| Listeria monocytogenes, a gram-positive bacillus with low G+C content, causes listeriosis in humans and ruminants. As a saprophytic bacterium thriving in diverse environments, L. monocytogenes can survive and grow over a wide range of environmental conditions, temperatures from-0.4 to 45 ℃, pH as low as 2.5 and high osmolarity (10%-20% NaCl). Human listeriosis results from consumption of contaminated foods, manifested as gastroenteritis, meningitis, encephalitis, mother-to-fetus infections and septicaemia with mortality as high as 30%.Stress resistance is particularly important for pathogenic bacteria to establish successful infection. As a foodborne pathogen, L. monocytogenes has to confront complicated food processing environments for its survival outside the host and defence mechanisms of the host upon entry. The general stress-responsive alternative sigma factor sigma B (σ~B), which was first identified in Bacillus subtilis, plays a pivotal role in its resistance to environmental stresses of L. monocytogenes by binding to RNA polymerase to form the holoenzyme to initiate stress-responsive genes. However, σ~B is also regulated by its regulator. Similar to σ~B of B. subtilis, L. monocytogenes σ~B is coexpressed with seven of its principal regulators (Regulator of.Sigma B:rsbR, rsbS, rsbT, rsbU, rsbV, rsbW and rsbX). Partner switching upon phosphorylation and dephosphorylation is the main regulatory mechanisms of this protein cluster in response to stresses. It is known that the dephosphorylated RsbV competes for RsbW resulting in σ~B liberation to turn-on the expression of stress-response genes. However, not so much is known about the σ~B turn-off mechanism.In this study, we attempted to(1) establish a reliable reporter plasmid containing the aB-promoter fused with gfp gene for analysis of σ~B activity of Listeria monocytogenes exposed to stresses; (2) explore stress-responsive genes and relationship between different stresses in L. monocytogenes; (3) investigate the role of RsbX in σ~B regulation of L. monocytogenes to different mode of stress and in stationary phase.SOE-PCR was used to construct recombinant plasmids on the pERL3 backbone carrying gfp fused with σ~B-dependent promoters of three L. monocytogenes genes. L. monocytogenes strains carrying the recombinant plasmids were exposed to acid or sodium stress and σ~B8 activity was measured and expreesed as relative fluorescence. We proved that PslgB of the sigB itself was more sensitive than the other two promoters P0880 å'Œ P1602 as an indicator of σ~B activity. Sodium stress was a more effective σ~B activator than acid stress. Stress with 5% NaCl for duration of 30 min could lead to effective aB activation. Thus, the GFP reporter plasmid is a reliable tool for measuring σ~B activity in L. monocytogenes as part of mechanistic studies of σ~B activation or regulation in response to stresses.RNA-seq was employed to examine the transcriptomic profiles of L. monocytogenes subjected to acid (pH 4.8), sodium salt (5% NaCl) or oxidative (5mM H2O2) stress. We obtained 26,29 and 55 differential expressed genes (DEGs) under acid, salt and oxidative stress respectively (>2-fold changes, P<0.05). The gene ontology (GO) analysis with DEGs revealed that most of the genes were closely related to metabolic processes. Principal component analysis (PC A) of expression magnitudes across all treatments indicated that osmotic and oxidative groups showed similar pattern of expression, but different from acidic stress. These results provide clues for selection of stress factors in further studies of the regulatory mechanisms of L. monocytogenes to stress.As a relatively independent σ~B regulator, RsbX is regarded as the only negative regulator among the seven σ~B operon genes. By genetic deletion and complementation, we found that RsbX did not affect growth in BHI medium at pH 4.8 or in BHI with 5% NaCl (mild acidic and sodium stresses), nor in BHI with high sodium (15% NaCl) stress although it did have some negative effect on listerial survival at sublethal acidic pH (3.5). We also found deletion of rsbX did not affect stress cross-protection. No matter the adaptive or protected stress was acid or sodium, the wild-type and the rsbX mutant showed similar survival after two rounds of continuous stress. In addition, there was virtually no difference in aB expression between the ArsbX mutant and its parent strain in response to 5% NaCl stress. The above findings suggest that RsbX does not have direct involvement in primary environmental stresses and stress-induced σ~B activation.In order to explore the additional role of RsbX in stress response, we initiated an approach of primary stress-recovery growth-secondary stress. We found that the â–³rsbX mutant had higher survival rate to the secondary stress than its parent strain after a 0.5-h or 1.5-h recovery period. Analyses by quantitative PCR, PsigB::gfp reporter assay and Western blot revealed that σ~B was significantly increased at the transcriptional and translational levels of the â–³rsbX mutant in the recovery period, as compared with its parent strain. Therefore, we come to the conclusion that increased survival of the â–³rsbX mutant in response to secondary stress is related to elevated σ~B expression in recovery period following primary stress. Moreover, we saw reduced intracellular and extracellular ATP levels in the â–³rsbX mutant cultures at the end of stress or during the recovery period, suggesting that RsbX might be acting to save energy by down-regulating σ~B. This is supported by the fact that the â–³sigB mutant had markedly higher level of intracellular ATP than the wild-type strain and the â–³rsbX mutant. The ATP results indicate that the σ~B turn-off mechanism is meant to save energy, which strengthen the role of RsbX as a σ~B negative regulator.We further compared the responses to high salt stress of L. monocytogenes â–³rsbX and its parent strain at the exponential and stationary phases of growth. Although RsbX did not affect σ~B activity in exponential cultures, we saw more stress resistance and higher σ~B expression in the â–³rsbX mutant than its parent strain when the stationary cultures were shifted to sodium stress. Therefore, we believe that RsbX negatively regulates σ~B of the stationary cultures of L. monocytogenes.In summary, a reliable reporter plasmid for analysis of σ~B activity is constructed. We provide clear evidence that RsbX is a negative regulator of L. monocytogenes σ~B during the recovery period after the primary stress or in the stationary phase, thus affecting its survival under secondary stress. Such negative regulation may be a strategy of the bacterium for energy conservation. Transcriptomic profiling has revealed that L. monocytogenes responded differentially to different stresses with both salt and peroxide stresses sharing more common responsive genes than acidic stress. Our findings could facilitate better understanding of the regulatory mechanisms of L. monocytogenes to stress. |
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