The non-pathogenic Escherichia coli strain W secretes SslE via the virulence-associated type II secretion system beta
© DeCanio et al.; licensee BioMed Central Ltd. 2013
Received: 11 March 2013
Accepted: 4 June 2013
Published: 12 June 2013
Many pathogenic E. coli strains secrete virulence factors using type II secretory systems, homologs of which are widespread in Gram-negative bacteria. Recently, the enteropathogenic Escherichia coli strain E2348/69 was shown to secrete and surface-anchor SslE, a biofilm-promoting virulence factor, via a type II secretion system. Genes encoding SslE and its associated secretion system are conserved in some non-pathogenic E. coli, including the commonly-used W (Waksman) strain.
We report here that E. coli W uses its type II secretion system to export a cognate SslE protein. SslE secretion is temperature- and nutrient-dependent, being robust at 37°C in rich medium but strongly repressed by lower temperatures or nutrient limitation. Fusing either of two glycosyl hydrolases to the C-terminus of SslE prevented it from being secreted or surface-exposed. We screened mutations that inactivated the type II secretion system for stress-related phenotypes and found that inactivation of the secretion system conferred a modest increase in tolerance to high concentrations of urea. Additionally, we note that the genes encoding this secretion system are present at a hypervariable locus and have been independently lost or gained in different lineages of E. coli.
The non-pathogenic E. coli W strain shares the extracellular virulence factor SslE, and its associated secretory system, with pathogenic E. coli strains. The pattern of regulation of SslE secretion we observed suggests that SslE plays a role in colonization of mammalian hosts by non-pathogenic as well as pathogenic E. coli. Our work provides a non-pathogenic model system for the study of SslE secretion, and informs future research into the function of SslE during host colonization.
KeywordsType II secretion Surface display Escherichia coli Colonization factor
Gram-negative bacteria use diverse type II secretion systems (T2SS) to deliver a wide variety of proteins into the extracellular milieu [1, 2]. Transport is effected by a membrane-spanning complex of 12–15 structural proteins, generically termed Gsp proteins (for general secretory pathway). Secreted substrates first cross the inner membrane by the Sec or Tat pathways; the Gsp proteins then recognize substrates and transport them across the outer membrane. T2SS function requires several proteins that have homologs in type IV pilus biogenesis systems, including an oligomerized secretin, a helical protein filament called the pseudopilus, and a prepilin peptidase essential for pseudopilus assembly [3, 4].
Secreted proteins serve many purposes, from electron transport to nutrient acquisition, and some are important pathogenicity factors for plant and animal pathogens in the Enterobacteraceae [5, 6]. Type II secretion has been extensively studied in pathogenic strains of Escherichia coli, which collectively are known to use two distinct disease-promoting T2SS: the StcE secreting system encoded by the pO157 virulence plasmid , and the heat-labile enterotoxin (LT) secreting system common to many pathogenic strains . Recently the latter T2SS was shown for the first time to additionally secrete a non-LT protein, known as SslE, from the enteropathogenic strain E2348/69, thereby promoting biofilm maturation and rabbit colonization by E2348/69 [9, 10]. The sslE gene sits immediately upstream of the T2SS-encoding secretory genes, and transcription of sslE and the gsp genes was shown to be co-regulated in E. coli strain H10407 . In E2348/69, SslE exists as a lipid-anchored, surface-exposed protein in the outer membrane and is also released into the culture supernatant. Strozen et al. termed the LT- and SslE-secreting system T2SSβ, to distinguish it from the chitinase-secreting T2SSα that co-occurs in several E. coli strains . Based on phylogenetic and structural analyses, Dunstan et al. recently determined that the E. coli T2SSβ is part of a larger group of T2SS that contain “Vibrio-type secretins”, making it a model for numerous type II secretion systems used to deliver toxic substrates by Vibrio and Escherichia species .
The SslE-secreting T2SSβ, unlike the StcE-secreting pO157 T2SS, is conserved in several non-pathogenic “safe” strains of E. coli (“safe” strains may colonize hosts, but have never been known to cause disease), including wild-type B and W isolates . To date, however, no report has described secretion of proteins by T2SSβ in any non-pathogenic strain. We were interested to determine whether non-pathogenic E. coli could also secrete the “virulence factor” SslE. Secretion of SslE by a safe strain would imply that SslE itself is not capable of promoting a disease state, and would invite comparisons of SslE function between pathogens and non-pathogens. Furthermore, if non-pathogenic E. coli could secrete SslE, the T2SSβ system could be studied using a non-pathogenic model organism.
