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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

    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

  3. 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

  4. A response regulator that represses transcription of several virulence operons in the group A streptococcus.

    PubMed

    Federle, M J; McIver, K S; Scott, J R

    1999-06-01

    A search for homologs of the Bacillus subtilis PhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified as csrR (J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209-219, 1998), caused the strain to produce mucoid colonies and to increase transcription of hasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription of ska (encoding streptokinase), sagA (streptolysin S), and speMF (mitogenic factor) but did not affect transcription of slo (streptolysin O), mga (multiple gene regulator of GAS), emm (M protein), scpA (complement C5a peptidase), or speB or speC (pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system in Escherichia coli whose mechanism is unrelated to that of these genes in GAS, the locus has been renamed covR, for "control of virulence genes" in GAS. Transcription of the covR operon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of the covR nonpolar insertion mutant were complemented by the covR gene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen. PMID:10368137

  5. A Response Regulator That Represses Transcription of Several Virulence Operons in the Group A Streptococcus

    PubMed Central

    Federle, Michael J.; McIver, Kevin S.; Scott, June R.

    1999-01-01

    A search for homologs of the Bacillus subtilis PhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified as csrR (J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209–219, 1998), caused the strain to produce mucoid colonies and to increase transcription of hasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription of ska (encoding streptokinase), sagA (streptolysin S), and speMF (mitogenic factor) but did not affect transcription of slo (streptolysin O), mga (multiple gene regulator of GAS), emm (M protein), scpA (complement C5a peptidase), or speB or speC (pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system in Escherichia coli whose mechanism is unrelated to that of these genes in GAS, the locus has been renamed covR, for “control of virulence genes” in GAS. Transcription of the covR operon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of the covR nonpolar insertion mutant were complemented by the covR gene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen. PMID:10368137

  6. 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

  7. Tandem attenuators control expression of the Salmonella mgtCBR virulence operon.

    PubMed

    Lee, Eun-Jin; Groisman, Eduardo A

    2012-10-01

    The mgtCBR operon from Salmonella enterica serovar Typhimurium specifies the virulence protein MgtC, the Mg(2+) transporter MgtB and the regulatory peptide MgtR. The mgtCBR transcript includes a long leader region harbouring two short open reading frames (ORFs). Translation of these ORFs is anticipated to impact the formation of particular stem-loop structures and control transcription of the coding region by an attenuation-like mechanism. We previously reported that ORF mgtM enables Salmonella to promote transcription of the mgtC and mgtB coding regions when experiencing a rise in cytoplasmic ATP levels. We now show that the proline codon-rich ORF mgtP mediates an increase in transcription of the mgtC and mgtB coding regions under conditions predicted to decrease the levels of proline-charged tRNA(Pro) . The high ATP and low proline signals act independently in an additive form. Replacing conserved mgtP proline codons with codons specifying other amino acids abolished the response to proline limitation but had no effect on the response to ATP. Substitution of conserved adenine nucleotides in mgtM abolished the response to ATP but had no effect in the response to proline limitation. This provides a singular example of a leader mRNA with tandem attenuators responding to different signals. PMID:22857388

  8. [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

  9. 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. PMID:24865652

  10. 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

  11. The Brucella suis Homologue of the Agrobacterium tumefaciens Chromosomal Virulence Operon chvE Is Essential for Sugar Utilization but Not for Survival in Macrophages

    PubMed Central

    Alvarez-Martinez, Maria-Teresa; Machold, Jan; Weise, Christoph; Schmidt-Eisenlohr, Heike; Baron, Christian; Rouot, Bruno

    2001-01-01

    Brucella strains possess an operon encoding type IV secretion machinery very similar to that coded by the Agrobacterium tumefaciens virB operon. Here we describe cloning of the Brucella suis homologue of the chvE-gguA-gguB operon of A. tumefaciens and characterize the sugar binding protein ChvE (78% identity), which in A. tumefaciens is involved in virulence gene expression. B. suis chvE is upstream of the putative sugar transporter-encoding genes gguA and gguB, also present in A. tumefaciens, but not adjacent to that of a LysR-type transcription regulator. Although results of Southern hybridization experiments suggested that the gene is present in all Brucella strains, the ChvE protein was detected only in B. suis and Brucella canis with A. tumefaciens ChvE-specific antisera, suggesting that chvE genes are differently expressed in different Brucella species. Analysis of cell growth of B. suis and of its chvE or gguA mutants in different media revealed that ChvE exhibited a sugar specificity similar to that of its A. tumefaciens homologue and that both ChvE and GguA were necessary for utilization of these sugars. Murine or human macrophage infections with B. suis chvE and gguA mutants resulted in multiplication similar to that of the wild-type strain, suggesting that virB expression was unaffected. These data indicate that the ChvE and GguA homologous proteins of B. suis are essential for the utilization of certain sugars but are not necessary for survival and replication inside macrophages. PMID:11514518

  12. 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. PMID:18284589

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

    PubMed Central

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

    1997-01-01

    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

  14. 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

  15. 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

  16. 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

  17. MprA and DosR coregulate a Mycobacterium tuberculosis virulence operon encoding Rv1813c and Rv1812c.

    PubMed

    Bretl, Daniel J; He, Hongjun; Demetriadou, Crystalla; White, Mark J; Penoske, Renee M; Salzman, Nita H; Zahrt, Thomas C

    2012-09-01

    Mycobacterium tuberculosis remains a significant global pathogen, causing extensive morbidity and mortality worldwide. This bacterium persists within granulomatous lesions in a poorly characterized, nonreplicating state. The two-component signal transduction systems MprAB and DosRS-DosT (DevRS-Rv2027c) are responsive to conditions likely to be present within granulomatous lesions and mediate aspects of M. tuberculosis persistence in vitro and in vivo. Here, we describe a previously uncharacterized locus, Rv1813c-Rv1812c, that is coregulated by both MprA and DosR. We demonstrate that MprA and DosR bind to adjacent and overlapping sequences within the promoter region of Rv1813c and direct transcription from an initiation site located several hundred base pairs upstream of the Rv1813 translation start site. We further show that Rv1813c and Rv1812c are cotranscribed, and that the genomic organization of this operon is specific to M. tuberculosis and Mycobacterium bovis. Although Rv1813c is not required for survival of M. tuberculosis in vitro, including under conditions in which MprAB and DosRST signaling are activated, an M. tuberculosis ΔRv1813c mutant is attenuated in the low-dose aerosol model of murine tuberculosis, where it exhibits a lower bacterial burden, delayed time to death, and decreased ability to stimulate proinflammatory cytokines interleukin-1β (IL-1β) and IL-12. Interestingly, overcomplementation of these phenotypes is observed in the M. tuberculosis ΔRv1813c mutant expressing both Rv1813c and Rv1812c, but not Rv1813c alone, in trans. Therefore, Rv1813c and Rv1812c may represent general stress-responsive elements that are necessary for aspects of M. tuberculosis virulence and the host immune response to infection. PMID:22689819

  18. The Divergently Transcribed Streptococcus parasanguis Virulence-Associated fimA Operon Encoding an Mn2+-Responsive Metal Transporter and pepO Encoding a Zinc Metallopeptidase Are Not Coordinately Regulated

    PubMed Central

    Oetjen, Joyce; Fives-Taylor, Paula; Froeliger, Eunice H.

    2002-01-01

    The study of how bacteria respond to and obtain divalent metal ions provides insight into the regulation of virulence factors in the host environment. Regulation of metal permease operons in gram-positive bacteria may involve the binding of metal-responsive repressors to palindromic domains in their control regions. The Streptococcus parasanguis fimA operon, which encodes an ATP-binding cassette (ABC) transporter system with sequence homology to the LraI family of metal transporters, possesses a palindromic regulatory region with high homology to that of the Streptococcus gordonii ScaR binding domain. Mapping of the promoter and regulatory regions of fimA and the divergently transcribed pepO gene, which encodes a zinc metalloendopeptidase, indicated that their promoter and regulatory elements overlap. fimA had one transcriptional start site, whereas pepO had three. Analysis of truncated versions of the pepO promoter suggested that all three transcriptional start sites are functional. Analysis of promoter activity under various environmental conditions indicated that the fimA operon promoter and the pepO promoter are not coordinately regulated. The fimA operon is responsive to changes in Mn2+ concentration, but the pepO promoter is not. A S. parasanguis fimA mutant showed a growth deficiency under conditions of limiting Mn2+. This deficiency was not alleviated by compensation with either Mg2+ or Fe3+. Wild-type S. parasanguis could take up Mn2+ and Fe3+, while the fimA mutant showed a marked reduction in this ability. These data suggested that FimA is a component of a metal transporter system capable of transporting both Mn2+ and Fe3+. FimA expression itself was shown to be responsive to Mn2+ concentration, but not to availability of Fe3+ or Mg2+. PMID:12228300

  19. Transcriptional analysis of the MrpJ network: modulation of diverse virulence-associated genes and direct regulation of mrp fimbrial and flhDC flagellar operons in Proteus mirabilis.

    PubMed

    Bode, Nadine J; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen; Pearson, Melanie M

    2015-06-01

    The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence. PMID:25847961

  20. Transcriptional Analysis of the MrpJ Network: Modulation of Diverse Virulence-Associated Genes and Direct Regulation of mrp Fimbrial and flhDC Flagellar Operons in Proteus mirabilis

    PubMed Central

    Bode, Nadine J.; Debnath, Irina; Kuan, Lisa; Schulfer, Anjelique; Ty, Maureen

    2015-01-01

    The enteric bacterium Proteus mirabilis is associated with a significant number of catheter-associated urinary tract infections (UTIs). Strict regulation of the antagonistic processes of adhesion and motility, mediated by fimbriae and flagella, respectively, is essential for disease progression. Previously, the transcriptional regulator MrpJ, which is encoded by the mrp fimbrial operon, has been shown to repress both swimming and swarming motility. Here we show that MrpJ affects an array of cellular processes beyond adherence and motility. Microarray analysis found that expression of mrpJ mimicking levels observed during UTIs leads to differential expression of 217 genes related to, among other functions, bacterial virulence, type VI secretion, and metabolism. We probed the molecular mechanism of transcriptional regulation by MrpJ using transcriptional reporters and chromatin immunoprecipitation (ChIP). Binding of MrpJ to two virulence-associated target gene promoters, the promoters of the flagellar master regulator flhDC and mrp itself, appears to be affected by the condensation state of the native chromosome, although both targets share a direct MrpJ binding site proximal to the transcriptional start. Furthermore, an mrpJ deletion mutant colonized the bladders of mice at significantly lower levels in a transurethral model of infection. Additionally, we observed that mrpJ is widely conserved in a collection of recent clinical isolates. Altogether, these findings support a role of MrpJ as a global regulator of P. mirabilis virulence. PMID:25847961

  1. A Natural Inactivating Mutation in the CovS Component of the CovRS Regulatory Operon in a Pattern D Streptococcal pyogenes Strain Influences Virulence-associated Genes*

    PubMed Central

    Liang, Zhong; Zhang, Yueling; Agrahari, Garima; Chandrahas, Vishwanatha; Glinton, Kristofor; Donahue, Deborah L.; Balsara, Rashna D.; Ploplis, Victoria A.; Castellino, Francis J.

    2013-01-01

    A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covSM) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covSM gene was mutated to WT-covS (AP53/covSWT) and a strain that contained an inactivated covR gene (AP53/ΔcovR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covSM, were also employed. SpeB was not measurably expressed in strains containing covRWT/covSM, whereas in strains with natural or engineered covRWT/covSWT, SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covSM, whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase. PMID:23316057

  2. Extended Virulence Genotype of Pathogenic Escherichia coli Isolates Carrying the afa-8 Operon: Evidence of Similarities between Isolates from Humans and Animals with Extraintestinal Infections

    PubMed Central

    Girardeau, Jean Pierre; Lalioui, Lila; Said, A. Mohamed Ou; De Champs, Christophe; Le Bouguénec, Chantal

    2003-01-01

    The afimbrial AfaE-VIII adhesin is common among Escherichia coli isolates from calves with intestinal and/or extraintestinal infections and from humans with sepsis or pyelonephritis. The virulence genotypes of 77 Escherichia coli afa-8 isolates from farm animals and humans were compared to determine whether any trait of commonality exists between isolates of the different host species. Over half of the extraintestinal afa-8 isolates were associated with pap and f17Ac adhesin genes and contained virulence genes (pap, hly, and cnf1) which are characteristic of human extraintestinal pathogenic E. coli (ExPEC). PapG, which occurs as three known variants (variants I to III), is encoded by the corresponding three alleles of papG. Among the pap-positive strains, new papG variants (papGrs) that differed from the isolates with genes for the three adhesin classes predominated over isolates with papG allele III, which in turn were more prevalent than those with allele II. The data showed the substantial prevalence of the enteroaggregative E. coli heat-stable enterotoxin gene (east1) among afa-8 isolates. Most of the afa-8 isolates harbored the high-pathogenicity island (HPI) present in pathogenic Yersinia; however, two-thirds of the HPI-positive strains shared a truncated HPI integrase gene. The presence of ExPEC-associated virulence factors (VFs) in extraintestinal isolates that carry genes typical of enteric strains and that express O antigens associated with intestinal E. coli is consistent with transfer of VFs and O-antigen determinants between ExPEC and enteric strains. The similarities between animal and human ExPEC strains support the hypothesis of overlapping populations, with members of certain clones or clonal groups including animal and human strains. The presence of multiple-antibiotic-resistant bovine afa-8 strains among such clones may represent a potential public health risk. PMID:12517852

  3. Development of a live, attenuated, potential vaccine strain of R. equi expressing vapA and the virR operon, and virulence assessment in the mouse.

    PubMed

    Whitehead, Ashley E; Parreira, Valeria R; Hewson, Joanne; Watson, Johanna L; Prescott, John F

    2012-01-15

    Pneumonia caused by Rhodococcus equi remains a significant problem in foals. The objective of this study was to develop a safe and efficacious attenuated strain of R. equi for eventual use in oral immunization of foals. The approach involved expression of vapA in a live, virulence plasmid-negative, strain of R. equi (strain 103-). PCR-amplified fragments of the vapA gene, with and without the upstream genes virR, orf5, vapH, orf7 and orf8 (orf4-8), were cloned into a shuttle vector pNBV1. These plasmids, named pAW48A and pAWVapA respectively, were electroporated into strain 103-. The presence of the recombinant vectors in the attenuated strain (103-) and the integrity of the inserted genes were confirmed, and both constructs expressed VapA. The virulence of the two strains was compared to that of wild type R. equi 103+ and negative controls by their intravenous inoculation into mice, followed by examination of liver clearance 4 days later. Mice inoculated with R. equi 103-, 103-/pAWVapA and 103-/pNBV1 completely cleared infection, whereas strain 103-/pAW48A persisted in 47% of mice. PMID:22088674

  4. A natural inactivating mutation in the CovS component of the CovRS regulatory operon in a pattern D Streptococcal pyogenes strain influences virulence-associated genes.

    PubMed

    Liang, Zhong; Zhang, Yueling; Agrahari, Garima; Chandrahas, Vishwanatha; Glinton, Kristofor; Donahue, Deborah L; Balsara, Rashna D; Ploplis, Victoria A; Castellino, Francis J

    2013-03-01

    A skin-tropic invasive group A Streptococcus pyogenes (GAS) strain, AP53, contains a natural inactivating mutation in the covS gene (covS(M)) of the two-component responder (CovR)/sensor (CovS) gene regulatory system. The effects of this mutation on specific GAS virulence determinants have been assessed, with emphasis on expression of the extracellular protease, streptococcal pyrogenic exotoxin B (SpeB), capsular hyaluronic acid, and proteins that allow host plasmin assembly on the bacterial surface, viz. a high affinity plasminogen (Pg)/plasmin receptor, Pg-binding group A streptococcal M protein (PAM), and the human Pg activator streptokinase. To further illuminate mechanisms of the functioning of CovRS in the virulence of AP53, two AP53 isogenic strains were generated, one in which the natural covS(M) gene was mutated to WT-covS (AP53/covS(WT)) and a strain that contained an inactivated covR gene (AP53/ΔcovR). Two additional strains that do not contain PAM, viz. WT-NS931 and NS931/covS(M), were also employed. SpeB was not measurably expressed in strains containing covR(WT)/covS(M), whereas in strains with natural or engineered covR(WT)/covS(WT), SpeB expression was highly up-regulated. Alternatively, capsule synthesis via the hasABC operon was enhanced in strain AP53/covS(M), whereas streptokinase expression was only slightly affected by the covS inactivation. PAM expression was not substantially influenced by the covS mutation, suggesting that covRS had minimal effects on the mga regulon that controls PAM expression. These results demonstrate that a covS inactivation results in virulence gene alterations and also suggest that the CovR phosphorylation needed for gene up- or down-regulation can occur by alternative pathways to CovS kinase. PMID:23316057

  5. Relationships between H-NS, sigma S, SpvR and growth phase in the control of spvR, the regulatory gene of the Salmonella plasmid virulence operon.

    PubMed

    Robbe-Saule, V; Schaeffer, F; Kowarz, L; Norel, F

    1997-10-01

    The sigma S-regulated gene spvR of Salmonella typhimurium encodes an autoregulatory protein required for transcriptional activation of the virulence operon spvABCD. A mutation in the histone-like protein H-NS, which negatively controls the sigma S level, has been reported to increase spv gene expression in S. typhimurium strain LT2. In agreement with this, we found that transcription of spvR and spvABCD was derepressed in hns strains of Escherichia coli and S. typhimurium. Moreover, levels of spv gene expression in hns rpoS double mutants were higher than expression levels in mutants deficient in rpoS alone, and were close to those measured in wild-type strains. This demonstrates that H-NS contributes to spv gene regulation independently of its function in controlling the sigma S level. Since the same start site was used for spvR gene transcription in wild-type as in hns and hns rpoS mutant strains, it is likely that the spvR promoter. spvRp1, can be recognized efficiently by an RNA polymerase containing sigma 70. The spvR promoter region shows an intrinsic DNA curvature that might be a determinant in H-NS- and/or sigma S-mediated control. A single amino acid substitution, Leu to Pro at position 265, abolished the regulatory function of SpvR in E. coli and Salmonella, implicating the C-terminal domain of SpvR in its structure and/or regulatory function. The spvR265 allele is not transcribed at detectable levels in hns or hns rpoS strains, suggesting that activation of spvRp1 in these strains remains dependent on SpvR. Thus, we propose a model for spvR gene regulation in which SpvR acts as a co-regulator of an RNA polymerase containing either sigma 70 (in the absence of H-NS) or sigma S, to induce transcriptional initiation at spvRp1. Moreover, growth-phase regulation of spv gene expression was maintained in hns and hns rpoS strains, indicating that an additional element, besides sigma S, is involved in the growth-phase regulation in rich medium. PMID:9393431

  6. 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

  7. 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

  8. 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

  9. 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.

