Andria M. Costello

Evidence that participate methane monooxygenase and ammonia monooxygenase may be evolutionarily related

Genes encoding particulate methane monooxygenase and ammonia monooxygenase share high sequence identity. Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase. A 525-bp internal DNA fragment of the genes encoding these polypeptides (pmoA and amoA) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced. Representatives of each of the phylogenetic groups of both methanotrophs (alpha- and gamma-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (beta- and gamma-Proteobacteria) were included. Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure. Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the gamma-Proteobacteria than to AmoA sequences. These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria.

Molecular characterization of methanotrophic isolates from freshwater lake sediment.

ABSTRACT Profiles of dissolved O2 and methane with increasing depth were generated for Lake Washington sediment, which suggested the zone of methane oxidation is limited to the top 0.8 cm of the sediment. Methane oxidation potentials were measured for 0.5-cm layers down to 1.5 cm and found to be relatively constant at 270 to 350 μmol/liter of sediment/h. Approximately 65% of the methane was oxidized to cell material or metabolites, a signature suggestive of type I methanotrophs. Eleven methanotroph strains were isolated from the lake sediment and analyzed. Five of these strains classed as type I, while six were classed as type II strains by 16S rRNA gene sequence analysis. Southern hybridization analysis with oligonucleotide probes detected, on average, one to two copies of pmoA and one to three copies of 16S rRNA genes. Only one restriction length polymorphism pattern was shown for pmoA genes in each isolate, and in cases where, sequencing was done, the pmoA copies were found to be almost identical. PCR primers were developed formmoX which amplified 1.2-kb regions from all six strains that tested positive for cytoplasmic soluble methane mono-oxygenase (sMMO) activity. Phylogenetic analysis of the translated PCR products with published mmoX sequences showed that MmoX falls into two distinct clusters, one containing the orthologs from type I strains and another containing the orthologs from type II strains. The presence of sMMO-containing Methylomonas strains in a pristine freshwater lake environment suggests that these methanotrophs are more widespread than has been previously thought.

Role of multiple gene copies in particulate methane monooxygenase activity in the methane-oxidizing bacterium Methylococcus capsulatus Bath.

Genes for the subunits of particulate methane monooxygenase, PmoABC, have been sequenced from the gamma-proteobacterial methanotroph Methylococcus capsulatus Bath. M. capsulatus Bath contains two complete copies of pmoCAB, as well as a third copy of pmoC. The two pmoCAB regions were almost identical at the nucleotide sequence level, differing in only 13 positions in 3183 bp. At the amino acid level, each translated gene product contained only one differing residue in each copy. However, the pmoC3 sequence was more divergent from the two other pmoC copies at both the far N-terminus and far C-terminus. Chromosomal insertion mutations were generated in all seven genes. Null mutants could not be obtained for pmoC3, suggesting that it may play an essential role in growth on methane. Null mutants were obtained for pmoC1, pmoC2, pmoA1, pmoA2, pmoB1 and pmoB2. All of these mutants grew on methane, demonstrating that both gene copies were functional. Copy 1 mutants showed about two-thirds of the wild-type whole-cell methane oxidation rate, while copy 2 mutants showed only about one-third of the wild-type rate, indicating that both gene copies were necessary for wild-type particulate methane monooxygenase activity. It was not possible to obtain double null mutants that were defective in both pmo copies, which may indicate that some expression of pMMO is important for growth.

The Biochemistry Of the Particulate Methane Monooxygenase

The particulate methane monooxygenase (pMMO) found in the intracytoplasmic membranes of methanotrophs has been known to be very difficult to study. Recent progress in our laboratory indicates that the pMMO is a novel copper-containing enzyme [1]. Metal/protein ratio data analysis clearly suggests that the pMMO is a multiple copper- containing enzyme. The pMMO-associated copper ions appear to be organized into trinuclear cluster units with rather defined-magnetic and redox properties. These copper clusters has been shown to be involved in dioxygen activation. The as-isolated pMMO enriched-membranes often contain a mixture of Cu(I) and Cu(II) ions in various proportions, depending on the history of the samples. The functional form of the enzyme has been found to be the reduced or partially reduced form.

Monitoring Genetic & Metabolic Potential for In Situ Bioremediation: Mass Spectrometry

A number of DOE sites are contaminated with dense non-aqueous phase liquids (DNAPLs) such as carbon tetrachloride and trichloroethylene. At many of these sites, microbial bioremediation is an attractive strategy for cleanup, since it has the potential to degrade DNAPLs in situ. A rapid screening method to determine the broad range potential of a sites microbial population for contaminant degradation would greatly facilitate assessment for in situ bioremediation, as well as for monitoring ongoing bioremediation treatment. Current laboratory based treatability methods are cumbersome and expensive. In this project, we are developing methods based on matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) for rapid and accurate detection of polymerase chain reaction (PCR) products from microbial genes involved in biodegradation of pollutants. PCR primers are being developed to amplify DNA sequences that are amenable to MALDI-MS detection. This work will lay the foundation for development of a field-portable MS-based technique for rapid on site assessment and monitoring of bioremediation processes.