No description available.

A recombinant phage that produced a polypeptide possessing the characteristics of pneumolysin, the membrane damaging toxin of the pneumococcus, was isolated from a bank of pneumococcal sequences in ?gt10. Subclones carrying the pneumolysin gene in various plasmids were haemolytic regardless of the orientation of the insert. The nucleotide sequence of a 5 kb fragment carrying the pneumolysin gene was determined. An open reading frame 1413 bp long was identified that when translated encoded a polypeptide with 471 amino acids and a molecular weight 52.8 kD. The N-terminal amino acid sequence of the predicted protein was identical to that of native pneumolysin. A single cysteine residue was present at position 428 in the amino acid sequence. Comparison of the DNA and amino acid sequences of pneumolysin with streptolysin O (SLO) revealed extensive homology in the amino acid sequence. The longest region of identity was a sequence of 12 amino acids surrounding the unique cysteine. A hybrid gene consisting of the 5' region of the pneumolysin gene and the 3' end of the SLO gene was constructed. The fusion polypeptide was made in E. coli, but possessed a very low haemolytic activity. Using the technique of oligonucleotide-mediated site-directed mutagenesis, two mutant genes were constructed in which the cysteine codon was changed to either a glycine or serine codon. Modified toxins when purified from E. coli had a specific activity of about 1-2 % that of wild type pneumolysin.

No description available.

No description available.

Bacterial protein export involves the translocation of precursor proteins across the inner cytoplasmic membrane. Over 100 proteins are exported from Escherichia coli. This study showed that energy in the form of ATP and membrane gradient energy is essential for the export of leucine specific binding protein and (3-lactamase precursors. Ionophores or combinations of ionophores (SF6847, valinomycin/nigericin and valinomycin/monensin) which dissipate protonmotive force inhibit protein export. Valinomycin alone also inhibits export, but not as well as reagents which dissipate protonmotive force. Nigericin or monensin alone slightly stimulate protein export. These results suggest that the transmembrane electrical potential ( DY) is the component of membrane gradient energy necessary for precursor protein export. ATP is necessary for the export of precursors. In the absence of ATP, in vitro export of leucine specific binding protein and B-lactamase precursors is not observed. An upper limit of l0μM was determined for the effective Km for ATP during in vitro protein export. It was also shown that ATP is consumed during the export process. The SecA protein was shown to contain an ATPase activity that is stimulated by the presence of inverted membranes and purified LSBP precursors. Vanadate and diethylstilbestrol, inhibitors of ATPases, inhibit in vitro protein export. Vanadate also inhibits SecA ATPase activity which depends on membranes and precursors. Vanadate is a specific inhibitor of P-type ion translocating ATPases. This study showed primary sequence homology between part of the SecA protein and the phosphorylation sequence of P-type ATPases. Sequence homology, vanadate inhibition of SecA ATPase activity, and vanadate inhibition of in vitro protein export suggest that SecA may function by a mechanism similar to the E1E2 mechanism found in P-type ATPases. Phosphorylation of two proteins with apparent sizes of 62 and 37 kDa is observed to occur in an export-associated fashion. This phosphorylation is dependent on membranes and precursors, is sensitive to hydroxylamine, and is sensitive to inhibitors of protein export, including valinomycin/nigericin and vanadate. Furthermore, phosphorylation of the 62 kDa protein is dependent on the presence of SecA. The phosphate linkage appears to be an acyl phosphate based on hydroxylamine sensitivity and reduction of the acyl phosphate linkage by NaCNBH3. Both proteins appear to be peripherally associated with the cytoplasmic face of the inner membrane, which is also consistent with a possible role in the bacterial protein export process.

