Publisher Summary This chapter discusses the metabolic aspects of aerobic obligate methanotrophy. Aerobic methanotrophs are a unique group of gram-negative bacteria that use methane as carbon and energy source. Methanotrophs have been studied intensively over the past 40 years since these bacteria possess significant metabolic potential for practical use in the biotransformation of a variety of organic substrates, bioremediation of pollutants the production of single-cell protein (SCP), and value-added products. They also play a vital role in the global methane cycle, mitigating the emissions and green-house effects of methane on the Earth's climate. Methanotrophs build all of their cell constituents from C1 compounds by employing special biosynthetic pathways for phosphotrioses, which are different from those of heterotrophic bacteria.

Metabolic aspects of aerobic obligate methanotrophy.

The complete genome sequence of Thiobacillus denitrificans ATCC 25259 is the first to become available for an obligately chemolithoautotrophic, sulfur-compound-oxidizing, β-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 sulfur-compound 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 sulfur-compound oxidation by β-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.

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The Genome Sequence of the Obligately Chemolithoautotrophic, Facultatively Anaerobic Bacterium Thiobacillus denitrificans

Biological principles for the effects of ethanol

Methane-oxidizing microorganisms.

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 α-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 α-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 α-ketoglutarate dehydrogenase. Comparing the predicted physicochemical properties of the polypeptides coded by the genes confirmed the putative gene products were similar to the active α-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 α-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.

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

SUMMARY: Growth of Methylococcus capsulatus on methane was inhibited by methanol (0.1%, v/v, and above), ethanol, n-propanol and n-butanol (0.01%, v/v, and above), but was unaffected by galactose, glucose, fructose, maltose, sucrose (at 0.1 M) or lactose (0.05 M). About one organism in 7 million grew well on solid medium using methanol vapour as a sole source of carbon and energy, but [14C]methanol was readily metabolized and assimilated by cultures growing on methane. Labelling patterns from [14C]methane and [14C]methanol were similar, indicating their assimilation by a common pathway. Dissimilarities between the labelling patterns obtained with 14CH4 and 14C-labelled formaldehyde, formate and carbonate indicated that the ribose phosphate cycle of formaldehyde assimilation may not account for all the carbon assimilated by M. capsulatus: significant incorporation of formate, carbon dioxide and possibly of intermediates of methane oxidation more reduced than formaldehyde may occur. 14C-Labelled ethanol and acetate showed restricted incorporation into lipid, leucine, glutamate, proline and arginine, indicating that M. capsulatus can produce acetyl coenzyme A from both compounds and introduce it into an incomplete biosynthetic tricarboxylic acid cycle. Methylococcus capsulatus was unable to assimilate more than trace amounts of [14C]glucose or sucrose.

M. Eccleston

Papers

Assimilation and Toxicity of Some Exogenous C1 Compounds, Alcohols, Sugars and Acetate in the Methane-oxidizing Bacterium Methylococcus capsulatus