  10. 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

  11. 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

  12. Structural analysis of the Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI) operon.

    PubMed Central

    Jansen, R; Briaire, J; Kamp, E M; Gielkens, A L; Smits, M A

    1993-01-01

    Actinobacillus pleuropneumoniae-RTX-toxin I (ApxI), an important virulence factor, is secreted by serotypes 1, 5, 9, 10, and 11 of A. pleuropneumoniae. However, sequences homologous to the secretion genes apxIBD of the ApxI operon are present in all 12 serotypes except serotype 3. The purpose of this study was to determine and compare the structures of the ApxI operons of the 12 A. pleuropneumoniae serotypes. We focused on the nucleotide sequence comparison of the ApxI-coding genes, the structures of the ApxI operons, and the transcription of the ApxI operons. We determined the nucleotide sequences of the toxin-encoding apxICA genes of serotype 9 and found that the gene for the structural toxin, apxIA, was almost identical to the apxIA gene of serotype 1. The toxin-encoding genes of the other serotypes are also similar for the main part; nevertheless, two variants were identified, one in serotypes 1, 9, and 11 and one in serotypes 5 and 10. The two apxIA variants differ mainly within the distal 110 nucleotides. Structural analysis demonstrated that intact ApxI operons, consisting of the four contiguous genes apxICABD, are present in serotypes 1, 5, 9, 10, and 11. ApxI operons with a major deletion in the apxICA genes are present in serotypes 2, 4, 6, 7, 8, and 12. Serotype 3 does not contain ApxI operon sequences. We found that all ApxI operons are transcriptionally active despite the partial deletion of the operon in some serotypes. The implications of these data for the expression and secretion of ApxI and the other Apx-toxins, ApxII and ApxIII, as well as for the development of a subunit vaccine against A. pleuropneumoniae will be discussed. Images PMID:8359891

  13. 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…

  14. 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.

  15. A mgl-like operon in Treponema pallidum, the syphilis spirochete.

    PubMed

    Porcella, S F; Popova, T G; Hagman, K E; Penn, C W; Radolf, J D; Norgard, M V

    1996-10-24

    A 38-kDa lipoprotein of Treponema pallidum subsp. pallidum (T. pallidum), the syphilis spirochete, previously was identified as a putative homolog of E. coli MglB [Becker et al. (1994) Infect. Immun. 62, 1381-1391]. In the present study, genome walking in regions adjacent to the T. pallidum 38-kDa lipoprotein gene has identified three contiguous genes (tp-mglB [formerly tpp38], tp-mglA, and tp-mglC) which appear to comprise a mgl-like operon in T. pallidum. A prominent transcript corresponding to tp-mglB, the first gene of the operon which encodes the carbohydrate receptor, is synthesized by T. pallidum along with lesser abundant transcript(s) corresponding to the entire T. pallidum mgl operon. An active promoter 135 bp upstream of tp-mglB is believed to direct mRNA synthesis for the operon. This is the first membrane protein-encoding operon of T. pallidum for which a putative function (glucose import) has been assigned. Furthermore, by analogy with E. coli MglB which interacts with the sensory transducer Trg to induce a chemotactic response, it is possible that T. pallidum also contains a homolog of E. coli Trg or other methyl-accepting chemotaxis proteins. The existence of a mgl operon in T. pallidum thus may have important implications with respect to T. pallidum survival, tissue dissemination, and sensory transduction during virulence expression. PMID:8921855

  16. Detecting uber-operons in prokaryotic genomes

    PubMed Central

    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: , the first of its kind. PMID:16682449

  17. 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

  18. The Bacillus subtilis sin Operon

    PubMed Central

    Voigt, Christopher A.; Wolf, Denise M.; Arkin, Adam P.

    2005-01-01

    The strategy of combining genes from a regulatory protein and its antagonist within the same operon, but controlling their activities differentially, can lead to diverse regulatory functions. This protein-antagonist motif is ubiquitous and present in evolutionarily unrelated regulatory pathways. Using the sin operon from the Bacillus subtilis sporulation pathway as a model system, we built a theoretical model, parameterized it using data from the literature, and used bifurcation analyses to determine the circuit functions it could encode. The model demonstrated that this motif can generate a bistable switch with tunable control over the switching threshold and the degree of population heterogeneity. Further, the model predicted that a small perturbation of a single critical parameter can bias this architecture into functioning like a graded response, a bistable switch, an oscillator, or a pulse generator. By mapping the parameters of the model to specific DNA regions and comparing the genomic sequences of Bacillus species, we showed that phylogenetic variation tends to occur in those regions that tune the switch threshold without disturbing the circuit function. The dynamical plasticity of the protein-antagonist operon motif suggests that it is an evolutionarily convergent design selected not only for particular immediate function but also for its evolvability. PMID:15466432

  19. 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....

  20. Ler Is a Negative Autoregulator of the LEE1 Operon in Enteropathogenic Escherichia coli

    PubMed Central

    Berdichevsky, Tatiana; Friedberg, Devorah; Nadler, Chen; Rokney, Assaf; Oppenheim, Amos; Rosenshine, Ilan

    2005-01-01

    Enteropathogenic Escherichia coli (EPEC) causes severe diarrhea in young children. Essential for colonization of the host intestine is the LEE pathogenicity island, which comprises a cluster of operons encoding a type III secretion system and related proteins. The LEE1 operon encodes Ler, which positively regulates many EPEC virulence genes in the LEE region and elsewhere in the chromosome. We found that Ler acts as a specific autorepressor of LEE1 transcription. We further show that Ler specifically binds upstream of the LEE1 operon in vivo and in vitro. A comparison of the Ler affinities to different DNA regions suggests that the autoregulation mechanism limits the steady-state level of Ler to concentrations that are just sufficient for activation of the LEE2 and LEE3 promoters and probably other LEE promoters. This mechanism may reflect the need of EPEC to balance maximizing the colonization efficiency by increasing the expression of the virulence genes and minimizing the immune response of the host by limiting their expression. In addition, we found that the autoregulation mechanism reduces the cell-to-cell variability in the levels of LEE1 expression. Our findings point to a new negative regulatory circuit that suppresses the noise and optimizes the expression levels of ler and other LEE1 genes. PMID:15601719

  1. 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

  2. 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

  3. 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

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

    PubMed

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

    2015-09-01

    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

  5. Expression of the ompATb operon accelerates ammonia secretion and adaptation of Mycobacterium tuberculosis to acidic environments.

    PubMed

    Song, Houhui; Huff, Jason; Janik, Katharine; Walter, Kerstin; Keller, Christine; Ehlers, Stefan; Bossmann, Stefan H; Niederweis, Michael

    2011-05-01

    Homeostasis of intracellular pH is a trait critical for survival of Mycobacterium tuberculosis in macrophages. However, mechanisms by which M. tuberculosis adapts to acidic environments are poorly understood. In this study, we analysed the physiological functions of OmpATb, a surface-accessible protein of M. tuberculosis. OmpATb did not complement the permeability defects of a Mycobacterium smegmatis porin mutant to glucose, serine and glycerol, in contrast to the porin MspA. Uptake rates of these solutes were unchanged in an ompATb operon mutant of M. tuberculosis indicating that OmpATb is not a general porin. Chemical analysis of low-pH culture filtrates showed that the proteins encoded by the ompATb operon are involved in generating a rapid ammonia burst, which neutralized medium pH and preceded exponential growth of M. tuberculosis. Addition of ammonia accelerated growth of the ompATb operon mutant demonstrating that ammonia secretion is indeed a mechanism by which M. tuberculosis neutralizes acidic environments. Infection experiments revealed that the ompATb operon was not required for full virulence in mice suggesting that M. tuberculosis has multiple mechanisms of resisting phagosomal acidification. Taken together, these results show that the ompATb operon is necessary for rapid ammonia secretion and adaptation of M. tuberculosis to acidic environments in vitro but not in mice. PMID:21410778

  6. 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

  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. Phylogenomics of Mycobacterium Nitrate Reductase Operon.

    PubMed

    Huang, Qinqin; Abdalla, Abualgasim Elgaili; Xie, Jianping

    2015-07-01

    NarGHJI operon encodes a nitrate reductase that can reduce nitrate to nitrite. This process enhances bacterial survival by nitrate respiration under anaerobic conditions. NarGHJI operon exists in many bacteria, especially saprophytic bacteria living in soil which play a key role in the nitrogen cycle. Most actinomycetes, including Mycobacterium tuberculosis, possess NarGHJI operons. M. tuberculosis is a facultative intracellular pathogen that expands in macrophages and has the ability to persist in a non-replicative form in granuloma lifelong. Nitrogen and nitrogen compounds play crucial roles in the struggle between M. tuberculosis and host. M. tuberculosis can use nitrate as a final electron acceptor under anaerobic conditions to enhance its survival. In this article, we reviewed the mechanisms regulating nitrate reductase expression and affecting its activity. Potential genes involved in regulating the nitrate reductase expression in M. tuberculosis were identified. The conserved NarG might be an alternative mycobacterium taxonomic marker. PMID:25980349

  9. 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…

  10. 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

  11. Genetic Analysis and Molecular Identification of Virulence in Xanthomonas oryzae pv. oryzae Isolates

    PubMed Central

    Onasanya, Amos; Onasanya, R. O.; Ojo, Abiodun A.; Adewale, B. O.

    2013-01-01

    Bacterial leaf blight (BLB) of rice is a very destructive disease worldwide and is caused by Xanthomonas oryzae pv. oryzae (Xoo). The aim of the present study was to examine if the Xoo virulence pathotypes obtained using phenotypic pathotyping could be confirmed using molecular approach. After screening of 60 Operon primers with genomic DNA of two Xoo isolates (virulent pathotype, Vr, and mildly virulent pathotype, MVr), 12 Operon primers that gave reproducible and useful genetic information were selected and used to analyze 50 Xoo isolates from 7 West African countries. Genetic analysis revealed two major Xoo virulence genotypes (Mta and Mtb) with Mta having two subgroups (Mta1 and Mta2). Mta1 (Vr1) subgroup genotype has occurrence in six countries and Mta2 (Vr2) in three countries while Mtb genotype characterized mildly virulence (MVr) Xoo isolates present in five countries. The study revealed possible linkage and correlation between phenotypic pathotyping and molecular typing of Xoo virulence. Xoo virulence genotypes were known to exist within country and there was evidence of Xoo pathogen migration between countries. Durable resistance rice cultivars would need to overcome both Mta and Mtb Xoo virulence genotypes in order to survive after their deployment into different rice ecologies in West Africa. PMID:27335673

  12. 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

  13. 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

  14. 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

  15. Transcriptome dynamics-based operon prediction in prokaryotes

    PubMed Central

    2014-01-01

    Background Inferring operon maps is crucial to understanding the regulatory networks of prokaryotic genomes. Recently, RNA-seq based transcriptome studies revealed that in many bacterial species the operon structure vary with the change of environmental conditions. Therefore, new computational solutions that use both static and dynamic data are necessary to create condition specific operon predictions. Results In this work, we propose a novel classification method that integrates RNA-seq based transcriptome profiles with genomic sequence features to accurately identify the operons that are expressed under a measured condition. The classifiers are trained on a small set of confirmed operons and then used to classify the remaining gene pairs of the organism studied. Finally, by linking consecutive gene pairs classified as operons, our computational approach produces condition-dependent operon maps. We evaluated our approach on various RNA-seq expression profiles of the bacteria Haemophilus somni, Porphyromonas gingivalis, Escherichia coli and Salmonella enterica. Our results demonstrate that, using features depending on both transcriptome dynamics and genome sequence characteristics, we can identify operon pairs with high accuracy. Moreover, the combination of DNA sequence and expression data results in more accurate predictions than each one alone. Conclusion We present a computational strategy for the comprehensive analysis of condition-dependent operon maps in prokaryotes. Our method can be used to generate condition specific operon maps of many bacterial organisms for which high-resolution transcriptome data is available. PMID:24884724

  16. Physiological regulation of a decontrolled lac operon.

    PubMed Central

    Wanner, B L; Kodaira, R; Neidhardt, F C

    1977-01-01

    The expression of the lac operon was studied under a variety of growth conditions in induced and in constitutive cells of Escherichia coli that carried different catabolite-insensitive lac promoters. Use of such "decontrolled" lac operons permitted a study of the expression of an operon that was presumably subject only to passive control. Since the use of toluenized cells was demonstrated not to be completely reliable, all enzyme assays were performed on sonic supernatant fluids. The cells contained different catabolite-insensitive promoters, which included the L1 and UV5 lac promoters, as well as others isolated in this study. There were three major observations. First, small but real carbon source effects were seen. Second, there was only a small change in beta-galactosidase specific activity with changes in the growth rate. This result implies a limited transcription and/or translation capacity within the cell. Third, at rapid growth rates, most promoters exhibited a decreased expression. The UV5 promoter, which was the "strongest" promoter, was an exception. A mechanism to explain this promoter-dependent control is discussed. PMID:323228

  17. 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

  18. Operons Are a Conserved Feature of Nematode Genomes

    PubMed Central

    Pettitt, Jonathan; Philippe, Lucas; Sarkar, Debjani; Johnston, Christopher; Gothe, Henrike Johanna; Massie, Diane; Connolly, Bernadette; Müller, Berndt

    2014-01-01

    The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and “spliced leader” (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing. PMID:24931407

  19. Operons are a conserved feature of nematode genomes.

    PubMed

    Pettitt, Jonathan; Philippe, Lucas; Sarkar, Debjani; Johnston, Christopher; Gothe, Henrike Johanna; Massie, Diane; Connolly, Bernadette; Müller, Berndt

    2014-08-01

    The organization of genes into operons, clusters of genes that are co-transcribed to produce polycistronic pre-mRNAs, is a trait found in a wide range of eukaryotic groups, including multiple animal phyla. Operons are present in the class Chromadorea, one of the two main nematode classes, but their distribution in the other class, the Enoplea, is not known. We have surveyed the genomes of Trichinella spiralis, Trichuris muris, and Romanomermis culicivorax and identified the first putative operons in members of the Enoplea. Consistent with the mechanism of polycistronic RNA resolution in other nematodes, the mRNAs produced by genes downstream of the first gene in the T. spiralis and T. muris operons are trans-spliced to spliced leader RNAs, and we are able to detect polycistronic RNAs derived from these operons. Importantly, a putative intercistronic region from one of these potential enoplean operons confers polycistronic processing activity when expressed as part of a chimeric operon in Caenorhabditis elegans. We find that T. spiralis genes located in operons have an increased likelihood of having operonic C. elegans homologs. However, operon structure in terms of synteny and gene content is not tightly conserved between the two taxa, consistent with models of operon evolution. We have nevertheless identified putative operons conserved between Enoplea and Chromadorea. Our data suggest that operons and "spliced leader" (SL) trans-splicing predate the radiation of the nematode phylum, an inference which is supported by the phylogenetic profile of proteins known to be involved in nematode SL trans-splicing. PMID:24931407

  20. Evolution of genes on the Salmonella Virulence plasmid phylogeny revealed from sequencing of the virulence plasmids of S. enterica serotype Dublin and comparative analysis.

    PubMed

    Chu, Chishih; Feng, Ye; Chien, An-Chi; Hu, Songnian; Chu, Chi-Hong; Chiu, Cheng-Hsun

    2008-11-01

    Salmonella enterica serotype Dublin harbors an approximately 80-kb virulence plasmid (pSDV), which mediates systemic infection in cattle. There are two types of pSDV: one is pSDVu (pOU1113) in strain OU7025 and the other pSDVr (pOU1115) in OU7409 (SD Lane) and many clinical isolates. Sequence analysis showed that pSDVr was a recombinant plasmid (co-integrate) of pSDVu and a plasmid similar to a 35-kb indigenous plasmid (pOU1114) of S. Dublin. Most of the F-transfer region in pSDVu was replaced by a DNA segment from the pOU1114-like plasmid containing an extra replicon and a pilX operon encoding for a type IV secretion system to form pSDVr. We reconstructed the particular evolutionary history of the seven virulence plasmids of Salmonella by comparative sequence analysis. The whole evolutionary process might begin with two different F-like plasmids (IncFI and IncFII), which then incorporated the spv operon and fimbriae operon from the chromosome to form the primitive virulence plasmids. Subsequently, these plasmids descended by deletion from a relatively large plasmid to smaller ones, with some recombination events occurring over time. Our results suggest that the phylogeny of virulence plasmids as a result of frequent recombination provides the opportunity for rapid evolution of Salmonella in response to the environmental cues. PMID:18718522

  1. 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

  2. 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

  3. 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

  4. 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

  5. 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

  6. 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

  7. 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

  8. Characterization of an orphan diterpenoid biosynthetic operon from Salinispora arenicola.

    PubMed

    Xu, Meimei; Hillwig, Matthew L; Lane, Amy L; Tiernan, Mollie S; Moore, Bradley S; Peters, Reuben J

    2014-09-26

    While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe-microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms. PMID:25203741

  9. Characterization of an Orphan Diterpenoid Biosynthetic Operon from Salinispora arenicola

    PubMed Central

    2015-01-01

    While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe–microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms. PMID:25203741

  10. Capsule depolymerase overexpression reduces Bacillus anthracis virulence.

    PubMed

    Scorpio, Angelo; Chabot, Donald J; Day, William A; Hoover, Timothy A; Friedlander, Arthur M

    2010-05-01

    Capsule depolymerase (CapD) is a gamma-glutamyl transpeptidase and a product of the Bacillus anthracis capsule biosynthesis operon. In this study, we examined the effect of modulating capD expression on B. anthracis capsule phenotype, interaction with phagocytic cells and virulence in guinea pigs. Transcriptional fusions of capD were made to the genes encoding heat-shock protein 60 (hsp60) and elongation factor Tu (EFTu), and to capA, a B. anthracis capsule biosynthesis gene. Translation signals were altered to improve expression of capD, including replacing the putative ribosome-binding site with a consensus sequence and the TTG start codon with ATG. CapD was not detected by immunoblotting in lysates from wild-type B. anthracis Ames but was detected in strains engineered with a consensus ribosome-binding site for capD. Strains overexpressing capD at amounts detected by immunoblotting were found to have less surface-associated capsule and released primarily lower-molecular-mass capsule into culture supernatants. Overexpression of capD increased susceptibility to neutrophil phagocytic killing and adherence to macrophages and resulted in reduced fitness in a guinea pig model of infection. These data suggest that B. anthracis may have evolved weak capD expression resulting in optimized capsule-mediated virulence. PMID:20110296

  11. Characterisation of the mgo operon in Pseudomonas syringae pv. syringae UMAF0158 that is required for mangotoxin production

    PubMed Central

    2012-01-01

    Background Mangotoxin is an antimetabolite toxin that is produced by strains of Pseudomonas syringae pv. syringae; mangotoxin-producing strains are primarily isolated from mango tissues with symptoms of bacterial apical necrosis. The toxin is an oligopeptide that inhibits ornithine N-acetyl transferase (OAT), a key enzyme in the biosynthetic pathway of the essential amino acids ornithine and arginine. The involvement of a putative nonribosomal peptide synthetase gene (mgoA) in mangotoxin production and virulence has been reported. Results In the present study, we performed a RT-PCR analysis, insertional inactivation mutagenesis, a promoter expression analysis and terminator localisation to study the gene cluster containing the mgoA gene. Additionally, we evaluated the importance of mgoC, mgoA and mgoD in mangotoxin production. A sequence analysis revealed an operon-like organisation. A promoter sequence was located upstream of the mgoB gene and was found to drive lacZ transcription. Two terminators were located downstream of the mgoD gene. RT-PCR experiments indicated that the four genes (mgoBCAD) constitute a transcriptional unit. This operon is similar in genetic organisation to those in the three other P. syringae pathovars for which complete genomes are available (P. syringae pv. syringae B728a, P. syringae pv. tomato DC3000 and P. syringae pv. phaseolicola 1448A). Interestingly, none of these three reference strains is capable of producing mangotoxin. Additionally, extract complementation resulted in a recovery of mangotoxin production when the defective mutant was complemented with wild-type extracts. Conclusions The results of this study confirm that mgoB, mgoC, mgoA and mgoD function as a transcriptional unit and operon. While this operon is composed of four genes, only the last three are directly involved in mangotoxin production. PMID:22251433

  12. 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

  13. 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.

