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It is well known that loss of aerobic respiration in Gram-negative bacteria can diminish the efficacy of a variety of bactericidal antibiotics, which has lead to subsequent demonstrations that the formation of reactive oxygen species (ROS) and the proton motive force (PMF) can both play a role in antibiotic toxicity. The susceptibility of Gram-negative bacteria to aminoglycoside antibiotics, particularly gentamicin, has previously been linked to both the production of ROS and the rate of antibiotic uptake that is mediated by the PMF, although the relative contributions of ROS and PMF to aminoglycoside toxicity has remained poorly understood. Herein, gentamicin was shown to elicit a very modest increase in ROS levels in an aerobically grown Escherichia coli clinical isolate. The well-characterised uncoupler 2,4-dinitrophenol (DNP) was used to disrupt the PMF, which resulted in a significant decrease in gentamicin lethality towards E. coli. DNP did not significantly alter respiratory oxygen consumption, supporting the hypothesis that this uncoupler does not increase ROS production via elevated respiratory oxidase activity. These observations support the hypothesis that maintenance of PMF rather than induction of ROS production underpins the mechanism for how the respiratory chain potentiates the toxicity of aminoglycosides. This was further supported by the demonstration that the uncoupler DNP elicits a dramatic decrease in gentamicin lethality under anaerobic conditions. Together, these data strongly suggest that maintenance of the PMF is the dominant mechanism for the respiratory chain in potentiating the toxic effects of aminoglycosides. Supplementary Information The online version contains supplementary material available at 10.1007/s00203-021-02710-y.

The development and advent of mutagenesis tools for solventogenic clostridial species in recent years has allowed for the increased refinement of industrially relevant strains. In this study we have utilised CLEAVE™, a CRISPR/Cas genome editing system developed by Green Biologics Ltd., to engineer a strain of Clostridium saccharoperbutylacetonicum N1-4(HMT) with potentially useful solvents titres and energy metabolism. As one of two enzymes responsible for the conversion of glyceraldehyde-3-phosphate (GAP) to 3-phosphoglyceric acid in glycolysis, it was hypothesised that deletion of gapN would increase ATP and NADH production that could in turn improve solvent production. Herein, whole genome sequencing has been used to evaluate CLEAVE™ and the successful knockout of gapN, demonstrating a clean knockout with no other detectable variations from the wild type sequence. Elevated solvent levels were detected during the first 24 h of batch fermentation, indicating an earlier shift to solventogenesis. A 2.4-fold increase in ATP concentration was observed, and quantitation of NAD(P)H derivatives revealed a more reducing cytoplasm for the gapN strain. These findings expand our understanding of clostridium carbon metabolism and report a new approach to optimising biofuel production.

Naegleria fowleri is both a pathogenic and a free-living microbial eukaryote, responsible for the development of primary amoebic meningoencephalitis (PAM) in humans. PAM is a rapid, severe and fatal underestimated infectious disease, which has been reported in countries with warmer climates. The major drawbacks with PAM are the lack of effective therapies and delay in diagnosis. The current frontline treatment presents a low rate of recovery (5%) and severe adverse effects. For example, many drug candidates lack efficacy, since they do not effectively cross the blood-brain-barrier. Consequently, more effective drugs are urgently needed. Herein, we report a new in vitro method suitable for medium- and high-throughput drug discovery assays, using the closely related Naegleria gruberi as a model. We have subsequently used this method to screen a library of 1175 Food and Drug Administration-approved drugs. As a result, we present three drugs (camptothecin, pyrimethamine, and terbinafine) that can be repurposed, and are anticipated to readily cross the blood-brain-barrier with activity against Naegleria species in therapeutically achievable concentrations. Successively, we integrated several in vitro assays that resulted in identifying fast-acting and high amoebicidal drugs. In conclusion, we present a new approach for the identification of anti-Naegleria drugs along with three potential drug candidates for further development for the treatment of PAM., Graphical abstract Image 1, Highlights • Novel in vitro approach for the identification of anti-Naegleria drugs • Modern drug discovery strategy by using a phenotypic screening against Naegleria • In vitro evaluation of 1175 FDA approved drugs against Naegleria • Camptothecin, pyrimethamine and terbinafine are promising drugs for the PAM • Higher activity, improved ADMET profile and bioavailability than amphotericin B

