The bacterial pathogen Campylobacter jejuni is a leading cause of foodborne gastroenteritis worldwide. Consumption of contaminated poultry meat is considered a major source of infection. Changes in cell morphology were demonstrated to occur spontaneously on entry in to stationary phase, with development of filamentous cells amongst short spiral morphotypes. The aim of this study was to investigate differences between the morphotypes of C. jejuni and C. coli and gain insights into their development. Using a minimal culture medium filament formation was observed to be wide spread amongst C. jejuni strains tested but was not universal in C. coli strains. Filamentation did not appear to arise due to the release of diffusible molecules or the accumulation of either toxic metabolites or oxidative stressors in the medium. Separated filaments exhibited greater intracellular ATP contents compared to spiral cells, and were able to survive longer in water at 4 and 37 C. C. jejuni 12661 was identified as producing long filaments but genome sequence analysis provided no clear explanation for the enhanced filament formation. Using RNA-Seq, transcriptome differences were examined between cells growing in exponential phase and separated cell morphotypes recovered from stationary phase cultures of the C. jejuni strains 12661 and PT14. These studies identified profound transcriptional differences between the cell morphotypes present at stationary phase, and highlighted problems of interpreting such data without separation of these sub-populations. Stationary phase cells of either morphotype were impaired in motility, which likely resulted from down-regulation of rpoN encoding sigma factor 54, and several key motility associated genes. The spoT gene of C. jejuni mediates the synthesis of ppp(G)pp as part of the stringent response to stress. The spoT gene was differentially regulated in the stationary phase morphotypes recovered in this study, as were the genes encoding the phosphohydrolases PPX1/PPX2 and polyphosphate kinases PPK1/PPK2 that control cellular (p) pp(G)pp pools. Prominent heat-shock and oxidative stress responses were evident in stationary phase cells compared to cells in exponential growth phase but transcription of the ribosomal proteins was not down-shifted. The transcript levels of several cell division associated genes were down-regulated in stationary phase spiral and filamentous cells. The formation of long filamentous cell morphotypes by C. jejuni 12661 corresponds with reduced expression of maf (inhibitor of septum formation), mreB (actin-like rod-shape determining protein), mreC (rod-shape determining protein), parA, parB (chromosome partitioning proteins), ftsA (actin-like function in cytokinesis), ftsH (ATP-dependent zinc metallopeptidase), ftsW (lipid II-linked peptidoglycan transporter) and ftsZ (tubulin-like z-ring formation) that collectively function in septum formation between daughter cells.

The bacterial genus Clostridium is composed of Gram-positive, spore-forming rods with widespread biotechnological applications. This study focused mainly on Clostridium pasteurianum DSM 525, a saccharolytic species which is able to convert glycerol, the by-product of the biodiesel industry, into the valuable chemical commodities n-butanol, ethanol and 1,3-propanediol. The aim was to formulate reproducible methods for the creation of mutants, both directed and random, and use the tools developed to investigate genes, and their products, important in solvent production. A prerequisite for the deployment of the envisaged genetic tools was a reproducible means for their introduction into the cell. Following the observation of low frequencies of plasmid transfer by electroporation it was hypothesised that the low level of transformants observed were a consequence of the presence of rare hypertransformable variants within the population. Accordingly, successfully transformed clonal populations were cured of their acquired plasmid and retransformed. In a number of instances the cured cell lines proved hypertransformable, with plasmid transformation frequencies obtained that were 5 orders of magnitude higher than those obtained with the progenitor strain. All of the hypertransformable strains isolated were shown by whole genome sequence to contain single nucleotide polymorphisms (SNPs) in one or more genes. In one instance, the single SNP present was shown to be directly responsible for the increased transformation frequency by its deliberate restoration to wild type using the allelic exchange procedures subsequently developed. Having established reproducible, high frequencies of plasmid transformation reverse genetics was employed to establish gene function. Accordingly, allelic exchange gene knock-out procedures were used to target genes coding for enzymes of the central energy metabolism in C. pasteurianum and the phenotypes of the mutants obtained were analysed in laboratory scale fermentations. Strains in which the genes encoding the redox response regulator (rex) and a hydrogenase (hyd) were deleted showed increased n-butanol titres, representing first steps towards utilisation of C. pasteurianum as a chassis for this important chemical. With the inactivation of the dhaBCE gene, encoding glycerol dehydratase, production of 1,3-propanediol was entirely eliminated, demonstrating the importance of the reductive pathway for growth and redox homeostasis of this organism when grown on glycerol. In order to allow forward genetic approaches, a mariner-transposon system previously exemplified in Clostridium difficile was adapted for use in alternative clostridial hosts. In the absence of an efficient transformation system for C. pasteurianum, the initial exemplification of the system was undertaken in Clostridium acetobutylicum and Clostridium sporogenes. Successful transposon delivery was demonstrated through the use of a plasmid conditional for replication and through the insertion of a gene encoding an alternate sigma-factor, TcdR, into their genomes. Transposition was shown to be entirely random and the libraries obtained of sufficient size to allow the isolation of both auxotrophic and sporulation/germination deficient mutants. Steps were taken to develop the same system in C. pasteurianum which was successful by using a suicide delivery plasmid, which was only possible with the high transformation efficiency achieved as part of this study. This study presents an essential forward genetics procedure for industrially important Clostridium species and a comprehensive genetic engineering approach for the important biofuel producer C. pasteurianum.

