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Sample records for iglabcd virulence operon

  1. Identification of an operon, Pil-Chp, that controls twitching motility and virulence in Xylella fastidiosa.

    PubMed

    Cursino, Luciana; Galvani, Cheryl D; Athinuwat, Dusit; Zaini, Paulo A; Li, Yaxin; De La Fuente, Leonardo; Hoch, Harvey C; Burr, Thomas J; Mowery, Patricia

    2011-10-01

    Xylella fastidiosa is an important phytopathogenic bacterium that causes many serious plant diseases, including Pierce's disease of grapevines. Disease manifestation by X. fastidiosa is associated with the expression of several factors, including the type IV pili that are required for twitching motility. We provide evidence that an operon, named Pil-Chp, with genes homologous to those found in chemotaxis systems, regulates twitching motility. Transposon insertion into the pilL gene of the operon resulted in loss of twitching motility (pilL is homologous to cheA genes encoding kinases). The X. fastidiosa mutant maintained the type IV pili, indicating that the disrupted pilL or downstream operon genes are involved in pili function, and not biogenesis. The mutated X. fastidiosa produced less biofilm than wild-type cells, indicating that the operon contributes to biofilm formation. Finally, in planta the mutant produced delayed and less severe disease, indicating that the Pil-Chp operon contributes to the virulence of X. fastidiosa, presumably through its role in twitching motility.

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

    PubMed Central

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

    2014-01-01

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

  4. Characterization of the Operon Encoding the Alternative ςB Factor from Bacillus anthracis and Its Role in Virulence

    PubMed Central

    Fouet, Agnès; Namy, Olivier; Lambert, Guillaume

    2000-01-01

    The operon encoding the general stress transcription factor ςB and two proteins of its regulatory network, RsbV and RsbW, was cloned from the gram-positive bacterium Bacillus anthracis by PCR amplification of chromosomal DNA with degenerate primers, by inverse PCR, and by direct cloning. The gene cluster was very similar to the Bacillus subtilis sigB operon both in the primary sequences of the gene products and in the order of its three genes. However, the deduced products of sequences upstream and downstream from this operon showed no similarity to other proteins encoded by the B. subtilis sigB operon. Therefore, the B. anthracis sigB operon contains three genes rather than eight as in B. subtilis. The B. anthracis operon is preceded by a ςB-like promoter sequence, the expression of which depends on an intact ςB transcription factor in B. subtilis. It is followed by another open reading frame that is also preceded by a promoter sequence similarly dependent on B. subtilis ςB. We found that in B. anthracis, both these promoters were induced during the stationary phase and induction required an intact sigB gene. The sigB operon was induced by heat shock. Mutants from which sigB was deleted were constructed in a toxinogenic and a plasmidless strain. These mutants differed from the parental strains in terms of morphology. The toxinogenic sigB mutant strain was also less virulent than the parental strain in the mouse model. B. anthracis ςB may therefore be a minor virulence factor. PMID:10960085

  5. 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

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

    PubMed

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

    2014-01-01

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

  7. The conserved nhaAR operon is drastically divergent between B2 and non-B2 Escherichia coli and is involved in extra-intestinal virulence.

    PubMed

    Lescat, Mathilde; Reibel, Florence; Pintard, Coralie; Dion, Sara; Glodt, Jérémy; Gateau, Cecile; Launay, Adrien; Ledda, Alice; Cruveiller, Stephane; Cruvellier, Stephane; Tourret, Jérôme; Tenaillon, Olivier

    2014-01-01

    The Escherichia coli species is divided in phylogenetic groups that differ in their virulence and commensal distribution. Strains belonging to the B2 group are involved in extra-intestinal pathologies but also appear to be more prevalent as commensals among human occidental populations. To investigate the genetic specificities of B2 sub-group, we used 128 sequenced genomes and identified genes of the core genome that showed marked difference between B2 and non-B2 genomes. We focused on the gene and its surrounding region with the strongest divergence between B2 and non-B2, the antiporter gene nhaA. This gene is part of the nhaAR operon, which is in the core genome but flanked by mobile regions, and is involved in growth at high pH and high sodium concentrations. Consistently, we found that a panel of non-B2 strains grew faster than B2 at high pH and high sodium concentrations. However, we could not identify differences in expression of the nhaAR operon using fluorescence reporter plasmids. Furthermore, the operon deletion had no differential impact between B2 and non-B2 strains, and did not result in a fitness modification in a murine model of gut colonization. Nevertheless, sequence analysis and experiments in a murine model of septicemia revealed that recombination in nhaA among B2 strains was observed in strains with low virulence. Finally, nhaA and nhaAR operon deletions drastically decreased virulence in one B2 strain. This effect of nhaAR deletion appeared to be stronger than deletion of all pathogenicity islands. Thus, a population genetic approach allowed us to identify an operon in the core genome without strong effect in commensalism but with an important role in extra-intestinal virulence, a landmark of the B2 strains.

  8. 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.

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

    PubMed Central

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

    2014-01-01

    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. LD50 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. The Haemophilus ducreyi Fis protein is involved in controlling expression of the lspB-lspA2 operon and other virulence factors.

    PubMed

    Labandeira-Rey, Maria; Dodd, Dana A; Brautigam, Chad A; Fortney, Kate R; Spinola, Stanley M; Hansen, Eric J

    2013-11-01

    Expression of the lspB-lspA2 operon encoding a virulence-related two-partner secretion system in Haemophilus ducreyi 35000HP is directly regulated by the CpxRA regulatory system (M. Labandeira-Rey, J. R. Mock, and E. J. Hansen, Infect. Immun. 77:3402-3411, 2009). In the present study, we show that this secretion system is also regulated by the small nucleoid-associated protein Fis. Inactivation of the H. ducreyi fis gene resulted in a reduction in expression of both the H. ducreyi LspB and LspA2 proteins. DNA microarray experiments showed that a H. ducreyi fis deletion mutant exhibited altered expression levels of genes encoding other important H. ducreyi virulence factors, including DsrA and Flp1, suggesting a possible global role for Fis in the control of virulence in this obligate human pathogen. While the H. ducreyi Fis protein has a high degree of sequence and structural similarity to the Fis proteins of other bacteria, its temporal pattern of expression was very different from that of enterobacterial Fis proteins. The use of a lacZ-based transcriptional reporter provided evidence which indicated that the H. ducreyi Fis homolog is a positive regulator of gyrB, a gene that is negatively regulated by Fis in enteric bacteria. Taken together, the Fis protein expression data and the observed regulatory effects of Fis in H. ducreyi suggest that this small DNA binding protein has a regulatory role in H. ducreyi which may differ in substantial ways from that of other Fis proteins.

  13. 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.

  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 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

  15. 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.

  16. MprA and DosR Coregulate a Mycobacterium tuberculosis Virulence Operon Encoding Rv1813c and Rv1812c

    PubMed Central

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

    2012-01-01

    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

  17. The flagellar master operon flhDC is a pleiotropic regulator involved in motility and virulence of the fish pathogen Yersinia ruckeri

    USDA-ARS?s Scientific Manuscript database

    Aims: To investigate the function of the master flagellar operon flhDC in the fish pathogen Yersinia ruckeri and compare the effect of flhD mutation to a naturally occurring mutation causing loss-of-motility in emergent biotype 2 (BT2) strains. Methods and Results: In this study isogenic Y. ruckeri ...

  18. 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.

  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 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

  20. The role of the phoPQ operon in the pathogenesis of the fully virulent CO92 strain of Yersinia pestis and the IP32953 strain of Yersinia pseudotuberculosis.

    PubMed

    Bozue, Joel; Mou, Sherry; Moody, Krishna L; Cote, Christopher K; Trevino, Sylvia; Fritz, David; Worsham, Patricia

    2011-06-01

    At the genomic level, Yersinia pestis and Yersinia pseudotuberculosis are nearly identical but cause very different diseases. Y. pestis is the etiologic agent of plague; whereas Y. pseudotuberculosis causes a gastrointestinal infection primarily after the consumption of contaminated food. In many gram-negative pathogenic bacteria, PhoP is part of a two-component global regulatory system in which PhoQ serves as the sensor kinase, and PhoP is the response regulator. PhoP is known to activate a number of genes in many bacteria related to virulence. To determine the role of the PhoPQ proteins in Yersinia infections, primarily using aerosol challenge models, the phoP gene was deleted from the chromosome of the CO92 strain of Y. pestis and the IP32953 strain of Y. pseudotuberculosis, leading to a polar mutation of the phoPQ operon. We demonstrated that loss of phoPQ from both strains leads to a defect in intracellular growth and/or survival within macrophages. These in vitro data would suggest that the phoPQ mutants would be attenuated in vivo. However, the LD(50) for the Y. pestis mutant did not differ from the calculated LD(50) for the wild-type CO92 strain for either the bubonic or pneumonic murine models of infection. In contrast, mice challenged by aerosol with the Y. pseudotuberculosis mutant had a LD(50) value 40× higher than the wild-type strain. These results demonstrate that phoPQ are necessary for full virulence by aerosol infection with the IP32953 strain of Y. pseudotuberculosis. However, the PhoPQ proteins do not play a significant role in infection with a fully virulent strain of Y. pestis. Published by Elsevier India Pvt Ltd.

  1. Characterization of the ebpfm pilus-encoding operon of Enterococcus faecium and its role in biofilm formation and virulence in a murine model of urinary tract infection

    PubMed Central

    Sillanpää, Jouko; Nallapareddy, Sreedhar R.; Singh, Kavindra V.; Prakash, Vittal P.; Fothergill, Timothy; Ton-That, Hung; Murray, Barbara E.

    2010-01-01

    We recently identified 15 genes encoding putative surface proteins with features of MSCRAMMs and/or pili in the Enterococcus faecium TX16 (DO) genome, including four predicted pilus-encoding gene clusters; we also demonstrated that one of these, ebpABCfm, is transcribed as an operon, that its putative major pilus subunit, EbpCfm (also called PilB), is polymerized into high molecular weight complexes, and that it is enriched among clinical E. faecium isolates. Here, we created a deletion of the ebpABCfm operon in an endocarditis-derived E. faecium strain (TX82) and showed, by a combination of whole-cell ELISA, flow cytometry, immunoblot and immunogold electron microscopy, that this deletion abolished EbpCfm expression and eliminated EbpCfm-containing pili from the cell surface. However, transcription of the downstream sortase, bpsfm, was not affected. Importantly, the ebpABCfm deletion resulted in significantly reduced biofilm formation (p < 0.0001) and initial adherence (p < 0.0001) versus the wild-type; both were restored by complementing ebpABCfm in trans, which also restored cell surface expression of EbpCfm and pilus production. Furthermore, the deletion mutant was significantly attenuated in two independent mixed infection mouse urinary tract experiments, i.e., outnumbered by the wild-type in kidneys (p = 0.0003 and < 0.0001, respectively) and urinary bladders (p = 0.0003 and = 0.002). In conclusion, we have shown that the ebpABCfm locus encodes pili on the E. faecium TX82 cell surface and provide the first evidence that pili of this emerging pathogen are important for its ability to form biofilm and to cause infection in an ascending UTI model. PMID:20676385

  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

    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.

  3. The Salmonella Spi1 virulence regulatory protein HilD directly activates transcription of the flagellar master operon flhDC.

    PubMed

    Singer, Hanna M; Kühne, Caroline; Deditius, Julia A; Hughes, Kelly T; Erhardt, Marc

    2014-04-01

    Infection of intestinal epithelial cells is dependent on the Salmonella enterica serovar Typhimurium pathogenicity island 1 (Spi1)-encoded type III injectisome system and flagellar motility. Thus, the expression of virulence and flagellar genes is subject to tight regulatory control mechanisms in order to ensure the correct spatiotemporal production of the respective gene products. In this work, we reveal a new level of cross-regulation between the Spi1 and flagellar regulatory systems. Transposon mutagenesis identified a class of mutants that prevented flhDC autorepression by overexpressing HilD. HilD, HilC, RtsA, and HilA comprise a positive regulatory circuit for the expression of the Spi1 genes. Here, we report a novel transcriptional cross talk between the Spi1 and flagellar regulons where HilD transcriptionally activates flhDC gene expression by binding to nucleotides -68 to -24 upstream from the P5 transcriptional start site. We additionally show that, in contrast to the results of a previous report, HilA does not affect flagellar gene expression. Finally, we discuss a model of the cross-regulation network between Spi1 and the flagellar system and propose a regulatory mechanism via the Spi1 master regulator HilD that would prime flagellar genes for rapid reactivation during host infection.

  4. Vulnerabilities in Yersinia pestis caf operon are unveiled by a Salmonella vector.

    PubMed

    Cao, Ling; Lim, Timothy; Jun, SangMu; Thornburg, Theresa; Avci, Recep; Yang, Xinghong

    2012-01-01

    During infection, Yersinia pestis uses its F1 capsule to enhance survival and cause virulence to mammalian host. Since F1 is produced in large quantities and secreted into the host tissues, it also serves as a major immune target. To hold this detrimental effect under proper control, Y. pestis expresses the caf operon (encoding the F1 capsule) in a temperature-dependent manner. However, additional properties of the caf operon limit its expression. By overexpressing the caf operon in wild-type Salmonella enterica serovar Typhimurium under a potent promoter, virulence of Salmonella was greatly attenuated both in vitro and in vivo. In contrast, expression of the caf operon under the regulation of its native promoter exhibited negligible impairment of Salmonellae virulence. In-depth investigation revealed all individual genes in the caf operon attenuated Salmonella when overexpressed. The deleterious effects of caf operon and the caf individual genes were further confirmed when they were overexpressed in Y. pestis KIM6+. This study suggests that by using a weak inducible promoter, the detrimental effects of the caf operon are minimally manifested in Y. pestis. Thus, through tight regulation of the caf operon, Y. pestis precisely balances its capsular anti-phagocytic properties with the detrimental effects of caf during interaction with mammalian host.

  5. A putative colR(XC1049)-colS(XC1050) two-component signal transduction system in Xanthomonas campestris positively regulates hrpC and hrpE operons and is involved in virulence, the hypersensitive response and tolerance to various stresses.

    PubMed

    Zhang, Sui-Sheng; He, Yong-Qiang; Xu, Li-Ming; Chen, Bo-Wen; Jiang, Bo-Le; Liao, Jie; Cao, Jin-Rui; Liu, Dan; Huang, Yan-Qiang; Liang, Xiao-Xia; Tang, Dong-Jie; Lu, Guang-Tao; Tang, Ji-Liang

    2008-01-01

    The ColR-ColS two-component signal transduction system was originally characterized as a regulatory system involved in the capacity of root-colonizing biocontrol bacterium Pseudomonas fluorescens to colonize plant roots. There are three pairs of putative colR-colS two-component regulatory systems annotated in the phytopathogen Xanthomonas campestris pathovar campestris. Mutational studies revealed that one of them, named colR(XC1049) and colS(XC1050), is a global regulatory system involved in various cellular processes, including virulence, hypersensitive response and stress tolerance. Growth rate determination showed that, although the colR(XC1049) and colS(XC1050) mutants are not auxotrophic, colR(XC1049) and colS(XC1050) are required for the pathogen to proliferate well in standard media and host plants. Assays of beta-glucuronidase activities of plasmid-driven promoter-gusA reporters and/or semi-quantitative RT-PCR demonstrated that colR(XC1049) and colS(XC1050) positively regulate expression of hrpC and hrpE operons, and that expression of colR(XC1049) and colS(XC1050) is not controlled by key hrp regulators HrpG and HrpX.

  6. The mbo operon is specific and essential for biosynthesis of mangotoxin in Pseudomonas syringae.

    PubMed

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

    2012-01-01

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

  7. Operon prediction in Pyrococcus furiosus

    PubMed Central

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

    2007-01-01

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

  8. The Life-cycle of Operons

    SciTech Connect

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

    2005-11-18

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

  9. Horizontally acquired glycosyltransferase operons drive salmonellae lipopolysaccharide diversity.

    PubMed

    Davies, Mark R; Broadbent, Sarah E; Harris, Simon R; Thomson, Nicholas R; van der Woude, Marjan W

    2013-06-01

    The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions.

  10. Computational analysis of Ciona intestinalis operons.

    PubMed

    Zeller, Robert W

    2010-07-01

    Operons are clusters of genes that are co-regulated from a common promoter. Operons are typically associated with prokaryotes, although a small number of eukaryotes have been shown to possess them. Among metazoans, operons have been extensively characterized in the nematode Caenorhabditis elegans in which ∼15% of the total genes are organized into operons. The most recent genome assembly for the ascidian Ciona intestinalis placed ∼20% of the genes (2909 total) into 1310 operons. The majority of these operons are composed of two genes, while the largest are composed of six. Here is reported a computational analysis of the genes that comprise the Ciona operons. Gene ontology (GO) terms were identified for about two-thirds of the operon-encoded genes. Using the extensive collection of public EST libraries, estimates of temporal patterns of gene expression were generated for the operon-encoded genes. Lastly, conservation of operons was analyzed by determining how many operon-encoded genes were present in the ascidian Ciona savignyi and whether these genes were organized in orthologous operons. Over 68% of the operon-encoded genes could be assigned one or more GO terms and 697 of the 1310 operons contained genes in which all genes had at least one GO term. Of these 697 operons, GO terms were shared by all of the genes within 146 individual operons, suggesting that most operons encode genes with unrelated functions. An analysis of operon gene expression from nine different EST libraries indicated that for 587 operons, all of the genes that comprise an individual operon were expressed together in at least one EST library, suggesting that these genes may be co-regulated. About 50% (74/146) of the operons with shared GO terms also showed evidence of gene co-regulation. Comparisons with the C. savignyi genome identified orthologs for 1907 of 2909 operon genes. About 38% (504/1310) of the operons are conserved between the two Ciona species. These results suggest that like

  11. Transcription termination Within the iron transport-biosynthesis operon of Vibrio anguillarum requires an antisense RNA

    USDA-ARS?s Scientific Manuscript database

    The iron transport-biosynthesis (ITB) operon in Vibrio anguillarum includes four genes for ferric-siderophore transport, fatD,C,B,A, and two genes for siderophorebiosynthesis, angR and angT and plays an important role in the virulence mechanism of this bacterium. Despite being part of the same polyc...

  12. Evolution of the capsular operon of Streptococcus iniae in response to vaccination.

    PubMed

    Millard, Candice M; Baiano, Justice C F; Chan, Candy; Yuen, Benedict; Aviles, Fabian; Landos, Matt; Chong, Roger S M; Benedict, Suresh; Barnes, Andrew C

    2012-12-01

    Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins.

  13. Evolution of the Capsular Operon of Streptococcus iniae in Response to Vaccination

    PubMed Central

    Millard, Candice M.; Baiano, Justice C. F.; Chan, Candy; Yuen, Benedict; Aviles, Fabian; Landos, Matt; Chong, Roger S. M.; Benedict, Suresh

    2012-01-01

    Streptococcus iniae causes severe septicemia and meningitis in farmed fish and is also occasionally zoonotic. Vaccination against S. iniae is problematic, with frequent breakdown of protection in vaccinated fish. The major protective antigens in S. iniae are the polysaccharides of the capsule, which are essential for virulence. Capsular biosynthesis is driven and regulated by a 21-kb operon comprising up to 20 genes. In a long-term study, we have sequenced the capsular operon of strains that have been used in autogenous vaccines across Australia and compared it with the capsular operon sequences of strains subsequently isolated from infected vaccinated fish. Intriguingly, strains isolated from vaccinated fish that subsequently become infected have coding mutations that are confined to a limited number of genes in the cps operon, with the remainder of the genes in the operon remaining stable. Mutations in strains in diseased vaccinated fish occur in key genes in the capsular operon that are associated with polysaccharide configuration (cpsG) and with regulation of biosynthesis (cpsD and cpsE). This, along with high ratios of nonsynonymous to synonymous mutations within the cps genes, suggests that immune response directed predominantly against capsular polysaccharide may be driving evolution in a very specific set of genes in the operon. From these data, it may be possible to design a simple polyvalent vaccine with a greater operational life span than the current monovalent killed bacterins. PMID:23001668

  14. 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.

  15. Allostery and the lac Operon.

    PubMed

    Lewis, Mitchell

    2013-07-10

    The ability to regulate gene expression is essential for controlling metabolic events in a cell. Proteins that function like molecular switches respond to fluctuations in the environment to maintain homeostasis. The operon model, proposed by Jacob and Monod, provides a cogent depiction for how gene expression is regulated. A molecular mechanism for the regulation followed shortly with the theory for allosteric transition. Over the past half-century, the details of the lac operon and the allosteric model have been tested using genetic, biochemical, and structural techniques. Remarkably, the principles originally put forward 50 years ago remain essentially unchanged. Models for the operon and the theory of allosteric transitions are two of the most profound discoveries of molecular biology.

  16. Computational identification of operons in microbial genomes.

    PubMed

    Zheng, Yu; Szustakowski, Joseph D; Fortnow, Lance; Roberts, Richard J; Kasif, Simon

    2002-08-01

    By applying graph representations to biochemical pathways, a new computational pipeline is proposed to find potential operons in microbial genomes. The algorithm relies on the fact that enzyme genes in operons tend to catalyze successive reactions in metabolic pathways. We applied this algorithm to 42 microbial genomes to identify putative operon structures. The predicted operons from Escherichia coli were compared with a selected metabolism-related operon dataset from the RegulonDB database, yielding a prediction sensitivity (89%) and specificity (87%) relative to this dataset. Several examples of detected operons are given and analyzed. Modular gene cluster transfer and operon fusion are observed. A further use of predicted operon data to assign function to putative genes was suggested and, as an example, a previous putative gene (MJ1604) from Methanococcus jannaschii is now annotated as a phosphofructokinase, which was regarded previously as a missing enzyme in this organism. GC content changes in the operon region and nonoperon region were examined. The results reveal a clear GC content transition at the boundaries of putative operons. We looked further into the conservation of operons across genomes. A trp operon alignment is analyzed in depth to show gene loss and rearrangement in different organisms during operon evolution.

  17. 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

  18. 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.

  19. 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…

  20. 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…

  1. Detecting uber-operons in prokaryotic genomes.

    PubMed

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

    2006-01-01

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

  2. 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

  3. Expression of a synthetic pertussis toxin operon in Escherichia coli.

    PubMed

    Pozza, T D; Yan, H; Walker, M J

    1997-06-01

    Bordetella pertussis is the causative agent of whooping cough, a severe disease of infants characterised by repeated of paroxysmal coughing. Pertussis toxin (PT) is a major virulence factor of B. pertussis and is a typical A/B bacterial toxin consisting of five subunits S1-S5 in a ratio of 1:1:1:2:1. The PT subunit genes are organized into an operon which is not expressed in Escherichia coli, thus hampering the use of this organism for vaccine production. We have expressed the five PT subunits individually in E. coli by replacing the wild-type transcriptional and translational signals, and in the case of the S4 subunit the leader peptide has been exchanged with a modified E. coli beta-lactamase leader sequence. We have developed a stepwise cloning method to construct a synthetic PT operon which simultaneously expresses the five PT subunits in E. coli. Western blot analysis indicated that in E. coli KS476 containing the synthetic PT operon, S4 and S5 were completely processed, S1 was partially processed, whilst the majority of S2 and S3 remained unprocessed. Periplasmic extracts contained soluble S1 and S3; however, the processed form of S2, S4 and S5 were not detected, suggesting that these subunits may be membrane associated or in an insoluble form. This work should allow an investigation of the potential of E. coli to produce detoxified PT in a background free of other pertussis virulence factors that may contribute to the side-effects of some vaccine preparations currently in use.

  4. Horizontally Acquired Glycosyltransferase Operons Drive Salmonellae Lipopolysaccharide Diversity

    PubMed Central

    Davies, Mark R.; Broadbent, Sarah E.; Harris, Simon R.; Thomson, Nicholas R.; van der Woude, Marjan W.

    2013-01-01

    The immunodominant lipopolysaccharide is a key antigenic factor for Gram-negative pathogens such as salmonellae where it plays key roles in host adaptation, virulence, immune evasion, and persistence. Variation in the lipopolysaccharide is also the major differentiating factor that is used to classify Salmonella into over 2600 serovars as part of the Kaufmann-White scheme. While lipopolysaccharide diversity is generally associated with sequence variation in the lipopolysaccharide biosynthesis operon, extraneous genetic factors such as those encoded by the glucosyltransferase (gtr) operons provide further structural heterogeneity by adding additional sugars onto the O-antigen component of the lipopolysaccharide. Here we identify and examine the O-antigen modifying glucosyltransferase genes from the genomes of Salmonella enterica and Salmonella bongori serovars. We show that Salmonella generally carries between 1 and 4 gtr operons that we have classified into 10 families on the basis of gtrC sequence with apparent O-antigen modification detected for five of these families. The gtr operons localize to bacteriophage-associated genomic regions and exhibit a dynamic evolutionary history driven by recombination and gene shuffling events leading to new gene combinations. Furthermore, evidence of Dam- and OxyR-dependent phase variation of gtr gene expression was identified within eight gtr families. Thus, as O-antigen modification generates significant intra- and inter-strain phenotypic diversity, gtr-mediated modification is fundamental in assessing Salmonella strain variability. This will inform appropriate vaccine and diagnostic approaches, in addition to contributing to our understanding of host-pathogen interactions. PMID:23818865

  5. 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.

  6. Transcriptional autoregulation of the Salmonella typhimurium phoPQ operon.

    PubMed

    Soncini, F C; Véscovi, E G; Groisman, E A

    1995-08-01

    The Salmonella typhimurium PhoP-PhoQ two-component regulatory system controls the expression of several genes, some of which are necessary for virulence. During a screening for PhoP-regulated genes, we identified the phoPQ operon as a PhoP-activated locus. beta-Galactosidase activity originating from phoPQ-lac transcriptional fusions required the presence of both the transcriptional regulator PhoP and its cognate sensor-kinase PhoQ. At low concentrations, PhoQ stimulated expression of phoPQ-lac transcriptional fusions. However, larger amounts of PhoQ protein without a concomitant increase in PhoP failed to activate phoPQ-lac fusions. Two different transcripts are produced from the phoPQ operon during exponential growth. These transcripts define two promoters: phoPp1, which requires both PhoP and PhoQ for activity and which is environmentally regulated, and phoPp2, which remains active in the absence of PhoP and PhoQ but which is slightly stimulated by these proteins. The pattern of transcriptional autoregulation was also observed at the protein level with anti-PhoP antibodies. In sum, autoregulation of the phoPQ operon provides several levels of control for the PhoP-PhoQ regulon. First, environmental signals would stimulate PhoQ to phosphorylate the PhoP protein that is produced at basal levels from the PhoP-PhoQ-independent promoter. Then, phospho-PhoP would activate transcription of phoPp1, resulting in larger amounts of PhoP and PhoQ and increased expression of PhoP-activated genes. A return to basal levels could be mediated by a posttranscriptional mechanism by which translation of the mRNA produced from phoPp1 is inhibited.

  7. The lac operon galactoside acetyltransferase.

    PubMed

    Roderick, Steven L

    2005-06-01

    Of the proteins encoded by the three structural genes of the lac operon, the galactoside acetyltransferase (thiogalactoside transacetylase, LacA, GAT) encoded by lacA is the only protein whose biological role remains in doubt. Here, we briefly note the classical literature that led to the identification and initial characterization of GAT, and focus on more recent results which have revealed its chemical mechanism of action and its membership in a large superfamily of structurally similar acyltransferases. The structural and sequence similarities of several members of this superfamily confirm the original claim for GAT as a CoA-dependent acetyltransferase specific for the 6-hydroxyl group of certain pyranosides, but do not yet point to the identity of the natural substrate(s) of the enzyme.

  8. Genetic map of the Actinobacillus pleuropneumoniae RTX-toxin (Apx) operons: characterization of the ApxIII operons.

    PubMed Central

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

    1994-01-01

    Actinobacillus pleuropneumoniae RTX-toxin III (ApxIII) is implicated as an important virulence factor of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia. Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized. The toxin appeared to be a member of the RTX-toxin family, as are the other two secreted toxins of A. pleuropneumoniae, i.e., ApxI and ApxII. In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8. This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively. Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB. We examined the presence of apxIIICABD sequences in the 12 serotypes of A. pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII. Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences. Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes. The differences were located between the glycine-rich repeats and the secretion signal. The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A. pleuropneumoniae. We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the toxins in pathogenesis. Images PMID:7927703

  9. Structural Insight into the Gene Expression Profiling of the hcn Operon in Pseudomonas aeruginosa.

    PubMed

    Chowdhury, Nilkanta; Bagchi, Angshuman

    2017-07-01

    Pseudomonas aeruginosa is a common opportunistic human pathogen. It generally attacks immunosuppressed patients like AIDS, cancer, cystic fibrosis, etc. The virulence of P. aeruginosa is mediated by various virulence factors. One of such potential virulence factors is HCN synthesized by HCN synthase enzyme, which is encoded by the hcnABC operon. The expressions of the genes of this operon are regulated by three transcriptional regulators, viz., LasR, ANR, and RhlR. In our previous work, we analyzed the molecular details of the functionalities of LasR. In this work, we focused on ANR. ANR is a regulatory protein which belongs to the FNR family and works in anaerobic condition. ANR binds to the promoter DNA, named ANR box, as a dimer. The dimerization of this ANR protein is regulated by Fe4S4, an iron-sulfur cluster. This dimer of ANR (ANR-Fe4S4/ANR-Fe4S4) recognizes and binds the promoter DNA sequence and regulates the transcription of this hcnABC operon. Till date, the biomolecular details of the interactions of ANR dimer with the promoter DNA are not fully understood. Thus, we built the molecular model of ANR-Fe4S4/ANR-Fe4S4. We docked the complex with the corresponding promoter DNA region. We analyzed the mode of interactions with the promoter DNA under different conditions. Thus, we tried to analyze the functionality of the ANR protein during the expressions of the genes of the hcnABC operon. So far, this is the first report to detail the molecular mechanism of the gene expression in P. aeruginosa.

  10. Identification of a Group 1-Like Capsular Polysaccharide Operon for Vibrio vulnificus

    PubMed Central

    Wright, Anita C.; Powell, Jan L.; Kaper, James B.; Morris, J. Glenn

    2001-01-01

    Virulence of Vibrio vulnificus correlates with changes in colony morphology that are indicative of a reversible phase variation for expression of capsular polysaccharide (CPS). Encapsulated variants are virulent with opaque colonies, whereas phase variants with reduced CPS expression are attenuated and are translucent. Using TnphoA mutagenesis, we identified a V. vulnificus CPS locus, which included an upstream ops element, a wza gene (wzaVv), and several open reading frames with homology to CPS biosynthetic genes. This genetic organization is characteristic of group 1 CPS operons. The wza gene product is required for transport of CPS to the cell surface in Escherichia coli. Polar transposon mutations in wzaVv eliminated expression of downstream biosynthetic genes, confirming operon structure. On the other hand, nonpolar inactivation of wzaVv was specific for CPS transport, did not alter CPS biosynthesis, and could be complemented in trans. Southern analysis of CPS phase variants revealed deletions or rearrangements at this locus. A survey of environmental isolates indicated a correlation between deletions in wzaVv and loss of virulent phenotype, suggesting a genetic mechanism for CPS phase variation. Full virulence in mice required surface expression of CPS and supported the essential role of capsule in the pathogenesis of V. vulnificus. PMID:11598064

  11. Virulence Determination

    USDA-ARS?s Scientific Manuscript database

    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....

  12. Operon and non-operon gene clusters in the C. elegans genome.

    PubMed

    Blumenthal, Thomas; Davis, Paul; Garrido-Lecca, Alfonso

    2015-04-28

    Nearly 15% of the ~20,000 C. elegans genes are contained in operons, multigene clusters controlled by a single promoter. The vast majority of these are of a type where the genes in the cluster are ~100 bp apart and the pre-mRNA is processed by 3' end formation accompanied by trans-splicing. A spliced leader, SL2, is specialized for operon processing. Here we summarize current knowledge on several variations on this theme including: (1) hybrid operons, which have additional promoters between genes; (2) operons with exceptionally long (> 1 kb) intercistronic regions; (3) operons with a second 3' end formation site close to the trans-splice site; (4) alternative operons, in which the exons are sometimes spliced as a single gene and sometimes as two genes; (5) SL1-type operons, which use SL1 instead of SL2 to trans-splice and in which there is no intercistronic space; (6) operons that make dicistronic mRNAs; and (7) non-operon gene clusters, in which either two genes use a single exon as the 3' end of one and the 5' end of the next, or the 3' UTR of one gene serves as the outron of the next. Each of these variations is relatively infrequent, but together they show a remarkable variety of tight-linkage gene arrangements in the C. elegans genome.

  13. Redundant phenazine operons in Pseudomonas aeruginosa exhibit environment-dependent expression and differential roles in pathogenicity

    PubMed Central

    Recinos, David A.; Sekedat, Matthew D.; Hernandez, Adriana; Cohen, Taylor Sitarik; Sakhtah, Hassan; Prince, Alice S.; Price-Whelan, Alexa; Dietrich, Lars E. P.

    2012-01-01

    Evolutionary biologists have postulated that several fitness advantages may be conferred by the maintenance of duplicate genes, including environmental adaptation resulting from differential regulation. We examined the expression and physiological contributions of two redundant operons in the adaptable bacterium Pseudomonas aeruginosa PA14. These operons, phzA1-G1 (phz1) and phzA2-G2 (phz2), encode nearly identical sets of proteins that catalyze the synthesis of phenazine-1-carboxylic acid, the precursor for several phenazine derivatives. Phenazines perform diverse roles in P. aeruginosa physiology and act as virulence factors during opportunistic infections of plant and animal hosts. Although reports have indicated that phz1 is regulated by the Pseudomonas quinolone signal, factors controlling phz2 expression have not been identified, and the relative contributions of these redundant operons to phenazine biosynthesis have not been evaluated. We found that in liquid cultures, phz1 was expressed at higher levels than phz2, although phz2 showed a greater contribution to phenazine production. In colony biofilms, phz2 was expressed at high levels, whereas phz1 expression was not detectable, and phz2 was responsible for virtually all phenazine production. Analysis of mutants defective in quinolone signal synthesis revealed a critical role for 4-hydroxy-2-heptylquinoline in phz2 induction. Finally, deletion of phz2, but not of phz1, decreased lung colonization in a murine model of infection. These results suggest that differential regulation of the redundant phz operons allows P. aeruginosa to adapt to diverse environments. PMID:23129634

  14. Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility.

    PubMed

    Huertas, Mónica G; Zárate, Lina; Acosta, Iván C; Posada, Leonardo; Cruz, Diana P; Lozano, Marcela; Zambrano, María M

    2014-12-01

    Klebsiella pneumoniae is an opportunistic pathogen important in hospital-acquired infections, which are complicated by the rise of drug-resistant strains and the capacity of cells to adhere to surfaces and form biofilms. In this work, we carried out an analysis of the genes in the K. pneumoniae yfiRNB operon, previously implicated in biofilm formation. The results indicated that in addition to the previously reported effect on type 3 fimbriae expression, this operon also affected biofilm formation due to changes in cellulose as part of the extracellular matrix. Deletion of yfiR resulted in enhanced biofilm formation and an altered colony phenotype indicative of cellulose overproduction when grown on solid indicator media. Extraction of polysaccharides and treatment with cellulase were consistent with the presence of cellulose in biofilms. The enhanced cellulose production did not, however, correlate with virulence as assessed using a Caenorhabditis elegans assay. In addition, cells bearing mutations in genes of the yfiRNB operon varied with respect to the WT control in terms of susceptibility to the antibiotics amikacin, ciprofloxacin, imipenem and meropenem. These results indicated that the yfiRNB operon is implicated in the production of exopolysaccharides that alter cell surface characteristics and the capacity to form biofilms--a phenotype that does not necessarily correlate with properties related with survival, such as resistance to antibiotics.

  15. 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

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

    PubMed

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

    2015-01-01

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

  17. 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

  18. 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.

  19. QapR (PA5506) Represses an Operon That Negatively Affects the Pseudomonas Quinolone Signal in Pseudomonas aeruginosa

    PubMed Central

    Tipton, Kyle A.; Coleman, James P.

    2013-01-01

    Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that can cause disease in varied sites within the human body and is a significant source of morbidity and mortality in those afflicted with cystic fibrosis. P. aeruginosa is able to coordinate group behaviors, such as virulence factor production, through the process of cell-to-cell signaling. There are three intercellular signaling systems employed by P. aeruginosa, and one of these systems utilizes the small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]). PQS is required for virulence in multiple infection models and has been found in the lungs of cystic fibrosis patients colonized by P. aeruginosa. In this study, we have identified an RpiR family transcriptional regulator, QapR, which is an autoregulatory repressor. We found that mutation of qapR caused overexpression of the qapR operon. We characterized the qapR operon to show that it contains genes qapR, PA5507, PA5508, and PA5509 and that QapR directly controls the transcription of these genes in a negative manner. We also show that derepression of this operon greatly reduces PQS concentration in P. aeruginosa. Our results suggest that qapR affects PQS concentration by repressing an enzymatic pathway that acts on PQS or a PQS precursor to lower the PQS concentration. We believe that this operon comprises a novel mechanism to regulate PQS concentration in P. aeruginosa. PMID:23708133

  20. QapR (PA5506) represses an operon that negatively affects the Pseudomonas quinolone signal in Pseudomonas aeruginosa.

    PubMed

    Tipton, Kyle A; Coleman, James P; Pesci, Everett C

    2013-08-01

    Pseudomonas aeruginosa is a Gram-negative, opportunistic pathogen that can cause disease in varied sites within the human body and is a significant source of morbidity and mortality in those afflicted with cystic fibrosis. P. aeruginosa is able to coordinate group behaviors, such as virulence factor production, through the process of cell-to-cell signaling. There are three intercellular signaling systems employed by P. aeruginosa, and one of these systems utilizes the small molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]). PQS is required for virulence in multiple infection models and has been found in the lungs of cystic fibrosis patients colonized by P. aeruginosa. In this study, we have identified an RpiR family transcriptional regulator, QapR, which is an autoregulatory repressor. We found that mutation of qapR caused overexpression of the qapR operon. We characterized the qapR operon to show that it contains genes qapR, PA5507, PA5508, and PA5509 and that QapR directly controls the transcription of these genes in a negative manner. We also show that derepression of this operon greatly reduces PQS concentration in P. aeruginosa. Our results suggest that qapR affects PQS concentration by repressing an enzymatic pathway that acts on PQS or a PQS precursor to lower the PQS concentration. We believe that this operon comprises a novel mechanism to regulate PQS concentration in P. aeruginosa.

  1. Stochastic simulations of the tetracycline operon

    PubMed Central

    2011-01-01

    Background The tetracycline operon is a self-regulated system. It is found naturally in bacteria where it confers resistance to antibiotic tetracycline. Because of the performance of the molecular elements of the tetracycline operon, these elements are widely used as parts of synthetic gene networks where the protein production can be efficiently turned on and off in response to the presence or the absence of tetracycline. In this paper, we investigate the dynamics of the tetracycline operon. To this end, we develop a mathematical model guided by experimental findings. Our model consists of biochemical reactions that capture the biomolecular interactions of this intriguing system. Having in mind that small biological systems are subjects to stochasticity, we use a stochastic algorithm to simulate the tetracycline operon behavior. A sensitivity analysis of two critical parameters embodied this system is also performed providing a useful understanding of the function of this system. Results Simulations generate a timeline of biomolecular events that confer resistance to bacteria against tetracycline. We monitor the amounts of intracellular TetR2 and TetA proteins, the two important regulatory and resistance molecules, as a function of intrecellular tetracycline. We find that lack of one of the promoters of the tetracycline operon has no influence on the total behavior of this system inferring that this promoter is not essential for Escherichia coli. Sensitivity analysis with respect to the binding strength of tetracycline to repressor and of repressor to operators suggests that these two parameters play a predominant role in the behavior of the system. The results of the simulations agree well with experimental observations such as tight repression, fast gene expression, induction with tetracycline, and small intracellular TetR2 amounts. Conclusions Computer simulations of the tetracycline operon afford augmented insight into the interplay between its molecular

  2. 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…

  3. 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…

  4. 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.

  5. Origin of Bistability in the lac Operon

    PubMed Central

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

    2007-01-01

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

  6. Indole and 7‐hydroxyindole diminish Pseudomonas aeruginosa virulence

    PubMed Central

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

    2009-01-01

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

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

    PubMed

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

    2009-01-01

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

  8. Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: relationship with expression of adhesins and production of toxins.

    PubMed

    Blanco, M; Blanco, J E; Alonso, M P; Mora, A; Balsalobre, C; Muñoa, F; Juárez, A; Blanco, J

    1997-12-01

    A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesin-encoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap+ and/or sfa+ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing alpha-haemolysin (Hly+) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesin-encoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.

  9. Transcription termination within the iron transport-biosynthesis operon of Vibrio anguillarum requires an antisense RNA.

    PubMed

    Stork, Michiel; Di Lorenzo, Manuela; Welch, Timothy J; Crosa, Jorge H

    2007-05-01

    The iron transport-biosynthesis (ITB) operon in Vibrio anguillarum includes four genes for ferric siderophore transport, fatD, -C, -B, and -A, and two genes for siderophore biosynthesis, angR and angT. This cluster plays an important role in the virulence mechanisms of this bacterium. Despite being part of the same polycistronic mRNA, the relative levels of transcription for the fat portion and for the whole ITB message differ profoundly, the levels of the fat transcript being about 17-fold higher. Using S1 nuclease mapping, lacZ transcriptional fusions, and in vitro studies, we were able to show that the differential gene expression within the ITB operon is due to termination of transcription between the fatA and angR genes, although a few transcripts proceeded beyond the termination site to the end of this operon. This termination process requires a 427-nucleotide antisense RNA that spans the intergenic region and acts as a novel transcriptional terminator.

  10. 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

  11. 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.

