Cultivable gut bacteria provide a pathway for adaptation of Chrysolina herbacea to Mentha aquatica volatiles.

BACKGROUND: A chemical cross-talk between plants and insects is required in order to achieve a successful co-adaptation. In response to herbivory, plants produce specific compounds, and feeding insects respond adequately7 to molecules produced by plants. Here we show the role of the gut microbial community of the mint beetle Chrysolina herbacea in the chemical cross-talk with Mentha aquatica (or watermint). RESULTS: By using two-dimensional gas chromatography-mass spectrometry we first evaluated the chemical patterns of both M. aquatica leaf and frass volatiles extracted by C. herbacea males and females feeding on plants, and observed marked differences between males and females volatiles. The sex-specific chemical pattern of the frass paralleled with sex-specific distribution of cultivable gut bacteria. Indeed, all isolated gut bacteria from females belonged to either α- or γ-Proteobacteria, whilst those from males were γ-Proteobacteria or Firmicutes. We then demonstrated that five Serratia marcescens strains from females possessed antibacterial activity against bacteria from males belonging to Firmicutes suggesting competition by production of antimicrobial compounds. By in vitro experiments, we lastly showed that the microbial communities from the two sexes were associated to specific metabolic patterns with respect to their ability to biotransform M. aquatica terpenoids, and metabolize them into an array of compounds with possible pheromone activity. CONCLUSIONS: Our data suggest that cultivable gut bacteria of Chrysolina herbacea males and females influence the volatile blend of herbivory induced Mentha aquatica volatiles in a sex-specific way.

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.

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.

Susceptibility to antibiotics of Vibrio sp. AO1 growing in pure culture or in association with its hydroid host Aglaophenia octodonta (Cnidaria, Hydrozoa).

Vibrio harveyi is the major causal organism of vibriosis, causing potential devastation to diverse ranges of marine invertebrates over a wide geographical area. These microorganisms, however, are phenotypically diverse, and many of the isolates are also resistant to multiple antibiotics. In a previous study, we described a previously unknown association between Vibrio sp. AO1, a luminous bacterium related to the species V. harveyi, and the benthic hydrozoan Aglaophenia octodonta. In this study, we analyzed the susceptibility to antibiotics (ampicillin, streptomycin, tetracycline, or co-trimoxazole = mix of sulfamethoxazole and trimetoprim) of Vibrio sp. AO1 growing in pure culture or in association with its hydroid host by using microcosm experiments. The results of minimum inhibitory concentration (MIC) experiments demonstrated that Vibrio sp. AO1 was highly resistant to ampicillin and streptomycin in pure culture. Nevertheless, these antibiotics, when used at sub-MIC values, significantly reduced the hydroid fluorescence. Co-trimoxazole showed the highest inhibitory effect on fluorescence of A. octodonta. However, in all treatments, the fluorescence was reduced after 48 h, but never disappeared completely around the folds along the hydrocaulus and at the base of the hydrothecae of A. octodonta when the antibiotic was used at concentration completely inhibiting growth in vitro. The apparent discrepancy between the MIC data and the fluorescence patterns may be due to either heterogeneity of the bacterial population in terms of antibiotic susceptibility or specific chemical-physical conditions of the hydroid microenvironment that may decrease the antibiotic susceptibility of the whole population. The latter hypothesis is supported by scanning electron microscope evidence for development of bacterial biofilm on the hydroid surface. On the basis of the results obtained, we infer that A. octodonta might behave as a reservoir of antibiotic multiresistant bacteria, increasing the risk of their transfer into aquaculture farms.

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

BACKGROUND: There 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. RESULTS: Spontaneous 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. CONCLUSION: Rifampicin 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.

Stimulatory Effects of Methyl-ß-cyclodextrin on Spiramycin Production and Physical-Chemical Characterization of Nonhost@Guest Complexes.

Spiramycin is a macrolide antibiotic and antiparasitic that is used to treat toxoplasmosis and various other infections of soft tissues. In the current study, we evaluated the effects of α-cyclodextrin, ß-cyclodextrin, or methyl-ß-cyclodextrin supplementation to a synthetic culture medium on biomass and spiramycin production by Streptomyces ambofaciens ATCC 23877. We found a high stimulatory effect on spiramycin production when the culture medium was supplemented with 0.5% (w/v) methyl-ß-cyclodextrin, whereas α-cyclodextrin or ß-cyclodextrin weakly enhanced antibiotic yields. As the stimulation of antibiotic production could be because of spiramycin complexation with cyclodextrins with effects on antibiotic stability and/or efflux, we analyzed the possible formation of complexes by physical-chemical methods. The results of Job plot experiment highlighted the formation of a nonhost@guest complex methyl-ß-cyclodextrin@spiramycin I in the stoichiometric ratio of 3:1 while they excluded the formation of complex between spiramycin I and α- or ß-cyclodextrin. Fourier-transform infrared spectroscopy measurements were then carried out to characterize the methyl-ß-cyclodextrin@spiramycin I complex and individuate the chemical groups involved in the binding mechanism. These findings may help to improve the spiramycin fermentation process, providing at the same time a new device for better delivery of the antibiotic at the site of infection by methyl-ß-cyclodextrin complexation, as it has been well-documented for other bioactive molecules.

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.

Clonothrix fusca Roze 1896, a filamentous, sheathed, methanotrophic gamma-proteobacterium.

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

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.