Bdellovibrio bacteriovorus is a naturally soil-dwelling, Gram-negative predatory bacterium that attaches to, invades, and replicates within a wide range of other Gram-negative bacterial species, killing such prey in the process. A small number of previous studies testing the effect of B. bacteriovorus against known Gram-negative plant pathogens have suggested that B. bacteriovorus has potential as a ‘food security agent’ against Gram-negative bacterial infections in crop plants. My project built on this knowledge by screening a range of known Gram-negative bacterial plant pathogens and Plant Growth-Promoting Rhizobacteria (PGPRs) for susceptibility to Bdellovibrio predation in vitro; testing predation-susceptible strains in a simple, semi-sterile in vivo system on the surface of Agaricus bisporus mushrooms; and finally testing the effect of Bdellovibrio addition in a more complex, natural Triticum aestivum (wheat) soil rhizosphere mesocosm. An in vitro prey strain growth assay showed that susceptibility to B. bacteriovorus predation varied between a range of 20 Gram-negative (mostly Pseudomonas) bacterial pathogen/PGPR species, isolated from a range of different host crops or soil environments. Four of these species (Pseudomonas avellanae 48, P. syringae pv. phaseolicola, P. tolaasii 2192T and P. agarici 2289) were highly susceptible to predation, and three species (B. vietnamiensis G4, P. marginalis 667, and Pectobacterium atrosepticum SCRI1143) showed apparent resistance to predation. P. tolaasii 2192T, causes dark, pathogenic lesions on post-harvest mushroom host crops; In vivo co-inoculation tests on the surface of A. bisporus mushrooms showed that lesions were significantly reduced with B. bacteriovorus treatment, which was due to B. bacteriovorus predatory killing and reduction of prey cell numbers, preventing symptoms. B. bacteriovorus also preyed upon and killed a putative pathogenic Pseudomonas species isolated from a grey lesion on an organic, garden mushroom, but some likely commensal species isolated from mushroom tissue showed resistance to predation. These data together suggest that B. bacteriovorus could be used commercially to prolong the shelf life of mushrooms, reducing crop losses through spoilage, with minimal negative effects on mushroom PGPR species. Finally, inoculating B. bacteriovorus into the soil around young winter wheat plants in a natural pot soil mesocosm was found to increase plant growth and grain yield at harvest; this was contrary to my initial hypothesis that B. bacteriovorus would reduce wheat plant growth, by preying upon and killing PGPR species such as P. fluorescens that reduce wheat plant infection with Gaeumannomyces graminis var. tritici, the yield-reducing take-all fungal pathogen of wheat. The soil was found to be low in nitrogen; thus B. bacteriovorus inoculation could have increased wheat growth due to B. bacteriovorus death in the soil and subsequent release of nutrients including nitrogen. However, some B. bacteriovorus cells survived in the soil where they could prey upon some Gram-negative bacterial species, reducing their numbers. Some of the wheat growth and yield-producing effects of B. bacteriovorus may be due to the predation of species that are associated with late flowering, and therefore grain development, in wheat, allowing time for more grain to develop. Alternatively, it could be due to processes performed by B. bacteriovorus in the soil that are not related to predation, such as production of the plant hormone IAA, or B. bacteriovorus colonisation of the roots and predation of root-associated pathogenic bacterial species. Further studies are required to identify the mechanisms behind these unexpected crop yield-promoting effects, and the extent of any nutrient ‘boost’ effect due to death of B. bacteriovorus in the wheat soil, to determine whether B. bacteriovorus could be used as a pre-harvest growth and yield-promoting agent. Although most studies of B. bacteriovorus so far have focussed on its predatory activity, it likely performs other functions in its natural soil habitat, which may underlie some of the growth and yield-promoting effects shown here. However, these data show that B. bacteriovorus could be used commercially as a ‘food security agent’ when used as a post-harvest treatment to prevent crop spoilage and loss (as for A. bisporus mushrooms).

