Frank T. Robb

Evolutionary divergence between sympatric species of southern African hakes, Merluccius capensis and M. paradoxus. II: Restriction enzyme analysis of mitochondrial DNA

We used 11 restriction endonucleases to measure nucleotide sequence variation in mitochondrial DNA (mtDNA) within and between two species of hake from the coastal waters of South Africa. A total of 14 different composite genotypes were observed among 26 individuals of Merluccius capensis, but only 6 composite genotypes were observed for 24 individuals of M. paradoxus. A parsimony network connecting the composite genotypes for these species did not correspond with the geographies of either set of samples. In M. capensis, the restriction patterns of three enzymes (Ava I, Xba I, Xho I) and the network of composite genotypes indicate that an addition of about 400 nucleotides in length and a deletion of 200 nucleotides have occurred in pathways leading from the most common genotype. The amount of nucleotide site polymorphism for M. capensis was 0·022 and was significantly greater than the level of polymorphism in M. paradoxus, which was 0·009. These results suggest that M. paradoxus may have experienced a population bottleneck in the past. The amount of sequence divergence between these species was 11·6 per cent (±0·036) and is typical for well differentiated species. Using the assumptions of the molecular clock, this represents a divergence time of 5·8 (±1·8) million years.

Regulation of exoprotease production by temperature and oxygen in Vibrio alginolyticus

The production of an extracellular collagenase and an alkaline protease by Vibrio alginolyticus during stationary phase was inhibited by a temperature shift from 30 to 37°C and by a lack of oxygen. The stability of the exoproteases was unaffected by incubation at 37°C and aeration. The optimum growth temperature for the V. alginolyticus strain was 33.5°C Aeration enhanced the rate of growth of exponential phase cells. Temperature and oxygen did not affect the growth of stationary phase cells when the exoproteases were being produced. Macromolecular synthesis in stationary phase cells was not affected by temperature. There was no rapid release of the exoproteases after temperature shift down and chloramphenicol inhibited the production of the enzymes when added at time of temperature shift down from 37 to 30°C. The regulation of exoprotease production by temperature and oxygen was specific and has implications regarding the ecology of V. alginolyticus. Cerulenin, quinacrine and O-phenanthroline inhibited the production of the exoproteases.

Nucleotide sequence and analysis of the Vibrio alginolyticus sucrase gene (scrB)

The nucleotide sequence of a 2.119-kb DNA fragment containing the Vibrio alginolyticus sucrase gene (scrB) was determined. The complete sequence (484 aa residues) of the sucrase was deduced and homology was detected between the sucrase enzymes from V. alginolyticus and the Gram-positive bacteria Bacillus subtilis and Streptococcus mutans. In Escherichia coli cells the cloned V. alginolyticus sucrase is translocated to the periplasm. Transposon phoA mutagenesis experiments strongly suggested that V. alginolyticus sucrase in E. coli is not exported across the cytoplasmic membrane by means of a typical signal sequence.

Isolation and Characterization of a Vibrio alginolyticus Mutant That Overproduces Extracellular Proteases

Summary: The Vibrio alginolyticus prot-T1 mutant was able to produce haloes of clearing on skim milk/peptone agar plates at 42°C whereas proteolysis by the wild-type strain was inhibited at 37°C. The prot-T1 mutant overproduced the three major alkaline proteases with apparent molecular masses of approximately 28000, 22500 and 19500 (proteases 1a, 2 and 3, respectively). Their synthesis was not markedly repressed by incubation at 37°C or by non-aeration. Both treatments inhibited protease synthesis in the wild-type strain, which only produced proteases during the stationary growth phase. The prot-T1 mutant synthesized proteases throughout the exponential and stationary growth phases in peptone medium. High protease activities were induced by glucose or glutamine in stationary phase prot-T1 that were pre-grown in peptone medium. Glucose or glutamine had the opposite effect on protease activities in stationary phase prot-T1 cultures that were pre-grown in minimal medium. Collagenase synthesis was not altered in the prot-T1 mutant and was repressed by growth at 37° or without aeration. The independent control of collagenase synthesis supports the conclusion that there are no regulatory proteins responsible for the overall control of extracellular protease synthesis by temperature, aeration and growth phase in V. alginolyticus.

Purification and regulation of glutamine synthetase in a collagenolytic Vibrio alginolyticus strain

Glutamine synthetase (EC 6.3.1.2) has been purified from a collagenolytic Vibrio alginolyticus strain. The apparent molecular weight of the glutamine synthetase subunit was approximately 62,000. This indicates a particle weight for the undissociated enzyme of 744,000, assuming the enzyme is the typical dodecamer. The glutamine synthetase enzyme had a sedimentation coefficient of 25.9 S and seems to be regulated by a denylylation and deadenylylation. The pH profiles assayed by the γ-glutamyltransferase method were similar for NH4-shocked and unshocked cell extracts and isoactivity point was not obtained from these eurves. The optimum pH for purified and crude cell extracts was 7.9. Cell-free glutamine synthetase was inhibited by some amino acids and AMP. The transferase activity of glutamine synthetase from mid-exponential phase cells varied greatly depending on the sources of nitrogen or carbon in the growth medium. Glutamine synthetase level was regulated by nitrogen catabolite repression by (NH4)2SO4 and glutamine, but cells grown, in the presence of proline, leucine, isoleucine, tryptophan, histidine, glutamic acid, glycine and arginine had enhanced levels of transferase activity. Glutamine synthetase was not subject to glucose, sucrose, fructose, glycerol or maltose catabolite repression and these sugars had the opposite effect and markedly enhanced glutamine synthetase activity.

