of the genetic basis for the microbial degradation of organic compounds.
Research in the Zylstra laboratory is directed toward understanding the mechanisms by which different bacterial strains utilize hydrocarbons as carbon and energy sources. Projects in the laboratory emphasize the use of molecular genetic tools in the analysis of gene (and protein) evolution, the regulation of gene expression, the identification of intermediate compounds in catabolic pathways, and the functional analysis of the enzymes involved. The primary theme for projects in the laboratory is the examination of microbial diversity and how this affects the degradation of hydrocarbons in the environment. Research thus focuses on a detailed biochemical, physiological, and molecular genetic investigation and comparison of different model catabolic pathways in several bacterial genera. Recent research follows two main themes: the identification of novel genes for known catabolic pathways and the discovery of new genes involved in hydrocarbon degradation. Below is a brief description of two of the laboratory's many current projects.
Research Example #1: Metagenomic Analysis of Hydrocarbon Degradation in the Environment
The degradation of hydrocarbons by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on hydrocarbons, limited information is available on the diversity of microbes involved in hydrocarbon degradation in the environment. We have been examining the underlying genetic diversity of hydrocarbon degradation directly in environmental samples using metagenomic technologies.
We have designed PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all polycyclic aromatic hydrocarbon (PAH) and chlorinated biphenyl (PCB) dioxygenase enzymes. Additional primers have been constructed that are specific for the Rieske site for the so-called angular dioxygenases which attack compounds such as dioxins and dibenzofurans. We have used these primers to examine the native populations of dioxygenase genes in natural environments and shifts in dioxygenase gene populations in response to an aromatic hydrocarbon challenge. Unsurprisingly, different sample sites have different starting populations of dioxygenase genes and exposure to different aromatic compounds results in different populations of dioxygenase genes appearing (and disappearing). Specific dioxygenase sequences increasing upon exposure to a chemical is the result of a variety of factors, including efficiency of the dioxygenase enzyme (and pathway) as well as physiological characteristics of the host microbe. The overall goal of these experiments is to develop molecular methods to predict whether biodegradation is actually occurring by monitoring DNA and RNA at contaminant sites. However, our general interest is in the discovery of novel dioxygenases (perhaps from as yet uncultured bacteria) that are involved in hydrocarbon degradation in the environment. So far we have applied this analysis technique to a comparison of PAH degradation, dibenzofuran degradation, and PCB degradation. In the PCB case, we examined the response of the microbial community to individual PCB congeners as well as to mixed PCB congeners to more accurately simulate what is found in the environment.
Of course, a major goal of metagenomics is to identify genes and pathways functional in environmental samples without culturing the microorganisms. Examining an anaerobic enrichment culture degrading 4-chlorobenzoate we were successful in extracting a metagenomic clone encoding the initial steps in the anaerobic dehalogenation of the growth substrate. Using stable isotope probing (SIP) we were able to extract from the DNA of a biphenyl degrading enrichment culture a metagenome clone encoding biphenyl dioxygenase. The clone is interesting because no other genes for biphenyl degradation are clustered nearby. In recent experiments we have identified and sequenced eleven metagenome clones encoding cytochrome P450 enzymes. All the clones were obtained from a single site (an alpine meadow in the mountains of Kyrgyzstan) and a single metagenome library. None of the clones are identical and a comparison of the cytochrome P450 sequences indicate that some are closely related to known enzymes and some represent new subfamilies of cytochrome P450 enzymes. Our future work in this area will be to utilize some of the more unique and prevalent sequences obtained through the experiments described in the last paragraph to fish out the corresponding full length gene sequences from metagenome libraries constructed from the same environmental samples.
Research Example #2: Physiological Analysis of Hydrocarbon Degradation in Pure Cultures
We have been examining the degradation of the three isomers of phthalate for many years. An extensive culture collection of gram negative and gram positive organisms that degrade phthalate, isophthalate, and/or terephthalate (and their esters) has been assembled. Our recent work has focused on comparative analysis of the genes for the degradative pathways in various organisms. This results in two interesting areas of investigation: (1) identifying novel genes for phthalate, isophthalate, or terephthalate degradation in genera not previously known to degrade these compounds and (2) comparing the acquisition of the three different gene clusters (for the three different isomers) by related strains of bacteria. We have also examined the physiology of phthalate degradation using our model strain Burkholderia cepacia DBO1 (closely related to strain G4 whose genome has been sequenced). The four genes encoding the three catabolic enzymes are arranged in at least three operons with the genes encoding the oxygenase and reductase components of the initial enzyme in the catabolic pathway, phthalate dioxygenase, located approximately seven kilobases away from each other. Interestingly, there are two transport mechanisms to get phthalate into the cell, a permease within this gene cluster and an ABC type transporter located elsewhere (but not all strains have both, an interesting evolutionary pattern yet to be explained). Phthalate is toxic to bacteria through inhibition of quinolinate phosphoribosyl transferase (involved in NAD synthesis) and many phthalate degrading strains have added a gene encoding this enzyme to the phthalate degradative gene cluster. Our future work with DBO1 and related strains will be to understand why some strains have two different mechanisms for transport of phthalate into the cell (when phthalate is known to be toxic), how the ability to degrade phthalate evolved (some strains have the genes in an operonic cluster while some don't), and enzyme engineering to investigate substrate range and catalysis by the initial oxygenase. Our work with isophthalate and terephthalate degradation in several different Comamonas strains isolated from the Passaic River showed that the sequences of the two degradative operons are highly identical in strains that have identical 16S rRNA sequences but that the regions flanking the operons are completely different. This indicates that Comamonas bacteria acquired the genes for isophthalate and terephthalate degradation at different times in the recent past. Using a Comamonas strain that degrades all three isomers of phthalate we examined cross-talk among the regulatory proteins. Quantitative PCR analysis of phthalate, isophthalate, or terephthalate oxygenase gene expression in the presence of each phthalate isomer (alone or in combination) showed little to none cross-regulation (or cross-repression) of gene expression. Our future work with phthalate isomer degradation will be to examine acquisition (and possible transposition) of the catabolic operons across species boundaries. We are also interested in acquisition and spread of the catabolic genes among bacterial populations around the world.