- Volume 1, Issue 1A, 2019

The genus Streptomyces is comprised of soil-dwelling Gram-positive Actinobacteria that are widely used for the industrial production of antibiotics. S. clavuligerus is used for the industrial production of clavulanic acid, which is a potent b-lactamase inhibitor, and is, therefore, able to restore the sensitivity of b-lactamase-producing bacteria penicillins and cephalosporins. In fermentations, the carbon sources available for utilisation by the producing organism have profoundly impact central carbon and specialised metabolic pathways. We have a long-term goal of using carbon sources from food waste to produce clavulanic acid with a view to developing more sustainable fermentations. To achieve this, the carbon utilisation profile of S. clavuligerus has to be diversified. Wildtype S. clavuligerus is a natural glucose auxotroph and has adapted to utilise glycerol most efficiently. It has been shown that the lack of glucose utilisation by S. clavuligerus is due to the insufficient expression of genes whose products are required for glucose uptake (glcP) and phosphorylation (glk). To enable glucose utilisation by S. clavuligerus strains, we have constructed strains for heterologous expression of either glcP or glk from different Streptomyces species. Further, the range of utilisable carbon sources for growth and clavulanic acid production has been investigated. Growth on solid media has revealed interplay between carbon and nitrogen metabolism, with extracellular protease production being regulated in a carbon source-dependent manner. Therefore, the role of protease secretion and its relationship with clavulanic acid production has been examined, revealing a complex role between carbon catabolite repression, protease production and clavulanic acid biosynthesis.

Acetogens are microbes which produce acetate as a fermentation by-product. They have diverse phylogeny but a metabolic feature in common called the Woods-Ljungdahl Pathway (WLP), which confers the ability to fix carbon dioxide via a non-photosynthetic route. Electrons for this process are derived from diverse substrates including molecular hydrogen and carbon monoxide. The ability of acetogens to utilise components of syngas (H₂, CO, CO₂) make them an attractive target for metabolic engineering for industrially relevant products. We have previously reported the construction of a genome-scale metabolic model of the model acetogen Acetobacterium woodii using a sequenced and annotated genome of strain DSM1030. The model consists of 836 metabolites, 909 reactions and 84 transporters and can account for growth on diverse substrates reported in the literature. We identified the reactions used to catabolise fifteen single substrates and 121 substrate pair combinations, and used this to construct a sub-model representing a core set of energy producing catabolic pathways. We then introduced heterologous reactions to allow for the production of chemical of interest. Elementary modes analysis of this extended sub-model was applied to further decompose it into unique sets of the smallest functioning sub-networks. With CO₂ and H₂ as substrates, we find routes for the production of several chemicals where small amounts of excess ATP are produced simultaneously. Repeated analysis with alternative renewable feedstocks such as methanol and formate, indicate a wider potential in producing compounds of interest while also maintaining energy generation and co-factor conservation.

The rapid expansion of global plastic production in the last number of decades (>355 million tonnes in 2017), coupled with poor waste management, has resulted in an estimated 5–12 million metric tonnes of plastic waste entering our oceans. Packaging applications account for much of the current waste production, and commonly include polyethylene- and polyethylene terephthalate (PET)-based materials that are resistant to natural degradation processes, particularly in marine environments. In response to this global marine pollution issue and the continuing demand for effective treatment of such plastics in terrestrial environments (e.g. 27.1 million tons of annual, post-consumer plastic waste in Europe alone), researchers have focused on addressing the biodegradation of recalcitrant plastic waste such as PET. To this end, we screened 20 Streptomyces spp. strains isolated from marine sponges for polycaprolactone (PCL)-degrading activity, which is considered a model substrate for PET. Although the Streptomyces genus is commonly explored for natural products discovery, little is still known about its potential for polymer degradation. Genomic analysis of two of the Streptomyces isolates which screened positive for PCL-degrading activity were found to have PET-hydrolase gene homologs that shared 41 % identity to the well-characterised PETase from Ideonella sakaiensis 201 F6. One of these genes was subsequently heterologous expressed in E. coli in order to further characterise the enzymatic activity and other biochemical properties of the enzyme.

