Investigations On The Role Of The Global Regulator H-NS In The Survival Of Escherichia Coli In Stationary-Phase
Studies on stationary phase cultures of microorganisms in the laboratory have helped us understand the genetic and physiological basis of adaptation in their natural habitats. Stasis or decline in bacterial populations due to nutrient depletion during stationary phase has been shown to lead to the selection of mutants that are able to survive better than their parent. This phenomenon, where a mutant exhibits relatively better growth than its immediate parent, under the conditions that prevailed during its appearance, is termed as Growth Advantage in Stationary Phase (GASP) and constitutes a general strategy to survive prolonged stationary phase. A mutation conferring growth advantage in one environment, however, can result in the loss of fitness in another environment, typifying the environment-specificity of the adaptation. Several GASP loci have been isolated over the last two decades and they have enhanced our understanding of the rapid evolution of microorganisms under nutrient starvation and the forces driving it. The results presented in this thesis detail the isolation and characterization of one such survivor from the culture of an E. coli strain ZK819 that was grown for 28 days without any additional nutrients. The strain ZK819 carries the rpoS819 allele that was isolated as the first GASP mutation resulting in attenuated activity of the stationary phase - specific sigma factor RpoS. Among the several possible mutations accumulating in the survivors over this long incubation period, one was identified to be within the hns gene encoding the global regulator H-NS. Expression of a majority of genes regulated by H-NS is environmentally modulated and their products are required under various stress conditions. The presence of the hns66 mutation was identified by the derepression of the bgl operon, one of the well-studied targets of H-NS mediated repression. The primary question being addressed in this study is whether this naturally isolated hns66 mutation has any role in the survival of its bearer during prolonged stationary phase. The hns66 mutation results in a partially active longer polypeptide due to a single nucleotide alteration within the stop codon of the H-NS ORF. Strains carrying the hns66 allele showed differential fitness under two different environments relative to the parent. In early stationary phase there is a strong disadvantage associated with the hns66 allele in the rpoS819 background, when competed against the parent, highlighted by the observation that there is a sharp fall in the number of mutants and a concomitant appearance of revertants that lead to the rapid displacement of the original mutant. The reversion is predominantly due to suppressor mutations within the ssrA locus encoding a tmRNA involved in the release of stalled ribosomes and degradation of aberrant proteins. Analysis of the suppression phenomenon revealed that the altered H-NS66 protein is a substrate for SsrA-mediated tagging for proteolysis. The H-NS66 protein is sub-optimally functional and can repress the bgl operon when its level is increased either by over-expression from a plasmid or a mutation in ssrA. Mutation in ssrA suppressed the strong growth disadvantage displayed by the original survivor in early stationary phase. The growth disadvantage of the hns66 survivor was also lost upon introduction of the wild type rpoS allele, indicating that the combined presence of the hns66 and rpoS819 alleles is detrimental in early stationary phase. In the presence of the rpoS+ allele, the hns66 mutant showed a modet GASP phenotype relative to the parent in early stationary phase. In contrast to the crash observed during early stationary phase, strains carryingthe hns66 allele fared better in late stationary phase conditions compared to the parent. When present in its original genetic context, the hns66 allele conferred growth advantage to the original survivor in late stationary phase conditions when competed against the parent grown for 21 days. The hns66 allele, when introduced in the parent strain ZK819 by transduction could still confer a strong GASP phenotype when competed against one-day old parent cells in medium derived from a 21 day old culture. These observations suggest that the hns66 allele enables the cells to scavenge for limited quantities of specific nutrients available in the aged medium. This is consistent with the observation that the advantage is seen when the mutant is in minority and is lost when the mutant is in majority. These studies highlight the fact that the conditions under stationary phase are constantly changing and to adapt under these rapidly changing conditions, modulation of an already existing function would be a preferred strategy than selection for a new function or the complete loss of a function. In this context, genetic modulation of a global regulator offers a better option due to the pleiotropic effects it may generate. This study adds hns to the list of genes, which upon mutation confer a GASP phenotype to cells. It also highlights that the age of the cells as well as the medium has an impact on the GASP phenotype. Elucidation of the mechanism of the H-NS mediated growth advantage and its relation to the status of the rpoS locus can contribute to our understanding of the dynamics of the stationary phase.