The type VII secretion system is a protein export pathway linked to diverse phenotypes in both Actinobacteria and Firmicutes. The Actinobacterial subtype of the T7SS, referred to as T7SSa, has been shown to play a critical role in various aspects of Mycobacterial life including virulence, conjugation, and metal homeostasis. The T7SSb of Firmicutes bacteria on the other hand has similarly been shown to influence virulence but by the direct growth inhibition of competitor bacteria. Structure-function analyses of the T7SSa apparatus as well as various effectors and chaperones have begun to build a more mechanistic understanding of how T7SSa functions. In contrast, we know little of how the T7SSb functions despite its noted importance to both pathogens and environmental bacteria such as Bacillus, Staphylococcus, Enterococcus, and Streptococcus. During my thesis work, I have addressed several gaps in our understanding of T7SSb function. The three major questions that I have studied are: (1) how do T7SSb immunity proteins inhibit the toxicity of their cognate toxins, (2) how does the T7SSb export effectors through the thick Gram-positive cell wall, and (3) what is the role of chaperone proteins in facilitating T7SSb effector export? / Thesis / Doctor of Philosophy (PhD) / Bacteria require space and various nutrients to survive and grow and must therefore compete against other bacteria for access to these resources. To gain advantage over their competitors, many bacteria have developed molecular weapons that target and kill other closely related bacteria. Some of these weapons take the form of protein secretion machines that export antibacterial toxins. Gram-positive bacteria use the type VIIb secretion system (T7SSb) to inhibit the growth of other Gram-positive bacteria. In this work, I explore several aspects of T7SSb including: (1) how toxins are inhibited by immunity proteins, (2) how toxins are secreted through the cell envelope, and (3) how toxins are recognized by the secretion apparatus. The goal of this work is to better understand how T7SSb functions at the molecular level.

As productive performance is improved by breed selection, amino acid requirements may change to support this higher performance in poultry. The first objective of this dissertation was to update the valine and tryptophan requirement of small-framed laying hens and the lysine requirement of young broilers using empirical dose-response methods. The tryptophan requirement was estimated as 155.8 mg/d for egg mass, 153.2 mg/d for egg production and 140.4 mg/d for feed conversion ratio using a linear broken line model. For valine, the requirement was highest for egg mass, 597.3 mg/d, followed by egg production, 591.9 mg/d and feed conversion ratio (FCR), 500.5 mg/d. The lysine requirement of young chicks was estimated by conducting four short term experiments from 1 to 3, 3 to 5, 5 to 8 and 8 to 11 days of age, respectively. The lysine requirement from 1 to 3, 3 to 5 and 5 to 8 days of age were not able to be estimated as no dose response was observed on growth performance most likely due to an overestimation of the lysine requirement. Digestible lysine requirement from 8 to 11 days of age was 1.057%, 1.050% and 1.016% based on body weight gain, FCR and pectoralis major weight using a linear broken line model, respectively. In addition to determining amino acid requirements, research was conducted to develop a new bacterial protein meal for use in laying hens diets. The data suggested that diets containing 7.5% of the bacterial protein meal was able to at least maintain egg production in laying hens, but 15% bacterial protein meal resulted in reduced performance. The second objective of this dissertation was to investigate the effects of various concentrations of dietary vitamin D3 on pullet and laying hen performance, eggshell quality and bone health in laying hens. Pullets/hens were randomly assigned to five dietary treatments containing vitamin D3 from 1,681 to 68,348 IU/kg diet from day of hatch until 68 weeks of age. These data suggested that dietary vitamin D3 fed at 68,348 IU/kg resulted in reduced egg production, but vitamin D from 8,348 to 35,014 IU/kg diet maintained egg production, increased egg vitamin D content in a dose dependent manner, and generally increased both eggshell quality and pullet and hen bone mineral status. / Ph. D. / The goal of the poultry industry is to increase the efficiency of meat and egg production. To achieve this goal, laying hens with higher egg production and broilers with faster growth rates are genetically selected over time. By breed selection, laying hens are able to produce 2-3 additional eggs every year. The body weight of a broiler chicken raised today is approximately four times greater than one raised to the same age in 1958. This Increased egg production and body growth requires a higher nutrient intake, especially amino acids, to support protein production. One objective of this dissertation was to update the requirement of three amino acids (valine, tryptophan and lysine) in poultry production to provide current and accurate information to poultry producers. Valine, tryptophan and lysine are essential amino acids that cannot be synthesized by poultry in sufficient quanities and needs to be ingested from the diet. Three experiments were conducted to determine the valine and tryptophan requirement in laying hens and lysine requirement in broilers. The results of the current experiment show that a laying hen require at least 156 mg tryptophan and 597 mg valine per day to maximize egg production from 41 to 60 weeks of age. The broiler chicks need to ingest rations containing at least 1.06% lysine to support growth from 8 to 11 days of age. Bacterial protein meal is a feed ingredient that has been proposed for use in poultry diets. It is usually produced via the fermentation process by converting various substrates such as methane, methanol, or agriculture by-products into protein-rich biomass. The advantage of using bacterial protein meal in the poultry industry is to decrease feed cost and alleviate the demand on croplands. A novel bacterial protein meal, generated from waste water purification, was evaluated as a feedstuff for laying hens. Two levels of bacterial protein meal, 7.5 or 15%, were added to a regular laying hen diet to replace soybean. The results indicated that replacing soybean meal with 7.5% bacterial protein meal was a feasible solution for egg production but a 15% inclusion rate may result in a decreased egg production. During egg production, bone structural health can be reduced as laying hens age. This loss of bone structural health is due to the loss of bone mineral content, especially calcium and phosphorus, as laying hens produce the calcium rich eggshell. With age, decreased bone mineral mass will induce a higher probability of bone structural failure. Vitamin D plays an important role on calcium absorption and bone mineral deposition. In addition to benefits to skeletal health, the addition of vitamin D₃ in the diet will result in increased vitamin D₃ content in eggs used for human consumption. An experiment was conducted to evaluate the use of high concentrations of vitamin D to increase egg vitamin D content, improve eggshell quatility and increase hen skeletal health. The data suggest that adding vitamin D₃ from 8,300 to 35,000 IU/kg diet will increase egg viatimn D content, and generally improve eggshell and bone quality; however, adding vitamin D₃ at 68,000 IU/kg diet resulted in negative effects on pullet growth and subsequent egg production of adult hens.

