Definitive assignment by multigenome analysis of the gammaproteobacterial genus Thermithiobacillus to the class Acidithiobacillia.

The class Acidithiobacillia was established using multiprotein phylogenetic analysis of all the available genomes of the genus Acidithiobacillus (comprising Family I, the Acidithiobacillaceae, of the Acidithiobacillales, the order created for Bergey's Manual of Systematic Bacteriology), and for representative genomes of all available bacterial orders. The Acidithiobacillales contain a second family, the Thermithiobacillaceae, represented by Thermithiobacillus tepidarius, and created on the basis of nearest neighbour 16S ribosomal RNA gene sequence similarities. This could not be included in the original multiprotein analysis as no genome sequence for Thermithio bacillus was available. The publication of the genome sequence of Thermithiobacillus tepidarius in 2013 has enabled phylogenetic assessment of this organism by comparative multigenome analysis. This shows definitively that Thermithiobacillus is a member of the class Acidithiobacillia, distinct from the Acidithiobacillus genus, and confirms it to represent a second family within the Acidithiobacillia.

Proposal for a new class within the phylum Proteobacteria, Acidithiobacillia classis nov., with the type order Acidithiobacillales, and emended description of the class Gammaproteobacteria.

The order Acidithiobacillales was previously assigned to the class Gammaproteobacteria. Recent analyses have indicated that this order actually lies outside all the proteobacterial classes, as a sister group to the combined classes Betaproteobacteria and Gammaproteobacteria. We now confirm this result with multiprotein phylogenetic analysis of all the available genomes of members of the order Acidithiobacillales and representatives of all available bacterial orders, and propose the new proteobacterial class, Acidithiobacillia, with the type order Acidithiobacillales, comprising the families Acidithiobacillaceae and Thermithiobacillaceae with the type genus Acidithiobacillus.

Phylogenetic assessment of culture collection strains of Thiobacillus thioparus, and definitive 16S rRNA gene sequences for T. thioparus, T. denitrificans, and Halothiobacillus neapolitanus.

The 16S rRNA gene sequences of 12 strains of Thiobacillus thioparus held by different culture collections have been compared. A definitive sequence for the reference type strain (Starkey; ATCC 8158(T)) was obtained. The sequences for four examples of the Starkey type strain were essentially identical, confirming their sustained identity after passage through different laboratories. One strain (NCIMB 8454) was reassigned as a strain of Halothiobacillus neapolitanus, and a second (NCIMB 8349) was a species of Thermithiobacillus. These two strains have been renamed in their catalog by the National Collection of Industrial and Marine Bacteria. The 16S rRNA gene sequence of the type strain of Halothiobacillus neapolitanus (NCIMB 8539(T)) was determined and used to confirm the identity of other culture collection strains of this species. The reference sequences for the type strains of Thiobacillus thioparus and Halothiobacillus neapolitanus have been added to the online List of Prokaryotic Names with Standing in Nomenclature. Comparison of the 16S rRNA gene sequences available for strains of Thiobacillus denitrificans indicated that the sequence for the type strain (NCIMB 9548(T)) should always be used as the reference sequence for new and existing isolates.

Design and evaluation of novel primers for the detection of genes encoding diverse enzymes of methylotrophy and autotrophy.

The phylogenetic significance of the diversity of key enzymes of methylotrophic and autotrophic metabolism is discussed. Primers for these key enzymes were designed using gene sequences encoding methanol dehydrogenase (mxaF; using subsets from database sequences for 22 Bacteria), hydroxypyruvate reductase (hpr; 36 sequences), methylamine dehydrogenase (mauA; 12 sequences), methanesulfonate monooxygenase (msmA; four sequences), and the ccbL and cbbM genes of ribulose bisphosphate carboxylase (26 and 23 sequences). These were effective in amplifying the correct gene products for the target genes in reference organisms and in test organisms not previously shown to contain the genes, as well as in some methylotrophic Proteobacteria isolated from the human mouth. The availability of the new primers increases the probability of detecting diverse examples of the genes encoding these key enzymes both in natural populations and in isolated bacterial strains.

Purification and characterization of dimethylsulfide monooxygenase from Hyphomicrobium sulfonivorans.

