The cell envelope of mycobacteria is critical for the survival and virulence of pathogenic species during infection, and its biosynthesis has been a proven drug target. Therefore, finding new targets in the biosynthetic pathway of cell envelope components is of great interest. Mycobacterium smegmatis is a model organism for the study of the devastating pathogen Mycobacterium tuberculosis. Previously, lipomannan elongation factor A (LmeA) has been identified as a cell envelope protein that is critical for the control of mannan chain length of lipomannan (LM) and lipoarabinomannan (LAM), lipoglycan components of the cell envelope. The deletion mutant, ∆lmeA, accumulates abnormal LM/LAM with fewer mannan residues. To understand the importance of this protein, the antibiotic sensitivity of ∆lmeA was tested using a resazurin-based viability assay. We found that the lmeA deletion leads to increased sensitivities to antibiotics such as vancomycin and erythromycin, and lmeA overexpression leads to increased antibiotic resistance. To directly test if the increased antibiotic sensitivity is due to the defective permeability barrier, we used an ethidium bromide uptake assay and found that ∆lmeA is more efficient in taking up ethidium bromide in the cell. We have also found that LmeA is important for protein stabilization under stress conditions. MptA is an α1,6-mannosyltransferase involved in elongation of LM and LAM mannan chain. During stress conditions in the ΔlmeA mutant, levels of MptA decrease significantly relative to wild-type. This also results in delayed doubling time after stress, a phenotype not seen in this mutant under normal growth conditions. In addition, the ΔlmeA mutant has differential protein expression during stress conditions relative to ΔlmeA in log phase, or to wild-type in either condition. To help elucidate the role of LmeA at the molecular level, binding behavior of this protein to membrane fractions was determined. In a subcellular fractionation analysis, LmeA localizes to fractions containing plasma membrane, which is tightly bound to cell wall layers. To test the binding of LmeA to membrane further, LmeA was heterologously expressed in Escherichia coli, purified, and mixed M. smegmatis cell lysate. LmeA localized to intracellular domain fractions (IMD), indicating that LmeA is capable of localizing to fractions containing only plasma membrane. Consistent with this finding, LmeA is capable of binding to spheroplasts in both an ELISA setting as well as in a sucrose gradient fractionation setting. It has also been determined that ΔlmeA has a defective capsular layer with a unique phenotype relative to other strains. We have concluded that LmeA is important for antibiotic resistance, cell envelope permeability, capsule formation, stress response, and have also determined its binding properties.

To investigate the bacterial composition of infected and non-infected dog bite wounds (DBW), a prospective study was performed on dogs with various grades of bite wounds presenting at the Onderstepoort Veterinary Academic Hospital, University of Pretoria, and a nearby animal shelter. Fifty dogs with bite wounds inflicted within the previous 72 hours were selected. This represented 104 wounds. Wounds were clinically graded according to severity. Swabs were collected from all wounds for bacterial culture and cytology. Infection was diagnosed if 2 of the following 3 criteria were met: macroscopic purulence, microscopic presence of phagocytosed bacteria, or pyrexia. Non-infected wounds were either classed as sterile (established by culture) or contaminated (culture positive but bacteria not phagocytosed on cytology). To determine the origin of the bacteria, swabs were collected from the skin near the wounds and gingiva of 15 bite victims. All swabs were cultured aerobically and anaerobically and all aerobic cultures were evaluated for antimicrobial susceptibility using the Kirby Bauer disk diffusion test. The victims were predominately male, uncastrated, small-breed dogs. Of the 104 wounds studied, 21 were judged to be infected and 83 non-infected. Infected wounds were significantly more likely to culture positive (Fisher's exact test: p = 0.02). Sixteen per cent of wounds did not culture bacteria, 67% grew aerobes only, 1% anaerobes only and 67% a mixture of aerobes and anaerobes. A total of 213 isolates were cultured representing a mean of 2 isolates per wound. Of the aerobe species cultured, 22%, 19% and 17% belonged to the genera of Pasteurella, Streptococcus and Staphylococcus respectively. The species of Pasteurella multocida (66%) and Staphylococcus intermedius (70%) were predominant. Pasteurella canis and pyogenic streptococci were common in infected wounds, whereas Bacillus spp., Actinomyces spp. and oral streptococci were usually found in contaminated wounds. Three anaerobic genera were cultured, namely, Prevotella, Clostridium and Peptostreptococcus, and were usually associated with wounds with dead space. This study also describes the first documented case of Capnocytophaga canimorsus in an infected dog bite wound. Notably clinical and cytological assessment was capable of establishing whether antimicrobials were required or not. Although no single antimicrobials was considered to be effective against all the bacteria, amoxycillin plus clavulanic acid, 1st and 3rd generation cephalosporins, ampicillin or amoxycillin and potentiated sulphonamides gave the best in vitro sensitivity results. / Dissertation (MMedVet(Surgery) Small Animal Surgery)--University of Pretoria, 2007. / Companion Animal Clinical Studies / unrestricted

