Revision for “History” created on 9 May, 2014 @ 16:41
<b>The Methanotrophs; a History. </b><span style="line-height: 1.5em;">By Chris Anthony, University of Southampton, Southampton, SO17 1BJ, UK. </span><a href="http:// www.chris-anthony.co.uk">www.chris-anthony.co.uk</a> <b>This history is in five sections: Introduction and carbon assimilation; Methane oxidation; Methanol oxidation; Electron transport; Genetics.</b> <b>Introduction</b> Of course, most reviews of any aspect of the subject will include some historical context. What I have tried to do is to be as thorough as possible when covering the beginnings. If there are any omissions, errors or insults please contact me to correct them. Much of the biochemistry of methanotrophs was first elucidated in methylotrophs growing on methanol, but unable to use methane. I will concentrate on methanotrophs when possible but use work with other methylotrophs where appropriate. Much of the history of each subject is covered in detail in my book The Biochemistry of Methylotrophs (1982) which covers the subject up to 1982 in 400 pages with more than 1000 references. This is not available in print but it can be downloaded as pdf from my website: <a href="http://www.chris-anthony.co.uk/methylotrophs.html">http://www.chris-anthony.co.uk/methylotrophs.html</a>. I own the copyright so downloading is free; help yourself. Methanotrophs, growing on methane as their sole source of carbon and energy, are a specific category of the more general category of Methylotrophs which grow on carbon compounds containing one or more carbon atoms but with no carbon-carbon bonds (eg methane, methanol, methylamine, trimethylamine). Methanotrophs are almost always obligate, growing only on methane or (sometimes) methanol which is the first intermediate in methane utilisation. All methylotrophs share two common challenges: they must obtain energy in the form of ATP and NAD(P)H by oxidation of their unusual substrates, and they must have special pathways for carbon assimilation, as all their carbon-carbon bonds must be made <i>de novo</i>. The ‘historical’ start of published work on methanotrophy is usually taken as the limited description in 1906 of an organism isolated on methane from aquatic plant material by Sohngen (1906, 1910) and named by him <i>Bacillus methanicus</i> (later amended by Orla-Jensen (1909) to <i>Methanomonas methanica</i>). It was only able to grow on methane or methanol. A firmer foundation was provided 50 years later by Dworkin and Foster (1956) with the full description and classification of “a pink methane-utilizing bacterium in all respects identical with <i>Methanomonas methanica</i> Sohngen” which they showed to be widely distributed in nature. They considered that “the genus <i>Pseudomonas</i> accommodates the methane-utilizing bacterium here under consideration perfectly satisfactorily” as it is “an aerobic, gram negative, polarly monoflagellated rod”. They proposed, therefore, “to rename Sohngen's organism <i>Pseudomonas methanica</i> (Sohngen) nov. comb”. “The distinctive physiological character of this species is its extremely limited substrate range: it cannot use any of a large number of conventional carbon sources and methane is the only saturated gaseous hydrocarbon which it can oxidize”. They rejected the name <i>Methanomonas</i> because the prefix Methano- was widely used for methanogenic bacteria. They then argued that “From the viewpoint of modern microbiology and systematics, generic designation on the basis of the utilization of particular compounds as a sole source of organic nourishment would hardly be tactful. If adopted in the case of all organic compounds arbitrarily regarded as unusual substrates, such a practice would produce taxonomic and nomenclatural chaos”. They considered that “recognition of physiological genera based on methane utilization would confer no advantage on systematics or diagnostics, and would imply a phylogenetic distinction more profound than is warranted. Furthermore, it might be a precedent for a whole series of trivial genera in connection with other individual