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Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum.

ABSTRACT: An Mn(2+) and Cd(2+) uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd(2+) sensitivity as well as energy-dependent Cd(2+) uptake activity. Both transcription and translation of mntA were induced by Mn(2+) starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd(2+) uptake systems have been identified in L. plantarum: one is a high-affinity Mn(2+) and Cd(2+) uptake system that is expressed in Mn(2+)-starved cells, and the other is a nonsaturable Cd(2+) uptake system that is expressed in Cd(2+)-sufficient cells (Z. Hao, H. R. Reiske, and D. B. Wilson, Appl. Environ. Microbiol. 65:592-99, 1999). MntA was not detected in an Mn(2+)-dependent mutant of L. plantarum which had lost high-affinity Mn(2+) and Cd(2+) uptake activity. The results suggest that mntA is the gene encoding the high-affinity Mn(2+) and Cd(2+) transporter. On the basis of its predicted amino acid sequence, MntA belongs to the family of P-type cation-translocating ATPases. The topology and potential Mn(2+)- and Cd(2+)-binding sites of MntA are discussed. A second clone containing a low-affinity Cd(2+) transport system was also isolated.

SUBMITTER: Hao Z

PROVIDER: S-EPMC91639 | biostudies-literature | 1999 Nov

REPOSITORIES: biostudies-literature

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Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum.

Hao Z Z Chen S S Wilson D B DB

Applied and environmental microbiology 19991101 11

An Mn(2+) and Cd(2+) uptake gene, mntA, was cloned from Lactobacillus plantarum ATCC 14917 into Escherichia coli. Its expression conferred on E. coli cells increased Cd(2+) sensitivity as well as energy-dependent Cd(2+) uptake activity. Both transcription and translation of mntA were induced by Mn(2+) starvation in L. plantarum, as indicated by reverse transcriptase PCR and immunoblotting. Two Cd(2+) uptake systems have been identified in L. plantarum: one is a high-affinity Mn(2+) and Cd(2+) up ...[more]

PMID: 10543781

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Project description:A 3-kb region, located downstream of the Lactobacillus brevis xylA gene (encoding D-xylose isomerase), was cloned in Escherichia coli TG1. The sequence revealed two open reading frames which could code for the D-xylulose kinase gene (xylB) and another gene (xylT) encoding a protein of 457 amino acids with significant similarity to the D-xylose-H+ symporters of E. coli, XylE (57%), and Bacillus megaterium, XylT (58%), to the D-xylose-Na+ symporter of Tetragenococcus halophila, XylE (57%), and to the L-arabinose-H+ symporter of E. coli, AraE (60%). The L. brevis xylABT genes showed an arrangement similar to that of the B. megaterium xylABT operon and the T. halophila xylABE operon. Southern hybridization performed with the Lactobacillus pentosus xylR gene (encoding the D-xylose repressor protein) as a probe revealed the existence of a xylR homologue in L. brevis which is not located with the xyABT locus. The existence of a functional XylR was further suggested by the presence of xylO sequences upstream of xylA and xylT and by the requirement of D-xylose for the induction of D-xylose isomerase, D-xylulose kinase, and D-xylose transport activities in L. brevis. When L. brevis was cultivated in a mixture of D-glucose and D-xylose, the D-xylose isomerase and D-xylulose kinase activities were reduced fourfold and the D-xylose transport activity was reduced by sixfold, suggesting catabolite repression by D-glucose of D-xylose assimilation. The xylT gene was functionally expressed in Lactobacillus plantarum 80, a strain which lacks proton motive force-linked D-xylose transport activity. The role of the XylT protein was confirmed by the accumulation of D-xylose in L. plantarum 80 cells, and this accumulation was dependent on the proton motive force generated by either malolactic fermentation or by the metabolism of D-glucose. The apparent affinity constant of XylT for D-xylose was approximately 215 microM, and the maximal initial velocity of transport was 35 nmol/min per mg (dry weight). Furthermore, of a number of sugars tested, only 6-deoxy-D-glucose inhibited the transport of D-xylose by XylT competitively, with a Ki of 220 microM.

Dataset Information

Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum.

Publications

Cloning, expression, and characterization of cadmium and manganese uptake genes from Lactobacillus plantarum.

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