We demonstrate here that the non-pathogenic E. coli strain W encodes a functional T2SSβ that secretes a cognate SslE protein. We found a strong effect of growth conditions on SslE secretion, which is relatively robust in rich medium at 37°C and undetectable when cells are cultured at 30°C or in minimal medium. Previous work suggested that the C-terminus of SslE might be a permissive site for sequence insertions with regards to T2SSβ recognition , but we found that C-terminal enzyme fusions to SslE blocked protein secretion and surface display.
As noted above, the T2SSβ was shown to promote mature biofilm formation in E. coli E2348/69. We searched for additional phenotypes in E. coli W by phenotypic microarray analysis of a mutant lacking T2SSβ-encoding genes on Biolog stress plates. The phenotypic microarray indicated a potential fitness effect of the mutation in high concentrations of urea. Using standard culture techniques, we found that deletion of T2SSβ-encoding genes, or the sslE gene, conferred a small survival advantage in medium containing high concentrations of urea.
Our findings make T2SSβ the only virulence-associated T2SS with shared functions in pathogenic and non-pathogenic E. coli. Considering our regulatory data and the clear homology between the T2SSβ-encoding operons of W and E2348/69, we propose that SslE is used by non-pathogenic as well as pathogenic strains of E. coli during host colonization.
E. coli W secretes SslE using T2SS β under specific temperature and nutrient conditions
Intracellular SslE did not appear abundant in wild-type E. coli W, even under conditions where secretion of SslE was detectable. We observed accumulation of SslE in the cell when SslE was expressed from a multicopy plasmid, however. We postulate that in wild-type cells, the intracellular concentration of SslE is maintained at a relatively low level, and that SslE release from cells over time results in accumulation in the supernatant.
Type II secretion systems require prepilin peptidases to produce the mature, functional forms of their prepilin proteins , and the prepilin peptidase PppA is required for secretion of LT by T2SSβ in E. coli H10407 . To determine whether PppA is similarly required for SslE secretion by E. coli W, we compared SslE secretion in WT to a ΔpppA strain. SslE secretion was not detectable in the ΔpppA background, and the mutation could be complemented by plasmid-encoded PppA (Figure 2C). These results confirm that a fully-functional T2SSβ is required to secrete SslE, and indicate that expression of the gspC-M genes alone is not sufficient to allow SslE secretion.
We hypothesized that SslE secretion in E. coli W might play a role in host colonization, and that secretion might be regulated such that more SslE is secreted under conditions that resemble the mammalian gut. We assessed this conditionality by examining SslE secretion from cultures grown at different temperatures and nutrient conditions: 30°C vs. 37°C, and minimal MOPS-glycerol broth vs. rich LB (Figure 2D). We observed secretion of SslE only in cultures grown in LB at 37°C, indicating that either reduced temperature or nutrient limitations are sufficient to block SslE secretion.
C-terminal fusions to SslE prevent secretion
In their initial characterization of SslE surface display and secretion, Baldi et al. found that C-terminal fusion of a small tetracysteine-containing motif to SslE did not interfere with localization of SslE . This result suggested that the C-terminus of SslE might not be important for the recognition of SslE by T2SSβ, and thus might be a permissive site for polypeptide fusions. We were interested in testing C-terminal permissiveness for two reasons: first, because it might provide information about the targeting of SslE for secretion (as there are no defined secretory signals for type II secretion substrates), and second, because SslE fusions might be useful to anchor other proteins to the cell surface. We therefore independently fused two plant cell wall degrading enzymes, Cel45A and Pel10A from Cellvibrio japonicus, to the C-terminus of E. coli W SslE and assessed the capacity of these fusion proteins to be secreted or displayed on the cell surface. Both fusions resulted in stable, enzymatically active proteins when expressed in E. coli W. We did not generate fusions to the potentially lipidated N-terminus of SslE to avoid changes in lipidation that could affect protein localization.
