Suppression mutations in the defective beta subunit of F1-ATPase from Escherichia coli

The Escherichia coli mutant of the proton-translocating ATPase KF11 (Kanazawa, H., Horiuchi, Y., Takagi, M., Ishino, Y., and Futai, M. (1980) J. Biochem. (Tokyo) 88, 695-703) has a defective beta subunit with serine being replaced by phenylalanine at codon 174. Four suppression mutants (RE10, RE17, RE18, and RE20) from this strain capable of growth on minimal plate agar supplemented by succinate were isolated. The original point mutation at codon 174 was intact in these strains. Additional point mutations, Ala-295 to Thr, Gly-149 to Ser, Leu-400 to Gln, Ala-295 to Pro, for RE10, RE17, RE18, and RE20, respectively, were identified by the polymerase chain reaction and sequencing. These mutations, except for RE10, were confirmed as a single mutation conferring a suppressive phenotype by genetic suppression assay using KF11 as the host cells. The results indicated that Ser-174 has functional interaction with Gly-149, Ala-295, and Leu-400. The residues are located within the previously estimated catalytic domain of the beta subunit, indicating that this domain is indeed folded for the active site of catalytic function. Growth rates of the revertants in the minimal medium with succinate increased compared with that of KF11, showing that ATP synthesis recovered to some extent. The ATP hydrolytic activity in the revertant membranes increased in RE17 and RE20 but did not in RE10 and RE18, suggesting that synthesis and hydrolysis are not necessarily reversible in the proton-translocating ATPase (F1F0).     

Isolation and characterization of plasmid-deletion derivatives from an Hfr made with Rts1 plasmid carrying a chromosomal mutation, plt

Summary Rts1 is a kanamycin-resistance plasmid and multiphenotypically thermosensitive. The detrimental host cell growth at 42° C is expressed only when it exitst autonomously but not in an integrated state. However, a cholate-resistant (plt) mutant of the Hfr with an integrated Rts1 plasmid was found to be thermosensitive like a strain with the same plasmid autonomoulsy. This thermosensitivity depends on the existence of the integrated plasmid. Deletion derivatives of integrated plasmid genome from this Hfr strain were isolated with or without thermal selective growth at 42° C and mapping of the plasmid was attempted by analyzing them. A total of 141 kanamycin-sensitive derivatives were independently isolated and examined for their thermosensitivity (genetic locus: tsg), incompatibility (genetic locus: incT), conjugal fertility (genetic locus: tra), restriction of T4 phase (two genetic loci: resA and resB) and for DNase activity (genetic locus: dns). On the basis of characterization of 141 deletion derivatives, they were divided into 15 patterns which would correspond to a linear map integrated into the chromosome: ... resA ... dns ... resB ... tra ... kan ... incT ... tsg ... It is noteworthy that restriction of T4D phage is determined by two distinct genes, resA and resB, intervened by dns and that propagation of T4D phage on a strain with a resA ⁺ resB ^- genome failed to produce modified progeny phages.     

An electron microscopic study on the mating tube formation in the heterobasidiomycete Tremella mesenterica

Ultrastructure of the mating tube formed in yeast haplont of the heterobasidiomycete Tremella mesenterica was studied by electron microscopy. Cell wall of the mating tube emerged as evagination of the inner layers, rupturing outer layers of the mother cell wall. Comparison with budding cells suggested that the tube emergence place at bud scar and the process of tube emergence was the same as that of bud emergence. Electron transparent vesicles of 0.1 m diameter were scattered in the cytoplasm of the mating tube. Nucleus-associated organelle was located at one side of the nuclear envelope which extended towards the mating tube. A few microtubules were detected in the mating tube, but their association with a nucleus was not clear. The cytoplasmic structure of the mating tube was discussed in comparison with that of hyphae of the filamentous fungi.     

Proton translocation coupled to trimethylamine N-oxide reduction in anaerobically grown Escherichia coli.

