Cloning in Escherichia coli K-12 of a Na+-dependent transport system from a marine bacterium

Abstract

The transport of D-alanine by Escherichia coli K-12 neither requires nor is stimulated by Na+. The transport of D-alanine by the marine bacterium Alteromonas haloplanklis 214 requires Na+ specifically. Mutants of E. coli which were unable to transport D-alanine were isolated by enrichment for D-cycloserine resistance. One of the mutants was transformed with a gene bank of A. haloplanktis chromosomal DNA. Two transformants, E. coli RMl(pPMl) and E. coli RM1(pPM2) were able to transport D-alanine by a Na+-dependent mechanism. Li' and K' were unable to replace Na+. Both transformants contained chimeric plasmids with inserts which hybridized with A. haloplanklis but not E. coli chromosomal DNA or each other. Despite the lack of homology between the inserts, Na+-dependent D-alanine transport in the two transformants could not be distinguished either by kinetic studies or by differences in the capacity of various amino acids to compete for D-alanine uptake.Alteromonas haloplanktis 214 accumulates solutes by a Na+-solute symport mechanism energized by an electrochemical potential of Na+ ions (17), while a Na+-proton antiporter maintains a downhill gradient of Na+ into the cells (16). In these respects, the organism is similar to a number of other bacteria possessing Na+-dependent transport systems (11). A. haloplanktis 214 transports D-alanine, D-serine, glycine, ax-aminoisobutyric acid, and to a lesser extent L-alanine by a pathway that requires Na+ (8). A transport system serving for the entry of D-alanine, D-serine, and glycine is also found in Escherichia coli (3,20,26), but in this organism the accumulation of these amino acids is energized by an electrochemical potential of protons (1, 15). In a previous report, MacLeod et al. (13) described the preparation of a gene bank of A. haloplanktis chromosomal DNA and showed that various biosynthetic genes in the gene bank could be expressed in E. coli. In the present study, we isolated mutants of E. coli defective for D-alanine transport and used the gene bank to obtain transformants of one of these which transport D-alanine by a Na+-dependent mechanism. This is the first report of the cloning in E. coli of genes for Na+-dependent transport from a marine bacterium. The melB gene which codes for the Na+-driven transport camer for melibiose in E. coli has been cloned (25) and sequenced (27). MATERIALS AND METHODSBacterial strains and culture conditions. E. coli K-12 E02717 (20) (F-hisGI purF) argHI thi-l dagA3 pyrBS9 ara-13 lacY) [ been investigated previously in this laboratory (10). E. coli strains were grown in either L broth (12) or M9 minimal medium (5) further supplemented with 5 mM adenine hydrochloride, 0.6 mM arginine hydrochloride, 0.1 mM histidine, 0.05 mM thiamine hydrochloride, and 0.01 mM * Corresponding author. 825 uracil and containing either 5 g of glycerol per liter (M9-glycerol medium) or 5 g of D-alanine per liter (M9-D-alanine medium) as carbon source. Ampicillin was also included at 50 ,ug/ml in L broth and at 25 pug/ml in M9 media when the media wer...

D-cycloserine uses an active transport mechanism in the human intestinal cell line Caco 2

“…2A); in fact, D-cycloserine is a modified amino acid. In bacterial cells, D-cycloserine uptake is known to be mediated by an amino acid permease (11,22). Since absorptive enterocytes express several amino acid carriers with overlapping specificities (17,33), the effects of amino acids on the transport of D-cycloserine were examined in competition experiments.…”

Expression and regulation of a Vibrio alginolyticus sucrose utilization system cloned in Escherichia coli

“…V. alginolyticus DNA was partially digested with nuclease Sau3A and fractionated on a sucrose density gradient. Fragments 10 Ci/mol). Samples (50 ,ul) were withdrawn at intervals, filtered through membrane filters (pore size, 0.45 ,um; Millipore Corp.), and rinsed once with 2 ml of minimal medium.…”

“…Samples (50 ,ul) of the E. coli cell suspension (2.5 mg of protein per ml) were added to 330 ,ul of the potassium phosphate buffer containing 12.5 mM glucose, 12.5 mM fructose, and either 0.1 M NaCl, KCl, or LiCl or no additional salt. Samples (25 ,ul) of the V. alginolyticus cell suspension (2.5 mg of protein per ml) were added to 365 ,ul of incubation medium (10) alginolyticus and E. coli JA221(pVS100) (Fig. 3).…”

“…V. alginolyticus cells were treated with choline chloride salts by the method of MacLeod and MacLeod (10). Samples (50 ,ul) of the E. coli cell suspension (2.5 mg of protein per ml) were added to 330 ,ul of the potassium phosphate buffer containing 12.5 mM glucose, 12.5 mM fructose, and either 0.1 M NaCl, KCl, or LiCl or no additional salt.…”

Antibiotics, peptidoglycan synthesis and genomics: the chlamydial anomaly revisited

“…trachomatis may need to acquire D-alanine. Regarding this point, it is interesting to note that C. trachomatis contains two homologues (ORFs D409 and D735) of dagA (encoding D-alanine glycine permease; synonym cycA), the product of which is a transport protein responsible for accumulation of Dalanine (and cycloserine) in E. cofi and other bacteria (Robbins & Oxender, 1973;MacLeod & MacLeod, 1986,1992. The presence of two similar genes encoding membrane proteins with D-alanine transport capacity in C. trachornatis may reflect dependency upon an exogenous source of D-alanine for peptidoglycan synthesis in the absence of a h which would be able to convert Lalanine to D-alanine.…”