When allyl alcohol was used as a suicide substrate, spontaneous mutants and UV light-and nitrous acid-generated mutants of Methylobacterium organophilum XX were selected which grew on methylamine but not on methanol. There was no detectable methanol dehydrogenase (MDH) activity in crude extracts of these mutants, yet Western blots revealed that some mutants still produced MDH protein. Complementation of 50 mutants by a cosmid gene bank of M. organophilum XX demonstrated that three major regions of the genome, each of which was separated by a minimum of 40 kilobases, were required for expression of active MDH. By subcloning and TnS insertion mutagenesis of subcloned fragments, at least 11 genes clustered within these three regions were subsequently identified. The identity of the MDH structural gene, which was initially determined by hybridization to the structural gene of Methylobacterium sp. strain AM1, was confirmed by Western blot analysis of an MDH-I-galactosidase fusion protein.Methylobacterium organophilum XX is a pink-pigmented facultative methylotroph that is capable of growth on both one-carbon compounds, such as methanol and methylamine, and a variety of heterotrophic substrates (21). The oxidation of methanol and methylamine to formaldehyde is effected by the periplasmic quinoproteins methanol dehydrogenase (MDH) and methylamine dehydrogenase, with formaldehyde being either oxidized to carbon dioxide or assimilated by the icl variant of the serine pathway (2). MDH is a dimer composed of identical 62-kilodalton subunits, each of which is associated with a single pyrroloquinoline quinone (PQQ) prosthetic group (27). During growth on methanol, MDH consists of 10 to 15% of the total cell protein, and this high level of expression is not affected by the presence of succinate or methylamine. Furthermore, even higher levels of MDH are produced by cells grown on methylamine (P. E. Tam and R. S. Hanson, unpublished data). Attention has therefore focused on the mechanisms which regulate the synthesis of MDH. The involvement of several gene products may be expected; for example, apoenzyme synthesis in the cytoplasm and subsequent transport across the membrane are two of the processes which may be subject to regulation.The recent isolation of new mutants that are unable to synthesize active MDH has revitalized genetic studies of methanol oxidation in Methylobacterium sp. strain AM1 and Paracoccus denitrificans (12,19). Nunn and Lidstrom (19,20) have shown that at least 10 different genes participate in the synthesis of active MDH in Methylobacterium sp. strain AML. Among the proposed functions, in addition to the MDH and cytochrome CL structural genes, are four positive regulatory proteins, three proteins that are involved in the periplasmic association of PQQ with the MDH subunits, and a gene product that is responsible for transport of MDH and cytochrome CL. The MDH structural gene of P. denitrificans has also been identified recently (11). Early attempts to mutagenize M. organophilum XX with UV light and Nmeth...
The hamster papovavirus (HapV) is associated with multiple skin epitheliomas of the Syrian hamster. We have sequenced its genome. It is a double-stranded circular DNA of 5366 bp. The hypothetical genomic organization deduced from this nucleotide sequence is clearly of the polyoma type with the two strands coding in the opposite directions from a noncoding region that shows some of the features of a replication origin and a transcription control region. The amino acid sequences predicted from the open reading frames show an average of 50% homology with polyoma-coded polypeptides.The HapV is, after polyoma, the second example of a papovavirus coding for a middle T antigen. The cloned DNA can immortalize primary rat embryo cells and transform an established rat cell line. The viral DNA is stably integrated into the host genome.
Restriction maps of genes required for the synthesis of active methanol dehydrogenase in Methylobacterium organophilum XX and Methylobacterium sp. strain AM1 have been completed and compared. In these two species of pink-pigmented, type II methylotrophs, 15 genes were identified that were required for the expression of methanol dehydrogenase activity. None of these genes were required for the synthesis of the prosthetic group of methanol dehydrogenase, pyrroloquinoline quinone. The structural gene required for the synthesis of cytochrome CL, an electron acceptor uniquely required for methanol dehydrogenase, and the genes encoding small basic peptides that copurffied with methanol dehydrogenases were closely linked to the methanol dehydrogenase structural genes. A cloned 22-kilobase DNA insert from Methylsporovibrio methanica 81Z, an obligate type II methanotroph, complemented mutants that contained lesions in four genes closely linked to the methanol dehydrogenase structural genes. The methanol dehydrogenase and cytochrome CL structural genes were found to be transcribed independently in M. organophilum XX. Only two of the genes required for methanol dehydrogenase synthesis in this bacterium were found to be cotranscribed.
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