scholarly journals SlyA and H-NS Regulate Transcription of the Escherichia coli K5 Capsule Gene Cluster, and Expression of slyA in Escherichia coli Is Temperature-dependent, Positively Autoregulated, and Independent of H-NS

2007 ◽  
Vol 282 (46) ◽  
pp. 33326-33335 ◽  
Author(s):  
David Corbett ◽  
Hayley J. Bennett ◽  
Hamdia Askar ◽  
Jeffrey Green ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Regulation Of Transcription ◽  
Temperature Dependent ◽  
Mobility Shift ◽  
E Coli ◽  
Dnase I Footprinting ◽  
Nucleoprotein Complex ◽  
Electrophoretic Mobility Shift Assays

In this paper, we present the first evidence of a role for the transcriptional regulator SlyA in the regulation of transcription of the Escherichia coli K5 capsule gene cluster and demonstrate, using a combination of reporter gene fusions, DNase I footprinting, and electrophoretic mobility shift assays, the dependence of transcription on the functional interplay between H-NS and SlyA. Both SlyA and H-NS bind to multiple overlapping sites within the promoter in vitro, but their binding is not mutually exclusive, resulting in a remodeled nucleoprotein complex. In addition, we show that expression of the E. coli slyA gene is temperature-regulated, positively autoregulated, and independent of H-NS.

scholarly journals The ArgP Protein Stimulates the Klebsiella pneumoniae gdhA Promoter in a Lysine-Sensitive Manner

2008 ◽  
Vol 190 (12) ◽  
pp. 4351-4359 ◽  
Author(s):  
Thomas J. Goss
Keyword(s):  
Klebsiella Pneumoniae ◽  
Upstream Region ◽  
Mobility Shift ◽  
Base Pairs ◽  
Dnase I Footprinting ◽  
Sensitive Factor ◽  
Electrophoretic Mobility Shift Assays ◽  
Knockout Mutation

ABSTRACT The lysine-sensitive factor that binds to the upstream region of the Klebsiella pneumoniae gdhA promoter and stimulates gdhA transcription during growth in minimal medium has been proposed to be the K. pneumoniae ArgP protein (M. R. Nandineni, R. S. Laishram, and J. Gowrishankar, J. Bacteriol. 186:6391-6399, 2004). A knockout mutation of the K. pneumoniae argP gene was generated and used to assess the roles of exogenous lysine and argP in the regulation of the gdhA promoter. Disruption of argP reduced the strength and the lysine-dependent regulation of the gdhA promoter. Electrophoretic mobility shift assays using crude extracts prepared from wild-type and argP-defective strains indicted the presence of an argP-dependent factor whose ability to bind the gdhA promoter was lysine sensitive. DNase I footprinting studies using purified K. pneumoniae ArgP protein indicated that ArgP bound the region that lies approximately 50 to 100 base pairs upstream of the gdhA transcription start site in a manner that was sensitive to the presence of lysine. Substitutions within the region bound by ArgP affected the binding of ArgP to the gdhA promoter region in vitro and the argP-dependent stimulation of the gdhA promoter in vivo. These observations suggest that elevated intracellular levels of lysine reduce the affinity of ArgP for its binding site at the gdhA promoter, preventing ArgP from binding to and stimulating transcription from the promoter in vivo.

scholarly journals HosA, a Member of the SlyA Family, Regulates Motility in Enteropathogenic Escherichia coli

