranges of artificially induced lac operon using the root locus method.

    PubMed

    Avcu, N; Alyürük, H; Demir, G K; Pekergin, F; Cavas, L; Güzeliş, C

    2015-06-01

    This paper employs the root locus method to conduct a detailed investigation of the parameter regions that ensure bistability in a well-studied gene regulatory network namely, lac operon of Escherichia coli (E. coli). In contrast to previous works, the parametric bistability conditions observed in this study constitute a complete set of necessary and sufficient conditions. These conditions were derived by applying the root locus method to the polynomial equilibrium equation of the lac operon model to determine the parameter values yielding the multiple real roots necessary for bistability. The lac operon model used was defined as an ordinary differential equation system in a state equation form with a rational right hand side, and it was compatible with the Hill and Michaelis-Menten approaches of enzyme kinetics used to describe biochemical reactions that govern lactose metabolism. The developed root locus method can be used to study the steady-state behavior of any type of convergent biological system model based on mass action kinetics. This method provides a solution to the problem of analyzing gene regulatory networks under parameter uncertainties because the root locus method considers the model parameters as variable, rather than fixed. The obtained bistability ranges for the lac operon model parameters have the potential to elucidate the appearance of bistability for E. coli cells in in vivo experiments, and they could also be used to design robust hysteretic switches in synthetic biology. PMID:25864166

  9. Bistable behavior in a model of the lac operon in Escherichia coli with variable growth rate.

    PubMed

    Santillán, M

    2008-03-15

    This work is a continuation from another study previously published in this journal. Both the former and the present works are dedicated to investigating the bistable behavior of the lac operon in Escherichia coli from a mathematical modeling point of view. In the previous article, we developed a detailed mathematical model that accounts for all of the known regulatory mechanisms in this system, and studied the effect of inducing the operon with lactose instead of an artificial inducer. In this article, the model is improved to account, in a more detailed way, for the interaction of the repressor molecules with the three lac operators. A recently discovered cooperative interaction between the CAP molecule (an activator of the lactose operon) and Operator 3 (which influences DNA folding) is also included in this new version of the model. The growth rate dependence on the rate of energy entering the bacteria (in the form of transported glucose molecules and of metabolized lactose molecules) is also considered. A large number of numerical experiments is carried out with this improved model. The results are discussed in regard to the bistable behavior of the lactose operon. Special attention is paid to the effect that a variable growth rate has on the system dynamics. PMID:18065471

  10. Lack of evidence for horizontal transfer of the lac operon into Escherichia coli.

    PubMed

    Stoebel, Daniel M

    2005-03-01

    The idea that Escherichia coli gained the lac operon via horizontal transfer, allowing it to invade a new niche and form a new species, has become a paradigmatic example of bacterial nonpathogenic adaptation and speciation catalyzed by horizontal transfer. Surprisingly, empirical evidence for this event is essentially nonexistent. To see whether horizontal transfer occurred, I compared a phylogeny of 14 Enterobacteriaceae based on two housekeeping genes to a phylogeny of a part of their lac operon. Although several species in this clade appear to have acquired some or all of the operon via horizontal transfer, there is no evidence of horizontal transfer into E. coli. It is not clear whether the horizontal transfer events for which there is evidence were adaptive because those species which have acquired the operon are not thought to live in high lactose environments. I propose that vertical transmission from the common ancestor of the Enterobacteriaceae, with subsequent loss of these genes in many species can explain much of the patchy distribution of lactose use in this clade. Finally, I argue that we need new, well-supported examples of horizontal transfer spurring niche expansion and speciation, particularly in nonpathogenic cases, before we can accept claims that horizontal transfer is a hallmark of bacterial adaptation. PMID:15563718

  11. Novel Functions and Regulation of Cryptic Cellobiose Operons in Escherichia coli

    PubMed Central

    Parisutham, Vinuselvi; Lee, Sung Kuk

    2015-01-01

    Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (β-glucosidase of asc operon) and nonsense mutation in yebK, (an uncharacterized transcription factor). Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolism. PMID:26121029

  12. Novel Functions and Regulation of Cryptic Cellobiose Operons in Escherichia coli.

    PubMed

    Parisutham, Vinuselvi; Lee, Sung Kuk

    2015-01-01

    Presence of cellobiose as a sole carbon source induces mutations in the chb and asc operons of Escherichia coli and allows it to grow on cellobiose. We previously engineered these two operons with synthetic constitutive promoters and achieved efficient cellobiose metabolism through adaptive evolution. In this study, we characterized two mutations observed in the efficient cellobiose metabolizing strain: duplication of RBS of ascB gene, (β-glucosidase of asc operon) and nonsense mutation in yebK, (an uncharacterized transcription factor). Mutations in yebK play a dominant role by modulating the length of lag phase, relative to the growth rate of the strain when transferred from a rich medium to minimal cellobiose medium. Mutations in ascB, on the other hand, are specific for cellobiose and help in enhancing the specific growth rate. Taken together, our results show that ascB of the asc operon is controlled by an internal putative promoter in addition to the native cryptic promoter, and the transcription factor yebK helps to remodel the host physiology for cellobiose metabolism. While previous studies characterized the stress-induced mutations that allowed growth on cellobiose, here, we characterize the adaptation-induced mutations that help in enhancing cellobiose metabolic ability. This study will shed new light on the regulatory changes and factors that are needed for the functional coupling of the host physiology to the activated cryptic cellobiose metabolism.

  13. Decreases in average bacterial community rRNA operon copy number during succession

    PubMed Central

    Nemergut, Diana R; Knelman, Joseph E; Ferrenberg, Scott; Bilinski, Teresa; Melbourne, Brett; Jiang, Lin; Violle, Cyrille; Darcy, John L; Prest, Tiffany; Schmidt, Steven K; Townsend, Alan R

    2016-01-01

    Trait-based studies can help clarify the mechanisms driving patterns of microbial community assembly and coexistence. Here, we use a trait-based approach to explore the importance of rRNA operon copy number in microbial succession, building on prior evidence that organisms with higher copy numbers respond more rapidly to nutrient inputs. We set flasks of heterotrophic media into the environment and examined bacterial community assembly at seven time points. Communities were arrayed along a geographic gradient to introduce stochasticity via dispersal processes and were analyzed using 16 S rRNA gene pyrosequencing, and rRNA operon copy number was modeled using ancestral trait reconstruction. We found that taxonomic composition was similar between communities at the beginning of the experiment and then diverged through time; as well, phylogenetic clustering within communities decreased over time. The average rRNA operon copy number decreased over the experiment, and variance in rRNA operon copy number was lowest both early and late in succession. We then analyzed bacterial community data from other soil and sediment primary and secondary successional sequences from three markedly different ecosystem types. Our results demonstrate that decreases in average copy number are a consistent feature of communities across various drivers of ecological succession. Importantly, our work supports the scaling of the copy number trait over multiple levels of biological organization, ranging from cells to populations and communities, with implications for both microbial ecology and evolution. PMID:26565722

  14. RNA polymerase supply and flux through the lac operon in Escherichia coli.

    PubMed

    Sendy, Bandar; Lee, David J; Busby, Stephen J W; Bryant, Jack A

    2016-11-01

    Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.This article is part of the themed issue 'The new bacteriology'. PMID:27672157

  15. RNA polymerase supply and flux through the lac operon in Escherichia coli

    PubMed Central

    Sendy, Bandar; Lee, David J.; Bryant, Jack A.

    2016-01-01

    Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli. By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase. This article is part of the themed issue ‘The new bacteriology’. PMID:27672157

  16. RNA polymerase supply and flux through the lac operon in Escherichia coli.

    PubMed

    Sendy, Bandar; Lee, David J; Busby, Stephen J W; Bryant, Jack A

    2016-11-01

    Chromatin immunoprecipitation, followed by quantification of immunoprecipitated DNA, can be used to measure RNA polymerase binding to any DNA segment in Escherichia coli By calibrating measurements against the signal from a single RNA polymerase bound at a single promoter, we can calculate both promoter occupancy levels and the flux of transcribing RNA polymerase through transcription units. Here, we have applied the methodology to the E. coli lactose operon promoter. We confirm that promoter occupancy is limited by recruitment and that the supply of RNA polymerase to the lactose operon promoter depends on its location in the E. coli chromosome. Measurements of RNA polymerase binding to DNA segments within the lactose operon show that flux of RNA polymerase through the operon is low, with, on average, over 18 s elapsing between the passage of transcribing polymerases. Similar low levels of flux were found when semi-synthetic promoters were used to drive transcript initiation, even when the promoter elements were changed to ensure full occupancy of the promoter by RNA polymerase.This article is part of the themed issue 'The new bacteriology'.

  17. ISOLATION OF AN OPERON INVOLVED IN XYLITOL METABOLISM FROM PANTOEA ANANATIS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An operon involved in xylitol metabolism in a xylitol-utilizing Pantoea ananatis mutant was cloned by the transposon tagging method. Sequencing analysis revealed that seven consecutive open reading frames (ORFs) are located in the same strand (xytA-G). Sequence homology search suggested that the o...

  18. Structure and regulation of the Salmonella typhimurium rnc-era-recO operon.

    PubMed

    Anderson, P E; Matsunaga, J; Simons, E L; Simons, R W

    1996-01-01

    The Escherichia coli rnc-era-recO operon encodes ribonuclease III (RNase III; a dsRNA endonuclease involved in rRNA and mRNA processing and decay), Era (an essential G-protein of unknown functions and RecO (involved in the RecF homologous recombination pathway). Expression of the rnc and era genes is negatively autoregulated: RNase III cleaves the rncO 'operator' in the untranslated leader, destabilizing the operon mRNA. As part of a larger effort to understand RNase III and Era structure and function, we characterized rnc operon structure, function and regulation in the closely related bacterium Salmonella typhimurium. Construction of a S typhimurium strain conditionally defective for RNase III and Era expression showed that Era is essential for cell growth. This mutant strain also enabled selection of recombinant clones containing the intact S typhimurium rnc-era-recO operon, whose nucleotide sequence, predicted protein sequence, and predicted rncO RNA secondary structure were all highly conserved with those of E coli. Furthermore, genetic and biochemical analysis revealed that S typhimurium rnc gene expression is negatively autoregulated by a mechanism very similar or identical to that in E coli, and that the cleavage specificities of RNase IIIs.t. and RNase IIIE.c. are indistinguishable with regard to rncO cleavage and S typhimurium 23S rRNA fragmentation in vivo. PMID:9150881

  19. Decreases in average bacterial community rRNA operon copy number during succession.

    PubMed

    Nemergut, Diana R; Knelman, Joseph E; Ferrenberg, Scott; Bilinski, Teresa; Melbourne, Brett; Jiang, Lin; Violle, Cyrille; Darcy, John L; Prest, Tiffany; Schmidt, Steven K; Townsend, Alan R

    2016-05-01

    Trait-based studies can help clarify the mechanisms driving patterns of microbial community assembly and coexistence. Here, we use a trait-based approach to explore the importance of rRNA operon copy number in microbial succession, building on prior evidence that organisms with higher copy numbers respond more rapidly to nutrient inputs. We set flasks of heterotrophic media into the environment and examined bacterial community assembly at seven time points. Communities were arrayed along a geographic gradient to introduce stochasticity via dispersal processes and were analyzed using 16 S rRNA gene pyrosequencing, and rRNA operon copy number was modeled using ancestral trait reconstruction. We found that taxonomic composition was similar between communities at the beginning of the experiment and then diverged through time; as well, phylogenetic clustering within communities decreased over time. The average rRNA operon copy number decreased over the experiment, and variance in rRNA operon copy number was lowest both early and late in succession. We then analyzed bacterial community data from other soil and sediment primary and secondary successional sequences from three markedly different ecosystem types. Our results demonstrate that decreases in average copy number are a consistent feature of communities across various drivers of ecological succession. Importantly, our work supports the scaling of the copy number trait over multiple levels of biological organization, ranging from cells to populations and communities, with implications for both microbial ecology and evolution. PMID:26565722

  20. Nonhemolytic Streptococcus pyogenes Isolates That Lack Large Regions of the sag Operon Mediating Streptolysin S Production▿

    PubMed Central

    Yoshino, Miho; Murayama, Somay Y.; Sunaoshi, Katsuhiko; Wajima, Takeaki; Takahashi, Miki; Masaki, Junko; Kurokawa, Iku; Ubukata, Kimiko

    2010-01-01

    Among nonhemolytic Streptococcus pyogenes (group A streptococcus) strains (n = 9) isolated from patients with pharyngitis or acute otitis media, we identified three deletions in the region from the epf gene, encoding the extracellular matrix binding protein, to the sag operon, mediating streptolysin S production. PMID:20018818

  1. Tryptophan auxotrophs were obtained by random transposon insertions in the Methanococcus maripaludis tryptophan operon.

    PubMed

    Porat, Iris; Whitman, William B

    2009-08-01

    Methanococcus maripaludis is an anaerobic, methane-producing archaeon that utilizes H(2) or formate for the reduction of CO(2) to methane. Tryptophan auxotrophs were constructed by in vitro insertions of the Tn5 transposon into the tryptophan operon, followed by transformation into M. maripaludis. This method could serve for rapid insertions into large cloned DNA regions. PMID:19566682

  2. Transcriptional and posttranscriptional regulation of Bacillus sp. CDB3 arsenic-resistance operon ars1

    PubMed Central

    Yu, Xuefei; Zheng, Wei; Bhat, Somanath; Aquilina, J. Andrew

    2015-01-01

    Bacillus sp. CDB3 possesses a novel eight-gene ars cluster (ars1, arsRYCDATorf7orf8) with some unusual features in regard to expression regulation. This study demonstrated that the cluster is a single operon but can also produce a short three-gene arsRYC transcript. A hairpin structure formed by internal inverted repeats between arsC and arsD was shown to diminish the expression of the full operon, thereby probably acting as a transcription attenuator. A degradation product of the arsRYC transcript was also identified. Electrophoretic mobility shift analysis demonstrated that ArsR interacts with the ars1 promoter forming a protein-DNA complex that could be impaired by arsenite. However, no interaction was detected between ArsD and the ars1 promoter, suggesting that the CDB3 ArsD protein may not play a regulatory role. Compared to other ars gene clusters, regulation of the Bacillus sp. CDB3 ars1 operon is more complex. It represents another example of specific mRNA degradation in the transporter gene region and possibly the first case of attenuator-mediated regulation of ars operons. PMID:26355338

  3. DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli

    PubMed Central

    Fulcrand, Geraldine; Dages, Samantha; Zhi, Xiaoduo; Chapagain, Prem; Gerstman, Bernard S.; Dunlap, David; Leng, Fenfei

    2016-01-01

    Escherichia coli lac repressor (LacI) is a paradigmatic transcriptional factor that controls the expression of lacZYA in the lac operon. This tetrameric protein specifically binds to the O1, O2 and O3 operators of the lac operon and forms a DNA loop to repress transcription from the adjacent lac promoter. In this article, we demonstrate that upon binding to the O1 and O2 operators at their native positions LacI constrains three (−) supercoils within the 401-bp DNA loop of the lac promoter and forms a topological barrier. The stability of LacI-mediated DNA topological barriers is directly proportional to its DNA binding affinity. However, we find that DNA supercoiling modulates the basal expression from the lac operon in E. coli. Our results are consistent with the hypothesis that LacI functions as a topological barrier to constrain free, unconstrained (−) supercoils within the 401-bp DNA loop of the lac promoter. These constrained (−) supercoils enhance LacI’s DNA-binding affinity and thereby the repression of the promoter. Thus, LacI binding is superhelically modulated to control the expression of lacZYA in the lac operon under varying growth conditions. PMID:26763930

  4. Efficient metabolic pathway engineering in transgenic tobacco and tomato plastids with synthetic multigene operons.

    PubMed

    Lu, Yinghong; Rijzaani, Habib; Karcher, Daniel; Ruf, Stephanie; Bock, Ralph

    2013-02-19

    The engineering of complex metabolic pathways requires the concerted expression of multiple genes. In plastids (chloroplasts) of plant cells, genes are organized in operons that are coexpressed as polycistronic transcripts and then often are processed further into monocistronic mRNAs. Here we have used the tocochromanol pathway (providing tocopherols and tocotrienols, collectively also referred to as "vitamin E") as an example to establish principles of successful multigene engineering by stable transformation of the chloroplast genome, a technology not afflicted with epigenetic variation and/or instability of transgene expression. Testing a series of single-gene constructs (encoding homogentisate phytyltransferase, tocopherol cyclase, and γ-tocopherol methyltransferase) and rationally designed synthetic operons in tobacco and tomato plants, we (i) confirmed previous results suggesting homogentisate phytyltransferase as the limiting enzymatic step in the pathway, (ii) comparatively characterized the bottlenecks in tocopherol biosynthesis in transplastomic leaves and tomato fruits, and (iii) achieved an up to tenfold increase in total tocochromanol accumulation. In addition, our results uncovered an unexpected light-dependent regulatory link between tocochromanol metabolism and the pathways of photosynthetic pigment biosynthesis. The synthetic operon design developed here will facilitate future synthetic biology applications in plastids, especially the design of artificial operons that introduce novel biochemical pathways into plants.

  5. An attenuated mutant of the Rv1747 ATP-binding cassette transporter of Mycobacterium tuberculosis and a mutant of its cognate kinase, PknF, show increased expression of the efflux pump-related iniBAC operon

    PubMed Central

    Spivey, Vicky L; Whalan, Rachael H; Hirst, Elizabeth M A; Smerdon, Stephen J; Buxton, Roger S

    2013-01-01

    The ATP-binding cassette transporter Rv1747 is required for the growth of Mycobacterium tuberculosis in mice and in macrophages. Its structure suggests it is an exporter. Rv1747 forms a two-gene operon with pknF coding for the serine/threonine protein kinase PknF, which positively modulates the function of the transporter. We show that deletion of Rv1747 or pknF results in a number of transcriptional changes which could be complemented by the wild type allele, most significantly up-regulation of the iniBAC genes. This operon is inducible by isoniazid and ethambutol and by a broad range of inhibitors of cell wall biosynthesis and is required for efflux pump functioning. However, neither the Rv1747 or pknF mutant showed increased susceptibility to a range of drugs and cell wall stress reagents including isoniazid and ethambutol, cell wall structure and cell division appear normal by electron microscopy, and no differences in lipoarabinomannan were found. Transcription from the pknF promoter was not induced by a range of stress reagents. We conclude that the loss of Rv1747 affects cell wall biosynthesis leading to the production of intermediates that cause induction of iniBAC transcription and implicates it in exporting a component of the cell wall, which is necessary for virulence. PMID:23915284

  6. Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli.

    PubMed

    Yanofsky, C; Horn, V; Gollnick, P

    1991-10-01

    Escherichia coli forms three permeases that can transport the amino acid tryptophan: Mtr, AroP, and TnaB. The structural genes for these permeases reside in separate operons that are subject to different mechanisms of regulation. We have exploited the fact that the tryptophanase (tna) operon is induced by tryptophan to infer how tryptophan transport is influenced by the growth medium and by mutations that inactivate each of the permease proteins. In an acid-hydrolyzed casein medium, high levels of tryptophan are ordinarily required to obtain maximum tna operon induction. High levels are necessary because much of the added tryptophan is degraded by tryptophanase. An alternate inducer that is poorly cleaved by tryptophanase, 1-methyltryptophan, induces efficiently at low concentrations in both tna+ strains and tna mutants. In an acid-hydrolyzed casein medium, the TnaB permease is most critical for tryptophan uptake; i.e., only mutations in tnaB reduce tryptophanase induction. However, when 1-methyltryptophan replaces tryptophan as the inducer in this medium, mutations in both mtr and tnaB are required to prevent maximum induction. In this medium, AroP does not contribute to tryptophan uptake. However, in a medium lacking phenylalanine and tyrosine the AroP permease is active in tryptophan transport; under these conditions it is necessary to inactivate the three permeases to eliminate tna operon induction. The Mtr permease is principally responsible for transporting indole, the degradation product of tryptophan produced by tryptophanase action. The TnaB permease is essential for growth on tryptophan as the sole carbon source. When cells with high levels of tryptophanase are transferred to tryptophan-free growth medium, the expression of the tryptophan (trp) operon is elevated. This observation suggests that the tryptophanase present in these cells degrades some of the synthesized tryptophan, thereby creating a mild tryptophan deficiency. Our studies assign roles to

  7. Tryptophan inhibits Proteus vulgaris TnaC leader peptide elongation, activating tna operon expression.

    PubMed

    Cruz-Vera, Luis R; Yang, Rui; Yanofsky, Charles

    2009-11-01

    Expression of the tna operon of Escherichia coli and of Proteus vulgaris is induced by L-tryptophan. In E. coli, tryptophan action is dependent on the presence of several critical residues (underlined) in the newly synthesized TnaC leader peptide, WFNIDXXL/IXXXXP. These residues are conserved in TnaC of P. vulgaris and of other bacterial species. TnaC of P. vulgaris has one additional feature, distinguishing it from TnaC of E. coli; it contains two C-terminal lysine residues following the conserved proline residue. In the present study, we investigated L-tryptophan induction of the P. vulgaris tna operon, transferred on a plasmid into E. coli. Induction was shown to be L-tryptophan dependent; however, the range of induction was less than that observed for the E. coli tna operon. We compared the genetic organization of both operons and predicted similar folding patterns for their respective leader mRNA segments. However, additional analyses revealed that L-tryptophan action in the P. vulgaris tna operon involves inhibition of TnaC elongation, following addition of proline, rather than inhibition of leader peptide termination. Our findings also establish that the conserved residues in TnaC of P. vulgaris are essential for L-tryptophan induction, and for inhibition of peptide elongation. TnaC synthesis is thus an excellent model system for studies of regulation of both peptide termination and peptide elongation, and for studies of ribosome recognition of the features of a nascent peptide. PMID:19767424

  8. Capsules, toxins and AtxA as virulence factors of emerging Bacillus cereus biovar anthracis.

    PubMed

    Brézillon, Christophe; Haustant, Michel; Dupke, Susann; Corre, Jean-Philippe; Lander, Angelika; Franz, Tatjana; Monot, Marc; Couture-Tosi, Evelyne; Jouvion, Gregory; Leendertz, Fabian H; Grunow, Roland; Mock, Michèle E; Klee, Silke R; Goossens, Pierre L

    2015-04-01

    Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d'Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged.

  9. Capsules, Toxins and AtxA as Virulence Factors of Emerging Bacillus cereus Biovar anthracis

    PubMed Central

    Corre, Jean-Philippe; Lander, Angelika; Franz, Tatjana; Monot, Marc; Couture-Tosi, Evelyne; Jouvion, Gregory; Leendertz, Fabian H.; Grunow, Roland; Mock, Michèle E.; Klee, Silke R.; Goossens, Pierre L.

    2015-01-01

    Emerging B. cereus strains that cause anthrax-like disease have been isolated in Cameroon (CA strain) and Côte d’Ivoire (CI strain). These strains are unusual, because their genomic characterisation shows that they belong to the B. cereus species, although they harbour two plasmids, pBCXO1 and pBCXO2, that are highly similar to the pXO1 and pXO2 plasmids of B. anthracis that encode the toxins and the polyglutamate capsule respectively. The virulence factors implicated in the pathogenicity of these B. cereus bv anthracis strains remain to be characterised. We tested their virulence by cutaneous and intranasal delivery in mice and guinea pigs; they were as virulent as wild-type B. anthracis. Unlike as described for pXO2-cured B. anthracis, the CA strain cured of the pBCXO2 plasmid was still highly virulent, showing the existence of other virulence factors. Indeed, these strains concomitantly expressed a hyaluronic acid (HA) capsule and the B. anthracis polyglutamate (PDGA) capsule. The HA capsule was encoded by the hasACB operon on pBCXO1, and its expression was regulated by the global transcription regulator AtxA, which controls anthrax toxins and PDGA capsule in B. anthracis. Thus, the HA and PDGA capsules and toxins were co-regulated by AtxA. We explored the respective effect of the virulence factors on colonisation and dissemination of CA within its host by constructing bioluminescent mutants. Expression of the HA capsule by itself led to local multiplication and, during intranasal infection, to local dissemination to the adjacent brain tissue. Co-expression of either toxins or PDGA capsule with HA capsule enabled systemic dissemination, thus providing a clear evolutionary advantage. Protection against infection by B. cereus bv anthracis required the same vaccination formulation as that used against B. anthracis. Thus, these strains, at the frontier between B. anthracis and B. cereus, provide insight into how the monomorphic B. anthracis may have emerged. PMID

  10. Molecular characterization of a genomic region associated with virulence in Dichelobacter nodosus.

    PubMed Central

    Katz, M E; Strugnell, R A; Rood, J I

    1992-01-01

    The major pathogen implicated in footrot, a highly contagious disease of sheep, is the strict anaerobe Dichelobacter nodosus (formerly Bacteroides nodosus). Sequence analysis of a 2,262-bp segment of the D. nodosus genome which is more prevalent in virulent isolates than in other isolates showed the presence of four open reading frames which appeared to have consensus transcriptional and translational start signals. These virulence-associated genes have been designated vapABCD. Two of the three copies of the vap region in the genome of the reference strain D. nodosus A198 were shown to carry all of the vap genes, whereas one copy contained only the vapD gene. The VapD protein was gel purified, shown to contain the predicted amino-terminal sequence, and used to raise rabbit antibodies. Western blots (immunoblots) showed that all of the D. nodosus strains tested that contained the vap region produced the VapD protein. The VapD protein had significant amino acid sequence identity with open reading frame 5 from the cryptic plasmid of Neisseria gonorrhoeae, and the vapBC operon had sequence similarity with the trbH region of the Escherichia coli F plasmid. It is proposed that these gene regions evolved from the integration of a conjugative plasmid from another bacterial species into the D. nodosus chromosome. Images PMID:1398971

  11. A Shigella flexneri virulence plasmid encoded factor controls production of outer membrane vesicles.

    PubMed

    Sidik, Saima; Kottwitz, Haila; Benjamin, Jeremy; Ryu, Julie; Jarrar, Ameer; Garduno, Rafael; Rohde, John R

    2014-12-01

    Shigella spp. use a repertoire of virulence plasmid-encoded factors to cause shigellosis. These include components of a Type III Secretion Apparatus (T3SA) that is required for invasion of epithelial cells and many genes of unknown function. We constructed an array of 99 deletion mutants comprising all genes encoded by the virulence plasmid (excluding those known to be required for plasmid maintenance) of Shigella flexneri. We screened these mutants for their ability to bind the dye Congo red: an indicator of T3SA function. This screen focused our attention on an operon encoding genes that modify the cell envelope including virK, a gene of partially characterized function. We discovered that virK is required for controlled release of proteins to the culture supernatant. Mutations in virK result in a temperature-dependent overproduction of outer membrane vesicles (OMVs). The periplasmic chaperone/protease DegP, a known regulator of OMV production in Escherichia coli (encoded by a chromosomal gene), was found to similarly control OMV production in S. flexneri. Both virK and degP show genetic interactions with mxiD, a structural component of the T3SA. Our results are consistent with a model in which VirK and DegP relieve the periplasmic stress that accompanies assembly of the T3SA. PMID:25378474

  12. A Shigella flexneri Virulence Plasmid Encoded Factor Controls Production of Outer Membrane Vesicles

    PubMed Central

    Sidik, Saima; Kottwitz, Haila; Benjamin, Jeremy; Ryu, Julie; Jarrar, Ameer; Garduno, Rafael; Rohde, John R.

    2014-01-01

    Shigella spp. use a repertoire of virulence plasmid-encoded factors to cause shigellosis. These include components of a Type III Secretion Apparatus (T3SA) that is required for invasion of epithelial cells and many genes of unknown function. We constructed an array of 99 deletion mutants comprising all genes encoded by the virulence plasmid (excluding those known to be required for plasmid maintenance) of Shigella flexneri. We screened these mutants for their ability to bind the dye Congo red: an indicator of T3SA function. This screen focused our attention on an operon encoding genes that modify the cell envelope including virK, a gene of partially characterized function. We discovered that virK is required for controlled release of proteins to the culture supernatant. Mutations in virK result in a temperature-dependent overproduction of outer membrane vesicles (OMVs). The periplasmic chaperone/protease DegP, a known regulator of OMV production in Escherichia coli (encoded by a chromosomal gene), was found to similarly control OMV production in S. flexneri. Both virK and degP show genetic interactions with mxiD, a structural component of the T3SA. Our results are consistent with a model in which VirK and DegP relieve the periplasmic stress that accompanies assembly of the T3SA. PMID:25378474

  13. The Escherichia coli K-12 cyn operon is positively regulated by a member of the lysR family.

    PubMed Central

    Sung, Y C; Fuchs, J A

    1992-01-01

    A regulatory gene, cynR, was found to be located next to the cyn operon but transcribed in the opposite direction. cynR encodes a positive regulatory protein that controls the cyn operon as well as its own synthesis. Positive regulation of the cyn operon requires cyanate and the cynR protein, but the negative autoregulation of the cynR gene appears to be independent of cyanate. The predicted amino acid sequence of the cynR protein derived from the DNA sequence was found to have significant homology to the predicted amino acid sequence of the lysR family of regulatory proteins. Images PMID:1592818

  14. Removal of the phage-shock protein PspB causes reduction of virulence in Salmonella enterica serovar Typhimurium independently of NRAMP1.

    PubMed

    Wallrodt, Inke; Jelsbak, Lotte; Thomsen, Line E; Brix, Lena; Lemire, Sébastien; Gautier, Laurent; Nielsen, Dennis S; Jovanovic, Goran; Buck, Martin; Olsen, John E

    2014-06-01

    The phage-shock protein (Psp) system is believed to manage membrane stress in all Enterobacteriaceae and has recently emerged as being important for virulence in several pathogenic species of this phylum. The core of the Psp system consists of the pspA-D operon and the distantly located pspG gene. In Salmonella enterica serovar Typhimurium (S. Typhimurium), it has recently been reported that PspA is essential for systemic infection of mice, but only in NRAMP1(+) mice, signifying that attenuation is related to coping with divalent cation starvation in the intracellular environment. In the present study, we investigated the contribution of individual psp genes to virulence of S. Typhimurium. Interestingly, deletion of the whole pspA-D set of genes caused attenuation in both NRAMP1(+) and NRAMP1(-) mice, indicating that one or more of the psp genes contribute to virulence independently of NRAMP1 expression in the host. Investigations of single gene mutants showed that knock out of pspB reduced virulence in both types of mice, while deletion of pspA only caused attenuation in NRAMP1(+) mice, and deletion of pspD had a minor effect in NRAMP1(-) mice, while deletions of either pspC or pspG did not affect virulence. Experiments addressed at elucidating the role of PspB in virulence revealed that PspB is dispensable for uptake to and intracellular replication in cultured macrophages and resistance to complement-induced killing. Furthermore, the Psp system of S. Typhimurium was dispensable during pIV-induced secretin stress. In conclusion, our results demonstrate that removal of PspB reduces virulence in S. Typhimurium independently of host NRAMP1 expression, demonstrating that PspB has roles in intra-host survival distinct from the reported contributions of PspA.

  15. The Abi-domain Protein Abx1 Interacts with the CovS Histidine Kinase to Control Virulence Gene Expression in Group B Streptococcus

    PubMed Central

    Firon, Arnaud; Tazi, Asmaa; Da Cunha, Violette; Brinster, Sophie; Sauvage, Elisabeth; Dramsi, Shaynoor; Golenbock, Douglas T.; Glaser, Philippe; Poyart, Claire; Trieu-Cuot, Patrick

    2013-01-01

    Group B Streptococcus (GBS), a common commensal of the female genital tract, is the leading cause of invasive infections in neonates. Expression of major GBS virulence factors, such as the hemolysin operon cyl, is regulated directly at the transcriptional level by the CovSR two-component system. Using a random genetic approach, we identified a multi-spanning transmembrane protein, Abx1, essential for the production of the GBS hemolysin. Despite its similarity to eukaryotic CaaX proteases, the Abx1 function is not involved in a post-translational modification of the GBS hemolysin. Instead, we demonstrate that Abx1 regulates transcription of several virulence genes, including those comprising the hemolysin operon, by a CovSR-dependent mechanism. By combining genetic analyses, transcriptome profiling, and site-directed mutagenesis, we showed that Abx1 is a regulator of the histidine kinase CovS. Overexpression of Abx1 is sufficient to activate virulence gene expression through CovS, overcoming the need for an additional signal. Conversely, the absence of Abx1 has the opposite effect on virulence gene expression consistent with CovS locked in a kinase-competent state. Using a bacterial two-hybrid system, direct interaction between Abx1 and CovS was mapped specifically to CovS domains involved in signal processing. We demonstrate that the CovSR two-component system is the core of a signaling pathway integrating the regulation of CovS by Abx1 in addition to the regulation of CovR by the serine/threonine kinase Stk1. In conclusion, our study reports a regulatory function for Abx1, a member of a large protein family with a characteristic Abi-domain, which forms a signaling complex with the histidine kinase CovS in GBS. PMID:23436996

  16. Evidence for Direct Control of Virulence and Defense Gene Circuits by the Pseudomonas aeruginosa Quorum Sensing Regulator, MvfR

    PubMed Central

    Maura, Damien; Hazan, Ronen; Kitao, Tomoe; Ballok, Alicia E.; Rahme, Laurence G.

    2016-01-01

    Pseudomonas aeruginosa defies eradication by antibiotics and is responsible for acute and chronic human infections due to a wide variety of virulence factors. Currently, it is believed that MvfR (PqsR) controls the expression of many of these factors indirectly via the pqs and phnAB operons. Here we provide strong evidence that MvfR may also bind and directly regulate the expression of additional 35 loci across the P. aeruginosa genome, including major regulators and virulence factors, such as the quorum sensing (QS) regulators lasR and rhlR, and genes involved in protein secretion, translation, and response to oxidative stress. We show that these anti-oxidant systems, AhpC-F, AhpB-TrxB2 and Dps, are critical for P. aeruginosa survival to reactive oxygen species and antibiotic tolerance. Considering that MvfR regulated compounds generate reactive oxygen species, this indicates a tightly regulated QS self-defense anti-poisoning system. These findings also challenge the current hierarchical regulation model of P. aeruginosa QS systems by revealing new interconnections between them that suggest a circular model. Moreover, they uncover a novel role for MvfR in self-defense that favors antibiotic tolerance and cell survival, further demonstrating MvfR as a highly desirable anti-virulence target. PMID:27678057

  17. TssB is essential for virulence and required for type VI secretion system in Ralstonia solanacearum.

    PubMed

    Zhang, Liqing; Xu, Jingsheng; Xu, Jin; Zhang, Hao; He, Liyuan; Feng, Jie

    2014-09-01

    The type VI secretion system (T6SS) is recently discovered machinery in Gram-negative bacteria for translocation of proteins and also is required for full virulence. TssB is a highly conserved protein among the T6SSs, and indispensable for composition and function of T6S. The plant pathogenic bacterium Ralstonia solanacearum also harbours T6SS gene clusters, and a homologue of TssB, hereafter designated as TssBRS, but up to date its characterization and function remain unclear. In this study, we showed that TssBRS of R. solanacearum was required for secretion of Hcp, the haemolysin coregulated protein and a hallmark of T6S pathway. Deletion of tssBRS in R. solanacearum GMI1000 strain resulted in defect of biofilm formation, and the expression of the flagella operon is decreased, leading to decreased motility. More importantly, tssBRS mutant strain had significantly attenuated its virulence on tomato plants. TssB is essential for virulence and required for type VI secretion system in R. solanacearum.

  18. Role of the FeoB Protein and Siderophore in Promoting Virulence of Xanthomonas oryzae pv. oryzae on Rice▿

    PubMed Central

    Pandey, Alok; Sonti, Ramesh V.

    2010-01-01

    Xanthomonas oryzae pv. oryzae causes bacterial blight, a serious disease of rice. Our analysis revealed that the X. oryzae pv. oryzae genome encodes genes responsible for iron uptake through FeoB (homolog of the major bacterial ferrous iron transporter) and a siderophore. A mutation in the X. oryzae pv. oryzae feoB gene causes severe virulence deficiency, growth deficiency in iron-limiting medium, and constitutive production of a siderophore. We identified an iron regulated xss gene cluster, in which xssABCDE (Xanthomonas siderophore synthesis) and xsuA (Xanthomonas siderophore utilization) genes encode proteins involved in biosynthesis and utilization of X. oryzae pv. oryzae siderophore. Mutations in the xssA, xssB, and xssE genes cause siderophore deficiency and growth restriction under iron-limiting conditions but are virulence proficient. An xsuA mutant displayed impairment in utilization of native siderophore, suggesting that XsuA acts as a specific receptor for a ferric-siderophore complex. Histochemical and fluorimetric assays with gusA fusions indicate that, during in planta growth, the feoB gene is expressed and that the xss operon is not expressed. This study represents the first report describing a role for feoB in virulence of any plant-pathogenic bacterium and the first functional characterization of a siderophore-biosynthetic gene cluster in any xanthomonad. PMID:20382771

  19. Role of the FeoB protein and siderophore in promoting virulence of Xanthomonas oryzae pv. oryzae on rice.

    PubMed

    Pandey, Alok; Sonti, Ramesh V

    2010-06-01

    Xanthomonas oryzae pv. oryzae causes bacterial blight, a serious disease of rice. Our analysis revealed that the X. oryzae pv. oryzae genome encodes genes responsible for iron uptake through FeoB (homolog of the major bacterial ferrous iron transporter) and a siderophore. A mutation in the X. oryzae pv. oryzae feoB gene causes severe virulence deficiency, growth deficiency in iron-limiting medium, and constitutive production of a siderophore. We identified an iron regulated xss gene cluster, in which xssABCDE (Xanthomonas siderophore synthesis) and xsuA (Xanthomonas siderophore utilization) genes encode proteins involved in biosynthesis and utilization of X. oryzae pv. oryzae siderophore. Mutations in the xssA, xssB, and xssE genes cause siderophore deficiency and growth restriction under iron-limiting conditions but are virulence proficient. An xsuA mutant displayed impairment in utilization of native siderophore, suggesting that XsuA acts as a specific receptor for a ferric-siderophore complex. Histochemical and fluorimetric assays with gusA fusions indicate that, during in planta growth, the feoB gene is expressed and that the xss operon is not expressed. This study represents the first report describing a role for feoB in virulence of any plant-pathogenic bacterium and the first functional characterization of a siderophore-biosynthetic gene cluster in any xanthomonad.

  20. Virulence and metabolic characteristics of Salmonella enterica serovar enteritidis strains with different sefD variants in hens.

    PubMed

    Morales, Cesar A; Guard, Jean; Sanchez-Ingunza, Roxana; Shah, Devendra H; Harrison, Mark

    2012-09-01

    Salmonella enterica serovar Enteritidis is one of a few Salmonella enterica serotypes that has SEF14 fimbriae encoded by the sef operon, which consists of 4 cotranscribed genes, sefABCD, regulated by sefR. A parental strain was used to construct a sefD mutant and its complement, and all 3 strains were compared for gene expression, metabolic properties, and virulence characteristics in hens. Transcription of sefD by wild type was suppressed at 42°C and absent for the mutant under conditions where the complemented mutant had 10(3) times higher transcription. Growth of the complemented mutant was restricted in comparison to that of the mutant and wild type. Hens infected with the wild type and mutant showed decreased blood calcium and egg production, but infection with the complemented mutant did not. Thus, the absence of sefD correlated with increased metabolic capacity and enhanced virulence of the pathogen. These results suggest that any contribution that sefD makes to egg contamination is either unknown or would be limited to early transmission from the environment to the host. Absence of sefD, either through mutation or by suppression of transcription at the body temperature of the host, may contribute to the virulence of Salmonella enterica by facilitating growth on a wide range of metabolites.

