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

Abstract

Listeria monocytogenes is a Gram-positive firmicute that causes foodborne infections, in part due to its ability to use multiple strategies, including biofilm formation, to survive adverse growth conditions. As a potential way to screen for genes required for biofilm formation, we harnessed the ability of bacteria to accumulate mutations in the genome over time, diverging the properties of seemingly identical strains. By sequencing the genomes of four laboratory reference strains of the commonly used L. monocytogenes EGDe, we showed that each isolate contains single nucleotide polymorphisms (SNPs) compared with the reference genome. We discovered that two SNPs, contained in two independent genes within one of the isolates, impacted biofilm formation. Using bacterial genetics and phenotypic assays, we confirmed that rsbU and rmlA influence biofilm formation. RsbU is the upstream regulator of the alternative sigma factor SigB, and mutation of either rsbU or sigB increased biofilm formation. In contrast, deletion of rmlA, which encodes the first enzyme for TDP-l-rhamnose biosynthesis, resulted in a reduction in the amount of biofilm formed. Further analysis of biofilm formation in a strain that still produces TDP-l-rhamnose but which cannot decorate the wall teichoic acid with rhamnose (rmlT mutant) showed that it is the decorated wall teichoic acid that is required for adhesion of the cells to surfaces. Together, these data uncover novel routes by which biofilm formation by L. monocytogenes can be impacted. IMPORTANCE Biofilms are an important mode of growth in many settings. Here, we looked at small differences in the genomes of the bacterium Listeria monocytogenes isolate EGDe and used them to find out how biofilms form. This important fundamental information may help new treatments to be developed and also highlights the fact that isolates of the same identity often diverge.

All Treatment Parameters Affect Environmental Surface Sanitation Efficacy, but Their Relative Importance Depends on the Microbial Target

“…Surface-adhered L. monocytogenes was determined to be 1.3 ± 0.2 μm by 0.6 ± 0.07 μm ( Fig. 1F , arrows) and modest extracellular polysaccharide (EPS) secretion was evident, as described previously ( 53 – 56 ). Surface adhered Exophiala spp.…”

Mild Stress Conditions during Laboratory Culture Promote the Proliferation of Mutations That Negatively Affect Sigma B Activity in Listeria monocytogenes

“…The data presented here suggest an explanation for the common detection of mutations in the sigB operon of L. monocytogenes (28,(33)(34)(35)(36)55), including an earlier study in our own laboratory where an rsbV missense mutation arose during routine subculture (31).…”

The σB-Mediated General Stress Response of Listeria monocytogenes: Life and Death Decision Making in a Pathogen

“…It has been proposed that the signal transduction cascade in L. monocytogenes can be inferred from the well-studied B. subtilis , since both species share a high level of conservation ( Ferreira et al, 2004 ). Many studies of σ B regulation in L. monocytogenes have confirmed that the signal transduction pathways likely function in a very similar way between these two microorganisms ( Chaturongakul and Boor, 2004 , 2006 ; Cosgrave, 2010 ; Shin et al, 2010a ; Utratna et al, 2014 ; O’Donoghue,, 2016 ; Guerreiro et al, 2020 ; Hsu et al, 2020 ).…”