Dimethylsulfide (DMS) is a volatile organosulfur compound which has been implicated in the biogeochemical cycling of sulfur and in climate control. Microbial degradation is a major sink for DMS. DMS metabolism in some bacteria involves its oxidation by a DMS monooxygenase in the first step of the degradation pathway; however, this enzyme has remained uncharacterized until now. We have purified a DMS monooxygenase from Hyphomicrobium sulfonivorans, which was previously isolated from garden soil. The enzyme is a member of the flavin-linked monooxygenases of the luciferase family and is most closely related to nitrilotriacetate monooxygenases. It consists of two subunits: DmoA, a 53-kDa FMNH2-dependent monooxygenase, and DmoB, a 19-kDa NAD(P)H-dependent flavin oxidoreductase. Enzyme kinetics were investigated with a range of substrates and inhibitors. The enzyme had a K(m) of 17.2 (± 0.48) µM for DMS (k(cat) = 5.45 s⁻¹) and a V(max) of 1.25 (± 0.01) µmol NADH oxidized min⁻¹ (mg protein⁻¹). It was inhibited by umbelliferone, 8-anilinonaphthalenesulfonate, a range of metal-chelating agents, and Hg²(+), Cd²(+), and Pb²(+) ions. The purified enzyme had no activity with the substrates of related enzymes, including alkanesulfonates, aldehydes, nitrilotriacetate, or dibenzothiophenesulfone. The gene encoding the 53-kDa enzyme subunit has been cloned and matched to the enzyme subunit by mass spectrometry. DMS monooxygenase represents a new class of FMNH2-dependent monooxygenases, based on its specificity for dimethylsulfide and the molecular phylogeny of its predicted amino acid sequence. The gene encoding the large subunit of DMS monooxygenase is colocated with genes encoding putative flavin reductases, homologues of enzymes of inorganic and organic sulfur compound metabolism, and enzymes involved in riboflavin synthesis.

Draft genome sequence of the chemolithoheterotrophic, halophilic methylotroph Methylophaga thiooxydans DMS010.

Methylophaga thiooxydans is a mesophilic, obligately halophilic bacterium that is capable of methylotrophic growth on a range of one-carbon compounds as well as chemolithoheterotrophic growth at the expense of thiosulfate. Here we present the draft genome sequence of Methylophaga thiooxydans DMS010 (DSM 22068(T), VKM B2586(T)), the type strain of the species, which has allowed prediction of the genes involved in one-carbon metabolism, nitrogen metabolism, and other aspects of central metabolism.

Facultative methylotrophs from the human oral cavity and methylotrophy in strains of Gordonia, Leifsonia, and Microbacterium.

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.

Proposal that the arsenite-oxidizing organisms Thiomonas cuprina and 'Thiomonas arsenivorans' be reclassified as strains of Thiomonas delicata, and emended description of Thiomonas delicata.

The three As(III)-oxidizing members of the class Betaproteobacteria Thiomonas delicata, Thiomonas cuprina and 'Thiomonas arsenivorans' were isolated from mining sites in geographically distinct areas, namely Japan, Germany and France, respectively. They are all able to oxidize As(III) but only 'T. arsenivorans' and T. cuprina show efficient autotrophic growth with As(III) and are able to grow on a sole carbon source. These two organisms are also motile, whereas T. delicata is not. Only T. cuprina can grow autotrophically on chalcopyrite. The three strains share >99% gene sequence similarity with each other based on their 16S rRNA genes and 16S-23S ITS regions. DNA-DNA hybridization results are above, or close to, the threshold value of 70% recommended for the definition of bacterial species. The three taxa show very similar fatty acid profiles with differences only in five minor fatty acid components. They possess phylogenetic and chemotaxonomic similarities supporting the reclassification of these taxa as a single species. We propose that 'T. arsenivorans' and T. cuprina be reassigned as strains of T. delicata (type strain DSM 17897(T)).

Oxidation of dimethylsulfide to tetrathionate by Methylophaga thiooxidans sp. nov.: a new link in the sulfur cycle.

