Characterization of the protocatechuic acid catabolic gene cluster from Streptomyces sp. strain 2065 - PubMed (original) (raw)

Characterization of the protocatechuic acid catabolic gene cluster from Streptomyces sp. strain 2065

S G Iwagami et al. Appl Environ Microbiol. 2000 Apr.

Abstract

Protocatechuate 3,4-dioxygenase (EC 1.13.11.3) catalyzes the ring cleavage step in the catabolism of aromatic compounds through the protocatechuate branch of the beta-ketoadipate pathway. A protocatechuate 3,4-dioxygenase was purified from Streptomyces sp. strain 2065 grown in p-hydroxybenzoate, and the N-terminal sequences of the beta- and alpha-subunits were obtained. PCR amplification was used for the cloning of the corresponding genes, and DNA sequencing of the flanking regions showed that the pcaGH genes belonged to a 6. 5-kb protocatechuate catabolic gene cluster; at least seven genes in the order pcaIJFHGBL appear to be transcribed unidirectionally. Analysis of the cluster revealed the presence of a pcaL homologue which encodes a fused gamma-carboxymuconolactone decarboxylase/beta-ketoadipate enol-lactone hydrolase previously identified in the pca gene cluster from Rhodococcus opacus 1CP. The pcaIJ genes encoded proteins with a striking similarity to succinyl-coenzyme A (CoA):3-oxoacid CoA transferases of eukaryotes and contained an indel which is strikingly similar between high-G+C gram-positive bacteria and eukaryotes.

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Figures

FIG. 1

FIG. 1

The protocatechuate and catechol branches of the β-ketoadipate pathway. Gene products catalyzing reactions in the pathway are given in parentheses. For the reactions common to both branches, some bacteria possess only a single set of genes, while others have separate pca and cat genes for one or more of the steps.

FIG. 2

FIG. 2

SDS-PAGE and subunit molecular weight determination of protocatechuate 3,4-dioxygenase. SDS-PAGE was performed as described in Materials and Methods. The α and β subunits of the streptomycete dioxygenase are represented as solid circles. A, bovine serum albumin (molecular size, 68 kDa); B, ovalbumin (43 kDa); C, carbonic anhydrase (29 kDa); D, β-lactoglobulin (18.4 kDa); E, lysozyme (14.3 kDa). The molecular sizes of the α and β subunits of the streptomycete dioxygenase are 24.2 and 33.7 kDa, respectively.

FIG. 3

FIG. 3

Native molecular mass determination of protocatechuate 3,4-dioxygenase by Superose 6 column chromatography. Gel filtration was performed as described in Materials and Methods. A, thyroglobulin (molecular mass, 670 kDa); B, immunoglobulin G (150 kDa); C, ovalbumin (44 kDa); D, myoglobin (17 kDa); E, vitamin B12 (1.35 kDa). The streptomycete dioxygenase is represented as a filled circle (158 kDa).

FIG. 4

FIG. 4

Effect of pH on the activity of protocatechuate 3,4-dioxygenase. The following buffers were used in each pH region by addition of protocatechuate 3,4-dioxygenase to the cuvette in the presence of the usual Tris-HCl (pH 8.5) buffer: pH 6.5 to 7.5, phosphate; pH 8.0 to 9.0, Tris-HCl; pH 9.5, carbonate-bicarbonate.

FIG. 5

FIG. 5

Gene organization of pca gene clusters from different bacteria. The gene cluster structures for Streptomyces sp. strain 2065 and Streptomyces coelicolor are identical; therefore, this gene cluster has been labeled with the genus name Streptomyces alone. Gene designations are given in Table 1. C refers to pcaC, encoding γ-carboxymuconolactone decarboxylase, while pcaD encodes β-ketpadipate enol-lactone hydrolase. pcaU, pcaR, and pcaQ are regulatory genes described in the text. pcaK is a proposed aromatic acid transport gene, and pcaT is proposed to encode a β-ketoadipate transporter. pobA encodes _p_-hydroxybenxzoate hydroxylase, while pobR is a regulatory gene.

FIG. 6

FIG. 6

Southern blot of streptomycete genomic digests hybridized with an 800-bp pcaH probe. All DNA was digested with _Sal_I. Lane 1, the cloned 4.5-kb _Sal_I fragment that contained pcaH; lane 2, Streptomyces sp. strain 2065; lane 3, S. viridosporus; lane 4, Streptomyces sp. strain D7; lane 5, S. setonii, lane 6, S. lavendulae; lane 7, S. hygroscopicus; lane 8, S. griseus; lane 9, S. griseolus; lane 10, S. coelicolor A(3)2; lane 11, S. baclius; lane 12, S. avermertilis; lane 13, S. lividans TK24. The blot was washed with 0.1× SSC (1× SSC is 0.15 M NaCl plus 0.015 M sodium citrate) at 60°C.

FIG. 7

FIG. 7

Dendrograms showing the relatedness of the α and β subunits of protocatechuate 3,4-dioxygenases. Accession numbers for the published sequences are L05770 (Acinetobacter sp. strain ADP1), M30791 (B. cepacia), U33634 (P. marginata), L14836 (P. putida), AF003947 (R. opacus), and AF109386 (Streptomyces sp. strain 2065).

FIG. 8

FIG. 8

Multiple alignment of SCOT homologues by ClustalW. The cloned pca genes were designated according to their functions in relation to previously characterized pca genes. Selected SCOT homologues representing major taxa are included in the alignments. The species and GenBank accession numbers are as follows: pig, 284562; human, 1519052; C. elegans (Ce), 3874069; Streptomyces sp. strain 2065 (Smp), AF109386 (this work); S. coelicolor (Smc), ALO79355; M. tuberculosis (Mt), 2113937; E. coli (Ec), 1788551; Bacillus subtilis (Bs), 666002; Clostridium acetobutylicum (Ca), 538947; Helicobacter pylori (Hp), 2313815. Highly conserved amino acids are marked by asterisks. Other abbreviations: E, eukaryote; HG+, high G+C gram-positive; LG+, low-G+C gram-positive; G−, gram-negative. Superscripts: a, sequence data for C. albicans (Ca) were obtained from the Stanford DNA Sequencing and Technology Center website at

http://www-sequence.stanford.edu/group/candida/

; b, preliminary sequence data for T. brucei (Tb) were obtained from The Institute for Genomic Research website at

http://www.tigr.org/

; c, the sequence data were produced by the Streptomyces coelicolor Sequencing Group at the Sanger Centre and can be obtained from

ftp://ftp.sanger.ac.uk/pub/S

coelicolor/sequences.

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References

    1. Aiba H. A 570-kb DNA sequence of the E. coli K-12 genome corresponding to the 28.0–40.1 min region on the linkage map. DNA Res. 1996;3:363–377. - PubMed
    1. Ausubel F M, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. Current protocols in molecular biology. New York, N.Y: Greene Publishing Associates; 1991.
    1. Baltz R H. Genetic manipulation of antibiotic-producing Streptomyces. Trends Microbiol. 1998;6:76–82. - PubMed
    1. Bull C, Ballou D P. Purification and properties of protocatechuate 3,4-dioxygenase from Pseudomonas putida. J Biol Chem. 1981;256:12673–12680. - PubMed
    1. Cain R B. Utilization of anthranilic and nitrobenzoic acids by Nocardia opaca and Flavobacterium. J Gen Microbiol. 1966;42:219–235. - PubMed

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