  12. Evolutionary conservation of bacterial operons: does transcriptional connectivity matter?

    PubMed

    Hazkani-Covo, Einat; Graur, Dan

    2005-07-01

    In the literature, it has been frequently suggested that the connectivity of a protein, i.e., the number of proteins with which it interacts, is inversely correlated with the rate of evolution. We attempted to extrapolate from proteins to operons by testing the hypothesis that operons with high transcriptional connectivity, i.e., operons that are controlled through interactions with many transcription factors, are evolutionarily more conserved at the structure and sequence levels than low-connectivity operons. With Escherichia coli used as reference, two structural- and two sequence-conservation measures were determined for 82 groups of homologous operons from 30 completely-sequenced bacterial genomes. In E. coli, large operons tend to be regulated by more transcription factors than either smaller operons or single genes. Large E. coli operons that are regulated by single transcription factors were found to be regulated by activators more frequently than by repressors. Levels of sequence conservation and structural conservation of operons were found to be independent of each other, i.e., structurally conserved operons may be divergent in sequence, and vice versa. Transcriptional connectivity was found to influence neither sequence nor structural conservation of operons. Although this finding seems to contradict the situation in genes, a critical review of the literature indicates that although gene connectivity is frequently touted as a factor in determining rates of evolution, only a very small fraction of the variability in degrees of evolutionary conservation is explainable by this factor.

  13. An operon for production of bioactive gibberellin A4 phytohormone with wide distribution in the bacterial rice leaf streak pathogen Xanthomonas oryzae pv. oryzicola.

    PubMed

    Nagel, Raimund; Turrini, Paula C G; Nett, Ryan S; Leach, Jan E; Verdier, Valérie; Van Sluys, Marie-Anne; Peters, Reuben J

    2017-05-01

    Phytopathogens have developed elaborate mechanisms to attenuate the defense response of their host plants, including convergent evolution of complex pathways for production of the GA phytohormones, which were actually first isolated from the rice fungal pathogen Gibberella fujikuroi. The rice bacterial pathogen Xanthomonas oryzae pv. oryzicola (Xoc) has been demonstrated to contain a biosynthetic operon with cyclases capable of producing the universal GA precursor ent-kaurene. Genetic (knock-out) studies indicate that the derived diterpenoid serves as a virulence factor for this rice leaf streak pathogen, serving to reduce the jasmonic acid-mediated defense response. Here the functions of the remaining genes in the Xoc operon are elucidated and the distribution of the operon in X. oryzae is investigated in over 100 isolates. The Xoc operon leads to production of the bioactive GA4 , an additional step beyond production of the penultimate precursor GA9 mediated by the homologous operons recently characterized from rhizobia. Moreover, this GA biosynthetic operon was found to be widespread in Xoc (> 90%), but absent in the other major X. oryzae pathovar. These results indicate selective pressure for production of GA4 in the distinct lifestyle of Xoc, and the importance of GA to both fungal and bacterial pathogens of rice. © 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.

  14. Overproduction of Type 8 Capsular Polysaccharide Augments Staphylococcus aureus Virulence

    PubMed Central

    Luong, Thanh T.; Lee, Chia Y.

    2002-01-01

    Type 8 capsular polysaccharide (CP8) is the most prevalent capsule type in clinical isolates of Staphylococcus aureus. However, its role in virulence has not been clearly defined. CP8 strains such as strain Becker produce a small amount of capsule on their surface in vitro. In contrast, CP1 strains such as strain M produce a large amount of capsule, which has been shown to be an important antiphagocytic virulence factor. The cap8 and cap1 operons, required for the synthesis of CP8 and CP1, respectively, have been cloned and sequenced. To test whether CP8 contributes to the pathogenesis of S. aureus, we replaced the weak native promoter of the cap8 operon in strain Becker with the strong constitutive promoter of the cap1 operon of strain M. The resultant strain, CYL770, synthesized cap8-specific mRNA at a level about sevenfold higher than that in the parent strain. Remarkably, the CYL770 strain produced about 80-fold more CP8. In a mouse infection model of bacteremia, the CP8-overproducing strain persisted longer in the bloodstream, the liver, and the spleen in mice than the parent strain. In addition, strain CYL770 was more resistant to ospsonophagocytosis in vitro by human polymorphonuclear leukocytes. These results indicate that CP8 is an antiphagocytic virulence factor of S. aureus. PMID:12065477

  15. A global analysis of adaptive evolution of operons in cyanobacteria.

    PubMed

    Memon, Danish; Singh, Abhay K; Pakrasi, Himadri B; Wangikar, Pramod P

    2013-02-01

    Operons are an important feature of prokaryotic genomes. Evolution of operons is hypothesized to be adaptive and has contributed significantly towards coordinated optimization of functions. Two conflicting theories, based on (i) in situ formation to achieve co-regulation and (ii) horizontal gene transfer of functionally linked gene clusters, are generally considered to explain why and how operons have evolved. Furthermore, effects of operon evolution on genomic traits such as intergenic spacing, operon size and co-regulation are relatively less explored. Based on the conservation level in a set of diverse prokaryotes, we categorize the operonic gene pair associations and in turn the operons as ancient and recently formed. This allowed us to perform a detailed analysis of operonic structure in cyanobacteria, a morphologically and physiologically diverse group of photoautotrophs. Clustering based on operon conservation showed significant similarity with the 16S rRNA-based phylogeny, which groups the cyanobacterial strains into three clades. Clade C, dominated by strains that are believed to have undergone genome reduction, shows a larger fraction of operonic genes that are tightly packed in larger sized operons. Ancient operons are in general larger, more tightly packed, better optimized for co-regulation and part of key cellular processes. A sub-clade within Clade B, which includes Synechocystis sp. PCC 6803, shows a reverse trend in intergenic spacing. Our results suggest that while in situ formation and vertical descent may be a dominant mechanism of operon evolution in cyanobacteria, optimization of intergenic spacing and co-regulation are part of an ongoing process in the life-cycle of operons.

  16. Multiple promoters control expression of the Yersinia enterocolitica phage-shock-protein A (pspA) operon.

    PubMed

    Maxson, Michelle E; Darwin, Andrew J

    2006-04-01

    The widely conserved phage-shock-protein A (pspA) operon encodes an extracytoplasmic stress response system that is essential for virulence in Yersinia enterocolitica, and has been linked to other important phenotypes in Escherichia coli, Salmonella enterica and Shigella flexneri. Regulation of pspA operon expression is mediated through a promoter upstream of pspA that depends on sigma factor RpoN (sigma(54)) and the enhancer binding protein PspF. PspA, PspB and PspC, encoded within the pspA operon, also regulate expression by participating in a putative signal transduction pathway that probably serves to modulate PspF activity. All of this suggests that appropriate expression of the pspA operon is critical. Previous genetic analysis of the Y. enterocolitica pspA operon suggested that an additional level of complexity might be mediated by PspF/RpoN-independent expression of some psp genes. Here, an rpoN null mutation and interposon analysis were used to confirm that PspF/RpoN-independent gene expression does originate within the psp locus. Molecular genetic approaches were used to systematically analyse the two large non-coding regions within the psp locus. Primer extension, control region deletion and site-directed mutagenesis experiments led to the identification of RpoN-independent promoters both upstream and downstream of pspA. The precise location of the PspF/RpoN-dependent promoter upstream of pspA was also determined. The discovery of these RpoN-independent promoters reveals yet another level of transcriptional complexity for the Y. enterocolitica pspA operon that may function to allow low-level constitutive expression of psp genes and/or additional regulation under some conditions.

  17. Escherichia coli fliAZY operon.

    PubMed Central

    Mytelka, D S; Chamberlin, M J

    1996-01-01

    We have cloned the Escherichia coli fliAZY operon, which contains the fliA gene (the alternative sigma factor sigma F) and two novel genes, fliZ and fliY. Transcriptional mapping of this operon shows two start sites, one of which is preceded by a canonical E sigma F-dependent consensus and is dependent on sigma F for expression in vivo and in vitro. We have overexpressed and purified sigma F and demonstrated that it can direct core polymerase to E sigma F-dependent promoters. FliZ and FliY are not required for motility but may regulate sigma F activity, perhaps in response to a putative cell density signal that may be detected by FliY, a member of the bacterial extracellular solute-binding protein family 3. PMID:8550423

  18. A Novel lux Operon in the Cryptically Bioluminescent Fish Pathogen Vibrio salmonicida Is Associated with Virulence▿

    PubMed Central

    Nelson, Eric J.; Tunsjø, Hege S.; Fidopiastis, Pat M.; Sørum, Henning; Ruby, Edward G.

    2007-01-01

    The cold-water-fish pathogen Vibrio salmonicida expresses a functional bacterial luciferase but produces insufficient levels of its aliphatic-aldehyde substrate to be detectably luminous in culture. Our goals were to (i) better explain this cryptic bioluminescence phenotype through molecular characterization of the lux operon and (ii) test whether the bioluminescence gene cluster is associated with virulence. Cloning and sequencing of the V. salmonicida lux operon revealed that homologs of all of the genes required for luminescence are present: luxAB (luciferase) and luxCDE (aliphatic-aldehyde synthesis). The arrangement and sequence of these structural lux genes are conserved compared to those in related species of luminous bacteria. However, V. salmonicida strains have a novel arrangement and number of homologs of the luxR and luxI quorum-sensing regulatory genes. Reverse transcriptase PCR analysis suggests that this novel arrangement of quorum-sensing genes generates antisense transcripts that may be responsible for the reduced production of bioluminescence. In addition, infection with a strain in which the luxA gene was mutated resulted in a marked delay in mortality among Atlantic salmon relative to infection with the wild-type parent in single-strain challenge experiments. In mixed-strain competition between the luxA mutant and the wild type, the mutant was attenuated up to 50-fold. It remains unclear whether the attenuation results from a direct loss of luciferase or a polar disturbance elsewhere in the lux operon. Nevertheless, these findings document for the first time an association between a mutation in a structural lux gene and virulence, as well as provide a new molecular system to study Vibrio pathogenesis in a natural host. PMID:17277225

  19. 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

  20. ProOpDB: Prokaryotic Operon DataBase.

    PubMed

    Taboada, Blanca; Ciria, Ricardo; Martinez-Guerrero, Cristian E; Merino, Enrique

    2012-01-01

    The Prokaryotic Operon DataBase (ProOpDB, http://operons.ibt.unam.mx/OperonPredictor) constitutes one of the most precise and complete repositories of operon predictions now available. Using our novel and highly accurate operon identification algorithm, we have predicted the operon structures of more than 1200 prokaryotic genomes. ProOpDB offers diverse alternatives by which a set of operon predictions can be retrieved including: (i) organism name, (ii) metabolic pathways, as defined by the KEGG database, (iii) gene orthology, as defined by the COG database, (iv) conserved protein domains, as defined by the Pfam database, (v) reference gene and (vi) reference operon, among others. In order to limit the operon output to non-redundant organisms, ProOpDB offers an efficient method to select the most representative organisms based on a precompiled phylogenetic distances matrix. In addition, the ProOpDB operon predictions are used directly as the input data of our Gene Context Tool to visualize their genomic context and retrieve the sequence of their corresponding 5' regulatory regions, as well as the nucleotide or amino acid sequences of their genes.

  1. Optimal gene partition into operons correlates with gene functional order

    NASA Astrophysics Data System (ADS)

    Zaslaver, Alon; Mayo, Avi; Ronen, Michal; Alon, Uri

    2006-09-01

    Gene arrangement into operons varies between bacterial species. Genes in a given system can be on one operon in some organisms and on several operons in other organisms. Existing theories explain why genes that work together should be on the same operon, since this allows for advantageous lateral gene transfer and accurate stoichiometry. But what causes the frequent separation into multiple operons of co-regulated genes that act together in a pathway? Here we suggest that separation is due to benefits made possible by differential regulation of each operon. We present a simple mathematical model for the optimal distribution of genes into operons based on a balance of the cost of operons and the benefit of regulation that provides 'just-when-needed' temporal order. The analysis predicts that genes are arranged such that genes on the same operon do not skip functional steps in the pathway. This prediction is supported by genomic data from 137 bacterial genomes. Our work suggests that gene arrangement is not only the result of random historical drift, genome re-arrangement and gene transfer, but has elements that are solutions of an evolutionary optimization problem. Thus gene functional order may be inferred by analyzing the operon structure across different genomes.

  2. Metazoan operons accelerate recovery from growth arrested states

    PubMed Central

    Zaslaver, Alon; Baugh, L. Ryan; Sternberg, Paul W.

    2011-01-01

    Summary Existing theories explain why operons are advantageous in prokaryotes, but their occurrence in metazoans is an enigma. Nematode operon genes, typically consisting of growth genes, are significantly up-regulated during recovery from growth-arrested states. This expression pattern is anti-correlated to non-operon genes consistent with a competition for transcriptional resources. We find that transcriptional resources are initially limiting during recovery, and that recovering animals are highly sensitive to any additional decrease in transcriptional resources. Operons become advantageous because by clustering growth genes into operons, fewer promoters compete for the limited transcriptional machinery, effectively increasing the concentration of transcriptional resources, and accelerating recovery. Mathematical modeling reveals how a moderate increase in transcriptional resources can substantially enhance transcription rate and recovery. This design principle occurs in different nematodes and the chordate C. intestinalis. As transition from arrest to rapid growth is shared by many metazoans, operons could have evolved to facilitate these processes. PMID:21663799

  3. Gene context conservation of a higher order than operons.

    PubMed

    Lathe, W C; Snel, B; Bork, P

    2000-10-01

    Operons, co-transcribed and co-regulated contiguous sets of genes, are poorly conserved over short periods of evolutionary time. The gene order, gene content and regulatory mechanisms of operons can be very different, even in closely related species. Here, we present several lines of evidence which suggest that, although an operon and its individual genes and regulatory structures are rearranged when comparing the genomes of different species, this rearrangement is a conservative process. Genomic rearrangements invariably maintain individual genes in very specific functional and regulatory contexts. We call this conserved context an uber-operon.

  4. Association of iss and iucA, but not tsh, with plasmid-mediated virulence of avian pathogenic Escherichia coli.

    PubMed

    Tivendale, Kelly A; Allen, Joanne L; Ginns, Carol A; Crabb, Brendan S; Browning, Glenn F

    2004-11-01

    Avian pathogenic Escherichia coli (APEC) is an economically important respiratory pathogen of chickens worldwide. Factors previously associated with the virulence of APEC include adhesins, iron-scavenging mechanisms, the production of colicin V (ColV), serum resistance, and temperature-sensitive hemagglutination, but virulence has generally been assessed by parenteral inoculation, which does not replicate the normal respiratory route of infection. A large plasmid, pVM01, is essential for virulence in APEC strain E3 in chickens after aerosol exposure. Here we establish the size of pVM01 to be approximately 160 kb and show that the putative virulence genes iss (increased serum survival) and tsh (temperature-sensitive hemagglutinin) and the aerobactin operon are on the plasmid. These genes were not clustered on pVM01 but, rather, were each located in quite distinct regions. Examination of APEC strains with defined levels of respiratory pathogenicity after aerosol exposure showed that both the aerobactin operon and iss were associated with high levels of virulence in APEC but that the possession of either gene was sufficient for intermediate levels of virulence. In contrast, the presence of tsh was not necessary for high levels of virulence. Thus, both the aerobactin operon and iss are associated with virulence in APEC after exposure by the natural route of infection. The similarities between APEC and extraintestinal E. coli infection in other species suggests that they may be useful models for definition of the role of these virulence genes and of other novel virulence genes that may be located on their virulence plasmids.

  5. Novel twin streptolysin S-like peptides encoded in the sag operon homologue of beta-hemolytic Streptococcus anginosus.

    PubMed

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

    2013-03-01

    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.

  6. 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

  7. Detection of virulence genes in Escherichia coli isolated from patients with cystitis and pyelonephritis.

    PubMed

    Firoozeh, Farzaneh; Saffari, Mahmood; Neamati, Foroogh; Zibaei, Mohammad

    2014-12-01

    Uropathogenic Escherichia coli (UPEC) is a common cause of ascending urinary tract infections including cystitis and pyelonephritis. The purpose of this study was to investigate virulence genes among Escherichia coli isolated from patients with cystitis and pyelonephritis. Between December 2012 and June 2013, 150 E. coli isolates from hospitalized patients with pyelonephritis (n = 72) and cystitis (n=78) were collected at Shahid Beheshti Hospital in Kashan. A PCR assay was used to evaluate the presence of virulence genes including pap, hly, aer, sfa, cnf, afa, traT, and pathogenicity island (PAI) markers in isolates. Of the total 150 UPEC isolates, 130 (86.7%) were found to carry the virulence genes studied. Nineteen different virulence patterns were identified. The most prevalent virulence pattern was UPEC including traT-PAI operons. The pap, traT, aer, hly, and PAI operons were more prevalent among patients with pyelonephritis than cystitis, and the sfa, afa, and cnf genes were not detected in any of the isolates. Higher virulence gene diversity was found among pyelonephritis UPEC isolates in comparison to cystitis UPEC isolates, showing that UPEC strains that cause pyelonephritis need more virulence factors. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  8. 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.

  9. Structure of the lac operon galactoside acetyltransferase.

    PubMed

    Wang, Xing-Guo; Olsen, Laurence R; Roderick, Steven L

    2002-04-01

    The galactoside acetyltransferase (thiogalactoside transacetylase) of Escherichia coli (GAT, LacA, EC 2.3.1.18) is a gene product of the classical lac operon. GAT may assist cellular detoxification by acetylating nonmetabolizable pyranosides, thereby preventing their reentry into the cell. The structure of GAT has been solved in binary complexes with acetyl-CoA or CoA and in ternary complexes with CoA and the nonphysiological acceptor substrates isopropyl beta-D-thiogalactoside (IPTG) or p-nitrophenyl beta-D-galactopyranoside (PNPbetaGal). A hydrophobic cleft that binds the thioisopropyl and p-nitrophenyl aglycones of IPTG and PNPbetaGal may discriminate against substrates with hydrophilic substituents at this position, such as lactose, or inducers of the lac operon. An extended loop projecting from the left-handed parallel beta helix domain contributes His115, which is in position to facilitate attack of the C6-hydroxyl group of the substrate on the thioester.

  10. Genomic rearrangements at rrn operons in Salmonella.

    PubMed

    Helm, R Allen; Lee, Alison G; Christman, Harry D; Maloy, Stanley

    2003-11-01

    Most Salmonella serovars are general pathogens that infect a variety of hosts. These "generalist" serovars cause disease in many animals from reptiles to mammals. In contrast, a few serovars cause disease only in a specific host. Host-specific serovars can cause a systemic, often fatal disease in one species yet remain avirulent in other species. Host-specific Salmonella frequently have large genomic rearrangements due to recombination at the ribosomal RNA (rrn) operons while the generalists consistently have a conserved chromosomal arrangement. To determine whether this is the result of an intrinsic difference in recombination frequency or a consequence of lifestyle difference between generalist and host-specific Salmonella, we determined the frequency of rearrangements in vitro. Using lacZ genes as portable regions of homology for inversion analysis, we found that both generalist and host-specific serovars of Salmonella have similar tolerances to chromosomal rearrangements in vitro. Using PCR and genetic selection, we found that generalist and host-specific serovars also undergo rearrangements at rrn operons at similar frequencies in vitro. These observations indicate that the observed difference in genomic stability between generalist and host-specific serovars is a consequence of their distinct lifestyles, not intrinsic differences in recombination frequencies.

  11. Pseudomonas aeruginosa ATCC 9027 is a non-virulent strain suitable for mono-rhamnolipids production.

    PubMed

    Grosso-Becerra, María-Victoria; González-Valdez, Abigail; Granados-Martínez, María-Jessica; Morales, Estefanía; Servín-González, Luis; Méndez, José-Luis; Delgado, Gabriela; Morales-Espinosa, Rosario; Ponce-Soto, Gabriel-Yaxal; Cocotl-Yañez, Miguel; Soberón-Chávez, Gloria

    2016-12-01

    Rhamnolipids produced by Pseudomonas aeruginosa are biosurfactants with a high biotechnological potential, but their extensive commercialization is limited by the potential virulence of P. aeruginosa and by restrictions in producing these surfactants in heterologous hosts. In this work, we report the characterization of P. aeruginosa strain ATCC 9027 in terms of its genome-sequence, virulence, antibiotic resistance, and its ability to produce mono-rhamnolipids when carrying plasmids with different cloned genes from the type strain PAO1. The genes that were expressed from the plasmids are those coding for enzymes involved in the synthesis of this biosurfactant (rhlA and rhlB), as well as the gene that codes for the RhlR transcriptional regulator. We confirm that strain ATCC 9027 forms part of the PA7 clade, but contrary to strain PA7, it is sensitive to antibiotics and is completely avirulent in a mouse model. We also report that strain ATCC 9027 mono-rhamnolipid synthesis is limited by the expression of the rhlAB-R operon. Thus, this strain carrying the rhlAB-R operon produces similar rhamnolipids levels as PAO1 strain. We determined that strain ATCC 9027 with rhlAB-R operon was not virulent to mice. These results show that strain ATCC 9027, expressing PAO1 rhlAB-R operon, has a high biotechnological potential for industrial mono-rhamnolipid production.

  12. Operons in Escherichia coli: genomic analyses and predictions.

    PubMed

    Salgado, H; Moreno-Hagelsieb, G; Smith, T F; Collado-Vides, J

    2000-06-06

    The rich knowledge of operon organization in Escherichia coli, together with the completed chromosomal sequence of this bacterium, enabled us to perform an analysis of distances between genes and of functional relationships of adjacent genes in the same operon, as opposed to adjacent genes in different transcription units. We measured and demonstrated the expected tendencies of genes within operons to have much shorter intergenic distances than genes at the borders of transcription units. A clear peak at short distances between genes in the same operon contrasts with a flat frequency distribution of genes at the borders of transcription units. Also, genes in the same operon tend to have the same physiological functional class. The results of these analyses were used to implement a method to predict the genomic organization of genes into transcription units. The method has a maximum accuracy of 88% correct identification of pairs of adjacent genes to be in an operon, or at the borders of transcription units, and correctly identifies around 75% of the known transcription units when used to predict the transcription unit organization of the E. coli genome. Based on the frequency distance distributions, we estimated a total of 630 to 700 operons in E. coli. This step opens the possibility of predicting operon organization in other bacteria whose genome sequences have been finished.

  13. Natural Selection for Operons Depends on Genome Size

    PubMed Central

    Nuñez, Pablo A.; Romero, Héctor; Farber, Marisa D.; Rocha, Eduardo P.C.

    2013-01-01

    In prokaryotes, genome size is associated with metabolic versatility, regulatory complexity, effective population size, and horizontal transfer rates. We therefore analyzed the covariation of genome size and operon conservation to assess the evolutionary models of operon formation and maintenance. In agreement with previous results, intraoperonic pairs of essential and of highly expressed genes are more conserved. Interestingly, intraoperonic pairs of genes are also more conserved when they encode proteins at similar cell concentrations, suggesting a role of cotranscription in diminishing the cost of waste and shortfall in gene expression. Larger genomes have fewer and smaller operons that are also less conserved. Importantly, lower conservation in larger genomes was observed for all classes of operons in terms of gene expression, essentiality, and balanced protein concentration. We reached very similar conclusions in independent analyses of three major bacterial clades (α- and β-Proteobacteria and Firmicutes). Operon conservation is inversely correlated to the abundance of transcription factors in the genome when controlled for genome size. This suggests a negative association between the complexity of genetic networks and operon conservation. These results show that genome size and/or its proxies are key determinants of the intensity of natural selection for operon organization. Our data fit better the evolutionary models based on the advantage of coregulation than those based on genetic linkage or stochastic gene expression. We suggest that larger genomes with highly complex genetic networks and many transcription factors endure weaker selection for operons than smaller genomes with fewer alternative tools for genetic regulation. PMID:24201372

  14. Electron microscopic visualization of trp operon expression in Salmonella typhimurium.

    PubMed

    French, S; Martin, K; Patterson, T; Bauerle, R; Miller, O L

    1985-07-01

    Transcriptional activity of plasmids carrying wild-type and mutant trp operons was visualized in cell lysates of Salmonella typhimurium. Plasmid and transcription-unit sizes varied with the size of the cloned operon. Following 3-(3-indolyl)acrylic acid derepression, all operons of a particular type exhibited the same high level of transcriptional activity. An estimated 11-14 transcripts must be initiated each minute to maintain the 190-base-pair spacing of RNA polymerases observed on the promoter-proximal half of the wild-type trp operon. A decline in RNA polymerase density was observed on promoter-distal portions of cloned trp operons, which may be attributable to premature transcription termination accompanying translation inhibition due to indolylacrylic acid's interference with normal tryptophanyl-tRNA synthetase activity.

  15. A long-term epigenetic memory switch controls bacterial virulence bimodality.

    PubMed

    Ronin, Irine; Katsowich, Naama; Rosenshine, Ilan; Balaban, Nathalie Q

    2017-02-07

    When pathogens enter the host, sensing of environmental cues activates the expression of virulence genes. Opposite transition of pathogens from activating to non-activating conditions is poorly understood. Interestingly, variability in the expression of virulence genes upon infection enhances colonization. In order to systematically detect the role of phenotypic variability in enteropathogenic E. coli (EPEC), an important human pathogen, both in virulence activating and non-activating conditions, we employed the ScanLag methodology. The analysis revealed a bimodal growth rate. Mathematical modeling combined with experimental analysis showed that this bimodality is mediated by a hysteretic memory-switch that results in the stable co-existence of non-virulent and hyper-virulent subpopulations, even after many generations of growth in non-activating conditions. We identified the per operon as the key component of the hysteretic switch. This unique hysteretic memory switch may result in persistent infection and enhanced host-to-host spreading.

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

    PubMed

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

    1994-05-01

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

  17. Expression, purification and functional characterization of AmiA of acetamidase operon of Mycobacterium smegmatis.

    PubMed

    Sundararaman, Balaji; Palaniyandi, Kannan; Venkatesan, Arunkumar; Narayanan, Sujatha

    2014-11-01

    Regulation of gene expression is one of the mechanisms of virulence in pathogenic organisms. In this context, we would like to understand the gene regulation of acetamidase enzyme of Mycobacterium smegmatis, which is the first reported inducible enzyme in mycobacteria. The acetamidase is highly inducible and the expression of this enzyme is increased 100-fold when the substrate acetamide is added. The acetamidase structural gene (amiE) is found immediately downstream of three predicted open reading frames (ORFs). Three of these genes along with a divergently expressed ORF are predicted to form an operon and involved in the regulation of acetamidase enzyme. Here we report expression, purification and functional characterization of AmiA which is one of these predicted ORFs. Electrophoretic mobility shift assays showed that AmiA binds to the region between the amiA and amiD near the predicted promoter (P2). Over-expression of AmiA significantly lowered the expression of acetamidase compared to the wild type as demonstrated by qRT-PCR and SDS-PAGE. We conclude that AmiA binds near P2 promoter and acts as a repressor in the regulation of acetamidase operon. The described work is a further step forward toward broadening the knowledge on understanding of the complex gene regulatory mechanism of Mycobacterium sp.

  18. 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/.

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

    PubMed Central

    Taboada, Blanca; Verde, Cristina; Merino, Enrique

    2010-01-01

    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

  20. A phylogenomic analysis of the Actinomycetales mce operons

    PubMed Central

    Casali, Nicola; Riley, Lee W

    2007-01-01

    Background The genome of Mycobacterium tuberculosis harbors four copies of a cluster of genes termed mce operons. Despite extensive research that has demonstrated the importance of these operons on infection outcome, their physiological function remains obscure. Expanding databases of complete microbial genome sequences facilitate a comparative genomic approach that can provide valuable insight into the role of uncharacterized proteins. Results The M. tuberculosis mce loci each include two yrbE and six mce genes, which have homology to ABC transporter permeases and substrate-binding proteins, respectively. Operons with an identical structure were identified in all Mycobacterium species examined, as well as in five other Actinomycetales genera. Some of the Actinomycetales mce operons include an mkl gene, which encodes an ATPase resembling those of ABC uptake transporters. The phylogenetic profile of Mkl orthologs exactly matched that of the Mce and YrbE proteins. Through topology and motif analyses of YrbE homologs, we identified a region within the penultimate cytoplasmic loop that may serve as the site of interaction with the putative cognate Mkl ATPase. Homologs of the exported proteins encoded adjacent to the M. tuberculosis mce operons were detected in a conserved chromosomal location downstream of the majority of Actinomycetales operons. Operons containing linked mkl, yrbE and mce genes, resembling the classic organization of an ABC importer, were found to be common in Gram-negative bacteria and appear to be associated with changes in properties of the cell surface. Conclusion Evidence presented suggests that the mce operons of Actinomycetales species and related operons in Gram-negative bacteria encode a subfamily of ABC uptake transporters with a possible role in remodeling the cell envelope. PMID:17324287

  1. Transcriptional and post-transcriptional regulation of pst2 operon expression in Vibrio cholerae O1.

    PubMed

    da C Leite, Daniel M; Barbosa, Livia C; Mantuano, Nathalia; Goulart, Carolina L; Veríssimo da Costa, Giovani C; Bisch, Paulo M; von Krüger, Wanda M A

    2017-02-27

    One of the most abundant proteins in V. cholerae O1 cells grown under inorganic phosphate (Pi) limitation is PstS, the periplasmic Pi-binding component of the high-affinity Pi transport system Pst2 (PstSCAB), encoded in pst2 operon (pstS-pstC2-pstA2-pstB2). Besides its role in Pi uptake, Pst2 has been also associated with V. cholerae virulence. However, the mechanisms regulating pst2 expression and the non-stoichiometric production of the Pst2 components under Pi-limitation are unknown. A computational-experimental approach was used to elucidate the regulatory mechanisms behind pst2 expression in V. cholerae O1. Bioinformatics analysis of pst2 operon nucleotide sequence revealed start codons for pstS and pstC genes distinct from those originally annotated, a regulatory region upstream pstS containing potential PhoB-binding sites and a pstS-pstC intergenic region longer than predicted. Analysis of nucleotide sequence between pstS-pstC revealed inverted repeats able to form stem-loop structures followed by a potential RNAse E-cleavage site. Another putative RNase E recognition site was identified within the pstA-pstB intergenic sequence. In silico predictions of pst2 operon expression regulation were subsequently tested using cells grown under Pi limitation by promoter-lacZ fusion, gel electrophoresis mobility shift assay and quantitative RT-PCR. The experimental and in silico results matched very well and led us to propose a pst2 promoter sequence upstream of pstS gene distinct from the previously annotated. Furthermore, V. cholerae O1 pst2 operon transcription is PhoB-dependent and generates a polycistronic mRNA molecule that is rapidly processed into minor transcripts of distinct stabilities. The most stable was the pstS-encoding mRNA, which correlates with PstS higher levels relative to other Pst2 components in Pi-starved cells. The relatively higher stability of pstS and pstB transcripts seems to rely on the secondary structures at their 3' untranslated regions

  2. 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.

  3. Bacterial cells carrying synthetic dual-function operon survived starvation.

    PubMed

    Matsumoto, Yuki; Ito, Yoichiro; Tsuru, Saburo; Ying, Bei-Wen; Yomo, Tetsuya

    2011-01-01

    A synthetic dual-function operon with a bistable structure was designed and successfully integrated into the bacterial genome. Bistability was generated by the mutual inhibitory structure comprised of the promoters P(tet) and P(lac) and the repressors LacI and TetR. Dual function essential for cell growth was introduced by replacing the genes (i.e., hisC and leuB) encoding proteins involved in the biosynthesis of histidine and leucine from their native chromosomal locations to the synthetic operon. Both colony formation and population dynamics of the cells carrying this operon showed that the cells survived starvation and the newly formed population transited between the two stable states, representing the induced hisC and leuB levels, in accordance with the nutritional status. The results strongly suggested that the synthetic design of proto-operons sensitive to external perturbations is practical and functional in native cells.

  4. yadBC of Yersinia pestis, a new virulence determinant for bubonic plague.

    PubMed

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

    2008-02-01

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

  5. Characterization of the rrnB operon of the plant pathogen Rhodococcus fascians and targeted integrations of exogenous genes at rrn loci.

    PubMed

    Pisabarro, A; Correia, A; Martín, J F

    1998-04-01

    A 6.0-kb SalI DNA fragment containing an entire rRNA operon (rrnB) was cloned from a cosmid gene bank of the phytopathogenic strain Rhodococcus fascians D188. The nucleotide sequence of the 6-kb fragment was determined and had the organization 16S rRNA-spacer-23S rRNA-spacer-5S rRNA without tRNA-encoding genes in the spacer regions. The 5' and 3' ends of the mature 16S, 23S, and 5S rRNAs were determined by alignment with the rrn operons of Bacillus subtilis and other gram-positive bacteria. Four copies of the rrn operons were identified by hybridization with an rrnB probe in R. fascians type strain ATCC 12974 and in the virulent strain R. fascians D188. However, another isolate, CECT 3001 (= NRRL B15096), also classified as R. fascians, produced five rrn-hybridizing bands. An integrative vector containing a 2.5-kb DNA fragment internal to rrnB was constructed for targeted integration of exogenous genes at the rrn loci. Transformants carrying the exogenous chloramphenicol resistance gene (cmr) integrated in different rrn operons were obtained. These transformants had normal growth rates in complex medium and minimal medium and were fully stable for the integrated marker.

  6. Structural basis for converting a general transcription factor into an operon-specific virulence regulator.

    PubMed

    Belogurov, Georgiy A; Vassylyeva, Marina N; Svetlov, Vladimir; Klyuyev, Sergiy; Grishin, Nick V; Vassylyev, Dmitry G; Artsimovitch, Irina

    2007-04-13

    RfaH, a paralog of the general transcription factor NusG, is recruited to elongating RNA polymerase at specific regulatory sites. The X-ray structure of Escherichia coli RfaH reported here reveals two domains. The N-terminal domain displays high similarity to that of NusG. In contrast, the alpha-helical coiled-coil C domain, while retaining sequence similarity, is strikingly different from the beta barrel of NusG. To our knowledge, such an all-beta to all-alpha transition of the entire domain is the most extreme example of protein fold evolution known to date. Both N domains possess a vast hydrophobic cavity that is buried by the C domain in RfaH but is exposed in NusG. We propose that this cavity constitutes the RNA polymerase-binding site, which becomes unmasked in RfaH only upon sequence-specific binding to the nontemplate DNA strand that triggers domain dissociation. Finally, we argue that RfaH binds to the beta' subunit coiled coil, the major target site for the initiation sigma factors.

  7. Structural Basis for Converting a General Transcription Factor into an Operon-Specific Virulence Regulator

    PubMed Central

    Belogurov, Georgiy A.; Vassylyeva, Marina N.; Svetlov, Vladimir; Klyuyev, Sergiy; Grishin, Nick V.; Vassylyev, Dmitry G.; Artsimovitch, Irina

    2011-01-01

    SUMMARY RfaH, a paralog of the general transcription factor NusG, is recruited to elongating RNA polymerase at specific regulatory sites. The X-ray structure of Escherichia coli RfaH reported here reveals two domains. The N-terminal domain displays high similarity to that of NusG. In contrast, the α-helical coiled-coil C domain, while retaining sequence similarity, is strikingly different from the β barrel of NusG. To our knowledge, such an all-β to all-α transition of the entire domain is the most extreme example of protein fold evolution known to date. Both N domains possess a vast hydrophobic cavity that is buried by the C domain in RfaH but is exposed in NusG. We propose that this cavity constitutes the RNA polymerase-binding site, which becomes unmasked in RfaH only upon sequence-specific binding to the nontemplate DNA strand that triggers domain dissociation. Finally, we argue that RfaH binds to the β′ subunit coiled coil, the major target site for the initiation σ factors. PMID:17434131

  8. Environmental temperature regulates transcription of a virulence pili operon in E. coli.

    PubMed Central

    Göransson, M; Uhlin, B E

    1984-01-01

    The expression in Escherichia coli K-12 of a pilus-adhesion determinant, obtained through molecular cloning from a pyelonephritic E. coli isolate, was studied at different temperatures. Strain HB101 carrying the recombinant plasmid pRHU845 agglutinated human erythrocytes after growth at 37 degrees C but not after growth at 22 degrees C. Quantitation of pilus subunit protein by an enzyme-linked immunosorbent assay (ELISA) for pilus antigen showed that synthesis of the pilus subunits was reduced at least 20-fold at 22 degrees C as compared with 37 degrees C. The 5' end of the pilus subunit structural gene, papA, was fused to the lacZ gene such that expression could be monitored at both translational and transcriptional levels. Measurements of beta-galactosidase production by the papA-lacZ hybrids provided evidence for thermoregulation of papA gene transcription. A regulatory determinant was localized to a 2-kb EcoRI-HindIII fragment encoding the papB gene and part of papA although none of the presently known pap gene products seem to be directly involved in a thermoregulatory mechanism. Comparison with other thermoregulatory systems in E. coli suggests that pap gene expression is regulated by a novel mechanism. PMID:6151898

  9. An ent-kaurene-derived diterpenoid virulence factor from Xanthomonas oryzae pv. oryzicola.

    PubMed

    Lu, Xuan; Hershey, David M; Wang, Li; Bogdanove, Adam J; Peters, Reuben J

    2015-04-01

    Both plants and fungi produce ent-kaurene as a precursor to the gibberellin plant hormones. A number of rhizobia contain functionally conserved, sequentially acting ent-copalyl diphosphate and ent-kaurene synthases (CPS and KS, respectively), which are found within a well-conserved operon that may lead to the production of gibberellins. Intriguingly, the rice bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc) contains a homologous operon. Here, we report biochemical characterization of the encoded CPS and KS, and the impact of insertional mutagenesis on virulence and the plant defense response for these genes, as well as that for one of the cytochromes P450 (CYP112) found in the operon. Activity of the CPS and KS found in this phytopathogen was verified - that is, Xoc is capable of producing ent-kaurene. Moreover, knocking out CPS, KS or CYP112 led to mutant Xoc that exhibited reduced virulence. Investigation of the effect on marker gene transcript levels suggests that the Xoc diterpenoid affects the plant defense response, most directly that mediated by jasmonic acid (JA). Xoc produces an ent-kaurene-derived diterpenoid as a virulence factor, potentially a gibberellin phytohormone, which is antagonistic to JA, consistent with the recent recognition of opposing effects for these phytohormones on the microbial defense response.

  10. Correlation of the virulence of Klebsiella pneumoniae K1 and K2 with the presence of a plasmid encoding aerobactin.

    PubMed

    Nassif, X; Sansonetti, P J

    1986-12-01

    Nine isolates of Klebsiella pneumoniae belonging to capsular serotypes K1 and K2 were assayed for virulence in mice. Virulent isolates (50% lethal dose of less than 10(3) microorganisms) and avirulent isolates (50% lethal dose of over 10(6) microorganisms) were selected. Supplementation of a defined minimal medium with transferrin markedly reduced the growth of avirulent strains but had no significant effect on the growth of virulent strains. All isolates produced enterochelin, but only production of aerobactin could be correlated with virulence. The genes encoding aerobactin and its receptor protein were located on a 180-kilobase plasmid. They were cloned into the mobilizable vector pSUP202. Homology was demonstrated with the aerobactin operon of the Escherichia coli plasmid pColV-K30. Transfer of the recombinant plasmid pKP4 into an avirulent recipient enhanced virulence by 100-fold. These experiments demonstrated that aerobactin is an essential factor of pathogenicity in K. pneumoniae.

  11. 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.

  12. Virulence factors and bacteriocins in faecal enterococci of wild boars.

    PubMed

    Poeta, Patricia; Igrejas, Gilberto; Costa, Daniela; Sargo, Roberto; Rodrigues, Jorge; Torres, Carmen

    2008-10-01

    The production of antimicrobial, haemolytic and gelatinase activities was tested in 67 enterococci (39 E. faecium, 24 E. hirae, 2 E. faecalis, and 2 Enterococcus spp.), recovered from faecal samples of wild boars. In addition, the presence of genes encoding bacteriocin and virulence factors was also analysed by PCR and sequencing. Production of antimicrobial activity was checked in all enterococci against 9 indicator bacteria and it was detected in 11 E. faecium isolates (16.5%); eight and two of them harboured the genes encoding enterocin A + enterocin B and enterocin L50A/B, respectively. Sixty-seven per cent of our enterococci harboured different combinations of genes of the cyl operon, but none of them contained the complete cyl L(L)L(S)ABM operon, necessary for cytolysin expression. The presence of gel E gene, associated with the fsr ABC locus, was identified in 4 E. faecium and two E. faecalis isolates, exhibiting all of them gelatinase activity. beta -hemolytic activity was not found in our isolates. Both cpd and ace genes, encoding respectively the accessory colonisation factor and pheromone, were detected in two E. faecalis isolates, and the hyl gene, encoding hyalorunidase, in two E. faecium isolates, one of them gelatinase-positive. Genes encoding bacteriocins and virulence factors are widely disseminated among faecal enterococci of wild boars and more studies should be carried out to know the global distribution of these determinants in enterococci of different ecosystems.

  13. 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.

  14. Staphylococcus aureus ArcR controls expression of the arginine deiminase operon.

    PubMed

    Makhlin, Julia; Kofman, Tzili; Borovok, Ilya; Kohler, Christian; Engelmann, Susanne; Cohen, Gerald; Aharonowitz, Yair

    2007-08-01

    We identified a single open reading frame that is strongly similar to ArcR, a member of the Crp/Fnr family of bacterial transcriptional regulators, in all sequenced Staphylococcus aureus genomes. The arcR gene encoding ArcR forms an operon with the arginine deiminase (ADI) pathway genes arcABDC that enable the utilization of arginine as a source of energy for growth under anaerobic conditions. In this report, we show that under anaerobic conditions, S. aureus growth is subject to glucose catabolic repression and is enhanced by arginine. Likewise, glucose and arginine have reciprocal effects on the transcription of the arcABDCR genes. Furthermore, we show using a mutant deleted for arcR that the transcription of the arc operon under anaerobic conditions depends strictly on a functional ArcR. These findings are supported by proteome analyses, which showed that under anaerobic conditions the expression of the ADI catabolic proteins depends on ArcR. Bioinformatic analysis of S. aureus ArcR predicts an N-terminal nucleotide binding domain and a C-terminal helix-turn-helix DNA binding motif. ArcR binds to a conserved Crp-like sequence motif, TGTGA-N(6)-TCACA, present in the arc promoter region and thereby activates the expression of the ADI pathway genes. Crp-like sequence motifs were also found in the regulatory regions of some 30 other S. aureus genes mostly encoding anaerobic enzymatic systems, virulence factors, and regulatory systems. ArcR was tested and found to bind to the regulatory regions of four such genes, adh1, lctE, srrAB, and lukM. In one case, for lctE, encoding l-lactate dehydrogenase, ArcR was able to bind only in the presence of cyclic AMP. These observations suggest that ArcR is likely to play an important role in the expression of numerous genes required for anaerobic growth.

