PEB3, a cell envelope protein, was identified as a target for use in an immunomagnetic isolation system (IMS). <i>Peb3</i> was cloned into <i>Escherichia coli</i> and expressed as a His-tagged construct (His.PEB3). Rabbits were then challenged with the purified construct to produce polyclonal antisera. Isolation of <i>C. jejuni </i>from a mixed culture (<i>E. coli</i> or <i>Arcobacter</i> spp. and <i>C. jejuni)</i> with polyclonal antisera was attempted but failed to capture whole <i>C. jejuni </i>cells. <i>C. jejuni</i> expresses two cell envelope associated polysaccharides: a short chain lipooligosaccharide (LOS) and a long chain lipid-linked polysaccharide, capsular polysaccharide (CPS). The Penner serotyping system has been shown to use CPS as its major discriminatory determinant. A novel molecular based typing system was devised based upon restriction fragments length polymorphism (RFLP) using the CPS locus as a discriminatory determinant. Primers for use in long template PCR (LT-PCR) were designed against the genes, <i>kpsD, kpsS, gmhA2</i> and <i>cj1430</i>. The theoretical maximum amplicon length obtainable in LT-PCR is ~40 kb; however, this is dependent on a number of factors including the %GC of the target amplicon. The low %GC of <i>C. jejuni</i> DNA prevented the amplification of the entire locus length (~41 kb) in one reaction and by experimentation the maximum amplifiable length was determined to be 25 kb. This maximum length was only achieved sporadically and the maximum length that could be reliably amplified was ~15 kb. Because of this limitation only the amplicon <i>kpsS-gmhA2 </i>was used in the typing scheme. Amplification was only achieved with the type strain, <i>C. jejuni</i> NCTC 11168. This is believed to be due to difficulties in amplifying low %GC targets with the Expand^TM 20 kb plus long template system (Roche).

No description available.

An aim of this work was to compare interactions between bacteria, and to correlate them with increased or decreased biofilm formation. A better understanding of the interactions occurring within biofilms may lead to more effective control strategies. As the strains used in this study were closely related Enteric species, considerable bacteriocin activity occurred. Bacteriocin-producing strains were found to have a competitive advantage over bacteriocin-sensitive strains, both in gaining a foothold into a new community, and discouraging the attachment of potential competitors. Bacteriocins and bacteriocin-producing strains may be used as a novel strategy to control biofilm growth, and discourage the attachment of pathogenic strains. In general, a decrease in biofilm size and stability, and an increase in sensitivity to disinfectants was exhibited by bacteriocin-producing mixed species biofilms. There were, however, exceptions: certain biofilms of <i>Enterobacter agglomerans/Ent </i>when antagonised with a second, competitive strain produced a signal to repress bacteriocin synthesis in the competing strain, leading to a co-operative state. These biofilms were thicker, more stable and demonstrated an increased resistance to disinfectants. There is also the possibility that bacteriophage can be used to control biofilm formation. Studies indicated that small titres of phage were more successful in the removal of <i>Enterobacter cloace/</i>5920 biofilms. However, infection of three phages, φ1.15, Winchburgh and Blackburn phage, was required to completely eradicate the biofilms. The triple-combination of phage was also found to selectively remove a single bacterial species form a mixed species biofilm. The role of EPS in biofilm resistance and the adaptation of biofilms to increasing concentrations of disinfectant were also investigated. While the involvement of EPS was found to be transient, it was thought that repeated exposure to an antimicrobial agent may select for a more resistant phenotype, leading to biofilm resistance. For example, biofilms responded to increasing concentrations of triclosan by producing a triclosan mutant, and it was thought that increasing concentrations of benzalkonium chloride selected for strains utilising increased expression of multi-drug efflux pumps.

Degenerate oligonucloetide primers designed from sequence alignments of a number of well characterised nitrite reductase enzymes were used to amplify a 350bp fragment from wild type <I>N. subflava</I> DNA using PCR. Sequence analysis and database comparisons on this DNA led to the conclusion that the nitrite reductase gene had been identified. Upon translation the enzyme was found to contain copper in its active site and was proposed to be unusually associated with the outer membrane. Sequence and probing analysis on a Tn<I>5 </I>insertion mutant of <I>N. subflava</I>, lacking the ability to reduce nitrite, determined that Tn<I>5</I> had not inserted into a structural or regulatory gene for nitrite reduction but was affecting the nitrite reductase gene distally. Further evidence from nitrite reductase activity assays on native polyacrylamide gels determined that the gene affected by the Tn<I>5</I> insertion was responsible for electron donation to the outer membrane nitrite reductase enzyme. An outer membrane location for the electron donor protein was also postulated. The presence of an azurin like protein attached to the outer membrane has been identified in <I>N. gonorrhoeae</I> which is thought to act as donor to the gonococcal membrane bound nitrite reductase. However it is anticipated that the donor protein in <I>N. subflava</I> will differ from this as attempts to PCR an azurin gene from <I>N. subflava</I> were repeatedly unsuccessful (Lambert, 1997). The presence of outer membrane associated nitrite reductase and electron donor proteins in <I>N. subflava</I> indicate that a novel arrangement of the denitrification pathway may exist in this organism which spans the periplasmic space. This periplasmic arrangement is proposed to be unique to <I>Neisseria </I>spp.

