The nature of the neutral Na+−Cl− coupled entry at the apical membrane of rabbit gallbladder epithelium: III. Analysis of transports on membrane vesicles

Summary In rabbit gallbladder epithelium, a Na⁺/H⁺, Cl^–/HCO ₃ ^– double exchange and a Na⁺–Cl^– symport are both present, but experiments on intact tissue cannot resolve whether the two transport systems operate simultaneously. Thus, isolated apical plasma membrane vesicles were prepared. After preloading with Na⁺, injection into a sodium-free medium caused a stable intravesicular acidification (monitored with the acridine orange fluorescence quenching method) that was reversed by Na⁺ addition to the external solution. Although to a lesser extent, acidification took place also in experiments with an electric potential difference (PD) equal to 0. If a preset pH difference (pH) was imposed ([H⁺]_in>[H⁺]_out, PD=0), the addition of Na-gluconate to the external solution caused pH dissipation at a rate that followed saturation kinetics. Amiloride (10^–4 m) reduced the pH dissipation rate. Taken together, these data indicate the presence of Na⁺ and H⁺ conductances in addition to an amiloride-sensitive, electroneutral Na⁺/H⁺ exchange.An inwardly directed [Cl^–] gradient (PD=0) did not induce intravesicular acidification. Therefore, in this preparation, there was no evidence for the presence of a Cl^–/OH^– exchange.When both [Na⁺] and [Cl^–] gradients (outwardly directed, PD=0) were present, fluorescence quenching reached a maximum 20–30 sec after vesicle injection and then quickly decreased. The decrease was not observed in the presence of a [Na⁺] gradient alone or the same [Na⁺] gradient with Cl^– at equal concentrations at both sides. Similarly, the decrease was abolished in the presence of both Na⁺ and Cl^– concentration gradients and hydrochlorothiazide (5×10^–4 m). The decrease was not influenced by an inhibitor of Cl^–/OH^– exchange (10^–4 m furosemide) or of Na⁺–K⁺–2Cl^– symport (10^–5 m bumetanide).We conclude that a Na⁺/H⁺ exchange and a Na⁺–Cl^– symport are present and act simultaneously. This suggests that in intact tissue the Na⁺–Cl^– symport is also likely to work in parallel with the Na⁺/H⁺ exchange and does not represent an induced homeostatic reaction of the epithelium when Na⁺/H⁺ exchange is inhibited.     

A limited number of Bacillus subtilis strains carry a tetracycline-resistance determinant at a site close to the origin of replication

Several strains of Bacillus subtilis, e.g., 168 derivatives and R, were found to carry a single copy of a tetracycline-resistance (TcR) determinant (named tetBS908) at a site close to the origin of replication on the chromosome. This gene is highly homologous (80% identical) to the TcR determinant of plasmids widely dispersed among aerobic spore-forming bacilli. B. subtilis RM125 (168 strain) transformants which carry a varying number of tetBS908 sequences in a tandem array on the chromosome were constructed and examined for their TcR level. A nearly proportional relationship between the TcR level and copy number of tetBS908 existed.     

Nucleotide sequence of the Bacillus subtilis ribosomal RNA operon, rrnB

The primary sequence of DNA covering a complete ribosomal RNA (rRNA) operon from Bacillus subtilis, designated rrnB has been elucidated. Following a set of tandem promoters, rrnB encodes: (i) a 16S and a 23S rRNA determinant with no tRNA spacer region in between; (ii) a 5S rRNA determinant; and (iii) 21 contiguous tRNA species; before (iv) two characteristic terminator hairpins are found. More than 7 kb are included within this operon, which maps between aroG and thr5 on the B. subtilis chromosome. This represents the first report of the entire sequence of an rRNA operon from B. subtilis or from any Gram-positive organism.     

Membrane thickness changes ion-selectivity of channel-proteins.

The plasma membrane is a highly dynamic cell-barrier if the nature and distribution of its constituents are considered. Ion channels are embedded in these double lipid bilayers, which modulate their 3D-structures. The structure modulations by the lipid bilayer can assume such a degree that channel activation depends on them, as was shown for the KcsA potassium channel. Here we show that the cation-over-anion selectivity of reconstituted ICln channels can be varied by the thickness of a bilayer build of phosphatidylcholines. The shorter the acyl-chains and therefore the thinner the bilayers of the membrane are, the more potassium selective the channels are. In contrast, the longer the acyl-chains and therefore the thicker the membranes are, the more chloride selective the channels become.     

