No description available.

<I>Erwinia carotovora </I>subspecies <I>caratovora</I> (<I>Ecc</I>) is a Gram-negative bacterial phytopathogen, belonging to the Enterbaceriaceae family. <I>Ecc </I>has a broad host range, including potato, causing soft rotting of tubers while in storage. This effect is due to the secretion of an arsenal of tissue-macerating extracellular enzymes, which includes multiple cellulases (Cel), pectate lyases (Pel) and proteases (Prt). The overall aim of this project was to learn more about the global regulatory mechanisms controlling the co-ordinate production of exoenzymes in <I>Ecc</I>. Global regulatory mutants were generated by λTn<I>phoA'-</I>2 mutagenesis. Both Rex (regulation of exoenzymes) and Hex (hyperproduction of exoenzymes) mutants were isolated. Rex and Hex mutants are down and up-regulated, respectively, for the synthesis of all the exoenzymes. This project concentrated on the analysis of the Hex mutants thereby identifying 'repressor' genes affecting exoenzyme synthesis. λTn<I>phoA</I>'-2 consists of a promoterless <I>lacZ</I> gene, such that an insertion of the transposon into a gene, in the correct orientation results in a transcriptional gene fusion. Of the 2500 gene fusions screened, 10 Hex and 22 Rex mutants were isolated. Southern hybridisation with a transposon-specific probe showed that the 10 Hex mutants carried a single transposon insertion in each case. Two of these Hex mutants were identified as defective in HexL. The other eight mutations from the Hex mutants were cloned and the DNA sequences flanking the transposons were sequenced. Only one of the disrupted genes encoded a product that showed significant sequence similarity with a known protein, HtrB (high temperature requirement) from <I>E. coli</I>.

Both fatty acid and erythromycin biosynthesis are anticipated to involve an acyl carrier protein (ACP) or an ACP domain within a larger protein. A series of oligonucleotides directed against a key amino acid sequence in <i>Escherichia coli</i> ACP were used to try and detect the gene or genes coding for ACP in '<i>Streptomyces erythreus</i>''. Two positively-hybridising fragments were detected and these were cloned into multicopy vectors in <i>E. coli</i>. The insert fragments were characterised by DNA sequencing and it was shown that neither in fact coded for an ACP. In an alternative approach, malonyl CoA-incorporating activity from '<i>Streptomyces erythreus</i>'' and <i>Streptomyces coelicolor</i> was characterised. In particular, this activity was found to be dependent on a heat stable factor which was subsequently identified as an authentic ACP. It was shown that ACP from <i>E. coli</i> could substitute for the '<i>Streptomyces erythreus</i>'' ACP. Evidently, the malonyl CoA-incorporating activities from <i>Streptomyces</i> are freely dissociable like that of <i>E. coli</i> fatty acid synthase. ACP from '<i>Streptomyces erythreus</i>'' was purified to homogeneity and the N-terminal portion of the protein sequence was determined. Also an ACP from <i>Propionibacterium shermanii</i>, (another Gram-positive actinomycete) was purified to homogeneity and sequenced to provide a comparison. The close similarity with <i>E. coli</i> fatty acid synthase, previously unexpected, should allow the assay and purification of the other components of fatty acid and erythronolide synthase. As an example, the condensing enzyme of fatty acid synthase has been fractionated and assayed.

<I>Serratia</I> sp. strain ATCC 39006 ('<I>39006</I>') produces a bright red pigment prodigiosin (Pig), and a carbapenem antibiotic, 1-carbapen-2-em-3-carboxylic acid (Car). Recently it was shown that <I>39006</I> also produced N-acylhomoserine lactones (AHL), which act as quorum sensing (QS) signalling molecules. The notion that <I>39006</I> used a QS system to regulate phenotypes according to cell-density was investigated and characterised here. <I>N</I>-butanoyl-L-homoserine lactone (BHL) and <I>N</I>-hexanoyl-L-homoserine lactone (HHL) were identified as the QS signalling molecules by thin-layer chromatography. Ahl<SUP>-</SUP> mutants carrying mini-transposon insertions in a single ORF (<I>smaI</I>) were concomitantly deficient in the production of Pig, Car, 'white-opaque phenotype' and the synthesis of the extracellular enzymes pectate lyase and cellulase, all of which were restored by the addition of synthetic AHLs. All five phenotypes were also restored in the <I>smaI</I><SUP>-</SUP> mutant when grown with or near a BHL/HHL-producing strain. It was therefore concluded that <I>39006</I> possessed a previously uncharacterised QS system that played a central role in exoenzyme and secondary metabolite production. The <I>smaI</I> gene was sequenced and found to encode a protein (SmaI) similar to members of the LuxI family of AHL synthases. DNA sequencing also revealed another ORF, <I>smaR</I>, which encoded a protein (SmaR) similar to the LuxR family of QS response regulator proteins. The regulation of Pig and Car production with respect to quorum sensing was investigated in this study using <I>lacZ</I> transcriptional fusions to <I>pigA, pigH</I> (<I>pig</I> biosynthetic gene cluster) and <I>carA</I> (<I>car</I> biosynthetic gene cluster). Data supported a model in which the <I>smaIR</I> locus formed a complete regulatory switch where SmaR acted as a repressor of gene transcription at low cell density. At high cell-density, in the presence of AHLs, SmaR-mediated repression was lifted thus allowing transcription.

No description available.

Strain-specific Klebsiella pneumoniae virulence determinants have been described but almost exclusively for hypervirulent liver abscess-associated strains. Island-tagging and fosmid-based marker rescue were used to capture and sequence KpGI-5, a novel genomic island integrated into the met56 tRNA gene of K. pneumoniae KR116. This 14.0 kb island exhibited a genome-anomalous G+C content, possessed near-perfect 46 bp direct repeats, and encoded a putative γ1-chaperone/usher fimbrial cluster (fim2). This island was shown to belong to a previously unknown KpGI-5-like island family and was hypothesized to have undergone substantial reductive evolution. Transcriptional analysis demonstrated expression of three fim2 genes and suggested that the eight-gene fim2A-fim2K cluster comprised an operon. In vivo models of urinary tract and lung infection, and large intestinal colonization, in addition to in vitro assays were used to examine the role of fim2 in pathogenesis by comparing KR2107, a streptomycin-resistant derivative of KR116, to three isogenic mutants (Δfim, Δfim2 and ΔfimΔfim2) constructed using optimized allelic exchange techniques. Although no statistically significant in vivo role for fim2 was demonstrable, liver and kidney CFU counts for lung and urinary tract infection models, respectively, hinted at an ordered gradation of virulence as fimbrial clusters were lost: KR2107 (most virulent), KR2107Δfim2, KR2107Δfim and KR2107ΔfimΔfim2 (least virulent). Despite using several methods the putative Fim2 fimbriae could not be visualised. Furthermore, the fim2-encoded putative phosphodiesterase Fim2K was determined to alter several c-di-GMP-dependent phenotypes, including biofilm formation and exopolysaccharide production. Additionally, fim2 was present in 14 % of Klebsiella strains studied and appeared intact in the majority of fim2-positive strains. Given the above findings, fim2 may confer a niche-specific evolutionary advantage, potentially related to its ability to encourage dissemination. This thesis is the first study of fim2 and KpGI-5-like islands and will aid further investigations into the full impact of these loci on the lifestyle of Klebsiella species.

No description available.

No description available.

No description available.

No description available.