The Escherichia coli haemolysin operon consists of four contiguous genes, hlyCABD on an approximately 7.5Kbp length of DNA. The 107KDa HlyA haemolysin is activated by the cytoplasmically localised 20KDa HlyC polypeptide and secreted to the culture medium by the cell envelope localised HlyB and HlyD (53KDa) polypeptides. Two proteins corresponding to HlyB have previously been identified with apparent molecular weights of 46KDa and 66KDa. Both the HlyB and HlyD polypeptides appear to be expressed at extremely low levels in vivo. Interestingly, the HlyB polypeptide appears to be a member of a novel family of ATP-dependent membrane localised transport proteins spanning the phylogenetic scale. In order to investigate the structure and function of the HlyB polypeptide(s) attempts were made to amplify the expression of hlyB, following the DNA sequencing of the E.coli LE2001 hlyB gene, using a variety of plasmid vectors designed for the high level expression of cloned inserts in order to isolate sufficient HlyB to raise antisera. These experiments identified potential levels of control in the regulation of hlyB expression that, although present in the original pathogenic strain, E.coli LE2001, appeared absent or attenuated in the plasmid subclones of hlyB. However, no over-expressed HlyB polypeptide(s) were identified. Therefore, antibodies were raised against synthetic penta-peptides selected from regions within HlyB and against LacZ-HlyB fusion proteins. Experiments using the anti-HlyB antisera resulted in the immunoprecipitation of HlyB from radiolabelled E.coli minicells and the identification of a 46KDa polypeptide expressed in the original wild-type pathogenic strain, E.coli LE2001. Further experiments demonstrated that the HlyA toxin was also secreted transiently during the growth of E.coli LE2001 and that the in vivo level of hlyB mRNA was restricted to a similar "secretion window". Therefore, the regulation of hlyB expression was considered to be a suitable candidate for the control of HlyA secretion during growth.

This thesis describes the characterisation of a set of six genes of the baker's yeast Saccharomyces cerevisiae, that are induced when a molasses-grown culture reaches the end of rapid fermentative growth. The rationale for the isolation of such genes was that their promoters may be useful in a biotechnological application, where the regulated expression of heterologous proteins is required. Additionally, their analysis should provide information about how yeast cells adapt to changing environmental situations. Therefore the aims of this project were to identify the unknown members of this set of genes and to determine the transcriptional regulatory mechanisms acting on all six. Six cDNA clones, which showed differential hybridisation to mRNAs of exponential and stationary phase cells, were used as probes to recover the cognate genes from a library of yeast genomic DNA fragments carried in the bacteriophage vector, EMBL3. Two had been identified as the previously described genes, HSP26 and HXKl encoding the 26kDa heat shock protein and the glucose-repressible hexokinase, respectively. A further two, previously undescribed genes, encode a second small, 12kDa, stress-induced protein (HSP12) and a thiamine biosynthetic enzyme (THI4). DNA sequence analysis carried out in this study has characterised the two remaining genes as HXT4=LGT1, a low affinity hexose transporter and a new thiamine regulated gene, THI5. Transcription of these six genes has been examined under a variety of growth conditions and in various mutant backgrounds. The two heat-shock genes, HSP12 and HSP26, are subject to glucose control via a cAMP-dependent protein kinase and become induced when the cells experience stress or starvation; presumably they have a role in maintaining the integrity of the cell under these conditions. The genes involved in hexose metabolism, HXKl and HXT4, are regulated by glucose repression through the TUP1/CYC8/SNF1 pathway and are derepressed by exhaustion of glucose from the growth medium; their products enable the cell to utilise alternative hexoses as growth substrates. Both of the thiamine genes, THI4 and THI5, are subject to repression by exogenous thiamine and become derepressed on depletion of the vitamin from the growth medium. Thus, although these genes show apparent coordinate induction they are regulated via different transcriptional control mechanisms. DNA sequence analysis revealed the THI5 gene to encode a second thiamine biosynthetic enzyme homologous to the product of the Schizosaccharomyces pombe gene nmtl. THI5 is present within the yeast genome in multiple copies located on chromosomes VI (THI5), X (THI11) and XIV (THI12). Evidence is presented to show that all three copies potentially express a functional product. Likely mechanisms by which the multiple copies of this gene have arisen and subsequent conclusions about the evolution of the yeast genome are discussed.

