Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation

Höfer, Bernd; Eltis, Lindsay D.; Dowling, David N.; Timmis, Kenneth N.

doi:10.1016/0378-1119(93)90345-4

Cited by 170 publications

(128 citation statements)

References 48 publications

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“…Resting cells were lysed with a cracking buffer (Tabor & Richardson, 1985) and proteins were separated by 0?1 % SDS-15 % PAGE as described previously (Hofer et al, 1993). Gels were stained with Coomassie brilliant blue R250 (Sambrook et al, 1989).…”

Section: Methodsmentioning

confidence: 99%

A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities

Kahl

Höfer

2003

Microbiology

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Aromatic-ring-hydroxylating dioxygenases (ARHDOs) are key enzymes in the aerobic bacterial metabolism of aromatic compounds. They are of biotechnological importance as they function as biocatalysts in the stereospecific synthesis of chiral synthons and the degradation of aromatic pollutants. This report describes the development and validation of a system for the rapid isolation and characterization of specific ARHDO activities. The system is based on the identification of ARHDO gene segments that encode the enzymes' major functional determinants, on consensus primers for the direct amplification of such partial genes and on a 'recipient' ARHDO gene cluster for the insertion of the amplified segments. Previously, it has been shown that neither the N-nor the C-terminal portions but only the core region of the large or a-subunit of a class II ARHDO significantly influence substrate and product spectra. On the basis of these observations, consensus primers were designed for the amplification of the gene segment encoding the catalytic core of the large subunit. These primers were tested on 11 bacterial isolates known to metabolize aromatic compounds. In 10 cases, a gene fragment of expected length was amplified. DNA sequencing confirmed similarity to ARHDO a-subunit gene cores. The heterologously well-expressible bphA gene cluster of Burkholderia sp. strain LB400 was modified to facilitate the in-frame insertion of amplified segments. It was used successfully to express the resulting hybrid gene clusters and to form catalytically active chimaeric ARHDOs. The metabolic properties of these enzymes differed significantly from each other and from the parental ARHDO of strain LB400. These results indicate that the system described here can be used to rapidly isolate and functionally characterize ARHDO activities, starting from isolated strains, mixtures of organisms or samples of nucleic acids. Applications of the system range from the recruitment of novel ARHDO activities to an improved characterization of natural ARHDO diversity.

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Section: Methodsmentioning

confidence: 99%

A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities

Kahl

Höfer

2003

Microbiology

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“…Recently, the N-terminal sequences of about ten testosterone-inducible proteins have been determined (Mo$ bus et al, 1997). Some of them show high homology with catabolic enzymes involved in degradation of aromatic compounds, such as BphC, a meta-cleavage enzyme involved in biphenyl degradation (for example see Furukawa et al, 1987 ;Kimbara et al, 1989 ;Hofer et al, 1993).…”

Section: Abbreviation : Km Kanamycinmentioning

confidence: 99%

“…BphC1 from Rhodococcus globerulus P6 (Asturias et al, 1994), Pseudomonas sp. strain KKS102 (Kimbara et al, 1989), Pseudomonas pseudoalcaligenes KF707 (Furukawa et al, 1987) and Burkholderia cepacia LB400 (Hofer et al, 1993) are meta-cleavage enzymes in the biphenyl degradation pathway which utilize 2,3-dihydroxybiphenyl as a sub- The extent of growth was determined at the beginning of the cultivation period and after about 24, 48 and 72 h. The data are the mean of more than three independent experiments.…”

Section: Nucleotide Sequence Of the Meta-cleavage Enzyme Genementioning

confidence: 99%

Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441

et al. 2001

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Comamonas testosteroni metabolizes testosterone as the sole carbon source via a meta-cleavage reaction. A meta-cleavage enzyme gene, tesB, was cloned from C. testosteroni TA441. The deduced N-terminal amino acid sequence of tesB matched that of the purified meta-cleavage enzyme which is induced in TA441 during growth on testosterone as the sole carbon source. The tesBdisrupted mutant did not show growth on testosterone, suggesting that tesB is necessary for TA441 to grow on testosterone. Downstream from tesB, three putative ORFs which encode products also necessary for growth of TA441 on testosterone were identified. The usual substrate of TesB is probably 3,4-dihydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione. Although this compound was not identified in the gene disrupted mutants, accumulation of upstream metabolites of testosterone degradation, 4-androstene-3,17-dione and 1,4-androstadiene-3,17-dione, was shown by TLC analysis.

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“…E. coli BL21(DE3) [pLysS] (Studier, 1991) and Pseudomonas putida KT2442 (Franklin et al, 1983) were used for gene expression. The recombinant expression vectors pAIA50 (Seeger et al, 1995a) and pDD372 (Hofer et al, 1993), harbouring bphA1A-2A3A4BC or bphBC of Burkholderia sp. strain LB400 (Bopp, 1986) respectively ; and pDM10 (McKay et al, 1997), harbouring bphA1A2A3A4 of R. globerulus P6 (Furukawa et al, 1978 ;Asturias et al, 1994), have been described previously.…”

Section: Methodsmentioning

confidence: 99%

“…2i cracking buffer and heated as above. Electrophoresis was carried out in 0n1 % SDS\12 % polyacrylamide gels as described previously (Hofer et al, 1993). Gels were stained with Coomassie brilliant blue R250 (Sambrook et al, 1989 deduced from the crystal structure of a naphthalene dioxygenase (Kauppi et al, 1998 ;Carredano et al, 2000) and from functional studies on BDO variants (Erickson & Mondello, 1993 ;Kimura et al, 1997 ;Mondello et al, 1997).…”

Section: Methodsmentioning

confidence: 99%

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

2002

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Protein engineering by segment exchange was used to distinguish between regions of major and minor influence on the structure of the substrate-binding pocket of a biphenyl dioxygenase (BDO). Eight chimaeric enzyme systems were generated that each consisted of a hybrid hydroxylase α subunit (BphA1) containing segments from Burkholderia sp. strain LB400 and Rhodococcus globerulus P6, and of a hydroxylase β subunit (BphA2), a ferredoxin (BphA3) and a ferredoxin reductase (BphA4) from strain LB400. All hybrid bphA1 genes were expressed at high levels. Seven of the resulting fusion subunits functionally interacted with the other polypeptides of the dioxygenase system to yield catalytically active enzymes. Changes in the regiospecificity of substrate attack, monitored by the formation of seventeen different dioxygenation products obtained from seven chlorobiphenyls, were used to monitor effects of segment exchanges on the structure of the BDO substratebinding site. Exchanges of neither the β subunit nor the N-and C-terminal regions of the α subunit exerted significant influences. All BDO regions that showed major effects on the substrate-binding pocket were located between approximately positions 165 and 395 of the α subunit. Within this part of the enzyme, in addition to segments identified previously, a subregion which is involved in ligation of the mononuclear iron significantly influenced the regiospecificity of substrate dioxygenation. Moreover, the results indicate that the construction of appropriate hybrid genes may be used as a general strategy to overcome problems in obtaining heterologous BDO activities in Escherichia coli or other host organisms.

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Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation

Cited by 170 publications

References 48 publications

A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities

A genetic system for the rapid isolation of aromatic-ring-hydroxylating dioxygenase activities

Meta-cleavage enzyme gene tesB is necessary for testosterone degradation in Comamonas testosteroni TA441

The principal determinants for the structure of the substrate-binding pocket are located within a central core of a biphenyl dioxygenase α subunit

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