Streptomyces are ubiquitous in soil and face a rapidly changing environment. Like other bacteria, they sense and respond to external stimuli via two component systems and Streptomyces species encode a particularly high number of these systems. One of these two component systems is called MtrAB-LpqB and it is highly conserved in the phylum Actinobacteria. Previous work in Mycobacterium tuberculosis, Corynebacterium glutamicum and Streptomyces coelicolor indicates that MtrAB-LpqB is involved in osmosensing and cell cycle progression. To investigate the function of MtrAB-LpqB I attempted to make single gene deletions in the new model organism Streptomyces venezuelae. I also performed chromatin immunoprecipitation and sequencing (ChIP-seq) against MtrA-3xFlag in S. venezuelae and S. coelicolor to identify the regulon of genes under its control. I present evidence that MtrA is essential in S. venezuelae whereas MtrB is dispensable. It was not possible to confirm deletion of lpqB. Deletion of mtrB activates MtrA and leads to the overproduction of cryptic secondary metabolite biosynthetic gene clusters (BGCs). The same effect was achieved by introducing a gain of function MtrA protein into the S. venezuelae wild-type strain. The cryptic BGCs are activated because MtrA binds to target genes spanning 85% of the BGCs in S. venezuelae and S. coelicolor. In Streptomyces, antibiotic production is linked to development and the MtrA regulon overlaps with the master regulator of development, BldD. The results presented here suggest that MtrAB senses external signals and modulates target gene expression to coordinate development with the production of secondary metabolites.

No description available.

The enterococcal surface protein (esp) gene has been proposed as a marker of human faecal contamination, as it appears to be restricted to surface waters receiving human inputs. However, it has been reported that the gene may also be present in non-human sources, and its geographical distribution has been questioned. This study investigated aspects of the ecology of the esp gene in human and non-human faeces and evaluated critically its potential as a microbial source tracking (MST) tool.

Pseudomonas aeruginosa is an opportunistic human pathogen that produces an extensive array of virulence determinants, many of which are regulated via quorum-sensing (QS) in a cell-density dependent manner and play an important role in the outcome of infection. QS controls virulence factor production via two signalling systems driven by either N-acylhomoserine lactone (AHL) or 2-alkyl-4-quinolone (AQ) signal molecules. The blocking of QS has been proposed as a potential future antibacterial drug strategy, therefore this study aimed to further the knowledge of QS in P. aeruginosa, investigating how QS is connected to other regulatory networks and virulence, and the potential of alternative methods of blocking QS. The use of probiotic bacteria to inhibit growth, QS and virulence factor production in P. aeruginosa was investigated. Nine probiotic species of bacteria were unable to inhibit QS, degrade QS signal molecules, or affect the production of virulence determinants, biofilm formation or attachment. Interestingly, the effects observed were mainly attributed to the MRS growth medium in which probiotics were grown as this medium showed significant antibacterial and quorum quenching properties. The function and the regulation of two genes, PA2383 and PA2384, which have been suggested to have roles in QS and iron-regulation, were investigated. PA2383 and PA2384, predicted to be in the same operon, were regulated at the transcriptional level both by a mechanism dependent upon iron availability and by AQ-like molecules with either QS or iron chelation abilities. Furthermore, these genes were involved in an autoregulatory feedback loop in which PA2384 either repressed or activated the transcription of the operon under iron-replete or iron-limited conditions, respectively. Additionally, positive post-transcriptional regulation was exerted on PA2384 by RsmA, itself under the control of GacS-GacA. PA2383 and PA2384 were found to regulate the expression of QS-dependent genes (including pqsH, pqsL, rhll, rhiR, last, phzA1) and genes required for iron uptake (pvds, pvdE, pirA and pfeA). PA2383 and PA2384 may therefore provide a link between QS, iron homeostasis and the GacS-GacA global regulatory system. Forty-nine P. aeruginosa clinical wound isolates from colonised patients were characterised to investigate variation and search for correlations in QS, virulence and antibiotic resistance. The isolates were found to produce varying levels of AHLs and AQs, and interestingly AHL proficient but AQ deficient isolates were also identified. Pyocyanin, LasB elastase and universal protease production in the isolates also varied. Some positive correlation between 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) and pyocyanin levels, and N-(3-oxo-dodeconyl)-homoserine lactone (3-oxo-C12-HSL) and LasB elastase/protease levels was also seen, however no correlation between QS signal production and swarming or attachment capabilities was found. High antibiotic susceptibility was seen in the isolates to aminoglycosides, but also high resistance to meropenem. The antibiotic susceptibility profile did not show great variability across the isolates and as such, no correlation between QS and antibiotic resistance was evident. In summary, studies have shown that the QS systems in P. aeruginosa may be a good target for novel antibacterial treatments. A proposed source for quorum quenching activity, probiotic bacteria, was investigated however was found to be ineffective against P. aeruginosa. This work further aimed to advance the knowledge of the QS systems to both discover how they are linked to other complex regulatory systems in P. aeruginosa, and to determine the characteristics and QS abilities of P. aeruginosa wound isolates for which quorum quenching treatments may one day act upon. This work gives new insight into the QS regulatory networks that will advance the knowledge needed for the development of quorum quenching agents.

