Salvatore Maurizio Tredici

Epibiotic Vibrio Luminous Bacteria Isolated from Some Hydrozoa and Bryozoa Species

Luminous bacteria are isolated from both Hydrozoa and Bryozoa with chitinous structures on their surfaces. All the specimens of the examined hydroid species (Aglaophenia kirchenpaueri, Aglaophenia octodonta, Aglaophenia tubiformis, Halopteris diaphana, Plumularia setacea, Ventromma halecioides), observed under blue light excitation, showed a clear fluorescence on the external side of the perisarc (chitinous exoskeleton) around hydrocladia. In the bryozoan Myriapora truncata, luminous bacteria are present on the chitinous opercula. All the isolated luminous bacteria were identified on the basis of both phenotypic and genotypic analysis. The isolates from A. tubiformis and H. diaphana were unambiguously assigned to the species Vibrio fischeri. In contrast, the isolates from the other hydroids, phenotypically assigned to the species Vibrio harveyi, were then split into two distinct species by phylogenetic analysis of 16S rRNA gene sequences and DNA–DNA hybridization experiments. Scanning electron microscopy analysis and results of culture-based and culture-independent approaches enabled us to establish that luminous vibrios represent major constituents of the bacterial community inhabiting the A. octodonta surface suggesting that the interactions between luminous bacteria and the examined hydrozoan and bryozoan species are highly specific. These interactions might have epidemiological as well as ecological implications because of the opportunistic pathogenicity of luminous Vibrio species for marine organisms and the wide-distribution of the hydrozoan and bryozoan functioning as carriers.

Clonothrix fusca Roze 1896, a Filamentous, Sheathed, Methanotrophic γ-Proteobacterium

ABSTRACT 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 γ-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 Cohns Crenothrix and Rozes 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 γ-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 γ-proteobacteria, and (iii) sequencing data demonstrating the presence in its genome of a methanol dehydrogenase α subunit-encoding gene (mxaF) and a conventional particulate methane mono-oxygenase α subunit-encoding gene (pmoA) that is different from the unusual pmoA (u-pmoA) of C. polyspora.

The microbial community of Vetiver root and its involvement into essential oil biogenesis.

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.

Phenotypes and gene expression profiles of Saccharopolyspora erythraea rifampicin-resistant (rif) mutants affected in erythromycin production

BackgroundThere is evidence from previous works that bacterial secondary metabolism may be stimulated by genetic manipulation of RNA polymerase (RNAP). In this study we have used rifampicin selection as a strategy to genetically improve the erythromycin producer Saccharopolyspora erythraea.ResultsSpontaneous rifampicin-resistant (rif) mutants were isolated from the parental strain NRRL2338 and two rif mutations mapping within rpoB, S444F and Q426R, were characterized. With respect to the parental strain, S444F mutants exhibited higher respiratory performance and up to four-fold higher final erythromycin yields; in contrast, Q426R mutants were slow-growing, developmental-defective and severely impaired in erythromycin production. DNA microarray analysis demonstrated that these rif mutations deeply changed the transcriptional profile of S. erythraea. The expression of genes coding for key enzymes of carbon (and energy) and nitrogen central metabolism was dramatically altered in turn affecting the flux of metabolites through erythromycin feeder pathways. In particular, the valine catabolic pathway that supplies propionyl-CoA for biosynthesis of the erythromycin precursor 6-deoxyerythronolide B was strongly up-regulated in the S444F mutants, while the expression of the biosynthetic gene cluster of erythromycin (ery) was not significantly affected. In contrast, the ery cluster was down-regulated (<2-fold) in the Q426R mutants. These strains also exhibited an impressive stimulation of the nitrogen regulon, which may contribute to lower erythromycin yields as erythromycin production was strongly inhibited by ammonium.ConclusionRifampicin selection is a simple and reliable tool to investigate novel links between primary and secondary metabolism and morphological differentiation in S. erythraea and to improve erythromycin production. At the same time genome-wide analysis of expression profiles using DNA microarrays allowed information to be gained about the mechanisms underlying the stimulatory/inhibitory effects of the rif mutations on erythromycin production.