A new pathway of dimethylsulfide (DMS) metabolism was identified in a novel species of Gammaproteobacteria, Methylophaga thiooxidans sp. nov., in which tetrathionate (S(4)O(6)(2-)) was the end-product of DMS oxidation. Inhibitor evidence indicated that DMS degradation was initiated by demethylation, catalysed by a corrinoid demethylase. Thiosulfate was an intermediate, which was oxidized to tetrathionate by a cytochrome-linked thiosulfate dehydrogenase. Thiosulfate oxidation was coupled to ATP synthesis, and M. thiooxidans could also use exogenous thiosulfate as an energy source during chemolithoheterotrophic growth on DMS or methanol. Cultures grown on a variety of substrates oxidized thiosulfate, indicating that thiosulfate oxidation was constitutive. The observations have relevance to interactions among sulfur-metabolizing bacteria in the marine environment. The production of tetrathionate from an organosulfur precursor is previously undocumented and represents a potential step in the biogeochemical sulfur cycle, providing a 'shunt' across the cycle.

Novel methylotrophic bacteria isolated from the River Thames (London, UK).

Enrichment and elective culture for methylotrophs from sediment of the River Thames in central London yielded a diversity of pure cultures representing several genera of Gram-negative and Gram-positive bacteria, which were mainly of organisms not generally regarded as typically methylotrophic. Substrates leading to successful isolations included methanol, monomethylamine, dimethylamine, trimethylamine, methanesulfonate and dimethylsulfone. Several isolates were studied in detail and shown by their biochemical and morphological properties and 16S rRNA gene sequencing to be Sphingomonas melonis strain ET35, Mycobacterium fluoranthenivorans strain DSQ3, Rhodococcus erythropolis strain DSQ4, Brevibacterium casei strain MSQ5, Klebsiella oxytoca strains MMA/F and MMA/1, Pseudomonas mendocina strain TSQ4, and Flavobacterium sp. strains MSA/1 and MMA/2. The results show that facultative methylotrophy is present across a wide range of Bacteria, suggesting that turnover of diverse C(1)-compounds is of much greater microbiological and environmental significance than is generally thought. The origins of the genes encoding the enzymes of methylotrophy in diverse heterotrophs need further study, and could further our understanding of the phylogeny and antiquity of methylotrophic systems.

Stable sulfur isotope fractionation and discrimination between the sulfur atoms of thiosulfate during oxidation by Halothiobacillus neapolitanus.

Growing cultures and nongrowing suspensions of Halothiobacillus neapolitanus selectively fractionated (32)S and (34)S during the oxidation of the sulfane- and sulfonate-sulfur atoms of thiosulfate. Sulfate was enriched in (32)S, with delta(34)S reaching -6.3 per thousand relative to the precursor sulfonate-sulfur of thiosulfate, which was progressively resynthesized from the thiosulfate-sulfane-sulfur during thiosulfate metabolism. Polythionates, principally trithionate, accumulated during thiosulfate oxidation and showed progressive increase in the relative (34)S content of their sulfonate groups, with delta(34)S values up to +20 per thousand, relative to the substrate sulfur. The origins of the sulfur in the sulfate and polythionate products of oxidation were tracked by the use thiosulfate labelled with (35)S in each of its sulfur atoms, enabling determination of the flow of the sulfur atoms into the oxidation products. The results confirm that highly significant fractionation of stable sulfur isotopes can be catalyzed by thiobacilli oxidizing thiosulfate, but that differences in the (34)S/(32)S ratios of the nonequivalent constituent sulfur atoms of the thiosulfate used as substrate mean that the oxidative fate of each atom needs separate determination. The data are very significant to the understanding of bacterial sulfur-compound oxidation and highly relevant to the origins of biogenic sulfate minerals.

Stable sulfur isotope fractionation by the green bacterium Chlorobaculum parvum during photolithoautotrophic growth on sulfide.

Growing cultures of the green obligate photolithotroph, Chlorobaculum parvum DSM 263T (formerly Chlorobium vibrioforme forma specialis thiosulfatophilum NCIB 8327), oxidized sulfide quantitatively to elemental sulfur, with no sulfate formation. In the early stages of growth and sulfide oxidation, the sulfur product became significantly enriched with 34S, with a maximum delta34S above +5 per thousand, while the residual sulfide was progressively depleted in 34S to delta34S values greater than -4 per thousand. As oxidation proceeded, the delta34S of the sulfur declined to approach that of the initial sulfide when most of the substrate sulfide had been converted to sulfur in this closed culture system. No significant formation of sulfate occurred, and the substrate sulfide and elemental sulfur product accounted for all the sulfur provided throughout oxidation. The mean isotope fractionation factors (epsilon) for sulfide and sulfur were equivalent at epsilon values of -2.4 per thousand and +2.4 per thousand respectively. The significance of the experimentally-observed fractionation to the 34S/32S ratios seen in natural sulfur-containing minerals is considered.

