De Novo Amino Acid Biosynthesis Contributes to Salmonella enterica Growth in Alfalfa Seedling Exudates

Abstract

Salmonella enterica is a member of the plant microbiome. Growth of S. enterica in sprouting-seed exudates is rapid; however, the active metabolic networks essential in this environment are unknown. To examine the metabolic requirements of S. enterica during growth in sprouting-seed exudates, we inoculated alfalfa seeds and identified 305 S. enterica proteins extracted 24 h postinoculation from planktonic cells. Over half the proteins had known metabolic functions, and they are involved in over onequarter of the known metabolic reactions. Ion and metabolite transport accounted for the majority of detected reactions. Proteins involved in amino acid transport and metabolism were highly represented, suggesting that amino acid metabolic networks may be important for S. enterica growth in association with roots. Amino acid auxotroph growth phenotypes agreed with the proteomic data; auxotrophs in amino acid-biosynthetic pathways that were detected in our screen developed growth defects by 48 h. When the perceived sufficiency of each amino acid was expressed as a ratio of the calculated biomass requirement to the available concentration and compared to growth of each amino acid auxotroph, a correlation between nutrient availability and bacterial growth was found. Furthermore, glutamate transport acted as a fitness factor during S. enterica growth in association with roots. Collectively, these data suggest that S. enterica metabolism is robust in the germinating-alfalfa environment; that single-amino-acid metabolic pathways are important but not essential; and that targeting central metabolic networks, rather than dedicated pathways, may be necessary to achieve dramatic impacts on bacterial growth. P lants can influence both the number and species of bacteria that colonize their surfaces. The availability and types of nutrients, secretion of chemoattractants or repellents, and production of antimicrobial compounds all impact the composition of the plant-associated bacterial community. In particular, germinating seeds and roots release exudates containing complex mixtures of amino acids, sugars, organic acids, and other plant metabolites that support the growth of microbial colonists in the spermosphere and rhizosphere (1-5). Successful colonization of and persistence in these plant niches requires that bacteria acquire and metabolize these plant-derived nutrients and biosynthesize any required metabolites not sufficiently available from the plant.The compositions of seed and root exudates are dynamic and vary based on seed and plant age, soil type, temperature, nutritional status, and crop (6, 7). The identity and quantity of nutrients in plant exudates have a profound impact on bacterial growth and metabolic activity (8). Seeds that released larger quantities of carbohydrates and amino acids supported higher Enterobacter cloacae populations, rates of bacterial growth, and metabolic activity (3,4,8). Nutrient identity (e.g., alanine, arginine, and aspartate), not just type (e.g., amino acids, sugars, and lipids), influen...

Salmonella Persistence in Tomatoes Requires a Distinct Set of Metabolic Functions Identified by Transposon Insertion Sequencing

“…It involves major changes in the bacterial transcriptome and requires genes involved in amino acid biosynthesis and transport, cellulose production, fimbriae, regulators, and surface structures (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). Some of the same mechanisms were involved in the colonization of both vegetative and reproductive tissues on different plant species.…”

“…The siderophore transporter fepDGC was required for Salmonella proliferation in tomato pericarps (16), and siderophore synthesis was necessary for the colonization of lettuce and alfalfa (10). Biosynthesis of amino acids was also a likely factor required for Salmonella growth in alfalfa root exudates (15). Plant colonization by Salmonella is affected by host factors; the genetic background can influence Salmonella colonization of sprouts (18), proliferation in the tomato phyllosphere (19,20), and pericarps (21).…”

“…The capacity of S. enterica (and other enteric pathogens) to readily multiply within plant tissues led to the hypothesis that persistence on plants is a part of the Salmonella life cycle, serving as reservoirs prior to reinfection of the preferred animal hosts (2)(3)(4)(5). Several studies have dissected the molecular basis of plant-Salmonella interactions (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16). However, the entire complement of genetic functions required for plant colonization by Salmonella is not yet known.…”

Diguanylate Cyclases AdrA and STM1987 Regulate Salmonella enterica Exopolysaccharide Production during Plant Colonization in an Environment-Dependent Manner

“…Except for the real-time PCR analysis described above, all statistical analyses were performed using R software (version 2.14.1; R Development Core Team, R Foundation for Statistical Computing, Vienna, Austria [http://www.R-project.org]) as described previously (31). Briefly, three biological replicates were performed for each experiment, and samples taken from one replicate were considered subsamples.…”

“…It is difficult to determine what natural environments either LB or ATM resemble and thus, what cues are important for biofilm regulation. The nutrients exuded by the germinating seeds change over time and impact S. enterica growth (31,45). Thus, the nutrients that are available at 24 or 48 hpi may influence the requirement for different biofilm components.…”