ABSTRACT
The product of the MTCY428.08 gene of Mycobacterium tuberculosis shows sequence homology with several NAD+ synthetases. The MTCY428.08 gene was cloned into the expression vectors pGEX-4T-1 and pET-15b. Expression in Escherichia coli led to overproduction of glutathione S-transferase fused and His6-tagged gene products, which were enzymatically assayed for NAD synthetase activity. Our results demonstrate that the MTCY428.08 gene of M. tuberculosis is the structural gene for NAD+ synthetase.
Subject(s)
Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/genetics , Genes, Bacterial , Mycobacterium tuberculosis/enzymology , Mycobacterium tuberculosis/genetics , Amino Acid Sequence , Carbon-Nitrogen Ligases with Glutamine as Amide-N-Donor/metabolism , Cloning, Molecular , Escherichia coli/genetics , Molecular Sequence Data , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Homology, Amino AcidABSTRACT
A continuous 75627 bp segment of the Mycobacterium leprae chromosome spanning the oriC region was sequenced. The gene order at this locus was similar to that found in the replication origin region of many other prokaryotes, particularly Mycobacterium tuberculosis and Streptomyces coelicolor. As in the case of several Gram-positive bacteria, essential genes involved in basic cellular functions, such as DNA or RNA metabolism (dnaA, dnaB, dnaN, gyrB, gyrA, pcnB, recF, rnpA, ssb), cell wall synthesis (ponA, pbpA) and probably cell division (gidB, rodA) were found. Strikingly, the gidA gene was absent from this part of the genome and there was no rRNA operon near oriC. The gyrA gene harbours an intein coding sequence indicating that protein splicing is required to produce the mature A subunit of DNA gyrase. Among the many other noteworthy features were ORFs encoding putative serine/threonine protein kinases and a protein phosphatase, three tRNA genes, one M. leprae-specific repetitive element and a glnQ pseudogene.
Subject(s)
Chromosomes, Bacterial/genetics , Genes, Bacterial , Mycobacterium leprae/genetics , Amino Acid Sequence , Bacterial Proteins/genetics , Cell Division/genetics , Cell Wall/metabolism , Chromosome Mapping , Cloning, Molecular , Cosmids , DNA Replication/genetics , DNA, Bacterial/genetics , DNA, Bacterial/metabolism , DNA-Binding Proteins/genetics , Evolution, Molecular , Molecular Sequence Data , Multigene Family , Mycobacterium leprae/metabolism , Open Reading Frames , Protein Biosynthesis , Protein Kinases/genetics , Pseudogenes , Replication Origin , Sequence Homology, Amino AcidABSTRACT
The complete nucleotide sequence of the gene encoding aspartokinase (Ask) II from thermophilic Bacillus stearothermophilus has been determined. Degenerate oligodeoxyribonucleotides primed the amplification of a 932-bp gene. This sequence was successively used for constructing new primers applied in inverse polymerase chain reaction using, as template, self-ligating DNA fragments. The deduced amino-acid sequence is 68.7% identical with the sequence of the Bacillus sp. strain MGA3 Ask II.