Insights into the obligate methanotroph Methylococcus capsulatus.

Completion of the genome sequence of Methylococcus capsulatus Bath is an important event in molecular microbiology, and an achievement for which the authors deserve congratulation. M. capsulatus, along with other methanotrophs, has been the subject of intense biochemical and molecular study because of its role in the global carbon cycle: the conversion of biogenic methane to carbon dioxide. The methane monooxygenase enzymes that are central to this process also have high biotechnological potential. Analysis of the genome sequence will potentially accelerate elucidation of the regulation of methane-dependent metabolism in obligate methanotrophs, and help explain the cause of obligate methanotrophy, the phenomenon making most methanotrophs unable to grow on any substrates other than methane and a very small number of other one-carbon compounds.

Inhibitory effects of sulfamic acid on three thiosulfate-oxidizing chemolithotrophs.

Sulfamate is an analogue of thiosulfate, and the sodium and potassium salts of sulfamic acid inhibited the chemolithoautotrophic growth on thiosulfate of Acidithiobacillus ferrooxidans and Halothiobacillus neapolitanus. The chemo-organotrophic growth of Paracoccus versutus on sucrose was similarly inhibited by sulfamate. Thiosulfate oxidation by suspensions of H. neapolitanus was, however, unaffected by sulfamate, showing that sulfamate did not directly affect thiosulfate uptake, activation or oxidation. Inhibition of P. versutus was not relieved by cysteine and methionine, indicating that sulfate uptake and sulfur amino acid biosynthesis were not directly affected by sulfamate. Sulfamate was not degraded by any of the bacteria, and so could not serve as an alternative to thiosulfate as an energy-yielding substrate. Sulfamate is also an analogue of ammonia and might act like hydrazine by inhibiting ammonium uptake or an essential enzyme activity.

A challenge for 21st century molecular biology and biochemistry: what are the causes of obligate autotrophy and methanotrophy?

We assess the use to which bioinformatics in the form of bacterial genome sequences, functional gene probes and the protein sequence databases can be applied to hypotheses about obligate autotrophy in eubacteria. Obligate methanotrophy and obligate autotrophy among the chemo- and photo-lithotrophic bacteria lack satisfactory explanation a century or more after their discovery. Various causes of these phenomena have been suggested, which we review in the light of the information currently available. Among these suggestions is the absence in vivo of a functional alpha-ketoglutarate dehydrogenase. The advent of complete and partial genome sequences of diverse autotrophs, methylotrophs and methanotrophs makes it possible to probe the reasons for the absence of activity of this enzyme. We review the role and evolutionary origins of the Krebs cycle in relation to autotrophic metabolism and describe the use of in silico methods to probe the partial and complete genome sequences of a variety of obligate genera for genes encoding the subunits of the alpha-ketoglutarate dehydrogenase complex. Nitrosomonas europaea and Methylococcus capsulatus, which lack the functional enzyme, were found to contain the coding sequences for the E1 and E2 subunits of alpha-ketoglutarate dehydrogenase. Comparing the predicted physicochemical properties of the polypeptides coded by the genes confirmed the putative gene products were similar to the active alpha-ketoglutarate dehydrogenase subunits of heterotrophs. These obligate species are thus genomically competent with respect to this enzyme but are apparently incapable of producing a functional enzyme. Probing of the full and incomplete genomes of some cyanobacterial and methanogenic genera and Aquifex confirms or suggests the absence of the genes for at least one of the three components of the alpha-ketoglutarate dehydrogenase complex in these obligate organisms. It is recognized that absence of a single functional enzyme may not explain obligate autotrophy in all cases and may indeed be only be one of a number of controls that impose obligate metabolism. Availability of more genome sequences from obligate genera will enable assessment of whether obligate autotrophy is due to the absence of genes for a few or many steps in organic compound metabolism. This problem needs the technologies and mindsets of the present generation of molecular microbiologists to resolve it.

