A single point mutation in the Listeria monocytogenes ribosomal gene rpsU enables SigB activation independently of the stressosome and the anti-sigma factor antagonist RsbV

Microbial population heterogeneity leads to different stress responses and growth behavior of individual cells in a population. Previously, a point mutation in the rpsU gene (rpsUG50C) encoding ribosomal protein S21 was identified in a Listeria monocytogenes LO28 variant, which leads to increased multi-stress resistance and a reduced maximum specific growth rate. However, the underlying mechanisms of these phenotypic changes remain unknown. In L. monocytogenes, the alternative sigma factor SigB regulates the general stress response, with its activation controlled by a series of Rsb proteins, including RsbR1 and anti-sigma factor RsbW and its antagonist RsbV. We combined a phenotype and proteomics approach to investigate the acid and heat stress resistance, growth rate, and SigB activation of L. monocytogenes EGDe wild type and the ΔsigB, ΔrsbV, and ΔrsbR1 mutant strains. While the introduction of rpsUG50C in the ΔsigB mutant did not induce a SigB-mediated increase in robustness, the presence of rpsUG50C in the ΔrsbV and the ΔrsbR1 mutants led to SigB activation and concomitant increased robustness, indicating an alternative signaling pathway for the SigB activation in rpsUG50C mutants. Interestingly, all these rpsUG50C mutants exhibited reduced maximum specific growth rates, independent of SigB activation, possibly attributed to compromised ribosomal functioning. In summary, the increased stress resistance in the L. monocytogenes EGDe rpsUG50C mutant results from SigB activation through an unknown mechanism distinct from the classical stressosome and RsbV/RsbW partner switching model. Moreover, the reduced maximum specific growth rate of the EGDe rpsUG50C mutant is likely unrelated to SigB activation and potentially linked to impaired ribosomal function

Divergent evolution of the activity and regulation of the glutamate decarboxylase systems in Listeria monocytogenes EGD-e and 10403S : roles in virulence and acid tolerance

The glutamate decarboxylase (GAD) system has been shown to be important for the survival of Listeria monocytogenes in low pH environments. The bacterium can use this faculty to maintain pH homeostasis under acidic conditions. The accepted model for the GAD system proposes that the antiport of glutamate into the bacterial cell in exchange for γ-aminobutyric acid (GABA) is coupled to an intracellular decarboxylation reaction of glutamate into GABA that consumes protons and therefore facilitates pH homeostasis. Most strains of L. monocytogenes possess three decarboxylase genes (gadD1, D2 & D3) and two antiporter genes (gadT1 & gadT2). Here, we confirm that the gadD3 encodes a glutamate decarboxylase dedicated to the intracellular GAD system (GADi), which produces GABA from cytoplasmic glutamate in the absence of antiport activity. We also compare the functionality of the GAD system between two commonly studied reference strains, EGD-e and 10403S with differences in terms of acid resistance. Through functional genomics we show that EGD-e is unable to export GABA and relies exclusively in the GADi system, which is driven primarily by GadD3 in this strain. In contrast 10403S relies upon GadD2 to maintain both an intracellular and extracellular GAD system (GADi/GADe). Through experiments with a murinised variant of EGD-e (EGDm) in mice, we found that the GAD system plays a significant role in the overall virulence of this strain. Double mutants lacking either gadD1D3 or gadD2D3 of the GAD system displayed reduced acid tolerance and were significantly affected in their ability to cause infection following oral inoculation. Since EGDm exploits GADi but not GADe the results indicate that the GADi system makes a contribution to virulence within the mouse. Furthermore, we also provide evidence that there might be a separate line of evolution in the GAD system between two commonly used reference strains

The role of stress and stress adaptations in determining the fate of the bacterial pathogen listeria monocytogenes in the food chain

