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Chaotropes are compounds which cause the disordering, unfolding and denaturation of biological macromolecules. It is the chaotropicity of fermentation products that often acts as the primary limiting factor in ethanol and butanol fermentations. Since ethanol is mildly chaotropic at low concentrations, it prevents the growth of the producing microbes via its impacts on a variety of macromolecular systems and their functions. Kosmotropes have the opposite effect to chaotropes and we hypothesised that it might be possible to use these to mitigate chaotrope-induced inhibition of Saccharomyces cerevisiae growth. We also postulated that kosmotrope-mediated mitigation of chaotropicity is not quantitatively predictable. The chaotropes ethanol and urea, and compatible solutes glycerol and betaine (kosmotrope), and the highly kosmotropic salt ammonium sulphate all inhibited the growth rate of Saccharomyces cerevisiae in the concentration range 5-15%. They resulted in increased lag times, decreased maximum specific growth rates, and decreased final optical densities. Surprisingly, neither the stress protectants nor ammonium sulphate reduced the inhibition of growth caused by ethanol. Whereas, in some cases, compatible solutes and kosmotropes mitigated against the inhibitory effects of urea. However, this effect was not mathematically additive from the quantification of chao-/kosmotropicity of each individual compound. The potential effects of glycerol, betaine and/or ammonium sulphate may have been reduced or masked by the metabolic production of compatible solutes. It may nevertheless be that the addition of kosmotropes to fermentations which produce chaotropic products can enhance metabolic activity, growth rate, and/or product formation.

Just as the expansion in genome sequencing has revealed and permitted the exploitation of phylogenetic signals embedded in bacterial genomes, the application of metagenomics has begun to provide similar insights at the ecosystem level for microbial communities. However, little is known regarding this aspect of bacteriophage associated with microbial ecosystems, and if phage encode discernible habitat-associated signals diagnostic of underlying microbiomes. Here we demonstrate that individual phage can encode clear habitat-related 'ecogenomic signatures', based on relative representation of phage-encoded gene homologues in metagenomic data sets. Furthermore, we show the ecogenomic signature encoded by the gut-associated ɸB124-14 can be used to segregate metagenomes according to environmental origin, and distinguish 'contaminated' environmental metagenomes (subject to simulated in silico human faecal pollution) from uncontaminated data sets. This indicates phage-encoded ecological signals likely possess sufficient discriminatory power for use in biotechnological applications, such as development of microbial source tracking tools for monitoring water quality.

Faeces are comprised of a wide array of metabolites arising from the circulatory system as well as the human microbiome. A global metabolite analysis (metabolomics) of faecal extracts offers the potential to uncover new compounds which may be indicative of the onset of bowel diseases such as colorectal cancer (CRC). To date, faecal metabolomics is still in its infancy and the compounds of low abundance present in faecal extracts poorly characterised. In this study, extracts of faeces from healthy subjects were profiled using a sensitive nanoflow-nanospray LC-MS platform which resulted in highly repeatable peak retention times (

Proteus mirabilis forms extensive crystalline biofilms on indwelling urethral catheters that block urine flow and lead to serious clinical complications. The Bcr/CflA efflux system has previously been identified as important for development of P. mirabilis crystalline biofilms, highlighting the potential for efflux pump inhibitors (EPIs) to control catheter blockage. Here we evaluate the potential for drugs already used in human medicine (fluoxetine and thioridazine) to act as EPIs in P. mirabilis, and control crystalline biofilm formation. Both fluoxetine and thioridazine inhibited efflux in P. mirabilis, and molecular modelling predicted both drugs interact strongly with the biofilm-associated Bcr/CflA efflux system. Both EPIs were also found to significantly reduce the rate of P. mirabilis crystalline biofilm formation on catheters, and increase the time taken for catheters to block. Swimming and swarming motilies in P. mirabilis were also significantly reduced by both EPIs. The impact of these drugs on catheter biofilm formation by other uropathogens (Escherichia coli, Pseudomonas aeruginosa) was also explored, and thioridazine was shown to also inhibit biofilm formation in these species. Therefore, repurposing of existing drugs with EPI activity could be a promising approach to control catheter blockage, or biofilm formation on other medical devices.

A series of 3α,5-cycloandrostane analogues with a range of functionality (6α and 6β alcohols and ketone) at carbon 6 were tested in the endogenous lactonization pathway in Aspergillus tamarii KITA. This metabolic route converts progesterone to testololactone in high yield through a four step enzymatic pathway. To date, no studies have looked at the effect of steroids devoid of polar functionality at carbon 3 and their subsequent metabolic fate by fungi which contain Baeyer-Villiger monooxygenases. Incubation of all of the cycloandrostane analogues resulted in lactonization of ring-D irrespective of C-6 stereochemistry or absence of C-3 functionality. Presence of 6β-hydroxy group and the C-17 ketone was required in order for these analogues to undergo hydroxylation at C-15β position. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data.

