Genes involved in meso-diaminopimelate synthesis in Bacillus subtilis: identification of the gene encoding aspartokinase I

Abstract

Thermosensitive mutants of Bacillus subtilis deficient in peptidoglycan synthesis were screened for mutations in the rneso-diaminopimelate ( L D -A~~~) metabolic pathway. Mutations in two out of five relevant linkage groups, lssB and IssD, were shown to induce, at the restrictive temperature, a deficiency in LD-A,pm synthesis and accumulation of UDP-MurNAc-dipeptide. Group IssB is heterogeneous; it encompasses mutations that confer deficiency in the deacylation of N-acetyI-~~-A,pm and accumulation of this precursor. Accordingly, these mutations are assigned to the previously identified locus dapE. Mutations in linkage group lssD entail a thermosensitive aspartokinase I. Therefore, they are most likely to affect the structural gene of this enzyme, which we propose to designate dapC. Mutation pyc-1476, previously reported to affect the pyruvate carboxylase, was shown to confer a deficiency in aspartokinase I, not in the carboxylase, and to belong to the dapC locus. dapG is closely linked to spoVF, the putative gene of dipicolinate synthase. In conclusion, mutations affecting only two out of eight steps known to be involved in LD-A,pm synthesis were uncovered in a large collection of thermosensitive mutants obtained by indirect selection. We propose that this surprisingly restricted distribution of the thermosensitive dap mutations isolated so far is due to the existence, in each step of the pathway, of isoenzymes encoded by separate genes. The biological role of different aspartokinases was investigated with mutants deficient in dapE and dapC genes. Growth characteristics of these mutants in the presence of various combinations of aspartate family amino acids allow a reassessment of a metabolic channel hypothesis, i.e. the proposed existence of multienzyme complexes, each specific for a given end product.

An early cytoplasmic step of peptidoglycan synthesis is associated to MreB in Bacillus subtilis

“…A). The ykuR gene of B. subtilis encodes a 374 aa (41 kDa) putative N ‐acetyl‐diaminopimelate deacetylase belonging to the peptidase M20 family and presumably involved in the DAP pathway (Roten et al ., ; Rodionov et al ., ). The MreB‐interacting fragment of YkuR originally isolated in the screen comprised aa 18–124.…”

“…In these two common steps, l ‐aspartate is converted to l ‐aspartate‐semialdehyde by enzymes with aspartokinase and aspartate semialdehyde dehydrogenase activities. B. subtilis encodes three monofunctional aspartokinase isoenzymes, DapG, LysC and YclM, and a single aspartate semialdehyde dehydrogenase, Asd (Rosner and Paulus, ; Chen et al ., 1989; 1993; Petricek et al ., ; Graves and Switzer, ; Zhang et al ., ; Roten et al ., ; Kobashi et al ., ). l ‐aspartate‐semialdehyde is then either fed into the methionine/threonine/isoleucine biosynthetic pathways or converted into m ‐DAP via six additional enzymatic steps, for which the corresponding B. subtilis genes remain to be characterized (Fig.…”

A conditional-lethal mutant of Bacillus subtilis 168 with a thermosensitive glycerol-3-phosphate cytidylyltransferase, an enzyme specific for the synthesis of the major wall teichoic acid

“…To assess if the increase in [ 3H]glycerol-containing pools is specific to tagB and tagF markers, incorporation of [ 2-3H]glycerol into four thermosensitive mutants bearing different Iss markers (Brandt & Karamata, 1987) was examined. The latter strains were shown to be affected in different steps of the synthesis of soluble precursors of peptidoglycan (PG) (Brandt & Karamata, 1987;Roten et al, 1991). It appeared (Table 2, and data not presented) that all the Iss loci were characterized by significantly increased soluble pools and a reduced incorporation of [2-3H]glycerol into the macromolecular fraction which were com parable to those associated with tagF markers (see below).…”

Plant peptidoglycan precursor biosynthesis: Conservation between moss chloroplasts and Gram negative bacteria

“…After 15 days tissue was harvested, weighed and ground in liquid nitrogen using a pestle and mortar before being frozen at −80°C. To extract TCA-soluble plant metabolites the tissue was ground again in 5 ml g -1 of ice cold 10% (w/v) TCA (Fisons AR grade) before being mixed gently in 50 ml Falcon tubes on a rolling shaker for 30 min at 4°C (Roten et al, 1991). Insoluble material was pelleted at 48,000 xg, 10 min, 2°C, the supernatant was retained and the pellet reextracted twice more, first with 2.5 ml.g -1 and then with 1.25 ml.g -1 (of the original pellet weight) of ice cold 10% (w/v) TCA.…”