  15. Operon Gene Order Is Optimized for Ordered Protein Complex Assembly.

    PubMed

    Wells, Jonathan N; Bergendahl, L Therese; Marsh, Joseph A

    2016-02-02

    The assembly of heteromeric protein complexes is an inherently stochastic process in which multiple genes are expressed separately into proteins, which must then somehow find each other within the cell. Here, we considered one of the ways by which prokaryotic organisms have attempted to maximize the efficiency of protein complex assembly: the organization of subunit-encoding genes into operons. Using structure-based assembly predictions, we show that operon gene order has been optimized to match the order in which protein subunits assemble. Exceptions to this are almost entirely highly expressed proteins for which assembly is less stochastic and for which precisely ordered translation offers less benefit. Overall, these results show that ordered protein complex assembly pathways are of significant biological importance and represent a major evolutionary constraint on operon gene organization.

  16. 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.

  17. 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

  18. Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

    PubMed Central

    Wong, H C; Fear, A L; Calhoon, R D; Eichinger, G H; Mayer, R; Amikam, D; Benziman, M; Gelfand, D H; Meade, J H; Emerick, A W

    1990-01-01

    An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum. Images PMID:2146681

  19. 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.

  20. The vapA co-expressed virulence plasmid gene vcgB (orf10) of the intracellular actinomycete Rhodococcus equi.

    PubMed

    Miranda-Casoluengo, Raúl; Miranda-Casoluengo, Aleksandra A; O'Connell, Enda P; Fahey, Ruth J; Boland, Clara A; Vázquez-Boland, Jose A; Meijer, Wim G

    2011-08-01

    The virulence plasmid of the pathogenic actinomycete Rhodococcus equi is essential for proliferation of this pathogen in macrophages and the development of disease. The pathogenicity island of this plasmid encodes a family of virulence-associated proteins (Vap), one of which (VapA) is a virulence factor. This paper describes the vcgAB operon (vapA co-expressed gene), located upstream of the vapA operon. Transcription of the vcgAB operon gave rise to transcripts with a half-life similar to those determined for other virulence plasmid genes (1.8 min). Transcription started at a promoter similar to the vapA promoter, and proceeded through an inefficient terminator into the downstream vcgC gene. In addition, vcgC is also transcribed from a promoter downstream of vcgB. The vcgAB and vapA operons were coordinately regulated by temperature and pH in a synergistic manner. The latter parameter only affected transcription at higher growth temperatures, indicating that temperature is the dominant regulatory signal. Transcription of the vcgAB operon increased 10-fold during the late exponential and stationary growth phases. Transcription was also upregulated during the initial hours following phagocytosis by phagocytic cells. In contrast to vcgA and vcgC, the vcgB gene is conserved in the porcine VapB-encoding plasmid, as well as in pathogenic mycobacteria. The coordinated regulation of vcgB and vapA, transcription of vcgB following phagocytosis and conservation of vcgB in pathogenic mycobacteria indicate a role for vcgB and the vcg genes in the virulence of R. equi.

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. Modeling network dynamics: the lac operon, a case study.

    PubMed

    Vilar, José M G; Guet, Călin C; Leibler, Stanislas

    2003-05-12

    We use the lac operon in Escherichia coli as a prototype system to illustrate the current state, applicability, and limitations of modeling the dynamics of cellular networks. We integrate three different levels of description (molecular, cellular, and that of cell population) into a single model, which seems to capture many experimental aspects of the system.

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

    PubMed

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

    2015-01-01

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

  7. Prevalence of the ica operon and insertion sequence IS256 among Staphylococcus epidermidis prosthetic joint infection isolates.

    PubMed

    Koskela, A; Nilsdotter-Augustinsson, A; Persson, L; Söderquist, B

    2009-06-01

    Joint replacement surgery has improved the quality of life for hundreds of thousands of patients. However, the infection of a joint implant is an important and serious complication, though the prevalence is low. Staphylococcus epidermidis is the most important pathogen involved in foreign-body infections. S. epidermidis is also a commensal that comprises a substantial part of the normal skin flora of humans. The possibility to demonstrate potential specific virulence markers may facilitate the interpretation of the bacteriological findings, as well as the clinical decision. The prevalence of the ica locus and insertion sequence IS256 by using polymerase chain reaction (PCR) among 32 clinical S. epidermidis isolates from prosthetic joint infections (PJIs) and 24 commensal isolates from nares and skin was investigated. Sixteen (50%) of the 32 PJI isolates harbored the ica operon compared with one-third of the commensal isolates obtained from the samples of the skin and nares of healthy individuals. The IS256 was demonstrated in 26 (81%) out of 32 PJI isolates. By contrast, IS256 was found in one of 24 commensal isolates. In conclusion, IS256 may be superior to the ica operon as a marker of the invasive capacity of S. epidermidis, since it was found in most of the PJI isolates, but rarely among commensals.

  8. 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

  9. Molecular Mechanism of Quorum-Sensing in Enterococcus faecalis: Its Role in Virulence and Therapeutic Approaches.

    PubMed

    Ali, Liaqat; Goraya, Mohsan Ullah; Arafat, Yasir; Ajmal, Muhammad; Chen, Ji-Long; Yu, Daojin

    2017-05-03

    Quorum-sensing systems control major virulence determinants in Enterococcusfaecalis, which causes nosocomial infections. The E. faecalis quorum-sensing systems include several virulence factors that are regulated by the cytolysin operon, which encodes the cytolysin toxin. In addition, the E. faecalis Fsr regulator system controls the expression of gelatinase, serine protease, and enterocin O16. The cytolysin and Fsr virulence factor systems are linked to enterococcal diseases that affect the health of humans and other host models. Therefore, there is substantial interest in understanding and targeting these regulatory pathways to develop novel therapies for enterococcal infection control. Quorum-sensing inhibitors could be potential therapeutic agents for attenuating the pathogenic effects of E. faecalis. Here, we discuss the regulation of cytolysin, the LuxS system, and the Fsr system, their role in E. faecalis-mediated infections, and possible therapeutic approaches to prevent E. faecalis infection.

  10. Molecular Mechanism of Quorum-Sensing in Enterococcus faecalis: Its Role in Virulence and Therapeutic Approaches

    PubMed Central

    Ali, Liaqat; Goraya, Mohsan Ullah; Arafat, Yasir; Ajmal, Muhammad; Chen, Ji-Long; Yu, Daojin

    2017-01-01

    Quorum-sensing systems control major virulence determinants in Enterococcus faecalis, which causes nosocomial infections. The E. faecalis quorum-sensing systems include several virulence factors that are regulated by the cytolysin operon, which encodes the cytolysin toxin. In addition, the E. faecalis Fsr regulator system controls the expression of gelatinase, serine protease, and enterocin O16. The cytolysin and Fsr virulence factor systems are linked to enterococcal diseases that affect the health of humans and other host models. Therefore, there is substantial interest in understanding and targeting these regulatory pathways to develop novel therapies for enterococcal infection control. Quorum-sensing inhibitors could be potential therapeutic agents for attenuating the pathogenic effects of E. faecalis. Here, we discuss the regulation of cytolysin, the LuxS system, and the Fsr system, their role in E. faecalis-mediated infections, and possible therapeutic approaches to prevent E. faecalis infection. PMID:28467378

  11. 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.

  12. 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

  13. 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

  14. 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

  15. CodY orchestrates the expression of virulence determinants in emetic Bacillus cereus by impacting key regulatory circuits.

    PubMed

    Frenzel, Elrike; Doll, Viktoria; Pauthner, Matthias; Lücking, Genia; Scherer, Siegfried; Ehling-Schulz, Monika

    2012-07-01

    Bacillus cereus causes gastrointestinal diseases and local and systemic infections elicited by the depsipeptide cereulide, enterotoxins, phospholipases, cytolysins and proteases. The PlcR-PapR quorum sensing system activates the expression of several virulence factors, whereas the Spo0A-AbrB regulatory circuit partially controls the plasmid-borne cereulide synthetase (ces) operon. Here, we show that CodY, a nutrient-responsive regulator of Gram-positive bacteria, has a profound effect on both regulatory systems, which have been assumed to operate independently of each other. Deletion of codY resulted in downregulation of virulence genes belonging to the PlcR regulon and a concomitant upregulation of the ces genes. CodY was found to be a repressor of the ces operon, but did not interact with the promoter regions of PlcR-dependent virulence genes in vitro, suggesting an indirect regulation of the latter. Furthermore, CodY binds to the promoter of the immune inhibitor metalloprotease InhA1, demonstrating that CodY directly links B. cereus metabolism to virulence. In vivo studies using a Galleria mellonella infection model, showed that the codY mutant was substantially attenuated, highlighting the importance of CodY as a key regulator of pathogenicity. Our results demonstrate that CodY profoundly modulates the virulence of B. cereus, possibly controlling the development of pathogenic traits in suitable host environments.

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

    PubMed

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

    2015-01-01

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

  17. Contribution of Salmonella typhimurium Virulence Factors to Diarrheal Disease in Calves

    PubMed Central

    Tsolis, Renée M.; Adams, L. Garry; Ficht, Thomas A.; Bäumler, Andreas J.

    1999-01-01

    Limited knowledge is available about the virulence mechanisms responsible for diarrheal disease caused by Salmonella typhimurium. To assess the contribution to diarrheal disease of virulence determinants identified in models of infection, we tested a collection of S. typhimurium mutants for their ability to cause enteritis in calves. S. typhimurium strains carrying mutations in the virulence plasmid (spvR), Salmonella pathogenicity island 2 (SPI-2) (spiB), or SPI-5 (sopB) caused mortality and acute diarrhea in calves. An S. typhimurium rfaJ mutant, which is defective for lipopolysaccharide outer core biosynthesis, was of intermediate virulence. Mutations in SPI-1 (hilA and prgH) or aroA markedly reduced virulence and the severity of diarrhea. Furthermore, histopathological examination of calves infected with SPI-1 or aroA mutants revealed a marked reduction or absence of intestinal lesions. These data suggest that virulence factors, such as SPI-1, which are required during intestinal colonization are more important for pathogenicity in calves than are genes required during the systemic phase of S. typhimurium infection, including SPI-2 or the spv operon. This is in contrast to the degree of attenuation caused by these mutations in the mouse. PMID:10456944

  18. 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

  19. Design and characterisation of synthetic operons for biohydrogen technology.

    PubMed

    Lamont, Ciaran M; Sargent, Frank

    2017-04-01

    Biohydrogen is produced by a number of microbial systems and the commonly used host bacterium Escherichia coli naturally produces hydrogen under fermentation conditions. One approach to engineering additional hydrogen production pathways is to introduce non-native hydrogenases into E. coli. An attractive candidate is the soluble [NiFe]-hydrogenase from Ralstonia eutropha, which has been shown to link NADH/NAD(+) biochemistry directly to hydrogen metabolism, an activity that E. coli does not perform. In this work, three synthetic operons were designed that code for the soluble hydrogenase and two different enzyme maturase systems. Interestingly, using this system, the recombinant soluble hydrogenase was found to be assembled by the native E. coli [NiFe]-hydrogenase assembly machinery, and, vice versa, the synthetic maturase operons were able to complement E. coli mutants defective in hydrogenase biosynthesis. The heterologously expressed soluble hydrogenase was found to be active and was shown to produce biohydrogen in vivo.

  20. [UV-inducibility of the LT-toxin operon].

    PubMed

    Tiganova, I G; Rusina, O Iu; Andreeva, I V; Demkin, V V; Brukhanskiĭ, G V; Aleshkin, G I; Skavronskaia, A G

    1989-07-01

    The plasmid elt-operon pVZ14 was constructed by fusing of the eltoperon of the plasmid pVZ357 with the lac-gene of the bacteriophage Mud1 (Amp, Lac). lacZ gene has been proven to be fused with an elt-promoter by the loss of toxin production coded by pVZ357 and acquiring of Lac+ phenotype by pVZ14 containing cells, as well as by HindIII fragments hybridization of pVZ357 and pVZ14 with the labelled elt-probe. The kinetics of beta-galactosidase synthesis in E. coli cells harboring pVZ14 shows an elt-operon promoter to have expressed constitutive activity and to be activated by a SOS-inducing agent, UV-light.

  1. 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.

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

    PubMed

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

    2011-02-01

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

  3. Structure of the E. coli hisT Operon.

    DTIC Science & Technology

    1985-01-01

    ELEMENT NO. NO. NO. ACCESSION NO ArintoV 221-50061153N IRRO41-05 ]RR041-O5-Oj NR204-123 11. TITLE (include Security Classification) (U) STRUCTURE OF THE E ... Coli hisT OPERON ritpe C., Arps, Peggy J. and Winkler, Malcolm E. 13 YE OF RAPORT 13b. TIME CO VEE 14. DATE OF REPORT (Year Month. Day) 15. PAGE

  4. Structural characterization of the Salmonella typhimurium LT2 umu operon

    SciTech Connect

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

    1990-09-01

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

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

    PubMed

    Zhou, Chuan; Ma, Qin; Li, Guojun

    2014-01-01

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

  6. Elucidation of Operon Structures across Closely Related Bacterial Genomes

    PubMed Central

    Li, Guojun

    2014-01-01

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

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

    PubMed

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

    2015-12-29

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

  8. 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

  9. The transcription of the cbb operon in Nitrosomonas europaea.

    PubMed

    Wei, Xueming; Sayavedra-Soto, Luis A; Arp, Daniel J

    2004-06-01

    Nitrosomonas europaea is an aerobic ammonia-oxidizing bacterium that participates in the C and N cycles. N. europaea utilizes CO(2) as its predominant carbon source, and is an obligate chemolithotroph, deriving all the reductant required for energy and biosynthesis from the oxidation of ammonia (NH(3)) to nitrite (). This bacterium fixes carbon via the Calvin-Benson-Bassham (CBB) cycle via a type I ribulose bisphosphate carboxylase/oxygenase (RubisCO). The RubisCO operon is composed of five genes, cbbLSQON. This gene organization is similar to that of the operon for 'green-like' type I RubisCOs in other organisms. The cbbR gene encoding the putative regulatory protein for RubisCO transcription was identified upstream of cbbL. This study showed that transcription of cbb genes was upregulated when the carbon source was limited, while amo, hao and other energy-harvesting-related genes were downregulated. N. europaea responds to carbon limitation by prioritizing resources towards key components for carbon assimilation. Unlike the situation for amo genes, NH(3) was not required for the transcription of the cbb genes. All five cbb genes were only transcribed when an external energy source was provided. In actively growing cells, mRNAs from the five genes in the RubisCO operon were present at different levels, probably due to premature termination of transcription, rapid mRNA processing and mRNA degradation.

  10. Analysis of the gluconate (gnt) operon of Bacillus subtilis.

    PubMed

    Reizer, A; Deutscher, J; Saier, M H; Reizer, J

    1991-05-01

    The gluconate (gnt) operon of Bacillus subtilis includes the gntR, gntK, gntP, and gntZ genes, respectively encoding the transcriptional repressor of the operon, gluconate kinase, the gluconate permease, and an unidentified open reading frame (Fujita and Fujita, 1987). We have compared the proteins encoded by the gnt operon of B.subtilis with published sequences and showed that (i) the gluconate repressor is homologous to several putative regulatory proteins in Escherichia coli, (ii) the gluconate kinase of B. subtilis is homologous to xylulose kinase, glycerol kinase and fucose kinase in E. coli (20-26% identity; 12-59 S.D.), (iii) the gluconate permease exhibits a C-terminal domain which is homologous to a hydrophobic protein encoded by an unidentified open reading frame (dsdAp) which precedes the dsdA gene of E. coli (39% identity; 19 S.D.), and (iv) the gntZ gene product is homologous to 6-phosphogluconate dehydrogenases of other bacteria and of animals (48-56%; 82-178 S.D.), thereby suggesting that the B. subtilis gntZ encodes 6-phosphogluconate dehydrogenase. Several conserved regions of the sequenced 6-phosphogluconate dehydrogenases can serve as signature patterns of this protein. Computer analyses have indicated that the previously reported sequences of the porcine and ovine 6-phosphogluconate dehydrogenases, as well as the hypothetical DsdAp protein, are probably erroneous. The probable reasons for the errors are reported along with the proposed revised sequences.

  11. Expression of an entire bacterial operon in plants.

    PubMed

    Mozes-Koch, Rita; Gover, Ofer; Tanne, Edna; Peretz, Yuval; Maori, Eyal; Chernin, Leonid; Sela, Ilan

    2012-04-01

    Multigene expression is required for metabolic engineering, i.e. coregulated expression of all genes in a metabolic pathway for the production of a desired secondary metabolite. To that end, several transgenic approaches have been attempted with limited success. Better success has been achieved by transforming plastids with operons. IL-60 is a platform of constructs driven from the geminivirus Tomato yellow leaf curl virus. We demonstrate that IL-60 enables nontransgenic expression of an entire bacterial operon in tomato (Solanum lycopersicum) plants without the need for plastid (or any other) transformation. Delivery to the plant is simple, and the rate of expressing plants is close to 100%, eliminating the need for selectable markers. Using this platform, we show the expression of an entire metabolic pathway in plants and delivery of the end product secondary metabolite (pyrrolnitrin). Expression of this unique secondary metabolite resulted in the appearance of a unique plant phenotype disease resistance. Pyrrolnitrin production was already evident 2 d after application of the operon to plants and persisted throughout the plant's life span. Expression of entire metabolic pathways in plants is potentially beneficial for plant improvement, disease resistance, and biotechnological advances, such as commercial production of desired metabolites.

  12. Prevalence of transcription promoters within archaeal operons and coding sequences

    PubMed Central

    Koide, Tie; Reiss, David J; Bare, J Christopher; Pang, Wyming Lee; Facciotti, Marc T; Schmid, Amy K; Pan, Min; Marzolf, Bruz; Van, Phu T; Lo, Fang-Yin; Pratap, Abhishek; Deutsch, Eric W; Peterson, Amelia; Martin, Dan; Baliga, Nitin S

    2009-01-01

    Despite the knowledge of complex prokaryotic-transcription mechanisms, generalized rules, such as the simplified organization of genes into operons with well-defined promoters and terminators, have had a significant role in systems analysis of regulatory logic in both bacteria and archaea. Here, we have investigated the prevalence of alternate regulatory mechanisms through genome-wide characterization of transcript structures of ∼64% of all genes, including putative non-coding RNAs in Halobacterium salinarum NRC-1. Our integrative analysis of transcriptome dynamics and protein–DNA interaction data sets showed widespread environment-dependent modulation of operon architectures, transcription initiation and termination inside coding sequences, and extensive overlap in 3′ ends of transcripts for many convergently transcribed genes. A significant fraction of these alternate transcriptional events correlate to binding locations of 11 transcription factors and regulators (TFs) inside operons and annotated genes—events usually considered spurious or non-functional. Using experimental validation, we illustrate the prevalence of overlapping genomic signals in archaeal transcription, casting doubt on the general perception of rigid boundaries between coding sequences and regulatory elements. PMID:19536208

  13. Tryptophan operon regulation in interspecific hybrids of enteric bacteria.

    PubMed Central

    Manson, M D; Yanofsky, C

    1976-01-01

    We examined tryptophan regulation in merodiploid hybrids in which a plasmid carrying the trp operon of Escherichia was introduced into Trp mutants of other enteric genera, or in which a plasmid carrying the trpR+ (repressor) gene of E. coli was transfered into fully constitutive trpR mutants of other genera. In these hybrids the trp operon of one species is controlled by the repressor of a different species. Similar investigations were possible in transduction hybrids in which either the trp operon or the trpR+ locus of Shigella dysenteriae was introduced into E. coli. Our measurements of trp enzymes levels in repressed and nonrepressed cells indicate that Trp regulation is normal, with only minor quantitative variations, in hybrids between E coli and Shigella dysenteriae, Salmonella typhimurium, Klebsiella aerogenes, Serratia marcescens, and Proteus mirabilis. Our results support the idea that a repressor-operator mechanism for regulating trp messenger ribonucleic acid production evolved in a common ancestor of the enteric bacteria, and that this repressor-operator recognition has been conversed during the evolutionary divergence of the Enterobacteriaceae. PMID:770450

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

    PubMed

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

    2016-01-01

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

  15. GapA and CrmA Coexpression Is Essential for Mycoplasma gallisepticum Cytadherence and Virulence

    PubMed Central

    Papazisi, L.; Frasca Jr., S.; Gladd, M.; Liao, X.; Yogev, D.; Geary, S. J.

    2002-01-01

    It was previously demonstrated that avirulent Mycoplasma gallisepticum strain Rhigh (passage 164) is lacking three proteins that are expressed in its virulent progenitor, strain Rlow (passage 15). These proteins were identified as the cytadhesin molecule GapA, the putative cytadhesin-related molecule CrmA, and a component of a high-affinity transporter system, HatA. Complementation of Rhigh with wild-type gapA restored expression in the transformant (GT5) but did not restore the cytadherence phenotype and maintained avirulence in chickens. These results suggested that CrmA might play an essential role in the M. gallisepticum cytadherence process. CrmA is encoded by the second gene in the gapA operon and shares significant sequence homology to the ORF6 gene of Mycoplasma pneumoniae, which has been shown to play an accessory role in the cytadherence process. Complementation of Rhigh with wild-type crmA resulted in the transformant (SDCA) that lacked the cytadherence and virulence phenotype comparable to that found in Rhigh and GT5. In contrast, complementation of Rhigh with the entire wild-type gapA operon resulted in the transformant (GCA1) that restored cytadherence to the level found in wild-type Rlow. In vivo pathogenesis trials revealed that GCA1 had regained virulence, causing airsacculitis in chickens. These results demonstrate that both GapA and CrmA are required for M. gallisepticum cytadherence and pathogenesis. PMID:12438360

  16. A trans-acting leader RNA from a Salmonella virulence gene.

    PubMed

    Choi, Eunna; Han, Yoontak; Cho, Yong-Joon; Nam, Daesil; Lee, Eun-Jin

    2017-09-05

    Bacteria use flagella to move toward nutrients, find its host, or retract from toxic substances. Because bacterial flagellum is one of the ligands that activate the host innate immune system, its synthesis should be tightly regulated during host infection, which is largely unknown. Here, we report that a bacterial leader mRNA from the mgtCBR virulence operon in the intracellular pathogen Salmonella enterica serovar Typhimurium binds to the fljB coding region of mRNAs in the fljBA operon encoding the FljB phase 2 flagellin, a main component of bacterial flagella and the FljA repressor for the FliC phase 1 flagellin, and degrades fljBA mRNAs in an RNase E-dependent fashion during infection. A nucleotide substitution of the fljB flagellin gene that prevents the mgtC leader RNA-mediated down-regulation increases the fljB-encoded flagellin synthesis, leading to a hypermotile phenotype inside macrophages. Moreover, the fljB nucleotide substitution renders Salmonella hypervirulent, indicating that FljB-based motility must be compromised in the phagosomal compartment where Salmonella resides. This suggests that this pathogen promotes pathogenicity by producing a virulence protein and limits locomotion by a trans-acting leader RNA from the same virulence gene during infection.

  17. The tryptophan pathway genes of the Sargasso Sea metagenome: new operon structures and the prevalence of non-operon organization

    PubMed Central

    Kagan, Juliana; Sharon, Itai; Beja, Oded; Kuhn, Jonathan C

    2008-01-01

    Background The enormous database of microbial DNA generated from the Sargasso Sea metagenome provides a unique opportunity to locate genes participating in different biosynthetic pathways and to attempt to understand the relationship and evolution of those genes. In this article, an analysis of the Sargasso Sea metagenome is made with respect to the seven genes of the tryptophan pathway. Results At least 5% of all the genes that are related to amino acid biosynthesis are tryptophan (trp) genes. Many contigs and scaffolds contain whole or split operons that are similar to previously analyzed trp gene organizations. Only two scaffolds discovered in this analysis possess a different operon organization of tryptophan pathway genes than those previously known. Many marine organisms lack an operon-type organization of these genes or have mini-operons containing only two trp genes. In addition, the trpB genes from this search reveal that the dichotomous division between trpB_1 and trpB_2 also occurs in organisms from the Sargasso Sea. One cluster was found to contain trpB sequences that were closely related to each other but distinct from most known trpB sequences. Conclusion The data show that trp genes are widely dispersed within this metagenome. The novel organization of these genes and an unusual group of trpB_1 sequences that were found among some of these Sargasso Sea bacteria indicate that there is much to be discovered about both the reason for certain gene orders and the regulation of tryptophan biosynthesis in marine bacteria. PMID:18221558

  18. 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

  19. The effector overlap between the lac and mel operons of Escherichia coli: Induction of the mel operon with β-galactosides.

    PubMed

    Narang, Atul; Oehler, Stefan

    2017-02-13

    The lac (lactose) operon (processes β-galactosides) and the mel (melibiose) operon (processes α-galactosides) of Escherichia coli have a close historical connection. A number of shared substrates and effectors of the permeases and regulatory proteins has been reported over the years. Up to now, β-thiogalactosides like TMG (methyl-β-D-thiogalactopyranoside) and IPTG (isopropyl-β-D-thiogalactopyranoside) are generally not considered inducers of the mel operon. The same is true for β-galactosides like lactose (β-D-galactopyranosyl-(1→4)-D-glucose), which is a substrate, but itself not an inducer of the lac operon. This report shows that all three sugars can induce the mel operon significantly, when they are accumulated in the cell by Lac permease. Strong induction by the gratuitous β-thiogalactosides is observed in the presence of Lac permease and strong induction by lactose (more than 200-fold) in the absence of β-galactosidase. This finding calls for re-evaluation of TMG uptake experiments as assays for Lac permease that were performed with mel(+) strains.IMPORTANCE The typical textbook picture of bacterial operons is that of stand-alone units of genetic information that perform, in a regulated manner, well-defined cellular functions. Less attention is given to the extensive interactions that can be found between operons. One well-described example of such interactions are the effector molecules shared by the lac and mel operons. It is here shown that this set has to be extended to include β-galactosides, which have been, until now, considered not to effect expression of the mel operon. That they can be inducers of the mel as well as the lac operon has not been noticed in decades of research, because of the E. coli genetic background used in previous studies.

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

    PubMed Central

    Island, M D; Mobley, H L

    1995-01-01

    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

  1. Transient virulence of emerging pathogens.

    PubMed

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

    2010-05-06

    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.

  2. Transient virulence of emerging pathogens

    PubMed Central

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

    2010-01-01

    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

  3. 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

  4. Activation of CpxRA in Haemophilus ducreyi primarily inhibits the expression of its targets, including major virulence determinants.

    PubMed

    Gangaiah, Dharanesh; Zhang, Xinjun; Fortney, Kate R; Baker, Beth; Liu, Yunlong; Munson, Robert S; Spinola, Stanley M

    2013-08-01

    Haemophilus ducreyi causes chancroid, a genital ulcer disease that facilitates the transmission of human immunodeficiency virus type 1. In humans, H. ducreyi is surrounded by phagocytes and must adapt to a hostile environment to survive. To sense and respond to environmental cues, bacteria frequently use two-component signal transduction (2CST) systems. The only obvious 2CST system in H. ducreyi is CpxRA; CpxR is a response regulator, and CpxA is a sensor kinase. Previous studies by Hansen and coworkers showed that CpxR directly represses the expression of dsrA, the lspB-lspA2 operon, and the flp operon, which are required for virulence in humans. They further showed that CpxA functions predominantly as a phosphatase in vitro to maintain the expression of virulence determinants. Since a cpxA mutant is avirulent while a cpxR mutant is fully virulent in humans, CpxA also likely functions predominantly as a phosphatase in vivo. To better understand the role of H. ducreyi CpxRA in controlling virulence determinants, here we defined genes potentially regulated by CpxRA by using RNA-Seq. Activation of CpxR by deletion of cpxA repressed nearly 70% of its targets, including seven established virulence determinants. Inactivation of CpxR by deletion of cpxR differentially regulated few genes and increased the expression of one virulence determinant. We identified a CpxR binding motif that was enriched in downregulated but not upregulated targets. These data reinforce the hypothesis that CpxA phosphatase activity plays a critical role in controlling H. ducreyi virulence in vivo. Characterization of the downregulated genes may offer new insights into pathogenesis.

  5. Virulent Clones of Klebsiella pneumoniae: Identification and Evolutionary Scenario Based on Genomic and Phenotypic Characterization

    PubMed Central

    Brisse, Sylvain; Fevre, Cindy; Passet, Virginie; Issenhuth-Jeanjean, Sylvie; Tournebize, Régis; Diancourt, Laure; Grimont, Patrick

    2009-01-01

    Klebsiella pneumoniae is found in the environment and as a harmless commensal, but is also a frequent nosocomial pathogen (causing urinary, respiratory and blood infections) and the agent of specific human infections including Friedländer's pneumonia, rhinoscleroma and the emerging disease pyogenic liver abscess (PLA). The identification and precise definition of virulent clones, i.e. groups of strains with a single ancestor that are associated with particular infections, is critical to understand the evolution of pathogenicity from commensalism and for a better control of infections. We analyzed 235 K. pneumoniae isolates of diverse environmental and clinical origins by multilocus sequence typing, virulence gene content, biochemical and capsular profiling and virulence to mice. Phylogenetic analysis of housekeeping genes clearly defined clones that differ sharply by their clinical source and biological features. First, two clones comprising isolates of capsular type K1, clone CC23K1 and clone CC82K1, were strongly associated with PLA and respiratory infection, respectively. Second, only one of the two major disclosed K2 clones was highly virulent to mice. Third, strains associated with the human infections ozena and rhinoscleroma each corresponded to one monomorphic clone. Therefore, K. pneumoniae subsp. ozaenae and K. pneumoniae subsp. rhinoscleromatis should be regarded as virulent clones derived from K. pneumoniae. The lack of strict association of virulent capsular types with clones was explained by horizontal transfer of the cps operon, responsible for the synthesis of the capsular polysaccharide. Finally, the reduction of metabolic versatility observed in clones Rhinoscleromatis, Ozaenae and CC82K1 indicates an evolutionary process of specialization to a pathogenic lifestyle. In contrast, clone CC23K1 remains metabolically versatile, suggesting recent acquisition of invasive potential. In conclusion, our results reveal the existence of important virulent

  6. Interplay of Noisy Gene Expression and Dynamics Explains Patterns of Bacterial Operon Organization

    NASA Astrophysics Data System (ADS)

    Igoshin, Oleg

    2011-03-01

    Bacterial chromosomes are organized into operons -- sets of genes co-transcribed into polycistronic messenger RNA. Hypotheses explaining the emergence and maintenance of operons include proportional co-regulation, horizontal transfer of intact ``selfish'' operons, emergence via gene duplication, and co-production of physically interacting proteins to speed their association. We hypothesized an alternative: operons can reduce or increase intrinsic gene expression noise in a manner dependent on the post-translational interactions, thereby resulting in selection for or against operons in depending on the network architecture. We devised five classes of two-gene network modules and show that the effects of operons on intrinsic noise depend on class membership. Two classes exhibit decreased noise with co-transcription, two others reveal increased noise, and the remaining one does not show a significant difference. To test our modeling predictions we employed bioinformatic analysis to determine the relationship gene expression noise and operon organization. The results confirm the overrepresentation of noise-minimizing operon architectures and provide evidence against other hypotheses. Our results thereby suggest a central role for gene expression noise in selecting for or maintaining operons in bacterial chromosomes. This demonstrates how post-translational network dynamics may provide selective pressure for organizing bacterial chromosomes, and has practical consequences for designing synthetic gene networks. This work is supported by National Institutes of Health grant 1R01GM096189-01.

  7. Operon Conservation and the Evolution of trans-Splicing in the Phylum Nematoda

    PubMed Central

    Guiliano, David B; Blaxter, Mark L

    2006-01-01

    The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage. PMID:17121468

  8. Operon conservation and the evolution of trans-splicing in the phylum Nematoda.

    PubMed

    Guiliano, David B; Blaxter, Mark L

    2006-11-24

    The nematode Caenorhabditis elegans is unique among model animals in that many of its genes are cotranscribed as polycistronic pre-mRNAs from operons. The mechanism by which these operonic transcripts are resolved into mature mRNAs includes trans-splicing to a family of SL2-like spliced leader exons. SL2-like spliced leaders are distinct from SL1, the major spliced leader in C. elegans and other nematode species. We surveyed five additional nematode species, representing three of the five major clades of the phylum Nematoda, for the presence of operons and the use of trans-spliced leaders in resolution of polycistronic pre-mRNAs. Conserved operons were found in Pristionchus pacificus, Nippostrongylus brasiliensis, Strongyloides ratti, Brugia malayi, and Ascaris suum. In nematodes closely related to the rhabditine C. elegans, a related family of SL2-like spliced leaders is used for operonic transcript resolution. However, in the tylenchine S. ratti operonic transcripts are resolved using a family of spliced leaders related to SL1. Non-operonic genes in S. ratti may also receive these SL1 variants. In the spirurine nematodes B. malayi and A. suum operonic transcripts are resolved using SL1. Mapping these phenotypes onto the robust molecular phylogeny for the Nematoda suggests that operons evolved before SL2-like spliced leaders, which are an evolutionary invention of the rhabditine lineage.

  9. 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.

  10. A long-term epigenetic memory switch controls bacterial virulence bimodality

    PubMed Central

    Ronin, Irine; Katsowich, Naama; Rosenshine, Ilan; Balaban, Nathalie Q

    2017-01-01

    When pathogens enter the host, sensing of environmental cues activates the expression of virulence genes. Opposite transition of pathogens from activating to non-activating conditions is poorly understood. Interestingly, variability in the expression of virulence genes upon infection enhances colonization. In order to systematically detect the role of phenotypic variability in enteropathogenic E. coli (EPEC), an important human pathogen, both in virulence activating and non-activating conditions, we employed the ScanLag methodology. The analysis revealed a bimodal growth rate. Mathematical modeling combined with experimental analysis showed that this bimodality is mediated by a hysteretic memory-switch that results in the stable co-existence of non-virulent and hyper-virulent subpopulations, even after many generations of growth in non-activating conditions. We identified the per operon as the key component of the hysteretic switch. This unique hysteretic memory switch may result in persistent infection and enhanced host-to-host spreading. DOI: http://dx.doi.org/10.7554/eLife.19599.001 PMID:28178445

  11. 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.

  12. Nucleotide sequence of the Rhodospirillum rubrum atp operon.

    PubMed Central

    Falk, G; Hampe, A; Walker, J E

    1985-01-01

    The nucleotide sequence was determined of a 8775-base-pair region of DNA cloned from the photosynthetic non-sulphur bacterium Rhodospirillum rubrum. It contains a cluster of five genes encoding F1-ATPase subunits. The genes are arranged in the same order as F1 genes in the Escherichia coli unc operon. However, as in the related organism Rhodopseudomonas blastica, neither genes for components of F0, the membrane sector of ATP synthase, nor a homologue of the E. coli uncI gene are associated with this locus, as they are in E. coli. Images Fig. 2. PMID:2861810

  13. 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.

  14. Operon required for fruiting body development in Myxococcus xanthus.

    PubMed

    Kim, Dohee; Chung, Jinwoo; Hyun, Hyesook; Lee, Chayul; Lee, Kyoung; Cho, Kyungyun

    2009-11-01

    We have used mutational analysis to identify four genes, MXAN3553, MXAN3554, MXAN3555, and MXAN3556, constituting an operon that is essential for normal fruiting body development in Myxococcus xanthus. Deletion of MXAN3553, which encoded a hypothetical protein, resulted in delayed fruiting body development. MXAN3554 was predicted to encode a metallopeptidase, and its deletion caused fruiting body formation to fail. Inactivation of MXAN3555, which encoded a putative NtrC-type response regulator, resulted in delayed aggregation and a severe reduction in sporulation. Fruiting bodies also failed to develop with the deletion of MXAN3556, another gene encoding a hypothetical protein.

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

    PubMed

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

    2014-07-08

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

  16. 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

  17. The molybdenum formylmethanofuran dehydrogenase operon and the tungsten formylmethanofuran dehydrogenase operon from Methanobacterium thermoautotrophicum. Structures and transcriptional regulation.

    PubMed

    Hochheimer, A; Linder, D; Thauer, R K; Hedderich, R

    1996-11-15

    Methanobacterium thermoautotrophicum contains a tungsten formylmethanofuran dehydrogenase (FwdABCD) and a molybdenum formylmethanofuran dehydrogenase (FmdABC). The fwdHFGDACB operon encoding the tungsten enzyme has recently been characterized. We report here on the structure and expression of the gene cluster encoding the molybdenum enzyme. This gene cluster is composed of three open reading frames (fmdECB). The fmdB gene was found to encode the molybdopterin-dinucleotide-binding subunit harboring the enzyme's active site; FmdB is thus functionally equivalent to FwdB. fmdC encodes a protein with sequence similarity to FwdC in its N-terminal part and with sequence similarity to FwdD in its C-terminal part; FmdC is thus functionally equivalent to FwdC and FwdD. Interestingly, the fmd operon lacks a gene fmdA encoding the subunit FmdA of the molybdenum enzyme. FmdA has the same apparent molecular mass and the same N-terminal amino acid sequence as FwdA and only one DNA sequence encoding for this N-terminal amino acid sequence was found in the M. thermoautotrophicum genome. It is therefore proposed that FmdA and FwdA are encoded by the same gene namely fwdA in the fwd operon. In agreement with this proposal is the finding that fwdA is expressed constitutively: northern-blot analysis of RNA from tungstate- and molybdate-grown cells of M. thermo-autotrophicum revealed that the fwdHFGDACB gene cluster is transcribed in the presence of either molybdate or tungstate in the growth medium whereas the fmdECB gene cluster was only transcribed when molybdate was present.

  18. 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

  19. 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

  20. The D-allose operon of Escherichia coli K-12.

    PubMed Central

    Kim, C; Song, S; Park, C

    1997-01-01

    Escherichia coli K-12 can utilize D-allose, an all-cis hexose, as a sole carbon source. The operon responsible for D-allose metabolism was localized at 92.8 min of the E. coli linkage map. It consists of six genes, alsRBACEK, which are inducible by D-allose and are under the control of the repressor gene alsR. This operon is also subject to catabolite repression. Three genes, alsB, alsA, and alsC, appear to be necessary for transport of D-allose. D-Allose-binding protein, encoded by alsB, is a periplasmic protein that has an affinity for D-allose, with a Kd of 0.33 microM. As was found for other binding-protein-mediated ABC transporters, the allose transport system includes an ATP-binding component (AlsA) and a transmembrane protein (AlsC). It was found that AlsE (a putative D-allulose-6-phosphate 3-epimerase), but not AlsK (a putative D-allose kinase), is necessary for allose metabolism. During this study, we observed that the D-allose transporter is partially responsible for the low-affinity transport of D-ribose and that strain W3110, an E. coli prototroph, has a defect in the transport of D-allose mediated by the allose permease. PMID:9401019

  1. Wrapped-around models for the lac operon complex.

    PubMed

    La Penna, Giovanni; Perico, Angelo

    2010-06-16

    The protein-DNA complex, involved in the lac operon of enteric bacteria, is paradigmatic in understanding the extent of DNA bending and plasticity due to interactions with protein assemblies acting as DNA regulators. For the lac operon, two classes of structures have been proposed: 1), with the protein tetramer lying away from the DNA loop (wrapped-away model); and 2), with the protein tetramer lying inside the DNA loop (wrapped-around model). A recently developed electrostatic analytical model shows that the size and net charge of the Lac protein tetramer allow the bending of DNA, which is consistent with another wrapped-around model from the literature. Coarse-grained models, designed based on this observation, are extensively investigated and show three kinds of wrapped-around arrangements of DNA and a lower propensity for wrapped-away configurations. Molecular dynamics simulations of an all-atom model, built on the basis of the most tightly collapsed coarse-grained model, show that most of the DNA double-helical architecture is maintained in the region between O3 and O1 DNA operators, that the DNA distortion is concentrated in the chain beyond the O1 operator, and that the protein tetramer can adapt the N-terminal domains to the DNA tension.

  2. Transcriptional regulation of Aggregatibacter actinomycetemcomitans lsrACDBFG and lsrRK operons and their role in biofilm formation.

    PubMed

    Torres-Escobar, Ascención; Juárez-Rodríguez, María Dolores; Lamont, Richard J; Demuth, Donald R

    2013-01-01

    Autoinducer-2 (AI-2) is required for biofilm formation and virulence of the oral pathogen Aggregatibacter actinomycetemcomitans, and we previously showed that lsrB codes for a receptor for AI-2. The lsrB gene is expressed as part of the lsrACDBFG operon, which is divergently transcribed from an adjacent lsrRK operon. In Escherichia coli, lsrRK encodes a repressor and AI-2 kinase that function to regulate lsrACDBFG. To determine if lsrRK controls lsrACDBFG expression and influences biofilm growth of A. actinomycetemcomitans, we first defined the promoters for each operon. Transcriptional reporter plasmids containing the 255-bp lsrACDBFG-lsrRK intergenic region (IGR) fused to lacZ showed that essential elements of lsrR promoter reside 89 to 255 bp upstream from the lsrR start codon. Two inverted repeat sequences that represent potential binding sites for LsrR and two sequences resembling the consensus cyclic AMP receptor protein (CRP) binding site were identified in this region. Using electrophoretic mobility shift assay (EMSA), purified LsrR and CRP proteins were shown to bind probes containing these sequences. Surprisingly, the 255-bp IGR did not contain the lsrA promoter. Instead, a fragment encompassing nucleotides +1 to +159 of lsrA together with the 255-bp IGR was required to promote lsrA transcription. This suggests that a region within the lsrA coding sequence influences transcription, or alternatively that the start codon of A. actinomycetemcomitans lsrA has been incorrectly annotated. Transformation of ΔlsrR, ΔlsrK, ΔlsrRK, and Δcrp deletion mutants with lacZ reporters containing the lsrA or lsrR promoter showed that LsrR negatively regulates and CRP positively regulates both lsrACDBFG and lsrRK. However, in contrast to what occurs in E. coli, deletion of lsrK had no effect on the transcriptional activity of the lsrA or lsrR promoters, suggesting that another kinase may be capable of phosphorylating AI-2 in A. actinomycetemcomitans. Finally, biofilm

  3. The effects of DNA supercoiling on the expression of operons of the ilv regulon of Escherichia coli suggest a physiological rationale for divergently transcribed operons.