The global brewing industry produces a large amount of waste, 85 % of this is composed of spent brewers’ grain. One use for this waste product is in the bioethanol industry where the yeast, S. cerevisiae uses the spent grain as a feedstock. Due to the nature of the feedstock, there is a lack of utilisable carbon for S. cerevisiae. To obtain optimum utilisation of the waste product in conjunction with high process efficiency, enhanced carbon metabolism of the production strain is required. As well as expanded nutrient utilisation there is also a requirement to maintain high ethanol production and ethanol tolerance that industrial strains have acquired in a preferred growth medium. We are using high-throughput phenotypic arrays to rapidly identify strains best able to grow in a wide range of conditions, including various carbon and nitrogen sources and multiple stress inducing conditions. This method has shown small but measurable differences between production strains in industrially relevant growth conditions. In collaboration with an industrial partner, both targeted and random chromosomal integration of transgenes have been made to multiple candidate production strains to improve recycled feedstock utilisation and process efficiency. In addition, whole genome sequencing is being utilised to interrogate the genetic basis for phenotypic differences between production strains. It has been found that some important null phenotypes are at the transcription level, this information is now in use to drive future rounds of genetic manipulation.

Municipal solid waste (MSW) production is projected to reach 3.4 billion tonnes per annum by 2050. The majority of MSW produced globally is incinerated or diverted to landfill, both methods which pollute the environment and contribute substantially to climate change. The organic fraction of MSW (OMSW) typically comprises ∼50 % lignocellulosic material and presents an abundant renewable feedstock for producing biofuels and chemicals. An important step toward OMSW valorisation is the identification of suitable microorganisms capable of fermenting this highly inconsistent, heterogeneous and complex feedstock. We have characterised the fermentation performance of eight biotechnologically relevant microorganisms (Clostridium saccharoperbutylacetonicum, Escherichia coli, Geobacillus thermoglucosidasius, Pseudomonas putida, Rhodococcus opacus, Saccharomyces cerevisiae, Schizosaccharomyces pombe and Zymomonas mobilis) on enzymatic hydrolysate of OMSW fibre produced by a commercial autoclave pre-treatment. S. cerevisiaewas the most efficient ethanol producer, followed closely by Z. mobilis. Both species produced ethanol to high titre within 24 h, but neither could ferment xylose. The most effective performance was demonstrated by R. opacus, which consumed all available glucose and xylose concurrently over 72 h and produced a remarkably large yield of triacylglycerol (a biodiesel and aviation fuel precursor). This work demonstrates that OMSW is a promising renewable feedstock capable of supporting the growth several industrially useful microorganisms to high product titres. The best performing species identified here are interesting candidates to study further for application in a MSW biorefinery.

Biosurfactants produced from microbial sources are increasingly viewed by industry as more sustainable and less toxic alternatives to their chemically derived counterparts. One major class of biosurfactant that has the potential for commercial exploitation are the rhamnolipids. Rhamnolipids are composed of one or two rhamnose monosaccharides covalently bonded to fatty acid chains of varying molecular weights. The major microbial producer of rhamnolipid is Pseudomonas aeruginosa, however as this is a known human pathogen many industries are reluctant to utilise rhamnolipids synthesised by this bacterium. In order to avoid this problem a consortium of both academic and industrial partners have been screening marine bacteria for their ability to synthesis biosurfactants in a project called MARISURF. Here we report our findings of rhamnolipid production by two marine bacterial strains. Rhamnolipid production by these strains was first identified via the phenotypic screening of surface tension reduction. Rhamnolipid synthesis was then confirmed and characterised via HPLC-MS and NMR. Both 16S rDNA and subsequent genomic sequencing revealed these strains to be Marinobacter sp. and Pseudomonas mendocina, both species where rhamnolipid production was previously un-reported. Finally, both strains were assessed for potential pathogenicity using the Galleria mellonella model. Importantly for commercial exploitation, neither strain was shown to be harmful to G. mellonella over a 72 h infection period. Confirmed identification of rhamnolipid production in bacterial strains isolated from the marine environment highlights global oceans as an untapped resource in the ocean for the discovery of novel sources of biosurfactants.

