Cross-regulation of biphenyl- and salicylate-catabolic genes by two regulatory systems in Pseudomonas pseudoalcaligenes KF707

Pseudomonas pseudoalcaligenes KF707 grows on biphenyl and salicylate as sole sources of carbon. The biphenyl-catabolic (bph) genes are organized as bphR1A1A2(orf3)A3A4BCX0X1X2X3D, encoding the enzymes for conversion of biphenyl to acetyl coenzyme A. In this study, the salicylate-catabolic (sal) gene cluster encoding the enzymes for conversion of salicylate to acetyl coenzyme A were identified 6.6-kb downstream of the bph gene cluster along with a second regulatory gene, bphR2. Both the bph and sal genes were cross-regulated positively and/or negatively by the two regulatory proteins, BphR1 and BphR2, in the presence or absence of the effectors. The BphR2 binding sequence exhibits homology with the NahR binding sequences in various naphthalene-degrading bacteria. Based on previous studies and the present study we propose a new regulatory model for biphenyl and salicylate catabolism in strain KF707.     

Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon).

bph operons coding for biphenyl-polychlorinated biphenyl degradation in Pseudomonas pseudoalcaligenes KF707 and Pseudomonas putida KF715 and tod operons coding for toluene-benzene metabolism in P. putida F1 are very similar in gene organization as well as size and homology of the corresponding enzymes (G. J. Zylstra and D. T. Gibson, J. Biol. Chem. 264:14940-14946, 1989; K. Taira, J. Hirose, S. Hayashida, and K. Furukawa, J. Biol. Chem. 267:4844-4853, 1992), despite their discrete substrate ranges for metabolism. The gene components responsible for substrate specificity between the bph and tod operons were investigated. The large subunit of the terminal dioxygenase (encoded by bphA1 and todC1) and the ring meta-cleavage compound hydrolase (bphD and todF) were critical for their discrete metabolic specificities, as shown by the following results. (i) Introduction of todC1C2 (coding for the large and small subunits of the terminal dioxygenase in toluene metabolism) or even only todC1 into biphenyl-utilizing P. pseudoalcaligenes KF707 and P. putida KF715 allowed them to grow on toluene-benzene by coupling with the lower benzoate meta-cleavage pathway. Introduction of the bphD gene (coding for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase) into toluene-utilizing P. putida F1 permitted growth on biphenyl. (ii) With various bph and tod mutant strains, it was shown that enzyme components of ferredoxin (encoded by bphA3 and todB), ferredoxin reductase (bphA4 and todA), and dihydrodiol dehydrogenase (bphB and todD) were complementary with one another. (iii) Escherichia coli cells carrying a hybrid gene cluster of todClbphA2A3A4BC (constructed by replacing bphA1 with todC1) converted toluene to a ring meta-cleavage 2-hydroxy-6-oxo-hepta-2,4-dienoic acid, indicating that TodC1 formed a functional multicomponent dioxygenase associated with BphA2 (a small subunit of the terminal dioxygenase in biphenyl metabolism), BphA3, and BphA4.     

Efficient degradation of trichloroethylene by a hybrid aromatic ring dioxygenase.

Engineering of hybrid gene clusters between the toluene metabolic tod operon and the biphenyl metabolic bph operon greatly enhanced the rate of biodegradation of trichloroethylene. Escherichia coli cells carrying a hybrid gene cluster composed of todC1 (the gene encoding the large subunit of toluene terminal dioxygenase in Pseudomonas putida F1), bphA2 (the gene encoding the small subunit of biphenyl terminal dioxygenase in Pseudomonas pseudoalcaligenes KF707), bphA3 (the gene encoding ferredoxin in KF707), and bphA4 (the gene encoding ferredoxin reductase in KF707) degraded trichloroethylene much faster than E. coli cells carrying the original toluene dioxygenase genes (todC1C2BA) or the original biphenyl dioxygenase genes (bphA1A2A3A4).     

Molecular cloning of salicylate hydroxylase genes from Pseudomonas cepacia and Pseudomonas putida

