|dc.description.abstract||In cold temperatures, the survival of Salmonella enterica serovar Typhimurium (S.
Typhimurium) requires the action of cold shock protein A (CspA) paralogues. These are
thought to melt misfolded ribonucleic acids, facilitating their translation at low temperatures.
However, through phenotypic analysis of our SL1344 csp null mutant (lacking all CspA
paralogues), it has been shown that CspA paralogues function during other environmental
stresses, outwith temperature reduction, and play an essential role in colony formation of an
SL1344 rpoS mutant at 37°C.
The general stress σ subunit, RpoS, plays an important role in adapting cells to a number of
stresses including oxidative stress, temperature changes, low pH and stationary phase. Under
such conditions, RpoS acts as an ‘emergency co-ordinator’, subsequently inducing the
transcription of necessary stress response genes. In Escherichia coli, RpoS is regulated posttranscriptionally
by at least three small RNAs (sRNAs): OxyS, DsrA and RprA; that require
interactions with the Sm-like RNA chaperone, Hfq. In S. Typhimurium, the stability of the
RpoS protein itself is regulated by ClpXP, an ATP-dependent protease responsible for RpoS
degradation, and a specific recognition factor that targets RpoS to this protease, MviA.
The present study has shown that the CspA paralogues of S. Typhimurium are involved in the
expression of RpoS and aims to elucidate the role of these proteins in RpoS production.
Comparative phenotypic tests were carried out in strains carrying mutations in rpoS, hfq and
the csp genes to gain insight into the interactions of Hfq and CspA paralogues, with respect to
RpoS expression. Both significant phenotypic overlaps, such as peroxide sensitivity, and
phenotypes unique to certain mutant strains, such as cold acclimation in the csp null strain,
were observed. CspA paralogues and Hfq are functionally distinct, not only in their
involvement in RpoS expression, but also in RpoS-independent processes, such as cold
acclimation, motility and to some extent, growth at 37°C.
The roles of Hfq and the CspA paralogues, in RpoS expression, were also assessed at the
molecular level. A combination of qRT-PCR analysis, transcriptional fusions and
immunoblotting (with anti-σ antibodies) has shown that DsrA and RprA are not essential for
RpoS expression in S. Typhimurium, during stationary phase or exponential cold shock, and
do not require Hfq under these conditions. Contrary to reports in E. coli, DsrA is not induced
upon cold shock in SL1344. Northern blots have shown that neither Hfq nor the CspA
paralogues are involved in regulating rpoS transcription during either stationary phase at 37°C
or cold shock in exponential phase. Immunoblotting and translational fusions have identified
different pathways for the regulation of RpoS during stationary phase at 37°C and cold shock
in exponential phase. Hfq is involved during the former condition only, whilst CspA
paralogues are involved in both. Protein stability experiments have shown that the CspA
paralogues do not play a major role in stabilising RpoS protein against degradation. Together,
these results have pointed to a role for both the CspA paralogues and Hfq in facilitating the
efficient translation of rpoS mRNA.
An SL1344 csp null rpoS mutant is unable to form colonies on LB agar at 37°C, a
phenomenon found when introducing combinations of mutations to SL1344 for phenotypic
assessment. A conditional rpoS mutant revealed that the SL1344 csp null rpoS strain is viable
but non-culturable. From the csp gene family, only cspA and cspB were able to restore colony
forming ability to the rpoS mutant. Further complementation experiments pointed to faulty
cell division, due to abnormal RNase E activity, as the cause.||en