The Cgl1281-encoding putative transporter of the cation diffusion facilitator family is responsible for alkali-tolerance in Corynebacterium glutamicum.
Mutants of Corynebacterium glutamicum that were unable to grow under mild alkaline pH conditions were isolated by mutagenesis. Strain AL-43 exhibiting the highest sensitivity to alkaline pH among the mutants was selected and used to clone a DNA fragment that could complement the phenotype. Sequencing and subcloning of the cloned 4.0-kb EcoRI DNA fragment showed that the Cgl1281 gene was responsible for the complementation. The deduced amino acid sequence of Cgl1281 was found to show significant sequence similarity with CzcD, a Me2+/H+(K+) antiporter, from Bacillus subtilis and also possess the features of the cation diffusion facilitator (CDF) family: the presence of 6 putative transmembrane segments and a signature sequence, indicating that the gene product is a member of the CDF family. Chromosomal disruption of the Cgl1281 rendered C. glutamicum cells sensitive to alkaline pH as well as cobalt, while expression of the gene from a plasmid restored alkali-tolerance to the wild-type level and also led to increased cobalt resistance. These results demonstrated that the putative transporter of the CDF family mediates resistance to cobalt and also plays a physiological role in alkaline pH tolerance in C. glutamicum.
Takeno
Seiki
S
Department of Bioscience and Biotechnology, Faculty of Agriculture, Shinshu University, Nagano 399-4598, Japan.
Nakamura
Mio
M
Fukai
Rie
R
Ohnishi
Junko
J
Ikeda
Masato
M
D000222:Adaptation, Physiological; D000468:Alkalies / Q000494:pharmacology*; D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003035:Cobalt / Q000494:pharmacology; D048230:Corynebacterium glutamicum / Q000187:drug effects / Q000235:genetics / Q000254:growth & development / Q000502:physiology*; D024881:Drug Resistance, Bacterial; D023061:Gene Order; D005816:Genetic Complementation Test; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009154:Mutation; D012097:Repressor Proteins / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000468:Alkalies; D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; C503087:CzcD protein, Bacillus subtilis; D026901:Membrane Transport Proteins; D012097:Repressor Proteins; D003035:Cobalt