Classification of a Violacein-Producing Psychrophilic Group of Isolates Associated with Freshwater in Antarctica and Description of Rugamonas violacea sp. nov.

A group of 11 bacterial strains was isolated from streams and lakes located in a deglaciated northern part of James Ross Island, Antarctica. They were rod-shaped, Gram-stain-negative, motile, and catalase-positive and produced blue-violet-pigmented colonies on R2A agar. A polyphasic taxonomic approach based on 16S rRNA gene sequencing, whole-genome sequencing, automated ribotyping, repetitive element sequence-based PCR (rep-PCR), MALDI-TOF MS, fatty acid profile, chemotaxonomy analyses, and extensive biotyping was applied in order to clarify the taxonomic position of these isolates. Phylogenetic analysis based on the 16S rRNA gene indicated that all the isolates constituted a coherent group belonging to the genus Rugamonas. The closest relatives to the representative isolate P5900T were Rugamonas rubra CCM 3730T, Rugamonas rivuli FT103WT, and Rugamonas aquatica FT29WT, exhibiting 99.2%, 99.1%, and 98.6% 16S rRNA pairwise similarity, respectively. The average nucleotide identity and digital DNA-DNA hybridization values calculated from the whole-genome sequencing data clearly proved that P5900T represents a distinct Rugamonas species. The G+C content of genomic DNAs was 66.1 mol%. The major components in fatty acid profiles were summed feature 3 (C16:1ω7c/C16:1ω6c), C 16:0, and C12:0. The cellular quinone content contained exclusively ubiquinone Q-8. The predominant polar lipids were diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. The polyamine pattern was composed of putrescine, 2-hydroxputrescine, and spermidine. IMPORTANCE Our polyphasic approach provides a new understanding of the taxonomy of novel pigmented Rugamonas species isolated from freshwater samples in Antarctica. The isolates showed considerable extracellular bactericidal secretions. The antagonistic activity of studied isolates against selected pathogens was proved by this study and implied the importance of such compounds' production among aquatic bacteria. The psychrophilic and violacein-producing species Roseomonas violacea may play a role in the diverse consortium among pigmented bacteria in the Antarctic water environment. Based on all the obtained results, we propose a novel species for which the name Rugamonas violacea sp. nov. is suggested, with the type strain P5900T (CCM 8940T; LMG 32105T). Isolates of R. violacea were obtained from different aquatic localities, and they represent the autochthonous part of the water microbiome in Antarctica.

New insight into poly (3-hydroxybutyrate) production by Azomonas macrocytogenes isolate KC685000: large scale production, kinetic modeling, recovery and characterization.

About 24 h incubation of Azomonas (A.) macrocytogenes isolate KC685000 in 14L fermenter produced 22% poly (3-hydroxybutyrate) (PHB) per cell dry weight (CDW) biopolymer using 1 vvm aeration, 10% inoculum size, and initial pH of 7.2. To control the fermentation process, Logistic and Leudeking-Piret models were used to describe the cell growth and PHB production, respectively. These two models were in good agreement with the experimental data confirming the growth associated nature of PHB production. The best method for recovery of PHB was chemical digestion using sodium hypochlorite alone. The characterization of the produced polymer was carried out using FT-IR, 1HNMR spectroscopy, gel permeation chromatography and transmission electron microscope. The analysis of the nucleotide sequences of PHA synthase enzyme revealed class III identity. The putative tertiary structure of PHA synthase enzyme was analyzed using Modular Approach to Structural class prediction software, Tied Mixture Hidden Markov Model server, and Swiss model software. It was deduced that PHA synthases' structural class was multidomain protein (α/ß) containing a conserved cysteine residue and lipase box as characteristic features of α/ß hydrolase super family. Taken together, all the results of molecular characterization and transmission electron microscope images supported that the PHB formation was attained by the micelle model. To the best of our knowledge, this is the first report on production of growth associated PHB polymer using A. macrocytogenes isolate KC685000, and its class III PHA synthase.

Sputum microbiota in severe asthma patients: Relationship to eosinophilic inflammation.

BACKGROUND: Altered composition of airway microbiota has been reported in subjects suffering from asthma but its relation to eosinophilic phenotype is unclear. OBJECTIVE: To examine the relationship between sputum microbiota, asthma severity and inflammatory type in asthmatic subjects from Guangzhou, China. METHODS: Induced sputum samples were obtained from 49 non-smoking asthma patients, 25 severe and 24 non-severe, and 15 healthy subjects. Total DNA was amplified using primers specific for the V3-V5 hypervariable region of bacterial 16s rRNA and sequenced using the 454 GS FLX sequencer. Sequences were assigned to bacterial taxa by comparing them with 16s rRNA sequences in the Ribosomal Database Project. RESULTS: Sputum eosinophil counts were higher and FEV1 (% predicted) was lower in severe compared to non-severe asthmatics. There were no significant differences in operational taxonomic unit (OTU) numbers at the phylum level and in diversity scores between non-severe asthmatics and severe asthmatics, and healthy subjects. At the family level, Porphyromonadaceae was most abundant in healthy subjects whereas Pseudomonadaceae and Enterobacteriaceae were higher in severe asthmatics compared to non-severe asthmatics (p < 0.05). Actinomycetaceae was particularly abundant in eosinophilic asthma patients compared to non-eosinophilic asthma (p = 0.011). Bacteroidaceae was positively correlated with FEV1 in all subjects (r = 0.335, p < 0.01), whereas body mass index was negatively associated with the number of species observed (r = -0.3, p < 0.05). Principal component analysis confirmed the positive association of Actinomycetaceae and Enterobacteriaceae abundance with eosinophilic asthma. CONCLUSION: Patients with asthma have an altered airway microbiota, with specific bacteria associated with severe asthma and the eosinophilic inflammatory phenotype.

