Biomedical subjects

M E Lidstrom

Publications and source records attributed to M E Lidstrom.

81 records · Page 5Linked to original sources

PubMed1985

Methanol dissimilation in Xanthobacter H4-14: activities, induction and comparison to Pseudomonas AM1 and Paracoccus denitrificans.

Methanol dissimilatory enzymes detected in the methanol autotroph Xanthobacter H4-14 were a typical phenazine methosulphate-linked methanol dehydrogenase, a NAD+-linked formate dehydrogenase, and a dye-linked formaldehyde dehydrogenase that could be assayed only by activity stains of polyacrylamide gels. This same methanol dehydrogenase activity was found in ethanol-grown cells and was apparently utilized for ethanol oxidation. Formaldehyde dehydrogenase activities were investigated in Paracoccus denitrificans, Xanthobacter H4-14, and Pseudomonas AM1. P. denitrificans contained a previously reported NAD+-linked, GSH-dependent activity, but both Xanthobacter H4-14 and Pseudomonas AM1 contained numerous activities detected by activity stains of polyacrylamide gels. Induction studies showed that in Xanthobacter H4-14, a 10 kDal polypeptide, probably a dehydrogenase-associated cytochrome c, was co-induced with methanol dehydrogenase, but the formaldehyde and formate dehydrogenases were not co-regulated. Analogous induction experiments revealed similar patterns in P. denitrificans, but no evidence for co-regulation of dissimilatory activities in Pseudomonas AM1.

Alcohol Dehydrogenase↗

PubMed1985

Organization of genes necessary for growth of the hydrogen-methanol autotroph Xanthobacter sp. strain H4-14 on hydrogen and carbon dioxide.

Mutants unable to grow on H2 and CO2 were isolated in the hydrogen-methanol autotroph Xanthobacter sp. strain H4-14 and complemented with a clone bank constructed in a broad-host-range cosmid vector. The mutants fell into two classes. Class I mutants (Cfx-) cannot grow on hydrogen or methanol and are deficient in one or more of the key enzymes of the Calvin Cycle. Class II mutants (Hox-) can grow on methanol but not on hydrogen and lack hydrogenase activity. Restriction maps of the complementing clones show that each class is not linked to the other. Subcloning and Tn5 mutagenesis have localized the regions of DNA complementing these mutants. The region complementing a class I mutant which is deficient in ribulosebisphosphate carboxylase activity is approximately 3.2 kilobase pairs in size. Expression of this enzyme activity from cloned DNA gave evidence for the orientation of an operon containing the structural genes for this enzyme. The region complementing most of the class II mutants is 3 to 4.5 kilobase pairs in size.

Carbon Dioxide↗

PubMed1984

Manipulation of methanotrophs.

Methanotrophs have interesting properties concerning the oxidation and dehalogenation of both straight-chain and aromatic hydrocarbons. However, the potential of these bacteria in the degradation of these compounds cannot be assessed until more experiments are carried out. It seems likely that genetic capabilities will play a major role in the exploitation of these bacteria. We have shown that it is possible to use recombinant DNA techniques to generate mutants and transfer genes in methanotrophic bacteria.

Hydrocarbons↗

PubMed1984

Plasmids in methanotrophic bacteria: isolation, characterization and DNA hybridization analysis.

Ten strains of obligate methanotrophs were screened for the presence of plasmid DNA using a variety of methods. Plasmids were detected in all strains except Methylococcus capsulatus Bath. No significant similarity between plasmids was observed with respect to size or restriction digest patterns except for three strains of Methylosinus trichosporium, which appeared to contain the same three plasmids. Nitrocellulose filter hybridization revealed that the plasmid DNA from the M. trichosporium strains shared a small region of homology with the plasmid DNA from Methylosinus sporium 5. All of the plasmids remain cryptic. As the first step in characterization, a restriction digest map of the 55 kb plasmid found in Methylomonas albus BG8 was constructed.

Collodion↗

PubMed1984

Nitrogen metabolism in a new obligate methanotroph, 'Methylosinus' strain 6.

