Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus.

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.

Salt tolerance of lactose-grown Vibrio parahaemolyticus carrying Escherichia coli lac genes.

Lac- strains of Vibrio parahaemolyticus were converted to Lac+ on receiving a hybrid plasmid containing the lactose utilization genes of Escherichia coli K-12. A V. parahaemolyticus strain containing this hybrid plasmid exhibited optimal growth rates on glucose and other carbon sources in the presence of 0.2 to 0.4 M NaCl. Growth of the same strain on lactose was inhibited at similar concentrations of NaCl. The altered growth rate responses in lactose medium appeared to be attributable to effects of NaCl on the activity of lactose permease, and possibly on that of beta-galactosidase, rather than on the levels of these enzymes in V. parahaemolyticus cells.

Shuttle cloning vectors for the marine bacterium Vibrio parahaemolyticus.

Two cosmid cloning vectors containing lambda cos sequences and a 42-base-pair multipurpose cloning sequence were constructed. pAD22 also contains a 1.4-kilobase TRP-ARS fragment from Saccharomyces cerevisiae. These cosmids transformed Escherichia coli and S. cerevisiae cells and could be mobilized into Vibrio parahaemolyticus strains with a conjugative plasmid, pRK2013. The cosmid pAD22 was genetically and structurally stable during passage through V. parahaemolyticus and E. coli strains.

Photorepair of ultraviolet-induced petite mutational damage in Saccharomyces cerevisiae requires the product of the PHR1 gene.

A wild-type (phr+) diploid yeast strain showed photorepair of petite mutational damage, whereas a photoreactivation-deficient (phr1/phr1) diploid strain did not, indicating that the PHR1 gene product was required for mitochondrial photorepair.

The Pasteur effect and catabolite repression in an oxidative yeast, Kluyveromyces lactis.

The presence of the Pasteur effect in Kluyveromyces lactis grown in glucose was shown by azide-stimulated glucose fermentation. Extracts from these cells contained ATP-sensitive phosphofructokinase activity. Cells grown on succinate oxidized glucose slowly at first without azide-stimulated rates of fermentation. Phosphofructokinase in these cells was ATP-insensitive. The activity of NAD+-isocitrate dehydrogenase in cell extracts did not require AMP activation. These results suggested the presence of a Pasteur effect in glucose-grown but not in succinate-grown K. lactis, mediated by (a) ATP inhibition of phosphofructokinase (b) possibly via feedback control of glucose transport, but not by AMP activation of isocitrate dehydrogenase. Azide inhibition of the Pasteur effect during growth of the cells did not lead to catabolite repression of respiratory activity. The results therefore suggest that the Pasteur effect does not inhibit the development of a Crabtree effect in oxidative yeasts.

The absence of DNA photoreactivation enzyme in yeast mitochondria.

Mitochondria isolated from haploid yeast cells by spheroplast lysis were purified by flotation on renografin gradients. Electron micrographs and respiratory control ratios revealed that the purified mitochondria were still intact and functional. Assays for photoreactivation enzyme using as substrate [3H]-thymine-labeled Escherichia coli DNA were performed on crude and purified mitochondrial preparations. While the crude preparation contained high amounts of photoreactivation enzyme, it appeared to be associated with contaminating nuclei. The purified mitochondria lacked any photoreactivation enzyme activity. We suggest that yeast mitochondria do not normally contain photoreactivation enzyme.

Evidence for an inducible glucose transport system in Kluyveromyces lactis.

To find the cause of delayed glucose oxidation in succinate-grown Kluyveromyces lactis, glucose transport was studied in glucose- and in succinate-grown cells. The initial rate of 2-deoxyglucose (2-dGlc) accumulation, as well as the appearance of 2-deoxyglucose 6-phosphate, was higher in the glucose-grown cells. In both cell types, 2-dGlc was apparently transported in the free form to be phosphorylated intracellularly. In glucose-grown cells the level of free 2-dGlc in the pool was always less than the external concentration. Exchange transport in starved, poisoned cells loaded with unlabeled 2-dGlc was 140-fold greater in glucose- than in succinate-grown cells, probably beacuse of the presence of an inducible transport component. The development of the increased rate of transport in a succinate-grown uracil-requiring auxotroph after transfer to glucose depends on the presence of uracil.

Dual effects of glucose on dicarboxylic acid transport in Kluyveromyces lactis.

The properties of succinate uptake in succinate-grown Kluyveromyces cells were examined. The rate of succinate transport at 15C exhibits an approximate V-max of 1.2 mumol times h-1 times mg-1 dry weight of cells and an apparent K-m of 18 muM. The uptake process appears to be tightly coupled to metabolism. L-Malate, fumarate, and alpha-ketoglutarate were the only other dicarboxylates tested, which were found to inhibit succinate transport. The aggreement between the order of inhibition of succinate transport by these dicarboxylates and their rates of uptake, as well as the competitive nature of the inhibition are all consistent with the existence of a common carrier system showing specificity for dicarboxylates of the TCA cycle. Cells transferred from succinate to glucose medium rapidly lose their ability to transport succinate. Glucose-grown cells also exhibit an inability to oxidize dicarboxylates or to use them for growth without a very long lag. The dicarboxylate uptake system, therefore, appears to be subject to a strong catabolite repression. The depression of the succinate transport system requires the presence of succinate, as well as low concentrations of glucose.

Glucose-induced crypticity toward succinate metabolism in Saccharomyces lactis.

Saccharomyces lactis grown on glucose adapted very slowly to growth on succinate. This initial inability of glucose-grown cells to grow on succinate was paralleled by their inability to oxidize succinate. The possibility that repression by glucose of respiratory chain components was responsible for these observations was examined. Glucose-grown cells were able to respire glucose, ethyl alcohol, and lactate and were able to initiate growth on ethyl alcohol as rapidly as succinate-grown cells. Respiratory enzyme levels were essentially the same in cells grown on succinate or on glucose. Spectroscopic analysis revealed that glucose-grown cells possessed a full complement of cytochrome bands. Since by these criteria glucose-grown S. lactis appears to possess a competent respiratory system, the penetration of succinate-2,3-(14)C into succinate- and glucose-grown cells was examined directly. Glucose-grown cells exhibited a strong permeability barrier to succinate. Comparison of glucose oxidation by S. lactis and by S. cerevisiae suggests that the crypticity to succinate does not depend upon a strong Crabtree effect in S. lactis.

Target analysis of mitochondrial genetic units in yeast.

Target analyses of the induction of rho(-) mutants indicated that the cytoplasm of an aerobic nonrepressed yeast contains approximately 20 mutable units, whereas repressed cells exhibit approximately 3. Aerobic adaptation of fully repressed cells brings about an increase in these cytoplasmic units which continues after maximal respiration has been reached. This increase can be correlated with the increase in numbers of stained mitochondria. The data are interpreted with reference to mitochondrial deoxyribonucleic acid.

Sequential replication of the chromosome of Bacillus licheniformis.

A sequential replication map of the chromosome of Bacillus licheniformis was constructed by employing the method of gene-frequency analysis presented by Yoshikawa and Sueoka. Our analysis of 11 genetic markers was based on the hypothesis that the chromosome initiated replication at a fixed origin and proceeded in a linear fashion to the terminus. The proposed locations of markers were validated by cotransformation and cotransduction analyses. Bacteriophage SP-15 cotransduced markers that failed to show linkage by transformation.