Understanding the mechanism of action of the glycosylated bacteriocin glycocin F : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Manawatu, New Zealand

Figures are re-used with their respective publisher's permission or under a Creative Commons Attribution 4.0 International (CC BY 4.0) license.With the increasing threat posed by antibiotic-resistant bacteria, efforts must be made to find new antimicrobial agents. One growing area of promise is the bacteriocins, which are a diverse group of antimicrobial peptides produced by bacteria. This thesis focuses on determining the mechanism of action of one of these peptides, glycocin F (GccF). GccF is produced by the bacterium Lactobacillus plantarum and is modified with two N-acetyl glucosamine (GlcNAc) sugar moieties, one located on an interhelical loop region, and the other at the end of a flexible C-terminal ‘tail’. It has also been shown to exhibit a unique effect on susceptible bacteria, putting them into a reversible state of hybernation as opposed to outright killing them. However, little is known about the roles of the structural features of GccF, how it triggers bacteriostasis in target cells, or even what part(s) of bacterial cells it targets. This work addresses these questions using three main approaches: studying the structure-function relationship of different parts of GccF with chemically synthesised analogues; looking at the transcriptional response of a pathogenic bacteria, Enterococcus faecalis, to GccF; and trying to identify binding partners of GccF and its respective immunity protein, GccH. The results presented here highlight different roles of the GlcNAcs attached to GccF, with both the interhelical loop and presence of GlcNAc on this loop being vital for activity, while the sugar at the C-terminal position is important, but not crucial for the peptide’s activity. Additionally, a role of the GlcNAc phosphotransferase system on the mechanism of GccF is strongly indicated, with evidence from both the transcriptional studies and the protein interaction studies of GccF’s immunity protein. Taken together, the results allow for two theoretical models of GccF’s mechanism of action to be proposed. These models presented here should serve to increase the understanding of other glycocin-class bacteriocins and their mechanisms of action, and possibly contribute towards the creation of a blueprint for a new class of antimicrobial agents

Characterization of A Ribose Metabolism Pathway in Bacteroides thetaiotaomicron and New Insights into the Nutrients Degraded by this Bacterium

Bacteria of the Bacteroidetes phylum, dominant members within the gut microbiota, devote large genomic capacity towards nutrient acquisition via gene clusters termed polysaccharide utilization loci (PULs). The model organism, Bacteroides thetaiotaomicron (Bt) contains 88 PULs that target complex polysaccharides of host- microbial- or dietary origin. However, many PULs remain uncharacterized in terms of cognate substrate, enzyme functionality, and regulation. I have expanded the known substrates targeted through characterizing the ribose utilization system (rus) PUL in Bt. I created gene deletions based on predicted functionality within rus. Using these strains allowed for in vitro characterization of the substrates (e.g. ribose, nucleosides and RNA) that are catabolized through this PUL. The ability to access these nutrients confers a competitive advantage in vivo on a fiber-rich diet containing nucleosides. Additionally, through biochemical and in vivo studies I have connected the actions of a genomically unlinked nucleoside phosphorylase (BT4554) and the rus ribokinases (RusK1/K2). Determining that these two enzymes work together by BT4554 cleaving nucleosides which produces ribose-1-phosphate (R1P), which is subsequently phosphorylated by RusK1/K2 yielding ribose-1,5-bisphosphate (PRibP). Further, RusK2 accepts ribose-5-phosphate (R5P) as a substrate and synthesizes PRibP by phosphorylating the 1’C position. The functions displayed by RusK1 and RusK2 are the first described in eubacteria generating PRibP from R1P or R5P, and represents new metabolism in Bt. Further, the ability of Bt to sense ribose transcriptionally alter genes located within other PULs and loci. Contrastingly, to the rus PUL, mucin-O¬-glycan (MOG) PULs are strongly upregulated in vivo on a fiber-free diet (FF diet); a condition where Bt relies on host-derived glycans for growth. This FF diet resembles Westernized human diets that have been implicated in inflammatory bowel disorders (IBD) leading to colitis by bacteria eroding the host mucosa. By deleting MOG-responsive, sulfatase-encoding PULs in Bt as single PUL deletions and sequentially, (up to a strain lacking 10 PULs), I abrogated growth of Bt on MOG. This approach has assisted in narrowing the gene-encoded functions responsible for disease. Additionally, using a transposon mutagenesis screen, I was able to discover a Bt-specific T-cell epitope recognized in vivo during disease. The expression of this epitope is affected both by glucose and salt concentrations, demonstrating even more the interesting and largely unknown regulatory strategies employed by Bt. The regulatory network in Bt is complicated, with each PUL encoding its own regulatory protein (ECF-σ/anti-σ proteins, hybrid two-component systems, etc.). Additionally, Bt encodes 22 ECF-σ proteins as well as 4 LacI-type regulators not associated with known metabolic loci, making them orphan regulatory proteins. I have deleted most of these genes, resulting in discovery of a single ECF-σ gene, BT2492, which when deleted, reduces growth on 12 of the polysaccharides Bt degrades. Further, two LacI deletion strains result in drastically improved growth on normally low-priority monosaccharides. Lastly, as suggested by in vitro RNAseq data of ribose growth, the presence of ribose affects priority of other nutrients. This phenomenon extends to other simple sugars as arabinose and xylose RNAseq data reveal that they also exert changes in gene expression for loci not associated with their catabolism, including orphan ECF-σ factors. Together these data point to a complex regulatory cascade through a multi-faceted system involving PUL-encoded activators, trans-encoded proteins, and sugar-dependent prioritization through these mechanisms.PHDMicrobiology & ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/155264/1/rwglowac_1.pd