When working with anaerobic bacteria it is important to have the capability to perform parallel bioreactor growth experiments that are both controllable and reproducible, although capital and consumables costs for commercially available systems are often prohibitively high. Hence, a three-vessel parallel bioreactor system was designed and constructed that has the capabilities for batch and fed batch processes and can also be set up for continuous culture at a fraction of the cost of commercial systems. This system carries over many of the same functionalities of those systems with a higher price point of entry, including in-line monitoring of temperature, pH, and redox poise. To validate the performance of this system Clostridium saccharoperbutylacetonicum was grown under conditions that promote ABE fermentation, an established industrial process used to produce the solvents acetone, butanol and ethanol. Measurements of cell density, pH, and redox poise all confirmed reproducible culture conditions for these parallel vessels, and solvent quantitation via GCMS verified consistent metabolic activities for the separate cultures. In future, this system will be of interest to researchers that require high performance parallel fermentation platforms but where commercial systems are not accessible.

Cryptosporidium spp. are microbial parasites that infect the gastrointestinal tract of humans and many animals, causing cryptosporidiosis, a disease characterised by acute watery diarrhoea. In dairy farms, C. parvum is the most common species in calves, leading to high mortality rate, stunted growth, and consequently high economic losses. Trade between farms and breeding centres is a major risk factor in the spread of such parasites, posing a threat to other farms worldwide as well as to human health. This problem is aggravated by the lack of good breeding practices, efficient detection tools, and lack of effective anti-cryptosporidial drugs. To address cryptosporidiosis in dairy farms, we have established the ‘Health For Dairy Cows (H4DC)’ consortium in order to tackle some of the aforementioned issues. Herein, we will present preliminary data from a 3-step strategy: 1)Dissemination of pilot farms in France, Belgium, The Netherlands and England. This collaboration will serve to test the effect of new husbandry practices in the occurrence of Cryptosporidium, which will aim to decrease Cryptosporidium incidence and ultimately decrease the economic burden of cryptosporidiosis. 2)These pilot farms will later be used as testing-grounds of the low-cost and easy-to-use in-situ C. parvum detection tool that will be developed during this project. 3)Development of a cell-based drug-screening system which will be used to screen various drugs and compounds for anti-Cryptosporidium activity. Finally, data from these findings will be used to establish model and strategies in order to transfer the developed technologies to both farmers and biotech/pharmaceutical companies.

The spread of multidrug-resistance in Gram-negative bacterial pathogens presents a major clinical challenge, and new approaches are required to combat these organisms. Nitric oxide (NO) is a well-known antimicrobial that is produced by the immune system in response to infection, and numerous studies have demonstrated that NO is a respiratory inhibitor with both bacteriostatic and bactericidal properties. However, given that loss of aerobic respiratory complexes is known to diminish antibiotic efficacy, it was hypothesised that the potent respiratory inhibitor NO would elicit similar effects. Indeed, the current work demonstrates that pre-exposure to NO-releasers elicits a > tenfold increase in IC50 for gentamicin against pathogenic E. coli (i.e. a huge decrease in lethality). It was therefore hypothesised that hyper-sensitivity to NO may have arisen in bacterial pathogens and that this trait could promote the acquisition of antibiotic-resistance mechanisms through enabling cells to persist in the presence of toxic levels of antibiotic. To test this hypothesis, genomics and microbiological approaches were used to screen a collection of E. coli clinical isolates for antibiotic susceptibility and NO tolerance, although the data did not support a correlation between increased carriage of antibiotic resistance genes and NO tolerance. However, the current work has important implications for how antibiotic susceptibility might be measured in future (i.e. ± NO) and underlines the evolutionary advantage for bacterial pathogens to maintain tolerance to toxic levels of NO.

