Cloning and expression in Escherichia coli of Acinetobacter calcoaceticus genes for benzoate degradation

Neidle, Ellen L.; Shapiro, Mary K.; Ornston, L N

doi:10.1128/jb.169.12.5496-5503.1987

Cited by 80 publications

(73 citation statements)

References 48 publications

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“…This microbe has genes in the order benP benK benM benABDE (4,26). Biochemical evidence indicates that the Acinetobacter benA, -B, and -C genes encode benzoate 1,2-dioxygenase and the benD gene encodes 2-hydro-1,2-dihydroxybenzoate dehydrogenase (39). The Acinetobacter benK gene encodes a benzoate transporter (5).…”

Section: Discussionmentioning

confidence: 99%

BenR, a XylS Homologue, Regulates Three Different Pathways of Aromatic Acid Degradation in Pseudomonas putida

2000

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Pseudomonas putida converts benzoate to catechol using two enzymes that are encoded on the chromosome and whose expression is induced by benzoate. Benzoate also binds to the regulator XylS to induce expression of the TOL (toluene degradation) plasmid-encodedmeta pathway operon for benzoate and methylbenzoate degradation. Finally, benzoate represses the ability of P. putida to transport 4-hydroxybenzoate (4-HBA) by preventing transcription of pcaK, the gene encoding the 4-HBA permease. Here we identified a gene, benR, as a regulator of benzoate, methylbenzoate, and 4-HBA degradation genes. AbenR mutant isolated by random transposon mutagenesis was unable to grow on benzoate. The deduced amino acid sequence of BenR showed high similarity (62% identity) to the sequence of XylS, a member of the AraC family of regulators. An additional seven genes located adjacent to benR were inferred to be involved in benzoate degradation based on their deduced amino acid sequences. ThebenABC genes likely encode benzoate dioxygenase, andbenD likely encodes 2-hydro-1,2-dihydroxybenzoate dehydrogenase. benK and benF were assigned functions as a benzoate permease and porin, respectively. The possible function of a final gene, benE, is not known.benR activated expression of a benA-lacZreporter fusion in response to benzoate. It also activated expression of a meta cleavage operon promoter-lacZ fusion inserted in an E. coli chromosome. Third, benRwas required for benzoate-mediated repression of pcaK-lacZfusion expression. The benA promoter region contains a direct repeat sequence that matches the XylS binding site previously defined for the meta cleavage operon promoter. It is likely that BenR binds to the promoter region of chromosomal benzoate degradation genes and plasmid-encoded methylbenzoate degradation genes to activate gene expression in response to benzoate. The action of BenR in repressing 4-HBA uptake is probably indirect.

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Section: Discussionmentioning

confidence: 99%

BenR, a XylS Homologue, Regulates Three Different Pathways of Aromatic Acid Degradation in Pseudomonas putida

2000

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“…In the wellcharacterized Acinetobacter sp. ADP1, a close physical association between the ben and cat gene clusters is evident, but the gene clusters are still distinctly separate (Collier et al, 1998 ;Neidle et al, 1987). Although the cbe and cat genes are also clustered in NK8, there are significant differences between ADP1 and NK8 in terms of the organization of the corresponding genes in the cluster.…”

Section: The Convergent Organization Of the Cbe-cat Gene Clustermentioning

confidence: 99%

“…(Haak et al, 1995), X79076 ; XylXYZ (toluate 1,2-dioxygenase) and XylL (toluate diol dehydrogenase) of Pseudomonas putida mt-2 TOL plasmid pWW0 , M64747 ; BenABC (benzoate 1,2-dioxygenase) and BenD (DHB dehydrogenase) of Acinetobacter, sp. ADP1 (Neidle et al, 1987), AF009224 ; AntABC (anthranilate dioxygenase) of Acinetobacter sp. ADP1 (Bundy et al, 1998), AF071556 ; and TftAB (2,4,5-trichlorophenoxyacetate oxygenase) of Burkholderia cepacia AC1100 (Danganan et al, 1994) Nucleotide sequence analysis of this 12n5 kb DNA region revealed the presence of 9 ORFs.…”

Section: Cloning and Sequencing Of The Chlorobenzoate Dioxygenase Genesmentioning

confidence: 99%

The chlorobenzoate dioxygenase genes of Burkholderia sp. strain NK8 involved in the catabolism of chlorobenzoates

