Some strains of Escherichia coli possess an iron uptake system, first found on a ColV plasmid, which utilises the hydroxamate siderophore aerobactin and which significantly enhances the virulence of host bacteria in experimental infections of mice. The aerobactin system of plasmid ColV-K30, cloned as the multicopy recombinant plasmid pABN1, was localised to a 7.6 kb segment of DNA by transposon mapping. In the maxicell and minicell expression systems, the cluster of genes was found to specify five polypeptides, four of which are involved in aerobactin biosynthesis, the fifth being the outer membrane receptor for ferric-aerobactin. The linear order of the genes specifying these polypeptides was determined, the gene for the receptor being at the 3' end of the cluster. The 5' and 3' transcription initiation and termination sites were located by S1 nuclease transcriptional mapping, and further transcription studies revealed a probable internal promoter within the cluster of genes, a minor unregulated promoter for the receptor gene, in addition to the major regulated promoter(s) at the 5' end of the system. Transcription of the system on ColV-K30 and subcloned plasmids, and production of the siderophore by ColV-K30, as measured by b-galactosidase production by ColV: :Mu(Ap lac) aerobactin-deficient strains, were found to be under the control of the level of freely available iron in the growth medium. The aerobactin system on pABN1, like that on ColV-K30, was shown to enhance the virulence of host bacteria in experimental infections of mice, and was found to be widespread among E. coli strains isolated from a number of extraintestinal infections of man and animals. The system was found not to be exclusively associated with ColV plasmids among these strains, and was apparently located on the chromosome of some isolates.

The microbial catabolism of two aromatic acids, phenylacetate and 3-hydroxybenzoate, has been studied. A variety of classical biochemical and genetic techniques have been used to eliminate several putative pathways for the catabolism of phenylacetate in Escherichia coli K10 and Pseudomonas putida U. A phenylacetate negative mutant of E.coli, previously isolated in this laboratory, was shown to accumulate 2-hydroxyphenylacetate when exposed to phenylacetate. The mutation responsible for the inability of this mutant to grow on phenylacetate has been mapped on the E.coli K10 chromosome to about minute 30.4. To facilitate the study of 3-hydroxybenzoate transport and degradation, a method was developed microsynthesis of radiolabelled [14c] 3-hydroxybenzoate of high specific activity. By a combination of substrate oxidation experiments by whole cells, enzyme assays and tracer experiments with radiolabelled 3-hydroxybenzoate, it has been demonstrated that K. pneumoniae degrades 3-hydroxybenzoate via an inducible pathway in which 2,5-dihydroxybenzoate, maleylpyruvate and fumarylpyruvate are intermediates. Experiments suggest that 2,5-dihydroxybenzoate and/or maleylpyruvate are the inducers of the pathway. The synthesis of [14c-carboxyl] 3-hydroxybenzoate also enabled the study of various aspects of the uptake of 3-hydroxybenzoate by K. pneumoniae. It was shown that the uptake of 3-hydroxybenzoate was mediated by a permease induced during growth on either 3-hydroxybenzoate or 2,5-dihydroxybenzoate. The permease displayed saturation kinetics with a Ks value of approximately 1,75 uM for 3-hydroxybenzoate and could transport 3-hydroxybenzoate against a concentration gradient. Uncouplers inhibited the uptake of 3-hydroxybenzoate demonstrating that the driving force for this active transport system was the proton motive force across the cellular membrane. No evidence was gained which would suggest that a general aromatic acid permease was induced during growth of K. pneumoniae on 3-hydroxybenzoate, but the 3-hydroxybenzoate permease may be capable of transporting some substrates of similar structure to 3-hydroxybenzoate.