Fundamental aspects of outer membrane vesicle (OMV) biogenesis and the engineering of producer strains have been major research foci for many in recent years. The focus of this study was OMV production in a variety of Escherichia coli strains including wild type (WT) (K12 and BW25113), mutants (from the Keio collection) and proprietary [BL21 and BL21 (DE3)] strains. The present study investigated the proteome and prospective mechanism that underpinned the key finding that the dominant protein present in E. coli K-12 WT OMVs was fimbrial protein monomer (FimA) (a polymerizable protein which is the key structural monomer from which Type 1 fimbriae are made). However, mutations in genes involved in fimbriae biosynthesis (ΔfimA, B, C, and F) resulted in the packaging of flagella protein monomer (FliC) (the major structural protein of flagella) into OMVs instead of FimA. Other mutations (ΔfimE, G, H, I, and ΔlrhA–a transcriptional regulator of fimbriation and flagella biosynthesis) lead to the packaging of both FimA and Flagellin into the OMVs. In the majority of instances shown within this research, the production of OMVs is considered in K-12 WT strains where structural appendages including fimbriae or flagella are temporally co-expressed throughout the growth curve as shown previously in the literature. The hypothesis, proposed and supported within the present paper, is that the vesicular packaging of the major FimA is reciprocally regulated with the major FliC in E. coli K-12 OMVs but this is abrogated in a range of mutated, non-WT E. coli strains. We also demonstrate, that a protein of interest (GFP) can be targeted to OMVs in an E. coli K-12 strain by protein fusion with FimA and that this causes normal packaging to be disrupted. The findings and underlying implications for host interactions and use in biotechnology are discussed.

The scsABCD (suppressor of copper sensitivity) locus of Salmonella encodes four proteins that resemble the disulphide-folding machinery of other bacteria. Previous work has shown that Salmonella encounters toxic levels of copper during infection and the Scs system provides protection against this copper-mediated toxicity. The current work reports that expression of the soluble periplasmic protein StScsC is induced by copper, and that intramacrophage survival in the presence of copper is diminished by the loss of StScsC. Using a combination of genetic and proteomics approaches, the abundance of various cysteine-containing periplasmic proteins were found to be elevated by StScsC in the Salmonella periplasm, implicating StScsC in the disulphide folding of superoxide dismutases and proteins involved in amino acid sensing and import. Co-purification and mass spectrometry approaches confirmed that the arginine-sensing periplasmic protein ArtI associates with StScsC via a disulphide interaction, and purified ArtI was shown to alter the thiol redox state of purified StScsC. This work reports the first demonstration of a redox partner for the Scs system of Salmonella, and provides insights into how this bacterial pathogen responds to copper stress during infection.

A detailed assessment of embryo development would assist biologists with selecting the most suitable embryos for transfer leading to higher pregnancy rates. Currently, only low resolution microscopy is employed to perform this assessment. Although this method delivers some information on the embryo surface morphology, no specific details are shown related to its inner structure. Using a Master-Slave Swept-Source Optical Coherence Tomography (SS-OCT), images of bovine embryos from day 7 after fertilization were collected from different depths. The dynamic changes inside the embryos were examined, in detail and in real-time from several depths. To prove our ability to characterize the morphology, a single embryo was imaged over 26 hours. The embryo was deprived of its life support environment, leading to its death. Over this period, clear morphological changes were observed.

his paper presents a theoretical study on a photonic crystal fiber (PCF) surface plasmon resonance biosensor. The proposed PCF sensor introduces the concept of simultaneous detection with H E11x and H E11x modes, which opens up some possibilities for multianalyte/multichannel sensing. Analysis was performed which considered the operation of the sensor in both amplitude and wavelength interrogation modes. Typical sensor resolutions of 4×10-5 RIU and 8×10-5 RIU with respect to H E11x and H E11y, respectively, are reported for the amplitude interrogation mode, while resolutions of 5 × 10-5 RIU and 6×10-5 RIU are reported for H E11x and H E11y, respectively, for the wavelength interrogation mode.

