Genome Sequence of Novosphingobium sp. Strain Rr 2-17, a Nopaline Crown Gall-Associated Bacterium Isolated from Vitis vinifera L. Grapevine

Novosphingobium sp. strain Rr 2-17 is an N-acyl homoserine lactone (AHL)-producing bacterium isolated from the crown gall tumor of a grapevine. To our knowledge, this is the first draft genome announcement of a plant-associated strain from the genus Novosphingobium.     

Functional genomics of dichloromethane utilization in Methylobacterium extorquens DM4

Dichloromethane (CH(2)Cl(2) , DCM) is a chlorinated solvent mainly produced by industry, and a common pollutant. Some aerobic methylotrophic bacteria are able to grow with this chlorinated methane as their sole carbon and energy source, using a DCM dehalogenase/glutathione S-transferase encoded by dcmA to transform DCM into two molecules of HCl and one molecule of formaldehyde, a toxic intermediate of methylotrophic metabolism. In Methylobacterium extorquens DM4 of known genome sequence, dcmA lies on a 126 kb dcm genomic island not found so far in other DCM-dechlorinating strains. An experimental search for the molecular determinants involved in specific cellular responses of strain DM4 growing with DCM was performed. Random mutagenesis with a minitransposon containing a promoterless reporter gfp gene yielded 25 dcm mutants with a specific DCM-associated phenotype. Differential proteomic analysis of cultures grown with DCM and with methanol defined 38 differentially abundant proteins. The 5.5 kb dcm islet directly involved in DCM dehalogenation is the only one of seven gene clusters specific to the DCM response to be localized within the dcm genomic island. The DCM response was shown to involve mainly the core genome of Methylobacterium extorquens, providing new insights on DCM-dependent adjustments of C1 metabolism and gene regulation, and suggesting a specific stress response of Methylobacterium during growth with DCM. Fatty acid, hopanoid and peptidoglycan metabolisms were affected, hinting at the membrane-active effects of DCM due to its solvent properties. A chloride-induced efflux transporter termed CliABC was also newly identified. Thus, DCM dechlorination driven by the dcm islet elicits a complex adaptive response encoded by the core genome common to dechlorinating as well as non-dechlorinating Methylobacterium strains.     

Methylobacterium soli sp. nov. a methanol-utilizing bacterium isolated from the forest soil

A Gram-negative, pink-pigmented, non-spore-forming rod shaped, methanol-utilizing bacterium, strain YIM 48816(T), was isolated from forest soil collected from Sichuan province, China. Strain YIM 48816(T) can grow at 4-37 °C, pH 5.0-7.0 and 0% NaCl (w/v). Based on 16S rRNA gene sequence similarity studies, it belonged to the genus Methylobacterium, and formed a phyletic line. The 16S rRNA gene sequence similarities were 96.2% to Methylobacterium mesophilicum DSM 1708(T) and 96.0% to Methylobacterium brachiatum DSM 19569(T), and the phylogenetic similarities to all other Methylobacterium species with validly published names were less than 96.0%. The major menaquinones detected were Q-10 (97.14%) and Q-9 (2.86%). The major fatty acids were C18:1 ω7c (80.84%). The DNA G + C content was 66.2 mol%. It is apparent from the genotypic and phenotypic data that strain YIM 48816(T) belongs to a novel species of the genus Methylobacterium, for which the name Methylobacterium soli sp. nov. is proposed. The type strain is YIM 48816(T) (CCTCC AA 208027(T) = KCTC 22810(T)).     

16S ribosomal RNA sequence analysis for determination of phylogenetic relationship among methylotrophs.

