SspA up-regulates gene expression of the LEE pathogenicity island by decreasing H-NS levels in enterohemorrhagic Escherichia coli
© Hansen and Jin; licensee BioMed Central Ltd. 2012
Received: 25 July 2012
Accepted: 9 October 2012
Published: 11 October 2012
Enterohemorrhagic Escherichia coli (EHEC) colonizes the intestinal epithelium and causes attaching and effacing (A/E) lesions. Expression of virulence genes, particularly those from the locus of the enterocyte effacement (LEE) pathogenicity island is required for the formation of a type three secretion system, which induces A/E lesion formation. Like other horizontally acquired genetic elements, expression of the LEE is negatively regulated by H-NS. In the non-pathogenic Escherichia coli K-12 strain the stringent starvation protein A (SspA) inhibits accumulation of H-NS, and thereby allows de-repression of the H-NS regulon during the stationary phase of growth. However, the effect of SspA on the expression of H-NS-controlled virulence genes in EHEC is unknown.
Here we assess the effect of SspA on virulence gene expression in EHEC. We show that transcription of virulence genes including those of the LEE is decreased in an sspA mutant, rendering the mutant strain defective in forming A/E lesions. A surface exposed pocket of SspA is functionally important for the regulation of the LEE and for the A/E phenotype. Increased expression of ler alleviates LEE expression in an sspA mutant, suggesting that the level of Ler in the mutant is insufficient to counteract H-NS-mediated repression. We demonstrate that the H-NS level is two-fold higher in an sspA mutant compared to wild type, and that the defects of the sspA mutant are suppressed by an hns null mutation, indicating that hns is epistatic to sspA in regulating H-NS repressed virulence genes.
SspA positively regulates the expression of EHEC virulence factors by restricting the intracellular level of H-NS. Since SspA is conserved in many bacterial pathogens containing horizontally acquired pathogenicity islands controlled by H-NS, our study suggests a common mechanism whereby SspA potentially regulates the expression of virulence genes in these pathogens.
KeywordsEnterohemorrhagic Escherichia coli LEE SspA H-NS
Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is an emerging food- and waterborne- enteric pathogen causing diarrhea, hemorrhagic colitis and the potentially fatal complication hemolytic uremic syndrome in humans [1, 2]. EHEC colonization of enterocytes of the large bowel is characterized by an intestinal attaching and effacing (A/E) histopathology, which is manifested by a localized degeneration of brush border microvilli and an intimate attachment of bacteria to actin-rich pedestal-like structures formed on the apical membrane directly beneath adherent bacteria . The A/E lesion is due to the activity of a type III secretion system (T3SS) mainly encoded by the 35–45 kb locus of enterocyte effacement pathogenicity island (hereafter named LEE), which is conserved in some EHEC isolates and other A/E pathogens such as enteropathogenic Escherichia coli (EPEC), atypical EPEC, rabbit EPEC, Escherichia albertii and Citrobacter rodentium[4–7]. The LEE pathogenicity island comprises at least 41 genes that mainly are located in five major operons (LEE1 5). The LEE encodes a TTSS, translocator proteins, secreted effectors, regulators, an intimin (adhesin) and a translocated intimin receptor. The LEE-encoded regulators Ler, Mpc, GrlR and GrlA are required for proper transcriptional regulation of both LEE- and non-LEE-encoded virulence genes in response to environmental cues [8–12].
The LEE was acquired by horizontal gene transfer  and is regulated by both generic E. coli- and pathogen-specific transcription factors. Consequently, the regulation of the LEE reflects characteristics of such genetic elements (For review see [11, 14]). Silencing of xenogeneic DNA in bacterial pathogens under conditions unfavorable for infection is important to ensure bacterial fitness . H-NS, which is an abundant pleiotropic negative modulator of genes involved in environmental adaptation and virulence [16–20], is a major silencing factor of horizontally acquired genes [21, 22]. H-NS silences genes in the H-NS regulon by various mechanisms. Binding of H-NS to regulatory regions of these genes prevents RNA polymerase from accessing and escaping from promoter DNA, which represents two different mechanisms used by H-NS to silence gene expression (see [23–25] and references therein). H-NS is also a major transcriptional modulator of the LEE pathogenicity island, where it negatively affects the expression of LEE1-5, map and grlRA[26–31]. Further, H-NS binds to regulatory sequences upstream of virulence-associated genes located outside of the LEE including those encoding the long polar fimbriae (lpf) required for intestine cell adherence and enterohemolysin (ehx) [32, 33].