A first attempt to attenuate the quorum sensing (QS) of a marine heterotroph microorganism, Vibrio fischeri , using signal molecule-sequestering polymers (SSPs) is presented. A set of rationally designed polymers with affinity toward a signal molecule of V. fischeri , N-(beta-ketocaproyl)-l-homoserine lactone (3-oxo-C6-AHL) was produced. It is reported that computationally designed polymers could sequester a signal molecule of V. fischeri and prevent QS-controlled phenotypes (in this case, bioluminescence) from being up-regulated. It was proven that the attenuation of bioluminescence of V. fischeri was due to sequestration of the signal molecule by specific polymers and not due to the toxicity of polymer or nonspecific depletion of nutrients. The ability to disrupt the bacterial communication using easy to synthesize and chemically inert polymers could provide a new concept for the development of pharmaceuticals and susceptible device coatings such as catheters.

A bacterium classified as Achromobacter xylosoxidans strain IR08 by phenotypic typing coupled with 16S rRNA gene analysis was isolated from a soil contaminated with electrical transformer fluid for over sixty years using Aroclor 1221 as an enrichment substrate. The substrate utilization profiles revealed that IR08 could grow on all three monochlorobiphenyls (CBs), 2,4'- and 4,4'-dichlorobiphenyl as well as 2-chlorobenzoate (2-CBA), 3-CBA, 4-CBA, and 2,3-dichlorobenzoate. Unusually, growth was poorly sustained on biphenyl and benzoate. In growth experiments, IR08 degraded all CBs (0.27 mmol/L) in less than 96 h with concomitant stoichiometric release of inorganic chloride and growth yields were 2-3 times higher than those observed on biphenyl. In contrast to most of the chlorobiphenyl-degrading strains described in the literature, which are reported to form CBA, no metabolite was identified in the culture broth by HPLC analysis. When co-incubated with respective CBs and biphenyl, strain IR08 preferentially utilized the chlorinated analogues in less than 96 h while it took another 264 h before 90% of the initially supplied biphenyl could be degraded. The promotion of co-metabolic transformation of halogenated substrates by the inclusion of their non-halogenated derivatives may not therefore, result in universal benefits.

Achromobacter xylosoxidans strain IR08 was isolated from soil contaminated with electrical transformer fluid by enrichment culture containing Aroclor 1221 as the sole carbon source. This strain was found to grow on all monochlorobiphenyls, 4,4'-dichlorobiphenyl (4,4'-diCB) and a wide range of other xenobiotic compounds. During growth on 4,4'-diCB, a near-stoichiometric amount of chloride was excreted into the culture fluid in less than 5 days and growth yield was more than twice that achieved on biphenyl. The production of 4-CBA or chlorocatechol as a metabolite was not observed. Quite unusually, coincubation of strain IR08 with 4,4'-diCB and biphenyl at relatively equal concentrations showed preferential utilization of the chlorobiphenyl: 4,4'-diCB was mineralized in less than 5 days concomitant with stoichiometric release of chloride, while biphenyl was poorly degraded. Growth on 2.5 mM CBA also resulted in complete disappearance of the substrate, however, inorganic chloride recovered from the culture broth was less than 65%. The isolation of a dichlorobiphenyl-mineralizing rather than transformation strain such as IR08 is an important advance in an effort to develop effective bioremediation strategy for polychlorinated biphenyl-contaminated soil.

In the present study, we compared the two-dimensional polypeptide maps of proteins extracts from Fusarium solani and Fusarium oxysporum, two telluric fungi previously isolated from contaminated soil and shown to have good and poor benzo [ a ] pyrene degrading abilities, respectively. Their protein compositions were analyzed when grown in the presence and absence of benzo [ a ] pyrene. We provide evidence that benzo [ a ] pyrene induced specific protein synthesis in F. solani. The induced polypeptides were mainly observed in the acidic part of gels at 50 , 45 and 30 kDa. In contrast, no additional polypeptides were detected in F. oxysporum when grown in the presence of benzo [ a ] pyrene. The polypeptides sequence of 45 and 30 kDa polypeptides have high similarity (44% and 67%, respectively) with a porphobilinogen deaminase (EC 2.5.1.61), a key enzyme in the heme and cytochrome P450 biosynthesis pathway.

