rpoS mutants in archival cultures of Salmonella enterica serovar Typhimurium

Long-term survival under limited growth conditions presents bacterial popul ations with unique environmental challenges. The existence of Salmonella en terica serovar Typhimurium cultures undisturbed in sealed nutrient agar sta b vials for 34 to 45 years offered a unique opportunity to examine genetic variability under natural conditions. We have initiated a study of genetic changes in these archival cultures, We chose to start with examination of t he rpoS gene since, among gram-negative bacteria, many genes needed for sur vival are regulated by RpoS, the stationary-phase sigma factor. In each of 27 vials examined, cells had the rpoS start codon UUG instead of the expect ed AUG of Salmonella and Escherichia coli strains recorded in GenBank. Ten of the 27 had additional mutations in the rpoS gene compared with the X7775 2 wild-type strain currently recorded in GenBank. The rpoS mutations in the 10 strains included two deletions as well as point mutations that altered amino acid sequences substantially, Since these stored strains were derived from ancestral cells inoculated decades ago and remained undisturbed, it i s assumed that the 10 rpoS mutations occurred during storage. Since the rem aining 17 sequences were wild type (other than in the start codon), it is o bvious that rpoS remained relatively stable during decades of sealed storag e.