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

    PubMed

    Jones, Megan M; Murphy, Timothy F

    2015-09-01

    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

  15. Effects of the ERES pathogenicity region regulator Ralp3 on Streptococcus pyogenes serotype M49 virulence factor expression.

    PubMed

    Siemens, Nikolai; Fiedler, Tomas; Normann, Jana; Klein, Johannes; Münch, Richard; Patenge, Nadja; Kreikemeyer, Bernd

    2012-07-01

    Streptococcus pyogenes (group A streptococcus [GAS]) is a highly virulent Gram-positive bacterium. For successful infection, GAS expresses many virulence factors, which are clustered together with transcriptional regulators in distinct genomic regions. Ralp3 is a central regulator of the ERES region. In this study, we investigated the role of Ralp3 in GAS M49 pathogenesis. The inactivation of Ralp3 resulted in reduced attachment to and internalization into human keratinocytes. The Δralp3 mutant failed to survive in human blood and serum, and the hyaluronic acid capsule was slightly decreased. In addition, the mutant showed a lower binding capacity to human plasminogen, and the SpeB activity was significantly decreased. Complementation of the Δralp3 mutant restored the wild-type phenotype. The transcriptome and quantitative reverse transcription-PCR analysis of the serotype M49 GAS strain and its isogenic Δralp3 mutant identified 16 genes as upregulated, and 43 genes were found to be downregulated. Among the downregulated genes, there were open reading frames encoding proteins involved in metabolism (e.g., both lac operons and the fru operon), genes encoding lantibiotics (e.g., the putative salivaricin operon), and ORFs encoding virulence factors (such as the whole Mga core regulon and further genes under Mga control). In summary, the ERES region regulator Ralp3 is an important serotype-specific transcriptional regulator for virulence and metabolic control. PMID:22544273

  16. The Yersinia pestis V antigen is a regulatory protein necessary for Ca2(+)-dependent growth and maximal expression of low-Ca2+ response virulence genes.

    PubMed Central

    Price, S B; Cowan, C; Perry, R D; Straley, S C

    1991-01-01

    The low-Ca2+ response is a multicomponent virulence regulon of the human-pathogenic yersiniae in which 12 known virulence genes are coordinately regulated in response to environmental cues of temperature, Ca2+, and nucleotides such as ATP. Yersinial growth also is regulated, with full growth yield being permitted at 37 degrees C only if Ca2+ or a nucleotide is present. In this study, we constructed and characterized a mutant Yersinia pestis specifically defective in the gene encoding the V antigen, one of the virulence genes of the low-Ca2+ response. An in-frame internal deletion-insertion mutation was made by removing bases 51 through 645 of lcrV and inserting 61 new bases. The altered lcrV was introduced into the low-Ca2+ response plasmid in Y. pestis by allelic exchange, and the resulting mutant was characterized for its two-dimensional protein profiles, growth, expression of an operon fusion to another low-Ca2+ response virulence operon, and virulence in mice. The mutant had lost its Ca2+ and nucleotide requirement for growth, showed diminished expression of Ca2(+)-and nucleotide-regulated virulence genes, and was avirulent in mice. The mutation could be complemented with respect to the growth property by supplying native V antigen operon sequences in trans in high copy number (on pBR322). Partial complementation of the growth defect and almost complete complementation of the virulence defect were seen with a lower-copy-number complementing replicon (a pACYC184 derivative). The data are consistent with the interpretation that V antigen is bifunctional, with a role in regulating growth and expression of low-Ca2+ response virulence genes in addition to its putative role as a secreted virulence protein. Images PMID:1901573

  17. 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

  18. The Success of Acinetobacter Species; Genetic, Metabolic and Virulence Attributes

    PubMed Central

    Peleg, Anton Y.; de Breij, Anna; Adams, Mark D.; Cerqueira, Gustavo M.; Mocali, Stefano; Galardini, Marco; Nibbering, Peter H.; Earl, Ashlee M.; Ward, Doyle V.; Paterson, David L.; Seifert, Harald; Dijkshoorn, Lenie

    2012-01-01

    An understanding of why certain Acinetobacter species are more successful in causing nosocomial infections, transmission and epidemic spread in healthcare institutions compared with other species is lacking. We used genomic, phenotypic and virulence studies to identify differences between Acinetobacter species. Fourteen strains representing nine species were examined. Genomic analysis of six strains showed that the A. baumannii core genome contains many genes important for diverse metabolism and survival in the host. Most of the A. baumannii core genes were also present in one or more of the less clinically successful species. In contrast, when the accessory genome of an individual A. baumannii strain was compared to a strain of a less successful species (A. calcoaceticus RUH2202), many operons with putative virulence function were found to be present only in the A. baumannii strain, including the csu operon, the acinetobactin chromosomal cluster, and bacterial defence mechanisms. Phenotype microarray analysis showed that compared to A. calcoaceticus (RUH2202), A. baumannii ATCC 19606T was able to utilise nitrogen sources more effectively and was more tolerant to pH, osmotic and antimicrobial stress. Virulence differences were also observed, with A. baumannii ATCC 19606T, A. pittii SH024, and A. nosocomialis RUH2624 persisting and forming larger biofilms on human skin than A. calcoaceticus. A. baumannii ATCC 19606T and A. pittii SH024 were also able to survive in a murine thigh infection model, whereas the other two species were eradicated. The current study provides important insights into the elucidation of differences in clinical relevance among Acinetobacter species. PMID:23144699

  19. Genes aroA and serC of Salmonella typhimurium constitute an operon.

    PubMed Central

    Hoiseth, S K; Stocker, B A

    1985-01-01

    Genetic analysis of aroA554::Tn10 derivatives of two mouse-virulent Salmonella typhimurium strains, "FIRN" and "WRAY," and of a nonreverting derivative of each constructed for use as a live vaccine, showed the site of the insertion among mapped aroA point mutants. The WRAY live-vaccine strain gave no aro+ recombinants in crosses with aroA point mutations to one side of the insertion, indicating a deletion from Tn10 through the sites of these point mutations. The FIRN live-vaccine strain gave wild-type recombinants with all tested point mutants; it probably has a deletion or inversion extending from Tn10 into aroA but not as far as the nearest point mutation. Some tetracycline-sensitive mutants of aroA554::Tn10 strains required serine and pyridoxine, indicating loss of serC function, and some that were found to be SerC- did not produce gas from glucose, indicating a loss of pfl function. These results show the gene order pfl-serC-aroA, as in Escherichia coli. Ampicillin enrichment applied to pools of tetracycline-sensitive mutants of strains with Tn10 insertions near aroA (i.e., zbj::Tn10 strains) yielded Aro- SerC- Pfl-, Aro- SerC+ Pfl+, and Aro- SerC- Pfl+ mutants but none which were Aro+ SerC-. All of the mutants are explicable by deletions or inversions extending clockwise from zbj::Tn10 into or through an operon comprising serC (promoter-proximal) and aroA. Such an operon was also shown by the identification of two Tn10 insertions causing phenotype Aro- SerC-, each able to revert to Aro+ SerC+ by precise excision. serC corresponds to the open reading frame promoter-proximal to aroA that was identified elsewhere by base sequencing of a cloned aroA segment of S. typhimurium (Comai et al., Science 221:370-371, 1983). Both serine and chorismate are precursors of enterochelin; this may be why serC and aroA are in a single operon. PMID:2989248

  20. 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

  1. Fusions of flagellar operons to lactose genes on a mu lac bacteriophage.

    PubMed Central

    Komeda, Y

    1982-01-01

    Previous studies have defined 29 genes necessary for synthesis of the Escherichia coli flagellar apparatus. This study analyzed the transcriptional control of flagellar genes, using Mu d (Apr lac) phage to generate flagellar mutants by insertion. These mutants contained operon fusions of flagellar genes to the lac genes of the Mu d phage and allowed the measurement of flagellar operon expression by detection of beta-galactosidase activity. These fusion mutants expressed the enzyme activity constitutively, and an autogenous regulation mechanism was not revealed. Lambda transducing phages carrying these chromosomal fla-lac fusions were also isolated and used to examine the effect of different fla mutations on expression of each flagellar operon. The results showed that flagellar operons are divided into six classes; (class 1) the flbB operon, which controls all of the other flagellar operons; (class 2) the flaU and flbC operons, which are controlled by the flbB operon gene products and are not required for the expression of other Fla operons; (class 3) the flbA, flaG, flaD, flaN, flaB, and flaA operons, which are under flbB operon control and are required for the expression of other fla operons; (class4) the flaZ operon, which is controlled by the gene products of the group 1 and 3 operons and is required for hag transcription; (class 5) the mocha and flaS operons, which are controlled by the gene products of the group 1 and 3 operons; and (class 6) the hag operon. These results are discussed with respect to the possible assembly sequence of the fla gene products. PMID:7037746

  2. 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

  3. 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

  4. 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

  5. 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

  6. Dynamic model of gene regulation for the lac operon

    NASA Astrophysics Data System (ADS)

    Angelova, Maia; Ben-Halim, Asma

    2011-03-01

    Gene regulatory network is a collection of DNA which interact with each other and with other matter in the cell. The lac operon is an example of a relatively simple genetic network and is one of the best-studied structures in the Escherichia coli bacteria. In this work we consider a deterministic model of the lac operon with a noise term, representing the stochastic nature of the regulation. The model is written in terms of a system of simultaneous first order differential equations with delays. We investigate an analytical and numerical solution and analyse the range of values for the parameters corresponding to a stable solution.

  7. New insights into the role of indole-3-acetic acid in the virulence of Pseudomonas savastanoi pv. savastanoi.

    PubMed

    Aragón, Isabel M; Pérez-Martínez, Isabel; Moreno-Pérez, Alba; Cerezo, Miguel; Ramos, Cayo

    2014-07-01

    Indole-3-acetic acid (IAA) is a widespread phytohormone among plant-associated bacteria, including the tumour-inducing pathogen of woody hosts, Pseudomonas savastanoi pv. savastanoi. A phylogenetic analysis of the iaaM/iaaH operon, which is involved in the biosynthesis of IAA, showed that one of the two operons encoded by Pseudomonas savastanoi pv. savastanoi NCPPB 3335, iaaM-1/iaaH-1, is horizontally transferred among bacteria belonging to the Pseudomonas syringae complex. We also show that biosynthesis of the phytohormone, virulence and full fitness of this olive pathogen depend only on the functionality of the iaaM-1/iaaH-1 operon. In contrast, the iaaM-2/iaaH-2 operon, which carries a 22-nt insertion in the iaaM-2 gene, does not contribute to the production of IAA by this bacterium. A residual amount of IAA was detected in the culture supernatants of a double mutant affected in both iaaM/iaaH operons, suggesting that a different pathway might also contribute to the total pool of the phytohormone produced by this pathogen. Additionally, we show that exogenously added IAA negatively and positively regulates the expression of genes related to the type III and type VI secretion systems, respectively. Together, these results suggest a role of IAA as a signalling molecule in this pathogen. PMID:24606017

  8. Complete DNA Sequence of a ColBM Plasmid from Avian Pathogenic Escherichia coli Suggests that It Evolved from Closely Related ColV Virulence Plasmids†

    PubMed Central

    Johnson, Timothy J.; Johnson, Sara J.; Nolan, Lisa K.

    2006-01-01

    Avian pathogenic Escherichia coli (APEC), an extraintestinal pathogenic E. coli causing colibacillosis in birds, is responsible for significant economic losses for the poultry industry. Recently, we reported that the APEC pathotype was characterized by possession of a set of genes contained within a 94-kb cluster linked to a ColV plasmid, pAPEC-O2-ColV. These included sitABCD, genes of the aerobactin operon, hlyF, iss, genes of the salmochelin operon, and the 5′ end of cvaB of the ColV operon. However, the results of gene prevalence studies performed among APEC isolates revealed that these traits were not always linked to ColV plasmids. Here, we present the complete sequence of a 174-kb plasmid, pAPEC-O1-ColBM, which contains a putative virulence cluster similar to that of pAPEC-O2-ColV. These two F-type plasmids share remarkable similarity, except that they encode the production of different colicins; pAPEC-O2-ColV contains an intact ColV operon, and pAPEC-O1-ColBM encodes the colicins B and M. Interestingly, remnants of the ColV operon exist in pAPEC-O1-ColBM, hinting that ColBM-type plasmids may have evolved from ColV plasmids. Among APEC isolates, the prevalence of ColBM sequences helps account for the previously observed differences in prevalence between genes of the “conserved” portion of the putative virulence cluster of pAPEC-O2-ColV and those genes within its “variable” portion. These results, in conjunction with Southern blotting and probing of representative ColBM-positive strains, indicate that this “conserved” cluster of putative virulence genes is primarily linked to F-type virulence plasmids among the APEC isolates studied. PMID:16885466

  9. The operon for cytokinin biosynthesis of Erwinia herbicola pv. gypsophilae contains two promoters and is plant induced.

    PubMed

    Guo, M; Manulis, S; Barash, I; Lichter, A

    2001-12-01

    The operon for cytokinin biosynthesis in the gall-forming bacterium Erwinia herbicola pv. gypsophilae (Ehg) has been previously shown to reside on an indigenous plasmid (pPATH(Ehg)) that is mandatory for pathogenicity. This operon consists of two genes: the first open reading frame (pre-etz) is of unknown function, whereas the second one (etz) encodes for isopentenyl transferase. Northern hybridization performed with the wild-type strain Ehg824-1 grown in Luria-Bertani broth demonstrated two transcripts of which an etz-specific transcript (1.0 kb) was predominant. Fusion of upstream DNA fragments of both pre-etz and etz to the ice nucleation reporter gene inaZ in pVSP61 showed high ice nucleation activity in both cultures, confirming the presence of two independent promoters. An increase of 1-1.5 orders in transcriptional activity of these promoters was observed following inoculation of gypsophila cuttings. Mutants of Ehg824-1 were generated by insertion of inaZ into pre-etz and etz using the transposon reporter Tn3-Spice. An increase of about two orders in transcriptional activity was recorded with both mutants following inoculation of gypsophila or bean cuttings. A similar induction was also observed when the bacteria were applied to the leaf surface of these plants. Unlike other virulence genes present on the pPATH(Ehg), neither pre-etz nor etz was regulated by the adjacent hrp gene cluster. PMID:11822839

  10. 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

  11. 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

  12. 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.

  13. 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.

  14. 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

  15. 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. PMID:25287924

  16. 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

  17. 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

  18. 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

  19. Exchange of Spacer Regions between Rrna Operons in Escherichia Coli

    PubMed Central

    Harvey, S.; Hill, C. W.

    1990-01-01

    The Escherichia coli rRNA operons each have one of two types of spacer separating the 16S and 23S coding regions. The spacers of four operons encode tRNA(Glu2) and the other three encode both tRNA(Ile) and tRNA(Ala 1 B). We have prepared a series of mutants in which the spacer region of a particular rrn operon has been replaced by the opposite type. Included among these were a mutant retaining only a single copy of the tRNA(Glu2) spacer (at rrnG) and another retaining only a single copy of the tRNA(Ile)-tRNA(Ala 1 B) spacer (at rrnA). While both mutants grew more slowly than controls, the mutant deficient in tRNA(Glu2) spacers was more severely affected. At a frequency of 6 X 10(-5), these mutants phenotypically reverted to faster growing types by increasing the copy number of the deficient spacer. In most of these phenotypic revertants, the deficient spacer type appeared in a rrn operon which previously contained the surplus type, bringing the ratio of spacer types closer to normal. In a few cases, these spacer changes were accompanied by an inversion of the chromosomal material between the donor and recipient rrn operons. Two examples of inversion of one-half of the E. coli chromosome between rrnG and rrnH were observed. The correlation of spacer change with inversion indicated that, in these particular cases, the change was due to an intrachromatid gene conversion event accompanied by a reciprocal crossover rather than reciprocal exchange between sister chromatids. PMID:2168847

  20. Transient virulence of emerging pathogens.

    PubMed

    Bolker, Benjamin M; Nanda, Arjun; Shah, Dharmini

    2010-05-01

    Should emerging pathogens be unusually virulent? If so, why? Existing theories of virulence evolution based on a tradeoff between high transmission rates and long infectious periods imply that epidemic growth conditions will select for higher virulence, possibly leading to a transient peak in virulence near the beginning of an epidemic. This transient selection could lead to high virulence in emerging pathogens. Using a simple model of the epidemiological and evolutionary dynamics of emerging pathogens, along with rough estimates of parameters for pathogens such as severe acute respiratory syndrome, West Nile virus and myxomatosis, we estimated the potential magnitude and timing of such transient virulence peaks. Pathogens that are moderately evolvable, highly transmissible, and highly virulent at equilibrium could briefly double their virulence during an epidemic; thus, epidemic-phase selection could contribute significantly to the virulence of emerging pathogens. In order to further assess the potential significance of this mechanism, we bring together data from the literature for the shapes of tradeoff curves for several pathogens (myxomatosis, HIV, and a parasite of Daphnia) and the level of genetic variation for virulence for one (myxomatosis). We discuss the need for better data on tradeoff curves and genetic variance in order to evaluate the plausibility of various scenarios of virulence evolution. PMID:19864267

  1. 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

  2. Genomic Subtractive Hybridization and Selective Capture of Transcribed Sequences Identify a Novel Salmonella typhimurium Fimbrial Operon and Putative Transcriptional Regulator That Are Absent from the Salmonella typhi Genome

    PubMed Central

    Morrow, Brian J.; Graham, James E.; Curtiss, Roy

    1999-01-01

    Salmonella typhi, the etiologic agent of typhoid fever, is adapted to the human host and unable to infect nonprimate species. The genetic basis for host specificity in S. typhi is unknown. The avirulence of S. typhi in animal hosts may result from a lack of genes present in the broad-host-range pathogen Salmonella typhimurium. Genomic subtractive hybridization was successfully employed to isolate S. typhimurium genomic sequences which are absent from the S. typhi genome. These genomic subtracted sequences mapped to 17 regions distributed throughout the S. typhimurium chromosome. A positive cDNA selection method was then used to identify subtracted sequences which were transcribed by S. typhimurium following macrophage phagocytosis. A novel putative transcriptional regulator of the LysR family was identified as transcribed by intramacrophage S. typhimurium. This putative transcriptional regulator was absent from the genomes of the human-adapted serovars S. typhi and Salmonella paratyphi A. Mutations within this gene did not alter the level of S. typhimurium survival within macrophages or virulence within mice. A subtracted genomic fragment derived from the ferrichrome operon also hybridized to the intramacrophage cDNA. Nucleotide sequence analysis of S. typhimurium and S. typhi chromosomal sequences flanking the ferrichrome operon identified a novel S. typhimurium fimbrial operon with a high level of similarity to sequences encoding Proteus mirabilis mannose-resistant fimbriae. The novel fimbrial operon was absent from the S. typhi genome. The absence of specific genes may have allowed S. typhi to evolve as a highly invasive, systemic human pathogen. PMID:10496884

  3. 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

  4. 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

  5. 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

  6. Positive and Negative Control of the Lac Operon

    NASA Astrophysics Data System (ADS)

    Qaddour, Jihad S.; Werman, Steven D.; Misra, Prasanta K.

