Molecular detection and isolation from antarctica of methylotrophic bacteria able to grow with methylated sulfur compounds

This study is the first demonstration that a diverse facultatively methylotrophic microbiota exists in some Antarctic locations. PCR amplification of genes diagnostic for methylotrophs was carried out with bacterial DNA isolated from 14 soil and sediment samples from ten locations on Signy Island, South Orkney Islands, Antarctica. Genes encoding the mxaF of methanol dehydrogenase, the fdxA for Afipia ferredoxin, the msmA of methanesulfonate monooxygenase, and the 16S rRNA gene of Methylobacterium were detected in all samples tested. The mxaF gene sequences corresponded to those of Hyphomicrobium, Methylobacterium, and Methylomonas. Over 30 pure cultures of methylotrophs were isolated on methanesulfonate, dimethylsulfone, or dimethylsulfide from ten Signy Island lakes. Some were identified from 16S rRNA gene sequences (and morphology) as Hyphomicrobium species, strains of Afipia felis, and a methylotrophic Flavobacterium strain. Antarctic environments thus contain diverse methylotrophic bacteria, growing on various C1-substrates, including C1-sulfur compounds.     

Isolation and molecular detection of methylotrophic bacteria occurring in the human mouth

Diverse methylotrophic bacteria were isolated from the tongue, and supra- and subgingival plaque in the mouths of volunteers and patients with periodontitis. One-carbon compounds such as dimethylsulfide in the mouth are likely to be used as growth substrates for these organisms. Methylotrophic strains of Bacillus, Brevibacterium casei, Hyphomicrobium sulfonivorans, Methylobacterium, Micrococcus luteus and Variovorax paradoxus were characterized physiologically and by their 16S rRNA gene sequences. The type strain of B. casei was shown to be methylotrophic. Enzymes of methylotrophic metabolism were characterized in some strains, and activities consistent with growth using known pathways of C1-compound metabolism demonstrated. Genomic DNA from 18 tongue and dental plaque samples from nine volunteers was amplified by the polymerase chain reaction using primers for the 16S rRNA gene of Methylobacterium and the mxaF gene of methanol dehydrogenase. MxaF was detected in all nine volunteers, and Methylobacterium was detected in seven. Methylotrophic activity is thus a feature of the oral bacterial community.     

1株甲基杆菌Methylobacterium sp.WGM16的鉴定及降解甲醇的最佳培养条件

从水稻根部土壤中筛选到1株粉红色、需氧的兼性甲基营养型菌株WGM16,该菌为革兰阴性杆菌。根据菌株16S rRNA基因序列比对分析及结合菌株常规形态特征、生理生化性状的鉴定,将该菌初步鉴定为Methylobacterium sp.PCR扩增到菌株WGM16编码甲醇脱氢酶α-亚基的mxaF基因,表明菌株WGM16中存在甲基营养代谢途径。在培养温度为32℃、以1%的甲醇作为碳源、pH值为8.0的培养条件下,其甲醇降解率可达75%。     

Analysis of 16S rRNA and mxaF genes revealing insights into Methylobacterium niche-specific plant association

The genus Methylobacterium comprises pink-pigmented facultative methylotrophic (PPFM) bacteria, known to be an important plant-associated bacterial group. Species of this group, described as plant-nodulating, have the dual capacity of producing cytokinin and enzymes, such as pectinase and cellulase, involved in systemic resistance induction and nitrogen fixation under specific plant environmental conditions. The aim hereby was to evaluate the phylogenetic distribution of Methylobacterium spp. isolates from different host plants. Thus, a comparative analysis between sequences from structural (16S rRNA) and functional mxaF (which codifies for a subunit of the enzyme methanol dehydrogenase) ubiquitous genes, was undertaken. Notably, some Methylobacterium spp. isolates are generalists through colonizing more than one host plant, whereas others are exclusively found in certain specific plant-species. Congruency between phylogeny and specific host inhabitance was higher in the mxaF gene than in the 16S rRNA, a possible indication of function-based selection in this niche. Therefore, in a first stage, plant colonization by Methylobacterium spp. could represent generalist behavior, possibly related to microbial competition and adaptation to a plant environment. Otherwise, niche-specific colonization is apparently impelled by the host plant.     

