Bibliography: pages 215-264. / The objective of this study has been to understand the metabolic interrelationship between yeast growth, regulation of glycolytic/gluconeogenic flux and accumulation of glycosyl donors for polysaccharide synthesis in brewing yeast (Saccharomyces uvarum) fermentations. Loss of fermenting power of a brewing yeast population may be created by a condition that inhibits growth by limiting amino acid formation and protein synthesis. In commercial strains of S. uvarum this loss may be transitory, or, if not corrected, may ultimately lead to yeast degeneration. The potential industrial impact is realised for fermentation systems which may limit yeast growth, eg. continuous systems, use of pressure and, particularly, systems utilizing immobilised cells.

Bibliography : pages 145-156. / This dissertation forms part of a fundamental investigation into the molecular biology of the industrially important bacterium, Thiobacillus ferrooxidans. The expression of T. ferrooxidans plasmid encoded functions, as well as the identification, cloning, sequencing and expression in a variety of heterotrophic bacteria and in vitro systems of the T. ferrooxidans nitrogenase structural genes were studied.

The aim of this study was to characterise the relationships between solventogenesis and endospore formation in Clostridium acetobutylicum strain P262. Growth and endospore formation was monitored in a number of complex and minimal media and as a result of these studies a new defined sporulation medium was developed. The defined system produced high levels of solvents and supported 60 80% sporulation in C. acetobutylicum P262. Endospore formation occurred near-synchronously, enabling this system to be used in correlative physiological and morphological studies. Five other Type Culture Clostridium strains grew and sporulated less well in the C. acetobutylicum minimal medium (CAMM). These variations emphasise the importance of strain differences amongst the Clostridia. Two well defined physiological phases, the acidogenic phase and the solventogenic phase, which characterise the industrial ABE fermentation process were observed in CAMM.

An atp gene cluster from the extreme acidophile Thiobacillus ferrooxidans ATCC 33020 was cloned by complementation of Escherichia coli unc mutants. Eight different E. coli unc mutants were screened with T. ferrooxidans ATCC 33020 pEcoR251 plasmid and pHC79 cosmid gene banks. The ability of the transformants/transductants to grow on succinate as the sole non-fermentable carbon source was used to select mutants with a functional F₁F₀ ATPsynthase. Many F₁ -complementing plasmids and cosmids were isolated from the four E. coli F₁ unc point mutants screened. No plasmids or cosmids which complemented an E. coli fΔunc strain or any of the three E. coli F₀ mutants screened, were isolated.

Bibliography: leaves 117-133. / Vibrio alginolyticus synthesized extracellular collagenase in a highly aerated peptone medium at the late-exponential and early-stationary phases of growth. Collagenase synthesis was subject to end-product repression and was repressed by various amino acids and ammonium ions. Glutamine caused severe repression of collagenase production. Collagenase synthesis was sensitive to catabolite repression by glucose and a number of carbon sources. Cyclic AMP, dibutyryl cyclic AMP and cyclic GMP did not relieve catabolite repression. Glucose and 2-deoxyglucose caused a severe transient repression. No intracellular preformed collagenase was detected and collagenase production ceased when induced cells were washed and resuspended in buffer.Trypsin and a-chymotrypsin had no effect on collagenase production by cells or sphaeroplasts. The inducers of collagenase production in peptone were shown to have abroad molecular weight range between 1, 000 and 60,000. The peptone inducers supported slow growth of V. alginolyticus when supplied as the sole nitrogen source in minimal medium. Digestion of the peptone inducers with purified V. alginolyticus collagenase resulted in a decrease in their inducing ability,whereas digestion with trypsin or a-chymotrypsin did not. Peptone acted as an inhibitor of collagenase.

Bibliography: pages 243-259. / Actinophage VCll is able to virulently infect 11 of the 20 Streptomyces strains tested. Examination of VCll infection of Streptomyces cattleya, Streptomyces olivaceus and Streptomyces lividans TC10 indicated the absence of restriction-modification systems which affect VCll infectivity of these Streptomyces strains.

Bibliography : pages 267-315. / An investigation of variability in hydrological and bacterial parameters at a fixed coastal station (Oudekraal. Cape Peninsula 33°59'S 17°21'E) showed that bacterial populations varied in numbers and biomass on both a short term (daily) and seasonal basis in response to changes in hydrological conditions which were largely wind induced.

Bibliography: leaves 166-191 / Bacteria were isolated from the kelp beds on the West Coast of South Africa. Strains isolated from the water column and kelp fronds were classified as Pseudomonas, Vibrio, Acinetobacter and Flavobacterium species. Bacterial diversity in adjacent kelp dominated habitats was examined using numerical analysis, and it was found that nearshore and offshore isolates were similar, whereas bacteria isolated from beached kelp and interstitial waters were dissimilar from them and from each other. Changes in numbers of bacteria able to form colonies on plates were monitored during upwelling and downwelling conditions.