Biological systems of bacteria were investigated at the single-cell and single-molecule level. Additionally, aspects of the techniques employed were studied. A unifying theme in each project is the reliance on optical imaging techniques coupled to microfluidic devices. Hypo-osmotic shock experiments with an Escherichia coli mechanosensitive channel deletion mutant were carried out at the single-cell level. E. coli MJF465 cells in which the three major mechanosensitive channel genes are deleted (∆mscL, ∆mscS, ∆mscK) show only 10% cell viability upon hypo-osmotic shock (from LB + 0.5 M NaCl into distilled water), compared to 90% viability of the wild-type strain. Bacterial cells were trapped with optical tweezers in microfluidic devices, enabling the first direct observation of single-cell behaviour upon hypo-osmotic shock. Phase-contrast microscopy revealed intra-population diversity in the cells response: Different features of lysis included cells bursting rapidly and leakage of ribosomes, DNA and protein from the cytoplasm. Fluorescence microscopy of hypo-osmotically-shocked GFP-expressing MJF465 cells showed either bursting of cells, which was a rare event, or fast leakage of GFP, indicating cell membrane ruptures. Data were analysed in terms of their kinetic behaviour and showed that lysis occurs on a timescale of milliseconds to seconds. The implications of these findings for the bacterial cell wall and cell membranes are discussed. Enzymes involved in homologous recombination and repair of double-stranded DNA (dsDNA) breaks are essential for maintaining genomic integrity in both eukaryotes and prokaryotes. RecBCD of E. coli and AddAB, found widely in bacteria, are involved in these processes, carrying out the same function. Both enzymes were studied kinetically with single-molecule total internal reflection fluorescence microscopy (TIRFM). Surface-tethered, hydrodynamically stretched lambda-DNA molecules, stained with YOYO-1, were imaged with TIRFM in a microfluidic flowcell. The RecBCD enzyme is a well characterised DNA helicase and was introduced to this system for method validation purposes. The AddAB enzyme of Bacteroides fragilis was then characterised as a helicase acting on lambda-DNA. It was found that AddAB helicase unwinds dsDNA with high processivity of on average 14,000 bp and up to 40,000 bp for individual enzyme complexes at an ATP-dependent rate ranging from 50-250 bp s−1 (for Mg2+-ATP concentrations larger or equal than 0.1 mM). This activity was detected by DNA binding dye (YOYO-1) displacement from the dsDNA and studied for different Mg2+-ATP concentrations, flow (shear) rates and different YOYO-1 staining ratios of DNA. Aspects of this last experimental setup were investigated. A kinetic analysis of intercalation of YOYO-1 into lambda-DNA is presented, occurring on a timescale of minutes. Different flow rates and staining ratios that influence the apparent (stretched) DNA molecule length were also examined. Several image analysis techniques were employed to enhance the data quality in images showing stretched lambda-DNA molecules. The Singular Value Decomposition was found to be the most effective technique which strongly reduces the noise in the obtained kymograph images.