  1. Evolution of viral virulence: empirical studies

    USGS Publications Warehouse

    Kurath, Gael; Wargo, Andrew R.

    2016-01-01

    The concept of virulence as a pathogen trait that can evolve in response to selection has led to a large body of virulence evolution theory developed in the 1980-1990s. Various aspects of this theory predict increased or decreased virulence in response to a complex array of selection pressures including mode of transmission, changes in host, mixed infection, vector-borne transmission, environmental changes, host vaccination, host resistance, and co-evolution of virus and host. A fundamental concept is prediction of trade-offs between the costs and benefits associated with higher virulence, leading to selection of optimal virulence levels. Through a combination of observational and experimental studies, including experimental evolution of viruses during serial passage, many of these predictions have now been explored in systems ranging from bacteriophage to viruses of plants, invertebrates, and vertebrate hosts. This chapter summarizes empirical studies of viral virulence evolution in numerous diverse systems, including the classic models myxomavirus in rabbits, Marek's disease virus in chickens, and HIV in humans. Collectively these studies support some aspects of virulence evolution theory, suggest modifications for other aspects, and show that predictions may apply in some virus:host interactions but not in others. Finally, we consider how virulence evolution theory applies to disease management in the field.

  2. Virulence Evolution Within the Ug99 Lineage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Race TTKSK (syn. Ug99) of Puccinia graminis f. sp. tritici, recognized for possessing virulence to the stem rust resistance gene Sr31, was first identified in Uganda in 1998. Since then, TTKSK has been identified in Kenya in 2005 and Yemen in 2006. In addition to virulence to Sr31, race TTKSK was ...

  3. Pathogenicity islands and virulence evolution in Listeria.

    PubMed

    Vázquez-Boland, J A; Domínguez-Bernal, G; González-Zorn, B; Kreft, J; Goebel, W

    2001-06-01

    As in other bacterial pathogens, the virulence determinants of Listeria species are clustered in genomic islands scattered along the chromosome. This review summarizes current knowledge about the structure, distribution and role in pathogenesis of Listeria virulence loci. Hypotheses about the mode of acquisition and evolution of these loci in this group of Gram-positive bacteria are presented and discussed.

  4. High Sensitivity Proteomics Assisted Discovery of a Novel Operon Involved in the Assembly of Photosystem II, a Membrane Protein Complex

    SciTech Connect

    Wegener, Kimberly M.; Welsh, Eric A.; Thornton, Leeann E.; Keren, Nir S.; Jacobs, Jon M.; Hixson, Kim K.; Monroe, Matthew E.; Camp, David G.; Smith, Richard D.; Pakrasi, Himadri B.

    2008-10-10

    Photosystem II (PSII) is a large membrane protein complex that performs the water oxidation reactions of the photosynthetic electron transport chain in plants, algae, and cyanobacteria. Utilizing a high-throughput proteomic analysis of isolated PSII complexes from the cyanobacterium Synechocystis sp. PCC 6803, we have identified four PSII associated proteins that are encoded by the cofactor integration operon (cio). This operon contains genes with putative binding domains for chlorophyll, iron-sulfur centers, and bilins. Protein levels of this operon are more abundant in several PSII lumenal mutants, suggesting an accumulation of cio products in partially assembled PSII complexes. This provides a rare example of a bacterial operon whose protein products are translationally coordinated and associated with a single protein complex. Genetic deletion of cio results in decreased oxygen evolution by PSII, suggesting that cio products may function as regulators of PSII complex assembly or degradation, maybe facilitating an uncharacterized step in PSII assembly.

  5. The different roles of tryptophan transfer RNA in regulating trp operon expression in E. coli versus B. subtilis.

    PubMed

    Yanofsky, Charles

    2004-08-01

    Escherichia coli and Bacillus subtilis use different mechanisms of sensing and responding to tryptophan and uncharged tRNA(Trp) as regulatory signals. In E. coli, tryptophan activates a repressor that binds to the trp promoter- operator, inhibiting transcription initiation. In B. subtilis, tryptophan activates an RNA-binding protein, TRAP, which binds to the trp operon leader RNA, causing transcription termination. In E. coli uncharged tRNA(Trp) accumulation stalls the ribosome attempting translation of tandem Trp codons in the leader-peptide coding region of the operon. This stalling permits the formation of an RNA antiterminator structure, preventing transcription termination. In B. subtilis uncharged tRNA(Trp) accumulation activates transcription and translation of the at operon. AT protein inhibits tryptophan-activated TRAP, thereby preventing TRAP-mediated transcription termination. These differences might reflect the unique organizational features of the respective trp operons and their ancestry. PMID:15262409

  6. Ni2+-based immobilized metal ion affinity chromatography of lactose operon repressor protein from Escherichia coli.

    PubMed

    Velkov, Tony; Jones, Alun; Lim, Maria L R

    2008-01-01

    A two-step chromatographic sequence is described for the purification of native lactose operon repressor protein from Escherichia coli cells. The first step involves Ni(2+)-based immobilized metal ion affinity chromatography of the soluble cytoplasmic extract. This method provides superior speed, resolution and yield than the established phosphocellulose cation-exchange chromatographic procedure. Anion-exchange chromatography is used for further purification to >95% purity. The identity and purity of the lactose repressor protein were demonstrated using sodium dodecylsulphate polyacrylamide electrophoresis, crystallization, tryptic finger-printing mass spectrometry, and inducer binding assays. The purified lac repressor exhibited inducer sensitivity for operator DNA binding and undergoes a conformational change upon inducer binding. By all these extensive biochemical criteria, the purified protein behaves exactly as that described for the Escherichia coli lactose operon repressor. PMID:18800304

  7. Crystal Structure of the Lactose Operon Repressor and Its Complexes with DNA and Inducer

    NASA Astrophysics Data System (ADS)

    Lewis, Mitchell; Chang, Geoffrey; Horton, Nancy C.; Kercher, Michele A.; Pace, Helen C.; Schumacher, Maria A.; Brennan, Richard G.; Lu, Ponzy

    1996-03-01

    The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor, a product of the lacI gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-β-D-1-thiogalactoside (IPTG) and the lac repressor complexed with a 21-base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in a stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quaternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites on the genomic DNA.

  8. Diverse galactooligosaccharides consumption by bifidobacteria: implications of β-galactosidase--LacS operon.

    PubMed

    Akiyama, Takuya; Kimura, Kazumasa; Hatano, Hiroshi

    2015-01-01

    Galactooligosaccharides (GOS) possess prebiotic properties that specifically increase the number of bifidobacteria in the human intestine, thus giving health benefits to the host. Although the bifidogenic effect of GOS has been demonstrated in numerous studies, the utilization of GOS by specific bifidobacteria remains unclear. The goal of our study was to elucidate GOS consumption by specific bifidobacteria and gain insights into the mechanism. First, we examined GOS consumption by 14 bifidobacterial strains belonging to seven different species by comparing growth rate, carbohydrate consumption, and acid production. We then performed a transcription analysis in the case of one strong GOS consumer, Bifidobacterium adolescentis YIT 4011(T), to predict the operon contributing to GOS use. The study indicated the contribution of an operon consisted of LacS symporter and β-galactosidase to bifidobacterial GOS consumption. PMID:25483279

  9. Characterization of the upstream region of the formate dehydrogenase operon of Methanobacterium formicicum.

    PubMed Central

    Patel, P S; Ferry, J G

    1988-01-01

    The fdhA and fdhB genes of Methanobacterium formicicum, which code for the alpha and beta subunits of formate dehydrogenase, were cotranscribed as part of a large transcript. By using Northern (RNA) gel blot analysis, the transcription start site was located within a 1.6-kilobase BglII-NcoI fragment 4.3 kilobases upstream from the fdhA gene. The precise transcription start site within the fragment was determined with the aid of primer extension analysis and S1 nuclease protection studies. A putative promoter sequence for structural genes of methanogenic archaebacteria is proposed based on a comparison of DNA sequences of the upstream region of methanogen operons for which transcription initiation sites are known. Comparison of the DNA sequence of the upstream region of the fdh operon of M. formicicum with the sequence upstream of the fdhF gene of Escherichia coli revealed regions of considerable identity. Images PMID:2457012

  10. Overexpression, purification and crystallization of the tetrameric form of SorC sorbitol operon regulator

    SciTech Connect

    Sanctis, Daniele de; Rêgo, Ana T.; Marçal, David; McVey, Colin E.; Carrondo, Maria A.; Enguita, Francisco J.

    2008-01-01

    The sorbitol operon regulator from K. pneumoniae has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 3.2 Å. The sorbitol operon regulator (SorC) regulates the metabolism of l-sorbose in Klebsiella pneumonia. SorC was overexpressed in Escherichia coli and purified, and crystals were obtained of a tetrameric form. A single crystal showed X-ray diffraction to 3.20 Å. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 91.6, b = 113.3, c = 184.1 Å. Analysis of the molecular-replacement solution indicates the presence of four SorC molecules in the asymmetric unit.

  11. Requirement of duplicated operons for maximal metabolism of phthalate by Rhodococcus sp. strain DK17.

    PubMed

    Choi, Ki Young; Kim, Dockyu; Chae, Jong-Chan; Zylstra, Gerben J; Kim, Eungbin

    2007-06-01

    The operons encoding the transformation of phthalate to protocatechuate are duplicated and present on two different megaplasmids [pDK2 (330 kb) and pDK3 (750 kb)] in Rhodococcus sp. strain DK17. RT-PCR experiments using gene-specific primers showed that both the pDK2- and the pDK3-encoded dihydroxyphthalate decarboxylase genes are simultaneously expressed during growth on phthalate. The doubling time of the pDK2-cured mutant strain DK176 in minimal liquid medium with 5mM phthalate is 52.5% of that of the wild-type strain DK17. The data indicate that both copies of the phthalate operon are equally functional in DK17, and gene dosage is the main reason for slower growth of DK176 on phthalate. PMID:17449009

  12. Diverse galactooligosaccharides consumption by bifidobacteria: implications of β-galactosidase--LacS operon.

    PubMed

    Akiyama, Takuya; Kimura, Kazumasa; Hatano, Hiroshi

    2015-01-01

    Galactooligosaccharides (GOS) possess prebiotic properties that specifically increase the number of bifidobacteria in the human intestine, thus giving health benefits to the host. Although the bifidogenic effect of GOS has been demonstrated in numerous studies, the utilization of GOS by specific bifidobacteria remains unclear. The goal of our study was to elucidate GOS consumption by specific bifidobacteria and gain insights into the mechanism. First, we examined GOS consumption by 14 bifidobacterial strains belonging to seven different species by comparing growth rate, carbohydrate consumption, and acid production. We then performed a transcription analysis in the case of one strong GOS consumer, Bifidobacterium adolescentis YIT 4011(T), to predict the operon contributing to GOS use. The study indicated the contribution of an operon consisted of LacS symporter and β-galactosidase to bifidobacterial GOS consumption.

  13. Streptococcus pyogenes malate degradation pathway links pH regulation and virulence.

    PubMed

    Paluscio, Elyse; Caparon, Michael G

    2015-03-01

    The ability of Streptococcus pyogenes to infect different niches within its human host most likely relies on its ability to utilize alternative carbon sources. In examining this question, we discovered that all sequenced S. pyogenes strains possess the genes for the malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised of four genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding a malate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression of maeP and maeE is induced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Furthermore, both maePE and maeKR are repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malate utilization requires the PTS transporter EI enzyme (PtsI), as a PtsI(-) mutant fails to express the ME genes and is unable to utilize malate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP(-), MaeK(-), and MaeR(-) mutants were attenuated for virulence, whereas a MaeE(-) mutant showed enhanced virulence compared to that of the wild type. Taken together, these data show that ME contributes to S. pyogenes' carbon source repertory, that malate utilization is a highly regulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptake and utilization contribute to the adaptive pH response, and ME can influence the outcome of infection.

  14. Streptococcus pyogenes Malate Degradation Pathway Links pH Regulation and Virulence

    PubMed Central

    Paluscio, Elyse

    2015-01-01

    The ability of Streptococcus pyogenes to infect different niches within its human host most likely relies on its ability to utilize alternative carbon sources. In examining this question, we discovered that all sequenced S. pyogenes strains possess the genes for the malic enzyme (ME) pathway, which allows malate to be used as a supplemental carbon source for growth. ME is comprised of four genes in two adjacent operons, with the regulatory two-component MaeKR required for expression of genes encoding a malate permease (maeP) and malic enzyme (maeE). Analysis of transcription indicated that expression of maeP and maeE is induced by both malate and low pH, and induction in response to both cues is dependent on the MaeK sensor kinase. Furthermore, both maePE and maeKR are repressed by glucose, which occurs via a CcpA-independent mechanism. Additionally, malate utilization requires the PTS transporter EI enzyme (PtsI), as a PtsI– mutant fails to express the ME genes and is unable to utilize malate. Virulence of selected ME mutants was assessed in a murine model of soft tissue infection. MaeP–, MaeK–, and MaeR– mutants were attenuated for virulence, whereas a MaeE– mutant showed enhanced virulence compared to that of the wild type. Taken together, these data show that ME contributes to S. pyogenes' carbon source repertory, that malate utilization is a highly regulated process, and that a single regulator controls ME expression in response to diverse signals. Furthermore, malate uptake and utilization contribute to the adaptive pH response, and ME can influence the outcome of infection. PMID:25583521

  15. Identification and Characterization of msf, a Novel Virulence Factor in Haemophilus influenzae.

    PubMed

    Kress-Bennett, Jennifer M; Hiller, N Luisa; Eutsey, Rory A; Powell, Evan; Longwell, Mark J; Hillman, Todd; Blackwell, Tenisha; Byers, Barbara; Mell, Joshua C; Post, J Christopher; Hu, Fen Z; Ehrlich, Garth D; Janto, Benjamin A

    2016-01-01

    Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi) emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs). Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf) and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4) (KO) resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites.

  16. Identification and Characterization of msf, a Novel Virulence Factor in Haemophilus influenzae.

    PubMed

    Kress-Bennett, Jennifer M; Hiller, N Luisa; Eutsey, Rory A; Powell, Evan; Longwell, Mark J; Hillman, Todd; Blackwell, Tenisha; Byers, Barbara; Mell, Joshua C; Post, J Christopher; Hu, Fen Z; Ehrlich, Garth D; Janto, Benjamin A

    2016-01-01

    Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi) emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs). Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf) and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4) (KO) resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites. PMID:26977929

  17. Identification and Characterization of msf, a Novel Virulence Factor in Haemophilus influenzae

    PubMed Central

    Kress-Bennett, Jennifer M.; Hiller, N. Luisa; Eutsey, Rory A.; Powell, Evan; Longwell, Mark J.; Hillman, Todd; Blackwell, Tenisha; Byers, Barbara; Mell, Joshua C.; Post, J. Christopher; Hu, Fen Z.; Ehrlich, Garth D.; Janto, Benjamin A.

    2016-01-01

    Haemophilus influenzae is an opportunistic pathogen. The emergence of virulent, non-typeable strains (NTHi) emphasizes the importance of developing new interventional targets. We screened the NTHi supragenome for genes encoding surface-exposed proteins suggestive of immune evasion, identifying a large family containing Sel1-like repeats (SLRs). Clustering identified ten SLR-containing gene subfamilies, each with various numbers of SLRs per gene. Individual strains also had varying numbers of SLR-containing genes from one or more of the subfamilies. Statistical genetic analyses of gene possession among 210 NTHi strains typed as either disease or carriage found a significant association between possession of the SlrVA subfamily (which we have termed, macrophage survival factor, msf) and the disease isolates. The PittII strain contains four chromosomally contiguous msf genes. Deleting all four of these genes (msfA1-4) (KO) resulted in a highly significant decrease in phagocytosis and survival in macrophages; which was fully complemented by a single copy of the msfA1 gene. Using the chinchilla model of otitis media and invasive disease, the KO strain displayed a significant decrease in fitness compared to the WT in co-infections; and in single infections, the KO lost its ability to invade the brain. The singly complemented strain showed only a partial ability to compete with the WT suggesting gene dosage is important in vivo. The transcriptional profiles of the KO and WT in planktonic growth were compared using the NTHi supragenome array, which revealed highly significant changes in the expression of operons involved in virulence and anaerobiosis. These findings demonstrate that the msfA1-4 genes are virulence factors for phagocytosis, persistence, and trafficking to non-mucosal sites. PMID:26977929

  18. Deterministic and stochastic simulation and analysis of biochemical reaction networks the lactose operon example.

    PubMed

    Yildirim, Necmettin; Kazanci, Caner

    2011-01-01

    A brief introduction to mathematical modeling of biochemical regulatory reaction networks is presented. Both deterministic and stochastic modeling techniques are covered with examples from enzyme kinetics, coupled reaction networks with oscillatory dynamics and bistability. The Yildirim-Mackey model for lactose operon is used as an example to discuss and show how deterministic and stochastic methods can be used to investigate various aspects of this bacterial circuit. PMID:21187231

  19. Intracellular induction of the Bartonella henselae virB operon by human endothelial cells.

    PubMed

    Schmiederer, M; Arcenas, R; Widen, R; Valkov, N; Anderson, B

    2001-10-01

    One of the more recently identified bacterial exportation systems is the type IV secretion mechanism, which is characterized by a multiprotein complex that spans the inner and outer bacterial membranes and contains a pilin component. The most thoroughly studied type IV secretion system is encoded by the virB operon of Agrobacterium tumefaciens. In Bartonella henselae, 8 of the 10 virB operon genes share extensive homology and arrangement with the virB operon of A. tumefaciens. Sequencing of the region upstream of the B. henselae virB2 gene revealed a region with sequence homology to the vir box of A. tumefaciens. This possible promoter region was cloned upstream of the green fluorescent protein reporter gene in the promoterless vector pANT3 and used to transform B. henselae. Minimal reporter gene expression was seen in the transformed bacteria cultivated in the absence of host cells, but expression was strongly induced in intracellular bacteria cultivated with human microvascular endothelial cells. Deletion of an 87-bp fragment, which contained the putative vir box from the 5' end of the promoter region, diminished intracellular induction of the reporter gene. Host cell induction of the 17-kDa antigen gene, which replaces virB5 in B. henselae, was also demonstrated at the protein level using specific antiserum. Thus, expression of the virB genes of B. henselae is induced in bacteria, which have invaded host cells, through a mechanism that may be similar to the environment-sensing mechanism found in the virB operon of A. tumefaciens. PMID:11553594

  20. Expression and regulation of the rnc and pdxJ operons of Escherichia coli.

    PubMed

    Matsunaga, J; Dyer, M; Simons, E L; Simons, R W

    1996-12-01

    Escherichia coli rnc-era-recO operon (rnc operon) expression is negatively autoregulated at the level of message stability by ribonuclease III (RNase III), which is encoded by the rnc gene. RNase III, a double-stranded RNA-specific endoribonuclease involved in rRNA and mRNA processing and degradation, cleaves a stemloop structure in the 5' untranslated leader, initiating rapid decay of the rnc operon mRNA. Here, we examine rnc operon expression and regulation in greater detail. Northern, primer extension, and lacZ fusion analyses show that a single promoter (rncP) specifies two principal mRNAs: the 1.9 kb rnc-era transcript and the less-abundant 3.7 kb RNA encoding rnc-era-recO and the downstream pdxJ and acpS genes. A 1.3 kb pdxJ-acpS RNA is transcribed from a promoter (pdxP) located within recO. About 70% of pdxJ transcription depends on transcription from rncP. Both promoters were characterized genetically. RNase III reduces 1.9 kb and 3.7 kb transcript levels and stability, and corresponding effects are seen with genetic fusions. These detailed studies enabled us to show that the first 378 nucleotides of the rnc transcript comprise a portable RNA stability element (rncO) that contains all of the cis-acting elements required for RNase III-initiated decay of the rnc mRNA as well as the heterologous lacZ transcript. Moreover, mutations in rncO that block RNase III cleavage also block control, showing that RNase III initiates mRNA decay by cleaving at a single site. PMID:8971718

  1. Proteomic Characterization of Yersinia pestis Virulence

    SciTech Connect

    Chromy, B; Murphy, G; Gonzales, A; Fitch, J P; McCutchen-Maloney, S L

    2005-01-05

    Yersinia pestis, the etiological agent of plague, functions via the Type III secretion mechanism whereby virulence factors are induced upon interactions with a mammalian host. Here, the Y. pestis proteome was studied by two-dimensional differential gel electrophoresis (2-D DIGE) under physiologically relevant growth conditions mimicking the calcium concentrations and temperatures that the pathogen would encounter in the flea vector and upon interaction with the mammalian host. Over 4100 individual protein spots were detected of which hundreds were differentially expressed in the entire comparative experiment. A total of 43 proteins that were differentially expressed between the vector and host growth conditions were identified by mass spectrometry. Expected differences in expression were observed for several known virulence factors including catalase-peroxidase (KatY), murine toxin (Ymt), plasminogen activator (Pla), and F1 capsule antigen (Caf1), as well as putative virulence factors. Chaperone proteins and signaling molecules hypothesized to be involved in virulence due to their role in Type III secretion were also identified. Other differentially expressed proteins not previously reported to contribute to virulence are candidates for more detailed mechanistic studies, representing potential new virulence determinants. For example, several sugar metabolism proteins were differentially regulated in response to lower calcium and higher temperature, suggesting these proteins, while not directly connected to virulence, either represent a metabolic switch for survival in the host environment or may facilitate production of virulence factors. Results presented here contribute to a more thorough understanding of the virulence mechanism of Y. pestis through proteomic characterization of the pathogen under induced virulence.

  2. Contribution of proton-translocating proteins to the virulence of Salmonella enterica serovars Typhimurium, Gallinarum, and Dublin in chickens and mice.

    PubMed

    Turner, A K; Barber, L Z; Wigley, P; Muhammad, S; Jones, M A; Lovell, M A; Hulme, S; Barrow, P A

    2003-06-01

    We investigated the attenuating effects of a range of respiratory chain mutations in three Salmonella serovars which might be used in the development of live vaccines. We tested mutations in nuoG, cydA, cyoA, atpB, and atpH in three serovars of Salmonella enterica: Typhimurium, Dublin, and Gallinarum. All three serovars were assessed for attenuation in their relevant virulence assays of typhoid-like infections. Serovar Typhimurium was assessed in 1-day-old chickens and the mouse. Serovar Gallinarum 9 was assessed in 3-week-old chickens, and serovar Dublin was assessed in 6-week-old mice. Our data show variation in attenuation for the nuoG, cydA, and cyoA mutations within the different serovar-host combinations. However, mutations in atpB and atpH were highly attenuating for all three serovars in the various virulence assays. Further investigation of the mutations in the atp operon showed that the bacteria were less invasive in vivo, showing reduced in vitro survival within phagocytic cells and reduced acid tolerance. We present data showing that this reduced acid tolerance is due to an inability to adapt to conditions rather than a general sensitivity to reduced pH. The data support the targeting of respiratory components for the production of live vaccines and suggest that mutations in the atp operon provide suitable candidates for broad-spectrum attenuation of a range of Salmonella serovars.

  3. Mycobacterium tuberculosis SigM positively regulates Esx secreted protein and nonribosomal peptide synthetase genes and down regulates virulence-associated surface lipid synthesis.

    PubMed

    Raman, Sahadevan; Puyang, Xiaoling; Cheng, Tan-Yun; Young, David C; Moody, D Branch; Husson, Robert N

    2006-12-01

    The Mycobacterium tuberculosis genome encodes 12 alternative sigma factors, several of which regulate stress responses and are required for virulence in animal models of acute infection. In this work we investigated M. tuberculosis SigM, a member of the extracytoplasmic function subfamily of alternative sigma factors. This sigma factor is expressed at low levels in vitro and does not appear to function in stress response regulation. Instead, SigM positively regulates genes required for the synthesis of surface or secreted molecules. Among these are genes encoding two pairs of Esx secreted proteins, a multisubunit nonribosomal peptide synthetase operon, and genes encoding two members of the proline-proline-glutamate (PPE) family of proteins. Genes up regulated in a sigM mutant strain include a different PPE gene, as well as several genes involved in surface lipid synthesis. Among these are genes involved in synthesis of phthiocerol dimycocerosate (PDIM), a surface lipid critical for virulence during acute infection, and the kasA-kasB operon, which is required for mycolic acid synthesis. Analysis of surface lipids showed that PDIM synthesis is increased in a sigM-disrupted strain and is undetectable in a sigM overexpression strain. These findings demonstrate that SigM positively and negatively regulates cell surface and secreted molecules that are likely to function in host-pathogen interactions. PMID:17028284

  4. Selfish Operons: Horizontal Transfer May Drive the Evolution of Gene Clusters

    PubMed Central

    Lawrence, J. G.; Roth, J. R.

    1996-01-01

    A model is presented whereby the formation of gene clusters in bacteria is mediated by transfer of DNA within and among taxa. Bacterial operons are typically composed of genes whose products contribute to a single function. If this function is subject to weak selection or to long periods with no selection, the contributing genes may accumulate mutations and be lost by genetic drift. From a cell's perspective, once several genes are lost, the function can be restored only if all missing genes were acquired simultaneously by lateral transfer. The probability of transfer of multiple genes increases when genes are physically proximate. From a gene's perspective, horizontal transfer provides a way to escape evolutionary loss by allowing colonization of organisms lacking the encoded functions. Since organisms bearing clustered genes are more likely to act as successful donors, clustered genes would spread among bacterial genomes. The physical proximity of genes may be considered a selfish property of the operon since it affects the probability of successful horizontal transfer but may provide no physiological benefit to the host. This process predicts a mosaic structure of modern genomes in which ancestral chromosomal material is interspersed with novel, horizontally transferred operons providing peripheral metabolic functions. PMID:8844169

  5. Artificial citrate operon confers mineral phosphate solubilization ability to diverse fluorescent pseudomonads.

    PubMed

    Adhikary, Hemanta; Sanghavi, Paulomi B; Macwan, Silviya R; Archana, Gattupalli; Naresh Kumar, G

    2014-01-01

    Citric acid is a strong acid with good cation chelating ability and can be very efficient in solubilizing mineral phosphates. Only a few phosphate solubilizing bacteria and fungi are known to secrete citric acids. In this work, we incorporated artificial citrate operon containing NADH insensitive citrate synthase (gltA1) and citrate transporter (citC) genes into the genome of six-plant growth promoting P. fluorescens strains viz., PfO-1, Pf5, CHAO1, P109, ATCC13525 and Fp315 using MiniTn7 transposon gene delivery system. Comprehensive biochemical characterization of the genomic integrants and their comparison with plasmid transformants of the same operon in M9 minimal medium reveals the highest amount of ∼7.6±0.41 mM citric and 29.95±2.8 mM gluconic acid secretion along with ∼43.2±3.24 mM intracellular citrate without affecting the growth of these P. fluorescens strains. All genomic integrants showed enhanced citric and gluconic acid secretion on Tris-Cl rock phosphate (TRP) buffered medium, which was sufficient to release 200-1000 µM Pi in TRP medium. This study demonstrates that MPS ability could be achieved in natural fluorescent pseudomonads by incorporation of artificial citrate operon not only as plasmid but also by genomic integration. PMID:25259527

  6. The Cytochrome c Maturation Operon Is Involved in Manganese Oxidation in Pseudomonas putida GB-1

    PubMed Central

    de Vrind, J. P. M.; Brouwers, G. J.; Corstjens, P. L. A. M.; den Dulk, J.; de Vrind-de Jong, E. W.

    1998-01-01

    A Pseudomonas putida strain, strain GB-1, oxidizes Mn2+ to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn2+ oxidation and/or secretion of the Mn2+-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn2+ oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn2+-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn2+ oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed. PMID:9758767

  7. Artificial Citrate Operon Confers Mineral Phosphate Solubilization Ability to Diverse Fluorescent Pseudomonads

    PubMed Central

    Adhikary, Hemanta; Sanghavi, Paulomi B.; Macwan, Silviya R.; Archana, Gattupalli; Naresh Kumar, G.

    2014-01-01

    Citric acid is a strong acid with good cation chelating ability and can be very efficient in solubilizing mineral phosphates. Only a few phosphate solubilizing bacteria and fungi are known to secrete citric acids. In this work, we incorporated artificial citrate operon containing NADH insensitive citrate synthase (gltA1) and citrate transporter (citC) genes into the genome of six-plant growth promoting P. fluorescens strains viz., PfO-1, Pf5, CHAO1, P109, ATCC13525 and Fp315 using MiniTn7 transposon gene delivery system. Comprehensive biochemical characterization of the genomic integrants and their comparison with plasmid transformants of the same operon in M9 minimal medium reveals the highest amount of ∼7.6±0.41 mM citric and 29.95±2.8 mM gluconic acid secretion along with ∼43.2±3.24 mM intracellular citrate without affecting the growth of these P. fluorescens strains. All genomic integrants showed enhanced citric and gluconic acid secretion on Tris-Cl rock phosphate (TRP) buffered medium, which was sufficient to release 200–1000 µM Pi in TRP medium. This study demonstrates that MPS ability could be achieved in natural fluorescent pseudomonads by incorporation of artificial citrate operon not only as plasmid but also by genomic integration. PMID:25259527

  8. The Rhizobium etli opt operon is required for symbiosis and stress resistance.

    PubMed

    Vos, K; Braeken, K; Fauvart, M; Ndayizeye, M; Verhaert, J; Zachurzok, S; Lambrichts, I; Michiels, J

    2007-07-01

    Rhizobium etli is a Gram-negative root-colonizing soil bacterium capable of fixing nitrogen while living in symbiosis with its leguminous host Phaseolus vulgaris. A genome-wide screening for R. etli symbiotic mutants revealed a R. etli operon encoding an oligopeptide ABC-transporter (Opt), two redA homologous genes and one redB gene. Expression analysis showed this opt operon to be transcribed both under free-living and symbiotic conditions and expression levels were demonstrated to be growth-phase-dependent. Plants nodulated by R. etli opt mutants showed a reduced symbiotic nitrogen fixation activity (approximately 50% reduction). Growth experiments with opt mutants in the presence of oligopeptides as the sole nitrogen source confirmed the involvement of the opt genes in oligopeptide uptake. Further phenotypic analysis of the opt mutants revealed them to display an enhanced resistance to the oligopeptide antibiotic bacitracin, an increased susceptibility to the beta-lactam antibiotic ampicillin and a decreased osmotolerance. In conclusion, our results demonstrate that the opt operon plays a crucial role during symbiosis and stress resistance. PMID:17564602

  9. Cloning and characterization of groESL operon in Acetobacter aceti.

    PubMed

    Okamoto-Kainuma, Akiko; Yan, Wang; Kadono, Sachiko; Tayama, Kenji; Koizumi, Yukimichi; Yanagida, Fujiharu

    2002-01-01

    The groESL operon of Acetobacter aceti was cloned and sequenced. We observed that GroES and GroEL of A. aceti had high amino acid sequence homologies to GroES and GroEL of Escherichia coli and Bacillus subtilis. The upstream region of the groESL operon contained the heat-shock promoter, which was previously reported in alpha-purple proteobacteria, and the highly conserved inverted repeat sequence. Phylogenetic analysis revealed that the A. aceti GroES and GroEL are very closely related to those of other alpha-purple proteobacteria. Transcription of this operon in A. aceti was induced by heat shock as well as by exposure to ethanol and acetic acid, which are present during fermentation of acetic acid. A. aceti that overexpressed the groESL was more resistant than the control strain to Stressors such as heat, ethanol, or acetic acid, indicating that GroES and GroEL are closely associated with the characteristic nature of Acetobacter and play an important role in acetic acid fermentation.

  10. Regulation of the pts operon in low G+C Gram-positive bacteria.

    PubMed

    Vadeboncoeur, C; Frenette, M; Lortie, L A

    2000-10-01

    The sugar transport system called phosphoenolpyruvate: sugar phosphotransferase (PTS) is widespread among eubacteria. Its is generally composed of two cytoplasmic proteins, HPr and El, which are found in all bacteria possessing a PTS, and a family of Ells whose number, specificity, and molecular structure in terms of domain arrangement vary from species to species. In low G+C Gram-positive bacteria, the genes coding for the general proteins HPr and El, designated ptsH and ptsl respectively, are organized into the pts operon. In this paper, we summarize current knowledge about the regulation of the pts operon in low G+C Gram-positive bacteria. Physiological data indicate that El and most particularly HPr make up a substantial proportion of cellular proteins. Their synthesis is not coordinated and is influenced by environmental factors. The principal DNA cis-elements involved in the regulation of pts operon transcription are a strong promoter whose sequence and structure are very similar to those of the canonical promoter recognized by the Escherichia coli and Bacillus subtilis major RNA polymerases, a 5'-untranslated region, a rho-dependent terminator located at the 5' end of ptsl, and an intrinsic terminator located downstream from ptsl. Analysis of ptsH and ptsl Shine-Dalgarno sequences as well as experimental results obtained with a Streptococcus salivarius mutant suggest that the expression of HPr and El is also controlled at the translation level.

  11. Transcriptional activity of the transposable element Tn10 in the Salmonella typhimurium ilvGEDA operon.

    PubMed Central

    Blazey, D L; Burns, R O

    1982-01-01

    Polarity of Tn10 insertion mutations in the Salmonella typhimurium ilvGEDA operon depends on both the location and the orientation of the Tn10 element. One orientation of Tn10 insertions in ilvG and ilvE permits low-level expression of the downstream ilvEDA and ilvDA genes, respectively. Our analysis of Salmonella ilv recombinant plasmids shows that this residual ilv expression must result from Tn10-directed transcription and does not reflect the presence of internal promoters in the ilvGEDA operon, as was previously suggested. The opposite orientation of Tn10 insertion in ilvE prevents ilvDA expression, indicating that only one end of Tn10 is normally active in transcribing adjacent genes. Both orientations of Tn10 insertion in ilvD exert absolute polarity on ilvA expression. Expression of ilvA is known to be dependent on effective translation of ilvD, perhaps reflecting the lack of a ribosome binding site proximal to the ilvA sequence. Therefore, recognition of the ability of Tn10 to promote transcription of contiguous genes in the ilvGEDA operon apparently requires the presence of associated ribosome binding sites. PMID:6289328

  12. Comparison of Deterministic and Stochastic Models of the lac Operon Genetic Network

    PubMed Central

    Stamatakis, Michail; Mantzaris, Nikos V.

    2009-01-01

    The lac operon has been a paradigm for genetic regulation with positive feedback, and several modeling studies have described its dynamics at various levels of detail. However, it has not yet been analyzed how stochasticity can enrich the system's behavior, creating effects that are not observed in the deterministic case. To address this problem we use a comparative approach. We develop a reaction network for the dynamics of the lac operon genetic switch and derive corresponding deterministic and stochastic models that incorporate biological details. We then analyze the effects of key biomolecular mechanisms, such as promoter strength and binding affinities, on the behavior of the models. No assumptions or approximations are made when building the models other than those utilized in the reaction network. Thus, we are able to carry out a meaningful comparison between the predictions of the two models to demonstrate genuine effects of stochasticity. Such a comparison reveals that in the presence of stochasticity, certain biomolecular mechanisms can profoundly influence the region where the system exhibits bistability, a key characteristic of the lac operon dynamics. For these cases, the temporal asymptotic behavior of the deterministic model remains unchanged, indicating a role of stochasticity in modulating the behavior of the system. PMID:19186128

  13. Pre-dispositions and epigenetic inheritance in the Escherichia coli lactose operon bistable switch.

    PubMed

    Robert, Lydia; Paul, Gregory; Chen, Yong; Taddei, François; Baigl, Damien; Lindner, Ariel B

    2010-04-13

    The lactose operon regulation in Escherichia coli is a primary model of phenotypic switching, reminiscent of cell fate determination in higher organisms. Under conditions of bistability, an isogenic cell population partitions into two subpopulations, with the operon's genes turned on or remaining off. It is generally hypothesized that the final state of a cell depends solely on stochastic fluctuations of the network's protein concentrations, particularly on bursts of lactose permease expression. Nevertheless, the mechanisms underlying the cell switching decision are not fully understood. We designed a microfluidic system to follow the formation of a transiently bimodal population within growing microcolonies. The analysis of genealogy and cell history revealed the existence of pre-disposing factors for switching that are epigenetically inherited. Both the pre-induction expression stochasticity of the lactose operon repressor LacI and the cellular growth rate are predictive factors of the cell's response upon induction, with low LacI concentration and slow growth correlating with higher switching probability. Thus, stochasticity at the local level of the network and global physiology are synergistically involved in cell response determination. PMID:20393577

  14. Cloning and properties of the Salmonella typhimurium tricarboxylate transport operon in Escherichia coli

    SciTech Connect

    Widenhorn, K.A.; Boos, W.; Somers, J.M.; Kay, W.W.

    1988-02-01

    The tricarboxylate transport operon (tctI) was cloned in Escherichia coli as a 12-kilobase (kb) fragment from an EcoRI library of the Salmonella typhimurium chromosome in lambdagtWES. It was further subcloned as a 12-kb fragment into pACYC184 and as an 8-kb fragment into pBR322. By insertional mutagenesis mediated by lambdaTn5, restriction mapping, and phenotypic testing, the tctI operon was localized to a 4.5-kb region. The tctC gene which encodes a periplasmic binding protein (C-protein) was located near the center of the insert. E. coli/tctI clones on either multicopy or single-copy vectors grew on the same tricarboxylates as S. typhimurium, although unusually long growth lags were observed. E. coli/tctI clones exhibited similar (/sup 14/C) fluorocitrate transport kinetics to those of S. typhimurium, whereas E. coli alone was virtually impermeable to (/sup 14/C) fluorocitrate. The periplasmic C proteins (C1 and C2 isoelectric forms) were produced in prodigious quantities from the cloned strains. Motile E. coli/tctI clones were not chemotactic toward citrate, whereas tctI deletion mutants of S. typhimurium were. Taken together, these observations indicate that tctI is not an operon involved in chemotaxis.

  15. The Mannitol Operon Repressor MTIR belongs to a new class of transcription regulators in bacteria.

    SciTech Connect

    Tan, K.; Borovilos, M.; Zhou, M; Horer, S; Clancy, S; Moy, S; Volkart, LL; Sassoon, J; Baumann, U; Joachimiak, A

    2009-12-25

    Many bacteria express phosphoenolpyruvate-dependent phosphotransferase systems (PTS). The mannitol-specific PTS catalyze the uptake and phosphorylation of d-mannitol. The uptake system comprises several genes encoded in the single operon. The expression of the mannitol operon is regulated by a proposed transcriptional factor, mannitol operon repressor (MtlR) that was first studied in Escherichia coli. Here we report the first crystal structures of MtlR from Vibrio parahemeolyticus (Vp-MtlR) and its homolog YggD protein from Shigella flexneri (Sf-YggD). MtlR and YggD belong to the same protein family (Pfam05068). Although Vp-MtlR and Sf-YggD share low sequence identity (22%), their overall structures are very similar, representing a novel all {alpha}-helical fold, and indicate similar function. However, their lack of any known DNA-binding structural motifs and their unfavorable electrostatic properties imply that MtlR/YggD are unlikely to bind a specific DNA operator directly as proposed earlier. This structural observation is further corroborated by in vitro DNA-binding studies of E. coli MtlR (Ec-MtlR), which detected no interaction of Ec-MtlR with the well characterized mannitol operator/promoter region. Therefore, MtlR/YggD belongs to a new class of transcription factors in bacteria that may regulate gene expression indirectly as a part of a larger transcriptional complex.