Haemolytic activity produced by E.coli strains carrying the recombinant plasmid pLG570 was investigated. This plasmid contains a haemolytic determinant subcloned from the wild-type E.coli LE2001, isolated from a human urinary tract infection. Quantitative assays for haemolytic activity and for the separation of soluble subcellular compartments were developed. These revealed the absence of any detectable periplasmic pool of haemolytic activity in strains expressing the intact haemolytic determinant. Restriction mapping the haemolytic determinant enabled the identification, subcloning and independent expression from separate plasmids of the hly genes. The production of secreted haemolytic activity was shown to require the expression of four contiguous genes, hlyC, hlyA (the haemolysin polypeptide), hlyB and hlyD. Quantitative assays of haemolytic activity were undertaken on subcellular fractions of strains expressing subsets of hly genes. This enabled the following observations. HlyC is required for activation, but not for secretion, of HlyA. Both HlyB and HlyD are required for secretion of HlyA. Absence of any combination of HlyC, HlyB and/or HlyD did not result in periplasmic accumulation of HlyA. Thus it was concluded that the secretion of haemolysin involves tightly coupled translocation through both bacterial membranes without release into the periplasm. Identification of a "nucleotide binding site" in HlyB implied that at least part of this protein was exposed at the cytoplasmic face of the bacterial inner membrane. A topogenic domain at the C-terminal of HlyA was identified. Truncation of hlyA, resulting in the deletion of 27 amino acids from the C-terminal of HlyA, was shown to produce a haemolytic polypeptide which was not secreted. In addition, HlyA derived polypeptides, expressed from subcloned 3' portions of the hlyA gene, were shown to be secreted. Thus it was concluded that the C-terminal 113 amino acids of HlyA apparently include all the information required for interaction with the hly secretion machinery.

Campylobacter jejuni/coli are the most common cause of acute diarrhoeal disease in man. The disease is worldwide, affects all age groups, and is mostly sporadic although common source outbreaks are frequently reported. The organism characteristically causes infection of the small intestine with extension into the colon and rectum, the disease being an acute enterocolitis. Illness may be inflammatory, with mucosal oedema and polymorphonuclear infiltration and blood in the faeces, or non-inflammatory with watery diarrhoea. The pathogenesis of the disease is unknown. Bacterial invasion of intestinal mucosa has been proposed as a mechanism of mucosal inflammation causing tissue damage. Investigation of the enterocyte-like Caco-2 and other epithelial cell lines for the ability of clinically characterised strains to adhere and invade showed that strains from colitic illness exhibited a greater tendency to invade than strains from non-inflammatory illness. Colitis and some non-inflammatory strains were also shown to transcytose from the apical to the basolateral cell membrane. Phosphorylation of mammalian cell proteins (such as ion channels) is important in diarrhoeal illness, mediated by bacterial cells and their secreted toxins. C. jejuni bacterial cells and a secreted toxin in culture supernatants caused phosphorylation of Caco-2 cell proteins, effects that mimic protein kinase C phosphorylation of myosin light chain. Culture supernatants increased intracellular calcium, an effect known to mediate fluid secretion. These effects are independent of the cholera-like toxin that is found in small amounts in culture supernatants. Colitis strains tested in rabbit ileal loops induced similar histological effects to those seen in man, caused fluid secretion, and white cell infiltrate consisting of polymorphonuclear leucocytes and macrophages. Villi were shortened and tissue was oedematous with submucosal bleeding. Tissue damage may prevent effective absorption of fluid and contribute to diarrhoea but biochemical analysis suggests a true secretory component to the diarrhoea. By contrast a non-inflammatory strain showed no histological changes in loops and elicited no fluid secretion. Large amounts of the host derived secretagogue prostaglandin E2 were induced in infected ileal loops and correlated with the tissue white cell infiltrate (along with leukotriene B4). In the absence of a cholera-like toxin produced by the bacteria PGE2 seems to be responsible for the increase in infected tissue cyclic AMP. PGE2 acts by binding to a cellular receptor and activating cell adenylate cyclase resulting in a rise in cAMP. Thus a host inflammatory mediator may contribute to fluid secretion in C. jejuni enterocolitis.