compounds”. With hindsight of course, in the case of methane and methanotrophs this sensible caution and tactfulness has proved to be unnecessary (note: <i>Pseudomonas methanica</i> is now known as <i>Methylomonas methanica</i>). In the next fourteen years a few more methanotrophs were described, all being strictly aerobic, and able to grow on methane and methanol but not on multicarbon compounds; these included <i>Methanomonas methanooxidans</i> (Brown et al., 1964) and <i>Methylococcus capsulatus</i> (Foster and Davis, 1966). A major landmark in the development of our knowledge of methanotrophs was the description in 1970 of more than 100 new isolates by Whittenbury, Wilkinson and their colleagues at Edinburgh (Whittenbury et al., 1970a,b). All were obligate methylotrophs able to grow only on methane (and sometimes methanol), but they were very diverse with respect to biochemistry and structural characteristics. This, together with the observation that no previously-described species of bacteria had been shown to grow on methane, made it obviously acceptable to discard Dworkin and Foster’s ‘tactful’ approach and to give the five ‘groups’ of isolates names that indicate their physiological and nutritional speciality, and these groups have subsequently been accepted as the genera <i>Methylocystis, Methylococcus, Methylosinus, Methylomonas and Methylobacter.</i> Some strains were considered to be identical with previously described methane-utilizing species, such as <i>Pseudomonas methanica</i> (Dworkin & Foster, 1956) and <i>Methylococcus capsulatus</i> (Foster & Davis, 1966). But none was identified as <i>Methanomonas methanooxidans</i> (Brown et al., 1964). <i></i> <b> </b> <b>Carbon assimilation pathways in methanotrophs</b> Ten years before Whittenbury’s great contribution to the study of methanotrophs, an extensive programme of study of methylotrophs growing on methanol was initiated by J. Rod Quayle in the University of Oxford (to be continued in the University of Sheffield, UK). He soon became established as an inspirational leader in the study of methylotrophs. He had worked in Calvin’s lab on the path of carbon in photosynthesis, and had published the first demonstration of the “Enzymatic carboxylation of ribulose diphosphate” by <i>Chlorella</i> extracts (by what we now know as ribulose bisphosphate carboxylase/oxygenase; RuBisCo) (Quayle et al., 1954). In 1961, with David Peel, he published a description of <i>Pseudomonas</i> AM1 (Peel & Quayle, 1961) which has become the ‘work-horse’ of methylotrophy, It is a pink facultative methylotroph that does not grow on methane but grows on methanol, methylamine, formate and also on multicarbon compounds. It is now called <i>Methylobacterium extorquens</i> AM1; other earlier names have included <i>Pseudomonas</i> AM1, <i>Pseudomonas </i>sp. M27, <i>Vibrio extorquens</i> and <i>Pseudomonas extorquens.</i> It had long been thought that reduced C1 compounds might first be oxidised to carbon dioxide and then assimilated by the Ribulose bisphosphate cycle or ‘Calvin cycle’as in plants and autotrophic bacteria , and Quayle had confirmed that this could be a possibility by showing that formate is assimilated by this cycle in <i>Pseudomonas oxalaticus</i> (Quayle & Keech, 1959a,b). However, that serine rather than phosphglycerate might be a possible<i> </i>first intermediate during methanol assimilations in a pink facultative methylotroph <i>Pseudomonas</i> PRL-W4 (similar<i> </i>to <i>Pseudomonas</i> AM1) had been suggested by Kaneda & Roxburgh (1959a,b). Subsequently, using the experimental approaches that had led to the elucidation of the ‘Calvin cycle’, Peter Large, David Peel and Rod Quayle, in a series of model papers, showed that methanol is assimilated in <i>Pseudomonas</i> AM1 at the levels of formaldehyde and carbon dioxide by a novel biosynthetic cycle, the Serine cycle (Large et al., 1961; 1962a,b; Large & Quayle, 1963). At this time a key step in the pathway was missing which, ten years later, was identified by Salem & Quayle (1973) as a cleavage reaction catalysed by malyl-coenzyme A lyase which