Physical and antibiotic stresses require activation of the RsbU phosphatase to induce the general stress response in Listeria monocytogenes

Among pathogenic strains of Listeria monocytogenes, the σB transcription factor has a pivotal role in the outcome of food-borne infections. This factor is activated by diverse stresses to provide general protection against multiple challenges, including those encountered during gastrointestinal passage. It also acts with the PrfA regulator to control virulence genes needed for entry into intestinal lumen cells. Environmental and nutritional signals modulate σB activity via a network that operates by the partner switching mechanism, in which protein interactions are controlled by serine phosphorylation. This network is well characterized in the related bacterium Bacillus subtilis. A key difference in Listeria is the presence of only one input phosphatase, RsbU, instead of the two found in B. subtilis. Here, we aim to determine whether this sole phosphatase is required to convey physical, antibiotic and nutritional stress signals, or if additional pathways might exist. To that end, we constructed L. monocytogenes 10403S strains bearing single-copy, σB-dependent opuCA–lacZ reporter fusions to determine the effects of an rsbU deletion under physiological conditions. All stresses tested, including acid, antibiotic, cold, ethanol, heat, osmotic and nutritional challenge, required RsbU to activate σB. This was of particular significance for cold stress activation, which occurs via a phosphatase-independent mechanism in B. subtilis. We also assayed the effects of the D80N substitution in the upstream RsbT regulator that activates RsbU. The mutant had a phenotype consistent with low and uninducible phosphatase activity, but nonetheless responded to nutritional stress. We infer that RsbU activity but not its induction is required for nutritional signalling, which would enter the network downstream from RsbU

Blue and Red Light Modulates SigB-Dependent Gene Transcription, Swimming Motility and Invasiveness in Listeria monocytogenes

Background: In a number of gram-positive bacteria, including Listeria, the general stress response is regulated by the alternative sigma factor B (SigB). Common stressors which lead to the activation of SigB and the SigB-dependent regulon are high osmolarity, acid and several more. Recently is has been shown that also blue and red light activates SigB in Bacillus subtilis. Methodology/Principal Findings: By qRT-PCR we analyzed the transcriptional response of the pathogen L. monocytogenes to blue and red light in wild type bacteria and in isogenic deletion mutants for the putative blue-light receptor Lmo0799 and the stress sigma factor SigB. It was found that both blue (455 nm) and red (625 nm) light induced the transcription of sigB and SigB-dependent genes, this induction was completely abolished in the SigB mutant. The blue-light effect was largely dependent on Lmo0799, proving that this protein is a genuine blue-light receptor. The deletion of lmo0799 enhanced the red-light effect, the underlying mechanism as well as that of SigB activation by red light remains unknown. Blue light led to an increased transcription of the internalin A/B genes and of bacterial invasiveness for Caco-2 enterocytes. Exposure to blue light also strongly inhibited swimming motility of the bacteria in a Lmo0799- and SigB-dependent manner, red light had no effect there. Conclusions/Significance: Our data established that visible, in particular blue light is an important environmental signal with an impact on gene expression and physiology of the non-phototrophic bacterium L. monocytogenes. In natural environments these effects will result in sometimes random but potentially also cyclic fluctuations of gene activity, depending on the light conditions prevailing in the respective habitat

A Small RNA Controls Expression of the Chitinase ChiA in Listeria monocytogenes

In recent years, more than 60 small RNAs (sRNAs) have been identified in the gram-positive human pathogen Listeria monocytogenes, but their putative roles and mechanisms of action remain largely unknown. The sRNA LhrA was recently shown to be a post-transcriptional regulator of a single gene, lmo0850, which encodes a small protein of unknown function. LhrA controls the translation and degradation of the lmo0850 mRNA by an antisense mechanism, and it depends on the RNA chaperone Hfq for efficient binding to its target. In the present study, we sought to gain more insight into the functional role of LhrA in L. monocytogenes. To this end, we determined the effects of LhrA on global-wide gene expression. We observed that nearly 300 genes in L. monocytogenes are either positively or negatively affected by LhrA. Among these genes, we identified lmo0302 and chiA as direct targets of LhrA, thus establishing LhrA as a multiple target regulator. Lmo0302 encodes a hypothetical protein with no known function, whereas chiA encodes one of two chitinases present in L. monocytogenes. We show here that LhrA acts as a post-transcriptional regulator of lmo0302 and chiA by interfering with ribosome recruitment, and we provide evidence that both LhrA and Hfq act to down-regulate the expression of lmo0302 and chiA. Furthermore, in vitro binding experiments show that Hfq stimulates the base pairing of LhrA to chiA mRNA. Finally, we demonstrate that LhrA has a negative effect on the chitinolytic activity of L. monocytogenes. In marked contrast to this, we found that Hfq has a stimulating effect on the chitinolytic activity, suggesting that Hfq plays multiple roles in the complex regulatory pathways controlling the chitinases of L. monocytogenes

Characterization of ecologically diverse viruses infecting co-occurring strains of cosmopolitan hyperhalophilic bacteroidetes

Hypersaline environments close to saturation harbor the highest density of virus-like particles reported for aquatic systems as well as low microbial diversity. Thus, they offer unique settings for studying virus-host interactions in nature. However, no viruses have been isolated so far infecting the two most abundant inhabitants of these systems (that is, the euryarchaeon Haloquadratum walsbyi and the bacteroidetes Salinibacter ruber). Here, using three different co-occurring strains, we have isolated eight viruses infecting the ubiquitous S. ruber that constitute three new different genera (named as 'Holosalinivirus', 'Kryptosalinivirus' and 'Kairosalinivirus') according to their genomic traits, different host range, virus-host interaction capabilities and abundances in natural systems worldwide. Furthermore, to get a more complete and comprehensive view of S. ruber virus assemblages in nature, a microcosm experiment was set with a mixture of S. ruber strains challenged with a brine virus concentrate, and changes of viral populations were monitored by viral metagenomics. Only viruses closely related to kairosalinivirus (strictly lytic and wide host range) were enriched, despite their low initial abundance in the natural sample. Metagenomic analyses of the mesocosms allowed the complete recovery of kairosalinivirus genomes using an ad hoc assembly strategy as common viral metagenomic assembly tools failed despite their abundance, which underlines the limitations of current approaches. The increase of this type of viruses was accompanied by an increase in the diversity of the group, as shown by contig recruitment. These results are consistent with a scenario in which host range, not only virus and host abundances, is a key factor in determining virus fate in nature.This research was supported by the Spanish Ministry of Economy projects CLG2015_66686-C3-1 (to JA) and CLG2015_66686-C3-3 (to RRM), which were also supported with European Regional Development Fund (FEDER) funds