The cadC gene product of alkaliphilic Bacillus firmus OF4 partially restores Na+ resistance to an Escherichia coli strain lacking an Na+/H+ antiporter (NhaA)

Abstract

A 5.6-kb fragment of alkaliphilic Bacilus firmus OF4 DNA was isolated by screening a library of total genomic DNA constructed in pGEM3Zf(+) for clones that reversed the Na+ sensitivity of Escherichia coli NM81, in which the gene encoding an Na+/H+ antiporter (NhaA) is deleted (E. Padan, N. Maisler, D. Taglicht, R. Karpel, and S. Schuldiner, J. Biol. Chem. 264:20297-20302, 1989 Alkaliphilic Bacillus species growing at a pH higher than 10 maintain a cytoplasmic pH that is much more acidic than that of the external medium by using an Na+ cycle consisting of Na+/H+ antiporters and a group of Na+/solute symporters (11,12). The antiporters catalyze the electrogenic exchange of extracellular H+ for intracellular Na+, driven by the proton motive force established by respiration (12,14). Solute uptake coupled to Na+ entry completes the cycle, which allows cells growing at pH 10.5 to maintain an intracellular pH of approximately 8.3 (7).The alkaliphile Na+ cycle has been extensively characterized physiologically, and our recent efforts are aimed at identifying and characterizing the genes encoding the antiporter activity. We have used a screen based on the observation of Padan et al. (21) alkaliphile gene distinct from nhaC into its genome by recombination with a plasmid from the alkaliphile DNA library (10).In this report, we describe a third transformant that confers Na+ resistance to E. coli NM81 without supplying a structural gene for an Na+/H+ antiporter. Rather, Na+ resistance is associated with the expression of a small protein normally involved in the efflux system bestowing cadmium resistance (33). MATERIALS AND METHODSBacterial strains and growth conditions. Alkaliphilic Bacillus firnus OF4 was grown routinely at pH 10.5 or 7.5 on highly buffered malate-containing media at 30°C as described previously (7). Tests of the cadmium resistance of B. firmus OF4 811M required the use of a modified medium to avoid precipitation of the cadmium. The basal medium for growth at pH 7.5 or 9.0 consisted of 50 mM Tris-HCl, 0.1% (NH4)2SO4, and 0.1 mM MgSO4. Cadmium resistance at pH 10.2 was monitored in a basal medium containing 50 mM sodium CAPS [3-(cyclohexylamino)-1-propanesulfonic acid] buffer instead of Tris-HCl. All media were supplemented with 50 mM DL-malate, 0.1% yeast extract, and 50 ,ug of methionine per ml. E. coli NM81 (21) was grown at 37°C in LBK medium (21) containing kanamycin (50 ,ug/ml). Transformants were selected on LBK-kanamycin medium plus ampicillin (100 ,ug/ml), and 0.4 to 0.7 M NaCl was added to screen for Na+-resistant transformants. E. coli JM109 (2) and DH5atMCR (GIBCO-BRL) were used for routine cloning procedures; transformants were selected and maintained on SOB medium (2) containing ampicillin. 4878on June 7, 2019 by guest

Electrogenic Antiport Activities of the Gram-positive Tet Proteins Include a Na+(K+)/K+ Mode That Mediates Net K+ Uptake

“…Possibly the truncated forms retain the capacity to bind Tc, Tc-metal, and monovalent cations, and this accounts for those effects. Such a basis for modest complementation in similar experiments has previously been noted (33).…”

Whole-Genome Transcriptional Analysis of Heavy Metal Stresses in Caulobacter crescentus

“…Sequence comparison shows that the metalbinding site of arsR resembles that of cadC from Staphylococcus aureas plasmid pI258 (76) and from Bacillus firmus OF4 (7). Cadmium ion is thought to relieve repression by cadC (20,74). Therefore, it is possible that the arsRBC operon in C. crescentus was up-regulated because cadmium binds to arsR and releases the repression.…”

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are similar to cadA and cadC of Staphylococcus aureus and are induced by cadmium

“…The 2,136-bp ORF encodes a 711-amino-acid polypeptide of 77.1 kDa, showing 67.5 and 65.8% amino acid sequence similarity with the CadA polypeptides of B. firmus (19) and S. aureus (37), respectively. CadA is member of the family of P-type ATPases, which includes bacterial Cu2,-, Mg2+-, and K+-ATPases, fungal H+-ATPases, and ATPases in higher eucaryotes.…”

“…monocytogenes cadmium resistance determinant does not confer zinc resistance. In S. aureus, the cadA4C efflux system confers cadmium and zinc resistance (61 (37), and B. firmus (19) with protein motifs involving metal ion binding (Cu2" [6,29,38,56] and Hg2e [18,31,32] (50). We investigated cadmium resistance inducibility by two different approaches.…”