  16. Distinct noise-controlling roles of multiple negative feedback mechanisms in a prokaryotic operon system.

    PubMed

    Nguyen, L K; Kulasiri, D

    2011-03-01

    Molecular fluctuations are known to affect dynamics of cellular systems in important ways. Studies aimed at understanding how molecular systems of certain regulatory architectures control noise therefore become essential. The interplay between feedback regulation and noise has been previously explored for cellular networks governed by a single negative feedback loop. However, similar issues within networks consisting of more complex regulatory structures remain elusive. The authors investigate how negative feedback loops manage noise within a biochemical cascade concurrently governed by multiple negative feedback loops, using the prokaryotic tryptophan (trp) operon system in Escherechia coli as the model system. To the authors knowledge, this is the first study of noise in the trp operon system. They show that the loops in the trp operon system possess distinct, even opposing, noise-controlling effects despite their seemingly analogous feedback structures. The enzyme inhibition loop, although controlling the last reaction of the cascade, was found to suppress noise not only for the tryptophan output but also for other upstream components. In contrast, the Repression (Rep) loop enhances noise for all systems components. Attenuation (Att) poses intermediate effects by attenuating noise for the upstream components but promoting noise for components downstream of its target. Regarding noise at the output tryptophan, Rep and Att can be categorised as noise-enhancing loops whereas Enzyme Inhibition as a noise-reducing loop. These findings suggest novel implications in how cellular systems with multiple feedback mechanisms control noise. [Includes supplementary material]. PMID:21405203

  17. OpWise: Operons aid the identification of differentially expressedgenes in bacterial microarray experiments

    SciTech Connect

    Price, Morgan N.; Arkin, Adam P.; Alm, Eric J.

    2005-11-23

    Differentially expressed genes are typically identified by analyzing the variation between replicate measurements. These procedures implicitly assume that there are no systematic errors in the data even though several sources of systematic error are known. Results-OpWise estimates the amount of systematic error in bacterial microarray data by assuming that genes in the same operon have matching expression patterns. OpWise then performs a Bayesian analysis of a linear model to estimate significance. In simulations, OpWise corrects for systematic error and is robust to deviations from its assumptions. In several bacterial data sets, significant amounts of systematic error are present, and replicate-based approaches overstate the confidence of the changers dramatically, while OpWise does not. Finally, OpWise can identify additional changers by assigning genes higher confidence if they are consistent with other genes in the same operon. Although microarray data can contain large amounts of systematic error, operons provide an external standard and allow for reasonable estimates of significance. OpWise is available at http://microbesonline.org/OpWise.

  18. Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome

    PubMed Central

    Anda, Mizue; Ohtsubo, Yoshiyuki; Okubo, Takashi; Sugawara, Masayuki; Nagata, Yuji; Tsuda, Masataka; Minamisawa, Kiwamu; Mitsui, Hisayuki

    2015-01-01

    rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the “main” chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general. PMID:26534993

  19. The cytochrome c maturation operon is involved in manganese oxidation in Pseudomonas putida GB-1

    SciTech Connect

    Vrind, J.P.M. de; Brouwers, G.J.; Corstijens, P.L.A.M.; Dulk, J. den; Vrind-de Jong, E.W. de

    1998-10-01

    A Pseudomonas putida strain, strain GB-1, oxidizes Mn{sup 2+} to Mn oxide in the early stationary growth phase. It also secretes a siderophore (identified as pyoverdine) when it is subjected to iron limitation. After transposon (Tn5) mutagenesis several classes of mutants with differences in Mn{sup 2+} oxidation and/or secretion of the Mn{sup 2+}-oxidizing activity were identified. Preliminary analysis of the Tn5 insertion site in one of the nonoxidizing mutants suggested that a multicopper oxidase-related enzyme is involved in Mn{sup 2+} oxidation. The insertion site in another mutant was preliminarily identified as a gene involved in the general protein secretion pathway. Two mutants defective in Mn{sup 2+}-oxidizing activity also secreted porphyrins into the medium and appeared to be derepressed for pyoverdine production. These strains were chosen for detailed analysis. Both mutants were shown to contain Tn5 insertions in the ccmF gene, which is part of the cytochrome c maturation operon. They were cytochrome oxidase negative and did not contain c-type cytochromes. Complementation with part of the ccm operon isolated from the wild type restored the phenotype of the parent strain. These results indicate that a functional ccm operon is required for Mn{sup 2+} oxidation in P. putida GB-1. A possible relationship between porphyrin secretion resulting from the ccm mutation and stimulation of pyoverdine production is discussed.

  20. The rec A operon: a novel stress response gene cluster in Bacteroides fragilis

    PubMed Central

    Nicholson, Samantha A; Smalley, Darren; Smith, C. Jeffrey; Abratt, Valerie R

    2014-01-01

    Bacteroides fragilis, an opportunistic pathogen of humans, is a leading cause of bacteraemias and anaerobic abscesses which are often treated with metronidazole, a drug which damages DNA. This study investigated the responses of the B. fragilis recA three gene operon to the stress experienced during metronidazole treatment and exposure to reactive oxygen species simulating those generated by the host immune system during infection. A transcriptionally regulated response was observed using quantitative RT-PCR after metronidazole and hydrogen peroxide treatment, with all three genes being upregulated under stress conditions. In vivo and in vitro analysis of the functional role of the second gene of the operon was done using heterologous complementation and protein expression (in Escherichia coli), with subsequent biochemical assay. This gene encoded a functional bacterioferritin co-migratory protein (BCP) which was thiol-specific and had antioxidant properties, including protection of the glutamine synthetase III enzyme. This in vitro data supports the hypothesis that the genes of the operon may be involved in protection of the bacteria from the oxidative burst during tissue invasion and may play a significant role in bacterial survival and metronidazole resistance during treatment of B. fragilis infections. PMID:24703997

  1. Comparative metabolic profiling of mce1 operon mutant vs wild-type Mycobacterium tuberculosis strains.

    PubMed

    Queiroz, Adriano; Medina-Cleghorn, Daniel; Marjanovic, Olivera; Nomura, Daniel K; Riley, Lee W

    2015-11-01

    Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. PMID:26319139

  2. Functional Analysis of the Magnetosome Island in Magnetospirillum gryphiswaldense: The mamAB Operon Is Sufficient for Magnetite Biomineralization

    PubMed Central

    Lohße, Anna; Ullrich, Susanne; Katzmann, Emanuel; Borg, Sarah; Wanner, Gerd; Richter, Michael; Voigt, Birgit; Schweder, Thomas; Schüler, Dirk

    2011-01-01

    Bacterial magnetosomes are membrane-enveloped, nanometer-sized crystals of magnetite, which serve for magnetotactic navigation. All genes implicated in the synthesis of these organelles are located in a conserved genomic magnetosome island (MAI). We performed a comprehensive bioinformatic, proteomic and genetic analysis of the MAI in Magnetospirillum gryphiswaldense. By the construction of large deletion mutants we demonstrate that the entire region is dispensable for growth, and the majority of MAI genes have no detectable function in magnetosome formation and could be eliminated without any effect. Only <25% of the region comprising four major operons could be associated with magnetite biomineralization, which correlated with high expression of these genes and their conservation among magnetotactic bacteria. Whereas only deletion of the mamAB operon resulted in the complete loss of magnetic particles, deletion of the conserved mms6, mamGFDC, and mamXY operons led to severe defects in morphology, size and organization of magnetite crystals. However, strains in which these operons were eliminated together retained the ability to synthesize small irregular crystallites, and weakly aligned in magnetic fields. This demonstrates that whereas the mamGFDC, mms6 and mamXY operons have crucial and partially overlapping functions for the formation of functional magnetosomes, the mamAB operon is the only region of the MAI, which is necessary and sufficient for magnetite biomineralization. Our data further reduce the known minimal gene set required for magnetosome formation and will be useful for future genome engineering approaches. PMID:22043287

  3. Cloning and molecular analysis of a mannitol operon of phosphoenolpyruvate-dependent phosphotransferase (PTS) type from Vibrio cholerae O395.

    PubMed

    Kumar, Sanath; Smith, Kenneth P; Floyd, Jody L; Varela, Manuel F

    2011-03-01

    A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395, and its activity was studied in Escherichia coli. The 3.9-kb operon comprising three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EII(Mtl)) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD), and a mannitol operon repressor (MtlR). The transport of [(3)H]mannitol by the cloned mannitol operon in E. coli was 13.8 ± 1.4 nmol/min/mg protein. The insertional inactivation of EII(Mtl) abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram-positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon.

  4. The role of Staphylococcal carotenogenesis in resistance to host defense peptides and in vivo virulence in experimental endocarditis model.

    PubMed

    Xiong, Yan Q; Yang, Soo-Jin; Tong, Steven Y C; Alvarez, Danya N; Mishra, Nagendra N

    2015-10-01

    The defining hallmark of the newly described species, Staphylococcus argenteus, in comparison to its sister species, S. aureus and S. schweitzeri, is the absence of production of the carotenoid pigment, staphyloxanthin. Staphylococcus argenteus lacks the responsible genetic locus crtOPQMN. We examined the impact of carotenoid synthesis in two non-pigmented S. argenteus strains, MSHR1132 and SCC1165. Following complementation with a plasmid containing the carotenoid operon (pTX-crtOPQMN), compared to wild type, both complemented strains showed substantial carotenoid production, with a resultant increase in cell membrane rigidity. Surprisingly, both crtOPQMN-complemented strains exhibited increased susceptibility to the host defense peptides, LL-37 and hNP-1 in vitro, and reduced virulence in an experimental rabbit endocarditis model.

  5. Virulence in malaria: an evolutionary viewpoint.

    PubMed Central

    Mackinnon, Margaret J; Read, Andrew F

    2004-01-01

    Malaria parasites cause much morbidity and mortality to their human hosts. From our evolutionary perspective, this is because virulence is positively associated with parasite transmission rate. Natural selection therefore drives virulence upwards, but only to the point where the cost to transmission caused by host death begins to outweigh the transmission benefits. In this review, we summarize data from the laboratory rodent malaria model, Plasmodium chabaudi, and field data on the human malaria parasite, P. falciparum, in relation to this virulence trade-off hypothesis. The data from both species show strong positive correlations between asexual multiplication, transmission rate, infection length, morbidity and mortality, and therefore support the underlying assumptions of the hypothesis. Moreover, the P. falciparum data show that expected total lifetime transmission of the parasite is maximized in young children in whom the fitness cost of host mortality balances the fitness benefits of higher transmission rates and slower clearance rates, thus exhibiting the hypothesized virulence trade-off. This evolutionary explanation of virulence appears to accord well with the clinical and molecular explanations of pathogenesis that involve cytoadherence, red cell invasion and immune evasion, although direct evidence of the fitness advantages of these mechanisms is scarce. One implication of this evolutionary view of virulence is that parasite populations are expected to evolve new levels of virulence in response to medical interventions such as vaccines and drugs. PMID:15306410

  6. Flavobacterium johnsoniae sprB Is Part of an Operon Spanning the Additional Gliding Motility Genes sprC, sprD, and sprF ▿ †

    PubMed Central

    Rhodes, Ryan G.; Nelson, Shawn S.; Pochiraju, Soumya; McBride, Mark J.

    2011-01-01

    Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as gliding motility. Gld proteins are thought to comprise the gliding motor that propels cell surface adhesins, such as the 669-kDa SprB. A novel protein secretion apparatus called the Por secretion system (PorSS) is required for assembly of SprB on the cell surface. Genetic and molecular analyses revealed that sprB is part of a seven-gene operon spanning 29.3 kbp of DNA. In addition to sprB, three other genes of this operon (sprC, sprD, and sprF) are involved in gliding. Mutations in sprB, sprC, sprD, and sprF resulted in cells that failed to form spreading colonies on agar but that exhibited some motility on glass in wet mounts. SprF exhibits some similarity to Porphyromonas gingivalis PorP, which is required for secretion of gingipain protease virulence factors via the P. gingivalis PorSS. F. johnsoniae sprF mutants produced SprB protein but were defective in localization of SprB to the cell surface, suggesting a role for SprF in secretion of SprB. The F. johnsoniae PorSS is involved in secretion of extracellular chitinase in addition to its role in secretion of SprB. SprF was not needed for chitinase secretion and may be specifically required for SprB secretion by the PorSS. Cells with nonpolar mutations in sprC or sprD produced and secreted SprB and propelled it rapidly along the cell surface. Multiple paralogs of sprB, sprC, sprD, and sprF are present in the genome, which may explain why mutations in sprB, sprC, sprD, and sprF do not result in complete loss of motility and suggests the possibility that semiredundant SprB-like adhesins may allow movement of cells over different surfaces. PMID:21131497

  7. The extinction differential induced virulence macroevolution

    NASA Astrophysics Data System (ADS)

    Zhang, Feng; Xu, Liufang; Wang, Jin

    2014-04-01

    We apply the potential-flux landscape theory to deal with the large fluctuation induced extinction phenomena. We quantify the most probable extinction pathway on the landscape and measure the extinction risk by the landscape topography. In this Letter, we investigate the disease extinction through an epidemic model described by a set of chemical reaction. We found the virulence-differential-dependent symbioses between mother and daughter pathogen species: mutualism and parasitism. The symbioses, whether mutualism or parasitism, benefit the higher virulence species. This implies that speciation towards lower virulence is an effective strategy for a pathogen species to reduce its extinction risk.

  8. Chromosomal islands of Streptococcus pyogenes and related streptococci: molecular switches for survival and virulence.

    PubMed

    Nguyen, Scott V; McShan, William M

    2014-01-01

    Streptococcus pyogenes is a significant pathogen of humans, annually causing over 700,000,000 infections and 500,000 deaths. Virulence in S. pyogenes is closely linked to mobile genetic elements like phages and chromosomal islands (CI). S. pyogenes phage-like chromosomal islands (SpyCI) confer a complex mutator phenotype on their host. SpyCI integrate into the 5' end of DNA mismatch repair (MMR) gene mutL, which also disrupts downstream operon genes lmrP, ruvA, and tag. During early logarithmic growth, SpyCI excise from the bacterial chromosome and replicate as episomes, relieving the mutator phenotype. As growth slows and the cells enter stationary phase, SpyCI reintegrate into the chromosome, again silencing the MMR operon. This system creates a unique growth-dependent and reversible mutator phenotype. Additional CI using the identical attachment site in mutL have been identified in related species, including Streptococcus dysgalactiae subsp. equisimilis, Streptococcus anginosus, Streptococcus intermedius, Streptococcus parauberis, and Streptococcus canis. These CI have small genomes, which range from 13 to 20 kB, conserved integrase and DNA replication genes, and no identifiable genes encoding capsid proteins. SpyCI may employ a helper phage for packaging and dissemination in a fashion similar to the Staphylococcus aureus pathogenicity islands (SaPI). Outside of the core replication and integration genes, SpyCI and related CI show considerable diversity with the presence of many indels that may contribute to the host cell phenotype or fitness. SpyCI are a subset of a larger family of streptococcal CI who potentially regulate the expression of other host genes. The biological and phylogenetic analysis of streptococcal chromosomal islands provides important clues as to how these chromosomal islands help S. pyogenes and other streptococcal species persist in human populations in spite of antibiotic therapy and immune challenges.

  9. Pathogenicity island sequences of pyelonephritogenic Escherichia coli CFT073 are associated with virulent uropathogenic strains.

    PubMed

    Kao, J S; Stucker, D M; Warren, J W; Mobley, H L

    1997-07-01

    Urinary tract infection is the most frequently diagnosed kidney and urologic disease, and Escherichia coli is by far the most common etiologic agent. Defined blocks of DNA termed pathogenicity islands have been found in uropathogenic strains to carry genes not generally found in fecal strains. We have identified one of these regions of DNA within the chromosome of the highly virulent E. coli CFT073, isolated from the blood and urine of a woman with acute pyelonephritis. This strain, which is cytotoxic for cultured renal cells and causes acute pyelonephritis in transurethrally infected CBA mice, contains two distinct copies of the pap operon and is hemolytic. One pap operon was localized on a cosmid clone which was used to identify three overlapping cosmid clones. By using restriction mapping, DNA hybridization, sequencing, and PCR amplification, a region of approximately 50 kb was found to be present in this uropathogenic strain and to have no corresponding sequences in E. coli K-12. This gene block also carries hemolysin genes hlyCABD. The pathogenicity island begins 7 bp downstream of dadX (catabolic alanine racemase; 26.55 min) and ends at a position in the K-12 genome 75 bp downstream of the metV tRNA gene (62.74 min); this suggests that a chromosomal rearrangement has occurred relative to the K-12 linkage map. The junctions of the pathogenicity island were verified by PCR amplification directly from the genomic DNA of strain CFT073. DNA sequencing within the boundaries of the junctions revealed genes not previously identified in E. coli or in some cases bearing no known homologs. When used as probes for DNA hybridization, these sequences were found significantly more often in strains associated with the clinical syndromes of cystitis (82%) and acute pyelonephritis (79%) than in fecal strains (19%; P < 0.001).

  10. Chromosomal islands of Streptococcus pyogenes and related streptococci: molecular switches for survival and virulence.

    PubMed

    Nguyen, Scott V; McShan, William M

    2014-01-01

    Streptococcus pyogenes is a significant pathogen of humans, annually causing over 700,000,000 infections and 500,000 deaths. Virulence in S. pyogenes is closely linked to mobile genetic elements like phages and chromosomal islands (CI). S. pyogenes phage-like chromosomal islands (SpyCI) confer a complex mutator phenotype on their host. SpyCI integrate into the 5' end of DNA mismatch repair (MMR) gene mutL, which also disrupts downstream operon genes lmrP, ruvA, and tag. During early logarithmic growth, SpyCI excise from the bacterial chromosome and replicate as episomes, relieving the mutator phenotype. As growth slows and the cells enter stationary phase, SpyCI reintegrate into the chromosome, again silencing the MMR operon. This system creates a unique growth-dependent and reversible mutator phenotype. Additional CI using the identical attachment site in mutL have been identified in related species, including Streptococcus dysgalactiae subsp. equisimilis, Streptococcus anginosus, Streptococcus intermedius, Streptococcus parauberis, and Streptococcus canis. These CI have small genomes, which range from 13 to 20 kB, conserved integrase and DNA replication genes, and no identifiable genes encoding capsid proteins. SpyCI may employ a helper phage for packaging and dissemination in a fashion similar to the Staphylococcus aureus pathogenicity islands (SaPI). Outside of the core replication and integration genes, SpyCI and related CI show considerable diversity with the presence of many indels that may contribute to the host cell phenotype or fitness. SpyCI are a subset of a larger family of streptococcal CI who potentially regulate the expression of other host genes. The biological and phylogenetic analysis of streptococcal chromosomal islands provides important clues as to how these chromosomal islands help S. pyogenes and other streptococcal species persist in human populations in spite of antibiotic therapy and immune challenges. PMID:25161960

  11. Salmonella virulence plasmid. Modular acquisition of the spv virulence region by an F-plasmid in Salmonella enterica subspecies I and insertion into the chromosome of subspecies II, IIIa, IV and VII isolates.

    PubMed Central

    Boyd, E F; Hartl, D L

    1998-01-01

    The spv operon is common to all Salmonella virulence plasmids. DNA hybridization analysis indicates that the spv region is limited in distribution to serovars of Salmonella enterica subspecies I, II, IIIa, IV, and VII and is absent from Salmonella bongori isolates. Among strains of subspecies II, IIIa, and VII, all isolates examined contained sequences that hybridized with the spv region. However, among isolates of subspecies I, DNA sequences capable of hybridizing with the spv region were found in some isolates of certain serovars. Furthermore, in isolates of subspecies I, the virulence plasmid was found in the same set of isolates as an F-related plasmid, as determined by the presence of the spv region of the virulence plasmid and the finO, traD, and repA sequences of the F-plasmid. The concordance of the virulence plasmid and all three F-plasmid sequences in subspecies I serovar Choleraesuis, Paratyphi, and Typhimurium is most easily explained if the spv region is carried in an F-related plasmid in these isolates. In contrast, among S. enterica subspecies II, IIIa, IV, and VII, the isolates that contain spv sequences did not hybridize with an F-related plasmid or any other identifiable plasmid. With the use of pulse-field gel electrophoresis, the spv region in subspecies II, IIIa, and VII was found to be encoded on the chromosome. Analysis of the phylogenetic distribution of spv among Salmonella isolates and comparative nucleotide sequence analysis of spvA and spvC suggests that the spv region was acquired very recently, after speciation of the salmonellae. PMID:9649513

  12. Complementation analysis of Agrobacterium tumefaciens Ti plasmid virB genes by use of a vir promoter expression vector: virB9, virB10, and virB11 are essential virulence genes.

    PubMed Central

    Ward, J E; Dale, E M; Christie, P J; Nester, E W; Binns, A N

    1990-01-01

    The virB gene products of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid have been proposed to mediate T-DNA transport through the bacterial cell wall into plant cells. Previous genetic analysis of the approximately 9.5-kilobase-pair virB operon has been limited to transposon insertion mutagenesis. Due to the polarity of the transposon insertions, only the last gene in the operon, virB11, is known to provide an essential virulence function. We have now begun to assess the contribution of the other virB genes to virulence. First, several previously isolated Tn3-HoHo1 insertions in the 3' end of the virB operon were precisely mapped by nucleotide sequence analysis. Protein extracts from A. tumefaciens strains harboring these insertions on the Ti plasmid were subjected to immunostaining analysis with VirB4-, VirB10-, and VirB11-specific antisera to determine the effect of the insertion on virB gene expression. In this manner, avirulent mutants containing polar insertions in the virB9 and virB10 genes were identified. To carry out a complementation analysis with these virB mutants, expression vectors were constructed that allow cloned genes to be expressed from the virB promoter in A. tumefaciens. These plasmids were used to express combinations of the virB9, virB10, and virB11 genes in trans in the virB insertion mutants, thereby creating strains lacking only one of these three virB gene products. Virulence assays on Kalanchoe daigremontiana demonstrated that in addition to virB11, the virB9 and virB10 genes are required for tumorigenicity. Images PMID:2203743

  13. Re-sequencing of a virulent strain of Campylobacter jejuni NCTC11168 reveals potential virulence factors.

    PubMed

    Cooper, Kerry K; Cooper, Margarethe A; Zuccolo, Andrea; Joens, Lynn A

    2013-01-01

    In vitro passage of Campylobacter jejuni strains results in phenotypic changes and a general loss of virulence, as is the case with the genome-sequenced strain C. jejuni NCTC11168. Re-sequencing of a virulent strain of NCTC11168 identified 41 SNPs or indels involving 20 genes, four intergenic regions and three pseudogenes. The genes include six motility genes, two chemotaxis genes, three hypothetical genes and a capsule biosynthesis gene, which might have a critical role in C. jejuni virulence. Additionally, we found an insertion in both Cj0676 and Cj1470c, pseudogenes in avirulent NCTC11168, but functional proteins in virulent NCTC11168.

  14. Neoparamoeba perurans loses virulence during clonal culture.

    PubMed

    Bridle, Andrew R; Davenport, Danielle L; Crosbie, Philip B B; Polinski, Mark; Nowak, Barbara F

    2015-08-01

    Amoebic Gill Disease affects farmed salmonids and is caused by Neoparamoeba perurans. Clonal cultures of this amoeba have been used for challenge experiments, however the effect of long-term culture on virulence has not been investigated. Here we show, using in vitro and in vivo methods, that a clone of N. perurans which was virulent 70 days after clonal culture lost virulence after 3 years in clonal culture. We propose that this is related either to the lack of attachment to the gills or the absence of an extracellular product, as shown by the lack of cytopathic effect on Chinook salmon embryo cells. The avirulent clonal culture of N. perurans allowed us to propose two potential virulence mechanisms/factors involved in Amoebic Gill Disease and is an invaluable tool for host-pathogen studies of Amoebic Gill Disease. PMID:26008963

  15. Horizontal gene transfer and diverse functional constrains within a common replication-partitioning system in Alphaproteobacteria: the repABC operon

    PubMed Central

    2009-01-01

    Background The repABC plasmid family, which is extensively present within Alphaproteobacteria, and some secondary chromosomes of the Rhizobiales have the particular feature that all the elements involved in replication and partitioning reside within one transcriptional unit, the repABC operon. Given the functional interactions among the elements of the repABC operon, and the fact that they all reside in the same operon, a common evolutionary history would be expected if the entire operon had been horizontally transferred. Here, we tested whether there is a common evolutionary history within the repABC operon. We further examined different incompatibility groups in terms of their differentiation and degree of adaptation to their host. Results We did not find a single evolutionary history within the repABC operon. Each protein had a particular phylogeny, horizontal gene transfer events of the individual genes within the operon were detected, and different functional constraints were found within and between the Rep proteins. When different repABC operons coexisted in the same genome, they were well differentiated from one another. Finally, we found different levels of adaptation to the host genome within and between repABC operons coexisting in the same species. Conclusion Horizontal gene transfer with conservation of the repABC operon structure provides a highly dynamic operon in which each member of this operon has its own evolutionary dynamics. In addition, it seems that different incompatibility groups present in the same species have different degrees of adaptation to their host genomes, in proportion to the amount of time the incompatibility group has coexisted with the host genome. PMID:19919719

  16. [Proteus bacilli: features and virulence factors].

    PubMed

    Rózalski, Antoni; Kwil, Iwona; Torzewska, Agnieszka; Baranowska, Magdalena; Staczek, Paweł

    2007-01-01

    In this article, different aspects of virulence factors of Proteus bacilii (P. mirabilis, P. vulgaris, P. penneri i P. hauseri) are presented. These are opportunistic pathogens that cause different kinds of infections, most frequently of the urinary tract. These bacteria have developed several virulence factors, such as adherence due to the presence of fimbriae or afimbrial adhesins, invasiveness, swarming phenomenon, hemolytic activity, urea hydrolysis, proteolysis, and endotoxicity. Below we focus on data concerning the molecular basis of the pathogenicity of Proteus bacilli.

  17. Spaceflight Effects on Virulence of Pseudomonas Aeruginosa

    NASA Astrophysics Data System (ADS)

    Broadway, S.; Goins, T.; Crandell, C.; Richards, C.; Patel, M.; Pyle, B.

    2008-06-01

    Pseudomonas aeruginosa is an opportunistic pathogen found in the environment. It is known to infect the immunocompromised. The organism has about 25 virulence genes that play different roles in disease processes. Several exotoxin proteins may be produced, including ExoA, ExoS, ExoT and ExoY, and other virulence factors. In spaceflight, possible increased expression of P. aeruginosa virulence proteins could increase health risks for spaceflight crews who experience decreased immunity. Cultures of P. aeruginosa strains PA01 and PA103 grown on orbit on Shuttle Endeavour flight STS-123 vs. static ground controls were used for analysis. The production of ETA was quantitated using an ELISA procedure. Results showed that while flight cultures of PA103 produced slightly more ETA than corresponding ground controls, the opposite was found for PA01. While it appears that spaceflight has little effect on ETA, stimulation of other virulence factors could cause increased virulence of this organism in space flight. Similar increased virulence in spaceflight has been observed for other bacteria. This is important because astronauts may be more susceptible to opportunistic pathogens including P. aeruginosa.

  18. Pathogenicity and virulence: another view.

    PubMed Central

    Isenberg, H D

    1988-01-01

    The concepts of pathogenicity and virulence have governed our perception of microbial harmfulness since the time of Pasteur and Koch. These concepts resulted in the recognition and identification of numerous etiological agents and provided natural and synthetic agents effective in therapy and prevention of diseases. However, Koch's postulates--the premier product of this view--place the onus of harmfulness solely on the microbial world. Our recent experiences with polymicrobic and nosocomial infections, legionellosis, and acquired immunodeficiency syndrome point to the host as the major determinant of disease. The principles of parasitism, enunciated by Theobold Smith, approximate more accurately the disturbances of the host-parasite equilibrium we designate as infection. Many complex attributes of microbial anatomy and physiology have been obscured by our dependency on the pure-culture technique. For example, bacterial attachment organelles and the production of exopolysaccharides enable microorganisms to interact with mammalian glycocalyces and specific receptors. In addition, selection, through the use of therapeutic agents, aids in the progression of environmental organisms to members of the intimate human biosphere, with the potential to complicate the recovery of patients. These factors emphasize further the pivotal significance of host reactions in infections. Parasitism, in its negative aspects, explains the emergence of "new" infections that involve harm to more than host organs and cells: we may encounter subtler infections that reveal parasitic and host cell nucleic acid interactions in a form of genomic parasitism. PMID:3060244

  19. Determinants of bistability in induction of the Escherichia coli lac operon.

    PubMed

    Dreisigmeyer, D W; Stajic, J; Nemenman, I; Hlavacek, W S; Wall, M E

    2008-09-01

    The authors have developed a mathematical model of regulation of expression of the Escherichia coli lac operon, and have investigated bistability in its steady-state induction behaviour in the absence of external glucose. Numerical analysis of equations describing regulation by artificial inducers revealed two natural bistability parameters that can be used to control the range of inducer concentrations over which the model exhibits bistability. By tuning these bistability parameters, the authors found a family of biophysically reasonable systems that are consistent with an experimentally determined bistable region for induction by thio-methylgalactoside (TMG) (in Ozbudak et al. Nature, 2004, 427; p. 737). To model regulation by lactose, the authors developed similar equations in which allolactose, a metabolic intermediate in lactose metabolism and a natural inducer of lac, is the inducer. For biophysically reasonable parameter values, these equations yield no bistability in response to induction by lactose - only systems with an unphysically small permease-dependent export effect can exhibit small amounts of bistability for limited ranges of parameter values. These results cast doubt on the relevance of bistability in the lac operon within the natural context of E. coli, and help shed light on the controversy among existing theoretical studies that address this issue. The results also motivate a deeper experimental characterisation of permease-independent transport of lac inducers, and suggest an experimental approach to address the relevance of bistability in the lac operon within the natural context of E. coli. The sensitivity of lac bistability to the type of inducer emphasises the importance of metabolism in determining the functions of genetic regulatory networks. PMID:19045824

  20. The effect of stochasticity on the lac operon: an evolutionary perspective.

    PubMed

    van Hoek, Milan; Hogeweg, Paulien

    2007-06-01

    The role of stochasticity on gene expression is widely discussed. Both potential advantages and disadvantages have been revealed. In some systems, noise in gene expression has been quantified, in among others the lac operon of Escherichia coli. Whether stochastic gene expression in this system is detrimental or beneficial for the cells is, however, still unclear. We are interested in the effects of stochasticity from an evolutionary point of view. We study this question in the lac operon, taking a computational approach: using a detailed, quantitative, spatial model, we evolve through a mutation-selection process the shape of the promoter function and therewith the effective amount of stochasticity. We find that noise values for lactose, the natural inducer, are much lower than for artificial, nonmetabolizable inducers, because these artificial inducers experience a stronger positive feedback. In the evolved promoter functions, noise due to stochasticity in gene expression, when induced by lactose, only plays a very minor role in short-term physiological adaptation, because other sources of population heterogeneity dominate. Finally, promoter functions evolved in the stochastic model evolve to higher repressed transcription rates than those evolved in a deterministic version of the model. This causes these promoter functions to experience less stochasticity in gene expression. We show that a high repression rate and hence high stochasticity increases the delay in lactose uptake in a variable environment. We conclude that the lac operon evolved such that the impact of stochastic gene expression is minor in its natural environment, but happens to respond with much stronger stochasticity when confronted with artificial inducers. In this particular system, we have shown that stochasticity is detrimental. Moreover, we demonstrate that in silico evolution in a quantitative model, by mutating the parameters of interest, is a promising way to unravel the functional

  1. A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon.

    PubMed

    Muhr, Enrico; Leicht, Oliver; González Sierra, Silvia; Thanbichler, Martin; Heider, Johann

    2015-01-01

    The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting. PMID:26858693

  2. A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon.

    PubMed

    Muhr, Enrico; Leicht, Oliver; González Sierra, Silvia; Thanbichler, Martin; Heider, Johann

    2015-01-01

    The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting.

  3. A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon

    PubMed Central

    Muhr, Enrico; Leicht, Oliver; González Sierra, Silvia; Thanbichler, Martin; Heider, Johann

    2016-01-01

    The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting. PMID:26858693

  4. Role of Tellurite Resistance Operon in Filamentous Growth of Yersinia pestis in Macrophages

    PubMed Central

    Ponnusamy, Duraisamy; Clinkenbeard, Kenneth D.

    2015-01-01

    Background Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections. Principal Findings In vitro treatment of Y. pestis culture with sodium tellurite (Na2TeO3) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype. Conclusions These findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a

  5. Regulation of the Pseudomonas sp. Strain ADP Cyanuric Acid Degradation Operon

    PubMed Central

    García-González, Vicente; Govantes, Fernando; Porrúa, Odil; Santero, Eduardo

    2005-01-01

    Pseudomonas sp. strain ADP is the model strain for studying bacterial degradation of the s-triazine herbicide atrazine. In this work, we focused on the expression of the atzDEF operon, involved in mineralization of the central intermediate of the pathway, cyanuric acid. Expression analysis of atzD-lacZ fusions in Pseudomonas sp. strain ADP and Pseudomonas putida showed that atzDEF is subjected to dual regulation in response to nitrogen limitation and cyanuric acid. The gene adjacent to atzD, orf99 (renamed here atzR), encoding a LysR-like regulator, was found to be required for both responses. Expression of atzR-lacZ was induced by nitrogen limitation and repressed by AtzR. Nitrogen regulation of atzD-lacZ and atzR-lacZ expression was dependent on the alternative σ factor σN and NtrC, suggesting that the cyanuric acid degradation operon may be subject to general nitrogen control. However, while atzR is transcribed from a σN-dependent promoter, atzDEF transcription appears to be driven from a σ70-type promoter. Expression of atzR from a heterologous promoter revealed that although NtrC regulation of atzD-lacZ requires the AtzR protein, it is not the indirect result of NtrC-activated AtzR synthesis. We propose that expression of the cyanuric acid degradation operon atzDEF is controlled by means of a complex regulatory circuit in which AtzR is the main activator. AtzR activity is in turn modulated by the presence of cyanuric acid and by a nitrogen limitation signal transduced by the Ntr system. PMID:15601699

  6. Opine-based Agrobacterium competitiveness: dual expression control of the agrocinopine catabolism (acc) operon by agrocinopines and phosphate levels.

    PubMed

    Kim, H Stanley; Yi, Hyojeong; Myung, Jaehee; Piper, Kevin R; Farrand, Stephen K

    2008-05-01

    Agrobacterium tumefaciens strain C58 can transform plant cells to produce and secrete the sugar-phosphate conjugate opines agrocinopines A and B. The bacterium then moves in response to the opines and utilizes them as exclusive sources of carbon, energy, and phosphate via the functions encoded by the acc operon. These privileged opine-involved activities contribute to the formation of agrobacterial niches in the environment. We found that the expression of the acc operon is induced by agrocinopines and also by limitation of phosphate. The main promoter is present in front of the first gene, accR, which codes for a repressor. This operon structure enables efficient repression when opine levels are low. The promoter contains two putative operators, one overlapping the -10 sequence and the other in the further upstream from it; two partly overlapped putative pho boxes between the two operators; and two consecutive transcription start sites. DNA fragments containing either of the operators bound purified repressor AccR in the absence of agrocinopines but not in the presence of the opines, demonstrating the on-off switch of the promoter. Induction of the acc operon can occur under low-phosphate conditions in the absence of agrocinopines and further increases when the opines also are present. Such opine-phosphate dual regulatory system of the operon may ensure maximum utilization of agrocinopines when available and thereby increase the chances of agrobacterial survival in the highly competitive environment with limited general food sources. PMID:18344359

  7. Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds

    PubMed Central

    2011-01-01

    Background The P27-P55 (lprG-Rv1410c) operon is crucial for the survival of Mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. P55 encodes an efflux pump that has been shown to provide Mycobacterium smegmatis and Mycobacterium bovis BCG with resistance to several drugs, while P27 encodes a mannosylated glycoprotein previously described as an antigen that modulates the immune response against mycobacteria. The objective of this study was to determine the individual contribution of the proteins encoded in the P27-P55 operon to the resistance to toxic compounds and to the cell wall integrity of M. tuberculosis. Method In order to test the susceptibility of a mutant of M. tuberculosis H37Rv in the P27-P55 operon to malachite green, sodium dodecyl sulfate, ethidium bromide, and first-line antituberculosis drugs, this strain together with the wild type strain and a set of complemented strains were cultivated in the presence and in the absence of these drugs. In addition, the malachite green decolorization rate of each strain was obtained from decolorization curves of malachite green in PBS containing bacterial suspensions. Results The mutant strain decolorized malachite green faster than the wild type strain and was hypersensitive to both malachite green and ethidium bromide, and more susceptible to the first-line antituberculosis drugs: isoniazid and ethambutol. The pump inhibitor reserpine reversed M. tuberculosis resistance to ethidium bromide. These results suggest that P27-P55 functions through an efflux-pump like mechanism. In addition, deletion of the P27-P55 operon made M. tuberculosis susceptible to sodium dodecyl sulfate, suggesting that the lack of both proteins causes alterations in the cell wall permeability of the bacterium. Importantly, both P27 and P55 are required to restore the wild type phenotypes in the mutant. Conclusions The results clearly indicate that P27 and P55 are functionally connected in

  8. The Mercury Resistance Operon: From an Origin in a Geothermal Environment to an Efficient Detoxification Machine

    PubMed Central

    Boyd, Eric S.; Barkay, Tamar

    2012-01-01

    Mercuric mercury (Hg[II]) is a highly toxic and mobile element that is likely to have had a pronounced and adverse effect on biology since Earth’s oxygenation ∼2.4 billion years ago due to its high affinity for protein sulfhydryl groups, which upon binding destabilize protein structure and decrease enzyme activity, resulting in a decreased organismal fitness. The central enzyme in the microbial mercury detoxification system is the mercuric reductase (MerA) protein, which catalyzes the reduction of Hg(II) to volatile Hg(0). In addition to MerA, mer operons encode for proteins involved in regulation, Hg binding, and organomercury degradation. Mer-mediated approaches have had broad applications in the bioremediation of mercury-contaminated environments and industrial waste streams. Here, we examine the composition of 272 individual mer operons and quantitatively map the distribution of mer-encoded functions on both taxonomic SSU rRNA gene and MerA phylogenies. The results indicate an origin and early evolution of MerA among thermophilic bacteria and an overall increase in the complexity of mer operons through evolutionary time, suggesting continual gene recruitment and evolution leading to an improved efficiency and functional potential of the Mer detoxification system. Consistent with a positive relationship between the evolutionary history and topology of MerA and SSU rRNA gene phylogenies (Mantel R = 0.81, p < 0.01), the distribution of the majority of mer functions, when mapped on these phylograms, indicates an overall tendency to inherit mer-encoded functions through vertical descent. However, individual mer functions display evidence of a variable degree of vertical inheritance, with several genes exhibiting strong evidence for acquisition via lateral gene transfer and/or gene loss. Collectively, these data suggest that (i) mer has evolved from a simple system in geothermal environments to a widely distributed and more complex and efficient

  9. Arsenic resistance operon structure in Leptospirillum ferriphilum and proteomic response to arsenic stress.

    PubMed

    Li, Bing; Lin, Jianqun; Mi, Shuang; Lin, Jianqiang

    2010-12-01

    The response of Leptospirillum ferriphilum ML-04 to arsenic stress was analyzed using two-dimensional electrophoresis (2-DE), matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). Thirty-eight of 65 significantly differentially expressed arsenic response proteins were identified, and 25 of them have known functions. Three proteins are arsenic resistance system (ARS) member proteins. Two ars operons appear to be present in this strain. In addition to the ARS system, phosphate regulation and glutathione (GSH) synthesis appear involved in As[V] and As[III] tolerance, respectively. These findings provide information potentially useful for the genetic engineering of arsenic resistant organisms. PMID:20696570

  10. Aromatic acid metabolites of Escherichia coli K-12 can induce the marRAB operon.

    PubMed

    Chubiz, Lon M; Rao, Christopher V

    2010-09-01

    MarR is a key regulator of the marRAB operon involved in antibiotic resistance and solvent stress tolerance in Escherichia coli. We show that two metabolic intermediates, 2,3-dihydroxybenzoate and anthranilate, involved in enterobactin and tryptophan biosynthesis, respectively, can activate marRAB transcription. We also found that a third intermediate involved in ubiquinone biosynthesis, 4-hydroxybenzoate, activates marRAB transcription in the absence of TolC. Of the three, however, only 2,3-dihydroxybenzoate directly binds MarR and affects its activity. PMID:20639340

  11. Promoter analysis of the Xanthomonas campestris pv. campestris gum operon directing biosynthesis of the xanthan polysaccharide.

