A systematic analysis of the role of GGDEF-EAL domain proteins in virulence and motility in Xanthomonas oryzae pv. oryzicola

The second messenger c-di-GMP is implicated in regulation of various aspects of the lifestyles and virulence of Gram-negative bacteria. Cyclic di-GMP is formed by diguanylate cyclases with a GGDEF domain and degraded by phosphodiesterases with either an EAL or HD-GYP domain. Proteins with tandem GGDEF-EAL domains occur in many bacteria, where they may be involved in c-di-GMP turnover or act as enzymatically-inactive c-di-GMP effectors. Here, we report a systematic study of the regulatory action of the eleven GGDEF-EAL proteins in Xanthomonas oryzae pv. oryzicola, an important rice pathogen causing bacterial leaf streak. Mutational analysis revealed that XOC_2335 and XOC_2393 positively regulate bacterial swimming motility, while XOC_2102, XOC_2393 and XOC_4190 negatively control sliding motility. The ΔXOC_2335/XOC_2393 mutant that had a higher intracellular c-di-GMP level than the wild type and the ΔXOC_4190 mutant exhibited reduced virulence to rice after pressure inoculation. In vitro purified XOC_4190 and XOC_2102 have little or no diguanylate cyclase or phosphodiesterase activity, which is consistent with unaltered c-di-GMP concentration in ΔXOC_4190. Nevertheless, both proteins can bind to c-di-GMP with high affinity, indicating a potential role as c-di-GMP effectors. Overall our findings advance understanding of c-di-GMP signaling and its links to virulence in an important rice pathogen.

Functional and structural dissection of the tape measure protein of lactococcal phage TP901-1

The tail tape measure protein (TMP) of tailed bacteriophages (also called phages) dictates the tail length and facilitates DNA transit to the cell cytoplasm during infection. Here, a thorough mutational analysis of the TMP from lactococcal phage TP901-1 (TMPTP901-1) was undertaken. We generated 56 mutants aimed at defining TMPTP901-1 domains that are essential for tail assembly and successful infection. Through analysis of the derived mutants, we determined that TP901-1 infectivity requires the N-terminal 154 aa residues, the C-terminal 60 residues and the first predicted hydrophobic region of TMPTP901-1 as a minimum. Furthermore, the role of TMPTP901-1 in tail length determination was visualized by electron microscopic imaging of TMP-deletion mutants. The inverse linear correlation between the extent of TMPTP901-1-encoding gene deletions and tail length of the corresponding virion provides an estimate of TMPTP901-1 regions interacting with the connector or involved in initiator complex formation. This study represents the most thorough characterisation of a TMP from a Gram-positive host-infecting phage and provides essential advances to understanding its role in virion assembly, morphology and infection.

The Rpf/DSF system and the regulation of virulence in the plant pathogen Xanthomonas campestris

The full virulence of Xanthomonas campestris pv. campestris (Xcc) to plants depends upon cell-to-cell signalling mediated by the signal molecule DSF (for diffusible signal factor), that has been characterised as cis-11-methyl-2-dodecenoic acid. DSF-mediated signalling regulates motility, biofilm dynamics and the synthesis of particular virulence determinants. The synthesis and perception of the DSF signal molecule involves products of the rpf (regulation of pathogenicity factors) gene cluster. DSF synthesis is fully dependent on RpfF, which encodes a putative enoyl-CoA hydratase. A two-component system, comprising the complex sensor histidine kinase RpfC and the HD-GYP domain regulator RpfG, is implicated in DSF perception. The HD-GYP domain of RpfG is a phosphodiesterase working on cyclic di-GMP; DSF perception is thereby linked to the turnover of this intracellular second messenger. The full range of regulatory influences of the Rpf/DSF system and of cyclic di-GMP in Xcc has yet to be established. In order to further characterise the Rpf/DSF regulatory network in Xcc, a proteomic approach was used to compare protein expression in the wildtype and defined rpf mutants. This work shows that the Rpf/DSF system regulates a range of biological functions that are associated with virulence and biofilm formation but also reveals new functions mediated by DSF regulation. These functions include antibiotic resistance, detoxification and stress tolerance. Mutational analysis showed that several of these regulated protein functions contribute to virulence in Chinese radish. Interestingly, it was demonstrated that different patterns of protein expression are associated with mutations of rpfF, rpfC and rpfG. This suggests that RpfG and RpfC have broader roles in regulation other than perception and transduction of DSF. Taken together, this analysis indicates the broad and complex regulatory role of Rpf/DSF system and identifies a number of new functions under Rpf/DSF control, which were shown to play a role in virulence.