Proton translocation coupled to trimethylamine N-oxide reduction was studied in Escherichia coli grown anaerobically in the presence of trimethylamine N-oxide. Rapid acidification of the medium was observed when trimethylamine N-oxide was added to anaerobic cell suspensions of E. coli K-10. Acidification was sensitive to the proton conductor 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile (SF6847). No pH change was shown in a strain deficient in trimethylamine N-oxide reductase activity. The apparent H+/trimethylamine N-oxide ratio in cells oxidizing endogenous substrates was 3 to 4 g-ions of H+ translocated per mol of trimethylamine N-oxide added. The addition of trimethylamine N-oxide and formate to ethylenediaminetetraacetic acid-treated cell suspension caused fluorescence quenching of 3,3'-dipropylthiacarbocyanine [diS-C3-(5)], indicating the generation of membrane potential. These results indicate that the reduction of trimethylamine N-oxide in E. coli is catalyzed by an anaerobic electron transfer system, resulting in formation of a proton motive force. Trimethylamine N-oxide reductase activity and proton extrusion were also examined in chlorate-resistant mutants. Reduction of trimethylamine N-oxide occurred in chlC, chlG, and chlE mutants, whereas chlA, chlB, and chlD mutants, which are deficient in the molybdenum cofactor, could not reduce it. Protons were extruded in chlC and chlG mutants, but not in chlA, chlB, and chlD mutants. Trimethylamine N-oxide reductase activity in a chlD mutant was restored to the wild-type level by the addition of 100 microM molybdate to the growth medium, indicating that the same molybdenum cofactor as used by nitrate reductase is required for the trimethylamine N-oxide reductase system.     

Corelike structures in a stable L-form ofStreptococcus pyogenes

Corelike structures, which were interpreted as straight, large cylinders containing ribosome-like particles surrounded by an amorphous substance of low electron density, were found in a stable L-form ofStreptococcus pyogenes grown in the absence of antibiotics.     

Plant growth-regulating activities of batatasin III analogues

Nine bibenzyls and 10 stilbenes were synthesized as analoguesof batatasin III, a growth inhibitor isolated from dormant yambulbils, and examined for their plant growth-regulating activities.The bioassays used were the elongation of dark-grown intactrice coleoptiles, auxin-induced elongation of excised oat coleoptiles,and germination of rape and barnyard grass seeds. In the elongationof intact rice coleoptiles, 3,3'-dihydroxy-5-methoxy- (batatasinIII), 3,5-dimethoxy-3'-nitro-, 4'-bromo-3-nitro-, 3-amino-3'-chloro-,3-amino-4'-chloro-bibenzyls and 3-benzyloxy-4'-bromo-5-methoxy-,3-benzyloxy-3',4'-dichloro-5-methoxy-stilbenes were inhibitory,and 4'-bromo-3-nitrostilbene was promotive at a concentrationof 100 mg/liter. The results obtained by the other bioassayswere qualitatively consistent with these findings, although3-amino-4'- chlorobibenzyl and 4'-bromo-3-nitrostilbene werenot tested in all the bioassays. In the seed germination, which was rather tolerant to the testanalogues, batatasin III was inactive but 3-benzyloxy-4'-bromo-5-methoxy-and 3-benzyloxy-3',4'-dichloro- 5-methoxystilbenes were veryactive. Thus, if substituted properly, bibenzyls and stilbenes are activewithout hydroxyl and methoxyl group(s) as the functional group. ³ Present address: The National Institute for EnvironmentalStudies, Yatabc, Ibaraki 300-21, Japan. (Received November 19, 1975; )     

A synthesis of 3′,4-́dideoxykanamycin B

3′,4′-Dideoxykanamycin B, the kanamycin B derivative that is active against resistant bacteria, was prepared from kanamycin B viaN-tosylation, 3′,4′-O-sulphonylation, 3′,4′-unsaturation, and hydrogenation. The unsaturated intermediate was obtained from the 3′,4′-di-O-sulphonyl derivatives by the action of sodium iodide in N,N-dimethylformamide; if zinc dust was added in this reaction, aziridine derivatives were formed, Removal of the tosyl group was successfully performed by using sodium in ammonia-ethylamine.