Extracellular and surface-displayed activity of SslE-Cel45A and SslE-Pel10A from liquid cultures
Inactivation of T2SSβmodestly increases urea tolerance
Viable cell counts for cultures grown with and without urea
2.8 ± 0.1 × 109
6.9 ± 0.3 × 109
2.0 ± 0.3 × 109
1.2 ± 0.1 × 109
2.6 ± 0.3 × 109
6.2 ± 0.2 × 109
2.4 ± 0.2 × 109
1.2 ± 0.1 × 109
2.7 ± 0.1 × 109
5.7 ± 0.2 × 109
2.3 ± 0.3 × 109
1.2 ± 0.1 × 109
5.8 ± 0.3 × 106
3.2 ± 0.1 × 106
1.6 ± 0.1 × 106
3.1 ± 0.1 × 105
7.9 ± 0.9 × 106
4.1 ± 0.2 × 106
2.2 ± 0.2 × 106
5.7 ± 0.3 × 105
6.3 ± 0.3 × 106
4.1 ± 0.3 × 106
2.1 ± 0.4 × 106
5.0 ± 0.6 × 105
Discussion and conclusions
Strains within the species Escherichia coli encode different combinations of type II secretion systems, each of which secrete different effectors and presumably provide specific advantageous phenotypes to their host organisms. To this point, the only T2SS shown to be functional in non-pathogenic E. coli strains is the chitinase-secreting T2SSα, which is the sole T2SS encoded by E. coli K-12 [13, 14] and whose role in natural environments is unknown. We demonstrate here that, surprisingly, the T2SSβ that promotes virulence of the enterotoxic strain H10407 and the enteropathogenic strain E2348/69 is conserved, and secretes a virulence factor homolog, in the non-pathogenic E. coli W strain. To our knowledge, this is the first time a virulence-associated type II secretion system has been shown to function in non-pathogenic E. coli. Deletion of sslE could be complemented in trans, indicating that an sslE disruption does not prevent expression or assembly of T2SSβ in E. coli W. We observed that E. coli W preferentially secretes SslE under nutrient-rich conditions at human body temperature (37°C), which suggests that SslE may be a colonization factor in non-pathogenic strains. The regulation of SslE secretion in other strains is unclear, but expression of genes encoding the LT-secreting T2SSβ in E. coli H10407 was also shown to be upregulated at host-associated temperatures . We hope that future experiments will elucidate the role of SslE in host colonization by non-pathogenic E. coli.
If secretion of SslE indeed aids diverse E. coli in gut colonization, it is perhaps surprising that some gut-derived isolates of E. coli, such as K-12 and O157:H7, lack the T2SS responsible for SslE secretion. Such strains may compensate for the loss of biofilm-forming propensity using other mechanisms; strains bearing the F plasmid (such as wild-type K-12) may rely on F pilus-mediated aggregation , for example. The genes encoding the SslE-secreting T2SSβ are present adjacent to the pheV tRNA gene, which appears to be a hypervariable locus in E. coli[16–18], so they may be randomly lost at a relatively high rate. Indeed, a comparison between phylogeny and T2SSα/T2SSβ presence suggests independent losses of T2SSβ in non-pathogenic strains (Figure 1). Notably, B and W encode the complete T2SSβ, while Crook’s and K-12 do not, in spite of the fact that Crook’s diverged from K-12 prior to the divergence of B. This indicates that either Crook’s and K-12 lost the T2SSβ-encoding genes independently, or that an ancestor of Crook’s, B, and K-12 lost the genes, which were subsequently re-acquired by strain B. An examination of the T2SSβ-encoding loci in Crook’s and K-12 strongly supports the former explanation. In K-12, the T2SSβ-encoding gsp operon clearly experienced an internal deletion that removed the gspD-K β genes, inactivating the T2SS. In Crook’s, however, the homologous genomic locus appears entirely different: all gsp genes are absent, and in their place is the fec operon (encoding a ferric citrate transport system) and a variety of putative ORFs. We infer that the most parsimonious explanation of the phylogenetic distribution of T2SSβ is that K-12 and Crook’s both lost the T2SS at different points in their evolutionary histories. It remains an open question what pattern of gene gains and losses best explains the distribution of T2SSβ across the diversity of E. coli strains not considered in our analysis.
