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.

Clostridium acetobutylicum P262 is an endospore-forming Gram-positive anaerobic bacterium, which has been used for the industrial production of acetone and butanol from carbohydrate substrates. This study forms part of a wider research effort into the genetics and molecular biology of C. acetobutylicum, which has as an ultimate goal the commercial improvement, and a fundamental understanding of the ABE fermentation. The aim of this study was to isolate and characterize genes involved in solventogenesis. The cloning, expression and characterization of the terminal solventogenic butanol dehydrogenase gene ( adhl), and the central pathway β-hydroxybutyryl-CoA dehydrogenase gene (hbd), which form part of a but operon are described.

Bibliography: pages 147-155. / In this study the conformational changes in beat stable and heat labile protein immunoprecipitin antigens of a typical mycobacterin, the culture filtrate of Mycobacterium bovis, strain BCG 172, during heating and cooling have been investigated and related to the more obvious manifestations of thermal denaturation which have been reported from studies of heated mycobacterins.

Plasmid pTF-FC2, a 12.4 kilobase pairs (kb) cryptic plasmid, was originally isolated from the acidophilic chemoautotroph Thiobacillus ferrooxidans and subsequently cloned into the pMB1-based vector, pBR325. Deletion of the pBR325 origin of replication revealed that pTF-FC2 was able to replicate autonomously in a number of Gram-negative bacteria besides T. ferrooxidans. Constructs carrying the pTF-FC2 origin were able to replicate independently of DNA polymerase I (Pol I) in Escherichia coli and this feature was used to distinguish between replication from the T. ferrooxidans origin and the pMB1-derived origins of the vectors. A 3.2 kb Sau3A partial fragment was obtained which had retained the ability to replicate in a E. coli polA- mutant and also in Pseudomonas aeruginosa. A series of deletions of this fragment was used to identify the minimal replicon, the vegetative origin of replication (orzV) and the areas determining plasmid incompatibility. The copy number of pTF-FC2 in E. coli was estimated at 12 - 15 copies per chromosome and a deletion plasmid was identi.fied which replicated at a reduced copy number. An area which affected the ability of the replicon to replicate in P. aeruginosa, was identified. The nucleotide sequence of the 3.2 kb minimal replicon of pTF-FC2 was determined from overlapping DNA sequence obtained from a series of sequential deletions from each end of the fragment. Analysis of the orzV sequence revealed three, tandemly repeated 22 base pairs (bp) DNA sequences and two sets of complementary inverted repeats. The 22 bp direct repeats appeared to be essential for replication and also for incompatibility. The role of one of the two sets of complementary inverted repeats was unclear. The deletion plasmid previously shown to replicate at reduced copy number was found to have half of the second set of repeats deleted.

Bibliography: pages 190-214. / This thesis describes investigations into the potential use of the Subgroup I geminivirus, maize streak virus (MSV), as a gene vector. These involved testing MSV-based replicons in transgenic cell lines, in transient expression assays in maize cells and in an infectious gene expression system in plants. MSV vectors which contained three different versions of a bar (bialaphos resistance) expression cassette in place of the viral movement and coat protein genes were used to generate transformed maize cell lines. A high proportion of these contained MSV-based episomes at high copy number. However, embryogenic maize tissue of the Hill line was not regenerable when an MSV-based replicon was present, possibly due to toxicity of the viral replication associated protein. In non-regenerable Black Mexican sweetcorn cell lines some of the MSV-bar episomes, which ranged in size from 3.15 kb to 4.78 kb, replicated for periods of over two years, and appeared structurally stable. The cellular levels of the bar gene product, phosphinothricin acetyl transferase (PAT), were significantly enhanced in lines where the gene was amplified by linkage to an MSV replicon in comparison with lines where the same gene was not amplified. Northern blot analysis also showed that higher levels of bar mRNA were produced in lines where the gene was amplified. However, the 3- to 5-fold enhancement in gene expression was less than was anticipated, based on results from similar Subgroup ill geminivirus-based transgene amplification systems. Several mutants of the MSV genome were generated to investigate the extent to which genome amplification contributes to the expression of the viral coat protein gene. The introduction of an Ncol restriction site at the start of the coat protein gene facilitated fusion of the gus marker gene with the coat protein upstream transcription and translation regulatory sequences. In one viral construct the plus strand origin of replication was inactivated by insertion of a short oligonucleotide; in another, the viral rep gene was inactivated by a frameshift mutation. These constructs were used to show that the MSV coat protein promoter has low, but measurable constitutive activity in the absence of genome amplification, but that viral replication enhances coat protein expression about 45-fold. I found no evidence for Rep-mediated transactivation of the coat protein promoter.

