Methyloversatilis universalis gen. nov., sp. nov., a novel taxon within the Betaproteobacteria represented by three methylotrophic isolates

Functional and genomic diversity of methylotrophic Rhodocyclaceae: description of Methyloversatilis discipulorum sp. nov.

Two novel genera of restricted facultative methylotrophs are described; both Methylosulfonomonas and Marinosulfonomonas are unique in being able to grow on methanesulfonic acid as their sole source of carbon and energy. Five identical strains of Methylosulfonomonas were isolated from diverse soil samples in England and were shown to differ in their morphology, physiology, DNA base composition, molecular genetics, and 16S rDNA sequences from the two marine strains of Marinosulfonomonas, which were isolated from British coastal waters. The marine strains were almost indistinguishable from each other and are considered to be strains of one species. Type species of each genus have been identified and named Methylosulfonomonas methylovora (strain M2) and Marinosulfonomonas methylotropha (strain PSCH4). Phylogenetic analysis using 16S rDNA sequencing places both genera in the alpha-Proteobacteria. Methylosulfonomonas is a discrete lineage within the alpha-2 subgroup and is not related closely to any other known bacterial genus. The Marinosulfonomonas strains form a monophyletic cluster in the alpha-3 subgroup of the Proteobacteria with Roseobacter spp. and some other partially characterized marine bacteria, but they are distinct from these at the genus level. This work shows that the isolation of bacteria with a unique biochemical character, the ability to grow on methanesulfonic acid as energy and carbon substrate, has resulted in the identification of two novel genera of methylotrophs that are unrelated to any other extant methylotroph genera.

Marinosulfonomonas methylotropha strain TR3 is a marine methylotroph that uses methanesulfonic acid (MSA) as a sole carbon and energy source. The genes from M. methylotropha strain TR3 encoding methanesulfonate monooxygenase, the enzyme responsible for the initial oxidation of MSA to formaldehyde and sulfite, were cloned and sequenced. They were located on two gene clusters on the chromosome of this bacterium. A 5.0-kbp HindIII fragment contained msmA, msmB, and msmC, encoding the large and small subunits of the hydroxylase component and the ferredoxin component, respectively, of the methanesulfonate monooxygenase, while a 6.5-kbp HindIII fragment contained duplicate copies of msmA and msmB, as well as msmD, encoding the reductase component of methanesulfonate. Both sets of msmA and msmB genes were virtually identical, and the derived msmA and msmB sequences of M. methylotropha strain TR3, compared with the corresponding hydroxylase from the terrestrial MSA utilizer Methylosulfonomonas methylovora strain M2 were found to be 82 and 69% identical. The msmA gene was investigated as a functional gene probe for detection of MSA-utilizing bacteria. PCR primers spanning a region of msmA which encoded a unique Rieske [2Fe-2S] binding region were designed. These primers were used to amplify the corresponding msmA genes from newly isolated Hyphomicrobium, Methylobacterium, and Pedomicrobium species that utilized MSA, from MSA enrichment cultures, and from DNA samples extracted directly from the environment. The high degree of identity of these msmA gene fragments, compared to msmA sequences from extant MSA utilizers, indicated the effectiveness of these PCR primers in molecular microbial ecology.

Methylosulfonomonas methylovora M2 is an unusual gram-negative methylotrophic bacterium that can grow on methanesulfonic acid (MSA) as the sole source of carbon and energy. Oxidation of MSA by this bacterium is carried out by a multicomponent MSA monooxygenase (MSAMO). Cloning and sequencing of a 7.5-kbp SphI fragment of chromosomal DNA revealed four tightly linked genes encoding this novel monooxygenase. Analysis of the deduced MSAMO polypeptide sequences indicated that the enzyme contains a two-component hydroxylase of the mononuclear-iron-center type. The large subunit of the hydroxylase, MsmA (48 kDa), contains a typical Rieske-type [2Fe–2S] center with an unusual iron-binding motif and, together with the small subunit of the hydroxylase, MsmB (20 kDa), showed a high degree of identity with a number of dioxygenase enzymes. However, the other components of the MSAMO, MsmC, the ferredoxin component, and MsmD, the reductase, more closely resemble those found in other classes of oxygenases. MsmC has a high degree of identity to ferredoxins from toluene and methane monooxygenases, which are enzymes characterized by possessing hydroxylases containing μ-oxo bridge binuclear iron centers. MsmD is a reductase of 38 kDa with a typical chloroplast-like [2Fe–2S] center and conserved flavin adenine dinucleotide- and NAD-binding motifs and is similar to a number of mono- and dioxygenase reductase components. Preliminary analysis of the genes encoding MSAMO from a marine MSA-degrading bacterium, Marinosulfonomonas methylotropha, revealed the presence of msm genes highly related to those found in Methylosulfonomonas, suggesting that MSAMO is a novel type of oxygenase that may be conserved in all MSA-utilizing bacteria.