Natural merodiploidy involving duplicated rpoB alleles affects secondary metabolism in a producer actinomycete

Actinomadura 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 µg ml−1), was isolated from the wild type strain that exhibited low resistance to rifampicin (MIC < 25 µg ml−1). 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, rpoBS and rpoBR, in both the wild type and the rif19. rpoBR 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 β‐chain. Transcript analysis revealed that rpoBR was expressed at a very low level in the wild type strain during the pseudo‐exponential growth phase, and that the amount of rpoBR mRNA increased during the transition to the stationary phase. In contrast, expression of rpoBR 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.

Design of mineral medium for growth of Actinomadura sp. ATCC 39727, producer of the glycopeptide A40926: effects of calcium ions and nitrogen sources.

Actinomadura sp. ATCC 39727 produces the glycopeptide antibiotic A40926, structurally similar to teicoplanin, with significant activity against Neisseria gonorrhoeae and precursor of the semi-synthetic antibiotic dalbavancin. In this study the production of A40926 by Actinomadura under a variety of growth conditions was investigated. The use of chemically defined mineral media allowed us to analyze the influence of carbon and nitrogen sources, phosphate, ammonium and calcium on the growth and the antibiotic productivity of Actinomadura. We confirm recent data [Gunnarsson et al. (2003) J Ind Microbiol Biotechnol 30:150–156] that low initial concentrations of phosphate and ammonium are beneficial for growth and A40926 production, and we provide new evidence that the production of A40926 is depressed by calcium, but promoted when l-glutamine or l-asparagine are used as nitrogen sources instead of ammonium salts.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) as a negative modulator of polynucleotide phosphorylase activity in a 'rare' actinomycete

With the beginning of the idiophase the highly phosphorylated guanylic nucleotides guanosine 5′‐diphosphate 3′‐diphosphate (ppGpp) and guanosine 5′‐triphosphate 3′‐diphosphate (pppGpp), collectively referred to as (p)ppGpp, activate stress survival adaptation programmes and trigger secondary metabolism in actinomycetes. The major target of (p)ppGpp is the RNA polymerase, where it binds altering the enzyme activity. In this study analysis of the polynucleotide phosphorylase (PNPase)‐encoding gene pnp mRNA, in Nonomuraea sp. ATCC 39727 wild‐type, constitutively stringent and relaxed strains, led us to hypothesize that in actinomycetes (p)ppGpp may modulate gene expression at the level of RNA decay also. This hypothesis was supported by: (i) in vitro evidence that ppGpp, at physiological levels, inhibited both polynucleotide polymerase and phosphorolytic activities of PNPase in Nonomuraea sp., but not in Escherichia coli, (ii) in vivo data showing that the pnp mRNA and the A40926 antibiotic cluster‐specific dpgA mRNA were stabilized during the idiophase in the wild‐type strain but not in a relaxed mutant and (iii) measurement of chemical decay of pulse‐labelled bulk mRNA. The results of biochemical tests suggest competitive inhibition of ppGpp with respect to nucleoside diphosphates in polynucleotide polymerase assays and mixed inhibition with respect to inorganic phosphate when the RNA phosphorolytic activity was determined.

Sphingomonas cynarae sp. nov., a proteobacterium that produces an unusual type of sphingan.

