Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle - PubMed (original) (raw)

Identification of two prpDBC gene clusters in Corynebacterium glutamicum and their involvement in propionate degradation via the 2-methylcitrate cycle

Wilfried A Claes et al. J Bacteriol. 2002 May.

Abstract

Genome sequencing revealed that the Corynebacterium glutamicum genome contained, besides gltA, two additional citrate synthase homologous genes (prpC) located in two different prpDBC gene clusters, which were designated prpD1B1C1 and prpD2B2C2. The coding regions of the two gene clusters as well as the predicted gene products showed sequence identities of about 70 to 80%. Significant sequence similarities were found also to the prpBCDE operons of Escherichia coli and Salmonella enterica, which are known to encode enzymes of the propionate-degrading 2-methylcitrate pathway. Homologous and heterologous overexpression of the C. glutamicum prpC1 and prpC2 genes revealed that their gene products were active as citrate synthases and 2-methylcitrate synthases. Growth tests showed that C. glutamicum used propionate as a single or partial carbon source, although the beginning of the exponential growth phase was strongly delayed by propionate for up to 7 days. Compared to growth on acetate, the specific 2-methylcitrate synthase activity increased about 50-fold when propionate was provided as the sole carbon source, suggesting that in C. glutamicum the oxidation of propionate to pyruvate occurred via the 2-methylcitrate pathway. Additionally, two-dimensional gel electrophoresis experiments combined with mass spectrometry showed strong induction of the expression of the C. glutamicum prpD2B2C2 genes by propionate as an additional carbon source. Mutational analyses revealed that only the prpD2B2C2 genes were essential for the growth of C. glutamicum on propionate as a sole carbon source, while the function of the prpD1B1C1 genes remains obscure.

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Figures

FIG. 1.

FIG. 1.

Physical and genetic maps of different bacterial prp gene regions. (a and b) Restriction maps of the C. glutamicum prpD1B1C1 and prpD2B2C2 gene fragments, subcloned DNA fragments, and deletion mutants. (c) Genetic organizations of the prp gene clusters of E. coli (GenBank accession no. U73857), S. enterica serovar Typhimurium (GenBank accession no. U51879), and R. eutropha (GenBank accession no. AF325554 and AF331923). Only the restriction sites used for cloning of both C. glutamicum prp loci are shown. The positions of the C. glutamicum ORFs and genes are indicated by arrows, and putative _rho_-independent termination structures are presented as hairpin structures. The deletions introduced into the C. glutamicum chromosome are described in detail in the Material and Methods section. Homologous genes within the prp regions of E. coli, S. enterica, R. eutropha, and C. glutamicum are shaded grey.

FIG. 2.

FIG. 2.

Growth of C. glutamicum RES167 on propionate as the sole or additional carbon source. An LB overnight culture of C. glutamicum RES167 was washed twice in 50 mM Tris-HCl (pH 7.5), and 2 × 107 cells were used as inoculum for 60 ml of the minimal medium CGXII. Each carbon source was added with a final concentration of 4 g liter of medium−1. Cultivations were done in 250-ml Erlenmeyer flasks at 30°C and 150 rpm, and aliquots for the determination of the optical density at 580 nm were taken at the time intervals indicated.

FIG. 3.

FIG. 3.

2D-PAGE of protein extracts of C. glutamicum during growth on acetate with or without propionate. Crude protein extracts of C. glutamicum RES167 were harvested in the late exponential growth phase after growth on minimal medium CGXII containing 4 g of acetate liter−1 (a) or 4 g of acetate plus 4 g propionate liter−1 (b) as sole carbon sources. Two hundred fifty micrograms of precipitated and resuspended proteins were subjected to 2D-PAGE. The pH range and the molecular masses of marker proteins are indicated on the gels. Areas of prpD2B2C2 protein spots of both gels are magnified for comparison.

FIG. 4.

FIG. 4.

Plate tests for the identification of prp genes essential for growth of C. glutamicum on propionate as sole carbon source. C. glutamicum RES167 was used as the control strain on each plate. Deletion mutant strains carrying mutations in the prpD1B1C1 region and the prpD2B2C2 region and the respective double mutant strains, corresponding to strains WAC1 to WAC12 (Table 1), were arranged on the plates by mutated prp genes. Cells (2 × 106) of each strain were spread onto a quarter of a minimal medium MM1 plate with 6 g of propionate liter−1 as the sole carbon source and incubated for 20 days at 30°C.

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