Nodulation and plant-growth promotion by methylotrophic bacteria isolated from tropical legumes

The nitrogen fixing methylotrophic bacteria were isolated from the nodules of tropical legumes. Two isolates CMCJ317 and CMSA322 isolated from Crotalaria juncea and Sesbania aculeata possessing high nitrogenase activities under pure culture conditions and able to form nodules under inoculated conditions were further characterized. The biochemical characteristics revealed their close relationship with Methylobacterium nodulans type strain ORS2060. The PCR amplification of nodA and mxaF genes showed the expected 584 and 555 bp products, respectively, similar to M. nodulans ORS2060 and digestion with restriction enzymes revealed that the two isolates differed. The strains showed significantly higher nitrogenase activity and also improved nodulation and shoot nitrogen of the plants when inoculated to Macroptilum atropurpureum. CMCJ317 and CMSA322 formed nodules on C. juncea and M. atropurpureum under green house conditions and also significantly increased the nitrogen concentration in shoots. These findings show that the ability to establish symbiosis with legumes is more widespread in Methylobacterium.     

Methylotrophic Methylobacterium bacteria nodulate and fix nitrogen in symbiosis with legumes

Rhizobia described so far belong to three distinct phylogenetic branches within the alpha-2 subclass of Proteobacteria. Here we report the discovery of a fourth rhizobial branch involving bacteria of the Methylobacterium genus. Rhizobia isolated from Crotalaria legumes were assigned to a new species, "Methylobacterium nodulans," within the Methylobacterium genus on the basis of 16S ribosomal DNA analyses. We demonstrated that these rhizobia facultatively grow on methanol, which is a characteristic of Methylobacterium spp. but a unique feature among rhizobia. Genes encoding two key enzymes of methylotrophy and nodulation, the mxaF gene, encoding the alpha subunit of the methanol dehydrogenase, and the nodA gene, encoding an acyltransferase involved in Nod factor biosynthesis, were sequenced for the type strain, ORS2060. Plant tests and nodA amplification assays showed that "M. nodulans" is the only nodulating Methylobacterium sp. identified so far. Phylogenetic sequence analysis showed that "M. nodulans" NodA is closely related to Bradyrhizobium NodA, suggesting that this gene was acquired by horizontal gene transfer.     

Methylotrophic metabolism is advantageous for Methylobacterium extorquens during colonization of Medicago truncatula under competitive conditions

Facultative methylotrophic bacteria of the genus Methylobacterium are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume Medicago truncatula. Competition experiments with Methylobacterium extorquens wild-type strain AM1 and methylotrophy mutants revealed that the ability to use methanol as a carbon and energy source provides a selective advantage during colonization of M. truncatula. Differences in the fitness of mutants defective in different stages of methylotrophic metabolism were found; whereas approximately 25% of the mutant incapable of oxidizing methanol to formaldehyde (deficient in methanol dehydrogenase) was recovered, 10% or less of the mutants incapable of oxidizing formaldehyde to CO2 (defective in biosynthesis of the cofactor tetrahydromethanopterin) was recovered. Interestingly, impaired fitness of the mutant strains compared with the wild type was found on leaves and roots. Single-inoculation experiments showed, however, that mutants with defects in methylotrophy were capable of plant colonization at the wild-type level, indicating that methanol is not the only carbon source that is accessible to Methylobacterium while it is associated with plants. Fluorescence microscopy with a green fluorescent protein-labeled derivative of M. extorquens AM1 revealed that the majority of the bacterial cells on leaves were on the surface and that the cells were most abundant on the lower, abaxial side. However, bacterial cells were also found in the intercellular spaces inside the leaves, especially in the epidermal cell layer and immediately underneath this layer.     

The rare occurrence of plant tissue culture contamination by Methylobacterium mesophilicum

A pink-pigmented, facultative methylotrophic (PPFM) bacterium, Methylobacterium mesophilicum, which is found on the leaf surface of most plants, has been reported to be a covert contaminant of tissue cultures initiated from Glycine max (soybean) leaves and seeds by Holland and Polacco (1992). The bacteria can be detected as pink colonies when leaves are pressed or tissue culture homogenates are plated on a medium with methanol as the sole carbon source. Since the presence of contaminating bacteria can confound any biochemical results obtained with such cultures (Holland and Polacco 1992), we wanted to determine the extent of the contamination of our tissue cultures of soybean and other species. No PPFMs were detected in any soybean culture we have, and previous results describing the biochemical characteristics of ureide utilization by one of our soybean suspension cultures (27C) also indicates that PPFM bacteria were not present. Analysis of about 200 other strains of 11 different species maintained in this lab showed that only three of about 160 callus cultures, recently initiated from Datura innoxia leaves, contained PPFMs. The D. innoxia leaves did have PPFMs on their surface but in most cases they did not survive the surface disinfestation and culture regimes. Thus PPFM bacterial contamination should not be a serious problem in most plant tissue cultures.Abbreviations AMS ammonium mineral salts medium - PPFM pink-pigmented facultative methylotrophic bacteria     

