Previously uncharacterized Salmonella enterica genes required for swarming play a role in seedling colonization

Abstract

Incidences of bacterial foodborne illness caused by ingestion of fresh produce are rising. Instead of this being due to incidental contamination, the animal pathogen Salmonella enterica utilizes specific molecular mechanisms to attach to and colonize plants. This work characterizes two S. enterica genes of unknown function: a putative periplasmic protein, STM0278, and a putative protein with a hydrolase in the C-terminus, STM0650. STM0278 and STM0650 are important for seedling colonization but appear to have different roles during the process of colonization. Mutants of either STM0278 or STM0650 showed reduced colonization of alfalfa seedlings at 24 h, and the STM0278 mutant also showed reduced colonization at 48 h. Both genes were expressed in planta at 4 h following inoculation of 3-day-old seedlings and at 72 h after seed inoculation. This suggests that the role of STM0650 in seedling colonization is less important later in the process or is duplicated by other mechanisms. Mutants of STM0278 and STM0650 were defective in swarming. The STM0278 mutant failed to swarm in 24 h, while swarming of the STM0650 mutant was delayed. Addition of surfactant restored swarming of the STM0278 mutant, suggesting that STM0278 is involved in surfactant or osmotic agent production or deployment. Alfalfa seed exudates as the sole nutrient source were capable of perpetuating S. enterica swarming. Sequence analysis revealed sequences homologous to STM0278 and STM0650 in plant-associated bacteria, but none in Escherichia coli. Phylogenetic analysis of STM0650 showed similar sequences from diverse classes of plant-associated bacteria. Bacteria that preferentially colonize roots, including S. enterica, may use a similar hydrolase for swarming or biofilm production on plants. Multicellular behaviours by S. enterica appear central to plant colonization. S. enterica genes involved in plant colonization and survival outside of a host are most likely among the ‘function unknown’ genes of this bacterium.