Identification of the 2-methylcitrate pathway involved in the catabolism of propionate in the polyhydroxyalkanoate-producing strain Burkholderia sacchari IPT101(T) and analysis of a mutant accumulating a copolyester with higher 3-hydroxyvalerate content

Abstract

Burkholderia sacchari IPT101(T) induced the formation of 2-methylcitrate synthase and 2-methylisocitrate lyase when it was cultivated in the presence of propionic acid. The prp locus of B. sacchari IPT101(T) is required for utilization of propionic acid as a sole carbon source and is relevant for incorporation of 3-hydroxyvalerate (3HV) into copolyesters, and it was cloned and sequenced. Five genes (prpR, prpB, prpC, acnM, and ORF5) exhibited identity to genes located in the prp loci of other gram-negative bacteria. prpC encodes a 2-methylcitrate synthase with a calculated molecular mass of 42,691 Da. prpB encodes a 2-methylisocitrate lyase. The levels of PrpC and PrpB activity were much lower in propionate-negative mutant IPT189 obtained from IPT101(T) and were heterologously expressed in Escherichia coli. The acnM gene (ORF4) and ORF5, which are required for conversion of 2-methylcitric acid to 2-methylisocitric acid in Ralstonia eutropha HF39, are also located in the prp locus. The translational product of ORF1 (prpR) had a calculated molecular mass of 70,598 Da and is a putative regulator of the prp cluster. Three additional open reading frames (ORF6, ORF7, and ORF8) whose functions are not known were located adjacent to ORF5 in the prp locus of B. sacchari, and these open reading frames have not been found in any other prp operon yet. In summary, the organization of the prp genes of B. sacchari is similar but not identical to the organization of these genes in other bacteria investigated recently. In addition, this study provided a rationale for the previously shown increased molar contents of 3HV in copolyesters accumulated by a B. sacchari mutant since it was revealed in this study that the mutant is defective in prpC.

FIG. 1.

Southern hybridization of E. coli S17-1 clones, harboring HindIII-digested genomic DNA of B. sacchari IPT101^T in cosmid pHC79, identified by colony hybridization by using the 2-kbp EcoRI fragment as a probe. Lane 1, PstI-digested λ DNA; lanes 2, HindIII-digested genomic DNA of B. sacchari IPT101^T; lanes 3, HindIII-restricted hybrid cosmids of the E. coli S17-1 clone.

FIG. 2.

(A) Arrangement of four EcoRI subfragments on the 25.5-kbp HindIII fragment of B. sacchari IPT101^T; (B) organization of the prp locus in B. sacchari IPT101^T.

FIG. 3.

Comparison of 2-methylcitrate synthase motifs with the corresponding citrate synthase signature. The shaded amino acids are the putative active sites (http://www.jgi.doe.gov/tempweb/JGI_microbial/html/index.html), which were identified on the basis of similarity. References, accession numbers (if available) and contig numbers are given in parentheses. Data for some of the organisms were obtained from the following websites: B. cepacia, R. metallidurans CH34, and Pseudomonas fluorescens, http://www.expasy.ch/cgi-bin/nicesite.pl?PS00161; and P. putida KT2440, P. syringae, and S. putrefaciens, http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi?organism.

FIG. 4.

Dendrogram based on aconitases and acnM translational products of several bacteria. The numbers in parentheses are accession numbers and references. Data for some of the organisms were obtained from the following websites: P. fluorescens and B. cepacia, http://www.expasy.ch/cgi-bin/nicesite.pl?PS00161; and S. putrefaciens and P. putida, http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi?organism.

FIG. 5.

Comparison of motifs of the acnM translational products of several organisms with the aconitase signature. The shaded amino acids are the putative cysteine residues which bind the Fe-S-cluster (identified on the basis of similarity) (http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi?organism). The boldface N is a highly conserved asparagine residue in acnM translational products; in aconitases in the tricarboxylic acid cycle this amino acid is isoleucine (indicated by boldface I). The references and accession numbers (if available), as well as contig numbers, are given in parentheses. Data for some of the organisms were obtained from the following websites: B. cepacia, R. metallidurans CH34, and P. fluorescens, http://www.expasy.ch/cgi-bin/nicesite.pl?PS00161; and P. putida KT2440, P. syringae, and S. putrefaciens, http://www.tigr.org/cgi-bin/BlastSearch/blast.cgi?organism.