2005 ◽  
Vol 73 (3) ◽  
pp. 1684-1694 ◽  
Author(s):  
Maria-José Ferrándiz ◽  
Keith Bishop ◽  
Paul Williams ◽  
Helen Withers
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Regulators ◽  
Positive Control ◽  
Broth Culture ◽  
Wild Type ◽  
Mobility Shift ◽  
E Coli ◽  
Protein Levels ◽  
K 12 ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT In enteropathogenic and enterohemorraghic Escherichia coli (EPEC and EHEC), two members of the SlyA family of transcriptional regulators have been identified as SlyA. Western blot analysis of the wild type and the corresponding hosA and slyA deletion mutants indicated that SlyA and HosA are distinct proteins whose expression is not interdependent. Of 27 different E. coli strains (EPEC, EHEC, enteroinvasive, enteroaggregative, uropathogenic, and commensal) examined, 14 were positive for both genes and proteins. To investigate hosA expression, a hosA::luxCDABE reporter gene fusion was constructed. hosA expression was significantly reduced in the hosA but not the slyA mutant and was influenced by temperature, salt, and pH. In contrast to SlyA, HosA did not activate the cryptic E. coli K-12 hemolysin ClyA. Mutation of hosA did not influence type III secretion, the regulation of the LEE1 and LEE4 operons, or the ability of E2348/69 to form attaching-and-effacing lesions on intestinal epithelial cells. HosA is, however, involved in the temperature-dependent positive control of motility on swim plates and regulates fliC expression and FliC protein levels. In electrophoretic mobility shift assays, purified HosA protein bound specifically to the fliC promoter, indicating that HosA directly modulates flagellin expression. While direct examination of flagellar structure and the motile behavior of individual hosA cells grown in broth culture at 30°C did not reveal any obvious differences, hosA mutants, unlike the wild type, clumped together, forming nonmotile aggregates which could account for the markedly reduced motility of the hosA mutant on swim plates at 30°C. We conclude that SlyA and HosA are independent transcriptional regulators that respond to different physicochemical cues to facilitate the environmental adaptation of E. coli.

scholarly journals Regulation of the Escherichia coli K5 Capsule Gene Cluster: Evidence for the Roles of H-NS, BipA, and Integration Host Factor in Regulation of Group 2 Capsule Gene Clusters in Pathogenic E. coli

2000 ◽  
Vol 182 (10) ◽  
pp. 2741-2745 ◽  
Author(s):  
Sonya Rowe ◽  
Nigel Hodson ◽  
Gary Griffiths ◽  
Ian S. Roberts
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Transcription Initiation ◽  
Gene Clusters ◽  
Integration Host Factor ◽  
Host Factor ◽  
Regulation Of Transcription ◽  
Global Regulators ◽  
E Coli ◽  
Group 2

ABSTRACT The expression of Escherichia coli group 2 capsules (K antigens) is temperature dependent, with capsules only being expressed at temperatures above 20°C. Thermoregulation is at the level of transcription, with no detectable transcription at 20°C. Using theE. coli K5 capsule gene cluster as a model system, we have shown that the nucleoid-associated protein H-NS plays a dual role in regulating transcription of group 2 capsule gene clusters at 37 and 20°C. At 37°C H-NS is required for maximal transcription of group 2 capsule gene clusters, whereas at 20°C H-NS functions to repress transcription. The BipA protein, previously identified as a tyrosine-phosphorylated GTPase and essential for virulence in enteropathogenic E. coli, was shown to play a similar role to H-NS in regulating transcription at 37 and 20°C. The binding of integration host factor (IHF) to the region 1 promoter was necessary to potentiate transcription at 37°C and IHF binding demonstrated by bandshift assays. The IHF binding site was 3′ to the site of transcription initiation, suggesting that sequences in the 5′ end of the first gene (kpsF) in region 1 may play a role in regulating transcription from this promoter at 37°C. Two additionalcis-acting sequences, conserved in both the region 1 and 3 promoters, were identified, suggesting a role for these sequences in the coordinate regulation of transcription from these promoters. These results indicate that a complex regulatory network involving a number of global regulators exists for the control of expression of group 2 capsules in E. coli.

scholarly journals Characterization of Stg Fimbriae from an Avian Pathogenic Escherichia coli O78:K80 Strain and Assessment of Their Contribution to Colonization of the Chicken Respiratory Tract

2006 ◽  
Vol 188 (18) ◽  
pp. 6449-6459 ◽  
Author(s):  
Maria H. Lymberopoulos ◽  
Sébastien Houle ◽  
France Daigle ◽  
Simon Léveillé ◽  
Annie Brée ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
Distinct Group ◽  
Immunogold Electron Microscopy ◽  
Carbohydrate Source ◽  
E Coli ◽  
Tract Infections ◽  
K 12