The sal gene encoding Pseudomonas cepacia salicylate hydroxylase was cloned and the sal encoding Pseudomonas putida salicylate hydroxylase was subcloned into plasmid vector pRO2317 to generate recombinant plasmids pTK3 and pTK1, respectively. Both cloned genes were expressed in the host Pseudomonas aeruginosa PAO1. The parental strain can utilize catechol, a product of the salicylate hydroxylase-catalyzed reaction, but not salicylate as the sole carbon source for growth due to a natural deficiency of salicylate hydroxylase. The pTK1- or pTK3-transformed P. aeruginosa PAO1, however, can be grown on salicylate as the sole carbon source and exhibited activities for the cloned salicylate hydroxylase in crude cell lysates. In wild-type P. cepacia as well as in pTK1- or pTK3-transformed P. aeruginosa PAO1, the presence of glucose in addition to salicylate in media resulted in lower efficiencies of sal expression P. cepacia apparently can degrade salicylate via the meta cleavage pathway which, unlike the plasmid-encoded pathway in P. putida, appears to be encoded on chromosome. As revealed by DNA cross hybridizations, the P. cepacia hsd and ht genes showed significant homology with the corresponding plasmid-borne genes of P. putida but the P. cepacia sal was not homologous to the P. putida sal. Furthermore, polyclonal antibodies developed against purified P. cepacia salicylate hydroxylase inactivated the cloned P. cepacia salicylate hydroxylase but not the cloned P. putida salicylate hydroxylase in P. aeruginosa PAO1. It appears that P. cepacia and P. putida salicylate hydroxylases, being structurally distinct, were probably derived through convergent evolution.     

Occurrence of Tn4371-related mobile elements and sequences in (chloro)biphenyl-degrading bacteria

Tn4371, a 55-kb transposable element involved in the degradation and biphenyl or 4-chlorobiphenyl identified in Ralstonia eutropha A5, displays a modular structure including a phage-like integrase gene (int), a Pseudomonas-like (chloro)biphenyl catabolic gene cluster (bph), and RP4- and Ti-plasmid-like transfer genes (trb) (C. Merlin, D. Springael, and A. Toussaint, Plasmid 41:40-54, 1999). Southern blot hybridization was used to examine the presence of different regions of Tn4371 in a collection of (chloro)biphenyl-degrading bacteria originating from different habitats and belonging to different bacterial genera. Tn4371-related sequences were never detected on endogenous plasmids. Although the gene probes containing only bph sequences hybridized to genomic DNA from most strains tested, a limited selection of strains, all beta-proteobacteria, displayed hybridization patterns similar to the Tn4371 bph cluster. Homology between Tn4371 and DNA of two of those strains, originating from the same area as strain A5, extended outside the catabolic genes and covered the putative transfer region of Tn4371. On the other hand, none of the (chloro)biphenyl degraders hybridized with the outer left part of Tn4371 containing the int gene. The bph catabolic determinant of the two strains displaying homology to the Tn4371 transfer genes and a third strain isolated from the A5 area could be mobilized to a R. eutropha recipient, after insertion into an endogenous or introduced IncP1 plasmid. The mobilized DNA of those strains included all Tn4371 homologous sequences previously identified in their genome. Our observations show that the bph genes present on Tn4371 are highly conserved between different (chloro)biphenyl-degrading hosts, isolated globally but belonging mainly to the beta-proteobacteria. On the other hand, Tn4371-related mobile elements carrying bph genes are apparently only found in isolates from the environment that provided the Tn4371-bearing isolate A5.     

Total degradation of pentachloroethane by an engineered Alcaligenes strain expressing a modified camphor monooxygenase and a hybrid dioxygenase

We engineered biphenyl-degrading Alcaligenes sp. strain KF711 for total degradation of pentachloroethane (PCA), which expresses a modified camphor monooxygenase and a hybrid dioxygenase consisting of TodC1 (a large subunit of toluene dioxygenase of Pseudomonas putida F1) and BphA2-BphA3-pbhA4 (a small subunit, ferredoxin and ferredoxin reductase of biphenyl dioxygenase, respectively, in strain KF707). Modified camphor monooxygenase genes (camCAB) were supplied as a plasmid and the todC1 gene was integrated within the chromosomal bph gene cluster by a single crossover recombination. The resultant strain KF711S-3cam dechlorinated PCA to trichloroethene by the action of the modified camphor monooxygenase under anaerobic conditions. The same strain subsequently degraded trichloroethene formed oxidatively by the action of the Tol-Bph hybrid dioxygenase under aerobic conditions. Thus sequential anaerobic and aerobic treatments of the KF711S-3cam resting cells resulted in efficient and total degradation of PCA.     

Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria.

We cloned the entire bphABCD genes encoding degradation of biphenyl and polychlorinated biphenyls to benzoate and chlorobenzoates from the chromosomal DNA of Pseudomonas putida KF715. The nucleotide sequence revealed two open reading frames corresponding to the bphC gene encoding 2,3-dihydroxybiphenyl dioxygenase and the bphD gene encoding 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (ring-meta-cleavage compound) hydrolase.     

Cloning and expression of the catA and catBC gene clusters from Pseudomonas aeruginosa PAO.

