Masayuki Kuroda

The construction of a cysteine-less melibiose carrier from E. coli

The melibiose carrier of E. coli is a cation-sugar cotransport system. This membrane protein contains four cysteine residues and the transport function is inhibited by sulfhydryl reagents. In order to investigate the importance of the cysteines, we have constructed a set of four melibiose transporters each of which has one cysteine replaced with serine or valine. The sensitivity of this set of carriers to N-ethylmaleimide was tested and Cys364 was identified as the target of the reagent. In addition, we constructed a melibiose transporter in which all 4 cysteines were replaced with either serine (Cys110, Cys310, and Cys364) or valine (Cys235) and we found that, as expected, the resulting cysteine-less transporter was resistant to the action of N-ethylmaleimide. The cysteine-less melibiose carrier had no significant decrease in ability to accumulate melibiose with cotransported sodium ions or protons. Thus, none of the 4 cysteines are necessary for the function of the melibiose carrier.

Cloning and sequencing of the meIB gene encoding the melibiose permease of Salmonella typhimurium LT2

SummaryThe nucleotide sequence of the melB gene coding for the Na+(Li+)/melibiose symporter of Salmonella typhimurium LT2 was determined, and its amino acid sequence was deduced. It consists of 1428 bp, corresponding to a protein of 476 amino acid residues (calculated molecular weight 52800). The amino acid sequence is homologous to that of the melibiose permease of Escherichia coli K12, with 85% identical residues. All, except one, of the amino acid residues that have been reported to be important for cation or substrate recognition in the melibiose permease of E. coli are conserved in the melibiose permease of S. typhimurium. In addition, part of the sequence resembles the lactose permease of Streptococcus thermophilus, the animal glucose transporter (GLUT1), the plasmid-coded raffinose permease (RafB), and the NADH-ubiquinone oxidoreductase chain 4 (Nuo4) of Aspergillus amstelodami.

A Major Li^+ Extrusion System NhaB of Pseudomonas aeruginosa : Comparison with the Major Na^+ Extrusion System NhaP

A gene encoding a Li+ extrusion system was cloned from the chromosomal DNA of Pseudomonas aeruginosa and expressed in Escherichia coli cells. The gene enabled growth of E. coli KNabc cells, which were unable to grow in the presence of 10 MM LiCl or 0.1 M NaCl because of the lack of major Na+(Li+)/H+ antiporters. We detected Li+/H+ and Na+/H+ antiport activities in membrane vesicles prepared from E. coli KNabc cells that harbored a plasmid carrying the cloned gene. Activity of this antiporter was pH‐dependent with an optimal pH activity between pH 7.5 and 8.5. These properties indicate that this antiporter is different from NhaP, an Na+/H+ antiporter from P. aeruginosa that we reported previously, and that is rather specific to Na+ but it cannot extrude Li+ effectively. The gene was sequenced and an open reading frame (ORF) was identified. The amino acid sequence deduced from the ORF showed homology (about 60% identity and 90% similarity) with that of the NhaB Na+/H+ antiporters of E. coli and Vibrio parahemolyticus. Thus, we designated the antiporter as NhaB of P. aeruginosa. E. coli KNabc carrying the nhaB gene from P. aeruginosa was able to grow in the presence of 10 to 50 MM LiCl, although KNabc carrying nhaP was unable to grow in these conditions. The antiport activity of NhaB from P. aeruginosa was produced in E. coli and showed apparent Km values for Li+ and Na+ of 2.0 MM and 1.3 MM, respectively. The antiport activity was inhibited by amiloride with a Ki value for Li+ and Na+ of 0.03 MM and 0.04 MM, respectively.

Characterization of a Novel Vibrio parahaemolyticus Phage, KVP241, and Its Relatives Frequently Isolated from Seawater

A vibriophage, KVP241, and six of its relatives were isolated independently from seawater using Vibrio parahaemolyticus as the host. All of the phages had the same morphology (a hexagonal head and a tail with a contractile sheath) and the same host range (specific for some V. parahaemolyticus strains). DNA‐DNA hybridization experiments elucidated that their genomes are highly homologous to each other. Analyses of amino acid sequences of putative major capsid proteins indicated that KVP241 may be weakly related to T4‐type phages having a more elongated head.

ARSENICAL PUMPS IN PROKARYOTES AND EUKARYOTES

Publisher Summary This chapter reviews the assays developed to measure the activities in cells, membrane preparations, and purified proteins. Whenever organisms are put under stress, they develop compensatory mechanisms. Bacteria, lower eukaryotes, and mammals have resistance mechanisms to arsenicals and antimonials. Bacteria have evolved a secondary oxyanion carrier that can convert to a primary adenosine triphospate (ATP)-coupled pump in association with an ATP-binding cassette (ABC) protein that is unrelated to members of the P-glycoprotein family. Leishrnania have evolved an ATP-coupled As-thiol pump. The substrates of the prokaryotic and eukaryotic systems are different but the results are the same: the lowering of intracellular metalloid concentration to produce resistance. Although the overall scheme is remarkably similar between kingdoms, the specific mechanisms and the proteins that produce them appear to be the results of independent, convergent evolution.

Characteristics of the melibiose transporter and its primary structure in Enterobacter aerogenes.

Cells of Enterobacter aerogenes can grow on melibiose as a sole source of carbon. This suggests the presence of melibiose operon in this organism. We found that E. aerogenes cells possess both alpha-galactosidase activity and melibiose transport activity, which were induced by melibiose. Neither Na+ nor Li+ stimulated the melibiose transport. However, transport of methyl-beta-thiogalactoside (TMG) was stimulated by Li+ but not by Na+. These findings suggest that the major coupling cation for the melibiose transporter in E. aerogenes is H+. In fact, we observed H+ entry into cells caused by an influx of melibiose and some of its analogs. We cloned the melB gene which encodes the melibiose transporter, and sequenced it. Deduced amino acid sequence of the transporter revealed that the melibiose transporter consists of 471 amino acid residues and the molecular weight was calculated to be 52214 Da. The sequence showed high homology with the sequences of the melibiose transporters of Escherichia coli, Salmonella typhimurium and Klebsiella pneumoniae. Higher homology was found with the melibiose transporter of K. pneumoniae than with that of E. coli and S. typhimurium.

Sequence of a melibiose transporter gene of Enterobacter cloacae

We cloned a fragment of the chromosomal DNA of Enterobacter cloacae, which enabled a melibiose-negative Escherichia coli mutant lacking melB to grow on melibiose as the sole source of carbon. Transformed cells harboring the hybrid plasmid carrying the cloned DNA showed melibiose transport activity. The nucleotide sequence of the DNA region was determined. One complete open reading frame (ORF) and a part of another ORF were found in the region, and the amino acid sequences were deduced. The complete ORF was found to encode a melibiose transporter which consisted of 425 amino acid residues. Hydropathy analysis revealed that there are about 12 hydrophobic domains in this transporter. The incomplete ORF which exists in the upstream region of the transporter gene seemed to encode an alpha-galactosidase.