Analysis of the tet gene of plasmid pCIS7 isolated from Bacillus subtilis

We have previously shown that plasmid pCIS7, which contains 11.5 kb of Bacillus subtilis DNA isolated from a tetracycline-sensitive (TcS) strain, confers Tc resistance when integrated and amplified in the chromosome of TcS B. subtilis 168trpC2 [Ives and Bott, J. Bacteriol. 171 (1989) 1801-1810]. Here, we report that the number of integrated plasmid sequences required to confer Tc resistance is greater than the 20 copies seen with increasing chloramphenicol selection and, by dot-blot analysis, exceeds 100 copies per cell. The amplification is accompanied by a corresponding increase in mRNA encoding the tet gene. The tet gene sequence of pCIS7 has been compared to B. subtilis tetGSY908 [Sakaguchi et al., Biochim. Biophys. Acta. 94 (1988) 49-57] and other Gram-positive tet genes. The tet gene of pCIS7 is a member of the class L TcR determinants, and probably confers Tc resistance by increasing the efflux of Tc from the bacterial cell.     

Acrylamide Gel Electrophoresis of Intracellular Proteins During Early Stages of Sporulation in Bacillus subtilis

Acrylamide gel electrophoresis of unfractionated cellular extracts of Bacillus subtilis is shown to be an effective method for characterizing many of the changes in protein composition, when coupled with specific histological-type staining reactions. The results obtained here by using extracts from cells at different stages of growth and sporulation are consistent with observations from other laboratories where extensively purified and highly characterized enzymes have been studied. In several instances, the histochemical reactions can be associated with a specific enzymatic function and appear to indicate the presence of multiple molecular forms. In other instances, the data cannot be evaluated in terms of known enzyme function because the specificity of the histochemical analysis is not certain. However, the assays described permit monitoring of electrophoretic changes at the level of individual proteins within sporulating cultures. The results suggest that B. subtilis may contain two "hexokinase-like" enzymes which cease to function before sporulation is initiated. Aldolase and alanine dehydrogenase are detectable as single bands of enzyme activity during vegetative growth but as multiple molecular forms once sporulation has been initiated. Reduced nicotinamide adenine dinucleotide dehydrogenase activity is represented by an entire family of reactive species in these crude extracts, which undergo multiple changes during the early stages of sporulation. Tricarboxylic acid cycle dehydrogenase enzymes and those bands having esterase activity on alpha-naphthyl acetate show detectable changes in specific activity after cessation of exponential growth. Glucose dehydrogenase is not detectable until the sequence of changes leading to spore formation has progressed for 4 or 5 hr.     

Coupling of carbon monoxide oxidation to CO2 and H2 with the phosphorylation of ADP in acetate-grown Methanosarcina barkeri

Cell suspensions of Methanosarcina barkeri, grown on acetate, catalyzed the conversion of carbon monoxide and H2O to CO2 and H2 in stoichiometric amounts when methane formation was inhibited by bromoethanesulfonate. The specific activity was 80-120 nmol min-1 mg protein-1 at 5% CO in the gas phase. CO oxidation was coupled with the phosphorylation of ADP as indicated by a rapid increase of the intracellular ATP level upon start of the reaction. At least 0.1 mol ATP was formed/mol CO consumed. The onset of CO oxidation was also accompanied by an increase of the proton motive force (delta p) from 100 mV to 150 mV (inside negative). Addition of the uncoupler tetrachlorosalicylanilide to CO-metabolizing cells led to a rapid decrease of the ATP level and of delta p, and to an increase of the CO oxidation rate up to 70%. In the presence of the proton-translocating ATPase inhibitor N,N'-dicyclohexylcarbodiimide the phosphorylation of ADP was inhibited and CO oxidation slowed down, whereas delta p was almost unaffected. Inhibition of CO oxidation under these conditions was relieved by the addition of the protonophore tetrachlorosalicylanilide. The results indicate that in acetate-grown M. barkeri the free-energy change associated with the formation of CO2 and H2 from CO and H2O (delta G degrees = -20 kJ/mol) can be used to drive the phosphorylation of ADP and that the coupling proceeds via a chemiosmotic mechanism. A possible role of the carbon monoxide oxidation reaction as an energy-conserving site in acetate fermentation to CH4 and CO2 is discussed.     

Mutation affecting expression of spectinomycin resistance in Bacillus subtilis.

A mutation that affects the expression of spectinomycin resistance in a spectinomycin-resistant (spcA), conditionally asporogenic strain of Bacillus subtilis has been designated srm (spectinomycin resistance modifier). This mutation resulted in altered colony morphology and increased growth rate and sporulation efficiency in the presence of spectinomycin.