The expression of EHEC virulence genes including those encoded by the LEE is derepressed from the H-NS-mediated transcriptional silencing under physiological conditions that EHEC encounters during infection. Also, LEE expression is growth phase-dependent with maximum expression in early stationary phase . H-NS-mediated silencing of transcription is overcome by the action of DNA-binding H-NS paralogues such as the LEE1-encoded global transcriptional regulator Ler (For review see ). Ler promotes the expression of many H-NS-repressed virulence genes including those of LEE1-5, grlRA and non-LEE-encoded virulence genes such as lpf and the virulence plasmid pO157-encoded mucinase stcE[26, 28, 31, 36–39]. Thus, Ler antagonizes H-NS in the regulation of many virulence genes, which belong to both the H-NS and Ler (H-NS/Ler) regulons.
The E. coli stringent starvation protein A (SspA) is a RNA polymerase-associated protein  that is required for transcriptional activation of bacteriophage P1 late genes and is important for survival of E. coli K-12 during nutrient depletion and prolonged stationary phase [41–43]. Importantly, SspA down-regulates the cellular H-NS level during stationary phase, and thereby derepress the H-NS regulon including genes for stationary phase induced acid tolerance in E. coli K-12 . A conserved surface-exposed pocket of SspA is important for its activity as a triple alanine substitution P84A/H85A/P86A in surface pocket residues abolishes SspA activity . SspA is highly conserved among Gram-negative pathogens , which suggests a role of SspA in bacterial pathogenesis. Indeed, SspA orthologs affect the virulence of Yersinia enterocolitica, Neisseria gonorrhoeae, Vibrio cholerae, Francisella tularensis and Francisella novicida[46–51]. Since E. coli K-12 SspA is conserved in EHEC where H-NS negatively modulates virulence gene expression, we asked the question of whether SspA-mediated regulation of H-NS affects EHEC virulence gene expression. Here we study the effect of SspA on the expression of LEE- and non-LEE-encoded virulence genes and its effect on H-NS accumulation in EHEC. Our results show that in an sspA mutant elevated levels of H-NS repress the expression of virulence genes encoding the T3SS system rendering the cells incapable of forming A/E lesions. Thus, our data indicate that SspA positively regulates stationary phase-induced expression of H-NS-controlled virulence genes in EHEC by restricting the H-NS level.
Results and discussion
SspA positively affects transcription of EHEC virulence genes
Increased expression of ler enhances expression of virulence genes in the sspAmutant
SspA activates virulence gene expression by reducing the H-NS level
SspA is required for cell adherence and A/E lesion formation
The correlation between the effects of sspA on the transcription of H-NS/Ler-regulated virulence genes and on A/E lesion formation upon infection of HEp-2 cells supports the conclusion that SspA upregulates the expression of LEE and other virulence genes by reducing the accumulation of H-NS in the cell. A reduced cellular H-NS level mediated by SspA will derepress the H-NS regulon and thereby allow the expression of transcriptional activators such as Ler and GrlA. These two activators then form a positive transcriptional regulatory loop partially by preventing H-NS-mediated repression . Accumulation of Ler will in turn antagonize H-NS function and with that enhance the expression of virulence genes controlled by Ler . At present, the molecular mechanism behind SspA-mediated regulation of the H-NS level during stationary phase and in infection to facilitate virulence gene expression in EHEC is unknown. Also, it remains to be determined whether SspA directly affects transcription of virulence genes as is the case for SspA in Francisella tularensis, where SspA along with two other transcription factors and ppGpp activates transcription to link the nutritional status to virulence gene expression [56, 57].
