A chemically-defined growth medium to support Lactobacillus-Acetobacter sp. community analysis.

Lactobacilli and Acetobacter sp. are commercially important bacteria that often form communities in natural fermentations, including food preparations, spoilage, and in the digestive tract of the fruit fly Drosophila melanogaster. Communities of these bacteria are widespread and prolific, despite numerous strain-specific auxotrophies, suggesting they have evolved nutrient interdependencies that regulate their growth. The use of a chemically-defined medium (CDM) supporting the growth of both groups of bacteria would facilitate the identification of the molecular mechanisms for the metabolic interactions between them. While numerous CDMs have been developed that support specific strains of lactobacilli or Acetobacter, there has not been a medium formulated to support both genera. We developed such a medium, based on a previous CDM designed for growth of lactobacilli, by modifying the nutrient abundances to improve growth yield. We further simplified the medium by substituting casamino acids in place of individual amino acids and the standard Wolfe's vitamins and mineral stocks in place of individual vitamins and minerals, resulting in a reduction from 40 to 8 stock solutions. These stock solutions can be used to prepare several CDM formulations that support robust growth of numerous lactobacilli and Acetobacters. Here, we provide the composition and several examples of its use, which is important for tractability in dissecting the genetic and metabolic basis of natural bacterial species interactions.

Listeria monocytogenes exopolysaccharide: origin, structure, biosynthetic machinery and c-di-GMP-dependent regulation.

Elevated levels of the second messenger c-di-GMP activate biosynthesis of an unknown exopolysaccharide (EPS) in the food-borne pathogen Listeria monocytogenes. This EPS strongly protects cells against disinfectants and desiccation, indicating its potential significance for listerial persistence in the environment and for food safety. We analyzed the potential phylogenetic origin of this EPS, determined its complete structure, characterized genes involved in its biosynthesis and hydrolysis and identified diguanylate cyclases activating its synthesis. Phylogenetic analysis of EPS biosynthesis proteins suggests that they have evolved within monoderms. Scanning electron microscopy revealed that L. monocytogenes EPS is cell surface-bound. Secreted carbohydrates represent exclusively cell-wall debris. Based on carbohydrate composition, linkage and NMR analysis, the structure of the purified EPS is identified as a ß-1,4-linked N-acetylmannosamine chain decorated with terminal α-1,6-linked galactose. All genes of the pssA-E operon are required for EPS production and so is a separately located pssZ gene. We show that PssZ has an EPS-specific glycosylhydrolase activity. Exogenously added PssZ prevents EPS-mediated cell aggregation and disperses preformed aggregates, whereas an E72Q mutant in the presumed catalytic residue is much less active. The diguanylate cyclases DgcA and DgcB, whose genes are located next to pssZ, are primarily responsible for c-di-GMP-dependent EPS production.

Cyclic di-GMP-dependent signaling pathways in the pathogenic Firmicute Listeria monocytogenes.

We characterized key components and major targets of the c-di-GMP signaling pathways in the foodborne pathogen Listeria monocytogenes, identified a new c-di-GMP-inducible exopolysaccharide responsible for motility inhibition, cell aggregation, and enhanced tolerance to disinfectants and desiccation, and provided first insights into the role of c-di-GMP signaling in listerial virulence. Genome-wide genetic and biochemical analyses of c-di-GMP signaling pathways revealed that L. monocytogenes has three GGDEF domain proteins, DgcA (Lmo1911), DgcB (Lmo1912) and DgcC (Lmo2174), that possess diguanylate cyclase activity, and three EAL domain proteins, PdeB (Lmo0131), PdeC (Lmo1914) and PdeD (Lmo0111), that possess c-di-GMP phosphodiesterase activity. Deletion of all phosphodiesterase genes (ΔpdeB/C/D) or expression of a heterologous diguanylate cyclase stimulated production of a previously unknown exopolysaccharide. The synthesis of this exopolysaccharide was attributed to the pssA-E (lmo0527-0531) gene cluster. The last gene of the cluster encodes the fourth listerial GGDEF domain protein, PssE, that functions as an I-site c-di-GMP receptor essential for exopolysaccharide synthesis. The c-di-GMP-inducible exopolysaccharide causes cell aggregation in minimal medium and impairs bacterial migration in semi-solid agar, however, it does not promote biofilm formation on abiotic surfaces. The exopolysaccharide also greatly enhances bacterial tolerance to commonly used disinfectants as well as desiccation, which may contribute to survival of L. monocytogenes on contaminated food products and in food-processing facilities. The exopolysaccharide and another, as yet unknown c-di-GMP-dependent target, drastically decrease listerial invasiveness in enterocytes in vitro, and lower pathogen load in the liver and gallbladder of mice infected via an oral route, which suggests that elevated c-di-GMP levels play an overall negative role in listerial virulence.

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus.

Vibrio parahaemolyticus isolates display variation in colony morphology, alternating between opaque (OP) and translucent (TR) cell types. Phase variation is the consequence of genetic alterations in the locus encoding the quorum sensing output regulator OpaR. Here, we show that both cell types form stable, but distinguishable biofilms that differ with respect to attachment and detachment profiles to polystyrene, pellicle formation and stability at the air/medium interface, and submerged biofilm architecture and dispersion at a solid/liquid interface. The pellicle, which is a cohesive mat of cells, was exploited to identify mutants having altered or defective biofilm formation. Transposon insertion mutants were obtained with defects in genes affecting multiple cell surface characteristics, including extracellular polysaccharide, mannose-sensitive haemagglutinin type 4 pili and polar (but not lateral) flagella. Other insertions disrupted genes coding for potential secreted proteins or transporters of secreted proteins, specifically haemolysin co-regulated protein and an RTX toxin-like membrane fusion transporter, as well as potential modifiers of cell surface molecules (nagAC operon). The pellicle screen also identified mutants with lesions in regulatory genes encoding H-NS, a CsgD-like repressor and an AraC-like protein. This work initiates the characterization of V. parahaemolyticus biofilm formation in the OP and TR cell types and identifies a diverse repertoire of cell surface elements that participate in determining multicellular architecture.