Nucleotide sequence of the Vibrio alginolyticus glnA region

The nucleotide sequence of a 4 kb fragment containing the Vibrio alginolyticus glnA, ntrB and ntrC genes was determined. The upstream region of the glnA gene contained tandem promoters. The upstream promoter resembled the consensus sequence for Escherichia coli σ70 promoters whereas the presumptive downstream promoter showed homology with nitrogen regulated promoters. Four putative NRI binding sites were located between the tandem promoters. The ntrB gene was preceded by a single presumptive NRI binding site. The ntrC gene was located 45 base pairs downstream from the ntrB gene. The V. alginolyticus ntrB and ntrC genes were able to complement ntrB, ntrC deletions in E. coli.

Temperature activation of foot muscle d-(−)-lactate dehydrogenase in the whelk Bullia digitalis

Abstract The rate of oxygen consumption of the sandy-beach whelk Bullia digitalis is virtually temperature-independent over the thermal range typical of its environment. This may be a physiological adaptation to sudden and unpredictable environmental temperature changes. Physiological adaptation mechanisms include the development of temperature-sensitive isozymes, often regulated by enzyme-substrate interactions. Examination of kinetics of d -(−)-lactate dehydrogenase (EC 1.1.1.28) in B. digitalis revealed differential temperature sensitivity of the two major isozymes. It is concluded that this mechanism may contribute to the successful adjustment of the whelks metabolism to its environmental temperature, without impairment of physical activity.

Temperature and oxygen regulated expression of a glutamine synthetase gene fromVibrio alginolyticus cloned inEscherichia coli

Glutamine synthetase (GS) synthesis inVibrio alginolyticus was regulated by temperature, oxygen and nitrogen levels. A GS gene,glnA fromV. alginolyticus was cloned on a 5.67 kb insert in the recombinant plasmid pRM210, which enabledEscherichia coli glnA, ntrB, ntrC deletion mutants to utilize (NH4)2SO4 as a sole source of nitrogen. TheV. alginolyticus glnA gene was expressed from a regulatory region contained within the cloned fragment.V. alginolyticus glnA expression from pRM210 was subject to regulation by temperature, oxygen and nitrogen levels. GS specific activity in anE. coli wild-type strain was not affected by temperature or oxygen. pRM211 was a deletion derivative of pRM210 and GS production by pRM211 was not regulated by temperature, oxygen or nitrogen levels inE. coli.

Physiological and Morphological Characteristics of Stationary Phase Vibrio Cells Able to Support Phage Growth

Summary: Growth of phage α3a on stationary phase Vibrio cultures requires micro-aerophilic conditions and is inhibited by aeration. Since pre-conditioning of the bacteria by allowing them to stand for 24 h after shaking for 3 d is an important aspect of the stationary phase phage growth system, various physiological and morphological characteristics of the stationary phase cells during the transition from shaking to standing were investigated. Shaken stationary phase cells were less viable and more sensitive to ultraviolet irradiation and heat than standing stationary phase cells. During pre-conditioning the small, non-flagellated cells present in shaken stationary phase cultures underwent morphological changes and became large, flagellated rods which resembled exponential phase cells. The transition of stationary phase cells from shaking to standing was associated with a marked increase in total RNA synthesis but a rapid and large decrease in total protein synthesis. Intracellular concentrations of ATP in shaken stationary phase cells were 53 % lower than those in standing stationary phase cells. Studies on leucine uptake indicated that its transport was inhibited by isoleucine and that the major part (90 %) of the total leucine uptake was due to a shared system for uptake of both amino acids. Shaken stationary phase cells transported less leucine than standing stationary phase cells. Inhibition of phage growth in aerated stationary phase cultures was not due to the prevention of phage adsorption by shaking. It is suggested that the observed differences between shaken and standing stationary phase cells could be due to aeration affecting the template specificity of the Vibrio RNA polymerase.

Cloning, Expression and Release of a Vibrio alginolyticus SDS-resistant Ca2+-dependent Exoprotease in Escherichia coli

SUMMARY: A Vibrio alginolyticus SDS-resistant, Ca2+-dependent serine exoprotease gene, cloned on a 7-1 kb DNA fragment in the recombinant plasmid pVP100, was expressed from its own promoter in Escherichia coli. Although active exoprotease was produced by late stationary phase E. coli(pVP100) cultures after 15 h incubation in proteinaceous medium containing Ca2+, transcription and translation of the exoprotease occurred before 6 h, during exponential growth. The cloned exoprotease was synthesized as a pool of inactive precursor molecules during exponential growth, and released as active exoprotease 8 h later by a process which did not require protein synthesis or involve cell lysis. Release of the exoprotease by E. coli(pVP100) was not inhibited by o-phenanthroline, quinacrine or cerulenin. Supernatant samples from E. coli(pVP100) cultures contained two SDS-resistant exoproteases with apparent M r values of approximately 54000 and 39000. The cloned exoprotease activity was inhibited by EDTA and a serine protease inhibitor, but was not affected by an inhibitor of trypsin-like enzymes.