16S ribosomal RNAs (rRNA) of 12 methylotrophic bacteria have been almost completely sequenced to establish their phylogenetic relationships. Methylotrophs that are physiologically related are phylogenetically diverse and are scattered among the purple eubacteria (class Proteobacteria). Group I methylotrophs can be classified in the beta- and the gamma-subdivisions and group II methylotrophs in the alpha-subdivision of the purple eubacteria, respectively. Pink-pigmented facultative and non-pigmented obligate group II methylotrophs form two distinctly separate branches within the alpha-subdivision. The secondary structures of the 16S rRNA sequences of 'Methylocystis parvus' strain OBBP, 'Methylosinus trichosporium' strain OB3b, 'Methylosporovibrio methanica' strain 81Z and Hyphomicrobium sp. strain DM2 are similar, and these non-pigmented obligate group II methylotrophs form one tight cluster in the alpha-subdivision. The pink-pigmented facultative methylotrophs, Methylobacterium extorquens strain AM1, Methylobacterium sp. strain DM4 and Methylobacterium organophilum strain XX form another cluster within the alpha-subdivision. Although similar in phenotypic characteristics, Methylobacterium organophilum strain XX and Methylobacterium extorquens strain AM1 are clearly distinguishable by their 16S rRNA sequences. The group I methylotrophs, Methylophilus methylotrophus strain AS1 and methylotrophic species DM11, which do not utilize methane, are similar in 16S rRNA sequence to bacteria in the beta-subdivision. The methane-utilizing, obligate group I methanotrophs, Methylococcus capsulatus strain BATH and Methylomonas methanica, are placed in the gamma-subdivision. The results demonstrate that it is possible to distinguish and classify the methylotrophic bacteria using 16S rRNA sequence analysis.     

Localization of methanol dehydrogenase in two strains of methylotrophic bacteria detected by immunogold labeling.

Antibodies to methanol dehydrogenase purified from Methylobacterium sp. strain AM1 and Methylomonas sp. strain A4 were raised. The antibody preparations were used in indirect immunogold labeling studies. With this approach, methanol dehydrogenase was found to be preferentially localized to the periplasmic region of the methylotroph Methylobacterium sp. strain AM1 and to the intracytoplasmic membrane of the methanotroph Methylomonas sp. strain A4. Antibody cross-reactivity to other methylotrophic bacteria was detected.     

Localization of methanol dehydrogenase in two strains of methylotrophic bacteria detected by immunogold labeling.

Antibodies to methanol dehydrogenase purified from Methylobacterium sp. strain AM1 and Methylomonas sp. strain A4 were raised. The antibody preparations were used in indirect immunogold labeling studies. With this approach, methanol dehydrogenase was found to be preferentially localized to the periplasmic region of the methylotroph Methylobacterium sp. strain AM1 and to the intracytoplasmic membrane of the methanotroph Methylomonas sp. strain A4. Antibody cross-reactivity to other methylotrophic bacteria was detected.     

<Emphasis Type="Italic">Methylobacterium dankookense</Emphasis> sp. nov., isolated from drinking water

A pink-pigmented bacterium, designated SW08-7^T was isolated from the drinking water of a water purifier. Cells were Gram-negative, rod-shaped, strictly aerobic, and non-spore-forming. It grew optimally at 25°C, pH 6∼7. Phylogenese analysis based on 16S rRNA gene sequence showed that strain SW08-7^T belongs to the genus Methylobacterium. The highest 16S rRNA gene sequence similarities were found to Methylobacterium mesophilicum JCM 2829^T (96.9%), Methylobacterium brachiatum B0021^T (96.9%), Methylobacterium phyllosphaerae CBMB27^T (96.6%), Methylobacterium radiotolerans JCM 2831^T (96.6%), and Methylobacterium hispanicum GP34^T (96.5%). DNA-DNA hybridization experiment revealed low-level (28.5%) of DNA-DNA relatedness between strain SW08-7^T and Methylobacterium hispanicum. The genomic DNA G+C content was 68.9 mol% and the major isoprenoid quinone was Q-10. The major cellular fatty acid of strain SW08-7^T was C_18:1 ω7c (79.8±2.1%). Results of phylogenetic, phenotypic, and biochemical analyses revealed that strain SW08-7^T could be classified as representing a novel species of genus Methylobacterium, for which the name Methylobacterium dankookense sp. nov. is proposed. The type strain is SW08-7^T (=KCTC 22512^T =DSM 22415¹).     