Microbially-induced calcite precipitation (MICP) is ubiquitous in nature and has become an area of interest for environmental, geotechnical, and civil engineering applications. These include bioremediation, soil engineering, and self-healing of cementitious materials. To date, ureolytic bacteria have been favoured due to their ability to rapidly increase the pH of the environment through the hydrolysis of urea and thereby induce precipitation of calcite. However, the requirement for urea can contribute to nitrogen-loading in the environment and prove to be incompatible in certain applications, such as in self-healing concrete where it delays setting. Non-ureolytic bacteria are thought to be less efficient at MICP as they lack the ability to hydrolyze urea and thus to induce rapid increases in pH. Profiling of environmental bacteria has revealed the fundamentally different mechanisms that ureolytic and non-ureolytic bacteria utilize to precipitate calcite. These affect the timing of MICP and morphology of the crystals, but not necessarily the overall quantity of calcite precipitated. Furthermore, we show that MICP facilitated by non-ureolytic bacteria results in precipitates that contain significant organic components. These precipitates appear to have increased volume and cohesiveness, which may prove advantageous in application. Our findings offer important new insights into the use of MICP for geotechnical and environmental engineering and will enable us to create a toolbox of microbial precipitators tailored for specific applications.

Antimicrobial resistant bacteria can become harboured in sediments of post-industrial estuaries. Subsequently, their resistance traits could be enriched by pollutants deposited in the sediments. Recent evidence strongly suggests this may pose hazards that not only affects the health care sector, but could also impact tourism and the aquaculture industries. The River Clyde, UK was chosen for this study due to its extensive industrial history, and three sites were chosen to sample from representing different levels and types of industrial activities—two highly polluted and one relatively ‘pristine’ site. We extracted and analysed for metal pollutants (or ‘potentially toxic elements’, PTE), and other geochemical characteristics for all sediment cores. Gram-negative, enteric bacteria were isolated from all sediment cores from the three sites. Their susceptibilities to antibiotics and metals were assayed—determining minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC). The results indicate that co-selection of PTEs and antibiotic resistance does occur, and this impacts bacteria that are potential human pathogens. Higher concentrations of metals in the environment correlated to antibiotic resistance and higher MICs to metals than among bacteria found in less polluted sites. To continue to protect human health, the interactions between environmental and human health must be fully understood. This study provides critical information behind the specific causes of antibiotic resistance due to a legacy of pollution.

Predatory myxobacteria have an antimicrobial nature that dominates their interactions with neighbouring organisms. They are abundantly found in soil, water, dung of herbivores, and have the potential to significantly affect the microbiome of an environment. In this project, we hypothesize that potential prey organisms evolve in response to the selective pressure exerted by predatory microbes. Using a variety of nutrient media, we isolated bacteria from soil samples to test their susceptibility and resistance to the laboratory strain Myxococcus xanthus DK1622 and its predatory secreted outer membrane vesicles (OMVs). Soil (with and without heat treatment) was spread onto plates which had been pre-inoculated with myxobacteria, OMVs, or no pre-treatment. Plates with myxobacteria lawns or OMVs exhibited reduced diversity of isolates compared to control plates. The yield and diversity of isolates obtained also depended on the media used. Heat pre-treated soil gave rise to distinct morphologies and fewer slime producers. Co-existence and competition were exhibited by soil isolates, which were identified using 16S rRNA gene sequencing and phylogenetic analysis. Purified isolates were also characterised for their resistance and/or susceptibility to predatory attack by a variety of myxobacteria. The bacterial isolates obtained varied when exposed to seven different myxobacteria predators. Our data suggest that the addition of myxobacteria to isolation plates biases isolation towards relatively predation-resistant prey organisms. Our next goal is to isolate myxobacterial predators from the same soil samples (on different prey isolates) and test predator-prey interactions quantitative using pure strains. The genetic basis of differential predatory activity and prey susceptibility can then be investigated.

