Occurrence of mutations impairing sigma factor B (SigB) function upon inactivation of Listeria monocytogenes genes encoding surface proteins

Abstract

Bacteria of the genus Listeria contain the largest family of LPXTG surface proteins covalently anchored to the peptidoglycan. The extent to which these proteins may function or be regulated cooperatively is at present unknown. Because of their unique cellular location, we reasoned that distinct LPXTG proteins could act as elements contributing to cell wall homeostasis or influencing the stability of other surface proteins bound to peptidoglycan. To test this hypothesis, we used proteomics to analyse mutants of the intracellular pathogen Listeria monocytogenes lacking distinct LPXTG proteins implicated in pathogen-host interactions, such as InlA, InlF, InlG, InlH, InlJ, LapB and Vip. Changes in the cell wall proteome were found in inlG and vip mutants, which exhibited reduced levels of the LPXTG proteins InlH, Lmo0610, Lmo0880 and Lmo2085, all regulated by the stress-related sigma factor SigB. The ultimate basis of this alteration was uncovered by genome sequencing, which revealed that these inlG and vip mutants carried lossof-function mutations in the rsbS, rsbU and rsbV genes encoding regulatory proteins that control SigB activity. Attempts to recapitulate this negative selection of SigB in a large series of new inlG or vip mutants constructed for this purpose were, however, unsuccessful. These results indicate that inadvertent secondary mutations affecting SigB functionality can randomly arise in L. monocytogenes when using common genetic procedures or during subculturing. Testing of SigB activity could be therefore valuable when manipulating genetically L. monocytogenes prior to any subsequent phenotypic analysis. This test may be even more justified when generating deletions affecting cell envelope components.

Comparison of Widely Used Listeria monocytogenes Strains EGD, 10403S, and EGD-e Highlights Genomic Differences Underlying Variations in Pathogenicity

“…Only five differences compared to the published sequence were found (Fig. 1A), confirming the high quality of the first published sequence (7, 28). As shown in Data Set S1 in the supplemental material, four of the five differences are in intergenic regions, where no small RNAs (sRNAs) have been identified so far, and only one difference induces an amino acid change, i.e., a glycine to a valine, in Lmo0247, a hypothetical protein.…”

Genomic Differences between Listeria monocytogenes EGDe Isolates Reveal Crucial Roles for SigB and Wall Rhamnosylation in Biofilm Formation

“…Differences in chitinase activity. Certain regions of the L. monocytogenes genome are prone to incorporating mutations during growth (15), including rsbS, rsbU, and rsbV (16). The products of these genes comprise part of the complex regulatory system that activates the alternative sigma factor sigma B (SigB) (17).…”

The alternative sigma factor σB plays a crucial role in adaptive strategies of Clostridium difficile during gut infection

“…This set includes genes encoding the S-layer precursor (slpA), putative autolysins (CD2767-cwp19 and CD2784-cwp6), a collagen-binding protein (cbpA), two adhesins (CD2831 and CD3246) anchored to peptidoglycan by a sortase (Peltier et al, 2015), and other cell wall-associated proteins of unknown function such as CD0440 (Cwp27), CD2518 (Cwp29), and CD2791 (Cwp2). This situation is reminiscent of the positive control by r B of cell wall adhesins and a fibronectin-binding protein in S. aureus (Entenza et al, 2005;Mitchell et al, 2008) and of the internalin InlA, of LPXTG-containing proteins anchored by sortase A, and potential collagen-binding proteins in L. monocytogenes (Oliver et al, 2009;Quereda et al, 2013). By contrast, genes encoding the lipoprotein CD0873 involved in adhesion of C. difficile to Caco-2 cells (Janoir, 2016), the fibronectin-binding protein FbpA, and a cell wall-binding protein of unknown function (cwp28/ CD1987) were up-regulated in the sigB mutant.…”