Research

Circular representation of the 2,812,094-bp genome of N. europaea ATCC 19718. (View larger version:)
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Description of Research

Nitrification

Ammonia-oxidizing bacteria and nitrite-oxidizing bacteria are participants in both the C and N cycles and together carry out the process of nitrification. Nitrification can influence both biogeochemical cycles through its effect on N availability to organisms. Nitrification influences the concentration of greenhouse gases in the atmosphere by production of NO and N₂O and by influencing consumption of CO₂. Most molecular investigations of these bacteria have focused on single genes and enzymes involved in the oxidation of ammonia or nitrite. However, with the sequencing of the genomes of representative nitrifiers, it is possible to examine the entire complement of genes involved in assimilating CO₂ and oxidizing NH₃ or NO₂^- and the coordination of their expression. We are exploiting the whole genome sequence information of Nitrosomonas europaea using genomic technology to understand its genetic structure and molecular regulatory mechanisms. The availability of complete genome sequences for Nitrosomonas europaea and Nitrobacter winogradskyi has enabled new approaches to studying interactions between the ammonia oxidizing bacteria and nitrite oxidizing bacteria. We are examining the interactions between these two bacteria.

We are generating selected knockout mutants in N. europaea through homologous recombination to further define regulatory networks and metabolic pathways. The research will contribute to our basic understanding of microbial physiology, especially as it applies to the basis of lithotrophy and autotrophy, and will also be relevant to efforts to mitigate the detrimental effects of these bacteria (e.g. in croplands fertilized with ammonia-based fertilizers) and to exploit their beneficial capabilities (e.g. wastewater treatment and bioremediation).

Alkane metabolism

Gaseous and liquid alkanes can serve as growth substrates for several bacteria. Alkane monooxygenases initiate the metabolism of alkanes by catalyzing the oxidation of the alkanes to alcohols. Alkane metabolism and the corresponding monooxygenases can conveniently be divided into three groups based on the number of carbon atoms in the alkane substrates: C₁, C₂-C₅, and C₆-C₂₀. The C₁ utilizers are the methanotrophs and their monooxygenases are of two types: soluble and particulate methane monooxygenases. For longer chain, liquid alkanes (C₆-C₂₀) a membrane associated diiron-oxo monooxygenase is well studied. For the gaseous, short-chain alkanes (C₂-C₅) the emerging view from our research is that a soluble diiron-containing butane monooxygenase functions in Pseudomonas butanovora while a particulate, Cu-containing enzyme functions in Nocardioides sp. strain CF8. The goal of this research is to further characterize butane metabolism in P. butanovora and to study the regulation of this inducible pathway.

We are characterizing the diiron containing butane monooxygenase from Pseudomonas butanovora. The physical and kinetic characteristics of the protein components are being determined and site specific and random amino acid substitutions are being introduced to interrogate the basis of alkane specificity with particular emphasis on the inability to oxidize methane. We are also evaluating the induction patterns of the operons in in the metabolism of butane in response to metabolites as well as to putative σ⁵⁴-dependent transcriptional activators via the use of promoter-reporter gene fusions.

This work will provide the detailed molecular description of a pathway of gaseous alkane metabolism (other than methane). Our focus is on butane metabolism, but we expect that the same pathway is utilized for ethane, propane and pentane (also gaseous alkanes) and thus the results would generally be applicable to the group of short chain alkanes. Our long-range plans, outside the scope of this work, are to continue to develop our knowledge of butane metabolism through examination of other butane oxidizing bacteria, determining the role of gaseous alkane utilizing bacteria in bioremediation, and completing detailed physical (spectroscopic and structural) and kinetic examinations of the purified enzymes.