Mutants of Escherichia coli with defects in triose phosphate isomerase (il.;i"), fructose diphosphate (FDP) aldolase(Fda") and pentose metabolism were used to investigate the role of glyceraldehyde 3-phosphate (G 3-.;) and dihydroxy ace tone phosphate (DHAP) in gluconeogenesis. A block in gluconeogenesis prevented growth of Q.;i -mutants on non-carbohydrates. Studies on glycogen synthesis suggested that both G and DHAP were required for the formation of the hexose moiety in gluconeogenesis. To explain the ability of Tpi -mutants to synthesize glycogen from glycerol, a novel route for the conversion of DHAP to G 3-P involving methyl glyoxal has been proposed. Both d.;i" and !I.;i.;strains of E. coli possessed an enzyme which convertedDHAP to methyl glyoxal. When the !I.;i"-mutants were exposed to compounds whichwere catabolized to DHAP, this accumulated and methyl glyoxal was excreted into the medium. Studies on gluconate and carbohydrate metabolism suggested that the impaired growth of Tpi - mutants on certain carbohydrates may be due to a deficiency in phosphoenolpyruvate and inhibition of glycolysis. The gene specifying triose phosphate isomerase was highly cotransducible with that for 6-phosphofructokinase. The presence of a small amount of aldolase in Fda -mutants probably explained their gluconeogenic ability. This enzyme showed a hi.;er ratio for FDP synthesis to FDP cleavage activity than the FDPaldolase in the parental strains. Further studies on FI3P cleavage activity shov;ed that these enzymes also differed in the pH-activity profile, the degree of activation by divalent metal ions and thiols and the K and V values m max A deficiency in idbose permease for one mutant and ribokinase for another, prevented their growth on ribose. CEhe chromosomal location of the gene specifying ribokinase was close to that for ribose permease.

This Thesis has investigated the genetic nature of the sensitivity of the bacterium Escherichia coli K-12 to the anti-bacterial protein, colicin E2. Particular attention has been paid to a group of E2-tolerant mutants, termed Cet-, since previous reports by other workers had shown that some cet mutations were pleiotropic, affecting a wide range of properties of the cell envelope. A genetic analysis has also been undertaken of Bfe- mutants, which differ in that they are resistant to all the E-group colicins because of a failure in colicin adsorption. The location of the cet gene has been determined, the gene order deduced being: dna-7D - dra - drm - serB - trpR - cet - thr, whereas previous reports located cet to the left of serB. Bfe mutations, comprising one complementation group, have been shown to map at a separate location, the gene order being metB-arg(ECBH)-bfe-thi-metA. In contrast to previous reports, none of the pleiotropic properties of Cet- mutants tested mapped near the cet locus. Thus, in two Cet- strains, UV-sensitivity derived from a Ion mutation; in another, UV-sensitivity mapped near the recA locus; in a third, resistance to phage T4 was shown to map at a position distinct from cet. The cet- allele was dominant to wild-type, whereas bfe- was recessive. The effect of different genetic backgrounds upon the expression of E2-tolerance in Cet- mutants has been examined. In particular, recA and recB mutations reduced E2-tolerance. An investigation of one of the pleiotropic properties of certain Cet- strains, resistance to phage ?, revealed it to be due to poor adsorption of the phage. Lastly, it has been foimd that E2-directed DNA degradation proceeds normally in some strains deficient in endonuclease I, and no evidence could be found that DNA degradation and inhibition of cell division induced by E2 are separate uncoupled processes.