We observed that SspA positively affects additional H-NS-controlled virulence traits of EHEC such as stationary phase-induced acid tolerance (data not shown), which enables survival of the pathogen during passage through the low pH environment of the human gastrointestinal tract, and thereby contributes to a low infectious dose [58, 59]. Also, sspA positively affects EHEC motility (data not shown), which could influence virulence as motility enables the pathogen to penetrate the intestinal mucus layer during colonization of host cells. This further supports an important role of sspA in EHEC virulence. Further experiments studying wild type and sspA mutant derivatives of the A/E pathogen Citrobacter rodentium in a mouse model could help determine whether sspA is required for virulence in vivo.
We established an important role of SspA in the regulation of LEE- and non-LEE-encoded virulence factors of a T3SS, which is important for A/E lesion formation by EHEC. SspA downregulates H-NS levels allowing the expression of EHEC virulence genes, which are part of the H-NS/Ler regulon. Virulence genes in many bacteria are horizontally acquired genetic elements and subject to repression by H-NS. Thus, our study indicates that SspA potentially plays an important role in the pathogenicity of many bacterial pathogens in general.
Standard DNA techniques, agar plates and liquid media were used as described . Restriction endonucleases, T4 DNA polynucleotide kinase- and ligase (New England Biolabs) and the Expand High Fidelity PCR System (Roche Applied Sciences) were used according to manufacturer’s instructions. DNA sequencing was performed by the National Cancer Institute DNA Sequencing MiniCore facility. Bacteria were grown at 37°C in LB or DMEM (Invitrogen #11885) media supplemented with ampicillin (100μg/ml), chloramphenicol (25 μg/ml) or kanamycin (25 μg/ml) as needed. HEp-2 cells (ATTC # CCL-23) were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin and 100 μg/ml streptomycin at 37°C in 5% CO2.
Strain and plasmid constructions
Oligonucleotide primers used in this study
Oligonucleotide sequence (5’ to 3’)
DMEM is known to enhance the expression of the T3SS, which was detrimental to growth of the hns mutant EHEC derivatives (data not shown) that already exhibit increased T3SS expression in the absence of H-NS-mediated repression. Therefore, virulence gene expression was monitored in cells grown in LB, where a mid-level expression of the T3SS occurs. Overnight cultures of wild type and mutant derivatives of EDL933 ATCC 700927 were diluted 1:1000 in LB, supplemented with antibiotics if necessary, and grown aerobically at 37°C to an optical density at 600 nm (OD600) of ~ 3.0 (stationary phase). Samples of the cultures corresponding to ~7.5 × 109 cells were collected and RNA was stabilized immediately by addition of RNAprotect bacteria reagent according to manufacturer’s protocol (QIAGEN). Total RNA was purified using MasterPure™ total RNA purification kit as recommended by the manufacturer (Epicentre). Contaminating DNA in the RNA preparations was removed by DNaseI treatment. Isolated RNA was quantified based on measurements of absorption at 260 nm. The quality of RNA was evaluated by determining the ratio of absorption at 260 nm and 280 nm, which was within the preferred range of 1.7 to 2.1, and by agarose gel electrophoresis.
Primer extension analysis
Primer extension reactions were carried out on 8 μg of total RNA using the AMV reverse transcriptase primer extension system according to manufacturer’s instructions (Promega). The 32P-labeled DNA oligos (1 pmol) were used to detect the transcripts of interest: lerPE (LEE1/ler), LEE2PE (LEE2/espZ), LEE3PE (LEE3/mpc), LEE4PE (LEE4/sepL), LEE5PE (LEE5/tir), mapPE (map), grlRPE (grlRA) and stcEPE (stcE). The 32P-labeled DNA oligo ompAPE was used as an internal control in each extension reaction to detect the transcripts from ompA P1 and P2. The DNA oligos were 5’-end labeled with (γ-32P) ATP (GE Healthcare) using T4 polynucleotide kinase (New England Biolabs). The cDNA products were separated on a 6 % denaturating gel along with a labeled φX174 hinfI DNA size marker (Promega) and visualized by autoradiography. The lengths of the cDNA transcripts ler, espZ, mpc, sepL, tir, map, grlRA, stcE, ompA P1, and ompA P2 were around 250, 120, 147, 122, 142, 135, 162, 125, 89 and 93 nt, respectively. No DNA product was detected in the absence of RNA. Transcript levels were quantified using ImageJ software  and normalized to ompA transcript levels. The primer extension experiments were carried out at least twice and similar results were obtained.