In order to better control nosocomial infections, and facilitate the most prudent and effective use of antibiotics, improved strategies for the rapid detection and identification of problematic bacterial pathogens are required. DNA aptamers have much potential in the development of diagnostic assays and biosensors to address this important healthcare need, but further development of aptamers targeting common pathogens, and the strategies used to obtain specific aptamers are required. Here we demonstrate the application of a quantitative PCR (qPCR) controlled Cell-SELEX process, coupled with downstream secondary-conformation-based aptamer profiling. We used this approach to identify and select DNA aptamers targeted against uropathogenic Escherichia coli, for which specific aptamers are currently lacking, despite the prevalence of these infections. The use of qPCR to monitor the Cell-SELEX process permitted a minimal number of SELEX cycles to be employed, as well as the cycle-by-cycle optimisation of standard PCR amplification of recovered aptamer pools at each round. Identification of useful aptamer candidates was also facilitated by profiling of secondary conformations and selection based on putative aptamer secondary structure. One aptamer selected this way (designated EcA5-27), displaying a guanine-quadruplex sequence motif, was shown to have high affinity and specificity for target cells, and the potential to discriminate between distinct strains of E. coli, highlighting the possibility for development of aptamers selectively recognising pathogenic strains. Overall, the identified aptamers hold much potential for the development of rapid diagnostic assays for nosocomial urinary tract infections caused by E. coli.

Proteus mirabilis is a common cause of catheter-associated urinary tract infections and frequently leads to blockage of catheters due to crystalline biofilm formation. Scanning electron microscopy (SEM) has proven to be a valuable tool in the study of these unusual biofilms, but entails laborious sample preparation that can introduce artefacts, undermining the investigation of biofilm development. In contrast, environmental scanning electron microscopy (ESEM) permits imaging of unprocessed, fully hydrated samples, which may provide much insight into the development of P. mirabilis biofilms. Here, we evaluate the utility of ESEM for the study of P. mirabilis crystalline biofilms in situ, on urinary catheters. In doing so, we compare this to commonly used conventional SEM approaches for sample preparation and imaging. Overall, ESEM provided excellent resolution of biofilms formed on urinary catheters and revealed structures not observed in standard SEM imaging or previously described in other studies of these biofilms. In addition, we show that energy-dispersive X-ray spectroscopy (EDS) may be employed in conjunction with ESEM to provide information regarding the elemental composition of crystalline structures and demonstrate the potential for ESEM in combination with EDS to constitute a useful tool in exploring the mechanisms underpinning crystalline biofilm formation.

Four isomers of 5α-androstan-3,17-diol have been transformed by the filamentous fungus Aspergillus tamarii, an organism which has the ability to convert progesterone to testololactone in high yield through an endogenous four step enzymatic pathway. The only diol handled within the lactonization pathway was 5α-androstan-3α,17β-diol which, uniquely underwent oxidation of the 17β-alcohol to the 17-ketone prior to its Baeyer-Villiger oxidation and the subsequent production of 3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-one. This demonstrated highly specific stereochemical requirements of the 17β-hydroxysteroid dehydrogenase for oxidation of this specific steroidal diol to occur. In contrast, the other three diols were transformed within the hydroxylation pathway resulting in functionalization at C-11β. Only 5α-androstan-3β,17α-diol could bind to the hydroxylase in multiple binding modes undergoing monohydroxylation in 6β and 7β positions. Evidence from this study has indicated that hydroxylation of saturated steroidal lactones may occur following binding of ring-D in its open form in which an α-alcohol is generated with close spatial parity to the C-17α hydroxyl position. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR and DEPT analysis and further characterized using infra-red, elemental analysis and accurate mass measurement.

