Pesticide fate in the environment is affected by microbial activity. Some pesticides are readily degraded by microorganisms, others have proven to be recalcitrant. A diverse group of bacteria, including members of the genera Alcaligenes, Flavobacterium, Pseudomonas and Rhodococcus, metabolize pesticides. Microbial degradation depends not only on the presence of microbes with the appropriate degradative enzymes, but also on a wide range of environmental parameters. This review describes recent advances in biodegradation of pesticides by addressing the biology and molecular characterization of some pesticide degrading bacteria.

Huhu grubs (Prionoplus reticularis) are wood-feeding beetle larvae endemic to New Zealand and belonging to the family Cerambycidae. Compared to the wood-feeding lower termites, very little is known about the diversity and activity of microorganisms associated with xylophagous cerambycid larvae. To address this, we used pyrosequencing to evaluate the diversity of metabolically active and inactive bacteria in the huhu larval gut. Our estimate, that the gut harbors at least 1,800 phylotypes, is based on 33,420 sequences amplified from genomic DNA and reverse-transcribed RNA. Analysis of genomic DNA-and RNA-derived data sets revealed that 71% of all phylotypes (representing 95% of all sequences) were metabolically active. Rare phylotypes contributed considerably to the richness of the community and were also largely metabolically active, indicating their participation in digestive processes in the gut. The dominant families in the active community (RNA data set) included Acidobacteriaceae (24.3%), Xanthomonadaceae (16.7%), Acetobacteraceae (15.8%), Burkholderiaceae (8.7%), and Enterobacteriaceae (4.1%). The most abundant phylotype comprised 14% of the active community and affiliated with Dyella ginsengisoli (Gammaproteobacteria), suggesting that a Dyella-related organism is a likely symbiont. This study provides new information on the diversity and activity of gutassociated microorganisms that are essential for the digestion of the nutritionally poor diet consumed by wood-feeding larvae. Many huhu gut phylotypes affiliated with insect symbionts or with bacteria present in acidic environments or associated with fungi.

Unculturable polycyclic aromatic hydrocarbon (PAH)-degrading bacteria are a significant reservoir of the microbial potential to catabolize low-molecular-weight PAHs. The population of these bacteria is larger than the population of nah-like bacteria that are the dominant organisms in culture-based studies. We used the recently described phn genes of Burkholderia sp. strain RP007, which feature only rarely in culture-based studies, as an alternative genotype for naphthalene and phenanthrene degradation and compared this genotype with the genotypically distinct but ubiquitous nah-like class in different soils. Competitive PCR quantification of phnAc and nahAc, which encode the iron sulfur protein large (␣) subunits of PAH dioxygenases in nah-like and phn catabolic operons, revealed that the phn genotype can have a greater ecological significance than the nah-like genotype.It could mistakenly be inferred from available nucleotide sequence data that highly conserved nah-like gene clusters, which are isolated from polycyclic aromatic hydrocarbon (PAH)-degrading pseudomonads obtained from diverse geographic areas, are the dominant gene clusters involved in degradation of the low-molecular-weight PAHs naphthalene and phenanthrene. The results of probing and PCR amplification of nah sequences from contaminated soils and sediments also indicate that these sequences are ubiquitous (7,17). Although PAH degraders with nah genotypes are easily isolated, it has been acknowledged that the nah catabolic cluster and closely related homologues may be present in only a small part of the PAH-degrading bacterial population (1,4,12,20). Recently, the phn genes of Burkholderia sp. strain RP007 provided evidence that there is a different genotype that exhibits a low level of sequence homology with nah and has a different gene order yet encodes enzymes for an identical PAH degradation pathway (9, 10).As increasingly diverse genes that encode enzymes for PAH catabolism are characterized (4-6, 14, 20), it is important not only to understand the function of these genes but also to determine their ecological significance in the context of environmental pollution. The objective of this study was, therefore, to compare the prevalence in aromatic hydrocarbon-contaminated soils of two distinct PAH catabolic genotypes. These genotypes were the divergent phn genes (9, 10), which are difficult to isolate by conventional microbiological methods (12), and the easily isolated nah-like genes (1,7,17). It has been shown that culture-based methods overemphasize nahlike genes and fail to detect bacteria with phn genotypes in contaminated soils in which both nahAc and phnAc are detected by PCR amplification and DNA hybridization (12). A molecular biological approach was required to overcome the disparity in the ease of culturing of host bacteria harboring these two genotypes. Highly specific primer combinations, which targeted genes that encode the iron sulfur protein large (␣) subunits of the nah-like and phn PAH initial dioxygenases, allowed us to deter...

Cloning and molecular ecological studies have underestimated the diversity of polycyclic aromatic hydrocarbon (PAH) catabolic genes by emphasizing classical nah-like (nah,ndo, pah, and dox) sequences. Here we report the description of a divergent set of PAH catabolic genes, the phn genes, which although isofunctional to the classical nah-like genes, show very low homology. Thisphn locus, which contains nine open reading frames (ORFs), was isolated on an 11.5-kb HindIII fragment from phenanthrene-degrading Burkholderia sp. strain RP007. The phn genes are significantly different in sequence and gene order from previously characterized genes for PAH degradation. They are transcribed by RP007 when grown at the expense of either naphthalene or phenanthrene, while in Escherichia coli the recombinant phn enzymes have been shown to be capable of oxidizing both naphthalene and phenanthrene to predicted metabolites. The locus encodes iron sulfur protein α and β subunits of a PAH initial dioxygenase but lacks the ferredoxin and reductase components. The dihydrodiol dehydrogenase of the RP007 pathway, PhnB, shows greater similarity to analogous dehydrogenases from described biphenyl pathways than to those characterized from naphthalene/phenanthrene pathways. An unusual extradiol dioxygenase, PhnC, shows no similarity to other extradiol dioxygenases for naphthalene or biphenyl oxidation but is the first member of the recently proposed class III extradiol dioxygenases that is specific for polycyclic arene diols. Upstream of the phn catabolic genes are two putative regulatory genes, phnR andphnS. Sequence homology suggests that phnS is a LysR-type transcriptional activator and that phnR, which is divergently transcribed with respect to phnSFECDAcAdB, is a member of the ς54-dependent family of positive transcriptional regulators. Reverse transcriptase PCR experiments suggest that this gene cluster is coordinately expressed and is under regulatory control which may involve PhnR and PhnS.

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