The concentration of GABA increases rapidly in wounded plant tissues, but the implication of this GABA pulse for plant-bacteria interactions is not known. Here we reveal that GABA stimulated the inactivation of the N-(3-oxooctanoyl)homoserine lactone (OC8-HSL) quorum-sensing signal (or ''quormone'') by the Agrobacterium lactonase AttM. GABA induced the expression of the attKLM operon, which was correlated to a decrease in OC8-HSL concentration in Agrobacterium tumefaciens cultures. The Agrobacterium GABA transporter Bra was required for this GABA-signaling pathway. Furthermore, transgenic tobacco plants with elevated GABA levels were less sensitive to A. tumefaciens C58 infection than were wild-type plants. These findings indicate that plant GABA may modulate quorum sensing in A. tumefaciens, thereby affecting its virulence on plants. Whereas GABA is an essential cell-to-cell signal in eukaryotes, here we provide evidence of GABA acting as a signal between eukaryotes and pathogenic bacteria. The GABA signal represents a potential target for the development of a strategy to control the virulence of bacterial pathogens.phytopathology ͉ plant signal ͉ lactonase ͉ quorum quenching

Agrobacterium tumefaciens C58 communicates using N-acyl-homoserine lactones (acyl-HSL) and contains two lactonase-encoding genes, attM and aiiB, the products of which are capable of inactivating the acyl-HSL signal. In A. tumefaciens A6, the expression of the attKLM operon is controlled by the transcriptional repressor encoded by an adjacent gene, attJ. An attJ::Tn5 mutant does not accumulate acyl-HSL because of the constitutive expression of the lactonase AttM, the activity of which inactivates acyl-HSL. In this work, the attKLM operon of A. tumefaciens C58 was shown to be involved in an assimilative pathway of gamma-butyrolactone (GBL), gamma-hydroxybutyrate (GHB), and succinate semialdehyde (SSA), in which AttM and AttL are key enzymes for GBL and GHB assimilation. The expression of the attKLM promoter was activated in the presence of GBL, GHB, and SSA. Under these conditions, A. tumefaciens C58 did not accumulate the acyl-HSL that it naturally synthesizes, and also became able to inactivate exogenous acyl-HSL signals. Therefore, in A. tumefaciens C58, the assimilative pathway of gamma-butyrolactone interferes with the acyl-HSL signaling.

Colistin is a mixture of polymyxin E1 and E2, bactericidal pentacationic lipopeptides used to treat infections caused by Gram-negative pathogens such as Pseudomonas aeruginosa and Klebsiella pneumoniae. Industrial production of colistin is obtained by a fermentation process of the natural producer Paenibacillus polymyxa var colistinus. NonRibosomal peptide synthetases (NRPS) coding the biosynthesis of polymyxins A, B and P have been recently described, rendering thereof the improvement of their production possible. However, the colistin biosynthesis pathway was not published so far. In this study, a Paenibacillus alvei has been identified by biochemical (Api 50 CH system) and molecular (16S rDNA sequencing) methods. Its culture supernatant displayed inhibitory activity against Gram-negative bacteria (P. aeruginosa, K. pneumoniae, Salmonella spp.). Two polymyxins, E1 and E2, were recovered from the supernatant and were characterized by high resolution LC-MS. A genomic library (960 clones) was constructed to identify the gene cluster responsible for biosynthesis of polymyxins. Selection of the clones harbouring the sequences of interest was obtained by a simple PCR-based screening. We used primers targeting NRPS sequences leading to the incorporation of amino acids present in polymyxins E. The sequences from three clones of interest were assembled on 50.4 kb. Thus, five open reading frames corresponding to a new NRPS gene cluster of 41 kb were identified. In silico, analyses revealed the presence of three NRPS implicated in the biosynthesis of polymyxins E. This work provides insightful information on colistin biosynthesis and might contribute to future drug developments in this group of antibiotics.

