Identification and Characterisation of Novel Phages of Pectobacterium and Erwinia

Losses in crop yields due to disease need to be reduced to meet increasing global food demands associated with growth in the human population. There is a well-recognised need to develop new environmentally-friendly control strategies to combat bacterial crop diseases. There are several crop diseases for which no effective bactericidal agents are currently available, such as potato blackleg and soft rot disease caused by Pectobacterium atrosepticum and other members of soft rot Enterobacteriaceae (Czajkowski et al., 2011). Furthermore, current control measures involving the use of traditional chemicals or antibiotics are losing their efficacy due to the natural development of bacterial resistance to these agents, as seen for fire blight of the pear and apple tree caused by Erwinia amylovora (de León Door et al., 2013; Mayerhofer et al., 2009; Ordax et al., 2006; Russo et al., 2008). Bacteriophages (phage), the viruses of bacteria, have received increased research interest in recent years as an environmentally friendly means of controlling bacterial diseases. However, not all phages possess the features that would enable them to be effective bactericidal agents. To this end, this thesis provides a detailed study of phages that infect Pectobacterium atrosepticum and Erwinia amylovora. The knowledge gained in the execution of this PhD thesis contributes to the pool knowledge about the lifestyles of the phages examined thus enabling a more informed choice with regard to the selection of suitable phages for biocontrol applications for the relevant phytopathogens

Characterisation of bacteriophage-encoded inhibitors of the bacterial RNA polymerase

RNA polymerase (RNAP) is an essential enzyme which catalyses transcription; a highly regulated process. Bacteriophage are viruses which infect bacteria and as a result have evolved a diverse range of mechanisms to regulate the bacterial RNAP to serve the needs of the virus. T7 Gp2 and Xp10 P7 are two bacteriophage-encoded transcription factors that inhibit the activity of the bacterial RNAP. The aim of this study is to investigate the molecular mechanisms of action of Gp2 and P7. Fluorescence anisotropy experiments proved Gp2 to bind to RNAP, independently of the σ- factor, with a 1:1 stoichiometry and a low nanomolar affinity. In vitro transcription assays demonstrated that a negatively charged strip in Gp2 is the major determinant for its inhibitory activity. Furthermore, it was shown that efficient Gp2-mediated inhibition of RNAP also depends upon the highly negatively charged and flexible σ70 specific domain, R1.1. Gp2 and R1.1 both bind in the downstream-DNA binding channel and exert long-range antagonistic effects on RNAP-promoter DNA interactions around the transcription start site. A systematic mutagenesis screen was used to identify residues in P7 necessary for binding to the RNAP; results were interpreted in the context of a newly resolved NMR structure of P7. Electrophoretic mobility shift assays revealed that P7 ‘traps’ a RNAP-promoter DNA complex en route to the transcriptionally-competent complex. Preliminary results from a fluorescence based RNAP-DNA interaction assay suggest that P7 may target RNAP interactions with the -35 promoter element and the ‘discriminator region’. This study has contributed to our understanding of how non-bacterial transcriptional factors can influence bacterial gene expression by modulating RNAP activity. This study has also uncovered vulnerabilities in RNAP, which have the potential to be exploited therapeutically. To this end, these structure-function studies of Gp2 and P7 have provided the basis for the rational design of novel anti-bacterial compounds

Bioinformatics

This book is divided into different research areas relevant in Bioinformatics such as biological networks, next generation sequencing, high performance computing, molecular modeling, structural bioinformatics, molecular modeling and intelligent data analysis. Each book section introduces the basic concepts and then explains its application to problems of great relevance, so both novice and expert readers can benefit from the information and research works presented here

Utilizing the zinc homeostasis system of Escherichia coli as a novel inducible promoter system