Strain SPC-1(T) was isolated from the phyllosphere of Cynara cardunculus L. var. sylvestris (Lamk) Fiori (wild cardoon), a Mediterranean native plant considered to be the wild ancestor of the globe artichoke and cultivated cardoon. This Gram-stain-negative, catalase-positive, oxidase-negative, non-spore-forming, rod-shaped and non-motile strain secreted copious amounts of an exopolysaccharide, formed slimy, viscous, orange-pigmented colonies and grew optimally at around pH 6.0-6.5 and 26-30 °C in the presence of 0-0.5 % NaCl. Phylogenetic analysis based on comparisons of 16S rRNA gene sequences demonstrated that SPC-1(T) clustered together with species of the genus Sphingomonas sensu stricto. The G+C content of the DNA (66.1 mol%), the presence of Q-10 as the predominant ubiquinone, sym-homospermidine as the predominant polyamine, 2-hydroxymyristic acid (C(14 : 0) 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the presence of sphingoglycolipid supported this taxonomic position. 16S rRNA gene sequence analysis showed that SPC-1(T) was most closely related to Sphingomonas hankookensis ODN7(T), Sphingomonas insulae DS-28(T) and Sphingomonas panni C52(T) (98.19, 97.91 and 97.11 % sequence similarities, respectively). However, DNA-DNA hybridization analysis did not reveal any relatedness at the species level. Further differences were apparent in biochemical traits, and fatty acid, quinone and polyamine profiles leading us to conclude that strain SPC-1(T) represents a novel species of the genus Sphingomonas, for which the name Sphingomonas cynarae sp. nov. is proposed; the type strain is SPC-1(T) ( = JCM 17498(T) = ITEM 13494(T)). A component analysis of the exopolysaccharide suggested that it represents a novel type of sphingan containing glucose, rhamnose, mannose and galactose, while glucuronic acid, which is commonly found in sphingans, was not detected.

Exposure to static magnetic field stimulates quorum sensing circuit in luminescent Vibrio strains of the Harveyi clade.

In this study, the evidence of electron-dense magnetic inclusions with polyhedral shape in the cytoplasm of Harveyi clade Vibrio strain PS1, a bioluminescent bacterium living in symbiosis with marine organisms, led us to investigate the behavior of this bacterium under exposure to static magnetic fields ranging between 20 and 2000 Gauss. When compared to sham-exposed, the light emission of magnetic field-exposed bacteria growing on solid medium at 18°C ±0.1°C was increased up to two-fold as a function of dose and growth phase. Stimulation of bioluminescence by magnetic field was more pronounced during the post-exponential growth and stationary phase, and was lost when bacteria were grown in the presence of the iron chelator deferoxamine, which caused disassembly of the magnetic inclusions suggesting their involvement in magnetic response. As in luminescent Vibrio spp. bioluminescence is regulated by quorum sensing, possible effects of magnetic field exposure on quorum sensing were investigated. Measurement of mRNA levels by reverse transcriptase real time-PCR demonstrated that luxR regulatory gene and luxCDABE operon coding for luciferase and fatty acid reductase complex were significantly up-regulated in magnetic field-exposed bacteria. In contrast, genes coding for a type III secretion system, whose expression was negatively affected by LuxR, were down-regulated. Up-regulation of luxR paralleled with down-regulation of small RNAs that mediate destabilization of luxR mRNA in quorum sensing signaling pathways. The results of experiments with the well-studied Vibrio campbellii strain BB120 (originally classified as Vibrio harveyi) and derivative mutants unable to synthesize autoinducers suggest that the effects of magnetic fields on quorum sensing may be mediated by AI-2, the interspecies quorum sensing signal molecule.

Nitrite metabolism in Debaryomyces hansenii TOB-Y7, a yeast strain involved in tobacco fermentation

The Italian cigar manufacturing process includes a fermentation step that leads to accumulation of nitrite and tobacco-specific nitrosamines (TSNA), undesirable by-products due to their negative impact on health. In this study, growth and biochemical properties of Debaryomyces hansenii TOB-Y7, a yeast strain that predominates during the early phase of fermentation, have been investigated. With respect to other D. hansenii collection strains (Y7426, J26, and CBS 1796), TOB-Y7 was characterized by the ability to tolerate very high nitrite levels and to utilize nitrite, but not nitrate, as a sole nitrogen source in a chemically defined medium, a property that was enhanced in microaerophilic environment. The ability to assimilate nitrite was associated to the presence of YNI1, the gene encoding the assimilatory NAD(P)H:nitrite reductase (NiR), absent in Y7426, J26, and CBS 1796 by Southern blot data. YNI1 from TOB-Y7 was entirely sequenced, and its expression was analyzed in different media by Northern blot and reverse transcriptase polymerase chain reaction. The evidence that, in D. hansenii TOB-Y7, YNI1 was transcriptional active also in the presence of high ammonia concentration typical of tobacco fermentation, stimulated the development of an improved process that, on a laboratory scale, was proved to be effective in minimizing nitrite and TSNA accumulation.