    PubMed

    Opel, M L; Arfin, S M; Hatfield, G W

    2001-03-01

    Transcriptional activities of closely spaced divergent promoters are affected by the accumulation of local negative superhelicity in the region between transcribing RNA polymerase molecules (transcriptional coupling). The effect of this transcription-induced DNA supercoiling on these promoters depends on their intrinsic properties. As the global superhelical density of the chromosome is controlled by the energy charge of the cell, which is affected by environmental stresses and transitions from one growth state to another, the transcriptional coupling that occurs between divergently transcribed promoters is likely to serve a physiological purpose. Here, we suggest that transcriptional coupling between the divergent promoters of the ilvYC operon of Escherichia coli serves to co-ordinate the expression of this operon with other operons of the ilv regulon during metabolic adjustments associated with growth state transitions. As DNA supercoiling-dependent transcriptional coupling between the promoters of other divergently transcribed operons is investigated, additional global gene regulatory mechanisms and physiological roles are sure to emerge.

  4. Molecular insight into the activity of LasR protein from Pseudomonas aeruginosa in the regulation of virulence gene expression by this organism.

    PubMed

    Chowdhury, Nilkanta; Bagchi, Angshuman

    2016-04-10

    Pseudomonas aeruginosa is an opportunistic human pathogen. This organism attacks human patients suffering from diseases like AIDS, cancer, cystic fibrosis, etc. One of the important virulent factors produced by this organism is Hydrogen Cyanide. This is expressed from the genes encoded by the hcnABC operon. The expressions of the genes encoded by hcnABC operon are mediated mainly by the interactions of LasR protein with the corresponding promoter region of the hcnABC operon. The LasR protein acts as a dimer and binds to the promoter DNA with the help of an autoinducer ligand. However, till date the detailed molecular mechanism of how the LasR protein interacts with the promoter DNA is not clearly known. Therefore, in this work, an attempt has been made to analyze the mode of interactions of the LasR protein with the promoter DNA region of the hcnABC operon. We analyzed the three dimensional structure of the LasR protein from Pseudomonas aeruginosa and docked the protein with the autoinducer ligand. We then docked the ligand-bound-LasR-protein as well the LasR-protein-without-the-autoinducer-ligand on to the promoter DNA region of hcnABC operon. We analyzed the details of the interaction profiles of LasR protein with the autoinducer ligand. We also deciphered the details of the LasR promoter-DNA interactions. We compared the modes of DNA bindings by the LasR protein in presence and absence of the autoinducer ligand and tried to analyze the molecular details of the binding of LasR protein with the promoter DNA region of hcnABC operon during hcnABC gene expression. This study may therefore pave the pathway for future experiments to determine the relative effects of the amino acid residues of LasR protein in DNA binding during the transcription of hcnABC operon.

  5. 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.

  6. Genomic Subtraction Identifies Salmonella typhimurium Prophages, F-Related Plasmid Sequences, and a Novel Fimbrial Operon, stf, Which Are Absent in Salmonella typhi

    PubMed Central

    Emmerth, Melanie; Goebel, Werner; Miller, Samuel I.; Hueck, Christoph J.

    1999-01-01

    Salmonella typhimurium causes systemic and fatal infection in inbred mice, while the related serotype Salmonella typhi is avirulent for mammals other than humans. In order to identify genes from the virulent strain S. typhimurium ATCC 14028 that are absent in S. typhi Ty2, and therefore might be involved in S. typhimurium mouse virulence, a PCR-supported genomic subtractive hybridization procedure was employed. We have identified a novel putative fimbrial operon, stfACDEFG, located at centisome 5 of the S. typhimurium chromosome, which is absent in S. typhi, Salmonella arizonae, and Salmonella bongori but was detected in several other Salmonella serotypes. The fimbrial genes represent a genomic insertion in S. typhimurium compared to the respective region between fhuB and hemL in Escherichia coli K-12. In addition, the subtraction procedure yielded F plasmid-related sequences from the S. typhimurium virulence plasmid, a number of DNA fragments representing parts of lambdoid prophages and putative sugar transporters, and several fragments with unknown sequences. The majority of subtracted chromosomal sequences map to three distinct locations, around centisomes 5, 27, and 57. PMID:10482505

  7. The distinctive signatures of promoter regions and operon junctions across prokaryotes

    PubMed Central

    Janga, Sarath Chandra; Lamboy, Warren F.; Huerta, Araceli M.; Moreno-Hagelsieb, Gabriel

    2006-01-01

    Here we show that regions upstream of first transcribed genes have oligonucleotide signatures that distinguish them from regions upstream of genes in the middle of operons. Databases of experimentally confirmed transcription units do not exist for most genomes. Thus, to expand the analyses into genomes with no experimentally confirmed data, we used genes conserved adjacent in evolutionarily distant genomes as representatives of genes inside operons. Likewise, we used divergently transcribed genes as representative examples of first transcribed genes. In model organisms, the trinucleotide signatures of regions upstream of these representative genes allow for operon predictions with accuracies close to those obtained with known operon data (0.8). Signature-based operon predictions have more similar phylogenetic profiles and higher proportions of genes in the same pathways than predicted transcription unit boundaries (TUBs). These results confirm that we are separating genes with related functions, as expected for operons, from genes not necessarily related, as expected for genes in different transcription units. We also test the quality of the predictions using microarray data in six genomes and show that the signature-predicted operons tend to have high correlations of expression. Oligonucleotide signatures should expand the number of tools available to identify operons even in poorly characterized genomes. PMID:16914446

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

    PubMed Central

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

    2015-01-01

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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  10. 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

  11. 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.

  12. 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

  13. 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.

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

    PubMed

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

    2014-01-07

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

  15. Bacterial proteases and virulence.

    PubMed

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell. These extracellular proteases are activated in complex cascades involving auto-processing and proteolytic maturation. Thus, proteolysis has been adopted by bacterial pathogens at multiple levels to ensure the success of the pathogen in contact with the human host.

  16. 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.

  17. 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

  18. narI region of the Escherichia coli nitrate reductase (nar) operon contains two genes.

    PubMed Central

    Sodergren, E J; DeMoss, J A

    1988-01-01

    In previous studies it has been established that in Escherichia coli the three known subunits of anaerobic nitrate reductase are encoded by the narGHI operon. From the nucleotide sequence of the narI region of the operon we conclude that, in addition to the narG and narH genes, the nar operon contains two other open reading frames (ORFs), ORF1 and ORF2, that encode proteins of 26.5 and 25.5 kilodaltons, respectively. Protein fusions to each of the genes in the operon showed that expression of all four genes was similarly regulated. The reading frames of ORF1 and ORF2 were verified, and the N-terminal sequence for the ORF1 fusion protein was determined. The nar operon therefore contains four genes designated and ordered as narGHJI. Images PMID:2832376

  19. Glycopeptide Resistance vanA Operons in Paenibacillus Strains Isolated from Soil

    PubMed Central

    Guardabassi, Luca; Perichon, Bruno; van Heijenoort, Jean; Blanot, Didier; Courvalin, Patrice

    2005-01-01

    The sequence and gene organization of the van operons in vancomycin (MIC of >256 μg/ml)- and teicoplanin (MIC of ≥32 μg/ml)-resistant Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B isolated from soil were determined. Both operons had regulatory (vanR and vanS), resistance (vanH, vanA, and vanX), and accessory (vanY, vanZ, and vanW) genes homologous to the corresponding genes in enterococcal vanA and vanB operons. The vanAPT operon in P. thiaminolyticus PT-2B1 had the same gene organization as that of vanA operons whereas vanAPA in P. apiarius PA-B2B resembled vanB operons due to the presence of vanW upstream from the vanHAX cluster but was closer to vanA operons in sequence. Reference P. apiarius strains NRRL B-4299 and NRRL B-4188 were found to harbor operons indistinguishable from vanAPA by PCR mapping, restriction fragment length polymorphism, and partial sequencing, suggesting that this operon was species specific. As in enterococci, resistance was inducible by glycopeptides and associated with the synthesis of pentadepsipeptide peptidoglycan precursors ending in d-Ala-d-Lac, as demonstrated by d,d-dipeptidase activities, high-pressure liquid chromatography, and mass spectrometry. The precursors differed from those in enterococci by the presence of diaminopimelic acid instead of lysine in the peptide chain. Altogether, the results are compatible with the notion that van operons in soil Paenibacillus strains and in enterococci have evolved from a common ancestor. PMID:16189102

  20. Glycopeptide resistance vanA operons in Paenibacillus strains isolated from soil.

    PubMed

    Guardabassi, Luca; Perichon, Bruno; van Heijenoort, Jean; Blanot, Didier; Courvalin, Patrice

    2005-10-01

    The sequence and gene organization of the van operons in vancomycin (MIC of >256 microg/ml)- and teicoplanin (MIC of > or =32 microg/ml)-resistant Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B isolated from soil were determined. Both operons had regulatory (vanR and vanS), resistance (vanH, vanA, and vanX), and accessory (vanY, vanZ, and vanW) genes homologous to the corresponding genes in enterococcal vanA and vanB operons. The vanA(PT) operon in P. thiaminolyticus PT-2B1 had the same gene organization as that of vanA operons whereas vanA(PA) in P. apiarius PA-B2B resembled vanB operons due to the presence of vanW upstream from the vanHAX cluster but was closer to vanA operons in sequence. Reference P. apiarius strains NRRL B-4299 and NRRL B-4188 were found to harbor operons indistinguishable from vanA(PA) by PCR mapping, restriction fragment length polymorphism, and partial sequencing, suggesting that this operon was species specific. As in enterococci, resistance was inducible by glycopeptides and associated with the synthesis of pentadepsipeptide peptidoglycan precursors ending in D-Ala-D-Lac, as demonstrated by D,D-dipeptidase activities, high-pressure liquid chromatography, and mass spectrometry. The precursors differed from those in enterococci by the presence of diaminopimelic acid instead of lysine in the peptide chain. Altogether, the results are compatible with the notion that van operons in soil Paenibacillus strains and in enterococci have evolved from a common ancestor.

  1. 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

  2. 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

  3. 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.

  4. Biochemical evidence for ToxR and ToxJ binding to the tox operons of Burkholderia glumae and mutational analysis of ToxR.

    PubMed

    Kim, Jinwoo; Oh, Jonghee; Choi, Okhee; Kang, Yongsung; Kim, Hongsup; Goo, Eunhye; Ma, Jun; Nagamatsu, Tomohisa; Moon, Jae Sun; Hwang, Ingyu

    2009-08-01

    Burkholderia glumae produces toxoflavin, a phytotoxin with a broad host range, which is a key virulence factor in bacterial rice grain rot. Based on genetic analysis, we previously reported that ToxR, a LysR-type regulator, activates both the toxABCDE (toxoflavin biosynthesis genes) and toxFGHI (toxoflavin transporter genes) operons in the presence of toxoflavin as a coinducer. Quorum sensing regulates the expression of the transcriptional activator ToxJ that is required for tox gene expression. Here, we used gel mobility shift and DNase I protection analyses to demonstrate that both ToxR and ToxJ bind simultaneously to the regulatory regions of both tox operons. ToxR and ToxJ both bound to the toxA and toxF regulatory regions, and the sequences for the binding of ToxR to the regulatory regions of both tox operons possessed T-N(11)-A motifs. Following random mutagenesis of toxR, 10 ToxR mutants were isolated. We constructed a reporter strain, S6K34 (toxR'A'::Omega toxF::Tn3-gusA34) to evaluate which amino acid residues are important for ToxR activity. Several single amino acid substitutions identified residues that might be important for ToxR binding to DNA and toxoflavin binding. When various toxoflavin derivatives were tested to determine whether toxoflavin is a specific coinducer of ToxR in the S6K34 strain, ToxR, together with toxoflavin, conferred toxF expression, whereas 4,8-dihydrotoxoflavin did so only slightly. With these results, we have demonstrated biochemically that B. glumae cells control toxoflavin production tightly by the requirement of both ToxJ and toxoflavin as coinducers of ToxR.

  5. Biochemical Evidence for ToxR and ToxJ Binding to the tox Operons of Burkholderia glumae and Mutational Analysis of ToxR▿

    PubMed Central

    Kim, Jinwoo; Oh, Jonghee; Choi, Okhee; Kang, Yongsung; Kim, Hongsup; Goo, Eunhye; Ma, Jun; Nagamatsu, Tomohisa; Moon, Jae Sun; Hwang, Ingyu

    2009-01-01

    Burkholderia glumae produces toxoflavin, a phytotoxin with a broad host range, which is a key virulence factor in bacterial rice grain rot. Based on genetic analysis, we previously reported that ToxR, a LysR-type regulator, activates both the toxABCDE (toxoflavin biosynthesis genes) and toxFGHI (toxoflavin transporter genes) operons in the presence of toxoflavin as a coinducer. Quorum sensing regulates the expression of the transcriptional activator ToxJ that is required for tox gene expression. Here, we used gel mobility shift and DNase I protection analyses to demonstrate that both ToxR and ToxJ bind simultaneously to the regulatory regions of both tox operons. ToxR and ToxJ both bound to the toxA and toxF regulatory regions, and the sequences for the binding of ToxR to the regulatory regions of both tox operons possessed T-N11-A motifs. Following random mutagenesis of toxR, 10 ToxR mutants were isolated. We constructed a reporter strain, S6K34 (toxR′A′::Ω toxF::Tn3-gusA34) to evaluate which amino acid residues are important for ToxR activity. Several single amino acid substitutions identified residues that might be important for ToxR binding to DNA and toxoflavin binding. When various toxoflavin derivatives were tested to determine whether toxoflavin is a specific coinducer of ToxR in the S6K34 strain, ToxR, together with toxoflavin, conferred toxF expression, whereas 4,8-dihydrotoxoflavin did so only slightly. With these results, we have demonstrated biochemically that B. glumae cells control toxoflavin production tightly by the requirement of both ToxJ and toxoflavin as coinducers of ToxR. PMID:19465657

  6. 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. Copyright © 2014 Elsevier Ltd. All rights reserved.

  7. RNA polymerase structure and function at lac operon.

    PubMed

    Borukhov, Sergei; Lee, Jookyung

    2005-06-01

    Transcription of E. coli lac operon by RNA polymerase (RNAP) is a classic example of how the basic functions of this enzyme, specifically the ability to recognize/bind promoters, melt the DNA and initiate RNA synthesis, is positively regulated by transcription activators, such as cyclic AMP-receptor protein, CRP, and negatively regulated by lac-repressor, LacI. In this review, we discuss the recent progress in structural and biochemical studies of RNAP and its binary and ternary complexes with CRP and lac promoter. With structural information now available for RNAP and models of binary and ternary elongation complexes, the interaction between these factors and RNAP can be modeled, and possible molecular mechanisms of their action can be inferred.

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

    PubMed Central

    2008-01-01

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

  9. A Novel cdsAB Operon Is Involved in the Uptake of l-Cysteine and Participates in the Pathogenesis of Yersinia ruckeri▿

    PubMed Central

    Méndez, Jessica; Reimundo, Pilar; Pérez-Pascual, David; Navais, Roberto; Gómez, Esther; Guijarro, José A.

    2011-01-01

    Application of in vivo expression technology (IVET) to Yersinia ruckeri, an important fish pathogen, allowed the identification of two adjacent genes that represent a novel bacterial system involved in the uptake and degradation of l-cysteine. Analysis of the translational products of both genes showed permease domains (open reading frame 1 [ORF1]) and amino acid position identities (ORF2) with the l-cysteine desulfidase from Methanocaldococcus jannaschii, a new type of enzyme involved in the breakdown of l-cysteine. The operon was named cdsAB (cysteine desulfidase) and is found widely in anaerobic and facultative bacteria. cdsAB promoter analysis using lacZY gene fusion showed highest induction in the presence of l-cysteine. Two cdsA and cdsB mutant strains were generated. The limited toxic effect and the low utilization of l-cysteine observed in the cdsA mutant, together with radiolabeled experiments, strongly suggested that CdsA is an l-cysteine permease. Fifty percent lethal dose (LD50) and competence index experiments showed that both the cdsA and cdsB loci were involved in the pathogenesis of the bacteria. In conclusion, this study has shown for the first time in bacteria the existence of an l-cysteine uptake system that together with an additional l-cysteine desulfidase-encoding gene constitutes a novel operon involved in bacterial virulence. PMID:21169490

  10. Two non-consensus Clp binding sites are involved in upregulation of the gum operon involved in xanthan polysaccharide synthesis in Xanthomonas campestris pv. campestris.

    PubMed

    Chen, Chih-Hua; Lin, Nien-Tsung; Hsiao, Yi-Min; Yang, Chiou-Ying; Tseng, Yi-Hsiung

    2010-09-01

    Biosynthesis of xanthan polysaccharide, a virulence factor of phytopathogenic Xanthomonas campestris pv. campestris (Xcc), involves the gum operon and the cyclic AMP receptor protein (CRP) homologue Clp. Clp was shown to have the same DNA binding specificity as the CRP at positions 5, 6, and 7 (GTG motif) of the left arm. Therefore, Clp binding sites (CBSs) have typically been identified by pattern searching of the Xcc genome using the consensus CRP binding sequence. Here, results of a reporter assay and electrophoretic mobility shift assay suggest that Clp upregulates the gum operon by binding to two non-consensus sites, in which a more conserved right arm may compensate for the lack of conservation in the left arm, a high GC content in the central region (6 bp) may be important for binding, and binding may be enhanced if the GC-rich central region is palindromic. These suggest that atypical CBSs exist in Xcc promoters and that Clp, while retaining the capacity to bind typical CBSs, has evolved to bind atypical CBS because: 1) Clp shares only moderate homology with the CRP and is modulated by cyclic di-GMP; and 2) Xcc has a higher GC content (65%) than Escherichia coli (50%).

  11. Engineering adherent bacteria by creating a single synthetic curli operon.

    PubMed

    Drogue, Benoît; Thomas, Philippe; Balvay, Laurent; Prigent-Combaret, Claire; Dorel, Corinne

    2012-11-16

    The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011). The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control. The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

  12. Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

    PubMed Central

    Drogue, Benoît; Thomas, Philippe; Balvay, Laurent; Prigent-Combaret, Claire; Dorel, Corinne

    2012-01-01

    The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011). The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control. The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon. PMID

  13. Identification and Characterization of the fis Operon in Enteric Bacteria

    PubMed Central

    Beach, Michael B.; Osuna, Robert

    1998-01-01

    The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage λ genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens, E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence for H. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) preceding fis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from −53 to +27 and the region around −116 containing an ihf binding site. Both β-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation. PMID:9811652

  14. Identification and characterization of the fis operon in enteric bacteria.

    PubMed

    Beach, M B; Osuna, R

    1998-11-01

    The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage lambda genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, and Proteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens, E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence for H. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) preceding fis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from -53 to +27 and the region around -116 containing an ihf binding site. Both beta-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.

  15. Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon).

    PubMed Central

    Furukawa, K; Hirose, J; Suyama, A; Zaiki, T; Hayashida, S

    1993-01-01

    bph operons coding for biphenyl-polychlorinated biphenyl degradation in Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715 and tod operons coding for toluene-benzene metabolism in P. putida F1 are very similar in gene organization as well as size and homology of the corresponding enzymes (G. J. Zylstra and D. T. Gibson, J. Biol. Chem. 264:14940-14946, 1989; K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphA1 and todC1) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todC1C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todC1 into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway. Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into toluene-utilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli cells carrying a hybrid gene cluster of todClbphA2A3A4BC (constructed by replacing bphA1 with todC1) converted toluene to a ring meta-cleavage 2-hydroxy-6-oxo-hepta-2,4-dienoic acid, indicating that TodC1 formed a functional multicomponent dioxygenase associated with BphA2 (a small subunit of the terminal dioxygenase in biphenyl metabolism), BphA3, and BphA4. PMID:8349562

  16. Virulence factors and genetic variability of uropathogenic Escherichia coli isolated from dogs and cats in Italy.

    PubMed

    Tramuta, Clara; Nucera, Daniele; Robino, Patrizia; Salvarani, Sara; Nebbia, Patrizia

    2011-03-01

    In this study, the association between virulence genotypes and phylogenetic groups among Escherichia (E.) coli isolates obtained from pet dogs and cats with cystitis was detected, and fingerprinting methods were used to explore the relationship among strains. Forty uropathogenic E. coli (UPEC) isolated from dogs (n = 30) and cats (n = 10) in Italy were analysed by polymerase chain reaction (PCR) for the presence of virulence factors and their classification into phylogenetic groups. The same strains were characterized by repetitive extragenic palindromic (REP)- and enterobacterial repetitive intergenic consensus (ERIC)-PCR techniques. We found a high number of virulence factors such as fimbriae A, S fimbriae (sfa) and cytotoxic necrotizing factor 1 (cnf1) significantly associated with phylogenetic group B2. We demonstrated a high correlation between α-hemolysin A and pyelonephritis C, sfa, and cnf1 operons, confirming the presence of pathogenicity islands in these strains. In addition, UPEC belonging to group B2 harboured a greater number of virulence factors than strains from phylogenetic groups A, B1, and D. REP- and ERIC-PCR grouped the UPEC isolates into two major clusters, the former grouping E. coli strains belonging to phylogenetic group B2 and D, the latter grouping those belonging to groups A and B1. Given the significant genetic variability among the UPEC strains found in our study, it can be hypothesized that no specific genotype is responsible for cystitis in cats or dogs.

  17. The Rcs regulon in Proteus mirabilis: implications for motility, biofilm formation, and virulence.

    PubMed

    Howery, Kristen E; Clemmer, Katy M; Rather, Philip N

    2016-11-01

    The overall role of the Rcs phosphorelay in Proteus mirabilis is largely unknown. Previous work had demonstrated that the Rcs phosphorelay represses the flhDC operon and activates the minCDE cell division inhibition system. To identify additional cellular functions regulated by the Rcs phosphorelay, an analysis of RNA-seq data was undertaken. In this report, the results of the RNA-sequencing are discussed with an emphasis on the predicted roles of the Rcs phosphorelay in swarmer cell differentiation, motility, biofilm formation, and virulence. RcsB is shown to activate genes important for differentiation and fimbriae formation, while repressing the expression of genes important for motility and virulence. Additionally, to follow up on the RNA-Seq data, we demonstrate that an rcsB mutant is deficient in its ability to form biofilm and exhibits enhanced virulence in a Galleria mellonella waxworm model. Overall, these results indicate the Rcs regulon in P. mirabilis extends beyond flagellar genes to include those involved in biofilm formation and virulence. Furthermore, the information presented in this study may provide clues to additional roles of the Rcs phosphorelay in other members of the Enterobacteriaceae.

  18. The Yersinia pestis caf1M1A1 fimbrial capsule operon promotes transmission by flea bite in a mouse model of bubonic plague.

    PubMed

    Sebbane, Florent; Jarrett, Clayton; Gardner, Donald; Long, Daniel; Hinnebusch, B Joseph

    2009-03-01

    Plague is a zoonosis transmitted by fleas and caused by the gram-negative bacterium Yersinia pestis. During infection, the plasmidic caf1M1A1 operon that encodes the Y. pestis F1 protein capsule is highly expressed, and anti-F1 antibodies are protective. Surprisingly, the capsule is not required for virulence after injection of cultured bacteria, even though it is an antiphagocytic factor and capsule-deficient Y. pestis strains are rarely isolated. We found that a caf-negative Y. pestis mutant was not impaired in either flea colonization or virulence in mice after intradermal inoculation of cultured bacteria. In contrast, absence of the caf operon decreased bubonic plague incidence after a flea bite. Successful development of plague in mice infected by flea bite with the caf-negative mutant required a higher number of infective bites per challenge. In addition, the mutant displayed a highly autoaggregative phenotype in infected liver and spleen. The results suggest that acquisition of the caf locus via horizontal transfer by an ancestral Y. pestis strain increased transmissibility and the potential for epidemic spread. In addition, our data support a model in which atypical caf-negative strains could emerge during climatic conditions that favor a high flea burden. Human infection with such strains would not be diagnosed by the standard clinical tests that detect F1 antibody or antigen, suggesting that more comprehensive surveillance for atypical Y. pestis strains in plague foci may be necessary. The results also highlight the importance of studying Y. pestis pathogenesis in the natural context of arthropod-borne transmission.

  19. Characterization of the groESL operon in Listeria monocytogenes: utilization of two reporter systems (gfp and hly) for evaluating in vivo expression.

    PubMed

    Gahan, C G; O'Mahony, J; Hill, C

    2001-06-01

    The ability of intracellular pathogens to sense and adapt to the hostile environment of the host is an important factor governing virulence. We have sequenced the operon encoding the major heat shock proteins GroES and GroEL in the gram-positive food-borne pathogen Listeria monocytogenes. The operon has a conserved orientation in the order groES groEL. Upstream of groES and in the opposite orientation is a gene encoding a homologue of the Bacillus subtilis protein YdiL, while downstream of groEL is a gene encoding a putative bile hydrolase. We used both reverse transcriptase-PCR (RT-PCR) and transcriptional fusions to the UV-optimized Aequorea victoria green fluorescent protein (GFP(UV)) to analyze expression of groESL under various environmental stress conditions, including heat shock, ethanol stress, and acid shock, and during infection of J774 mouse macrophage cells. Strains harboring GFP(UV) transcriptional fusions to the promoter region of groESL demonstrated a significant increase in fluorescence following heat shock that was detected by both fluorimetry and fluorescence microscopy. Using both RT-PCR and GFP technology we detected expression of groESL following internalization by J774 cells. Increased intracellular expression of dnaK was also determined using RT-PCR. We have recently described a system which utilizes L. monocytogenes hemolysin as an in vivo reporter of gene expression within the host cell phagosome (C. G. M. Gahan and C. Hill, Mol. Microbiol. 36:498-507, 2000). In this study a strain was constructed in which hemolysin expression was placed under the control of the groESL promoter. In this strain hemolysin expression during infection also confirms transcription from the groESL promoter during J774 and murine infection, albeit at lower levels than the known virulence factor plcA.

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

    PubMed Central

    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

  1. 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.

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

    PubMed Central

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

    2016-01-01

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

  3. 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.

  4. An insight into the regulation of mce4 operon of Mycobacterium tuberculosis.

    PubMed

    Rathor, Nisha; Chandolia, Amita; Saini, Neeraj Kumar; Sinha, Rajesh; Pathak, Rakesh; Garima, Kushal; Singh, Satendra; Varma-Basil, Mandira; Bose, Mridula

    2013-07-01

    The mce4 operon is reported to be involved in cholesterol utilization and intracellular survival of Mycobacterium tuberculosis (M. tuberculosis). The regulatory mechanism of this important operon was unknown so far. Here we report detection of the promoter region and regulatory factors of the mce4 operon. The in silico analyzed putative promoter region was cloned in promoter selection vector and promoter strength was measured by O-Nitrophenyl-β-D-galactopyranosidase (ONPG) assay. The transcription start site was determined by 5' Rapid amplification of C terminal end (5'RACE). Surface stress, hypoxia and presence of cholesterol, were found to be stimulatory for mce4 operon promoter induction. Pull down assay coupled with 2D gel electrophoresis resolved many proteins; few prominent spots were processed for identification. MALDI TOF-TOF identified proteins of M. tuberculosis which supported the regulatory function of the identified promoter region and cholesterol utilization of mce4 operon. Since mce4 operon is involved in cholesterol utilization and intracellular survival of M. tuberculosis in the later phase of infection, identification of the promoter sequence as reported in the present communication may facilitate development of effective inhibitors to regulate expression of mce4 operon which may prove to be a good drug target to prevent latency in tuberculosis.

  5. Interplay of gene expression noise and ultrasensitive dynamics affects bacterial operon organization.

    PubMed

    Ray, J Christian J; Igoshin, Oleg A

    2012-01-01

    Bacterial chromosomes are organized into polycistronic cotranscribed operons, but the evolutionary pressures maintaining them are unclear. We hypothesized that operons alter gene expression noise characteristics, resulting in selection for or against maintaining operons depending on network architecture. Mathematical models for 6 functional classes of network modules showed that three classes exhibited decreased noise and 3 exhibited increased noise with same-operon cotranscription of interacting proteins. Noise reduction was often associated with a decreased chance of reaching an ultrasensitive threshold. Stochastic simulations of the lac operon demonstrated that the predicted effects of transcriptional coupling hold for a complex network module. We employed bioinformatic analysis to find overrepresentation of noise-minimizing operon organization compared with randomized controls. Among constitutively expressed physically interacting protein pairs, higher coupling frequencies appeared at lower expression levels, where noise effects are expected to be dominant. Our results thereby suggest an important role for gene expression noise, in many cases interacting with an ultrasensitive switch, in maintaining or selecting for operons in bacterial chromosomes.

  6. Virulence Markers of Dengue Viruses

    DTIC Science & Technology

    1988-06-10

    AD VIRULENCE MARKERS OF DENGUE VIRUSES 00 ANNUAL REPORT 0 James L. Hardy and Srisakul C. Kliks June 10, 1988 Supported by U.S. ARMY MEDICAL RESEARCH...Virulence Markers of Dengue Viruses (U) 12. PERSONAL AUTHOR(S) James L. Hardy ind Sriqakul.C. Klik,,q 13a. TYPE OF REPORT 13b. TIME COVERED 14. DATE OF...TERMS (Continue on reverse it necessary and identify by block number) FIELD GROUP SUB-GROUP Dengue viruses, dengue hemorrhagic fever, virulence, U3

  7. 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

  8. Cloning and sequencing of part of the S10 operon from Actinobacillus actinomycetemcomitans FDC Y4.

    PubMed

    Hayashida, H; Hotokezaka, H; Ohara, N; Kimura, M; Takagi, O; Yamada, T

    1997-06-01

    We have cloned and sequenced the 5.2 kb EcoRI fragment that contained part of the S10 operon from Actinobacillus actinomycetemcomitans FDC Y4. The order of the ribosomal protein genes was identical to that of the S10 operon of Haemophilus influenzae and Escherichia coli. The deduced amino acid sequences of ribosomal proteins in this operon displayed significant homologies (65.3%-100%) to those of H. influenzae, E. coli, Yersinia enterocolitica and Yersinia pseudotuberculosis. Phylogenetic trees obtained for these ribosomal proteins were similar to that obtained for 16S rRNA.

  9. Ancient Origin of the Tryptophan Operon and the Dynamics of Evolutionary Change†

    PubMed Central

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

    2003-01-01

    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

  10. Quorum sensing in bacterial virulence.

    PubMed

    Antunes, L Caetano M; Ferreira, Rosana B R; Buckner, Michelle M C; Finlay, B Brett

    2010-08-01

    Bacteria communicate through the production of diffusible signal molecules termed autoinducers. The molecules are produced at basal levels and accumulate during growth. Once a critical concentration has been reached, autoinducers can activate or repress a number of target genes. Because the control of gene expression by autoinducers is cell-density-dependent, this phenomenon has been called quorum sensing. Quorum sensing controls virulence gene expression in numerous micro-organisms. In some cases, this phenomenon has proven relevant for bacterial virulence in vivo. In this article, we provide a few examples to illustrate how quorum sensing can act to control bacterial virulence in a multitude of ways. Several classes of autoinducers have been described to date and we present examples of how each of the major types of autoinducer can be involved in bacterial virulence. As quorum sensing controls virulence, it has been considered an attractive target for the development of new therapeutic strategies. We discuss some of the new strategies to combat bacterial virulence based on the inhibition of bacterial quorum sensing systems.

  11. Physiological control and regulation of the Rhodobacter capsulatus cbb operons.

    PubMed

    Paoli, G C; Vichivanives, P; Tabita, F R

    1998-08-01

    The genes encoding enzymes of the Calvin-Benson-Bassham (CBB) reductive pentose phosphate pathway in Rhodobacter capsulatus are organized in at least two operons, each preceded by a separate cbbR gene, encoding potential LysR-type transcriptional activators. As a prelude to studies of cbb gene regulation in R. capsulatus, the nucleotide sequence of a 4,537-bp region, which included cbbRII, was determined. This region contained the following open reading frames: a partial pgm gene (encoding phosphoglucomutase) and a complete qor gene (encoding NADPH:quinone oxidoreductase), followed by cbbRII, cbbF (encoding fructose 1,6-bisphosphatase), cbbP (encoding phosphoribulokinase), and part of cbbT (encoding transketolase). Physiological control of the CBB pathway and regulation of the R. capsulatus cbb genes were studied by using a combination of mutant strains and promoter fusion constructs. Characterization of mutant strains revealed that either form I or form II ribulose 1, 5-bisphosphate carboxylase/oxygenase (RubisCO), encoded by the cbbLS and cbbM genes, respectively, could support photoheterotrophic and autotrophic growth. A strain with disruptions in both cbbL and cbbM could not grow autotrophically and grew photoheterotrophically only when dimethyl sulfoxide was added to the culture medium. Disruption of cbbP resulted in a strain that did not synthesize form II RubisCO and had a phenotype similar to that observed in the RubisCO-minus strain, suggesting that there is only one cbbP gene in R. capsulatus and that this gene is cotranscribed with cbbM. Analysis of RubisCO activity and synthesis in strains with disruptions in either cbbRI or cbbRII, and beta-galactosidase determinations from wild-type and mutant strains containing cbbIp- and cbbIIp-lacZ fusion constructs, indicated that the cbbI and cbbII operons of R. capsulatus are within separate CbbR regulons.

  12. 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

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

    PubMed Central

    Santillán, Moisés; Mackey, Michael C.

    2008-01-01

    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

  14. Incorporation of a horizontally transferred gene into an operon during cnidarian evolution.

    PubMed

    Dana, Catherine E; Glauber, Kristine M; Chan, Titus A; Bridge, Diane M; Steele, Robert E

    2012-01-01

    Genome sequencing has revealed examples of horizontally transferred genes, but we still know little about how such genes are incorporated into their host genomes. We have previously reported the identification of a gene (flp) that appears to have entered the Hydra genome through horizontal transfer. Here we provide additional evidence in support of our original hypothesis that the transfer was from a unicellular organism, and we show that the transfer occurred in an ancestor of two medusozoan cnidarian species. In addition we show that the gene is part of a bicistronic operon in the Hydra genome. These findings identify a new animal phylum in which trans-spliced leader addition has led to the formation of operons, and define the requirements for evolution of an operon in Hydra. The identification of operons in Hydra also provides a tool that can be exploited in the construction of transgenic Hydra strains.

  15. Incorporation of a Horizontally Transferred Gene into an Operon during Cnidarian Evolution

    PubMed Central

    Dana, Catherine E.; Glauber, Kristine M.; Chan, Titus A.; Bridge, Diane M.; Steele, Robert E.

    2012-01-01

    Genome sequencing has revealed examples of horizontally transferred genes, but we still know little about how such genes are incorporated into their host genomes. We have previously reported the identification of a gene (flp) that appears to have entered the Hydra genome through horizontal transfer. Here we provide additional evidence in support of our original hypothesis that the transfer was from a unicellular organism, and we show that the transfer occurred in an ancestor of two medusozoan cnidarian species. In addition we show that the gene is part of a bicistronic operon in the Hydra genome. These findings identify a new animal phylum in which trans-spliced leader addition has led to the formation of operons, and define the requirements for evolution of an operon in Hydra. The identification of operons in Hydra also provides a tool that can be exploited in the construction of transgenic Hydra strains. PMID:22328943

  16. 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

  17. An overlap between operons involved in carotenoid and bacteriochlorophyll biosynthesis in Rhodobacter capsulatus.

    PubMed

    Young, D A; Rudzik, M B; Marrs, B L

    1992-08-15

    A new example of superoperonal gene arrangement has been documented in the Rhodobacter capsulatus photosynthetic gene cluster. The promoter for the operon initiated by the bchI gene is embedded within an upstream operon for carotenoid synthesis. The stop codon for the crtA gene, the only gene in the first operon, overlaps the start codon of the downstream bchI gene. As a consequence of this overlap, the promoter(s) for the bch operon must be located within the crtA structural gene. The bchI gene is shown here for the first time to be required for the conversion of protoporphyrin IX to subsequent intermediates in bacteriochlorophyll biosynthesis.

  18. 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

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

    PubMed

    Santillán, Moisés; Mackey, Michael C

    2008-08-06

    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.

  20. 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.

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

    PubMed Central

    Choe, M; Reznikoff, W S

    1991-01-01

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

  2. Upgrading bioluminescent bacterial bioreporter performance by splitting the lux operon.

    PubMed

    Yagur-Kroll, Sharon; Belkin, Shimshon

    2011-05-01

    Bioluminescent bacterial bioreporters harbor a fusion of bacterial bioluminescence genes (luxCDABE), acting as the reporting element, to a stress-response promoter, serving as the sensing element. Upon exposure to conditions that activate the promoter, such as an environmental stress or the presence of an inducing chemical, the promoter::reporter fusion generates a dose-dependent bioluminescent signal. In order to improve bioluminescent bioreporter performance we have split the luxCDABE genes of Photorhabdus luminescens into two smaller functional units: luxAB, that encode for the luciferase enzyme, which catalyzes the luminescence reaction, and luxCDE that encode for the enzymatic complex responsible for synthesis of the reaction's substrate, a long-chain aldehyde. The expression of each subunit was put under the control of either an inducible stress-responsive promoter or a synthetic constitutive promoter, and different combinations of the two units were tested for their response to selected chemicals in Escherichia coli. In all cases tested, the split combinations proved to be superior to the native luxCDABE configuration, suggesting an improved efficiency in the transcription and/or translation of two small gene units instead of a larger one with the same genes. The best combination was that of an inducible luxAB and a constitutive luxCDE, indicating that aldehyde availability is limited when the five genes are expressed together in E. coli, and demonstrating that improved biosensor performance may be achieved by rearrangement of the lux operon genes.

  3. Evolution of a tRNA operon in gamma purple bacteria.

    PubMed Central

    Giroux, S; Cedergren, R

    1989-01-01

    Genomic DNA from eubacteria belonging to the gamma-3 subdivision of purple bacteria, as classified by Woese (C.R. Woese, Microbiol. Rev. 51:221-271, 1987), were probed with the argT operon of Escherichia coli encoding 5'-tRNA(Arg)-tRNA(His)-tRNA(Leu)-tRNA(Pro)-3'. The homologous operon from Vibrio harveyi was isolated and sequenced. Comparison of the five available sequences of this tRNA cluster from members of the families Enterobacteriaceae, Aeromonadaceae, and Vibrionaceae led to the conclusion that variations in different versions of this operon arose not only by point mutations but also by duplication and addition-deletion of entire tRNA genes. This data base permitted the formulation of a proposal dealing with the evolutionary history of this operon and suggested that DNA regions containing tRNA genes are active centers (hot spots) of recombination. Finally, since the operon from V. harveyi was not highly repetitive and did not contain tRNA pseudogenes, as in the Photobacterium phosphoreum operon, hybridization of genomic DNAs from different photobacterial strains with probes specific for the repeated pseudogene element was performed. We conclude that the phylogenetic distribution of the repetitive DNA is restricted to strains of P. phosphoreum. Images PMID:2687235

  4. 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.

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

    PubMed

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

    2014-01-01

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

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

    PubMed Central

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

    2014-01-01

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

  7. 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.

  8. Nucleotide sequence and functional analysis of regulatory region of the lumP and the lux operon from Photobacterium leiognathi.

    PubMed

    Lin, J W; Chao, Y F; Weng, S F

    1995-05-25

    The lumP gene is linked to the lux operon, but runs in the opposite direction in Photobacterium leiognathi PL741. The gene order of the lumP and the lux operon is < -lumP-R & R-luxC-luxD-luxA-luxB-luxN-luxE- > (R & R: regulatory region). The nucleotide sequence of the regulatory region (827-bp) between the lumP and the lux operon was determined. Sequence analysis illustrates that the regulatory region includes two divergent promoter systems, PR-promoter system for the lux operon (R-operon) and PL-promoter system for the lumP or lum operon (L-operon). Functional analysis of the regulatory region shows that the PR- and PL-promoter systems both are able to lead the gene expression. The deletion experiment result elicits that the PR- and PL-promoter are coordinatively and negatively regulated; the PR- and PL-promoter might be competing for recognition by RNA polymerase to initiate transcription. The fact of the LumP responsible for the spectral blue shift in P. leiognathi implied that the lumP gene closedly linked to the lux operon is for coordinative regulation with the lux operon. In addition, the glucose repression on the PR-promoter system shows that the expression of the lux operon is regulated by cAMP-CRP induction in E. coli.

  9. Deciphering the regulon of a GntR family regulator via transcriptome and ChIP-exo analyses and its contribution to virulence in Xanthomonas citri.

    PubMed

    Zhou, Xiaofeng; Yan, Qing; Wang, Nian

    2017-02-01

    Xanthomonas contains a large group of plant-associated species, many of which cause severe diseases on important crops worldwide. Six gluconate-operon repressor (GntR) family transcriptional regulators are predicted in Xanthomonas, one of which, belonging to the YtrA subfamily, plays a prominent role in bacterial virulence. However, the direct targets and comprehensive regulatory profile of YtrA remain unknown. Here, we performed microarray and high-resolution chromatin immunoprecipitation-exonuclease (ChIP-exo) experiments to identify YtrA direct targets and its DNA binding motif in X. citri ssp. citri (Xac), the causal agent of citrus canker. Integrative microarray and ChIP-exo data analysis revealed that YtrA directly regulates three operons by binding to a palindromic motif GGTG-N16 -CACC at the promoter region. A similar palindromic motif and YtrA homologues were also identified in many other bacteria, including Stenotrophomonas, Pseudoxanthomonas and Frateuria, indicating a widespread phenomenon. Deletion of ytrA in Xac abolishes bacterial virulence and induction of the hypersensitive response (HR). We found that YtrA regulates the expression of hrp/hrc genes encoding the bacterial type III secretion system (T3SS) and controls multiple biological processes, including motility and adhesion, oxidative stress, extracellular enzyme production and iron uptake. YtrA represses the expression of its direct targets in artificial medium or in planta. Importantly, over-expression of yro3, one of the YtrA directly regulated operons which contains trmL and XAC0231, induced weaker canker symptoms and down-regulation of hrp/hrc gene expression, suggesting a negative regulation in Xac virulence and T3SS. Our study has significantly advanced the mechanistic understanding of YtrA regulation and its contribution to bacterial virulence. © 2016 BSPP AND JOHN WILEY & SONS LTD.

  10. 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

  11. 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

    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.

  12. 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

  13. 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.