It is of interest to note that a non-polar deletion of the pppA gene, encoding a prepilin peptidase, prevents secretion of SslE by E. coli W. This result agrees with a similar experiment performed by Strozen et al. to assess effects of PppA on LT secretion in H10407 . Both W and H10407 also encode a second prepilin peptidase (GspO) whose homolog is functional in facilitating ChiA secretion via T2SSα in K-12 . Whether the GspO peptidase is not expressed under conditions associated with SslE secretion in both W and H10407, or whether the two peptidases display different substrate specificities, remains to be determined.
Strikingly, in the presence of the otherwise intact gsp operon, deletion of sslE was effective in promoting modest urea tolerance. When we first observed the urea-tolerant phenotype of the Δgsp strain, we hypothesized that the mutant’s advantage stemmed from lacking the transmembrane components of the T2SS, particularly the secretin pore in the outer membrane, which might be denatured by urea. The urea tolerance of the ΔsslE mutant rules out this hypothesis, however, and indicates that secretion of SslE by T2SSβ renders cells modestly more sensitive to urea. Relative urea sensitivity is likely due to indirect effects on cell physiology of bearing surface-displayed SslE or of releasing of SslE into the culture medium.
We report here that enzymatic fusions to the C-terminus of SslE interfere with its targeting to the T2SS, as measured by release of fusion proteins and by display of fusion proteins on the outer leaflet of the outer membrane. Previously, Baldi et al. fused a tetracysteine motif to the C-terminus of E2348/69 SslE and saw that the fusion protein was still displayed on the cell surface . We do not think these results contradict ours, due to the significant structural differences between the fusion proteins in question. We propose that the six amino acids appended to the C-terminus of SslE in the study by Baldi et al. did not affect secretion of SslE, but that our fusions of SslE to large tightly-folded proteins (plant cell wall degrading enzymes from Cellvibrio japonicus) occluded important targeting motifs recognized by the T2SS. The uncharacterized nature of T2SS recognition of substrates  unfortunately limits our ability to speculate further as to what these motifs might be. Future dissection of the SslE protein with internal deletions and protein fusions may yield new insights into the targeting motif(s) of SslE, and determine whether SslE fusions can be used in the surface display of other proteins.
Growth media, strains and plasmids
Strains and plasmids used in this study
E. colistrain or plasmid
Reference or sourceb
Wild-type E. coli W
W ΔgspC-M::FRT, derived by FLP recombination from W Δgsp::Kan
W ΔpppA::FRT, derived by FLP recombination from W ΔpppA::Kan
W ΔsslE::FRT, derived by FLP recombination from W ΔsslE::Kan
pACYC184-derived; trc promoter; lacIq
pTrc99A-derived; trc promoter; lacIq
pRH21 with sslE cloned into the MCS
pRH21 with sslE lacking the signal peptide-encoding sequence cloned into the MCS
pRH21 with pppA cloned into the MCS
pRH31 with an sslE-cel45A fusion cloned into the MCS
pRH31 with an sslE-pel10A fusion cloned into the MCS
Primers used in this study
sslE cloning for fusions
All synthetic DNA for plasmid constructions described below was provided by Geneart (Regensburg, Germany). Plasmid pRH21 was constructed from pEG100  by replacing the multiple cloning site (MCS) with a synthetic variant including a tandem His-FLAG tag and by adding the rrnB-derived terminators from pTrc99A downstream of the MCS. pRH31 was constructed from pTrc99A  by replacing the MCS with the same synthetic variant as in pRH21. pMSD6 was constructed using sslE amplified from E. coli W genomic DNA with the sslE-up and sslE-dn primers (Table 4). pMSD7 was constructed using sslE similarly amplified with the sslE-noSP-up and sslE-dn primers. pMSD8 was constructed using pppA similarly amplified with the pppA-up and pppA-dn primers. For construction of pMSD6, pMSD7, and pMSD8, the PCR products were digested with Acc65I and BamHI and ligated into the large Acc65I/BamHI fragment of pRH21.
For construction of pRH153, sslE was amplified from E. coli W genomic DNA using primers sslE-up and sslE-dn-nostop, and the PCR product was digested with Acc65I and BamHI. A gene encoding the mature form of Cel45A from Cellvibrio japonicus Ueda107 was synthesized and codon optimized for E. coli expression, then amplified using the cel45A-noSP-up and cel45A-dn primers, and the PCR product was digested with BamHI and HindIII. The two digested PCR products (sslE and cel45A) were ligated into the large Acc65I/HindIII fragment of pRH31. pRH154 was constructed as pRH153, with a synthetic gene encoding mature Pel10A from C. japonicus Ueda107 (with altered codon usage for expression in E. coli) being amplified using the pel10A-noSP-up and pel10A-dn primers prior to digestion and ligation.
