Cloning and expression of Acinetobacter calcoaceticus catBCDE genes in Pseudomonas putida and Escherichia coli

Shanley, Mark S.; Neidle, Ellen L.; Parales, Rebecca E.; Ornston, L N

doi:10.1128/jb.165.2.557-563.1986

Cited by 64 publications

(62 citation statements)

References 39 publications

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“…Cell pellets were suspended in 200 l of breaking buffer [50 mM Tris-HCl, 10% glycerol, 5 mM (NH 4 ) 2 SO 4 , 2.5 mM EDTA, 1 mM dithiothreitol (pH 7.5)]. Cell extracts were prepared as described previously (42), and P3,4DO activity was determined spectrophotometrically by measuring the decrease in absorbance at 290 nm (44). Protein concentrations were determined by the method of Bradford (2).…”

Section: Methodsmentioning

confidence: 99%

Diverse Organization of Genes of the β-Ketoadipate Pathway in Members of the Marine Roseobacter Lineage

Buchan¹,

Neidle

Moran³

2004

Appl Environ Microbiol

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Members of the Roseobacter lineage, an ecologically important marine clade within the class ␣-Proteobacteria, harbor genes for the protocatechuate branch of the ␤-ketoadipate pathway, a major catabolic route for lignin-related aromatic compounds. The genes of this pathway are typically clustered, although gene order varies among organisms. Here we characterize genes linked to pcaH and -G, which encode protocatechuate 3,4-dioxygenase, in eight closely related members of the Roseobacter lineage (pairwise 16S rRNA gene sequence identities, 92 to 99%). Sequence analysis of genomic fragments revealed five unique pca gene arrangements. Identical gene organization was found for isolates demonstrating species-level identity (i.e., >99% 16S rRNA gene similarity). In one isolate, six functionally related genes were clustered: pcaQ, pobA, pcaD, pcaC, pcaH, and pcaG. The remaining seven isolates lacked at least one of these genes in their clusters, although the relative order of the remaining genes was preserved. Three genes (pcaC, -H, and -G) were physically linked in all isolates. A highly conserved open reading frame (ORF) was found immediately downstream of pcaG in all eight isolates. Reverse transcription-PCR analysis of RNA from one isolate, Silicibacter pomeroyi DSS-3, provides evidence that this ORF is coexpressed with upstream pca genes. The absence of this ORF in similar bacterial pca gene clusters from diverse microbes suggests a niche-specific role for its protein product in Roseobacter group members. Collectively, these comparisons of bacterial pca gene organization illuminate a complex evolutionary history and underscore the widespread ecological importance of the encoded ␤-ketoadipate pathway.

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Section: Methodsmentioning

confidence: 99%

Diverse Organization of Genes of the β-Ketoadipate Pathway in Members of the Marine Roseobacter Lineage

Buchan¹,

Neidle

Moran³

2004

Appl Environ Microbiol

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“…Acinetobacter sp. strain ADP1 (BD413) and Escherichia coli strains and growth conditions have been described (2,19,20). A benM deletion was made by allelic replacement methods (2).…”

Section: Methodsmentioning

confidence: 99%

Synergistic transcriptional activation by one regulatory protein in response to two metabolites

Bundy

Collier

Hoover

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

129

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BenM is a LysR-type bacterial transcriptional regulator that controls aromatic compound degradation in Acinetobacter sp. ADP1. Here, in vitro transcription assays demonstrated that two metabolites of aromatic compound catabolism, benzoate and cis,cismuconate, act synergistically to activate gene expression. The level of BenM-regulated benA transcription was significantly higher in response to both compounds than the combined levels due to each alone. These compounds also were more effective together than they were individually in altering the DNase I footprint patterns of BenM-DNA complexes. This type of dual-inducer synergy provides great potential for rapid and large modulations of gene expression and may represent an important, and possibly widespread, feature of transcriptional control.

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“…Escherichia coli DH5␣ (GIBCO BRL) was used as a plasmid host. Bacteria were cultured in Luria-Bertani (LB) broth or minimal medium at 37°C as previously described (23,25). Succinate, benzoate, or anthranilate was added as a carbon source to minimal medium at a final concentration of 10, 3, or 2.5 mM, respectively.…”

Section: Methodsmentioning

confidence: 99%

Mutations in catB , the Gene Encoding Muconate Cycloisomerase, Activate Transcription of the Distal ben Genes and Contribute to a Complex Regulatory Circuit in Acinetobacter sp. Strain ADP1

Cosper

Collier²,

Clark³

et al. 2000

J Bacteriol

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Mutants of the bacterium Acinetobacter sp. strain ADP1 were selected to grow on benzoate without the BenM transcriptional activator. In the wild type, BenM responds to benzoate andcis,cis-muconate to activate expression of thebenABCDE operon, which is involved in benzoate catabolism. This operon encodes enzymes that convert benzoate to catechol, a compound subsequently degraded by cat gene-encoded enzymes. In this report, four spontaneous mutants were found to carrycatB mutations that enabled BenM-independent growth on benzoate. catB encodes muconate cycloisomerase, an enzyme required for benzoate catabolism. Its substrate,cis,cis-muconate, is enzymatically produced from catechol by the catA-encoded catechol 1,2-dioxygenase. Muconate cycloisomerase was purified to homogeneity from the wild type and the catB mutants. Each purified enzyme was active, although there were differences in the catalytic properties of the wild type and variant muconate cycloisomerases. Strains with a chromosomalbenA::lacZ transcriptional fusion were constructed and used to investigate how catB mutations affect growth on benzoate. All of the catB mutations increased cis,cis-muconate-activatedben gene expression in strains lacking BenM. A model is presented in which the catB mutations reduce muconate cycloisomerase activity during growth on benzoate, thereby increasing intracellular cis, cis-muconate concentrations. This, in turn, may allow CatM, an activator similar to BenM in sequence and function, to activate ben gene transcription. CatM normally responds to cis,cis-muconate to activate cat gene expression. Consistent with this model, muconate cylcoisomerase specific activities in cell extracts of benzoate-grown catB mutants were low relative to that of the wild type. Moreover, the catechol 1,2-dioxygenase activities of the mutants were elevated, which may result from CatM responding to the altered intracellular levels ofcis,cis-muconate and increasingcatA expression. Collectively, these results support the important role of metabolite concentrations in controlling benzoate degradation via a complex transcriptional regulatory circuit.

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Cloning and expression of Acinetobacter calcoaceticus catBCDE genes in Pseudomonas putida and Escherichia coli

Abstract: This report describes the isolation and preliminary characterization of a 5.0-kilobase-pair (kbp) EcoRI DNA restriction fragment carrying the catBCDE genes from Acinetobacter calcoaceticus. The

Cited by 64 publications

References 39 publications

Diverse Organization of Genes of the β-Ketoadipate Pathway in Members of the Marine Roseobacter Lineage

Diverse Organization of Genes of the β-Ketoadipate Pathway in Members of the Marine Roseobacter Lineage

Synergistic transcriptional activation by one regulatory protein in response to two metabolites

Mutations in catB , the Gene Encoding Muconate Cycloisomerase, Activate Transcription of the Distal ben Genes and Contribute to a Complex Regulatory Circuit in Acinetobacter sp. Strain ADP1

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