  14. Energetic methods to study bifunctional biotin operon repressor.

    PubMed

    Beckett, D

    1998-01-01

    measurements. The results of quantitative studies of the biotin regulatory system can be interpreted in the context of the biological function of the system. The biotin holoenzyme ligases are a class of enzymes found across the evolutionary spectrum. Only a subset of these enzymes, including BirA, also function as transcriptional repressors. The tight binding of the allosteric effector may be understood in light of the bifunctional nature of the BirA-bio-5'-AMP complex. It is possible that the unusually high thermodynamic and kinetic stability of the complex ensures that the most probable state of the protein in vivo is the adenylate-bound form. This complex, not the unliganded protein, is active in both enzymatic transfer of biotin and site-specific DNA binding. This ensures that on depletion of the intracellular pool of apoBCCP, BirA-bio-5'-AMP accumulates and binds to bioO to repress transcription of the biotin biosynthesis operon. The intracellular demand for and synthesis of biotin are, consequently, tightly coupled in the system. The dimerization that accompanies adenylate binding to BirA appears to be significant for site-specific binding of the protein to bioO. Functionally, the simultaneous binding of the two monomers to the two operator half-sites, regardless of the kinetic mechanism by which it occurs, ensures coordinate regulation of transcription initiation from both biotin operon promoters. The multifaceted approach utilized in studies of the biotin regulatory system can serve as a model for studies of any complex transcriptional regulatory system. It is critical in elucidating the functional energetics of any of these systems that the assembly first be dissected into the constituent interactions and that each of these interactions be studied in isolation. This is not only critical for understanding the physicochemical properties of each individual contributing interaction, but is also a necessary precursor to studies of thermodynamic linkage in the system. (AB

  15. The nif Gene Operon of the Methanogenic Archaeon Methanococcus maripaludis

    PubMed Central

    Kessler, Peter S.; Blank, Carrine; Leigh, John A.

    1998-01-01

    Nitrogen fixation occurs in two domains, Archaea and Bacteria. We have characterized a nif (nitrogen fixation) gene cluster in the methanogenic archaeon Methanococcus maripaludis. Sequence analysis revealed eight genes, six with sequence similarity to known nif genes and two with sequence similarity to glnB. The gene order, nifH, ORF105 (similar to glnB), ORF121 (similar to glnB), nifD, nifK, nifE, nifN, and nifX, was the same as that found in part in other diazotrophic methanogens and except for the presence of the glnB-like genes, also resembled the order found in many members of the Bacteria. Using transposon insertion mutagenesis, we determined that an 8-kb region required for nitrogen fixation corresponded to the nif gene cluster. Northern analysis revealed the presence of either a single 7.6-kb nif mRNA transcript or 10 smaller mRNA species containing portions of the large transcript. Polar effects of transposon insertions demonstrated that all of these mRNAs arose from a single promoter region, where transcription initiated 80 bp 5′ to nifH. Distinctive features of the nif gene cluster include the presence of the six primary nif genes in a single operon, the placement of the two glnB-like genes within the cluster, the apparent physical separation of the cluster from any other nif genes that might be in the genome, the fragmentation pattern of the mRNA, and the regulation of expression by a repression mechanism described previously. Our study and others with methanogenic archaea reporting multiple mRNAs arising from gene clusters with only a single putative promoter sequence suggest that mRNA processing following transcription may be a common occurrence in methanogens. PMID:9515920

  16. act Operon Control of Developmental Gene Expression in Myxococcus xanthus

    PubMed Central

    Gronewold, Thomas M. A.; Kaiser, Dale

    2002-01-01

    Cell-bound C-signal guides the building of a fruiting body and triggers the differentiation of myxospores. Earlier work has shown that transcription of the csgA gene, which encodes the C-signal, is directed by four genes of the act operon. To see how expression of the genes encoding components of the aggregation and sporulation processes depends on C-signaling, mutants with loss-of-function mutations in each of the act genes were investigated. These mutations were found to have no effect on genes that are normally expressed up to 3 h into development and are C-signal independent. Neither the time of first expression nor the rate of expression increase was changed in actA, actB, actC, or actD mutant strains. Also, there was no effect on A-signal production, which normally starts before 3 h. By contrast, the null act mutants have striking defects in C-signal production. These mutations changed the expression of four gene reporters that are related to aggregation and sporulation and are expressed at 6 h or later in development. The actA and actB null mutations substantially decreased the expression of all these reporters. The other act null mutations caused either premature expression to wild-type levels (actC) or delayed expression (actD), which ultimately rose to wild-type levels. The pattern of effects on these reporters shows how the C-signal differentially regulates the steps that together build a fruiting body and differentiate spores within it. PMID:11807078

  17. The nif gene operon of the methanogenic archaeon Methanococcus maripaludis.

    PubMed

    Kessler, P S; Blank, C; Leigh, J A

    1998-03-01

    Nitrogen fixation occurs in two domains, Archaea and Bacteria. We have characterized a nif (nitrogen fixation) gene cluster in the methanogenic archaeon Methanococcus maripaludis. Sequence analysis revealed eight genes, six with sequence similarity to known nif genes and two with sequence similarity to glnB. The gene order, nifH, ORF105 (similar to glnB), ORF121 (similar to glnB), nifD, nifK, nifE, nifN, and nifX, was the same as that found in part in other diazotrophic methanogens and except for the presence of the glnB-like genes, also resembled the order found in many members of the Bacteria. Using transposon insertion mutagenesis, we determined that an 8-kb region required for nitrogen fixation corresponded to the nif gene cluster. Northern analysis revealed the presence of either a single 7.6-kb nif mRNA transcript or 10 smaller mRNA species containing portions of the large transcript. Polar effects of transposon insertions demonstrated that all of these mRNAs arose from a single promoter region, where transcription initiated 80 bp 5' to nifH. Distinctive features of the nif gene cluster include the presence of the six primary nif genes in a single operon, the placement of the two glnB-like genes within the cluster, the apparent physical separation of the cluster from any other nif genes that might be in the genome, the fragmentation pattern of the mRNA, and the regulation of expression by a repression mechanism described previously. Our study and others with methanogenic archaea reporting multiple mRNAs arising from gene clusters with only a single putative promoter sequence suggest that mRNA processing following transcription may be a common occurrence in methanogens.

  18. 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.

  19. Response regulator, VemR, positively regulates the virulence and adaptation of Xanthomonas campestris pv. campestris.

    PubMed

    Tao, Jun; He, Chaozu

    2010-03-01

    Xanthomonas campestris pv. campestris (Xcc) is the causal agent of black rot disease in cruciferous plants. The synthesis of known virulence factors in this organism, such as extracellular enzymes and biofilm, is strictly regulated in response to environmental stimuli. Two-component signal transduction systems sense environmental signals and alter bacterial behavior by regulating gene expression. Here, we identified a response regulator, VemR, that regulates Xcc pathogenesis. The vemR gene encodes an atypical response regulator that only contains a receiver domain. Deletion of vemR resulted in decreased virulence, exopolysaccharide production and motility of Xcc. The vemR gene is located in an operon flanked by genes fleQ and rpoN2. Genetic analysis indicated that deletion of fleQ does not affect motility significantly. However, a double mutant DeltavemR/DeltafleQ reversed the phenotype of DeltavemR, indicating that fleQ is epistatic to vemR in the regulation of virulence and adaptation.

  20. Diverse pathways for salicin utilization in Shigella sonnei and Escherichia coli carrying an impaired bgl operon.

    PubMed

    Desai, Stuti K; Nandimath, Krithi; Mahadevan, S

    2010-10-01

    Utilization of the aryl-β-glucosides salicin or arbutin in most wild-type strains of E. coli is achieved by a single-step mutational activation of the bgl operon. Shigella sonnei, a branch of the diverse E. coli strain tree, requires two sequential mutational steps for achieving salicin utilization as the bglB gene, encoding the phospho-β-glucosidase B, harbors an inactivating insertion. We show that in a natural isolate of S. sonnei, transcriptional activation of the gene SSO1595, encoding a phospho-β-glucosidase, enables salicin utilization with the permease function being provided by the activated bgl operon. SSO1595 is absent in most commensal strains of E. coli, but is present in extra-intestinal pathogens as bgcA, a component of the bgc operon that enables β-glucoside utilization at low temperature. Salicin utilization in an E. coli bglB laboratory strain also requires a two-step activation process leading to expression of BglF, the PTS-associated permease encoded by the bgl operon and AscB, the phospho-β-glucosidase B encoded by the silent asc operon. BglF function is needed since AscF is unable to transport β-glucosides as it lacks the IIA domain involved in phopho-relay. Activation of the asc operon in the Sal(+) mutant is by a promoter-up mutation and the activated operon is subject to induction. The pathway to achieve salicin utilization is therefore diverse in these two evolutionarily related organisms; however, both show cooperation between two silent genetic systems to achieve a new metabolic capability under selection.

  1. The alr-groEL1 operon in Mycobacterium tuberculosis: an interplay of multiple regulatory elements

    PubMed Central

    Bhat, Aadil H.; Pathak, Deepika; Rao, Alka

    2017-01-01

    Threonylcarbamoyladenosine is a universally conserved essential modification of tRNA that ensures translational fidelity in cellular milieu. TsaD, TsaB and TsaE are identified as tRNA-A37-threonylcarbamoyl (t6A)-transferase enzymes that have been reconstituted in vitro, in few bacteria recently. However, transcriptional organization and regulation of these genes are not known in any of these organisms. This study describes the intricate architecture of a complex multicistronic alr-groEL1 operon, harboring essential genes, namely tsaD, tsaB, tsaE, groES, groEL1, and alr (required for cell wall synthesis), and rimI encoding an N-α- acetyltransferase in Mycobacterium tuberculosis. Using northern blotting, RT-PCR and in vivo fluorescence assays, genes alr to groEL1 were found to constitute an ~6.3 kb heptacistronic operon with multiple internal promoters and an I-shaped intrinsic hairpin-like cis-regulatory element. A strong promoter PtsaD within the coding sequence of rimI gene is identified in M. tuberculosis, in addition. The study further proposes an amendment in the known bicistronic groESL1 operon annotation by providing evidence that groESL1 is co-transcribed as sub-operon of alr-groEL1 operon. The architecture of alr-groEL1 operon, conservation of the genetic context and a mosaic transcriptional profile displayed under various stress conditions convincingly suggest the involvement of this operon in stress adaptation in M. tuberculosis. PMID:28256563

  2. 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

  3. 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. © 2010 John Wiley & Sons A/S.

  4. Differences in virulence of Naegleria fowleri.

    PubMed

    De Jonckheere, J

    1979-10-01

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

  5. The subcutaneous inoculation of pH 6 antigen mutants of Yersinia pestis does not affect virulence and immune response in mice.

    PubMed

    Anisimov, Andrey P; Bakhteeva, Irina V; Panfertsev, Evgeniy A; Svetoch, Tat'yana E; Kravchenko, Tat'yana B; Platonov, Mikhail E; Titareva, Galina M; Kombarova, Tat'yana I; Ivanov, Sergey A; Rakin, Alexander V; Amoako, Kingsley K; Dentovskaya, Svetlana V

    2009-01-01

    Two isogenic sets of Yersinia pestis strains were generated, composed of wild-type strains 231 and I-1996, their non-polar pH 6(-) mutants with deletions in the psaA gene that codes for its structural subunit or the whole operon, as well as strains with restored ability for temperature- and pH-dependent synthesis of adhesion pili or constitutive production of pH 6 antigen. The mutants were generated by site-directed mutagenesis of the psa operon and subsequent complementation in trans. It was shown that the loss of synthesis or constitutive production of pH 6 antigen did not influence Y. pestis virulence or the average survival time of subcutaneously inoculated BALB/c naïve mice or animals immunized with this antigen.

  6. Ralstonia solanacearum iron scavenging by the siderophore staphyloferrin B is controlled by PhcA, the global virulence regulator.

    PubMed

    Bhatt, Garima; Denny, Timothy P

    2004-12-01

    PhcA is a transcriptional regulator that activates expression of multiple virulence genes in the plant pathogen Ralstonia solanacearum. Relative to their wild-type parents, phcA mutants overproduced iron-scavenging activity detected with chrome azurol S siderophore detection medium. Transposon mutagenesis of strain AW1-PC (phcA1) generated strain GB6, which was siderophore negative but retained weak iron-scavenging activity. The ssd gene inactivated in GB6 encodes a protein similar to group IV amino acid decarboxylases, and its transcription was repressed by iron(III) and PhcA. ssd is the terminal gene in a putative operon that also appears to encode three siderophore synthetase subunits, a integral membrane exporter, and three genes with no obvious role in siderophore production. A homologous operon was found in the genomes of Ralstonia metallidurans and Staphylococcus aureus, both of which produce the polycarboxylate siderophore staphyloferrin B. Comparison of the siderophores present in culture supernatants of R. solanacearum, R. metallidurans, and Bacillus megaterium using chemical tests, a siderophore utilization bioassay, thin-layer chromatography, and mass spectroscopy indicated that R. solanacearum produces staphyloferrin B rather than schizokinen as was reported previously. Inactivation of ssd in a wild-type AW1 background resulted in a mutant almost incapable of scavenging iron but normally virulent on tomato plants. AW1 did not produce siderophore activity when cultured in tomato xylem sap, suggesting that the main location in tomato for R. solanacearum during pathogenesis is iron replete.

  7. 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

  8. 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

    2016-08-23

    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.

  9. Insights into Entamoeba histolytica virulence modulation.

    PubMed

    Padilla-Vaca, F; Anaya-Velázquez, F

    2010-08-01

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

  10. Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes

    PubMed Central

    Semenov, Andrey N.; Gintsburg, Alexandr L.

    2016-01-01

    Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A “lacking biofilm production” (LBP) strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR) gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg) has a key role in biofilm formation. The relative location (i.e., by being separated by another gene) of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents. PMID:28070515

  11. Burkholderia contaminans Biofilm Regulating Operon and Its Distribution in Bacterial Genomes.

    PubMed

    Voronina, Olga L; Kunda, Marina S; Ryzhova, Natalia N; Aksenova, Ekaterina I; Semenov, Andrey N; Romanova, Yulia M; Gintsburg, Alexandr L

    2016-01-01

    Biofilm formation by Burkholderia spp. is a principal cause of lung chronic infections in cystic fibrosis patients. A "lacking biofilm production" (LBP) strain B. contaminans GIMC4587:Bct370-19 has been obtained by insertion modification of clinical strain with plasposon mutagenesis. It has an interrupted transcriptional response regulator (RR) gene. The focus of our investigation was a two-component signal transduction system determination, including this RR. B. contaminans clinical and LBP strains were analyzed by whole genome sequencing and bioinformatics resources. A four-component operon (BiofilmReg) has a key role in biofilm formation. The relative location (i.e., by being separated by another gene) of RR and histidine kinase genes is unique in BiofilmReg. Orthologs were found in other members of the Burkholderiales order. Phylogenetic analysis of strains containing BiofilmReg operons demonstrated evidence for earlier inheritance of a three-component operon. During further evolution one lineage acquired a fourth gene, whereas others lost the third component of the operon. Mutations in sensor domains have created biodiversity which is advantageous for adaptation to various ecological niches. Different species Burkholderia and Achromobacter strains all demonstrated similar BiofilmReg operon structure. Therefore, there may be an opportunity to develop a common drug which is effective for treating all these causative agents.

  12. Discovery of an operon that participates in agmatine metabolism and regulates biofilm formation in Pseudomonas aeruginosa.

    PubMed

    Williams, Bryan J; Du, Rui-Hong; Calcutt, M Wade; Abdolrasulnia, Rasul; Christman, Brian W; Blackwell, Timothy S

    2010-04-01

    Agmatine is the decarboxylation product of arginine and a number of bacteria have devoted enzymatic pathways for its metabolism. Pseudomonas aeruginosa harbours the aguBA operon that metabolizes agmatine to putrescine, which can be subsequently converted into other polyamines or shunted into the TCA cycle for energy production. We discovered an alternate agmatine operon in the P. aeruginosa strain PA14 named agu2ABCA' that contains two genes for agmatine deiminases (agu2A and agu2A'). This operon was found to be present in 25% of clinical P. aeruginosa isolates. Agu2A' contains a twin-arginine translocation signal at its N-terminus and site-directed mutagenesis and cell fractionation experiments confirmed this protein is secreted to the periplasm. Analysis of the agu2ABCA' promoter demonstrates that agmatine induces expression of the operon during the stationary phase of growth and during biofilm growth and agu2ABCA' provides only weak complementation of aguBA, which is induced during log phase. Biofilm assays of mutants of all three agmatine deiminase genes in PA14 revealed that deletion of agu2ABCA', specifically its secreted product Agu2A', reduces biofilm production of PA14 following addition of exogenous agmatine. Together, these findings reveal a novel role for the agu2ABCA' operon in the biofilm development of P. aeruginosa.

  13. 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.

  14. A novel marRAB operon contributes to the rifampicin resistance in Mycobacterium smegmatis.

    PubMed

    Zhang, Haiwei; Gao, Long; Zhang, Jiaoling; Li, Weihui; Yang, Min; Zhang, Hua; Gao, Chunhui; He, Zheng-Guo

    2014-01-01

    The multiple-antibiotic resistance regulator (MarR) plays an important role in modulating bacterial antibiotic resistance. However, the regulatory model of the marRAB operon in mycobacteria remains to be characterized. Here we report that a MarR, encoded by Ms6508, and its marRAB operon specifically contribute to rifampicin (RIF) resistance in Mycobacterium smegmatis. We show that the MarR recognizes a conserved 21-bp palindromic motif and negatively regulates the expression of two ABC transporters in the operon, encoded by Ms6509-6510. Unlike other known drug efflux pumps, overexpression of these two ABC transporters unexpectedly increased RIF sensitivity and deletion of these two genes increased mycobacterial resistance to the antibiotic. No change can be detected for the sensitivity of recombinant mycobacterial strains to three other anti-TB drugs. Furthermore, HPLC experiments suggested that Ms6509-Ms6510 could pump RIF into the mycobacterial cells. These findings indicated that the mycobacterial MarR functions as a repressor and constitutively inhibits the expression of the marRAB operon, which specifically contributes to RIF resistance in M. smegmatis. Therefore, our data suggest a new regulatory mechanism of RIF resistance and also provide the new insight into the regulatory model of a marRAB operon in mycobacteria.

  15. Virulence Mechanisms of Enteroinvasive Pathogens

    DTIC Science & Technology

    1988-01-01

    of Plasmid Gene Products otactic response facilitates the establishment of in Virulence a Salmonella infection (47). The action of pan- Anucleate...1967. Electron microscope studies of 36:615-620. experimental salmonella infection . 1. Penetration into 35. Rout, W. R., S. B. Formal, G. J. Dammin

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

    PubMed

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

    2003-11-01

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

  17. Salmonella enteritidis agfBAC operon encoding thin, aggregative fimbriae.

    PubMed

    Collinson, S K; Clouthier, S C; Doran, J L; Banser, P A; Kay, W W

    1996-02-01

    Salmonella enteritidis produces thin, aggregative fimbriae, named SEF17, which are composed of polymerized AgfA fimbrin proteins. DNA sequence analysis of a 2-kb region of S. enteritidis DNA revealed three contiguous genes, agfBAC. The 453-bp agfA gene encodes the AgfA fimbrin, which was predicted to be 74% identical and 86% similar in primary sequence to the Escherichia coli curli structural protein, CsgA. pHAG, a pUC18 derivative containing a 3.0-kb HindIII fragment encoding agfBAC, directed the in vitro expression of the major AgfA fimbrin, with an M(r) of 17,000, and a minor AgfB protein, with an M(r) of 16,000, encoded by the 453-bp agfB gene. AgfA was not expressed from pDAG, a pUC18 derivative containing a 3.1-kb DraI DNA fragment encoding agfA but not agfB. Primer extension analysis identified two adjacent transcription start sites located immediately upstream of agfB in positions analogous to those of the E. coli curlin csgBA operon. No transcription start sites were located immediately upstream of agfA or agfC. Northern (RNA) blot analysis confirmed that transcription of agfA was initiated from the agfB promoter region. Secondary-structure analysis of the putative mRNA transcript for agfBAC predicted the formation of a stem-loop structure (delta Gzero, -22 kcal/mol [-91 kJ/mol]) in the intercistronic region between agfA and agfC, which may be involved in stabilization of the agfBA portion of the agfBAC transcript. agfBAC and flanking regions had a high degree of sequence similarity with those counterparts of the E. coli curlin csgBA region for which sequence data are available. These data are demonstrative of the high degree of similarity between S. enteritidis SEF17 fimbriae and E. coli curli with respect to fimbrin amino acid sequence and genetic organization and, therefore, are indicative of a common and relatively recent ancestry.

  18. Transcriptional activation of virulence genes of Rhizobium etli.

    PubMed

    Wang, Luyao; Lacroix, Benoît; Guo, Jianhua; Citovsky, Vitaly

    2017-01-09

    Recently, Rhizobium etli has emerged, in addition to Agrobacterium spp., as a prokaryotic species that encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we showed that the signal phenolic molecule acetosyringone (AS) induced R. etli vir gene expression both in R. etli and in A. tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter T-DNA. Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium, but not from non-host plants. Early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside of the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a versus pTiC58 of A. tumefaciens IMPORTANCE: The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into plant cell genome. In this study, we explore the transcriptional regulation and induction of virulence genes in R. etli and show similarities and differences

  19. 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.

  20. Paralogous Outer Membrane Proteins Mediate Uptake of Different Forms of Iron and Synergistically Govern Virulence in Francisella tularensis tularensis*

    PubMed Central

    Ramakrishnan, Girija; Sen, Bhaswati; Johnson, Richard

    2012-01-01

    Francisella tularensis subsp. tularensis is a highly infectious bacterium causing acute disease in mammalian hosts. Mechanisms for the acquisition of iron within the iron-limiting host environment are likely to be critical for survival of this intracellular pathogen. FslE (FTT0025) and FupA (FTT0918) are paralogous proteins that are predicted to form β-barrels in the outer membrane of virulent strain Schu S4 and are unique to Francisella species. Previous studies have implicated both FupA, initially identified as a virulence factor and FslE, encoded by the siderophore biosynthetic operon, in iron acquisition. Using single and double mutants, we demonstrated that these paralogs function in concert to promote growth under iron limitation. We used a 55Fe transport assay to demonstrate that FslE is involved in siderophore-mediated ferric iron uptake, whereas FupA facilitates high affinity ferrous iron uptake. Optimal replication within J774A.1 macrophage-like cells required at least one of these uptake systems to be functional. In a mouse model of tularemia, the ΔfupA mutant was attenuated, but the ΔfslE ΔfupA mutant was significantly more attenuated, implying that the two systems of iron acquisition function synergistically to promote virulence. These studies highlight the importance of specific iron acquisition functions, particularly that of ferrous iron, for virulence of F. tularensis in the mammalian host. PMID:22661710

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

    PubMed Central

    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

    2016-01-01

    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

  2. Analysis of the Rickettsia africae genome reveals that virulence acquisition in Rickettsia species may be explained by genome reduction

    PubMed Central

    Fournier, Pierre-Edouard; El Karkouri, Khalid; Leroy, Quentin; Robert, Catherine; Giumelli, Bernadette; Renesto, Patricia; Socolovschi, Cristina; Parola, Philippe; Audic, Stéphane; Raoult, Didier

    2009-01-01

    Background The Rickettsia genus includes 25 validated species, 17 of which are proven human pathogens. Among these, the pathogenicity varies greatly, from the highly virulent R. prowazekii, which causes epidemic typhus and kills its arthropod host, to the mild pathogen R. africae, the agent of African tick-bite fever, which does not affect the fitness of its tick vector. Results We evaluated the clonality of R. africae in 70 patients and 155 ticks, and determined its genome sequence, which comprises a circular chromosome of 1,278,540 bp including a tra operon and an unstable 12,377-bp plasmid. To study the genetic characteristics associated with virulence, we compared this species to R. prowazekii, R. rickettsii and R. conorii. R. africae and R. prowazekii have, respectively, the less and most decayed genomes. Eighteen genes are present only in R. africae including one with a putative protease domain upregulated at 37°C. Conclusion Based on these data, we speculate that a loss of regulatory genes causes an increase of virulence of rickettsial species in ticks and mammals. We also speculate that in Rickettsia species virulence is mostly associated with gene loss. The genome sequence was deposited in GenBank under accession number [GenBank: NZ_AAUY01000001]. PMID:19379498

  3. 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

  4. The Polyamine N-Acetyltransferase-Like Enzyme PmvE Plays a Role in the Virulence of Enterococcus faecalis

    PubMed Central

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

    2014-01-01

    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 N1-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

  5. A proteomic analysis of the iron response of Photobacterium damselae subsp. damselae reveals metabolic adaptations to iron levels changes and novel potential virulence factors.

    PubMed

    Puentes, Beatriz; Balado, Miguel; Bermúdez-Crespo, José; Osorio, Carlos R; Lemos, Manuel L

    2017-03-01

    Photobacterium damselae subsp. damselae (Pdd) is a marine bacterium that can infect numerous species of marine fish as well as other species including humans. Low iron availability is one of the signs that bacterial pathogens can detect in order to begin colonizing their host, and the reduction of iron levels is a nonspecific host defense strategy that prevents bacterial proliferation. In this work a proteomic approach was used to study the gene expression adaptations of a Pdd strain in response to iron availability. A comparative analysis of induced proteins in both high- and low-iron conditions showed profound cellular metabolic adaptations that result, for instance, in amino acid requirement. It also provided important information about the changes that occur in the energetic metabolism induced by the surrounding iron levels, allowing for the identification of novel potential virulence factors. Among others, genes involved in the synthesis and transport of a vibrioferrin-like siderophore were identified for the first time. In addition to plasmid pPHDD1-encoded Dly and HlyA hemolysins, a pPHDD1-borne operon, which may encode a transferrin receptor, was also found. This operon identification suggests that this virulence plasmid could encode so-far unknown additional virulence factors other than hemolysins. Copyright © 2017 Elsevier B.V. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

  7. Compensatory role of PspA, a member of the phage shock protein operon, in rpoE mutant Salmonella enterica serovar Typhimurium.

    PubMed

    Becker, Lynne A; Bang, Iel-Soo; Crouch, Marie-Laure; Fang, Ferric C

    2005-05-01

    Sigma(E) is an alternative sigma factor that responds to and ameliorates extracytoplasmic stress. In Salmonella enterica serovar Typhimurium (S. Typhimurium), sigma(E) is required for oxidative stress resistance, stationary-phase survival and virulence in mice. Microarray analysis of stationary-phase gene expression in rpoE mutant bacteria revealed a dramatic increase in expression of pspA, a member of the phage shock protein (psp) operon. The psp operon can be induced by filamentous bacteriophages or by perturbations of protein secretion, and is believed to facilitate the maintenance of proton motive force (PMF). We hypothesized that increased pspA expression may represent a compensatory response to the loss of sigma(E) function. Increased pspA expression was confirmed in rpoE mutant Salmonella and also observed in a mutant lacking the F(1)F(0) ATPase. Alternatively, expression of pspA could be induced by exposure to CCCP, a protonophore that disrupts PMF. An rpoE pspA double mutant strain was found to have a stationary-phase survival defect more pronounced than that of isogenic strains harbouring single mutations. The double mutant strains were also more susceptible to killing by CCCP or by a bactericidal/permeability-increasing protein (BPI)-derived anti-microbial peptide. Using fluorescence ratio imaging, differences were observed in the Deltapsi of wild-type and rpoE or pspA mutant bacteria. These findings suggest that pspA expression in S. Typhimurium is induced by alterations in PMF and a functional sigma(E) regulon is essential for the maintenance of PMF.

  8. Enterococcal cytolysin: activities and association with other virulence traits in a pathogenicity island.

    PubMed

    Shankar, Nathan; Coburn, Phillip; Pillar, Chris; Haas, Wolfgang; Gilmore, Michael

    2004-04-01

    Enterococcal cytolysin is a structurally novel bacterial toxin expressed by some strains of E. faecalis and is distantly related to the class of bacteriocins known as lantibiotics. The cytolysin can be encoded by large pheromone-responsive plasmids, or on the chromosome within pathogenicity island. It is produced by a complex process that involves the products of eight genes, designated cylR1, cylR2, cylLL, cylLS, cylM, cylB, cylA, and cylI. The cytolysin toxin, maturation and regulatory genes are organized into two divergent transcripts: a structural transcript cylLLLSMBAI, and a regulatory transcript cylR1R2. The active cytolysin subunits, CylLL" and CylLS", are synthesized ribosomally as non-identical peptides, post-translationally modified, then secreted and activated. The cytolysin operon is repressed by the activities of two proteins, CylR1 and CylR2, and derepressed by a quorum-sensing process involving secreted autoinducer CylLS". The cytolysin operon within the E. faecalis pathogenicity island is associated with other virulence determinants, including aggregation substance and enterococcal surface protein, Esp.

  9. 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

  10. Phylogeny and virulence of naturally occurring type III secretion system-deficient Pectobacterium strains.

    PubMed

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

    2009-07-01

    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.

  11. The Mycoplasma gallisepticum Virulence Factor Lipoprotein MslA Is a Novel Polynucleotide Binding Protein

    PubMed Central

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

    2013-01-01

    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

  12. An Escherichia coli chromosomal ars operon homolog is functional in arsenic detoxification and is conserved in gram-negative bacteria.

    PubMed

    Diorio, C; Cai, J; Marmor, J; Shinder, R; DuBow, M S

    1995-04-01

    Arsenic is a known toxic metalloid, whose trivalent and pentavalent ions can inhibit many biochemical processes. Operons which encode arsenic resistance have been found in multicopy plasmids from both gram-positive and gram-negative bacteria. The resistance mechanism is encoded from a single operon which typically consists of an arsenite ion-inducible repressor that regulates expression of an arsenate reductase and inner membrane-associated arsenite export system. Using a lacZ transcriptional gene fusion library, we have identified an Escherichia coli operon whose expression is induced by cellular exposure to sodium arsenite at concentrations as low as 5 micrograms/liter. This chromosomal operon was cloned, sequenced, and found to consist of three cistrons which we named arsR, arsB, and arsC because of their strong homology to plasmid-borne ars operons. Mutants in the chromosomal ars operon were found to be approximately 10- to 100-fold more sensitive to sodium arsenate and arsenite exposure than wild-type E. coli, while wild-type E. coli that contained the operon cloned on a ColE1-based plasmid was found to be at least 2- to 10-fold more resistant to sodium arsenate and arsenite. Moreover, Southern blotting and high-stringency hybridization of this operon with chromosomal DNAs from a number of bacterial species showed homologous sequences among members of the family Enterobacteriaceae, and hybridization was detectable even in Pseudomonas aeruginosa. These results suggest that the chromosomal ars operon may be the evolutionary precursor of the plasmid-borne operon, as a multicopy plasmid location would allow the operon to be amplified and its products to confer increased resistance to this toxic metalloid.

  13. Unusual organization, complexity and redundancy at the Escherichia coli hcp-hcr operon promoter.

    PubMed

    Chismon, David L; Browning, Douglas F; Farrant, Gregory K; Busby, Stephen J W

    2010-08-15

    Expression from the Escherichia coli hcp-hcr operon promoter is optimally induced during anaerobic conditions in the presence of nitrite. This expression depends on transcription activation by FNR (fumarate and nitrate reduction regulator), which binds to a target centred at position -72.5 upstream of the transcript start site. Mutational analysis was exploited to identify the corresponding -10 and -35 hexamer elements. A DNA site for NarL and NarP, located at position -104.5, plays only a minor role, whereas NsrR binding to a DNA target centred at position +6 plays a major role in induction of the hcp-hcr operon promoter. Electrophoretic mobility-shift assays show that NsrR binds to this target. The consequences of this for the kinetics of induction of the hcp-hcr operon are discussed.

  14. Identification and characterization of an operon in Salmonella typhimurium involved in thiamine biosynthesis.

    PubMed

    Petersen, L A; Downs, D M

    1997-08-01

    Thiamine pyrophosphate (TPP) is synthesized de novo in Salmonella typhimurium and is a required cofactor for many enzymes in the cell. Five kinase activities have been implicated in TPP synthesis, which involves joining a 4-methyl-5-(beta-hydroxyethyl)thiazole (THZ) moiety and a 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety. We report here identification of a 2-gene operon involved in thiamine biosynthesis and present evidence that the genes in this operon, thiMD, encode two previously identified kinases, THZ kinase and HMP phosphate (HMP-P) kinase, respectively. We further show that this operon belongs to the growing class of genes involved in TPP synthesis that are transcriptionally regulated by TPP. Our data are consistent with ThiM being a salvage enzyme and ThiD being a biosynthetic enzyme involved in TPP synthesis, as previously suggested.

  15. Two Mutations in the First Gene of the Histidine Operon of Salmonella typhimurium Affecting Control

    PubMed Central

    Rothman-Denes, Lucia; Martin, Robert G.

    1971-01-01

    Two strains with mutations in the first structural gene of the histidine operon of Salmonella typhimurium were characterized. (The first structural gene specifies the first enzyme of histidine biosynthesis, phosphoribosyltransferase, which is sensitive to feedback inhibition by histidine.) One mutation, hisG3934, results in a phosphoribosyltransferase which is no longer sensitive to feedback inhibition by histidine but is instead subject to inhibition by aspartic acid. The other mutation, hisG3935, allows the histidine operon to be partially repressed by several amino acids, including aspartic acid. Analysis of hisG3935 is consistent with the hypothesis that phosphoribosyltransferase is directly involved in the regulation of the histidine operon. PMID:4928009

  16. Molecular analysis of the UV-inducible pili operon from Sulfolobus acidocaldarius.

    PubMed

    van Wolferen, Marleen; Ajon, Małgorzata; Driessen, Arnold J M; Albers, Sonja-Verena

    2013-12-01

    Upon ultraviolet (UV) stress, hyperthermophilic Sulfolobus species show a highly induced transcription of a gene cluster responsible for pili biogenesis: the UV-inducible pili operon (ups operon). This operon is involved in UV-induced pili assembly, cellular aggregation, and subsequent DNA exchange between cells. As the system increases the fitness of Sulfolobus cells after UV light exposure, we assume that transfer of DNA takes place in order to repair UV-induced DNA damages via homologous recombination. Here, we studied all genes present in the ups cluster via gene deletion analysis with a focus on UpsX, a protein that shows no identifiable functional domains. UspX does not seem to be structurally essential for UV-induced pili formation and cellular aggregation, but appears to be important for efficient DNA transfer. In addition, we could show that pilin subunits UpsA and UpsB probably both function as major pilin subunits in the ups pili.

  17. Footprints of Optimal Protein Assembly Strategies in the Operonic Structure of Prokaryotes

    PubMed Central

    Ewald, Jan; Kötzing, Martin; Bartl, Martin; Kaleta, Christoph

    2015-01-01

    In this work, we investigate optimality principles behind synthesis strategies for protein complexes using a dynamic optimization approach. We show that the cellular capacity of protein synthesis has a strong influence on optimal synthesis strategies reaching from a simultaneous to a sequential synthesis of the subunits of a protein complex. Sequential synthesis is preferred if protein synthesis is strongly limited, whereas a simultaneous synthesis is optimal in situations with a high protein synthesis capacity. We confirm the predictions of our optimization approach through the analysis of the operonic organization of protein complexes in several hundred prokaryotes. Thereby, we are able to show that cellular protein synthesis capacity is a driving force in the dissolution of operons comprising the subunits of a protein complex. Thus, we also provide a tested hypothesis explaining why the subunits of many prokaryotic protein complexes are distributed across several operons despite the presumably less precise co-regulation. PMID:25927816

  18. Cloning and Analysis of the rnc-era-recO Operon from Pseudomonas aeruginosa

    PubMed Central

    Powell, Bradford; Peters, Howard K.; Nakamura, Yoshikazu; Court, Donald

    1999-01-01

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

  19. Growth rate regulation of lac operon expression in Escherichia coli is cyclic AMP dependent.

    PubMed

    Kuo, Jong-Tar; Chang, Yu-Jen; Tseng, Ching-Ping

    2003-10-23

    In contrast to the ribosomal RNA gene expression increasing with growth rate, transcription of the lac operon is downregulated by cell growth rate. In continuous culture, growth rate regulation of lac promoter was independent of carbon substrate used and its location on the chromosome. Since the lac operon is activated by cyclic adenosine monophosphate (cAMP), which decreases with increasing cell growth rate, expression of plac-lacZ reporter fusion was analyzed in cya mutant under various growth conditions. The results demonstrated that expression of plac-lacZ in cya mutant was both lower and growth rate independent. In addition, ppGpp (guanosine tetraphosphate) was not involved in the mechanism of growth rate regulation of the lac promoter. Thus, the results of this study indicate that cAMP mediates the growth rate-dependent regulation of lac operon expression in Escherichia coli.

  20. Footprints of optimal protein assembly strategies in the operonic structure of prokaryotes.

    PubMed

    Ewald, Jan; Kötzing, Martin; Bartl, Martin; Kaleta, Christoph

    2015-04-28

    In this work, we investigate optimality principles behind synthesis strategies for protein complexes using a dynamic optimization approach. We show that the cellular capacity of protein synthesis has a strong influence on optimal synthesis strategies reaching from a simultaneous to a sequential synthesis of the subunits of a protein complex. Sequential synthesis is preferred if protein synthesis is strongly limited, whereas a simultaneous synthesis is optimal in situations with a high protein synthesis capacity. We confirm the predictions of our optimization approach through the analysis of the operonic organization of protein complexes in several hundred prokaryotes. Thereby, we are able to show that cellular protein synthesis capacity is a driving force in the dissolution of operons comprising the subunits of a protein complex. Thus, we also provide a tested hypothesis explaining why the subunits of many prokaryotic protein complexes are distributed across several operons despite the presumably less precise co-regulation.

  1. Characterization of the petI and res Operons of Acidithiobacillus ferrooxidans

    PubMed Central

    Levicán, Gloria; Bruscella, Patrice; Guacunano, Maritza; Inostroza, Carolina; Bonnefoy, Violaine; Holmes, David S.; Jedlicki, Eugenia

    2002-01-01

    DNA sequence analysis and bioinformatic interpretations have identified two adjacent clusters of genes potentially involved in the formation of a bc1 complex and in the maturation of a cytochrome c-type protein in two strains (ATCC 19859 and ATCC 33020) of the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans (formerly Thiobacillus ferrooxidans). Reverse transcriptase-PCR experiments suggest that the two clusters are organized as operons, and +1 start sites of transcription for the operons have been determined by primer extension experiments. Potential promoters have been identified. The presence of these operons lends support to a recent model of reverse electron flow and is consistent with previous reports of phenotypic switching in this bacterium. PMID:11844787

  2. Positive and negative regulation of the bgl operon in Escherichia coli.

    PubMed Central

    Mahadevan, S; Reynolds, A E; Wright, A

    1987-01-01

    We have analyzed the functions encoded by the bgl operon in Escherichia coli K-12. Based on the ability of cloned regions of the operon to complement a series of Bgl- point mutations, we show that the three bgl structural genes, bglC, bglS, and bglB, are located downstream of the regulatory locus bglR in the order indicated. Using a bgl-lacZ transcriptional fusion, we show that bglC and bglS are involved in regulating operon expression. The presence of the bglC gene in trans is absolutely required for the expression of the fusion, which is constitutive when only the bglC gene is present. When the bglC and the bglS genes are both present in the cell, expression of the fusion requires a beta-glucoside inducer. From these observations, we conclude that (i) the bglC gene encodes a positive regulatory of bgl operon expression and (ii) the bglS gene encodes a negative regulator of operon expression, causing the requirement for a beta-glucoside inducer. These conclusions are supported by our observations that (i) a majority of bglC mutants exhibits a Bgl- phenotype, whereas rare trans-dominant mutations in bglC result in constitutive expression of the bgl operon and the fusion, and (ii) mutations in the bglS gene lead to constitutive expression of the fusion. Based on several lines of evidence presented, we propose that the bglS gene product has an additional role as a component of the beta-glucoside transport system. PMID:3294798

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

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

    2016-01-01

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

  5. Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species.

    PubMed

    Peters, M; Heinaru, E; Talpsep, E; Wand, H; Stottmeister, U; Heinaru, A; Nurk, A

    1997-12-01

    Horizontal transfer of genes of selective value in an environment 6 years after their introduction into a watershed has been observed. Expression of the gene pheA, which encodes phenol monooxygenase and is linked to the pheBA operon (A. Nurk, L. Kasak, and M. Kivisaar, Gene 102:13-18, 1991), allows pseudomonads to use phenol as a growth substrate. Pseudomonas putida strains carrying this operon on a plasmid were used for bioremediation after an accidental fire in the Estonia oil shale mine in Estonia in 1988. The water samples used for studying the fate of the genes introduced were collected in 1994. The same gene cluster was also detected in Pseudomonas strains isolated from water samples of a nearby watershed which has been continuously polluted with phenols due to oil shale industry leachate. Together with the more frequently existing counterparts of the dmp genes (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), the pheA gene was also represented in the phenol-degrading strains. The area where the strains containing the pheA gene were found was restricted to the regular route of phenolic leachate to the Baltic Sea. Nine Pseudomonas strains belonging to four different species (P. corrugata, P. fragi, P. stutzeri, and P. fluorescens biotypes B, C, and F) and harboring horizontally transferred pheBA operons were investigated. The phe genes were clustered in the same manner in these nine phe operons and were connected to the same promoter as in the case of the original pheBA operon. One 10.6-kb plasmid carrying a pheBA gene cluster was sequenced, and the structure of the rearranged pheBA operon was described. This data indicates that introduced genetic material could, if it encodes a beneficial capability, enrich the natural genetic variety for biodegradation.

  6. Acquisition of a deliberately introduced phenol degradation operon, pheBA, by different indigenous Pseudomonas species.

