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.

Pseudomonas aeruginosa clinical blood isolates display significant phenotypic variability.

Pseudomonas aeruginosa is a significant threat in healthcare settings where it deploys a wide host of virulence factors to cause disease. Many virulence-related phenotypes such as pyocyanin production, biofilm formation, and twitching motility have been implicated in causing disease in a number of hosts. In this study, we investigate these three virulence factors in a collection of 22 clinical strains isolated from blood stream infections. Despite the fact that all 22 strains caused disease and came from the same body site of different patients, they show significant variability in assays for each of the three specific phenotypes examined. There was no significant correlation between the strength of the three phenotypes across our collection, suggesting that they can be independently modulated. Furthermore, strains deficient in each of the virulence-associated phenotypes examined could be identified. To understand the genetic basis of this variability we sequenced the genomes of the 22 strains. We found that the majority of genes responsible for pyocyanin production, biofilm formation, and twitching motility were highly conserved among the strains despite their phenotypic variability, suggesting that the phenotypic variability is likely due to regulatory changes. Our findings thus demonstrate that no one lab-assayed phenotype of pyocyanin production, biofilm production, and twitching motility is necessary for a P. aeruginosa strain to cause blood stream infection and that additional factors may be needed to fully predict what strains will lead to specific human diseases.

Pseudomonas aeruginosa detachment from surfaces via a self-made small molecule.

Pseudomonas aeruginosa is a significant threat in both healthcare and industrial biofouling. Surface attachment of P. aeruginosa is particularly problematic as surface association induces virulence and is necessary for the ensuing process of biofilm formation, which hampers antibiotic treatments. Previous efforts have searched for dispersal agents of mature biofilm collectives, but there are no known factors that specifically disperse individual surface-attached P. aeruginosa. In this study, we develop a quantitative single-cell surface-dispersal assay and use it to show that P. aeruginosa itself produces factors that can stimulate its dispersal. Through bioactivity-guided fractionation, mass spectrometry, and nuclear magnetic resonance, we elucidated the structure of one such factor, 2-methyl-4-hydroxyquinoline (MHQ). MHQ is an alkyl quinolone with a previously unknown activity and is synthesized by the PqsABC enzymes. Pure MHQ is sufficient to disperse P. aeruginosa, but the dispersal activity of natural P. aeruginosa conditioned media requires additional factors. Whereas other alkyl quinolones have been shown to act as antibiotics or membrane depolarizers, MHQ lacks these activities and known antibiotics do not induce dispersal. In contrast, we show that MHQ inhibits the activity of Type IV Pili (TFP) and that TFP targeting can explain its dispersal activity. Our work thus identifies single-cell surface dispersal as a new activity of P. aeruginosa-produced small molecules, characterizes MHQ as a promising dispersal agent, and establishes TFP inhibition as a viable mechanism for P. aeruginosa dispersal.

Nutritional control of antibiotic production by Streptomyces platensis MA7327: importance of l-aspartic acid.

Streptomyces platensis MA7327 is a bacterium producing interesting antibiotics, which act by the novel mechanism of inhibiting fatty acid biosynthesis. The antibiotics produced by this actinomycete are platensimycin and platencin plus some minor related antibiotics. Platensimycin and platencin have activity against antibiotic-resistant bacteria such as methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus; they also lack toxicity in animal models. Platensimycin also has activity against diabetes in a mouse model. We have been interested in studying the effects of primary metabolites on production of these antibiotics in our chemically defined production medium. In the present work, we tested 32 primary metabolites for their effect. They included 20 amino acids, 7 vitamins and 5 nucleic acid derivatives. Of these, only l-aspartic acid showed stimulation of antibiotic production. We conclude that the stimulatory effect of aspartic acid is due to its role as a precursor involved in the biosynthesis of aspartate-4-semialdehyde, which is the starting point for the biosynthesis of the 3-amino-2,4-dihydroxy benzoic acid portion of the platensimycin molecule.

The Vibrio cholerae mannitol transporter is regulated posttranscriptionally by the MtlS small regulatory RNA.

Vibrio cholerae continues to pose a health threat in many developing nations and regions of the world struck by natural disasters. It is a pathogen that rapidly adapts to aquatic environments and the human small intestine. Small regulatory RNAs (sRNAs) may contribute to this adaptability. Specifically, the mannitol operon sRNA (MtlS sRNA; previously designated the IGR7 sRNA) is transcribed antisense to the 5' untranslated region of the mtl operon, encoding the mannitol-specific phosphotransferase system. Mannitol is a six-carbon sugar alcohol that accumulates in the human small intestine, the primary site of V. cholerae colonization. To better understand the V. cholerae mtl operon at a molecular level, we investigated mtlA expression in the presence of various carbon sources and the role of the MtlS sRNA. We observed that MtlA protein is present only in cells grown on mannitol sugar, whereas MtlS sRNA is expressed during growth on all sugars other than mannitol. In contrast, mtlA mRNA is expressed in similar amounts regardless of the carbon source used for bacterial growth. These observations suggest that the regulation of MtlA protein expression is a posttranscriptional event. We further demonstrate that MtlS sRNA overexpression repressed MtlA synthesis without affecting the stability of the messenger and that this process is largely independent of Hfq. We propose a model in which, when carbon sources other than mannitol are present, MtlS sRNA is transcribed, base pairs with the 5' untranslated region of the mtlA mRNA, occluding the ribosome binding site, and inhibits the synthesis of the mannitol-specific phosphotransferase system.