An alkali-sensitive mutant, 38154, of the alkalophilic Bacillus sp. strain C-125 could not grow at an alkaline pH. The nucleotide sequence of a 3.7 kb parental DNA fragment that recovers the growth of 38154 at alkaline pH has four open reading frames (ORF1-4). By subcloning the fragment, we demonstrated that a 0.25 kb DNA region is responsible for the recovery. Direct sequencing of the mutant's corresponding region revealed a G to A substitution. The mutation resulted in an amino acid substitution from Gly-393 to Arg of the putative ORF1 product, which was deduced to be an 804-amino-acid polypeptide with a molecular weight of 89,070. The N-terminal part of the putative ORF1 product showed amino acid similarity to those of the chain-5 products of eukaryotic NADH quinone oxidoreductases. Membrane vesicles prepared from 38154 did not show membrane potential (delta psi)-driven Na+/H+ antiporter activity. Antiporter activity was resumed by introducing a parental DNA fragment which recovered the mutant's alkalophily. These results indicate that the mutation in 38154 affects, either directly or indirectly, the electrogenic Na+/H+ antiporter activity. This is the first report which shows that a gene responsible for the Na+/H+ antiporter system is important in the alkalophily of alkalophilic microorganisms.

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Sha (also known as Mrp/Mnh/Pha) is a Na؉ /H ؉ antiporter encoded by a cluster of six or seven genes that probably form a multisubunit transport complex. The Sha system is important for the homeostasis of H ؉ , Na ؉ , and other monovalent cations and plays a critical role in various functions, including alkaliphily, sporulation, and symbiosis. Here, we characterized the sha homologue genes from the opportunistic pathogen Pseudomonas aeruginosa, which exist as a cluster of six genes (PA1054 to PA1059). The gene cluster PA1054 to PA1059, but not the cluster with a deletion of PA1054, complemented a growth defect in the presence of 0.2 M NaCl and a defect in Na ؉ /H ؉ antiport activity of the Escherichia coli TO114 mutant lacking the three major Na ؉ /H ؉ antiporters, indicating that genes PA1054 to PA1059 are responsible for Na ؉ /H ؉ antiport activity. We disrupted PA1054 (a shaA homologue gene) and determined its effect on Na ؉ tolerance during growth, Na ؉ efflux, and pathogenicity in mice. Disruption of PA1054 resulted in severe Na ؉ sensitivity during growth and decreased Na ؉ efflux activity. In mice, the deletion mutant of PA1054 also exhibited an attenuated virulence in systemic, pulmonary, and urinary tract infections and also a decrease in colonization of the infected organs. From these results, we conclude that the genes PA1054 to PA1059 encode a Na ؉ /H ؉ antiporter that is largely responsible for Na ؉ extrusion in P. aeruginosa and has a role in the infection of the pathogen. We propose to designate PA1054 to PA1059 as the sha (sodium hydrogen antiporter) genes, shaABCDEFG.

Two alkali-sensitive mutants of alkalophilic Bacillus sp. strain C-125 were obtained. Mutant 38154 showed defective regulation of internal pH. Plasmids pALKi and pALK2, containing DNA fragments different from those of the parent strain, were able to recover alkalophily in mutants 18224 and 38154, respectively. DNA analysis suggested that the two fragments overlapped on the chromosomal DNA of strain C-125.

Using the characteristic morphological changes of mammaliancells, we screened novel antimitotic substances and found that a strain of Streptomyces sp. No.9885 produced FR182877. This substance was isolated from the culture broth by ethyl acetate extraction, silica gel column chromatography and ODScolumn chromatography Structural studies on FR1 82877 suggested that it had a unique hexacyclic structure encompassing its highly strained double bond. FR182877 exhibited potent antitumor activities against murine ascitic tumor and solid tumor in vivo.