A new obligate methanotroph was isolated and characterized. It was classified as a 'Methylosinus' species and named 'Methylosinus' sp. strain 6. Nitrogen metabolism in 'Methylosinus' 6 was found to be similar to other Type II methanotrophs, including the assimilation of nitrogen exclusively by the glutamine synthetase/glutamate synthase system. However, unlike other Type II methanotrophs, it appeared that glutamine synthetase activity was regulated by adenylylation in this organism. 'Methylosinus' 6 was grown in continuous culture with either dinitrogen or nitrate as sole nitrogen source under various dissolved oxygen tensions. Higher rates of methane oxidation and a more developed intracytoplasmic membrane system were found at lower oxygen tensions with nitrate as the nitrogen source but at higher oxygen tensions with dinitrogen as the nitrogen source. This suggested that carbon metabolism was influenced by nitrogen metabolism in this organism.

Acetylene↗

PubMed1984

Seasonal study of methane oxidation in lake washington.

The distribution of methane and methane-oxidizing bacteria in the water column of Lake Washington was determined monthly for 1 year. The methane profiles were relatively constant, with little stratification and low concentrations (0.05 to 0.5 muM). The number of methane-oxidizing bacteria detected by a filter-plating method was routinely <1/ml throughout the water column, and no incorporation or oxidation of methane was detected by radioisotopic labeling, even after methane was added. However, samples taken from the sediment-water interface contained as much as 3 muM methane and 50 CFU of methane-oxidizing bacteria per ml and showed significant rates of methane oxidation and incorporation. To define the region of maximum activity more precisely, vertical profiles of the sediment were examined. The concentration of methane increased with depth to a maximum of 150 to 325 muM at 2.5 cm, and significant rates of methane oxidation were found within the top 2.5 cm. The apparent K(m)s for methane and oxygen were determined for samples from the top 1.0 cm of the sediment and found to be ca. 10 and 20 muM, respectively. Projected values for methane oxidation rates suggested that maximum methane oxidation occurred in the top 0.5 cm of the sediment.

Journal Article↗

PubMed1984

DNA hybridization analysis of the nif region of two methylotrophs and molecular cloning of nif-specific DNA.

DNA isolated from two diazotrophic methylotrophs, the obligate methanotroph Methylosinus sp. strain 6 and the methanol autotroph Xanthobacter sp. H4-14, hybridized to DNA fragments encoding nitrogen fixation (nif) genes from Klebsiella pneumoniae. This interspecific nif homology was limited to DNA fragments encoding the nitrogenase structural proteins (nifH, nifD, and nifK) and specific methylotroph DNA sequences. The hybridization patterns obtained with the two methylotrophs were dissimilar, indicating that the nif region of methylotrophs is not physically conserved. By using the K. pneumoniae nif structural genes as a probe, a fragment of nif DNA from each methylotroph was cloned and characterized. The DNA fragment from Methylosinus sp. 6 encoded two polypeptides of 57,000 and 34,000 molecular weight.

Bacteria↗

PubMed1984

Molecular construction and characterization of nif mutants of the obligate methanotroph Methylosinus sp. strain 6.

We describe here a method for constructing mutants in bacteria that are not amenable to mutant isolation by conventional means. A one-step marker exchange procedure was used to construct nitrogen fixation (nif) mutants of the obligate methane-utilizing bacterium Methylosinus sp. strain 6, using transposon 5 (Tn5)-containing nif genes cloned into pBR325. The resultant mutants appeared to contain defects in nif structural genes, and DNA hybridization analysis showed that although one out of five had apparently been produced as a result of double-crossover homologous recombination, a variety of molecular events had led to the production of the other four mutants.

DNA Transposable Elements↗

PubMed1984

Molecular cloning of a malyl coenzyme A lyase gene from Pseudomonas sp. strain AM1, a facultative methylotroph.

A genomic library containing HindIII partial digests of Pseudomonas sp. strain AM1 DNA was constructed in the broad-host-range cosmid pVK100. PCT57, a Pseudomonas sp. strain AM1 methanol mutant deficient in malyl coenzyme A lyase activity, was complemented to a methanol-positive phenotype by mobilization of the pVK100 library into PCT57 recipients with the ColE1/RK2 mobilizing plasmid pRK2013. Six different complemented isolates all contained a recombinant plasmid carrying the same 19.6-kilobase-pair Pseudomonas sp. strain AM1 DNA insert. Subcloning and complementation analysis demonstrated that the gene deficient in PCT57 (mcl-1) was located in a 1.6-kilobase-pair region within a 7.4-kilobase-pair EcoRI-HindIII fragment.

Cloning, Molecular↗