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

Abstract

In Listeria monocytogenes, the full details of how stress signals are integrated into the σB regulatory pathway are not yet available. To help shed light on this question, we investigated a collection of transposon mutants that were predicted to have compromised activity of the alternative sigma factor B (σB). These mutants were tested for acid tolerance, a trait that is known to be under σB regulation, and they were found to display increased acid sensitivity, similar to a mutant lacking σB (ΔsigB). The transposon insertions were confirmed by whole-genome sequencing, but in each case, the strains were also found to carry a frameshift mutation in the sigB operon. The changes were predicted to result in premature stop codons, with negative consequences for σB activation, independently of the transposon location. Reduced σB activation in these mutants was confirmed. Growth measurements under conditions similar to those used during the construction of the transposon library revealed that the frameshifted sigB operon alleles conferred a growth advantage at higher temperatures, during late exponential phase. Mixed-culture experiments at 42°C demonstrated that the loss of σB activity allowed mutants to take over a population of parental bacteria. Together, our results suggest that mutations affecting σB activity can arise during laboratory culture because of the growth advantage conferred by these mutations under mild stress conditions. The data highlight the significant cost of stress protection in this foodborne pathogen and emphasize the need for whole-genome sequence analysis of newly constructed strains to confirm the expected genotype. IMPORTANCE In the present study, we investigated a collection of Listeria monocytogenes strains that all carried sigB operon mutations. The mutants all had reduced σB activity and were found to have a growth advantage under conditions of mild heat stress (42°C). In mixed cultures, these mutants outcompeted the wild type when mild heat stress was present but not at an optimal growth temperature. An analysis of 22,340 published L. monocytogenes genome sequences found a high rate of premature stop codons present in genes positively regulating σB activity. Together, these findings suggest that the occurrence of mutations that attenuate σB activity can be favored under conditions of mild stress, probably highlighting the burden on cellular resources that stems from deploying the general stress response.

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

“…Both regulators are modulated by complex multi-layered control circuitry, highlighting the importance to fitness of deploying these systems only when the prevailing conditions are suitable. We have seen clear evidence that there is a significant burden on resources associated with deploying the GSR (Guerreiro et al, 2020) and a similar cost has been reported for inappropriate activation of PrfA (Bruno and Freitag, 2010). Clarification of the nature of the regulatory crosstalk between these systems will give new insights into the biology of this human pathogen as well as suggesting new approaches to control it.…”

“…Following the release of RsbT and consequent activation of σ B , a negative feedback mechanism controlled by the phosphatase RsbX allows the stressosome to be reset to its non-stressed dephosphorylated state, which leads to the recapture of RsbT ( Voelker et al, 1997 ; Chen et al, 2004 ; Eymann et al, 2011 ). Deletion of rsbX produces a constitutive σ B activation and consequently increased survival in acidic conditions ( Xia et al, 2016 ) and a reduced competitiveness against a WT strain, a consequence of the reduced growth rate associated with increased σ B activity ( Guerreiro et al, 2020 ) (see section “σ B Deployment Is a Double-Edged Sword”).…”

Understanding How Microorganisms Respond to Acid pH Is Central to Their Control and Successful Exploitation

“…Metagenomic studies can give information on many levels starting from species composition down to specific pathways and genes (Quatrini and Johnson, 2018). Linking genomics tools with phenotypical studies will definitely bring more insight into genetic mechanisms behind microbial responses to acidic environment (Guerreiro et al, 2020). Mathematical modeling and computational approaches will also play an important role in understanding microbial physiological processes during acid adaptation (Mancini et al, 2020) and interactions between bacteria (Ratzke and Gore, 2017).…”

Redefining the Clostridioides difficile σ B Regulon: σ B Activates Genes Involved in Detoxifying Radicals That Can Result from the Exposure to Antimicrobials and Hydrogen Peroxide

“…In C. difficile , the phosphatase RsbZ is responsible for RsbV dephosphorylation ( 11 ). The tight regulation of σ B activity by a partner-switching mechanism is necessary, as the energy burden associated with σ B activity was found to be disadvantageous in several different organisms ( 12 , 13 ).…”