    1997-03-01

    We present a mathematical model for the positive and negative control of lac operon. We investigate a steady state solution for the coupled nonlinear differential equations representing the dynamic behaviors of the repressor-inducer components of negative control as well as the cyclic AMP receptor components of the positive control. A dimensionless derivation of the lac operon system is employed to produce singularly perturbed models. The first model represents the dynamical behavior of the operator while the slow model represents the dynamical behaviors of the inducer and the repressor. We use the singular perturbation theory to show that the behavior of the system can be described as a rapid on-off switch of structural gene transformation.

  7. Direct involvement of IS26 in an antibiotic resistance operon.

    PubMed Central

    Lee, K Y; Hopkins, J D; Syvanen, M

    1990-01-01

    The plasmid pBWH77, originally found in an isolate of Klebsiella pneumoniae, harbors a new antibiotic resistance operon containing two resistance genes transcribed from an IS26-hybrid promoter, as shown by nucleotide sequencing, mRNA mapping, and the effect of inserting a transcription terminator within the promoter-proximal gene. The nucleotide sequence of this region revealed that the operon (IAB) is made up of three sections that are closely related to previously described genetic elements. The -35 region of the promoter, together with the adjacent sequence, is identical to sequences of the IS26 element. One of the resistance genes, aphA7, which is located next to the hybrid promoter, confers assistance to neomycin and structurally related aminoglycosides. This aphA7 gene is highly homologous to aphA1 of Tn903, with five nucleotide differences. The second gene, blaS2A, encodes an evolved SHV-type beta-lactamase with a pI of 7.6 that confers resistance to the broad-spectrum cephalosporins cefotaxime and ceftizoxime. The deduced amino acid sequence of SHV-2A shows that amino acid 238 is a serine, a residue reported to confer resistance to cefotaxime. We discuss how the operon may have evolved by a combination of insertion sequence-mediated genetic rearrangements and acquisitive evolution. Using phylogenetic parsimony, we show that aphA7 in the IAB operon evolved from an ancestral form similar to aphA1 in Tn903 and that blaS2A evolved from an ancestral form similar to blaS1. Images PMID:2160941

  8. 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

  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. Proteus mirabilis urease: operon fusion and linker insertion analysis of ure gene organization, regulation, and function.

    PubMed

    Island, M D; Mobley, H L

    1995-10-01

    Urease is an inducible virulence factor of uropathogenic Proteus mirabilis. Although eight contiguous genes necessary for urease activity have been cloned and sequenced, the transcriptional organization and regulation of specific genes within the Proteus gene cluster has not been investigated in detail. The first gene, ureR, is located 400 bp upstream and is oriented in the direction opposite the other seven genes, ureDABCEFG. The structural subunits of urease are encoded by ureABC. Previously, UreR was shown to contain a putative helix-turn-helix DNA-binding motif 30 residues upstream of a consensus sequence which is a signature for the AraC family of positive regulators; this polypeptide is homologous to other DNA-binding regulatory proteins. Nested deletions of ureR linked to either ureD-lacZ or ureA-lacZ operon fusions demonstrated that an intact ureR is required for urea-induced synthesis of LacZ from either ureA or ureD and identified a urea-regulated promoter in the ureR-ureD intergenic region. However, lacZ operon fusions to fragments encompassing putative promoter regions upstream of ureA and ureF demonstrated that no urea-regulated promoters occur upstream of these open reading frames; regions upstream of ureR, ureE, and ureG were not tested. These data suggest that UreR acts as a positive regulator in the presence of urea, activating transcription of urease structural and accessory genes via sequences upstream of ureD. To address the role of the nonstructural regulatory and accessory genes, we constructed deletion, cassette, and linker insertion mutations throughout the ure gene cluster and determined the effect of these mutations on production and regulation of urease activity in Escherichia coli. Mutations were obtained, with locations determine by DNA sequencing, in all genes except ureA and ureE. In each case, the mutation resulted in a urease-negative phenotype. PMID:7559355

  11. Characterization of genes in the cellulose-synthesizing operon (acs operon) of Acetobacter xylinum: implications for cellulose crystallization.

    PubMed Central

    Saxena, I M; Kudlicka, K; Okuda, K; Brown, R M

    1994-01-01

    The synthesis of an extracellular ribbon of cellulose in the bacterium Acetobacter xylinum takes place from linearly arranged, membrane-localized, cellulose-synthesizing and extrusion complexes that direct the coupled steps of polymerization and crystallization. To identify the different components involved in this process, we isolated an Acetobacter cellulose-synthesizing (acs) operon from this bacterium. Analysis of DNA sequence shows the presence of three genes in the acs operon, in which the first gene (acsAB) codes for a polypeptide with a molecular mass of 168 kDa, which was identified as the cellulose synthase. A single base change in the previously reported DNA sequence of this gene, resulting in a frameshift and synthesis of a larger protein, is described in the present paper, along with the sequences of the other two genes (acsC and acsD). The requirement of the acs operon genes for cellulose production was determined using site-determined TnphoA/Kanr GenBlock insertion mutants. Mutant analysis showed that while the acsAB and acsC genes were essential for cellulose production in vivo, the acsD mutant produced reduced amounts of two cellulose allomorphs (cellulose I and cellulose II), suggesting that the acsD gene is involved in cellulose crystallization. The role of the acs operon genes in determining the linear array of intramembranous particles, which are believed to be sites of cellulose synthesis, was investigated for the different mutants; however, this arrangement was observed only in cells that actively produced cellulose microfibrils, suggesting that it may be influenced by the crystallization of the nascent glucan chains. Images PMID:8083166

  12. 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. PMID:24968349

  13. 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

  14. 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

  15. 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

  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. Engineered ribosomal RNA operon copy-number variants of E. coli reveal the evolutionary trade-offs shaping rRNA operon number.

    PubMed

    Gyorfy, Zsuzsanna; Draskovits, Gabor; Vernyik, Viktor; Blattner, Frederick F; Gaal, Tamas; Posfai, Gyorgy

    2015-02-18

    Ribosomal RNA (rrn) operons, characteristically present in several copies in bacterial genomes (7 in E. coli), play a central role in cellular physiology. We investigated the factors determining the optimal number of rrn operons in E. coli by constructing isogenic variants with 5-10 operons. We found that the total RNA and protein content, as well as the size of the cells reflected the number of rrn operons. While growth parameters showed only minor differences, competition experiments revealed a clear pattern: 7-8 copies were optimal under conditions of fluctuating, occasionally rich nutrient influx and lower numbers were favored in stable, nutrient-limited environments. We found that the advantages of quick adjustment to nutrient availability, rapid growth and economic regulation of ribosome number all contribute to the selection of the optimal rrn operon number. Our results suggest that the wt rrn operon number of E. coli reflects the natural, 'feast and famine' life-style of the bacterium, however, different copy numbers might be beneficial under different environmental conditions. Understanding the impact of the copy number of rrn operons on the fitness of the cell is an important step towards the creation of functional and robust genomes, the ultimate goal of synthetic biology. PMID:25618851

  19. Brucella, nitrogen and virulence.

    PubMed

    Ronneau, Severin; Moussa, Simon; Barbier, Thibault; Conde-Álvarez, Raquel; Zuniga-Ripa, Amaia; Moriyon, Ignacio; Letesson, Jean-Jacques

    2016-08-01

    The brucellae are α-Proteobacteria causing brucellosis, an important zoonosis. Although multiplying in endoplasmic reticulum-derived vacuoles, they cause no cell death, suggesting subtle but efficient use of host resources. Brucellae are amino-acid prototrophs able to grow with ammonium or use glutamate as the sole carbon-nitrogen source in vitro. They contain more than twice amino acid/peptide/polyamine uptake genes than the amino-acid auxotroph Legionella pneumophila, which multiplies in a similar vacuole, suggesting a different nutritional strategy. During these two last decades, many mutants of key actors in nitrogen metabolism (transporters, enzymes, regulators, etc.) have been described to be essential for full virulence of brucellae. Here, we review the genomic and experimental data on Brucella nitrogen metabolism and its connection with virulence. An analysis of various aspects of this metabolism (transport, assimilation, biosynthesis, catabolism, respiration and regulation) has highlighted differences and similarities in nitrogen metabolism with other α-Proteobacteria. Together, these data suggest that, during their intracellular life cycle, the brucellae use various nitrogen sources for biosynthesis, catabolism and respiration following a strategy that requires prototrophy and a tight regulation of nitrogen use. PMID:25471320

  20. 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

  1. 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

  2. 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

  3. An inducible tellurite-resistance operon in Proteus mirabilis.

    PubMed

    Toptchieva, Anna; Sisson, Gary; Bryden, Louis J; Taylor, Diane E; Hoffman, Paul S

    2003-05-01

    Tellurite resistance (Te(r)) is widespread in nature and it is shown here that the natural resistance of Proteus mirabilis to tellurite is due to a chromosomally located orthologue of plasmid-borne ter genes found in enteric bacteria. The P. mirabilis ter locus (terZABCDE) was identified in a screen of Tn5lacZ-generated mutants of which one contained an insertion in terC. The P. mirabilis terC mutant displayed increased susceptibility to tellurite (Te(s)) and complementation with terC carried on a multicopy plasmid restored high-level Te(r). Primer extension analysis revealed a single transcriptional start site upstream of terZ, but only with RNA harvested from bacteria grown in the presence of tellurite. Northern blotting and reverse transcriptase-PCR (RT-PCR) analyses confirmed that the ter operon was inducible by tellurite and to a lesser extent by oxidative stress inducers such as hydrogen peroxide and methyl viologen (paraquat). Direct and inverted repeat sequences were identified in the ter promoter region as well as motifs upstream of the -35 hexamer that resembled OxyR-binding sequences. Finally, the 390 bp intergenic promoter region located between orf3 and terZ showed no DNA sequence identity with any other published ter sequences, whereas terZABCDE genes exhibited 73-85 % DNA sequence identity. The ter operon was present in all clinical isolates of P. mirabilis and Proteus vulgaris tested and is inferred for Morganella and Providencia spp. based on screening for high level Te(r) and preliminary PCR analysis. Thus, a chromosomally located inducible tellurite resistance operon appears to be a common feature of the genus Proteus. PMID:12724390

  4. The Virulence Plasmid of Yersinia, an Antihost Genome

    PubMed Central

    Cornelis, Guy R.; Boland, Anne; Boyd, Aoife P.; Geuijen, Cecile; Iriarte, Maite; Neyt, Cécile; Sory, Marie-Paule; Stainier, Isabelle

    1998-01-01

    The 70-kb virulence plasmid enables Yersinia spp. (Yersinia pestis, Y. pseudotuberculosis, and Y. enterocolitica) to survive and multiply in the lymphoid tissues of their host. It encodes the Yop virulon, an integrated system allowing extracellular bacteria to disarm the cells involved in the immune response, to disrupt their communications, or even to induce their apoptosis by the injection of bacterial effector proteins. This system consists of the Yop proteins and their dedicated type III secretion apparatus, called Ysc. The Ysc apparatus is composed of some 25 proteins including a secretin. Most of the Yops fall into two groups. Some of them are the intracellular effectors (YopE, YopH, YpkA/YopO, YopP/YopJ, YopM, and YopT), while the others (YopB, YopD, and LcrV) form the translocation apparatus that is deployed at the bacterial surface to deliver the effectors into the eukaryotic cells, across their plasma membrane. Yop secretion is triggered by contact with eukaryotic cells and controlled by proteins of the virulon including YopN, TyeA, and LcrG, which are thought to form a plug complex closing the bacterial secretion channel. The proper operation of the system also requires small individual chaperones, called the Syc proteins, in the bacterial cytosol. Transcription of the genes is controlled both by temperature and by the activity of the secretion apparatus. The virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis also encodes the adhesin YadA. The virulence plasmid contains some evolutionary remnants including, in Y. enterocolitica, an operon encoding resistance to arsenic compounds. PMID:9841674

  5. 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

  6. Economy of operon formation: cotranscription minimizes shortfall in protein complexes.

    PubMed

    Sneppen, Kim; Pedersen, Steen; Krishna, Sandeep; Dodd, Ian; Semsey, Szabolcs

    2010-01-01

    Genes of prokaryotes and Archaea are often organized in cotranscribed groups, or operons. In contrast, eukaryotic genes are generally transcribed independently. Here we show that there is a substantial economic gain for the cell to cotranscribe genes encoding protein complexes because it synchronizes the fluctuations, or noise, in the levels of the different components. This correlation substantially reduces the shortfall in production of the complex. This benefit is relatively large in small cells such as bacterial cells, in which there are few mRNAs and proteins per cell, and is diminished in larger cells such as eukaryotic cells. PMID:20877578

  7. 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.

  8. 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

  9. 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

  10. 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

  11. Chromosomal Organization of Rrna Operons in Bacillus Subtilis

    PubMed Central

    Jarvis, E. D.; Widom, R. L.; LaFauci, G.; Setoguchi, Y.; Richter, I. R.; Rudner, R.

    1988-01-01

    Integrative mapping with vectors containing ribosomal DNA sequences were used to complete the mapping of the 10 rRNA gene sets in the endospore forming bacterium Bacillus subtilis. Southern hybridizations allowed the assignment of nine operons to distinct BclI restriction fragments and their genetic locus identified by transductional crosses. Nine of the ten rRNA gene sets are located between 0 and 70° on the genomic map. In the region surrounding cysA14, two sets of closely spaced tandem clusters are present. The first (rrnJ and rrnW) is located between purA16 and cysA14 closely linked to the latter; the second (rrnI, rrnH and rrnG) previously mapped within this area is located between attSPO2 and glpT6. The operons at or near the origin of replication (rrnO,rrnA and rrnJ,rrnW) represent ``hot spots'' of plasmid insertion. PMID:2465199

  12. Regulation of Francisella Tularensis Virulence

    PubMed Central

    Dai, Shipan; Mohapatra, Nrusingh P.; Schlesinger, Larry S.; Gunn, John S.

    2011-01-01

    Francisella tularensis is one of the most virulent bacteria known and a Centers for Disease Control and Prevention Category A select agent. It is able to infect a variety of animals and insects and can persist in the environment, thus Francisella spp. must be able to survive in diverse environmental niches. However, F. tularensis has a surprising dearth of sensory and regulatory factors. Recent advancements in the field have identified new functions of encoded transcription factors and greatly expanded our understanding of virulence gene regulation. Here we review the current knowledge of environmental adaptation by F. tularensis, its transcriptional regulators and their relationship to animal virulence. PMID:21687801

  13. 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. PMID:21051341

  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. 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

  16. A predictive biophysical model of translational coupling to coordinate and control protein expression in bacterial operons

    PubMed Central

    Tian, Tian; Salis, Howard M.

    2015-01-01

    Natural and engineered genetic systems require the coordinated expression of proteins. In bacteria, translational coupling provides a genetically encoded mechanism to control expression level ratios within multi-cistronic operons. We have developed a sequence-to-function biophysical model of translational coupling to predict expression level ratios in natural operons and to design synthetic operons with desired expression level ratios. To quantitatively measure ribosome re-initiation rates, we designed and characterized 22 bi-cistronic operon variants with systematically modified intergenic distances and upstream translation rates. We then derived a thermodynamic free energy model to calculate de novo initiation rates as a result of ribosome-assisted unfolding of intergenic RNA structures. The complete biophysical model has only five free parameters, but was able to accurately predict downstream translation rates for 120 synthetic bi-cistronic and tri-cistronic operons with rationally designed intergenic regions and systematically increased upstream translation rates. The biophysical model also accurately predicted the translation rates of the nine protein atp operon, compared to ribosome profiling measurements. Altogether, the biophysical model quantitatively predicts how translational coupling controls protein expression levels in synthetic and natural bacterial operons, providing a deeper understanding of an important post-transcriptional regulatory mechanism and offering the ability to rationally engineer operons with desired behaviors. PMID:26117546

  17. Characterization of the RRN Operons in the Channel Catfish Pathogen Edwardsiella ictaluri

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: To advance diagnostics and phylogenetics of Edwardsiella ictaluri by sequencing and characterizing its rrn operons. Methods and Results: The Edw. ictaluri rrn operons were identified from a 5-7 kb insert lambda library and from Edw. ictaluri fosmid clones. We present the complete sequences...

