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.     

Synthesis of D-cysteine from 3-chloro-D-alanine and hydrogen sulfide by 3-chloro-D-alanine hydrogen chloride-lyase (deaminating) of Pseudomonasputida

Synthesis of D-cysteine from 3-chloro-D-alanine and hydrogen sulfide is catalyzed by highly purified 3-chloro-D-alanine hydrogen chloride-lyase from $\text{Pseudomonas}\text{putida}$. The synthetic reaction proceeds optimally at pH 8.5, as a function of enzyme concentration and incubation time. The enzymatically synthesized D-cysteine was isolated from the large scale reaction mixture and identified by physicochemical means.     

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.     

Effects of the anti-lipogenic antibiotic cerulenin on growth and fatty acid composition of n-alkane-utilizingCandida lipolytica

Summary The effects of cerulenin, an anti-lipogenic antibiotic, on the growth and cellular fatty acid composition ofCandida lipolytica were investigated by changing the chain length of n-alkane, the growth substrate. The antibiotic inhibited almost completely the growth of the yeast on glucose, n-undecane and n-dodecane, but partly that on n-tridecane. The yeast growth on longer alkanes, e.g., from n-tetradecane to n-octadecane, was not affected by this antibiotic, indicating that a chain elongation system and/or intact incorporation system predominantly operate in the formation of cellular fatty acids from such longer chain n-alkanes. Comparison of the fatty acid profiles between the cells grown on n-alkanes of different chain lengths, especially on n-pentadecane, in the presence and absence of cerulenin, supported the supposition that only the de novo synthesis system of the yeast would be affected by the antibiotic, whereas the chain elongation system would not.     

Thermodynamics of equilibria of hemoglobins M Milwaukee-I and Saskatoon and isolated chains of hemoglobin a with carbon monoxide

The thermodynamic parameters of the CO-equilibria of isolated chains of hemoglobin A and of two α-chains in hemoglobins M Milwaukee-I and Saskatoon at 25°, pH 7.0 were determined. The parameters for the binding of the first CO molecule to the hemoglobins M were ΔH′=?17 and ?18 kcal/mole heme and ΔS′=?30 and ?29 e.u. for hemoglobins M Milwaukee-I and Saskatoon, respectively. In contrast to this the characteristics of the second step of the binding were ΔH′=+5.9^· and +4.3 kcal/mole and ΔS′=+51 and +49 e.u. These values for the second step were also significantly different from those of the isolated α-chain (ΔH′=?15 kcal/mole and ΔS′=?11 e.u.).     

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.