Coppercontaining Membraneassociated Enzyme Complex

Until recently, the dogma was that all methanotrophs possess the membrane-bound or particulate methane monooxygenase (pMMO). The only exception to this may be an acidophilic methanotroph, strain K, recently named Methylocella, isolated from northern wetland peat. This Methylocella species is a member of the a-subclass of Proteobacteria and most closely related to Beijerinckia indica (23). This strain has been shown to contain sMMO similar to those of type-II methanotrophs but does not appear to contain pMMO (Dedysh and Semrau, personal communication.). The pMMO in methanotrophs is expressed under growth conditions where the copper-to-biomass ratio is high (greater than 1 pM). Removing all trace amounts of copper ions from the growth medium results in poor growth of methanotrophs which do not possess sMMO, such as M. album BG8 or M. parvus OBBP.

Unlike sMMO, the membrane-bound enzyme pMMO has a relatively narrow substrate specificity, oxidizing alkanes and alkenes of up to five carbons in length. It does not oxidize longer alkanes or alkenes nor does it oxidize aromatic compounds such as naphthalene. However, it can still be useful for bioremediation (24). Partial purification of pMMO was first achieved by solubilizing the enzyme from membranes of M. capsulatus Bath using dodecyl maltoside (25). Subsequent studies by several groups have concentrated on the pMMO from this organism, although the pMMO from M. trichosporium has also been investigated (26) and its properties appear to be similar to those of the pMMO from Methylococcus.

The pMMO from M. capsulatus Bath consists of three subunits of approx 45, 27, and 23 kDa in a stoichiometry of 1:1:1. The 45- and 27-kDa subunits probably constitute the active site as they can be labeled by the suicide substrate acetylene (27). Active pMMO contains 2 iron atoms and approx 15 copper atoms per mole, both of which are probably involved in catalysis. Duroquinol and NADH2 can be used as artificial reductants for pMMO, but the physiological reductant is not known; potential candidates could include cytochromes-b559/569, -c, or -c553. The exact nature of the copper in pMMO is not known, but it has been suggested that trinuclear copper clusters are involved in catalysis (26). In addition to copper ions associated with the active-site polypeptides of pMMO, small copper-binding compounds (CBC) of 1.218 and 0.779 kDa are also associated with pMMO. High concentrations of these CBCs can be isolated from the culture medium when M. trichosporium OB3b and M. capsulatus Bath are expressing pMMO under "copper-starved" growth conditions (i.e., when the majority of free-copper ions had been removed by the organism from the growth medium) (27,29). These CBCs have a high affinity for copper ions and appear to bind a large proportion of the copper associated with the membrane fractions in cells expressing pMMO. Studies assessing the bioavailability of copper to M. trichosporium OB3b in different soil matrices suggest the CBCs may play a role in the environment (30). Although their exact function is not known, they could be important for sequestering copper ions, stabilizing pMMO, or maintaining the appropriate redox state of the enzyme (29). Interestingly, sMMO-constitutive mutant of M. trichosporium OB3b (19,41), believed to be defective in copper uptake, constitutively produce but cannot reinternalize these CBCs (29). However, experiments showed that functional copper-containing terminal oxidases still persist in these mutants, suggesting that this means of copper uptake is a pMMO-specific or secondary system (29).

7. MOLECULAR BIOLOGY OF PMMO

In the chromosome of M. capsulatus Bath, there are two virtually identical copies of the genes encoding pMMO (pmoCAB) (13 bp changes over 3183 bp of pmoCAB) (Fig. 2). In addition, a third, separate copy of pmoC has also been identified in the chromosome of M. capsulatus. Lidstrom and colleagues have constructed chromosomal insertion mutants in all seven pmo genes in M. capsulatus Bath. With the exception of the lone third copy of pmoC, for which no null mutants could be obtained, all other mutants grew on methane, indicating that both sets of genes were probably functionally equivalent. Copy 1 mutants showed about two-thirds of the wild-type methane oxidation activity, whereas copy 2 mutants had about one-third of the activity of the wild-type strain. No double null mutants defective in both copies of pmoCAB were obtained, which suggests that the cells require pMMO for normal growth (31). Copy-specific analysis of pmo transcripts has revealed that copy 2 predominates at 5 pM copper, but at higher concentrations (50 pM), expression of copy 1 reaches the same levels as copy 2 (32). Interestingly, low levels of pmo-specific transcripts were detected under all conditions tested.

