SurA is a molecular chaperone in the periplasm of E.coli and has been implicated in the maturation of outer membrane proteins (OMPs). SurA consists of four domains, but only two of them, namely the N and C-­terminal domains, are necessary for chaperone function. Very little is known about which residues drive the interaction between the N and C-­termini that facilitates normal activity. We mutated several conserved residues on the C-terminus and generated additive truncations to observe the effects of each on the fitness of the cell. We found one mutation E(408):A was sufficient to reduce SurA activity by 3-­fold, but structural characterization of the mutated protein revealed little variation from wild-­‐type SurA. Most notable, we found that when at least 10 residues are removed from the C-­‐terminus, the protein is completely non-­‐functional. We introduced a random peptide library to substitute these 10 residues and found that ~1.5% of all possible sequences in the library can restore SurA function to at least 50% activity. Moreover, we observed no pattern in the sequences of 26 different variants that were chosen and characterized. Here we show for the first time that SurA can tolerate many mutations at the C-­terminus and still be active.

The viable but non-culturable (VBNC) state is outstanding among bacterial stress responses as being completely uncharacterised at the molecular level. The aim of this investigation was to gain an insight into the molecular basis of the condition by identifying genes whose expression was up-regulated in response to VBNC-inducing stimuli. First, a model experimental system was established where bacteria were induced to enter the state in a routine and predictable manner. <I>Escherichia coli </I>HB101 exhibited a partial viable but non-culturable phenotype when inoculated into microcosms of artificial seawater at 37°C, <I>Pseudomonas fluorescens </I>10586 became viable but non-culturable in microcosms of drinking water incubated at 37°C, and <I>Vibrio vulnificus </I>MO6-24/T entered a viable, non-culturable state in artificial seawater at 5°C. A transposon mutagenesis strategy utilising a promoter-less bioluminescent reporter cassette, <I>lux</I>AB, was employed in the search for VBNC-associated genes. The mini-Tn<I>5 lux</I>AB transposon was induced to transform into arbitrary positions of the <I>P. fluorescens </I>10586 chromosome, thus creating a library of <I>P. fluorescens lux</I>AB mutants. This library (consisting of over 1200 transformants) was screened for those which were dark under normal circumstances, but luminesced in response to VBNC stimuli, indicating that the transposon had integrated downstream of a gene up-regulated during the VBNC response. Unfortunately, no mutant examined exhibited such a bioluminescence profile. Differential display of RNA technology was employed subsequently and resulted in the cloning and sequencing of several <I>V. vulnificus </I>transcripts thought to be associated with the VBNC state. Although absolute verification of the involvement of these transcripts was not achieved, hints as to what mechanisms lay at the basis of the VBNC state were gained. Some findings indicated that VBNC cells experience considerable levels of oxidative stress, and it was proposed that this physiological state may lie at the crux of the VBNC phenotype.

To enhance outbreak investigations of Shiga toxin-producing Escherichia coli O157:H7, PulseNet Canada has recently applied Multiple Locus Variable Number Tandem Repeat Analysis (MLVA) as a supplemental subtyping tool in combination with the gold standard subtyping method Pulsed-field Gel Electrophoresis (PFGE) for enhanced resolution of isolates exhibiting indistinguishable/highly similar PFGE patterns. The objective was to assess the discriminatory power and level of specificity MLVA offers for outbreak detection and response. Results demonstrate that MLVA provides a statistically significant increase in discriminatory power for outbreak investigations (0.998) compared to PFGE alone (0.993). MLVA was able to provide additional resolution over PFGE analysis and generally agreed with PFGE when isolates were identical and epidemiologically linked. MLVA shows great promise as a molecular epidemiological tool to complement PFGE, as it improves case categorization during outbreak investigations, and the greatest benefits of MLVA may be realized during routine surveillance, when epidemiological information is not available.

Bacterial biofilms are microbial communities that adhere to abiotic or biotic surfaces. Biofilm formation (BF) studies in E. coli have primarily concentrated on uropathogenic E. coli, commensal K-12 and enterohemorrhagic E. coli O157:H7. This does not include the vast diversity of environmental strains. Quorum sensing (QS) is a means by which bacteria can communication with one another through the production of signalling molecules. The autoinducer 2 (AI-2) QS system is utilized by E. coli and several other bacterial species for controlling gene expression. The role of AI-2 in E. coli BF varies among different strains. For example in the K-12 strain, AI-2 regulates motility, and thus can affect BF; whereas in O157:H7, AI-2 has a more metabolic role. Interestingly, in strain O157:H7, motility is controlled by a newly discovered QS system regulated by the autoinducer 3 (AI-3) molecule plus the mammalian hormones epinephrine (Epi) and norepinephrine (Ne). The purpose of this study was to investigate the ability of a panel of environmental E. coli strains to form biofilms and to determine whether QS is involved in the process. A new pathotype of E. coli, adherent invasive E. coli (AIEC) which is associated with Crohn’s disease was included in the investigation. Study 1 sought to determine whether BF under different media conditions correlated with the presence of genes involved in the AI-2 QS system or adhesin factors. Media conditions were the principal variable affecting the BF. Study 2 examined the role of the AI-2 and AI-3/Epi/Ne QS systems in motility and BF by the AIEC strain. It was discovered that the AI-3 system is involved in motility; whereas the AI-2 system had no effect on BF or motility. In Study 3, microarray gene expression analysis and invasion assays were performed using qseB or qseC mutants. These genes encode the two-component regulatory system recognizing AI-3 or its cognate, epinephrine. Our findings indicate that alternative pathways likely account for the BF observed for the qseB and qseC mutants. It was concluded that the AI-3/Epi/Ne QS system partially controls AIEC motility and the invasion of epithelial cells.