Plasmid analysis in pink facultative methylotrophic bacteria using a modified acetone-alkaline hydrolysis method

Routine screening of indigenous and recombinant plasmids in pink facultative methylotrophic bacteria has been difficult, time-consuming, and yields variable results. We report a modified alkaline hydrolysis method for rapid plasmid isolation from these organisms that reproducibly results in good yields of closed circular plasmid DNA which can be readily digested with restriction enzymes. This method greatly facilitates direct screening of indigenous and introduced recombinant plasmids in the methylotrophic host strain. We have confirmed earlier findings that the original NCIB wild-type strain of Methylobacterium sp. strain AM1 (NCIB 9133) contains three cryptic plasmids. However, sizing of these plasmids by comparison to standards and by restriction fragment analysis suggests that they are larger than previously reported. We have designated these plasmids pAM1-1 (65 kb), pAM1-2 (40 kb) and pAM1-3 (33 kb). We have also shown that a rifamycin-resistant strain of Methylobacterium sp. strain AM1 used routinely in our laboratory lacks pAM1-2, although no phenotype has been associated with its loss. Finally, we have shown that another pink facultative methylotroph, Methylobacterium isolate (#YK1), contains three cryptic plasmids of approximately 43, 37 and 22 kb, respectively.     

Intercellular colonization and growth promoting effects of Methylobacterium sp. with plant-growth regulators on rice (Oryza sativa L. Cv CO-43)

The Methylobacterium sp. strain NPFM-SB3, isolated from Sesbania rostrata stem nodules possessed nitrogenase activity and nodA genes. Pure culture of NPFM-SB3 strain produced indole-3-acetic acid, cytokinins and on inoculation to rice plants resulted in numerous lateral roots. Inoculation of synthetic auxins 2,4-dichlorophenoxy acetic acid, naphthalene acetic acid or flavonoids naringenin and dihydroxy-4-methoxyisoflavone individually or to bacterial inoculated rice seedlings improved the plant growth and lateral root formation under hydroponic condition. The formation of nodule-like structure and nitrogenase activity which is purely auxin dependent was observed in 2,4-dichlorophenoxy acetic acid treatments to Methylobacterium sp. NPFM-SB3 inoculated rice plants. The rhizobia entered through fissures formed due to lateral root emergence and spread intercellularly in the nodular structures concluded that the effect of 2,4-dichlorophenoxy acetic acid treatment for rice seedlings grown under gnotobiotic conditions is to create a niche in which these bacteria can grow.     

Comparative analyses of the complete genome sequences of Pierce's disease and citrus variegated chlorosis strains of Xylella fastidiosa

Xylella fastidiosa is a xylem-dwelling, insect-transmitted, gamma-proteobacterium that causes diseases in many plants, including grapevine, citrus, periwinkle, almond, oleander, and coffee. X. fastidiosa has an unusually broad host range, has an extensive geographical distribution throughout the American continent, and induces diverse disease phenotypes. Previous molecular analyses indicated three distinct groups of X. fastidiosa isolates that were expected to be genetically divergent. Here we report the genome sequence of X. fastidiosa (Temecula strain), isolated from a naturally infected grapevine with Pierce's disease (PD) in a wine-grape-growing region of California. Comparative analyses with a previously sequenced X. fastidiosa strain responsible for citrus variegated chlorosis (CVC) revealed that 98% of the PD X. fastidiosa Temecula genes are shared with the CVC X. fastidiosa strain 9a5c genes. Furthermore, the average amino acid identity of the open reading frames in the strains is 95.7%. Genomic differences are limited to phage-associated chromosomal rearrangements and deletions that also account for the strain-specific genes present in each genome. Genomic islands, one in each genome, were identified, and their presence in other X. fastidiosa strains was analyzed. We conclude that these two organisms have identical metabolic functions and are likely to use a common set of genes in plant colonization and pathogenesis, permitting convergence of functional genomic strategies.     

Methylopila helvetica sp. nov. and Methylobacterium dichloromethanicum sp. nov.--novel aerobic facultatively methylotrophic bacteria utilizing dichloromethane

