glnA Truncation in Salmonella enterica Results in a Small Colony Variant Phenotype, Attenuated Host Cell Entry, and Reduced Expression of Flagellin and SPI-1-Associated Effector Genes

Many pathogenic bacteria use sophisticated survival strategies to overcome harsh environmental conditions. One strategy is the formation of slow-growing subpopulations termed small colony variants (SCVs). Here we characterize an SCV that spontaneously emerged from an axenicSalmonella enterica serovar Typhimurium water culture. We found that the SCV harbored a frameshift mutation in the glutamine synthetase geneglnA , leading to an ∼90% truncation of the corresponding protein. Glutamine synthetase, a central enzyme in nitrogen assimilation, converts glutamate and ammonia to glutamine. Glutamine is an important nitrogen donor that is required for the synthesis of cellular compounds. The internal glutamine pool serves as an indicator of nitrogen availability inSalmonella . In our study, the SCV and a constructedglnA knockout mutant showed reduced growth rates, compared to the wild type. Moreover, the SCV and theglnA mutant displayed attenuated entry into host cells and severely reduced levels of exoproteins, including flagellin and severalSalmonella pathogenicity island 1 (SPI-1)-dependent secreted virulence factors. We found that these proteins were also depleted in cell lysates, indicating their diminished synthesis. Accordingly, the SCV and theglnA mutant had severely decreased expression of flagellin genes, several SPI-1 effector genes, and a class 2 motility gene (flgB ). However, the expression of a class 1 motility gene (flhD ) was not affected. Supplementation with glutamine or genetic reversion of theglnA truncation restored growth, cell entry, gene expression, and protein abundance. In summary, our data show thatglnA is essential for the growth ofS. enterica and controls important motility- and virulence-related traits in response to glutamine availability.IMPORTANCE Salmonella enterica serovar Typhimurium is a significant pathogen causing foodborne infections. Here we describe anS . Typhimurium small colony variant (SCV) that spontaneously emerged from a long-term starvation experiment in water. It is important to study SCVs because (i) SCVs may arise spontaneously upon exposure to stresses, including environmental and host defense stresses, (ii) SCVs are slow growing and difficult to eradicate, and (iii) only a few descriptions ofS. enterica SCVs are available. We clarify the genetic basis of the SCV described here as a frameshift mutation in the glutamine synthetase geneglnA , leading to glutamine auxotrophy. InSalmonella , internal glutamine limitation serves as a sign of external nitrogen deficiency and is thought to regulate cell growth. In addition to exhibiting impaired growth, the SCV showed reduced host cell entry and reduced expression of SPI-1 virulence and flagellin genes.