produced acetyl-Coenzyme A. A final step was to elucidate the route for oxidation of this product to glyoxylate and the elucidation of this part of the pathway required almost another 40 years (see Anthony, 2011). In their first paper on the Serine cycle in methanol-utilisers, Quayle and colleagues said that “It remains to be seen whether microbial growth on <i>methane</i> involves a metabolism broadly similar to that involved in growth on <i>methanol</i>” (that is, not involving the RuBP pathway of carbon dioxide fixation) (Large et al., 1961). That this was a possibility was indicated by the observation by Leadbetter & Foster (1958) that during growth of <i>Pseudomonas methanica</i> on methane a considerable proportion of the carbon incorporated by the cell does not pass through a stage readily exchangeable with carbon dioxide making it “unlikely that an autotrophic type of metabolism is involved”. That a novel pathway was indeed operating during growth on methane in <i>Pseudomonas methanica </i>was confirmed by showing that ribulose bisphosphate carboxylase was absent, and that the <sup>14</sup>C-labelled compounds accumulating at early times during incubation with <sup>14</sup>CH<sub>4</sub> or <sup>14</sup>CH<sub>3</sub>OH were mainly glucose and fructose phosphates, and not the first product of the ribulose bisphosphate pathway of CO<sub>2</sub> fixation (phosphoglycerate) (Johnson & Quayle, 1965). A formaldehyde-condensing enzyme was then demonstrated in crude extracts of <i>Methylomonas methanica</i> (previously called <i>Pseudomonas methanica</i>) by Kemp and Quayle (1965,1966), suggesting a novel pentose phosphate cycle for formaldehyde assimilation, in which the product of condensation with formaldeyde was tentatively identified as allulose phosphate (Kemp & Quayle, 1966). Thorough examination of the labelling patterns confirmed the proposed pathway (Kemp & Quayle, 1967). The pentose phosphate was subsequently shown to be ribulose monophosphate and the product of the condensation reaction is the novel sugar hexulose 6-phosphate) (Kemp, 1974). This pathway is now known as the ribulose monophosphate pathway (RuMP pathway). That this, or a similar, pathway also operates in <i>Methylococcus capsulatus</i>, was shown by demonstrating that <sup>14</sup>CH<sub>3</sub>OH was rapidly incorporated into hexose phosphates, and that an enzyme system is present which is able to condense formaldehyde with pentose phosphate to give a mixture of hexose phosphates (Lawrence et al., 1970). The Ribulose monophosphate cycle of formaldehyde fixation (RuMP pathway) in methanotrophs was eventually completed by the purification and characterisation of the hexulose phosphate synthase and isomerase from <i>Methylococcus capsulatus</i> (Ferenci et al., 1974); and by demonstrating the presence of essential cleavage and rearrangement enzymes in <i>Methylomonas methanica</i> and <i>Methylococcus capsulatus</i> (Strom et al., 1974). That all methanotrophs do not assimilate methane by the RuMP pathway was shown using the same short-term labelling techniques as were used with <i>Methylococcus capsulatus</i> but using <i>Methanomonas methano-oxidans</i> (a type II methanotroph – see below) (Lawrence et al., 1970). In this case, early label was found in serine and carboxylic acids (rather than in sugar phosphates) as had been found during methanol assimilation in <i>Pseudomonas</i> AM1. Furthermore, a key enzyme of the Serine cycle (hydroxypyruvate reductase) was present at high specific activity while the key formaldehyde-fixing enzyme of the RuMP pathway (hexulose phosphate synthase) was absent. The presence or absence of these two key enzymes was subsequently used for allocation of putative pathways for methane assimilation by Whittenbury’s new isolates. Lawrence & Quayle (1970) showed that division of the organisms into two classes based on their assimilation pathways as described above corresponds exactly with their subdivision based on membrane systems. Whittenbury had divided his new isolates into two Types based mainly on the nature of their characteristic internal membrane