Molecular Biology And Genetics Of Smmo

The genes encoding sMMO have been cloned and sequenced from several methanotrophs, including M. capsulatus (Bath) (type I) and the type-II representatives Methylocystis strain M and M. trichosporium OB3b. sMMO genes are clustered on the chromosome of all methanotrophs studied to date. mmoX, mmoY, and mmoZare the genes that encode the a, p, and y subunits of the hydroxylase, respectively; mmoB and mmoC code for protein B and protein C, respectively. An open reading frame orfY with a coding capacity of 12 kDa separates mmoY and mmoZ in all methanotrophs exam-

Fig. 2. Organization of the sMMO and pMMO gene clusters in methanotrophs.

ined to date (15) (Fig. 2). The exact function of this ORF is unknown and attempts to delete this gene from the cluster to examine effects on methane oxidation have proved unsuccessful. In addition, orfY has been expressed as a glutathione-S-transferase fusion polypeptide, purified by affinity chromatography and polypeptides raised to this polypeptide in order to probe cell-free extracts of M. capsulatus Bath grown under a variety of different growth conditions, including in high- and low-copper media (see Section 5.). However, no crossreactivity was observed with this antibody and polypeptides from M. capsulatus Bath and, therefore, the function of orfY (if any) remains to be elucidated (Murrell, et al., unpublished) (see Section 10.).

As might be expected for such a unique enzyme system, sMMO genes are highly conserved in all methanotrophs examined, with sequence identities of 55-95% and 47-96% being observed between the corresponding DNA and amino acid sequences, respectively. Upstream (5') of the sMMO gene cluster of M. trichosporium OB3b lie genes encoding the chaperones Hsp60 and Hsp10 (interestingly they are also found 3' of the corresponding sMMO gene cluster in Methylococcus) and it is tempting to speculate that they may be involved in assembly of the sMMO enzyme complex, although there is no proof for this yet. Directly upstream of the chaperone genes lies a homolog of the acoR gene from Ralstonia eutrophus (16; Murrell et al., unpublished). This is a member of the nifA family of tran-scriptional activators. These are absolutely required for activation of transcription from sigma 54-type promoters (17) and it is possible that this gene product is involved in positive regulation of the sMMO gene cluster.

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