Aspergillus tamarii contains an endogenous lactonization pathway which can transform progesterone to testololactone in high yield through a sequential four step enzymatic pathway. In this pathway testosterone is formed which primarily undergoes oxidation of the C-17beta-alcohol to a C-17 ketone but, can also enter a minor hydroxylation pathway where 11beta-hydroxytestosterone is produced. It was recently demonstrated that this hydroxylase could monohydroxylate 3beta-hydroxy substituted saturated steroidal lactones in all four possible binding orientations (normal, reverse, inverted normal, inverted reverse) on rings B and C of the steroid nucleus. It was therefore of interest to determine the fate of a series of 3alpha-substituted steroidal analogues to determine stereochemical effect on transformation. Hydroxylation on the central rings was found to be restricted to the 11beta-position (normal binding), indicating that the 3alpha-stereochemistry removes freedom of binding orientation within the hydroxylase. The only other hydroxylation observed was at the 1beta-position. Interestingly the presence of this functional group did not prevent lactonization of the C-17 ketone. In contrast the presence of the 11beta-hydroxyl completely inhibited Baeyer-Villiger oxidation, a result which again demonstrates that single functional groups can exert significant control over metabolic handling of steroids in this organism. This may also explain why lactonization of 11beta-hydroxytestosterone does not occur. Lactonization of the C-17 ketone was not significantly affected by the 3alpha-alcohol with significant yields achieved (53%). Interestingly a time course experiment demonstrated that the presence of the 3alpha-acetate inhibited the Baeyer-Villiger monooxygenase with its activity being observed 24h later than non-acetate containing analogues. Apart from oxidative transformations observed a minor reductive pathway was revealed with the C-17 ketone being reduced to a C-17beta-alcohol for the first time in this organism.

A series of steroids (progesterone, testosterone acetate, 17beta-acetoxy-5 alpha-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17beta side-chain. This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17beta-acetoxy-5 alpha-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17beta-alcohol of testosterone. All steroidal metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.

The fungus Aspergillus tamarii metabolizes progesterone to testololactone in high yield through a sequential four step enzymatic pathway which, has demonstrated flexibility in handling a range of steroidal probes. These substrates have revealed that subtle changes in the molecular structure of the steroid lead to significant changes in route of metabolism. It was therefore of interest to determine the metabolism of a range of 5-ene containing steroidal substrates. Remarkably the primary route of 5-ene steroid metabolism involved a 3beta-hydroxy-steroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD/isomerase) enzyme(s), generating 3-one-4-ene functionality and identified for the first time in a fungus with the ability to handle both dehydroepiansdrosterone (DHEA) as well as C-17 side-chain containing compounds such as pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one. Uniquely in all the steroids tested, 3beta-HSD/isomerase activity only occurred following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibited 3beta-HSD/isomerase activity and of the substrates tested, was only observed with DHEA and its 13alpha-methyl analogue. In contrast to previous studies of fungi with 3beta-HSD/isomerase activity DHEA could also enter a minor hydroxylation pathway. Pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one were metabolized solely through the putative 3beta-HSD/isomerase pathway, indicating that a 17beta-methyl ketone functionality inhibits allylic oxidation at C-7. The presence of the 3beta-HSD/isomerase in A. tamarii and the transformation results obtained in this study highlight an important potential role that fungi may have in the generation of environmental androgens.

This paper demonstrates for the first time transformation of a series of steroids (progesterone, androst-4-en-3,17-dione, testosterone, pregnenolone and dehydroepiandrosterone) by the thermophilic fungus Rhizomucor tauricus. All transformations were found to be oxidative (monohydroxylation and dihydroxylation) with allylic hydroxylation the predominant route of attack functionalizing the steroidal skeleta. Timed experiments demonstrated that dihydroxylation of progesterone, androst-4-en-3,17-dione and pregnenolone all initiated with hydroxylation on ring-B followed by attack on ring-C. Similar patterns of steroidal transformation to those observed with R. tauricus have been observed with some species of thermophilic Bacilli and mesophilic fungi. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data. The application of thermophilic fungi to steroid transformation may represent a potentially rich source for the generation of new steroidal compounds as well as for uncovering inter and intraspecies similarities and differences in steroid metabolism.

Accumulating evidence shows indigenous gut microbes can interact with the human host through modulation of serotonin (5-HT) signaling. Here we investigate the impact of the probiotic Escherichia coli Nissle 1917 (EcN) on 5-HT signalling in gut tissues. Ex-vivo mouse ileal tissue sections were treated with either EcN or the human gut commensal MG1655 and effects on levels of 5-HT, precursors and metabolites, were evaluated using amperometry and high performance liquid chromatography with electrochemical detection (HPLC-EC). Exposure of tissue to EcN cells, but not MG1655 cells, was found to increase levels of extra-cellular 5-HT. These effects were not observed when tissues were treated with cell-free supernatant from bacterial cultures. In contrast, when supernatant recovered from untreated ileal tissue was pre-incubated with EcN, the derivative cell-free supernatant was able to elevate 5-HT overflow when used to treat fresh ileal tissue. Measurement of 5-HT precursors and metabolites indicated EcN also increases intracellular 5-HTP and reduces 5-HIAA. The former pointed to modulation of tryptophan hydroxylase-1 to enhance 5-HT synthesis, while the latter indicates an impact on clearance into enterocytes through SERT. Taken together, these findings show EcN is able to enhance 5-HT bioavailability in ileal tissues through interaction with compounds secreted from host tissues.