Protein expression and detection
For assessing secretion of wild-type SslE, cultures of indicated strains (mutants were all kan-marked, except ΔpppA mutants, which were unmarked) were grown in liquid media (LB at 37°C unless otherwise noted) with aeration for 16–20 hours. For complementation of the ΔsslE mutation, gene expression from plasmids was induced with 1 μM isopropyl-β-d-galactopyranoside (IPTG). Cells were harvested by centrifugation and resuspended in SDS sample buffer (SSB)  according to the following formula: resuspension volume (in μl) = 100 × A600 × vol harvested (in ml). These concentrated cell lysates were diluted 1:100 in SSB for SDS-PAGE. Cell-free supernatants were concentrated ~10-fold by filtration using Centricon spin columns (Millipore, Billerica, MA, USA), and added to concentrated SSB for SDS-PAGE. Samples were separated on 4-12% SDS-polyacrylamide gels and stained with silver to visualize protein bands . SslE secretion experiments were repeated 2–4 times, and single representative gels are shown.
To produce the images in Figure 2, the stained gels were digitally photographed and gel images were enhanced using Adobe Photoshop software. Linear transformations (contrast and brightness adjustments) were applied to the images for clarity; such transformations were applied uniformly across any given gel image.
Fusion protein localization by enzyme activity
To measure secretion and surface display of SslE-enzyme fusions, cultures of WT and ΔpppA::FRT strains bearing the indicated plasmids were grown in LB at 37°C with aeration for 16–20 hours. Cells were harvested by centrifugation, and cell-free supernatants were removed; an aliquot of collected cells was removed and lysed using the PopCulture reagent from Novagen (Madison, WI, USA). Enzymatic activities associated with intact cells, lysed cells, and cell-free supernatants were then immediately measured. SslE-Cel45A activity was measured using the CRACC assay , and SslE-Pel10A activity was measured using the pectate lyase assay described by Collmer .
Phenotypic microarray experiments were performed using an OmniLog reader (Biolog, Hayward, CA, USA) as per the manufacturer’s instructions using plate types PM-9 and PM-10. Cultures were grown at 37°C for 48 hours, and respiration data were analyzed using the PM software provided with the OmniLog reader. Strains used were wild-type W and Δgsp::FRT (unmarked deletion of gspC-M).
To compare urea tolerances in 96-well plates, wild-type, Δgsp::FRT, and ΔpppA::FRT strains were cultured in 200 μl aliquots of LB containing 0, 0.9 M, or 1.15 M urea in 96-well plates (inoculated as 1:100 dilutions from LB overnight cultures). Plates were grown with shaking at 37°C in a Tecan M1000 plate reader (Durham, NC, USA). Growth and survival were followed by regular measurement of A595 for each culture.
To compare urea tolerances in glass culture tubes, wild-type, Δgsp::FRT, and ΔsslE::FRT strains were cultured in 8 ml volumes of LB containing no urea or 1.15 M urea on a rolling wheel at 37°C. Biological duplicate cultures of each strain were inoculated with 1:1000 dilutions from LB overnight cultures after verification that all overnight cultures grew to equivalent A600 turbidity readings. Turbidity in growing cultures was measured by reading A600 using a Spectronic 20D digital spectrophotometer; for cultures with high densities (A600 > 1.5), aliquots of the culture were diluted 1:10 or 1:20 prior to measurement of A600. Viable cells were enumerated by 10-fold serial dilution of cultures into sterile 0.9% NaCl followed by plating of dilutions on non-selective media and colony counting.
Availability of supporting data
Biolog cultivation data are included as Additional file 1. Data from microtiter plate growth experiments of cells under urea stress are included as in Additional file 2: Figure S1. The sequences of all plasmids described in this study are included as Additional file 3.
Type II secretion systems
General secretory pathway
Multiple cloning site.