1. Strains of E. co1i have been characterised which are galactose-positive despite their failure to express, any of the active transport systems for galactose during growth on this sugar. The growth of such strains on galactose occurs at rates that are a function of the galactose concentration of the medium: half-maximal growth rates require more than 2mM galactose to be present. The introduction of a mutation in the glucose phosphotransferase Enzyme II specified by the gene umg severely impairs the ability of these strains to grow on galactose; it has been established by a variety of means that this Enzyme II, or a component of it, provides the means of galactose entry into these organisms. However, the uptake of galactose does not require phosphotransferase activity, but occurs by facilitated diffusion on this carrier. The implications of this finding on the current understanding of tine mechanism of glucose uptake by the phosphotransferase system are discussed. 2. Although the Umg-system provides the major route of glucose uptake in many strains of E. co1i, this is not true for all strains. Evidence is presented that suggests the existence of a fourth Enzyme II for glucose, in addition to those specified by the genes umg, ptsX and bgl. 3. The galactose permease specified by the gene galP can transport glucose in addition to galactose. A screening procedure for distinguishing GalP+ from GalP- strains is described which makes use of this property and which has been used to locate the galP lesion close to minute 55 on the E. coli chromosome, between the genetic markers fda and lysA.

In this project enhancement of poly(3-hydroxybutyrate), P(3HB) production using a Gram positive bacteria, Bacillus cereus SPV and sucrose as the main carbon source was successfully achieved. Different modes of fermentation including shaken flask, batch, fedbatch and two-stage fermentation were investigated in the study. A modified G-medium was formulated and used throughout the study. Potassium and sulphate were identified as the main limiting factor for P(3HB) accumulation in Bacillus cereus SPV. By limiting the potassium phosphate concentration to 0.5 g/L K2HPO4 in the production medium, the dry cell weight, P(3HB) yield and P(3HB) concentration improved to, 7.21 g/L, 82 % dcw and 5.95 g/L respectively (i.e. 236, 115.8 and 830 % increase in dry cell weight, P(3HB) yield and P(3HB) concentration respectively). In addition, economic production of P(3HB) using agricultural/industrial waste (molasses) as the main carbon substrate was achieved. The study was also carried out in both shaken flask and 2L fermenter. The maximum P(3HB) yield achieved was 61.07 % dcw in 1L shaken flask and 51.37 % dcw in 2L fermenter. A novel wet cell PHA extraction was successfully developed in this project leading to high purity of the PHA produced with reduced crystallinity and efficient recovery. This is expected to save time, cost and enhanced continuous PHA production. Furthermore, a novel inexpensive and sustainable ‘compression moulding/particulate leaching’ technique for tissue engineering scaffold fabrication was developed. The novel technique enabled the production of biodegradable composite scaffolds of P(3HB)/microfibrillated cellulose and magnetic P(3HB) nanocomposite for possible applications in cartilage and bone tissue engineering, respectively. Detailed studies on the 2D and 3D composites showed that the inclusion of microfibrillated cellulose into the P(3HB) matrix enhanced the mechanical properties, hydrophilicity, introduced microtopography features, enhanced surface chemistry and biocompatibility of the composite material while inclusion of magnetic nanoparticles and ferrofluid, in addition to the above features added magnetic properties and microhardness to the composite materials. A unique controlled drug delivery system was developed with potential application in multiple drug delivery. The release study confirmed that the delivery system was able to control the release of BSA, a model protein. Finally, composite magnetic microspheres were also produced and characterised for their efficient use in the delivery of cancer drug and analyses performed showed that the composite constructs have superparamagnetic properties which would be useful for targeted delivery.

