Pyruvate:quinone oxidoreductase in Corynebacterium glutamicum: molecular analysis of the pqo gene, significance of the enzyme, and phylogenetic aspects

Abstract

Corynebacterium glutamicum recently has been shown to possess pyruvate:quinone oxidoreductase (PQO), catalyzing the oxidative decarboxylation of pyruvate to acetate and CO2 with a quinone as the electron acceptor. Here, we analyze the expression of the C. glutamicum pqo gene, investigate the relevance of the PQO enzyme for growth and amino acid production, and perform phylogenetic studies. Expression analyses revealed that transcription of pqo is initiated 45 bp upstream of the translational start site and that it is organized in an operon together with genes encoding a putative metal-activated pyridoxal enzyme and a putative activator protein. Inactivation of the chromosomal pqo gene led to the absence of PQO activity; however, growth and amino acid production were not affected under either condition tested. Introduction of plasmid-bound pqo into a pyruvate dehydrogenase complex-negative C. glutamicum strain partially relieved the growth phenotype of this mutant, indicating that high PQO activity can compensate for the function of the pyruvate dehydrogenase complex. To investigate the distribution of PQO enzymes in prokaryotes and to clarify the relationship between PQO, pyruvate oxidase (POX), and acetohydroxy acid synthase enzymes, we compiled and analyzed the phylogeny of respective proteins deposited in public databases. The analyses revealed a wide distribution of PQOs among prokaryotes, corroborated the hypothesis of a common ancestry of the three enzymes, and led us to propose that the POX enzymes of Lactobacillales were derived from a PQO.

FIG. 1.

Genomic locus of the pqo gene in C. glutamicum. The arrows represent the computer-predicted coding regions of pqo and the adjacent genes, designated qacB, mapA, and tipA and putatively encoding a multidrug efflux protein, a metal-activated pyridoxal enzyme, and a thiostreptone-induced transcriptional activator, respectively. The Δ symbol indicates the deleted region in C. glutamicum WT Δpqo, WT ΔaceEΔpqo, and MH20-22B. The transcriptional start site is indicated as Ts. The double-headed arrows indicate the fragments used for Southern blot hybridizations and for the construction of the pqo deletion mutant.

FIG. 2.

Primer extension analysis of the transcriptional start site of the C. glutamicum pqo gene. The primer extension product is shown in lane 1. Lanes A, C, G, and T represent the products of sequencing reactions with the same primer used for the primer extension reaction. The relevant DNA sequence (coding strand) is shown on the right; the transcriptional start site and the predicted −10 region are indicated. Note that the sequence given to the right represents the noncoding strand and thus is complementary to the sequence of the sequencing reactions.

FIG. 3.

Growth of C. glutamicum WT (open squares), WT ΔpqoΔaceE (pEKEx-pqo) (open circles), and C. glutamicum WT ΔpqoΔaceE (pEKEx2) (open triangles) on TY complex medium.

FIG. 4.

Phylogenetic tree of PQO, POX, and AHAS enzymes deposited in public databases. Clusters I and II represent PQO/POX enzymes, cluster III AHAS enzymes. Numbers at the nodes give the bootstrap values. The arrow indicates the proposed separation of the PQO and POX enzymes. Organisms are listed in Materials and Methods.