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

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.     

Mapping of the lipoprotein signal peptidase gene (lsp)

A pBR322 plasmid which contains a fragment of Escherichia coli DNA encoding the lipoprotein signal peptidase gene was used to transform Hfr polA1 strains. Ampr transformants were used as donors in conjugation experiments, and the location of the plasmid amp gene adjacent to the chromosomal lsp gene was determined to be near the thr ara loci of the E. coli chromosome. P1 transduction experiments established that the location of the lsp gene is closely linked to that of dapB , at 0.5 to 0.6 min on the E. coli genetic map. The position of the lsp gene was further determined to be between ileS and dapB by complementation analysis of an E. coli mutant showing temperature-sensitive prolipoprotein signal peptidase activity.     

Genetic determination of the meso-diaminopimelate biosynthetic pathway of mycobacteria.

The increasing incidence of multiple-drug-resistant mycobacterial infections indicates that the development of new methods for treatment of mycobacterial diseases should be a high priority. meso-Diaminopimelic acid (DAP), a key component of a highly immunogenic subunit of the mycobacterial peptidoglycan layer, has been implicated as a potential virulence factor. The mycobacterial DAP biosynthetic pathway could serve as a target for design of new antimycobacterial agents as well as the construction of in vivo selection systems. We have isolated the asd, dapA, dapB, dapD, and dapE genes involved in the DAP biosynthetic pathway of Mycobacterium bovis BCG. These genes were isolated by complementation of Escherichia coli mutations with an expression library of BCG DNA. Our analysis of these genes suggests that BCG may use more than one pathway for biosynthesis of DAP. The nucleotide sequence of the BCG dapB gene was determined. The activity of the product of this gene in Escherichia coli provided evidence that the gene may encode a novel bifunctional dihydrodipicolinate reductase and DAP dehydrogenase.     

Sequence of the regulatory region of omp T, the gene specifying major outer membrane protein a (3b) of Escherichia coli K-12: implications for regulation and processing

Summary The DNA of the promoter region of ompT, including the putative start for the pro-OmpT protein (proprotein a), has been sequenced. Previous studies showed that trypsin inhibitors prevent the processing of pro-OmpT to OmpT protein which led to the prediction that the processing site would be a lysine or an arginine. The deduced amino acid sequence contains a lysine at amino acid 12 and an arginine at amino acid 17 from the N terminus. Chou-Fassman analysis would predict processing at the lysine (but not the arginine) to remove a 1389 dalton peptide, consistent with the fact that the estimated molecular masses of pro-OmpT and OmpT are 42 kd and 40 kd respectively. In addition, the predicted mRNA of the promoter region can form a stable secondary structure (-17.1 kcal) that sequesters the Shine-Dalgarno (SD) sequence as well as the initiator AUG codon. There is evidence that the perA (tpo, envZ) gene product is required for synthesis of OmpT protein (as well as several outer membrane and periplasmic proteins). The perA gene product could be activating translation of OmpT protein by disrupting the mRNA secondary structure that sequesters the SD sequence. OmpT protein synthesis is reduced at temperatures below 32°C and this may also be related to the greater stability of the sequestered SD sequence of the mRNA at low temperature.     

Nucleotide sequence of the asd gene of Escherichia coli: absence of a typical attenuation signal.

The asd gene of escherichia coli encodes aspartic semialdehyde dehydrogenase, an enzyme involved in lysine, threonine, and methionine biosynthesis; its synthesis is controlled by a multivalent repression mechanism. It was cloned in plasmid pBR322 and its complete nucleotide sequence determined. The sequence predicts a polypeptide chain of 367 amino acids, in good agreement with results obtained for the purified protein ( Biellmann et al., 1980a ). Our data indicate a Cys residue instead of a His residue, which was proposed after covalent labeling of the active center of the enzyme; this is more in line with the catalytic site of glyceraldehyde-3-phosphate dehydrogenase, an enzyme which carries out a similar reaction. The nucleotide sequence that precedes the translational start does not display any of the characteristic features of an attenuation signal. Hence the expression of the asd gene is probably not controlled in the same way as other multivalently repressed operons such as ilva and thr.