MAP is the causative agent of a wasting disease in ruminants and other animals called Johne’s disease. Culture of the organism can take months and in the case of some sheep strains of MAP, culture can take up to a year. It can take several years for an animal infected with MAP to show clinical symptoms of disease. During this subclinical stage of infection, MAP can be shed into the environment contaminating their surroundings and infecting other animals. As well as this Johne’s disease is particularly difficult to diagnose during the subclinical stage of infection. Culture is very difficult and takes too long to be a viable method to diagnose Johne’s disease. Microscopic methods can be used on histological samples to detect MAP, however common acid-fast stains used are not specific for MAP and other mycobacteria and acid-fast organisms can be detected. Molecular methods, such as PCR, exist to rapidly detect the signature DNA sequences of these organisms, however they have the disadvantage of not being able to distinguish between live and dead organisms. Other immunological methods, such as ELISA tests, exist and are routinely used to diagnose Johne’s disease, however their sensitivity is very poor especially during the subclinical stage of disease. The aim of these studies was to develop novel rapid methods of detecting MAP to act as an alternative to methods already available. Sample processing using magnetic separation was carried out to allow good capture of MAP cells and to allow efficient phage infection. Using the phage assay, a specific, sensitive phage based method was developed that could detect approximately 10 cells per ml of blood within 24 h in the laboratory with a sensitive, specific plaque-PCR. This optimised detection method was then used to determine whether MAP cells could be detected in clinical blood samples of cattle suffering from Johne’s disease. The results suggest that animals experimentally and naturally infected with MAP harboured cells in their blood during subclinical and clinical stages of infection. A novel high-throughput method of detecting mycobacteria was also developed. Using phage D29 as a novel mycobacterial DNA extraction tool, viable MAP cells were detected within 8 h and the format of the assay means that it can be adapted to be used in a high-throughput capacity. Factors affecting phage infection and phage-host interactions were investigated to make sure the phage based methods of detection were as efficient as possible. It was found that periods of recovery were often necessary to not only make sure the phage were not inhibited but to also allow the host cells to be metabolically active as it was found that phage D29 can only infect mycobacteria cells that are metabolically active. A fluorescent fusion-peptide capable of specifically labelling MAP cells was also developed to be used as an alternative to acid-fast staining. Peptides that were found to specifically bind to MAP cells were fused with green fluorescent protein and cells mounted on slides were specifically labelled with the fluorescent fusion protein. This resulted in a good alternative to the generic acid-fast staining methods. The blood phage assay has shown that viable MAP cells can be found in the blood of animals suffering from Johne’s disease within 24 h and this can be confirmed using a MAP specific plaque-PCR protocol. A novel faster method to detect MAP was also developed, to cut down the time to detection of viable MAP cells to 8 h, which can be formatted to be used in a high-throughput capacity. The phage assay was used as a tool to determine different metabolic states of mycobacteria, and helped investigate optimal detection conditions when using the phage assay. Finally a novel fluorescent label was developed to detect MAP as an alternative to insensitive acid-fast staining. The development of these novel methods to rapidly, specifically and sensitively detect MAP will push further the understanding of Johne’s disease and help control it.