  18. RNase III cleavage of Escherichia coli beta-galactosidase and tryptophan operon mRNA.

    PubMed Central

    Shen, V; Imamoto, F; Schlessinger, D

    1982-01-01

    Purified RNase III of Escherichia coli cleaved the initial 479-nucleotide sequence of lac operon mRNA at four specific sites and also gave limited cleavage of trp operon mRNA. This action explains the inactivation of mRNA coding capacity by RNase III in vitro. Images PMID:6176575

  19. Induction of group A Streptococcus virulence by a human antimicrobial peptide

    PubMed Central

    Gryllos, Ioannis; Tran-Winkler, Hien J.; Cheng, Ming-Fang; Chung, Hachung; Bolcome, Robert; Lu, Wuyuan; Lehrer, Robert I.; Wessels, Michael R.

    2008-01-01

    Group A streptococci (Streptococcus pyogenes or GAS) freshly isolated from individuals with streptococcal sore throat or invasive (“flesh-eating”) infection often grow as mucoid colonies on primary culture but lose this colony appearance after laboratory passage. The mucoid phenotype is due to abundant production of the hyaluronic acid capsular polysaccharide, a key virulence determinant associated with severe GAS infections. These observations suggest that signal(s) from the human host trigger increased production of capsule and perhaps other virulence factors during infection. Here we show that subinhibitory concentrations of the human antimicrobial cathelicidin peptide LL-37 stimulate expression of the GAS capsule synthesis operon (hasABC). Up-regulation is mediated by the CsrRS 2-component regulatory system: it requires a functional CsrS sensor protein and can be antagonized by increased extracellular Mg2+, the other identified environmental signal for CsrS. Up-regulation was also evident for other CsrRS-regulated virulence genes, including the IL-8 protease PrtS/ScpC and the integrin-like/IgG protease Mac/IdeS, findings that suggest a coordinated GAS virulence response elicited by this antimicrobial immune effector peptide. LL-37 signaling through CsrRS led to a marked increase in GAS resistance to opsonophagocytic killing by human leukocytes, an in vitro measure of enhanced GAS virulence, consistent with increased expression of the antiphagocytic capsular polysaccharide and Mac/IdeS. We propose that the human cathelicidin LL-37 has the paradoxical effect of stimulating CsrRS-regulated virulence gene expression, thereby enhancing GAS pathogenicity during infection. The ability of GAS to sense and respond to LL-37 may explain, at least in part, the unique susceptibility of the human species to streptococcal infection. PMID:18936485

  20. Large-scale isolation of candidate virulence genes of Pseudomonas aeruginosa by in vivo selection.

    PubMed Central

    Wang, J; Mushegian, A; Lory, S; Jin, S

    1996-01-01

    Pseudomonas aeruginosa, an opportunistic human pathogen, is a major causative agent of mortality and morbidity in immunocompromised patients and those with cystic fibrosis genetic disease. To identify new virulence genes of P. aeruginosa, a selection system was developed based on the in vivo expression technology (IVET) that was first reported in Salmonella system. An adenine-requiring auxotrophic mutant strain of P. aeruginosa was isolated and found avirulent on neutropenic mice. A DNA fragment that can complement the mutant strain, containing purEK operon that is required for de novo biosynthesis of purine, was sequenced and used in the IVET vector construction. By applying the IVET selection system to a neutropenic mouse infection model, genetic loci that are specifically induced in vivo were identified. Twenty-two such loci were partially sequenced and analyzed. One of them was a well-studied virulence factor, pyochelin receptor (FptA), that is involved in iron acquisition. Fifteen showed significant homology to reported sequences in GenBank, while the remaining six did not. One locus, designated np20, encodes an open reading frame that shares amino acid sequence homology to transcriptional regulators, especially to the ferric uptake regulator (Fur) proteins of other bacteria. An insertional np20 null mutant strain of P. aeruginosa did not show a growth defect on laboratory media; however, its virulence on neutropenic mice was significantly reduced compared with that of a wild-type parent strain, demonstrating the importance of the np20 locus in the bacterial virulence. The successful isolation of genetic loci that affect bacterial virulence demonstrates the utility of the IVET system in identification of new virulence genes of P. aeruginosa. Images Fig. 2 Fig. 4 PMID:8816818

  1. 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.

  2. [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

  3. Insights into arsenic multi-operons expression and resistance mechanisms in Rhodopseudomonas palustris CGA009

    PubMed Central

    Zhao, Chungui; Zhang, Yi; Chan, Zhuhua; Chen, Shicheng; Yang, Suping

    2015-01-01

    Arsenic (As) is widespread in the environment and causes numerous health problems. Rhodopseudomonas palustris has been regarded as a good model organism for studying arsenic detoxification since it was first demonstrated to methylate environmental arsenic by conversion to soluble or gaseous methylated species. However, the detailed arsenic resistance mechanisms remain unknown though there are at least three arsenic-resistance operons (ars1, ars2, and ars3) in R. palustris. In this study, we investigated how arsenic multi-operons contributed to arsenic detoxification in R. palustris. The expression of ars2 or ars3 operons increased with increasing environmental arsenite (As(III)) concentrations (up to 1.0 mM) while transcript of ars1 operon was not detected in the middle log-phase (55 h). ars2 operon was actively expressed even at the low concentration of As(III) (0.01 μM), whereas the ars3 operon was expressed at 1.0 μM of As(III), indicating that there was a differential regulation mechanism for the three arsenic operons. Furthermore, ars2 and ars3 operons were maximally transcribed in the early log-phase where ars2 operon was 5.4-fold higher than that of ars3 operon. A low level of ars1 transcript was only detected at 43 h (early log-phase). Arsenic speciation analysis demonstrated that R. palustris could reduce As(V) to As(III). Collectively, strain CGA009 detoxified arsenic by using arsenic reduction and methylating arsenic mechanism, while the latter might occur with the presence of higher concentrations of arsenic. PMID:26441915

  4. 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

  5. Detection of a putative hemolysin operon, hhdBA, of Haemophilus parasuis from pigs with Glässer disease.

    PubMed

    Assavacheep, Pornchalit; Assavacheep, Anongnart; Turni, Conny

    2012-03-01

    The aim of the current study was to investigate whether polymerase chain reaction amplification of 16S ribosomal (r)RNA and a putative hemolysin gene operon, hhdBA, can be used to monitor live pigs for the presence of Haemophilus parasuis and predict the virulence of the strains present. Nasal cavity swabs were taken from 30 live, healthy, 1- to 8-week-old pigs on a weekly cycle from a commercial Thai nursery pig herd. A total of 27 of these pigs (90%) tested positive for H. parasuis as early as week 1 of age. None of the H. parasuis-positive samples from healthy pigs was positive for the hhdBA genes. At the same pig nursery, swab samples from nasal cavity, tonsil, trachea, and lung, and exudate samples from pleural/peritoneal cavity were taken from 30 dead pigs displaying typical pathological lesions consistent with Glässer disease. Twenty-two of 140 samples (15.7%) taken from 30 diseased pigs yielded a positive result for H. parasuis. Samples from the exudate (27%) yielded the most positive results, followed by lung, tracheal swab, tonsil, and nasal swab, respectively. Out of 22 positive samples, 12 samples (54.5%) harbored hhdA and/or hhdB genes. Detection rates of hhdA were higher than hhdB. None of the H. parasuis-positive samples taken from nasal cavity of diseased pigs tested positive for hhdBA genes. More work is required to determine if the detection of hhdBA genes is useful for identifying the virulence potential of H. parasuis field isolates. PMID:22379049

  6. 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. PMID:24478084

  7. The RpoE Stress Response Pathway Mediates Reduction of the Virulence of Enteropathogenic Escherichia coli by Zinc

    PubMed Central

    Xue, Yuan; Osborn, Jossef; Panchal, Anand

    2015-01-01

    Zinc supplements are an effective clinical treatment for infantile diarrheal disease caused by enteric pathogens. Previous studies demonstrated that zinc acts on enteropathogenic Escherichia coli (EPEC) bacteria directly to suppress several virulence-related genes at a concentration that can be achieved by oral delivery of dietary zinc supplements. Our in vitro studies showed that a micromolar concentration of zinc induced the envelope stress response and suppressed virulence in EPEC, providing a possible mechanistic explanation for zinc's therapeutic action. In this report, we investigated the molecular and physiological changes in EPEC induced by zinc. We found that micromolar concentrations of zinc reduced the bacterial growth rate without affecting viability. We observed increased membrane permeability caused by zinc. Zinc upregulated the RpoE-dependent envelope stress response pathway and suppressed EPEC virulence gene expression. RpoE alone was sufficient to inhibit virulence factor expression and to attenuate attaching and effacing lesion formation on human host cells. By mutational analysis we demonstrate that the DNA-binding motif of RpoE is necessary for suppression of the LEE1, but not the LEE4, operon. Predictably, inhibition of the RpoE-mediated envelope stress response in combination with micromolar concentrations of zinc reduced EPEC viability. In conclusion, zinc induces the RpoE and stress response pathways in EPEC, and the alternate sigma factor RpoE downregulates EPEC LEE and non-LEE virulence genes by multiple mechanisms. PMID:25819956

  8. Analysis of Genome Sequences from Plant Pathogenic Rhodococcus Reveals Genetic Novelties in Virulence Loci

    PubMed Central

    Davis, Edward W.; Putnam, Melodie L.; Hu, Erdong; Swader-Hines, David; Mol, Adeline; Baucher, Marie; Prinsen, Els; Zdanowska, Magdalena; Givan, Scott A.; Jaziri, Mondher El; Loper, Joyce E.; Mahmud, Taifo; Chang, Jeff H.

    2014-01-01

    Members of Gram-positive Actinobacteria cause economically important diseases to plants. Within the Rhodococcus genus, some members can cause growth deformities and persist as pathogens on a wide range of host plants. The current model predicts that phytopathogenic isolates require a cluster of three loci present on a linear plasmid, with the fas operon central to virulence. The Fas proteins synthesize, modify, and activate a mixture of growth regulating cytokinins, which cause a hormonal imbalance in plants, resulting in abnormal growth. We sequenced and compared the genomes of 20 isolates of Rhodococcus to gain insights into the mechanisms and evolution of virulence in these bacteria. Horizontal gene transfer was identified as critical but limited in the scale of virulence evolution, as few loci are conserved and exclusive to phytopathogenic isolates. Although the fas operon is present in most phytopathogenic isolates, it is absent from phytopathogenic isolate A21d2. Instead, this isolate has a horizontally acquired gene chimera that encodes a novel fusion protein with isopentyltransferase and phosphoribohydrolase domains, predicted to be capable of catalyzing and activating cytokinins, respectively. Cytokinin profiling of the archetypal D188 isolate revealed only one activate cytokinin type that was specifically synthesized in a fas-dependent manner. These results suggest that only the isopentenyladenine cytokinin type is synthesized and necessary for Rhodococcus phytopathogenicity, which is not consistent with the extant model stating that a mixture of cytokinins is necessary for Rhodococcus to cause leafy gall symptoms. In all, data indicate that only four horizontally acquired functions are sufficient to confer the trait of phytopathogenicity to members of the genetically diverse clade of Rhodococcus. PMID:25010934

  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. PMID:24582794

  10. 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

  11. Mycobacterium tuberculosis Co-operonic PE32/PPE65 Proteins Alter Host Immune Responses by Hampering Th1 Response

    PubMed Central

    Khubaib, Mohd; Sheikh, Javaid A.; Pandey, Saurabh; Srikanth, Battu; Bhuwan, Manish; Khan, Nooruddin; Hasnain, Seyed E.; Ehtesham, Nasreen Z.

    2016-01-01

    PE/PPE genes, present in cluster with ESAT-6 like genes, are suspected to have a role in antigenic variation and virulence of Mycobacterium tuberculosis. Their roles in immune evasion and immune modulation of host are also well documented. We present evidence that PE32/PPE65 present within the RD8 region are co-operonic, co-transcribed, and co-translated, and play role in modulating host immune responses. Experiments with macrophage cell lines revealed that this protein complex suppresses pro-inflammatory cytokines such as TNF-α and IL-6 whereas also inducing high expression of anti-inflammatory IL-10. Immunization of mice with these recombinant proteins dampens an effective Th1 response as evident from reduced frequency of IFN-γ and IL-2 producing CD4+ and CD8+ T cells. IgG sub-typing from serum of immunized mice revealed high levels of IgG1 when compared with IgG2a and IgG2b. Further IgG1/IgG2a ratio clearly demonstrated that the protein complex manipulates the host immune response favorable to the pathogen. Our results demonstrate that the co-transcribed and co-translated PE32 and PPE65 antigens are involved specifically in modulating anti-mycobacterial host immune response by hampering Th1 response. PMID:27242739

  12. 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

  13. The role of FIS in trans activation of stable RNA operons of E. coli.

    PubMed

    Nilsson, L; Vanet, A; Vijgenboom, E; Bosch, L

    1990-03-01

    The thrU(tufB) operon of Escherichia coli is endowed with a cis-acting region upstream of the promoter, designated UAS for Upstream Activator Sequence. A protein fraction has been isolated that binds specifically to DNA fragments of the UAS, thus forming three protein-DNA complexes corresponding to three binding sites on the UAS. It stimulates in vitro transcription of the operon by facilitating the binding of the RNA polymerase to the promoter. All three protein-DNA complexes contain one and the same protein. Dissociation constants for the three complexes have been determined, the lowest being in the sub-nanomolar range. The protein also binds to the UAS of the tyrT operon and to the UAS upstream of the P1 promoter of the rrnB operon, suggesting that transcription of the three operons, if not of more stable RNA operons, is activated by a common trans activator. We demonstrate that the E.coli protein FIS (Factor for Inversion Stimulation) also binds to the UAS of the thrU(tufB) operon forming three protein-DNA complexes. A burst of UAS- and FIS-dependent promoter activity is observed after reinitiation of growth of stationary cultures in fresh medium. PMID:1690124

  14. 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

  15. When experimental selection for virulence leads to loss of virulence.

    PubMed

    Rafaluk, Charlotte; Jansen, Gunther; Schulenburg, Hinrich; Joop, Gerrit

    2015-09-01

    Information on virulence evolution is critical for understanding disease dynamics. Theory predicts that under certain evolutionary conditions virulence should increase; for example, during host-parasite coevolution. Although these theoretical predictions are supported by natural observations, tests of these hypotheses using experimental evolution have yielded confounding and contradictory results, with discrepancies often being seen among experiments. Here we provide a critical overview of experimental tests of hypotheses regarding virulence evolution and provide potential explanations for the contradictory results. We emphasise the key role of parasite transmission mechanisms that can explain many of the observed discrepancies among evolution experiments. Finally, we make suggestions for how evolution experiments could be conducted in the future to avoid potentially confounding factors. PMID:26166750

  16. 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

  17. The HD-GYP Domain Protein RpfG of Xanthomonas oryzae pv. oryzicola Regulates Synthesis of Extracellular Polysaccharides that Contribute to Biofilm Formation and Virulence on Rice

    PubMed Central

    Zhang, Yuanbao; Wei, Chao; Jiang, Wendi; Wang, Lei; Li, Churui; Wang, Yunyue; Dow, John Maxwell; Sun, Wenxian

    2013-01-01

    Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions. PMID:23544067

  18. 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

  19. TrxR, a new CovR-repressed response regulator that activates the Mga virulence regulon in group A Streptococcus.

    PubMed

    Leday, Temekka V; Gold, Kathryn M; Kinkel, Traci L; Roberts, Samantha A; Scott, June R; McIver, Kevin S

    2008-10-01

    Coordinate regulation of virulence factors by the group A streptococcus (GAS) Streptococcus pyogenes is important in this pathogen's ability to cause disease. To further elucidate the regulatory network in this human pathogen, the CovR-repressed two-component system (TCS) trxSR was chosen for further analysis based on its homology to a virulence-related TCS in Streptococcus pneumoniae. In a murine skin infection model, an insertion mutation in the response regulator gene, trxR, led to a significant reduction in lesion size, lesion severity, and lethality. Curing the trxR mutation restored virulence comparable to the wild-type strain. The trxSR operon was defined in vivo, and CovR was found to directly repress its promoter in vitro. DNA microarray analysis established that TrxR activates transcription of Mga-regulated virulence genes, which may explain the virulence attenuation of the trxR mutant. This regulation appears to occur by activation of the mga promoter, Pmga, as demonstrated by analysis of a luciferase reporter fusion. Complementation of the trxR mutant with trxR on a plasmid restored expression of Mga regulon genes and restored virulence in the mouse model to wild-type levels. TrxR is the first TCS shown to regulate Mga expression. Because it is CovR repressed, TrxR defines a new pathway by which CovR can influence Mga to affect pathogenesis in the GAS. PMID:18678666

  20. TrxR, a New CovR-Repressed Response Regulator That Activates the Mga Virulence Regulon in Group A Streptococcus▿ †

    PubMed Central

    Leday, Temekka V.; Gold, Kathryn M.; Kinkel, Traci L.; Roberts, Samantha A.; Scott, June R.; McIver, Kevin S.