Total protein was prepared from cultures grown in LB at 37°C to OD600 ~ 3.0. Samples containing equal amounts of total protein equivalent to 0.03 OD600 units of cell culture were prepared and analyzed essentially as previously described . Polyclonal antibodies against H-NS or Fis were used to detect the respective proteins. The western blots were developed using ECL plus reagents (GE Healthcare) and quantified with a FluorChem imaging system (Alpha Innotech). The western analysis was carried out at least twice, and similar results were obtained.
Assay for the presence of A/E lesions on HEp-2 cells
The ability of EHEC EDL933 (ATCC 700927) wild type and its mutant derivatives to adhere and form A/E lesions on HEp-2 cell monolayers was evaluated using the fluorescent actin staining assay as described . Bacterial cells were grown without aeration for 16–18 h at 37°C in tryptic soy broth that was supplemented with antibiotics if needed. Prior to infection cells were diluted 1:5 in infection medium (DMEM supplemented with 2% FBS and 0.5% mannose) and incubated at 37°C 5% CO2 for 2 h. About 2 × 106 bacteria (M.O.I. ~ 10) in 100 μl were added to semi-confluent HEp-2 cell monolayers grown on glass coverslips in a 6-well plate (Multiwell™ Falcon #353046). After infection for 4–5 h, monolayers were fixed with 4% formamide in PBS, washed three times with PBS, permeabilized with 0.1% Triton X-100 in PBS, and then stained with Alexa Fluor 488 phalloidin (Invitrogen). Coverslips were mounted on slides using Prolong Gold antifade reagent (Invitrogen) and the edges of the coverslip were sealed with cytoseal-60 (Richard-Allan Scientific). The samples were visualized using a Zeiss Axiophot II microscope equipped with a 40X objective, epifluorescence filters and a 1.25 optovar (Carl Zeiss MicroImaging Inc.). Images were captured with a charge-coupled device camera (Micromax) using IPL lab software. For each bacterial strain the assay was carried out independently at least three times and at least 50 HEp-2 cells were visually examined.
We thank Darren Sledjeski for the antiserum against H-NS. We also thank lab members for interaction and discussion during the course of the study. This work was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.
- Nataro JP, Kaper JB: Diarrheagenic Escherichia coli. Clin Microbiol Rev. 1998, 11: 142-201.PubMedPubMed Central
- Karmali MA: Infection by Shiga toxin-producing Escherichia coli: an overview. Mol Biotechnol. 2004, 26: 117-122. 10.1385/MB:26:2:117.PubMedView Article
- Frankel G, Phillips AD, Rosenshine I, Dougan G, Kaper JB, Knutton S: Enteropathogenic and enterohaemorrhagic Escherichia coli: more subversive elements. Mol Microbiol. 1998, 30: 911-921. 10.1046/j.1365-2958.1998.01144.x.PubMedView Article
- Jerse AE, Yu J, Tall BD, Kaper JB: A genetic locus of enteropathogenic Escherichia coli necessary for the production of attaching and effacing lesions on tissue culture cells. Proc Natl Acad Sci USA. 1990, 87: 7839-7843. 10.1073/pnas.87.20.7839.PubMedPubMed CentralView Article
- McDaniel TK, Jarvis KG, Donnenberg MS, Kaper JB: A genetic locus of enterocyte effacement conserved among diverse enterobacterial pathogens. Proc Natl Acad Sci USA. 1995, 92: 1664-1668. 10.1073/pnas.92.5.1664.PubMedPubMed CentralView Article
- Huys G, Cnockaert M, Janda JM, Swings J: Escherichia albertii sp. nov., a diarrhoeagenic species isolated from stool specimens of Bangladeshi children. Int J Syst Evol Microbiol. 2003, 53: 807-810. 10.1099/ijs.0.02475-0.PubMedView Article