We would like to thank David Keating for thoughtful discussions and critical review of the manuscript. This work was funded by the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-FC02-07ER64494). Sequencing of E. coli W by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
- Korotkov KV, Sandkvist M, Hol WG: The type II secretion system: biogenesis, molecular architecture and mechanism. Nat Rev Microbiol. 2012, 10: 336-351.PubMedPubMed CentralGoogle Scholar
- McLaughlin LS, Haft RJF, Forest KT: Structural insights into the type II secretion nanomachine. Curr Opin Struct Biol. 2012, 22: 208-216. 10.1016/j.sbi.2012.02.005.PubMedPubMed CentralView ArticleGoogle Scholar
- Peabody CR, Chung YJ, Yen MR, Vidal-Ingigliardi D, Pugsley AP, Saier MH: Type II protein secretion and its relationship to bacterial type IV pili and archaeal flagella. Microbiology. 2003, 149: 3051-3072. 10.1099/mic.0.26364-0.PubMedView ArticleGoogle Scholar
- Hobbs M, Mattick JS: Common components in the assembly of type 4 fimbriae, DNA transfer systems, filamentous phage and protein-secretion apparatus: a general system for the formation of surface-associated protein complexes. Mol Microbiol. 1993, 10: 233-243. 10.1111/j.1365-2958.1993.tb01949.x.PubMedView ArticleGoogle Scholar
- Cianciotto NP: Type II secretion: a protein secretion system for all seasons. Trends Microbiol. 2005, 13: 581-588. 10.1016/j.tim.2005.09.005.PubMedView ArticleGoogle Scholar
- Sandkvist M: Type II secretion and pathogenesis. Infect Immun. 2001, 69: 3523-3535. 10.1128/IAI.69.6.3523-3535.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- Lathem WW, Grys TE, Witowski SE, Torres AG, Kaper JB, Tarr PI, Welch RA: StcE, a metalloprotease secreted by Escherichia coli O157:H7, specifically cleaves C1 esterase inhibitor. Mol Microbiol. 2002, 45: 277-288. 10.1046/j.1365-2958.2002.02997.x.PubMedView ArticleGoogle Scholar
- Tauschek M, Gorrell RJ, Strugnell RA, Robins-Browne RM: Identification of a protein secretory pathway for the secretion of heat-labile enterotoxin by an enterotoxigenic strain of Escherichia coli. Proc Natl Acad Sci USA. 2002, 99: 7066-7071. 10.1073/pnas.092152899.PubMedPubMed CentralView ArticleGoogle Scholar
- Baldi DL, Higginson EE, Hocking DM, Praszkier J, Cavaliere R, James CE, Bennett-Wood V, Azzopardi KI, Turnbull L, Lithgow T, et al: The type II secretion system and its ubiquitous lipoprotein substrate, SslE, are required for biofilm formation and virulence of enteropathogenic Escherichia coli. Infect Immun. 2012, 80: 2042-2052. 10.1128/IAI.06160-11.PubMedPubMed CentralView ArticleGoogle Scholar
- Dunstan RA, Heinz E, Wijeyewickrema LC, Pike RN, Purcell AW, Evans TJ, Praszkier J, Robins-Browne RM, Strugnell RA, Korotkov KV, Lithgow T: Assembly of the type II secretion system such as found in Vibrio cholerae depends on the novel pilotin AspS. PLoS Pathog. 2013, 9: e1003117-10.1371/journal.ppat.1003117.PubMedPubMed CentralView ArticleGoogle Scholar
- Yang J, Baldi DL, Tauschek M, Strugnell RA, Robins-Browne RM: Transcriptional regulation of the yghJ-pppA-yghG-gspCDEFGHIJKLM cluster, encoding the type II secretion pathway in enterotoxigenic Escherichia coli. J Bacteriol. 2007, 189: 142-150. 10.1128/JB.01115-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Strozen TG, Li G, Howard SP: YghG (GspSβ) is a novel pilot protein required for localization of the GspSβ type II secretion system secretin of enterotoxigenic Escherichia coli. Infect Immun. 2012, 80: 2608-2622. 10.1128/IAI.06394-11.PubMedPubMed CentralView ArticleGoogle Scholar