This rather unusual type of gene duplication with pmoCAB has also been observed with the genes encoding the analogous enzyme ammonia monooxygenase (amoCAB) in ammonia-oxidizing bacteria (33). Comparison of pmo and amo gene sequences suggests that pMMO and AMO could be evo-lutionarily related (34,35). The pmo gene clusters have also been sequenced from the type-II methanotrophs M. trichosporium OB3b and Methylocystis sp. strain M (36). These methanotrophs also have two copies of pmoCAB and there is a high degree of similarity (80-94%) at the derived amino-acid sequence level with pMMO polypeptides from different methanotrophs, again perhaps not surprising in bacteria that rely solely on methane as a carbon and energy source.

PmoC and PmoA are predicted to be highly hydrophobic and consist mainly of putative trans-membrane-spanning helices, whereas PmoB only has two putative transmembrane regions. It has only been possible to clone these pmo gene clusters on overlapping DNA fragments because parts of these genes appear to be toxic to the E. coli host and, therefore, expression of pMMO in heterologous hosts may be difficult, if not impossible. We are currently expressing pmoA gene/glutathione-S-trans-ferase fusions in E. coli in order to raise antibody to pMMO for immunogold-labeling experiments designed to localize pMMO on the membranes of methanotrophs (Murrell et al., unpublished).

8. REGULATION OF MMO EXPRESSION BY A UNIQUE "COPPER SWITCH"

In the type-I methanotroph M. capsulatus Bath and the type-II methanotroph M. trichosporium OB3b, which possess both pMMO and sMMO, a novel metabolic switch mediated by copper ions occurs. When cells are essentially starved for copper, and the copper-to-biomass ratio of the cell is low, sMMO is expressed. These experiments are best done in a chemostat culture where it is possible to exert fine control over the availability of nutrients and maintain steady-state growth conditions. Cells grown under excess copper (i.e., with a high copper-to-biomass ratio) express pMMO and there is no detectable sMMO expression (37-39). It is interesting that no other metal ion effects this metabolic switch and this is exclusively copper-regulated gene expression.

Although it has been difficult to isolate good intact mRNA from methanotrophs, Northern blotting and primer extension analysis experiments with M. capsulatus Bath have shown that the six ORFs of the sMMO gene cluster are organized as an operon. Under low-copper growth conditions, three transcripts have been identified, one of which is probably the full-length 5.5-kb transcript encoding the entire sMMO operon. The only putative promoter identified was one with rather weak identity to E. coli -35, -10 consensus sequences located 5' of mmoX and the only primer extension product obtained was 37 bp upstream of the mmoX start codon. These transcripts were not detected 15 min after the addition of CuSO4 to steady-state chemostat cultures of M. capsulatus Bath expressing sMMO (38).

Similar experiments with M. trichosporium OB3b have also shown that transcription of the sMMO cluster is switched off within 10 min of the addition of 50 pM CuSO4. Three major sMMO transcripts were identified, corresponding to (a) mmoX, (b) mmoY, mmoB, and mmoZ, and (c) mmoY, mmoB, mmoZ, orfY, and mmoC. An unstable full-length transcript of 5.5 kb was also observed (39). Primer extension analysis has shown that transcription of mmoX is directed from a o54-like promoter immediately upstream of its transcription start site. -12, -24 consensus sequences are also present in the

Fig. 3. Hypothetical model for regulation of sMMO and pMMO genes in M. trichosporium OB3b. In cells growing under low copper-to-biomass ratios, a positive regulator binds to an upstream activating sequence (UAS) and directly interacts with the sigma 54 protein bound at P054. This directs the formation of an open complex and transcription of the sMMO genes occurs. At the same time, transcription of the pMMO genes from Po70 is repressed by an unknown repressor. On the addition of copper sMMO gene transcription becomes deactivated and pMMO gene transcription is derepressed.

