Due to their importance for scientific and technological purposes, interactions between swimming microorganisms and solid boundaries is raising a growing interest in the scientific community but, despite recent efforts, many of their aspects are still unknown. In this thesis we investigate through experiments carried out in PDMS microfluidic devices and simulations the effect that the presence of solid walls produce on the motility of a well known puller-type microswimmer Chlamydomonas reinhardtii (CR). We present the study of the scattering of CR against circular pillars in a microfluidic device. Exploiting the curvature of the pillar surface, we are able to determine the contribution of hydrodynamic and contact forces to the scattering interaction. We find that lubrication forces tend to make algae swim parallel to the pillar surface and flagellar contact break this entrapment, making CR to scatter away at a fixed angle determined by the ratio between flagellar and body length independently of their original swimming direction. We also report the existence of random scattering, which, as the name suggests, results in an outgoing trajectory probability uniformly distributed across all possible directions. We find that these interaction rules also apply flat and concave surfaces. Through these results we are able to describe the effect of geometrical confinement in narrow straight channels and circular pools. We find that in channels it is possible to change the cellular transport efficiency simply by varying the distance between channel walls and we discover the existence of an optimal width for the transport of wild type CR. We also describe the escape dynamics of cells from circular pools through narrow apertures; once again we find that the attraction to solid boundaries is essential in describing the results, as cells can find the exit on the perimeter by sliding out of the pools while swimming along its perimeter.

Campylobacter jejuni is one the leading causes of food poisoning in the UK. Motility in C. jejuni is key to its survival within the gut and other environments. Without the two flagella, one at either end of the cell, it is highly attenuated to cause disease. As the flagellar structure is imperative to Campylobacters lifestyle, studying the regulation of flagella synthesis is important. Unlike other bacteria, Campylobacter lacks a master regulator atop the flagella synthesis pathway. It does however, have an FlhF protein with a signal recognition particle (SRP) domain and GTPase activity whose role is still enigmatic. Previous studies and observations have found that in a C. jejuni strain lacking FlhF, loss of motility and an aflagellate phenotype are observed. However under selective pressure, C. jejuni ΔflhF is able to revert to have partial motility. These pseudorevertants have an increasing unipolar phenotype when transitioning from exponential to stationary growth phase and have 50-70% of the wild-type motility. These pseudorevertant strains have provided a novel way of studying the FlhF protein. Sequencing of the pseudorevertants identified mutations in the fliF and fliG genes. FliF forms an oligomeric structure called the MS ring, which sits at the base of the flagella structure and surrounds the T3SS. FliG interacts with FliF and forms part of the C ring, placed below the MS ring. Analyses of these mutations suggest that they cause changes that make the structure more rigid, potentially forcing an interaction between the rings and T3SS. In the pseudorevertants, unlike the ΔflhF strain, transcription levels of σ54 and σ28 genes reach wild-type levels. The data in this thesis suggest that the role of FlhF is to activate a conformational change within the flagella base, locking it into the correct position and thereby creating the signal for the FlgSR cascade and σ54 transcription. The global analysis conducted in this thesis also provides many other insights into the C. jejuni flagellar cascade.

The Gram positive anaerobic bacterium, Clostridioides difficile, is one of the leading causes of hospital associated diarrhoea world-wide. An opportunistic pathogen, C. difficile colonises the gut during intestinal microbial dysbiosis, causing Clostridioides difficile infection (CDI). Treatment of CDI is complicated by the increasing numbers of recurrent infections. C. difficile has demonstrated its ability to produce composite, adherent multicellular communities, or biofilms, in vitro. In vitro biofilms offer the bacteria within increased resistance to a range of environmental stresses including antibiotics and oxygen stress. However the mechanisms underlying C. difficile community formation are poorly understood. In other bacteria, quorum sensing, a process mediated by small signalling molecules that accumulate in the extracellular environment, coordinates biofilm formation. In several bacteria, the metabolic enzyme LuxS, produces the signalling molecule autoinducer-2 (AI-2), which plays a key role in quorum sensing. AI-2 is considered to be a cross-species signalling molecule and for many bacterial species AI-2 has been shown to have a signalling role during biofilm formation and development. Here we show C. difficile luxS mutants (LuxS) are defective in biofilm formation and demonstrate the ability for chemically synthesised AI-2 to partially restore the biofilm defect of LuxS. Through RNA-seq analysis we show that LuxS/AI-2 quorum sensing likely influences C. difficile prophage expression, affecting levels of extracellular DNA present within the biofilm. Additionally we show that Bacterioides fragilis has an inhibitory effect on C. difficile, with increased levels of inhibition observed in WT compared to LuxS. By utilising dual species RNA-sequencing we propose a number of possible mechanisms responsible for the observed inhibition.

