Stx₁prophage excision inEscherichia colistrain PA20 confers strong curli and biofilm formation by restoring nativemlrA

Abstract

Prophage insertions in Escherichia coli O157:H7 mlrA contribute to the low expression of curli fimbriae and biofilm observed in many clinical isolates. Varying levels of CsgD-dependent curli/biofilm expression are restored to strains bearing prophage insertions in mlrA by mutation of regulatory genes affecting csgD Our previous study identified strong biofilm- and curli-producing variants in O157:H7 cultures that had lost the mlrA-imbedded prophage characteristic of the parent population, suggesting prophage excision as a mechanism for restoring biofilm properties. In this study, we compared genomic, transcriptomic and phenotypic properties of parent strain PA20 (stx1, stx2) and its prophage-cured variant, 20R2R (stx2), and confirmed the mechanism underlying the differences in biofilm formation.

Sulfamethoxazole – Trimethoprim represses csgD but maintains virulence genes at 30°C in a clinical Escherichia coli O157:H7 isolate

“…Spontaneous prophage excisions from mlrA restore a small percentage of PCR-detectable unoccupied insertion sites within growing O157:H7 populations at either host (37°C) or environmental (30°C) temperatures, and prophage-inducing agents such as SMX-TM can increase the level of cells with restored mlrA [ 35 , 37 ]. However, plating of the induced or un-induced populations rarely identifies individuals that have lost the prophage, suggesting that prophage excisions are transient or detrimental [ 38 ]. It has also been shown that the mlrA portion distal to the prophage insertion site encodes truncated mlrA proteins that could be driven by run-off transcription [ 38 ].…”

“…However, plating of the induced or un-induced populations rarely identifies individuals that have lost the prophage, suggesting that prophage excisions are transient or detrimental [ 38 ]. It has also been shown that the mlrA portion distal to the prophage insertion site encodes truncated mlrA proteins that could be driven by run-off transcription [ 38 ]. When expressed as recombinant forms, these proteins restored some CR affinity, but not biofilm formation.…”

“…We previously showed that E . coli serotype O157:H7 clinical isolate PA20 carried a stx 1 prophage in mlrA but that curli/biofilm production might be influenced by prophage activation and excision [ 35 , 38 ]. In this study, we used transcriptomics to study the effect of sub-lethal SMX-TM concentrations on prophage induction, biofilm regulation, and virulence gene expression under stress conditions.…”

New candidates for regulated gene integrity revealed through precise mapping of integrative genetic elements

“…That is, such cases need not inactivate the gene; thus, simply detecting that an IGE has invaded a CDS is not sufficient to show that gene function has been inactivated. More rarely, IGEs are known to inactivate genes within conserved peptideencoding regions, in two situations: 1) accidental targeting of the gene by a promiscuous integrase or by an off-target event from a site-specific integrase, or 2) regulatory gene inactivation as described at comK, sigK, spsM, mutL, gerE, mlrA, hlb, nifD, fdxN and hupL (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). We devised a stringent test for gene inactivation, namely, Pfam domain disruption, by any non-tRNA IGE, comparing top Pfam scores for possible peptides encoded in the attB region to those for the attLR regions (Table 1).…”

“…Likewise, at an attB target in a proteincoding sequence (CDS), the IGE typically integrates innocuously, preserving target gene function using a similar attP fragment strategy, or perhaps targeting an extreme tail of the protein sequence that does not affect protein function. However certain IGEs are known to control gene activity, inactivating the target CDS upon integration and reactivating it upon excision (4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). This regulated gene integrity (RGI) can occur irreversibly in the development of nonreproducing cells of multicellular bacteria, where integrases catalyze specific IGE deletions in the chromosome that restore the sigK or spsM genes in spore mother cells (5,10,12), or the nifD, hupL, or fdxN genes in cyanobacterial heterocysts (6,8,9).…”