Eight strains of Gram-negative, aerobic, asporogenous, neutrophilic, mesophilic, facultatively methylotrophic bacteria are taxonomically described. These icl- serine pathway methylobacteria utilize dichloromethane, methanol and methylamine as well as a variety of polycarbon compounds as the carbon and energy source. The major cellular fatty acids of the non-pigmented strains DM1, DM3, and DM5 to DM9 are C18:1, C16:0, C18:0, Ccy19:0 and that of the pink-pigmented strain DM4 is C18:1. The main quinone of all the strains is Q-10. The non-pigmented strains have similar phenotypic properties and a high level of DNA-DNA relatedness (81-98%) as determined by hybridization. All strains belong to the alpha-subgroup of the alpha-Proteobacteria. 16S rDNA sequence analysis led to the classification of these dichloromethane-utilizers in the genus Methylopila as a new species - Methylopila helvetica sp.nov. with the type strain DM9 (=VKM B-2189). The pink-pigmented strain DM4 belongs to the genus Methylobacterium but differs from the known members of this genus by some phenotypic properties, DNA-DNA relatedness (14-57%) and 16S rDNA sequence. Strain DM4 is named Methylobacterium dichloromethanicum sp. nov. (VKM B-2191 = DSMZ 6343).     

Morphological variation and relationships of Georgian populations of Vitis vinifera L. subsp. sylvestris (C.C. Gmel.) Hegi

Georgia is considered as one of the regions where for the first time wild growing grapevine plants were used and taken into cultivation by men. An ampelographic and ampelometric study of leaf characters and flower landmark morphometrics was carried out in seven populations of wild grapevine located in four river basins of three geographic regions – Western (1 popul.), Eastern (5 popul.) and Southern (1 popul.) Georgia, and then compared using principal component analysis and multivariate discriminant analysis allowing the populations to be distinguished. Lengths of main leaf veins and nectaries in male flowers were the variables, which allowed the best discrimination among populations. A mutation leading to white-fruited grapevine is found in one population. Pubescence varies within populations and even within an individual genome and could be used as a discriminant trait only in combination with shapes and sizes of leaves and other morphological characters. The results revealed significant differences among populations located in different geographic regions of the Southern Caucasus area, but the five investigated populations from Eastern Georgia differ only insignificantly. The high morphological variability of wild grapevine in Georgia might be considered as characterizing the large gene pool that contributed to the domestication process of grapevine.     

Balneomonas flocculans gen. nov., sp. nov., a new cellulose-producing member of the alpha-2 subclass of Proteobacteria

A new bacterial strain capable of producing cellulose was isolated from a hot spring. The isolate was Gram-negative, aerobic, and rod-shaped. The optimum temperature for growth was 40-45 degrees C. Methanol, glucose and other common carbohydrates were not utilized as sole growth substrates. Thiosulfate was not oxidized. The G+C content of the DNA was determined to be 64.0 mol%. Comparative 16S rDNA analysis indicated that Bosea thiooxidans and some strains of the genus Methylobacterium were the nearest relatives. The isolate can be distinguished from these relatives by its defectiveness in methanol utilization and thiosulfate oxidation. On the basis of its phenotypic properties and phylogeny, it is proposed that the isolate be designated Balneomonas flocculans gen. nov., sp. nov. The type strain is TFBT (= JCM 11936T, = KCTC 12101T, = IAM 15034T, = ATCC BAA-817T).     

Molecular detection and isolation of facultatively methylotrophic bacteria, including Methylobacterium podarium sp. nov., from the human foot microflora

This is the first study to demonstrate that diverse methylotrophic bacteria occur in the human foot microflora. Polymerase chain reaction (PCR) amplification of DNA from the soles and toe clefts of feet of five subjects indicated Methylobacterium strains to be present in all cases. Polymerase chain reaction amplification also showed the gene for the alpha-subunit of methanol dehydrogenase (mxaF) to be present in all samples. Two types of mxaF were recovered, one closest to that of Methylobacterium extorquens and the other most similar to that of Hyphomicrobium methylovorum. Numerous methylotrophic strains able to grow on methylamine were isolated with ease from the feet of nine volunteers. These were found by 16S rRNA analysis to be most closely related to Methylobacterium species, Brevibacterium casei, Pseudomonas strain NZ099 and P. migulae. Three strains from two subjects were of a novel species, Methylobacterium podarium sp. nov. This facultatively methylotrophic, obligately aerobic, pink-pigmented, non-motile rod grew with a wide range of multicarbon and one-carbon compounds including citrate, xylose, mono-, di-, and trimethylamine, dimethylsulphide, methanethiol, dimethylsulphoxide, dimethylsulphone and methanol.     

