No abstract

The degradation of polycyclic aromatic hydrocarbons (PAHs) by bacteria has been widely studied. While many pure cultures have been isolated and characterized for their ability to grow on PAHs, limited information is available on the diversity of microbes involved in PAH degradation in the environment. We have designed generic PCR primers targeting the gene fragment encoding the Rieske iron sulfur center common to all PAH dioxygenase enzymes. These Rieske primers were employed to track dioxygenase gene population shifts in soil enrichment cultures following exposure to naphthalene, phenanthrene, or pyrene. PAH degradation was monitored by gas chromatograph with flame ionization detection. DNA was extracted from the enrichment cultures following PAH degradation. 16S rRNA and Rieske gene fragments were PCR amplified from DNA extracted from each enrichment culture and an unamended treatment. The PCR products were cloned and sequenced. Molecular monitoring of the enrichment cultures before and after PAH degradation using denaturing gradient gel electrophoresis and 16S rRNA gene libraries suggests that specific phylotypes of bacteria were associated with the degradation of each PAH. Sequencing of the cloned Rieske gene fragments showed that different suites of genes were present in soil microbe populations under each enrichment culture condition. Many of the Rieske gene fragment sequences fell into clades which are distinct from the reference dioxygenase gene sequences used to design the PCR primers. The ability to profile not only the bacterial community but also the dioxygenases which they encode provides a powerful tool for both assessing bioremediation potential in the environment and for the discovery of novel dioxygenase genes.

Relatively little is known about the number, diversity, and function of chitinases produced by bacteria, even though chitin is one of the most abundant polymers in nature. Because of the importance of chitin, especially in marine environments, we examined chitin-degrading proteins in the marine bacterium Vibrio harveyi. This bacterium had a higher growth rate and more chitinase activity when grown on ␤-chitin (isolated from squid pen) than on ␣-chitin (isolated from snow crab), probably because of the more open structure of ␤-chitin. When exposed to different types of chitin, V. harveyi excreted several chitin-degrading proteins into the culture media. Some chitinases were present with all of the tested chitins, while others were unique to a particular chitin. We cloned and identified six separate chitinase genes from V. harveyi. These chitinases appear to be unique based on DNA restriction patterns, immunological data, and enzyme activity. This marine bacterium and probably others appear to synthesize separate chitinases for efficient utilization of different forms of chitin and chitin by-products.

No abstract

The contamination of polar regions due to the global distribution of anthropogenic pollutants is of great concern because it leads to the bioaccumulation of toxic substances, methylmercury among them, in Arctic food chains. Here we present the first evidence that microbes in the high Arctic possess and express diverse merA genes, which specify the reduction of ionic mercury [Hg(II)] to the volatile elemental form [Hg(0)]. The sampled microbial biomass, collected from microbial mats in a coastal lagoon and from the surface of marine macroalgae, was comprised of bacteria that were most closely related to psychrophiles that had previously been described in polar environments. We used a kinetic redox model, taking into consideration photoredox reactions as well as mer-mediated reduction, to assess if the potential for Hg(II) reduction by Arctic microbes can affect the toxicity and environmental mobility of mercury in the high Arctic. Results suggested that mer-mediated Hg(II) reduction could account for most of the Hg(0) that is produced in high Arctic waters. At the surface, with only 5% metabolically active cells, up to 68% of the mercury pool was resolved by the model as biogenic Hg(0). At a greater depth, because of incident light attenuation, the significance of photoredox transformations declined and merA-mediated activity could account for up to 90% of Hg(0) production. These findings highlight the importance of microbial redox transformations in the biogeochemical cycling, and thus the toxicity and mobility, of mercury in polar regions.

The reduction of ionic mercury to elemental mercury by the mercuric reductase (MerA) enzyme plays an important role in the biogeochemical cycling of mercury in contaminated environments by partitioning mercury to the atmosphere. This activity, common in aerobic environments, has rarely been examined in anoxic sediments where production of highly toxic methylmercury occurs. Novel degenerate PCR primers were developed which span the known diversity of merA genes in Gram-negative bacteria and amplify a 285 bp fragment at the 3' end of merA. These primers were used to create a clone library and to analyse merA diversity in an anaerobic sediment enrichment collected from a mercury-contaminated site in the Meadowlands, New Jersey. A total of 174 sequences were analysed, representing 71 merA phylotypes and four novel MerA clades. This first examination of merA diversity in anoxic environments suggests an untapped resource for novel merA sequences.

No abstract

Sphingobium yanoikuyae B1 utilizes both polycyclic aromatic hydrocarbons (biphenyl, naphthalene, and phenanthrene) and monocyclic aromatic hydrocarbons (toluene, m- and p-xylene) as its sole source of carbon and energy for growth. The majority of the genes for these intertwined monocyclic and polycyclic aromatic pathways are grouped together on a 39 kb fragment of chromosomal DNA. However, this gene cluster is missing several genes encoding essential enzymatic steps in the aromatic degradation pathway, most notably the genes encoding the oxygenase component of the initial polycyclic aromatic hydrocarbon (PAH) dioxygenase. Transposon mutagenesis of strain B1 yielded a mutant blocked in the initial oxidation of PAHs. The transposon insertion point was sequenced and a partial gene sequence encoding an oxygenase component of a putative PAH dioxygenase identified. A cosmid clone from a genomic library of S. yanoikuyae B1 was identified which contains the complete putative PAH oxygenase gene sequence. Separate clones expressing the genes encoding the electron transport components (ferredoxin and reductase) and the PAH dioxygenase were constructed. Incubation of cells expressing the dioxygenase enzyme system with biphenyl or naphthalene resulted in production of the corresponding cis-dihydrodiol confirming PAH dioxygenase activity. This demonstrates that a single multicomponent dioxygenase enzyme is involved in the initial oxidation of both biphenyl and naphthalene in S. yanoikuyae B1.