- Rasko DA, Rosovitz MJ, Myers GS, Mongodin EF, Fricke WF, Gajer P, Crabtree J, Sebaihia M, Thomson NR, Chaudhuri R, et al: The pangenome structure of Escherichia coli: comparative genomic analysis of E. coli commensal and pathogenic isolates. J Bacteriol. 2008, 190: 6881-6893. 10.1128/JB.00619-08.PubMedPubMed CentralView Article
- Jarvis KG, Giron JA, Jerse AE, McDaniel TK, Donnenberg MS, Kaper JB: Enteropathogenic Escherichia coli contains a putative type III secretion system necessary for the export of proteins involved in attaching and effacing lesion formation. Proc Natl Acad Sci USA. 1995, 92: 7996-8000. 10.1073/pnas.92.17.7996.PubMedPubMed CentralView Article
- Elliott SJ, Wainwright LA, McDaniel TK, Jarvis KG, Deng YK, Lai LC, McNamara BP, Donnenberg MS, Kaper JB: The complete sequence of the locus of enterocyte effacement (LEE) from enteropathogenic Escherichia coli E2348/69. Mol Microbiol. 1998, 28: 1-4.PubMedView Article
- Garmendia J, Frankel G, Crepin VF: Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation. Infect Immun. 2005, 73: 2573-2585. 10.1128/IAI.73.5.2573-2585.2005.PubMedPubMed CentralView Article
- Mellies JL, Barron AM, Carmona AM: Enteropathogenic and enterohemorrhagic Escherichia coli virulence gene regulation. Infect Immun. 2007, 75: 4199-4210. 10.1128/IAI.01927-06.PubMedPubMed CentralView Article
- Tsai NP, Wu YC, Chen JW, Wu CF, Tzeng CM, Syu WJ: Multiple functions of l0036 in the regulation of the pathogenicity island of enterohaemorrhagic Escherichia coli O157:H7. Biochem J. 2006, 393: 591-599. 10.1042/BJ20051201.PubMedPubMed CentralView Article
- Perna NT, Mayhew GF, Posfai G, Elliott S, Donnenberg MS, Kaper JB, Blattner FR: Molecular evolution of a pathogenicity island from enterohemorrhagic Escherichia coli O157:H7. Infect Immun. 1998, 66: 3810-3817.PubMedPubMed Central
- Kaper JB, Nataro JP, Mobley HL: Pathogenic Escherichia coli. Nat Rev Microbiol. 2004, 2: 123-140. 10.1038/nrmicro818.PubMedView Article
- Navarre WW, McClelland M, Libby SJ, Fang FC: Silencing of xenogeneic DNA by H-NS-facilitation of lateral gene transfer in bacteria by a defense system that recognizes foreign DNA. Genes Dev. 2007, 21: 1456-1471. 10.1101/gad.1543107.PubMedView Article
- Atlung T, Ingmer H: H-NS: a modulator of environmentally regulated gene expression. Mol Microbiol. 1997, 24: 7-17. 10.1046/j.1365-2958.1997.3151679.x.PubMedView Article
- Falconi M, Colonna B, Prosseda G, Micheli G, Gualerzi CO: Thermoregulation of Shigella and Escherichia coli EIEC pathogenicity. A temperature-dependent structural transition of DNA modulates accessibility of virF promoter to transcriptional repressor H-NS. EMBO J. 1998, 17: 7033-7043. 10.1093/emboj/17.23.7033.PubMedPubMed CentralView Article
- Rowe S, Hodson N, Griffiths G, Roberts IS: 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. J Bacteriol. 2000, 182: 2741-2745. 10.1128/JB.182.10.2741-2745.2000.PubMedPubMed CentralView Article
- Muller CM, Dobrindt U, Nagy G, Emody L, Uhlin BE, Hacker J: Role of histone-like proteins H-NS and StpA in expression of virulence determinants of uropathogenic Escherichia coli. J Bacteriol. 2006, 188: 5428-5438. 10.1128/JB.01956-05.PubMedPubMed CentralView Article
- Erol I, Jeong KC, Baumler DJ, Vykhodets B, Choi SH, Kaspar CW: H-NS controls metabolism and stress tolerance in Escherichia coli O157:H7 that influence mouse passage. BMC Microbiol. 2006, 6: 72-10.1186/1471-2180-6-72.PubMedPubMed CentralView Article
