Identification of a catabolic transposon, Tn4371, carrying biphenyl and 4-chlorobiphenyl degradation genes in Alcaligenes eutrophus A5

Abstract

Alcaligenes eutrophus A5 catabolizes biphenyl to CO2 via benzoate and 4-chlorobiphenyl to 4-chlorobenzoate. In curing and conjugation experiments, the A5 endogenous 51-kb IncP1 plasmid pSS50 was found to be dispensable for biphenyl and 4-chlorobiphenyl catabolism. Transfer of the biphenyl-and 4-chlorobiphenyldegrading phenotype by means of pSS50 was observed at a frequency of 10-5 per transferred plasmid in matings of A5 with other A. eutrophus strains. Transconjugants harbor enlarged pSS50 derivatives which contain additional genetic information governing the oxidation of biphenyl and 4-chlorobiphenyl to benzoate and 4-chlorobenzoate and originating from the chromosome of strain A5. Genes encoding enzymes that degrade polychlorinated biphenyls (PCBs) have been found on both bacterial chromosomes and plasmids. The molecular cloning of chromosomal genes involved in PCB metabolism has been reported previously (1,8,10,17,18). The genetic basis of plasmidmediated degradation has yet to be developed (9,14,29), and this genetic basis may provide information on how such catabolic pathways evolved and spread in the environment (22).Alcaligenes eutrophus A5 was isolated from PCB-contaminated river sediment at Fort Loundoun Reservoir, Knoxville, Tenn., for its ability to utilize 4-chlorobiphenyl (4CBP) as a growth substrate (29). A5 catabolizes 4CBP to 4-chlorobenzoate (4CBA) and biphenyl (BP) to CO2 via benzoate (BA). The appearance of a yellow cleavage intermediate (A437) during 4CBP and BP degradation (31) suggests that A5 metabolizes 4CBP and BP to 4CBA and BA via the catabolic pathway described by Furukawa et al., which includes metacleavage of a 2,3-dihydroxybiphenyl compound (Fig. 1) (11). Strain A5 carries the 51-kb large IncP1 plasmid pSS50, whose characteristics and restriction map are described elsewhere (2,14,19). pSS50 is a remnant of a 75-kb plasmid which lost a 4-CBA dehalogenase activity converting the compound top-hydroxybenzoate and originally enabling A5 to mineralize 4CBP to CO2 (19).So far, it is not clear whether pSS50 still plays a role in the catabolism of 4CBP and BP to 4CBA and BA. We found that transfer of the ability to use 4CBP and BP as the sole sources of carbon and energy (Bph+ Cbp+) from A5 to other A. eutrophus strains occurred at a low frequency and was accompanied by the appearance of an enlarged pSS50 plasmid in the transconjugants (32). The present article reports the genetic analysis of these enlarged pSS50 derivatives and shows that the genes involved in the degradation of BP and * Corresponding author.4CBP to BA and 4CBA in A5 are carried on a chromosomally located element which is able to transpose on pSS50 and other plasmids. MATERIALS AND METHODSBacterial strains, plasmids, and culture media. Bacterial strains and plasmids used in this study and their characteristics are listed in Table 1 All strains were grown at 30°C in Tris minimal medium (21) supplemented with the appropriate carbon source (gluconate or lactate, 0.5%, wt/vol). BP and 4CBP were added as crystals to the lid of...

Versatile Transcription of Biphenyl Catabolic bphOperon in Pseudomonas pseudoalcaligenes KF707

“…Such elements permit microorganisms to degrade aromatic compounds by horizontal gene transfer. It was reported that the bph gene cluster involved in the metabolism of biphenyl/4-chlorobiphenyl is located in the 55-kb transposon Tn4371 (17,18,33,34). Our recent study also revealed that the 90-kb bph-sal element coding for biphenyl and salicylate metabolism behaves like a conjugative transposon in Pseudomonas putida KF715 whose bph gene cluster is nearly identical to that of KF707 except that the bphX0X1X2X3 genes were deleted (35).…”

Biochemical and genetic bases of microbial degradation of polychlorinated biphenyls(PCBs).

“…KKS102 was induced by the ring meta-cleavage product (Ohtsubo et al, 2000). Recently, the bph gene cluster involved in the metabolism of biphenyl and 4-chlorobiphenyl was found in the 55-kb transposon Tn4371 in Ralstonia eutopha A5 (Merlin et al, 1997;Springael et al, 1993). It was suggested that the bphEFGA1A2A3BCD operon in Tn4371 is transcribed from a s70 promoter and the bphS gene procuct negatively regulates the transcription of the bph gene cluster as a repressor (Mouz et al, 1999).…”

Ralstonia metallidurans CH34 RpoN sigma factor and the control of nitrogen metabolism and biphenyl utilization The GenBank accession number for the sequence determined in this work is AJ131690.

“…Transposon Tn4371 carries bph genes responsible for the mineralization of BP (Springael et al, 1993 ;Merlin et al, 1997). Merlin et al (1997) observed that in contrast to R. eutropha H16\RP4 : : Tn4371, which grows on minimal medium supplemented with BP as a sole carbon source, HF09, an rpoN mutant of H16 (Friedrich et al, 1981) strains also displayed different growth patterns when streaked (or spread ; not shown) on TRIS-agar plates, where BP was provided as the sole carbon source in the form of crystals stuck to the plate lid.…”

“…which allow Ralstonia metallidurans (formerly Alcaligenes eutrophus ; see Goris et al, 2001) strains such as CH34, and other Ralstonia species, to use biphenyl (BP) as sole carbon and energy source (Springael et al, 1993 ;and unpublished results). The BP catabolic pathway consists of an upper and a lower pathway.…”