The foodborne pathogen Listeria monocytogenes is a highly adaptable organism that can persist in a wide range of environmental and food-related niches. The consumption of contaminated ready-to-eat foods can cause infections, termed listeriosis, in vulnerable humans, particularly those with weakened immune systems. Although these infections are comparatively rare they are associated with high mortality rates and therefore this pathogen has a significant impact on food safety. L. monocytogenes can adapt to and survive a wide range of stress conditions including low pH, low water activity, and low temperature, which makes it problematic for food producers who rely on these stresses for preservation. Stress tolerance in L. monocytogenes can be explained partially by the presence of the general stress response (GSR), a transcriptional response under the control of the alternative sigma factor sigma B (GB) that reconfigures gene transcription to provide homeostatic and protective functions to cope with the stress. Within the host GB also plays a key role in surviving the harsh conditions found in the gastrointestinal tract. As the infection progresses beyond the GI tract L, monocytogenes uses an intracellular infectious cycle to propagate, spread and remain protected from the host\u27s humoral immunity. Many of the virulence genes that facilitate this infectious cycle are under the control of a master transcriptional regulator called PrIA. In this review we consider the environmental reservoirs that enable L. monocytogenes to gain access to the food chain and discuss the stresses that the pathogen must overcome to survive and grow in these environments. The overlap that exists between stress tolerance and virulence is described. We review the principal measures that are used to control the pathogen and point to exciting new approaches that might provide improved means of control in the future

The role of stress and stress adaptations in determining the fate of the bacterial pathogen listeria monocytogenes in the food chain

The foodborne pathogen Listeria monocytogenes is a highly adaptable organism that can persist in a wide range of environmental and food-related niches. The consumption of contaminated ready-to-eat foods can cause infections, termed listeriosis, in vulnerable humans, particularly those with weakened immune systems. Although these infections are comparatively rare they are associated with high mortality rates and therefore this pathogen has a significant impact on food safety. L. monocytogenes can adapt to and survive a wide range of stress conditions including low pH, low water activity, and low temperature, which makes it problematic for food producers who rely on these stresses for preservation. Stress tolerance in L. monocytogenes can be explained partially by the presence of the general stress response (GSR), a transcriptional response under the control of the alternative sigma factor sigma B (GB) that reconfigures gene transcription to provide homeostatic and protective functions to cope with the stress. Within the host GB also plays a key role in surviving the harsh conditions found in the gastrointestinal tract. As the infection progresses beyond the GI tract L, monocytogenes uses an intracellular infectious cycle to propagate, spread and remain protected from the host's humoral immunity. Many of the virulence genes that facilitate this infectious cycle are under the control of a master transcriptional regulator called PrIA. In this review we consider the environmental reservoirs that enable L. monocytogenes to gain access to the food chain and discuss the stresses that the pathogen must overcome to survive and grow in these environments. The overlap that exists between stress tolerance and virulence is described. We review the principal measures that are used to control the pathogen and point to exciting new approaches that might provide improved means of control in the future

Effects of growth phase and temperature onσbactivity within alisteria monocytogenespopulation: evidence for rsbv-independent activation ofσbat refrigeration temperatures

The alternative sigma factor sigma(B) of Listeria monocytogenes is responsible for regulating the transcription of many of the genes necessary for adaptation to both food-related stresses and to conditions found within the gastrointestinal tract of the host. The present study sought to investigate the influence of growth phase and temperature on the activation of sigma(B) within populations of L. monocytogenes EGD-e wild-type, Delta sigB, and Delta rsbV throughout growth at both 4 degrees C and 37 degrees C, using a reporter fusion that couples expression of EGFP to the strongly sigma(B)-dependent promoter of lmo2230. A similar sigma(B) activation pattern within the population was observed in wt-egfp at both temperatures, with the highest induction of sigma(B) occurring in the early exponential phase of growth when the fluorescent population rapidly increased, eventually reaching the maximum in early stationary phase. Interestingly, induction of sigma(B) activity was heterogeneous, with only a proportion of the cells in the wt-egfp population being fluorescent above the background autofluorescence level. Moreover, significant RsbV-independent activation of sigma(B) was observed during growth at 4 degrees C. This result suggests that an alternative route to sigma(B) activation exists in the absence of RsbV, a finding that is not explained by the current model for sigma(B) regulation

Effects of growth phase and temperature on sigma(B) Activity within a Listeria monocytogenes population: evidence for RsbV-independent activation of sigma(B) at refrigeration temperatures