- Navarre WW, Porwollik S, Wang Y, McClelland M, Rosen H, Libby SJ, Fang FC: Selective silencing of foreign DNA with low GC content by the H-NS protein in Salmonella. Science. 2006, 313: 236-238. 10.1126/science.1128794.PubMedView Article
- Lucchini S, Rowley G, Goldberg MD, Hurd D, Harrison M, Hinton JC: H-NS mediates the silencing of laterally acquired genes in bacteria. PLoS Pathog. 2006, 2: e81-10.1371/journal.ppat.0020081.PubMedPubMed CentralView Article
- Fang FC, Rimsky S: New insights into transcriptional regulation by H-NS. Curr Opin Microbiol. 2008, 11: 113-120. 10.1016/j.mib.2008.02.011.PubMedPubMed CentralView Article
- Ali SS, Xia B, Liu J, Navarre WW: Silencing of foreign DNA in bacteria. Curr Opin Microbiol. 2012, 15: 175-181. 10.1016/j.mib.2011.12.014.PubMedView Article
- Dorman CJ: H-NS: a universal regulator for a dynamic genome. Nat Rev Microbiol. 2004, 2: 391-400. 10.1038/nrmicro883.PubMedView Article
- Bustamante VH, Santana FJ, Calva E, Puente JL: Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression. Mol Microbiol. 2001, 39: 664-678. 10.1046/j.1365-2958.2001.02209.x.PubMedView Article
- Haack KR, Robinson CL, Miller KJ, Fowlkes JW, Mellies JL: Interaction of Ler at the LEE5 (tir) operon of enteropathogenic Escherichia coli. Infect Immun. 2003, 71: 384-392. 10.1128/IAI.71.1.384-392.2003.PubMedPubMed CentralView Article
- Barba J, Bustamante VH, Flores-Valdez MA, Deng W, Finlay BB, Puente JL: A positive regulatory loop controls expression of the locus of enterocyte effacement-encoded regulators Ler and GrlA. J Bacteriol. 2005, 187: 7918-7930. 10.1128/JB.187.23.7918-7930.2005.PubMedPubMed CentralView Article
- Umanski T, Rosenshine I, Friedberg D: Thermoregulated expression of virulence genes in enteropathogenic Escherichia coli. Microbiology. 2002, 148: 2735-2744.PubMedView Article
- Laaberki MH, Janabi N, Oswald E, Repoila F: Concert of regulators to switch on LEE expression in enterohemorrhagic Escherichia coli O157:H7: interplay between Ler, GrlA, HNS and RpoS. Int J Med Microbiol. 2006, 296: 197-210. 10.1016/j.ijmm.2006.02.017.PubMedView Article
- Sanchez-SanMartin C, Bustamante VH, Calva E, Puente JL: Transcriptional regulation of the orf19 gene and the tir-cesT-eae operon of enteropathogenic Escherichia coli. J Bacteriol. 2001, 183: 2823-2833. 10.1128/JB.183.9.2823-2833.2001.PubMedPubMed CentralView Article
- Torres AG, Slater TM, Patel SD, Popov VL, renas-Hernandez MM: Contribution of the Ler- and H-NS-regulated long polar fimbriae of Escherichia coli O157:H7 during binding to tissue-cultured cells. Infect Immun. 2008, 76: 5062-5071. 10.1128/IAI.00654-08.PubMedPubMed CentralView Article
- Rogers MT, Zimmerman R, Scott ME: Histone-like nucleoid-structuring protein represses transcription of the ehx operon carried by locus of enterocyte effacement-negative Shiga toxin-expressing Escherichia coli. Microb Pathog. 2009, 47: 202-211. 10.1016/j.micpath.2009.07.003.PubMedView Article
- Roe AJ, Yull H, Naylor SW, Woodward MJ, Smith DG, Gally DL: Heterogeneous surface expression of EspA translocon filaments by Escherichia coli O157:H7 is controlled at the posttranscriptional level. Infect Immun. 2003, 71: 5900-5909. 10.1128/IAI.71.10.5900-5909.2003.PubMedPubMed CentralView Article