We report here the identification, characterization and culture of a Gram-negative to Gramvariable, rod-shaped, non-spore-forming anaerobic bacterium (strain FM1025 T ) isolated from the caecum of a duck. Phylogenetic analysis based on comparative 16S rRNA gene sequencing showed that this strain clustered with species of the family 'Acidaminococcaceae', with 94.9 % similarity to Megamonas hypermegale DSM 1672 T and less than 91 % similarity with type strains of Pectinatus species. Sequence similarities of at least 98-99 % were observed with numerous sequences deposited in GenBank of uncultured strains from human and chicken caecal contents, but this strain is the first isolate of this taxon to be cultivated and described. On the basis of morphological, physiological and phylogenetic features, this strain should be assigned to a novel species in the genus Megamonas, for which the name Megamonas rupellensis sp. nov. is proposed. The type strain is strain FM1025 T (5DSM 19944 T 5CIP 109788 T ).The microbial composition of the chicken intestine evolves with age and varies in its different sections. It is estimated that 60-90 % of the intestinal bacterial community can not currently be cultivated (Lu et al., 2003). The dominant micro-organisms in the gut include bacteria related to Clostridium, Lactobacillus, Bacteroides and Megamonas (Lan et al., 2002;Zhu et al., 2002). This complex microflora is known to play a key role in the health of birds. For instance, young chickens are very sensitive to enteropathogen infections because their intestinal micro-organisms are not fully established (Nurmi & Rantala, 1973). In 2000, searching for bacteria that produce anti-enteropathogen molecules, Portrait et al. (2000) isolated an anaerobic strain (FM1025 T ) from the caecum of a duck. This strain was preliminarily identified as Fusobacterium mortiferum using API galleries. In the present work, we have characterized this bacterium further and, using 16S rRNA gene sequence analysis, we have shown that strain FM1025 T represents a novel species belonging to the genus Megamonas (Shah & Collins, 1982). Strain FM1025 T was grown routinely at 37 u C on TGY broth (w/v; 3 % tryptone, 2 % yeast extract, 0.5 % glucose, 0.05 % L-cysteine hydrochloride) using either anaerobic jars (AnaeroGen; Oxoid) or an anaerobic chamber containing 10 % H 2 , 10 % CO 2 and 80 % N 2 (MACS DG500; Don Whitley Scientific). Strain FM1025 T was restreaked weekly onto TGY agar [TGY broth solidified with 1.2 % (w/v) agar] and could be stored for several years after mixing 0.5 ml of an overnight culture with 0.5 ml 30 % (w/v) glycerol. Unless otherwise specified, growth of strain FM1025 T was tested in 250 ml TGY broth incubated at 37 u C under continuous stirring (160 r.p.m.) in the anaerobic chamber. Strain FM1025 T grew optimally at 37 uC and the pH range for growth was pH 5-9 with an optimum at pH 7. On solid M63 minimal medium (Miller, 1972) supplemented with the reducing amino acid L-cysteine hydrochloride (0.05 % w/v), no colonies were observed after 72 h ...

Nonribosomal peptide synthetases (NRPS) are actively sought out, due to pharmacologically important activities of their metabolites. In marine environment, the most prevalent nonribosomal peptide antibiotic producers are sponges inhabiting microorganisms. Conversely, strains from marine sediments and more especially from intertidal mudflats have not been extensively screened for the presence of new NRPS. In this study, for the first time, a collection of one hundred intertidal mudflat bacterial isolates (Marennes-Oléron Bay, France) was assessed for (1) the presence of NRPS genes by degenerated PCR targeting conserved adenylation domains and (2) for their production of antimicrobial molecules. (1) Bacteria with adenylation domains (14 strains) were identified by 16S rRNA gene sequence analysis and grouped into Firmicutes (one strain) and Proteobacteria (13 strains). In silico analysis of the NRPS amino acid sequences (n = 7) showed 41-58% ID with sequences found in the NCBI database. Three new putative adenylation domain signatures were found. (2) The culture supernatant of one of these strains, identified as a Bacillus, was shown to strongly inhibit the growth of Staphylococcus aureus, S. epidermidis, and Enterococcus faecalis. This study portends that the intertidal mudflat niche could be of interest for the discovery of new NRPS genes and antimicrobial producing strains.

A simple and efficient microwave-assisted methodology for regioselective alkylation of exocyclic nitrogen of cyclic amidines was developed and novel N-alkylated 3,4-dihydropyrazino[2,1-b]quinazolin-6-ones were prepared. Although none of the molecules tested have any specific anti-quorum sensing ( -QS) activity, our result validates the growth tests devised to control the bias of the anti-QS tests. Among the molecules studied, compound 2b exhibits interesting activity against the Gramnegative bacteria Escherichia coli and Shigella sonnei.

The undefined mixed starter culture (UMSC) is used in the manufacture of cheeses. Deciphering UMSC microbial diversity is important to optimize industrial processes. The UMSC was studied using culture-dependent and culture-independent based methods. MALDI-TOF MS enabled identification of species primarily from the Lactococcus genus. Comparisons of carbohydrate metabolism profiles allowed to discriminate five phenotypes of Lactococcus (n = 26/1616). The 16S sequences analysis (V1–V3, V3–V4 regions) clustered the UMSC microbial diversity into two Lactococcus operational taxonomic units (OTUs). These clustering results were improved with the DADA2 algorithm on the housekeeping purR sequences. Five L. lactis variants were detected among the UMSC. The whole-genome sequencing of six isolates allowed for the identification of the lactis subspecies using Illumina® (n = 5) and Pacbio® (n = 1) technologies. Kegg analysis confirmed the L. lactis species-specific niche adaptations and highlighted a progressive gene pseudogenization. Then, agar spot tests and agar well diffusion assays were used to assess UMSC antimicrobial activities. Of note, isolate supernatants (n = 34/1616) were shown to inhibit the growth of Salmonella ser. Typhimurium CIP 104115, Lactobacillus sakei CIP 104494, Staphylococcus aureus DSMZ 13661, Enterococcus faecalis CIP103015 and Listeria innocua CIP 80.11. Collectively, these results provide insightful information about UMSC L. lactis diversity and revealed a potential application as a bio-protective starter culture.

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