Currently, the surface of Mars cannot sustain liquid water, but there is evidence suggesting that water was present in the Noachian. Although water might exist in the subsurface of Mars, it could not sustain in the present day unless it was highly saline. Thus, saline springs in polar desert environments are analogues with which to investigate martian conditions. An example of this is Axel Heiberg Island, located in a region of continuous permafrost in the Canadian High Arctic, which hosts sulfidic and highly saline springs. In this study, cultivation-dependant and independent techniques were used to investigate the microbial diversity of a sediment sample collected from a saline cold (3–8 °C) pool at Colour Peak Springs on Axel Heiberg. Both DNA and RNA were extracted from the samples, and the microbial community was characterised using the 16S rRNA gene from the extracted nucleic acids. The metabolic profile was characterised by screening DNA and cDNA for functional genes relating to the cycling of carbon (coxL, xoxF, cbbL), nitrogen (nifH, nosZ, nod) and sulfur (dsrB, soxB). The community profiles were used to inform enrichment strategies, allowing for the isolation and characterisation of several halophilic isolates including strains of Marinobacter, Halomonas, Halanaerobium and Loktanella. Through this work we have been able to develop an in-depth characterisation of the metabolic and phylogenetic diversity that is present and viable within this analogue site. This allows us to start building an understanding of the underlying mechanisms and strategies that enable organisms to persist in these environments.

Understanding distribution patterns at various spatial scales is a central issue in microbial ecology. Beyond the lone identification of biogeographical patterns, understanding the environmental drivers behind community diversity and structure is key. While many studies identify pH as a major parameter structuring microbial communities at large spatial scales, many other variables impact distribution patterns on smaller scales. Here, we investigated the biogeographical patterns of Arctic soil microbial communities from 1 m to 500 m, within Adventdalen, Svalbard, using 16S sequencing, gravimetric measurements and X-ray fluorescence. Multivariate analyses identified key environmental variables shaping microbial communities and revealed the importance of soil moisture, organic carbon and elements such as aluminium, calcium and potassium in structuring distribution patterns. The indicator species analyses identified key associations between environmental variables and OTUs. Using geostatistical kriging, we mapped the biodiversity and distribution of key OTUs across the landscape. Overall, our results highlight the spatial heterogeneity in Arctic soils and identifies the sampling scale needed to characterize microbial communities within an area of interest with seemingly homogeneous landscape.

Commercial mushroom crops (Agaricus bisporus) are susceptible to a disease causedby a complex of 18 viruses known collectively as mushroom virus X (MVX). Symptoms of MVX infection vary from bare patches in crops to mushroom discolouration (browning). To understand the dynamic interaction between A. bisporus and MVX, we have studied five strains; including the globally-cultivated commercial strain, one wild strain, and one commercial-wild hybrid strain. Our transmission experiments using ‘healthy’ mycelium challenged with MVX-infected mycelium, detected MVX in the first day of hyphal anastomosis in all five strains. However, our commercial-scale crop experiment revealed varying degrees of disease tolerance in the fruiting body, the commercial strain being most susceptible and the hybrid strain most resistant to MVX. LC-MS/MS proteomics and RNA-seq analyses have elucidated key differences in response to both early and late crop inoculation of MVX. Quantitative shotgun proteomics of the susceptible commercial strain at late MVX inoculation revealed striking levels of proteins relating to mechanical membrane damage via detection of myo-inositol-associated biosynthetic proteins. Defense proteins relating to chalcone isomerase activity were also detected exclusively in the MVX-infected commercial strain. MVX-infected wild strain isolates showed significantly greater abundance of proteins related to fundamental cellular processes such as RNA polymerase activity and cell-redox homeostasis. Our findings show that although vegetative transmission of MVX is prevalent in all five strains, the fruiting body may oppose the infection in certain strains. Our findings of dynamic host response of A. bisporus to MVX, provides novel insights for this economically important, globally cultivated crop.