    PubMed Central

    Katzen, F; Becker, A; Zorreguieta, A; Pühler, A; Ielpi, L

    1996-01-01

    The Xanthomonas campestris gum gene cluster is composed of 12 genes designated gumB, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, and -M. The transcriptional organization of this gene cluster was analyzed by the construction of gum-lacZ transcriptional fusions in association with plasmid integration mutagenesis. This analysis, coupled with primer extension assays, indicated that the gum region was mainly expressed as an operon from a promoter located upstream of the first gene, gumB. PMID:8763965

  12. Identification of Secreted Exoproteome Fingerprints of Highly-Virulent and Non-Virulent Staphylococcus aureus Strains

    PubMed Central

    Bonar, Emilia; Wojcik, Iwona; Jankowska, Urszula; Kedracka-Krok, Sylwia; Bukowski, Michal; Polakowska, Klaudia; Lis, Marcin W.; Kosecka-Strojek, Maja; Sabat, Artur J.; Dubin, Grzegorz; Friedrich, Alexander W.; Miedzobrodzki, Jacek; Dubin, Adam; Wladyka, Benedykt

    2016-01-01

    Staphylococcus aureus is a commensal inhabitant of skin and mucous membranes in nose vestibule but also an important opportunistic pathogen of humans and livestock. The extracellular proteome as a whole constitutes its major virulence determinant; however, the involvement of particular proteins is still relatively poorly understood. In this study, we compared the extracellular proteomes of poultry-derived S. aureus strains exhibiting a virulent (VIR) and non-virulent (NVIR) phenotype in a chicken embryo experimental infection model with the aim to identify proteomic signatures associated with the particular phenotypes. Despite significant heterogeneity within the analyzed proteomes, we identified alpha-haemolysin and bifunctional autolysin as indicators of virulence, whereas glutamylendopeptidase production was characteristic for non-virulent strains. Staphopain C (StpC) was identified in both the VIR and NVIR proteomes and the latter fact contradicted previous findings suggesting its involvement in virulence. By supplementing NVIR, StpC-negative strains with StpC, and comparing the virulence of parental and supplemented strains, we demonstrated that staphopain C alone does not affect staphylococcal virulence in a chicken embryo model. PMID:27242969

  13. A conserved UDP-glucose dehydrogenase encoded outside the hasABC operon contributes to capsule biogenesis in group A Streptococcus.

    PubMed

    Cole, Jason N; Aziz, Ramy K; Kuipers, Kirsten; Timmer, Anjuli M; Nizet, Victor; van Sorge, Nina M

    2012-11-01

    Group A Streptococcus (GAS) is a human-specific bacterial pathogen responsible for serious morbidity and mortality worldwide. The hyaluronic acid (HA) capsule of GAS is a major virulence factor, contributing to bloodstream survival through resistance to neutrophil and antimicrobial peptide killing and to in vivo pathogenicity. Capsule biosynthesis has been exclusively attributed to the ubiquitous hasABC hyaluronan synthase operon, which is highly conserved across GAS serotypes. Previous reports indicate that hasA, encoding hyaluronan synthase, and hasB, encoding UDP-glucose 6-dehydrogenase, are essential for capsule production in GAS. Here, we report that precise allelic exchange mutagenesis of hasB in GAS strain 5448, a representative of the globally disseminated M1T1 serotype, did not abolish HA capsule synthesis. In silico whole-genome screening identified a putative HasB paralog, designated HasB2, with 45% amino acid identity to HasB at a distant location in the GAS chromosome. In vitro enzymatic assays demonstrated that recombinant HasB2 is a functional UDP-glucose 6-dehydrogenase enzyme. Mutagenesis of hasB2 alone slightly decreased capsule abundance; however, a ΔhasB ΔhasB2 double mutant became completely acapsular. We conclude that HasB is not essential for M1T1 GAS capsule biogenesis due to the presence of a newly identified HasB paralog, HasB2, which most likely resulted from gene duplication. The identification of redundant UDP-glucose 6-dehydrogenases underscores the importance of HA capsule expression for M1T1 GAS pathogenicity and survival in the human host.

  14. SPI-9 of Salmonella enterica serovar Typhi is constituted by an operon positively regulated by RpoS and contributes to adherence to epithelial cells in culture.

    PubMed

    Velásquez, Juan C; Hidalgo, Alejandro A; Villagra, Nicolás; Santiviago, Carlos A; Mora, Guido C; Fuentes, Juan A

    2016-08-01

    The genomic island 9 (SPI-9) from Salmonella enterica serovar Typhi (S. Typhi) carries three ORFs (STY2876, STY2877, STY2878) presenting 98 % identity with a type 1 secretory apparatus (T1SS), and a single ORF (STY2875) similar to a large RTX-like protein exhibiting repeated Ig domains. BapA, the Salmonella enterica serovar Enteritidis orthologous to S. Typhi STY2875, has been associated with biofilm formation, and is described as a virulence factor in mice. Preliminary in silico analyses revealed that S. Typhi STY2875 ORF has a 600 bp deletion compared with S. Enteritidis bapA, suggesting that S. Typhi STY2875 might be non-functional. At present, SPI-9 has not been studied in S. Typhi. We found that the genes constituting SPI-9 are arranged in an operon whose promoter was up-regulated in high osmolarity and low pH in a RpoS-dependent manner. All the proteins encoded by S. Typhi SPI-9 were located at the membrane fraction, consistent with their putative role as T1SS. Furthermore, SPI-9 contributed to adherence of S. Typhi to epithelial cells when bacteria were grown under high osmolarity or low pH. Under the test conditions, S. Typhi SPI-9 did not participate in biofilm formation. SPI-9 is functional in S. Typhi and encodes an adhesin induced under conditions normally found in the intestine, such as high osmolarity. Hence, this is an example of a locus that might be designated a pseudogene by computational approaches but not by direct biological assays.

  15. The fruRBA Operon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood.

    PubMed

    Valdes, Kayla M; Sundar, Ganesh S; Vega, Luis A; Belew, Ashton T; Islam, Emrul; Binet, Rachel; El-Sayed, Najib M; Le Breton, Yoann; McIver, Kevin S

    2016-04-01

    Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogen Streptococcus pyogenes(the group A Streptococcus[GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where the frulocus (fruRBA) was the most induced. Reverse transcriptase PCR showed that fruRBA formed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entire fruoperon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, and N-acetyl-D-galactosamine. The inactivation of sloR, a fruA homolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ΔfruR and ΔfruB mutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ΔfruA mutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment.

  16. The fruRBA Operon Is Necessary for Group A Streptococcal Growth in Fructose and for Resistance to Neutrophil Killing during Growth in Whole Human Blood.

    PubMed

    Valdes, Kayla M; Sundar, Ganesh S; Vega, Luis A; Belew, Ashton T; Islam, Emrul; Binet, Rachel; El-Sayed, Najib M; Le Breton, Yoann; McIver, Kevin S

    2016-04-01

    Bacterial pathogens rely on the availability of nutrients for survival in the host environment. The phosphoenolpyruvate-phosphotransferase system (PTS) is a global regulatory network connecting sugar uptake with signal transduction. Since the fructose PTS has been shown to impact virulence in several streptococci, including the human pathogen Streptococcus pyogenes(the group A Streptococcus[GAS]), we characterized its role in carbon metabolism and pathogenesis in the M1T1 strain 5448. Growth in fructose as a sole carbon source resulted in 103 genes affected transcriptionally, where the frulocus (fruRBA) was the most induced. Reverse transcriptase PCR showed that fruRBA formed an operon which was repressed by FruR in the absence of fructose, in addition to being under carbon catabolic repression. Growth assays and carbon utilization profiles revealed that although the entire fruoperon was required for growth in fructose, FruA was the main transporter for fructose and also was involved in the utilization of three additional PTS sugars: cellobiose, mannitol, and N-acetyl-D-galactosamine. The inactivation of sloR, a fruA homolog that also was upregulated in the presence of fructose, failed to reveal a role as a secondary fructose transporter. Whereas the ability of both ΔfruR and ΔfruB mutants to survive in the presence of whole human blood or neutrophils was impaired, the phenotype was not reproduced in murine whole blood, and those mutants were not attenuated in a mouse intraperitoneal infection. Since the ΔfruA mutant exhibited no phenotype in the human or mouse assays, we propose that FruR and FruB are important for GAS survival in a human-specific environment. PMID:26787724

  17. Organizational requirements of the SaeR binding sites for a functional P1 promoter of the sae operon in Staphylococcus aureus.

    PubMed

    Cho, Hoonsik; Jeong, Do-Won; Li, Chunling; Bae, Taeok

    2012-06-01

    In Staphylococcus aureus, the SaeRS two-component system controls the expression of multiple virulence factors. Of the two promoters in the sae operon, P1 is autoinduced and has two binding sites for the response regulator SaeR. In this study, we examined the organizational requirements of the SaeR binding sites in P1 for transcription activation. Mutational studies showed that both binding sites are essential for binding to phosphorylated SaeR (P-SaeR) and transcription activation. When the 21-bp distance between the centers of the two SaeR binding sites was altered to 26 bp, 31 bp, 36 bp, or 41 bp, only the 31-bp mutant retained approximately 40% of the original promoter activity. When the -1-bp spacing (i.e.,1-bp overlap) between the primary SaeR binding site and the -35 promoter region was altered, all mutant P1 promoters failed to initiate transcription; however, when the first nucleotide of the -35 region was changed from A to T, the mutants with 0-bp or 22-bp spacing showed detectable promoter activity. Although P-SaeR was essential for the binding of RNA polymerase to P1, it was not essential for the binding of the enzyme to the alpha-hemolysin promoter. When the nonoptimal spacing between promoter elements in P1 or the coagulase promoter was altered to the optimal spacing of 17 bp, both promoters failed to initiate transcription. These results suggest that SaeR binding sites are under rather strict organizational restrictions and provide clues for understanding the molecular mechanism of sae-mediated transcription activation.

  18. Structure of wild-type and mutant repressors and of the control region of the rbt operon of Klebsiella aerogenes.

    PubMed

    Wu, J; Anderton-Loviny, T; Smith, C A; Hartley, B S

    1985-05-01

    Pentitol metabolism in Klebsiella aerogenes is encoded by continuous ribitol (rbt) and D-arabitol (dal) operons transcribed in bipolar fashion and sandwiched between long stretches of homologous DNA. The operons are separated by a central control region (2.2 kb) which encodes both the repressors and all the control sequences. The rbt repressor (270 amino acids) shows homology to the Escherichia coli lac repressor and other DNA-binding proteins. It is transcribed from the strand opposite the rbt operon and the intervening control region (254-bp) contains features which reflect the complex regulation. A rbt-constitutive mutant strain used in previous studies of experimental enzyme evolution encodes a truncated rbt-peptide of 133 residues due to a frameshift mutation. PMID:3891331

  19. Cation-Induced Transcriptional Regulation of the dlt Operon of Staphylococcus aureus

    PubMed Central

    Koprivnjak, Tomaz; Mlakar, Vid; Swanson, Lindsey; Fournier, Benedicte; Peschel, Andreas; Weiss, Jerrold P.

    2006-01-01

    Lipoteichoic and wall teichoic acids (TA) are highly anionic cell envelope-associated polymers containing repeating polyglycerol/ribitol phosphate moieties. Substitution of TA with d-alanine is important for modulation of many cell envelope-dependent processes, such as activity of autolytic enzymes, binding of divalent cations, and susceptibility to innate host defenses. d-Alanylation of TA is diminished when bacteria are grown in medium containing increased NaCl concentrations, but the effects of increased salt concentration on expression of the dlt operon encoding proteins mediating d-alanylation of TA are unknown. We demonstrate that Staphylococcus aureus transcriptionally represses dlt expression in response to high concentrations of Na+ and moderate concentrations of Mg2+ and Ca2+ but not sucrose. Changes in dlt mRNA are induced within 15 min and sustained for several generations of growth. Mg2+-induced dlt repression depends on the ArlSR two-component system. Northern blotting, reverse transcription-PCR, and SMART-RACE analyses suggest that the dlt transcript begins 250 bp upstream of the dltA start codon and includes an open reading frame immediately upstream of dltA. Chloramphenicol transacetylase transcriptional fusions indicate that a region encompassing the 171 to 325 bp upstream of dltA is required for expression and Mg2+-induced repression of the dlt operon in S. aureus. PMID:16672616

  20. Dynamics and bistability in a reduced model of the lac operon.

    PubMed

    Yildirim, Necmettin; Santillan, Moises; Horike, Daisuke; Mackey, Michael C

    2004-06-01

    It is known that the lac operon regulatory pathway is capable of showing bistable behavior. This is an important complex feature, arising from the nonlinearity of the involved mechanisms, which is essential to understand the dynamic behavior of this molecular regulatory system. To find which of the mechanisms involved in the regulation of the lac operon is the origin of bistability, we take a previously published model which accounts for the dynamics of mRNA, lactose, allolactose, permease and beta-galactosidase involvement and simplify it by ignoring permease dynamics (assuming a constant permease concentration). To test the behavior of the reduced model, three existing sets of data on beta-galactosidase levels as a function of time are simulated and we obtain a reasonable agreement between the data and the model predictions. The steady states of the reduced model were numerically and analytically analyzed and it was shown that it may indeed display bistability, depending on the extracellular lactose concentration and growth rate. PMID:15189056

  1. Corepressor system for catabolite repression of the lac operon in Escherichia coli.

    PubMed

    Dobrogosz, W J

    1969-03-01

    Acetylated amino sugars, normally used in the biosynthesis of cell walls and cell membranes, were found to play a role as corepressors for catabolite repression of the lac operon in Escherichia coli. This conclusion was derived from studies conducted on mutants of E. coli that were able to assimilate an exogenous source of N-acetylglucosamine (AcGN) but were unable to dissimilate or grow on this compound. At concentrations less than 10(-4)m, AcGN caused severe catabolite repression of beta-galactosidase synthesis in cultures grown under either nonrepressed or partially repressed conditions. This repression occurred in the absence of any effect of AcGN on either the carbon and energy metabolism or the growth of the organism. In addition, this repression by AcGN occurred in a mutant strain that is constitutive for beta-galactosidase production, demonstrating that the AcGN effect does not involve the uptake of inducer. This model for the corepressor system of catabolite repression is discussed in relation to the existing theories on repression of the lac operon. PMID:4887497

  2. Paradoxical Effect of Weak Inducers on the lac Operon of Escherichia coli

    PubMed Central

    Williams, Beverly; Paigen, Kenneth

    1968-01-01

    Previously, we reported the existence of a group of compounds whose function in the regulation of the lac operon was “paradoxical” in that they acted as either inducers or repressors depending on the circumstances. We now show that this group of compounds does not repress the lac operon by catabolite repression, transient repression, or by preventing the uptake of inducers. A model is presented which shows that “paradoxical” behavior is to be expected if a weak inducer is present at a concentration that is high relative to its binding affinity for the regulatory macromolecule. This model depends on the assumptions that the regulatory macromolecule is an allosteric protein which undergoes a transition between two conformational states and that the rate of enzyme synthesis depends on the fraction of protein molecules in each state. The previous observations on the responses of lac regulatory mutants to weak inducers have been extended to a series of such mutants. Weak inducers repress β-galactosidase synthesis in several i− mutants. When this happens, enzyme synthesis can be reinduced by using a strong inducer such as isopropyl-β-d-thiogalactoside. These compounds induce operator constitutives and the it mutant more easily than they induce a wild-type strain. PMID:4882023

  3. Structure of Intergenic Spacer IGS1 of Ribosomal Operon from Schistidium Mosses.

    PubMed

    Milyutina, I A; Ignatova, E A; Ignatov, M S; Goryunov, D V; Troitsky, A V

    2015-11-01

    The structure of the intergenic spacer 1 (IGS1) of the ribosomal operon from 12 species of Schistidium mosses was studied. In the IGS1 sequences of these species, three conserved regions and two areas of GC- and A-enriched repeats were identified. All of the studied mosses have a conserved pyrimidine-enriched motif at the 5'-end of IGS1. Species-specific nucleotide substitutions and insertions were found in the conserved areas. The repeated units contain single nucleotide substitutions that make unique the majority of repeated units. The positions of such repeats in IGS1 are species-specific, but their number can vary within the species and among operons of the same specimen. The comparison of IGS1 sequences from the Schistidium species and from representatives of ten other moss genera revealed the presence of common conserved motifs with similar localization. Presumably, these motifs are elements of termination of the pre-rRNA transcription and processing of rRNA. PMID:26615440

  4. Toward Bioremediation of Methylmercury Using Silica Encapsulated Escherichia coli Harboring the mer Operon

    PubMed Central

    Kane, Aunica L.; Al-Shayeb, Basem; Holec, Patrick V.; Rajan, Srijay; Le Mieux, Nicholas E.; Heinsch, Stephen C.; Psarska, Sona; Aukema, Kelly G.; Sarkar, Casim A.; Nater, Edward A.; Gralnick, Jeffrey A.

    2016-01-01

    Mercury is a highly toxic heavy metal and the ability of the neurotoxin methylmercury to biomagnify in the food chain is a serious concern for both public and environmental health globally. Because thousands of tons of mercury are released into the environment each year, remediation strategies are urgently needed and prompted this study. To facilitate remediation of both organic and inorganic forms of mercury, Escherichia coli was engineered to harbor a subset of genes (merRTPAB) from the mercury resistance operon. Protein products of the mer operon enable transport of mercury into the cell, cleavage of organic C-Hg bonds, and subsequent reduction of ionic mercury to the less toxic elemental form, Hg(0). E. coli containing merRTPAB was then encapsulated in silica beads resulting in a biological-based filtration material. Performing encapsulation in aerated mineral oil yielded silica beads that were smooth, spherical, and similar in diameter. Following encapsulation, E. coli containing merRTPAB retained the ability to degrade methylmercury and performed similarly to non-encapsulated cells. Due to the versatility of both the engineered mercury resistant strain and silica bead technology, this study provides a strong foundation for use of the resulting biological-based filtration material for methylmercury remediation. PMID:26761437

  5. Crystal structure of the lactose operon repressor and its complexes with DNA and inducer

    SciTech Connect

    Lewis, M.; Chang, G.; Horton, N.C.

    1996-03-01

    The lac operon of Escherichia coli is the paradigm for gene regulation. Its key component is the lac repressor a product of the lacl gene. The three-dimensional structures of the intact lac repressor, the lac repressor bound to the gratuitous inducer isopropyl-B-D-1thiogalactoside (IPTG) and the lac repressor complexed with a 21 base pair symmetric operator DNA have been determined. These three structures show the conformation of the molecule in both the induced and the repressed states and provide a framework for understanding a wealth of biochemical and genetic information. The DNA sequence of the lac operon has three lac repressor recognition sites in stretch of 500 base pairs. The crystallographic structure of the complex with DNA suggests that the tetrameric repressor functions synergistically with catabolite gene activator protein (CAP) and participates in the quarternary formation of repression loops in which one tetrameric repressor interacts simultaneously with two sites in the genomic DNA. 76 refs., 11 figs., 1 tab.

  6. The mechanistic-holistic divide revisited: The case of the lac operon.

    PubMed

    Racine, Valérie

    2016-10-01

    In this paper, I revisit the development of the repression model of genetic regulation in the lac operon to challenge a common application of a conceptual framework in the history of biology. I take Allen's (1978) account of the changes in the life sciences during the early and mid-twentieth century as an example of a common application of a framework based on the dichotomy between a mechanistic, or reductionist, approach to science and a holistic one. From this conceptual framework, Allen infers two general claims about the process of science and its goals: (1) that "mechanistic materialism" has often presented a more practical way to begin the study of complex phenomena in the life sciences, and (2) that the approach described as "holistic materialism" provides a more complete or accurate description of the natural world. The development of the lac operon model does not fit Allen's generalizations about scientific developments, and it can be used to cast some doubt on the scope of application of that conceptual framework. I argue that a better framework to interpret particular episodes in the history of molecular biology is to consider the ways in which biologists prioritize and track different aspects of the phenomena under study, rather than to focus on whether certain scientific practices are best described as developing from mechanistic to more holistic approaches. I end with some implications for the historiography of science by considering the appropriateness of different conceptual frameworks for different grains of resolution in the history of biology.

  7. The Brucella suis virB operon is induced intracellularly in macrophages.

    PubMed

    Boschiroli, Maria Laura; Ouahrani-Bettache, Safia; Foulongne, Vincent; Michaux-Charachon, Sylvie; Bourg, Gisele; Allardet-Servent, Annick; Cazevieille, Chantal; Liautard, Jean Pierre; Ramuz, Michel; O'Callaghan, David

    2002-02-01

    A type IV secretion system similar to the VirB system of the phytopathogen Agrobacterium tumefaciens is essential for the intracellular survival and multiplication of the mammalian pathogen Brucella. Reverse transcriptase-PCR showed that the 12 genes encoding the Brucella suis VirB system form an operon. Semiquantitative measurements of virB mRNA levels by slot blotting showed that transcription of the virB operon, but not the flanking genes, is regulated by environmental factors in vitro. Flow cytometry used to measure green fluorescent protein expression from the virB promoter confirmed the data from slot blots. Fluorescence-activated cell sorter analysis and fluorescence microscopy showed that the virB promoter is induced in macrophages within 3 h after infection. Induction only occurred once the bacteria were inside the cells, and phagosome acidification was shown to be the major signal inducing intracellular expression. Because phagosome acidification is essential for the intracellular multiplication of Brucella, we suggest that it is the signal that triggers the secretion of unknown effector molecules. These effector molecules play a role in the remodeling of the phagosome to create the unique intracellular compartment in which Brucella replicates. PMID:11830669

  8. Relationship between the persistence of mer operon sequences in Escherichia coli and their resistance to mercury.

    PubMed

    Murtaza, Imtiyaz; Dutt, Amit; Ali, Arif

    2002-03-01

    Studies related to geographic distribution of E. coli carrying mer operon sequences were carried out on the Indian subcontinent. Out of the 80 E. coli isolates, collected from five geographically distinct regions of India, 68 were found to be resistant to one or the other heavy metal used in the study. Among these isolates, 36 were found to be resistant to the inorganic form (HgCl2) and only 5 to resist both the inorganic and organic forms of mercury. Colony hybridization studies revealed 35 isolates out of 68 to hybridize with the probe. Interestingly, some of the mercury-sensitive isolates (Hgs), especially from the Dal Lake, were found positive in hybridization studies. These findings, supported by mercury volatilization studies, indicate the presence of nonfunctional/vestigial mer sequences in the isolates collected from different environments. On the other hand, few of the mercury-resistant isolates (Hgr) from the Yamuna River did not show any sign of hybridization. Further, volatilization studies also indicated an alternate mode of resistance mechanism operating in them. The studies demonstrate that the mer operon sequences share very high homology among the E. coli isolates collected from different geographical locations, and this metal resistance may be a genetic character that arose from a common ancestral background. PMID:11821925

  9. Multiple antibiotic resistance in Pseudomonas aeruginosa: evidence for involvement of an efflux operon.

    PubMed Central

    Poole, K; Krebes, K; McNally, C; Neshat, S

    1993-01-01

    An outer membrane protein of 50 kDa (OprK) was overproduced in a siderophore-deficient mutant of Pseudomonas aeruginosa capable of growth on iron-deficient minimal medium containing 2,2'-dipyridyl (0.5 mM). The expression of OprK in the mutant (strain K385) was associated with enhanced resistance to a number of antimicrobial agents, including ciprofloxacin, nalidixic acid, tetracycline, chloramphenicol, and streptonigrin. OprK was inducible in the parent strain by growth under severe iron limitation, as provided, for example, by the addition of dipyridyl or ZnSO4 to the growth medium. The gene encoding OprK (previously identified as ORFC) forms part of an operon composed of three genes (ORFABC) implicated in the secretion of the siderophore pyoverdine. Mutants defective in ORFA, ORFB, or ORFC exhibited enhanced susceptibility to tetracycline, chloramphenicol, ciprofloxacin, streptonigrin, and dipyridyl, consistent with a role for the ORFABC operon in multiple antibiotic resistance in P. aeruginosa. Sequence analysis of ORFC (oprK) revealed that its product is homologous to a class of outer membrane proteins involved in export. Similarly, the products of ORFA and ORFB exhibit homology to previously described bacterial export proteins located in the cytoplasmic membrane. These data suggest that ORFA-ORFB-oprK (ORFC)-dependent drug efflux contributes to multiple antibiotic resistance in P. aeruginosa. We propose, therefore, the designation mexAB (multiple efflux) for ORFAB. Images PMID:8226684

  10. Toward Bioremediation of Methylmercury Using Silica Encapsulated Escherichia coli Harboring the mer Operon.

    PubMed

    Kane, Aunica L; Al-Shayeb, Basem; Holec, Patrick V; Rajan, Srijay; Le Mieux, Nicholas E; Heinsch, Stephen C; Psarska, Sona; Aukema, Kelly G; Sarkar, Casim A; Nater, Edward A; Gralnick, Jeffrey A

    2016-01-01

    Mercury is a highly toxic heavy metal and the ability of the neurotoxin methylmercury to biomagnify in the food chain is a serious concern for both public and environmental health globally. Because thousands of tons of mercury are released into the environment each year, remediation strategies are urgently needed and prompted this study. To facilitate remediation of both organic and inorganic forms of mercury, Escherichia coli was engineered to harbor a subset of genes (merRTPAB) from the mercury resistance operon. Protein products of the mer operon enable transport of mercury into the cell, cleavage of organic C-Hg bonds, and subsequent reduction of ionic mercury to the less toxic elemental form, Hg(0). E. coli containing merRTPAB was then encapsulated in silica beads resulting in a biological-based filtration material. Performing encapsulation in aerated mineral oil yielded silica beads that were smooth, spherical, and similar in diameter. Following encapsulation, E. coli containing merRTPAB retained the ability to degrade methylmercury and performed similarly to non-encapsulated cells. Due to the versatility of both the engineered mercury resistant strain and silica bead technology, this study provides a strong foundation for use of the resulting biological-based filtration material for methylmercury remediation. PMID:26761437

  11. IHF is required for the transcriptional regulation of the Desulfovibrio vulgaris Hildenborough orp operons.

    PubMed

    Fiévet, Anouchka; Cascales, Eric; Valette, Odile; Dolla, Alain; Aubert, Corinne

    2014-01-01

    Transcriptional activation of σ(54)-dependent promoters is usually tightly regulated in response to environmental cues. The high abundance of potential σ(54)-dependent promoters in the anaerobe bacteria, Desulfovibrio vulgaris Hildenborough, reflects the high versatility of this bacteria suggesting that σ(54) factor is the nexus of a large regulatory network. Understanding the key players of σ(54)-regulation in this organism is therefore essential to gain insights into the adaptation to anaerobiosis. Recently, the D. vulgaris orp genes, specifically found in anaerobe bacteria, have been shown to be transcribed by the RNA polymerase coupled to the σ(54) alternative sigma factor. In this study, using in vitro binding experiments and in vivo reporter fusion assays in the Escherichia coli heterologous host, we showed that the expression of the divergent orp promoters is strongly dependent on the integration host factor IHF. Bioinformatic and mutational analysis coupled to reporter fusion activities and mobility shift assays identified two functional IHF binding site sequences located between the orp1 and orp2 promoters. We further determined that the D. vulgaris DVU0396 (IHFα) and DVU1864 (IHFβ) subunits are required to control the expression of the orp operons suggesting that they form a functionally active IHF heterodimer. Interestingly results obtained from the in vivo inactivation of DVU0396, which is required for orp operons transcription, suggest that several functionally IHF active homodimer or heterodimer are present in D. vulgaris.

  12. Dynamics and bistability in a reduced model of the lac operon

    NASA Astrophysics Data System (ADS)

    Yildirim, Necmettin; Santillán, Moisés; Horike, Daisuke; Mackey, Michael C.

    2004-06-01

    It is known that the lac operon regulatory pathway is capable of showing bistable behavior. This is an important complex feature, arising from the nonlinearity of the involved mechanisms, which is essential to understand the dynamic behavior of this molecular regulatory system. To find which of the mechanisms involved in the regulation of the lac operon is the origin of bistability, we take a previously published model which accounts for the dynamics of mRNA, lactose, allolactose, permease and β-galactosidase involvement and simplify it by ignoring permease dynamics (assuming a constant permease concentration). To test the behavior of the reduced model, three existing sets of data on β-galactosidase levels as a function of time are simulated and we obtain a reasonable agreement between the data and the model predictions. The steady states of the reduced model were numerically and analytically analyzed and it was shown that it may indeed display bistability, depending on the extracellular lactose concentration and growth rate.

  13. The structure of a ribosomal protein S8/spc operon mRNA complex.

    PubMed

    Merianos, Helen J; Wang, Jimin; Moore, Peter B

    2004-06-01

    In bacteria, translation of all the ribosomal protein cistrons in the spc operon mRNA is repressed by the binding of the product of one of them, S8, to an internal sequence at the 5' end of the L5 cistron. The way in which the first two genes of the spc operon are regulated, retroregulation, is mechanistically distinct from translational repression by S8 of the genes from L5 onward. A 2.8 A resolution crystal structure has been obtained of Escherichia coli S8 bound to this site. Despite sequence differences, the structure of this complex is almost identical to that of the S8/helix 21 complex seen in the small ribosomal subunit, consistent with the hypothesis that autogenous regulation of ribosomal protein synthesis results from conformational similarities between mRNAs and rRNAs. S8 binding must repress the translation of its own mRNA by inhibiting the formation of a ribosomal initiation complex at the start of the L5 cistron.

  14. Functional and comparative genomic analyses of an operon involved in fructooligosaccharide utilization by Lactobacillus acidophilus

    PubMed Central

    Barrangou, Rodolphe; Altermann, Eric; Hutkins, Robert; Cano, Raul; Klaenhammer, Todd R.

    2003-01-01

    Lactobacillus acidophilus is a probiotic organism that displays the ability to use prebiotic compounds such as fructooligosaccharides (FOS), which stimulate the growth of beneficial commensals in the gastrointestinal tract. However, little is known about the mechanisms and genes involved in FOS utilization by Lactobacillus species. Analysis of the L. acidophilus NCFM genome revealed an msm locus composed of a transcriptional regulator of the LacI family, a four-component ATP-binding cassette (ABC) transport system, a fructosidase, and a sucrose phosphorylase. Transcriptional analysis of this operon demonstrated that gene expression was induced by sucrose and FOS but not by glucose or fructose, suggesting some specificity for nonreadily fermentable sugars. Additionally, expression was repressed by glucose but not by fructose, suggesting catabolite repression via two cre-like sequences identified in the promoter–operator region. Insertional inactivation of the genes encoding the ABC transporter substrate-binding protein and the fructosidase reduced the ability of the mutants to grow on FOS. Comparative analysis of gene architecture within this cluster revealed a high degree of synteny with operons in Streptococcus mutans and Streptococcus pneumoniae. However, the association between a fructosidase and an ABC transporter is unusual and may be specific to L. acidophilus. This is a description of a previously undescribed gene locus involved in transport and catabolism of FOS compounds, which can promote competition of beneficial microorganisms in the human gastrointestinal tract. PMID:12847288

  15. Structure of Intergenic Spacer IGS1 of Ribosomal Operon from Schistidium Mosses.

    PubMed

    Milyutina, I A; Ignatova, E A; Ignatov, M S; Goryunov, D V; Troitsky, A V

    2015-11-01

    The structure of the intergenic spacer 1 (IGS1) of the ribosomal operon from 12 species of Schistidium mosses was studied. In the IGS1 sequences of these species, three conserved regions and two areas of GC- and A-enriched repeats were identified. All of the studied mosses have a conserved pyrimidine-enriched motif at the 5'-end of IGS1. Species-specific nucleotide substitutions and insertions were found in the conserved areas. The repeated units contain single nucleotide substitutions that make unique the majority of repeated units. The positions of such repeats in IGS1 are species-specific, but their number can vary within the species and among operons of the same specimen. The comparison of IGS1 sequences from the Schistidium species and from representatives of ten other moss genera revealed the presence of common conserved motifs with similar localization. Presumably, these motifs are elements of termination of the pre-rRNA transcription and processing of rRNA.

  16. Derepression and repression of the histidine operon: role of the feedback site of the first enzyme.

    PubMed Central

    Fernández, V M; Martíndelrío, R; Tébar, A R; Guisán, J M; Ballesteros, A O

    1975-01-01

    Thiazolealanine, a false feedback inhibitor, causes transient repression of the his operon previously derepressed by a severe histidine limitation in strains with a wild-type or feedback-hypersensitive first enzyme but not in feedback-resistant mutants. Since experiments reported here clearly demonstrate that thiazolealanine is not transferred to tRNAHis, it is proposed that this "transient repression" is effected through the interaction of thiazolealanine with the feedback site of the enzyme. Experiments in the presence of rifampin indicate that this thiazolealanine-mediated effect is exerted at the level of translation. We conclude that histidine (free), in addition to forming co-repressor, also represses the operon at the level of translation through feedback interaction with the first enzyme of the pathway (adenosine 5'-triphosphate phosphoribosyltransferase). Rates of derepression in feedback-resistant strains are roughly half of those observed in controls, suggesting a positive role played by a first enzyme with a normal but unoccupied feedback site. Some feedback-resistant mutants, in contrast to the wild type, were unable to exhibit derepression under histidine limitation caused by aminotriazole. PMID:1104584

  17. Carbonic anhydrase in Escherichia coli. A product of the cyn operon.

    PubMed

    Guilloton, M B; Korte, J J; Lamblin, A F; Fuchs, J A; Anderson, P M

    1992-02-25

    The product of the cynT gene of the cyn operon in Escherichia coli has been identified as a carbonic anhydrase. The cyn operon also includes the gene cynS, encoding the enzyme cyanase. Cyanase catalyzes the reaction of cyanate with bicarbonate to give ammonia and carbon dioxide. The carbonic anhydrase was isolated from an Escherichia coli strain overexpressing the cynT gene and characterized. The purified enzyme was shown to contain 1 Zn2+/subunit (24 kDa) and was found to behave as an oligomer in solution; the presence of bicarbonate resulted in partial dissociation of the oligomeric enzyme. The kinetic properties of the enzyme are similar to those of carbonic anhydrases from other species, including inhibition by sulfonamides and cyanate. The amino acid sequence shows a high degree of identity with the sequences of two plant carbonic anhydrases. but not with animal and algal carbonic anhydrases. Since carbon dioxide formed in the bicarbonate-dependent decomposition of cyanate diffuses out of the cell faster than it would be hydrated to bicarbonate, the apparent function of the induced carbonic anhydrase is to catalyze hydration of carbon dioxide and thus prevent depletion of cellular bicarbonate.

  18. Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon

    SciTech Connect

    Zylstra, G.J.; McCombie, W.R.; Gibson, D.T.; Finette, B.A.

    1988-06-01

    Pseudomonas putida PpF1 degrades toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is metabolized through the well-established meta pathway for catechol degradation. The first four steps in the pathway involve the sequential action of toluene dioxygenase (todABC1C2), cis-toluene, dihydrodiol dehydrogenase (todD), 3-methylcatechol 2,3-dioxygenase (todE), and 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase (todF). The genes for these enzymes form part of the tod operon which is responsible for the degradation of toluene by this organism. A combination of transposon mutagenesis of the PpF1 chromosome, was well as the analysis of cloned chromosomal fragments, was used to determine the physical order of the genes in the tod operon. The genes were determined to be transcribed in the order todF, todC1, todC2, todB, todA, todD, todE.

  19. Relationship between the persistence of mer operon sequences in Escherichia coli and their resistance to mercury.

    PubMed

    Murtaza, Imtiyaz; Dutt, Amit; Ali, Arif

    2002-03-01

    Studies related to geographic distribution of E. coli carrying mer operon sequences were carried out on the Indian subcontinent. Out of the 80 E. coli isolates, collected from five geographically distinct regions of India, 68 were found to be resistant to one or the other heavy metal used in the study. Among these isolates, 36 were found to be resistant to the inorganic form (HgCl2) and only 5 to resist both the inorganic and organic forms of mercury. Colony hybridization studies revealed 35 isolates out of 68 to hybridize with the probe. Interestingly, some of the mercury-sensitive isolates (Hgs), especially from the Dal Lake, were found positive in hybridization studies. These findings, supported by mercury volatilization studies, indicate the presence of nonfunctional/vestigial mer sequences in the isolates collected from different environments. On the other hand, few of the mercury-resistant isolates (Hgr) from the Yamuna River did not show any sign of hybridization. Further, volatilization studies also indicated an alternate mode of resistance mechanism operating in them. The studies demonstrate that the mer operon sequences share very high homology among the E. coli isolates collected from different geographical locations, and this metal resistance may be a genetic character that arose from a common ancestral background.

  20. Function of RNA secondary structures in transcriptional attenuation of the Bacillus subtilis pyr operon.

    PubMed

    Lu, Y; Turner, R J; Switzer, R L

    1996-12-10

    The Bacillus subtilis pyr operon is regulated by exogenous pyrimidines by a transcriptional attenuation mechanism. Transcription in vitro from pyr DNA templates specifying attenuation regions yielded terminated and read-through transcripts of the expected lengths. Addition of the PyrR regulatory protein plus UMP led to greatly increased termination. Synthetic antisense deoxyoligonucleotides were used to probe possible secondary structures in the pyr mRNA that were proposed to play roles in controlling attenuation. Oligonucleotides predicted to disrupt terminator structures suppressed termination, whereas oligonucleotides predicted to disrupt the stem of antiterminator stem-loops strongly promoted termination at the usual termination site. Oligonucleotides that disrupt a previously unrecognized stem-loop structure, called the anti-antiterminator, the formation of which interferes with formation of the downstream antiterminator, suppressed termination. We propose that transcriptional attenuation of the pyr operon is governed by switching between alternative antiterminator versus anti-antiterminator plus terminator structures, and that PyrR acts by UMP-dependent binding to and stabilization of the anti-antiterminator.

  1. Genomics of pyrrolnitrin biosynthetic loci: evidence for conservation and whole-operon mobility within gram-negative bacteria.

