Methanol Oxidation Genes in the Marine Methanotroph Methylomonas sp. Strain A4

Methanol dehydrogenase has been purified from the type I marine methanotroph Methylomonas sp. strain A4 and found to be similar to other methanol dehydrogenase enzymes in subunit composition, molecular mass, and N-terminal sequence of the two subunits. A heterologous gene probe and a homologous oligonucleotide have been used to identify a DNA fragment from Methylomonas sp. strain A4 which contains moxF, the gene encoding the large subunit of methanol dehydrogenase. Protein expression experiments with Escherichia coli, immunoblotting of expression extracts, and partial DNA sequence determination have confirmed the presence of moxF on this DNA fragment. In addition, expression and immunoblot experiments have shown the presence of the genes for the small subunit of methanol dehydrogenase (moxI) and for the methanol dehydrogenase-specific cytochrome c (moxG). The moxG gene product has been shown to be cytochrome c552. The expression experiments have also shown that two other genes are present on this DNA fragment, and our evidence suggests that these are the homologs of moxJ and moxR, whose functions are unknown. Our data suggest that the order of these genes in Methylomonas sp. strain A4 is moxFJGIR, the same as in the facultative methylotrophs. The transcriptional start site for moxF was mapped. The sequence 5' to the transcriptional start does not resemble other promoter sequences, including the putative moxF promoter sequence of facultative methylotrophs. These results suggest that although the order of these genes and the N-terminal amino acid sequence of MoxF and MoxI are conserved between distantly related methylotrophs, the promoters for this gene cluster differ substantially

An Analysis of Bacteriophage Lambda Site-Specific Recombination Using IHF Binding Site Mutants

213 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.Integration Host Factor (IHF) is an Escherichia coli protein required for integration and excision of bacteriophage lambda. Mutations of the three sites to which this protein binds within the lambda attachment site were constructed using oligonucleotide-directed site-specific mutagenesis and were ananlysed for their effects on site-specific recombination. One group of mutations had alterations across one site; in a second group, a pattern of changes was made in each of the three sites. Certain base changes affected some sites more severely than others; some changes had more effect in vitro than in vivo. All mutations depress integrative recombination in vivo and depress or eliminate it in vitro. In excision, mutations of the H1 site raise the frequency of excisive recombination, while mutations of the H2 or H\sp\prime sites lower the frequency of excisive recombination. These results indicate an involvement of all three sites in both integration and excision; occupancy by IHF of the H2 and H\sp\prime sites is required for either reaction, while the presence of IHF at H1 favors integration and its absence favors excision.Two other projects concerning site-specific recombination between lambda and its host are presented. The isolation and analysis of a novel secondary attachment site for lambda is described. The DNA sequence of this site, located within the deoD gene of E. coli, is compared to other published primary and secondary attachment site sequences to identify the point of exchange and regions resembling protein binding sites. The structure of secondary attachment sites is discussed in terms of current models for lambda site-specific recombination.A new protocol for the purification of gpxis is detailed. This procedure yields abundant protein in fewer, easier steps than previously published procedures and removes a Mg-dependent endonuclease activity which co-purified with Xis activity in one procedure. The amino acid length (72 amino acids) of the Xis protein agrees with the value predicted from the DNA sequence. The predominant species (>90%) of protein present is gpxis, according to Northern blot analysis.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD