A molecular genetic investigation into stress sensing in the food-borne pathogen Listeria Monocytogenes: roles for RsbR and its paralogues

The alternative sigma factor σB is conserved across several Gram-positive bacteria species as the major general stress response (GSR) regulator. For the food-borne pathogen Listeria monocytogenes, σB enables the bacterium to persist in growth-limiting environments, thus posing a serious concern for food processing industries. Activation of σB is dependent on signalling from a multiprotein stress-sensing complex known as the stressosome. Here, we investigate the individual roles of the five identified stress sensor proteins: RsbR, the blue-light photoreceptor Lmo0799, Lmo0161, Lmo1642 and Lmo1842 which are thought to form a multi-protein stressosome complex. Mutant strains were constructed or obtained, each lacking RsbR or one of its-paralogues and were subjected to a number of phenotypic tests. Blue (460-470 nm) light was found to have a clear inhibitory effect on growth. Removal of the Lmo0799 protein, whose light-sensing function was proven to be dependent on a conserved cysteine residue at position 56, did not affect σB-mediated survival in the presence of higher intensity blue light. Phenotypic test results suggested that there may be redundancy in stress sensing between several of the RsbR paralogues but the RsbR protein plays a core structural role in stressosome formation. Novel phenotypes initially observed for the Δlmo1842 and Δlmo1642 strains were found to be caused by the presence of an RsbV I23T amino acid replacement which prevented σB activation at 37 °C but not 30 °C. Additionally, we identified that at low levels of stress, the ΔsigB mutant displayed a growth advantage over the wild-type, highlighting the energy cost associated with activating the GSR. These findings provide new insight into the mechanisms by which L. monocytogenes senses and responds to its environment and may have potential implications for control of this pathogen in food environments.2018-02-0

Loss of sigb in listeria monocytogenes strains egd-e and 10403s confers hyperresistance to hydrogen peroxide in stationary phase under aerobic conditions

SigB is the main stress gene regulator in Listeria monocytogenes affecting the expression of more than 150 genes and thus contributing to multiple-stress resistance. Despite its clear role in most stresses, its role in oxidative stress is uncertain, as results accompanying the loss of sigB range from hyperresistance to hypersensitivity. Previously, these differences have been attributed to strain variation. In this study, we show conclusively that unlike for all other stresses, loss of sigB results in hyperresistance to H2O2 (more than 8 log CFU ml(-1) compared to the wild type) in aerobically grown stationary-phase cultures of L. monocytogenes strains 10403S and EGD-e. Furthermore, growth at 30 degrees C resulted in higher resistance to oxidative stress than that at 37 degrees C. Oxidative stress resistance seemed to be higher with higher levels of oxygen. Under anaerobic conditions, the loss of SigB in 10403S did not affect survival against H2O2, while in EGD-e, it resulted in a sensitive phenotype. During exponential phase, minor differences occurred, and this result was expected due to the absence of sigB transcription. Catalase tests were performed under all conditions, and stronger catalase results corresponded well with a higher survival rate, underpinning the important role of catalase in this phenotype. Furthermore, we assessed the catalase activity in protein lysates, which corresponded with the catalase tests and survival. In addition, reverse transcription-PCR (RT-PCR) showed no differences in transcription between the wild type and the Delta sigB mutant in various oxidative stress genes. Further investigation of the molecular mechanism behind this phenotype and its possible consequences for the overall phenotype of L. monocytogenes are under way. IMPORTANCE SigB is the most important stress gene regulator in L. monocytogenes and other Gram-positive bacteria. Its increased expression during stationary phase results in resistance to multiple stresses. However, despite its important role in general stress resistance, its expression is detrimental for the cell in the presence of oxidative stress, as it promotes hypersensitivity against hydrogen peroxide. This peculiar phenotype is an important element of the physiology of L. monocytogenes, and it might help us explain the behavior of this organism in environments where oxidative stress is present