    PubMed Central

    Peters, M; Heinaru, E; Talpsep, E; Wand, H; Stottmeister, U; Heinaru, A; Nurk, A

    1997-01-01

    Horizontal transfer of genes of selective value in an environment 6 years after their introduction into a watershed has been observed. Expression of the gene pheA, which encodes phenol monooxygenase and is linked to the pheBA operon (A. Nurk, L. Kasak, and M. Kivisaar, Gene 102:13-18, 1991), allows pseudomonads to use phenol as a growth substrate. Pseudomonas putida strains carrying this operon on a plasmid were used for bioremediation after an accidental fire in the Estonia oil shale mine in Estonia in 1988. The water samples used for studying the fate of the genes introduced were collected in 1994. The same gene cluster was also detected in Pseudomonas strains isolated from water samples of a nearby watershed which has been continuously polluted with phenols due to oil shale industry leachate. Together with the more frequently existing counterparts of the dmp genes (V. Shingler, J. Powlowski, and U. Marklund, J. Bacteriol. 174:711-724, 1992), the pheA gene was also represented in the phenol-degrading strains. The area where the strains containing the pheA gene were found was restricted to the regular route of phenolic leachate to the Baltic Sea. Nine Pseudomonas strains belonging to four different species (P. corrugata, P. fragi, P. stutzeri, and P. fluorescens biotypes B, C, and F) and harboring horizontally transferred pheBA operons were investigated. The phe genes were clustered in the same manner in these nine phe operons and were connected to the same promoter as in the case of the original pheBA operon. One 10.6-kb plasmid carrying a pheBA gene cluster was sequenced, and the structure of the rearranged pheBA operon was described. This data indicates that introduced genetic material could, if it encodes a beneficial capability, enrich the natural genetic variety for biodegradation. PMID:9406411

  7. Regulation of microcin C51 operon expression: the role of global regulators of transcription.

    PubMed

    Fomenko, D; Veselovskii, A; Khmel, I

    2001-06-01

    Expression of the microcin C51 operon in Escherichia coli cells is regulated as a function of the phase of growth; it is stimulated during the decelerating phase of growth. Using single-copy P(mcc)-lac transcriptional fusion (the promoter region of the microcin C51 operon fused to a promoterless lac operon in lambda phage), we showed that transcription from the microcin operon promoter is dependent on sigma(s) (RpoS) factor. However, some level of P(mcc)-lac expression is possible in rpoS null mutants, indicating that another sigma factor might be involved in transcription of the microcin C51 operon. Overproduction of sigma70 decreased Pmcc-directed transcription, presumably as a result of competition of sigma factors for the limited amount of core RNA polymerase. The cyclic AMP-CRP complex was shown to stimulate transcription from Pmcc: the absence of CRP or cAMP in crp or cya mutant cells strongly decreased the level of P(mcc)-lac expression. The production of C51 microcin decreased or was absent in rpoS, crp and cya mutant cells. Leucine-responsive protein Lrp and histone-like protein H-NS repressed P(mcc)-lac expression in the exponential and decelerating phases of growth. In studies of P(mcc)-lac expression in double mutant cells, we showed that proteins CRP, Lrp and H-NS acted in rpoS-dependent and rpoS-independent ways in transcription of the microcin C51 operon. Mutation hns(-) resulted in an increase in P(mcc)-lac expression in crp, rpoS and lrp mutant cells, as in wild-type cells.

  8. HP0197 contributes to CPS synthesis and the virulence of Streptococcus suis via CcpA.

    PubMed

    Zhang, Anding; Chen, Bo; Yuan, Zhengzhi; Li, Ran; Liu, Cheng; Zhou, Hongbo; Chen, Huanchun; Jin, Meilin

    2012-01-01

    Streptococcus suis serotype 2 (SS2), a major swine pathogen and an emerging zoonotic agent, has greatly challenged global public health. The encoding proteins with unknown functions the bacterium encodes are an obstruction to studies of the pathogenesis. A novel surface protective antigen HP0197 is one of these proteins which have no sequence homology to any known protein. In the present study, the protein was determined to be involved in bacterial virulence through an evaluation of the isogenic mutant (Δhp0197) in both mice and pigs. The experimental infection also indicated that Δhp0197 could be cleared easily during infection, which could be attributed to the reduced thickness of the capsular polysaccharides (CPS) and the significantly reduced phagocytotic resistance. Microarrays-based comparative transcriptome analysis suggested that the suppressed expression of the operon responsible for CPS synthesis might be reversed by CcpA activity, which controlled global regulation of carbon catabolite through the binding of the CcpA and HPr-Ser-46-P to the catabolite-responsive elements (cre) of the target operons. The hypothesis was approved by the fact that the purified FLAG-tagged HPr from WT stain exhibited a higher binding activity to cre with CcpA compared to the Δhp0197 by the Electrophoretic Mobility Shift Assay, suggesting lower level of phosphorylation of the phosphocarrier protein HPr at residue Ser-46 (HPr-Ser-46P) in Δhp0197. These indicated that HP0197 could enhance CcpA activity to control the expression of genes involved in carbohydrate utilization and CPS synthesis, thus contributing to the virulence of S. suis.

  9. Massive horizontal gene transfer, strictly vertical inheritance and ancient duplications differentially shape the evolution of Bacillus cereus enterotoxin operons hbl, cytK and nhe.

    PubMed

    Böhm, Maria-Elisabeth; Huptas, Christopher; Krey, Viktoria Magdalena; Scherer, Siegfried

    2015-11-10

    Bacillus cereus sensu lato comprises eight closely related species including the human pathogens Bacillus anthracis and Bacillus cereus. Within B. cereus sensu lato, chromosomally and plasmid-encoded toxins exist. While plasmid-mediated horizontal gene transfer of the emetic toxin, anthrax and insecticidal toxins is known, evolution of enterotoxin genes within the group has not been studied. We report draft genome assemblies of 25 strains, a phylogenetic network of 142 strains based on ANI derived from genome sequences and a phylogeny based on whole-genome SNP analysis. The data clearly support subdivision of B. cereus sensu lato into seven phylogenetic groups. While group I, V and VII represent B. pseudomycoides, B. toyonensis and B. cytotoxicus, which are distinguishable at species level (ANI border ≥ 96 %), strains ascribed to the other five species do not match phylogenic groups. The chromosomal enterotoxin operons nheABC and hblCDAB are abundant within B. cereus both isolated from infections and from the environment. While the duplicated hbl variant hbl a is present in 22 % of all strains investigated, duplication of nheABC is extremely rare (0.02 %) and appears to be phylogenetically unstable. Distribution of toxin genes was matched to a master tree based on seven concatenated housekeeping genes, which depicts species relationships in B. cereus sensu lato as accurately as whole-genome comparisons. Comparison to the phylogeny of enterotoxin genes uncovered ample evidence for horizontal transfer of hbl, cytK and plcR, as well as frequent deletion of both toxins and duplication of hbl. No evidence for nhe deletion was found and stable horizontal transfer of nhe is rare. Therefore, evolution of B. cereus enterotoxin operons is shaped unexpectedly different for yet unknown reasons. Frequent exchange of the pathogenicity factors hbl, cytK and plcR in B. cereus sensu lato appears to be an important mechanism of B. cereus virulence evolution, including so

  10. Characterization of virulence factor regulation by SrrAB, a two-component system in Staphylococcus aureus.

    PubMed

    Pragman, Alexa A; Yarwood, Jeremy M; Tripp, Timothy J; Schlievert, Patrick M

    2004-04-01

    Workers in our laboratory have previously identified the staphylococcal respiratory response AB (SrrAB), a Staphylococcus aureus two-component system that acts in the global regulation of virulence factors. This system down-regulates production of agr RNAIII, protein A, and toxic shock syndrome toxin 1 (TSST-1), particularly under low-oxygen conditions. In this study we investigated the localization and membrane orientation of SrrA and SrrB, transcription of the srrAB operon, the DNA-binding properties of SrrA, and the effect of SrrAB expression on S. aureus virulence. We found that SrrA is localized to the S. aureus cytoplasm, while SrrB is localized to the membrane and is properly oriented to function as a histidine kinase. srrAB has one transcriptional start site which results in either an srrA transcript or a full-length srrAB transcript; srrB must be cotranscribed with srrA. Gel shift assays of the agr P2, agr P3, protein A (spa), TSST-1 (tst), and srr promoters revealed SrrA binding at each of these promoters. Analysis of SrrAB-overexpressing strains by using the rabbit model of bacterial endocarditis demonstrated that overexpression of SrrAB decreased the virulence of the organisms compared to the virulence of isogenic strains that do not overexpress SrrAB. We concluded that SrrAB is properly localized and oriented to function as a two-component system. Overexpression of SrrAB, which represses agr RNAIII, TSST-1, and protein A in vitro, decreases virulence in the rabbit endocarditis model. Repression of these virulence factors is likely due to a direct interaction between SrrA and the agr, tst, and spa promoters.

  11. [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.

  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. The mangotoxin biosynthetic operon (mbo) is specifically distributed within Pseudomonas syringae genomospecies 1 and was acquired only once during evolution.

    PubMed

    Carrión, Víctor J; Gutiérrez-Barranquero, José A; Arrebola, Eva; Bardaji, Leire; Codina, Juan C; de Vicente, Antonio; Cazorla, Francisco M; Murillo, Jesús

    2013-02-01

    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.

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

    PubMed Central

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

    2014-01-01

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

  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. Novel genetic tools for diaminopimelic acid selection in virulence studies of Yersinia pestis.

    PubMed

    Bland, David M; Eisele, Nicholas A; Keleher, Lauren L; Anderson, Paul E; Anderson, Deborah M

    2011-03-02

    Molecular studies of bacterial virulence are enhanced by expression of recombinant DNA during infection to allow complementation of mutants and expression of reporter proteins in vivo. For highly pathogenic bacteria, such as Yersinia pestis, these studies are currently limited because deliberate introduction of antibiotic resistance is restricted to those few which are not human treatment options. In this work, we report the development of alternatives to antibiotics as tools for host-pathogen research during Yersinia pestis infections focusing on the diaminopimelic acid (DAP) pathway, a requirement for cell wall synthesis in eubacteria. We generated a mutation in the dapA-nlpB(dapX) operon of Yersinia pestis KIM D27 and CO92 which eliminated the expression of both genes. The resulting strains were auxotrophic for diaminopimelic acid and this phenotype was complemented in trans by expressing dapA in single and multi-copy. In vivo, we found that plasmids derived from the p15a replicon were cured without selection, while selection for DAP enhanced stability without detectable loss of any of the three resident virulence plasmids. The dapAX mutation rendered Y. pestis avirulent in mouse models of bubonic and septicemic plague which could be complemented when dapAX was inserted in single or multi-copy, restoring development of disease that was indistinguishable from the wild type parent strain. We further identified a high level, constitutive promoter in Y. pestis that could be used to drive expression of fluorescent reporters in dapAX strains that had minimal impact to virulence in mouse models while enabling sensitive detection of bacteria during infection. Thus, diaminopimelic acid selection for single or multi-copy genetic systems in Yersinia pestis offers an improved alternative to antibiotics for in vivo studies that causes minimal disruption to virulence.

  17. The evolution of tuberculosis virulence.

    PubMed

    Basu, Sanjay; Galvani, Alison P

    2009-07-01

    The evolution of Mycobacterium tuberculosis presents several challenges for public health. HIV and resistance to antimycobacterial medications have evolutionary implications for how Mycobacterium tuberculosis will evolve, as these factors influence the host environment and transmission dynamics of tuberculosis strains. We present an evolutionary invasion analysis of tuberculosis that characterizes the direction of tuberculosis evolution in the context of different natural and human-driven selective pressures, including changes in tuberculosis treatment and HIV prevalence. We find that the evolution of tuberculosis virulence can be affected by treatment success rates, the relative transmissibility of emerging strains, the rate of reactivation from latency among hosts, and the life expectancy of hosts. We find that the virulence of tuberculosis strains may also increase as a consequence of rising HIV prevalence, requiring faster case detection strategies in areas where the epidemics of HIV and tuberculosis collide.

  18. Manganese uptake and streptococcal virulence.

    PubMed

    Eijkelkamp, Bart A; McDevitt, Christopher A; Kitten, Todd

    2015-06-01

    Streptococcal solute-binding proteins (SBPs) associated with ATP-binding cassette transporters gained widespread attention first as ostensible adhesins, next as virulence determinants, and finally as metal ion transporters. In this mini-review, we will examine our current understanding of the cellular roles of these proteins, their contribution to metal ion homeostasis, and their crucial involvement in mediating streptococcal virulence. There are now more than 35 studies that have collected structural, biochemical and/or physiological data on the functions of SBPs across a broad range of bacteria. This offers a wealth of data to clarify the formerly puzzling and contentious findings regarding the metal specificity amongst this group of essential bacterial transporters. In particular we will focus on recent findings related to biological roles for manganese in streptococci. These advances will inform efforts aimed at exploiting the importance of manganese and manganese acquisition for the design of new approaches to combat serious streptococcal diseases.

  19. Campylobacter virulence and survival factors.

    PubMed

    Bolton, Declan J

    2015-06-01

    Despite over 30 years of research, campylobacteriosis is the most prevalent foodborne bacterial infection in many countries including in the European Union and the United States of America. However, relatively little is known about the virulence factors in Campylobacter or how an apparently fragile organism can survive in the food chain, often with enhanced pathogenicity. This review collates information on the virulence and survival determinants including motility, chemotaxis, adhesion, invasion, multidrug resistance, bile resistance and stress response factors. It discusses their function in transition through the food processing environment and human infection. In doing so it provides a fundamental understanding of Campylobacter, critical for improved diagnosis, surveillance and control. Copyright © 2014 Elsevier Ltd. All rights reserved.

  20. Activation of Bvg-repressed genes in Bordetella pertussis by RisA requires cross-talk from a non co-operonic histidine kinase RisK.

    PubMed

    Chen, Qing; Ng, Victoria; Warfel, Jason M; Merkel, Tod J; Stibitz, Scott

    2017-08-21

    The two-component response regulator RisA, encoded by BP3554 in the Bordetella pertussis Tohama I genomic sequence, is a known activator of vrgs, a set of genes whose expression is increased under the same environmental conditions (known as modulation) that result in repression of the bvgAS virulence regulon. Here we demonstrate that RisA is phosphorylated in vivo and that RisA phosphorylation is required for activation of vrgs. An adjacent histidine kinase gene, risS, is truncated by frameshift mutation in B. pertussis, but not in B. bronchiseptica or B. parapertussis Neither deletion of risS' or bvgAS, nor phenotypic modulation with MgSO4, affected levels of RisA∼P in B. pertussis However, RisA phosphorylation did require the histidine kinase encoded by BP3223, here named RisK (cognate histidine kinase of RisA). RisK was also required for expression of the vrgs. This requirement could be obviated by the introduction of the phosphorylation-mimicking RisA(D60E) mutant, indicating that an active conformation of RisA, but not phosphorylation per se, is crucial for vrg activation. Interestingly, expression of vrgs is still modulated by MgSO4 in cells harboring the RisA(D60E) mutation, suggesting that the activated RisA senses additional signals to control vrg expression in response to environmental stimuli.IMPORTANCE In B. pertussis, the BvgAS two-component system activates the expression of virulence genes by binding of BvgA∼P to their promoters. Expression of the reciprocally-regulated vrgs requires RisA and is also repressed by the Bvg-activated BvgR. RisA is an OmpR-like response regulator, but RisA phosphorylation was not expected because the gene for its presumed, co-operonic, histidine kinase is inactivated by mutation. In this study, we demonstrate phosphorylation of RisA in vivo by a non co-operonic histidine kinase. We also show that RisA phosphorylation is necessary but not sufficient for vrg activation, but, importantly, is not affected by BvgAS status

  1. 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.

  2. Polyphasic characterization and genetic relatedness of low-virulence and virulent Listeria monocytogenes isolates

    PubMed Central

    2012-01-01

    Background Currently, food regulatory authorities consider all Listeria monocytogenes isolates as equally virulent. However, an increasing number of studies demonstrate extensive variations in virulence and pathogenicity of L. monocytogenes strains. Up to now, there is no comprehensive overview of the population genetic structure of L. monocytogenes taking into account virulence level. We have previously demonstrated that different low-virulence strains exhibit the same mutations in virulence genes suggesting that they could have common evolutionary pathways. New low-virulence strains were identified and assigned to phenotypic and genotypic Groups using cluster analysis. Pulsed-field gel electrophoresis, virulence gene sequencing and multi-locus sequence typing analyses were performed to study the genetic relatedness and the population structure between the studied low-virulence isolates and virulent strains. Results These methods showed that low-virulence strains are widely distributed in the two major lineages, but some are also clustered according to their genetic mutations. These analyses showed that low-virulence strains initially grouped according to their lineage, then to their serotypes and after which, they lost their virulence suggesting a relatively recent emergence. Conclusions Loss of virulence in lineage II strains was related to point mutation in a few virulence genes (prfA, inlA, inlB, plcA). These strains thus form a tightly clustered, monophyletic group with limited diversity. In contrast, low-virulence strains of lineage I were more dispersed among the virulence strains and the origin of their loss of virulence has not been identified yet, even if some strains exhibited different mutations in prfA or inlA. PMID:23267677

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

    PubMed Central

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

    2009-01-01

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

  4. Lines of evidence for horizontal gene transfer of a phenazine producing operon into multiple bacterial species.

    PubMed

    Fitzpatrick, David A

    2009-02-01

    Phenazines are secondary metabolites with broad-spectrum antibiotic activity against bacteria, fungi, and eukaryotes. In pseudomonad species, a conserved seven-gene phenazine operon (phzABCDEFG) is required for the conversion of chorismic acid to the broad-spectrum antibiotic phenazine-1-carboxylate. Previous analyses of genes involved in phenazine production from nonpseudomonad species uncovered a high degree of sequence similarity to pseudomonad homologues. The analyses undertaken in this study wished to eluciadate the evolutionary history of genes involved in the production of phenazines. Furthermore, I wanted to determine if the phenazine operon has been transferred through horizontal gene transfer. Analyses of GC content, codon usage patterns, frequency of 3:1 dinucleotides, sequence similarities, and phylogenetic reconstructions were undertaken to map the evolutionary history of phenazine genes from multiple bacterial species. Patchy phyletic distribution, high sequence similarities, and phylogenetic evidence infer that pseudomonad, Streptomyces cinnamonensis, Pantoea agglomerans, Burkholderia cepacia, Pectobacterium atrosepticum, Brevibacterium linens, and Mycobacterium abscessus species all contain a phenazine operon which has most likely been transferred among these species through horizontal gene transfer. The acquisition of an antibiotic-associated operon is significant, as it may increase the relative fitness of the recipient species.

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

    PubMed

    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

    2015-03-01

    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 knockout 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 open up further interest on this operon, which might be of importance to success of B. anthracis as pathogen.

  6. Gene expression of the arsenic resistance operon in Chromobacterium violaceum ATCC 12472.

    PubMed

    Azevedo, Juliana Simão Nina de; Silva-Rocha, Rafael; Silva, Artur; Peixe Carepo, Marta Sofia; Cruz Schneider, Maria Paula

    2008-02-01

    Chromobacterium violaceum ATCC 12472 presents an arsRCB-type operon, which is involved in arsenic resistance. The regulating protein of this resistance system (ArsR) does not have the small conserved site (ELCVDCL) to link to the metalloid, as observed in Escherichia coli, and is thus considered to be an atypical ArsR protein, like that observed in Acidithiobacillus ferrooxidans. In the present study, the gene expression profile of the ars operon under induction at different concentrations of arsenite - As(III) - was obtained via real-time PCR (TaqMan), by correlating the threshold cycle (Ct) values of induced and uninduced (control) samples. Through linear regression analysis (R2 = 0.9926), the gene expression profile of the ars operon showed clearly that the 0.125 micromol/L concentration of As(III) was sufficient to provoke a 4-fold increase in the resistance system, and a further increase in concentration resulted in an increase of up to 53-fold in transcription rates. The relation between resistance and induction of the ars operon indicates that the increased resistance to As(III) is associated with the increase in the number of transcripts.

  7. Modeling feedback loops in the H-NS-mediated regulation of the Escherichia coli bgl operon.

    PubMed

    Radde, Nicole; Gebert, Jutta; Faigle, Ulrich; Schrader, Rainer; Schnetz, Karin

    2008-01-21

    The histone-like nucleoid-associated protein H-NS is a global transcriptional repressor that controls approximately 5% of all genes in Escherichia coli and other enterobacteria. H-NS binds to DNA with low specificity. Nonetheless, repression of some loci is exceptionally specific. Experimental data for the E. coli bgl operon suggest that highly specific repression is caused by regulatory feedback loops. To analyze whether such feedback loops can account for the observed specificity of repression, here a model was built based on expression data. The model includes several regulatory interactions, which are synergy of repression by binding of H-NS to two regulatory elements, an inverse correlation of the rate of repression by H-NS and transcription, and a threshold for positive regulation by anti-terminator BglG, which is encoded within the operon. The latter two regulatory interactions represent feedback loops in the model. The resulting system of equations was solved for the expression level of the operon and analyzed with respect to different promoter activities. This analysis demonstrates that a small (3-fold) increase of the bgl promoter activity results in a strong (80-fold) enhancement of bgl operon expression. Thus, the parameters included into the model are sufficient to simulate specific repression by H-NS.

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

    PubMed

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

    2015-02-01

    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.

  9. The lumazine protein-encoding gene in Photobacterium leiognathi is linked to the lux operon.

    PubMed

    Lin, J W; Chao, Y F; Weng, S F

    1993-04-15

    The nucleotide (nt) sequence of the lumP (EMBL accession No. X65612) gene of Photobacterium leiognathi PL741 was determined and the amino acid (aa) sequence deduced. The encoded aa sequence of lumP was identified as that of the lumazine protein (LumP) by homology with that of Photobacterium phosphoreum (56%). This small protein has a calculated M(r) of 19,997 and comprises 186 aa residues. Biochemical studies suggested that LumP is the protein which, when combined with luciferase, is responsible for the bioluminescent spectrum shift from blue-green light (490-505 nm) to blue (470 nm) in P. leiognathi. The nt sequence of the flanking region showed that lumP is linked to the lux operon but runs in the opposite direction. The gene order of the lumP and lux operon is as follows: <--lumP-R&R-luxC-luxD-luxA-luxB-luxN-lu xE-->; the R&R regulatory region sequence included two promoter systems, PR for the lux operon and PL for the lumP or the lum operon.

  10. 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

  11. 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

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

    USDA-ARS?s Scientific Manuscript database

    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...

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

    PubMed Central

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

    2016-01-01

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

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

    USDA-ARS?s Scientific Manuscript database

    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...

  15. Interruptions in gene expression drive highly expressed operons to the leading strand of DNA replication

    PubMed Central

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

    2005-01-01

    In bacteria, most genes are on the leading strand of replication, a phenomenon attributed to collisions between the DNA and RNA polymerases. In Escherichia coli, these collisions slow the movement of the replication fork through actively transcribed genes only if they are coded on the lagging strand. For genes on both strands, however, these collisions sever nascent transcripts and interrupt gene expression. Based on these observations, we propose a new theory to explain strand bias: genes whose expression is important for fitness are selected to the leading strand because this reduces the duration of these interruptions. Our theory predicts that multi-gene operons, which are subject to longer interruptions, should be more strongly selected to the leading strand than singleton transcripts. We show that this is true even after controlling for the tendency for essential genes, which are strongly biased to the leading strand, to occur in operons. Our theory also predicts that other factors that are associated with strand bias should have stronger effects for genes that are in operons. We find that expression level and phylogenetic ubiquity are correlated with strand bias for both essential and non-essential genes, but only for genes in operons. PMID:15942025

  16. Interruptions in gene expression drive highly expressed operons to the leading strand of DNA replication.

    PubMed

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

    2005-01-01

    In bacteria, most genes are on the leading strand of replication, a phenomenon attributed to collisions between the DNA and RNA polymerases. In Escherichia coli, these collisions slow the movement of the replication fork through actively transcribed genes only if they are coded on the lagging strand. For genes on both strands, however, these collisions sever nascent transcripts and interrupt gene expression. Based on these observations, we propose a new theory to explain strand bias: genes whose expression is important for fitness are selected to the leading strand because this reduces the duration of these interruptions. Our theory predicts that multi-gene operons, which are subject to longer interruptions, should be more strongly selected to the leading strand than singleton transcripts. We show that this is true even after controlling for the tendency for essential genes, which are strongly biased to the leading strand, to occur in operons. Our theory also predicts that other factors that are associated with strand bias should have stronger effects for genes that are in operons. We find that expression level and phylogenetic ubiquity are correlated with strand bias for both essential and non-essential genes, but only for genes in operons.

  17. The Pyrimidine Operon pyrRPB-carA from Lactococcus lactis

    PubMed Central

    Martinussen, Jan; Schallert, Jette; Andersen, Birgit; Hammer, Karin

    2001-01-01

    The four genes pyrR, pyrP, pyrB, and carA were found to constitute an operon in Lactococcus lactis subsp. lactis MG1363. The functions of the different genes were established by mutational analysis. The first gene in the operon is the pyrimidine regulatory gene, pyrR, which is responsible for the regulation of the expression of the pyrimidine biosynthetic genes leading to UMP formation. The second gene encodes a membrane-bound high-affinity uracil permease, required for utilization of exogenous uracil. The last two genes in the operon, pyrB and carA, encode pyrimidine biosynthetic enzymes; aspartate transcarbamoylase (pyrB) is the second enzyme in the pathway, whereas carbamoyl-phosphate synthetase subunit A (carA) is the small subunit of a heterodimeric enzyme, catalyzing the formation of carbamoyl phosphate. The carA gene product is shown to be required for both pyrimidine and arginine biosynthesis. The expression of the pyrimidine biosynthetic genes including the pyrRPB-carA operon is subject to control at the transcriptional level, most probably by an attenuator mechanism in which PyrR acts as the regulatory protein. PMID:11292797

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

    PubMed Central

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

    2016-01-01

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

  19. 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.

  20. 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.

  1. Bistable Behavior in a Model of the lac Operon in Escherichia coli with Variable Growth Rate

    PubMed Central

    Santillán, M.

    2008-01-01

    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

  2. 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.

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

    USDA-ARS?s Scientific Manuscript database

    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. The inhibition of Escherichia coli lac operon gene expression by antigene oligonucleotides-mathematical modeling.

    PubMed

    Cheng, B; Fournier, R L; Relue, P A

    2000-11-20

    Gene transcription is regulated by transcription factors that can bind to specific regions on DNA. Antigene oligonucleotides (oligos) can bind to specific regions on DNA and form a triplex with the double-stranded DNA. The triplex can competitively inhibit the binding of transcription factors and, as a result, transcription can be inhibited. A genetically structured model has been developed to quantitatively describe the inhibition of the Escherichia coli lac operon gene expression by triplex-forming oligos. The model predicts that the effect of triplex-forming oligos on the lac operon gene expression depends on their target sites. Oligonucleotides targeted to the operator are much more effective than those targeted to other regulatory sites on the lac operon. In some cases, the effect of oligo binding is similar to that of a mutation in the lac operon. The model provides insight as to the specific binding site to be targeted to achieve the most effective inhibition of gene expression. The model is also capable of predicting the oligo concentration needed to inhibit gene expression, which is in general agreement with results reported by other investigators.

  5. 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.

  6. 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.

  7. 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

    2016-01-14

    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.

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

    PubMed

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

    2015-06-01

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

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

    PubMed

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

    2016-11-05

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

  10. 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

  11. Helicobacter pylori virulence and cancer pathogenesis.

    PubMed

    Yamaoka, Yoshio; Graham, David Y

    2014-06-01

    Helicobacter pylori is human gastric pathogen that causes chronic and progressive gastric mucosal inflammation and is responsible for the gastric inflammation-associated diseases, gastric cancer and peptic ulcer disease. Specific outcomes reflect the interplay between host-, environmental- and bacterial-specific factors. Progress in understanding putative virulence factors in disease pathogenesis has been limited and many false leads have consumed scarce resources. Few in vitro-in vivo correlations or translational applications have proved clinically relevant. Reported virulence factor-related outcomes reflect differences in relative risk of disease rather than specificity for any specific outcome. Studies of individual virulence factor associations have provided conflicting results. Since virulence factors are linked, studies of groups of putative virulence factors are needed to provide clinically useful information. Here, the authors discuss the progress made in understanding the role of H. pylori virulence factors CagA, vacuolating cytotoxin, OipA and DupA in disease pathogenesis and provide suggestions for future studies.

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

    PubMed Central

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

    2013-01-01

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

  13. 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

  14. 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.

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

    PubMed

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

    2009-11-01

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

  16. ppGpp Conjures Bacterial Virulence

    PubMed Central

    Dalebroux, Zachary D.; Svensson, Sarah L.; Gaynor, Erin C.; Swanson, Michele S.

    2010-01-01

    Summary: Like for all microbes, the goal of every pathogen is to survive and replicate. However, to overcome the formidable defenses of their hosts, pathogens are also endowed with traits commonly associated with virulence, such as surface attachment, cell or tissue invasion, and transmission. Numerous pathogens couple their specific virulence pathways with more general adaptations, like stress resistance, by integrating dedicated regulators with global signaling networks. In particular, many of nature's most dreaded bacteria rely on nucleotide alarmones to cue metabolic disturbances and coordinate survival and virulence programs. Here we discuss how components of the stringent response contribute to the virulence of a wide variety of pathogenic bacteria. PMID:20508246

  17. Functions of the Duplicated hik31 Operons in Central Metabolism and Responses to Light, Dark, and Carbon Sources in Synechocystis sp. Strain PCC 6803

    PubMed Central

    Nagarajan, Sowmya; Sherman, Debra M.; Shaw, Isaac

    2012-01-01

    There are two closely related hik31 operons involved in signal transduction on the chromosome and the pSYSX plasmid in the cyanobacterium Synechocystis sp. strain PCC 6803. We studied the growth, cell morphology, and gene expression in operon and hik mutants for both copies, under different growth conditions, to examine whether the duplicated copies have the same or different functions and gene targets and whether they are similarly regulated. Phenotype analysis suggested that both operons regulated common and separate targets in the light and the dark. The chromosomal operon was involved in the negative control of autotrophic events, whereas the plasmid operon was involved in the positive control of heterotrophic events. Both the plasmid and double operon mutant cells were larger and had division defects. The growth data also showed a regulatory role for the chromosomal hik gene under high-CO2 conditions and the plasmid operon under low-O2 conditions. Metal stress experiments indicated a role for the chromosomal hik gene and operon in mediating Zn and Cd tolerance, the plasmid operon in Co tolerance, and the chromosomal operon and plasmid hik gene in Ni tolerance. We conclude that both operons are differentially and temporally regulated. We suggest that the chromosomal operon is the primarily expressed copy and the plasmid operon acts as a backup to maintain appropriate gene dosages. Both operons share an integrated regulatory relationship and are induced in high light, in glucose, and in active cell growth. Additionally, the plasmid operon is induced in the dark with or without glucose. PMID:22081400

  18. DNA Adenine Methylation Regulates Virulence Gene Expression in Salmonella enterica Serovar Typhimurium▿

    PubMed Central

    Balbontín, Roberto; Rowley, Gary; Pucciarelli, M. Graciela; López-Garrido, Javier; Wormstone, Yvette; Lucchini, Sacha; García-del Portillo, Francisco; Hinton, Jay C. D.; Casadesús, Josep

    2006-01-01

    Transcriptomic analyses during growth in Luria-Bertani medium were performed in strain SL1344 of Salmonella enterica serovar Typhimurium and in two isogenic derivatives lacking Dam methylase. More genes were repressed than were activated by Dam methylation (139 versus 37). Key genes that were differentially regulated by Dam methylation were verified independently. The largest classes of Dam-repressed genes included genes belonging to the SOS regulon, as previously described in Escherichia coli, and genes of the SOS-inducible Salmonella prophages ST64B, Gifsy-1, and Fels-2. Dam-dependent virulence-related genes were also identified. Invasion genes in pathogenicity island SPI-1 were activated by Dam methylation, while the fimbrial operon std was repressed by Dam methylation. Certain flagellar genes were repressed by Dam methylation, and Dam− mutants of S. enterica showed reduced motility. Altered expression patterns in the absence of Dam methylation were also found for the chemotaxis genes cheR (repressed by Dam) and STM3216 (activated by Dam) and for the Braun lipoprotein gene, lppB (activated by Dam). The requirement for DNA adenine methylation in the regulation of specific virulence genes suggests that certain defects of Salmonella Dam− mutants in the mouse model may be caused by altered patterns of gene expression. PMID:16997949

  19. An antimicrobial peptide-resistant minor subpopulation of Photorhabdus luminescens is responsible for virulence

    PubMed Central

    Mouammine, Annabelle; Pages, Sylvie; Lanois, Anne; Gaudriault, Sophie; Jubelin, Gregory; Bonabaud, Maurine; Cruveiller, Stéphane; Dubois, Emeric; Roche, David; Legrand, Ludovic; Brillard, Julien; Givaudan, Alain

    2017-01-01

    Some of the bacterial cells in isogenic populations behave differently from others. We describe here how a new type of phenotypic heterogeneity relating to resistance to cationic antimicrobial peptides (CAMPs) is determinant for the pathogenic infection process of the entomopathogenic bacterium Photorhabdus luminescens. We demonstrate that the resistant subpopulation, which accounts for only 0.5% of the wild-type population, causes septicemia in insects. Bacterial heterogeneity is driven by the PhoPQ two-component regulatory system and expression of pbgPE, an operon encoding proteins involved in lipopolysaccharide (LPS) modifications. We also report the characterization of a core regulon controlled by the DNA-binding PhoP protein, which governs virulence in P. luminescens. Comparative RNAseq analysis revealed an upregulation of marker genes for resistance, virulence and bacterial antagonism in the pre-existing resistant subpopulation, suggesting a greater ability to infect insect prey and to survive in cadavers. Finally, we suggest that the infection process of P. luminescens is based on a bet-hedging strategy to cope with the diverse environmental conditions experienced during the lifecycle. PMID:28252016

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

    PubMed Central

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

    1992-01-01

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

  1. The roles of AtxA orthologs in virulence of anthrax-like Bacillus cereus G9241.

    PubMed

    Scarff, Jennifer M; Raynor, Malik J; Seldina, Yuliya I; Ventura, Christy L; Koehler, Theresa M; O'Brien, Alison D

    2016-11-01

    AtxA is a critical transcriptional regulator of plasmid-encoded virulence genes in Bacillus anthracis. Bacillus cereus G9241, which caused an anthrax-like infection, has two virulence plasmids, pBCXO1 and pBC210, that each harbor toxin genes and a capsule locus. G9241 also produces two orthologs of AtxA: AtxA1, encoded on pBCXO1, and AtxA2, encoded on pBC210. The amino acid sequence of AtxA1 is identical to that of AtxA from B. anthracis, while the sequences of AtxA1 and AtxA2 are 79% identical and 91% similar to one another. We found by qRT-PCR that AtxA1 and AtxA2 function as positive regulators of toxin (AtxA1) and capsule operon (both) transcription in G9241 and that a ΔatxA1 mutant produced lower levels of the anthrax toxins and no hyaluronic acid capsule. Deletion of atxA1 or atxA2 decreased the virulence of spores administered intranasally or subcutaneously to C57BL/6 mice but not to A/J mice, and deletion of both genes rendered spores avirulent in A/J mice. In addition, unlike AtxA1, AtxA2 did not form stable homomultimers in vitro, although AtxA1 and AtxA2 formed heterodimers. Our data show that AtxA1 is the primary regulator of G9241 virulence factor expression and that AtxA1 and AtxA2 are both required for full virulence. © 2016 John Wiley & Sons Ltd.

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

    PubMed

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

    2014-06-01

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

  3. Co-regulation of Iron Metabolism and Virulence Associated Functions by Iron and XibR, a Novel Iron Binding Transcription Factor, in the Plant Pathogen Xanthomonas.

    PubMed

    Pandey, Sheo Shankar; Patnana, Pradeep Kumar; Lomada, Santosh Kumar; Tomar, Archana; Chatterjee, Subhadeep

    2016-11-01

    Abilities of bacterial pathogens to adapt to the iron limitation present in hosts is critical to their virulence. Bacterial pathogens have evolved diverse strategies to coordinately regulate iron metabolism and virulence associated functions to maintain iron homeostasis in response to changing iron availability in the environment. In many bacteria the ferric uptake regulator (Fur) functions as transcription factor that utilize ferrous form of iron as cofactor to regulate transcription of iron metabolism and many cellular functions. However, mechanisms of fine-tuning and coordinated regulation of virulence associated function beyond iron and Fur-Fe2+ remain undefined. In this study, we show that a novel transcriptional regulator XibR (named Xanthomonas iron binding regulator) of the NtrC family, is required for fine-tuning and co-coordinately regulating the expression of several iron regulated genes and virulence associated functions in phytopathogen Xanthomonas campestris pv. campestris (Xcc). Genome wide expression analysis of iron-starvation stimulon and XibR regulon, GUS assays, genetic and functional studies of xibR mutant revealed that XibR positively regulates functions involved in iron storage and uptake, chemotaxis, motility and negatively regulates siderophore production, in response to iron. Furthermore, chromatin immunoprecipitation followed by quantitative real-time PCR indicated that iron promoted binding of the XibR to the upstream regulatory sequence of operon's involved in chemotaxis and motility. Circular dichroism spectroscopy showed that purified XibR bound ferric form of iron. Electrophoretic mobility shift assay revealed that iron positively affected the binding of XibR to the upstream regulatory sequences of the target virulence genes, an effect that was reversed by ferric iron chelator deferoxamine. Taken together, these data revealed that how XibR coordinately regulates virulence associated and iron metabolism functions in Xanthomonads in

  4. Insightful directed evolution of Escherichia coli quorum sensing promoter region of the lsrACDBFG operon: a tool for synthetic biology systems and protein expression

    PubMed Central

    Hauk, Pricila; Stephens, Kristina; Mckay, Ryan; Virgile, Chelsea Ryan; Ueda, Hana; Ostermeier, Marc; Ryu, Kyoung-Seok; Sintim, Herman O.; Bentley, William E.

    2016-01-01

    Quorum sensing (QS) regulates many natural phenotypes (e.q. virulence, biofilm formation, antibiotic resistance), and its components, when incorporated into synthetic genetic circuits, enable user-directed phenotypes. We created a library of Escherichia coli lsr operon promoters using error-prone PCR (ePCR) and selected for promoters that provided E. coli with higher tetracycline resistance over the native promoter when placed upstream of the tet(C) gene. Among the fourteen clones identified, we found several mutations in the binding sites of QS repressor, LsrR. Using site-directed mutagenesis we restored all p-lsrR-box sites to the native sequence in order to maintain LsrR repression of the promoter, preserving the other mutations for analysis. Two promoter variants, EP01rec and EP14rec, were discovered exhibiting enhanced protein expression. In turn, these variants retained their ability to exhibit the LsrR-mediated QS switching activity. Their sequences suggest regulatory linkage between CytR (CRP repressor) and LsrR. These promoters improve upon the native system and exhibit advantages over synthetic QS promoters previously reported. Incorporation of these promoters will facilitate future applications of QS-regulation in synthetic biology and metabolic engineering. PMID:27915294

  5. Genetic analysis of an incomplete bio operon in a biotin auxotrophic strain of Bacillus subtilis natto OK2.

    PubMed

    Sasaki, Mayumi; Kawamura, Fujio; Kurusu, Yasurou

    2004-03-01

    We describe the genetic analysis of the bio operon of the biotin auxotrophic Bacillus subtilis natto OK2 strain. The OK2 strain would only cross-feed with the Escherichia coli bioB mutant and also grew well in medium containing dethiobiotin. Sequencing analysis revealed two significant genetic alterations in the bioW and bioF genes within the bio operon of the OK2 strain. Complementation analysis with B. subtilis 168 bio mutants demonstrated that only the bioB gene could complement, but other bio operon genes could not. A bio(+) transformant, isolated from an OK2 strain, has biotin autotrophy.

  6. rRNA (rrn) Operon-Engineered Bacillus subtilis as a Feasible Test Organism for Antibiotic Discovery

    PubMed Central

    Tanaka, Yukinori; Nanamiya, Hideaki; Yano, Koichi; Kakugawa, Koji; Kawamura, Fujio

    2013-01-01

    Bacillus subtilis contains 10 rRNA (rrn) operons. We found that rRNA operon-engineered B. subtilis strain RIK543, with only the rrnO operon, is specifically hypersensitive to RNA polymerase inhibitors such as rifamycin SV and rifampin (80-fold and 20-fold, respectively). In pilot screening experiments, we found actinomycete isolates successfully at an incidence of 1.9% (18/945) that produced antibacterials that were detectable only with RIK543 as the test organism. Strain RIK543 may be a feasible test organism for the discovery of novel RNA polymerase inhibitors. PMID:23335737

  7. [UV-induction of the LT-toxin operon depending on genes lexA, recA, and umuD].

    PubMed

    Tiganova, I G; Rusina, O Iu; Andreeva, I V; Brukhanskiĭ, G V; Skavronskaia, A G

    1994-06-01

    UV induction of the elt operon (the LT-toxin operon in Escherichia coli) was demonstrated in experiments using fusion of elt::lac operons with the help of Mud1(Ap lac) phage. UV induction of the elt operon is lexA-dependent; thus, the possibility of SOS regulation of this process may be assumed. However, UV induction of the elt operon turned out to be recA-independent, which makes it impossible to consider this induction as a typical SOS response. UV induction of the elt operon is also observed in Salmonella typhimurium, which differs from E. coli in the product of umuD, which suggests that the UV induction of the elt operon is umuD independent.

  8. Ectopic expression of Ralstonia solanacearum effector protein PopA early in invasion results in loss of virulence.

    PubMed

    Kanda, Ayami; Yasukohchi, Masahiko; Ohnishi, Kouhei; Kiba, Akinori; Okuno, Tetsuro; Hikichi, Yasufumi

    2003-05-01

    Ralstonia solanacearum OE1-1 (OE1-1) is pathogenic to tobacco. The type III-secreted effector protein popA of OE1-1 showed 97.6% identity to popA of R. solanacearum GMI1000, which is not pathogenic to tobacco. Reverse transcription-polymerase chain reaction analysis showed that popA in OE1-1 was expressed at 3 h after inoculation (HAI), but not before, in infiltrated-tobacco leaves. Pathogenicity analysis using a popABC operon-deleted mutant of OE1-1 (deltaABC) showed that popABC is not directly involved in the pathogenicity of OE1-1. When Papa, which constitutively expresses popA, was infiltrated into tobacco leaves, popA was expressed by 0.5 HAI. Papa could no longer multiply or spread in tobacco leaves and was no longer virulent. Moreover, the hypersensitive response (HR) and expression of HR-related genes were not induced in Papa-infiltrated leaves. Papa was also avirulent in a tobacco root-dipping inoculation assay. These results suggest that the expression of popA in Papa immediately after invasion triggers the suppression of bacterial proliferation and movement, resulting in loss of virulence. However, Papa retained its virulence when directly inoculated into xylem vessels. This result suggests that tobacco plants can recognize PopA when it is expressed early in disease development, and respond with an effective defense in the intercellular spaces.