ABSTRACT In a previous study, ecs-3, a sequence from avian pathogenic Escherichia coli (APEC) O78:K80 strain χ7122, was found to be expressed in vivo in infected chicken tissues. The region encompassing ecs-3 carries a fimbrial gene cluster that is a putative ortholog of the stg fimbrial gene cluster of Salmonella enterica serovar Typhi. This APEC fimbrial gene cluster, which we have termed stg, is a member of a distinct group of related fimbriae that are located in the glmS-pstS intergenic region of certain E. coli and S. enterica strains. Under the control of the pBAD promoter, the production of Stg fimbriae was demonstrated by Western blotting and immunogold electron microscopy with E. coli K-12. Transcriptional fusions suggest that stg expression is influenced by the carbohydrate source and decreased by the addition of iron and that Fur plays a role in the regulation of stg expression. stg sequences were associated with APEC O78 isolates, and stg was phylogenetically distributed among E. coli reference strains and clinical isolates from human urinary tract infections. Stg fimbriae contributed to the adherence of a nonfimbriated E. coli K-12 strain to avian lung sections and human epithelial cells in vitro. Coinfection experiments with APEC strain χ7122 and an isogenic Δstg mutant demonstrated that compared to the wild-type parent, the Δstg mutant was less able to colonize air sacs, equally able to colonize lungs, and able to more effectively colonize tracheas of infected chickens. Stg fimbriae, together with other adhesins, may therefore contribute to the colonization of avian respiratory tissues by certain APEC strains.

scholarly journals Gene Products Required for De Novo Synthesis of Polysialic Acid in Escherichia coli K1

2006 ◽  
Vol 188 (5) ◽  
pp. 1786-1797 ◽  
Author(s):  
Ekaterina N. Andreishcheva ◽  
Willie F. Vann
Keyword(s):  
Escherichia Coli ◽  
Gene Cluster ◽  
De Novo ◽  
Polysialic Acid ◽  
Neonatal Meningitis ◽  
Gene Products ◽  
De Novo Synthesis ◽  
E Coli ◽  
Tract Infections

ABSTRACT Escherichia coli K1 is responsible for 80% of E. coli neonatal meningitis and is a common pathogen in urinary tract infections. Bacteria of this serotype are encapsulated with the α(2-8)-polysialic acid NeuNAc(α2-8), common to several bacterial pathogens. The gene cluster encoding the pathway for synthesis of this polymer is organized into three regions: (i) kpsSCUDEF, (ii) neuDBACES, and (iii) kpsMT. The K1 polysialyltransferase, NeuS, cannot synthesize polysialic acid de novo without other products of the gene cluster. Membranes isolated from strains having the entire K1 gene cluster can synthesize polysialic acid de novo. We designed a series of plasmid constructs containing fragments of regions 1 and 2 in two compatible vectors to determine the minimum number of gene products required for de novo synthesis of the polysialic acid from CMP-NeuNAc in K1 E. coli. We measured the ability of the various combinations of region 1 and 2 fragments to restore polysialyltransferase activity in vitro in the absence of exogenously added polysaccharide acceptor. The products of region 2 genes neuDBACES alone were not sufficient to support de novo synthesis of polysialic acid in vitro. Only membrane fractions harboring NeuES and KpsCS could form sialic polymer in the absence of exogenous acceptor at the concentrations formed by wild-type E. coli K1 membranes. Membrane fractions harboring NeuES and KpsC together could form small quantities of the sialic polymer de novo.

scholarly journals Regulatory Interactions among Adhesin Gene Systems of Uropathogenic Escherichia coli

2007 ◽  
Vol 76 (2) ◽  
pp. 771-780 ◽  
Author(s):  
Stina Lindberg ◽  
Yan Xia ◽  
Berit Sondén ◽  
Mikael Göransson ◽  
Jörg Hacker ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Regulatory Protein ◽  
Mobility Shift ◽  
E Coli ◽  
Fimbrial Adhesins ◽  
Gel Mobility Shift

ABSTRACT Uropathogenic Escherichia coli strain J96 carries multiple determinants for fimbrial adhesins. The regulatory protein PapB of P fimbriae has previously been implicated in potential coregulatory events. The focB gene of the F1C fimbria determinant is highly homologous to papB; the translated sequences share 81% identity. In this study we investigated the role of PapB and FocB in regulation of the F1C fimbriae. By using gel mobility shift assays, we showed that FocB binds to sequences in both the pap and foc operons in a somewhat different manner than PapB. The results of both in vitro cross-linking and in vivo oligomerization tests indicated that FocB could function in an oligomeric fashion. Furthermore, our results suggest that PapB and FocB can form heterodimers and that these complexes can repress expression of the foc operon. The effect of FocB on expression of type 1 fimbriae was also tested. Taken together, the results that we present expand our knowledge about a regulatory network for different adhesin gene systems in uropathogenic E. coli and suggest a hierarchy for expression of the fimbrial adhesins.

scholarly journals Characterization of the Organic Hydroperoxide Resistance System of Brucella abortus 2308