Pathogenic bacteria are causative agents of epidemics and deaths worldwide; water is a vector for the transport of bacteria and their consequent consumption by humans. The accepted "gold standard" for assessing bacterial concentrations in water is by traditional plate counts. However, this globally accepted technique takes longer than 24 hours. An alternative method to rapidly detect and to continuously assess bacterial concentration is required. A biosensor that provides near real-time information on the bacterial loading of a waterbody is proposed. This thesis describes the development of a microfluidic-magnetophoretic device to detect bacteria in water. Escherichia coli is a well-reported bacterium found in the intestinal tract of mammals. Particular strains of E. coli are pathogenic to humans. E. coli is used as an "indicator" for water contamination and was chosen as the target for the biosensor. Magnetic particles (MPs) were functionalised with an anti-E. coli antibody. The functionalised MPs were used to capture E. coli from drinking water by forming MP-bacterial complexes. A microfluidic device was designed, incorporating magnetophoresis, to separate and concentrate MPs suspended within a sample. A separation efficiency of approximately 80% of the MPs from a sample flow was achieved. Using a magnetic gradient and video tracking of the MPs, the magnetic-drift velocities of unbound and bound MP-bacterial complexes was determined. It was found that viable and non-viable MP-bacterial complexes had different magnetically-induced drift velocities. A single viable E. coli bound to an MP travelled at 0.49 times the velocity of an unbound MP, and a single non-viable E. coli travelled at 0.75 that of an unbound MP. As such, this novel magnetophoretic separation technique can be used to determine the viability of bacteria. The developed biosensor provides the ability to concentrate, separate and enumerate viable E. coli from drinking water, with the potential for automation and sampling from continuous flow.

Escherichia coli O157 is a strain of coliform bacteria associated with serious human disease; infection by the strain, particularly in children and the elderly, may lead to death. Although human infection is often associated with food poisoning, other routes of infection have been identified. These include contact with animals, particularly livestock, bathing in infected water, and mere wandering in rural areas. Aside from the human cost in terms of distress to the afflicted and to their families, there is an economic cost. Although there is extensive literature on E. coli O157, much remains uncertain. The present thesis addresses some of these uncertainties. It specifically addresses livestock (cattle and sheep) infection within North Wales. Issues addressed comprise livestock faecal E. coli and other coliform bacteria counts within the area; the effects of grazing on the said counts, and differences in land type (upland or lowland) on them; seasonal variations; the effects of physical and chemical soil properties on E. coli and other coliform bacteria populations; the differential effects of grazing system and crop type on the bacterial populations; and transmission of E. coli O157 to different types of location by yellow dung flies (Scathophaga stercoraria). Investigation of these issues comprises field studies and, in the case of transmission of the bacteria by dung flies, laboratory experiments. The majority of these studies were successful insofar as they suggest robust conclusions. The most important of these are that sheep faeces host more non-pathogenic E. coli than does cattle faeces, that E. coli populations within the environment vary by season, that lowland farms have higher populations of E. coli, that intensive grazing adds to risk of infection, that yellow dung flies can carry substantial populations of E. coli O157, and that the flies may transmit the bacteria to the environment, particularly to water and to vegetation (grass).