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

    PubMed

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

    2014-09-01

    The type VI secretion system (T6SS) is recently discovered machinery in Gram-negative bacteria for translocation of proteins and also is required for full virulence. TssB is a highly conserved protein among the T6SSs, and indispensable for composition and function of T6S. The plant pathogenic bacterium Ralstonia solanacearum also harbours T6SS gene clusters, and a homologue of TssB, hereafter designated as TssBRS, but up to date its characterization and function remain unclear. In this study, we showed that TssBRS of R. solanacearum was required for secretion of Hcp, the haemolysin coregulated protein and a hallmark of T6S pathway. Deletion of tssBRS in R. solanacearum GMI1000 strain resulted in defect of biofilm formation, and the expression of the flagella operon is decreased, leading to decreased motility. More importantly, tssBRS mutant strain had significantly attenuated its virulence on tomato plants. TssB is essential for virulence and required for type VI secretion system in R. solanacearum. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    PubMed Central

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

    2016-01-01

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

  11. 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.

  12. Virulence Evolution Within the Ug99 Lineage

    USDA-ARS?s Scientific Manuscript database

    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 ...

  13. Productive steps toward an antimicrobial targeting virulence

    PubMed Central

    Barczak, Amy K.; Hung, Deborah T.

    2009-01-01

    Summary Targeting virulence factors has gained increasing attention as a potential approach to new antibiotics. Small molecule inhibitors of virulence have been shown to change the course of disease in whole organism infection models. Recently, key advances in the field include the identification of novel targets within cell signaling pathways, a new class of anti-virulence compounds that target bacterial defenses against host immunity, and a growing body of in vivo data to support the general approach of anti-virulence therapies. Additionally, there has been a distinct trend toward developing broader spectrum anti-virulence compounds, in particular agents with activity against diverse Gram-negative organisms. Herein we provide an update on the status of the field with a focus on recent advancements. PMID:19631578

  14. 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

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

    PubMed Central

    Paluscio, Elyse

    2015-01-01

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

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

    PubMed

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

    2014-04-01

    We hypothesized that capsular switch event, in which pneumococcus acquires a new capsule operon by horizontal gene transfer, may result in emergence of strains with increased virulence in acute otitis media. Using serotype 6A strain from a patient with invasive pneumococcal disease and clonally distant serotype 6C strain isolated from asymptomatic carrier we created 6A:6C (6A background with 6C capsule) capsular transformants and applied whole genome macro-restriction analysis to assess conservation of the 6A chassis. Next, we assessed complement (C3) and antibodies deposition on surface of pneumococcal cells and tested capsule recipient, capsule donor and two 6A:6C transformants for virulence in chinchilla experimental otitis media model. Both 6A:6C(1 or 2) transformants bound less C3 compared to 6C capsule-donor strain but more compared to serotype 6A capsule-recipient strain. Pneumococci were present in significantly higher proportion of ears among animals challenged with either of two 6A:6C(1 or 2) transformants compared to chinchillas infected with 6C capsule-donor strain [p < 0.001] whereas a significantly decreased proportion of ears were infected with 6A:6C(1 or 2) transformants as compared to 6A capsule-recipient strain. Our observations though limited to two serotypes demonstrate that capsular switch events can result in Streptococcus pneumoniae strains of enhanced virulence for respiratory tract infection. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  17. 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.

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

    SciTech Connect

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

    2008-10-10

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

  19. NSR from Streptococcus agalactiae confers resistance against nisin and is encoded by a conserved nsr operon.

    PubMed

    Khosa, Sakshi; AlKhatib, Zainab; Smits, Sander H J

    2013-11-01

    Nisin is a lantibiotic produced by Lactococcus lactis (L. lactis), which is active against many Gram-positive bacteria. However, in various pathogenic and nonpathogenic bacteria, the presence of a nisin resistance protein (NSR) confers resistance against nisin. Here, we show that NSR from Streptococcus agalactiae (SaNSR) confers 20-fold resistance when expressed in L. lactis. We also show that SaNSR is encoded by an operon structure comprising of a lipoprotein and an ATP-binding cassette transporter as well as a two-component system that is putatively involved in expression and regulation. This organization of the operon is conserved in several (non)pathogenic strains that do not produce nisin themselves.

  20. In Vitro Repression of Transcription of the Trytophan Operon by trp Repressor

    PubMed Central

    Shimizu, Yoshiko; Shimizu, Nobuyoshi; Hayashi, Masaki

    1973-01-01

    The in vitro repression of transcription of the tryptophan operon by the trp repressor from Escherichia coli was studied. By measuring the inhibitory effect for trp-specific RNA synthesis in an in vitro transcription system directed by DNA of trp-transducing phage, we have detected and concentrated a trp repressor in an eluate of a Φ80 ptED native DNA-cellulose column. The repression of transcription of trp operon required the addition of L-tryptophan in the system, and when several tryptophan analogues were added, the repression or derepression was similar to that observed in vivo. The repressor fraction was separated from the majority of tryptophanyl-tRNA synthetase activity by Bio-gel P60 column chromatography. PMID:4579009

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

    NASA Astrophysics Data System (ADS)

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

    1996-03-01

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

  2. [Heterologous genes expression on Escherichia coli chromosome lac operon using Red recombination].

    PubMed

    Li, Shanhu; Shi, Qingguo; Huang, Cuifen; Zhou, Jianguang

    2008-04-01

    To achieve efficient and stable expression of heterologous exogenetic protein or antigen in E. coli chromosome, the luciferase report gene was knocked in lacZ site of chromosome lac operon by using Red recombination system and selection-counterselection kan/sacB technology. The quantitative analysis of exogenous gene expression indicated that the target gene could be efficiently expressed at lacZ site of lac operon. The results confirmed the efficient screening and stable expression of heterologous protein or antigen on chromosome by using the recombinant engineering technique. This study demonstrated that the chromosome could be used as a vector for heterologous protein or antigen and the stable expression of exogenous gene on E. coli chromosome had no side effect on the bacterial growth and propagation.

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

    SciTech Connect

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

    2008-01-01

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

  4. ExsB Is Required for Correct Assembly of the Pseudomonas aeruginosa Type III Secretion Apparatus in the Bacterial Membrane and Full Virulence In Vivo

    PubMed Central

    Perdu, Caroline; Huber, Philippe; Bouillot, Stéphanie; Blocker, Ariel; Elsen, Sylvie; Attrée, Ina

    2015-01-01

    Pseudomonas aeruginosa is responsible for high-morbidity infections of cystic fibrosis patients and is a major agent of nosocomial infections. One of its most potent virulence factors is a type III secretion system (T3SS) that injects toxins directly into the host cell cytoplasm. ExsB, a lipoprotein localized in the bacterial outer membrane, is one of the components of this machinery, of which the function remained elusive until now. The localization of the exsB gene within the exsCEBA regulatory gene operon suggested an implication in the T3SS regulation, while its similarity with yscW from Yersinia spp. argued in favor of a role in machinery assembly. The present work shows that ExsB is necessary for full in vivo virulence of P. aeruginosa. Furthermore, the requirement of ExsB for optimal T3SS assembly and activity is demonstrated using eukaryotic cell infection and in vitro assays. In particular, ExsB promotes the assembly of the T3SS secretin in the bacterial outer membrane, highlighting the molecular role of ExsB as a pilotin. This involvement in the regulation of the T3S apparatus assembly may explain the localization of the ExsB-encoding gene within the regulatory gene operon. PMID:25690097

  5. Putative promoter region of rRNA operon from archaebacterium Halobacterium halobium.

    PubMed Central

    Mankin, A S; Teterina, N L; Rubtsov, P M; Baratova, L A; Kagramanova, V K

    1984-01-01

    The 100 bp sequence from the beginning of the 16S rRNA gene of archaebacterium Halobacterium halobium and the adjacent 800 bp upstream sequence were determined. Four long (80 bp) direct repeats were found in the region preceeding the structural gene of the 16S rRNA. These repeats are proposed to constitute the promoter region of the rRNA operon of H. halobium. PMID:6089119

  6. Identification of nah-1 genes of the Pseudomonas putida naphthalene-degrading NPL-41 plasmid operon.

    PubMed

    Serebriiskaya, T S; Lenets, A A; Goldenkova, I V; Kobets, N S; Piruzian, E S

    1999-01-01

    Pseudomonas putida BS202 degrades naphthalene via a plasmid-encoded catabolic pathway. The nucleotide sequence of the nahC gene encoding one of this pathway enzymes, 1,2-dihydroxynaphthalene dioxygenase, has been determined. Analysis of nucleotide sequence of its flanking regions identified partially the nahF and putative nahQ genes. Comparison of these three genes with corresponding ones in the NAH7 plasmid and DOX operon showed a high degree of homology.

  7. Silencing of Essential Genes within a Highly Coordinated Operon in Escherichia coli

    PubMed Central

    Hohmeier, Angela; Stone, Timothy C.; Offord, Victoria; Sarabia, Francisco; Garcia-Ruiz, Cristina; Good, Liam

    2015-01-01

    Essential bacterial genes located within operons are particularly challenging to study independently because of coordinated gene expression and the nonviability of knockout mutants. Essentiality scores for many operon genes remain uncertain. Antisense RNA (asRNA) silencing or in-frame gene disruption of genes may help establish essentiality but can lead to polar effects on genes downstream or upstream of the target gene. Here, the Escherichia coli ribF-ileS-lspA-fkpB-ispH operon was used to evaluate the possibility of independently studying an essential gene using expressed asRNA and target gene overexpression to deregulate coupled expression. The gene requirement for growth in conditional silencing strains was determined by the relationship of target mRNA reduction with growth inhibition as the minimum transcript level required for 50% growth (MTL50). Mupirocin and globomycin, the protein inhibitors of IleS and LspA, respectively, were used in sensitization assays of strains containing both asRNA-expressing and open reading frame-expressing plasmids to examine deregulation of the overlapping ileS-lspA genes. We found upstream and downstream polar silencing effects when either ileS or lspA was silenced, indicating coupled expression. Weighted MTL50 values (means and standard deviations) of ribF, ileS, and lspA were 0.65 ± 0.18, 0.64 ± 0.06, and 0.76 ± 0.10, respectively. However, they were not significantly different (P = 0.71 by weighted one-way analysis of variance). The gene requirement for ispH could not be determined due to insufficient growth reduction. Mupirocin and globomycin sensitization experiments indicated that ileS-lspA expression could not be decoupled. The results highlight the inherent challenges associated with genetic analyses of operons; however, coupling of essential genes may provide opportunities to improve RNA-silencing antimicrobials. PMID:26070674

  8. Identification of regulatory elements that control expression of the tbpBA operon in Neisseria gonorrhoeae.

    PubMed

    Vélez Acevedo, Rosuany N; Ronpirin, Chalinee; Kandler, Justin L; Shafer, William M; Cornelissen, Cynthia Nau

    2014-08-01

    Iron is an essential nutrient for survival and establishment of infection by Neisseria gonorrhoeae. The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is the TonB-dependent transporter that accomplishes iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA and separated from it by an inverted repeat. The operon is under the control of the ferric uptake regulator (Fur); however, promoter elements necessary for regulated expression of the genes have not been experimentally defined. In this study, putative regulatory motifs were identified and confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of several novel repeats. We hypothesized that these repeats are involved in additional regulation of the operon. Insertional mutagenesis of regions upstream of the characterized promoter region resulted in decreased tbpB and tbpA transcript levels but increased protein levels for both TbpA and TbpB. Using RNA sequencing (RNA-Seq) technology, we determined that a long RNA was produced from the region upstream of tbpB. We localized the 5' endpoint of this transcript to between the two upstream insertions by qualitative RT-PCR. We propose that expression of this upstream RNA leads to optimized expression of the gene products from within the tbpBA operon.

  9. Influence of catabolite repression and inducer exclusion on the bistable behavior of the lac operon.

    PubMed

    Santillán, Moisés; Mackey, Michael C

    2004-03-01

    A mathematical model of the lac operon which includes all of the known regulatory mechanisms, including external-glucose-dependent catabolite repression and inducer exclusion, as well as the time delays inherent to transcription and translation, is presented. With this model we investigate the influence of external glucose, by means of catabolite repression and the regulation of lactose uptake, on the bistable behavior of this system.

  10. Characterization of the Vibrio cholerae vceCAB Multiple-Drug Resistance Efflux Operon in Escherichia coli

    PubMed Central

    Woolley, Robin C.; Vediyappan, Govindsamy; Anderson, Matthew; Lackey, Melinda; Ramasubramanian, Bhagavathi; Jiangping, Bai; Borisova, Tatyana; Colmer, Jane A.; Hamood, Abdul N.; McVay, Catherine S.; Fralick, Joe A.

    2005-01-01

    Herein, we identify vceC as a component of a vceCAB operon, which codes for the Vibrio cholerae VceAB multiple-drug resistance (MDR) efflux pump, and vceR, which codes for a transcriptional autoregulatory protein that negatively regulates the expression of the vceCAB operon and is modulated by some of the substrates of this MDR efflux pump. PMID:16030246

  11. rRNA Operon Copy Number Can Explain the Distinct Epidemiology of Hospital-Associated Methicillin-Resistant Staphylococcus aureus

    PubMed Central

    Jansen, M. D.; Bosch, T.; Jansen, W. T. M.; Schouls, L.; Jonker, M. J.; Boel, C. H. E.

    2016-01-01

    The distinct epidemiology of original hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and early community-associated MRSA (CA-MRSA) is largely unexplained. S. aureus carries either five or six rRNA operon copies. Evidence is provided for a scenario in which MRSA has adapted to the hospital environment by rRNA operon loss (six to five copies) due to antibiotic pressure. Early CA-MRSA, in contrast, results from wild-type methicillin-susceptible S. aureus (MSSA) that acquired mecA without loss of an rRNA operon. Of the HA-MRSA isolates (n = 77), 67.5% had five rRNA operon copies, compared to 23.2% of the CA-MRSA isolates (n = 69) and 7.7% of MSSA isolates (n = 195) (P < 0.001). In addition, 105 MSSA isolates from cystic fibrosis patients were tested, because these patients are repeatedly treated with antibiotics; 32.4% of these isolates had five rRNA operon copies. For all subsets, a correlation between resistance profile and rRNA copy number was found. Furthermore, we showed that in vitro antibiotic pressure may result in rRNA operon copy loss. We also showed that without antibiotic pressure, S. aureus isolates containing six rRNA copies are more fit than isolates with five copies. We conclude that HA-MRSA and cystic fibrosis isolates most likely have adapted to an environment with high antibiotic pressure by the loss of an rRNA operon copy. This loss has facilitated resistance development, which promoted survival in these niches. However, strain fitness decreased, which explains their lack of success in the community. In contrast, CA-MRSA isolates retained six rRNA operon copies, rendering them fitter and thereby able to survive and spread in the community. PMID:27671073

  12. rRNA Operon Copy Number Can Explain the Distinct Epidemiology of Hospital-Associated Methicillin-Resistant Staphylococcus aureus.

    PubMed

    Fluit, A C; Jansen, M D; Bosch, T; Jansen, W T M; Schouls, L; Jonker, M J; Boel, C H E

    2016-12-01

    The distinct epidemiology of original hospital-associated methicillin-resistant Staphylococcus aureus (HA-MRSA) and early community-associated MRSA (CA-MRSA) is largely unexplained. S. aureus carries either five or six rRNA operon copies. Evidence is provided for a scenario in which MRSA has adapted to the hospital environment by rRNA operon loss (six to five copies) due to antibiotic pressure. Early CA-MRSA, in contrast, results from wild-type methicillin-susceptible S. aureus (MSSA) that acquired mecA without loss of an rRNA operon. Of the HA-MRSA isolates (n = 77), 67.5% had five rRNA operon copies, compared to 23.2% of the CA-MRSA isolates (n = 69) and 7.7% of MSSA isolates (n = 195) (P < 0.001). In addition, 105 MSSA isolates from cystic fibrosis patients were tested, because these patients are repeatedly treated with antibiotics; 32.4% of these isolates had five rRNA operon copies. For all subsets, a correlation between resistance profile and rRNA copy number was found. Furthermore, we showed that in vitro antibiotic pressure may result in rRNA operon copy loss. We also showed that without antibiotic pressure, S. aureus isolates containing six rRNA copies are more fit than isolates with five copies. We conclude that HA-MRSA and cystic fibrosis isolates most likely have adapted to an environment with high antibiotic pressure by the loss of an rRNA operon copy. This loss has facilitated resistance development, which promoted survival in these niches. However, strain fitness decreased, which explains their lack of success in the community. In contrast, CA-MRSA isolates retained six rRNA operon copies, rendering them fitter and thereby able to survive and spread in the community. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

  13. Regulating Bacterial Virulence with RNA.

    PubMed

    Quereda, Juan J; Cossart, Pascale

    2017-09-08

    Noncoding RNAs (ncRNAs) regulating virulence have been identified in most pathogens. This review discusses RNA-mediated mechanisms exploited by bacterial pathogens to successfully infect and colonize their hosts. It discusses the most representative RNA-mediated regulatory mechanisms employed by two intracellular [Listeria monocytogenes and Salmonella enterica serovar Typhimurium (S. Typhimurium)] and two extracellular (Vibrio cholerae and Staphylococcus aureus) bacterial pathogens. We review the RNA-mediated regulators (e.g., thermosensors, riboswitches, cis- and trans-encoded RNAs) used for adaptation to the specific niches colonized by these bacteria (intestine, blood, or the intracellular environment, for example) in the framework of the specific pathophysiological aspects of the diseases caused by these microorganisms. A critical discussion of the newest findings in the field of bacterial ncRNAs shows how examples in model pathogens could pave the way for the discovery of new mechanisms in other medically important bacterial pathogens.

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

    SciTech Connect

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

    2005-11-23

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

  15. Organization and post-transcriptional processing of the psb B operon from chloroplasts of Populus deltoides.

    PubMed

    Dixit, R; Trivedi, P K; Nath, P; Sane, P V

    1999-09-01

    Chloroplast genes are typically organized into polycistronic transcription units that give rise to complex sets of mono- and oligo-cistronic overlapping RNAs through a series of processing steps. The psbB operon contains genes for the PSII (psbB, psbT, psbH) and cytochrome b(6)f (petB and petD) complexes which are needed in different amounts during chloroplast biogenesis. The functional significance of gene organization in this polycistronic unit, containing information for two different complexes, is not known and is of interest. To determine the organization and expression of these complexes, studies have been carried out on crop plants by different groups, but not much information is known about trees. We present the nucleotide sequences of PSII genes and RNA profiles of the genes located in the psbB operon from Populus deltoides, a tree species. Although the gene organization of this operon in P. deltoides is similar to that in other species, a few variations have been observed in the processing scheme.

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

    SciTech Connect

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

    1988-02-01

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

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

    PubMed

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

    2015-11-01

    Mycobacterium tuberculosis disrupted in a 13-gene operon (mce1) accumulates free mycolic acids (FM) in its cell wall and causes accelerated death in mice. Here, to more comprehensively analyze differences in their cell wall lipid composition, we used an untargeted metabolomics approach to compare the lipid profiles of wild-type and mce1 operon mutant strains. By liquid chromatography-mass spectrometry, we identified >400 distinct lipids significantly altered in the mce1 mutant compared to wild type. These lipids included decreased levels of saccharolipids and glycerophospholipids, and increased levels of alpha-, methoxy- and keto mycolic acids (MA), and hydroxyphthioceranic acid. The mutant showed reduced expression of mmpL8, mmpL10, stf0, pks2 and papA2 genes involved in transport and metabolism of lipids recognized to induce proinflammatory response; these lipids were found to be decreased in the mutant. In contrast, the transcripts of mmpL3, fasI, kasA, kasB, acpM and RV3451 involved in MA transport and metabolism increased; MA inhibits inflammatory response in macrophages. Since the mce1 operon is known to be regulated in intracellular M. tuberculosis, we speculate that the differences we observed in cell wall lipid metabolism and composition may affect host response to M. tuberculosis infection and determine the clinical outcome of such an infection. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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

    PubMed Central

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

    2014-01-01

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

  19. Amplification of the groESL operon in Pseudomonas putida increases siderophore gene promoter activity.

    PubMed

    Venturi, V; Wolfs, K; Leong, J; Weisbeek, P J

    1994-10-17

    Pseudobactin 358 is the yellow-green fluorescent siderophore [microbial iron(III) transport agent] produced by Pseudomonas putida WCS358 under iron-limiting conditions. The genes encoding pseudobactin 358 biosynthesis are iron-regulated at the level of transcription. In this study, the molecular characterization is reported of a cosmid clone of WCS358 DNA that can stimulate, in an iron-dependent manner, the activity of a WCS358 siderophore gene promoter in the heterologous Pseudomonas strain A225. The functional region in the clone was identified by subcloning, transposon mutagenesis and DNA sequencing as the groESL operon of strain WCS358. This increase in promoter activity was not observed when the groESL genes of strain WCS358 were integrated via a transposon vector into the genome of Pseudomonas A225, indicating that multiple copies of the operon are necessary for the increase in siderophore gene promoter activity. Amplification of the Escherichia coli and WCS358 groESL genes also increased iron-regulated promoter activity in the parent strain WCS358. The groESL operon codes for the chaperone proteins GroES and GroEL, which are responsible for mediating the folding and assembly of many proteins.

  20. Structure and regulation of the Escherichia coli ruv operon involved in DNA repair and recombination.

    PubMed Central

    Shinagawa, H; Makino, K; Amemura, M; Kimura, S; Iwasaki, H; Nakata, A

    1988-01-01

    The ruv gene of Escherichia coli, which is involved in DNA repair and recombination, was cloned on a plasmid vector. The DNA of the ruv region was sequenced; it had two open reading frames in tandem that could code for 22- and 37-kilodalton proteins. The proteins encoded by these open reading frames were identified by the maxicell method. The two genes were aligned in the same orientation and regulated by the SOS system, so the two genes probably constitute an operon. The distal one complemented the ruv mutations. Transcription of the operon was studied both in vivo and in vitro. Two transcription initiation sites were identified upstream of the coding frames, and the transcription from both sites was repressed by the LexA repressor. A DNA sequence that is homologous to the SOS box and bound by LexA protein was found in the regulatory region of the operon. The amino acid sequence of Ruv protein deduced from the DNA sequence shows a high degree of homology to the consensus sequence shared by ATP-binding proteins. Images PMID:2842314

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

    PubMed Central

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

    1996-01-01

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

  2. Structural and physiological studies of the Escherichia coli histidine operon inserted into plasmid vectors.

    PubMed Central

    Bruni, C B; Musti, A M; Frunzio, R; Blasi, F

    1980-01-01

    A fragment of deoxyribonucleic acid 5,300 base paris long and containing the promoter-proximal portion of the histidine operon of Escherichia coli K-12, has been cloned in plasmid pBR313 (plasmids pCB2 and pCB3). Restriction mapping, partial nucleotide sequencing, and studies on functional expression in vivo and on protein synthesis in minicells have shown that the fragment contains the regulatory region of the operon, the hisG, hisD genes, and part of the hisC gene. Another plasmid (pCB5) contained the hisG gene and part of the hisD gene. Expression of the hisG gene in the latter plasmid was under control of the tetracycline promoter of the pBR313 plasmid. The in vivo expression of the two groups of plasmids described above, as well as their effect on the expression of the histidine genes not carried by the plasmids but present on the host chromosome, has been studied. The presence of multiple copies of pCB2 or pCB3, but not of pCB5, prevented derepression of the chromosomal histidine operon. Possible interpretations of this phenomenon are discussed. Images PMID:6246067

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

    PubMed Central

    Stamatakis, Michail; Mantzaris, Nikos V.

    2009-01-01

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

  4. The recA operon: A novel stress response gene cluster in Bacteroides fragilis.

    PubMed

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

    2014-05-01

    Bacteroides fragilis, an opportunistic pathogen of humans, is a leading cause of bacteraemias and anaerobic abscesses which are often treated with metronidazole, a drug which damages DNA. This study investigated the responses of the B. fragilis recA three gene operon to the stress experienced during metronidazole treatment and exposure to reactive oxygen species simulating those generated by the host immune system during infection. A transcriptionally regulated response was observed using quantitative RT-PCR after metronidazole and hydrogen peroxide treatment, with all three genes being upregulated under stress conditions. In vivo and in vitro analysis of the functional role of the second gene of the operon was done using heterologous complementation and protein expression (in Escherichia coli), with subsequent biochemical assay. This gene encoded a functional bacterioferritin co-migratory protein (BCP) which was thiol-specific and had antioxidant properties, including protection of the glutamine synthetase III enzyme. This in vitro data supports the hypothesis that the genes of the operon may be involved in protection of the bacteria from the oxidative burst during tissue invasion and may play a significant role in bacterial survival and metronidazole resistance during treatment of B. fragilis infections. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  5. Aerobic anoxygenic photosynthesis genes and operons in uncultured bacteria in the Delaware River.

    PubMed

    Waidner, Lisa A; Kirchman, David L

    2005-12-01

    Photosynthesis genes and operons of aerobic anoxygenic photosynthetic (AAP) bacteria have been examined in a variety of marine habitats, but genomic information about freshwater AAP bacteria is lacking. The goal of this study was to examine photosynthesis genes of AAP bacteria in the Delaware River. In a fosmid library, we found two clones bearing photosynthesis gene clusters with unique gene content and organization. Both clones contained 37 open reading frames, with most of those genes encoding known AAP bacterial proteins. The genes in one fosmid were most closely related to those of AAP bacteria in the Rhodobacter genus. The genes of the other clone were related to those of freshwater beta-proteobacteria. Both clones contained the acsF gene, which is required for aerobic bacteriochlorophyll synthesis, suggesting that these bacteria are not anaerobes. The beta-proteobacterial fosmid has the puf operon B-A-L-M-C and is the first example of an uncultured bacterium with this operon structure. The alpha-3-proteobacterial fosmid has a rare gene order (Q-B-A-L-M-X), previously observed only in the Rhodobacter genus. Phylogenetic analyses of photosynthesis genes revealed a possible freshwater cluster of AAP beta-proteobacteria. The data from both Delaware River clones suggest there are groups of freshwater or estuarine AAP bacteria distinct from those found in marine environments.

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

    SciTech Connect

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

    1998-10-01

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

  7. Expression of an Entire Bacterial Operon in Plants1[W][OA

    PubMed Central

    Mozes-Koch, Rita; Gover, Ofer; Tanne, Edna; Peretz, Yuval; Maori, Eyal; Chernin, Leonid; Sela, Ilan

    2012-01-01

    Multigene expression is required for metabolic engineering, i.e. coregulated expression of all genes in a metabolic pathway for the production of a desired secondary metabolite. To that end, several transgenic approaches have been attempted with limited success. Better success has been achieved by transforming plastids with operons. IL-60 is a platform of constructs driven from the geminivirus Tomato yellow leaf curl virus. We demonstrate that IL-60 enables nontransgenic expression of an entire bacterial operon in tomato (Solanum lycopersicum) plants without the need for plastid (or any other) transformation. Delivery to the plant is simple, and the rate of expressing plants is close to 100%, eliminating the need for selectable markers. Using this platform, we show the expression of an entire metabolic pathway in plants and delivery of the end product secondary metabolite (pyrrolnitrin). Expression of this unique secondary metabolite resulted in the appearance of a unique plant phenotype disease resistance. Pyrrolnitrin production was already evident 2 d after application of the operon to plants and persisted throughout the plant's life span. Expression of entire metabolic pathways in plants is potentially beneficial for plant improvement, disease resistance, and biotechnological advances, such as commercial production of desired metabolites. PMID:22353575

  8. Role of a Tannerella forsythia exopolysaccharide synthesis operon in biofilm development.

    PubMed

    Honma, Kiyonobu; Inagaki, Satoru; Okuda, Katsuji; Kuramitsu, Howard K; Sharma, Ashu

    2007-04-01

    Tannerella forsythia is a Gram-negative oral anaerobe implicated in the development of periodontitis, a chronic inflammatory disease induced by bacterial infections which leads to tooth loss if untreated. Since biofilms formed by periodontal bacteria are considered important in disease progression and pose difficulties in treatment, we sought to investigate the underlying mechanisms of T. forsythia biofilm formation. This was carried out by screening random insertion mutants of T. forsythia for alterations in biofilm development. This approach lead to the identification of an operon involved in exopolysaccharide (EPS) synthesis. An isogenic mutant of one of the genes, wecC, contained within the operon was constructed. The isogenic wecC mutant showed increased ability to form biofilms as compared to the parent strain. The wecC mutant also formed aggregated microcolonies and showed increased cell-surface associated hydrophobicity as compared to the parent strain. Moreover, biochemical characterization of the wecC mutant indicated that glycosylation of surface glycoproteins was reduced. Therefore, our results suggest that the wecC operon is associated with glycosylation of surface-glycoprotein expression and likely plays an inhibitory role in T. forsythia biofilm formation.

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

    PubMed

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

    2014-01-01

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

  10. Regulation of nrf operon expression in pathogenic enteric bacteria: sequence divergence reveals new regulatory complexity

    PubMed Central

    Godfrey, Rita E.; Lee, David J.; Busby, Stephen J. W.

    2017-01-01

    Summary The Escherichia coli K‐12 nrf operon encodes a periplasmic nitrite reductase, the expression of which is driven from a single promoter, pnrf. Expression from pnrf is activated by the FNR transcription factor in response to anaerobiosis and further increased in response to nitrite by the response regulator proteins, NarL and NarP. FNR‐dependent transcription is suppressed by the binding of two nucleoid associated proteins, IHF and Fis. As Fis levels increase in cells grown in rich medium, the positioning of its binding site, overlapping the promoter −10 element, ensures that pnrf is sharply repressed. Here, we investigate the expression of the nrf operon promoter from various pathogenic enteric bacteria. We show that pnrf from enterohaemorrhagic E. coli is more active than its K‐12 counterpart, exhibits substantial FNR‐independent activity and is insensitive to nutrient quality, due to an improved −10 element. We also demonstrate that the Salmonella enterica serovar Typhimurium core promoter is more active than previously thought, due to differences around the transcription start site, and that its expression is repressed by downstream sequences. We identify the CsrA RNA binding protein as being responsible for this, and show that CsrA differentially regulates the E. coli K‐12 and Salmonella nrf operons. PMID:28211111

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

    PubMed

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

    2015-11-17

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

  12. Comparison of deterministic and stochastic models of the lac operon genetic network.

    PubMed

    Stamatakis, Michail; Mantzaris, Nikos V

    2009-02-01

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

  13. A powerful hybrid puc operon promoter tightly regulated by both IPTG and low oxygen level.

    PubMed

    Hu, Zongli; Zhao, Zhiping; Pan, Yu; Tu, Yun; Chen, Guoping

    2010-04-01

    Rhodobacter sphaeroides has been intensively studied and provides an excellent model for studying both photosynthesis and membrane development. The photosynthetic apparatus (LH2 and LH1-RC complexes) can be synthesized in large scale and integrated into the intracytoplasmic membrane system under specific conditions, which thus provides us insight to utilize the puc or(and) puf operon to heterologously express recombinant proteins in the intracytoplasmic membrane using Rb. sphaeroides as a novel expression system. However, basal level of expression of puc and puf promoter is uncontrolled. We report the construction of LH2 polypeptide expression vector that contains a reengineered lacI(q)-puc promoter-lac operator hybrid promoter, which allows the puc operon to be regulated by both IPTG and low oxygen level. Synthesis of LH2 complexes was completely repressed in the absence of isopropyl beta-D-thiogalactoside (IPTG), and the degree of induction was controlled by varying the concentration of IPTG. The optimal concentration of IPTG was determined. SDS-PAGE and Western blot were employed for further analysis. Our results suggest that the reengineered hybrid promoter is efficient to tightly regulate the expression of the puc operon, and our strategy can open up a new approach in the study of the membrane protein expression system.

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

    SciTech Connect

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

    2009-12-25

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

  15. The Legionella pneumophila GIG operon responds to gold and copper in planktonic and biofilm cultures

    PubMed Central

    Jwanoswki, Kathleen; Wells, Christina; Bruce, Terri; Rutt, Jennifer; Banks, Tabitha; McNealy, Tamara L.

    2017-01-01

    Legionella pneumophila contaminates man-made water systems and creates numerous exposure risks for Legionnaires’ Disease. Because copper/silver ionization is commonly used to control L. pneumophila, its mechanisms of metal response and detoxification are of significant interest. Here we describe an L. pneumophila operon with significant similarity to the GIG operon of Cupriavidus metallidurans. The Legionella GIG operon is present in a subset of strains and has been acquired as part of the ICE-βox 65-kB integrative conjugative element. We assessed GIG promoter activity following exposure of L. pneumophila to multiple concentrations of HAuCl4, CuSO4 and AgNO3. At 37°C, control stationary phase cultures exhibited GIG promoter activity. This activity increased significantly in response to 20 and 50uM HAuCl4 and CuSO4 but not in response to AgNO3. Conversely, at 26°C, cultures exhibited decreased promoter response to copper. GIG promoter activity was also induced by HAuCl4 or CuSO4 during early biofilm establishment at both temperatures. When an L. pneumophila GIG promoter construct was transformed into E. coli DH5α, cultures showed baseline expression levels that did not increase following metal addition. Analysis of L. pneumophila transcriptional regulatory mutants suggested that GIG up-regulation in the presence of metal ions may be influenced by the stationary phase sigma factor, RpoS. PMID:28463986

  16. The Xis2d protein of CTnDOT binds to the intergenic region between the mob and tra operons

    PubMed Central

    Hopp, Crystal M.; Gardner, Jeffrey F.; Salyers, Abigail A.

    2015-01-01

    CTnDOT is a 65kbp integrative and conjugative element (ICE) that carries genes encoding both tetracycline and erythromycin resistances. The Excision operon of this element encodes Xis2c, Xis2d, and Exc proteins involved in the excision of CTnDOT from host chromosomes. These proteins are also required in the complex transcriptional regulation of the divergently transcribed transfer (tra) and mobilization (mob) operons of CTnDOT. Transcription of the tra operon is positively regulated by Xis2c and Xis2d, whereas, transcription of the mob operon is positively regulated by Xis2d and Exc. Xis2d is the only protein that is involved in the excision reaction, as well as the transcriptional regulation of both the mob and tra operons. This paper helps establish how Xis2d binds the DNA in the mob and tra region. Unlike other excisionase proteins, Xis2d binds a region of dyad symmetry. The binding site is located in the intergenic region between the mob and tra promoters, and once bound Xis2d induces a bend in the DNA. Xis2d binding to this region could be the preliminary step for the activation of both operons. Then the other proteins, like Exc, can interact with Xis2d and form higher order complexes. PMID:26212728

  17. The Xis2d protein of CTnDOT binds to the intergenic region between the mob and tra operons.

    PubMed

    Hopp, Crystal M; Gardner, Jeffrey F; Salyers, Abigail A

    2015-09-01

    CTnDOT is a 65kbp integrative and conjugative element (ICE) that carries genes encoding both tetracycline and erythromycin resistances. The excision operon of this element encodes Xis2c, Xis2d, and Exc proteins involved in the excision of CTnDOT from host chromosomes. These proteins are also required in the complex transcriptional regulation of the divergently transcribed transfer (tra) and mobilization (mob) operons of CTnDOT. Transcription of the tra operon is positively regulated by Xis2c and Xis2d, whereas, transcription of the mob operon is positively regulated by Xis2d and Exc. Xis2d is the only protein that is involved in the excision reaction, as well as the transcriptional regulation of both the mob and tra operons. This paper helps establish how Xis2d binds the DNA in the mob and tra region. Unlike other excisionase proteins, Xis2d binds a region of dyad symmetry. The binding site is located in the intergenic region between the mob and tra promoters, and once bound Xis2d induces a bend in the DNA. Xis2d binding to this region could be the preliminary step for the activation of both operons. Then the other proteins, like Exc, can interact with Xis2d and form higher order complexes. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Nucleotide sequence and functional analysis of cbbR, a positive regulator of the Calvin cycle operons of Rhodobacter sphaeroides.

    PubMed Central

    Gibson, J L; Tabita, F R

    1993-01-01

    Structural genes encoding Calvin cycle enzymes in Rhodobacter sphaeroides are duplicated and organized within two physically distinct transcriptional units, the form I and form II cbb operons. Nucleotide sequence determination of the region upstream of the form I operon revealed a divergently transcribed open reading frame, cbbR, that showed significant similarity to the LysR family of transcriptional regulatory proteins. Mutants containing an insertionally inactivated cbbR gene were impaired in photoheterotrophic growth and completely unable to grow photolithoautotrophically with CO2 as the sole carbon source. In the cbbR strain, expression of genes within the form I operon was completely abolished and that of the form II operon was reduced to about 30% of the wild-type level. The cloned cbbR gene complemented the mutant for wild-type growth characteristics, and normal levels of ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) were observed. However, rocket immunoelectrophoresis revealed that the wild-type level of RubisCO was due to overexpression of the form II enzyme, whereas expression of the form I RubisCO was 10% of that of the wild-type strain. The cbbR insertional inactivation did not appear to affect aerobic expression of either CO2 fixation operon, but preliminary evidence suggests that the constitutive expression of the form II operon observed in the cbbR strain may be subject to repression during aerobic growth. PMID:8376325

  19. Cloning and Molecular Analysis of a Mannitol Operon of Phosphoenolpyruvate-dependent Phosphotransferase (PTS) type From Vibrio cholerae O395

    PubMed Central

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

    2010-01-01

    A putative mannitol operon of the phosphoenolpyruvate phosphotransferase (PTS) type was cloned from Vibrio cholerae O395 and its activity studied in Escherichia coli. The 3.9 kb operon comprising of three genes is organized as mtlADR. Based on the sequence analysis, these were identified as genes encoding a putative mannitol-specific enzyme IICBA (EIIMtl) component (MtlA), a mannitol-1-phosphate dehydrogenase (MtlD) and a mannitol operon repressor (MtlR). The transport of [3H]mannitol by the cloned mannitol operon in E. coli was 13.8±1.4 nmol/min/mg protein. The insertional inactivation of EIIMtl abolished mannitol and sorbitol transport in V. cholerae O395. Comparison of the mannitol utilization apparatus of V. cholerae with those of Gram-negative and Gram positive bacteria suggests highly conserved nature of the system. MtlA and MtlD exhibit 75% similarity with corresponding sequences of E. coli mannitol operon genes, while MtlR has 63% similarity with MtlR of E. coli. The cloning of V. cholerae mannitol utilization system in an E. coli background will help in elucidating the functional properties of this operon. PMID:21184218

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

    PubMed Central

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

    2011-01-01

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

  1. Nebulon: a system for the inference of functional relationships of gene products from the rearrangement of predicted operons

    PubMed Central

    Janga, Sarath Chandra; Collado-Vides, Julio; Moreno-Hagelsieb, Gabriel

    2005-01-01

    Since operons are unstable across Prokaryotes, it has been suggested that perhaps they re-combine in a conservative manner. Thus, genes belonging to a given operon in one genome might re-associate in other genomes revealing functional relationships among gene products. We developed a system to build networks of functional relationships of gene products based on their organization into operons in any available genome. The operon predictions are based on inter-genic distances. Our system can use different kinds of thresholds to accept a functional relationship, either related to the prediction of operons, or to the number of non-redundant genomes that support the associations. We also work by shells, meaning that we decide on the number of linking iterations to allow for the complementation of related gene sets. The method shows high reliability benchmarked against knowledge-bases of functional interactions. We also illustrate the use of Nebulon in finding new members of regulons, and of other functional groups of genes. Operon rearrangements produce thousands of high-quality new interactions per prokaryotic genome, and thousands of confirmations per genome to other predictions, making it another important tool for the inference of functional interactions from genomic context. PMID:15867197

  2. Kin selection and the evolution of virulence.

    PubMed

    Buckling, A; Brockhurst, M A

    2008-05-01

    Social interactions between conspecific parasites are partly dependent on the relatedness of interacting parasites (kin selection), which, in turn, is predicted to affect the extent of damage they cause their hosts (virulence). High relatedness is generally assumed to favour less competitive interactions, but the relationship between relatedness and virulence is crucially dependent on the social behaviour in question. Here, we discuss the rather limited body of experimental work that addresses how kin-selected social behaviours affect virulence. First, if prudent use of host resources (a form of cooperation) maximizes the transmission success of the parasite population, decreased relatedness is predicted to result in increased host exploitation and virulence. Experimental support for this well-established theoretical result is surprisingly limited. Second, if parasite within-host growth rate is a positive function of cooperation (that is, when individuals need to donate public goods, such as extracellular enzymes), virulence is predicted to increase with increasing relatedness. The limited studies testing this hypothesis are broadly consistent with this prediction. Finally, there is some empirical evidence supporting theory that suggests that spiteful behaviours are maximized at intermediate degrees of relatedness, which, in turn, leads to minimal virulence because of the reduced growth rate of the infecting population. We highlight the need for further thorough experimentation on the role of kin selection in the evolution of virulence and identify additional biological complexities to these simple frameworks.

  3. Virulence in malaria: an evolutionary viewpoint.

    PubMed Central

    Mackinnon, Margaret J; Read, Andrew F

    2004-01-01

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

  4. Serine/Threonine Protein Kinase Stk Is Required for Virulence, Stress Response, and Penicillin Tolerance in Streptococcus pyogenes▿

    PubMed Central

    Bugrysheva, Julia; Froehlich, Barbara J.; Freiberg, Jeffrey A.; Scott, June R.

    2011-01-01

    Genes encoding one or more Ser/Thr protein kinases have been identified recently in many bacteria, including one (stk) in the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). We report that in GAS, stk is required to produce disease in a murine myositis model of infection. Using microarray and quantitative reverse transcription-PCR (qRT-PCR) studies, we found that Stk activates genes for virulence factors, osmoregulation, metabolism of α-glucans, and fatty acid biosynthesis, as well as genes affecting cell wall synthesis. Confirming these transcription studies, we determined that the stk deletion mutant is more sensitive to osmotic stress and to penicillin than the wild type. We discuss several possible Stk phosphorylation targets that might explain Stk regulation of expression of specific operons and the possible role of Stk in resuscitation from quiescence. PMID:21788381

  5. The extinction differential induced virulence macroevolution

    NASA Astrophysics Data System (ADS)

    Zhang, Feng; Xu, Liufang; Wang, Jin

    2014-04-01

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

  6. Virulence factors of uropathogenic Escherichia coli.

    PubMed

    Emody, L; Kerényi, M; Nagy, G

    2003-10-01

    Virulence factors of Escherichia coli are of two main types; those produced on the surface of the cell and those produced within the cell and then exported to the site of action. Those on the surface include different sorts of fimbriae that have a role in adhesion to the surface of host cells but may also have additional roles such as tissue invasion, biofilm formation or cytokine induction. The activities of cell wall components are discussed and several exported virulence factors are described that have anti host cell activities. Others virulence factors enable the bacteria to grow in an environment of iron restriction.