In this thesis, we aimed to identify HJ resolvases through protein sequence alignment. We identified <i>Saccharomyces cerevisiae</i> Yol093wp by the PSI-BLAST search with the sequence of the yeast mitochondrial resolvase Cce1p or Ydc2p. Yol093wp homologs are widely found in eukaryotes. These homologs of the Yol093wp family are poorly related to each other and have small protein sizes; similar to those observed among the families of known resolvases. In this work, a variety of general and specific approaches were used to characterize the functions of <i>YOL093W</i>. We have shown that His-tagged Yol093wp expressed and enriched from <i>E. coli</i>, and the Yol093wp extracts might have DNA binding properties. Interestingly, <i>in vitro</i> studies revealed the preferential binding of Yol093wp extracts to a mobile four-way junction than to a 60 nt-ssDNA or a 60 bp-dsDNA. The basis of this binding activity is still unclear. Further analysis will be required to elucidate the specificity of this DNA binding. Cells lacking Yol093wp have no obvious growth defects under standard growth conditions. Meiotic recombination assays showed that <i>YOL093W </i>deletion has not effect on meiotic recombination. A yeast two-hybrid screen and TAP tag purification were utilised to identify Yol093wp-interacting proteins. In the two-hybrid screen, only one protein, Mtl1p, was retrieved with relatively low significance. The TAP purification of Yol093wp complex showed that the Yol093w protein was synthesized in the TAP-tagged strain, and could be purified followed the TAP purification procedure. However, the identities of the potential interacting proteins identified did not throw much light on the function of Yol093wp. Finally, the immunofluorescence micrographs of Yol093wp-GFP and Yol093wp-13myc in conjunction with DAPI straining suggested that the Yol093w fusion proteins are located in the nucleus. All of these data may be indicative of the function of Yol093wp.

It is well known that baker's yeast (<I>Saccharomyces cerevisiae</I>) can be used as a reagent for the reduction of a variety of functional groups, especially the enantioselective reduction of carbonyl groups to the corresponding optically active alcohols. In the context of this thesis it has been shown that aromatic nitro compounds undergo reduction with <I>S. cerevisiae </I>to the corresponding anilines under conditions of neutral pH, room temperature and in an aqueous medium. Mechanistic studies employing putative intermediates, suggest that nitroso and hydroxylamino species are involved on the reduction pathway. Further insights into the mechanism have been gained by investigating the reduction of a series of dicyanonitroaromatic substrates. In all cases, in addition to reduction of the nitro group, the nitrile group <I>ortho</I> to the nitro group undergoes conversion to the amide, <I>via</I> a proposed heterocyclic intermediate. The broad range of nitroaromatic substrates reduced by the baker's yeast prompted us to commence purification of the enzyme(s) responsible for the biotransformations. Ten litre fermentations were used in order to provide a sufficient quantity of cells for the subsequent purification steps. The use of size exclusion, anion exchange, hydrophobic interaction and affinity chromatography led to partial purification of the nitroaryl reductase enzymes. The reduction of 1,4-dinitrobenzene to 4-nitroaniline provided an ideal enzyme assay for the production of kinetic data, the enzyme demonstrating Michaelis-Menten kinetics. Subsequent to the successful optimisation of the purification protocol. Marina Alexeeva (University of Edinburgh) has completed the purification of three nitroaryl reductases with molecular weights of approximately 50 KDa, 45 KDa as judged by SDS-PAGE.

A two-fold strategy was adopted to identify factors which functionally interact with Prp2p: 1) the yeast two-hybrid system was utilised to identify Prp2p interacting proteins and, 2) the technique of high copy-number suppression screening was used to search for suppressors of dominant negative <i>PRP2</i> mutations. An exhaustive two-hybrid screen with Prp2p as the bait isolated Spp2p, another step 1 splicing factor that was originally identified as a high copy-number suppressor of a <i>prp2-1</i> mutation. Subsequently the reciprocal interaction was observed in a two-hybrid screen with Spp2p as the bait. Surprisingly the most statistically significant result of the Prp2p screen was Sec59p, an endoplasmic reticulum membrane protein involved in core glycosylation. The functional significance of this interaction was investigated by a variety of biochemical and genetic techniques but no evidence implicating Sec59p in pre-mRNA splicing was obtained. An interesting finding from the Spp2p screen was a novel protein Yor093p, which had also been isolated from a two-hybrid screen with Prp17p (a step 2 splicing factor) as the bait. Deletion of the entire open reading frame encoding Yor093p revealed that <i>YOR093c</i> is dispensable for cell growth under all conditions tested. A yeast strain was constructed in which an HA-tagged Yor093p fusion protein was expressed from a chromosomal locus. This strain was subsequently used in coimmunoprecipitation experiments to demonstrate that Yor093p does not stably associate with spliceosomes. A two-hybrid screen with Yor093p as the bait failed to identify any interactions with known splicing factors. The potential role of Yor093p as the bait failed to identify any interactions with known splicing factors. The potential role of Yor093p in pre-mRNA splicing therefore remains unclear. High copy-number suppression screens were performed to isolate putative suppressors of dominant negative <i>PRP2 </i>alleles.