Microbes inhabiting surface waters of the Earth’s oceans are exquisitely adapted to their nutrient-poor environment. Marine phytoplankton, for example, are able to reduce their requirements for phosphorus by replacing membrane phospholipids with alternative non-phosphorus lipids. Heterotrophic bacteria, which can also thrive when phosphorus is scarce, had not, however, been shown to carry out this process – seemingly placing these organisms at a competitive disadvantage. In this thesis, I show that substitution of membrane phospholipids for a variety of non-phosphorus lipids is a conserved response to phosphorus deficiency amongst phylogenetically diverse marine heterotrophic bacteria. By deletion mutagenesis and complementation in the model marine bacterium Phaeobacter sp. MED193 and heterologous expression in recombinant Escherichia coli, I confirmed the roles of a phospholipase C (PlcP) and a glycosyltransferase in lipid remodelling. Analyses of two large collections of marine metagenomes, the Global Ocean Sampling (GOS) and Tara datasets, demonstrate that PlcP is particularly abundant in areas characterised by low phosphate concentrations. To better understand the lipids that potentially replace phospholipids during this remodeling process, I investigated a number of poorly-characterised aminolipids that are prevalent in the globally important marine Roseobacter group. I was able to identify two genes involved in the synthesis of one of these lipids, a glutamine lipid. Subsequent phylogenetic analysis of one of these genes revealed that the capacity to synthesise glutamine lipid appears to be virtually ubiquitous within the Roseobacter group. A further class of aminolipids was present in many Roseobacter strains, which I identified as a novel class of homotaurine-containing lipids using high-resolution, accurate mass spectrometry. These homotaurine lipids were detected in a battery of Roseobacter strains, enabling me to employ a comparative genomics approach to identify genes potentially involved in their biosynthesis. Surprisingly, neither of these aminolipids appeared to play an important role in the response to phosphorus limitation in the Roseobacter strains tested. Together, these results point to a key role for lipid substitution as an adaptive strategy enabling heterotrophic bacteria to thrive in vast, phosphorus-depleted areas of the ocean. Although phosphorus-free lipids play a crucial role in this process of adaptation, my work emphasises that many of these lipids are not simply substitutes for phospholipids but rather appear to have important roles in the cell in their own right.