- Stoebel DM, Free A, Dorman CJ: Anti-silencing: overcoming H-NS-mediated repression of transcription in Gram-negative enteric bacteria. Microbiology. 2008, 154: 2533-2545. 10.1099/mic.0.2008/020693-0.PubMedView Article
- Mellies JL, Elliott SJ, Sperandio V, Donnenberg MS, Kaper JB: The Per regulon of enteropathogenic Escherichia coli: identification of a regulatory cascade and a novel transcriptional activator, the locus of enterocyte effacement (LEE)-encoded regulator (Ler). Mol Microbiol. 1999, 33: 296-306. 10.1046/j.1365-2958.1999.01473.x.PubMedView Article
- Elliott SJ, Sperandio V, Giron JA, Shin S, Mellies JL, Wainwright L, Hutcheson SW, McDaniel TK, Kaper JB: The locus of enterocyte effacement (LEE)-encoded regulator controls expression of both LEE- and non-LEE-encoded virulence factors in enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun. 2000, 68: 6115-6126. 10.1128/IAI.68.11.6115-6126.2000.PubMedPubMed CentralView Article
- Sperandio V, Mellies JL, Delahay RM, Frankel G, Crawford JA, Nguyen W, Kaper JB: Activation of enteropathogenic Escherichia coli (EPEC) LEE2 and LEE3 operons by Ler. Mol Microbiol. 2000, 38: 781-793. 10.1046/j.1365-2958.2000.02168.x.PubMedView Article
- Mellies JL, Larabee FJ, Zarr MA, Horback KL, Lorenzen E, Mavor D: Ler interdomain linker is essential for anti-silencing activity in enteropathogenic Escherichia coli. Microbiology. 2008, 154: 3624-3638. 10.1099/mic.0.2008/023382-0.PubMedPubMed CentralView Article
- Ishihama A, Saitoh T: Subunits of RNA polymerase in function and structure. IX. Regulation of RNA polymerase activity by stringent starvation protein (SSP). J Mol Biol. 1979, 129: 517-530. 10.1016/0022-2836(79)90466-2.PubMedView Article
- Williams MD, Fuchs JA, Flickinger MC: Null mutation in the stringent starvation protein of Escherichia coli disrupts lytic development of bacteriophage P1. Gene. 1991, 109: 21-30. 10.1016/0378-1119(91)90584-X.PubMedView Article
- Williams MD, Ouyang TX, Flickinger MC: Starvation-induced expression of SspA and SspB: the effects of a null mutation in sspA on Escherichia coli protein synthesis and survival during growth and prolonged starvation. Mol Microbiol. 1994, 11: 1029-1043. 10.1111/j.1365-2958.1994.tb00381.x.PubMedView Article
- Hansen AM, Lehnherr H, Wang X, Mobley V, Jin DJ: Escherichia coli SspA is a transcription activator for bacteriophage P1 late genes. Mol Microbiol. 2003, 48: 1621-1631. 10.1046/j.1365-2958.2003.03533.x.PubMedView Article
- Hansen AM, Qiu Y, Yeh N, Blattner FR, Durfee T, Jin DJ: SspA is required for acid resistance in stationary phase by downregulation of H-NS in Escherichia coli. Mol Microbiol. 2005, 56: 719-734. 10.1111/j.1365-2958.2005.04567.x.PubMedView Article
- Hansen AM, Gu Y, Li M, Andrykovitch M, Waugh DS, Jin DJ, Ji X: Structural basis for the function of stringent starvation protein a as a transcription factor. J Biol Chem. 2005, 280: 17380-17391. 10.1074/jbc.M501444200.PubMedView Article
- De Reuse H, Taha MK: RegF, an SspA homologue, regulates the expression of the Neisseria gonorrhoeae pilE gene. Res Microbiol. 1997, 148: 289-303. 10.1016/S0923-2508(97)81585-9.PubMedView Article
- Badger JL, Young BM, Darwin AJ, Miller VL: Yersinia enterocolitica ClpB affects levels of invasin and motility. J Bacteriol. 2000, 182: 5563-5571. 10.1128/JB.182.19.5563-5571.2000.PubMedPubMed CentralView Article
- Baron GS, Nano FE: MglA and MglB are required for the intramacrophage growth of Francisella novicida. Mol Microbiol. 1998, 29: 247-259. 10.1046/j.1365-2958.1998.00926.x.PubMedView Article
