The number of food borne illnesses associated with the consumption of fresh fruits and vegetables and their minimally processed products (juices) has increased over the past years. Of particular interest is the ability of microbial pathogens to internalize and survive in fresh produce that are commonly used for juices. This research project addresses the issue of the ability of Escherichia coli O157:H7 to internalize and survive in whole apples before and after harvest. Four cultivars of apples, Redfree, Red Delicious, Golden Delicious, and York, were inoculated under field conditions with a surrogate strain of E. coli, Escherichia coli ATCC 25922. The Redfree cultivar was inoculated at the beginning of its growth stage (day 0), and again 30 days later, and sampled for two weeks, until E. coli was not recoverable through microbiological methods after three successive sampling days. Red Delicious, Golden Delicious, and York cultivars were spray inoculated with the surrogate strain two weeks before their anticipated harvest date and sampled every other day until E. coli was not recoverable for three successive sampling days. For each cultivar, the presence of E. coli ATCC 25922 was not detectable after 7 to 9 days. In the laboratory study the Red Delicious, Golden Delicious, Rome, and York cultivars received one of three treatments; unblemished control, bruising, or puncturing. The apples were inoculated by immersion in cold water containing E. coli O157:H7 GFP, incubated for three days then microbiologically analyzed for presence of the bacteria. In all cases, the punctured apples of each cultivar showed the greatest uptake of E. coli O157:H7 GFP. Escherichia coli O157:H7 GFP was visualized in flesh and core sections of untreated, bruised, and punctured apples of all cultivars. The microbe was found in between cells, but not within cells of the apple. Internalization of Escherichia coli in whole apples on the tree is not likely, and leads to the conclusion that internalization is a post-harvest problem. Internalization may occur before pressing or processing of apples, leading to an increased risk of infection with E. coli for consumers of apple products that are not properly treated to destroy pathogens. Internalization does occur when apples are immersed in solutions containing the pathogen Escherichia coli O157:H7, and better post harvest controls need to be implemented in order to prevent this in whole apples that are used for cider and juice production. / Master of Science

No / Enteroaggregative Escherichia coli (EAEC), increasingly recognized as an important cause of infant and travelers' diarrhoea, exhibits an aggregative, stacked-brick pattern of adherence to epithelial cells. Adherence is mediated by aggregative adherence fimbriae (AAFs), which are encoded on the pAA virulence plasmid. We recently described a highly prevalent pAA plasmid-borne gene, aap, which encodes a protein (nicknamed dispersin) that is secreted to the bacterial cell surface. Dispersin-null mutants display a unique hyper-aggregating phenotype, accompanied by collapse of AAF pili onto the bacterial cell surface. To study the mechanism of this effect, we solved the structure of dispersin from EAEC strain 042 using solution NMR, revealing a stable beta-sandwich with a conserved net positive surface charge of +3 to +4 among 23 dispersin alleles. Experimental data suggest that dispersin binds non-covalently to lipopolysaccharide on the surface of the bacterium. We also show that the AAF organelles contribute positive charge to the bacterial surface, suggesting that dispersin's role in fimbrial function is to overcome electrostatic attraction between AAF and the bacterial surface

No description available.

Call number: LD2668 .T4 1933 B33 / Master of Science

BE [(1,4)-a-D-glucan:(I,4)-a-D-glucan 6-glucosyltransferase, EC 2.4.1.18] catalyses a transglycosylation reaction where a branch-point is created by the cleavage of an a-l,4 glycosidic bond to form an a-l,6 glycosidic bond. Branching enzyme (BE) from baker's yeast was purified to near homogeneity by chromatography on DEAE-cellulose, Sephacryl S-200 and Protein Pak Q. Electrophoresis on SDS-PAGE revealed one major band of molecular weight 74 kDa. Three distinct methods for determining BE activity (Phosphorylase Stimulation, Iodine- Binding and Branch-Linkage Assays) were used to characterise the purified protein. The enzyme displayed a temperature optimum between 15-25°C and a broad pH optimum of 6.5-7.5 with maximum activity occurring in phosphate buffer. The enzyme was fully stable after incubation at 20°C for 5 hours. A Km value of 1474 Jlg/ ml for amylose was obtained. Primary structural analysis involving N-terminal sequencing and amino acid composition suggested that yeast BE may share some homology with BEs isolated from other sources. Immunological comparisons between yeast, maize (BEll) and Escherichia coli BE using yeast polyclonal antiserum indicated that the enzymes may share antigenic determinants. However, similar comparisons between yeast BE and E.coli antiserum revealed that the antibody only recognised yeast BE in its denatured conformation. Yeast BE was used to modify potato amylose and amylopectin and wheat starch. The enzyme was capable of introducing additional branch points to these substrates resulting in a displacement of the iodine Amax from 629 nm to 568 nm, from 543 nm to 411 nm and from 632 nm to 568 nm for amylose, amylopectin and wheat starch, respectively. HPAEC-PAD analysis of the branched products produced by yeast BE revealed that predominantly short chains of dp 2 to I? were transferred. At least three BE fractions of higher specific activities were isolated from brewer's yeast hatyested at the late exponential phase, suggesting the expression of more than one BE in Saccharomyces cerevisiae.