Novel formaldehyde-activating enzyme in Methylobacterium extorquens AM1 required for growth on methanol

Formaldehyde is toxic for all organisms from bacteria to humans due to its reactivity with biological macromolecules. Organisms that grow aerobically on single-carbon compounds such as methanol and methane face a special challenge in this regard because formaldehyde is a central metabolic intermediate during methylotrophic growth. In the alpha-proteobacterium Methylobacterium extorquens AM1, we found a previously unknown enzyme that efficiently catalyzes the removal of formaldehyde: it catalyzes the condensation of formaldehyde and tetrahydromethanopterin to methylene tetrahydromethanopterin, a reaction which also proceeds spontaneously, but at a lower rate than that of the enzyme-catalyzed reaction. Formaldehyde-activating enzyme (Fae) was purified from M. extorquens AM1 and found to be one of the major proteins in the cytoplasm. The encoding gene is located within a cluster of genes for enzymes involved in the further oxidation of methylene tetrahydromethanopterin to CO(2). Mutants of M. extorquens AM1 defective in Fae were able to grow on succinate but not on methanol and were much more sensitive toward methanol and formaldehyde. Uncharacterized orthologs to this enzyme are predicted to be encoded by uncharacterized genes from archaea, indicating that this type of enzyme occurs outside the methylotrophic bacteria.     

XoxF is required for expression of methanol dehydrogenase in Methylobacterium extorquens AM1

In Gram-negative methylotrophic bacteria, the first step in methylotrophic growth is the oxidation of methanol to formaldehyde in the periplasm by methanol dehydrogenase. In most organisms studied to date, this enzyme consists of the MxaF and MxaI proteins, which make up the large and small subunits of this heterotetrameric enzyme. The Methylobacterium extorquens AM1 genome contains two homologs of MxaF, XoxF1 and XoxF2, which are ～50% identical to MxaF and ～90% identical to each other. It was previously reported that xoxF is not required for methanol growth in M. extorquens AM1, but here we show that when both xoxF homologs are absent, strains are unable to grow in methanol medium and lack methanol dehydrogenase activity. We demonstrate that these defects result from the loss of gene expression from the mxa promoter and suggest that XoxF is part of a complex regulatory cascade involving the 2-component systems MxcQE and MxbDM, which are required for the expression of the methanol dehydrogenase genes.     

Diversity and biogeography of selected phyllosphere bacteria with special emphasis on Methylobacterium spp

On the basis of cultivation-dependent (isolation on mineral salt medium supplemented with 0.5% methanol) and -independent (DGGE analysis) methods, we investigated the influence of the host plant species Trifolium repens and Cerastium holosteoides, three geographic locations and the land-use types meadow, mown pasture and pasture on the abundance and community composition of selected phyllosphere bacteria with emphasis on Methylobacterium species. Methylobacterium abundance was significantly higher on leaves of T. repens (mean value 2.0×10(7) CFU PPFM per g leaf) than on leaves of C. holosteoides (mean value 2.0×10(6) CFU per g leaf). Leaves from the sampling site Schorfheide-Chorin showed slightly lower Methylobacterium numbers than leaves of the other sampling sites. Land-use and sampling period had no consistent influence on Methylobacterium community size. Methylobacterium community composition was very similar over both sampling periods, all three sampling sites, all land-use types and both plant species. Moreover, no relationship between geographic and genetic distance was observed. Community composition of selected Proteobacteria was influenced by plant species, geographic location and land-use. Often, differences in community composition could be observed between meadows, mown pastures and pastures but not between different kinds of meadows (cutted once versus three times) and mown pastures (fertilized versus non-fertilized). The results also indicate, that whether there are differences between land-use types or not strongly depends on the investigated host plant species and ecosystem. Besides Methylobacterium, representatives of Methylophilus were detected. The results indicate that Methylobacterium species are generally abundant and stable members of the phyllosphere community whereas other genera occur more occasionally, and that Methylobacterium clearly dominates the methylotrophic phyllosphere community.     