Gram-negative bacteria are the most common cause of the sepsis and lipopolysaccharide (LPS) a major component of Gram-negative bacteria is known to be of major importance in the development of sepsis. Human infection with Salmonella, a Gram-negative bacterium, is associated with a number of cases of sepsis and is particularly important in childhood sepsis. During salmonellosis, monocytes and macrophages produce a number of different pro-inflammatory mediators such as TNF-α, IL-1α, IL-12, IL-18, IFN-γ, reactive nitrogen species and oxygen species. Although the production of these inflammatory mediators is required for resolution of bacterial infections, they are contraindicated in diseases such as sepsis. In the initial (acute) phase of sepsis a Systemic Inflammatory Response Syndrome (SIRS) occurs in which inflammatory mediators are produced in high concentration, which can lead to organ failure and death. The SIRS phase is then replaced by a Compensatory Anti-inflammatory Response Syndrome (CARS) phase which leads to immunosuppression. The CARS phase can lead to secondary infection and subsequent mortality within 28 days of hospital admission. To date, several studies have evaluated the role of vasoactive intestinal peptide (VIP) as an anti-inflammatory agent that may have therapeutic potential in septic patients both in vitro and in vivo. VIP has been shown to inhibit production of inflammatory mediators produced by human monocytes in response to LPS. The aim of the work described in this thesis was to investigate the therapeutic potential of VIP in sepsis using an ex vivo human monocytes model infected with viable Salmonella Typhimurium 4/74 (rather than LPS). The study shows that VIP (10-7 M) stimulates an increase in the numbers of Salmonella recovered from infected human monocytes (MOI = 10). In addition, VIP also increases the survival rate of human monocytes infected with Salmonella. These two results may suggest a detrimental effect of VIP during bacteraemia and sepsis, since monocyte death may be beneficial during sepsis and bacterial overgrowth could lead to further increased LPS (and other antigen) stimulation of the immune system. However, VIP did significantly decrease Salmonella and LPS-induced TNF-α, IL-1β and IL-6 in monocyte supernatants. VIP also had a positive effect on IL-10 production in human monocytes infected with Salmonella or stimulated with LPS. Whether this suggests a possible detrimental effect of VIP is unknown but septic patients with high serum IL-10/ low TNF-α concentration ratio have previously been shown to have a poor prognosis. Higher IL-10 concentrations in infected monocytes (due to VIP) could also increase the CARS phase of disease with increased immunosuppression. Flow cytometry and qPCR analyses showed that of all of the VIP receptors, VPAC1 was expressed most highly during Salmonella infection, or LPS stimulation, of human monocytes. Administration of VIP inhibited VPAC1 has been shown by many studies to be the most important receptor by which VIP inhibits production of inflammatory immune mediators, or increases IL-10 production from murine macrophages. Results in this thesis, therefore, suggested that Salmonella infection may promote VPAC1 expression and so provide a mechanism of inhibiting the production of inflammatory mediators in infected cells. This could then increase intracellular survival of Salmonella and provide a means of greater dissemination of the infection. To ascertain how increased VPAC1 expression on the surface of monocytes may be achieved, analysis of the expression of known intracellular endosomal and exosomal constituents was performed. Confocal laser microscopy, using specific antibodies, showed that VPAC1 on the monocyte cell membrane was internalised within early endosomes (measured by co-localisation of VPAC1 and EEA1) rather than being degraded within lysosomes (measured by immunoreactivity to LAMP1). VPAC1 is then transported via a Rab11A recycling endosome and packaged in the Trans-Golgi network (TGN), shown by co-localisation of VPAC1/Rab11A and the TGN marker (TGN46). VPAC1 was then associated with Rab3a and calmodulin. The function of these latter two proteins in the docking of exosomes to the cell membrane is well known, thus suggesting that Salmonella induced VPAC1 was also recycled to the cell membrane within exosomes. VIP inhibited the expression of both Rab3a and calmodulin but not the co-localisation of VPAC1 with these two proteins. Further studies then showed that a calmodulin agonist (CALP1) increased VPAC1 expression on the surface of monocytes, while a calmodulin antagonist (W-7) decreased expression of VPAC1 on the surface of monocytes. In conclusion, this thesis does present hitherto unknown data regarding Salmonella infection of human monocytes and the effects of VIP on infected monocytes. VIP has potential as an anti-sepsis therapy since it reduces the production of inflammatory mediators by Salmonella-infected and LPS-stimulated monocytes. However, the fact that VIP increases survival of infected human monocyte and increased growth of Salmonella in human monocytes may preclude its use in sepsis.

