The biphenyl dioxygenases (BP Dox) of strains Pseudomonas pseudoalcaligenes KF707 and Pseudomonas cepacia LB400 exhibit a distinct difference in substrate ranges of polychlorinated biphenyls (PCB) despite nearly identical amino acid sequences. The range of congeners oxidized by LB400 BP Dox is much wider than that oxidized by KF707 BP Dox. The PCB degradation abilities of these BP Dox were highly dependent on the recognition of the chlorinated rings and the sites of oxygen activation. The KF707 BP Dox recognized primarily the 4-chlorinated ring (97%) of 2,5,4-trichlorobiphenyl and introduced molecular oxygen at the 2,3 position. The LB400 BP Dox recognized primarily the 2,5-dichlorinated ring (95%) of the same compound and introduced O 2 at the 3,4 position. It was confirmed that the BphA1 subunit (iron-sulfur protein of terminal dioxygenase encoded by bphA1) plays a crucial role in determining the substrate selectivity. We constructed a variety of chimeric bphA1 genes by exchanging four common restriction fragments between the KF707 bphA1 and the LB400 bphA1. Observation of Escherichia coli cells expressing various chimeric BP Dox revealed that a relatively small number of amino acids in the carboxy-terminal half (among 20 different amino acids in total) are involved in the recognition of the chlorinated ring and the sites of dioxygenation and thereby are responsible for the degradation of PCB. The site-directed mutagenesis of Thr-376 (KF707) to Asn-376 (LB400) in KF707 BP Dox resulted in the expansion of the range of biodegradable PCB congeners.Recent studies revealed that oxygenases involved in the initial oxidation of aromatic hydrocarbons are multicomponent enzymes and that their corresponding subunits show various degrees of homology (14,26,27,31). This implies that soil bacteria have adaptively evolved, by modifying key enzymes, to utilize a variety of aromatic compounds which are present in the environment. Biphenyl-utilizing bacteria are ubiquitously distributed and can be isolated from various environmental samples (3,8,29) including intestine of termite (5), suggesting that they are involved in the degradation of plant lignin at the final stage, together with other aromatic degraders (24). Biphenyl-utilizing bacteria have been extensively studied with respect to the degradation of polychlorinated biphenyls (PCB), which are known to be serious environmental pollutants. These studies revealed considerable differences in the congener selectivity patterns and in the range of activity of various PCBdegrading bacteria (9). It was also demonstrated that both the relative rates of primary degradation of PCB and the choice of the ring attacked were dependent on the bacterial strains (1, 2, 4, 16). Pseudomonas pseudoalcaligenes KF707 was isolated from soil near a biphenyl-producing factory in Kitakyushu, Japan (12,15). This strain showed a narrow range of degradable PCB congeners (7, 17). Pseudomonas sp. strain LB400 (referred to as Pseudomonas cepacia LB400 in a recent publication [19]) was isolated from a PCB...

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The biphenyl and salicylate metabolic pathways in Pseudomonas putida KF715 are chromosomally encoded. The bph gene cluster coding for the conversion of biphenyl to benzoic acid and the sal gene cluster coding for the salicylate meta-pathway were obtained from the KF715 genomic cosmid libraries. These two gene clusters were separated by 10-kb DNA and were highly prone to deletion when KF715 was grown in nutrient medium. Two types of deletions took place at the region including only the bph genes (ca. 40 kb) or at the region including both the bph and sal genes (ca. 70 kb). A 90-kb DNA region, including both the bph and sal genes (termed the bph-sal element), was transferred by conjugation from KF715 to P. putida AC30. Such transconjugants gained the ability to grow on biphenyl and salicylate as the sole sources of carbon. The bph and sal element was located on the chromosome of the recipient. The bph-sal element in strain AC30 was also highly prone to deletion; however, it could be mobilized to the chromosome of P. putida KT2440 and the two deletion mutants of KF715.A number of biphenyl-utilizing bacteria have been isolated to date. They include gram-negative species of Pseudomonas, Achromobacter, Sphingomonas, Comamonas, Burkholderia, Ralstonia, and Alcaligenes and gram-positive Rhodococcus spp. (1,3,11,37). Because these biphenyl-utilizing strains cometabolize polychlorinated biphenyls (PCB), the biochemistry of PCB degradation has been extensively studied (12). A gene cluster coding for biphenyl-PCB degradation (termed bph) was first cloned from Pseudomonas pseudoalcaligenes KF707 (14). Since then, a number of bph genes have been cloned and sequenced. Southern and sequence analyses of the bph genes revealed that some biphenyl-utilizing strains possess bph genes that are very similar, if not identical, to one another, but some share various degrees of homology (13).The bph genes are present on bacterial chromosomes (8, 14, 16, 35), plasmids (18, 39), and transposons (22, 28, 34). The presence of similar genes in different strains implies that even chromosomal bph genes have or used to have a mechanism for mobilization to other strains. The bph genes of Pseudomonas sp. strain CB406 were mobilized following the construction in vivo of a cointegrate plasmid inserted into the broad-hostrange plasmid RP4 (22). Springael and coworkers (23, 34) identified a transposon, Tn4371, carrying the bph genes encoding conversion of biphenyl to benzoic acid from Ralstonia eutrophus A5 (formerly Alcaligenes eutrophus A5) in which Tn4371 first transposed from the chromosome to indigenous IncP1 plasmid, and the plasmid carrying Tn4371 can be transferred to other strains by conjugation. A recent study shows that Tn4371 is a kind of conjugative transposon of 55 kb (24, 29). Interestingly, another smaller conjugative transposon Tnbph coding for biphenyl catabolism resides within Tn4371. This can be transferred to the recipient strain by conjugation independently. Divergence of the bph genes among various biphenyl-utilizing strains in...

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