Corynebacterium glutamicum Utilizes both Transsulfuration and Direct Sulfhydrylation Pathways for Methionine Biosynthesis

A direct sulfhydrylation pathway for methionine biosynthesis in Corynebacterium glutamicum was found. The pathway was catalyzed by metY encoding O-acetylhomoserine sulfhydrylase. The gene metY, located immediately upstream of metA, was found to encode a protein of 437 amino acids with a deduced molecular mass of 46,751 Da. In accordance with DNA and protein sequence data, the introduction of metY into C. glutamicum resulted in the accumulation of a 47-kDa protein in the cells and a 30-fold increase in O-acetylhomoserine sulfhydrylase activity, showing the efficient expression of the cloned gene. Although disruption of the metB gene, which encodes cystathionine γ-synthase catalyzing the transsulfuration pathway of methionine biosynthesis, or the metY gene was not enough to lead to methionine auxotrophy, an additional mutation in the metY or the metB gene resulted in methionine auxotrophy. The growth pattern of the metY mutant strain was identical to that of the metB mutant strain, suggesting that both methionine biosynthetic pathways function equally well. In addition, an Escherichia coli metB mutant could be complemented by transformation of the strain with a DNA fragment carrying corynebacterial metY and metA genes. These data clearly show that C. glutamicum utilizes both transsulfuration and direct sulfhydrylation pathways for methionine biosynthesis. Although metY and metA are in close proximity to one another, separated by 143 bp on the chromosome, deletion analysis suggests that they are expressed independently. As with metA, methionine could also repress the expression of metY. The repression was also observed with metB, but the degree of repression was more severe with metY, which shows almost complete repression at 0.5 mM methionine in minimal medium. The data suggest a physiologically distinctive role of the direct sulfhydrylation pathway in C. glutamicum

Interaction of GlxR with SprA in the two-hybrid system.

<p>The interaction was assayed by monitoring the growth of cells harboring bait and target proteins. Positive control cells harbored well-established interacting proteins such as LGF2 as bait and Gal11p as the target. Negative control cells harbored empty pBT (bait) and pTRG (target) vectors. NCgl0550p, NCgl0550-encoded protein; and CFU, colony-forming units.</p

Effects of cAMP on <i>in vitro</i> digestion of GlxR by His₆-SprA.

<p>Protein purification and proteolytic assays were performed as described in the Materials and Methods. Proteins were incubated at 4°C or 30°C for 20 h, analyzed on a 15% SDS-PAGE gel, and then visualized by staining with Coomassie brilliant blue G250. Molecular weights are shown in kDa. M, molecular weight markers; and MBP, maltose-binding protein.</p

Growth characteristics of <i>C. glutamicum</i> wild type and mutants.

<p>Cells were grown on glucose (A) or acetate (B) MCGC minimal media. The data represent three independent experiments. Symbols: •, wild-type <i>C. glutamicum</i>; ○, <i>C. glutamicum</i> Δ<i>sprA</i>; ▾, <i>C. glutamicum</i> harboring pSL509 (P₁₈₀-<i>sprA</i>); ▵, <i>C. glutamicum</i> harboring pSL535 (pMT1-<i>sprA</i>).</p

Transcription of genes in <i>C. glutamicum</i> strains.

<p>Cells were grown in glucose MCGC media, and mRNA levels were measured by RT-qPCR as described in the Materials and Methods. The data represent three independent experiments. Bars indicate mRNA levels in wild-type (empty bars), Δ<i>sprA</i> (light grey bars), and P₁₈₀-<i>sprA</i> (dark grey bars) strains.</p

Identification of protein affected in <i>sprA</i>-overexpressing <i>C. glutamicum</i> (P₁₈₀-<i>sprA</i>).

<p>2D-PAGE was performed as described in the Materials and Methods. A total of 150 μg of protein was loaded onto each gel. The identity of each protein spot was determined by electrospray ionization mass spectroscopy.</p