Patterns of Gene Duplication and Their Contribution to Expansion of Gene Families in Grapevine

Grapevine is an important fruit crop that has undergone a long history of evolution. Analysis of the whole genome sequence of grapevine has revealed presence of an early palaeo-hexaploid along with three complements. Thus, gene duplication and genome expansion are common in this genome. In this study, we identified 17,922 duplicated genes in the whole grapevine genome. Among these, 2,039; 628; 1,428; 722; and 2,942 were identified respectively as produced by genome-wide, tandem, proximal, retrotransposed, and DNA-based transposed duplications. Analyses of the evolutionary patterns for different types of duplication using non-synonymous and synonymous substitution rates uncovered a series of underlying rules. Thereafter, all the grapevine genes were classified into families, and the contributions of different types of duplication to the expansion of large families were revealed. No duplication type was solely responsible for the formation of any large gene family, but some families showed enrichment of a special type of duplication. On the basis of this study, we believe that uncovering the underlying rules for gene duplications, expansions of gene families, and their evolutionary styles will contribute significantly to a comprehensive understanding of the features of the grapevine genome.     

Construction of physical map and mapping of chromosomal virulence genes of the biovar 3 Agrobacterium (Rhizobium vitis) strain K-Ag-1

Most plant pathogenic Agrobacterium strains have been classified into three biovars, "biovar 1 (A. tumefaciens; Rhizobium radiobacter), biovar 2 (A. rhizogenes; R. rhizogenes) and biovar 3 (A. vitis; R. vitis)". The bacteria possess diverse types of genomic organization depending on the biovar. Previous genomic physical maps indicated difference in location of rDNA and chromosomally-coded virulence genes between biovar 1 and 2 genomes. In order to understand biovar 3 genome and its evolution in relation to the biovar 1, 2 and 3 genomes, we constructed physical map of a pathogenic biovar 3 strain K-Ag-1 in this study. Its genome consisted of two circular chromosomes (3.6 and 1.1 Mbp in length), and three plasmids (560, 230 and 70 kbp). Gene mapping based on the physical map showed presence of two rDNA loci in the larger chromosome and at least one rDNA locus in the smaller chromosome. Six chromosomal virulence genes, namely chvA, chvD, chvE, glgP, exoC and ros were found in the larger chromosome and not in the smaller chromosome. The location of rDNA loci is similar with that of biovar 1 genome, whereas the location of chromosomal virulence genes is similar with that of biovar 2 genome despite of the closer 16S-rRNA based phylogenetic relation of biovar 3 with biovar 1 than with biovar 2. Genomic PFGE RFLP analysis revealed that the K-Ag-1 strain, which was isolated on a kiwifruit plant in Japan, has the closest intra-species relation with two strains isolated from grapevine plants in Japan among eight biovar 3 strains examined. This datum suggests that the line of the strain is a major one in biovar 3 in Japan. Evolution of the genome of the strain is discussed based on the data.     

Characterization of genome-wide long terminal repeat retrotransposons provide insights into trait evolution of four grapevine species

Grapevine is one of the most economically important crops in the world. Although long terminal repeat (LTR) retrotransposons are thought to have played an important role in plants, its distribution in grapevine is not clear. Here, we identified genome-wide intact LTR retrotransposons in a total of six high-quality grapevine genomes from Vitis vinifera L., Vitis sylvestris C.C. Gmel., Vitis riparia Michx. and Vitis amurensis Rupr. with an average of 2938 per genome. Among them, the Copia superfamily (particularly for Ale) is a major component of the LTR retrotransposon in grapevine. Insertion time and copy number analysis revealed that the expansion of 70% LTR retrotransposons concentrating on approximately 2.5 Ma was able to drive genome size variation. Phylogenetic tree and syntenic analyses showed that most LTR retrotransposons in these genomes formed and evolved after species divergence. Furthermore, the function and expression of genes inserted by LTR retrotransposons in V. vinifera (Pinot noir) and V. riparia were explored. The length and expression of genes related to starch metabolism and quinone synthesis pathway in Pinot noir and environmental adaptation pathway in V. riparia were significantly affected by LTR retrotransposon insertion. The results improve the understanding of LTR retrotransposons in grapevine genomes and provide insights for its potential contribution to grapevine trait evolution.