Zn(ii) is an essential post-transition metal found in all life, however, at high concentrations Zn(ii) can become toxic, causing oxidative stress and inactivation of essential enzymes by replacing other metals in catalytic centres of proteins. Escherichia coli cells can control their internal zinc to a femtomolar concentration, equivalent to one to two free Zn(ii) ions per cell. This tightly controlled zinc homeostasis system is regulated by two transcription factors, ZntR and Zur, which regulated the expression of the major zinc export and zinc acquisition genes. This zinc homeostasis system of E. coli has the potential to be developed as a novel inducible promoter system. The ZntR regulated promoter (PzntA) demonstrated a strong correlation between increasing zinc concentrations and promoter induction, as well as showing a comparable induction level to the IPTG inducible promoter, Ptrc. Six Zur regulated promoters showed a variation of induction level when induced with the zinc chelator TPEN (Pc1265>PykgM>PznuA>PznuCB>PpliG>PzinT). Promoters Pc1265 and PykgM demonstrated more desirable characteristics than the IPTG inducible Ptrc; showing lower basal expression, higher induced expression and higher fold induction. Both ZntR and Zur regulated promoters show strong potential to be utilized as either a zinc or TPEN inducible promoter for use in both research and biotechnology. Flow cytometry data of E. coli zntA:rfp in combination with Bayesian analysis demonstrated that E. coli mounts a heterogenous gene expression of zntA. This analysis further showed that with increasing zinc concentration, E. coli shifts the heterogenous zntA expression, reducing low level gene expression and increasing high level gene expression. In silico analysis of the uncharacterised Zur regulated C1265-7 suggested that C1265 is a TonB-dependent receptor which translocates zinc, C1266 may be involved in bacteria-host adhesion, and C1276 is likely a COG0523 protein. In vitro analysis did not suggest a phenotype and it is likely that the true phonotype of C1265-7 can only be observed in an infection model

Genetic diversity and its consequences for light adaptation in Prochlorococcus

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2011.Cataloged from PDF version of thesis.Includes bibliographical references (p. 215-223).When different cells thrive across diverse environments, their genetic differences can reveal what genes are essential to survival in a particular environment. Prochlorococcus, a cyanobacterium that dominates the open ocean, offers an opportunity to explore such differences. Its diversity is examined here, beginning with an overview and comparison of 12 fully sequenced Prochlorococcus genomes. The Prochlorococcus core genome, that set of genes shared by all cultured Prochlorococcus, appears to be well defined by the set shared by these isolates. The flexible genome, that set of genes found in some isolates but not shared by all Prochlorococcus, was found to be much larger and open-ended. Most laterally-acquired genes were found to be located in highly variable islands such as those described in previous studies of Prochlorococcus. Those lateral gene transfer events can also be placed on the Prochlorococcus phylogenetic tree: each Prochlorococcus isolate possesses a significant number of genes that even its closest sequenced cousin does not. A particular gene family may define a Prochlorococcus ecotype if those genes are possessed by all members of that ecotype, and if their presence gives that ecotype a selective advantage in some circumstance, thus contributing to the determination of its niche. One gene family is conspicuous for appearing in many copies per genome in one Prochlorococcus clade, referred to as eNATL. The sequenced strains belonging to this clade each possess over 40 copies of genes encoding high light inducible proteins (HLIPs), compared to only 9-24 in the other Prochlorococcus genomes. Other studies suggest these genes may be involved in resistance to sudden increases in light intensity, among other stresses. This becomes especially interesting as recent field studies also found that eNATL cells may survive changes in light intensity more easily than other lowlight adapted Prochlorococcus. Here, the effects of light shocks on an eNATL strain and on other Prochlorococcus strains are studied. eNATL cultures do recover from light shock conditions that are lethal to other low light-adapted Prochlorococcus. Measurements of bulk in vivo chlorophyll fluorescence, fluorescence per cell, and variable fluorescence, along with preliminary gene expression data, suggest that the early, rapid response of high light-adapted cells and of eNATL cells distinguish them from other low light-adapted cells, possibly explaining their subsequent survival. The possible role of HLIPs in this response is discussed. The discussion of HLIPs and eNATL is based on the complete sequences of only two eNATL genomes, both sampled from the same part of the ocean at the same time. That dataset is expanded by the inclusion of Global Ocean Survey environmental shotgun reads, from which are identified several thousand HLIP genes. Past work has shown that HLIPs are divided into two distinct clades: the core, freshwater cyanobacteria-like HLIPs and the flexible, phage-like, island-bound copies. That distinction is examined in the metagenomic data, demonstrating that the separate types are consistently found in distinct chromosomal neighborhoods.(cont.) The evolution of HLIPs is also explored by the analysis of large-insert environmental clones containing islands from a variety of eNATL cells. Here, not even all island-bound, HLIP-encoding genes appear to be alike, as only a subset are consistently found in the same locations across the whole eNATL clade. Ecotype-defining genes are those genes, shared by all members of an ecotype, that provide an ecologically significant advantage, thus helping to define the ecotype's niche. It can be expected that, as environmental data accumulates (including additional measurements of Prochlorococcus abundance and newly sequenced genomes from uncultured cells), additional such genes can be identified. This work should represent a model for searching for and examining such genes. Hopefully, future experiments will be able to test the physiological significance of candidate ecotype-defining genes, while feeding back to the environmental data to verify their importance in the open ocean.by Gregory C. Kettler.Ph.D