Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria

Selvaraj, Gopalan; Iyer, V. N.

doi:10.1128/jb.156.3.1292-1300.1983

Cited by 203 publications

(73 citation statements)

References 39 publications

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“…1) by the strain DOC21 (Merino et al, 2013). To identify steroid degradation (std) genes we used the Tn5 to generate a library of transposon mutants (Selvaraj and Iyer, 1983;Herrero et al, 1990), from which we isolated 54 mutants (Fig. 2) unable to grow on cholate or other bile acids as the sole organic sub-strate.…”

Section: Identification Of Genes Involved In Steroid Degradation In Pmentioning

confidence: 99%

Functional analyses of three acyl‐CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21

Barrientos

Merino

Casabon

et al. 2014

Environmental Microbiology

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Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

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Section: Identification Of Genes Involved In Steroid Degradation In Pmentioning

confidence: 99%

Functional analyses of three acyl‐CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21

Barrientos

Merino

Casabon

et al. 2014

Environmental Microbiology

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“…The isolation of mutants of P: putidu unable to catabolize PhAcOH or 4HOPhAcOH was carried out by the mating procedure as previously reported [20,211. Cells grown on the mating filters were resuspended in 3 ml Luria-Bertani medium and seeded on plates containing Luria-Bertani medium 20 pg/ml rifampicin and 25 pg/ml kanamycin monosulfate and incubated 36-48 h at 30°C.…”

Section: Mutagenesis With Transposon Tn5mentioning

confidence: 99%

Catabolism of aromatics in Pseudomonas putida U

Olivera

Reglero

Martı́nez-Blanco

et al. 1994

European Journal of Biochemistry

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Phenylacetic acid (PhAcOH) and 4‐hydroxyphenylacetic acid (4HOPhAcOH) are catabolized in Pseudomonas putida U through two different pathways. Mutation carried out with the transposon Tn5 has allowed the isolation of several mutants which, unlike the parental strain, are unable to grow in chemically defined medium containing either PhAcOH or 4HOPhAcOH as the sole carbon source. Analysis of these strains showed that the ten mutants unable to grow in PhAcOH medium grew well in the one containing 4HOPhAcOH, whereas four mutants handicapped in the degradation of 4HOPhAcOH were all able to utilize PhAcOH. These results show that the degradation of these two aromatic compounds in P. putida U is not carried out as formerly believed through a single linear and common pathway, but by two unrelated routes. Identification of the blocked point in the catabolic pathway and analysis of the intermediate accumulated, showed that the mutants unable to utilize 4HOPhAcOH corresponded to two different groups: those blocked in the gene encoding 4‐hydroxyphenylacetic acid‐3‐hydroxylase; and those blocked in the gene encoding homoprotocatechuate‐2,3‐dioxygenase. Mutants unable to use PhAcOH as the sole carbon source have been also classified into two different groups: those which contain a functional PhAc‐CoA ligase protein; and those lacking this enzyme activity.

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“…P. acidovorans strain M3GY [4] and Escherichia coli C600 pGS9: :Tn5 [6] have been described elsewhere.…”

Section: Strainsmentioning

confidence: 99%

The use of mutants to discern the degradation pathway of 3,4′-dichlorobiphenyl in Pseudomonas acidovorans M3GY

Cullar¹

2002

FEMS Microbiology Ecology

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Pseudomonas acidovorans strain M3GY is a recombinant bacterium with the novel ability to utilize 3,4P-dichlorobiphenyl (3,4P-DCBP) as a growth substrate. This strain was previously shown to oxidize the 3P-ring and produce 4-chlorobenzoate (4-CBa) through the standard biphenyl pathway. Although 4-CBa was metabolized through the meta-fission pathway, the genes encoding the orthochlorocatechol pathway were retained. Nevertheless, neither 3-CBa nor 3-chlorocatechol (3-CC) were detected as intermediates during metabolism of 3,4P-DCBP, nor was 4-CBa utilized as a sole carbon source, by this strain. Two mutant strains were produced to resolve these anomalies. Mutant strain M3GY-9 was obtained by Tn5 insertion and selection for growth on biphenyl, and was unable to grow on 3-CBa. It accumulated 3-CC from 3,4P-DCBP when grown on biphenyl. Thus, M3GY attacks both rings, and the failure to isolate 3-CBa or 3-CC is due to rapid turnover by the enzymes of the ortho-chlorocatechol pathway in the wild-type strain. Mutant strain M3GY-1 grew on 4-CBa, unlike the wild-type strain. Washed cell suspensions of mutant strain MEGY-1 consumed 4-fluorobenzoate, 4-bromobenzoate, and, to a lesser extent 4-iodobenzoate. The mutation that resulted in the ability of mutant strain M3GY-I to effectively utilize 4-CBa as a sole carbon source was associated with a transport mechanism. ß 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Microbiological Societies.

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Suicide plasmid vehicles for insertion mutagenesis in Rhizobium meliloti and related bacteria

Cited by 203 publications

References 39 publications

Functional analyses of three acyl‐CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21

Functional analyses of three acyl‐CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21

Catabolism of aromatics in Pseudomonas putida U

The use of mutants to discern the degradation pathway of 3,4′-dichlorobiphenyl in Pseudomonas acidovorans M3GY

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