Generation of a restriction minus enteropathogenic Escherichia coli E2348/69 strain that is efficiently transformed with large, low copy plasmids

<p>Abstract</p> <p>Background</p> <p>Many microbes possess restriction-modification systems that protect them from parasitic DNA molecules. Unfortunately, the presence of a restriction-modification system in a given microbe also hampers genetic analysis. Although plasmids can be successfully conjugated into the enteropathogenic <it>Escherichia coli </it>strain E2348/69 and optimized protocols for competent cell preparation have been developed, we found that a large, low copy (~15) bioluminescent reporter plasmid, pJW15, that we modified for use in EPEC, was exceedingly difficult to transform into E2348/69. We reasoned that a restriction-modification system could be responsible for the low transformation efficiency of E2348/69 and sought to identify and inactivate the responsible gene(s), with the goal of creating an easily transformable strain of EPEC that could complement existing protocols for genetic manipulation of this important pathogen.</p> <p>Results</p> <p>Using bioinformatics, we identified genes in the unfinished enteropathogenic <it>Escherichia coli </it>(EPEC) strain E2348/69 genome whose predicted products bear homology to the HsdM methyltransferases, HsdS specificity subunits, and HsdR restriction endonucleases of type I restriction-modification systems. We constructed a strain carrying a deletion of the conserved enzymatic domain of the EPEC HsdR homologue, NH4, and showed that its transformation efficiency was up to four orders of magnitude higher than that of the parent strain. Further, the modification capacity of NH4 remained intact, since plasmids that were normally recalcitrant to transformation into E2348/69 could be transformed upon passage through NH4. NH4 was unaffected in virulence factor production, since bundle forming pilus (BFP) subunits and type III secreted (T3S) proteins were present at equivalent levels to those seen in E2348/69. Further, NH4 was indistinguishable from E2348/69 in tissue culture infection model assays of localized adherence and T3S.</p> <p>Conclusion</p> <p>We have shown that EPEC strain E2348/69 utilizes a type I restriction-modification system to limit entry of new DNA. This restriction-modification system does not appear to be involved in virulence determinant expression or infection phenotypes. The <it>hsdR </it>mutant strain should prove useful in genetic analysis of the important diarrheal pathogen EPEC.</p

Characterization of the Cpx Regulon in Escherichia coli Strain MC4100 ▿

The Cpx two-component signal transduction pathway of Escherichia coli mediates adaptation to envelope protein misfolding. However, there is experimental evidence that at least 50 genes in 34 operons are part of the Cpx regulon and many have functions that are undefined or unrelated to envelope protein maintenance. No comprehensive analysis of the Cpx regulon has been presented to date. In order to identify strongly Cpx-regulated genes that might play an important role(s) in envelope protein folding and/or to further define the role of the Cpx response and to gain insight into what makes a gene subject to strong Cpx regulation, we have carried out a uniform characterization of a Cpx-regulated lux reporter library in a single-strain background. Strongly Cpx-regulated genes encode proteins that are directly linked to envelope protein folding, localized to the envelope but uncharacterized, or involved in limiting the cellular concentration of noxious molecules. Moderately Cpx-regulated gene clusters encode products implicated in biofilm formation. An analysis of CpxR binding sites in strongly regulated genes indicates that while neither a consensus match nor their orientation predicts the strength of Cpx regulation, most genes contain a CpxR binding site within 100 bp of the transcriptional start site. Strikingly, we found that while there appears to be little overlap between the Cpx and Bae envelope stress responses, the σE and Cpx responses reciprocally regulate a large group of strongly Cpx-regulated genes, most of which are uncharacterized

The Cpx envelope stress response modifies peptidoglycan cross-linking via the L,D-transpeptidase LdtD and the novel protein YgaU

