NOTEReclassification of some species of Thiobacillus to the newly designated genera Acidithiobacillus gen. nov., Halothiobacillus gen. nov. and Thermithiobacillus gen. nov.

The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, ␤-proteobacterium. Analysis of the 2,909,809-bp genome will facilitate our molecular and biochemical understanding of the unusual metabolic repertoire of this bacterium, including its ability to couple denitrification to sulfur-compound oxidation, to catalyze anaerobic, nitrate-dependent oxidation of Fe(II) and U(IV), and to oxidize mineral electron donors. Notable genomic features include (i) genes encoding c-type cytochromes totaling 1 to 2 percent of the genome, which is a proportion greater than for almost all bacterial and archaeal species sequenced to date, (ii) genes encoding two [NiFe]hydrogenases, which is particularly significant because no information on hydrogenases has previously been reported for T. denitrificans and hydrogen oxidation appears to be critical for anaerobic U(IV) oxidation by this species, (iii) a diverse complement of more than 50 genes associated with sulfurcompound oxidation (including sox genes, dsr genes, and genes associated with the AMP-dependent oxidation of sulfite to sulfate), some of which occur in multiple (up to eight) copies, (iv) a relatively large number of genes associated with inorganic ion transport and heavy metal resistance, and (v) a paucity of genes encoding organic-compound transporters, commensurate with obligate chemolithoautotrophy. Ultimately, the genome sequence of T. denitrificans will enable elucidation of the mechanisms of aerobic and anaerobic sulfurcompound oxidation by ␤-proteobacteria and will help reveal the molecular basis of this organism's role in major biogeochemical cycles (i.e., those involving sulfur, nitrogen, and carbon) and groundwater restoration.Thiobacillus denitrificans, first isolated by Beijerinck over a century ago (4), was one of the first nonfilamentous bacteria ever described to be capable of growth on inorganic sulfur compounds as sole energy sources (47, 49). Characterized by its ability to conserve energy from the oxidation of inorganic sulfur compounds under either aerobic or denitrifying conditions, T. denitrificans is the best studied of the very few obligate chemolithoautotrophic species known to couple denitrification to sulfur-compound oxidation (Thiomicrospira denitrificans and Thioalkalivibrio thiocyanodenitrificans also have this ability [76,85]). Despite many years of work on the biochemistry of inorganic sulfur-compound oxidation by Thiobacillus thioparus and T. denitrificans, the mechanisms of oxidation and how they are coupled to energy conservation are still not well understood in these ␤-proteobacteria, relative to the advances made with facultatively chemolithotrophic ␣-proteobacterial genera, such as Paracoccus and Starkeya (28,39,45,50). The availability of the complete genome sequence should enable elucidation of the sulfur-oxidation pathway(s) and lead to specifically focused biochemical investigations to resolve these knowledge gaps.Rec...

A re-evaluation of the taxonomy of Paracoccus denitrificans and a proposal for the combination Paracoccus pantotrophus comb. nov.Frederick A. Rainey,' Donovan P. Kelly Comparison of both 16s rRNA coding sequences and DNA-DNA hybridization of ten strains of a-subclass of Proteobacteria currently classified as strains of Paracoccus denitrificans has shown that they fall into two groups which are distinct from each other a t the species level. Comparison with published data on the cytochrome c profiles and other 16s rRNA coding sequences in the literature has confirmed these observations and enabled several other strains also to be assigned to these two groups. Group A comprises strains ATCC 1774IT (the type strain of P. denitrificans), LMD 22.2IT, DSM 413T, ATCC 19367, ATCC 13543, DSM 1404, DSM 1405, Pd 1222 (a genetic modification of DSM 4133 and NCIMB 8944. Group B comprises ATCC 35512T (the original type strain of Thiosphaera pantotropha), LMD 82.5T, LMD 92.63, DSM 65, LMG 4218, IAM 12479, JCM 6892, DSM 11072, DSM I1073 and DSM 11104. In light of these findings, it is proposed that: (1) strains of group A are retained as P. denitrificans, with ATCC 1774IT as the type strain of the type species; and (2) all strains of group B are assigned to the new species combination Paracoccus pantotrophus comb. nov., with strain ATCC 35512T as the type strain. Comparative 16s rRNA sequence analysis and DNA-DNA hybridization of strains of Paracoccus versutus confirm that this species is distinct from both P. denitrificans and P. pantotrophus, but that its nearest phylogenetic neighbour is P. pantotrophus.

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.

Background-Despite major developments in community mental health services, inpatient care remains an important yet costly part of the service system and patients who are admitted frequently spend a long period of time in hospital. It is, therefore, crucial to have a good understanding of activities that take place on inpatient wards.

NOTE Confirmation of Thiobacillus denitrificans as a species of the genus

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. 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 FMNH 2 -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.Dimethylsulfide (DMS) is a volatile organosulfur compound, important in the biogeochemical cycling of sulfur and global climate regulation (4, 9). Bacterial metabolism of DMS is an important sink of the compound in nature and is thought to account for degradation of over 80% of the DMS produced in the marine environment. Although bacterial pathways of DMS degradation have been studied previously in Hyphomicrobium spp. and in Thiobacillus spp. (12, 36), they remain poorly characterized, and few enzymes of DMS metabolism have been purified (see reference 32). DMS monooxygenase was first reported from an assay of NADH-dependent oxygen uptake in the presence of DMS by cell extracts of Hyphomicrobium S (12), an activity also demonstrated in cell extracts of other Hyphomicrobium, Thiobacillus, and Arthrobacter isolates (6,7,34), with specific activities around 30 nmol NADH oxidized min Ϫ1 mg protein Ϫ1 . The enzyme has not previously been purified or characterized.The aims of this study were to purify and characterize the DMS monooxygenase enzyme from a member of the genus Hyphomicrobium. Since Hyphomicrobium S is no longer available, studies were undertaken using the type strain of H. sulfonivorans. The strain was originally isolated from garden soil and grows on DMS, as well as the related compounds dimethyl sulfoxide (DMSO) and dimethylsulfone (DMSO 2 ). During growth on DMSO 2 , H. sulfonivorans first reduces DMSO 2 to DMSO by a dimethylsulfone reductase, and subsequently a DMSO reductase converts DMSO to DMS, which is further oxidized to methanethiol and formaldehyde by a DMS mono-

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