systems: Type I have a series of bundles of disc-shaped vesicles distributed throughout the organism whereas Type II have a system of paired membranes running throughout the organism or aggregated at the periphery. It was shown that methanotrophs using the RuMP pathway have the Type I membrane system (<i>Methylococcus</i>, <i>Methylomonas</i> and <i>Methylobacter</i>), whereas those using the Serine cycle have a Type II membrane system (<i>Methylosinus</i> and <i>Methylocystis</i>), Lawrence and Quayle (1970) pointed out that “it therefore appears likely that the methane-utilizing bacteria, far from being a closely similar group of organisms as might be anticipated from their striking nutritional similarity of being able to grow only on methane or methanol, may in fact be divided into at least two major groups possessing fundamental differences in their mode of carbon assimilation and system of internal membranes. These differences are wide enough to suggest that the two groups of organisms may have evolved quite separately as methane utilizers. If this were so, it raises the question as to why both groups have convergently evolved as obligate methylotrophs”. <b>The oxidation of methane to methanol</b> The most obvious suggestion for a system for oxidation of methane is that a monooxygenase is involved, catalysing this reaction: CH<sub>4</sub> + NADH + H<sup>+</sup> + O<sub>2</sub> <b>→</b> CH<sub>3</sub>OH + NAD<sup>+</sup> + H<sub>2</sub>O That such a mixed function monooxygenase is involved was first implied by the work of Leadbetter & Foster (1959) on <i>Pseudomonas methanica</i> oxidising ethane and other alkanes (they did not investigate methane itself). This was later confirmed by Higgins and Quayle (1970) who showed that whole cells of <i>Methylomonas methanica</i> and <i>Methanomonas methanooxidans</i> incubated with methane incorporate <sup>18</sup>O into methanol from <sup>18</sup>O<sub>2</sub> but not from water containing <sup>18</sup>O. Further understanding of the nature of the methane monooxygenase (MMO) has depended on isolation of cell-free systems able to catalyse methane oxidation. This first depended on measuring NADH oxidation and O<sub>2</sub> consumption in the presence of methane by particles prepared from <i>Methylococcus capsulatus</i> (Ribbons and Michalover, 1970) but as these preparations also oxidise NADH, in the absence of methane (Ribbons, 1975), interpretation presented considerable difficulties. These difficulties were eventually overcome by John Colby, Howard Dalton and colleagues at Warwick who developed alternative assay methods that depended on the wide substrate specificity of the MMO; they measured oxidation of bromomethane or ethene or propene, their products being analysed by gas-liquid chromatography (Colby et al., 1975, 1977). This led them to provide the first definitive description of a soluble NADH-requiring methane monooxygenase, consisting of three proteins, present in <i>Methylococcus capsulatus</i> and in <i>Methylosinus trichosporium</i> OB3b (Colby & Dalton, 1978,1979) . This work has led to much of our modern understanding of methane oxidation in methanotrophs and is summarised in Howard Dalton’s Leeuwenhoek Lecture: The Natural and Unnatural History of Methane-Oxidizing Bacteria (Dalton, 1975), and in a published tribute to Howard after his death (Anthony, 2008). That acquiring an understanding of methane oxidation in methanotrophs was not straightforward, is illustrated by the work of John Higgins and his group at Canterbury (Kent) who described a completely different <i>particulate</i> system from <i>Methylosinus trichosporium</i> which was said to use ascorbate or reduced cytochrome <i>c</i>, but not NADH, in solubilised, purified preparations (Tonge et al., 1977). However, Stirling & Dalton (1979) in a comparison of substrate and electron donor specificities in <i>Methylococcus capsulatus, Methylomonas methanica</i> and <i>Methylosinus trichosporium</i> concluded that NAD(P)H is the electron donor. The debate was further confused by difficulties in repeating experiments, some of the conflict