Ruminants depend on the highly diverse microbial community that resides in the rumen, the first and largest chamber of their digestive system, to gain nutrients from their herbivorous diet. The Hyper Ammonia Producers (HAPs) are obligate amino acid fermenting bacteria found in low numbers in this community. This break down of amino acids and peptides results in excessive ammonia production, as well as hydrogen and carbon dioxide, resulting in loss of nitrogen from the host and contribution to environmental emissions from enteric fermentation. Despite their large impact, little is known about the genomic underpinnings of the HAP phenotype. Our study employed comparative genomics and transcriptomics approaches to address this question. A phylogenetic tree of 498 rumen prokaryotic microbial genomes from the Hungate 1000 project (including 12 known HAPs) identified the HAP phenotype as polyphyletic, indicating independent origins or a result of horizontal gene transfer (HGT). However, following construction of sequence similarity networks for all genomes, few uniquely shared homologous genes families were apparent in the HAPs, suggesting that HGT did not drive their evolution. Instead, independent evolution of the phenotype is supported by similar functional analog profiles in the genomes of organisms with the HAP phenotype. Genome-wide characterisation and expression of functional analogs in known HAPS will allow in silico prediction of novel HAPs from the rumen which can be confirmed in vitro.

It has been well documented that antibiotic resistance (AR) is a clinical concern that affects both human and animal health but AR in the environment and food-chain is not as well understood. AR bacteria can occur naturally in soil, water and organic fertilizers used in agriculture so there is a risk that AR can pass to humans via the food-chain. This study focuses on lettuce cultivation undergoing four treatments (Normal irrigation water+normal soil, normal irrigation water+manure, UV irrigation water+normal soil, UV irrigation water+manure)to determine the mechanisms by which the AR is transferred to the plants over the growth period of the lettuce (7 time-points – week 0 to week 6). Plasmids (n=318) have been isolated from irrigation water (n=36), soil (n=45) and lettuce (n=42) samples using the exogenous isolation method for week 0 and week 6 initially. Antibiotic susceptibility testingto amikacin, cefotaxime, ciprofloxacin, imipenem, kanamycin, tetracycline has been carried out. Multi-drug resistance profiles were established for soil taken at timepoint 0 and lettuce taken at timepoint 6. Extracted plasmid DNA was sent for metagenomic analysis to determine which genes are involved in the transfer of AR at the interfaces. The results of the sequencing showed that there are multiple AR genespresent, including Tet, Sme, Cmy, Oxa and ANT(4’)-Ib, that confer resistance to bacteria. The identification of multi-drug resistance in soil and lettuce samples is concerning and highlights the need to determine the mechanisms leading to antibiotic resistance in food.

One potential design for a geological disposal facility (GDF) for intermediate level radioactive waste (ILW) involves the use of a cement base grout which will establish a highly alkaline environment for extended time periods [1]. Methane generation by colonising microbes could impact the long-term performance of the facility by influencing gas pressures and potentially leading to the migration of 14C to the biosphere [1]. Sediments acquired from a wide-range of anthropogenic alkaline sites in the UK were used to develop acetoclastic and hydrogenotrophic methanogen enrichment cultures over a broad range of pH values (7.0–12.0). The generation of methane from hydrogen and acetate was assessed to determine the dominant methanogenic pathways. Archaeal community analysis via Illumina MiSeq was employed to describe the populations involved and the acetoclastic inhibitor methyl fluoride was utilised to confirm the lack of acetate-dependent methane generation under alkaline conditions. High pH (pH>9.0) microcosms employing alkaline sediments were dominated by hydrogen-consuming methanogens of the orders Methanobacteriales and Methanomicrobiales, with no acetate consumption detected under these conditions. In contrast, neutral pH microcosms employing control sediments were dominated by acetoclastic methanogens of the order Methanosarcinales and demonstrated high acetate consumption rates. The rate of acetate consumption and proportion of acetoclastic methanogens decreased in a linear fashion as the pH within cultures was increased, however hydrogen consumption rates remained stable up to pH 11.0. The data shown suggests hydrogenotrophic methanogenesis is the dominant methanogenic pathway at high pH which could have important consequences on gas pressures within a GDF.