    2008-01-01

    Coordinate regulation of virulence factors by the group A streptococcus (GAS) Streptococcus pyogenes is important in this pathogen's ability to cause disease. To further elucidate the regulatory network in this human pathogen, the CovR-repressed two-component system (TCS) trxSR was chosen for further analysis based on its homology to a virulence-related TCS in Streptococcus pneumoniae. In a murine skin infection model, an insertion mutation in the response regulator gene, trxR, led to a significant reduction in lesion size, lesion severity, and lethality. Curing the trxR mutation restored virulence comparable to the wild-type strain. The trxSR operon was defined in vivo, and CovR was found to directly repress its promoter in vitro. DNA microarray analysis established that TrxR activates transcription of Mga-regulated virulence genes, which may explain the virulence attenuation of the trxR mutant. This regulation appears to occur by activation of the mga promoter, Pmga, as demonstrated by analysis of a luciferase reporter fusion. Complementation of the trxR mutant with trxR on a plasmid restored expression of Mga regulon genes and restored virulence in the mouse model to wild-type levels. TrxR is the first TCS shown to regulate Mga expression. Because it is CovR repressed, TrxR defines a new pathway by which CovR can influence Mga to affect pathogenesis in the GAS. PMID:18678666

  1. PTS phosphorylation of Mga modulates regulon expression and virulence in the group A streptococcus.

    PubMed

    Hondorp, Elise R; Hou, Sherry C; Hause, Lara L; Gera, Kanika; Lee, Ching-En; McIver, Kevin S

    2013-06-01

    The ability of a bacterial pathogen to monitor available carbon sources in host tissues provides a clear fitness advantage. In the group A streptococcus (GAS), the virulence regulator Mga contains homology to phosphotransferase system (PTS) regulatory domains (PRDs) found in sugar operon regulators. Here we show that Mga was phosphorylated in vitro by the PTS components EI/HPr at conserved PRD histidines. A ΔptsI (EI-deficient) GAS mutant exhibited decreased Mga activity. However, PTS-mediated phosphorylation inhibited Mga-dependent transcription of emm in vitro. Using alanine (unphosphorylated) and aspartate (phosphomimetic) mutations of PRD histidines, we establish that a doubly phosphorylated PRD1 phosphomimetic (D/DMga4) is completely inactive in vivo, shutting down expression of the Mga regulon. Although D/DMga4 is still able to bind DNA in vitro, homo-multimerization of Mga is disrupted and the protein is unable to activate transcription. PTS-mediated regulation of Mga activity appears to be important for pathogenesis, as bacteria expressing either non-phosphorylated (A/A) or phosphomimetic (D/D) PRD1 Mga mutants were attenuated in a model of GAS invasive skin disease. Thus, PTS-mediated phosphorylation of Mga may allow the bacteria to modulate virulence gene expression in response to carbohydrate status. Furthermore, PRD-containing virulence regulators (PCVRs) appear to be widespread in Gram-positive pathogens. PMID:23651410

  2. Identification and sequence analysis of lpfABCDE, a putative fimbrial operon of Salmonella typhimurium.

    PubMed Central

    Bäumler, A J; Heffron, F

    1995-01-01

    A chromosomal region present in Salmonella typhimurium but absent from related species was identified by hybridization. A DNA probe originating from 78 min on the S. typhimurium chromosome hybridized with DNA from Salmonella enteritidis, Salmonella heidelberg, and Salmonella dublin but not with DNA from Salmonella typhi, Salmonella arizonae, Escherichia coli, and Shigella serotypes. Cloning and sequence analysis revealed that the corresponding region of the S. typhimurium chromosome encodes a fimbrial operon. Long fimbriae inserted at the poles of the bacterium were observed by electron microscopy when this fimbrial operon was introduced into a nonpiliated E. coli strain. The genes encoding these fimbriae were therefore termed lpfABCDE, for long polar fimbriae. Genetically, the lpf operon was found to be most closely related to the fim operon of S. typhimurium, both in gene order and in conservation of the deduced amino acid sequences. PMID:7721701

  3. 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

  4. Parallel Evolution and Horizontal Gene Transfer of the pst Operon in Firmicutes from Oligotrophic Environments

    PubMed Central

    Moreno-Letelier, Alejandra; Olmedo, Gabriela; Eguiarte, Luis E.; Martinez-Castilla, Leon; Souza, Valeria

    2011-01-01

    The high affinity phosphate transport system (pst) is crucial for phosphate uptake in oligotrophic environments. Cuatro Cienegas Basin (CCB) has extremely low P levels and its endemic Bacillus are closely related to oligotrophic marine Firmicutes. Thus, we expected the pst operon of CCB to share the same evolutionary history and protein similarity to marine Firmicutes. Orthologs of the pst operon were searched in 55 genomes of Firmicutes and 13 outgroups. Phylogenetic reconstructions were performed for the pst operon and 14 concatenated housekeeping genes using maximum likelihood methods. Conserved domains and 3D structures of the phosphate-binding protein (PstS) were also analyzed. The pst operon of Firmicutes shows two highly divergent clades with no correlation to the type of habitat nor a phylogenetic congruence, suggesting horizontal gene transfer. Despite sequence divergence, the PstS protein had a similar 3D structure, which could be due to parallel evolution after horizontal gene transfer events. PMID:21461370

  5. Parallel Evolution and Horizontal Gene Transfer of the pst Operon in Firmicutes from Oligotrophic Environments.

    PubMed

    Moreno-Letelier, Alejandra; Olmedo, Gabriela; Eguiarte, Luis E; Martinez-Castilla, Leon; Souza, Valeria

    2011-01-01

    The high affinity phosphate transport system (pst) is crucial for phosphate uptake in oligotrophic environments. Cuatro Cienegas Basin (CCB) has extremely low P levels and its endemic Bacillus are closely related to oligotrophic marine Firmicutes. Thus, we expected the pst operon of CCB to share the same evolutionary history and protein similarity to marine Firmicutes. Orthologs of the pst operon were searched in 55 genomes of Firmicutes and 13 outgroups. Phylogenetic reconstructions were performed for the pst operon and 14 concatenated housekeeping genes using maximum likelihood methods. Conserved domains and 3D structures of the phosphate-binding protein (PstS) were also analyzed. The pst operon of Firmicutes shows two highly divergent clades with no correlation to the type of habitat nor a phylogenetic congruence, suggesting horizontal gene transfer. Despite sequence divergence, the PstS protein had a similar 3D structure, which could be due to parallel evolution after horizontal gene transfer events. PMID:21461370

  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. Lineage-specific Virulence Determinants of Haemophilus influenzae Biogroup aegyptius

    PubMed Central

    Strouts, Fiona R.; Power, Peter; Croucher, Nicholas J.; Corton, Nicola; van Tonder, Andries; Quail, Michael A.; Langford, Paul R.; Hudson, Michael J.; Parkhill, Julian; Bentley, Stephen D.

    2012-01-01

    An emergent clone of Haemophilus influenzae biogroup aegyptius (Hae) is responsible for outbreaks of Brazilian purpuric fever (BPF). First recorded in Brazil in 1984, the so-called BPF clone of Hae caused a fulminant disease that started with conjunctivitis but developed into septicemic shock; mortality rates were as high as 70%. To identify virulence determinants, we conducted a pan-genomic analysis. Sequencing of the genomes of the BPF clone strain F3031 and a noninvasive conjunctivitis strain, F3047, and comparison of these sequences with 5 other complete H. influenzae genomes showed that >77% of the F3031 genome is shared among all H. influenzae strains. Delineation of the Hae accessory genome enabled characterization of 163 predicted protein-coding genes; identified differences in established autotransporter adhesins; and revealed a suite of novel adhesins unique to Hae, including novel trimeric autotransporter adhesins and 4 new fimbrial operons. These novel adhesins might play a critical role in host–pathogen interactions. PMID:22377449

  8. ROLE OF NA+ IN TRANSPORT OF HG2+ AND INDUCTION OF THE TN21 "MER" OPERON

    EPA Science Inventory

    The effects of sodium ions on the uptake of Hg 2 + and induction of the TN21 mer operon were studied using E. coli HMS174 harboring the reporter plasmids pRB28 and pOS14. lasmid pRB28 carries merRT' and pOS14 carries merRTPC of the mer operon, both cloned upstream of a promoterle...

  9. Escherichia coli tryptophan operon directs the in vivo synthesis of a leader peptide.

    PubMed Central

    Dekel-Gorodetsky, L; Schoulaker-Schwarz, R; Engelberg-Kulka, H

    1986-01-01

    Here we report the identification of the Escherichia coli trp leader peptide synthesized in vivo. We identified the peptide in UV-irradiated maxicells by selective labeling with radioactive amino acids which are included in the predicted sequence of this peptide. Our results support the hypothesis that translation of the peptide-coding region of the leader RNA has a role in the mechanism of attenuation of biosynthetic operons in general and in the E. coli trp operon in particular. Images PMID:2419306

  10. 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.

  11. A universally applicable method of operon map prediction on minimally annotated genomes using conserved genomic context

    PubMed Central

    Edwards, Martin T.; Rison, Stuart C. G.; Stoker, Neil G.; Wernisch, Lorenz

    2005-01-01

    An important step in understanding the regulation of a prokaryotic genome is the generation of its transcription unit map. The current strongest operon predictor depends on the distributions of intergenic distances (IGD) separating adjacent genes within and between operons. Unfortunately, experimental data on these distance distributions are limited to Escherichia coli and Bacillus subtilis. We suggest a new graph algorithmic approach based on comparative genomics to identify clusters of conserved genes independent of IGD and conservation of gene order. As a consequence, distance distributions of operon pairs for any arbitrary prokaryotic genome can be inferred. For E.coli, the algorithm predicts 854 conserved adjacent pairs with a precision of 85%. The IGD distribution for these pairs is virtually identical to the E.coli operon pair distribution. Statistical analysis of the predicted pair IGD distribution allows estimation of a genome-specific operon IGD cut-off, obviating the requirement for a training set in IGD-based operon prediction. We apply the method to a representative set of eight genomes, and show that these genome-specific IGD distributions differ considerably from each other and from the distribution in E.coli. PMID:15942028

  12. Eukaryotic operon-like transcription of functionally related genes in Drosophila

    PubMed Central

    Ben-Shahar, Yehuda; Nannapaneni, Kishore; Casavant, Thomas L.; Scheetz, Todd E.; Welsh, Michael J.

    2007-01-01

    Complex biological processes require coordinated function of many genes. One evolutionary solution to the problem of coordinately expressing functionally related genes in bacteria and nematodes is organization of genes in operons. Surprisingly, eukaryotic operons are considered rare outside the nematode lineage. In Drosophila melanogaster, we found lounge lizard (llz), which encodes a degenerin/ENaC cation channel, cotranscribed with CheB42a, a nonhomologous gene of unknown function residing <100 bp upstream. These two genes were transcribed from a single promoter as one primary transcript and were processed posttranscriptionally to generate individual mRNAs. The mechanism did not involve alternative splicing, and it differed from the trans splicing used in nematode operons. Both genes were expressed in the same tissues, and previous work suggested that both may be involved in courtship behavior. A bioinformatic approach identified numerous additional loci as potential Drosophila operons. These data reveal eukaryotic operon-like transcription of functionally related genes in Drosophila. The results also suggest that operon-based transcription may be more common in eukaryotes than previously appreciated. PMID:17190802

  13. 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.

  14. Analysis of a ribosomal RNA operon in the actinomycete Frankia.

    PubMed

    Normand, P; Cournoyer, B; Simonet, P; Nazaret, S

    1992-02-01

    The organisation of ribosomal RNA-encoding (rrn) genes has been studied in Frankia sp. strain ORS020606. The two rrn clusters present in Frankia strain ORS020606 were isolated from genomic banks in phage lambda EMBL3 by hybridization with oligodeoxyribonucleotide probes. The 5'-3' gene order is the usual one for bacteria: 16S-23S-5S. The two clusters are not distinguishable by restriction enzyme mapping inside the coding section, but vary considerably outside it. Sequencing showed that the 16S-rRNA-encoding gene of ORS020606 is very closely related to that of another Alnus-infective Frankia strain (Ag45/Mut15) and highly homologous to corresponding genes of Streptomyces spp. Two possible promoter sequences were detected upstream from the 16S gene, while no tRNA-encoding gene was detected in the whole operon. Regions with a high proportion of divergence for the study of phylogenetic relationships within the genus were looked for and found in the first intergenic spacer, in the 23S and in the 16S gene. PMID:1372279

  15. Characterization of fig operon mutants of Francisella novicida U112

    PubMed Central

    Kiss, Katalin; Liu, Wei; Huntley, Jason F.; Norgard, Michael V.; Hansen, Eric J.

    2009-01-01

    Francisella species secrete a polycarboxylate siderophore that resembles rhizoferrin to acquire ferric iron. Several of the Francisella siderophore synthesis genes are contained in a Fur-regulated operon (designated fig or fsl) comprised of at least seven open reading frames (ORFs) including fur. Reverse transcriptase-PCR showed transcriptional linkage between figD and figE and between figE and figF. Mutations were constructed in four of these ORFs (figB, figC, figD, and figE) in F. novicida U112. All four of these new mutants and a F. novicida figA mutant grew at rates comparable to that of wild-type under iron-replete conditions but growth of all five mutants was stunted in iron-limiting media. When ferric rhizoferrin was added to the iron-limited media, growth of the figA, figB, figC, and figD mutants was restored to levels similar to those obtained in iron-replete media. However, this exogenously added siderophore could not rescue the figE mutant. When Chrome Azurol S assays were used to measure siderophore production, the figA, figB, and figC mutants were markedly deficient in their ability to synthesize siderophore whereas the figD and figE mutants produced siderophore at levels equivalent to the wild-type parent strain. PMID:18564336

  16. Transcriptional regulation of a Bacillus subtilis dipeptide transport operon.

    PubMed

    Slack, F J; Mueller, J P; Strauch, M A; Mathiopoulos, C; Sonenshein, A L

    1991-08-01

    The Bacillus subtilis dciA operon, which encodes a dipeptide transport system, was induced rapidly by several conditions that caused the cells to enter stationary phase and initiate sporulation. The in vivo start point of transcription was mapped precisely and shown to correspond to a site of transcription initiation in vitro by the major vegetative form of RNA polymerase. Post-exponential expression was prevented by a mutation in the spo0A gene (whose product is a known regulator of early sporulation genes) but was restored in a spo0A abrB double mutant. This implicated AbrB, another known regulator, as a repressor of dciA. In fact, purified AbrB protein bound to a portion of the dciA promoter region, protecting it against DNase I digestion. Expression of dciA in growing cells was also repressed independently by glucose and by a mixture of amino acids; neither of these effects was mediated by AbrB. PMID:1766371

  17. 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. PMID:26889782

  18. 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

  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 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

  2. A Mutation in the 16S rRNA Decoding Region Attenuates the Virulence of Mycobacterium tuberculosis.

    PubMed

    Watanabe, Shinya; Matsumura, Kazunori; Iwai, Hiroki; Funatogawa, Keiji; Haishima, Yuji; Fukui, Chie; Okumura, Kayo; Kato-Miyazawa, Masako; Hashimoto, Masahito; Teramoto, Kanae; Kirikae, Fumiko; Miyoshi-Akiyama, Tohru; Kirikae, Teruo

    2016-08-01

    Mycobacterium tuberculosis contains a single rRNA operon that encodes targets for antituberculosis agents, including kanamycin. To date, only four mutations in the kanamycin binding sites of 16S rRNA have been reported in kanamycin-resistant clinical isolates. We hypothesized that another mutation(s) in the region may dramatically decrease M. tuberculosis viability and virulence. Here, we describe an rRNA mutation, U1406A, which was generated in vitro and confers resistance to kanamycin while highly attenuating M. tuberculosis virulence. The mutant showed decreased expression of 20% (n = 361) of mycobacterial proteins, including central metabolic enzymes, mycolic acid biosynthesis enzymes, and virulence factors such as antigen 85 complexes and ESAT-6. The mutation also induced three proteins, including KsgA (Rv1010; 16S rRNA adenine dimethyltransferase), which closely bind to the U1406A mutation site on the ribosome; these proteins were associated with ribosome maturation and translation initiation processes. The mutant showed an increase in 17S rRNA (precursor 16S rRNA) and a decrease in the ratio of 30S subunits to the 70S ribosomes, suggesting that the U1406A mutation in 16S rRNA attenuated M. tuberculosis virulence by affecting these processes. PMID:27245411

  3. The catabolite control protein E (CcpE) affects virulence determinant production and pathogenesis of Staphylococcus aureus.

    PubMed

    Hartmann, Torsten; Baronian, Grégory; Nippe, Nadine; Voss, Meike; Schulthess, Bettina; Wolz, Christiane; Eisenbeis, Janina; Schmidt-Hohagen, Kerstin; Gaupp, Rosmarie; Sunderkötter, Cord; Beisswenger, Christoph; Bals, Robert; Somerville, Greg A; Herrmann, Mathias; Molle, Virginie; Bischoff, Markus

    2014-10-24

    Carbon metabolism and virulence determinant production are often linked in pathogenic bacteria, and several regulatory elements have been reported to mediate this linkage in Staphylococcus aureus. Previously, we described a novel protein, catabolite control protein E (CcpE) that functions as a regulator of the tricarboxylic acid cycle. Here we demonstrate that CcpE also regulates virulence determinant biosynthesis and pathogenesis. Specifically, deletion of ccpE in S. aureus strain Newman revealed that CcpE affects transcription of virulence factors such as capA, the first gene in the capsule biosynthetic operon; hla, encoding α-toxin; and psmα, encoding the phenol-soluble modulin cluster α. Electrophoretic mobility shift assays demonstrated that CcpE binds to the hla promoter. Mice challenged with S. aureus strain Newman or its isogenic ΔccpE derivative revealed increased disease severity in the ΔccpE mutant using two animal models; an acute lung infection model and a skin infection model. Complementation of the mutant with the ccpE wild-type allele restored all phenotypes, demonstrating that CcpE is negative regulator of virulence in S. aureus. PMID:25193664

  4. The Catabolite Control Protein E (CcpE) Affects Virulence Determinant Production and Pathogenesis of Staphylococcus aureus*

    PubMed Central

    Hartmann, Torsten; Baronian, Grégory; Nippe, Nadine; Voss, Meike; Schulthess, Bettina; Wolz, Christiane; Eisenbeis, Janina; Schmidt-Hohagen, Kerstin; Gaupp, Rosmarie; Sunderkötter, Cord; Beisswenger, Christoph; Bals, Robert; Somerville, Greg A.; Herrmann, Mathias; Molle, Virginie; Bischoff, Markus

    2014-01-01

    Carbon metabolism and virulence determinant production are often linked in pathogenic bacteria, and several regulatory elements have been reported to mediate this linkage in Staphylococcus aureus. Previously, we described a novel protein, catabolite control protein E (CcpE) that functions as a regulator of the tricarboxylic acid cycle. Here we demonstrate that CcpE also regulates virulence determinant biosynthesis and pathogenesis. Specifically, deletion of ccpE in S. aureus strain Newman revealed that CcpE affects transcription of virulence factors such as capA, the first gene in the capsule biosynthetic operon; hla, encoding α-toxin; and psmα, encoding the phenol-soluble modulin cluster α. Electrophoretic mobility shift assays demonstrated that CcpE binds to the hla promoter. Mice challenged with S. aureus strain Newman or its isogenic ΔccpE derivative revealed increased disease severity in the ΔccpE mutant using two animal models; an acute lung infection model and a skin infection model. Complementation of the mutant with the ccpE wild-type allele restored all phenotypes, demonstrating that CcpE is negative regulator of virulence in S. aureus. PMID:25193664

  5. The bcp gene in the bcp-recA-vimA-vimE-vimF operon is important in oxidative stress resistance in Porphyromonas gingivalis W83.