2012 ◽  
Vol 194 (18) ◽  
pp. 5065-5072 ◽  
Author(s):  
Clayton C. Caswell ◽  
John E. Baumgartner ◽  
Daniel W. Martin ◽  
R. Martin Roop
Keyword(s):  
Brucella Abortus ◽  
Sequence Similarity ◽  
Mobility Shift ◽  
Organic Hydroperoxide ◽  
Content Type ◽  
Dnase I Footprinting ◽  
Organic Hydroperoxides ◽  
Electrophoretic Mobility Shift Assays

ABSTRACTThe organic hydroperoxide resistance protein Ohr has been identified in numerous bacteria where it functions in the detoxification of organic hydroperoxides, and expression ofohris often regulated by a MarR-type regulator called OhrR. The genes annotated as BAB2_0350 and BAB2_0351 in theBrucella abortus2308 genome sequence are predicted to encode OhrR and Ohr orthologs, respectively. Using isogenicohrandohrRmutants andlacZpromoter fusions, it was determined that Ohr contributes to resistance to organic hydroperoxide, but not hydrogen peroxide, inB. abortus2308 and that OhrR represses the transcription of bothohrandohrRin this strain. Moreover, electrophoretic mobility shift assays and DNase I footprinting revealed that OhrR binds directly to a specific region in the intergenic region betweenohrandohrRthat shares extensive nucleotide sequence similarity with so-called “OhrR boxes” described in other bacteria. While Ohr plays a prominent role in protectingB. abortus2308 from organic hydroperoxide stress inin vitroassays, this protein is not required for the wild-type virulence of this strain in cultured murine macrophages or experimentally infected mice.

scholarly journals New Insight into the Molecular Mechanism of the FUT2 Regulating Escherichia coli F18 Resistance in Weaned Piglets

2018 ◽  
Vol 19 (11) ◽  
pp. 3301 ◽  
Author(s):  
Zhengchang Wu ◽  
Haiyue Feng ◽  
Yue Cao ◽  
Yanjie Huang ◽  
Chaohui Dai ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Mechanism ◽  
Function Analysis ◽  
Differentially Expressed Gene ◽  
Mrna Levels ◽  
Weaned Piglets ◽  
Mobility Shift ◽  
E Coli ◽  
Promoter Dna ◽  
Electrophoretic Mobility Shift Assays

Escherichia coli (E. coli) F18 is the main pathogen responsible for post-weaning diarrhea (PWD) in piglets. Resistance to E. coli F18 depends on the expression of the cognate receptors in the intestinal epithelial cells. However, the molecular mechanism of E. coli F18 resistance in weaned piglets remains unclear. Here, we performed a comparative transcriptome study of the duodenal tissue from Sutai E. coli F18 sensitive and resistant pigs by RNA-seq, and pig α(1,2) fucosyltransferase 2 (FUT2) was identified as a host differentially expressed gene controlling the E. coli F18 infection. Function analysis showed that the FUT2 expression was high in the duodenum and jejunum, with higher levels detected in sensitive individuals than in resistant individuals (p < 0.01). Expression levels of FUT2 were upregulated in IPEC-J2 cells after lipopolysaccharide (LPS)-induction or E. coli stimulation. FUT2 knockdown decreased the adhesion of E. coli F18 to IPEC-J2 cells (p < 0.05). FUT2 overexpression markedly increased the adhesion of E. coli F18 to IPEC-J2 cells (p < 0.05 or p < 0.01). Furthermore, the FUT2 mRNA levels correlated with methylation levels of the mC-22 site in the specificity protein 1 (Sp1) transcription factor (p < 0.05). Electrophoretic mobility shift assays (EMSA) showed that Sp1 interacts with the wild-type FUT2 promoter DNA, but not with methylated DNA. Our data suggested that FUT2 methylation at the mC-22 site inhibits Sp1 binding to the FUT2 promoter, thereby reducing FUT2 expression and enhancing E. coli F18 resistance in weaned piglets. These observations highlight FUT2 as a promising new target for combating E. coli F18 susceptibility in weaned piglets.

scholarly journals Kinetics of Polynucleotide Phosphorylase: Comparison of Enzymes from Streptomyces and Escherichia coli and Effects of Nucleoside Diphosphates

