Alternative sigma factor σB has previously been shown to contribute to the ability of L.
monocytogenes, as well as other related organism such as B. subtilis and S. aureus, to survive under various environmental stress conditions including osmotic stress (Becker et al., 1998; Chan et al., 1998; Kazmierczak et al., 2003; Moorhead & Dykes, 2003; Raengpradub et al., 2008; Volker et al.,
152 1999). The sigB gene (lmo0895) itself showed no significant change in transcription during
hyperosmotic shock from the control state. It is possible that the σB activation is almost
instantaneous (occurs within seconds following stress) and has been missed in this experiment. Genes that have been shown to be positively up-regulated either directly or indirectly by this alternative sigma factor in L. monocytogenes (Abram et al., 2008; Hain et al., 2008; Raengpradub et al., 2008; Wemekamp-Kamphuis et al., 2004b)showed significant up-regulation (T-value score of 6.54, Table 4.3) and those genes shown to be more actively expressed in a sigB null mutant were down-regulated (T-value score of -3.37, Table 4.3).
Table 4.3Response of L. monocytogenes strain ATCC19115 genes to NaCl shock and adapted stresses based on regulons.
Regulon Shock Response Adapted Response
T-value* P-value# T-value* P-value#
HrcA+ -5.07 1.4E-05 5.50 3.9E-06
HrcA- 1.11 0.45 SigB- -3.31 0.0012 4.87 3.2E-06 SigB+ 6.54 6.0E-10 -7.79 CodY+ 1.25 -1.63 4.6E-13 CodY- 1.61 -7.02 5.9E-10 CtsR- 2.19 -2.60 0.0407 RpoN+ 3.22 0.0045 -0.56 RpoN- 4.80 1.1E-05 -3.26 0.0018
PrfA+ 5.65 4.0E-07 -4.24 7.4E-05
PrfA- 2.70 0.0354 -1.55
VirR+ 4.98 0.0002 0.29
*T-value scores were determined from the expression data using the approach of Boorsma et al. (2005).
# P-values not shown are >0.05
+ indicate +ve regulation by a given regulon, - indicate –ve regulation by a given regulon.
¥ The gene sets for the given regulatory proteins are designated from empirical deletion studies on the basis of what genes are positively
influenced (“+”) or negatively influence (“-“), The influence of the regulators in many cases are not direct thus T-profile data only gives a general trend in terms of expression responses
PrfA functions as a virulence regulator in L. monocytogenes which itself is positively regulated by σB
(Chakraborty et al., 1992; Kazmierczak et al., 2006; Nadon et al., 2002; Schwab et al., 2005). This transcriptional activator showed no significant change during hyperosmotic shock. Genes positively controlled or influenced indirectly by prfA on the other hand showed up-regulation (T-value score 5.65, Table 4.3). Additional virulence regulator VirR also showed activation in cold shocked cells with
T-value score of 4.98 (Table 4.3). A number of studies have previously shown the link between stress response and virulence in this pathogen. In order to cause disease the pathogen has to overcome a number of environmental obstacles, such as acid stress in the stomach, oxidative stress inside
153 macrophages and osmotic stress inside the gastrointestinal lumen. The regulatory mechanisms for stress response and virulence have evolved to be closely interrelated, thus activating one stimulates the other and vice versa. Interestingly genes suppressed by PrfA also showed overall activation (T- value score of 2.70, Table 4.3). This small group of genes mainly encodes for ABC transporters for sugars, it is possible that the influence of other regulatory proteins or non-coding RNAs influences the expression of this set of genes.
There was no evident direct correlation between CodY regulon and the salt shock response (Table 4.3) most likely due to interactions with other regulators as not all genes in its regulon are under sole direct control of CodY. In B. subtilis CodY helps to regulate transition from rapid exponential growth to the stationary growth phase (Molle et al., 2003; Shivers & Sonenshein, 2004; Sonenshein,
2005). As L. monocytogenes undergoes rapid transition from exponential growth in control medium
to suppressed growth (a temporary lag phase) due to encountered NaCl stress, CodY regulon
appears to not be directly involved in overcoming the imposed environmental stress, its involvement appears to be more apparent in the adaptive response to hyperosmotic stress with CodY repressed genes showing a strong down-regulation of -7.02 (as discussed previously in section 3.3.2.11). L. monocytogenes CodY represses genes associated with metabolism and transport of carbohydrates as well as amino acid biosynthesis (Bennett et al., 2007). CodY also has a major role in monitoring the energetic capacity and nutritional state of the cell (Sonenshein, 2005). During hyperosmotic
shock L. monocytogenes showed increased demand for energy in terms of carbohydrate uptake and
metabolism, which is re-repressed by inactive CodY regulator.
Hyperosmotic shock induced genes activated by rpoN regulator, also known as an alternative sigma factor σN or σ54. The σ54–dependent genes have a wide variety of cellular functions in bacteria from
nitrogen and carbon utilization to flagella synthesis and virulence (Arous et al., 2004; Buck et al., 2000; Studholme & Buck, 2000). In L. monocytogenesσ54 has been shown to assist in regulation of
mainly carbohydrate metabolism genes (Arous et al., 2004).The mpt operon, which is under positive
control of σ54, showed up-regulation of over 3.3-fold. This further emphasizes the involvement of alternative sigma factor σ54 in carbohydrate metabolism and subsequently its involvement in adaptation to hyperosmotic shock. Negative control of gene expression by σ54 was not very straight
forward. Some genes showed down-regulation such as rplF (lmo2617) which was repressed 2.6-fold.
154
σ54 control of gene expression is somewhat indirect (Arous et al., 2004) and interaction with other
regulatory factors during hyperosmotic shock is evident, at least in L. monocytogenes ATCC19115.
4.3.1.13 Genes with analogous responses during hyperosmotic shock and