Rhizobacter profundi sp. nov., isolated from freshwater sediment.

Three bacterial strains, designated DS48-6-5T, DS48-6-7 and DS48-6-9, were isolated from a sediment sample taken from Daechung Reservoir (Republic of Korea) at a water depth of 48 m. Cells of the strains were Gram-stain-negative, aerobic, rod-shaped and motile with a single polar flagellum. Comparative 16S rRNA gene sequence studies showed that the three isolates had clear affiliation with Betaproteobacteria and the closest relatives were Rhizobacter bergeniae KCTC 32299T, Rhizobacter dauci DSM 11587T and Rhizobacter fulvus KCTC 12591T with 97.2-97.9 % 16S rRNA gene sequence similarities; the 16S rRNA gene sequence similarities between the three strains were 99.5-100 %. The only isoprenoid quinone of the three strains was ubiquinone-8, and the major fatty acids were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c) and C16 : 0. The G+C content of the genomic DNA of strains DS48-6-5T, DS48-6-7 and DS48-6-9 was 66.7, 67.0 and 66.8 mol%, respectively. DNA-DNA hybridization values of the novel strains with R. bergeniae KCTC 32299T, R. dauci DSM 11587T and R. fulvus KCTC 12591T were 19.3-48.5 %. Based on the evidence from this taxonomic study using a polyphasic approach, it is proposed that strains, DS48-6-5T, DS48-6-7 and DS48-6-9, represent a novel species of the genus Rhizobacter, for which the name Rhizobacter profundi sp. nov. is proposed. The type strain is DS48-6-5T ( = KCTC 42645T = NBRC 111169T).

Permianibacter aggregans gen. nov., sp. nov., a bacterium of the family Pseudomonadaceae capable of aggregating potential biofuel-producing microalgae.

A novel bacterial strain, capable of aggregating potential biofuel-producing microalgae, was isolated from the phycosphere of an algal culture and designated HW001(T). The novel bacterial strain was identified on the basis of its phylogenetic, genotypic, chemotaxonomic and phenotypic characteristics in this study. Cells were aerobic, Gram-negative rods. 16S rRNA gene-based phylogenetic analysis revealed that strain HW001(T) is affiliated with the family Pseudomonadaceae in the phylum Proteobacteria, but forms a distinct clade within this family. The DNA G+C content of strain HW001(T) was 55.4 mol%. The predominant cellular fatty acids were iso-C15:0, summed feature 9 (iso-C17:1ω9c), C16:0 and summed feature 3 (C16:1ω7c/C16:1ω6c). Q-8 was the main respiratory quinone. The polar lipid profile contained phosphatidylethanolamine, an unidentified aminophospholipid and some unidentified lipids. Based on the extensive polyphasic analysis, strain HW001(T) represents a novel species of a new genus in the family Pseudomonadaceae, for which the name Permianibacter aggregans gen. nov., sp. nov., is proposed. The type strain of the type species is HW001(T) ( = CICC 10856(T) = KCTC 32485(T)).

Effect of ferritin overexpression in tobacco on the structure of bacterial and pseudomonad communities associated with the roots.

The genetic structures of total bacterial and pseudomonad communities were characterized in rhizosphere soil and rhizoplane+root tissues of tobacco wild type and a ferritin overexpressor transgenic line (P6) by a cultivation-independent method using directly extracted DNA at the end of three consecutive plant cultures. The structure of total bacterial communities was characterized by automated ribosomal intergenic spacer analysis (A-RISA), and that of pseudomonad communities was characterized by PCR-restriction fragment length polymorphism (PCR-RFLP) from DNA amplified with specific primers. The structure of total bacterial communities was significantly modified in the rhizosphere soil by the overaccumulation of iron in the tobacco transgenic P6 line at the first culture, to a lesser extent at the second culture, and not at all at the third culture. No significant difference was recorded between the total communities associated with the roots (rhizoplane+root tissues) of the two plant genotypes in any of the cultures. In contrast, the difference in pseudomonad structure between the two plant genotypes increased with successive culture at the root level, but was not detected at a significant level in the rhizosphere soil. The impact of iron overaccumulation by the tobacco transgenic P6 line on pseudomonads supports previous findings on the importance of iron competition among fluorescent pseudomonads.

