Isolation of a mutant auxotrophic for L-alanine and identification of three major aminotransferases that synthesize L-alanine in Escherichia coli.

For Escherichia coli, it has been assumed that L-alanine is synthesized by alanine-valine transaminase (AvtA) in conjunction with an unknown alanine aminotransferase(s). We isolated alanine auxotrophs from a prototrophic double mutant deficient in AvtA and YfbQ, a novel alanine aminotransferase, by chemical mutagenesis. A shotgun cloning experiment identified two genes, uncharacterized yfdZ and serC, that complemented the alanine auxotrophy. When the yfdZ- or serC-mutation was introduced into the double mutant, one triple mutant (avtA yfbQ yfdZ) showed alanine auxotrophy, and another (avtA yfbQ serC), prototrophy. In addition, we found that four independent alanine auxotrophs possessed a point mutation in yfdZ but not in serC. We also found that yfdZ expression was induced in minimal medium. Furthermore, yfbQ-bearing plasmid conferred the ability to excrete alanine on the mutant lacking D-amino acid dehydrogenase-encoding gene, dadA. From these results, we concluded that E. coli synthesizes L-alanine by means of three aminotransferases, YfbQ, YfdZ, and AvtA.

Inducible L-alanine exporter encoded by the novel gene ygaW (alaE) in Escherichia coli.

We previously isolated a mutant hypersensitive to L-alanyl-L-alanine from a non-L-alanine-metabolizing Escherichia coli strain and found that it lacked an inducible l-alanine export system. Consequently, this mutant showed a significant accumulation of intracellular L-alanine and a reduction in the L-alanine export rate compared to the parent strain. When the mutant was used as a host to clone a gene(s) that complements the dipeptide-hypersensitive phenotype, two uncharacterized genes, ygaW and ytfF, and two characterized genes, yddG and yeaS, were identified. Overexpression of each gene in the mutant resulted in a decrease in the intracellular l-alanine level and enhancement of the L-alanine export rate in the presence of the dipeptide, suggesting that their products function as exporters of L-alanine. Since ygaW exhibited the most striking impact on both the intra- and the extracellular L-alanine levels among the four genes identified, we disrupted the ygaW gene in the non-L-alanine-metabolizing strain. The resulting isogenic mutant showed the same intra- and extracellular L-alanine levels as observed in the dipeptide-hypersensitive mutant obtained by chemical mutagenesis. When each gene was overexpressed in the wild-type strain, which does not intrinsically excrete alanine, only the ygaW gene conferred on the cells the ability to excrete alanine. In addition, expression of the ygaW gene was induced in the presence of the dipeptide. On the basis of these results, we concluded that YgaW is likely to be the physiologically most relevant exporter for L-alanine in E. coli and proposed that the gene be redesignated alaE for alanine export.

Identification of an L-alanine export system in Escherichia coli and isolation and characterization of export-deficient mutants.

An Escherichia coli strain that exhibits a double auxotrophy for L-alanine and D-alanine was constructed. During growth in the presence of the dipeptide L-alanyl-L-alanine (Ala-Ala), this was fully consumed with concomitant extracellular accumulation of l-alanine in a twofold molar concentration compared with the dipeptide. This finding indicates that the strain not only can hardly degrade L-alanine but has an export system(s) for L-alanine. To obtain access to the system, we chemically mutagenized the L-alanine-nonmetabolizing strain and isolated mutants with increased Ala-Ala sensitivity. Two such mutants accumulated L-alanine up to 150-190 mM in the cytoplasm with a reduced rate of L-alanine export relative to the parent strain in the presence of Ala-Ala. Furthermore, when chloramphenicol was added together with Ala-Ala, the parent strain accumulated L-alanine in the cytoplasm to a level similar to that observed in the mutants in the absence of chloramphenicol. In contrast, the intracellular l-alanine level in the mutants did not change irrespective of chloramphenicol treatment. From these results, we conclude that E. coli has an inducible l-alanine export carrier, together with a second, as yet unidentified, mechanism of alanine export.

Antibiotic resistance in bacteria and its future for novel antibiotic development.

Since the first introduction of the sulfa drugs and penicillin into clinical use, large numbers of antibiotics have been developed and hence contributed to human health. But extensive use of antibiotics has raised a serious public health problem due to multiantibiotic resistant bacterial pathogens that inevitably develop resistance to every new drug launched in the clinic. Consequently, there is a pressing need to develop new antibiotics to keep pace with bacterial resistance. Recent advances in microbial genomics and X-ray crystallography provide opportunities to identify novel antibacterial targets for the development of new classes of antibiotics and to design more potent antimicrobial compounds derived from existing antibiotics respectively. To prevent and control infectious diseases caused by multiantibiotic resistant bacteria, we need to understand more about the molecular aspects of the pathogens' physiology and to pursue ways to prolong the life of precious antibiotics.

Identification and cloning of the gene involved in the final step of chlortetracycline biosynthesis in Streptomyces aureofaciens.

For chlortetracycline biosynthesis in Streptomyces aureofaciens, the final reduction step is essential to give an antibiotic activity to its intermediate, which is catalyzed by tetracycline dehydrogenase with 7,8-dedimethyl-8-hydroxy-5-deazariboflavin (FO) as a cofactor. We identified and cloned the gene, which is essential for the biosynthesis of 6-demethyltetracycline and participates in the final step of its biosynthesis, from the genomic DNA of the 6-demethyltetracycline producer S. aureofaciens HP77. DNA sequence analysis revealed that the gene (tchA) had an open reading frame of 455 amino acids with an estimated molecular mass of 48.1 kDa. Southern hybridization analysis revealed that the tchA gene was located external to the chlortetracycline biosynthetic gene cluster in the genome. A conserved domain search of protein sequence databases indicated that TchA showed a similarity to FbiB, which is involved in the modification of FO in Mycobacterium bovis.

A novel system with positive selection for the chromosomal integration of replicative plasmid DNA in Corynebacterium glutamicum.

A simple system has been developed for generating Corynebacterium glutamicum strains containing stable replicative plasmids integrated into the chromosome via homologous recombination. The system is based upon extremely strong incompatibility between two plasmids, which cannot be co-maintained even under antibiotic selective pressure. Integration of the resident plasmid that contained the trpD gene of C. glutamicum was achieved by introduction of a second plasmid and subsequent selection for the maintenance of both plasmids. Plasmid integrates positive for both plasmid markers were obtained at a frequency about 10(-3) of the normal transformation frequency with selection for the maintenance of only the second plasmid. Southern analysis revealed that the integration had occurred through a single-crossover homologous recombination between the trpD regions of the host genome and the plasmid. On the basis of the Campbell-type integration, chromosome walking was attempted by using Escherichia coli replication origins that were also present in the integrated plasmid. The chromosomal DNA was digested, ligated, and used to transform E. coli, which enabled recovery of the expected adjacent genomic DNA regions. The plasmid integrate was stably maintained for 30 generations under non-selective culture conditions, suggesting that the integrated sequences carrying a replicon active in the host were maintained as a stable chromosomal insert in C. glutamicum.