being related to the subsequent observation that whether or not soluble or particulate MMO is produced is determined by the growth conditions. It is now known that copper stress underlies the intracellular location of MMO (Stanley et al., 1983). The soluble, NADH-requiring sMMO is produced when copper/biomass ratio is low, and the (completely different) membrane pMMO is produced when this ratio is high. It is now thought that the electron donor to the membrane enzyme (pMMO) is probably not NADH but membrane ubiquinol which may be produced by reverse electron transport from methanol by way of methanol dehydrogenase, its electron acceptor cytochrome <i>c</i><sub>L</sub> and the cytochrome <i>bc</i><sub>1</sub> complex, driven by the protonmotive force provided by the oxidation of cytochrome <i>c</i><sub>L.</sub> The details of the early confusing story of the development of understanding of methane oxidation are more fully covered by Anthony (1982). <b>Methanol oxidation in methanotrophs</b> The first step in the oxidation of methane is its oxidation to methanol which consumes energy in the form of NADH or ubiquinol. This must be provided by the subsequent oxidation of formaldehyde or formate, produced by the oxidation of methanol. Methanol is oxidised to formaldehyde by an unusual methanol dehydrogenase. During methane and methanol all carbon passes through this enzyme to produce formaldehyde which is either assimilated or oxidised. I hope I will be forgiven for making the description of this part of the history of methanotrophy relatively personal. In 1960 I started working for my PhD in the Microbiology Department of the University of Reading, UK with Len J. Zatman as supervisor. He had previously worked on NAD-linked alcohol dehydrogenases and had been impressed that none of these is able to oxidise methanol. Hearing that some bacteria can grow on methanol he concluded that they must have an unusual enzyme to oxidise their growth substrate, and to find this enzyme was my PhD project. In my youthful ignorance I thought this might not be sufficiently interesting to occupy me for 3 years. But our first paper on the unusual methanol dehydrogenase was published in 1964 (Anthony & Zatman, 1964b) and my last paper, published on the atomic resolution of the same enzyme, was more than 40 years later (Williams et al., 2005). This history is dedicated to the memory of my hero and friend Len Zatman. I started my project by isolating a pink facultative methanol-utilising (<i>Pseudomonas</i> sp. M27; Anthony & Zatman, 1964a). A year after we started, a description of a very similar organism was described by Peel & Quayle (1961) (<i>Pseudomonas</i> AM1). The first reported investigation of methanol oxidation, in a similar organism, was by Kaneda & Roxburgh (1959a) who described methanol-dependent reduction of NAD by extracts of <i>Pseudomonas</i> PRL-W4, but this has never again been demonstrated. Harrington & Kallio (1960) subsequently suggested that methanol is oxidised in the methanotroph <i>Pseudomonas methanica</i> (Iowa strain) by the peroxidative action of catalase with methanol as substrate. Evidence against this was obtained from our inhibitor studies with methanol-grown <i>Pseudomonas</i> M 27 (Anthony & Zatman, 1964a). Complete inhibition of catalase activity had no effect on methanol oxidation; and conversely, methanol oxidation was completely inhibited by 5 mM-EDTA and the phenylhydrazines (0.1 mM) which had no effect on catalase activity. Harrington & Kallio (1960) suggested that the inhibition of methanol oxidation in whole cells by EDTA was due to inhibition of the iron-requiring catalase, this inhibition being relieved by addition of ferrous sulphate. But we showed this was an artefact caused by the aerobic inorganic chemical oxidation of Fe<sup>2+</sup> to Fe<sup>3+</sup> ion which occurs more rapidly in the presence of EDTA (Anthony & Zatman, 1964a). It was much later shown that EDTA inhibits methanol oxidation in whole cells by competing with the electron acceptor cytochrome <i>c</i><sub>L</sub> for the same binding site on the methanol dehydrogenase (Chan & Anthony, 1992). Cell extracts of <i>Pseudomonas</i> M27 had, as expected, no NAD(P)H-linked methanol dehydrogenase activity. Eventually we demonstrated methanol-dependent reduction of the redox dye 2,6-dichlorophenolindophenol in the presence of an essential mediator phenazine methosulphate, using whole cells incubated anaerobically in Thunberg tubes. Initially this activity was not seen in spectrophotometric measurements with extracts because lab buffers contain tiny amounts of alcohols and so the rates were the same with or without added substrate. Furthermore, activity was never seen when smaller amounts of enzyme were used because we showed later that the dye-linked methanol dehydrogenase requires ammonia (base) as activator, provided by addition of ammonium salts (Anthony & Zatman, 1964b). The dye-linked methanol dehydrogenase (MDH) was soluble; it had a wide substrate specificity, was inhibited by none of the usual inhibitors, was very stable with a high isoelectric point and a molecular weight of about 120 kDa, being made up of 2 identical subunits of about 60 kDa (Anthony & Zatman, 1965, 1967a). Neither we ourselves, nor the dozens of later studies of MDH saw the other two tiny (8.4 kDa) subunits at that time; we found these more than 20 years later (Nunn et al., 1989). MDH was not very active which meant that a lot of enzyme had to be prepared for any study; but it also meant that, fortunately, the bacteria made a lot of it (about 10% of the soluble protein). Johnson & Quayle (1964) showed that the same enzyme is present in the very closely related <i>Pseudomonas</i> AM1 (<i>Methylobacterium extorquens</i> AM1). Methanotrophs have the same (or similar) MDH as in <i>Methylobacterium extorquens</i>, as first demonstrated in <i>Methylomonas methanica</i> by Johnson & Quayle (1964), The MDH was later purified and characterised from <i>Methylococcus capsulatus</i> and shown to be almost identical to that in <i>Methylobacterium extorquens</i> (Patel et al., 1972, 1973; Wadzinsky & Ribbons, 1975) and the MDHs from <i>M. extorquens </i>and methanotrophs (<i>Methylococcus,</i> <i>Methylobacter</i> and <i>Methylosinus</i>) were shown to be immunologically similar (Patel <i>et al</i>., 1973). The most different of the MDHs was that from <i>Methylosinus sporium</i> which was purified and crystallized by Patel & Felix (1976) who showed that it was less stable at low pH values and appeared to be monomeric (molecular weight 60kDa). In 1967 we showed that MDH has a novel (unidentified) prosthetic group (Anthony & Zatman 1967b) which we purified (as a tiny amount of brick-red powder) and characterised by its fluorescence characteristics. We found out later that the same prosthetic group had been described in glucose dehydrogenase in <i>Acinetobacter calcoaceticus</i> by Hauge (1964). It was thirteen years after we first described the novel prosthetic group of methanol dehydrogenase that its structure was determined, by X-ray crystallography in Olga Kennard’s lab at Cambridge, to be pyrroloquinoline quinone (PQQ) (Salisbury et al., 1979). Methanol dehydrogenase was the first extensively studied example of what have become known as quinoproteins, a major new category of bacterial enzymes responsible for the first step in the oxidation of their growth substrates. (For reviews of this enzyme and the many related quinoproteins see Anthony, 1986, 2000, 2004; Goodwin & Anthony, 1998; Anthony & Ghosh, 1998; and <a href="http://www.chris-anthony.co.uk/">www.chris-anthony.co.uk</a>). <b> </b> <b>Electron transport</b> David O’Keefe and Sandy Cross in 1980 showed that <i>Methylobacterium extorquens</i> and <i>Methylophilus </i>methylotrophus have two types of cytochrome <i>c</i>, named after their high and low isoelectric points – cytochrome <i>c</i><sub>H</sub> and cytochrome c<sub>L</sub> (O’Keeffe & Anthony, 1980; Cross & Anthony, 1980), and this is a notable feature of all methylotrophs (Anthony, 1992). We now know that cytochrome <i>c</i><sub>L</sub> is a completely unique cytochrome with no sequence identity whatsoever with any other cytochrome <i>c</i> (except for the CXXCH haem-binding motif). Remarkably, however, its X-ray structure shows a strong similarity to other cytochromes <i>c</i> (Williams et al., 2006). This novel cytochrome c is the physiological electron acceptor for MDH (Duine et al., 1979; Beardmore-Gray et al., 1983; Anthony 1992). In an investigation of proton translocation during operation of the electron transport chain for methanol oxidation we showed that this must be occurring in the periplasm (O’Keeffe & Anthony (1978), a conclusion supported by a direct demonstration of the periplasmic location of MDH by Alefounder & Ferguson (1983). The cytochrome <i>c</i><sub>L</sub> is oxidised by a typical small cytochrome <i>c</i> (in these bacteria called cytochrome <i>c</i><sub>H</sub>) which is the substrate for the oxidase, cytochrome <i>aa</i><sub>3</sub>. This remarkable periplasmic electron transport chain leads to the release of 2 protons on the outside and consumption of 2 protons at the internal active site of the oxidase, leading to a protonmotive force sufficient to produce 0.7 ATP (O’Keeffe & Anthony,1978; Anthony, 1992). There has been relatively little work on the electron transport chain of methanotrophs but later studies of their genomes indicates that these are essentially the same as in <i>Methylobacterium extorquens</i>. <b>Genetics of methanotrophs</b> <i>‘Facultative methanotrophs’; genetic analysis</i> This topic is included to facilitate the following brief account of the history of genetics in methanotrophs, most of which are not able to grow on multicarbon compounds; that is, they are obligate methanotrophs, which makes work on mutant isolation and genetics more difficult. The isolation of <i>Methylobacterium</i> <i>organophilum</i> appeared to offer some hope, because it was first described as a facultative methanotroph and some preliminary work was done using genetic transformation to show linkage of C1-related genes (O’Connor & Hanson, 1977, 1978); it should be noted that none of this work depended on growth on methane, all experiments being done on methanol media. It is quite possible that this was not a true facultative methanotroph. Theisen & Murrell (2005) nicely summarised the situation: “Claims for the existence of facultative methanotrophs have a long and somewhat checkered history dating back almost 40 years, when Patt and colleagues first isolated and later described <i>Methylobacterium organophilum</i>, which was able to grow on methane or glucose (Patt et al., 1974, 1976). This was followed by other reports of facultative methanotrophs, notably strain R6, <i>Methylobacterium ethanolicum</i>, and <i>Methylomonas</i> sp. strain 761M (Lynch et al., 1980; Patel et al., 1978; Zhao et al., 1984). In most cases, either the methane-oxidizing capacity was lost or the results were not substantiated in other laboratories. For example, for <i>M. organophilum</i> it was hypothesized that methane oxidation genes were plasmid encoded and that the loss of growth on methane was attributable to loss of a plasmid. In the case of <i>M. ethanolicum</i>, the culture was shown to be a very tight syntrophic association between a methanotroph and a <i>Xanthobacter</i> species (Lidstrom-O’Connor et al., 1983)”. More recently, bona fide facultative methanotrophs have been described, together with the meticulous techniques required to confirm their special status. The general problems associated with the study of facultative methanotrophs have been usefully reviewed by Theisen & Murrell (2005) and Semrau et al., (2011). <i>Genetics of obligate methanotrophs.