We have characterized a biphenyl degrader, Rhodococcus sp. strain RHA1. Biphenyl-grown cells of strain RHA1 efficiently transformed 45 components in the 62 major peaks of a polychlorinated biphenyl (PCB) mixture of Kanechlors 200, 300, 400, and 500 within 3 days, which includes mono-to octachlorobiphenyls. Among the intermediate metabolites of PCB transformation, di-and trichlorobenzoic acids were identified. The gradual decrease of these chlorobenzoic acids during incubation indicated that these chlorobenzoic acids would also be degraded by this strain. The effect of the position of chlorine substitution was determined by using PCB mixtures that have chlorine substitutions mainly at either the ortho or the meta position. This strain transformed both types of congeners, and strong PCB transformation activity of RHA1 was indicated. RHA1 accumulated 4-chlorobenzoic acid temporally during the transformation of 4-chlorobiphenyl. The release of most chloride in the course of 2,2-dichlorobiphenyl degradation was observed. These results suggested that RHA1 would break down at least some PCB congeners into smaller molecules to a considerable extent.

Bacillus sp. strain JF8, which was isolated from compost, utilizes naphthalene and biphenyl as carbon sources at 60³C. Biphenyl grown cells of strain JF8 barely degraded naphthalene while naphthalene grown cells did not degrade pchlorobiphenyl, suggesting the existence of two independent degradation pathways. Isolation of JF8N, a mutant strain which can not utilize biphenyl as a carbon source while retaining the ability to utilize naphthalene, supports this hypothesis. Biphenyl grown cells of strain JF8 can degrade several polychlorinated biphenyl congeners including tetra-and pentachlorobiphenyl. bph and nah probes from mesophilic organisms failed to hybridize to strain JF8 DNA. ß

Bacillus sp. strain JF8, which was isolated from compost, utilizes naphthalene and biphenyl as carbon sources at 60 degrees C. Biphenyl grown cells of strain JF8 barely degraded naphthalene while naphthalene grown cells did not degrade p-chlorobiphenyl, suggesting the existince of two independent degradation pathways. Isolation of JF8N, a mutant strain which can not utilize biphenyl as a carbon source while retaining the ability to utilize naphthalene, supports this hypothesis. Biphenyl grown cells of strain JF8 can degrade several polychlorinated biphenyl congeners including tetra- and pentachlorobiphenyl. bph and nah probes from mesophilic organisms failed to hybridize to strain JF8 DNA.

Bacillus sp. JF8 is a thermophilic polychlorinated biphenyl (PCB) degrader, which utilizes biphenyl and naphthalene. A thermostable, Mn-dependent 2,3-dihydroxybiphenyl 1,2-dioxygenase, BphC_JF8, has been characterized previously. Upstream of bphC are five ORFs exhibiting low homology with, and a different gene order from, previously characterized bph genes. From the 59 to 39 direction the genes are: a putative regulatory gene (bphR), a hydrolase (bphD), the large and small subunits of a ring-hydroxylating dioxygenase (bphA1A2), and a cis-diol dehydrogenase (bphB). Hybridization studies indicate that the genes are located on a plasmid. Ring-hydroxylating activity of recombinant BphA1A2_JF8 towards biphenyl, PCB, naphthalene and benzene was observed in Escherichia coli cells, with complementation of non-specific ferredoxin and ferredoxin reductase by host cell proteins. PCB degradation by recombinant BphA1A2_JF8 showed that the congener specificity of the recombinant enzyme was similar to Bacillus sp. JF8. BphD_JF8, with an optimum temperature of 85 6C, exhibited a narrow substrate preference for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid. The Arrhenius plot of BphD_JF8 was biphasic, with two characteristic energies of activation and a break point at 47 6C.

To improve the capabilities of microorganisms relevant for biodegradation, we developed a new genetic approach and applied it to the bph operon (bphEGF[orf4]A1A2A3CD[orf1]A4R) of Pseudomonas sp. strain KKS102 to enhance its biphenyl-and polychlorinated biphenyl (PCB)-degrading activity. A native promoter of the bph operon, which was under control, was replaced through homologous recombination by a series of promoters that had constitutive activity. By testing a series of promoters with various strengths, we were able to obtain strains that have enhanced degradation activity for biphenyl and PCBs. This strategy removes the rate-limiting factor associated with transcription and has the potential to improve the degradation activity of a wide variety of microorganisms involved in biodegradation.The use of microbial metabolic potential for elimination of environmental pollutants is a promising technology. Although various factors such as pathway enzyme specificity, substrate availability, incomplete degradation pathways, and the transcription and translation of genes for bioconversion can limit efficient biodegradation, genetic engineering can be used to overcome such factors and improve degradation (3,9,20,22,30).Among pollutants, polychlorinated biphenyls (PCBs) are the most serious pollutants, and their degradation by microorganisms has been studied extensively (9, 10). Pseudomonas sp. strain KKS102 is one of the well-characterized PCB and biphenyl degraders, and its bph gene organization, catabolic route, and regulatory mechanisms have been characterized. The bph genes are organized into an operon in the following order: bphEGF(orf4)A1A2A3CD(orf1) A4R (8, 13-15). The transcription of the bph operon is dependent on the pE promoter, which is located upstream of the bphE gene and is controlled by a negative regulator, BphS. The bphS gene is divergently orientated upstream of bphE and is separated from bphE by an insertion sequence (17). The repression mediated by BphS protein is counteracted by a meta-cleaved intermediate of biphenyl degradation (17,18).It is generally recognized that microorganisms can be genetically engineered to increase the rate of pollutant removal. The design of improved microorganisms includes various optimization strategies among which altering the level of transcription is a good target. In general, genes encoding catalytic activities are organized into an operon and transcription of the operon is under the control of activator(s) (6). Various efforts have been made to increase the level of transcription, e.g., creation of mutant regulators that mediate a higher level of transcription or recognize new substrates (2, 19, 21) and construction of plasmids or transposons that carry degradative genes under the control of constitutive promoters (11,16). The transcription level should be optimized through trials with a series of promoters of different strengths if we are to exclude the ratelimiting steps associated with transcription. It can be expected that application of too strong a promoter re...