Fluorinated compounds are known to be more resistant to microbial degradation than other halogenated chemicals. A microbial consortium capable of aerobic biodegradation of fluorobenzene (FB) as the sole source of carbon and energy was isolated by selective enrichment from sediments collected in a drain near an industrial site. A combination of three microbial strains recovered from the enriched consortium was shown to be necessary for complete FB mineralization. Two of the strains (F1 and F3) were classified by 16S rRNA analysis as belonging to the Sphingobacterium/Flavobacterium group, while the third (F4) falls in the ␤-Proteobacteria group, clustering with Alcaligenes species. Strain F4 was consistently found in the liquid cultures in a much greater proportion than strains F1 and F3 (86:8:6 for F4, F1, and F3, respectively). Stoichiometric release of fluoride ions was measured in batch and fed-batch cultures. In batch cultures, the consortium was able to use FB up to concentrations of 400 mg liter ؊1 and was able to utilize a range of other organic compounds, including 4-fluorophenol and 4-fluorobenzoate. To our knowledge this is the first time biodegradation of FB as a sole carbon source has been reported.The advances in organic synthesis have led to the introduction of numerous new organic compounds into the environment, whose susceptibilities to biotreatment processes are unknown. Fluoroaromatics are being increasingly used in a wide range of agrochemical and pharmaceutical products, due to the need to find environmentally acceptable alternatives to chlorinated compounds (17). The diversity of structures and the chemical inertness of many halogenated organics pose particular problems and challenges for microbial degradation (10). Some authors propose that the recalcitrance of a halogenated organic compound usually becomes greater with the increase of the electronegativity of the substituents; thus, the recalcitrance of F-C is greater than that of Cl-C, Br-C, and I-C (9).The biodegradation of a vast range of halogenated aromatic compounds, especially chlorinated compounds, has been described (13,22), but scant information is available on the metabolic and cometabolic fate of fluorinated aromatic compounds in bacteria. Examples of biodegradation of fluorinated compounds most commonly found in the literature involve fluorobenzoic acids (7,15,20,21,24) and fluorophenols (1, 2, 23). Although degradation under aerobic conditions is usually reported, anaerobic degradation of fluorobenzoates under denitrifying conditions has also been reported (26). The existence of various metabolic pathways, some of which may lead to the formation of inhibitor metabolites, has been reported (15,24,25). In some cases, as in the degradation of fluoroacetate, a specific enzyme is responsible for the cleavage of the C-F bond (12). Studies on the metabolism of 2-flurobenzoate have shown that cleavage of the C-F bond occurs incidentally during oxygenase attack on the aromatic ring (20). It has been reported that biodegradation of fluo...

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A pure bacterial strain capable of aerobic biodegradation of fluorobenzene (FB) as the sole carbon and energy source was isolated by selective enrichment from sediments collected from a polluted site. 16S rRNA and fatty acid analyses support that strain F11 belongs to a novel genus within the alpha-2 subgroup of the Proteobacteria, possibly within a new clade related to the order Rhizobiales. In batch cultures, growth of strain F11 on FB led to stoichiometric release of fluoride ion. Maximum experimental growth rate of 0.04 h-1 was obtained at FB concentration of 0.4 mM. Growth kinetics were described by the Luong model. An inhibitory effect with increasing FB concentrations was observed, with no growth occurring at concentrations higher than 3.9 mM. Strain F11 was shown to be able to use a range of other organic compounds, including other fluorinated compounds such as 2-fluorobenzoate, 4-fluorobenzoate and 4-fluorophenol. To our knowledge, this is the first time biodegradation of FB, as the sole carbon and energy source, by a pure bacterium has been reported.