Use of tRNA consensus primers to indicate subgroups of Pseudomonas solanacearum by polymerase chain reaction amplification.

Polymerase chain reaction amplification of DNA from 112 Pseudomonas solanacearum strains with the tRNA consensus primers T3A and T5A divided the species into three fingerprint groups. These groups correspond well with previous divisions made by restriction fragment length polymorphism analysis. This polymerase chain reaction test is a facile method for rapidly classifying P. solanacearum strains.     

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.     

Production of acyl-homoserine lactone quorum-sensing signals is widespread in gram-negative Methylobacterium

Members of Methylobacterium, referred as pink-pigmented facultative methylotrophic bacteria, are frequently associated with terrestrial and aquatic plants, tending to form aggregates on the phyllosphere. We report here that the production of autoinducer molecules involved in the cell-to-cell signaling process, which is known as quorum sensing, is common among Methylobacterium species. Several strains of Methylobacterium were tested for their ability to produce N-acyl-homoserine lactone (AHL) signal molecules using different indicators. Most strains of Methylobacterium tested could elicit a positive response in Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. The synthesis of these compounds was cell-density dependent, and the maximal activity was reached during the late exponential to stationary phases. The bacterial extracts were separated by thin-layer chromatography and bioassayed with A. tumefaciens NT1 (traR, tra::lacZ749). They revealed the production of various patterns of the signal molecules, which are strain dependent. At least two signal molecules could be detected in most of the strains tested, and comparison of their relative mobilities suggested that they are homologs of N-octanoyl-DL-homoserine lactone (C8-HSL) and N-decanoyl-DL-homoserine lactone (C10-HSL).     

Characterization and heterologous gene expression of a novel esterase from Lactobacillus casei CL96

A novel esterase gene (estI) of Lactobacillus casei CL96 was localized on a 3.3-kb BamHI DNA fragment containing an open reading frame (ORF) of 1,800 bp. The ORF of estI was isolated by PCR and expressed in Escherichia coli, the methylotrophic bacterium Methylobacterium extorquens, and the methylotrophic yeast Pichia pastoris under the control of T7, methanol dehydrogenase (P(mxaF)), and alcohol oxidase (AOX1) promoters, respectively. The amino acid sequence of EstI indicated that the esterase is a novel member of the GHSMG family of lipolytic enzymes and that the enzyme contains a lipase-like catalytic triad, consisting of Ser325, Asp516, and His558. E. coli BL21(DE3)/pLysS containing estI expressed a novel 67.5-kDa protein corresponding to EstI in an N-terminal fusion with the S. tag peptide. The recombinant L. casei CL96 EstI protein was purified to electrophoretic homogeneity in a one-step affinity chromatography procedure on S-protein agarose. The optimum pH and temperature of the purified enzyme were 7.0 and 37 degrees C, respectively. Among the pNP (p-nitrophenyl) esters tested, the most selective substrate was pNP-caprylate (C(8)), with K(m) and k(cat) values of 14 +/- 1.08 microM and 1,245 +/- 42.3 S(-1), respectively.     

High-throughput identification and screening of novel Methylobacterium species using whole-cell MALDI-TOF/MS analysis

Methylobacterium species are ubiquitous α-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing.     

Facultative methylotrophs from the human oral cavity and methylotrophy in strains of <Emphasis Type="Italic">Gordonia,Leifsonia</Emphasis>, and <Emphasis Type="Italic">Microbacterium</Emphasis>

We show that bacteria with methylotrophic potential are ubiquitous in the human mouth microbiota. Numerous strains of Actinobacteria (Brevibacterium, Gordonia, Leifsonia, Microbacterium, Micrococcus, Rhodococcus) and Proteobacteria (Achromobacter, Klebsiella, Methylobacterium, Pseudomonas, Ralstonia) were isolated, and one strain of each of the eleven genera was studied in detail. These strains expressed enzymes associated with methylotrophic metabolism (methanol, methylamine, and formate dehydrogenases), and the assimilation of one-carbon compounds by the serine pathway (hydroxypyruvate reductase). Methylotrophic growth of the strains was enhanced by the addition of glass beads to cultures, suggesting that they may naturally occur in biofilms in the mouth. This is the first report of Gordonia, Leifsonia, and Rhodococcus being present in the mouth and of the unequivocal demonstration for the first time of the methylotrophic potential of strains of Gordonia, Leifsonia, and Microbacterium.