</i> Earlier studies of obligate methanotrophs proved them to be refractory to classical genetic analysis (Williams and Bainbridge 1971; Harwood et al. 1972; Williams et al. 1977). Although methods for mutagenesis of methane oxidation systems and for conjugal transfer of broad host range plasmids (R300B and pULB113) were successfully applied to <i>Methylomonas albus</i> and <i>Methylosinus trichosporium</i> (A1-Taho and Warner 1987; Nicolaidis and Sargent 1987; McPheat et al. 1987a, b), these were not further exploited at that time. One of the first systems to prove amenable to genetic analysis in the methanotrophs was the nitrogen fixation system. A <i>Klebsiella pneumoniae</i> DNA fragment containing the <i>nif</i> structural genes was used to identify a clone from a <i>Methylosinus</i> 6 genomic bank that contained the <i>nifD</i> gene (Toukdarian & Lidstrom, 1984a), the identity of this gene being confirmed by construction of a chromosomal Tn5 mutant by homologous recombination. This mutant contained no detectable nitrogenase activity and was missing the <i>nifD</i> polypeptide on two-dimensional polyacrylamide gels (Toukdarian & Lidstrom, (1984b). The nitrogen fixation genes were similarly some of the first to be involved in genetic analysis by Murrell and colleagues in the University of Warwick, UK (Oakley & Murrell, 1988) who used the <i>nifH</i> gene from <i>Klebsiella pneumoniae</i>, which codes for the Fe protein component of nitrogenase, as a probe to detect <i>nifH</i> homologues in total cellular DNA from 13 obligate methane oxidizing bacteria. All but one of those strains that had previously been shown capable of fixing dinitrogen contained sequences homologous to <i>nifH</i>. Two key papers in the further development of methylotroph genetics were published in 1986 by David Nunn and Mary Lidstrom (at the University of Washington, Seattle, USA), who reported the genetic analysis of functions necessary for methanol oxidation (Mox functions) in the facultative methanol-utilizer <i>Methylobacterium</i> AM1, which suggested that at least ten different gene products are involved (Nunn & Lidstrom, 1986a,b). The ten <i>mox</i> genes included two encoding the methanol dehydrogenase MDH) and its specific electron acceptor cytochrome <i>c</i><sub>L</sub>, and eight were involved in assembly and regulation. The genetics of the assimilation pathway was initiated in this same organism (Stone & Goodwin, 1989) and transposon mutagenesis of methanol oxidation genes were described (Lee et al., 1991). Lidstrom’s work on the methanol-utiliser <i>Methylobacterium </i>AM1<i> </i>was then extended by her group to the obligate methanotrophs, using a portion of the <i>Methylobacterium </i>AM1 MDH structural gene (<i>moxF</i>) as a hybridization probe to isolate and characterize the <i>moxF</i> genes from two obligate methanotrophs, <i>Methylococcus capsulatus </i>and <i>Methylomonas alba </i>(Stephens et al., 1988). This work on the methanol oxidation system in methanotrophs was later extended to investigate the genetics of methanol oxidation in <i>Methylomonas</i> sp. A4 (Waechter-Brulla et al., 1993) and to identify the promoter for expression of the gene for methanol dehydrogenase (<i>moxF</i>) in the Type I methanotroph, <i>Methylobacter albus </i>BG (Chistoserdova et al., 1994). At about the time that Mary Lidstrom’s group started their investigations of the genetics of the methanol oxidation system, work started in the University of Warwick (UK) in the groups of Murrell and Dalton on the genetics of methanotrophs, especially in relation to the methane monooxygenase (MMO) (Mullens & Dalton, 1987; McPheat et al., 1987a,b). In this work, oligonucleotide probes specific to the N-terminal amino acid sequence of individual polypeptides of the soluble MMO from <i>MethyIococcus capsuIatus </i>(Bath) were used to identify MMO genes. Subsequently, the genes encoding the β and γ subunits of MMO were shown to be linked in this organism, and analysis of a recombinant plasmid containing 12 kilobases of <i>Methylococcus </i>DNA provided the first evidence for the localization and linkage of genes encoding the methane monooxygenase enzyme complex, DNA sequence analysis providing the complete sequence of the genes encoding the β and γ subunits of MMO (Stainthorpe et al., 1989). These studies were extended and expanded in Murrell’s group, in collaboration with Lidstrom’s group, mainly concentrating on the genetics of the two MMOs and their regulation (Stainthorpe et al., 1990; Cardy et al., 1991a,b; Semrau et al., 1995; Murrell, 1992, 1994). 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