    PubMed

    Costa, Rodrigo; van Aarle, Ingrid M; Mendes, Rodrigo; van Elsas, Jan Dirk

    2009-01-01

    Pyrrolnitrin (PRN) is a tryptophan-derived secondary metabolite produced by a narrow range of gram-negative bacteria. The PRN biosynthesis by rhizobacteria presumably has a key role in their life strategies and in the biocontrol of plant diseases. The biosynthetic operon that encodes the pathway that converts tryptophan to PRN is composed of four genes, prnA through D, whose diversity, genomic context and spread over bacterial genomes are poorly understood. Therefore, we launched an endeavour aimed at retrieving, by in vitro and in silico means, diverse bacteria carrying the prnABCD biosynthetic loci in their genomes. Analysis of polymorphisms of the prnD gene sequences revealed a high level of conservation between Burkholderia, Pseudomonas and Serratia spp. derived sequences. Whole-operon- and prnD-based phylogeny resulted in tree topologies that are incongruent with the taxonomic status of the evaluated strains as predicted by 16S rRNA gene phylogeny. The genomic composition of c. 20 kb DNA fragments containing the PRN operon varied in different strains. Highly conserved and distinct transposase-encoding genes surrounding the PRN biosynthetic operons of Burkholderia pseudomallei strains were found. A prnABCD-deprived genomic region in B. pseudomallei strain K96243 contained the same gene composition as, and shared high homology with, the flanking regions of the PRN operon in B. pseudomallei strains 668, 1106a and 1710b. Our results strongly suggest that the PRN biosynthetic operon is mobile. The extent, frequency and promiscuity of this mobility remain to be understood. PMID:18793314

  2. Learning virulent proteins from integrated query networks

    PubMed Central

    2012-01-01

    Background Methods of weakening and attenuating pathogens’ abilities to infect and propagate in a host, thus allowing the natural immune system to more easily decimate invaders, have gained attention as alternatives to broad-spectrum targeting approaches. The following work describes a technique to identifying proteins involved in virulence by relying on latent information computationally gathered across biological repositories, applicable to both generic and specific virulence categories. Results A lightweight method for data integration is used, which links information regarding a protein via a path-based query graph. A method of weighting is then applied to query graphs that can serve as input to various statistical classification methods for discrimination, and the combined usage of both data integration and learning methods are tested against the problem of both generalized and specific virulence function prediction. Conclusions This approach improves coverage of functional data over a protein. Moreover, while depending largely on noisy and potentially non-curated data from public sources, we find it outperforms other techniques to identification of general virulence factors and baseline remote homology detection methods for specific virulence categories. PMID:23198735

  3. Genetically manipulated virulence of Yersinia enterocolitica.

    PubMed Central

    Heesemann, J; Algermissen, B; Laufs, R

    1984-01-01

    Mobilizable virulence plasmids of Yersinia enterocolitica of serotypes O:3 and O:9 were constructed by cointegration of a mobilizable vector into the virulence plasmids. The obtained cointegrates were mobilized into plasmidless Y. enterocolitica strains of serotypes O:3, O:5, O:8, and O:9. The transfer experiments revealed the existence of two different subgroups of plasmid-associated traits. (i) Animal virulence functions (mouse lethality and conjuctivitis provocation) were only transferable to plasmid-cured derivatives of virulent parent strains (serotypes O:3, O:8, and O:9), but they were not transferable to Y. enterocolitica antigen reference strains (serotypes O:3 and O:8) or to a plasmidless clinical isolate of serotype O:5. A further striking result was that a serotype O:8 strain regained the mouse lethality trait after receipt of a plasmid from a strain not lethal to mice. These results demonstrate that plasmid-mediated animal virulence functions are not uniformly expressed within Y. enterocolitica. (ii) The second subgroup of plasmid-mediated traits (calcium dependency, surface agglutinogens, HEp-2 cell adherence, and protein release) were transferable to all Y. enterocolitica recipient strains tested (serotypes O:3, O:5, O:8, and O:9 of different origin). For the first time HEp-2 cell adherence and temperature-induced release of five major protein species are described as transferable traits. Images PMID:6480101

  4. Salmonella promotes virulence by repressing cellulose production.

    PubMed

    Pontes, Mauricio H; Lee, Eun-Jin; Choi, Jeongjoon; Groisman, Eduardo A

    2015-04-21

    Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a trade-off between acute virulence and transmission.

  5. Plant Natural Products Targeting Bacterial Virulence Factors.

    PubMed

    Silva, Laura Nunes; Zimmer, Karine Rigon; Macedo, Alexandre José; Trentin, Danielle Silva

    2016-08-24

    Decreased antimicrobial efficiency has become a global public health issue. The paucity of new antibacterial drugs is evident, and the arsenal against infectious diseases needs to be improved urgently. The selection of plants as a source of prototype compounds is appropriate, since plant species naturally produce a wide range of secondary metabolites that act as a chemical line of defense against microorganisms in the environment. Although traditional approaches to combat microbial infections remain effective, targeting microbial virulence rather than survival seems to be an exciting strategy, since the modulation of virulence factors might lead to a milder evolutionary pressure for the development of resistance. Additionally, anti-infective chemotherapies may be successfully achieved by combining antivirulence and conventional antimicrobials, extending the lifespan of these drugs. This review presents an updated discussion of natural compounds isolated from plants with chemically characterized structures and activity against the major bacterial virulence factors: quorum sensing, bacterial biofilms, bacterial motility, bacterial toxins, bacterial pigments, bacterial enzymes, and bacterial surfactants. Moreover, a critical analysis of the most promising virulence factors is presented, highlighting their potential as targets to attenuate bacterial virulence. The ongoing progress in the field of antivirulence therapy may therefore help to translate this promising concept into real intervention strategies in clinical areas. PMID:27437994

  6. Salmonella promotes virulence by repressing cellulose production

    PubMed Central

    Pontes, Mauricio H.; Lee, Eun-Jin; Choi, Jeongjoon; Groisman, Eduardo A.

    2015-01-01

    Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a trade-off between acute virulence and transmission. PMID:25848006

  7. Salmonella promotes virulence by repressing cellulose production.

    PubMed

    Pontes, Mauricio H; Lee, Eun-Jin; Choi, Jeongjoon; Groisman, Eduardo A

    2015-04-21

    Cellulose is the most abundant organic polymer on Earth. In bacteria, cellulose confers protection against environmental insults and is a constituent of biofilms typically formed on abiotic surfaces. We report that, surprisingly, Salmonella enterica serovar Typhimurium makes cellulose when inside macrophages. We determine that preventing cellulose synthesis increases virulence, whereas stimulation of cellulose synthesis inside macrophages decreases virulence. An attenuated mutant lacking the mgtC gene exhibited increased cellulose levels due to increased expression of the cellulose synthase gene bcsA and of cyclic diguanylate, the allosteric activator of the BcsA protein. Inactivation of bcsA restored wild-type virulence to the Salmonella mgtC mutant, but not to other attenuated mutants displaying a wild-type phenotype regarding cellulose. Our findings indicate that a virulence determinant can promote pathogenicity by repressing a pathogen's antivirulence trait. Moreover, they suggest that controlling antivirulence traits increases long-term pathogen fitness by mediating a trade-off between acute virulence and transmission. PMID:25848006

  8. Virulence and prodigiosin antibiotic biosynthesis in Serratia are regulated pleiotropically by the GGDEF/EAL domain protein, PigX.

    PubMed

    Fineran, Peter C; Williamson, Neil R; Lilley, Kathryn S; Salmond, George P C

    2007-11-01

    Gram-negative bacteria of the genus Serratia are opportunistic human, plant, and insect pathogens. Serratia sp. strain ATCC 39006 secretes pectinases and cellulases and produces the secondary metabolites carbapenem and prodigiosin. Mutation of a gene (pigX) resulted in an extremely pleiotropic phenotype: prodigiosin antibiotic biosynthesis, plant virulence, and pectinase production were all elevated. PigX controlled secondary metabolism by repressing the transcription of the target prodigiosin biosynthetic operon (pigA-pigO). The transcriptional start site of pigX was determined, and pigX expression occurred in parallel with Pig production. Detailed quantitative intracellular proteome analyses enabled the identification of numerous downstream targets of PigX, including OpgG, mutation of which reduced the production of the plant cell wall-degrading enzymes and virulence. The highly pleiotropic PigX regulator contains GGDEF and EAL domains with noncanonical motifs and is predicted to be membrane associated. Genetic evidence suggests that PigX might function as a cyclic dimeric GMP phosphodiesterase. This is the first characterization of a GGDEF and EAL domain protein in Serratia and the first example of the regulation of antibiotic production by a GGDEF/EAL domain protein.

  9. Organization, structure, and variability of the rRNA operon of the Whipple's disease bacterium (Tropheryma whippelii).

    PubMed

    Maiwald, M; von Herbay, A; Lepp, P W; Relman, D A

    2000-06-01

    Whipple's disease is a systemic disorder associated with a cultivation-resistant, poorly characterized actinomycete, Tropheryma whippelii. We determined a nearly complete rRNA operon sequence of T. whippelii from specimens from 3 patients with Whipple's disease, as well as partial operon sequences from 43 patients. Variability was observed in the 16S-23S rRNA spacer sequences, leading to the description of five distinct sequence types. One specimen contained two spacer sequence types, raising the possibility of a double infection. Secondary structure models for the primary rRNA transcript and mature rRNAs revealed rare or unique features.

  10. 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

  11. Stabilization of the Virulence Plasmid pSLT of Salmonella Typhimurium by Three Maintenance Systems and Its Evaluation by Using a New Stability Test

    PubMed Central

    Lobato-Márquez, Damián; Molina-García, Laura; Moreno-Córdoba, Inma; García-del Portillo, Francisco; Díaz-Orejas, Ramón

    2016-01-01

    Certain Salmonella enterica serovars belonging to subspecies I carry low-copy-number virulence plasmids of variable size (50–90 kb). All of these plasmids share the spv operon, which is important for systemic infection. Virulence plasmids are present at low copy numbers. Few copies reduce metabolic burden but suppose a risk of plasmid loss during bacterial division. This drawback is counterbalanced by maintenance modules that ensure plasmid stability, including partition systems and toxin-antitoxin (TA) loci. The low-copy number virulence pSLT plasmid of Salmonella enterica serovar Typhimurium encodes three auxiliary maintenance systems: one partition system (parAB) and two TA systems (ccdABST and vapBC2ST). The TA module ccdABST has previously been shown to contribute to pSLT plasmid stability and vapBC2ST to bacterial virulence. Here we describe a novel assay to measure plasmid stability based on the selection of plasmid-free cells following elimination of plasmid-containing cells by ParE toxin, a DNA gyrase inhibitor. Using this new maintenance assay we confirmed a crucial role of parAB in pSLT maintenance. We also showed that vapBC2ST, in addition to contribute to bacterial virulence, is important for plasmid stability. We have previously shown that ccdABST encodes an inactive CcdBST toxin. Using our new stability assay we monitored the contribution to plasmid stability of a ccdABST variant containing a single mutation (R99W) that restores the toxicity of CcdBST. The “activation” of CcdBST (R99W) did not increase pSLT stability by ccdABST. In contrast, ccdABST behaves as a canonical type II TA system in terms of transcriptional regulation. Of interest, ccdABST was shown to control the expression of a polycistronic operon in the pSLT plasmid. Collectively, these results show that the contribution of the CcdBST toxin to pSLT plasmid stability may depend on its role as a co-repressor in coordination with CcdAST antitoxin more than on its toxic activity. PMID

  12. Identification of a novel operon in Lactococcus lactis encoding three enzymes for lactic acid synthesis: phosphofructokinase, pyruvate kinase, and lactate dehydrogenase.

    PubMed Central

    Llanos, R M; Harris, C J; Hillier, A J; Davidson, B E

    1993-01-01

    The discovery of a novel multicistronic operon that encodes phosphofructokinase, pyruvate kinase, and lactate dehydrogenase in the lactic acid bacterium Lactococcus lactis is reported. The three genes in the operon, designated pfk, pyk, and ldh, contain 340, 502, and 325 codons, respectively. The intergenic distances are 87 bp between pfk and pyk and 117 bp between pyk and ldh. Plasmids containing pfk and pyk conferred phosphofructokinase and pyruvate kinase activity, respectively, on their host. The identity of ldh was established previously by the same approach (R. M. Llanos, A. J. Hillier, and B. E. Davidson, J. Bacteriol. 174:6956-6964, 1992). Each of the genes is preceded by a potential ribosome binding site. The operon is expressed in a 4.1-kb transcript. The 5' end of the transcript was determined to be a G nucleotide positioned 81 bp upstream from the pfk start codon. The pattern of codon usage within the operon is highly biased, with 11 unused amino acid codons. This degree of bias suggests that the operon is highly expressed. The three proteins encoded on the operon are key enzymes in the Embden-Meyerhoff pathway, the central pathway of energy production and lactic acid synthesis in L. lactis. For this reason, we have called the operon the las (lactic acid synthesis) operon. Images PMID:8478320

  13. The gut bacterium Bacteroides thetaiotaomicron influences the virulence potential of the enterohemorrhagic Escherichia coli O103:H25.

    PubMed

    Iversen, Hildegunn; Lindbäck, Toril; L'Abée-Lund, Trine M; Roos, Norbert; Aspholm, Marina; Stenfors Arnesen, Lotte

    2015-01-01

    Enterohemorrhagic E. coli (EHEC) is associated with severe gastrointestinal disease. Upon entering the gastrointestinal tract, EHEC is exposed to a fluctuating environment and a myriad of other bacterial species. To establish an infection, EHEC strains have to modulate their gene expression according to the GI tract environment. In order to explore the interspecies interactions between EHEC and an human intestinal commensal, the global gene expression profile was determined of EHEC O103:H25 (EHEC NIPH-11060424) co-cultured with B. thetaiotaomicron (CCUG 10774) or grown in the presence of spent medium from B. thetaiotaomicron. Microarray analysis revealed that approximately 1% of the EHEC NIPH-11060424 genes were significantly up-regulated both in co-culture (30 genes) and in the presence of spent medium (44 genes), and that the affected genes differed between the two conditions. In co-culture, genes encoding structural components of the type three secretion system were among the most affected genes with an almost 4-fold up-regulation, while the most affected genes in spent medium were involved in chemotaxis and were more than 3-fold up-regulated. The operons for type three secretion system (TTSS) are located on the Locus of enterocyte effacement (LEE) pathogenicity island, and qPCR showed that genes of all five operons (LEE1-LEE5) were up-regulated. Moreover, an increased adherence to HeLa cells was observed in EHEC NIPH-11060424 exposed to B. thetaiotaomicron. Expression of stx2 genes, encoding the main virulence factor of EHEC, was down-regulated in both conditions (co-culture/spent medium). These results show that expression of EHEC genes involved in colonization and virulence is modulated in response to direct interspecies contact between cells, or to diffusible factors released from B. thetaiotaomicron. Such interspecies interactions could allow the pathogen to recognize its predilection site and modulate its behaviour accordingly, thus increasing the

  14. The Streptococcus pyogenes serotype M49 Nra-Ralp3 transcriptional regulatory network and its control of virulence factor expression from the novel eno ralp3 epf sagA pathogenicity region.

    PubMed

    Kreikemeyer, Bernd; Nakata, Masanobu; Köller, Thomas; Hildisch, Hendrikje; Kourakos, Vassilios; Standar, Kerstin; Kawabata, Shigetada; Glocker, Michael O; Podbielski, Andreas

    2007-12-01

    Many Streptococcus pyogenes (group A streptococcus [GAS]) virulence factor- and transcriptional regulator-encoding genes cluster together in discrete genomic regions. Nra is a central regulator of the FCT region. Previous studies exclusively described Nra as a transcriptional repressor of adhesin and toxin genes. Here transcriptome and proteome analysis of a serotype M49 GAS strain and an isogenic Nra mutant of this strain revealed the complete Nra regulon profile. Nra is active in all growth phases tested, with the largest regulon in the transition phase. Almost exclusively, virulence factor-encoding genes are repressed by Nra; these genes include the GAS pilus operon, the capsule synthesis operon, the cytolysin-mediated translocation system genes, all Mga region core virulence genes, and genes encoding other regulators, like the Ihk/Irr system, Rgg, and two additional RofA-like protein family regulators. Surprisingly, our experiments revealed that Nra additionally acts as a positive regulator, mostly for genes encoding proteins and enzymes with metabolic functions. Epidemiological investigations revealed strong genetic linkage of one particular Nra-repressed regulator, Ralp3 (SPy0735), with a gene encoding Epf (extracellular protein factor from Streptococcus suis). In a serotype-specific fashion, this ralp3 epf gene block is integrated, most likely via transposition, into the eno sagA virulence gene block, which is present in all GAS serotypes. In GAS serotypes M1, M4, M12, M28, and M49 this novel discrete genetic region is therefore designated the eno ralp3 epf sagA (ERES) pathogenicity region. Functional experiments showed that Epf is a novel GAS plasminogen-binding protein and revealed that Ralp3 activity counteracts Nra and MsmR regulatory activity. In addition to the Mga and FCT regions, the ERES region is the third discrete chromosomal pathogenicity region. All of these regions are transcriptionally linked, adding another level of complexity to the known

  15. Virulence factors of the Mycobacterium tuberculosis complex

    PubMed Central

    Forrellad, Marina A.; Klepp, Laura I.; Gioffré, Andrea; Sabio y García, Julia; Morbidoni, Hector R.; Santangelo, María de la Paz; Cataldi, Angel A.; Bigi, Fabiana

    2013-01-01

    The Mycobacterium tuberculosis complex (MTBC) consists of closely related species that cause tuberculosis in both humans and animals. This illness, still today, remains to be one of the leading causes of morbidity and mortality throughout the world. The mycobacteria enter the host by air, and, once in the lungs, are phagocytated by macrophages. This may lead to the rapid elimination of the bacillus or to the triggering of an active tuberculosis infection. A large number of different virulence factors have evolved in MTBC members as a response to the host immune reaction. The aim of this review is to describe the bacterial genes/proteins that are essential for the virulence of MTBC species, and that have been demonstrated in an in vivo model of infection. Knowledge of MTBC virulence factors is essential for the development of new vaccines and drugs to help manage the disease toward an increasingly more tuberculosis-free world. PMID:23076359

  16. Viral population dynamics and virulence thresholds.

    PubMed

    Lancaster, Karen Z; Pfeiffer, Julie K

    2012-08-01

    Viral factors and host barriers influence virally induced disease, and asymptomatic versus symptomatic infection is governed by a 'virulence threshold'. Understanding modulation of virulence thresholds could lend insight into disease outcome and aid in rational therapeutic and vaccine design. RNA viruses are an excellent system to study virulence thresholds in the context of quasispecies population dynamics. RNA viruses have high error frequencies and our understanding of viral population dynamics has been shaped by quasispecies evolutionary theory. In turn, research using RNA viruses as replicons with short generation times and high mutation rates has been an invaluable tool to test models of quasispecies theory. The challenge and new frontier of RNA virus population dynamics research is to combine multiple theoretical models and experimental data to describe viral population behavior as it changes, moving within and between hosts, to predict disease and pathogen emergence. Several excellent studies have begun to undertake this challenge using novel approaches.

  17. Potential drivers of virulence evolution in aquaculture.

    PubMed

    Kennedy, David A; Kurath, Gael; Brito, Ilana L; Purcell, Maureen K; Read, Andrew F; Winton, James R; Wargo, Andrew R

    2016-02-01

    Infectious diseases are economically detrimental to aquaculture, and with continued expansion and intensification of aquaculture, the importance of managing infectious diseases will likely increase in the future. Here, we use evolution of virulence theory, along with examples, to identify aquaculture practices that might lead to the evolution of increased pathogen virulence. We identify eight practices common in aquaculture that theory predicts may favor evolution toward higher pathogen virulence. Four are related to intensive aquaculture operations, and four others are related specifically to infectious disease control. Our intention is to make aquaculture managers aware of these risks, such that with increased vigilance, they might be able to detect and prevent the emergence and spread of increasingly troublesome pathogen strains in the future.

  18. The primary transcriptome of the Escherichia coli O104:H4 pAA plasmid and novel insights into its virulence gene expression and regulation

    PubMed Central

    Berger, Petya; Knödler, Michael; Förstner, Konrad U.; Berger, Michael; Bertling, Christian; Sharma, Cynthia M.; Vogel, Jörg; Karch, Helge; Dobrindt, Ulrich; Mellmann, Alexander

    2016-01-01

    Escherichia coli O104:H4 (E. coli O104:H4), which caused a massive outbreak of acute gastroenteritis and hemolytic uremic syndrome in 2011, carries an aggregative adherence fimbriae I (AAF/I) encoding virulence plasmid, pAA. The importance of pAA in host-pathogen interaction and disease severity has been demonstrated, however, not much is known about its transcriptional organization and gene regulation. Here, we analyzed the pAA primary transcriptome using differential RNA sequencing, which allows for the high-throughput mapping of transcription start site (TSS) and non-coding RNA candidates. We identified 248 TSS candidates in the 74-kb pAA and only 21% of them could be assigned as TSS of annotated genes. We detected TSS for the majority of pAA-encoded virulence factors. Interestingly, we mapped TSS, which could allow for the transcriptional uncoupling of the AAF/I operon, and potentially regulatory antisense RNA candidates against the genes encoding dispersin and the serine protease SepA. Moreover, a computational search for transcription factor binding sites suggested for AggR-mediated activation of SepA expression, which was additionally experimentally validated. This work advances our understanding of the molecular basis of E. coli O104:H4 pathogenicity and provides a valuable resource for further characterization of pAA virulence gene regulation. PMID:27748404

  19. Biofilm formation by virulent and non-virulent strains of Haemophilus parasuis.

    PubMed

    Bello-Ortí, Bernardo; Deslandes, Vincent; Tremblay, Yannick D N; Labrie, Josée; Howell, Kate J; Tucker, Alexander W; Maskell, Duncan J; Aragon, Virginia; Jacques, Mario

    2014-01-01

    Haemophilus parasuis is a commensal bacterium of the upper respiratory tract of healthy pigs. It is also the etiological agent of Glässer's disease, a systemic disease characterized by polyarthritis, fibrinous polyserositis and meningitis, which causes high morbidity and mortality in piglets. The aim of this study was to evaluate biofilm formation by well-characterized virulent and non-virulent strains of H. parasuis. We observed that non-virulent strains isolated from the nasal cavities of healthy pigs formed significantly (p < 0.05) more biofilms than virulent strains isolated from lesions of pigs with Glässer's disease. These differences were observed when biofilms were formed in microtiter plates under static conditions or formed in the presence of shear force in a drip-flow apparatus or a microfluidic system. Confocal laser scanning microscopy using different fluorescent probes on a representative subset of strains indicated that the biofilm matrix contains poly-N-acetylglucosamine, proteins and eDNA. The biofilm matrix was highly sensitive to degradation by proteinase K. Comparison of transcriptional profiles of biofilm and planktonic cells of the non-virulent H. parasuis F9 strain revealed a significant number of up-regulated membrane-related genes in biofilms, and genes previously identified in Actinobacillus pleuropneumoniae biofilms. Our data indicate that non-virulent strains of H. parasuis have the ability to form robust biofilms in contrast to virulent, systemic strains. Biofilm formation might therefore allow the non-virulent strains to colonize and persist in the upper respiratory tract of pigs. Conversely, the planktonic state of the virulent strains might allow them to disseminate within the host. PMID:25428823

  20. Quorum sensing-mediated regulation of staphylococcal virulence and antibiotic resistance.

    PubMed

    Singh, Rachna; Ray, Pallab

    2014-01-01

    Accessory gene regulator (agr)-mediated quorum sensing plays a central role in staphylococcal pathogenesis. It primarily upregulates secreted virulence factors and downregulates cell surface proteins, thereby governing invasiveness of staphylococci and cell dispersal from biofilms. Except for α- and β-PSMs, which are directly controlled by AgrA, the effector functions of agr are primarily mediated by RNAIII, a regulatory RNA encoded by this operon. agr phenotype and expression considerably influence the chronicity of an infection. It has also been linked with altered susceptibility of Staphylococcus aureus against antibiotics. Four classes of S. aureus and Staphylococcus epidermidis AIPs exist based on sequence variation, and lead to inter-strain and species cross-inhibition. Certain agr classes have been associated with specific clonal complexes, disease syndromes and intermediate-susceptibility to glycopeptides. It is also being investigated as a prophylactic and therapeutic target. This article describes the presently available literature regarding the role of agr in S. aureus and S. epidermidis infections.

  1. The Twin Arginine Translocation System Is Essential for Aerobic Growth and Full Virulence of Burkholderia thailandensis

    PubMed Central

    Wagley, Sariqa; Hemsley, Claudia; Thomas, Rachael; Moule, Madeleine G.; Vanaporn, Muthita; Andreae, Clio; Robinson, Matthew; Goldman, Stan; Wren, Brendan W.; Butler, Clive S.

    2014-01-01

    The twin arginine translocation (Tat) system in bacteria is responsible for transporting folded proteins across the cytoplasmic membrane, and in some bacteria, Tat-exported substrates have been linked to virulence. We report here that the Tat machinery is present in Burkholderia pseudomallei, B. mallei, and B. thailandensis, and we show that the system is essential for aerobic but not anaerobic growth. Switching off of the Tat system in B. thailandensis grown anaerobically resulted in filamentous bacteria, and bacteria showed increased sensitivity to some β-lactam antibiotics. In Galleria mellonella and zebrafish infection models, the Tat conditional mutant was attenuated. The aerobic growth-restricted phenotype indicates that Tat substrates may play a functional role in oxygen-dependent energy conservation. In other bacteria, aerobic growth restriction in Tat mutants has been attributed to the inability to translocate PetA, the Rieske iron-sulfur protein which forms part of the quinol-cytochrome c oxidoreductase complex. Here, we show that PetA is not responsible for aerobic growth restriction in B. thailandensis. However, we have identified an operon encoding 2 proteins of unknown function (BTH_I2176 and BTH_I2175) that play a role in aerobic growth restriction, and we present evidence that BTH_I2176 is Tat translocated. PMID:24214943

  2. Cranberry impairs selected behaviors essential for virulence in Proteus mirabilis HI4320.

    PubMed

    McCall, Jennifer; Hidalgo, Gabriela; Asadishad, Bahareh; Tufenkji, Nathalie

    2013-06-01

    Proteus mirabilis is an etiological agent of complicated urinary tract infections. North American cranberries (Vaccinium macrocarpon) have long been considered to have protective properties against urinary tract infections. This work reports the effects of cranberry powder (CP) on the motility of P. mirabilis HI4320 and its expression of flaA, flhD, and ureD. Our results show that swimming and swarming motilities and swarmer-cell differentiation were inhibited by CP. Additionally, transcription of the flagellin gene flaA and of flhD, the first gene of the flagellar master operon flhDC, decreased during exposure of P. mirabilis to various concentrations of CP. Moreover, using ureD-gfp, a fusion of the urease accessory gene ureD with gfp, we show that CP inhibits urease expression. Because we demonstrate that CP does not inhibit the growth of P. mirabilis, the observed effects are not attributable to toxicity. Taken together, our results demonstrate that CP hinders motility of P. mirabilis and reduces the expression of important virulence factors.

  3. Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1

    PubMed Central

    Jiang, Bei; Xiao, Bo; Liu, Linde; Ge, Yihe; Hu, Xiaomei

    2016-01-01

    Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1. PMID:26735915

  4. Cross-Regulation between the phz1 and phz2 Operons Maintain a Balanced Level of Phenazine Biosynthesis in Pseudomonas aeruginosa PAO1.

    PubMed

    Cui, Qinna; Lv, Huinan; Qi, Zhuangzhuang; Jiang, Bei; Xiao, Bo; Liu, Linde; Ge, Yihe; Hu, Xiaomei

    2016-01-01

    Gene duplication often provides selective advantages for the survival of microorganisms in adapting to varying environmental conditions. P. aeruginosa PAO1 possesses two seven-gene operons [phz1 (phzA1B1C1D1E1F1G1) and phz2 (phzA2B2C2D2E2F2G2)] that are involved in the biosynthesis of phenazine-1-carboxylic acid and its derivatives. Although the two operons are highly homologous and their functions are well known, it is unclear how the two phz operons coordinate their expressions to maintain the phenazine biosynthesis. By constructing single and double deletion mutants of the two phz operons, we found that the phz1-deletion mutant produced the same or less amount of phenazine-1-carboxylic acid and pyocyanin in GA medium than the phz2-knockout mutant while the phz1-phz2 double knockout mutant did not produce any phenazines. By generating phzA1 and phzA2 translational and transcriptional fusions with a truncated lacZ reporter, we found that the expression of the phz1 operon increased significantly at the post-transcriptional level and did not alter at the transcriptional level in the absence of the phz2 operon. Surprisingly, the expression the phz2 operon increased significantly at the post-transcriptional level and only moderately at the transcriptional level in the absence of the phz1 operon. Our findings suggested that a complex cross-regulation existed between the phz1 and phz2 operons. By mediating the upregulation of one phz operon expression while the other was deleted, this crosstalk would maintain the homeostatic balance of phenazine biosynthesis in P. aeruginosa PAO1. PMID:26735915

  5. Effect of DNA looping on the induction kinetics of the lac operon.

    PubMed

    Narang, Atul

    2007-08-21

    The induction of the lac operon follows cooperative kinetics. The first mechanistic model of these kinetics is the de facto standard in the modeling literature [Yagil, G., Yagil, E., 1971. On the relation between effector concentration and the rate of induced enzyme synthesis. Biophys. J. 11, 11-17]. Yet, subsequent studies have shown that the model is based on incorrect assumptions. Specifically, the repressor is a tetramer with four (not two) inducer-binding sites, and the operon contains two auxiliary operators (in addition to the main operator). Furthermore, these structural features are crucial for the formation of DNA loops, the key determinants of lac repression and induction. Indeed, the repression is determined almost entirely (>95%) by the looped complexes [Oehler, S., Eismann, E.R., Krämer, H., Müller-Hill, B., 1990. The three operators of the lac operon cooperate in repression. EMBO J. 9(4), 973-979], and the pronounced cooperativity of the induction curve hinges upon the existence of the looped complexes [Oehler, S., Alberti, S., Müller-Hill, B., 2006. Induction of the lac promoter in the absence of DNA loops and the stoichiometry of induction. Nucleic Acids Res. 34(2), 606-612]. Here, we formulate a model of lac induction taking due account of the tetrameric structure of the repressor and the existence of looped complexes. We show that: (1) The kinetics are significantly more cooperative than those predicted by the Yagil and Yagil model. The cooperativity is higher because the formation of looped complexes is easily abolished by repressor-inducer binding. (2) The model provides good fits to the repression data for cells containing wild-type tetrameric or mutant dimeric repressor, as well as the induction curves for 6 different strains of Escherichia coli. It also implies that the ratios of certain looped and non-looped complexes are independent of inducer and repressor levels, a conclusion that can be rigorously tested by gel electrophoresis. (3

  6. Complex processing patterns of mRNAs of the large ATP synthase operon in Arabidopsis chloroplasts.

    PubMed

    Malik Ghulam, Mustafa; Ghulam, Mustafa Malik; Courtois, Florence; Lerbs-Mache, Silva; Merendino, Livia

    2013-01-01

    Chloroplasts are photosynthetic cell organelles which have evolved from endosymbiosis of the cyanobacterial ancestor. In chloroplasts, genes are still organized into transcriptional units as in bacteria but the corresponding poly-cistronic mRNAs undergo complex processing events, including inter-genic cleavage and 5' and 3' end-definition. The current model for processing proposes that the 3' end of the upstream cistron transcripts and the 5' end of the downstream cistron transcripts are defined by the same RNA-binding protein and overlap at the level of the protein-binding site. We have investigated the processing mechanisms that operate within the large ATP synthase (atp) operon, in Arabidopsis thaliana chloroplasts. This operon is transcribed by the plastid-encoded RNA polymerase starting from two promoters, which are upstream and within the operon, respectively, and harbors four potential sites for RNA-binding proteins. In order to study the functional significance of the promoters and the protein-binding sites for the maturation processes, we have performed a detailed mapping of the atp transcript ends. Our data indicate that in contrast to maize, atpI and atpH transcripts with overlapping ends are very rare in Arabidopsis. In addition, atpA mRNAs, which overlap with atpF mRNAs, are even truncated at the 3' end, thus representing degradation products. We observe, instead, that the 5' ends of nascent poly-cistronic atp transcripts are defined at the first protein-binding site which follows either one of the two transcription initiation sites, while the 3' ends are defined at the subsequent protein-binding sites or at hairpin structures that are encountered by the progressing RNA polymerase. We conclude that the overlapping mechanisms of mRNA protection have only a limited role in obtaining stable processed atp mRNAs in Arabidopsis. Our findings suggest that during evolution of different plant species as maize and Arabidopsis, chloroplasts have evolved multiple

  7. Role of the ganSPQAB Operon in Degradation of Galactan by Bacillus subtilis.

    PubMed

    Watzlawick, Hildegard; Morabbi Heravi, Kambiz; Altenbuchner, Josef

    2016-10-15

    Bacillus subtilis possesses different enzymes for the utilization of plant cell wall polysaccharides. This includes a gene cluster containing galactan degradation genes (ganA and ganB), two transporter component genes (ganQ and ganP), and the sugar-binding lipoprotein-encoding gene ganS (previously known as cycB). These genes form an operon that is regulated by GanR. The degradation of galactan by B. subtilis begins with the activity of extracellular GanB. GanB is an endo-β-1,4-galactanase and is a member of glycoside hydrolase (GH) family 53. This enzyme was active on high-molecular-weight arabinose-free galactan and mainly produced galactotetraose as well as galactotriose and galactobiose. These galacto-oligosaccharides may enter the cell via the GanQP transmembrane proteins of the galactan ABC transporter. The specificity of the galactan ABC transporter depends on the sugar-binding lipoprotein, GanS. Purified GanS was shown to bind galactotetraose and galactotriose using thermal shift assay. The energy for this transport is provided by MsmX, an ATP-binding protein. The transported galacto-oligosaccharides are further degraded by GanA. GanA is a β-galactosidase that belongs to GH family 42. The GanA enzyme was able to hydrolyze short-chain β-1,4-galacto-oligosaccharides as well as synthetic β-galactopyranosides into galactose. Thermal shift assay as well as electrophoretic mobility shift assay demonstrated that galactobiose is the inducer of the galactan operon regulated by GanR. DNase I footprinting revealed that the GanR protein binds to an operator overlapping the -35 box of the σ(A)-type promoter of Pgan, which is located upstream of ganS IMPORTANCE: Bacillus subtilis is a Gram-positive soil bacterium that utilizes different types of carbohydrates, such as pectin, as carbon sources. So far, most of the pectin degradation systems and enzymes have been thoroughly studied in B. subtilis Nevertheless, the B. subtilis utilization system of galactan, which is

  8. The effect of cell growth phase on the regulatory cross-talk between flagellar and Spi1 virulence gene expression.

    PubMed

    Mouslim, Chakib; Hughes, Kelly T

    2014-03-01

    The flagellar regulon controls Salmonella biofilm formation, virulence gene expression and the production of the major surface antigen present on the cell surface: flagellin. At the top of a flagellar regulatory hierarchy is the master operon, flhDC, which encodes the FlhD₄C₂ transcriptional complex required for the expression of flagellar, chemotaxis and Salmonella pathogenicity island 1 (Spi1) genes. Of six potential transcriptional start-sites within the flhDC promoter region, only two, P1(flhDC) and P5(flhDC), were functional in a wild-type background, while P6(flhDC) was functional in the absence of CRP. These promoters are transcribed differentially to control either flagellar or Spi1 virulent gene expression at different stages of cell growth. Transcription from P1(flhDC) initiates flagellar assembly and a negative autoregulatory loop through FlhD₄C₂-dependent transcription of the rflM gene, which encodes a repressor of flhDC transcription. Transcription from P1(flhDC) also initiates transcription of the Spi1 regulatory gene, hilD, whose product, in addition to activating Spi1 genes, also activates transcription of the flhDC P5 promoter later in the cell growth phase. The regulators of flhDC transcription (RcsB, LrhA, RflM, HilD, SlyA and RtsB) also exert their control at different stages of the cell growth phase and are also subjected to cell growth phase control. This dynamic of flhDC transcription separates the roles of FlhD₄C₂ transcriptional activation into an early cell growth phase role for flagellar production from a late cell growth phase role in virulence gene expression.

  9. Comparative Proteomic Analyses of Avirulent, Virulent, and Clinical Strains of Mycobacterium tuberculosis Identify Strain-specific Patterns*

    PubMed Central

    Jhingan, Gagan Deep; Kumari, Sangeeta; Jamwal, Shilpa V.; Kalam, Haroon; Arora, Divya; Jain, Neharika; Kumaar, Lakshmi Krishna; Samal, Areejit; Rao, Kanury V. S.; Kumar, Dhiraj; Nandicoori, Vinay Kumar

    2016-01-01

    Mycobacterium tuberculosis is an adaptable intracellular pathogen, existing in both dormant as well as active disease-causing states. Here, we report systematic proteomic analyses of four strains, H37Ra, H37Rv, and clinical isolates BND and JAL, to determine the differences in protein expression patterns that contribute to their virulence and drug resistance. Resolution of lysates of the four strains by liquid chromatography, coupled to mass spectrometry analysis, identified a total of 2161 protein groups covering ∼54% of the predicted M. tuberculosis proteome. Label-free quantification analysis of the data revealed 257 differentially expressed protein groups. The differentially expressed protein groups could be classified into seven K-means cluster bins, which broadly delineated strain-specific variations. Analysis of the data for possible mechanisms responsible for drug resistance phenotype of JAL suggested that it could be due to a combination of overexpression of proteins implicated in drug resistance and the other factors. Expression pattern analyses of transcription factors and their downstream targets demonstrated substantial differential modulation in JAL, suggesting a complex regulatory mechanism. Results showed distinct variations in the protein expression patterns of Esx and mce1 operon proteins in JAL and BND strains, respectively. Abrogating higher levels of ESAT6, an important Esx protein known to be critical for virulence, in the JAL strain diminished its virulence, although it had marginal impact on the other strains. Taken together, this study reveals that strain-specific variations in protein expression patterns have a meaningful impact on the biology of the pathogen. PMID:27151218

  10. Virulence gene profiling and pathogenicity characterization of non-typhoidal Salmonella accounted for invasive disease in humans.

    PubMed

    Suez, Jotham; Porwollik, Steffen; Dagan, Amir; Marzel, Alex; Schorr, Yosef Ilan; Desai, Prerak T; Agmon, Vered; McClelland, Michael; Rahav, Galia; Gal-Mor, Ohad

    2013-01-01

    Human infection with non-typhoidal Salmonella serovars (NTS) infrequently causes invasive systemic disease and bacteremia. To understand better the nature of invasive NTS (iNTS), we studied the gene content and the pathogenicity of bacteremic strains from twelve serovars (Typhimurium, Enteritidis, Choleraesuis, Dublin, Virchow, Newport, Bredeney, Heidelberg, Montevideo, Schwarzengrund, 9,12:l,v:- and Hadar). Comparative genomic hybridization using a Salmonella enterica microarray revealed a core of 3233 genes present in all of the iNTS strains, which include the Salmonella pathogenicity islands 1-5, 9, 13, 14; five fimbrial operons (bcf, csg, stb, sth, sti); three colonization factors (misL, bapA, sinH); and the invasion gene, pagN. In the iNTS variable genome, we identified 16 novel genomic islets; various NTS virulence factors; and six typhoid-associated virulence genes (tcfA, cdtB, hlyE, taiA, STY1413, STY1360), displaying a wider distribution among NTS than was previously known. Characterization of the bacteremic strains in C3H/HeN mice showed clear differences in disease manifestation. Previously unreported characterization of serovars Schwarzengrund, 9,12:l,v:-, Bredeney and Virchow in the mouse model showed low ability to elicit systemic disease, but a profound and elongated shedding of serovars Schwarzengrund and 9,12:l,v:- (as well as Enteritidis and Heidelberg) due to chronic infection of the mouse. Phenotypic comparison in macrophages and epithelial cell lines demonstrated a remarkable intra-serovar variation, but also showed that S. Typhimurium bacteremic strains tend to present lower intracellular growth than gastroenteritis isolates. Collectively, our data demonstrated a common core of virulence genes, which might be required for invasive salmonellosis, but also an impressive degree of genetic and phenotypic heterogeneity, highlighting that bacteremia is a complex phenotype, which cannot be attributed merely to an enhanced invasion or intracellular

  11. Identification and characterization of novel Salmonella mobile elements involved in the dissemination of genes linked to virulence and transmission.