<I>Escherichia coli</I> mutants impaired in growth and survival at low external pH in minimal medium were selected and attempts made to identify the disrupted genes. This study suggested that <I>clpX</I>, encoding a heat-shock induced protease and molecular chaperone, was functional in survival of <I>E. coli </I>at pH 3.3. Promoter probe plasmid libraries of <I>Salmonella typhimurium </I>LT2 DNA were created in <I>E. coli </I>and screened for acid-inducible transcriptional elements, and transcriptionally active fragments of degradative amino-acid decarboxylase genes recovered. Chromosomal gene fusions to the reporter gene <I>lacZ </I>in <I>E. coli</I> generated by Mu DII 1734 insertion were screened in a similar way and suggested that the gene encoding adenylate cyclase (<I>cya</I>) could be induced by mild cytoplasmic acidification. The sequence of a gene known to be inducible by cytoplasmic acidification, <I>inaA, </I>became available during the course of this study. The 5' region of this gene was used to generate a set of plasmids carrying fragments of the acid-inducible promoter transcriptionally fused to a luciferase based reporter system. Elements of the sequence required for induction by cytoplasmic acidification were identified. One of these reporter constructs was used to screen an <I>E. coli </I>Tn<I>10</I> chromosomal insertional mutant library for genes involved in the regulation of <I>inaA. </I>One such mutant had a multiple antibiotic resistant (<I>mar</I>) phenotype. The disrupted loci in 2 other mutants were identified by inverse PCR, sequence analysis and database searches. Both were known only as open reading frames (ORFs) discovered during the sequencing of the entire <I>E. coli</I> genome, and were tentatively identified as <I>yddB </I>(closely linked to <I>gadB </I>and <I>gadC</I>; required for glutamate dependent acid resistance) and <I>f300</I> (closely linked to <I>pldA; </I>required for detergent resistance). The promoter of <I>f300</I> was shown to be sensitive to cytoplasmic acidification. The <I>inaA</I> promoter was also demonstrated to be induced at the onset of stationary phase, and to be independent of the stationary phase and weak-acid inducible σ factor RpoS and also of cAMP levels.

Bacterial contamination due to excessive levels of bacteria is a confounding problem and remediation of impaired watersheds relies on the detection of fecal indicator bacteria and then assessing the source of said bacteria. Bacterial source tracking (BST) is an approach for assessing potential sources of this contamination. The purpose of this study was to utilize both cultivation-independent and –dependent methods to improve the ability to track sources of fecal contamination. First, E. coli community composition was assessed across three standard water quality assessments including USEPA Methods 1603 and 1604, and Colilert®, to determine their impact on BST library-based performance. Results indicate that the three assessed methods of enumeration and isolation may select for different populations of E. coli and standardized methods may be warranted if library-dependent BST is part of a research plan. Next, BST techniques were used to enumerate and characterize E. coli communities across various dairy manure management techniques used in the Leon River watershed in central Texas to determine effectiveness of BST efforts in tracking contamination from dairy manure. Results of this study indicated that manure and effluent management strategies which employed means to remove solids from the manure tended to decrease the levels of E. coli in the effluent. Some E. coli genotypes were found across the managerial treatments even though there were no clear seasonal trends or site groupings among the dataset. The vast majority of the isolates classified using the Texas E. coli BST library were correctly classified back to their major source class, thus increasing confidence in the methods currently being utilized to track dairy fecal contributions in this Central Texas watershed. Finally, deer bacterial fecal communities from south and central Texas were analyzed using 454-pyrosequencing to assess the potential for the development of a deer-specific BST marker. Microbial communities did not cluster by site or year suggesting that deer fecal communities in these Texas regions are stable over time and could be amenable to marker development.

Graduation date: 1969

Typescript. / Thesis (Ph. D.)--University of Hawaii at Manoa, 1972. / Bibliography: leaves 91-96. / ix, 96 l illus

Integral membrane proteins perform a wide range of biological processes, including respiration, signal transduction and molecular transport. Structural information is necessary for a full understanding of the mechanisms by which integral membrane proteins work. Ferrous iron transporter protein B (FeoB) is an integral membrane protein of Escherichia coli which is considered to transport ferrous iron in to bacteria. But there are no definite proofs or clear indications of the precise mechanism of ferrous transport. By expressing and crystallizing the G-protein domain (FeoGP) and FeoB, it will be helpful to know about the iron transport system. In order to express FeoB and FeoGP, expression vector pFeoB (FeoB in pGEX-4T-1) and pFeoGP (FeoB in pGEX-4T-1) were made. FeoB and FeoGP proteins were expressed and purified. Using vapour diffusion method crystallization trials of FeoB and FeoGP were done. Crystals of FeoGP are observed and no crystal formation for FeoB. Native crystals of FeoGP diffracted to 2.2 ?? resolution, and mant-GMPPNP crystals to 2.6 ??. Preliminary data processing indicate space group P212121 for native crystals, with cell dimensions 46 x 119 x 146 ??. The data set is 100% complete, Rmerge 0.08, and I/ ?? 3.2.

vii, 185 leaves : ill., tables ; 29 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology, 1977?