- Lauriano CM, Barker JR, Yoon SS, Nano FE, Arulanandam BP, Hassett DJ, Klose KE: MglA regulates transcription of virulence factors necessary for Francisella tularensis intraamoebae and intramacrophage survival. Proc Natl Acad Sci USA. 2004, 101: 4246-4249. 10.1073/pnas.0307690101.PubMedPubMed CentralView Article
- Merrell DS, Hava DL, Camilli A: Identification of novel factors involved in colonization and acid tolerance of Vibrio cholerae. Mol Microbiol. 2002, 43: 1471-1491. 10.1046/j.1365-2958.2002.02857.x.PubMedView Article
- Xu Q, Dziejman M, Mekalanos JJ: Determination of the transcriptome of Vibrio cholerae during intraintestinal growth and midexponential phase in vitro. Proc Natl Acad Sci USA. 2003, 100: 1286-1291. 10.1073/pnas.0337479100.PubMedPubMed CentralView Article
- Perna NT, Plunkett G, Burland V, Mau B, Glasner JD, Rose DJ, Mayhew GF, Evans PS, Gregor J, Kirkpatrick HA, et al: Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature. 2001, 409: 529-533. 10.1038/35054089.PubMedView Article
- Knutton S, Baldwin T, Williams PH, McNeish AS: Actin accumulation at sites of bacterial adhesion to tissue culture cells: basis of a new diagnostic test for enteropathogenic and enterohemorrhagic Escherichia coli. Infect Immun. 1989, 57: 1290-1298.PubMedPubMed Central
- Torres AG, Giron JA, Perna NT, Burland V, Blattner FR, velino-Flores F, Kaper JB: Identification and characterization of lpfABCC'DE, a fimbrial operon of enterohemorrhagic Escherichia coli O157:H7. Infect Immun. 2002, 70: 5416-5427. 10.1128/IAI.70.10.5416-5427.2002.PubMedPubMed CentralView Article
- Torres AG, Lopez-Sanchez GN, Milflores-Flores L, Patel SD, Rojas-Lopez M, Martinez de la Pena CF, renas-Hernandez MM, Martinez-Laguna Y: Ler and H-NS, regulators controlling expression of the long polar fimbriae of Escherichia coli O157:H7. J Bacteriol. 2007, 189: 5916-5928. 10.1128/JB.00245-07.PubMedPubMed CentralView Article
- Charity JC, Costante-Hamm MM, Balon EL, Boyd DH, Rubin EJ, Dove SL: Twin RNA polymerase-associated proteins control virulence gene expression in Francisella tularensis. PLoS Pathog. 2007, 3: e84-10.1371/journal.ppat.0030084.PubMedPubMed CentralView Article
- Charity JC, Blalock LT, Costante-Hamm MM, Kasper DL, Dove SL: Small molecule control of virulence gene expression in Francisella tularensis. PLoS Pathog. 2009, 5: e1000641-10.1371/journal.ppat.1000641.PubMedPubMed CentralView Article
- Peterson WL, Mackowiak PA, Barnett CC, Marling-Cason M, Haley ML: The human gastric bactericidal barrier: mechanisms of action, relative antibacterial activity, and dietary influences. J Infect Dis. 1989, 159: 979-983. 10.1093/infdis/159.5.979.PubMedView Article
- Lin J, Lee IS, Frey J, Slonczewski JL, Foster JW: Comparative analysis of extreme acid survival in Salmonella typhimurium, Shigella flexneri, and Escherichia coli. J Bacteriol. 1995, 177: 4097-4104.PubMedPubMed Central
- Sambrook J, Russell DW: Molecular cloning: a laboratory manual. 2001, Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory
- Datsenko KA, Wanner BL: One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. Proc Natl Acad Sci USA. 2000, 97: 6640-6645. 10.1073/pnas.120163297.PubMedPubMed CentralView Article
- Abramoff MD, Magalhaes PJ, Ram SJ: Image Processing with ImageJ. Biophoton Int. 2004, 11 (7): 36-42.
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