    PubMed

    Moreno Switt, Andrea I; den Bakker, Henk C; Cummings, Craig A; Rodriguez-Rivera, Lorraine D; Govoni, Gregory; Raneiri, Matthew L; Degoricija, Lovorka; Brown, Stephanie; Hoelzer, Karin; Peters, Joseph E; Bolchacova, Elena; Furtado, Manohar R; Wiedmann, Martin

    2012-01-01

    The genetic diversity represented by >2,500 different Salmonella serovars provides a yet largely uncharacterized reservoir of mobile elements that can contribute to the frequent emergence of new pathogenic strains of this important zoonotic pathogen. Currently, our understanding of Salmonella mobile elements is skewed by the fact that most studies have focused on highly virulent or common serovars. To gain a more global picture of mobile elements in Salmonella, we used prediction algorithms to screen for mobile elements in 16 sequenced Salmonella genomes representing serovars for which no prior genome scale mobile element data were available. From these results, selected mobile elements underwent further analyses in the form of validation studies, comparative analyses, and PCR-based population screens. Through this analysis we identified a novel plasmid that has two cointegrated replicons (IncI1-IncFIB); this plasmid type was found in four genomes representing different Salmonella serovars and contained a virulence gene array that had not been previously identified. A Salmonella Montevideo isolate contained an IncHI and an IncN2 plasmid, which both encoded antimicrobial resistance genes. We also identified two novel genomic islands (SGI2 and SGI3), and 42 prophages with mosaic architecture, seven of them harboring known virulence genes. Finally, we identified a novel integrative conjugative element (ICE) encoding a type IVb pilus operon in three non-typhoidal Salmonella serovars. Our analyses not only identified a considerable number of mobile elements that have not been previously reported in Salmonella, but also found evidence that these elements facilitate transfer of genes that were previously thought to be limited in their distribution among Salmonella serovars. The abundance of mobile elements encoding pathogenic properties may facilitate the emergence of strains with novel combinations of pathogenic traits.

  12. Cyanobacterial flv4-2 Operon-Encoded Proteins Optimize Light Harvesting and Charge Separation in Photosystem II.

    PubMed

    Chukhutsina, Volha; Bersanini, Luca; Aro, Eva-Mari; van Amerongen, Herbert

    2015-05-01

    Photosystem II (PSII) complexes drive the water-splitting reaction necessary to transform sunlight into chemical energy. However, too much light can damage and disrupt PSII. In cyanobacteria, the flv4-2 operon encodes three proteins (Flv2, Flv4, and Sll0218), which safeguard PSII activity under air-level CO2 and in high light conditions. However, the exact mechanism of action of these proteins has not been clarified yet. We demonstrate that the PSII electron transfer properties are influenced by the flv4-2 operon-encoded proteins. Accelerated secondary charge separation kinetics was observed upon expression/overexpression of the flv4-2 operon. This is likely induced by docking of the Flv2/Flv4 heterodimer in the vicinity of the QB pocket of PSII, which, in turn, increases the QB redox potential and consequently stabilizes forward electron transfer. The alternative electron transfer route constituted by Flv2/Flv4 sequesters electrons from QB(-) guaranteeing the dissipation of excess excitation energy in PSII under stressful conditions. In addition, we demonstrate that in the absence of the flv4-2 operon-encoded proteins, about 20% of the phycobilisome antenna becomes detached from the reaction centers, thus decreasing light harvesting. Phycobilisome detachment is a consequence of a decreased relative content of PSII dimers, a feature observed in the absence of the Sll0218 protein.

  13. Regulation of Internal Promoters in a Zinc-Responsive Operon Is Influenced by Transcription from Upstream Promoters

    PubMed Central

    Napolitano, Mauro; Rubio, Miguel Ángel; Camargo, Sergio

    2013-01-01

    In the cyanobacterium Anabaena sp. strain PCC 7120 (also known as Nostoc sp. strain PCC 7120), a zinc-responsive operon (all4725-all4721) has been described, which contains 4 distinct promoters. The two most upstream ones bind Zur with high affinity, whereas the other two do not or do so with a very low affinity. In this paper, a detailed characterization of the four promoters is presented, showing that all four were induced by metal depletion, and they were constitutively derepressed in a zur mutant, despite the two downstream promoters not being direct targets for this regulator. Crucially, induction by metal depletion of the two downstream promoters was abrogated when transcription initiated at the upstream promoters was interrupted by a polar insertion midway in the operon. In contrast, insertion of a nitrogen-responsive promoter at a roughly similar position provoked the two downstream promoters to adopt a regulatory pattern mimicking that of the inserted promoter. Thus, regulation of the two downstream promoters is apparently influenced by transcription from promoters upstream. Evidence is presented indicating that the activity of the two downstream promoters is kept basal in Anabaena by repression. A regulatory model compatible with these results is proposed, where promoters controlled by repression in bacterial operons may be subjected to a hierarchical regulation depending on their position in the operon. According to this model, internal promoters may respond to stimuli governing the activity of promoters upstream by an indirect regulation and to specific stimuli by a direct regulation. PMID:23316045

  14. Influence of the feedback loops in the trp operon of B. subtilis on the system dynamic response and noise amplitude.

    PubMed

    Zamora-Chimal, Criseida; Santillán, Moisés; Rodríguez-González, Jesús

    2012-10-01

    In this paper we introduce a mathematical model for the tryptophan operon regulatory pathway in Bacillus subtilis. This model considers the transcription-attenuation, and the enzyme-inhibition regulatory mechanisms. Special attention is paid to the estimation of all the model parameters from reported experimental data. With the aid of this model we investigate, from a mathematical-modeling point of view, whether the existing multiplicity of regulatory feedback loops is advantageous in some sense, regarding the dynamic response and the biochemical noise in the system. The tryptophan operon dynamic behavior is studied by means of deterministic numeric simulations, while the biochemical noise is analyzed with the aid of stochastic simulations. The model feasibility is tested comparing its stochastic and deterministic results with experimental reports. Our results for the wildtype and for a couple of mutant bacterial strains suggest that the enzyme-inhibition feedback loop, dynamically accelerates the operon response, and plays a major role in the reduction of biochemical noise. Also, the transcription-attenuation feedback loop makes the trp operon sensitive to changes in the endogenous tryptophan level, and increases the amplitude of the biochemical noise.

  15. Functional Conservation of the Capacity for ent-Kaurene Biosynthesis and an Associated Operon in Certain Rhizobia

    PubMed Central

    Hershey, David M.; Lu, Xuan; Zi, Jiachen

    2014-01-01

    Bacterial interactions with plants are accompanied by complex signal exchange processes. Previously, the nitrogen-fixing symbiotic (rhizo)bacterium Bradyrhizobium japonicum was found to carry adjacent genes encoding two sequentially acting diterpene cyclases that together transform geranylgeranyl diphosphate to ent-kaurene, the olefin precursor to the gibberellin plant hormones. Species from the three other major genera of rhizobia were found to have homologous terpene synthase genes. Cloning and functional characterization of a representative set of these enzymes confirmed the capacity of each genus to produce ent-kaurene. Moreover, comparison of their genomic context revealed that these diterpene synthases are found in a conserved operon which includes an adjacent isoprenyl diphosphate synthase, shown here to produce the geranylgeranyl diphosphate precursor, providing a critical link to central metabolism. In addition, the rest of the operon consists of enzymatic genes that presumably lead to a more elaborated diterpenoid, although the production of gibberellins was not observed. Nevertheless, it has previously been shown that the operon is selectively expressed during nodulation, and the scattered distribution of the operon via independent horizontal gene transfer within the symbiotic plasmid or genomic island shown here suggests that such diterpenoid production may modulate the interaction of these particular symbionts with their host plants. PMID:24142247

  16. Influence of the feedback loops in the trp operon of B. subtilis on the system dynamic response and noise amplitude.

    PubMed

    Zamora-Chimal, Criseida; Santillán, Moisés; Rodríguez-González, Jesús

    2012-10-01

    In this paper we introduce a mathematical model for the tryptophan operon regulatory pathway in Bacillus subtilis. This model considers the transcription-attenuation, and the enzyme-inhibition regulatory mechanisms. Special attention is paid to the estimation of all the model parameters from reported experimental data. With the aid of this model we investigate, from a mathematical-modeling point of view, whether the existing multiplicity of regulatory feedback loops is advantageous in some sense, regarding the dynamic response and the biochemical noise in the system. The tryptophan operon dynamic behavior is studied by means of deterministic numeric simulations, while the biochemical noise is analyzed with the aid of stochastic simulations. The model feasibility is tested comparing its stochastic and deterministic results with experimental reports. Our results for the wildtype and for a couple of mutant bacterial strains suggest that the enzyme-inhibition feedback loop, dynamically accelerates the operon response, and plays a major role in the reduction of biochemical noise. Also, the transcription-attenuation feedback loop makes the trp operon sensitive to changes in the endogenous tryptophan level, and increases the amplitude of the biochemical noise. PMID:22713856

  17. Cyanobacterial flv4-2 Operon-Encoded Proteins Optimize Light Harvesting and Charge Separation in Photosystem II.

    PubMed

    Chukhutsina, Volha; Bersanini, Luca; Aro, Eva-Mari; van Amerongen, Herbert

    2015-05-01

    Photosystem II (PSII) complexes drive the water-splitting reaction necessary to transform sunlight into chemical energy. However, too much light can damage and disrupt PSII. In cyanobacteria, the flv4-2 operon encodes three proteins (Flv2, Flv4, and Sll0218), which safeguard PSII activity under air-level CO2 and in high light conditions. However, the exact mechanism of action of these proteins has not been clarified yet. We demonstrate that the PSII electron transfer properties are influenced by the flv4-2 operon-encoded proteins. Accelerated secondary charge separation kinetics was observed upon expression/overexpression of the flv4-2 operon. This is likely induced by docking of the Flv2/Flv4 heterodimer in the vicinity of the QB pocket of PSII, which, in turn, increases the QB redox potential and consequently stabilizes forward electron transfer. The alternative electron transfer route constituted by Flv2/Flv4 sequesters electrons from QB(-) guaranteeing the dissipation of excess excitation energy in PSII under stressful conditions. In addition, we demonstrate that in the absence of the flv4-2 operon-encoded proteins, about 20% of the phycobilisome antenna becomes detached from the reaction centers, thus decreasing light harvesting. Phycobilisome detachment is a consequence of a decreased relative content of PSII dimers, a feature observed in the absence of the Sll0218 protein. PMID:25704162

  18. Co2+-dependent gene expression in Streptococcus pneumoniae: opposite effect of Mn2+ and Co2+ on the expression of the virulence genes psaBCA, pcpA, and prtA

    PubMed Central

    Manzoor, Irfan; Shafeeq, Sulman; Kloosterman, Tomas G.; Kuipers, Oscar P.

    2015-01-01

    Manganese (Mn2+)-, zinc (Zn2+)- and copper (Cu2+) play significant roles in transcriptional gene regulation, physiology, and virulence of Streptococcus pneumoniae. So far, the effect of the important transition metal ion cobalt (Co2+) on gene expression of S. pneumoniae has not yet been explored. Here, we study the impact of Co2+ stress on the transcriptome of S. pneumoniae strain D39. BLAST searches revealed that the genome of S. pneumoniae encodes a putative Co2+-transport operon (cbi operon), the expression of which we show here to be induced by a high Co2+ concentration. Furthermore, we found that Co2+, as has been shown previously for Zn2+, can cause derepression of the genes of the PsaR virulence regulon, encoding the Mn2+-uptake system PsaBCA, the choline binding protein PcpA and the cell-wall associated serine protease PrtA. Interestingly, although Mn2+ represses expression of the PsaR regulon and Co2+ leads to derepression, both metal ions stimulate interaction of PsaR with its target promoters. These data will be discussed in the light of previous studies on similar metal-responsive transcriptional regulators. PMID:26257722

  19. Tight-binding repressors of the lac operon: selection system and in vitro analysis.

    PubMed

    Pfahl, M

    1979-01-01

    The isolation and characterization of altered repressors of the lac operon which have an increased affinity for an operator should give useful clues about the molecular basis for the very tight and specific interaction between repressor and operator. A selection system has been devised which allows the isolation of such repressor mutants. This system selects for mutant repressors which can overcome lac operator-constitutive (Oc) mutations. By using in vivo assays, 24 candidates were obtained which, compared with wild type, have an increased trans effect of their repressor on one or several Oc operators. Three of these candidates have been investigated in vitro; the affinity of their repressor for inducer was unchanged, whereas the affinity for wild-type operator was increased 15-, 86-, and 262-fold, respectively. PMID:104955

  20. Analysis of the complete nucleotide sequence of the Agrobacterium tumefaciens virB operon.

    PubMed

    Thompson, D V; Melchers, L S; Idler, K B; Schilperoort, R A; Hooykaas, P J

    1988-05-25

    The complete nucleotide sequence of the virB locus, from the octopine Ti plasmid of Agrobacterium tumefaciens strain 15955, has been determined. In the large virB-operon (9600 nucleotides) we have identified eleven open reading frames, designated virB1 to virB11. From DNA sequence analysis it is proposed that nearly all VirB products, i.e. VirB1 to VirB9, are secreted or membrane associated proteins. Interestingly, both a membrane protein (VirB4) and a potential cytoplasmic protein (VirB11) contain the consensus amino acid sequence of ATP-binding proteins. In view of the conjugative T-DNA transfer model, the VirB proteins are suggested to act at the bacterial surface and there play an important role in directing T-DNA transfer to plant cells. PMID:2837739

  1. Optimal performance of the tryptophan operon of E. coli: a stochastic, dynamical, mathematical-modeling approach.

    PubMed

    Salazar-Cavazos, Emanuel; Santillán, Moisés

    2014-02-01

    In this work, we develop a detailed, stochastic, dynamical model for the tryptophan operon of E. coli, and estimate all of the model parameters from reported experimental data. We further employ the model to study the system performance, considering the amount of biochemical noise in the trp level, the system rise time after a nutritional shift, and the amount of repressor molecules necessary to maintain an adequate level of repression, as indicators of the system performance regime. We demonstrate that the level of cooperativity between repressor molecules bound to the first two operators in the trp promoter affects all of the above enlisted performance characteristics. Moreover, the cooperativity level found in the wild-type bacterial strain optimizes a cost-benefit function involving low biochemical noise in the tryptophan level, short rise time after a nutritional shift, and low number of regulatory molecules. PMID:24307084

  2. Construction of metabolic operons catalyzing the de novo biosynthesis of indigo in Escherichia coli.

    PubMed

    Murdock, D; Ensley, B D; Serdar, C; Thalen, M

    1993-03-01

    The efficient production of the textile dye indigo by fermentation has been a goal since the early 1980's when the first bacterial strains capable of this synthesis were constructed. We report here the development of a recombinant microorganism that directly synthesizes indigo from glucose. This construction involved the cloning and genetic manipulation of at least 9 genes and modifications of the fermentation medium to help stabilize the biosynthetic activity. Directed genetic changes in two operons caused significant increases in reaction rates and in the stability of the catalytic enzymes. This example of whole cell catalysis by a recombinant Escherichia coli represents a novel and environmentally sound approach to the synthesis of a high value specialty chemical.

  3. Operon structure and cotranslational subunit association direct protein assembly in bacteria.

    PubMed

    Shieh, Yu-Wei; Minguez, Pablo; Bork, Peer; Auburger, Josef J; Guilbride, D Lys; Kramer, Günter; Bukau, Bernd

    2015-11-01

    Assembly of protein complexes is considered a posttranslational process involving random collision of subunits. We show that within the Escherichia coli cytosol, bacterial luciferase subunits LuxA and LuxB assemble into complexes close to the site of subunit synthesis. Assembly efficiency decreases markedly if subunits are synthesized on separate messenger RNAs from genes integrated at distant chromosomal sites. Subunit assembly initiates cotranslationally on nascent LuxB in vivo. The ribosome-associated chaperone trigger factor delays the onset of cotranslational interactions until the LuxB dimer interface is fully exposed. Protein assembly is thus directly coupled to the translation process and involves spatially confined, actively chaperoned cotranslational subunit interactions. Bacterial gene organization into operons therefore reflects a fundamental cotranslational mechanism for spatial and temporal regulation that is vital to effective assembly of protein complexes. PMID:26405228

  4. The presence of the glycolysis operon in SAR11 genomes is positively correlated with ocean productivity.

    PubMed

    Schwalbach, M S; Tripp, H J; Steindler, L; Smith, D P; Giovannoni, S J

    2010-02-01

    Bacteria in the SAR11 clade are highly abundant in marine surface waters, but currently little is known about the carbon compounds that support these large heterotrophic populations. To better understand the carbon requirements of these organisms, we conducted a multiphasic exploration of carbohydrate utilization among SAR11 isolates from the Northeast Pacific Ocean and the Sargasso Sea. A comparison of three SAR11 genomes showed they all lacked a recognizable PTS system, the oxidative portion of the pentose phosphate shunt (zwf-, pgl-), genes for the Embden-Meyerhoff-Parnas (pfk-, pyk-) and Entner-Doudoroff (eda-) pathways of glycolysis. Strain HTCC7211, isolated from an ocean gyre, was missing other glycolysis genes as well. Growth assays, radioisotopes, metagenomics and microarrays were used to test the hypothesis that these isolates might be limited in their abilities to transport and oxidize exogenous carbohydrates. Galactose, fucose, rhamnose, arabinose, ribose and mannose could not serve as carbon sources for the isolates tested. However, differences in glucose utilization were detected between coastal and ocean gyre isolates, with the coastal isolates capable of transporting, incorporating and oxidizing glucose while the open ocean isolate could not. Subsequent microarray analysis of a coastal isolate suggested that an operon encoding a variant of the Entner-Doudoroff pathway is likely responsible for the observed differences in glucose utilization. Metagenomic analysis indicated this operon is more commonly found in coastal environments and is positively correlated with chlorophyll a concentrations. Our results indicated that glycolysis is a variable metabolic property of SAR11 metabolism and suggest that glycolytic SAR11 are more common in productive marine environments.

  5. The presence of the glycolysis operon in SAR11 genomes is positively correlated with ocean productivity.

    PubMed

    Schwalbach, M S; Tripp, H J; Steindler, L; Smith, D P; Giovannoni, S J

    2010-02-01

    Bacteria in the SAR11 clade are highly abundant in marine surface waters, but currently little is known about the carbon compounds that support these large heterotrophic populations. To better understand the carbon requirements of these organisms, we conducted a multiphasic exploration of carbohydrate utilization among SAR11 isolates from the Northeast Pacific Ocean and the Sargasso Sea. A comparison of three SAR11 genomes showed they all lacked a recognizable PTS system, the oxidative portion of the pentose phosphate shunt (zwf-, pgl-), genes for the Embden-Meyerhoff-Parnas (pfk-, pyk-) and Entner-Doudoroff (eda-) pathways of glycolysis. Strain HTCC7211, isolated from an ocean gyre, was missing other glycolysis genes as well. Growth assays, radioisotopes, metagenomics and microarrays were used to test the hypothesis that these isolates might be limited in their abilities to transport and oxidize exogenous carbohydrates. Galactose, fucose, rhamnose, arabinose, ribose and mannose could not serve as carbon sources for the isolates tested. However, differences in glucose utilization were detected between coastal and ocean gyre isolates, with the coastal isolates capable of transporting, incorporating and oxidizing glucose while the open ocean isolate could not. Subsequent microarray analysis of a coastal isolate suggested that an operon encoding a variant of the Entner-Doudoroff pathway is likely responsible for the observed differences in glucose utilization. Metagenomic analysis indicated this operon is more commonly found in coastal environments and is positively correlated with chlorophyll a concentrations. Our results indicated that glycolysis is a variable metabolic property of SAR11 metabolism and suggest that glycolytic SAR11 are more common in productive marine environments. PMID:19889000

  6. The stb Operon Balances the Requirements for Vegetative Stability and Conjugative Transfer of Plasmid R388

    PubMed Central

    Guynet, Catherine; Cuevas, Ana; Moncalián, Gabriel; de la Cruz, Fernando

    2011-01-01

    The conjugative plasmid R388 and a number of other plasmids carry an operon, stbABC, adjacent to the origin of conjugative transfer. We investigated the role of the stbA, stbB, and stbC genes. Deletion of stbA affected both conjugation and stability. It led to a 50-fold increase in R388 transfer frequency, as well as to high plasmid loss. In contrast, deletion of stbB abolished conjugation but provoked no change in plasmid stability. Deletion of stbC showed no effect, neither in conjugation nor in stability. Deletion of the entire stb operon had no effect on conjugation, which remained as in the wild-type plasmid, but led to a plasmid loss phenotype similar to that of the R388ΔstbA mutant. We concluded that StbA is required for plasmid stability and that StbA and StbB control conjugation. We next observed the intracellular positioning of R388 DNA molecules and showed that they localize as discrete foci evenly distributed in live Escherichia coli cells. Plasmid instability of the R388ΔΔstbA mutant correlated with aberrant localization of the plasmid DNA molecules as clusters, either at one cell pole, at both poles, or at the cell center. In contrast, plasmid molecules in the R388ΔΔstbB mutant were mostly excluded from the cell poles. Thus, results indicate that defects in both plasmid maintenance and transfer are a consequence of variations in the intracellular positioning of plasmid DNA. We propose that StbA and StbB constitute an atypical plasmid stabilization system that reconciles two modes of plasmid R388 physiology: a maintenance mode (replication and segregation) and a propagation mode (conjugation). The consequences of this novel concept in plasmid physiology will be discussed. PMID:21625564

  7. A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

    PubMed

    Zhu, Y; Lin, E C

    1988-05-01

    L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose. PMID:2834341

  8. The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex.

    PubMed Central

    Rossi, M; Pollock, W B; Reij, M W; Keon, R G; Fu, R; Voordouw, G

    1993-01-01

    The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases. Images PMID:8335628

  9. Synthesis and degradation of the mRNA of the Tn21 mer operon.

    PubMed

    Gambill, B D; Summers, A O

    1992-05-20

    The mercury resistance locus encoded by Tn21 on the monocopy IncFII plasmid R100 (merTn21) consists of a metal-responsive activator/repressor, merR, which controls initiation of a polycistronic message that includes genes for the uptake (merTPC) and reduction (merA) of Hg2+ and merD, which may also play a minor regulatory role. Comparison of the relative abundance of the 5' and 3' ends of the merTPCAD transcript revealed a strong transcriptional gradient in the operon, consistent with previous observations of lower relative abundance of the more promoter-distal gene products. In vivo mRNA degradation rates varied only slightly for the different genes: however, the rates of mRNA synthesis varied considerably from the beginning to the end of the operon. Specifically, mRNA corresponding to the promoter-proximal genes, merTPC, achieved a maximum in vivo synthesis rate between 60 and 120 seconds after induction; this rate was maintained for approximately ten minutes. In contrast, the synthesis rates of mRNA corresponding to the promoter-distal genes merA and merD, were initially fivefold lower than the rates of the promoter-proximal genes for the first five minutes after induction, and then rose gradually to approximately 50% of the merTPC synthesis rates. These data suggested that early after induction only 20% of the transcripts initiating at merT proceed beyond merC. At later times after induction approximately 50% of the transcripts proceed beyond merC. Nuclease end mapping did not reveal any discrete termination events in the merPCA region, thus, premature termination may occur at many sites.

  10. Entamoeba histolytica. Phagocytosis as a virulence factor

    PubMed Central

    1983-01-01

    In this paper, we attempted to define the role of phagocytosis in the virulence of Entamoeba histolytica. We have isolated, from a highly phagocytic and virulent strain, a clone deficient in phagocytosis. Trophozoites of wild-type strain HM1:IMSS were fed with Escherichia coli strain CR34-Thy- grown on 5-bromo,2'-deoxyuridine. The trophozoites that had incorporated the base analog through phagocytosis of the bacteria were killed by irradiation with 310 nm light. The survivors, presumably trophozoites defective in phagocytosis, were grown until log phase and submitted two more times to the selection procedure. Clone L-6, isolated from a subpopulation resulting from this selection procedure, showed 75-85% less erythrophagocytic activity than the wild-type strain. The virulence of clone L-6 and strain HM1:IMSS was measured. The inoculum required to induce liver abscesses in 50% of the newborn hamsters inoculated (AD50) of HM1:IMSS was 1.5 X 10(4) trophozoites. Clone L-6 trophozoites failed to induce liver abscesses in newborn hamsters even with inocula of 5 X 10(5) trophozoites. Virulence revertants were obtained by successive passage of L-6 trophozoites through the liver of young hamsters. The trophozoites that recovered the ability to produce liver abscesses simultaneously recuperate high erythrophagocytic rates. These results show that phagocytosis is involved in the aggressive mechanisms of E. histolytica. PMID:6313842

  11. Entamoeba histolytica: oxygen resistance and virulence.

    PubMed

    Ramos-Martínez, Espiridión; Olivos-García, Alfonso; Saavedra, Emma; Nequiz, Mario; Sánchez, Ernesto C; Tello, Eusebio; El-Hafidi, Mohamed; Saralegui, Andrés; Pineda, Erika; Delgado, José; Montfort, Irmgard; Pérez-Tamayo, Ruy

    2009-05-01

    Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E. histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O(2). The only difference observed was a higher in vitro susceptibility of nvEh to O(2). The molecular mechanism of that difference was analyzed in both groups of amoebae after high O(2) exposure. vEh O(2) resistance correlated with: (i) higher O(2) reduction (O(2)(-) and H(2)O(2) production); (ii) increased H(2)O(2) resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nvEh after O(2) exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O(2) and H(2)O(2) as well as PFOR reactivation, whereas nvEh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death.

  12. Molecular nature of virulence in Entamoeba histolytica.

    PubMed

    Olivos-García, Alfonso; Saavedra, Emma; Ramos-Martínez, Espiridión; Nequiz, Mario; Pérez-Tamayo, Ruy

    2009-12-01

    For many years virulence of pathogenic Entamoeba histolytica has been attributed to the capacity of the parasite to destroy tissues through the expression and/or secretion of various molecules. Such view is supported mainly by in vitro experimentation, whereas data obtained by using animal models of the disease have clearly demonstrated that the host's inflammatory response is primarily responsible for tissue damage. This review analyzes the content and/or activity of some of the presumed toxic amebic molecules present in amebic strains with different degrees of virulence compared to various parasite in vitro functions that are supposed to correlate with in vivo virulence. The analysis suggests that amebic virulence is primarily determined by the parasite's capacity to adapt and survive the aerobic conditions present in animal tissues. This initial episode in the host-parasite relationship is an absolute requirement for the further development of tissue lesions, which result from the concerted action of many molecules derived from both, the host and the parasite.

  13. Rare Helicobacter pylori Virulence Genotypes in Bhutan

    PubMed Central

    Matsunari, Osamu; Miftahussurur, Muhammad; Shiota, Seiji; Suzuki, Rumiko; Vilaichone, Ratha-korn; Uchida, Tomohisa; Ratanachu-ek, Thawee; Tshering, Lotay; Mahachai, Varocha; Yamaoka, Yoshio

    2016-01-01

    Both the prevalence of Helicobacter pylori infection and the incidence of gastric cancer are high in Bhutan. The high incidence of atrophic gastritis and gastric cancer suggest the phylogeographic origin of an infection with a more virulent strain of H. pylori. More than 90% of Bhutanese strains possessed the highly virulent East Asian-type CagA and all strains had the most virulent type of vacA (s1 type). More than half also had multiple repeats in East Asian-type CagA, which are rare in other countries and are reported characteristictly found in assciation with atrophic gastritis and gastric cancer consistent with Bhutanese strains having multiple H. pylori virulence factors associated with an increase in gastric cancer risk. Phylogeographic analyses showed that most Bhutanese strains belonged to the East Asian population type with some strains (17.5%) sharing East Asian and Amerindian components. Only 9.5% belonged to the European type consistant with H. pylori in Bhutan representing an intermediate evolutionary stage between H. pylori from European and East Asian countries. PMID:26931643

  14. Entamoeba histolytica: oxygen resistance and virulence.

    PubMed

    Ramos-Martínez, Espiridión; Olivos-García, Alfonso; Saavedra, Emma; Nequiz, Mario; Sánchez, Ernesto C; Tello, Eusebio; El-Hafidi, Mohamed; Saralegui, Andrés; Pineda, Erika; Delgado, José; Montfort, Irmgard; Pérez-Tamayo, Ruy

    2009-05-01

    Entamoeba histolytica virulence has been attributed to several amoebic molecules such as adhesins, amoebapores and cysteine proteinases, but supporting evidence is either partial or indirect. In this work we compared several in vitro and in vivo features of both virulent E. histolytica (vEh) and non-virulent E. histolytica (nvEh) axenic HM-1 IMSS strains, such as complement resistance, proteinase activity, haemolytic, phagocytic and cytotoxic capacities, survival in mice caecum, and susceptibility to O(2). The only difference observed was a higher in vitro susceptibility of nvEh to O(2). The molecular mechanism of that difference was analyzed in both groups of amoebae after high O(2) exposure. vEh O(2) resistance correlated with: (i) higher O(2) reduction (O(2)(-) and H(2)O(2) production); (ii) increased H(2)O(2) resistance and thiol peroxidase activity, and (iii) reversible pyruvate: ferredoxin oxidoreductase (PFOR) inhibition. Despite the high level of carbonylated proteins in nvEh after O(2) exposure, membrane oxidation by reactive oxygen species was not observed. These results suggest that the virulent phenotype of E. histolytica is related to the greater ability to reduce O(2) and H(2)O(2) as well as PFOR reactivation, whereas nvEh undergoes irreversible PFOR inhibition resulting in metabolic failure and amoebic death. PMID:19073188

  15. Virulence factors of medically important fungi.

    PubMed Central

    Hogan, L H; Klein, B S; Levitz, S M

    1996-01-01

    Human fungal pathogens have become an increasingly important medical problem with the explosion in the number of immunocompromised patients as a result of cancer, steroid therapy, chemotherapy, and AIDS. Additionally, the globalization of travel and expansion of humankind into previously undisturbed habitats have led to the reemergence of old fungi and new exposure to previously undescribed fungi. Until recently, relatively little was known about virulence factors for the medically important fungi. With the advent of molecular genetics, rapid progress has now been made in understanding the basis of pathogenicity for organisms such as Aspergillus species and Cryptococcus neoformans. The twin technologies of genetic transformation and "knockout" deletion construction allowed for genetic tests of virulence factors in these organisms. Such knowledge will prove invaluable for the rational design of antifungal therapies. Putative virulence factors and attributes are reviewed for Aspergillus species, C. neoformans, the dimorphic fungal pathogens, and others, with a focus upon a molecular genetic approach. Candida species are excluded from coverage, having been the subject of numerous recent reviews. This growing body of knowledge about fungal pathogens and their virulence factors will significantly aid efforts to treat the serious diseases they cause. PMID:8894347

  16. Novel Strategies to Combat Bacterial Virulence

    PubMed Central

    Lynch, S.V.; Wiener-Kronish, J.P.

    2010-01-01

    Purpose of review Incidences of antimicrobial resistant infections have increased dramatically over the past several decades and are associated with adverse patient outcomes. Alternative approaches to combat infection are critical, and have led to the development of more specific drugs targeted at particular bacterial virulence systems or essential regulatory pathways. The purpose of this review is to highlight the recent developments in anti-bacterial therapy and the novel approaches toward increasing our therapeutic armory against bacterial infection. Recent findings Although classic antibiotic development is not occurring rapidly, alternative therapeutics that target specific bacterial virulence systems are progressing from the discovery stage through the FDA approval process. Here we review novel antibodies that target specific virulence systems as well as a variety of newly discovered small molecules that block bacterial attachment, communication systems (quorum sensing) or important regulatory processes associated with virulence gene expression. Summary The success of novel therapeutics could significantly change clinical practice. Furthermore, the complications of collateral damage due to antibiotic administration e.g. suprainfections or decreased host immunity due to loss of synergistic bacterial communities, may be minimized using therapeutics that specifically target pathogenic behavior. PMID:18787455

  17. Bacterial Reductionism: Host Thiols Enhance Virulence

    PubMed Central

    Sperandio, Vanessa

    2016-01-01

    Intracellular bacteria exploit host cytosolic signals to upregulate virulence genes. In this issue of Cell Host & Microbe, Wong et al. (2015) show that Burkholderia pseudomallei senses host cytosolic glutathione, a low-molecular-weight thiol, through the membrane-bound histidine sensor kinase VirA, highlighting the importance of inter-kingdom signaling in bacterial pathogenesis. PMID:26159714

  18. Virulence Factor-activity Relationships: Workshop Summary

    EPA Science Inventory

    The concept or notion of virulence factor–activity relationships (VFAR) is an approach for identifying an analogous process to the use of qualitative structure–activity relationships (QSAR) for identifying new microbial contaminants. In QSAR, it is hypothesized that, for new chem...

  19. Virulent Aeromonas hydrophila in channel catfish

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In this study, we investigated factors that predisposed catfish to motile aeromonas septicemia (MAS) caused by virulent Aeromonas hydrophila (vAh). Our results revealed that wounding on fish body surface was a prerequisite for vAh infection and disease development. A reproducible waterborne challeng...

  20. Are secondary metabolites dispensable for virulence?

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The production of toxins by conidial fungal pathogens and their association with virulence has been assumed to occur in vivo and is widely accepted as dogma, but this association has yet to be definitively proven by either genetic or chemical means. Several studies from our labs have used targeted g...

  1. Characterization of the primary starch utilization operon in the obligate anaerobe Bacteroides fragilis: Regulation by carbon source and oxygen.

    PubMed

    Spence, Cheryl; Wells, W Greg; Smith, C Jeffrey

    2006-07-01

    The opportunistic pathogen Bacteroides fragilis is a commensal organism in the large intestine, where it utilizes both dietary and host-derived polysaccharides as a source of carbon and energy. In this study, a four-gene operon required for starch utilization was identified. The operon also was found to be oxygen responsive and thus was designated osu for oxygen-induced starch utilization. The first three genes in the operon were predicted to encode outer membrane proteins involved in starch binding, and a fourth gene, osuD, encoded an amylase involved in starch hydrolysis. Insertional mutation of the osuA gene (Omega osuA) resulted in the inability to utilize starch or glycogen and an insertional mutation into the osuD gene (Omega osuD) was severely impaired for growth on starch media. Transcriptional studies indicated that maltose, maltooligosaccharides, and starch were inducers of osu expression and that maltose was the strongest inducer. A transcriptional activator of osuABCD, OsuR, was identified and found to mediate maltose induction. The Omega osuA and Omega osuD mutants were able to grow on maltose but not starch, whereas a mutation in osuR abolished growth on both substrates, indicating that additional genes under the control of OsuR are needed for maltose utilization. The osuABCD operon also was induced by exposure to oxygen and was shown to be part of the oxidative stress response important for aerotolerance of B. fragilis. Transcriptional analyses showed that osuA was induced 20-fold by oxygen, but OsuR was not required for this activation. Analysis of osu mutants suggested that expression of the operon was important for survival during oxygen exposure but not to hydrogen peroxide stress.

  2. The use of amino sugars by Bacillus subtilis: presence of a unique operon for the catabolism of glucosamine.

    PubMed

    Gaugué, Isabelle; Oberto, Jacques; Putzer, Harald; Plumbridge, Jacqueline

    2013-01-01

    B. subtilis grows more rapidly using the amino sugar glucosamine as carbon source, than with N-acetylglucosamine. Genes for the transport and metabolism of N-acetylglucosamine (nagP and nagAB) are found in all the sequenced Bacilli (except Anoxybacillus flavithermus). In B. subtilis there is an additional operon (gamAP) encoding second copies of genes for the transport and catabolism of glucosamine. We have developed a method to make multiple deletion mutations in B. subtilis employing an excisable spectinomycin resistance cassette. Using this method we have analysed the contribution of the different genes of the nag and gam operons for their role in utilization of glucosamine and N-acetylglucosamine. Faster growth on glucosamine is due to the presence of the gamAP operon, which is strongly induced by glucosamine. Although the gamA and nagB genes encode isozymes of GlcN6P deaminase, catabolism of N-acetylglucosamine relies mostly upon the gamA gene product. The genes for use of N-acetylglucosamine, nagAB and nagP, are repressed by YvoA (NagR), a GntR family regulator, whose gene is part of the nagAB yvoA(nagR) operon. The gamAP operon is repressed by YbgA, another GntR family repressor, whose gene is expressed divergently from gamAP. The nagAB yvoA synton is found throughout the Bacilli and most firmicutes. On the other hand the ybgA-gamAP synton, which includes the ybgB gene for a small protein of unknown provenance, is only found in B. subtilis (and a few very close relatives). The origin of ybgBA-gamAP grouping is unknown but synteny analysis suggests lateral transfer from an unidentified donor. The presence of gamAP has enabled B. subtilis to efficiently use glucosamine as carbon source.

  3. Comparative analysis of the mechanisms of sulfur anion oxidation and reduction by dsr operon to maintain environmental sulfur balance.

    PubMed

    Ghosh, Semanti; Bagchi, Angshuman

    2015-12-01

    Sulfur metabolism is one of the oldest known redox geochemical cycles in our atmosphere. These redox processes utilize different sulfur anions and the reactions are performed by the gene products of dsr operon from phylogenetically diverse sets of microorganisms. The operon is involved in the maintenance of environmental sulfur balance. Interestingly, the dsr operon is found to be present in both sulfur anion oxidizing and reducing microorganisms and in both types of organisms DsrAB protein complex plays a vital role. Though there are various reports regarding the genetics of dsr operon there are practically no reports dealing with the structural aspects of sulfur metabolism by dsr operon. In our present study, we tried to compare the mechanisms of sulfur anion oxidation and reduction by Allochromatium vinosum and Desulfovibrio vulgaris respectively through DsrAB protein complex. We analyzed the modes of bindings of sulfur anions to the DsrAB protein complex and observed that for sulfur anion oxidizers, sulfide and thiosulfate are the best substrates whereas for reducers sulfate and sulfite have the best binding abilities. We analyzed the binding interaction pattern of the DsrA and DsrB proteins while forming the DsrAB protein complexes in Desulfovibrio vulgaris and Allochromatium vinosum. To our knowledge this is the first report that analyzes the differences in binding patterns of sulfur substrates with DsrAB protein from these two microorganisms. This study would therefore be essential to predict the biochemical mechanism of sulfur anion oxidation and reduction by these two microorganisms i.e., Desulfovibrio vulgaris (sulfur anion reducer) and Allochromatium vinosum (sulfur anion oxidizer). Our observations also highlight the mechanism of sulfur geochemical cycle which has important implications in future study of sulfur metabolism as it has a huge application in waste remediation and production of industrial bio-products viz. vitamins, bio-polyesters and bio

  4. Comparative analysis of the mechanisms of sulfur anion oxidation and reduction by dsr operon to maintain environmental sulfur balance.