2007 ◽  
Vol 190 (1) ◽  
pp. 98-106 ◽  
Author(s):  
Samantha A. Chang ◽  
Madeline Cozad ◽  
George A. Mackie ◽  
George H. Jones
Keyword(s):  
Escherichia Coli ◽  
Streptomyces Coelicolor ◽  
Loop Structure ◽  
Polynucleotide Phosphorylase ◽  
Mobility Shift ◽  
Stem Loop ◽  
Streptomyces Antibioticus ◽  
E Coli ◽  
Stem Loop Structure ◽  
Electrophoretic Mobility Shift Assays

ABSTRACT We examined the activity of polynucleotide phosphorylase (PNPase) from Streptomyces coelicolor, Streptomyces antibioticus, and Escherichia coli in phosphorolysis using substrates derived from the rpsO-pnp operon of S. coelicolor. The Streptomyces and E. coli enzymes were both able to digest a substrate with a 3′ single-stranded tail although E. coli PNPase was more effective in digesting this substrate than were the Streptomyces enzymes. The k cat for the E. coli enzyme was ca. twofold higher than that observed with the S. coelicolor enzyme. S. coelicolor PNPase was more effective than its E. coli counterpart in digesting a substrate possessing a 3′ stem-loop structure, and the Km for the E. coli enzyme was ca. twice that of the S. coelicolor enzyme. Electrophoretic mobility shift assays revealed an increased affinity of S. coelicolor PNPase for the substrate possessing a 3′ stem-loop structure compared with the E. coli enzyme. We observed an effect of nucleoside diphosphates on the activity of the S. coelicolor PNPase but not the E. coli enzyme. In the presence of a mixture of 20 μM ADP, CDP, GDP, and UDP, the Km for the phosphorolysis of the substrate with the 3′ stem-loop was some fivefold lower than the value observed in the absence of nucleoside diphosphates. No effect of nucleoside diphosphates on the phosphorolytic activity of E. coli PNPase was observed. To our knowledge, this is the first demonstration of an effect of nucleoside diphosphates, the normal substrates for polymerization by PNPase, on the phosphorolytic activity of that enzyme.

scholarly journals PglB function and glycosylation efficiency is temperature dependent when the pgl locus is integrated in the Escherichia coli chromosome

2022 ◽  
Vol 21 (1) ◽  
Author(s):  
Vanessa S. Terra ◽  
Marta Mauri ◽  
Thippeswamy H. Sannasiddappa ◽  
Alexander A. Smith ◽  
Mark P. Stevens ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rapid Identification ◽  
Effect Of Temperature ◽  
Limiting Factor ◽  
Transcriptional Level ◽  
Coding Region ◽  
Temperature Dependent ◽  
Live Attenuated Vaccine ◽  
E Coli

Abstract Background Campylobacter is an animal and zoonotic pathogen of global importance, and a pressing need exists for effective vaccines, including those that make use of conserved polysaccharide antigens. To this end, we adapted Protein Glycan Coupling Technology (PGCT) to develop a versatile Escherichia coli strain capable of generating multiple glycoconjugate vaccine candidates against Campylobacter jejuni. Results We generated a glycoengineering E. coli strain containing the conserved C. jejuni heptasaccharide coding region integrated in its chromosome as a model glycan. This methodology confers three advantages: (i) reduction of plasmids and antibiotic markers used for PGCT, (ii) swift generation of many glycan-protein combinations and consequent rapid identification of the most antigenic proteins or peptides, and (iii) increased genetic stability of the polysaccharide coding-region. In this study, by using the model glycan expressing strain, we were able to test proteins from C. jejuni, Pseudomonas aeruginosa (both Gram-negative), and Clostridium perfringens (Gram-positive) as acceptors. Using this pgl integrant E. coli strain, four glycoconjugates were readily generated. Two glycoconjugates, where both protein and glycan are from C. jejuni (double-hit vaccines), and two glycoconjugates, where the glycan antigen is conjugated to a detoxified toxin from a different pathogen (single-hit vaccines). Because the downstream application of Live Attenuated Vaccine Strains (LAVS) against C. jejuni is to be used in poultry, which have a higher body temperature of 42 °C, we investigated the effect of temperature on protein expression and glycosylation in the E. coli pgl integrant strain. Conclusions We determined that glycosylation is temperature dependent and that for the combination of heptasaccharide and carriers used in this study, the level of PglB available for glycosylation is a step limiting factor in the glycosylation reaction. We also demonstrated that temperature affects the ability of PglB to glycosylate its substrates in an in vitro glycosylation assay independent of its transcriptional level.

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