The genus Streptomyces is known to be responsible for the production of more than two-thirds of the world’s antibiotics, through complex specialised metabolic pathways. However, given the high frequency of rediscovery of known antibiotics and the challenge of producing novel analogues via chemical synthesis, biosynthetic engineering has emerged as an attractive approach to optimising antibiotic natural products for clinical use. This technique utilises enzymes from antibiotic biosynthetic pathways to create novel antibiotic derivatives. However, its application requires an understanding of how antibiotics are biosynthesised. This work is focused on the methylenomycin antibiotics produced by Streptomyces coelicolor A 3 (2), a model Actinobacterium. The cluster of genes directing methylenomycin production and its regulation are carried on the giant linear plasmid SCP1. The sequencing of the entire 356-kb SCP1 plasmid allowed bioinformatics analyses to be applied to the assignment of putative roles in methylenomycin biosynthesis for several of the enzymes encoded within the methylenomycin biosynthetic gene cluster. However, experimental evidence to support the proposed roles of several of these enzymes has yet to be obtained, while the roles of some of the proteins encoded by the cluster remain unclear. Here, work towards understanding the biosynthesis as well as the mode of action of the methylenomycin antibiotics is reported. In particular, the roles of MmyO and MmyF in the epoxidation of methylenomycin C to produce methylenomycin A are demonstrated via feeding of methylenomycin C to a methylenomycin-resistant derivative of S. coelicolor M145 expressing mmyO and mmyF. A putative butenolide intermediate in the pathway, believed to derive from a MmyD-catalysed condensation of acetoacetyl-MmyA with a pentulose, was identified in S. coelicolor strains expressing the methylenomycin biosynthetic gene cluster. The pattern of incorporation of [U-13C]-D-ribose into the putative butenolide intermediate was similar to that observed for methylenomycin C, indicating the former could indeed be a precursor to the latter. A putative intermediate of the pathway, pre-methylenomycin C, accumulating in a mmyE mutant strain, and its lactone form, pre-methylenomycin C lactone, were shown to be 16 and 256 times, respectively, more potent than methylenomycin A, against methicillin-resistant Staphylococcus aureus (MRSA). Expression of the methylenomycin resistance determinant (mmr) in Streptomyces species also confers no resistance against these two putative intermediates unlike methylenomycin A, the final antibiotic product of the pathway. Investigations into the mechanism of action of methylenomycin antibiotics with luciferase and β-galactosidase pathway-specific promoter-reporter fusion strains strongly suggest that the methylenomycins exert their antibiotic effects in bacteria primarily by targeting the biosynthesis of cell wall peptidoglycan, consistent with their activities mainly against Gram-positive strains. This is the first report of the mode of action of methylenomycin family of antibiotics.

Natural products with antimicrobial activity have played an important role in the treatment of infection since their discovery. The increasing emergence of pathogens resistant to multiple antibiotics has raised awareness of the urgent need for novel antibiotics. Soil microorganisms are the major source of antibiotics and Actinobacteria in particular have an impressive capacity for production of diverse bioactive secondary metabolites. However, culture-independent studies have shown a greater microbial diversity present in soil with potential for novel chemical structures and these can be explored further using metagenomic approaches capturing genes without the need to cultivate the host. Different metagenomic tools were used to study and explore microbial secondary metabolite diversity in soil. In particular, amplicon sequencing of 16S rRNA gene, NRPS and PKS biosynthetic genes allowed the identification of novel potential phylogenetic drivers of secondary metabolite diversity in the less characterized phyla Verrucomicrobia and Bacteroidetes and potential geographic hotspots harbouring unique biosynthetic diversity such as Antarctica and Cuba. The exploitation of these hotspots presented some bottlenecks in the form of DNA extraction efficiency, library creation, screening and heterologous expression. These were overcome by comparative analysis of different eDNA extraction methods to optimise fragment size and purity combined with development of new cloning tools for both DNA capture and expression. Modification of the microbial community through the amendment of the soil with chitin, highlighted the beneficial effect of microbial enrichment allowing a higher recovery of eDNA and higher detection of the biosynthetic gene of interest related to secondary metabolite production. Further additions were made to the metagenomic molecular toolbox in the form of BAC vectors (pBCaBAC and pBCkBAC) which were tested with suitable heterologous host systems (Streptomyces sp. and the engineered Pseudomonas putida species) potentially facilitating heterologous expression. In conclusion this is the first study to identify the drivers of microbial secondary metabolite diversity in situ and provided a comparative analysis of a range of diverse soil types. This approach paired with new developments in metagenomic technologies will make a substantial contribution to improving the likelihood for discovery and exploitation of new drugs for treating multi-resistant pathogenic bacteria.