Journal articleThe alternative sigma factor sigma(B) of Listeria monocytogenes is responsible for regulating the transcription of many of the genes necessary for adaptation to both food-related stresses and to conditions found within the gastrointestinal tract of the host. The present study sought to investigate the influence of growth phase and temperature on the activation of sigma(B) within populations of L. monocytogenes EGD-e wild-type, Delta sigB, and Delta rsbV throughout growth at both 4 degrees C and 37 degrees C, using a reporter fusion that couples expression of EGFP to the strongly sigma(B)-dependent promoter of lmo2230. A similar sigma(B) activation pattern within the population was observed in wt-egfp at both temperatures, with the highest induction of sigma(B) occurring in the early exponential phase of growth when the fluorescent population rapidly increased, eventually reaching the maximum in early stationary phase. Interestingly, induction of sigma(B) activity was heterogeneous, with only a proportion of the cells in the wt-egfp population being fluorescent above the background autofluorescence level. Moreover, significant RsbV-independent activation of sigma(B) was observed during growth at 4 degrees C. This result suggests that an alternative route to sigma(B) activation exists in the absence of RsbV, a finding that is not explained by the current model for sigma(B) regulation.Science Foundation Ireland Research Frontiers Programme Grant no. 05/RFP/GEN0044; SFI Grant no. SRC 09/SRC/B1794; SFI Stokes ProfessorshipPeer reviewe

Effects of growth phase and temperature on sigma(B) Activity within a Listeria monocytogenes population: evidence for RsbV-independent activation of sigma(B) at refrigeration temperatures

Journal articleThe alternative sigma factor sigma(B) of Listeria monocytogenes is responsible for regulating the transcription of many of the genes necessary for adaptation to both food-related stresses and to conditions found within the gastrointestinal tract of the host. The present study sought to investigate the influence of growth phase and temperature on the activation of sigma(B) within populations of L. monocytogenes EGD-e wild-type, Delta sigB, and Delta rsbV throughout growth at both 4 degrees C and 37 degrees C, using a reporter fusion that couples expression of EGFP to the strongly sigma(B)-dependent promoter of lmo2230. A similar sigma(B) activation pattern within the population was observed in wt-egfp at both temperatures, with the highest induction of sigma(B) occurring in the early exponential phase of growth when the fluorescent population rapidly increased, eventually reaching the maximum in early stationary phase. Interestingly, induction of sigma(B) activity was heterogeneous, with only a proportion of the cells in the wt-egfp population being fluorescent above the background autofluorescence level. Moreover, significant RsbV-independent activation of sigma(B) was observed during growth at 4 degrees C. This result suggests that an alternative route to sigma(B) activation exists in the absence of RsbV, a finding that is not explained by the current model for sigma(B) regulation.Science Foundation Ireland Research Frontiers Programme Grant no. 05/RFP/GEN0044; SFI Grant no. SRC 09/SRC/B1794; SFI Stokes ProfessorshipPeer reviewe

Effects of growth phase and temperature onσbactivity within alisteria monocytogenespopulation: evidence for rsbv-independent activation ofσbat refrigeration temperatures

The alternative sigma factor sigma(B) of Listeria monocytogenes is responsible for regulating the transcription of many of the genes necessary for adaptation to both food-related stresses and to conditions found within the gastrointestinal tract of the host. The present study sought to investigate the influence of growth phase and temperature on the activation of sigma(B) within populations of L. monocytogenes EGD-e wild-type, Delta sigB, and Delta rsbV throughout growth at both 4 degrees C and 37 degrees C, using a reporter fusion that couples expression of EGFP to the strongly sigma(B)-dependent promoter of lmo2230. A similar sigma(B) activation pattern within the population was observed in wt-egfp at both temperatures, with the highest induction of sigma(B) occurring in the early exponential phase of growth when the fluorescent population rapidly increased, eventually reaching the maximum in early stationary phase. Interestingly, induction of sigma(B) activity was heterogeneous, with only a proportion of the cells in the wt-egfp population being fluorescent above the background autofluorescence level. Moreover, significant RsbV-independent activation of sigma(B) was observed during growth at 4 degrees C. This result suggests that an alternative route to sigma(B) activation exists in the absence of RsbV, a finding that is not explained by the current model for sigma(B) regulation