    PubMed

    Ghosh, Semanti; Bagchi, Angshuman

    2015-12-01

    Sulfur metabolism is one of the oldest known redox geochemical cycles in our atmosphere. These redox processes utilize different sulfur anions and the reactions are performed by the gene products of dsr operon from phylogenetically diverse sets of microorganisms. The operon is involved in the maintenance of environmental sulfur balance. Interestingly, the dsr operon is found to be present in both sulfur anion oxidizing and reducing microorganisms and in both types of organisms DsrAB protein complex plays a vital role. Though there are various reports regarding the genetics of dsr operon there are practically no reports dealing with the structural aspects of sulfur metabolism by dsr operon. In our present study, we tried to compare the mechanisms of sulfur anion oxidation and reduction by Allochromatium vinosum and Desulfovibrio vulgaris respectively through DsrAB protein complex. We analyzed the modes of bindings of sulfur anions to the DsrAB protein complex and observed that for sulfur anion oxidizers, sulfide and thiosulfate are the best substrates whereas for reducers sulfate and sulfite have the best binding abilities. We analyzed the binding interaction pattern of the DsrA and DsrB proteins while forming the DsrAB protein complexes in Desulfovibrio vulgaris and Allochromatium vinosum. To our knowledge this is the first report that analyzes the differences in binding patterns of sulfur substrates with DsrAB protein from these two microorganisms. This study would therefore be essential to predict the biochemical mechanism of sulfur anion oxidation and reduction by these two microorganisms i.e., Desulfovibrio vulgaris (sulfur anion reducer) and Allochromatium vinosum (sulfur anion oxidizer). Our observations also highlight the mechanism of sulfur geochemical cycle which has important implications in future study of sulfur metabolism as it has a huge application in waste remediation and production of industrial bio-products viz. vitamins, bio-polyesters and bio-hydrogen.

  5. Insights on Evolution of Virulence and Resistance from the Complete Genome Analysis of an Early Methicillin-Resistant Staphylococcus aureus Strain and a Biofilm-Producing Methicillin-Resistant Staphylococcus epidermidis Strain†

    PubMed Central

    Gill, Steven R.; Fouts, Derrick E.; Archer, Gordon L.; Mongodin, Emmanuel F.; DeBoy, Robert T.; Ravel, Jacques; Paulsen, Ian T.; Kolonay, James F.; Brinkac, Lauren; Beanan, Mauren; Dodson, Robert J.; Daugherty, Sean C.; Madupu, Ramana; Angiuoli, Samuel V.; Durkin, A. Scott; Haft, Daniel H.; Vamathevan, Jessica; Khouri, Hoda; Utterback, Terry; Lee, Chris; Dimitrov, George; Jiang, Lingxia; Qin, Haiying; Weidman, Jan; Tran, Kevin; Kang, Kathy; Hance, Ioana R.; Nelson, Karen E.; Fraser, Claire M.

    2005-01-01

    Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the ∼2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the ∼2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis. PMID:15774886

  6. Sample collection of virulent and non-virulent B. anthracis and Y. pestis for bioforensics analysis

    SciTech Connect

    Hong-geller, Elizabeth; Valdez, Yolanda E; Shou, Yulin; Yoshida, Thomas M; Marrone, Babetta L; Dunbar, John

    2009-01-01

    Validated sample collection methods are needed for recovery of microbial evidence in the event of accidental or intentional release of biological agents into the environment. To address this need, we evaluated the sample recovery efficiencies of two collection methods -- swabs and wipes -- for both non-virulent and virulent strains of B. anthracis and Y. pestis from four types of non-porous surfaces: two hydrophilic surfaces, stainless steel and glass, and two hydrophobic surfaces, vinyl and plastic. Sample recovery was quantified using Real-time qPCR to assay for intact DNA signatures. We found no consistent difference in collection efficiency between swabs or wipes. Furthermore, collection efficiency was more surface-dependent for virulent strains than non-virulent strains. For the two non-virulent strains, B. anthracis Sterne and Y. pestis A1122, collection efficiency was approximately 100% and 1 %, respectively, from all four surfaces. In contrast, recovery of B. anthracis Ames spores and Y. pestis C092 from vinyl and plastic was generally lower compared to collection from glass or stainless steel, suggesting that surface hydrophobicity may playa role in the strength of pathogen adhesion. The surface-dependent collection efficiencies observed with the virulent strains may arise from strain-specific expression of capsular material or other cell surface receptors that alter cell adhesion to specific surfaces. These findings contribute to validation of standard bioforensics procedures and emphasize the importance of specific strain and surface interactions in pathogen detection.

  7. Microevolution and virulence of dengue viruses.

    PubMed

    Rico-Hesse, Rebeca

    2003-01-01

    The evolution of dengue viruses has had a major impact on their virulence for humans and on the epidemiology of dengue disease around the world. Although antigenic and genetic differences in virus strains had become evident, it is mainly due to the lack of animal models of disease that has made it difficult to detect differences in virulence of dengue viruses. However, phylogenetic studies of many different dengue virus samples have led to the association between specific genotypes (within serotypes) and the presentation of more or less severe disease. Currently, dengue viruses can be classified as being of epidemiologically low, medium, or high impact; i.e., some viruses may remain in sylvatic cycles of little or low transmissibility to humans, others produce dengue fever (DF) only, and some genotypes have been associated with the potential to cause the more severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) in addition to DF. Although the factors that contribute to dengue virus epidemiology are complex, studies have suggested that specific viral structures may contribute to increased replication in human target cells and to increased transmission by the mosquito vector; however, the immune status and possibly the genetic background of the host are also determinants of virulence or disease presentation. As to the question of whether dengue viruses are evolving toward virulence as they continue to spread throughout the world, phylogenetic and epidemiological analyses suggest that the more virulent genotypes are now displacing those that have lower epidemiological impact; there is no evidence for the transmission of antigenically aberrant, new strains.

  8. Deletion of the Bacillus anthracis capB homologue in Francisella tularensis subspecies tularensis generates an attenuated strain that protects mice against virulent tularaemia.

    PubMed

    Michell, Stephen L; Dean, Rachel E; Eyles, Jim E; Hartley, Margaret Gill; Waters, Emma; Prior, Joann L; Titball, Richard W; Oyston, Petra C F

    2010-11-01

    As there is currently no licensed vaccine against Francisella tularensis, the causative agent of tularaemia, the bacterium is an agent of concern as a potential bioweapon. Although F. tularensis has a low infectious dose and high associated mortality, it possesses few classical virulence factors. An analysis of the F. tularensis subspecies tularensis genome sequence has revealed the presence of a region containing genes with low sequence homology to part of the capBCADE operon of Bacillus anthracis. We have generated an isogenic capB mutant of F. tularensis subspecies tularensis SchuS4 and shown it to be attenuated. Furthermore, using BALB/c mice, we have demonstrated that this capB strain affords protection against significant homologous challenge with the wild-type strain. These data have important implications for the development of a defined and efficacious tularaemia vaccine.

  9. DNA sequence analysis of the imp UV protection and mutation operon of the plasmid TP110: identification of a third gene.

    PubMed Central

    Lodwick, D; Owen, D; Strike, P

    1990-01-01

    The sequence of the imp operon of the plasmid TP110 (which belongs to the Incl1 incompatibility group) has been determined, and is shown to contain three open reading frames. This operon, involved in UV protection and mutation, is functionally analogous to the umuDC operon of E. coli and the mucAB operon of the plasmid pKM101, which belongs to the quite unrelated IncN incompatibility group. The umu and muc operons however contain only two open reading frames, coding for proteins of approximately 16kD and 46kD. The high degree of homology between the two 16kD proteins (UmuD and MucA) and between the two 46kD proteins (UmuC and MucB) clearly shows their relatedness. This is shown also to extend to the imp gene products, with ImpA sharing homology with UmuD and MucA, and ImpB sharing homology with UmuC and MucB. However, the two imp genes are preceded in the operon by a third gene, impC, which encodes a small protein of 9.5kD and which has no equivalent in the umu and muc operons. Images PMID:2129552

  10. The spc ribosomal protein operon of Escherichia coli: sequence and cotranscription of the ribosomal protein genes and a protein export gene.

    PubMed

    Cerretti, D P; Dean, D; Davis, G R; Bedwell, D M; Nomura, M

    1983-05-11

    The genes encoding the 52 ribosomal proteins (r-proteins) of Escherichia coli are organized into approximately 19 operons scattered throughout the chromosome. One of these, the spc operon, contains the genes for ten ribosomal proteins: L14, L24, L5, S14, S8, L6, L18, S5, L30 and L15 (rp1N, rp1X, rp1E, rpsN, rpsH, rp1F, rp1R, rpsE, rpmD, and rp1O). We now report the entire 5.9 kb nucleotide sequence of the spc operon. DNA sequence analysis has confirmed the genetic organization and refined the amino acid sequence of the ten r-proteins in this operon. It has also revealed the presence of two open reading frames past the last known gene (L15) of the spc operon. One of these corresponds to a gene (pr1A or secY) which recently has been shown by others to be involved in protein export. In addition, S1 mapping experiments indicate that a significant proportion of transcription initiated from the spc operon continues not only into the two putative genes, but also without termination into the downstream alpha r-protein operon.

  11. icaR encodes a transcriptional repressor involved in environmental regulation of ica operon expression and biofilm formation in Staphylococcus epidermidis.

    PubMed

    Conlon, Kevin M; Humphreys, Hilary; O'Gara, James P

    2002-08-01

    Biofilm formation in Staphylococcus epidermidis is dependent upon the ica operon-encoded polysaccharide intercellular adhesin, which is subject to phase-variable and environmental regulation. The icaR gene, located adjacent to the ica operon, appears to be a member of the tetR family of transcriptional regulators. In the reference strain RP62A, reversible inactivation of the ica operon by IS256 accounts for 25 to 33% of phase variants. In this study, icaA and icaR regulation were compared in RP62A and a biofilm-forming clinical isolate, CSF41498, in which IS256 is absent. Predictably, ica operon expression was detected only in wild-type CSF41498 and RP62A but not in non-IS256-generated phase variants. In contrast, the icaR gene was not expressed in RP62A phase variants but was expressed in CSF41498 variants. An icaR::Em(r) insertion mutation in CSF41498 resulted in an at least a 5.8-fold increase in ica operon expression but did not significantly alter regulation of the icaR gene itself. Activation of ica operon transcription by ethanol in CSF41498 was icaR dependent. In contrast, a small but significant induction of ica by NaCl and glucose (NaCl-glucose) was observed in the icaR::Em(r) mutant. In addition, transcription of the icaR gene itself was not significantly affected by NaCl-glucose but was repressed by ethanol. Expression of the ica operon was induced by ethanol or NaCl-glucose in phase variants of CSF41498 (icaR+) but not in RP62A variants (icaR deficient). These data indicate that icaR encodes a repressor of ica operon transcription required for ethanol but not NaCl-glucose activation of ica operon expression and biofilm formation.

  12. The napF and narG Nitrate Reductase Operons in Escherichia coli Are Differentially Expressed in Response to Submicromolar Concentrations of Nitrate but Not Nitrite

    PubMed Central

    Wang, Henian; Tseng, Ching-Ping; Gunsalus, Robert P.

    1999-01-01

    Escherichia coli synthesizes two biochemically distinct nitrate reductase enzymes, a membrane-bound enzyme encoded by the narGHJI operon and a periplasmic cytochrome c-linked nitrate reductase encoded by the napFDAGHBC operon. To address why the cell makes these two enzymes, continuous cell culture techniques were used to examine napF and narG gene expression in response to different concentrations of nitrate and/or nitrite. Expression of the napF-lacZ and narG-lacZ reporter fusions in strains grown at different steady-state levels of nitrate revealed that the two nitrate reductase operons are differentially expressed in a complementary pattern. The napF operon apparently encodes a “low-substrate-induced” reductase that is maximally expressed only at low levels of nitrate. Expression is suppressed under high-nitrate conditions. In contrast, the narGHJI operon is only weakly expressed at low nitrate levels but is maximally expressed when nitrate is elevated. The narGHJI operon is therefore a “high-substrate-induced” operon that somehow provides a second and distinct role in nitrate metabolism by the cell. Interestingly, nitrite, the end product of each enzyme, had only a minor effect on the expression of either operon. Finally, nitrate, but not nitrite, was essential for repression of napF gene expression. These studies reveal that nitrate rather than nitrite is the primary signal that controls the expression of these two nitrate reductase operons in a differential and complementary fashion. In light of these findings, prior models for the roles of nitrate and nitrite in control of narG and napF expression must be reconsidered. PMID:10464201

  13. icaR encodes a transcriptional repressor involved in environmental regulation of ica operon expression and biofilm formation in Staphylococcus epidermidis.

    PubMed

    Conlon, Kevin M; Humphreys, Hilary; O'Gara, James P

    2002-08-01

    Biofilm formation in Staphylococcus epidermidis is dependent upon the ica operon-encoded polysaccharide intercellular adhesin, which is subject to phase-variable and environmental regulation. The icaR gene, located adjacent to the ica operon, appears to be a member of the tetR family of transcriptional regulators. In the reference strain RP62A, reversible inactivation of the ica operon by IS256 accounts for 25 to 33% of phase variants. In this study, icaA and icaR regulation were compared in RP62A and a biofilm-forming clinical isolate, CSF41498, in which IS256 is absent. Predictably, ica operon expression was detected only in wild-type CSF41498 and RP62A but not in non-IS256-generated phase variants. In contrast, the icaR gene was not expressed in RP62A phase variants but was expressed in CSF41498 variants. An icaR::Em(r) insertion mutation in CSF41498 resulted in an at least a 5.8-fold increase in ica operon expression but did not significantly alter regulation of the icaR gene itself. Activation of ica operon transcription by ethanol in CSF41498 was icaR dependent. In contrast, a small but significant induction of ica by NaCl and glucose (NaCl-glucose) was observed in the icaR::Em(r) mutant. In addition, transcription of the icaR gene itself was not significantly affected by NaCl-glucose but was repressed by ethanol. Expression of the ica operon was induced by ethanol or NaCl-glucose in phase variants of CSF41498 (icaR+) but not in RP62A variants (icaR deficient). These data indicate that icaR encodes a repressor of ica operon transcription required for ethanol but not NaCl-glucose activation of ica operon expression and biofilm formation. PMID:12142410

  14. Targeted deletion of the ara operon of Salmonella typhimurium enhances L-arabinose accumulation and drives PBAD-promoted expression of anti-cancer toxins and imaging agents.

    PubMed

    Hong, Hyun; Lim, Daejin; Kim, Geun-Joong; Park, Seung-Hwan; Sik Kim, Hyeon; Hong, Yeongjin; Choy, Hyon E; Min, Jung-Joon

    2014-01-01

    Tumor-specific expression of antitumor drugs can be achieved using attenuated Salmonella typhimurium harboring the PBAD promoter, which is induced by L-arabinose. However, L-arabinose does not accumulate because it is metabolized to D-xylulose-5-P by enzymes encoded by the ara operon in Salmonellae. To address this problem, we developed an engineered strain of S. typhimurium in which the ara operon is deleted. Linear DNA transformation was performed using λ red recombinase to exchange the ara operon with linear DNA carrying an antibiotic-resistance gene with homology to regions adjacent to the ara operon. The ara operon-deleted strain and its parental strain were transformed with a plasmid encoding Renilla luciferase variant 8 (RLuc8) or cytolysin A (clyA) under the control of the PBAD promoter. Luciferase assays demonstrated that RLuc8 expression was 49-fold higher in the ara operon-deleted S. typhimurium than in the parental strain after the addition of L-arabinose. In vivo bioluminescence imaging showed that the tumor tissue targeted by the ara operon-deleted Salmonella had a stronger imaging signal (~30-fold) than that targeted by the parental strain. Mice with murine colon cancer (CT26) that had been injected with the ara operon-deleted S. typhimurium expressing clyA showed significant tumor suppression. The present report demonstrates that deletion of the ara operon of S. typhimurium enhances L-arabinose accumulation and thereby drives PBAD-promoted expression of cytotoxic agents and imaging agents. This is a promising approach for tumor therapy and imaging.

  15. Candida Virulence Properties and Adverse Clinical Outcomes in Neonatal Candidiasis

    PubMed Central

    Bliss, Joseph M.; Wong, Angela Y.; Bhak, Grace; Laforce-Nesbitt, Sonia S.; Taylor, Sarah; Tan, Sylvia; Stoll, Barbara J.; Higgins, Rosemary D.; Shankaran, Seetha; Benjamin, Daniel K.

    2012-01-01

    Objective To determine if premature infants with invasive Candida infection caused by strains with increased virulence properties have worse clinical outcomes than those infected with less virulent strains. Study design Clinical isolates were studied from 2 populations; premature infants colonized with Candida (commensal, n=27), and those with invasive candidiasis (n=81). Individual isolates of C. albicans and C. parapsilosis were tested for virulence in each of 3 assays: phenotypic switching, adhesion, and cytotoxicity. Invasive isolates were considered to have enhanced virulence if they measured more than 1 SD above the mean for the commensal isolates in at least 1 assay. Outcomes of patients with invasive isolates with enhanced virulence were compared with those with invasive isolates lacking enhanced virulence characteristics. Results 61% of invasive isolates of C. albicans and 42% of invasive isolates of C. parapsilosis had enhanced virulence. All C. albicans cerebrospinal fluid (CSF) isolates (n=6) and 90% of urine isolates (n=10) had enhanced virulence, compared with 48% of blood isolates (n=40). Infants with more virulent isolates were younger at the time of positive culture and had higher serum creatinine. Conclusions Individual isolates of Candida species vary in their virulence properties. Strains with higher virulence are associated with certain clinical outcomes. PMID:22504098

  16. Toward an improved laboratory definition of Listeria monocytogenes virulence.

    PubMed

    Liu, Dongyou; Lawrence, Mark L; Ainsworth, A Jerald; Austin, Frank W

    2007-09-15

    Listeria monocytogenes is an opportunistic foodborne pathogen that encompasses a diversity of strains with varied virulence. The ability to rapidly determine the pathogenic potential of L. monocytogenes strains is integral to the control and prevention campaign against listeriosis. Early methods for assessing L. monocytogenes virulence include in vivo bioassays and in vitro cell assays. While in vivo bioassays provide a measurement of all virulence determinants of L. monocytogenes, they are not applied routinely due to their reliance on experimental animals whose costs have become increasingly prohibitive. As a low cost alternative, in vitro cell assays are useful for estimating the virulence of L. monocytogenes strains. However, these assays are often slow, and at times variable. Prior attempts to ascertain L. monocytogenes virulence by targeting virulence-associated proteins and genes have been largely unsuccessful, since many of the assay targets are present in both virulent and avirulent strains. Recent identification of novel virulence-specific genes (particularly internalin gene inlJ) has opened a new avenue for rapid, sensitive, and precise differentiation of virulent L. monocytogenes strains from avirulent strains. The application of DNA sequencing technique also offers an additional tool for assessing L. monocytogenes virulence potential. By providing an update on the laboratory methods that have been reported for the determination of L. monocytogenes pathogenicity, this review discusses future research needs that may help achieve an improved laboratory definition of L. monocytogenes virulence.

  17. Systematic Targeted Mutagenesis of Brucella melitensis 16M Reveals a Major Role for GntR Regulators in the Control of Virulence

    PubMed Central

    Haine, Valérie; Sinon, Audrey; Van Steen, Frédéric; Rousseau, Stéphanie; Dozot, Marie; Lestrate, Pascal; Lambert, Christophe; Letesson, Jean-Jacques; De Bolle, Xavier

    2005-01-01

    In order to identify transcriptional regulators involved in virulence gene control in Brucella melitensis, we generated a collection of 88 mutants in the AraC, ArsR, Crp, DeoR, GntR, IclR, LysR, MerR, RpiR, and TetR families of regulators. This collection was named LiMuR (library of mutants for regulators). We developed a method to test several mutants simultaneously in one animal in order to identify those unable to survive. This method, called the plasmid-tagged mutagenesis method, was used to test the residual virulence of mutants after 1 week in a mouse model of infection. Ten attenuated mutants, of which six and three belong to the GntR and LysR families, respectively, were identified and individually confirmed to replicate at lower rates in mice. Among these 10 mutants, only gntR10 and arsR6 are attenuated in cellular models. The LiMuR also allows simple screenings to identify regulators of a particular gene or operon. As a first example, we analyzed the expression of the virB operon in the LiMuR mutants. We carried out Western blottings of whole-cell extracts to analyze the production of VirB proteins using polyclonal antisera against VirB proteins. Four mutants produced small amounts of VirB proteins, and one mutant overexpressed VirB proteins compared to the wild-type strain. In these five mutants, reporter analysis using the virB promoter fused to lacZ showed that three mutants control virB at the transcriptional level. The LiMuR is a resource that will provide straightforward identification of regulators involved in the control of genes of interest. PMID:16113274

  18. Expression and secretion of Salmonella pathogenicity island-2 virulence genes in response to acidification exhibit differential requirements of a functional type III secretion apparatus and SsaL.

    PubMed

    Coombes, Brian K; Brown, Nat F; Valdez, Yanet; Brumell, John H; Finlay, B Brett

    2004-11-26

    Salmonella pathogenicity island (SPI)-2 is pivotal to the intracellular survival of Salmonella and for virulence in mammals. SPI-2 encodes virulence factors (called effectors) that are translocated into the host cell, a type III secretion apparatus and a two-component regulatory system that regulates intracellular expression of SPI-2. Salmonella SPI-2 secretion activity appears to be induced in response to acidification of the vacuole in which it replicates. Here we show that the expression of the SPI-2 proteins, SseB and SseD (filament and pore forming components of the secretion apparatus, respectively) in response to acidification requires an intact secretion system and SsaL, a Salmonella homologue of SepL, a regulator required for type III-dependent secretion of translocators but not effectors in attaching and effacing gastrointestinal pathogens. We show that the expression of SPI-2-encoded effectors is acid-regulated but can be uncoupled from the expression of filament and translocon components, thus showing a differential requirement of SsaL for expression. The secretion and translocation of SPI-2-encoded effectors requires SsaL, but SsaL is dispensable for the secretion of SPI-2 effectors encoded in other pathogenicity loci, suggesting a secretion regulation function for SsaL. Further, we demonstrate that the differential expression of adjacent genes within the sseA operon (sseD and sseE) occurs at the transcriptional level. These data indicate that a Salmonella SPI-2 activation state is achieved by an acidregulated response that requires SsaL. These data also suggest the existence of a previously unrecognized regulatory element within SPI-2 for the "effector operon" region downstream of sseD that might demarcate the expression of translocators and effectors.

  19. AraC protein, regulation of the l-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action.

    PubMed

    Schleif, Robert

    2010-09-01

    This review covers the physiological aspects of regulation of the arabinose operon in Escherichia coli and the physical and regulatory properties of the operon's controlling gene, araC. It also describes the light switch mechanism as an explanation for many of the protein's properties. Although many thousands of homologs of AraC exist and regulate many diverse operons in response to many different inducers or physiological states, homologs that regulate arabinose-catabolizing genes in response to arabinose were identified. The sequence similarities among them are discussed in light of the known structure of the dimerization and DNA-binding domains of AraC.

  20. 'Ready made' virulence and 'dual use' virulence factors in pathogenic environmental fungi--the Cryptococcus neoformans paradigm.

    PubMed

    Casadevall, Arturo; Steenbergen, Judith N; Nosanchuk, Joshua D

    2003-08-01

    Environmental pathogenic fungi present a paradox in that they are virulent in animals without requiring animal hosts for replication or survival, a phenomenon we call 'ready-made' virulence. In the human pathogenic fungus Cryptococcus neoformans, the capacity for virulence in animals may originate from environmental selective pressures imposed by such organisms as amoeboid and nematode predators. Many C. neoformans virulence factors appear to have 'dual use' capabilities that confer survival advantages in both animal hosts and in the environment. The findings with C. neoformans may provide a paradigm for understanding the origin and maintenance of virulence in other pathogenic environmental fungi.

  1. Genomic and Functional Portrait of a Highly Virulent, CTX-M-15-Producing H30-Rx Subclone of Escherichia coli Sequence Type 131

    PubMed Central

    Ranjan, Amit; Shaik, Sabiha; Hussain, Arif; Nandanwar, Nishant; Semmler, Torsten; Jadhav, Savita; Wieler, Lothar H.

    2015-01-01

    Escherichia coli sequence type 131 (ST131) is a pandemic clone associated with multidrug-resistant, extraintestinal infections, attributable to the presence of the CTX-M-15 extended-spectrum β-lactamase gene and mutations entailing fluoroquinolone resistance. Studies on subclones within E. coli ST131 are critically required for targeting and implementation of successful control efforts. Our study comprehensively analyzed the genomic and functional attributes of the H30-Rx subclonal strains NA097 and NA114, belonging to the ST131 lineage. We carried out whole-genome sequencing, comparative analysis, phenotypic virulence assays, and profiling of the antibacterial responses of THP1 cells infected with these subclones. Phylogenomic analysis suggested that the strains were clonal in nature and confined entirely to a single clade. Comparative genomic analysis revealed that the virulence and resistance repertoires were comparable among the H30-Rx ST131 strains except for the commensal ST131 strain SE15. Similarly, seven phage-specific regions were found to be strongly associated with the H30-Rx strains but were largely absent in the genome of SE15. Phenotypic analysis confirmed the virulence and resistance similarities between the two strains. However, NA097 was found to be more robust than NA114 in terms of virulence gene carriage (dra operon), invasion ability (P < 0.05), and antimicrobial resistance (streptomycin resistance). RT2 gene expression profiling revealed generic upregulation of key proinflammatory responses in THP1 cells, irrespective of ST131 lineage status. In conclusion, our study provides comprehensive, genome-inferred insights into the biology and immunological properties of ST131 strains and suggests clonal diversification of genomic and phenotypic features within the H30-Rx subclone of E. coli ST131. PMID:26195517

  2. Copper tolerance and virulence in bacteria.

    PubMed

    Ladomersky, Erik; Petris, Michael J

    2015-06-01

    Copper (Cu) is an essential trace element for all aerobic organisms. It functions as a cofactor in enzymes that catalyze a wide variety of redox reactions due to its ability to cycle between two oxidation states, Cu(I) and Cu(II). This same redox property of copper has the potential to cause toxicity if copper homeostasis is not maintained. Studies suggest that the toxic properties of copper are harnessed by the innate immune system of the host to kill bacteria. To counter such defenses, bacteria rely on copper tolerance genes for virulence within the host. These discoveries suggest bacterial copper intoxication is a component of host nutritional immunity, thus expanding our knowledge of the roles of copper in biology. This review summarizes our current understanding of copper tolerance in bacteria, and the extent to which these pathways contribute to bacterial virulence within the host.

  3. Regulation of virulence of Entamoeba histolytica.

    PubMed

    Marie, Chelsea; Petri, William A

    2014-01-01

    Entamoeba histolytica is the third-leading cause of parasitic mortality globally. E. histolytica infection generally does not cause symptoms, but the parasite has potent pathogenic potential. The origins, benefits, and triggers of amoebic virulence are complex. Amoebic pathogenesis entails depletion of the host mucosal barrier, adherence to the colonic lumen, cytotoxicity, and invasion of the colonic epithelium. Parasite damage results in colitis and, in some cases, disseminated disease. Both host and parasite genotypes influence the development of disease, as do the regulatory responses they govern at the host-pathogen interface. Host environmental factors determine parasite transmission and shape the colonic microenvironment E. histolytica infects. Here we highlight research that illuminates novel links between host, parasite, and environmental factors in the regulation of E. histolytica virulence.

  4. Riboregulators: Fine-Tuning Virulence in Shigella

    PubMed Central

    Fris, Megan E.; Murphy, Erin R.

    2016-01-01

    Within the past several years, RNA-mediated regulation (ribo-regulation) has become increasingly recognized for its importance in controlling critical bacterial processes. Regulatory RNA molecules, or riboregulators, are perpetually responsive to changes within the micro-environment of a bacterium. Notably, several characterized riboregulators control virulence in pathogenic bacteria, as is the case for each riboregulator characterized to date in Shigella. The timing of virulence gene expression and the ability of the pathogen to adapt to rapidly changing environmental conditions is critical to the establishment and progression of infection by Shigella species; ribo-regulators mediate each of these important processes. This mini review will present the current state of knowledge regarding RNA-mediated regulation in Shigella by detailing the characterization and function of each identified riboregulator in these pathogens. PMID:26858941

  5. Copper tolerance and virulence in bacteria

    PubMed Central

    Ladomersky, Erik; Petris, Michael J.

    2015-01-01

    Copper (Cu) is an essential trace element for all aerobic organisms. It functions as a cofactor in enzymes that catalyze a wide variety of redox reactions due to its ability to cycle between two oxidation states, Cu(I) and Cu(II). This same redox property of copper has the potential to cause toxicity if copper homeostasis is not maintained. Studies suggest that the toxic properties of copper are harnessed by the innate immune system of the host to kill bacteria. To counter such defenses, bacteria rely on copper tolerance genes for virulence within the host. These discoveries suggest bacterial copper intoxication is a component of host nutritional immunity, thus expanding our knowledge of the roles of copper in biology. This review summarizes our current understanding of copper tolerance in bacteria, and the extent to which these pathways contribute to bacterial virulence within the host. PMID:25652326

  6. [Genetic virulence markers of opportunistic bacteria].

    PubMed

    Bondarenko, V M

    2011-01-01

    The analysis of opportunistic bacteria phenotypic and genetic virulence markers indicates that pathogenicity formation is based on a structural modification of bacterial DNA which is linked with migration of interbacterial pathogenicity "islands" genetic determinants. Structural organization features of these mobile genetic elements determine high expression probability, and PCR detection of pathogenicity "islands" determinants that control adhesins, invasins, cytotoxic and cytolitic toxines synthesis may indicate etiopathogenetic significance of clinical isolates.

  7. Virulence Factors of Erwinia amylovora: A Review.

    PubMed

    Piqué, Núria; Miñana-Galbis, David; Merino, Susana; Tomás, Juan M

    2015-06-05

    Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3'-5')-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them.

  8. Virulence Factors of Erwinia amylovora: A Review

    PubMed Central

    Piqué, Núria; Miñana-Galbis, David; Merino, Susana; Tomás, Juan M.

    2015-01-01

    Erwinia amylovora, a Gram negative bacteria of the Enterobacteriaceae family, is the causal agent of fire blight, a devastating plant disease affecting a wide range of host species within Rosaceae and a major global threat to commercial apple and pear production. Among the limited number of control options currently available, prophylactic application of antibiotics during the bloom period appears the most effective. Pathogen cells enter plants through the nectarthodes of flowers and other natural openings, such as wounds, and are capable of rapid movement within plants and the establishment of systemic infections. Many virulence determinants of E. amylovora have been characterized, including the Type III secretion system (T3SS), the exopolysaccharide (EPS) amylovoran, biofilm formation, and motility. To successfully establish an infection, E. amylovora uses a complex regulatory network to sense the relevant environmental signals and coordinate the expression of early and late stage virulence factors involving two component signal transduction systems, bis-(3′-5′)-cyclic di-GMP (c-di-GMP) and quorum sensing. The LPS biosynthetic gene cluster is one of the relatively few genetic differences observed between Rubus- and Spiraeoideae-infecting genotypes of E. amylovora. Other differential factors, such as the presence and composition of an integrative conjugative element associated with the Hrp T3SS (hrp genes encoding the T3SS apparatus), have been recently described. In the present review, we present the recent findings on virulence factors research, focusing on their role in bacterial pathogenesis and indicating other virulence factors that deserve future research to characterize them. PMID:26057748

  9. Bistability of the lac operon during growth of Escherichia coli on lactose and lactose+glucose.

    PubMed

    Narang, Atul; Pilyugin, Sergei S

    2008-05-01

    The lac operon of Escherichia coli can exhibit bistability. Early studies showed that bistability occurs during growth on TMG/succinate and lactose+glucose, but not during growth on lactose. More recently, studies with lacGFP-transfected cells show bistability during growth on TMG/succinate, but not during growth on lactose and lactose+glucose. In the literature, these results are invariably attributed to variations in the destabilizing effect of the positive feedback generated by induction. Specifically, during growth on TMG/succinate, lac induction generates strong positive feedback because the permease stimulates the accumulation of intracellular TMG, which in turn, promotes the synthesis of even more permease. This positive feedback is attenuated during growth on lactose because hydrolysis of intracellular lactose by beta-galactosidase suppresses the stimulatory effect of the permease. It is attenuated even more during growth on lactose + glucose because glucose inhibits the uptake of lactose. But it is clear that the stabilizing effect of dilution also changes dramatically as a function of the medium composition. For instance, during growth on TMG/succinate, the dilution rate of lac permease is proportional to its activity, e, because the specific growth rate is independent of e (it is completely determined by the concentration of succinate). However, during growth on lactose, the dilution rate of the permease is proportional to e2 because the specific growth rate is proportional to the specific lactose uptake rate, which in turn, proportional to e. We show that: (a) This dependence on e2 creates such a strong stabilizing effect that bistability is virtually impossible during growth on lactose, even in the face of the intense positive feedback generated by induction. (b) This stabilizing effect is weakened during growth on lactose+glucose because the specific growth rate on glucose is independent of e, so that the dilution rate once again contains a term that

  10. The Q gene of Rhodobacter sphaeroides: its role in puf operon expression and spectral complex assembly.

    PubMed Central

    Gong, L; Lee, J K; Kaplan, S

    1994-01-01

    The Q gene of the facultative photoheterotroph Rhodobacter sphaeroides, localized immediately upstream of the oxygen- and light-regulated puf operon, encodes a 77-amino-acid polypeptide. The 5' and 3' ends of the 561-bp Q transcript were determined. To gain insight into the role of the Q gene product, a number of Q mutations were constructed by oligonucleotide-directed mutagenesis and subsequent substitution of the mutated form of the gene in single copy for the chromosomal copy via homologous recombination. The resulting mutants can grow photosynthetically, with the exception of QSTART, in which the initiation codon for the Q protein was altered. Spectral analysis of the intracytoplasmic membranes showed that one of the missense mutants (QdA) was deficient in the formation of detectable B875 light-harvesting complex (LHC), whereas deletion of the stem-loop structure (Qloop) failed to form B800-850 LHC when grown anaerobically either in the dark or under light intensity of 100 W/m2. Other missense mutants (QuA and QuB) contained either more B800-850 LHC or more B875 LHC, respectively, than the wild type. Although the levels of puf and puc transcripts isolated from QSTART grown anaerobically on succinate-dimethyl sulfoxide in the dark were comparable to wild-type levels, no B875 spectral complex was detected and there was a greater than 90% reduction in the level of the B800-850 pigment-protein complex. It has also been confirmed that the ultimate cellular levels of either the B875 or B800-850 spectral complexes can vary over wide limits without any change in the level(s) of complex specific transcripts. When the wild-type Q gene was reintroduced in trans into the Q mutations, QSTART was able to grow photosynthetically and both B800-850 and B875 spectral complexes were formed in either QdA or Qloop. Finally, we demonstrated that the level of each puf-specific mRNA behaves independently of one another as well as independently of the level(s) of Q gene-specific m

  11. Hantavirus interferon regulation and virulence determinants.

    PubMed

    Mackow, Erich R; Dalrymple, Nadine A; Cimica, Velasco; Matthys, Valery; Gorbunova, Elena; Gavrilovskaya, Irina

    2014-07-17

    Hantaviruses predominantly replicate in primary human endothelial cells and cause 2 diseases characterized by altered barrier functions of vascular endothelium. Most hantaviruses restrict the early induction of interferon-β (IFNβ) and interferon stimulated genes (ISGs) within human endothelial cells to permit their successful replication. PHV fails to regulate IFN induction within human endothelial cells which self-limits PHV replication and its potential as a human pathogen. These findings, and the altered regulation of endothelial cell barrier functions by pathogenic hantaviruses, suggest that virulence is determined by the ability of hantaviruses to alter key signaling pathways within human endothelial cells. Our findings indicate that the Gn protein from ANDV, but not PHV, inhibits TBK1 directed ISRE, kB and IFNβ induction through virulence determinants in the Gn cytoplasmic tail (GnT) that inhibit TBK1 directed IRF3 phosphorylation. Further studies indicate that in response to hypoxia induced VEGF, ANDV infection enhances the permeability and adherens junction internalization of microvascular and lymphatic endothelial cells. These hypoxia/VEGF directed responses are rapamycin sensitive and directed by mTOR signaling pathways. These results demonstrate the presence of at least two hantavirus virulence determinants that act on endothelial cell signaling pathways: one that regulates antiviral IFN signaling responses, and a second that enhances normal hypoxia-VEGF-mTOR signaling pathways to facilitate endothelial cell permeability. These findings suggest signaling pathways as potential targets for therapeutic regulation of vascular deficits that contribute to hantavirus diseases and viral protein targets for attenuating pathogenic hantaviruses.

  12. Tracking bacterial virulence: global modulators as indicators

    PubMed Central

    Prieto, Alejandro; Urcola, Imanol; Blanco, Jorge; Dahbi, Ghizlane; Muniesa, Maite; Quirós, Pablo; Falgenhauer, Linda; Chakraborty, Trinad; Hüttener, Mário; Juárez, Antonio

    2016-01-01

    The genomes of Gram-negative bacteria encode paralogues and/or orthologues of global modulators. The nucleoid-associated H-NS and Hha proteins are an example: several enterobacteria such as Escherichia coli or Salmonella harbor H-NS, Hha and their corresponding paralogues, StpA and YdgT proteins, respectively. Remarkably, the genome of the pathogenic enteroaggregative E. coli strain 042 encodes, in addition to the hha and ydgT genes, two additional hha paralogues, hha2 and hha3. We show in this report that there exists a strong correlation between the presence of these paralogues and the virulence phenotype of several E. coli strains. hha2 and hha3 predominate in some groups of intestinal pathogenic E. coli strains (enteroaggregative and shiga toxin-producing isolates), as well as in the widely distributed extraintestinal ST131 isolates. Because of the relationship between the presence of hha2/hha3 and some virulence factors, we have been able to provide evidence for Hha2/Hha3 modulating the expression of the antigen 43 pathogenic determinants. We show that tracking global modulators or their paralogues/orthologues can be a new strategy to identify bacterial pathogenic clones and propose PCR amplification of hha2 and hha3 as a virulence indicator in environmental and clinical E. coli isolates. PMID:27169404

  13. A cis/trans Test of the Effect of the First Enzyme for Histidine Biosynthesis on Regulation of the Histidine Operon

    PubMed Central

    Kovach, John S.; Ballesteros, Antonio O.; Meyers, Marilyn; Soria, Marco; Goldberger, Robert F.

    1973-01-01

    Previous studies showed that when triazolalanine was added to a derepressed culture of a histidine auxotroph, repression of the histidine operon occurred as though histidine had been added (6). However, when triazolalanine was added to a derepressed culture of a strain with a mutation in the first gene of the histidine operon which rendered the first enzyme for histidine biosynthesis resistant to inhibition by histidine, repression did not occur. The studies reported here represent a cis/trans test of this effect of mutations to feedback resistance. Using specially constructed merodiploid strains, we were able to show that the wild-type allele is dominant to the mutant (feedback resistant) allele and that the effect operates in trans. We conclude that the enzyme encoded by the first gene of the histidine operon exerts its regulatory effect on the operon not by acting locally at its site of synthesis, but by acting as a freely diffusible protein. PMID:4572718

  14. Bistable behavior of the lac operon in E. coli when induced with a mixture of lactose and TMG.

    PubMed

    Díaz-Hernández, Orlando; Santillán, Moisés

    2010-01-01

    In this work we investigate multistability in the lac operon of Escherichia coli when it is induced by a mixture of lactose and the non-metabolizable thiomethyl galactoside (TMG). In accordance with previously published experimental results and computer simulations, our simulations predict that: (1) when the system is induced by TMG, the system shows a discernible bistable behavior while, (2) when the system is induced by lactose, bistability does not disappear but excessively high concentrations of lactose would be required to observe it. Finally, our simulation results predict that when a mixture of lactose and TMG is used, the bistability region in the extracellular glucose concentration vs. extracellular lactose concentration parameter space changes in such a way that the model predictions regarding bistability could be tested experimentally. These experiments could help to solve a recent controversy regarding the existence of bistability in the lac operon under natural conditions. PMID:21423364

  15. Constructing a recombinant hyaluronic acid biosynthesis operon and producing food-grade hyaluronic acid in Lactococcus lactis.