The Cpx envelope stress response mediates a complex adaptation to conditions that cause protein misfolding in the periplasm. A recent microarray study demonstrated that Cpx response activation led to changes in the expression of genes known, or predicted, to be involved in cell wall remodeling. We sought to characterize the changes that the cell wall undergoes during activation of the Cpx pathway in Escherichia coli. Luminescent reporters of gene expression confirmed that LdtD, a putative L,D-transpeptidase; YgaU, a protein of unknown function; and Slt, a lytic transglycosylase, are upregulated in response to Cpx-inducing conditions. Phosphorylated CpxR binds to the upstream regions of these genes, which contain putative CpxR binding sites, suggesting that regulation is direct. We show that the activation of the Cpx response causes an increase in the abundance of diaminopimelic acid (DAP)-DAP cross-links that involves LdtD and YgaU. Altogether, our data indicate that changes in peptidoglycan structure are part of the Cpx-mediated adaptation to envelope stress and indicate a role for the uncharacterized gene ygaU in regulating cross-linking.This work was supported by Canadian Institute of Health Research operating grant 97819 and Natural Science and Engineering Research Council grant RGPIN 238422-2013. T.L.R. was supported by a senior scholar award from Alberta Innovates Health Solutions. We acknowledge financial support (project number BFU2009-09200) from the Spanish Ministry of Economy and Competitiveness to J.A.A.Peer Reviewe

The Cpx Envelope Stress Response Affects Expression of the Type IV Bundle-Forming Pili of Enteropathogenic Escherichia coli

The Cpx envelope stress response mediates adaptation to potentially lethal envelope stresses in Escherichia coli. The two-component regulatory system consisting of the sensor kinase CpxA and the response regulator CpxR senses and mediates adaptation to envelope insults believed to result in protein misfolding in this compartment. Recently, a role was demonstrated for the Cpx response in the biogenesis of P pili, attachment organelles expressed by uropathogenic E. coli. CpxA senses misfolded P pilus assembly intermediates and initiates increased expression of both assembly and regulatory factors required for P pilus elaboration. In this report, we demonstrate that the Cpx response is also involved in the expression of the type IV bundle-forming pili of enteropathogenic E. coli (EPEC). Bundle-forming pili were not elaborated from an exogenous promoter in E. coli laboratory strain MC4100 unless the Cpx pathway was constitutively activated. Further, an EPEC cpxR mutant synthesized diminished levels of bundle-forming pili and was significantly affected in adherence to epithelial cells. Since type IV bundle-forming pili are very different from chaperone-usher-type P pili in both form and biogenesis, our results suggest that the Cpx envelope stress response plays a general role in the expression of envelope-localized organelles with diverse structures and assembly pathways

Cpx Signal Transduction Is Influenced by a Conserved N-Terminal Domain in the Novel Inhibitor CpxP and the Periplasmic Protease DegP

In Escherichia coli, envelope stress can be overcome by three different envelope stress responses: the σ(E) stress response and the Bae and Cpx two-component systems. The Cpx envelope stress response is controlled by the sensor kinase CpxA, the response regulator CpxR, and the novel periplasmic protein CpxP. CpxP mediates feedback inhibition of the Cpx pathway through a hypothetical interaction with the sensing domain of CpxA. No informative homologues of CpxP are known, and thus it is unclear how CpxP exerts this inhibition. Here, we identified six cpxP loss-of-function mutations using a CpxP-β-lactamase (CpxP′-′Bla) translational fusion construct. These loss-of-function mutations identified a highly conserved, predicted α-helix in the N-terminal domain of CpxP that affects both the function and the stability of the protein. In the course of this study, we also found that CpxP′-′Bla stability is differentially controlled by the periplasmic protease DegP in response to inducing cues and that mutation of degP diminishes Cpx pathway activity. We propose that the N-terminal α-helix is an important functional domain for inhibition of the Cpx pathway and that CpxP is subject to DegP-dependent proteolysis

Cpx signaling pathway monitors biogenesis and affects assembly and expression of P pili

P pili are important virulence factors in uropathogenic Escherichia coli. The Cpx two-component signal transduction system controls a stress response and is activated by misfolded proteins in the periplasm. We have discovered new functions for the Cpx pathway, indicating that it may play a critical role in pathogenesis. P pili are assembled via the chaperone/usher pathway. Subunits that go ‘OFF-pathway’ during pilus biogenesis generate a signal. This signal is derived from the misfolding and aggregation of subunits that failed to come into contact with the chaperone in the periplasm. In response, Cpx not only controls the stress response, but also controls genes necessary for pilus biogenesis, and is involved in regulating the phase variation of pap expression and, potentially, the expression of a panoply of other virulence factors. This study demonstrates how the prototypic chaperone/usher pathway is intricately linked and dependent upon a signal transduction system