Comparison of ATPase-encoding type III secretion system hrcN genes in biocontrol fluorescent Pseudomonads and in phytopathogenic proteobacteria.

Type III protein secretion systems play a key role in the virulence of many pathogenic proteobacteria, but they also occur in nonpathogenic, plant-associated bacteria. Certain type III protein secretion genes (e.g., hrcC) have been found in Pseudomonas sp. strain SBW25 (and other biocontrol pseudomonads), but other type III protein secretion genes, such as the ATPase-encoding gene hrcN, have not been found. Using both colony hybridization and a PCR approach, we show here that hrcN is nevertheless present in many biocontrol fluorescent pseudomonads. The phylogeny of biocontrol Pseudomonas strains based on partial hrcN sequences was largely congruent with the phylogenies derived from analyses of rrs (encoding 16S rRNA) and, to a lesser extent, biocontrol genes, such as phlD (for 2,4-diacetylphloroglucinol production) and hcnBC (for HCN production). Most biocontrol pseudomonads clustered separately from phytopathogenic proteobacteria, including pathogenic pseudomonads, in the hrcN tree. The exception was strain KD, which clustered with phytopathogenic pseudomonads, such as Pseudomonas syringae, suggesting that hrcN was acquired from the latter species. Indeed, strain KD (unlike strain SBW25) displayed the same organization of the hrpJ operon, which contains hrcN, as P. syringae. These results indicate that the occurrence of hrcN in most biocontrol pseudomonads is not the result of recent horizontal gene transfer from phytopathogenic bacteria, although such transfer might have occurred for a minority of biocontrol strains.

The genome sequence of the plant pathogen Xylella fastidiosa. The Xylella fastidiosa Consortium of the Organization for Nucleotide Sequencing and Analysis.

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis--a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to 47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

Regulation of the iron uptake genes in Pseudomonas fluorescens M114 by pseudobactin M114: the pbrA sigma factor gene does not mediate the siderophore regulatory response.

The iron-regulated PbrA sigma factor dictates the production of the siderophore, pseudobactin M114, and its cognate outer membrane receptor, PbuA, in Pseudomonas fluorescens M114. However, the siderophore molecule also has a role in regulating the expression of the siderophore biosynthetic and siderophore receptor genes in P. fluorescens M114. This is based on the fact that beta-galactosidase levels from lacZ fusions of M114 siderophore promoters (biosynthetic and receptor) were reduced in M114 siderophore biosynthetic mutants compared to wild-type M114. Expression of both promoters was increased by the addition of pseudobactin M114 to the growth medium. This effect was widespread and applicable to all but one of the siderophore negative strains of M114 tested. Furthermore, it was demonstrated that transcription of the pbr A sigma factor gene was not reduced in the siderophore biosynthetic mutants. This excludes the possibility that reduced expression of the siderophore biosynthetic and receptor promoters in the siderophore biosynthetic mutants is mediated at the level of expression of the pbr A gene itself. In addition, it was noted that the siderophore regulated response was applicable to promoters with and without the DNA sequence motif, (G/C)CTAAATCCC, which is required for iron-regulated expression of some pseudomonad promoters.

The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera. Characterization and mechanistic studies of the cloned enzyme over-produced in Escherichia coli.

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hydroxybutyryl-CoA for poly(D-3-hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/mg). The enzyme is an alpha 4 homotetramer of four 25-kDa subunits. It has a Km of 2 microM and a kcat/Km of 1.8 X 10(8) M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyl-CoA above 10 microM. K is 10(-10) M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3',5'-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase.

Effect of oxygen on levels of mRNA coding for reaction-centre and light-harvesting polypeptides of Rhodobacter sphaeroides.

The relative levels of mRNA for the reaction-centre L and M subunits, B875 (LH1) alpha and beta polypeptides and B800-850 (LH2) alpha and beta polypeptides, have been measured during pigment induction of Rhodobacter sphaeroides. Over the 6 h of the experiment, bacteriochlorophyll levels increased by at least 100-fold. No transcripts for photosynthetic components were detectable at the start of induction; after 2 h the levels of transcripts from the puf operon (encoding reaction-centre and B875 subunits) had reached the maximum; these transcripts were 2.7 and 0.5 kb respectively. The transcript for the puc operon (B800-850 complex) was estimated to be 0.55 kb and reached a maximum level after 6 h. These results are consistent with the proposal that, during the assembly of the photosynthetic apparatus, the synthesis of B875 reaction-centre aggregates precedes that of the major antenna, B800-850.

Isolation of mutants of Caulobacter crescentus deficient in ATP-dependent deoxyribonuclease activity.

Caulobacter crescentus mutants sensitive to UV radiation, mitomycin C and methyl methane-sulfonate were isolated and tested for ATP-dependent deoxyribonuclease activity. Two mutants were identified of which one had less than 5 per cent of the wild-type level of the nuclease activity. This mutant in cross with another auxotrophic partner gave highly reproducible two-fold reduction in number of recombinants compared to a control cross. The suggested causes for reduced recombination frequency when one of the partners has residual ATP-dependent deoxyribonuclease activity are discussed.