    PubMed

    Sheng, Juzheng; Ling, Peixue; Wang, Fengshan

    2015-02-01

    Hyaluronic acid (HA), a natural high molecular weight polysaccharide, is produced by Streptococcus zooepidemicus. However, Streptococcus has several drawbacks including its potential to produce exotoxins, so there is demand for an alternative HA source. Here, a recombinant HA biosynthesis operon, as well as the HA biosynthesis operon of S. zooepidemicus were introduced into L. lactis using the nisin-controlled expression system, respectively. HA was successfully synthesized by recombinant L. lactis. Furthermore, overexpression of the endogenous enzymes directing the synthesis of precursor sugars was effective at increasing HA production, and increasing the supply of UDP-activated monosaccharide donors aided synthesis of monodisperse HA polysaccharides. Besides GRAS host strain (L. lactis) and NICE system, the selecting marker (lacF gene) of the recombinant strain is also food grade. Therefore, HA produced by recombinant L. lactis overcomes the problems associated with Streptococcus and provides a source of food-grading HA appropriate for widespread biotechnological applications. PMID:25447786

  16. The R-Operon: A Model of Repetitive DNA-Organized Transcriptional Compartmentation of Eukaryotic Chromosomes for Coordinated Gene Expression

    PubMed Central

    Tang, Shao-Jun

    2016-01-01

    In eukaryotic genomes, it is essential to coordinate the activity of genes that function together to fulfill the same biological processes. Genomic organization likely plays a key role in coordinating transcription of different genes. However, little is known about how co-regulated genes are organized in the cell nucleus and how the chromosomal organization facilitates the co-regulation of different genes. I propose that eukaryotic genomes are organized into repeat assembly (RA)-based structural domains (“R-operons”) in the nuclear space. R-operons result from the interaction of homologous DNA repeats. In an R-operon, genes in different loci of the linear genome are brought into spatial vicinity and co-regulated by the same pool of transcription factors. This type of large-scale chromosomal organization may provide a mechanism for functional compartmentation of chromosomes to facilitate the transcriptional coordination of gene expression. PMID:27110825

  17. Sigma-G RNA polymerase controls forespore-specific expression of the glucose dehydrogenase operon in Bacillus subtilis.

    PubMed Central

    Nakatani, Y; Nicholson, W L; Neitzke, K D; Setlow, P; Freese, E

    1989-01-01

    The gene encoding glucose dehydrogenase (gdh) is part of an operon whose expression is transcriptionally activated specifically in the developing forespore of Bacillus subtilis at stage III of sporulation. The in vivo startpoint of gdh transcription was determined using primer extension analysis. Deletion mapping and site-specific mutagenesis experiments indicated that the region responsible for regulated expression of gdh in vivo was limited to the "-35" and "-10" regions preceding the transcriptional start site. RNA polymerase containing omega G (E omega G) transcribed gdh in vitro with a start site identical to that found in vivo, and transcription of gdh by E omega G in vitro also did not require any specific sequences upstream from "-35" region. These results suggest that the appearance of E omega G in the forespore at stage III of sporulation is sufficient to cause temporal and compartment-specific expression of the gdh operon. Images PMID:2493633

  18. Virulence factors of the family Legionellaceae.

    PubMed Central

    Dowling, J N; Saha, A K; Glew, R H

    1992-01-01

    Whereas bacteria in the genus Legionella have emerged as relatively frequent causes of pneumonia, the mechanisms underlying their pathogenicity are obscure. The legionellae are facultative intracellular pathogens which multiply within the phagosome of mononuclear phagocytes and are not killed efficiently by polymorphonuclear leukocytes. The functional defects that might permit the intracellular survival of the legionellae have remained an enigma until recently. Phagosome-lysosome fusion is inhibited by a single strain (Philadelphia 1) of Legionella pneumophila serogroup 1, but not by other strains of L. pneumophila or other species. It has been found that following the ingestion of Legionella organisms, the subsequent activation of neutrophils and monocytes in response to both soluble and particulate stimuli is profoundly impaired and the bactericidal activity of these cells is attenuated, suggesting that Legionella bacterial cell-associated factors have an inhibitory effect on phagocyte activation. Two factors elaborated by the legionellae which inhibit phagocyte activation have been described. First, the Legionella (cyto)toxin blocks neutrophil oxidative metabolism in response to various agonists by an unknown mechanism. Second, L. micdadei bacterial cells contain a phosphatase which blocks superoxide anion production by stimulated neutrophils. The Legionella phosphatase disrupts the formation of critical intracellular second messengers in neutrophils. In addition to the toxin and phosphatase, several other moieties that may serve as virulence factors by promoting cell invasion or intracellular survival and multiplication are elaborated by the legionellae. Molecular biological studies show that a cell surface protein named Mip is necessary for the efficient invasion of monocytes. A possible role for a Legionella phospholipase C as a virulence factor is still largely theoretical. L. micdadei contains an unusual protein kinase which catalyzes the phosphorylation of

  19. Characterization of PaxA and Its Operon: a Cohemolytic RTX Toxin Determinant from Pathogenic Pasteurella aerogenes

    PubMed Central

    Kuhnert, Peter; Heyberger-Meyer, Bénédicte; Nicolet, Jacques; Frey, Joachim

    2000-01-01

    Pasteurella aerogenes is known as a commensal bacterium or as an opportunistic pathogen, as well as a primary pathogen found to be involved in abortion cases of humans, swine, and other mammals. Using broad-range DNA probes for bacterial RTX toxin genes, we cloned and subsequently sequenced a new operon named paxCABD encoding the RTX toxin PaxA in P. aerogenes. The pax operon is organized analogous to the classical RTX operons containing the activator gene paxC upstream of the structural toxin gene paxA, which is followed by the secretion protein genes paxB and paxD. The highest sequence similarity of paxA with known RTX toxin genes is found with apxIIIA (82%). PaxA is structurally similar to ApxIIIA and also shows functional analogy to ApxIIIA, since it shows cohemolytic activity with the sphingomyelinase of Staphylococcus aureus, known as the CAMP effect, but is devoid of direct hemolytic activity. In addition, it shows to some extent immunological cross-reactions with ApxIIIA. P. aerogenes isolated from various specimens showed that the pax operon was present in about one-third of the strains. All of the pax-positive strains were specifically related to swine abortion cases or septicemia of newborn piglets. These strains were also shown to produce the PaxA toxin as determined by the CAMP phenomenon, whereas none of the pax-negative strains did. This indicated that the PaxA toxin is involved in the pathogenic potential of P. aerogenes. The examined P. aerogenes isolates were phylogenetically analyzed by 16S rRNA gene (rrs) sequencing in order to confirm their species. Only a small heterogeneity (<0.5%) was observed between the rrs genes of the strains originating from geographically distant farms and isolated at different times. PMID:10603361

  20. Study of factors which negatively affect expression of the phenol degradation operon pheBA in Pseudomonas putida.

    PubMed

    Putrins, Marta; Tover, Andres; Tegova, Radi; Saks, Ulle; Kivisaar, Maia

    2007-06-01

    Transcription of the plasmid-borne phenol catabolic operon pheBA in Pseudomonas putida is activated by the LysR-family regulator CatR in the presence of the effector molecule cis,cis-muconate (CCM), which is an intermediate of the phenol degradation pathway. In addition to the positive control of the operon, several factors negatively affect transcription initiation from the pheBA promoter. First, the activation of the pheBA operon depends on the extracellular concentration of phenol. The pheBA promoter is rapidly activated in the presence of micromolar concentrations of phenol in minimal growth medium, but the initiation of transcription from this promoter is severely delayed after sudden exposure of bacteria to 2.5 mM phenol. Second, the transcriptional activation from this promoter is impeded when the growth medium of bacteria contains amino acids. The negative effects of amino acids can be suppressed either by overproducing CatR or by increasing, the intracellular amount of CCM. However, the intracellular amount of CCM is a major limiting factor for the transcriptional activation of the pheBA operon, as accumulation of CCM in a P. putida catB-defective strain, unable to metabolize CCM (but expressing CatR at a natural level), almost completely relieves the negative effects of amino acids. The intracellular amount of CCM is negatively affected by the catabolite repression control protein via downregulating at the post-transcriptional level the expression of the pheBA-encoded catechol 1,2-dioxygenase and the phenol monooxygenase, the enzymes needed for CCM production. PMID:17526843

  1. Characterization of the sat Operon in Streptococcus mutans: Evidence for a Role of Ffh in Acid Tolerance

    PubMed Central

    Kremer, Bas H. A.; van der Kraan, Marieke; Crowley, Paula J.; Hamilton, Ian R.; Brady, L. Jeannine; Bleiweis, Arnold S.

    2001-01-01

    An essential protein translocation pathway in Escherichia coli and Bacillus subtilis involves the signal recognition particle (SRP), of which the 54-kDa homolog (Ffh) is an essential component. In a previous study, we found that a transposon insertion in the ylxM-ffh intergenic region of the designated secretion and acid tolerance (sat) operon of Streptococcus mutans resulted in an acid-sensitive phenotype. In the present study, we further characterized this genomic region in S. mutans after construction of bonafide sat operon mutants and confirmed the role of the SRP pathway in acid resistance. Northern blot and primer extension analyses identified an acid-inducible promoter upstream of ylxM that was responsible for upregulating the coordinate expression of all five genes of the sat operon when cells were grown at acid pH. Two constitutive promoters, one immediately upstream of satD and one just 3′ to the acid-inducible promoter, were also identified. Except for Ffh, the functions of the sat operon gene products are unknown. SatC, SatD, and SatE have no homology to proteins with known functions, although YlxM may function as a transcriptional regulator linked to genes encoding SRP pathway proteins. Nonpolar mutations created in each of the five genes of the sat locus resulted in viable mutants. Most striking, however, was the finding that a mutation in ffh did not result in loss of cell viability, as is the case in all other microbial species in which this pathway has been described. This mutant also lacked immunologically detectable Ffh and was severely affected in resistance to acid. Complementation of the mutation resulted in restoration of acid tolerance and reappearance of cytoplasmic Ffh. These data provide evidence that the SRP pathway plays an important role in acid tolerance in S. mutans. PMID:11274114

  2. Structure and copy number of gene clusters related to the pap P-adhesin operon of uropathogenic Escherichia coli.

    PubMed

    Arthur, M; Campanelli, C; Arbeit, R D; Kim, C; Steinbach, S; Johnson, C E; Rubin, R H; Goldstein, R

    1989-02-01

    The structurally related pap and prs operons of the uropathogenic Escherichia coli isolate J96 encode a P and an F adhesin that mediate bacterial attachment to the human P blood group antigen and the Forssman antigen, respectively. Using probes prepared from different segments of the pap operon, Southern blot hybridizations were performed to characterize pap-related sequences of 30 E. coli clinical isolates expressing different adhesin phenotypes. Gene clusters encoding P and F adhesins displayed no restriction site polymorphism in sequences homologous to the papH, papC, and papD genes that encode proteins essential to the transport and polymerization of the subunits of the P-pilus adhesin. In contrast, pap-related genetic elements associated with a null phenotype either lacked homology to the papH, papC, and papD genes or displayed a restriction site polymorphism in this region. Sequences within and surrounding the J96 papG and prsG adhesin genes that determine the binding specificities to the P and F antigens, respectively, were not conserved. However, gene clusters encoding different binding specificities could not be distinguished based on such restriction site polymorphisms. The majority of clinical isolates had more than one copy of pap-related sequences that involved gene clusters similar to the J96 pap operon, as well as genetic elements that were related only to a part of this operon. The implications of this unexpected copy number polymorphism with respect to possible recombination events involving pap-related sequences are discussed.

  3. The Spl Serine Proteases Modulate Staphylococcus aureus Protein Production and Virulence in a Rabbit Model of Pneumonia

    PubMed Central

    Salgado-Pabon, Wilmara; Meyerholz, David K.; White, Mark J.; Schlievert, Patrick M.

    2016-01-01

    ABSTRACT The Spl proteases are a group of six serine proteases that are encoded on the νSaβ pathogenicity island and are unique to Staphylococcus aureus. Despite their interesting biochemistry, their biological substrates and functions in virulence have been difficult to elucidate. We found that an spl operon mutant of the community-associated methicillin-resistant S. aureus USA300 strain LAC induced localized lung damage in a rabbit model of pneumonia, characterized by bronchopneumonia observed histologically. Disease in the mutant-infected rabbits was restricted in distribution compared to that in wild-type USA300-infected rabbits. We also found that SplA is able to cleave the mucin 16 glycoprotein from the surface of the CalU-3 lung cell line, suggesting a possible mechanism for wild-type USA300 spreading pneumonia to both lungs. Investigation of the secreted and surface proteomes of wild-type USA300 and the spl mutant revealed multiple alterations in metabolic proteins and virulence factors. This study demonstrates that the Spls modulate S. aureus physiology and virulence, identifies a human target of SplA, and suggests potential S. aureus targets of the Spl proteases. IMPORTANCE Staphylococcus aureus is a versatile human pathogen that produces an array of virulence factors, including several proteases. Of these, six proteases called the Spls are the least characterized. Previous evidence suggests that the Spls are expressed during human infection; however, their function is unknown. Our study shows that the Spls are required for S. aureus to cause disseminated lung damage during pneumonia. Further, we present the first example of a human protein cut by an Spl protease. Although the Spls were predicted not to cut staphylococcal proteins, we also show that an spl mutant has altered abundance of both secreted and surface-associated proteins. This work provides novel insight into the function of Spls during infection and their potential ability to degrade

  4. Regulation of the nitrate reductase operon: effect of mutations in chlA, B, D and E genes.

    PubMed

    Pascal, M C; Burini, J F; Ratouchniak, J; Chippaux, M

    1982-01-01

    Introduction of chlA, B or E mutant alleles into strains carrying fusions between the lac structural genes and the promoter of the nitrate reductase operon led to the partial or total constitutive expression of the fusion. Presence of chlD mutated alleles in the same strains did not result in constitutive expression of the fusion and allowed full induction by nitrate only in the presence of molybdenum. It is proposed that the molybdenum cofactor, Mo-X, of the nitrate reductase is also corepressor of the operon. The chlA, B and E genes would be involved in the biosynthesis of the X-moity. Mutations in these genes would give an altered X-moity which still binds to molybdenum but leads to a less efficient repressor complex; chlD gene would code for an enzyme inserting molybdenum in the X-moity of the cofactor. Mutations in chlD give an empty cofactor leading to a complex which permanently represses the operon unless molybdenum is added. PMID:6757667

  5. Synthetic operon for (R,R)-2,3-butanediol production in Bacillus subtilis and Escherichia coli.

    PubMed

    de Oliveira, Rafael R; Nicholson, Wayne L

    2016-01-01

    To reduce dependence on petroleum, an alternative route to production of the chemical feedstock 2,3-butanediol (2,3-BD) from renewable lignocellulosic sources is desirable. In this communication, the genes encoding the pathway from pyruvate to 2,3-BD (alsS, alsD, and bdhA encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase, respectively) from Bacillus subtilis were engineered into a single tricistronic operon under control of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible Pspac promoter in a shuttle plasmid capable of replication and expression in either B. subtilis or Escherichia coli. We describe the construction and performance of a shuttle plasmid carrying the IPTG-inducible synthetic operon alsSDbdhA coding for 2,3-BD pathway capable of (i) expression in two important representative model microorganisms, the gram-positive B. subtilis and the gram-negative E. coli; (ii) increasing 2,3-BD production in B. subtilis; and (iii) successfully introducing the B. subtilis 2,3-BD pathway into E. coli. The synthetic alsSDbdhA operon constructed using B. subtilis native genes not only increased the 2,3-BD production in its native host but also efficiently expressed the pathway in the heterologous organism E. coli. Construction of an efficient shuttle plasmid will allow investigation of 2,3-BD production performance in related organisms with industrial potential for production of bio-based chemicals. PMID:26454865

  6. Synthetic operon for (R,R)-2,3-butanediol production in Bacillus subtilis and Escherichia coli.

    PubMed

    de Oliveira, Rafael R; Nicholson, Wayne L

    2016-01-01

    To reduce dependence on petroleum, an alternative route to production of the chemical feedstock 2,3-butanediol (2,3-BD) from renewable lignocellulosic sources is desirable. In this communication, the genes encoding the pathway from pyruvate to 2,3-BD (alsS, alsD, and bdhA encoding acetolactate synthase, acetolactate decarboxylase, and butanediol dehydrogenase, respectively) from Bacillus subtilis were engineered into a single tricistronic operon under control of the isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible Pspac promoter in a shuttle plasmid capable of replication and expression in either B. subtilis or Escherichia coli. We describe the construction and performance of a shuttle plasmid carrying the IPTG-inducible synthetic operon alsSDbdhA coding for 2,3-BD pathway capable of (i) expression in two important representative model microorganisms, the gram-positive B. subtilis and the gram-negative E. coli; (ii) increasing 2,3-BD production in B. subtilis; and (iii) successfully introducing the B. subtilis 2,3-BD pathway into E. coli. The synthetic alsSDbdhA operon constructed using B. subtilis native genes not only increased the 2,3-BD production in its native host but also efficiently expressed the pathway in the heterologous organism E. coli. Construction of an efficient shuttle plasmid will allow investigation of 2,3-BD production performance in related organisms with industrial potential for production of bio-based chemicals.

  7. Expression of the Escherichia coli malPQ operon remains unaffected after drastic alteration of its promoter.

    PubMed Central

    Débarbouillé, M; Raibaud, O

    1983-01-01

    The malPQ operon, one of the three operons of the maltose regulon, is positively controlled by the product of gene malT. The starting point for malPQ transcription was deduced from experiments which involved a hybridization of in vivo-synthesized malPQ mRNA with adequate DNA probes, followed either by a digestion of nonhybridized DNA (S1 nuclease mapping) or by an extension of the hybridized probe (reverse transcriptase mapping). In the wild-type strain, this starting point was 37 nucleotides upstream from the initiation codon for malP. This analysis was also performed on a double mutant which contained both a 13-base pair deletion and a 3-base pair insertion in the promoter region. This double mutant expressed the malPQ operon exactly as the wild-type strain did, in a maltose-inducible manner. In this strain, the starting point for malPQ transcription was shifted 11 nucleotides downstream from the wild-type location. An analysis of these results suggests that (i) the binding site for the malT product is located upstream from the region which is severely altered in the double mutant, i.e., upstream from position -31; and (ii) the 30-base pair sequence which precedes the transcription starting point contains very few positions which are essential for promoter activity. Images PMID:6186658

  8. A distinct alleles and genetic recombination of pmrCAB operon in species of Acinetobacter baumannii complex isolates.

    PubMed

    Kim, Dae Hun; Ko, Kwan Soo

    2015-07-01

    To investigate pmrCAB sequence divergence in 5 species of Acinetobacter baumannii complex, a total of 80 isolates from a Korean hospital were explored. We evaluated nucleotide and amino acid polymorphisms of pmrCAB operon, and phylogenetic trees were constructed for each gene of prmCAB operon. Colistin and polymyxin B susceptibility was determined for all isolates, and multilocus sequence typing was also performed for A. baumannii isolates. Our results showed that each species of A. baumannii complex has divergent pmrCAB operon sequences. We identified a distinct pmrCAB allele allied with Acinetobacter nosocomialis in gene trees. Different grouping in each gene tree suggests sporadic recombination or emergence of pmrCAB genes among Acinetobacter species. Sequence polymorphisms among Acinetobacter species might not be associated with colistin resistance. We revealed that a distinct pmrCAB allele may be widespread across the continents such as North America and Asia and that sporadic genetic recombination or emergence of pmrCAB genes might occur.

  9. Identification of the virB operon genes encoding the type IV secretion system, in Colombian Brucella canis isolates.

    PubMed

    de la Cuesta-Zuluaga, Juan Jacobo; Sánchez-Jiménez, Miryan Margot; Martínez-Garro, Juliana; Olivera-Angel, Martha

    2013-04-12

    Canine brucellosis is a zoonotic disease caused by Brucella canis. The establishment of intracellular replicative niches of B. canis is mediated by proteins secreted by the type IV secretion system, which is encoded by the virB operon. The characterization of such genes has been conducted in other species of the genus, but not in B. canis. We report the design of a multiplex PCR test for the detection of the virB operon genes of B. canis. Primers for each of the 12 genes were designed and evaluated using bioinformatics tools. A multiplex PCR assay was standardized and applied to 36 isolates obtained from infected dogs of Aburrá Valley kennels, as well to the Brucella abortus, Brucella melitensis, Brucella suis and Brucella ovis DNA strains. As a result of the in silico design, a pair of primers for each gene was selected. All species and isolates evaluated showed evidence of the presence of the entire virB operon. PMID:23290573

  10. Proteus mirabilis MR/P fimbrial operon: genetic organization, nucleotide sequence, and conditions for expression.

    PubMed Central

    Bahrani, F K; Mobley, H L

    1994-01-01

    Proteus mirabilis, an agent of urinary tract infection, expresses at least four fimbrial types. Among these are the MR/P (mannose-resistant/Proteus-like) fimbriae. MrpA, the structural subunit, is optimally expressed at 37 degrees C in Luria broth cultured statically for 48 h by each of seven strains examined. Genes encoding this fimbria were isolated, and the complete nucleotide sequence was determined. The mrp gene cluster encoded by 7,293 bp predicts eight polypeptides: MrpI (22,133 Da), MrpA (17,909 Da), MrpB (19,632 Da), MrpC (96,823 Da), MrpD (27,886 Da), MrpE (19,470 Da), MrpF (17,363 Da), and MrpG (13,169 Da). mrpI is upstream of the gene encoding the major structural subunit gene mrpA and is transcribed in the direction opposite to that of the rest of the operon. All predicted polypeptides share > or = 25% amino acid identity with at least one other enteric fimbrial gene product encoded by the pap, fim, smf, fan, or mrk gene clusters. Images PMID:7910820

  11. Bacteriophage 933W encodes a functional esterase downstream of the Shiga toxin 2a operon.

    PubMed

    Nübling, Simone; Eisele, Thomas; Stöber, Helen; Funk, Joschua; Polzin, Sabrina; Fischer, Lutz; Schmidt, Herbert

    2014-05-01

    In this study, the 1938bp open reading frame z1466, which is encoded directly downstream the Shiga toxin 2a (Stx2a) operon in E. coli O157:H7 phage 933W was cloned and expressed recombinantly. Purification with Ni-NTA agarose beads with subsequent SDS-PAGE revealed a 68kDa protein, designated 933Wp42-His. Analysis of 933Wp42-His demonstrated an esterase activity by activity staining of native gels using triacetin as a substrate. Purified 933Wp42-His demonstrated a Km value of about 10mM and a Vmax value of 1.667nkat/ml for 4-methylumbelliferyl-acetate (4-MUF-Ac) as a substrate. The enzyme was most active in the pH-range of 7.0-8.0, and at 50°C. Furthermore, 933Wp42-His was able to hydrolyze acetic acid from mucin, and 5-N-acetyl-9-O-acetyl neuraminic acid (Neu5,9Ac2). This is the first description of an enzymatic activity of the Stx-phage-encoded protein 933Wp42. Its role in substrate utilization during colonization and human infection is discussed.

  12. Effect of salt bridge on transcription activation of CRP-dependent lactose operon in Escherichia coli.

    PubMed

    Tutar, Yusuf; Harman, James G

    2006-09-15

    Expression of catabolite-sensitive operons in Escherichia coli is cAMP-dependent and mediated through the CRP:cAMP complex binding to specific sequences in DNA. Five specific ionic or polar interactions occur in cAMP binding pocket of CRP. E72 interacts with the cAMP 2' OH, R82 and S83 interact with the negatively charged phosphate moiety, and T127 and S128 interact with the adenine ring. There is evidence to suggest that E72 and R82 may mediate an essential CRP molecular switch mechanism. Therefore, stimulation of CRP transcription activation was examined by perturbing these residues. Further, CRP:cAMP complex was treated with a specific DNA sequence containing the lac CRP binding site along with RNA polymerase to mimic in vivo conditions. Biochemical and biophysical results revealed that regulation of transcription activation depends on alignment of CRP tertiary structure through inter-domain communication and it was concluded that positions 72 and 82 are essential in the activation of CRP by cAMP. PMID:16934214

  13. Elements involved in catabolite repression and substrate induction of the lactose operon in Lactobacillus casei.

    PubMed

    Gosalbes, M J; Monedero, V; Pérez-Martínez, G

    1999-07-01

    In Lactobacillus casei ATCC 393, the chromosomally encoded lactose operon, lacTEGF, encodes an antiterminator protein (LacT), lactose-specific phosphoenolpyruvate-dependent phosphotransferase system (PTS) elements (LacE and LacF), and a phospho-beta-galactosidase. lacT, lacE, and lacF mutant strains were constructed by double crossover. The lacT strain displayed constitutive termination at a ribonucleic antiterminator (RAT) site, whereas lacE and lacF mutants showed an inducer-independent antiterminator activity, as shown analysis of enzyme activity obtained from transcriptional fusions of lac promoter (lacp) and lacpDeltaRAT with the Escherichia coli gusA gene in the different lac mutants. These results strongly suggest that in vivo under noninducing conditions, the lactose-specific PTS elements negatively modulate LacT activity. Northern blot analysis detected a 100-nucleotide transcript starting at the transcription start site and ending a consensus RAT sequence and terminator region. In a ccpA mutant, transcription initiation was derepressed but no elongation through the terminator was observed in the presence of glucose and the inducing sugar, lactose. Full expression of lacTEGF was found only in a man ccpA double mutant, indicating that PTS elements are involved in the CcpA-independent catabolite repression mechanism probably via LacT. PMID:10383959

  14. Cu(I)-mediated Allosteric Switching in a Copper-sensing Operon Repressor (CsoR)*

    PubMed Central

    Chang, Feng-Ming James; Coyne, H. Jerome; Cubillas, Ciro; Vinuesa, Pablo; Fang, Xianyang; Ma, Zhen; Ma, Dejian; Helmann, John D.; García-de los Santos, Alejandro; Wang, Yun-Xing; Dann, Charles E.; Giedroc, David P.

    2014-01-01

    The copper-sensing operon repressor (CsoR) is representative of a major Cu(I)-sensing family of bacterial metalloregulatory proteins that has evolved to prevent cytoplasmic copper toxicity. It is unknown how Cu(I) binding to tetrameric CsoRs mediates transcriptional derepression of copper resistance genes. A phylogenetic analysis of 227 DUF156 protein members, including biochemically or structurally characterized CsoR/RcnR repressors, reveals that Geobacillus thermodenitrificans (Gt) CsoR characterized here is representative of CsoRs from pathogenic bacilli Listeria monocytogenes and Bacillus anthracis. The 2.56 Å structure of Cu(I)-bound Gt CsoR reveals that Cu(I) binding induces a kink in the α2-helix between two conserved copper-ligating residues and folds an N-terminal tail (residues 12–19) over the Cu(I) binding site. NMR studies of Gt CsoR reveal that this tail is flexible in the apo-state with these dynamics quenched upon Cu(I) binding. Small angle x-ray scattering experiments on an N-terminally truncated Gt CsoR (Δ2–10) reveal that the Cu(I)-bound tetramer is hydrodynamically more compact than is the apo-state. The implications of these findings for the allosteric mechanisms of other CsoR/RcnR repressors are discussed. PMID:24831014

  15. Genetic analysis of the stationary phase-induced mcb operon promoter in Escherichia coli.

    PubMed

    Mao, W; Siegele, D A

    1998-01-01

    A combination of deletion analysis and random mutagenesis was used to identify regulatory elements in Pmcb, the stationary phase-induced promoter of the mcb operon. Our results indicate that Pmcb is controlled by at least three different factors, two previously identified and at least one unknown factor, which act at four different sites in the promoter. Sequences between -344 and -164 upstream of the transcriptional start site were required for wild-type levels of mcb transcription in stationary phase. More dramatic reductions in both exponential and stationary phase expression were observed when sequences from -164 to -54 were deleted. Point mutations located between -105 and -138 decreased both exponential and stationary phase expression. All but one of these mutations decreased OmpR-dependent activation of Pmcb transcription. EmrR, also known as MprA, acts directly or indirectly at sequences downstream of -54 to repress Pmcb. A minimal promoter containing sequences from -34 to +79 was still induced > or = 10-fold in stationary phase. Point mutations within this region identified sequences at -8, -11, -30, -31 and -32 as important for Pmcb activity. These bases are in the gearbox sequence, present in Pmcb and several other stationary phase-induced Escherichia coli promoters.

  16. The Rhodomonas salina mitochondrial genome: bacteria-like operons, compact gene arrangement and complex repeat region.

    PubMed

    Hauth, Amy M; Maier, Uwe G; Lang, B Franz; Burger, Gertraud

    2005-01-01

    To gain insight into the mitochondrial genome structure and gene content of a putatively ancestral group of eukaryotes, the cryptophytes, we sequenced the complete mitochondrial DNA of Rhodomonas salina. The 48 063 bp circular-mapping molecule codes for 2 rRNAs, 27 tRNAs and 40 proteins including 23 components of oxidative phosphorylation, 15 ribosomal proteins and two subunits of tat translocase. One potential protein (ORF161) is without assigned function. Only two introns occur in the genome; both are present within cox1 belong to group II and contain RT open reading frames. Primitive genome features include bacteria-like rRNAs and tRNAs, ribosomal protein genes organized in large clusters resembling bacterial operons and the presence of the otherwise rare genes such as rps1 and tatA. The highly compact gene organization contrasts with the presence of a 4.7 kb long, repeat-containing intergenic region. Repeat motifs approximately 40-700 bp long occur up to 31 times, forming a complex repeat structure. Tandem repeats are the major arrangement but the region also includes a large, approximately 3 kb, inverted repeat and several potentially stable approximately 40-80 bp long hairpin structures. We provide evidence that the large repeat region is involved in replication and transcription initiation, predict a promoter motif that occurs in three locations and discuss two likely scenarios of how this highly structured repeat region might have evolved.

  17. Autonomous expression of the slo gene of the bicistronic nga-slo operon of Streptococcus pyogenes.

    PubMed

    Savic, Dragutin J; McShan, William M; Ferretti, Joseph J

    2002-05-01

    A recent model for cytolysin-mediated translocation in Streptococcus pyogenes proposes that NAD-glycohydrolase is translocated through streptolysin O-generated pores into a host cell (J. Madden, N. Ruiz, and M. Caparon, Cell 104:143-152, 2001). This model also assumes that the NAD-glycohydrolase (nga) and streptolysin O (slo) genes that code for these products are organized in an operon-like structure expressed from a single promoter only (nga). We expand this model by showing that slo possesses its own autonomous promoter, which is located 155 bp upstream of the slo gene. Under experimental conditions in which S. pyogenes is grown in THY medium, the strength of the slo promoter, as measured by the activity of a lacZ reporter gene, resulted in low but highly reproducible values. Finally, we demonstrated that sloR, a S. pyogenes gene that closely resembles the Clostridium perfringens pfoR gene, exerts a negative effect on the expression of the slo gene. PMID:11953421

  18. Cloning and Expression of Poly 3-Hydroxybutyrate Operon Into Escherichia coli

    PubMed Central

    Jari, Maryam; Khatami, Saeid Reza; Galehdari, Hamid; Shafiei, Mohammad

    2015-01-01

    Background: Poly 3-Hydroxybutyrate (PHB), a class of Poly Hydroxyalkanoates (PHAs), is a group of bacterial storage polymers, produced by various microorganisms in response to nutrient limitation. PHAs are biodegradable polymers which could be a good substitute for current petrochemical plastics. PHB has been synthesized during three enzymatic steps including three genes. Objectives: Our aim was PHB production from recombinant bacteria. Materials and Methods: Ralstonia eutropha was cultured and its genomic DNA was extracted. The phbCAB operon was amplified using designed primers. The fragment was cloned into pET-28a expression vector and then transformed into Escherichia coli BL21. Sudan black staining was used to show the production of PHB. Results: The extracted recombinant plasmid was digested with restriction enzymes. Separation of the desired fragment from the vector was performed to prove the correct insertion of the PCR products into the vector. The colony PCR and sequencing results confirmed the successful transformation. The production of PHB was confirmed by Sudan Black B staining under a light microscope. Conclusions: Various metabolic and fermentation methods have been used in some bacterial strains for PHB production. The use of a recombinant system harboring PHB synthesis genes can produce PHB in higher concentrations compare to natural PHA-producing bacteria. The present study was one of the most important and basic steps of designing a recombinant E. coli that can produce PHB. PMID:25834710

  19. Characterization of the glnK-amtB operon of Azotobacter vinelandii.

    PubMed

    Meletzus, D; Rudnick, P; Doetsch, N; Green, A; Kennedy, C

    1998-06-01

    To determine whether in Azotobacter vinelandii the PII protein influences the regulation of nif gene expression in response to fluxes in the ammonium supply, the gene encoding PII was isolated and characterized. Its deduced translation product was highly similar to PII proteins from other organisms, with the greatest degree of relatedness being exhibited to the Escherichia coli glnK gene product. A gene designated amtB was found downstream of and was contranscribed with glnK as in E. coli. The AmtB protein is similar to functionally characterized ammonium transport proteins from a few other eukaryotes and one other prokaryote. glnK and amtB comprise an operon. Attempts to isolate a stable glnK mutant strain were unsuccessful, suggesting that glnK, like glnA, is an essential gene in A. vinelandii. amtB mutants were isolated, and although growth on limiting amounts of ammonium was similar in the mutant and wild-type strains, the mutants were unable to transport [14C]methylammonium.

  20. The Legionella pneumophila kai operon is implicated in stress response and confers fitness in competitive environments

    PubMed Central

    Loza-Correa, Maria; Sahr, Tobias; Rolando, Monica; Daniels, Craig; Petit, Pierre; Skarina, Tania; Valero, Laura Gomez; Dervins-Ravault, Delphine; Honoré, Nadine; Savchenko, Aleksey; Buchrieser, Carmen

    2014-01-01

    Summary Legionella pneumophila uses aquatic protozoa as replication niche and protection from harsh environments. Although L. pneumophila is not known to have a circadian clock, it encodes homologues of the KaiBC proteins of Cyanobacteria that regulate circadian gene expression. We show that L. pneumophila kaiB, kaiC and the downstream gene lpp1114, are transcribed as a unit under the control of the stress sigma factor RpoS. KaiC and KaiB of L. pneumophila do not interact as evidenced by yeast and bacterial two-hybrid analyses. Fusion of the C-terminal residues of cyanobacterial KaiB to Legionella KaiB restores their interaction. In contrast, KaiC of L. pneumophila conserved autophosphorylation activity, but KaiB does not trigger the dephosphorylation of KaiC like in Cyanobacteria. The crystal structure of L. pneumophila KaiB suggests that it is an oxidoreductase-like protein with a typical thioredoxin fold. Indeed, mutant analyses revealed that the kai operon-encoded proteins increase fitness of L. pneumophila in competitive environments, and confer higher resistance to oxidative and sodium stress. The phylogenetic analysis indicates that L. pneumophila KaiBC resemble Synechosystis KaiC2B2 and not circadian KaiB1C1. Thus, the L. pneumophila Kai proteins do not encode a circadian clock, but enhance stress resistance and adaption to changes in the environments. PMID:23957615

  1. Post-transcriptional processing of the LEE4 operon in Enterohemorrhagic Escherichia coli

    PubMed Central

    Lodato, Patricia B.; Kaper, James B.

    2009-01-01

    SUMMARY Enterohemorrhagic Escherichia coli (EHEC) employs a type III secretion system (T3SS) to export translocator and effector proteins required for mucosal colonization. The T3SS is encoded in a pathogenicity island called the locus of enterocyte effacement (LEE) that is organized in five major operons, LEE1 to LEE5. LEE4 encodes a regulator of secretion (SepL), translocators (EspA, D and B), two chaperones (CesD2 and L0017), a T3SS component (EscF), and an effector protein (EspF). It was originally proposed that the esp transcript is transcribed from a promoter located at the end of sepL but other authors suggested that this transcript is the result of a post-transcriptional processing event. In this study, we established that the espADB mRNA is generated by post-transcriptional processing at the end of the sepL coding sequence. RNase E is the endonuclease involved in the cleavage, but the interaction of this enzyme with other proteins through its C-terminal half is dispensable. A putative transcription termination event in the cesD2 coding region would generate the 3’ end of the transcript. Similar to what has been described for other processed transcripts, the cleavage of LEE4 seems a mechanism to differentially regulate SepL and Esp protein production. PMID:19019141

  2. Biofilm formation of ica operon-positive Staphylococcus epidermidis from different sources.

    PubMed

    Argudín, Maria Angeles; Vanderhaeghen, Wannes; Vandendriessche, Stien; Vandecandelaere, Ilse; Denis, Olivier; Coenye, Tom; Butaye, Patrick

    2015-12-01

    Information on the prevalence of biofilm-related factors (PIA, Bhp, Aap, Embp) in Staphylococcus epidermidis of animal origin is scarce. In this study, 263 S. epidermidis isolates of diverse origin (animal, farmers, patients, and laboratory staff) were investigated for the presence of the ica operon (icaRADBC). The icaRADBC-positive isolates were further characterized by means of biofilm formation, presence of other biofilm-related genes, antimicrobial resistance, and population structure. Of all isolates, 28.5% (n = 75) were icaRADBC-positive, including 16.5% of animal origin, 29.1% farmer isolates, and 44.6% hospital-associated isolates (including patients and laboratory staff isolates). Most icaRADBC-positive isolates carried embp (n = 73), aap (n = 57), bhp (n = 22), and IS256 (n = 29). Statistical differences were found between animal and patient isolates for the presence of icaRADBC, bhp, and aap. No statistically significant relation was found between the presence of one or more genes and the level of biofilm formation. Most icaRADBC-positive isolates belonged to the clonal complex 5 (formerly 2) and most sequence types corresponded to types previously observed in community and nosocomial S. epidermidis populations. Although the prevalence of S. epidermidis in the nasal cavity of bovines and poultry is low, some isolates belong to STs related to ica-positive clinical strains. PMID:26547374

  3. Gene position in a long operon governs motility development in Bacillus subtilis

    PubMed Central

    Cozy, Loralyn M.; Kearns, Daniel B.

    2010-01-01

    Growing cultures of Bacillus subtilis bifurcate into subpopulations of motile individuals and non-motile chains of cells that are differentiated at the level of gene expression. The motile cells are ON and the chaining cells are OFF for transcription that depends on RNA polymerase and the alternative sigma factor σD. Here we show that chaining cells were OFF for σD-dependent gene expression because σD levels fell below a threshold, and σD activity was inhibited by the anti-sigma factor FlgM. The probability that σD exceeded the threshold was governed by the position of the sigD genes. The proportion of ON cells increased when sigD was artificially moved forward in the 27kb fla/che operon. In addition, we identified a new σD-dependent promoter that increases sigD expression and may provide positive feedback to stabilize the ON state. Finally, we demonstrate that ON/OFF motility states in B. subtilis are a form of development because mosaics of stable and differentiated epigenotypes were evident when the normally dispersed bacteria were forced to grow in one dimension. PMID:20233303

  4. Biofilm formation of ica operon-positive Staphylococcus epidermidis from different sources.

    PubMed

    Argudín, Maria Angeles; Vanderhaeghen, Wannes; Vandendriessche, Stien; Vandecandelaere, Ilse; Denis, Olivier; Coenye, Tom; Butaye, Patrick

    2015-12-01

    Information on the prevalence of biofilm-related factors (PIA, Bhp, Aap, Embp) in Staphylococcus epidermidis of animal origin is scarce. In this study, 263 S. epidermidis isolates of diverse origin (animal, farmers, patients, and labora