- Archer CT, Kim JF, Jeong H, Park JH, Vickers CE, Lee SY, Nielsen LK: The genome sequence of E. coli W (ATCC 9637): comparative genome analysis and an improved genome-scale reconstruction of E. coli. BMC Genomics. 2011, 12: 9-10.1186/1471-2164-12-9.PubMedPubMed CentralView ArticleGoogle Scholar
- Blattner FR, Plunkett G, Bloch CA, Perna NT, Burland V, Riley M, Collado-Vides J, Glasner JD, Rode CK, Mayhew GF, et al: The complete genome sequence of Escherichia coli K-12. Science. 1997, 277: 1453-1462. 10.1126/science.277.5331.1453.PubMedView ArticleGoogle Scholar
- Lawley TD, Wilkins BM, Frost L: Bacterial conjugation in Gram-negative bacteria. Plasmid biology. Edited by: Phillips G, Funnell BE. 2004, Washington, D.C: ASM Press, 203-226.View ArticleGoogle Scholar
- Rumer L, Jores J, Kirsch P, Cavignac Y, Zehmke K, Wieler LH: Dissemination of pheU- and pheV-located genomic islands among enteropathogenic (EPEC) and enterohemorrhagic (EHEC) E. coli and their possible role in the horizontal transfer of the locus of enterocyte effacement (LEE). Int J Med Microbiol. 2003, 292: 463-475. 10.1078/1438-4221-00229.PubMedView ArticleGoogle Scholar
- Vimr ER, Steenbergen SM: Mobile contingency locus controlling Escherichia coli K1 polysialic acid capsule acetylation. Mol Microbiol. 2006, 60: 828-837. 10.1111/j.1365-2958.2006.05158.x.PubMedView ArticleGoogle Scholar
- Schneider G, Dobrindt U, Bruggemann H, Nagy G, Janke B, Blum-Oehler G, Buchrieser C, Gottschalk G, Emody L, Hacker J: The pathogenicity island-associated K15 capsule determinant exhibits a novel genetic structure and correlates with virulence in uropathogenic Escherichia coli strain 536. Infect Immun. 2004, 72: 5993-6001. 10.1128/IAI.72.10.5993-6001.2004.PubMedPubMed CentralView ArticleGoogle Scholar
- Francetic O, Pugsley AP: The cryptic general secretory pathway (gsp) operon of Escherichia coli K-12 encodes functional proteins. J Bacteriol. 1996, 178: 3544-3549.PubMedPubMed CentralGoogle Scholar
- Filloux A: Secretion signal and protein targeting in bacteria: a biological puzzle. J Bacteriol. 2010, 192: 3847-3849. 10.1128/JB.00565-10.PubMedPubMed CentralView ArticleGoogle Scholar
- Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Short protocols in molecular biology. 2002, New York: Wiley, 5Google Scholar
- Neidhardt FC, Bloch PL, Smith DF: Culture medium for enterobacteria. J Bacteriol. 1974, 119: 736-747.PubMedPubMed CentralGoogle Scholar
- Thomason L, Court DL, Bubunenko M, Costantino N, Wilson H, Datta S, Oppenheim A: Recombineering: genetic engineering in bacteria using homologous recombination. Curr Protoc Mol Biol. 2007, Chapter 1 (Unit 1): 1.16.1-1.16.24.Google Scholar
- Baba T, Ara T, Hasegawa M, Takai Y, Okumura Y, Baba M, Datsenko KA, Tomita M, Wanner BL, Mori H: Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol Syst Biol. 2006, 2: 2006.0008-PubMedPubMed CentralView ArticleGoogle Scholar
- Haft RJ, Palacios G, Nguyen T, Mally M, Gachelet EG, Zechner EL, Traxler B: General mutagenesis of F plasmid TraI reveals its role in conjugative regulation. J Bacteriol. 2006, 188: 6346-6353. 10.1128/JB.00462-06.PubMedPubMed CentralView ArticleGoogle Scholar
- Amann E, Ochs B, Abel KJ: Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988, 69: 301-315. 10.1016/0378-1119(88)90440-4.PubMedView ArticleGoogle Scholar
- Haft RJF, Gardner JG, Keating DH: Quantitative colorimetric measurement of cellulose degradation under microbial culture conditions. Appl Microbiol Biotechnol. 2012, 94: 223-229. 10.1007/s00253-012-3968-5.PubMedView ArticleGoogle Scholar
- Collmer A, Ried JL, Mount MS: Assay methods for pectic enzymes. Methods Enzymol. 1988, 161: 329-335.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.