The carbon and energy metabolism of the human pathogen Clostridium difficile is poorly understood. Amino acid metabolism by the Stickland reactions has previously been described as a primary source of energy in a number of Clostridium species, especially when grown in a medium containing only amino acids. Deeper insights may be gained by metabolic analyses using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) platforms, but such experiments are best performed in well-defined growth media. A number of C. difficile strains have been successfully cultivated on defined media but these media provide an excess of nutrients, particularly in terms of amino acid provision, resulting in undesirable background growth in the absence of glucose. To overcome these challenges, three variants of a defined medium were developed that contain the essential nutrients that support the growth of this bacterium, in the presence of a carbon and energy source such as glucose together with an LC-MS/MS method that will simultaneously measure all twenty amino acids. Since the focus in this study was amino acid utilisation, a comprehensive and most effective technique that will provide as much information as possible for understanding the metabolic requirements of eight Clostridium difficile strains (CD630∆erm, DH196, R20291, EK15, EK28, R12801, L26, O17 Serotype F) and two transposon mutants (CRG-2979, CRG-3887) was required. Due to high water solubility and the range of ionic characteristics of amino acids, an aqueous normal phase chromatographic method was considered to simultaneously separate a mixture of amino acids without derivatisation. Aqueous normal phase chromatographic method represents an important new technology with a capability of the silica-hydride-base stationary phases, offering a distinct advantage for practical application with a high degree of reproducibility and long-term stability of polar and non-polar compounds. The developed methods were subsequently adapted to study amino acid utilisation in the presence or absence of a fermentable carbohydrate and/or selenium. OD determinations were performed by measuring absorbance at 600 nm (or OD600) using a Biomate 3 spectrophotometer. The concentration of individual amino acids remaining in the spent C. difficile culture media was measured after 24 h and/or 48 h using the developed LC-MS/MS method in order to determine which amino acids were being utilised by these organisms and in which order. This analysis should give further insight on the importance of amino acids for the survival of this bacterium in the gut, which may possibly lead to discovery of novel fermentable products and metabolic pathways and/or eventually aid in the successful control of the disease. Data obtained for CD630∆erm, DH196, R20291, EK15, EK28, R12801, L26, O17 Serotype F strains showed that they all grew on the fully defined medium, with different growth profiles in terms of lag phase, growth rate, and maximum OD reached. The LC-MS/MS data generated suggest that cysteine, glutamine, isoleucine, leucine, serine, threonine and tyrosine are preferentially utilised, both in the presence and absence of glucose. Several other amino acids, including asparagine, glycine, phenylalanine, proline and valine were also utilised but to a lesser extent. Notable in this study was the lack of glutamate utilisation, except by strain L26, and the excretion of alanine after its initial uptake by most of the tested stains. The excretion of alanine may be due to the use of pyruvate as an amino acceptor during the degradation of preferentially fermented amino acids, whereas, glutamate is not a substrate for most C. difficile strains. Thus, LC-MS/MS profiling confirmed that these organisms derive most of their carbon and energy from the fermentation of a selected range of amino acids. Given the importance of selenium-dependent Stickland reactions to the growth of this bacterium, further studies were undertaken to evaluate the metabolism of amino acids by two different C. difficile strains (630∆erm, R20291) and two transposon mutants CRG-2979 (defective in hadB, encoding one of the two subunits of hydroxyisocaproyl-CoA dehydratase required for reductive degradation of leucine) and CRG-3887 (defective in selA encoding selenocysteine synthase) in the presence or absence of glucose/selenium. LC-MS/MS data reveal that amino acid utilisation was affected by the presence of selenite, notably proline utilisation, which could be explained by the presence of the enzyme proline reductase and the lack of glycine consumption, known to be selenium-dependent. The non-utilisation of glycine could be explained by the presence of proline which represses the formation of the necessary enzyme systems required for glycine degradation. Data generated for CRG-2979 reveals that this mutant could only thrive in the presence of selenium when glucose is present, possibly due to the presence of proline replacing leucine as the major Stickland acceptor. The results of the transposon mutant CRG-3887, were much of a surprise too, because this mutant was predicted to be deficient in proline and glycine breakdown which is also reliant on selenoenzymes. This suggests the presence of selenium independent proline fermentation pathway although this hypothesis is not supported by the existing literature. Experimental data provided further evidence about the ability of this bacterium to obtain its carbon and energy in the absence of a fermentable carbohydrate; by Stickland reactions and that the presence or absence of certain amino acids could repress the utilisation or biosynthesis of other amino acids.