Accumulation and elimination of polycyclic aromatic hydrocarbons (PAHs) were studied in the fungus Fusarium solani. When the fungus was grown on a synthetic medium containing benzo[a]pyrene, hyphae of F. solani contained numerous lipid vesicles which could be stained by the lipid-specific dyes: Sudan III and Rhodamine B. The fluorescence produced by Rhodamine B and PAH benzo[a]pyrene were at the same locations in the fungal hyphae, indicating that F. solani stored PAH in pre-existing lipid vesicles. A passive temperature-independent process is involved in the benzo[a]pyrene uptake and storage. Sodium azide, a cytochrome c oxidation inhibitor, and the two cytoskeleton inhibitors colchicine and cytochalasin did not prevent the transport and accumulation of PAH in lipid vesicles of F. solani hyphae. F. solani degraded a large range of PAHs at different rates. PAH intracellular storage in lipid vesicles was not necessarily accompanied by degradation and was common to numerous other fungi.

Biocatalysis inherently offers the prospect of clean industrial processing and has become an accepted technology throughout most sectors. The convergence of biology and chemistry has enabled a plethora of industrial opportunities to be targeted, while discoveries in biodiversity and the impact of molecular biology and computational science are extending the range of natural and engineered biocatalysts that can be customised for clean industrial requirements.

Aromatic and heterocyclic aldehydes may be produced by the mandelate pathway of Pseudomonas putida ATCC 12633 via the biotransformation of benzoyl formate and substrate analogues. Under optimised biotransformation conditions (37 °C, pH 5.4) and with benzoyl formate as a substrate, benzaldehyde may be accumulated with yields above 85%. Benzaldehyde is toxic to P. putida ATCC 12633; levels above 0.5 g/l (5 mM) reduce the biotransformation activity. Total activity loss occurs at an aldehyde concentration of 2.1 g/l (20 mM). To overcome this limitation, the rapid removal of the aldehyde is desirable via in situ product removal. The biotransformation of benzoyl formate (working volume 1 l) without in situ product removal accumulates 2.1 g/l benzaldehyde. Benzaldehyde removal by gas stripping produces a total of 3.5 g/l before inhibition. However, the most efficient method is solid-phase adsorption using activated charcoal as the sorbant, this allows the production of over 4.1 g/l benzaldehyde. Addition of bisulphite as a complexing agent causes inhibition of the biotransformation and bisulphite is therefore is not suitable for in situ product removal.

Aromatic aldehydes may be produced by Pseudomonas putida ATCC 12633 via biotransformation of benzoylformate and substrate analogues using the mandelic acid pathway. A reduction in pH to 5.4 causes the partial inactivation of the benzaldehyde dehydrogenase isoenzymes within this pathway, thereby allowing the accumulation of the aldehyde. The reduction in dehydrogenase activity with pH is a general phenomenon affecting all dehydrogenases within the cell and is not restricted to the benzaldehyde dehydrogenases of the mandelate pathway. Both benzaldehyde and thiophene-2-carboxaldehyde have been produced with yields of over 85%.