Methane oxidizing bacteria (MOB) use methane as their main source of carbon and energy. The main methane oxidizing enzyme in MOB is the copper-containing particulate methane monooxygenase (pMMO), a rare example of cytoplasmic copper enzyme. Some ‘switchover’ strains are capable of differentially expressing pMMO as well as a soluble iron-containing form (sMMO), and the switchover is regulated by copper. MOB secrete methanobactin (mb) which mediates copper uptake and is internalized in the cytoplasm. Despite this pathway for copper import, as well as copper regulating components such as CopA, CopZ and CueR being present in MOB, little is known on how these bacteria handle the large amounts of copper required for methane oxidation by pMMO. Through metalloproteomic analysis of soluble extracts from the switchover MOB M. trichosporium OB3b a large number of soluble copper pools were visualized and a novel copper protein, Csp1, was identified. Two more homologues, Csp2 and Csp3, were identified in M. trichosporium OB3b through bioinformatics. In vitro characterization of Csp1 and the homologue Csp3 showed these proteins are tetramers of 4-helix bundles that bind 13 and 18 Cu(I) ions per monomer, respectively, all of which are stored inside the core of the 4-helix bundle and are coordinated mostly by Cys residues. Csp1 binds tightly at least 10 Cu(I) ions whereas Csp3 has an average Cu(I) affinity at the order of 1017 M-1. Csp1 and Csp3 do not remove Cu(I) from Cu(I)-mb, however it is likely that apo-mb, which removes Cu(I) from these proteins, notably at very different rates, transports Cu(I) to pMMO. Csp1 is thought to be exported from the cytosol potentially to the intra-cytoplasmic membranes, where pMMO is localised, to store copper for the enzyme. Csp3 is thought to be cytosolic and either sequesters copper to prevent copper-induced toxicity or, more likely, supplies copper to unknown cytosolic copper enzymes, consistent with the large number of soluble copper pools visualized in the organism. Csp1 protein homologues are present in other bacteria, including MOB, while homologues of the cytosolic Csp3 are widely distributed in members of all major bacterial phyla. The presence of Csp3 in the bacterial cytosol implies either a function as a defense mechanism against copper-induced toxicity or more likely copper storage for supply to cytosolic copper enzymes, yet to be identified. The latter possibility challenges the present model according to which bacteria do not have a cytosolic requirement for copper.

Pneumolysin is an important virulence factor produced by the human pathogen Streptococcus pneumoniae. It belongs to the family of cholesterol dependent cytolysins (CDCs) that damage the target cell membrane, by forming large oligomeric pores of 30 to 80 toxin monomers, where each monomer is thought to contribute at least two p-hairpins. A panel of mutations was done in the two putative transmembrane region of pneumolysin TMH1 and TMH2 located in domain 3 of the toxin monomer, and believed to be lining the pore lumen. The generated mutants exhibited different levels of haemolytic activity, particularly the single mutation W278F, W278D, and the triple mutation (D257N-E258Q-E260Q), largely impaired the haemolytic activity of the wild-type toxin. These mutant toxins along with a previously made lytic deficient mutant W433F were subjected to further studies. Circular dichroism analysis done with those mutants showed that the secondary structure of the native toxin was conserved. The kinetics of release of calcein from liposomes along with the kinetics of lysis of erythrocytes exposed to these mutants was substantially slower than that of the wild-type toxin. Pneumolysin and other CDCs induced pores were studied on model systems like lipid bilayer and liposomes. In this thesis, I demonstrated the formation of pores by pneumolysin on the membrane of a 'real' cell by using the patch-clamp technique. Pneumolysin induced heterogeneous pore on either side of the membrane, of different conductance states, classified as small, medium and large. A stepwise increase in current was observed with early appearance of small conductance channels followed by larger ones. The mutant toxins generated in this work and W433F were also tested with patch clamping. They formed pores of various conductance states with a decrease in the occurrence of large channels, in comparison to the wild-type.