Clostridium difficile is the leading cause of health care associated diarrhoea and remains a burden for the NHS. Disease symptoms can range from mild diarrhoea through to fulminant pseudomembranous colitis, resulting in mortality for some patients. Recurrence is a major problem and estimates are that 20% of all patients with disease will either relapse (with the same strain) or have a re-infection (with a different strain). Arguably, the main virulence factors are toxins A (TcdA) and toxin B (TcdB) which cause disease symptoms. The genes encoding TcdA and TcdB are located within the pathogenicity locus (PaLoc) along with three accessory genes; tcdR, tcdE and tcdC. The regulatory network has been studied but we aimed to add to this knowledge by using two under investigated strains R20291 a so-called hypervirulent strain and VPI 10463 a strain known to produce higher levels of toxin. Two different methods of investigation were employed during this study to improve our understanding of both the regulation of TcdA / TcdB but also the genetic mechanisms behind clinical relapse. These methods were; using forward and reverse genetic analysis to assess phenotypic differences and using bioinformatics to identify genes and / or single nucleotide variants (SNP) that may play a role. Using a combination these methods we have identified potential regulators of toxin production in both strains. We have also identified unique genes and SNPs that might provide a fitness benefit to strains of C. difficile that were isolated from patients who had suffered relapse episodes.

Currently, fossil fuels contribute a large number of high value chemicals that are used on a daily basis. Crude oil is cracked to give a number of high value chemicals, including vehicle fuels as well as chemicals and solvents that are used daily both commercially and industrially. However, fossil fuel reserves are in decline, with research going into alternatives to obtain these useful chemicals, one of which is biofuels. Biofuels can be generated in a number of ways, one of which is the fermentation of acetogenic bacteria, microorganisms that generate acetate as a product of anaerobic metabolism. Clostridium autoethanogenum is an acetogen that can grow on one carbon gases as its feedstock, and can be used to generate valuable chemicals, with scope to develop the range of metabolic products further. One aim of this project was to investigate the metabolic flux through pathways of the bacterium using isotopically labelled compounds, which would be assessed by mass spectrometry and NMR. Following on from this, design of inhibitors for the enzymes of the pathways with a view to drive the metabolic processes towards higher value chemical compounds by ‘switching off’ other branches of the pathway. Putative small molecule mimics of acetyl-CoA, SNAC thioesters, were synthesised and tested for uptake and activity in whole cell growth experiments with C. autoethanogenum, and determined to be unsuccessful. Further to this, compounds were designed and synthesised to replace pantothenic acid in the growth media, which were not tested in growth experiments. A library of inhibitor compounds was synthesised and tested against recombinantly purified acetate kinase. A number of compounds were shown to inhibit the enzyme, and the mode of inhibition was determined, as well as IC50 and Ki values for each. This project operated as part of a larger GASCHEM project in the Synthetic Biology Research Centre at the University of Nottingham.