Bibliography: leaves 221-[234]. / [The work described here had multiple aims: to create a promoter probe that was suitable for the isolation of developmentally regulated Strepcomyces promoters, to isolate such promoters, to develop a computer assisted analysis system whereby potential promoter sequences could be determined and to use this in the analysis of the cloned promoters. Initially the suitability of the Streptomyces antibioticus me1C operon for use as a reporter system in Streptomyces was investigated. It was established that late-expressed promoters could be identified and that it was possible to use the me1C2 gene alone for this purpose. However, it was shown that the use of both me1C1 and me1C2 resulted in a more sensitive reporter system. High copy number promoter probe vectors were constructed and tested. A low copy number promoter probe (which used the Streptomyces penemefaciens pSPN1 origin of replication) was also constructed. The characteristics (copy number, stability and mobility) of the probe were established. The conditions in which sporulation was induced by phosphate limitation were identified. Under such conditions late expressing, phosphate dependent promoters were isolated, using the promoter probes previously developed. The expression of these promoters was tested in Streptomyces coelicolor bldA mutants, and the bldA dependent promoters identified. These were sequenced. Computer assisted analysis of DNA sequence bias was conducted, with the intention of using bias patterns to identify potential regulatory regions. The initial approach of using the sequence bias of protein coding regions (based on the premise that regulatory sites are likely to be under represented in these regions) was unsuccessful. Further analysis in which the positional preference of sequences that were over represented in regulatory regions was conducted. Based on this the known promoters of Streptomyces were partially classified. The sequence bias of protein coding DNA regions was used to develop a novel method to identify the protein coding regions of Streptomyces DNA. The computer programs were then used to identify protein coding and potential regulatory regions.

An electron microscope study of normal and L-forms of a marine luminous bacterium was carried out. In addition to the features found normally in Gram-negative bacilli, observed in cells from solid medium, cells from actively luminescent liquid culture were found to possess certain peculiar features. The most notable of these were tetrads, cells in which ingrowth of the cytoplasmic membrane causes the contents to be divided up into four protoplasts within the cell wall. Small cytoplasmic spherules which arise from the cytoplasmic membrane were also seen to occur. The cell walls which were three-layered and typical of Gram-negative bacteria often had blebs on the surface. The cytoplasmic membrane, typical of a 7.5 nm unit biological membrane in appearance in normal cells seemed to be composed of a number of laminations in some L-form cells. These laminations consisting of alternate dark and light bands may consist of numbers of unit membranes with adjacent protein layers fused. The occurrence of L-form cells was shown to be associated with luminescence and aeration in liquid sea-water medium.

Bibliography: pages 256-363. / This thesis deals with a study of the effects of far-UV radiation, oxygen and hydrogen peroxide on macromolecular synthesis and viability in the obligate anaerobe, Bacteroides fragilis, as well as the specific proteins induced in this organism by these different DNA damaging agents.

Bibliography: pages 135-150. / This dissertation represents a continuation of the research on the sucrose utilization system of the aerobic, collagenolytic, halotolerant, Gram-negative bacterium Vibrio alginolyticus. The V. alginolyticus sucrose utilization system originally cloned into Escherichia coli on plasmid pVS100 involves a sucrase enzyme (gene scrB), and a sucrose uptake system. Synthesis of the sucrase and sucrose uptake system in V. alginolyticus and E. coli(pVS100) is regulated. The nucleotide sequence and analysis of DNA regions encoding the sucrose uptake and regulatory functions are presented here. An investigation of the expression of the. V. alginolyticus sucrose utilization system in Bacillus subtilis is also presented.

Bibliography: p. 155-177. / V. cholerae 569B is a bacterium infamous for its role as the causative agent of the diarrhoeal disease cholera. Although the bacterium occurs naturally in brackish waters and estuaries, cholera outbreaks are closely linked to specific environmental conditions. For example, most outbreaks occur during the summer months when the bacterium experiences an increase in water temperature, play a role in activating virulence in V. cholerae 569B, the exact mechanism remains to be elucidated. Previously it was observed that when V. cholerae is exposed to elevated temperature and salinity, the bacterium initiates the synthesis of a brown-black pigment known as melanin. The function of the pigment and the genes involved in its synthesis was unknown. We therefore set out to determine the function of pigmentation in V. cholerae 569B, since pigmentation could significantly enhance the survival of the bacterium during adverse conditions and therefore aid in the persistence of the organism in the environment.