Previous work in this laboratory has indicated that in thymine low requiring strains of Escherichia coli, the transit time of a replication fork can be varied without an appreciable effect on the growth rate, simply by varying the thymine concentration in the growth medium. This results in a change in the DNA concentration and therefore changes the dosage of most genes. I have defined three more precise parameters which have previously been encompassed in the term gene dosage. These I have called gene concentration, relative gene dosage, and gene dosage. On theoretical grounds, these three parameters change in separate and distinct ways as a function of the replication velocity and as a function of the relative position of the gene on the chromosome. In order to distinguish the relative importance of each of them in determining the overall rate of protein synthesis, I have compared the rate of enzyme synthesis from five genes in a fully repressed or induced state and one in an autogenous state in cultures growing at the same rate but having different transit times. The data permit the following conclusions: (1) The initiation mass is independent of the transit time of a replication fork at one growth rate. (2) Replication is bidirectional. The chromosome origin is near the gene coding for tryptophanase and the terminus near the gene specifying tryptophan synthetase. The data place the origin in the region of 67 minutes on the E.coli linkage map. (3) The output of a gene under conditions when it is subject to regulation by a cytoplasmic effector decreases as its concentration is increased. In addition, I have used the observed differential effect of changes in replication velocity on gene concentration to determine if the replication of the sex factor F is directly coupled to any stage of the chromosome replication cycle. The data preclude the possibility that F replication is coupled to any part of the chromosome replication cycle other than to initiation (or to the replication of genes close to the origin) or, at the growth rate used here, that it occurs at a constant cell mass.

The Incil plasmid Colib-P9 was found to carry a single-stranded DNA-binding protein gene (ssb), and the cloned gene was able to suppress the UV and temperature-sensitivity of an ssb-l strain of Escherichia coli K-12. Determination of the nucleotide sequence of Colib ssb demonstrated that the gene shows considerable homology to the ssb gene of plasmid F. In contrast, Southern hybridization techniques indicated that the IncP plasmid RP4 lacks a gene with any extensive homology to F ssb. It was shown that the direction of transfer of Colib-P9 is such that the Colib ssb gene, which lies approximately 11 kb from the origin of transfer, is located within the region transferred early during conjugation. The Colib and F ssb genes are therefore similarly located on their respective plasmids. The Colib ssb gene was shown to be coordinately expressed with the transfer (tra) genes, suggesting that the Colib SSB protein may participate in the conjugative process. However, a mutant Colibdrd-1 derivative carrying a Tn903-derived insertion in ssb showed no defect in tests of conjugative efficiency and was apparently maintained stably both following mating and during vegetative growth. Thus no biological role for the Colib SSB protein was detected. However, unlike the parental plasmid, the Colib ssb mutants conferred a marked Psi- (plasmid- mediated SOS inhibition) phenotype on recA441 and recA730 strains. This may result from high level expression of a psi gene due to readthrough from the Tn903 insertion. It is now apparent that many conjugative plasmids previously thought to be unrelated may be derived from a common ancestral plasmid which possessed both ssb and psi genes. It is speculated that the function of the SSB proteins of conjugative plasmids such as Colib and F may subsequently have been duplicated by analogues derived from newly aquired conjugation systems.

No description available.

A gene (VRPl) encoding a novel proline-rich protein (verprolin) has been isolated from the yeast Saccharomyces cerevisiae as a result of its hybridisation to a chick vinculin cDNA probe. The deduced protein sequence contains 24% proline residues present as proline-rich motifs throughout the verprolin sequence. Several of these motifs resemble recently identified sequences shown to bind Src Homology 3 (SH3) domains in vitro. Replacement of the wild-type VRP1 allele with a mutant allele results in strains that grow slower than wild-type strains and are temperature-sensitive. The vrpl mutants are impaired in both cell shape and size and display aberrant chitin and actin localisation. These phenotypic characteristics resemble those observed in yeast cytoskeletal mutants. Using the data generated in this study, together with the rapidly accumulating data on SH3 domains, the cytoskeleton and signal- transduction, it is possible to speculate as to the in vivo role of verprolin.