Molecular studies of Escherichia coli capsule gene clusters C. Rachel Drake Escherichia coli can produce a large number (over 70) of structurally distinct capsular polysaccharide (K antigens) which have previously been divided into two groups. Group II K antigens are encoded by kps near serA. The kps genes are homologous between group II capsule gene clusters which encode chemically distinct K antigens. Genes encoding the K4 antigen (group II), an unusual substituted polymer (a fructo-sylated chondroitin), were cloned and expressed in E. coli K-12 and shown to contain the group II kps determinants. By nucleotide sequence analysis it was shown that the 3' end of one group II capsule gene, kpsS, is different in the K1, K4 and K5 capsule gene clusters. It was demonstrated that strains expressing a group I capsule do not carry the group II kps determinants on the chromosome. The K3, K10, K11 and K54 capsules have characteristics of both groups I and II and were also shown to lack the group II capsule genes despite the K10 and K54 capsule genes having previously been mapped near serA. The existence of a third capsule group, group I/II, encoded by genes distinct from the group II capsule genes yet located in the same region of the chromosome is formally proposed. An unsuccessful attempt was made to clone the K9 antigen (group I) biosynthesis genes. DNA flanking the K1 and K4 antigen gene clusters was used as probes in Southern blot analysis of different E. coli isolates and was shown to be a common component of the E. coli chromosome. The K1, K4 and K5 capsule gene clusters appear to be located at the same chromosomal location. DNA adjacent to one end of the K4 capsule gene cluster was highly polymorphic and present in more than one copy in some strains. The organisation and variability of the serA region of the E. coli chromosome is discussed.

The studies concern the mechanism that primes the synthesis of DNA complementary to the strand of DNA that is transferred during IncIalpha plasmid-mediated conjugation. This DNA synthesis was found to be independent of the two defined priming enzymes of the host bacterium, RNA polymerase and primase. It is proposed that the synthesis is initiated by a novel priming enzyme which is specified by the plasmid and supplied by the donor parent. It was found that rifampicin, an inhibitor of RNA polymerase, has complex effects on mating bacteria. Rifampicin treatment of recipients enhanced plasmid transfer from drug-resistant donors, implying that the amount of DNA transferred is normally limited by the synthesis of a plasmid-specified product made in newly infected recipients. Enhanced transfer occurring in rifampicin-treated matings has been exploited to search for intermediates of transfer. The results show that the transferred material, isolated from recipients, is the same size as the parental plasmid. In previous studies it was claimed that transfer of IncIalpha plasmids was inhibited by rifampicin. It is shown that rifampicin-treatment of donors allowed transfer to proceed normally for about 10 min when it stopped abruptly. Cessation of transfer correlated with an abrupt disruption of conjugal DNA synthesis in donor cells. The inhibitory effect of rifampicin on Incl plasmid transfer may result from a side effect of the drug on DNA metabolism, rather than a direct result of inhibiting RNA polymerase. Previous models for IncI plasmid transfer, involving a requirement for rifampicin-sensitive synthesis of an untranslated RNA molecule in the donor, have been reevaluated. In an attempt to understand the molecular bases of these phenomena, a genetic analysis of IncIalpha plasmids was initiated.

The inhibition of cell division after UV in wild-type bacteria is considered to be an "SOS" function under the control of recA; this control was the subject of the investigation. A kinetic study of division inhibition and recA synthesis after a low dose of UV indicated that recA did hot act directly to inhibit division. Further, recA synthesis was uncoupled from the division block in the presence of low levels of rifampicin. Nevertheless, recA is essential for division inhibition after UV. A Ion mutant, which unlike Ion strains did not recover from the division block, showed no alteration in the kinetics of recA induction. A mutation in a second gene, sulB, suppressed the lethal effect of UV on Ion mutants by promoting recovery after a period of division inhibition; possible mechanisms of suppression are considered. To investigate .the interaction between sulB, recA and Ion at the molecular level, a transducing phage was obtained carrying the proposed sulB region of the chromosome. The transducing DNA was recloned into a plasmid vector and a strain containing the recombinant plasmid showed an altered division response after UV, suggesting that a division control gene was present in the cloned DNA. Gene products coded by the transducing phage and the recombinant plasmids were demonstrated by in vitro and semi-in vivo techniques and the m vitro systems was developed further with the object of detecting possible transcriptional control of cloned genes by recA. The results are considered in the light of some recent developments having important implication's for the project.

No description available.