Synthetic bacterial communities for plant growth promotion

PhD ThesisIncreasing food demands have driven the adoption of new global strategies to intensify productivity without relying on heavy chemical treatments. In the last decades, plant-growth promoting rhizobacteria (PGPR) have emerged as potential biofertilisers and biopesticides in agriculture. The overall aim of this study was to research and develop approaches to genetically engineer PGPR to improve their beneficial activities toward the plant partner. A simplified PGPR community, a Bacillus consortium of three strains, was adopted to study the complexity of the interactions occurring within the consortium and the plant microbiome. Firstly, the comparative genomic analysis of the consortium highlighted the unique and shared features responsible for plant promotion, microbial interaction and cooperation among the strains (niche partitioning, organisation in biofilms with cooperative mechanisms of quorum sensing, cell density control and antibiotic detoxification). Flux balance analysis identified cross-feeding interactions among the strains and the metabolic capability of the consortium to provide nitrogen to the plant, transforming it into forms available for plant utilisation. The consortium PGP potential was then investigated in vitro (LEAP mesocosm assay) and in vivo (pot experiment) on the vegetable crop Brassica rapa. These tests show increased plant growth when the strains were inoculated together rather than individually and when the consortium was used as a supplement of the natural bulk soil microbiome. The in silico study and the plant experiments highlighted areas for genetic improvement of the consortium genomes. Lastly, this work describes the development of a conjugation system that could be used to efficiently engineer non-domesticated bacteria and bacterial communities, such as rhizobacteria and plant microbiomes. The system, based on the plasmid pLS20, was developed in Bacillus subtilis 168 and successfully tested on twenty-three wild type Bacillus strains and three rhizobacillus communities. The research presented here provides tools and approaches for the genetic manipulation of rhizobacterial communities, with the ultimate aim of generating sustainable agricultural bioformulations and sheds light on the complex interactions that can occur in a model microbial PGPR consortia

Understand biological regulatory systems using computational models: Reconstruction, Analysis and Integration

Biological regulatory system is complex and involves many types of interactions, including transcriptional regulations, protein interactions, metabolic reactions and etc., to ensure the regulations of biological organisms. These regulations forms complex networks and play important roles in living organisms to adapt to the environment, control the rate of growth, and develop different phenotypes accordingly to its life cycle and the surrounding environment. Many of mechanisms and interactions of these networks are still not clear. Although better understanding of the regulatory systems is very important for biological research and engineering, to systematically reconstruct, analyze and integrate the complex regulatory systems is always challenging. At first, a novel method to reconstruct gene regulatory networks (GRNs) was developed, implemented, tested, and applied to experimental data. This method introduced a hidden transcription factor activity (TFA) layer to the conventional GRN reconstruction methods. The testing results showed significantly improved network reconstruction precision and recall comparing to conventional methods. The Application to E. coli transcriptome experimental data demonstrated the potential biological significance of the reconstructed network. A three level analysis framework to analyze TFAs and GRNs under different experimental conditions was followed up. The first level analyzes TFA patterns of individual transcription factors. The second level uses enrichment test and summarizes TFA behaviors by groups and their properties. The third level identifies key TFs of each experimental condition using network based analysis approach on effective regulatory network (ERN), a newly proposed differencial regulatory network model between experimental conditions. This analysis framework expands the traditional transcriptome data analysis to TFA and GRN level. The application to E. coli data showed the biological meaningfulness and helpfulness of analyzing transcriptome data on TFA and GRN level. At last, a comprehensive regulatory focused regulatory system model for E. coli had been constructed by integrating transcriptional regulatory networks, protein interaction networks, metabolic reaction networks, and all other related regulations. Statistical tests and network property analysis of this constructed network revealed the connection between biological functions and the special network properties of the constructed network. And simulations of the regulatory signal response of this constructed network verified the biological meaningfulness of this network