et al. 2001

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Burkholderia sp. NK8 grows abundantly on 3-chlorobenzoate (3CB), 4-chlorobenzoate (4CB) and benzoate. The genes encoding the oxidation of (chloro)benzoates (cbeABCD) and catechol (catA, catBC), the LysR-type regulatory gene cbeR and the gene cbeE with unknown function, all of which form a single cluster in NK8, were cloned and analysed. The protein sequence of chlorobenzoate 1,2-dioxygenase (CbeABC) is 50-65 % identical to the benzoate dioxygenase (BenABC) of Acinetobacter sp. ADP1, toluate dioxygenase (XylXYZ) of the TOL plasmid pWW0 and 2-halobenzoate dioxygenase (CbdABC) of Burkholderia cepacia 2CBS. Disruption of the cbeA gene resulted in the simultaneous loss of the ability to grow on benzoate and monochlorobenzoates, indicating the involvement of the cbeABCD genes in the degradation of these aromatics. The cbeABCD genes are preceded by catA, the gene for catechol dioxygenase. lacZ transcriptional fusion studies in Pseudomonas putida showed that catA and cbeA are co-expressed under the positive control of cbeR, a LysR-type transcriptional regulatory gene. The cbeA ::lacZ transcriptional fusion studies showed that the inducers of the genes are 3CB, 4CB, benzoate and probably cis,cis-muconate. On the other hand, 2-chlorobenzoate (2CB) did not activate the expression of the genes. The chlorobenzoate dioxygenase was able to transform 2CB, 3CB, 4CB and benzoate at considerable rates. 2CB yielded both catechol and 3-chlorocatechol (3CC), and 3CB gave rise to 4-chlorocatechol and 3CC as the major and minor intermediate products, respectively, indicating that the NK8 dioxygenase lacks absolute regiospecificity. The absence of growth of NK8 on 2CB, despite its considerable degradation activity against 2CB, is apparently due to the inability of CbeR to recognize 2CB as an inducer of the expression of the cbe genes.

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“…In the following plasmid descriptions, the restriction endonuclease site positions correspond to the 15,923-nucleotide sequence of the ben-cat region in GenBank, accession number M76991. A. calcoaceticus DNA on plasmid pIB1381 (22) was deleted between the SmaI and SspI sites, positions 4,416 and 5,983, respectively, to generate pIGG21. This deletion eliminated the 5Ј end of the catA gene and part of an open reading frame upstream of catA (25).…”

Section: Methodsmentioning

confidence: 99%

Novel nuclear magnetic resonance spectroscopy methods demonstrate preferential carbon source utilization by Acinetobacter calcoaceticus

Gaines¹,

Smith²,

Neidle³

1996

J Bacteriol

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Novel nuclear magnetic resonance spectroscopy techniques, designated metabolic observation, were used to study aromatic compound degradation by the soil bacterium Acinetobacter calcoaceticus. Bacteria which had been rendered spectroscopically invisible by growth with deuterated ( 2 H) medium were used to inoculate cultures in which natural-abundance 1 H hydrogen isotopes were provided solely by aromatic carbon sources in an otherwise 2 H medium. Samples taken during the incubation of these cultures were analyzed by proton nuclear magnetic resonance spectroscopy, and proton signals were correlated with the corresponding aromatic compounds or their metabolic descendants. This approach allowed the identification and quantitation of metabolites which accumulated during growth. This in vivo metabolic monitoring facilitated studies of catabolism in the presence of multiple carbon sources, a topic about which relatively little is known. A. calcoaceticus initiates aromatic compound dissimilation by forming catechol or protocatechuate from a variety of substrates. Degradation proceeds via the ␤-ketoadipate pathway, comprising two discrete branches that convert catechol or protocatechuate to tricarboxylic acid cycle intermediates. As shown below, when provided with several carbon sources simultaneously, all degraded via the ␤-ketoadipate pathway, A. calcoaceticus preferentially degraded specific compounds. For example, benzoate, degraded via the catechol branch, was consumed in preference to p-hydroxybenzoate, degraded via the protocatechuate branch, when both compounds were present. To determine if this preference were governed by metabolites unique to catechol degradation, pathway mutants were constructed. Studies of these mutants indicated that the product of catechol ring cleavage, cis,cis-muconate, inhibited the utilization of p-hydroxybenzoate in the presence of benzoate. The accumulation of high levels of cis,cis-muconate also appeared to be toxic to the cells.Despite extensive research on bacterial hydrocarbon metabolism, little is known about how the presence of multiple carbon sources influences the degradation of an individual compound. In these studies, a new nuclear magnetic resonance (NMR) spectroscopy technique was used to monitor the catabolism of aromatic compound mixtures by the soil bacterium Acinetobacter calcoaceticus. This technique, designated metabolic observation NMR (MO-NMR), is based on the principle that NMR spectrometers will not detect deuterated ( 2 H) compounds when tuned to proton ( 1 H) frequencies (11,14,20,32). As described in this report, A. calcoaceticus was grown in fully deuterated medium, thereby rendering the bacterial cultures spectroscopically invisible. Mixtures of 1 H aromatic compounds were added to the deuterated cultures as sources of carbon and energy, and samples were taken at specific times. NMR spectra of the samples were used to monitor the metabolic fates of the 1 H compounds over time; these spectra provided information about the identity and the accumulation of meta...

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Cloning and expression in Escherichia coli of Acinetobacter calcoaceticus genes for benzoate degradation

Cited by 80 publications

References 48 publications

BenR, a XylS Homologue, Regulates Three Different Pathways of Aromatic Acid Degradation in Pseudomonas putida

BenR, a XylS Homologue, Regulates Three Different Pathways of Aromatic Acid Degradation in Pseudomonas putida

The chlorobenzoate dioxygenase genes of Burkholderia sp. strain NK8 involved in the catabolism of chlorobenzoates

Novel nuclear magnetic resonance spectroscopy methods demonstrate preferential carbon source utilization by Acinetobacter calcoaceticus

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