Haemophilus influenzae type b (Hib) was a main cause of bacterial meningitis. Together with Neisseria meningitidis and Streptococcus pneumoniae, it occurred with comparable frequency prior to the introduction of conjugated polysaccharide vaccines against Hib. Despite the fact that the introduction of conjugated vaccines against Hib has virtually eradicated infection in many areas of the globe, this pathogen still causes many infections in developing countries and a number of cases of infection have been reported in fully vaccinated children in developed countries. Recent studies have focused on the mechanisms by which these pathogens invade the central nervous system through the blood brain barrier (BBB). One study revealed that the outer membrane protein OmpP2 of H. influenzae interacted with the 37/67-kDa non-integrin laminin receptor (LamR) of human brain microvascular endothelial cells (HBMECs). In this study, OmpP2 of H. influenzae was expressed and purified and its interactions with purified recombinant LamR (rLamR) were analysed. OmpP2, which is predicted to contain eight extracellular loops, was expressed in two parts: OmpP2Δ1-4, lacking loops 1-4, and OmpP2Δ5-8, lacking loops 5-8. The protein fragments were purified and their interaction with rLamR was investigated by ELISA. The LamR binding site of OmpP2 was found to be restricted to loops 1-4. Therefore, ompP2 derivatives encoding OmpP2 lacking loops 1, 2, 3 and 4 individually, were constructed. The OmpP2 derivatives were expressed and tested in rLamR-binding assays to determine which of these loops is required for LamR binding. Only OmpP2∆L2 showed dramatically decreased binding to rLamR. Accordingly, loop 2 of OmpP2 may play an important role in the interaction of H. influenzae with LamR. Therefore, ompP2 and ompP2ΔL2 null mutants were generated in H. influenzae strain Rd KW20 and utilized in subsequent characterization experiments to facilitate a study of the potential role of OmpP2 and its second loop in the pathogenesis of H. influenzae. The interaction of H. influenzae and its derivative mutants to LamR was investigated using a combination of molecular and immunological techniques, including ELISA, whole cell lysate pull-down assays, invasion and association assays and flow cytometry. H. influenzae cells either lacking OmpP2 or expressing OmpP2∆L2 showed significantly reduced rLamR-binding compared to the wild type. Furthermore, synthetic peptides based on the loop 2 sequence coupled to micro-beads mediated adherence of the beads to HBMECs. The amino acid sequence of OmpP2 loop 2 was found to be highly conserved in Hib isolates, but not in non-typeable H. influenzae, which rarely cause invasive disease. The potential roles of OmpP2 and Loop 2 in H. influenzae pathogenesis are discussed.

A C. perfringens type A genomic library was constructed in E. coli by banking overlapping 6-10 kbp Hind III fragments of chromosomal DNA from the enterotoxin (CPE) positive strain NCTC 8239 into the pUC derived vector pHG165. The library was screened by colony hybridization with a degenerate 26 bp oligonucleotide probe, derived from the amino acid sequence CPE9_17A. complex mixture of plasmid DNA was isolated from the only hybridization positive clone. A second round of screening picked out a single plasmid, with an apparently altered copy number, pLWl, that carried the CPE gene, cpe, on a 6.8 kbp insert. A sequence deduced primer strategy for direct plasmid sequencing was initiated using a primer deduced in a similar manner to the 26 bp probe, obviating the need for prior mapping and subcloning of the insert. The amino acid sequence for the conceptual gene product of the single open reading frame differed only slightly from the known CPE sequence but lacked the C terminal residues. The biased cpe codon usage reflected the low %G+C content of the DNA. The %G+C content was even lower in the upstream region and possessed properties characteristic of bent DNA. The region 5' to the ATG translational start codon contained a Shine-Dalgarno sequence and several sequences with significant homology to the putative transcriptional control regions for the tetanus toxin gene. The N-terminal coding region contained a direct repeat of an upstream sequence that shared considerable homologies with the crossover point in site 1 of the Tn3 res region. Southern blot analyses of chromosomal and plasmid DNAs from several isolates indicated that the majority of strains were cpe-. The chromosomal location and architecture of cpe appeared identical in all cpe+ strains. A second copy, pLW2, of the 5' end of cpe, on a 4.5 kbp Pst I/Eco RI restriction fragment, was cloned during one of many unsuccessful attempts to clone the 3' end. A separate re-cloning experiment isolated several different clones that contained the 0.6 kbp Hind III located = 2.5 kbp 5' to the ATG codon of both cloned copies of cpe but none of them carried the CPE gene. The fragment was used as a DNA probe to show that it was present in high copy number in some strains of C. perfringens but completely absent from others. An hypothesis describing the possible involvement of a mobile genetic element in C. perfringens enterotoxin production offers explanations for the cloning of a complex mixture of plasmids, the apparent alteration in plasmid copy number, the identification of putative DNA crossover points, the failure to clone the 3' end of cpe and the isolation of a novel DNA fragment.