Bacteria elicit a phage tolerance response subsequent to infection of their neighbors.

Appearance of plaques on a bacterial lawn is a sign of successive rounds of bacteriophage infection. Yet, mechanisms evolved by bacteria to limit plaque spread have been hardly explored. Here, we investigated the dynamics of plaque development by lytic phages infecting the bacterium Bacillus subtilis. We report that plaque expansion is followed by a constriction phase owing to bacterial growth into the plaque zone. This phenomenon exposed an adaptive process, herein termed "phage tolerance response", elicited by non-infected bacteria upon sensing infection of their neighbors. The temporary phage tolerance is executed by the stress-response RNA polymerase sigma factor σX (SigX). Artificial expression of SigX prior to phage attack largely eliminates infection. SigX tolerance is primarily conferred by activation of the dlt operon, encoding enzymes that catalyze D-alanylation of cell wall teichoic acid polymers, the major attachment sites for phages infecting Gram-positive bacteria. D-alanylation impedes phage binding and hence infection, thus enabling the uninfected bacteria to form a protective shield opposing phage spread.

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

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.

Wall teichoic acid-dependent phagocytosis of intact cell walls of Lactiplantibacillus plantarum elicits IL-12 secretion from macrophages.

Selected lactic acid bacteria can stimulate macrophages and dendritic cells to secrete IL-12, which plays a key role in activating innate and cellular immunity. In this study, we investigated the roles of cell wall teichoic acids (WTAs) displayed on whole intact cell walls (ICWs) of Lactiplantibacillus plantarum in activation of mouse macrophages. ICWs were prepared from whole bacterial cells of several lactobacilli without physical disruption, and thus retaining the overall shapes of the bacteria. WTA-displaying ICWs of several L. plantarum strains, but not WTA-lacking ICWs of strains of other lactobacilli, elicited IL-12 secretion from mouse bone marrow-derived macrophages (BMMs) and mouse macrophage-like J774.1 cells. The ability of the ICWs of L. plantarum to induce IL-12 secretion was abolished by selective chemical elimination of WTAs from ICWs, but was preserved by selective removal of cell wall glycopolymers other than WTAs. BMMs prepared from TLR2- or TLR4-deficient mouse could secret IL-12 upon stimulation with ICWs of L. plantarum and a MyD88 dimerization inhibitor did not affect ICW-mediated IL-12 secretion. WTA-displaying ICWs, but not WTA-lacking ICWs, were ingested in the cells within 30 min. Treatment with inhibitors of actin polymerization abolished IL-12 secretion in response to ICW stimulation and diminished ingestion of ICWs. When overall shapes of ICWs of L. plantarum were physically disrupted, the disrupted ICWs (DCWs) failed to induce IL-12 secretion. However, DCWs and soluble WTAs inhibited ICW-mediated IL-12 secretion from macrophages. Taken together, these results show that WTA-displaying ICWs of L. plantarum can elicit IL-12 production from macrophages via actin-dependent phagocytosis but TLR2 signaling axis independent pathway. WTAs displayed on ICWs are key molecules in the elicitation of IL-12 secretion, and the sizes and shapes of the ICWs have an impact on actin remodeling and subsequent IL-12 production.

Complete Genome of Bacillus subtilis subsp. subtilis KCTC 3135T and Variation in Cell Wall Genes of B. subtilis Strains.

The type strain Bacillus subtilis subsp. subtilis KCTC 3135T was deeply sequenced and annotated, replacing a previous draft genome in this study. The tar and tag genes were involved in synthesizing wall teichoic acids (WTAs), and these genes and their products were previously regarded as the distinguishing difference between B. s. subtilis and B. s. spizizenii. However, a comparative genomic analysis of B. subtilis spp. revealed that both B. s. subtilis and B. s. spizizenii had various types of cell walls. These tar and tag operons were mutually exclusive and the tar genes from B. s. spizizenii were very similar to the genes from non-Bacillus bacteria, unlike the tag genes from B. s. subtilis. The results and previous studies suggest that the tar genes and the tag genes are not inherited after subspecies speciation. The phylogenetic tree based on whole genome sequences showed that each subspecies clearly formed a monophyletic group, while the tree based on tar genes showed that monophyletic groups were formed according to the cell wall type rather than the subspecies. These findings indicate that the tar genes and the presence of ribitol as a cell-wall constituent were not the distinguishing difference between the subspecies of B. subtilis and that the description of subspecies B. s. spizizenii should be updated.

A rapid NMR-based method for discrimination of strain-specific cell wall teichoic acid structures reveals a third backbone type in Lactobacillus plantarum.

The lactic acid bacterium Lactobacillus plantarum is capable of producing strain-specific structures of cell wall teichoic acid (WTA), an anionic polysaccharide found in the Gram-positive bacterial cell wall. In this study, we established a rapid, NMR-based procedure to discriminate WTA structures in this species, and applied it to 94 strains of L. plantarum. Six previously reported glycerol- and ribitol-containing WTA subtypes were successfully identified from 78 strains, suggesting that these were the dominant structures. However, the level of structural variety differed markedly among bacterial sources, possibly reflecting differences in strain-level microbial diversity. WTAs from eight strains were not identified based on NMR spectra and were classified into three groups. Structural analysis of a partial degradation product of an unidentified WTA produced by strain TUA 1496L revealed that the WTA was 1-O-ß-d-glucosylglycerol. Two-dimensional NMR analysis of the polymer structure showed phosphodiester bonds between C-3 and C-6 of the glycerol and glucose residues, suggesting a polymer structure of 3,6΄-linked poly(1-O-ß-d-glucosyl-sn-glycerol phosphate). This is the third WTA backbone structure in L. plantarum, following 3,6΄-linked poly(1-O-α-d-glucosyl-sn-glycerol phosphate) and 1,5-linked poly(ribitol phosphate).