Human intestinal microbiota and metabolites they produce in relation to host health

The aim of this thesis was to identify selected potential probiotic characteristics of Bifidobacterium longum strains isolated from human sources, and to examine these characteristics in detail using genomic and phenotypic techniques. One strain in particular Bifidobacterium longum DPC 6315 was the main focus of the thesis and this strain was used in both the manufacture of yoghurt and an animal study. In total, 38 B. longum strains, obtained from infants and adults, were assessed in vitro for the selected probiotic traits using a combined phenotypic and molecular approach. Differentiation of the 38 strains using amplified ribosomal DNA restriction analysis (ARDRA) into subspecies indicated that of the 38 bifidobacterial strains tested, 34 were designated B. longum subsp. longum and four B. longum subsp. infantis.

Molecular genetic analysis of DNA alterations in Irish colorectal cancer

Colorectal cancer is the most common cause of death due to malignancy in nonsmokers in the western world. In 1995 there were 1,757 cases of colon cancer in Ireland. Most colon cancer is sporadic, however ten percent of cases occur where there is a previous family history of the disease. In an attempt to understand the tumorigenic pathway in Irish colon cancer patients, a number of genes associated with colorectal cancer development were analysed in Irish sporadic and HNPCC colon cancer patients. The hereditary forms of colon cancer include Familial adenomatous polyposis coli (FAP) and Hereditary Non-Polyposis Colon Cancer (HNPCC). Genetic analysis of the gene responsible for FAP, (the APC gene) has been previously performed on Irish families, however the genetic analysis of HNPCC families is limited. In an attempt to determine the mutation spectrum in Irish HNPCC pedigrees, the hMSH2 and hMLHl mismatch repair genes were screened in 18 Irish HNPCC families. Using SSCP analysis followed by DNA sequencing, five mutations were identified, four novel and a previously reported mutation. In families where a mutation was detected, younger asyptomatic members were screened for the presence of the predisposing mutation (where possible). Detection of mutations is particularly important for the identification of at risk individuals as the early diagnosis of cancer can vastly improve the prognosis. The sensitive and efficient detection of multiple different mutations and polymorphisms in DNA is of prime importance for genetic diagnosis and the identification of disease genes. A novel mutation detection technique has recently been developed in our laboratory. In order to assess the efficacy and application of the methodology in the analysis of cancer associated genes, a protocol for the analysis of the K-ras gene was developed and optimised. Matched normal and tumour DNA from twenty sporadic colon cancer patients was analysed for K-ras mutations using the Glycosylase Mediated Polymorphism Detection technique. Five mutations of the K-ras gene were detected using this technology. Sequencing analysis verified the presence of the mutations and SSCP analysis of the same samples did not identify any additional mutations. The GMPD technology proved to be highly sensitive, accurate and efficient in the identification of K-ras gene mutations. In order to investigate the role of the replication error phenomenon in Irish colon cancer, 3 polyA tract repeat loci were analysed. The repeat loci included a 10 bp intragenic repeat of the TGF-β-RII gene. TGF-β-RII is involved in the TGF-β epithelial cell growth pathway and mutation of the gene is thought to play a role in cell proliferation and tumorigenesis. Due to the presence of a repeat sequence within the gene, TGFB-RII defects are associated with tumours that display the replication error phenomenon. Analysis of the TGF-β-RII 10 bp repeat failed to identify mutations in any colon cancer patients. Analysis of the Bat26 and Bat 40 polyA repeat sequences in the sporadic and HNPCC families revealed that instability is associated with HNPCC tumours harbouring mismatch repair defects and with 20 % of sporadic colon cancer tumours. No correlation between K-ras gene mutations and the RER+ phenotype was detected in sporadic colon cancer tumours.