Iron is extremely important for many organisms. Despite its abundancy, it exists in insoluble forms that makes its usability difficult. Some organisms secrete siderophores, low molecular weight compounds, that can chelate iron and convert it into usable forms for cells. One such organism, Rhizobium leguminosarum, is a nitrogen fixing symbiont proteobacteria that infects leguminous plants. The genome of Rhizobium leguminosarum ATCC 14479, which infects the red clover, Trifoli pratense, has previously been completely sequenced in our lab. Our lab has identified several genes in this strain involved in the biosynthesis of a siderophore, vicibactin. The protein product of one of those genes, VbsS, is hypothesized to be a non-ribosomal peptide synthase. It has been attempted to knockout the VbsS gene utilizing the ‘splicing by overlap extension’ method. Additionally, an in-silico analysis of the genome revealed the Vbs genes in R. leguminosarum ATCC 14479 strain were similar to genes in found in the proteobacterium Phyllobacterium sp. 628 and the fungi Aspergillus fumigatus Af293.

A study was conducted in Rich County, Utah to determine the extent of avian tuberculosis in livestock, poultry and wild birds. Sensitivity was elicited in cattle, swine and chickens by tuberculin testing. The Mycobacterium avium organism was obtained from tissues and specimens of cattle, swine, goats, chickens, magpies and sparrows. Methods of eradication of avian tuberculosis and the relationship of avian tuberculosis to mammalian tuberculosis are discussed.

Chapter I reviews current literature on gene transfer systems in lactic acid bacteria, how genetically altered microorganisms for food are presently regulated, and how nisin is used as a food preservative. Chapter II investigates previous reports which linked genes for nisin biosynthesis and sucrose utilization (Nip+Suc+) to plasmid DNA in two well characterized L· lactis subsp. lactis strains. Plasmid curing studies, conjugations, and DNA-DNA hybridizations indicated that these genes were encoded by chromosomal loci in all Nip+Suc+ strains examined. Similar results were noted in nisin-sucrose transconjugants of L. lactis subsp. cremoris and S. salivarius subsp. thermophilus in Chapters III and IV. Chapter III describes the use of conjugation to construct nisin-producing Lactococcus lactis subsp. cremoris strains. The direct-plate conjugation method was developed to facilitate transfer of Nip+Suc+ to L. lactis subsp. cremoris recipients. DNA-DNA hybridizations to transconjugant DNAs with an oligonucleotide that detected the nisin structural gene, nisA, demonstrated that this gene was transferred during conjugation. Lactococcus lactis subsp. cremoris Nip+Suc+ transconjugants retained the recipient strain phenotype with respect to bacteriophage resistance and acid production in milk. These results indicated that it would be feasible to construct nisin-producing L. lactis subsp. cremoris strains for mixed and multiple starter systems. Chapter IV investigates features of Nip+Suc+ transfer using a Lactococcus lactis subsp. lactis model system. Intergeneric transfer of nisin-sucrose genes was also achieved between lactococcal Nip+Suc+ donors and Streptococcus salivarius subsp. thermophilus recipients. Streptococcal transconjugants acquired Suc+ and nisin immunity but did not produce nisin. DNA-DNA hybridizations, however, demonstrated that nisA was present in these transconjugants. To investigate whether nisA was involved in nisin immunity, this gene was cloned and electro-transformed into Lactococcus lactis subsp. lactis LM0230. Electro-transformants did not express nisin immunity or any other trait linked to nisin production in lactococci. Results presented in Chapter V indicate that nisin may have application for control or prevention of bovine mastitis. Gram-positive pathogens which cause bovine mastitis were examined for their susceptibility to nisin. Disc diffusion assays indicated that minimum inhibitory concentrations of nisin ranged from 10 to 250 ug per ml. In addition, 50 ug of nisin per ml in milk inhibited all gram-positive pathogens tested.