Constantly changing environments in nature have led to bacteria evolving regulatory strategies that result in differential gene expression. A novel and understudied aspect of these networks are regulatory RNAs. The Gram-positive model organism Bacillus subtilis not only modulates gene expression to survive a variety of stresses, but also can form endospores to ensure its survival. Sporulation is an essential survival mechanism for many species, allowing them to enter a state of dormancy with resistance to various harsh conditions. This, in turn, ensures survival of not only the population, but also the species. The process of sporulation requires the controlled expression of approximately a quarter of the genes encoded by B. subtilis. Previous large-scale studies have identified that many transcripts do not encode proteins, but exhibited expression profiles similar to genes already known to be part of the sporulation network. Many of these transcripts were selected to likely function as small regulatory RNAs (sRNAs). This study has shown that many putative sRNAs are active during sporulation, three of which show specific phenotypes that alter germination capabilities in the presence of specific germinants. Cells lacking the necessary components to reverse this process are at a strong disadvantage. Detection of favorable growth conditions is key, but how is this conveyed during metabolic inactivity? Initial selection of putative sRNAs was done by in silico characterization. Prediction of transcriptional control and regulatory regions combined with tiling array profiling was used to select putative sRNAs for confirmation in vivo. Transcriptional fusion constructs were generated to confirm compartmental specific expression during sporulation. Spore specific sRNAs were further characterized with phenotypic studies, which suggested a role in endospore formation. This study explored some of the global analysis methods to identify sRNA targets. Whilst no targets for the four chosen sRNAs could be identified, this study produced the most comprehensive data set of proteins to be identified from a B. subtilis endospore.

Quaternary amines such as choline and carnitine are essential nutrients for humans supplied from daily food; however, quaternary amines metabolism by gut microbiota can lead to the development of various diseases, including non-alcoholic fatty liver disease and cardiovascular disease. It is hypothesized that both diseases are promoted by microbial catabolism of choline and carnitine to trimethylamine (TMA). Proteus mirabilis is a Gram-negative gut proteobacterium, which can metabolize choline anaerobically to form TMA. I demonstrated that the identified cutC gene is essential for choline degradation and subsequent TMA production in this bacterium. Using P. mirabilis as the model, I investigated the physiological role of quaternary amine metabolism from the bacterial perspective and demonstrated that P. mirabilis can rapidly uptake and degrade choline to enhance growth rate, cell yield and swarming speed under anaerobic and microaerophilic conditions. I also provide the first evidence of a novel choline-metabolizing microcompartment, which is present in both vegetative and swarming cells supplemented with choline. Another important dietary source of TMA in human gut is carnitine. I used two model proteobacteria Acinetobacter baumannii and Escherichia coli in this project to investigate the role of carnitine metabolism to TMA in health and disease. A. baumannii and E. coli can use carnitine as a growth substrate to produce TMA. To better understand the role of quaternary amine metabolism in host health and disease, I used Caenorhabditis elegans model to investigate carnitine metabolism on the life span of the worm. My data suggest that malate, the degradation product of carnitine, extends the life span of C. elegans fed on A. baumannii or E. coli. Together, my study reveals that choline and carnitine metabolism as an adaptation strategy for gut proteobacteria and contributes to better understand the ecology of these TMA-forming gut bacteria in health and disease.

No description available.

In this work, we aim to understand the behaviour of filaments and bundles of filaments in the presence of diffusible cross-linkers; we posit that these are analogues of the structures present in the Z-ring, with special focus on E. coli. Then, we study these structures by constructing a mathematical model based on statistical mechanics, and analysing its behaviour under different ranges of parameters. We show that the ring-like geometry of the division plane is conducive to constriction, and that this effect can be potentialised by the dynamics of depolymerisation. Furthermore, we show that percolating bundles of filaments are also capable of constriction, and that percolation is necessary but not sufficient for a contractile force to arise. Finally, we investigate how such a model can originate percolating bundles of filaments and how to maximise the contractile potential of such bundles. Concomitantly, we investigate the mutant ftsZ84, an E. coli mutant with uncommon properties that might help us understand the relationship between FtsZ assembly and bacterial cell division, by using a new experimental approach. Though the results prove inconclusive, we were able to confirm that the activity of this mutant agrees with the published literature.