Salmonella’s Desiccation Survival and Thermal Tolerance: Genetic, Physiological, and Metabolic Factors

University of Minnesota Ph.D. dissertation. July 2017. Major: Food Science. Advisors: Francisco Diez-Gonzalez, Ryan Fink. 1 computer file (PDF); xi, 265 pages.Salmonella can survive for long periods under extreme desiccation and low water activity conditions (aw < 0.6) while becoming tolerant to heat. This stress tolerance poses a risk for food safety, but relatively little is known about the molecular and cellular processes involved in this adaptation mechanism and its potential for cross-protection. This dissertation consists of three distinct studies focused on elucidating this mechanism. The objective of the first study was to identify the genes involved in Salmonella’s resistance to desiccation. A global transcriptomic analysis comparing S. enterica serovar Typhimurium cells equilibrated to low aw (aw 0.11) and cells equilibrated to high aw (aw 1.0) determined that 719 genes (16% of the total number of genes in the genome) were differentially expressed between the two conditions. The genes that were up-regulated at aw 0.11 (290) were mostly involved in metabolic pathways, DNA replication/repair, regulation of transcription and translation, and virulence. Based on the transcriptomic analysis, we created deletion mutants for two virulence genes, sseD and sopD, and tested their ability to survive desiccation and low aw on glass beads. The two mutants exhibited significant cell viability reductions after desiccation compared to the wild-type and additional decrease after exposure to aw 0.11 for 7 days. Under scanning electron microscopy, the mutants displayed a different cell morphology and extracellular matrix production when compared to the wild-type under the same conditions. The findings of this study suggested that sopD and sseD are required for Salmonella’s survival during desiccation. The objective of the second study was to determine the effect of food and inert matrices, nutrient availability, and growth conditions on desiccation survival and thermal tolerance of S. enterica serovar Typhimurium. Salmonella was grown in LBglc and M9 media, in the presence or absence of EDTA and dipyridyl. Cultures were inoculated on toasted oat cereal (TOC) or glass beads, dried, and equilibrated for a week at aw 0.11 and 1.0, before being thermally treated at 75, 85, 90, and 95oC. For all growth conditions and temperatures tested, cells exposed to aw 0.11 had inactivation rates (δ-values) at least 10-fold longer than cells equilibrated at aw 1.0. Our results showed that growth in the presence of EDTA or Dipyridyl did not have any effect on Salmonella’s thermal tolerance at either aw on TOC. In control conditions, recovery after drying and thermal tolerance was higher on TOC than on glass beads, suggesting that the food matrix was protective for desiccation and thermal treatment. Growth in M9 resulted in lower survival to drying and exposure to low aw on glass beads, compared to LBglc. On the contrary, thermal tolerance increased in cells grown in M9 compared to LBglc at both aw. Cells grown in LBglc and M9 displayed differences in the production of extracellular matrix, in particular during equilibration to aw 0.11 and after thermal treatment at both aw. Additionally, when Salmonella was grown on glass beads in LBglc as biofilm, the thermal tolerance was greater than free cells dried on beads. Our observations suggest that the presence of nutrients during growth and before exposure to desiccation and thermal treatment influenced Salmonella’s ability to survive desiccation and develop thermal tolerance. The objective of the third study was to identify proteins involved in Salmonella’s resistance to desiccation and thermal treatment using iTRAQ. Proteins were extracted from S. enterica servorar Typhimurium cells dried, equilibrated at high aw (1.0) and low aw (0.11), and thermally treated at 75°C. Our analysis determined that 734 proteins were differentially expressed among samples, and of these 175 proteins were the most significant in determining differences in the proteomic profiles among treatments. Based on their proteomic expression profiles, the samples were clustered in two main groups by PCA analysis, “dry” samples and “wet” samples, while we did not observe significant differences between the thermally treated samples and the non-heated samples, at both aw. Protein profiles indicated shifts in cell metabolism in both samples, as well as a strict regulation of DNA repair, replication, transcription, and translation. “Dry” samples had higher levels of 50S and 30S ribosomal proteins, indicating that ribosomal proteins might be important for extra-ribosomal regulation of cellular response even when the synthesis of proteins is slowed down. Stress response proteins were more frequently present in “wet” samples compared to “dry” samples, including SspA, GorA, and Dps, suggesting that “wet” cells were activating stress systems in response to rehydration. In conclusion, our study indicated that pre-adaptation to dry conditions was linked to increased thermal tolerance, while reversion from a dry state into a wet state implied a significant change in protein expression that is linked with reduced thermal tolerance