The lexA protein in E. coli is a specific repressor of the recA gene. The lexA protein is cleaved by the recA protein in response to DNA damage. Cleavage derepresses the recA gene resulting in high level synthesis of recA protein and the expression of other DNA damage inducible functions (SOS functions). The lexA3 mutation makes the lexA protein resistant to cleavage and thus inhibits expression of DNA damage inducible functions. A mutant of E. coli has been isolated which exhibits many of the properties expected of a strain carrying an operator-constitutive mutation in the recA gene. The mutation partially suppresses the UV sensitivity of lexA3 strains, maps near the recA structural gene, allows constitutive synthesis of the recA protein and the recA message, and is cis-acting. Strains carrying the recAo('c) mutation were used to study the role of amplified levels of recA protein in the expression of certain SOS functions. The recAo('c) mutation did not suppress the UV inhibitory effect of the lexA3 mutation on the expression of UV induced cellular mutagenesis, and the reactivation and mutagenesis of UV irradiated phage (lamda). The expression of these functions in lexA('+) strains was not enhanced by the recAo('c) mutation. Constitutive recA synthesis did not result in lethal filamentous growth. These results are consistent with those reported elsewhere that the expression of SOS function is not dependent on high levels of recA protein and that the various "SOS genes" are repressed by the lexA protein as is the recA gene. Thus, recA protein is required in SOS expression for the inactivation of lexA protein and recA amplification is a consequence, not a cause of SOS expression. The DNA sequence of the recA operator region from a (lamda)precA transducing phage thought to carry the recAo('c) mutation isolated here, was determined. No difference was detected between the supposed mutant DNA and wild type controls. The significance of these results and the possibility that the recAo('c) mutation was not transferred to the phage are discussed.

The product of the Escherichia coli lexA gene has been identified, and the regulation of lexA gene expression in vivo has been examined. A series of specialized transducing phages carring lexA⁺ and 3 different amber lexA alleles was constructed by in vivo recombination between λlexA3 and host lexA alleles. These phages were characterized extensively to confirm that they carried the appropriate lexA allele. The lexA gene product was identified by comparison of the polypeptides encoded by λlexA3 and the amber lexA phages. A 24,000 dalton polypeptide, synthesized after infection of both amber-suppressor and non-suppressor hosts by λlexA3 was not synthesized following amber lexA phage infection of non-suppressor hosts. Synthesis of this polypeptide following amber lexA phage infection was restored by the presence of an amber suppressor mutation in the host. On the basis of these data, the 24,000 dalton polypeptide was identified as the lexA gene product. Regulation of lexA gene expression in vivo was examined by hybridization experiments to measure lexA mRNA levels. The basla level of lexA mRNA in wild type E. coli was found to be .006% of total mRNA. Treatment of the bacteria with 100 erglmm² ultraviolet irradiation (UV) led to an eight-fold increase in lexA mRNA levels within 10 minutes, the lexA mRNA remained elevated until 70 minutes after irradiation, then slowly declined. By comparison, the level of recA mRNA increased from .05% to .51% of total mRNA within 10 minutes following UV irradiation, then declined. Both lexA and recA genes were induced by nalidixic acid treatment; the induction was not as rapid as UV induction and different relative induction kinetics of the two genes were seen. The levels of lexA and recA mRNAS were measured in several mutant strains following UV-irradiation.

published_or_final_version / Microbiology / Doctoral / Doctor of Philosophy

No description available.

Under conditions of phosphate starvation Escherichia coli can solubilize mineral phosphates, such as dicalcium phosphate, to orthophosphate which is then available for uptake and cell growth processes. lac operon fusions were created using MudX phage, and mineral phosphate solubilization (Mps) mutants were identified by their inability to solubilize mineral phosphate on plate assays. Four of these mutants have been mapped on the E. coli chromosome via Hfr matings and are located at two distinct portions of the chromosome; between 23 and 50 minutes and between 60 and 90 minutes. One mutant in each region has phosphate starvation inducible (Psi) promoter activity. One of these mutants (DB1047) was mapped to between 69 and 75 minutes via F' matings, and fine structure mapped to 75 minutes by hybridization with λ clones from a genomic library of Escherichia coli. DB1047 was characterized more closely and found to exhibit pleiotropy with regard to several membrane related traits. Evidence that this is a single insertional event comes from the simultaneous loss of all traits tested in spontaneous revertants. Additionally, a Tn5 mutant was identified that was identical for these traits. Our data strongly support the hypothesis that the mutation carried by DB1047 is in the ompB locus. This locus consists of the two regulatory cotranscribed genes, ompR and envZ. This locus is involved in regulation of transcription of the ompC and ompF genes for outer membrane porin proteins, and is located at 75 minutes on the chromosome as is the DB1047 mutation. DB1047 lacks the outer membrane porin OmpF, a phenotype previously attributed to envZ mutants. However, the ompR321 mutant resembles DB1047 in reduced ability to solubilize phosphate. Additional supporting evidence for the DB1047 mutation belonging to the ompB locus comes from the most recent report that mutations in the himA gene, which I found to be deficient in the ability to solubilize phosphate, also affect the regulation of production of the outer membrane porin OmpF.