    PubMed

    Johnson, N A; McKenzie, R M E; Fletcher, H M

    2011-02-01

    The ability of Porphyromonas gingivalis to overcome oxidative stress in the inflammatory environment of the periodontal pocket is critical for its survival. We have previously demonstrated that the recA locus, which carries the bacterioferritin co-migratory protein (bcp) gene and has a unique genetic architecture, plays a role in virulence regulation and oxidative stress resistance in P. gingivalis. To further characterize the bcp gene, which was confirmed to be part of the bcp-recA-vimA-vimE-vimF operon, we created a P. gingivalis bcp-defective isogenic mutant (FLL302) by allelic exchange. Compared with the wild-type, FLL302 had a similar growth rate, black pigmentation, β-hemolysis and UV sensitivity. Although there was no change in the distribution of gingipain activity, there was a 30% reduction in both Arg-X and Lys-X activities in the mutant strain compared with the wild-type. When exposed to 0.25 mm hydrogen peroxide, P. gingivalis FLL302 was more sensitive than the wild-type. In addition, the cloned P. gingivalis bcp gene increased resistance to 0.25 mm hydrogen peroxide in a bcp-defective Escherichia coli mutant. The mutant also demonstrated decreased aerotolerance when compared with the wild-type. Porphyromonas gingivalis FLL302 and the wild-type strain had similar virulence profiles in a mouse model of virulence. These observations suggest that the bcp gene may play a role in oxidative stress resistance but has a decreased functional significance in the pathogenic potential of P. gingivalis. PMID:21214873

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

    PubMed

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

    1992-02-01

    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

  7. 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

  8. Identification of pTiC58 plasmid-encoded proteins for virulence in Agrobacterium tumefaciens.

    PubMed Central

    Hagiya, M; Close, T J; Tait, R C; Kado, C I

    1985-01-01

    Analyses were made of the host-dependent-variation (hdv) locus of the virulence (vir) region of the pTiC58 plasmid of Agrobacterium tumefaciens. The hdv locus is comprised of at least four genes that encode polypeptides of 13, 15, 29, and 28 kDa. Insertion of transposon Tn5 in the first gene abolishes the expression of all four genes in vitro and in vivo. Nucleotide sequence analysis of the hdv locus revealed four open reading frames tandemly arranged with spacer sequences having no promoter-like sequences and lacking the ability to bind A. tumefaciens RNA polymerase. These studies suggest that the hdv locus is comprised of at least four genes arranged in an operon in the vir region. The protein products of these genes are likely to function in some aspect of the host-range determination of A. tumefaciens. Images PMID:2986128

  9. The Mycoplasma gallisepticum virulence factor lipoprotein MslA is a novel polynucleotide binding protein.

    PubMed

    Masukagami, Yumiko; Tivendale, Kelly A; Mardani, Karim; Ben-Barak, Idan; Markham, Philip F; Browning, Glenn F

    2013-09-01

    Although lipoproteins of mycoplasmas are thought to play a crucial role in interactions with their hosts, very few have had their biochemical function defined. The gene encoding the lipoprotein MslA in Mycoplasma gallisepticum has recently been shown to be required for virulence, but the biochemical function of this gene is not known. Although this gene has no significant sequence similarity to any gene of known function, it is located within an operon in M. gallisepticum that contains a homolog of a gene previously shown to be a nonspecific exonuclease. We mutagenized both genes to facilitate expression in Escherichia coli and then examined the functions of the recombinant proteins. The capacity of MslA to bind polynucleotides was examined, and we found that the protein bound single- and double-stranded DNA, as well as single-stranded RNA, with a predicted binding site of greater than 1 nucleotide but less than or equal to 5 nucleotides in length. Recombinant MslA cleaved into two fragments in vitro, both of which were able to bind oligonucleotides. These findings suggest that the role of MslA may be to act in concert with the lipoprotein nuclease to generate nucleotides for transport into the mycoplasma cell, as the remaining genes in the operon are predicted to encode an ABC transporter. PMID:23798535

  10. 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

  11. Phylogeny and Virulence of Naturally Occurring Type III Secretion System-Deficient Pectobacterium Strains▿

    PubMed Central

    Kim, Hye-Sook; Ma, Bing; Perna, Nicole T.; Charkowski, Amy O.

    2009-01-01

    Pectobacterium species are enterobacterial plant-pathogenic bacteria that cause soft rot disease in diverse plant species. Previous epidemiological studies of Pectobacterium species have suffered from an inability to identify most isolates to the species or subspecies level. We used three previously described DNA-based methods, 16S-23S intergenic transcribed spacer PCR-restriction fragment length polymorphism analysis, multilocus sequence analysis (MLSA), and pulsed-field gel electrophoresis, to examine isolates from diseased stems and tubers and found that MLSA provided the most reliable classification of isolates. We found that strains belonging to at least two Pectobacterium clades were present in each field examined, although representatives of only three of five Pectobacterium clades were isolated. Hypersensitive response and DNA hybridization assays revealed that strains of both Pectobacterium carotovorum and Pectobacterium wasabiae lack a type III secretion system (T3SS). Two of the T3SS-deficient strains assayed lack genes adjacent to the T3SS gene cluster, suggesting that multiple deletions occurred in Pectobacterium strains in this locus, and all strains appear to have only six rRNA operons instead of the seven operons typically found in Pectobacterium strains. The virulence of most of the T3SS-deficient strains was similar to that of T3SS-encoding strains in stems and tubers. PMID:19411432

  12. 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

  13. [Virulence determinant of Chromobacterium violaceum].

    PubMed

    Miki, Tsuyoshi

    2014-01-01

    Chromobacterium violaceum is a Gram-negative bacterium that infects humans and animals with fatal sepsis. The infection with C. violaceum is rare in case of those who are healthy, but once established, C. violaceum causes sever disease accompanied by abscess formation in the lungs, liver and spleen. Furthermore, C. violaceum is resistant to a broad range of antibiotics, which in some cases renders the antimicrobial therapy for this infection difficult. Thus, the infection with C. violaceum displays high mortality rates unless initial proper antimicrobial therapy. In contrast, the infection mechanism had completely remained unknown. To this end, we have tried to identify virulence factors-associated with C. violaceum infection. Two distinct type III secretion systems (TTSSs) were thought to be one of the most important virulence factors, which are encoded by Chromobacterium pathogenicity island 1/1a and 2 (Cpi-1/-1a and -2) respectively. Our results have shown that Cpi-1/-1a-encoded TTSS, but not Cpi-2, is indispensable for the virulence in a mouse infection model. C. violaceum caused fulminant hepatitis in a Cpi-1/-1a-encoded TTSS-dependent manner. We next have identified 16 novel effectors secreted from Cpi-1/-1a-encoded TTS machinery. From these effectors, we found that CopE (Chromobacterium outer protein E) has similarities to a guanine nucleotide exchange factor (GEF) for Rho GTPases. CopE acts as GEF for Rac1 and Cdc42, leading to induction of actin cytoskeletal rearrangement. Interestingly, C. violaceum invades cultured human epithelial cells in a CopE-dependent manner. Finally, an inactivation of CopE by disruption of copE gene or amino acid point mutation leading to loss of GEF activity attenuates significantly the mouse virulence of C. violaceum. These results suggest that Cpi-1/-1a-encoded TTSS is a major virulence determinant for C. violaceum infection, and that CopE contributes to the virulence in part of this pathogen. PMID:25447983

  14. 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

  15. 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.

  16. Surface attachment induces Pseudomonas aeruginosa virulence

    PubMed Central

    Siryaporn, Albert; Kuchma, Sherry L.; O’Toole, George A.; Gitai, Zemer

    2014-01-01

    Pseudomonas aeruginosa infects every type of host that has been examined by deploying multiple virulence factors. Previous studies of virulence regulation have largely focused on chemical cues, but P. aeruginosa may also respond to mechanical cues. Using a rapid imaging-based virulence assay, we demonstrate that P. aeruginosa activates virulence in response to attachment to a range of chemically distinct surfaces, suggesting that this bacterial species responds to mechanical properties of its substrates. Surface-activated virulence requires quorum sensing, but activating quorum sensing does not induce virulence without surface attachment. The activation of virulence by surfaces also requires the surface-exposed protein PilY1, which has a domain homologous to a eukaryotic mechanosensor. Specific mutation of the putative PilY1 mechanosensory domain is sufficient to induce virulence in non–surface-attached cells, suggesting that PilY1 mediates surface mechanotransduction. Triggering virulence only when cells are both at high density and attached to a surface—two host-nonspecific cues—explains how P. aeruginosa precisely regulates virulence while maintaining broad host specificity. PMID:25385640

  17. 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.

  18. 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. PMID:25016342

  19. Divergent Operons and the Genetic Structure of the Maltose B Region in ESCHERICHIA COLI K12

    PubMed Central

    Hofnung, Maurice

    1974-01-01

    Complementation and polarity suppression data are interpreted in terms of the genetic structure of the maltose B region. It is proposed that this region comprises two divergent operons. One operon includes malK, a cistron involved in maltose permeation, and lamB the only known cistron specifically involved in λ receptor synthesis. The other operon includes malJ1 and malJ2 which are most probably two different cistrons, both involved in maltose permeation*. It is further assumed that expression of the two operons is controlled by malT, the positive regulatory gene of the maltose system, located in the malA region. The target(s) for the action of the malT product is (are) most likely to be located between malJ1 and malK. There is an indication that the two operons might overlap in the region of their promoters. The structure of such an overlap as well as the possible function of the products of the different cistrons in malB are briefly discussed. PMID:4595640

  20. 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

  1. [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

  2. Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246

    PubMed Central

    2013-01-01

    Background Streptococcus equi ssp. zooepidemicus (S. zooepidemicus) is an important pathogen causing swine streptococcosis in China. Pathogenicity islands (PAIs) of S. zooepidemicus have been transferred among bacteria through horizontal gene transfer (HGT) and play important roles in the adaptation and increased virulence of S. zooepidemicus. The present study used comparative genomics to examine the different pathogenicities of S. zooepidemicus. Results Genome of S. zooepidemicus ATCC35246 (Sz35246) comprises 2,167,264-bp of a single circular chromosome, with a GC content of 41.65%. Comparative genome analysis of Sz35246, S. zooepidemicus MGCS10565 (Sz10565), Streptococcus equi. ssp. equi. 4047 (Se4047) and S. zooepidemicus H70 (Sz70) identified 320 Sz35246-specific genes, clustered into three toxin-antitoxin (TA) systems PAIs and one restriction modification system (RM system) PAI. These four acquired PAIs encode proteins that may contribute to the overall pathogenic capacity and fitness of this bacterium to adapt to different hosts. Analysis of the in vivo and in vitro transcriptomes of this bacterium revealed differentially expressed PAI genes and non-PAI genes, suggesting that Sz35246 possess mechanisms for infecting animals and adapting to a wide range of host environments. Analysis of the genome identified potential Sz35246 virulence genes. Genes of the Fim III operon were presumed to be involved in breaking the host-restriction of Sz35246. Conclusion Genome wide comparisons of Sz35246 with three other strains and transcriptome analysis revealed novel genes related to bacterial virulence and breaking the host-restriction. Four specific PAIs, which were judged to have been transferred into Sz35246 genome through HGT, were identified for the first time. Further analysis of the TA and RM systems in the PAIs will improve our understanding of the pathogenicity of this bacterium and could lead to the development of diagnostics and vaccines. PMID:23742619

  3. Transcriptome dynamics-based operon prediction and verification in Streptomyces coelicolor

    PubMed Central

    Charaniya, Salim; Mehra, Sarika; Lian, Wei; Jayapal, Karthik P.; Karypis, George; Hu, Wei-Shou

    2007-01-01

    Streptomyces spp. produce a variety of valuable secondary metabolites, which are regulated in a spatio-temporal manner by a complex network of inter-connected gene products. Using a compilation of genome-scale temporal transcriptome data for the model organism, Streptomyces coelicolor, under different environmental and genetic perturbations, we have developed a supervised machine-learning method for operon prediction in this microorganism. We demonstrate that, using features dependent on transcriptome dynamics and genome sequence, a support vector machines (SVM)-based classification algorithm can accurately classify >90% of gene pairs in a set of known operons. Based on model predictions for the entire genome, we verified the co-transcription of more than 250 gene pairs by RT-PCR. These results vastly increase the database of known operons in S. coelicolor and provide valuable information for exploring gene function and regulation to harness the potential of this differentiating microorganism for synthesis of natural products. PMID:17959654

  4. Escherichia coli mutant with altered respiratory control of the frd operon.

    PubMed Central

    Iuchi, S; Kuritzkes, D R; Lin, E C

    1985-01-01

    In wild-type Escherichia coli, fumarate reductase encoded by the frd operon is inducible by its substrate in the absence of molecular oxygen and nitrate. Synthesis of this enzyme under permissive conditions requires the fnr+ gene product, which is believed to be a pleiotropic regulatory protein that activates transcription. A spontaneous mutant was isolated in which the expression of the frd operon no longer depended on the presence of fumarate or the fnr+ gene product. Aerobic repression of the operon was abolished, but nitrate repression remained intact. Transductional analysis showed that the mutation was closely linked to the frd locus. The mutant phenotype strongly suggests that repression by molecular oxygen and nitrate is mediated by different mechanisms. PMID:3882660

  5. 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

  6. 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. PMID

  7. 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

  8. Characterization and Nucleotide Sequence of the Cryptic Cel Operon of Escherichia Coli K12

    PubMed Central

    Parker, L. L.; Hall, B. G.

    1990-01-01

    Wild-type Escherichia coli are not able to utilize β-glucoside sugars because the genes for utilization of these sugars are cryptic. Spontaneous mutations in the cel operon allow its expression and enable the organism to ferment cellobiose, arbutin and salicin. In this report we describe the structure and nucleotide sequence of the cel operon. The cel operon consists of five genes: celA, whose function is unknown; celB and celC which encode phosphoenolpyruvate-dependent phosphotransferase system enzyme II(cel) and enzyme III(cel), respectively, for the transport and phosphorylation of β-glucoside sugars; celD, which encodes a negative regulatory protein; and celF, which encodes a phospho-β-glucosidase that acts on phosphorylated cellobiose, arbutin and salicin. The mutationally activated cel operon is induced in the presence of its substrates, and is repressed in their absence. A comparison of proteins encoded by the cel operon with functionally equivalent proteins of the bgl operon, another cryptic E. coli gene system responsible for the catabolism of β-glucoside sugars, revealed no significant homology between these two systems despite common functional characteristics. The celD and celF encoded repressor and phospho-β-glucosidase proteins are homologous to the melibiose regulatory protein and to the melA encoded α-galactosidase of E. coli, respectively. Furthermore, the celC encoded PEP-dependent phosphotransferase system enzyme III(cel) is strikingly homologous to an enzyme III(lac) of the Gram-positive organism Staphylococcus aureus. We conclude that the genes for these two enzyme IIIs diverged much more recently than did their hosts, indicating that E. coli and S. aureus have undergone relatively recent exchange of chromosomal genes. PMID:2179047

  9. Sequencing and Characterization of the xyl Operon of a Gram-Positive Bacterium, Tetragenococcus halophila

    PubMed Central

    Takeda, Yasuo; Takase, Kazuma; Yamato, Ichiro; Abe, Keietsu

    1998-01-01

    The xyl operon of a gram-positive bacterium, Tetragenococcus halophila (previously called Pediococcus halophilus), was cloned and sequenced. The DNA was about 7.7 kb long and contained genes for a ribose binding protein and part of a ribose transporter, xylR (a putative regulatory gene), and the xyl operon, along with its regulatory region and transcription termination signal, in this order. The DNA was AT rich, the GC content being 35.8%, consistent with the GC content of this gram-positive bacterium. The xyl operon consisted of three genes, xylA, encoding a xylose isomerase, xylB, encoding a xylulose kinase, and xylE, encoding a xylose transporter, with predicted molecular weights of 49,400, 56,400, and 51,600, respectively. The deduced amino acid sequences of the XylR, XylA, XylB, and XylE proteins were similar to those of the corresponding proteins in other gram-positive and -negative bacteria, the similarities being 37 to 64%. Each polypeptide of XylB and XylE was expressed functionally in Escherichia coli. XylE transported d-xylose in a sodium ion-dependent manner, suggesting that it is the first described xylose/Na+ symporter. The XylR protein contained a consensus sequence for binding catabolites of glucose, such as glucose-6-phosphate, which has been discovered in glucose and fructose kinases in bacteria. Correspondingly, the regulatory region of this operon contained a putative binding site of XylR with a palindromic structure. Furthermore, it contained a consensus sequence, CRE (catabolite-responsive element), for binding CcpA (catabolite control protein A). We speculate that the transcriptional regulation of this operon resembles the regulation of catabolite-repressible operons such as the amy, lev, xyl, and gnt operons in various gram-positive bacteria. We discuss the significance of the regulation of gene expression of this operon in T. halophila. PMID:9647823

  10. Sequencing and characterization of the xyl operon of a gram-positive bacterium, Tetragenococcus halophila.

    PubMed

    Takeda, Y; Takase, K; Yamato, I; Abe, K

    1998-07-01

    The xyl operon of a gram-positive bacterium, Tetragenococcus halophila (previously called Pediococcus halophilus), was cloned and sequenced. The DNA was about 7.7 kb long and contained genes for a ribose binding protein and part of a ribose transporter, xylR (a putative regulatory gene), and the xyl operon, along with its regulatory region and transcription termination signal, in this order. The DNA was AT rich, the GC content being 35.8%, consistent with the GC content of this gram-positive bacterium. The xyl operon consisted of three genes, xylA, encoding a xylose isomerase, xylB, encoding a xylulose kinase, and xylE, encoding a xylose transporter, with predicted molecular weights of 49,400, 56,400, and 51,600, respectively. The deduced amino acid sequences of the XylR, XylA, XylB, and XylE proteins were similar to those of the corresponding proteins in other gram-positive and -negative bacteria, the similarities being 37 to 64%. Each polypeptide of XylB and XylE was expressed functionally in Escherichia coli. XylE transported D-xylose in a sodium ion-dependent manner, suggesting that it is the first described xylose/Na+ symporter. The XylR protein contained a consensus sequence for binding catabolites of glucose, such as glucose-6-phosphate, which has been discovered in glucose and fructose kinases in bacteria. Correspondingly, the regulatory region of this operon contained a putative binding site of XylR with a palindromic structure. Furthermore, it contained a consensus sequence, CRE (catabolite-responsive element), for binding CcpA (catabolite control protein A). We speculate that the transcriptional regulation of this operon resembles the regulation of catabolite-repressible operons such as the amy, lev, xyl, and gnt operons in various gram-positive bacteria. We discuss the significance of the regulation of gene expression of this operon in T. halophila. PMID:9647823

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

    PubMed

    Berg, C M; Shaw, K J

    1981-02-01

    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. PMID:7007356

  12. The Mangotoxin Biosynthetic Operon (mbo) Is Specifically Distributed within Pseudomonas syringae Genomospecies 1 and Was Acquired Only Once during Evolution

    PubMed Central

    Carrión, Víctor J.; Gutiérrez-Barranquero, José A.; Arrebola, Eva; Bardaji, Leire; Codina, Juan C.; de Vicente, Antonio

    2013-01-01

    Mangotoxin production was first described in Pseudomonas syringae pv. syringae strains. A phenotypic characterization of 94 P. syringae strains was carried out to determine the genetic evolution of the mangotoxin biosynthetic operon (mbo). We designed a PCR primer pair specific for the mbo operon to examine its distribution within the P. syringae complex. These primers amplified a 692-bp DNA fragment from 52 mangotoxin-producing strains and from 7 non-mangotoxin-producing strains that harbor the mbo operon, whereas 35 non-mangotoxin-producing strains did not yield any amplification. This, together with the analysis of draft genomes, allowed the identification of the mbo operon in five pathovars (pathovars aptata, avellanae, japonica, pisi, and syringae), all of which belong to genomospecies 1, suggesting a limited distribution of the mbo genes in the P. syringae complex. Phylogenetic analyses using partial sequences from housekeeping genes differentiated three groups within genomospecies 1. All of the strains containing the mbo operon clustered in groups I and II, whereas those lacking the operon clustered in group III; however, the relative branching order of these three groups is dependent on the genes used to construct the phylogeny. The mbo operon maintains synteny and is inserted in the same genomic location, with high sequence conservation around the insertion point, for all the strains in groups I and II. These data support the idea that the mbo operon was acquired horizontally and only once by the ancestor of groups I and II from genomospecies 1 within the P. syringae complex. PMID:23144138

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. Genetic organization of the Pantoea stewartii subsp. stewartii hrp gene cluster and sequence analysis of the hrpA, hrpC, hrpN, and wtsE operons.