Escherichia coli Nissle 1917 (EcN) is among the best characterised probiotics, with a proven clinical impact in a range of conditions. Despite this, the mechanisms underlying these "probiotic effects" are not clearly defined. Here we applied random transposon mutagenesis to identify genes relevant to the interaction of EcN with intestinal epithelial cells. This demonstrated mutants disrupted in the kfiB gene, of the K5 capsule biosynthesis cluster, to be significantly enhanced in attachment to Caco-2 cells. However, this phenotype was distinct from that previously reported for EcN K5 deficient mutants (kfiC null mutants), prompting us to explore further the role of kfiB in EcN:Caco-2 interaction. Isogenic mutants with deletions in kfiB (EcNΔkfiB), or the more extensively characterised K5 capsule biosynthesis gene kfiC (EcNΔkfiC), were both shown to be capsule deficient, but displayed divergent phenotypes with regard to impact on Caco-2 cells. Compared with EcNΔkfiC and the EcN wild-type, EcNΔkfiB exhibited significantly greater attachment to Caco-2 cells, as well as apoptotic and cytotoxic effects. In contrast, EcNΔkfiC was comparable to the wild-type in these assays, but was shown to induce significantly greater COX-2 expression in Caco-2 cells. Distinct differences were also apparent in the pervading cell morphology and cellular aggregation between mutants. Overall, these observations reinforce the importance of the EcN K5 capsule in host-EcN interactions, but demonstrate that loss of distinct genes in the K5 pathway can modulate the impact of EcN on epithelial cell health.

Bacteriophage associated with the human gut microbiome are likely to have an important impact on community structure and function, and provide a wealth of biotechnological opportunities. Despite this, knowledge of the ecology and composition of bacteriophage in the gut bacterial community remains poor, with few well characterized gut-associated phage genomes currently available. Here we describe the identification and in-depth (meta)genomic, proteomic, and ecological analysis of a human gut-specific bacteriophage (designated φB124-14). In doing so we illuminate a fraction of the biological dark matter extant in this ecosystem and its surrounding eco-genomic landscape, identifying a novel and uncharted bacteriophage gene-space in this community. φB124-14 infects only a subset of closely related gut-associated Bacteroides fragilis strains, and the circular genome encodes functions previously found to be rare in viral genomes and human gut viral metagenome sequences, including those which potentially confer advantages upon phage and/or host bacteria. Comparative genomic analyses revealed φB124-14 is most closely related to φB40-8, the only other publically available Bacteroides sp. phage genome, whilst comparative metagenomic analysis of both phage failed to identify any homologous sequences in 136 non-human gut metagenomic datasets searched, supporting the human gut-specific nature of this phage. Moreover, a potential geographic variation in the carriage of these and related phage was revealed by analysis of their distribution and prevalence within 151 human gut microbiomes and viromes from Europe, America and Japan. Finally, ecological profiling of φB124-14 and φB40-8, using both gene-centric alignment-driven phylogenetic analyses, as well as alignment-free gene-independent approaches was undertaken. This not only verified the human gut-specific nature of both phage, but also indicated that these phage populate a distinct and unexplored ecological landscape within the human gut microbiome.

Corynespora cassiicola has a unique but unexplored ability amongst fungi, in that it can hydroxylate 17α-hydroxyprogesterone at the highly hindered C-8 position of the steroid nucleus. In order to gain greater understanding of the mechanistic basis and capability of the 8β-hydroxylase we have transformed a range of structurally diverse androgens and progestogens with this organism. This has revealed that both steroid types can be hydroxylated at the 8β-position. The collective data has demonstrated the first time that 8β-hydroxylation occurs through inverted binding within a 9α-hydroxylase of the fungus. In the case of the progestogens, for this to occur, the presence of 17α-oxygen functionality (alcohol or epoxide) was essential. Remarkably monohydroxylation of 17α-hydroxyprogesterone at carbons 8β and 15β has strongly indicated that the responsible hydroxylase has 2 different binding sites for the ring-A ketone. Unusually, with one exception, all hydroxylation occurred at axial protons and in the case of the progestogens, all above the plane of the ring system. In general all maximally oxidised metabolites contained four oxygen atoms. The importance of these findings in relation to 8β-hydroxylation of these steroids is discussed.