A 9.9-kilobase (kb) BamHI restriction endonuclease fragment encoding the catA and catBC gene clusters was selected from a gene bank of the Pseudomonas aeruginosa PAO1c chromosome. The catA, catB, and catC genes encode enzymes that catalyze consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catA, catechol-1,2-dioxygenase (EC 1.13.11.1); catB, muconate lactonizing enzyme (EC 5.5.1.1); and catC, muconolactone isomerase (EC 5.3.3.4). A recombinant plasmid, pRO1783, which contains the 9.9-kb BamHI restriction fragment complemented P. aeruginosa mutants with lesions in the catA, catB, or catC gene; however, this fragment of chromosomal DNA did not contain any other catabolic genes which had been placed near the catA or catBC cluster based on cotransducibility of the loci. Restriction mapping, deletion subcloning, and complementation analysis showed that the order of the genes on the cloned chromosomal DNA fragment is catA, catB, catC. The catBC genes are tightly linked and are transcribed from a single promoter that is on the 5' side of the catB gene. The catA gene is approximately 3 kb from the catBC genes. The cloned P. aeruginosa catA, catB, and catC genes were expressed at basal levels in blocked mutants of Pseudomonas putida and did not exhibit an inducible response. These observations suggest positive regulation of the P. aeruginosa catA and catBC cluster, the absence of a positive regulatory element from pRO1783, and the inability of the P. putida regulatory gene product to induce expression of the P. aeruginosa catA, catB, and catC genes.     

Adrenoleukodystrophy: a complex chromosomal rearrangement in the Xq28 red/green-color-pigment gene region indicates two possible gene localizations.

We have characterized a complex chromosomal rearrangement in band Xq28, in an adrenoleukodystrophy patient who also has blue-cone monochromacy. A 130-kb region upstream from the color-vision pigment genes was isolated as yeast artificial chromosome or cosmid clones. Another Xq28 sequence, not included in the above region, was obtained by cloning a deletion breakpoint from the patient. Using probes derived from the cloned sequences, we have shown that the rearrangement affects the color-pigment genes and includes two deletions, most likely separated by a large (greater than 110-kb) inversion. One deletion encompasses part of the pigment gene cluster and 33 kb of upstream sequences and accounts for the patient's blue-cone monochromacy. If this rearrangement also caused ALD, the disease gene would be expected to lie within or close to one of the deletions. However, deletions were not detected in a 50-kb region upstream of the red-color-pigment gene in 81 other ALD patients. Two CpG islands were mapped, at 46 and 115 kb upstream from the pigment genes.     

Characterization and functional analysis of a novel gene cluster involved in biphenyl degradation in Rhodococcus sp. strain R04

AIMS: Isolation of the genes relative to PCB biodegradation and identification of the bph gene function in Rhodococcus sp. R04. METHODS AND RESULTS: A 8.7-kb fragment carrying the biphenyl catabolic genes bphABCD was isolated from the gene library in Rhodococcus sp. R04. Based on the deduced amino acid sequence homology, seven bph genes, bphA1A2A3A4, bphB, bphC and bphD, were thought to be responsible for the initial four steps of biphenyl degradation. In Escherichia coli, BphA exhibited poor activity for biphenyl transformation, and BphB, BphC and BphD were found to be catalytically active towards 2,3-dihydro-2,3-dihydroxybiphenyl, 2,3-dihydroxybiphenyl and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate, respectively (activities of 50, 8.1 and 2.4 micromol l(-1) min(-1) mg(-1)). SDS-PAGE analysis indicated that the sizes of bphA1A2A3A4, bphB, bphC and bphD gene products were 49, 19, 14, 47, 32, 30 and 31 kDa, respectively. After disruption of bph genes, the bphA1 mutants lost the ability to grow on biphenyl, the bphB and bphD mutants were able to transform a little of biphenyl, but hardly grew on biphenyl. CONCLUSION: The cloned bph genes indeed play an important role in the biphenyl catabolism in this strain. SIGNIFICANCE AND IMPACT OF THE STUDY: This bph gene organization in Rhodococcus sp. R04 differs from that of other biphenyl degraders reported previously, indicating it is a novel type of bph gene cluster. Analysis of the phylogenetic tree suggested that BphA1 and BphA2 in Rhodococcus sp. R04 had a different evolutionary relationship with those in the other PCB degraders.     

Molecular cloning and characterization of catechol 2,3-dioxygenases from biphenyl/polychlorinated biphenyls-degrading bacteria.