Kms1 and Kms2 are important for integrity of the SPB. To find the biological significance of the interactions between Plo1 and these SPB proteins, attempts were made to disrupt the interaction by mutations. For this purpose, firstly regions responsible for the interaction were identified, and then mutations were made in the SPB proteins by random mutagenesis of this region. For Sid4, I isolated two point mutations, which had greatly weakened interaction with Plo1. To study the effect of disrupting the interaction <i>in vivo, </i>the two <i>sid4</i> mutants were expressed from a fission yeast promoter in the <i>sid4</i> temperature-sensitive mutant. Both point mutants of Sid4 that had weakened interaction with Plo1 were able to rescue the temperature-sensitive <i>sid </i>mutant with a similar strength as that of wild-type <i>sid4</i> under the same promoter. The identification of potential Plo1 interacting proteins and mutants defective in these interactions will be an important step to understand cell cycle control by Plo1.

The GroE heat shock proteins (GroEL and GroES) of <i>Escherichia coli</i> represent major molecular chaperones that participate in folding and assembly of a variety of proteins and are essential for cell growth at all temperatures. From <i>in vitro</i> studies, GroEL is thought to be highly promiscuous in substrate binding, interacting with almost any non-native model protein. However, <i>in vivo</i>, GroEL is involved in the folding of only 10-15% of newly translated polypeptides, suggesting specificity for a defined set of substrates. In an attempt to identify GroE substrates, a strain of <i>E. coli</i> has been constructed in our laboratory in which expression of GroE can be turned off. Previous work from our laboratory had suggested that DapA, the first enzyme on the lysine biosynthetic pathway, was affected by GroE depletion in <i>Escherichia coli</i>. Native DapA is present as a homotetramer; in this study, work is described that suggests that DapA achieves a homotetrameric state in GroE depleted cells but lacks enzymatic activity. Work is also described that suggests that native DapA cannot be overproduced in GroE depleted cells as it becomes highly susceptible to aggregation. Work in chapter 4 suggests that the <i>lac</i> inhibitor protein, LacI, is also affected by GroE depletion.  <i>In vitro</i> footprinting studies in GroE depleted cells suggest that LacI remains bound to the <i>lac</i> operator sequence in the presence of the gratuitous inducer IPTG. Previous work in other laboratories had suggested that <i>dapA </i>is not regulated at the level of expression. In this work data is presented that suggests that <i>dapA </i>is regulated at the level of expression, possibly by a transcriptional activator, in response to intracellular levels of the molecule diaminopimelic acid.

The inheritance pattern of extranuclear DNA in malaria parasites has been investigated in a cross between two different clones of the human malaria parasite Plasmodium falciparum. A 1:1 mixture of gametocytes from the two clones, 3D7 and HB3, had been fed to mosquitoes. In this project, a polymorphism between clones 3D7 and HB3 was identified in the highly conserved mitochrondiral cytochrome b gene of the extranuclear DNA. When this allelic marker was examined in the oocysts from the cross, each hybrid oocyst showed only one form, and never both forms, of the parental alleles. This indicated that extranuclear DNA in P. falciparum is uniparentally inherited. Unexpectedly 58 out of the 59 hybrid oocysts examined showed the 3D7 form and only one oocyst showed the HB3 form of the allele. The sex of the gamete transmitting the extranuclear DNA was investigated using purified preparations of male and of female gametes of the avian malaria parasite P. gallinaceum. Probes for the two extranuclear DNA elements in malaria, the 6kb mitochondrial element and the 35kb plastid-like element, hybridised to the female gamete DNA but not to the male gamete DNA. This led to the conclusion that inheritance of extranuclear DNA is through the female gamete, and implies that the majority of the hybrid oocysts in the 3D7xHB3 cross were of the 3D7female/HB3 male type. Analysis of cross fertilization and self fertilization events in this cross, using nuclear gene alleles, was shown to be in accordance with random mating, falling within the Hardy-Weinberg equilibrium predictions. The existence of the bias in parental contribution to the hybrids revealed by the extranuclear markers in this present work is difficult to reconcile with such random mating.