Nuclear power is an important energy source that can compensate for carbon emissions from fossil fuel power plants. However, processing of radioactive waste from nuclear plants is a significant challenge. The current treatment prior to final geological disposal involves wet storage of spent fuel in designated ponds, and microbial colonisation of these ponds can complicate plant operation. To help identify the key microbes that colonise hydraulically interlinked spent fuel storage ponds at Sellafield, UK, a series of samples were collected and analysed using next generation (Illumina) sequencing. Samples were taken from the facility’s indoor Fuel Handling Plant (FHP) pond (feeding head tank, main and subponds), and also from the open-air First Generation Magnox Storage Pond (FGMSP). 16S rRNA gene sequencing revealed that the FHP is colonized mainly by Bacteria (99%), affiliated with species of Curvibacter, Rhodoferax, Sphingomonas and Roseococcus, in addition to the hydrogen-oxidising bacterium Hydrogenophaga. In contrast the open-air FGSMP pond contained species of Hydrogenophaga, Nevskia, and Roseococcus, and also photosynthetic cyanobacteria (Pseudanabaena). Biological function was also assessed by metagenomic sequencing and analyses. The most abundant genes were associated with carbohydrate and protein metabolism, cell wall and capsule synthesis, stress responses and respiration. Genes involved in respiration were also more abundant in the indoor pond microbiome, including genes underpinning hydrogen metabolism, whilst photosynthesis genes were more abundant in the open-air ponds. These datasets give valuable insight into the microbial communities inhabiting nuclear storage facilities, the metabolic processes that underpin their colonisation and can help inform appropriate control strategies.

The type VI secretion system (T6SS) is a bacterial nanomachine utilised by many Gram-negative bacteria, including Acinetobacter baumannii, to deliver toxic effectors for microbial warfare. These toxic effectors are often delivered via specific non-covalent interactions with cognate VgrG proteins, which form part of the T6SS tip. In A. baumannii, each vgrG gene is usually located in the same locus as two other genes, one encoding the cognate effector and one encoding an immunity protein that protects against self-intoxication. Bioinformatic searches of ninety seven A. baumannii genomes using a highly conserved domain found within the VgrG proteins, enabled the identification of more than 250 genes encoding putative effectors and, in most cases, the gene encoding the corresponding immunity protein. Phylogenetic analysis revealed that the predicted effectors clustered into 33 distinct groups, some of which contained predicted amidases, chitinases, lipases, nucleases and deaminases. Two effectors, Tse5Ab, containing no toxic domains and Tse6Ab, containing a Tox-GHH nuclease domain characteristic of nucleases, were chosen for functional analysis. The C-terminal region encoding the predicted toxic domain of each effector was cloned and expressed in E. coli. Expression of this region of Tse5Ab did not perturb E. coli growth. In contrast, expression of Tse6Ab was toxic but toxicity could be neutralised by the co-expression of the cognate immunity protein. However, Tse6Ab did not exhibit DNase activity and instead may function as an RNase. Further characterisation of the diverse A. baumannii T6SS effectors may lead to the identification of antibacterial molecules with novel activities.

The microbiota is crucial for gut homeostasis by aiding in nutrient uptake, and protecting against pathogens. Recent evidence suggests the benefits provided by the microbiota are not restricted to the intestine but also extend to systemic sites. Systemic benefits are hypothesized to be mediated by bacterial products, derived from the microbiota, such as peptidoglycan and lipopolysaccharide, entering the bloodstream and acting as novel signalling molecules at distal sites. However the precise way in which these microbial products enter the bloodstream remains largely unclear. Our data suggest bacterial products can cross the intestinal epithelium, and that routes across may vary between different bacterial products. Using in vitro and in vivo models, we find that host processing of cell wall molecules, by host antimicrobial lysozyme, promotes their translocation across the epithelium. Once they have traversed the intestinal barrier our preliminary data provide support that the liver plays a role in clearing bacterial products from the blood, as here we see a reservoir of peptidoglycan. This increased dissemination of cell wall molecules additionally enhances resistance to pulmonary infection. Therefore lysozyme treatment enhances bacterial product migration and increases host protection against systemic pathogens. Our work provides mechanistic insight into how the gut microbiota exerts systemic effects. Furthermore it provides a basis on which to launch further investigations, including examining the influence these aggregated cell wall proteins have over innate immune cells at these sites. Giving us greater insight into how the host controls microbial signalling and the benefits provided to our innate immune system.