Catechol 2,3-dioxygenases were cloned from Alcaligenes sp. KF711, Pseudomonas putida KF715, and Achromobacter xylosoxidans KF701 which are biphenyl/polychlorinated biphenyls-degrading bacteria. All of the cloned enzymes were purified by preparative polyacrylamide gel electrophoresis (PAGE). The purified catechol 2,3-dioxygenases were significantly different from one another in ring-fission activities to catechol and its derivatives. The catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited higher ring-fission activity to 4-chlorocatechol than those from P. putida KF715 and A. xylosoxidans KF701. In electrophoretic mobilities, the three enzymes were different from one another on nondenaturing PAGE but the same on SDS-PAGE.     

The biphenyl- and 4-chlorobiphenyl-catabolic transposon Tn4371, a member of a new family of genomic islands related to IncP and Ti plasmids

The nucleotide sequence of the biphenyl catabolic transposon Tn4371 has been completed and analyzed. It confirmed that the element has a mosaic structure made of several building blocks. In addition to previously identified genes coding for a tyrosine recombinase related to phage integrases and for biphenyl degradation enzymes very similar to those of Achromobacter georgiopolitanum KKS102, Tn4371 carries many plasmid-related genes involved in replication, partition, and other, as-yet-unknown, plasmid functions. One gene cluster contains most of the genes required to express a type IV secretion-mating pair formation apparatus coupled with a TraG ATPase, all of which are related to those found on IncP and Ti plasmids. Orthologues of all Tn4371 plasmid-related genes and of the tyrosine recombinase gene were found, with a very similar organization, in the chromosome of Ralstonia solanacearum and on the yet-to-be-determined genomic sequences of Erwinia chrysanthemi and Azotobacter vinelandii. In each of these chromosomal segments, conserved segments were separated by different groups of genes, which also differed from the Tn4371 bph genes. The conserved blocks of genes were also identified, in at least two copies, in the chromosome of Ralstonia metallidurans CH34. Tn4371 thus appears to represent a new family of potentially mobile genomic islands with a broad host range since they reside in a wide range of soil proteobacteria, including plant pathogens.     

Polychlorinated biphenyl/biphenyl degrading gene clusters in Rhodococcus sp. K37, HA99, and TA431 are different from well-known bph gene clusters of Rhodococci

Four kinds of polychlorinated biphenyl (PCB)-degrading Rhodococcus sp. (TA421, TA431, HA99, and K37) have been isolated from termite ecosystem and under alkaline condition. The bph gene cluster involved in the degradation of PCB/biphenyl has been analyzed in strain TA421. This gene cluster was highly homologous to bph gene clusters in R. globerulus P6 and Rhodococcus sp. RHA1. In this study, we cloned and analyzed the bph gene cluster essential to PCB/biphenyl degradation from R. rhodochrous K37. The order of the genes and the sequence were different in K37 than in P6, RHA1, and TA421. The bphC8(K37) gene was more homologous to the meta-cleavage enzyme involved in phenanthrene metabolism than bphC genes involved in biphenyl metabolism. Two other Rhodococcus strains (HA99 and TA431) had PCB/biphenyl degradation gene clusters similar to that in K37. These findings suggest that these bph gene clusters evolved separately from the well-known bph gene clusters of PCB/biphenyl degraders.     

Gene-specific transposon mutagenesis of the biphenyl/polychlorinated biphenyl-degradation-controlling bph operon in soil bacteria.

A transposon, Tn5-B21, was gene-specifically inserted into the chromosomal biphenyl/polychlorinated biphenyl-catabolic operon (bph operon) of soil bacteria. The cloned bphA, bphB and bphC genes of Pseudomonas pseudoalcaligenes KF707, coding for conversion of biphenyl into a ring meta-cleavage product (2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid), carried random insertions of Tn5-B21. The mutagenized bphABC DNA, carried by a suicide plasmid, was introduced back into the parent strain KF707, resulting in the appearance of gene-specific transposon mutants by double crossover homologous recombination: the bphA::Tn5-B21 mutant did not attack 4-chlorobiphenyl, the bphB::Tn5-B21 mutant accumulated dihydrodiol, and the bphC::Tn5-B21 mutant produced dihydroxy compound. Gene-specific transposon mutants of the bph operon were also obtained for some other biphenyl-utilizing strains which possess bph operons nearly identical to that of KF707.     

Molecular Cloning and Physical Mapping of the Daptomycin Gene Cluster from Streptomyces roseosporus

The daptomycin biosynthetic gene cluster of Streptomyces roseosporus was analyzed by Tn5099 mutagenesis, molecular cloning, partial DNA sequencing, and insertional mutagenesis with cloned segments of DNA. The daptomycin biosynthetic gene cluster spans at least 50 kb and is located about 400 to 500 kb from one end of the ~7,100-kb linear chromosome. We identified two peptide synthetase coding regions interrupted by a 10- to 20-kb region that may encode other functions in lipopeptide biosynthesis.