    PubMed

    Frederick, R D; Ahmad, M; Majerczak, D R; Arroyo-Rodríguez, A S; Manulis, S; Coplin, D L

    2001-10-01

    The hrp/wts gene cluster of Pantoea stewartii subsp. stewartii is required for pathogenicity on sweet corn and the ability to elicit a hypersensitive response (HR) in tobacco. Site-directed transposon mutagenesis and nucleotide sequencing were used to identify hrp/wts genes within the left 20 kb of this cluster. Seventeen open reading frames (ORFs) comprise seven genetic complementation groups. These ORFs share homology with hrp and dsp genes from Erwinia amylovora, Erwinia chrysanthemi, and Pseudomonas syringae pathovars and have been designated, in map order, wtsF, wtsE, hrpN, hrpV, hrpT, hrcC, hrpG, hrpF, hrpE, hrpD, hrcJ, hrpB, hrpA, hrpS, hrpY, hrpX, and hrpL. Putative hrp consensus promoter sequences were identified upstream of hrpA, hrpF, hrpN, and wtsE. Expression of the hrpA, hrpC, and wtsE operons was regulated by HrpS. Transposon mutations in all of the hrp operons abolished pathogenicity and HR elicitation, except for the hrpN and hrpV mutants, which were still pathogenic. hrpS, hrpXY, and hrpL regulatory mutations abolished HrpN synthesis, whereas secretory mutations in the hrpC, hrpA, and hrpJ operons permitted intracellular HrpN synthesis. wtsEF mutants were not pathogenic but still produced HrpN and elicited the HR. wtsE encodes a 201-kDa protein that is similar to DspE in E. amylovora and AvrE in P. syringae pv. tomato, suggesting that this protein is a major virulence factor involved in the elicitation of water-soaked lesions. PMID:11605961

  19. Genomic islands of uropathogenic Escherichia coli contribute to virulence.

    PubMed

    Lloyd, Amanda L; Henderson, Tiffany A; Vigil, Patrick D; Mobley, Harry L T

    2009-06-01

    Uropathogenic Escherichia coli (UPEC) strain CFT073 contains 13 large genomic islands ranging in size from 32 kb to 123 kb. Eleven of these genomic islands were individually deleted from the genome, and nine isogenic mutants were tested for their ability to colonize the CBA/J mouse model of ascending urinary tract infection. Three genomic island mutants (Delta PAI-aspV, Delta PAI-metV, and Delta PAI-asnT) were significantly outcompeted by wild-type CFT073 in the bladders and/or kidneys following transurethral cochallenge (P operon (Deltac0294-97 Delta c2518-15) was significantly outcompeted by wild-type CFT073 in cochallenge. Strains with mutations in a type VI secretion system within the PAI-metV island did not show attenuation. The attenuation of the PAI-metV island was localized to genes c3405-10, encoding a putative phosphotransferase transport system, which is common to UPEC and avian pathogenic E. coli strains but absent from E. coli K-12. We have shown that, in addition to encoding virulence genes, genomic islands contribute to the overall fitness of UPEC strain CFT073 in vivo. PMID:19329634

  20. The Abi-domain protein Abx1 interacts with the CovS histidine kinase to control virulence gene expression in group B Streptococcus.

    PubMed

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

    2013-02-01

    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

  1. 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

  2. Dissecting the Multiple Roles of PqsE in Pseudomonas aeruginosa Virulence by Discovery of Small Tool Compounds.

    PubMed

    Zender, Michael; Witzgall, Florian; Drees, Steffen L; Weidel, Elisabeth; Maurer, Christine K; Fetzner, Susanne; Blankenfeldt, Wulf; Empting, Martin; Hartmann, Rolf W

    2016-06-17

    Pseudomonas aeruginosa uses quorum sensing (QS) as a cell-to-cell communication system to orchestrate the expression of virulence determinants. The biosynthesis of the important Pseudomonas quinolone signal (PQS) requires the pqsABCDE operon. Here, PqsE acts as a pathway-specific thioesterase, but it also contributes to the regulation of bacterial virulence via an unknown mechanism. In this manuscript, we report the discovery of PqsE inhibitors as tool compounds to gain further insights into its different functions. Differential scanning fluorimetry (DSF) was used to screen a fragment library, and isothermal titration calorimetry (ITC) was employed as a secondary filter. As proven by X-ray crystallography, hit molecules bound to the active center inhibiting PqsE's thioesterase activity in cell-based and in vitro assays. Notably, the ligands did not affect the levels of the PqsE-regulated virulence factor pyocyanin. These findings indicate that the regulatory function of PqsE is not linked to its thioesterase activity and must be encoded outside of the active center. This study highlights the potential of fragment-based screening for the discovery of tool compounds. This approach provided novel insight into complex biological systems, which could not be obtained by knockout studies. PMID:27082157

  3. Cloning and characterization of two groESL operons of Rhodobacter sphaeroides: transcriptional regulation of the heat-induced groESL operon.

    PubMed Central

    Lee, W T; Terlesky, K C; Tabita, F R

    1997-01-01

    The nonsulfur purple bacterium Rhodobacter sphaeroides was found to contain two groESL operons. The groESL1 heat shock operon was cloned from a genomic library, and a 2.8-kb DNA fragment was sequenced and found to contain the groES and groEL genes. The deduced amino acid sequences of GroEL1 (cpn60) and GroES1 (cpn10) were in agreement with N-terminal sequences previously obtained for the isolated proteins (K. C. Terlesky and F. R. Tabita, Biochemistry 30:8181-8186, 1991). These sequences show a high degree of similarity to groESL genes isolated from other bacteria. Northern analysis indicated that the groESL1 genes were expressed as part of a 2.2-kb polycistronic transcript that is induced 13-fold after heat shock. Transcript size was not affected by heat shock; however, the amount of transcript was induced to its greatest extent 15 to 30 min after a 40 degrees C heat shock, from an initial temperature of 28 degrees C, and remained elevated up to 120 min. The R. sphaeroides groESL1 operon contains a putative hairpin loop at the start of the transcript that is present in other bacterial heat shock genes. Primer extension of the message showed that the transcription start site is at the start of this conserved hairpin loop. In this region were also found putative -35 and -10 sequences that are conserved upstream from other bacterial heat shock genes. Transcription of the groESL1 genes was unexpectedly low under photoautotrophic growth conditions. Thus far, it has not been possible to construct a groESL1 deletion strain, perhaps indicating that these genes are essential for growth. A second operon (groESL2) was also cloned from R. sphaeroides, using a groEL1 gene fragment as a probe; however, no transcript was observed for this operon under several different growth conditions. A groESL2 deletion strain was constructed, but there was no detectable change in the phenotype of this strain compared to the parental strain. PMID:8990302

  4. Blastocystis: Taxonomy, biology and virulence

    PubMed Central

    Parija, Subhash Chandra; Jeremiah, SS

    2013-01-01

    The unicellular protist Blastocystis has long been an unsolved puzzle for taxonomists, microbiologists and clinicians. Over the years, the organism has been bounced on and off the different branches of the tree of life due the possession of unique phenotypic characters intermediary to different organisms. The organism is polymorphic with only few of forms such as vacuolar, granular, amoeboid, and the cyst form being commonly known. However it could exist in other forms much more frequently than the widely known forms which could be missed by the unaware observer. Certain older concepts in the life cycle of Blastocystis although has been proven wrong are still being followed in various textbooks and other trustworthy internet sources. The causal role of Blastocystis in human disease has long been a subject of controversy. It is widely believed that certain subtypes of the organism are virulent. But this is not so as other factors are also involved in the clinical outcome of the infection. In these contexts, this review intends to shed light on the past misconceptions and the recent findings on the taxonomy, biology and the virulence of this organism. PMID:23961437

  5. 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 ...

  6. 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.

  7. Analysis of a ribosomal RNA operon (rrn) from “Candiatus Liberibacter asiaticus”

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 5,005 bp DNA sequence containing a nearly complete rrn operon of “Candidatus Liberibacter asiaticus”, a bacterium associated with citrus Huanglongbing (yellow shoot disease), was obtained by PCR using sequences conserved for Rhizobiaceae in the alpha-proteobacteria as primers. The rrn locus consis...

  8. Identification and characterization of an iron ABC transporter operon in Gluconacetobacter diazotrophicus Pal 5.

    PubMed

    Urzúa, Lucia Soto; Vázquez-Candanedo, Ada P; Sánchez-Espíndola, Adriana; Ramírez, Carlos Ávila; Baca, Beatriz E

    2013-06-01

    Gluconacetobacter diazotrophicus is a nitrogen-fixing bacterium and endophyte of sugarcane. We have cloned and sequenced the genes coding for the components of the iron ABC-type acquisition system of G. diazotrophicus. Sequence analysis revealed three ORFs, (feuA, feuB, and feuC) organized as an operon and encoding polypeptides of 346 (38 kDa), 342 (34.2 kDa), and 240 (26 kDa) amino acids, respectively. The deduced translation products of the feu operon showed similarity with a periplasmic solute-binding protein (FeuA), permease (FeuB), and ATPase (FeuC) involved in Fe transport. The role of FeuB in the survival of G. diazotrophicus under iron depletion was evaluated by comparing the ability of wild-type and FeuB-Km(R) -mutant strains in a medium without iron supplementation and in a medium containing 2, 2'-dipyridyl (DP). Growth of the mutant was affected in the medium containing DP. The operon was expressed at higher levels in cells depleted for iron than in those that contained the metal. A decrease in nitrogenase activity was observed with the FeuB-Km(R) -mutant strain that with the wild-type under iron deficiency conditions, suggesting that the Feu operon play role in Fe nutrition of G. diazotrophicus. PMID:23624722

  9. 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

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

    PubMed

    Yu, Xuefei; Zheng, Wei; Bhat, Somanath; Aquilina, J Andrew; Zhang, Ren

    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

  11. In Vitro Activation of the Transcription of araBAD Operon by araC Activator

    PubMed Central

    Lee, Nancy; Wilcox, Gary; Gielow, William; Arnold, John; Cleary, Paul; Englesberg, Ellis

    1974-01-01

    The transcription of araBAD operon requires araC activator and cyclic AMP. D-Fucose inhibits ara mRNA synthesis. Our results indicate that the positive control by araC activator is exerted at the level of transcription. PMID:4362626

  12. 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

  13. 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

  14. 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

  15. 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

  16. 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

  17. 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

  18. 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

  19. clpC operon regulates cell architecture and sporulation in Bacillus anthracis

    PubMed Central

    Singh, Lalit K.; Dhasmana, Neha; Sajid, Andaleeb; Kumar, Prasun; Bhaduri, Asani; Bharadwaj, Mitasha; Gandotra, Sheetal; Kalia, Vipin C.; Das, Taposh K.; Goel, Ajay K.; Pomerantsev, Andrei P.; Misra, Richa; Gerth, Ulf; Leppla, Stephen H.; Singh, Yogendra

    2014-01-01

    The clpC operon is known to regulate several processes such as genetic competence, protein degradation and stress survival in bacteria. Here, we describe the role of clpC operon in Bacillus anthracis. We generated knock-out strains of the clpC operon genes to investigate the impact of CtsR, McsA, McsB and ClpC deletion on essential processes of B. anthracis. We observed that growth, cell division, sporulation and germination were severely affected in mcsB and clpC deleted strains, while none of deletions affected toxin secretion. Growth defect in these strains was pronounced at elevated temperature. The growth pattern gets restored on complementation of mcsB and clpC in respective mutants. Electron microscopic examination revealed that mcsB and clpC deletion also causes defect in septum formation leading to cell elongation. These vegetative cell deformities were accompanied by inability of mutant strains to generate morphologically intact spores. Higher levels of polyhydroxybutyrate granules accumulation were also observed in these deletion strains, indicating a defect in sporulation process. Our results demonstrate, for the first time, the vital role played by McsB and ClpC in physiology of B. anthracis and opens up further interest on this operon, which might be of importance to success of B. anthracis as pathogen PMID:24947607

  20. 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

  1. Mechanisms of Activation of the Cryptic Cel Operon of Escherichia Coli K12

    PubMed Central

    Parker, L. L.; Hall, B. G.

    1990-01-01

    The cel (cellobiose utilization) operon of Escherichia coli K12 is not expressed in the wild-type organism. However, mutants that can express the operon and thereby utilize the β-glucoside sugars cellobiose, arbutin and salicin are easily isolated. Two kinds of mutations are capable of activating the operon. The first involves mutations that allow the repressor to recognize the substrates cellobiose, arbutin and salicin as inducers. We have identified the sequence changes in five different active alleles and found those differences to be single base pair changes at one of two lysine codons in the repressor gene. The second kind of mutation involves the integration of the insertion sequences IS1, IS2 or IS5 into a 108-bp region 72-180 bp upstream of the start of transcription. Integration occurs at several different sites and in different orientations. Transcription of the cel operon begins at the same base pair in all mutants examined. Of 44 independent cel(+) mutants, 27 were activated by point mutations and 17 were activated by insertion sequences. The preferred mechanism of activation appears to be strain dependent, since one of the parents yielded 94% insertionally activated alleles, while another yielded 100% point mutation activated alleles. PMID:2179048

  2. msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus

    PubMed Central

    Sahukhal, Gyan S.; Batte, Justin L.; Elasri, Mohamed O.

    2015-01-01

    Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm. PMID:25724778

  3. 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...

  4. Using the TxtAB Operon to Quantify Pathogenic Streptomyces in Potato Tubers and Soil

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The phytotoxin thaxtomin, produced by plant pathogenic Streptomyces species, is a pathogenicity determinant for common scab. In this study a SYBR Green quantitative real-time PCR assay using primers targeted on the txtAB operon of Streptomyces was developed to quantify pathogenic bacterial populati...

  5. The PQQ biosynthetic operons and their transcriptional regulation in Pseudomonas aeruginosa.

    PubMed

    Gliese, Nicole; Khodaverdi, Viola; Görisch, Helmut

    2010-01-01

    Gene PA1990 of Pseudomonas aeruginosa, located downstream of pqqE and encoding a putative peptidase, was shown to be involved in excretion of PQQ into the culture supernatant. This gene is cotranscribed with the pqqABCDE cluster and was named pqqH. A PA1990::Km(r) mutant (VK3) did not show any effect in growth behaviour; however, in contrast to the wild-type, no excretion of PQQ into the culture supernatant was observed. The putative pqqF gene of P. aeruginosa was shown to be essential for PQQ biosynthesis. A pqqF::Km(r) mutant did not grow aerobically on ethanol, because of its inability to produce PQQ. Transcription of the pqqABCDEH operon was induced upon aerobic growth on ethanol, 1-propanol, 1,2-propanediol and 1-butanol, while on glycerol, succinate and acetate, transcription was low. Transcription of the pqqABCDEH operon was also found upon anoxic growth on ethanol with nitrate as electron acceptor, but no PQQ was produced. Expression of the pqqABCDEH operon is regulated at the transcriptional level. In contrast, the pqqF operon appeared to be transcribed constitutively at a very low level under all growth conditions studied. PMID:19902179

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

    PubMed

    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

  7. 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.

  8. 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

  9. Subtle genetic changes enhance virulence of methicillin resistant and sensitive Staphylococcus aureus

    PubMed Central

    Highlander, Sarah K; Hultén, Kristina G; Qin, Xiang; Jiang, Huaiyang; Yerrapragada, Shailaja; Mason, Edward O; Shang, Yue; Williams, Tiffany M; Fortunov, Régine M; Liu, Yamei; Igboeli, Okezie; Petrosino, Joseph; Tirumalai, Madhan; Uzman, Akif; Fox, George E; Cardenas, Ana Maria; Muzny, Donna M; Hemphill, Lisa; Ding, Yan; Dugan, Shannon; Blyth, Peter R; Buhay, Christian J; Dinh, Huyen H; Hawes, Alicia C; Holder, Michael; Kovar, Christie L; Lee, Sandra L; Liu, Wen; Nazareth, Lynne V; Wang, Qiaoyan; Zhou, Jianling; Kaplan, Sheldon L; Weinstock, George M

    2007-01-01

    Background Community acquired (CA) methicillin-resistant Staphylococcus aureus (MRSA) increasingly causes disease worldwide. USA300 has emerged as the predominant clone causing superficial and invasive infections in children and adults in the USA. Epidemiological studies suggest that USA300 is more virulent than other CA-MRSA. The genetic determinants that render virulence and dominance to USA300 remain unclear. Results We sequenced the genomes of two pediatric USA300 isolates: one CA-MRSA and one CA-methicillin susceptible (MSSA), isolated at Texas Children's Hospital in Houston. DNA sequencing was performed by Sanger dideoxy whole genome shotgun (WGS) and 454 Life Sciences pyrosequencing strategies. The sequence of the USA300 MRSA strain was rigorously annotated. In USA300-MRSA 2658 chromosomal open reading frames were predicted and 3.1 and 27 kilobase (kb) plasmids were identified. USA300-MSSA contained a 20 kb plasmid with some homology to the 27 kb plasmid found in USA300-MRSA. Two regions found in US300-MRSA were absent in USA300-MSSA. One of these carried the arginine deiminase operon that appears to have been acquired from S. epidermidis. The USA300 sequence was aligned with other sequenced S. aureus genomes and regions unique to USA300 MRSA were identified. Conclusion USA300-MRSA is highly similar to other MRSA strains based on whole genome alignments and gene content, indicating that the differences in pathogenesis are due to subtle changes rather than to large-scale acquisition of virulence factor genes. The USA300 Houston isolate differs from another sequenced USA300 strain isolate, derived from a patient in San Francisco, in plasmid content and a number of sequence polymorphisms. Such differences will provide new insights into the evolution of pathogens. PMID:17986343

  10. 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