Crenothrix polyspora Cohn 1870 and Clonothrix fusca Roze 1896 are two filamentous, sheathed microorganisms exhibiting complex morphological differentiation, whose phylogeny and physiology have been obscure for a long time due to the inability to cultivate them. Very recently, DNA sequencing data from uncultured C. polyspora-enriched material have suggested that Crenothrix is a methane-oxidizing ␥-proteobacterium (39). In contrast, the possible ecological function of C. fusca, originally considered a developmental stage of C. polyspora, is unknown. In this study, temporal succession of two filamentous, sheathed microorganisms resembling Cohn's Crenothrix and Roze's Clonothrix was observed by analyzing the microbial community of an artesian well by optical microscopy. Combined culture-based and culture-independent approaches enabled us to assign C. fusca to a novel subgroup of methane-oxidizing ␥-proteobacteria distinct from that of C. polyspora. This assignment was supported by (i) methane uptake and assimilation experiments, (ii) ultrastructural data showing the presence in C. fusca cytoplasm of an elaborate membrane system resembling that of methanotrophic ␥-proteobacteria, and (iii) sequencing data demonstrating the presence in its genome of a methanol dehydrogenase ␣ subunit-encoding gene (mxaF) and a conventional particulate methane mono-oxygenase ␣ subunit-encoding gene (pmoA) that is different from the unusual pmoA (u-pmoA) of C. polyspora.

Vetiver is the only grass cultivated worldwide for the root essential oil, which is a mixture of sesquiterpene alcohols and hydrocarbons, used extensively in perfumery and cosmetics. Light and transmission electron microscopy demonstrated the presence of bacteria in the cortical parenchymatous essential oil-producing cells and in the lysigen lacunae in close association with the essential oil. This finding and the evidence that axenic Vetiver produces in vitro only trace amounts of oil with a strikingly different composition compared with the oils from in vivo Vetiver plants stimulated the hypothesis of an involvement of these bacteria in the oil metabolism. We used culture-based and culture-independent approaches to analyse the microbial community of the Vetiver root. Results demonstrate a broad phylogenetic spectrum of bacteria, including alpha-, beta- and gamma-Proteobacteria, high-G+C-content Gram-positive bacteria, and microbes belonging to the Fibrobacteres/Acidobacteria group. We isolated root-associated bacteria and showed that most of them are able to grow by using oil sesquiterpenes as a carbon source and to metabolize them releasing into the medium a large number of compounds typically found in commercial Vetiver oils. Several bacteria were also able to induce gene expression of a Vetiver sesquiterpene synthase. These results support the intriguing hypothesis that bacteria may have a role in essential oil biosynthesis opening the possibility to use them to manoeuvre the Vetiver oil molecular structure.

SummaryActinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin. Production of A40926 is governed by the stringent response at the transcriptional level. In fact, addition of an amino acid pool prevented the transcription of dbv cluster genes involved in the A40926 biosynthesis and the antibiotic production in chemically defined media, and a thiostrepton-resistant relaxed mutant was severely impaired in its ability to produce the antibiotic. The derivative strain rif19 , highly resistant to rifampicin (minimal inhibitory concentration, MIC > 200 m m m m g ml), was isolated from the wild type strain that exhibited low resistance to rifampicin (MIC < 25 m m m m g ml). In this strain A40926 production started earlier than in the wild type, and reached higher final levels. Moreover, the antibiotic production was not subjected to the stringent control. Molecular analysis led to the identification of two distinct rpoB alleles, rpoB S and rpoB R , in both the wild type and the rif19 . rpoB R harboured the H426N missense which is responsible for rifampicin-resistance in bacteria, in addition to other nucleotide substitutions affecting the primary structure of the RNA polymerase b b b b -chain. Transcript analysis revealed that rpoB R was expressed at a very low level in the wild type strain during the pseudo-exponential growth phase, and that the amount of rpoB R mRNA increased during the transition to the stationary phase. In contrast, expression of rpoB R was constitutive in the rif19 . The results of mRNA half-life analysis did not support the hypothesis that post-transcriptional events are responsible for the different rpoB expression patterns in the two strains, suggesting a role of transcriptional mechanisms.