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 is the formation of slow-growing subpopulations termed small colony variants (SCVs). Here we characterize an SCV, which spontaneously emerged from an axenic Salmonella enterica serovar Typhimurium water culture. We found that the SCV harbors a frameshift mutation in the glutamine synthetase gene glnA leading to a ∼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 required for the synthesis of cellular compounds. The internal glutamine pool serves as an indicator for nitrogen availability in Salmonella In our study, the SCV and a constructed glnA knockout mutant showed reduced growth rates compared to the wild type. Moreover, the SCV and glnA mutant displayed attenuated entry into host cells and severely reduced levels of exoproteins, including flagellin and several Salmonella 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 glnA mutant had severely decreased expression of flagellin genes, several SPI-1 effector genes and a class-2 ( flgB ) motility gene. However, the expression of a class 1 motility gene ( flhD ) was not affected. Supplementation with glutamine or genetic reversion of glnA truncation restored growth, cell entry, gene expression and protein abundance. In summary, our data show that glnA is essential for growth of S. enterica and controls important motility- and virulence-related traits in response to glutamine availability. IMPORTANCE Salmonella enterica serovar Typhimurium is a significant pathogen causing food-borne infections. Here we describe an S Typhimurium small colony variant (SCV) that spontaneously emerged from a long-term starvation experiment in water. It is important to study SCVs because they a) may arise spontaneously upon stress exposure including environmental and host-defense stresses, b) are slow growing and difficult to eradicate, and c) only few descriptions of S enterica SCVs are available. We clarify the genetic basis of the SCV described here as frameshift mutation in the gene of glutamine synthetase, glnA, leading to glutamine auxotrophy. In Salmonella , internal glutamine limitation serves as a signal for external nitrogen deficiency and is thought to regulate cell growth. In addition to its impaired growth, the SCV showed reduced host cell entry and reduced expression of SPI-1 virulence and flagellin genes.