  7. Transcriptional regulation of the Aggregatibacter actinomycetemcomitans ygiW-qseBC operon by QseB and integration host factor proteins.

    PubMed

    Juárez-Rodríguez, María Dolores; Torres-Escobar, Ascención; Demuth, Donald R

    2014-12-01

    The QseBC two-component system plays a pivotal role in regulating virulence and biofilm growth of the oral pathogen Aggregatibacter actinomycetemcomitans. We previously showed that QseBC autoregulates the ygiW-qseBC operon. In this study, we characterized the promoter that drives ygiW-qseBC expression. Using lacZ transcriptional fusion constructs and 5'-rapid amplification of cDNA ends, we showed that ygiW-qseBC expression is driven by a promoter that initiates transcription 53 bases upstream of ygiW and identified putative cis-acting promoter elements, whose function was confirmed using site-specific mutagenesis. Using electrophoretic mobility shift assays, two trans-acting proteins were shown to interact with the ygiW-qseBC promoter. The QseB response regulator bound to probes containing the direct repeat sequence CTTAA-N6-CTTAA, where the CTTAA repeats flank the -35 element of the promoter. The ygiW-qseBC expression could not be detected in A. actinomycetemcomitans ΔqseB or ΔqseBC strains, but was restored to WT levels in the ΔqseBC mutant when complemented by single copy chromosomal insertion of qseBC. Interestingly, qseB partially complemented the ΔqseBC strain, suggesting that QseB could be activated in the absence of QseC. QseB activation required its phosphorylation since complementation did not occur using qseB(pho-), encoding a protein with the active site aspartate substituted with alanine. These results suggest that QseB is a strong positive regulator of ygiW-qseBC expression. In addition, integration host factor (IHF) bound to two sites in the promoter region and an additional site near the 5' end of the ygiW ORF. The expression of ygiW-qseBC was increased by twofold in ΔihfA and ΔihfB strains of A. actinomycetemcomitans, suggesting that IHF is a negative regulator of the ygiW-qseBC operon.

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

    PubMed Central

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

    2011-01-01

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

  9. Vibrio parahaemolyticus has a homolog of the Vibrio cholerae toxRS operon that mediates environmentally induced regulation of the thermostable direct hemolysin gene.

    PubMed Central

    Lin, Z; Kumagai, K; Baba, K; Mekalanos, J J; Nishibuchi, M

    1993-01-01

    In an effort to identify the regulatory gene controlling the expression of the tdh gene, encoding the thermostable direct hemolysin of Vibrio parahaemolyticus, we examined total DNA of AQ3815 (a Kanagawa phenomenon-positive strain) for sequences homologous to that of the toxR gene of Vibrio cholerae. The extracted DNA gave a weak hybridization signal under reduced-stringency conditions with a toxR-specific DNA probe. Cloning and sequence analysis of the probe-positive sequence revealed an operon (Vp-toxRS) which was highly similar to the toxRS operon of V. cholerae (Vc-toxRS) (52 and 62% similarities in the two genes, respectively). The deduced amino acid sequences of the Vp-toxRS gene products (Vp-ToxRS) contained regions similar to the proposed transmembrane and activity domains of the Vc-toxRS gene products (Vc-ToxRS). All clinical and environmental strains of V. parahaemolyticus examined possessed the Vp-toxRS genes. In the presence of Vp-ToxS, Vp-ToxR promoted expression of the tdh2 gene, one of two tdh genes (tdh1 and tdh2) carried by Kanagawa phenomenon-positive strains. The DNA sequence located 144 bp upstream of the tdh2 coding region was shown to be important for the Vp-ToxR-stimulated expression of the tdh2 gene in an Escherichia coli background. Comparative analysis of AQ3815 and its isogenic Vp-toxR null mutant gave the following results: (i) Vp-ToxR promoted, in an AQ3815 background, expression of the tdh gene to different degrees in various culture media, with KP broth (2% peptone, 0.5% NaCl, 0.03 M KH2PO4, pH 6.2) being most effective (12-fold); (ii) the promotion of tdh gene expression in KP broth was at the level of transcription; and (iii) Vp-ToxR was essential for demonstration of enterotoxic activity of AQ3815 in the rabbit ileal loop, a model previously used to demonstrate thermostable direct hemolysin-mediated enterotoxic activity of AQ3815. These results demonstrate that Vp-ToxR and Vc-ToxR share a strikingly similar function, i.e., direct

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

    PubMed

    Nguyen, Scott V; McShan, William M

    2014-01-01

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

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

    PubMed Central

    Nguyen, Scott V.; McShan, William M.

    2014-01-01

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

  12. TcpP protein is a positive regulator of virulence gene expression in Vibrio cholerae.

    PubMed

    Häse, C C; Mekalanos, J J

    1998-01-20

    The production of several virulence factors in Vibrio cholerae O1, including cholera toxin and the pilus colonization factor TCP (toxin-coregulated pilus), is strongly influenced by environmental conditions. To specifically identify membrane proteins involved in these signal transduction events, we examined a transposon library of V. cholerae generated by Tnbla mutagenesis for cells that produce TCP when grown under various nonpermissive conditions. To select for TCP-producing cells we used the recently described bacteriophage CTX phi-Kan, which uses TCP as its receptor and carries a gene encoding resistance to kanamycin. Among the isolated mutants was a transposon insertion in a gene homologous to nqrB from Vibrio alginolyticus, which encodes a subunit of a Na(+)-translocating NADH:ubiquinone oxidoreductase, and tcpI, encoding a chemo-receptor previously implicated in the negative regulation of TCP production. A third transposon mutant had an insertion in tcpP, which is in an operon with tcpH, a known positive regulator of TCP production. However, TcpP was shown to be essential for TCP production in V. cholerae, as a tcpP-deletion strain was deficient in pili production. The amino-terminal region of TcpP shows sequence homology to the DNA-binding domains of several regulatory proteins, including ToxR from V. cholerae and PsaE from Yersinia pestis. Like ToxR, TcpP activates transcription of the toxT gene, an essential activator of tcp operon transcription. Furthermore, TcpH, with its large periplasmic domain and inner membrane anchor, has a structure similar to that of ToxS and was shown to enhance the activity of TcpP. We propose that TcpP/TcpH constitute a pair of regulatory proteins functionally similar to ToxR/ToxS and PsaE/PsaF that are required for toxT transcription in V. cholerae.

  13. The Burkholderia pseudomallei BpeAB-OprB Efflux Pump: Expression and Impact on Quorum Sensing and Virulence

    PubMed Central

    Chan, Ying Ying; Chua, Kim Lee

    2005-01-01

    BpeAB-OprB is a multidrug efflux pump of the bacterial pathogen Burkholderia pseudomallei and is responsible for conferring antimicrobial resistance to aminoglycosides and macrolides. Expression of bpeAB-oprB is inducible by its substrate erythromycin and upon entry into stationary phase. BpeR, a member of the TetR family, functions as a repressor of the bpeAB-oprB operon. bpeR expression was similarly induced at stationary phase but lagged behind the induction of bpeAB-oprB expression. The induction of bpeAB-oprB expression could be advanced to the early exponential phase by exogenous addition of the B. pseudomallei autoinducers N-octanoyl-homoserine lactone (C8HSL) and N-decanoyl-homoserine lactone (C10HSL), suggesting that the bpeAB-oprB operon may be quorum regulated. On the other hand, acyl-homoserine lactone (acyl-HSL) production was undetectable in the bpeAB-null mutant and strains which overexpress bpeR. The failure of these strains to produce acyl-HSLs seemed to be at the level of synthesis of acyl-HSLs, as growth-phase-dependent expression of the autoinducer synthase BpsI was abolished in the bpeAB-null mutant. bpsI expression remained growth phase dependent in the bpeR mutant which had functional BpeAB-OprB. BpeAB-OprB function is likewise necessary for optimal production of quorum-sensing-controlled virulence factors such as siderophore and phospholipase C and for biofilm formation. Cell invasion and cytotoxicity towards human lung epithelial (A549) and human macrophage (THP-1) cells were also significantly attenuated in both the bpeAB mutant and bpeR-overexpressing strains, thus suggesting the possibility of attenuating B. pseudomallei virulence using inhibitors of the BpeAB-OprB efflux pump. PMID:15995185

  14. The Burkholderia pseudomallei BpeAB-OprB efflux pump: expression and impact on quorum sensing and virulence.

    PubMed

    Chan, Ying Ying; Chua, Kim Lee

    2005-07-01

    BpeAB-OprB is a multidrug efflux pump of the bacterial pathogen Burkholderia pseudomallei and is responsible for conferring antimicrobial resistance to aminoglycosides and macrolides. Expression of bpeAB-oprB is inducible by its substrate erythromycin and upon entry into stationary phase. BpeR, a member of the TetR family, functions as a repressor of the bpeAB-oprB operon. bpeR expression was similarly induced at stationary phase but lagged behind the induction of bpeAB-oprB expression. The induction of bpeAB-oprB expression could be advanced to the early exponential phase by exogenous addition of the B. pseudomallei autoinducers N-octanoyl-homoserine lactone (C8HSL) and N-decanoyl-homoserine lactone (C10HSL), suggesting that the bpeAB-oprB operon may be quorum regulated. On the other hand, acyl-homoserine lactone (acyl-HSL) production was undetectable in the bpeAB-null mutant and strains which overexpress bpeR. The failure of these strains to produce acyl-HSLs seemed to be at the level of synthesis of acyl-HSLs, as growth-phase-dependent expression of the autoinducer synthase BpsI was abolished in the bpeAB-null mutant. bpsI expression remained growth phase dependent in the bpeR mutant which had functional BpeAB-OprB. BpeAB-OprB function is likewise necessary for optimal production of quorum-sensing-controlled virulence factors such as siderophore and phospholipase C and for biofilm formation. Cell invasion and cytotoxicity towards human lung epithelial (A549) and human macrophage (THP-1) cells were also significantly attenuated in both the bpeAB mutant and bpeR-overexpressing strains, thus suggesting the possibility of attenuating B. pseudomallei virulence using inhibitors of the BpeAB-OprB efflux pump.

  15. Role of Ribosome Release in Regulation of tna Operon Expression in Escherichia coli

    PubMed Central

    Konan, Kouacou Vincent; Yanofsky, Charles

    1999-01-01

    Expression of the degradative tryptophanase (tna) operon of Escherichia coli is regulated by catabolite repression and tryptophan-induced transcription antitermination. In cultures growing in the absence of added tryptophan, transcription of the structural genes of the tna operon is limited by Rho-dependent transcription termination in the leader region of the operon. Tryptophan induction prevents this Rho-dependent termination, and requires in-frame translation of a 24-residue leader peptide coding region, tnaC, that contains a single, crucial, Trp codon. Studies with a lacZ reporter construct lacking the spacer region between tnaC and the first major structural gene, tnaA, suggested that tryptophan induction might involve cis action by the TnaC leader peptide on the ribosome translating the tnaC coding region. The leader peptide was hypothesized to inhibit ribosome release at the tnaC stop codon, thereby blocking Rho’s access to the transcript. Regulatory studies with deletion constructs of the tna operon of Proteus vulgaris supported this interpretation. In the present study the putative role of the tnaC stop codon in tna operon regulation in E. coli was examined further by replacing the natural tnaC stop codon, UGA, with UAG or UAA in a tnaC-stop codon-tnaA′-′lacZ reporter construct. Basal level expression was reduced to 20 and 50% when the UGA stop codon was replaced by UAG or UAA, respectively, consistent with the finding that in E. coli translation terminates more efficiently at UAG and UAA than at UGA. Tryptophan induction was observed in strains with any of the stop codons. However, when UAG or UAA replaced UGA, the induced level of expression was also reduced to 15 and 50% of that obtained with UGA as the tnaC stop codon, respectively. Introduction of a mutant allele encoding a temperature-sensitive release factor 1, prfA1, increased basal level expression 60-fold when the tnaC stop codon was UAG and 3-fold when this stop codon was UAA; basal level

  16. Influence of cAMP receptor protein (CRP) on bacterial virulence and transcriptional regulation of allS by CRP in Klebsiella pneumoniae.

    PubMed

    Xue, Jian; Tan, Bin; Yang, Shiya; Luo, Mei; Xia, Huiming; Zhang, Xian; Zhou, Xipeng; Yang, Xianxian; Yang, Ruifu; Li, Yingli; Qiu, Jingfu

    2016-11-15

    cAMP receptor protein (CRP) is one of the most important transcriptional regulators, which can regulate large quantities of operons in different bacteria. The gene allS was well-known as allantoin-utilizing capability and involving in bacterial virulence in Klebsiella pneumoniae (K. pneumoniae). The specific DNA recognition motif of transcription regulator CRP was found in allS promoter region. Therefore, this study is aimed to investigate the function of CRP on virulence and its transcriptional regulation mechanism to gene allS in K. pneumoniae. The wild-type (WT) K. pneumoniae NTUH-2044, crp knockout (Kp-Δcrp) and the complemented knockout (KpC-Δcrp) strains were used to determine the function of crp gene. The lacZ fusion, qRT-PCR, electrophoretic mobility shift and DNase I footprinting assays were performed to study the transcriptional regulation of CRP on allS. The result showed a decreased virulence in crp knockout strain. Complement through supplementing crp fragment in expression plasmid partially restore virulence of knockout bacteria. The CRP could bind to the allS promoter-proximal region and the binding site was further refined to be located from 60bp to 94bp upstream of the allS promoter. Based on these results, we proposed that CRP is an essential virulence regulator and knock out of crp gene will result in reduced virulence in K. pneumoniae. In the meantime, the transcription of gene allS is positively regulated by CRP via directly binding to upstream of allS promoter.

  17. Listeriolysin S Is a Streptolysin S-Like Virulence Factor That Targets Exclusively Prokaryotic Cells In Vivo

    PubMed Central

    Quereda, Juan J.; Nahori, Marie A.; Meza-Torres, Jazmín; Sachse, Martin; Titos-Jiménez, Patricia; Gomez-Laguna, Jaime; Dussurget, Olivier; Cossart, Pascale

    2017-01-01

    ABSTRACT Streptolysin S (SLS)-like virulence factors from clinically relevant Gram-positive pathogens have been proposed to behave as potent cytotoxins, playing key roles in tissue infection. Listeriolysin S (LLS) is an SLS-like hemolysin/bacteriocin present among Listeria monocytogenes strains responsible for human listeriosis outbreaks. As LLS cytotoxic activity has been associated with virulence, we investigated the LLS-specific contribution to host tissue infection. Surprisingly, we first show that LLS causes only weak red blood cell (RBC) hemolysis in vitro and neither confers resistance to phagocytic killing nor favors survival of L. monocytogenes within the blood cells or in the extracellular space (in the plasma). We reveal that LLS does not elicit specific immune responses, is not cytotoxic for eukaryotic cells, and does not impact cell infection by L. monocytogenes. Using in vitro cell infection systems and a murine intravenous infection model, we actually demonstrate that LLS expression is undetectable during infection of cells and murine inner organs. Importantly, upon intravenous animal inoculation, L. monocytogenes is found in the gastrointestinal system, and only in this environment LLS expression is detected in vivo. Finally, we confirm that LLS production is associated with destruction of target bacteria. Our results demonstrate therefore that LLS does not contribute to L. monocytogenes tissue injury and virulence in inner host organs as previously reported. Moreover, we describe that LlsB, a putative posttranslational modification enzyme encoded in the LLS operon, is necessary for murine inner organ colonization. Overall, we demonstrate that LLS is the first SLS-like virulence factor targeting exclusively prokaryotic cells during in vivo infections. PMID:28377528

  18. A prl Mutation in SecY Suppresses Secretion and Virulence Defects of Listeria monocytogenes secA2 Mutants

    PubMed Central

    Durack, Juliana; Burke, Thomas P.

    2014-01-01

    The bulk of bacterial protein secretion occurs through the conserved SecY translocation channel that is powered by SecA-dependent ATP hydrolysis. Many Gram-positive bacteria, including the human pathogen Listeria monocytogenes, possess an additional nonessential specialized ATPase, SecA2. SecA2-dependent secretion is required for normal cell morphology and virulence in L. monocytogenes; however, the mechanism of export via this pathway is poorly understood. L. monocytogenes secA2 mutants form rough colonies, have septation defects, are impaired for swarming motility, and form small plaques in tissue culture cells. In this study, 70 spontaneous mutants were isolated that restored swarming motility to L. monocytogenes secA2 mutants. Most of the mutants had smooth colony morphology and septated normally, but all were lysozyme sensitive. Five representative mutants were subjected to whole-genome sequencing. Four of the five had mutations in proteins encoded by the lmo2769 operon that conferred lysozyme sensitivity and increased swarming but did not rescue virulence defects. A point mutation in secY was identified that conferred smooth colony morphology to secA2 mutants, restored wild-type plaque formation, and increased virulence in mice. This secY mutation resembled a prl suppressor known to expand the repertoire of proteins secreted through the SecY translocation complex. Accordingly, the ΔsecA2prlA1 mutant showed wild-type secretion levels of P60, an established SecA2-dependent secreted autolysin. Although the prl mutation largely suppressed almost all of the measurable SecA2-dependent traits, the ΔsecA2prlA1 mutant was still less virulent in vivo than the wild-type strain, suggesting that SecA2 function was still required for pathogenesis. PMID:25535272

  19. CcpA and LacD.1 Affect Temporal Regulation of Streptococcus pyogenes Virulence Genes ▿ †

    PubMed Central

    Kietzman, Colin C.; Caparon, Michael G.

    2010-01-01

    Production of H2O2 follows a growth phase-dependent pattern that mimics that of many virulence factors of Streptococcus pyogenes. To gain greater insight into mechanisms coupling virulence factor expression to growth phase, we investigated the molecular basis for H2O2 generation and its regulation. Deletion of the gene encoding lactate oxidase (lctO) or culture in the presence of glucose eliminated H2O2 production, implicating carbohydrate regulation of lctO as a key element of growth phase control. In examining known carbohydrate-responsive regulators, deletion of the gene encoding CcpA but not that encoding LacD.1 resulted in both derepression and an uncoupling of lctO transcription from its growth phase pattern. Expanding this analysis to additional virulence factors demonstrated both negative (cfa, encoding CAMP factor) and positive (speB, encoding a cysteine protease) regulation by CcpA and that CcpA mutants were highly cytotoxic for cultured macrophages. This latter property resulted from enhanced transcription of the streptolysin S biogenesis operon. Examination of CcpA-promoter interactions using a DNA pull-down assay mimicking physiological conditions showed direct binding to the promoters of lctO and speB but not those of sagA. CcpA but not LacD.1 mutants were attenuated in a murine model of soft-tissue infection, and analysis of gene expression in infected tissue indicated that CcpA mutants had altered expression of lctO, cfa, and speB but not the indirectly regulated sagA gene. Taken together, these data show that CcpA regulates virulence genes via at least three distinct mechanisms and that disruption of growth phase regulation alters transcriptional patterns in infected tissues. PMID:19841076

  20. Plaque assay for virulent Legionella pneumophila.

    PubMed Central

    Fernandez, R C; Lee, S H; Haldane, D; Sumarah, R; Rozee, K R

    1989-01-01

    Methods of assessing virulence of Legionella pneumophila, the etiologic agent of Legionnaires disease, include the infection of guinea pigs, fertile chicken eggs, and mammalian and protozoan cell cultures. Guinea pig assays, in particular, are expensive, laborious, or unsuitable for routine screening of Legionella isolates. We have developed a virulence assay that requires the enumeration of viruslike plaques which are the result of virulent L. pneumophila infecting mouse L929 cells. Each plaque is the consequence of the initial infection of an L cell with a single bacterium. A nonvirulent mutant derived from the serial passage of virulent L. pneumophila on Mueller-Hinton agar fails to survive within L cells and consequently fails to produce plaques. Images PMID:2674192

  1. Correlation of autoagglutination and virulence of yersiniae.

    PubMed Central

    Laird, W J; Cavanaugh, D C

    1980-01-01

    Virulent strains of Yersinia pestis, Y. pseudotuberculosis and Yersinia enterocolitica invariably autoagglutinated in tissue culture media when grown at 36 degrees C. Avirulent strains did not possess this property. PMID:7372804

  2. Mechanisms and evolution of virulence in oomycetes.

    PubMed

    Jiang, Rays H Y; Tyler, Brett M

    2012-01-01

    Many destructive diseases of plants and animals are caused by oomycetes, a group of eukaryotic pathogens important to agricultural, ornamental, and natural ecosystems. Understanding the mechanisms underlying oomycete virulence and the genomic processes by which those mechanisms rapidly evolve is essential to developing effective long-term control measures for oomycete diseases. Several common mechanisms underlying oomycete virulence, including protein toxins and cell-entering effectors, have emerged from comparing oomycetes with different genome characteristics, parasitic lifestyles, and host ranges. Oomycete genomes display a strongly bipartite organization in which conserved housekeeping genes are concentrated in syntenic gene-rich blocks, whereas virulence genes are dispersed into highly dynamic, repeat-rich regions. There is also evidence that key virulence genes have been acquired by horizontal transfer from other eukaryotic and prokaryotic species.

  3. Regulation and secretion of Xanthomonas virulence factors.

    PubMed

    Büttner, Daniela; Bonas, Ulla

    2010-03-01

    Plant pathogenic bacteria of the genus Xanthomonas cause a variety of diseases in economically important monocotyledonous and dicotyledonous crop plants worldwide. Successful infection and bacterial multiplication in the host tissue often depend on the virulence factors secreted including adhesins, polysaccharides, LPS and degradative enzymes. One of the key pathogenicity factors is the type III secretion system, which injects effector proteins into the host cell cytosol to manipulate plant cellular processes such as basal defense to the benefit of the pathogen. The coordinated expression of bacterial virulence factors is orchestrated by quorum-sensing pathways, multiple two-component systems and transcriptional regulators such as Clp, Zur, FhrR, HrpX and HpaR. Furthermore, virulence gene expression is post-transcriptionally controlled by the RNA-binding protein RsmA. In this review, we summarize the current knowledge on the infection strategies and regulatory networks controlling secreted virulence factors from Xanthomonas species.

  4. The effect of iatrogenic Staphylococcus epidermidis intercellar adhesion operon on the formation of bacterial biofilm on polyvinyl chloride surfaces.

    PubMed

    Lianhua, Ye; Yunchao, Huang; Guangqiang, Zhao; Kun, Yang; Xing, Liu; Fengli, Guo

    2014-12-01

    The intercellular adhesion gene (ica) of Staphylococcus epidermidis is a key factor for bacterial aggregation. This study explored the effect of ica on the formation of bacterial biofilm on polyvinyl chloride (PVC) surfaces. Genes related to bacterial biofilm formation, including 16S rRNA, autolysin (atlE), fibrinogen binding protein gene (fbe), and ica were identified and sequenced from 112 clinical isolates of iatrogenic S. epidermidis by polymerase chain reaction (PCR) and gene sequencing. Based on the sequencing result, ica operon-positive (icaADB+/atlE+/fbe+) and ica operon-negative (icaADB-/atlE+/fbe+) strains were separated and co-cultivated with PVC material. After 6, 12, 18, 24, and 30 h of co-culture, the thickness of the bacterial biofilm and quantity of bacterial colony on the PVC surface were measured under the confocal laser scanning microscope and scanning electron microscope. The positive rate of S. epidermidis-specific 16SrRNA in 112 iatrogenic strains was 100% (112/112). The genotype of ica-positive (icaADB+/atlE+/fbe+) strains accounted for 57.1% (64/112), and genotype of ica-negative (icaADB-/atlE+/fbe+) strains accounted for 37.5% (42/112). During 30 h of co-culture, no obvious bacterial biofilm formed on the surface of PVC in the ica-positive group, however, mature bacterial biofilm structure formed after 24 h. For all time points, thickness of bacterial biofilm and quantity of bacterial colony on PVC surfaces in the ica operon-positive group were significantly higher than those in ica operon-negative group (p<0.01). Iatrogenic S. epidermidis can be categorized into ica operon-negative and ica operon-positive strains. The ica operon plays an important role in bacterial biofilm formation and bacterial multiplication on PVC material.

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

    PubMed

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

    2007-01-01

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

  6. Dimorphism and virulence in fungi

    PubMed Central

    Klein, Bruce S.; Tebbets, Brad

    2012-01-01

    The signature feature of systemic dimorphic fungi – a family of six primary fungal pathogens of humans – is a temperature-induced phase transition. These fungi grow as a mold in soil at ambient temperature and convert to yeast after infectious spores are inhaled into the lungs of a mammalian host. Seminal work 20 years ago established that a temperature-induced phase transition from mold to yeast is required for virulence. Several yeast-phase specific genes, identified one-by-one and studied by reverse genetics, have revealed mechanisms by which the phase transition promotes disease pathogenesis. Transcriptional profiling of microarrays built with genomic elements of Histoplasma capsulatum and ESTs of Paracoccidioides brasiliensis that represent partial genomes has identified 500 genes and 328 genes, respectively, that are differentially expressed upon the phase transition. The genomes of most of the dimorphic fungi are now in varying stages of being sequenced. The creation of additional microarrays and the application of new reverse genetic tools promise fresh insight into genes and mechanisms that regulate pathogenesis and morphogenesis. The use of insertional mutagenesis by Agrobacterium has uncovered a hybrid histidine kinase that regulates dimorphism and pathogenicity in B. dermatitidis and H. capsulatum. Two-component signaling appears to be a common strategy for model and pathogenic fungi to sense and respond to environmental stresses. PMID:17719267

  7. Dimorphism and virulence in fungi.

    PubMed

    Klein, Bruce S; Tebbets, Brad

    2007-08-01

    The signature feature of systemic dimorphic fungi - a family of six primary fungal pathogens of humans - is a temperature-induced phase transition. These fungi grow as a mold in soil at ambient temperature and convert to yeast after infectious spores are inhaled into the lungs of a mammalian host. Seminal work 20 years ago established that a temperature-induced phase transition from mold to yeast is required for virulence. Several yeast-phase specific genes, identified one-by-one and studied by reverse genetics, have revealed mechanisms by which the phase transition promotes disease pathogenesis. Transcriptional profiling of microarrays built with genomic elements of Histoplasma capsulatum and ESTs of Paracoccidioides brasiliensis that represent partial genomes has identified 500 genes and 328 genes, respectively, that are differentially expressed upon the phase transition. The genomes of most of the dimorphic fungi are now in varying stages of being sequenced. The creation of additional microarrays and the application of new reverse genetic tools promise fresh insight into genes and mechanisms that regulate pathogenesis and morphogenesis. The use of insertional mutagenesis by Agrobacterium has uncovered a hybrid histidine kinase that regulates dimorphism and pathogenicity in Blastomyces dermatitidis and H. capsulatum. Two-component signaling appears to be a common strategy for model and pathogenic fungi to sense and respond to environmental stresses.

  8. Pathogenicity and virulence: another view.

    PubMed Central

    Isenberg, H D

    1988-01-01

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

  9. Spaceflight Effects on Virulence of Pseudomonas Aeruginosa

    NASA Astrophysics Data System (ADS)

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

    2008-06-01

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

  10. Xanthoferrin, the α-hydroxycarboxylate-type siderophore of Xanthomonas campestris pv. campestris, is required for optimum virulence and growth inside cabbage.

    PubMed

    Pandey, Sheo Shankar; Patnana, Pradeep Kumar; Rai, Rikky; Chatterjee, Subhadeep

    2016-06-27

    Xanthomonas campestris pv. campestris causes black rot, a serious disease of crucifers. Xanthomonads encode a siderophore biosynthesis and uptake gene cluster xss (Xanthomonas siderophore synthesis) involved in the production of a vibrioferrin-type siderophore. However, little is known about the role of the siderophore in the iron uptake and virulence of X. campestris pv. campestris. In this study, we show that X. campestris pv. campestris produces an α-hydroxycarboxylate-type siderophore (named xanthoferrin), which is required for growth under low-iron conditions and for optimum virulence. A mutation in the siderophore synthesis xssA gene causes deficiency in siderophore production and growth under low-iron conditions. In contrast, the siderophore utilization ΔxsuA mutant is able to produce siderophore, but exhibits a defect in the utilization of the siderophore-iron complex. Our radiolabelled iron uptake studies confirm that the ΔxssA and ΔxsuA mutants exhibit defects in ferric iron (Fe(3+) ) uptake. The ΔxssA mutant is able to utilize and transport the exogenous xanthoferrin-Fe(3+) complex; in contrast, the siderophore utilization or uptake mutant ΔxsuA exhibits defects in siderophore uptake. Expression analysis of the xss operon using a chromosomal gusA fusion indicates that the xss operon is expressed during in planta growth and under low-iron conditions. Furthermore, exogenous iron supplementation in cabbage leaves rescues the in planta growth deficiency of ΔxssA and ΔxsuA mutants. Our study reveals that the siderophore xanthoferrin is an important virulence factor of X. campestris pv. campestris which promotes in planta growth by the sequestration of Fe(3+) .

  11. The Effect of Stochasticity on the Lac Operon: An Evolutionary Perspective

    PubMed Central

    van Hoek, Milan; Hogeweg, Paulien

    2007-01-01

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

  12. Functional characterization and evolution of the isotuberculosinol operon in Mycobacterium tuberculosis and related Mycobacteria

    PubMed Central

    Mann, Francis M.; Xu, Meimei; Davenport, Emily K.; Peters, Reuben J.

    2012-01-01

    Terpenoid metabolites are important to the cellular function, structural integrity, and pathogenesis of the human-specific pathogen Mycobacterium tuberculosis (Mtb). Genetic and biochemical investigations have indicated a role for the diterpenoid isotuberculosinol (isoTb) early in the infection process. There are only two genes (Rv3377c and Rv3378c) required for production of isoTb, yet these are found in what appears to be a five-gene terpenoid/isoprenoid biosynthetic operon. Of the three remaining genes (Rv3379c, Rv3382c, and Rv3383c), previous work has indicated that Rv3379c is an inactive pseudo-gene. Here we demonstrate that Rv3382c and Rv3383c encode biochemically redundant machinery for isoprenoid metabolism, encoding a functional 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB) for isoprenoid precursor production and a geranylgeranyl diphosphate (GGPP) synthase, respectively, for which the Mtb genome contains other functional isozymes (Rv1110 and Rv0562, respectively). These results complete the characterization of the isoTb biosynthetic operon, as well as further elucidating isoprenoid metabolism in Mtb. In addition, we have investigated the evolutionary origin of this operon, revealing Mtb-specific conservation of the diterpene synthase genes responsible for isoTb biosynthesis, which supports our previously advanced hypothesis that isoTb acts as a human-specific pathogenic metabolite and is consistent with the human host specificity of Mtb. Intriguingly, our results revealed that many mycobacteria contain orthologs for both Rv3383c and Rv0562, suggesting a potentially important role for these functionally redundant GGPP synthases in the evolution of terpenoid/isoprenoid metabolism in the mycobacteria. PMID:23091471

  13. arc-dependent thermal regulation and extragenic suppression of the Escherichia coli cytochrome d operon.

    PubMed

    Wall, D; Delaney, J M; Fayet, O; Lipinska, B; Yamamoto, T; Georgopoulos, C

    1992-10-01

    In a screen for Escherichia coli genes whose products are required for high-temperature growth, we identified and characterized a mini-Tn10 insertion that allows the formation of wild-type-size colonies at 30 degrees C but results in microcolony formation at 36 degrees C and above (Ts- phenotype). Mapping, molecular cloning, and DNA sequencing analyses showed that the mini-Tn10 insertion resides in the cydB gene, the distal gene of the cydAB operon (cytochrome d). The Ts- growth phenotype was also shown to be associated with previously described cyd alleles. In addition, all cyd mutants were found to be extremely sensitive to hydrogen peroxide. Northern (RNA) blot analysis showed that cyd-specific mRNA levels accumulate following a shift to high temperature. Interestingly, this heat shock induction of the cyd operon was not affected in an rpoH delta background but was totally absent in an arcA or arcB mutant background. Extragenic suppressors of the Cyd Ts- phenotype are found at approximately 10(-3). Two extragenic suppressors were shown to be null alleles in either arcA or arcB. One interpretation of our results is that in the absence of ArcA or ArcB, which are required for the repression of the cyo operon (cytochrome o), elevated levels of Cyo are produced, thus compensating for the missing cytochrome d function. Consistent with this interpretation, the presence of the cyo gene on a multicopy plasmid suppressed the Ts- and hydrogen peroxide-sensitive phenotypes of cyd mutants.

  14. A Fluorescent Bioreporter for Acetophenone and 1-Phenylethanol derived from a Specifically Induced Catabolic Operon

    PubMed Central

    Muhr, Enrico; Leicht, Oliver; González Sierra, Silvia; Thanbichler, Martin; Heider, Johann

    2016-01-01

    The β-proteobacterium Aromatoleum aromaticum degrades the aromatic ketone acetophenone, a key intermediate of anaerobic ethylbenzene metabolism, either aerobically or anaerobically via a complex ATP-dependent acetophenone carboxylase and a benzoylacetate-CoA ligase. The genes coding for these enzymes (apcABCDE and bal) are organized in an apparent operon and are expressed in the presence of the substrate acetophenone. To study the conditions under which this operon is expressed in more detail, we constructed a reporter strain by inserting a gene fusion of apcA, the first gene of the apc-bal operon, with the gene for the fluorescent protein mCherry into the chromosome of A. aromaticum. The fusion protein indeed accumulated consistently with the expression pattern of the acetophenone-metabolic enzymes under various growth conditions. After evaluating and quantifying the data by fluorescence microscopy, fluorescence-based flow cytometry and immunoblot analysis, mCherry production was found to be proportional to the applied acetophenone concentrations. The reporter strain allowed quantification of acetophenone within a concentration range of 50 μM (detection limit) to 250 μM after 12 and 24 h. Moreover, production of the Apc-mCherry fusion protein in the reporter strain was highly specific and responded to acetophenone and both enantiomers of 1-phenylethanol, which are easily converted to acetophenone. Other analogous substrates showed either a significantly weaker response or none at all. Therefore, the reporter strain provides a basis for the development of a specific bioreporter system for acetophenone with an application potential reaching from environmental monitoring to petroleum prospecting. PMID:26858693

  15. Role of Tellurite Resistance Operon in Filamentous Growth of Yersinia pestis in Macrophages.

    PubMed

    Ponnusamy, Duraisamy; Clinkenbeard, Kenneth D

    2015-01-01

    Yersinia pestis initiates infection by parasitism of host macrophages. In response to macrophage infections, intracellular Y. pestis can assume a filamentous cellular morphology which may mediate resistance to host cell innate immune responses. We previously observed the expression of Y. pestis tellurite resistance proteins TerD and TerE from the terZABCDE operon during macrophage infections. Others have observed a filamentous response associated with expression of tellurite resistance operon in Escherichia coli exposed to tellurite. Therefore, in this study we examine the potential role of Y. pestis tellurite resistance operon in filamentous cellular morphology during macrophage infections. In vitro treatment of Y. pestis culture with sodium tellurite (Na2TeO3) caused the bacterial cells to assume a filamentous phenotype similar to the filamentous phenotype observed during macrophage infections. A deletion mutant for genes terZAB abolished the filamentous morphologic response to tellurite exposure or intracellular parasitism, but without affecting tellurite resistance. However, a terZABCDE deletion mutant abolished both filamentous morphologic response and tellurite resistance. Complementation of the terZABCDE deletion mutant with terCDE, but not terZAB, partially restored tellurite resistance. When the terZABCDE deletion mutant was complemented with terZAB or terCDE, Y. pestis exhibited filamentous morphology during macrophage infections as well as while these complemented genes were being expressed under an in vitro condition. Further in E. coli, expression of Y. pestis terZAB, but not terCDE, conferred a filamentous phenotype. These findings support the role of Y. pestis terZAB mediation of the filamentous response phenotype; whereas, terCDE confers tellurite resistance. Although the beneficial role of filamentous morphological responses by Y. pestis during macrophage infections is yet to be fully defined, it may be a bacterial adaptive strategy to macrophage

  16. Characterization of heterologous multigene operons in transgenic chloroplasts: transcription, processing, and translation.

    PubMed

    Quesada-Vargas, Tania; Ruiz, Oscar N; Daniell, Henry

    2005-07-01

    The first characterization of transcriptional, posttranscriptional, and translational processes of heterologous operons expressed via the tobacco (Nicotiana tabacum) chloroplast genome is reported here. Northern-blot analyses performed on chloroplast transgenic lines harboring seven different heterologous operons revealed that polycistronic mRNA was the predominant transcript produced. Despite the lack of processing of such polycistrons, large amounts of foreign protein accumulation was observed in these transgenic lines, indicating abundant translation of polycistrons. This is supported by polysome fractionation assays, which allowed detection of polycistronic RNA in lower fractions of the sucrose gradients. These results show that the chloroplast posttranscriptional machinery can indeed detect and translate multigenic sequences that are not of chloroplast origin. In contrast to native transcripts, processed and unprocessed heterologous polycistrons were stable, even in the absence of 3' untranslated regions (UTRs). Unlike native 5'UTRs, heterologous secondary structures or 5'UTRs showed efficient translational enhancement independent of cellular control. Abundant read-through transcripts were observed in the presence of chloroplast 3'UTRs but they were efficiently processed at introns present within the native operon. Heterologous genes regulated by the psbA (the photosystem II polypeptide D1) promoter, 5' and 3'UTRs have greater abundance of transcripts than the endogenous psbA gene because transgenes were integrated into the inverted repeat region. Addressing questions about polycistrons, and the sequences required for their processing and transcript stability, are essential in chloroplast metabolic engineering. Knowledge of such factors would enable engineering of foreign pathways independent of the chloroplast complex posttranscriptional regulatory machinery.

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

    PubMed

    van Hoek, Milan; Hogeweg, Paulien

    2007-06-01

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

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

    PubMed

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

    2008-09-01

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

  19. Interactome analyses of Salmonella pathogenicity islands reveal SicA indispensable for virulence.

    PubMed

    Lahiri, Chandrajit; Pawar, Shrikant; Sabarinathan, Radhakrishnan; Ashraf, Md Izhar; Chand, Yamini; Chakravortty, Dipshikha

    2014-12-21

    Serovars of Salmonella enterica, namely Typhi and Typhimurium, reportedly, are the bacterial pathogens causing systemic infections like gastroenteritis and typhoid fever. To elucidate the role and importance in such infection, the proteins of the Type III secretion system of Salmonella pathogenicity islands and two component signal transduction systems, have been mainly focused. However, the most indispensable of these virulent ones and their hierarchical role has not yet been studied extensively. We have adopted a theoretical approach to build an interactome comprising the proteins from the Salmonella pathogeneicity islands (SPI) and two component signal transduction systems. This interactome was then analyzed by using network parameters like centrality and k-core measures. An initial step to capture the fingerprint of the core network resulted in a set of proteins which are involved in the process of invasion and colonization, thereby becoming more important in the process of infection. These proteins pertained to the Inv, Org, Prg, Sip, Spa, Ssa and Sse operons along with chaperone protein SicA. Amongst them, SicA was figured out to be the most indispensable protein from different network parametric analyses. Subsequently, the gene expression levels of all these theoretically identified important proteins were confirmed by microarray data analysis. Finally, we have proposed a hierarchy of the proteins involved in the total infection process. This theoretical approach is the first of its kind to figure out potential virulence determinants encoded by SPI for therapeutic targets for enteric infection. A set of responsible virulent proteins was identified and the expression level of their genes was validated by using independent, published microarray data. The result was a targeted set of proteins that could serve as sensitive predictors and form the foundation for a series of trials in the wet-lab setting. Understanding these regulatory and virulent proteins would

  20. An Operon for a Putative ATP-Binding Cassette Transport System Involved in Acetoin Utilization of Bacillus subtilis

    PubMed Central

    Yoshida, Ken-Ichi; Fujita, Yasutaro; Ehrlich, S. Dusko

    2000-01-01

    The ytrABCDEF operon of Bacillus subtilis was deduced to encode a putative ATP-binding cassette (ABC) transport system. YtrB and YtrE could be the ABC subunits, and YtrC and YtrD are highly hydrophobic and could form a channel through the cell membrane, while YtrF could be a periplasmic lipoprotein for substrate binding. Expression of the operon was examined in cells grown in a minimal medium. The results indicate that the expression was induced only early in the stationary phase. The six ytr genes form a single operon, transcribed from a putative ςA-dependent promoter present upstream of ytrA. YtrA, which possesses a helix-turn-helix motif of the GntR family, acts probably as a repressor and regulates its own transcription. Inactivation of the operon led to a decrease in maximum cell yield and less-efficient sporulation, suggesting its involvement in the growth in stationary phase and sporulation. It is known that B. subtilis produces acetoin as an external carbon storage compound and then reuses it later during stationary phase and sporulation. When either the entire ytr operon or its last gene, ytrF, was inactivated, the production of acetoin was not affected, but the reuse of acetoin became less efficient. We suggest that the Ytr transport system plays a role in acetoin utilization during stationary phase and sporulation. PMID:10986249

  1. An operon for a putative ATP-binding cassette transport system involved in acetoin utilization of Bacillus subtilis.

    PubMed

    Yoshida, K I; Fujita, Y; Ehrlich, S D

    2000-10-01

    The ytrABCDEF operon of Bacillus subtilis was deduced to encode a putative ATP-binding cassette (ABC) transport system. YtrB and YtrE could be the ABC subunits, and YtrC and YtrD are highly hydrophobic and could form a channel through the cell membrane, while YtrF could be a periplasmic lipoprotein for substrate binding. Expression of the operon was examined in cells grown in a minimal medium. The results indicate that the expression was induced only early in the stationary phase. The six ytr genes form a single operon, transcribed from a putative sigma(A)-dependent promoter present upstream of ytrA. YtrA, which possesses a helix-turn-helix motif of the GntR family, acts probably as a repressor and regulates its own transcription. Inactivation of the operon led to a decrease in maximum cell yield and less-efficient sporulation, suggesting its involvement in the growth in stationary phase an