Background: Acinetobacter baumannii and methicillin-resistant Staphylococcus aureus (MRSA) represent the most worrying Gram-negative and Gram-positive nosocomial pathogens of the present age. They are of increasing concern in the clinical environment due to their multi-drug resistance and the dwindling therapeutic options available. A. baumannii is the most frequently isolated clinical species of the genus, and is able to rapidly acquire resistance. Hypermutators, most frequently deficient in mismatch repair (MMR) via defects in the mutS gene, have been associated with antimicrobial resistance in several bacterial populations. To date, however, the potential role of MMR-deficient mutators in the development of resistance in clinical Acinetobacter spp. has not been investigated. Biocides, most notably chlorhexidine (CHX), are increasingly used in the hospital environment to prevent bacterial spread. This has led to concerns about the development of reduced biocide susceptibility and associated antibiotic resistance in hospital bacterial populations, where there is frequent exposure to both of these factors. The effect of CHX upon defined clinical MRSA isolates is examined here. Methods: The mutS gene of clinical Acinetobacter spp. isolates with varying sensitivities was sequenced and compared to establish whether any variations were present. Mutation studies were performed on isolates by challenging them with ciprofloxacin to determine whether different mutS types correlated with any variation in their ability to develop significant fluoroquinolone resistance. The response of clinical MRSA isolates to a range of CHX concentrations was examined with susceptibility testing methods, and effects were compared with standard strains. Determination of post-exposure minimum inhibitory concentrations (MICs) of a range of antibiotics enabled evaluation of whether exposure to CHX had an effect on susceptibility to antibiotics. Results: Variation was observed in the mutS gene of clinical Acinetobacter spp. isolates, with greater homology observed as resistance increased. A highly conserved and previously unreported amino acid sequence was discovered in resistant isolates. Nonresistant isolates with this ‘R-type’ mutS sequence appeared to have a greater ability to develop significant ciprofloxacin resistance. Clinical MRSA isolates had varying susceptibility to CHX, and there were differences in the susceptibility of standard strains compared to clinical isolates. CHX residues exerted a prolonged minimal inhibitory effect, and several increases in antibiotic MICs following CHX exposure were observed. Conclusions: The correlation of the mutS sequence with mutation ability suggests that defects in the mutS gene may have a role to play in the ability of certain Acinetobacter spp. to rapidly acquire resistance. This could have implications for the treatment of Acinetobacter spp. infections, and may enable quick determination of which clinical isolates have the potential to develop clinically significant resistance. Incomplete eradication due to the prolonged minimal effect of CHX residues may act as a selective pressure in the hospital environment, allowing survival of reduced susceptibility MRSA isolates. Increases in antibiotic MICs following CHX exposure is of grave concern for the future of biocide usage.

The opportunistic bacterial pathogen Pseudomonas aeruginosa uses type IV pili (T4P) for adherence to, and rapid colonization of, surfaces via twitching motility. T4P are formed from thousands of pilin (PilA) subunits. Two groups of P. aeruginosa pilins were described previously (I and II), distinguished by protein length and sequence. PilA_I was glycosylated with an O-antigen subunit through the action of PilO/TfpO, encoded downstream of pilA_I. To determine if additional pilin variants existed, analysis of the pilin locus of >300 P. aeruginosa strains from a variety of environments was conducted. Three additional pilin alleles were discovered, each of which was invariantly associated with a unique, previously unidentified, downstream gene(s): pilA_III+tfpY, pilAIV+tfpW+tfpX, pilA_V+tfpZ. This survey also revealed that strains with group I T4P were more commonly associated with respiratory infections than strains with other pilins, suggesting that glycosylated T4P may confer a colonization advantage in this environment. The newly identified group IV pilin, represented by strain Pa5196, migrated aberrantly through SDS-PA gels, suggesting it was also glycosylated, a hypothesis confirmed by periodic acid-Schiff staining and mass spectrometry (MS) analyses. Disruption of Pa5196 O-antigen biosynthesis did not prevent the production of glycosylated pilins, demonstrating that these pilins were modified in a novel manner, unlike group I pilins. Using MS, nuclear magnetic resonance spectroscopy and site-directed mutagenesis, the Pa5196 pilins were shown to be uniquely modified with homo-oligosaccharides of mycobacterial-like α-1,5-D-arabinofuranose at multiple locations. Residues Thr64 and Thr66, located on the αβ-loop region of the protein, appear to be the preferred, but not exclusive sites of modification, each being modified with up to four D-Araf sugars. This region of the pilin is partially surface-exposed in the pilus, therefore modification of these sites may influence the surface chemistry of the fibre. Residues Ser81, Ser82, Ser85 and Ser89, located in the β-strand region, were also modified, mainly with mono- and disaccharides. Bioinformatic analyses and mutagenesis of TfpW suggest that this novel protein is an arabinosyltransferase necessary for PilA_IV modification. This research has increased our understanding of the complexity of this virulence factor, and may aid in development of new therapeutics for P. aeruginosa and mycobacterial infections.