Halothiobacillus neapolitanus strain OSWA isolated from "The Old Sulphur Well" at Harrogate (Yorkshire, England).

The "Old Sulphur Well" has a subterranean input of water containing 5.5mM total sulfide, which would be inhibitory to the growth of most bacteria. The obligately chemolithoautotrophic Halothiobacillus neapolitanus is a sulfur bacterium known to tolerate and metabolize high sulfide concentrations, and we report the isolation of H. neapolitanus strain OSWA from this source. Strain OSWA grows well on thiosulfate and tetrathionate as energy sources, and tolerates at least 5mM sulfide. Its specific growth rates and yields in batch culture were 0.22h(-1) and 5.3 gmol(-1) (thiosulfate), and 0.23 h(-1) and 9.5 gmol(-1) (tetrathionate). Its 16S rRNA gene sequence shows >99% identity to reference sequences of H. neapolitanus, and it shares morphological and physiological characteristics typical of the species. It is one of a very small number of strains of H. neapolitanus described to date, and the first to be isolated from an ancient sulfide-rich natural spa.

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.

Further evidence to justify reassignment of Mycoplasma mycoides subspecies mycoides Large Colony type to Mycoplasma mycoides subspecies capri.

Analysis, using the polymerase chain reaction (PCR), restriction enzyme endonuclease analysis (REA), protein profile patterns, random amplification of polymorphic DNA (RAPD) fingerprinting, 16S rRNA gene sequencing and antisera growth inhibition tests, of 22 strains of Mycoplasma mycoides subsp. mycoides Large Colony type (MmmLC) and eight strains of M. mycoides subsp. capri (Mmc) are presented, along with a summary of comparative data from the literature for over 100 strains, all of which supports the reclassification of the MmmLC and Mmc strains into the single subspecies, M. mycoides subspecies capri.

Kinetics of substrate oxidation and hydrogen peroxide production by Mycoplasma mycoides subsp. mycoides Large Colony (LC) type and Mycoplasma mycoides subsp. capri.

Mycoplasma mycoides subsp. mycoides Large Colony (LC) type is a pathogen of goats causing contagious agalactia and respiratory disease, found on all continents where small ruminants are kept. It shares close genetic characteristics with M. mycoides subsp. capri. Substrate oxidation by 22 strains of M. mycoides subsp. mycoides LC from nine countries was compared with that of eight strains of M. mycoides subsp. capri from five countries. There was considerable similarity in the substrates used, but substrate saturation coefficients (K(s)) varied for different substrates. Substrate utilization patterns and K(s) values did not (1) significantly differentiate the LC strains from each other, (2) show any correlation with geographical origin, or (3) distinguish the LC strains from the capri strains. These results support previous studies justifying the reclassification of these subspecies as a single species.

Proposal that the strains of the Mycoplasma ovine/caprine serogroup 11 be reclassified as Mycoplasma bovigenitalium.

This proposal is our response to the recommendation of the International Committee on Systematics of Prokaryotes (Subcommittee on the taxonomy of Mollicutes) that we 'write a proposal to classify Mycoplasma bovigenitalium and ovine/caprine serogroup 11 as a single species'. Physiological and phylogenetic comparisons between 27 strains of M. bovigenitalium and Mycoplasma serogroup 11 showed that (i) growth and patterns of organic acid substrate use completely overlapped among strains; (ii) all had lipase and phosphatase activities; (iii) the strains were indistinguishable in their SDS-PAGE whole-cell protein profiles, which differed from five other species; (iv) strains were indistinguishable in immunoblotting of cell proteins and cross-reactivity in ELISA, but differed from other Mycoplasma species; (v) DNA-DNA hybridization did not distinguish between the two groups, and (vi) comparison of 16S and 23S rRNA gene sequences of ten strains of Mycoplasma serogroup 11 and six strains of M. bovigenitalium showed that they shared 98-100% similarity across all strains tested, but only 86-95% to other Mycoplasma species. Strains of the Mycoplasma ovine/caprine serogroup 11 must therefore be reassigned as Mycoplasma bovigenitalium.