A study was made of the interactions between the element silicon, mainly as silicic acid, and various microbial processes. The effect of silicon compounds on fungal growth was determined under both oligotrophic and nutrient-rich (copiotrophic) conditions. Mycelium of Aspergillus oryzae was grown from a spore inoculum added to ultra-pure water (upw) containing silicon compounds, but not in upw alone. Growth of other fungi also only occurred in upw when silicon compounds were added. Increased growth of fungi also followed the addition of silicon compounds to Czapek Dox medium. Silicic acid also increased the protein content of fungi grown under such nutrient-rich conditions. The fungi solubilised the insoluble silicon compounds under both oligotrophic and copiotrophic conditions. Silicon was not however, accumulated by fungi as electron-dense hyphal bodies. Addition of silicic acid to nutrient rich media also increased the growth of species of Streptomyces but decreased the chlorophyll content of the alga, Dunaliella parva; the growth of two yeasts and the bacteria, E. colt and S. aureus also was not affected by silicon addition; the observed stimulatory effect therefore appears to be restricted to filamentous microorganisms. The effect of silicon compounds on various microbial processes was also investigated. Silicic acid stimulated the production of citric acid by Aspergillus niger, but decreased nitrification and sulphur oxidation in this fungus. Silicic acid addition also led to a reduction in antibiotic production by species of Streptomyces. Studies were initiated to study the possibility that fungi and bacteria can erode the surface of both bulk and porous silicon wafers. While no such surface erosion was evident, we observed that E. coif underwent extensive extreme pleomorphism when growing under starvation conditions for up to 14 days. Such pleomorphism consisted of the formation of bulbous protrusions from the normal rod, dumbbell-shaped cells and long filaments, these were up to 50g in length (compared to the normal 1-3μ, rods). Such filamentation was clearly caused by the inability of the bacterial cells (rods) to separate on division. The observed bacterial pleomorphism was not however, silicon-specific, as it was also found to occur on titanium and glass surfaces. Such extreme pleomorphism may have important implications in relation to the growth of E. coli in low nutrient environments and may influence the bacterium's ability to affect pathogenesis. While the microbiology of silicon has largely been neglected the results of this thesis show that there is considerable interaction between this element and microbial growth. Future studies should in particular be directed towards determining if silicon can be used as an energy source by microorganisms. Additionally, the observed phenomenon of extreme pleomorphism in E. coil is clearly worthy of further study.

Bacillus spores are widely used as commercial oral probiotic preparations, both for human and farm animals use. Nevertheless, the mechanisms underlying the probiotic effects are not yet known, although they are likely to be the result of a complex network of interconnected microbiological and immunological factors. The broad aim of the research work carried out for this thesis was to gain a better understanding of the immunological and probiotic properties of spores, exploring the application of these findings in the design of spore-based mucosal vaccine vehicles and assessing their potential for novel probiotic applications. Characterization studies on the immunomodulatory activity of B. subtilis spores at the mucosal and systemic level in a mouse and a rabbit model as well as studies on the immune recognition and surface display of spore coat proteins are described. Heterologous antigen delivery was achieved both at the level of the spore surface and spore interior. The OG, 6H loop of the VP1 protein of Foot-and- Mouth Disease virus (FMDV) and the recombinant adjuvant molecule CTA1- OVA-DD were chosen as model heterologous antigens. Immunization studies showed a specific anti-FMDV mucosal immune response upon intranasal delivery. The use of B. subtilis as a competitive exclusion agent in an E. coli 078: K80 chick model of infection was explored. Results showed that a single oral dose of B. subtilis spores could suppress all aspects of E. coli 078: K80 infection, along with a substantial reduction of dissemination and colonization. Finally, in order to determine the life stage of the Bacillus life cycle responsible for its probiotic action, an RT-PCR study of spore germination was carried out in a mouse model to identify loci and timing of this response at the intestinal level. Results showed that Bacillus subtilis spores are able to germinate in the jejunum and the ileum. These findings provide relevant insights for future studies on the probiotic activity of Bacillus subtilis as well as for the development of improved Bacillus mucosal delivery systems for heterologous antigens

No description available.

No description available.