Bibliography: pages 202-212. / The binding of divalent cations (particularly calcium and magnesium) to strains of tobacco mosaic virus (TMV) and their isolated proteins was investigated, using equilibrium dialysis and potentiometric titration, in an attempt to elucidate the role of divalent cations in virus stabilisation. It was found that dissociation of bound calcium ions from TMV is apparently a necessary but insufficient condition for in vitro virus disassembly. TMV and the closely related strain, Y-TAMV, possessed three groups per protein subunit which titrated near neutral pH and which showed significant metal ion binding. The tightest of the three calcium binding sites, which was absent on the RNA-free protein, had a computed pKH of 8.3 and pKCa of 5.2 and had a significantly higher affinity for Ca⁺² over Mg⁺² This group thus had some of the characteristics to be expected for a calcium-mediated switch controlling in vivo virus disassembly, and possibly controlled the in vitro alkaline degradation of TMV as well. Both the U2 and cowpea strains of TMV bound one additional metal ion per protein subunit relative to vulgare, this binding site being retained by the polymerised proteins. However, calcium ions stabilised the polymerised forms of the proteins of all four TMV strains at pH values where depolymerisation would normally have occurred. Both bromegrass mosaic virus and turnip crinkle virus bound calcium ions, which stabilised compact forms of these viruses. The phenomenon of cation binding is thus not limited to TMV. In the light of published evidence, it appears that most if not all plant viruses are able to bind divalent cations, which thus represent a hitherto disregarded stabilising element.

Bibliography: pages 146-162. / The regulation of nitrogen (N) metabolism is being investigated in the Gram positive anaerobic bacterium Clostridium acetobutylicum, which has been utilized in industrial fermentations to produce acetone, butanol and ethanol. The C. acetobutylicum glutamine synthetase (GS) glnA gene region originally cloned in Escherichia coli, is characterized by the glnA structural gene, the upstream promoter sequences P₁ and P₂, a downstream DNA sequence complementary to the 5'end of the glnA gene and the downstream promoter P₃. Transcription of the glnA gene is controlled by the upstream promoters P₁ and P₂. Transcription from the downstream promoter P₃ in the opposite orientation of the glnA gene was demonstrated to express the 43-base glnA antisense (AS) RNA in E. coli and C. acetobutylicum cells. The expression of GS activity or the glnA AS RNA were not regulated by N in the heterologous E. coli host, but the expression of the antisense RNA in these cells was associated with decreased levels of GS activity. The regulation of the glnA gene expression was studied in C. acetobutylicum cells after suitable media for the growth of this bacterium was developed. C. acetobutylicum GS activity, the transcription of glnA mRNA and the glnA AS RNA were regulated by N. In cells grown in N-rich medium GS activity and glnA mRNA were repressed. Repression ratios for GS activity varied from 1.6 to 9.0 depending on the sampling time. The relative number of glnA transcripts was approximately 25%-28% lower in cells grown in N-limiting medium. The expression of the glnA AS RNA was differentially regulated relative to the. GS activity and glnA mRNA levels. The glnA AS RNA was repressed in N-limiting medium and induced in N-rich medium. The relative. number of AS RNA transcripts was approximately 1.5-fold in excess of glnA mRNA transcripts under conditions that repressed GS activity. Under conditions that induced GS activity, glnA mRNA transcripts exceeded AS RNA transcripts. Since differential regulation by N levels of the glnA AS RNA expression relative to the glnA gene was demonstrated in C. acetobutylicum, additional regulatory element(s) affecting the C. acetobutylicum glnA system were investigated. C. acetobutylicum gene libraries were cotransformed in trans with an in-frame glnA-lacZ fusion construct and plasmids from the C. acetobutylicum gene libraries were tested for flgalactosidase expression. No alteration of the lacZ gene expression was detected in the cotransformed colonies. However, DNA sequencing of the region situated downstream of the C. acetobutylicum glnA gene revealed the presence of an open reading frame (ORF) located 199 to 766bp from the 3'end of the glnA structural gene. The glnA AS coding region is located on the putative ribosome binding site and the 5'region of this C. acetobutylicum ORF. The protein encoded by this ORF showed 30% similarity with the carboxy terminus of the Pseudomonas aeruginosa aliphatic amidase regulator encoded by the amiE gene. The amino terminus of this protein also has 28% and 26% similarity with the amino terminal region of the DegU and CheB response regulators, respectively. These regulators belong to the family of the response regulators involving two component signal transduction systems, suggesting that the protein encoded by this ORF may play a role in the mechanism of regulation of the C. acetobutylicum glnA gene in response to nitrogen.