Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis

Abstract

The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis.Pseudomonas syringae pv. tabaci BR2.024, a plasmid-free derivative of strain BR2, is a tabtoxin-producing bacterium that causes wildfire disease on green beans (27, 31). Tabtoxin, a dipeptide, is not biologically active, but hydrolysis by peptidases produces tabtoxinine-␤-lactam (T␤L), the active toxin (22). Feeding studies with labeled precursors (26,34,40) indicated that carbons 1 to 6 of T␤L ( Fig. 1) are derived from aspartate and pyruvate, and labelling patterns led to speculation that the T␤L and lysine biosynthetic pathways may have common initial steps. Although lysine and diaminopimelic acid (DAP) are structurally related to T␤L (Fig. 1), label from these amino acids is not effectively incorporated into T␤L. The methyl group of methionine can provide the ␤-lactam carbonyl carbon. Unkefer et al. (40) suggested that the two pathways might diverge after L-dihydrodipicolinate (DHDPA). Reduction to L-2,3,4,5-tetrahydrodipicolinate (THDPA) by DapB (5) would lead to DAP, while hydration would place a hydroxyl group on the carbon that becomes carbon 5 of T␤L.All of the genes required for T␤L biosynthesis and resistance are located in a region of about 32 kb of the BR2 chromosome (19). Approximately 3 kb of that region has been sequenced, and two genes (tabA and tblA), essential for T␤L production but with unknown functions, have been identified. The deduced tabA product has an amino acid sequence homologous to meso-DAP decarboxylase (the lysA gene product) characterized in other bacteria (9, 32); however, tabA is unable to complement an Escherichia coli lysA mutant (9). The second gene, tblA, encodes a product with an amino acid sequence unrelated to those of known polypeptides (2). The transcription of tblA is positively regulated by ...

A possible role for acetylated intermediates in diaminopimelate and tabtoxinine-beta-lactam biosynthesis in Pseudomonas syringae pv. tabaci BR2.024

“…We considered three possible explanations for these observations. (i) BR2.024 contains a second gene encoding a protein with THDPA-ST activity; however, attempts to detect such a gene by complementing E. coli dapD mutant ␤274 with DNA from a genomic library of BR11.000, a mutant in which the region containing tabB had been deleted (21), were unsuccessful (data not shown). (ii) tabB is expressed at low levels in BR2.024 under our growth conditions.…”

“…DNA isolation, restriction digestion, ligation reactions, transformation, and hybridization were carried out as previously described (21). The dideoxy-chain termination method (33) and Deaza sequencing kit (Pharmacia) were used for double-stranded sequencing.…”

“…Among other gram-negative bacteria, the only lysine biosynthetic genes that, to our knowledge, have been characterized are lysA (22) from Pseudomonas aeruginosa and dapB from Pseudomonas syringae pv. tabaci (21).…”

“…T␤L is structurally related to lysine, and labeling studies (23,31,39) indicated some commonality in the biosynthetic pathways of those two compounds. Genetic studies (21) showed that DapB is essential for both pathways and that THDPA may be an intermediate in both pathways. Two other genes, tblA (1) and tabA (8), required for T␤L biosynthesis are located within a 31-kb region that contains all of the genes needed for T␤L biosynthesis and tabtoxin resistance (17).…”

Advances in Molecular Biology of Phytotoxin Production and Its Involvement in Plant Pathogenesis

“…tabaci which conferred tabtoxin production suggesting all genes necessary for tabtoxin synthesis were clustered in this region. Three genes, tabA, tabB and tblA were involved in tabtoxin synthesis and self-resistance are characterised (Engst and Shaw, 1992;Liu and Shaw, 1997;Barta et aI., 1993). All these genes showed relatedness to the genes in lysine biosynthetic pathway.…”