Fig. 3. Hypothetical model for regulation of sMMO and pMMO genes in M. trichosporium OB3b. In cells growing under low copper-to-biomass ratios, a positive regulator binds to an upstream activating sequence (UAS) and directly interacts with the sigma 54 protein bound at P054. This directs the formation of an open complex and transcription of the sMMO genes occurs. At the same time, transcription of the pMMO genes from Po70 is repressed by an unknown repressor. On the addition of copper sMMO gene transcription becomes deactivated and pMMO gene transcription is derepressed.

same regions upstream of mmoX in the gene cluster of Methylocystis sp. strain M (15) and Methylomonas sp. KSWIII (4). The -24 region and the distance between the conserved motifs match the consensus sequence exactly, and only the last base pair of the -12 region differs in two of the three gene sequences; however, this is the most variable position in the consensus sequence. Transcription is initiated approx 110 to 150 bp upstream of the start codon of mmoX, and there is no similarity among the three sequences in this region until the 5' end of the mmoX gene, except for the Shine-Dalgarno sequence immediately upstream of mmoX. A second transcript initiating between mmoX and mmoY has been observed for M. trichosporium OB3b, upstream of which putative -35, -10 sequences of o70 promoters have been identified. Concomitant with the loss of sMMO activity and repression of sMMO transcription during the copper switch experiment was the appearance of pmo transcripts of 4.0 and 1.2 kb.

Similar transcript analysis has been carried out with pmo gene clusters from the type-II methanotrophs M. trichosporium OB3b and Methylocystis sp. strain M. In these methanotrophs, pmoCAB clusters are probably transcribed from a single transcriptional start site located 300 bp upstream of pmoC, initiating at a putative o70 promoter that is negatively regulated under low-copper conditions, when these bacteria are expressing sMMO (36, Murrell et al., unpublished).

The exact mechanism for the reciprocal regulation of the sMMO and pMMO gene clusters by copper ions is not clear at present. Cu2+ could mediate transcription of sMMO gene promoters via a regulatory protein that binds Cu2+ and is then able to bind to operator regions on the promoter, thereby interfering with RNA polymerase binding or shielding promoter sequences (i.e., the classic repressor model). An alternative model is that sMMO gene expression is subject to positive control, with Cu2+ binding to and thereby inactivating an "activator protein." There is more evidence for the second model because of the presence of a a54 promoter upstream of mmoX in three genera of methanotrophs that contain sMMO, because all known a54 promoters rely on activator proteins for the formation of a transcription-competent open complex (16,40). We are currently cloning the rpoN from M. trichosporium OB3b in order to mutate this gene and then examine the effects on sMMO expression in this methanotroph (Stafford and Murrell, unpublished).

Activation of pmo genes in M. trichosporium OB3b probably involves a copper-binding activator protein. However, an alternative hypothesis is that pmo genes could be subject to negative control in the absence of copper ions. A hypothetical model summarizing one possible mechanism for copper-dependent transcriptional regulation of MMO genes is outlined in Fig. 3.

9. FUTURE STUDIES

A considerable amount is now known about the physiology, biochemistry, and molecular biology of methane oxidation systems in bacteria. Recently, several new methanotrophs have been isolated from more extreme environments (e.g., acidic and alkaline environments, high pH, and also environments where there are extremes of temperature). It will be interesting to learn how the physiology of these new isolates allows them to grow under harsher conditions in these environments. A number of unanswered questions remain concerning the MMO systems in methanotrophs. For example, why are there duplicate copies of pMMO genes and are they differentially regulated in response to environmental changes? What is the exact nature of the pMMO in terms of its molecular structure and copper/iron centers? How is it assembled in the membranes of methanotrophs? How is the sMMO di-iron center inserted into the active-site pocket of this enzyme and what accessory genes and chaperonins are required for its assembly? Another area for further study is the regulation of expression of MMO enzymes by copper ions. At present, virtually nothing is known about the uptake of metal ions into methanotrophs and how they sequester and distribute metal ions. The transcriptional machinery that controls MMO expression is also not fully understood yet and it will be intriguing to learn the exact molecular mechanisms by which copper ions exert their effect. The genome sequence of M. capsulatus Bath is nearing completion and this information, coupled with proteomics and transcriptomics technology, should rapidly advance our current understanding of the unique copper switch in methanotrophs.

10. NOTE ADDED IN PROOF

Merkx and Lippard have recently expressed orfY as a fusion protein in Escherichia coli and their experiments show that this protein, designated MMOD, is a component of the sMMO system. They discuss the possible functions of MMOD in a recent paper (see ref. 42).

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