The nature of the action of penicillin on the cell walls of bacteria is reviewed. The composition of bacterial cell walls is compared to cell walls of blue-green algae. The test organisms used were thermophilic: Anacystis nidulans grew optimally at 35⁰C, whereas Synechococcus lividus grew best at 45⁰ C. Growth was recorded by reading optical densities. Cells of these two thermophilic blue-green algae were treated with varying dosages of penicillin and streptomycin. Penicillin inhibited growth of Anacystis nidulans and Synechococcus lividus in concentrations of 3.0 Mg/ml and 0.03 Mg/ml respectively. However, when 0.3 Mg/ml of penicillin was added to Anacystis nidulans, an initial lag phase of growth was observed. Possible reasons for this lag are suggested and results of pertinent experiments are discussed. No similar lag was noted in Synechococcus lividus. Streptomycin inhibited growth of both species when present in 0.03 Mg/ml.

Salicylic acid, and to a lesser extent aspirin, have been shown to repress β-galactosidase synthesis in Escherichia coli. The repression is not due to decreased inducer uptake, nor does it result from competition with inducer for repressor. Dinitrophenol does not exert similar effects on β-galactosidase synthesis. Cyclic adenosine 31, 51-monophosphate partially relieves the repression. The extent or the relief by cyclic adenosine monophosphate seems to depend on the concentration of salicylic acid. This indicates there may be direct interaction between the actions of cyclic adenosine monophosphate and salicylic acid in E. coli cells.

Fish of the genus Cottus were found infected with heterophyid metacercariae. Laboratory animals were infected with the metacercariae, and adult heterophid trematodes were recovered. These flukes were found to represent an undescribed species of the genus Euryhelmis. Various streams in the Willamette Valley and coastal areas were sampled for infected Cottus sp. Snails of the genera Oxytrema and Fluminicola were collected. Several possible definitive hosts were examined. A partial review of the subfamily Apophallinae and a complete review of the genus Euryhelmis are presented. Euryhelmis cotti n. sp. is placed in the subfamily Apophallinae, and its life cycle is partially described.

Salmonella enterica serovar Typhimurium is one of the most prevalent food-borne pathogens, affecting millions around the world every year, making it a threat to global health. Salmonella possesses the ability to survive the normally lethal condition of desiccation, however, discovery of the genes and mechanisms behind this phenomenon are still ongoing. Using a transposon mutagenesis approach to construct a broad transposon library, this study aimed to uncover genes that may be contributing to changes in Salmonella’s survivability under desiccation, particularly when exposed to the antimicrobial molecule salicylic acid. Building on previous findings showing salicylic acid can alter cell viability through differential gene regulation, transposon mutants were exposed to salicylic acid and subsequently desiccated to screen for mutants that displayed an alteration in survival phenotypes. This work identified a transposon mutant with an interruption of the porin-coding gene ompC that displayed an augmented survivability phenotype under these conditions, leading to further exploration into the origin of this phenomenon.

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado. / During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP. / National Institutes of Health / Revisión por pares