Plasmid analysis, comparative genomics and transcriptomics of beer-spoilage lactic acid bacteria emphasizing the role of dissolved carbon dioxide and traditional beer-spoilage markers

Specific isolates of lactic acid bacteria (LAB) are capable of growing in and spoiling beer, and are the cause of product and process contamination, and financial loss for brewers the world over. To date, our understanding of how these contaminants are able to grow in beer is limited to analysis of hop-tolerance mechanisms, with a limited number of putative hop-tolerance genes having been described. In order to demonstrate that these hop-tolerance genes are incomplete descriptors of overall beer-spoilage ability, the transcriptional activity of these genes in two different beer-spoilage related (BSR) LAB isolates, and the prevalence and sequence conservation of hop-tolerance gene horC in BSR LAB with varying beer-spoilage ability is examined. This analysis is followed by work demonstrating that the total plasmid profile of a beer-spoilage LAB, and not just plasmids harboring hop-tolerance genes, contributes to the isolate’s overall beer-spoilage phenotype and highlights redundancy in potential beer-spoilage mechanisms. The next chapter provides evidence that the presence of dissolved CO2 (dCO2) in beer selects for the ability of LAB to spoil packaged beer, and that tolerance to this stress is not correlated with hop-tolerance, indicating that dCO2 stress is an important part of the total beer environment. This is followed by the presentation and analysis of the genome of the rapid beer-spoiling isolate Lactobacillus brevis BSO 464 and subsequent RNA sequencing for this isolate when grown in degassed and gassed beer so as to elucidate which genes are active when grown in beer, and when grown specifically in the presence of dCO2. Global transcriptome sequencing of this L. brevis isolate and Pediococcus claussenii ATCC BAA-344T when each were grown in growth-limiting concentrations of hops was also performed in order to clarify the hop-specific transcriptional response from that of the response when these isolates grow in the total beer environment. Lastly, comparison is made between available genomes of BSR LAB to reveal that the specific brewery environment a BSR LAB is recovered from, influences genetic variability and that comparison within a given LAB species reveals genetic differences that can be exploited as beer-spoilage genetic markers. This comparative analysis reveals that the total plasmid-coding capacity strongly influences individual BSR LAB beer-spoilage phenotype and the environment they are able to grow in. Overall, beer-spoilage ability is shown to be adaptive and acquired incrementally and not solely as a result of the presence of hop-tolerance genes

Facultative bacterial symbionts from European Orius species: Evidence for an ancestral symbiotic association

Pest control in agriculture employs diverse strategies, among which the use of predatory insects has steadily increased. The use of several species within the genus Orius in pest control is widely spread, particularly in Mediterranean Europe. The use of predatory insects in pest control in agriculture has spread worldwide and increased significantly, especially in the use of various Orius species. Currently, most studies about Orius species have been focused on the diet manipulation or selective breeding methods to reduce the rearing costs and improve the efficiency, only a few studies were associated to their Wolbachia symbionts. The characterisation and contribution of microbial symbionts to Orius sp. fitness, behaviour, and potential impact on human health has been neglected. Therefore, there is a lack of knowledge regarding Orius’ symbionts such as their taxonomic characterisation, the functions of the symbionts and potential influences on human health. This project was focused on the first comparative genomics report of genome sequences level description of the predominant culturable facultative bacterial symbionts associated with the analyses of draft genomes of facultative symbionts using Next Generation Sequencing (NGS) technique related to five Orius species (Orius laevigatus, Orius niger, Orius pallidicornis, Orius majusculus and Orius albidipennis) and collected from various European countries (Greece, Italy, and Spain). Initially, coxl (COI) based taxonomic classification of the Orius species used was performed, followed by the isolation of culturable bacteria from live insects. The whole genome sequences of the bacterial isolates were generated and assembled into draft genomes using NGS. The isolates of two predominant bacteria belong to Serratia and Leucobacter genera, the third predominant bacteria are most likely to be a new genus within the Erwiniaceae. Orius sp. Serratia isolates genomes are more similar to Serratia sp. SCBI. Pan-genome analysis of Serratia sp. Orius isolates evidenced an open pan-genome, and 279 accessory genes were related to the insect symbiosis trait. Additionally, pan-genome analyses of the Serratia sp. isolates offered clues linking Type VI secretion system effector–immunity proteins from the Tai4 sub-family to the symbiotic lifestyle. These symbionts were found to colonise all the insect specimens tested, which evidenced an ancestral symbiotic association between these bacteria and the genus Orius. Additionally, plasmid sequence analyses suggest sequence exchanges between Serratia sp. Orius isolates and pathogenic Serratia species, which may have implications for food safety and human health