Chlorobenzoates (CBA) arise as intermediates during the degradation of polychlorinated biphenyls (PCBs) and some chlorinated herbicides. Since PCBs were produced as complex mixtures, a range of mono-, di-, and possibly trichloro-substituted benzoates would be formed. Chlorobenzoate degradation has been proposed to be one of the rate-limiting steps in the overall PCB-degradation process. Three hybrid bacteria constructed to have the ability to completely mineralise 2-, 3-, or 4-monochlorobiphenyl respectively, have been studied to establish the range of mono- and diCBAs that can be utilised. The three strains were able to mineralise one or more of the following CBAs: 2-, 3-, and 4-monochlorobenzoate and 3,5-dichlorobenzoate. No utilisation of 2,3-, 2,5-, 2,6-, or 3,4-diCBA was observed, and only a low concentration (0.11 mM) of 2,4-diCBA was mineralised. When the strain with the widest substrate range (Burkholderia cepacia JHR22) was simultaneously supplied with two CBAs, one that it could utilise plus one that it was unable to utilise, inhibitory effects were observed. The utilisation of 2-CBA (2.5 mM) by this strain was inhibited by 2,3-CBA (200 microM) and 3,4-CBA (50 microM). Although 2,5-cba and 2,6-cba were not utilised as carbon sources by strain jhr22, they did not inhibit 2-cba utilisation at the concentrations studied, whereas 2,4-cba was co-metabolised with 2-cba. The utilisation of 2-, 3-, and 4-chlorobiphenyl by strain JHR22 was also inhibited by the presence of 2,3- or 3,4-diCBA. We conclude that the effect of the formation of toxic intermediates is an important consideration when designing remediation strategies.

Titanocene dichloride, in conjunction with sodium borohydride and pyridine, was shown to dechlorinate Aroclor 1242 to give biphenyl. This was observed as a shift in the congener profile from more heavily chlorinated congeners to the lower chlorinated congeners over the time course of the reaction. The dechlorination process resulted in a transient increase in concentration of the monochlorinated biphenyls, particularly 3-chlorobiphenyl. Dechlorination in the presence of alternative amines or in PCB-contaminated soils proceeded at reduced rates and yielded mono- and dichlorobiphenyls in the final product. Since three hybrid bacteria had previously been produced for the mineralisation of biphenyl and lower chlorinated biphenyls, chemical dechlorination of the PCB mixture (Aroclor 1242) or of contaminated soils produces a potential feed for an aerobic microbial process for complete chlorobiphenyl mineralisation.

Surface plasmon resonance (SPR) sensors provide high sensitivity without the use of molecular labels (Homola, Yee, and Gauglitz 1999). They been widely used in the analysis of biomolecular interactions (BIA) and detection of chemical and biological analytes (Homola, Yee, and Gauglitz 1999), where they provide benefits of real-time, sensitive and label-free technology. They have also been used for the detection of various chemical and biological compounds in areas such as environmental protection, food safety and medical diagnostics (Mouvet et al. 1997; Nooke et al. 2010).

Factors affecting the enzymatic intramolecular lactonization of 16-hydroxyhexadecanoic acid are presented. A screen of 33 enzymes, predominantly lipases, showed that only a proportion were able to catalyze the synthetic reaction in a microaqueous environment. Certain of these enzymes showed no observable formation of the oligolactones by the favored intermolecular esterification reaction, as reported by other workers. Indeed, the immobilized lipase from Candida antarctica showed formation of the intramolecular monolactone product, hexadecanolide, which could be produced continuously for 55 hs. Many variables were considered, including the choice of enzyme (source and preformulation), substrate concentration, product concentration, solvent, temperature, pH, and water content of the system. The effect of these variables on hexadecanolide yield was analyzed statistically using a Plackett-Burman matrix. Significant effects were only demonstrated for the product hexadecanolide (stimulatory) and the substrate 16-hydroxyhexadecanoic acid (inhibitory).

When colonising host-niches or non-animated medical devices, individual cells of the fungal pathogen Candida albicans expand into significant biomasses. Here we show that within such biomasses, fungal metabolically generated CO2 acts as a communication molecule promoting the switch from yeast to filamentous growth essential for C. albicans pathology. We find that CO2-mediated intra-colony signalling involves the adenylyl cyclase protein (Cyr1p), a multi-sensor recently found to coordinate fungal responses to serum and bacterial peptidoglycan. We further identify Lys 1373 as essential for CO2/bicarbonate regulation of Cyr1p. Disruption of the CO2/bicarbonate receptor-site interferes selectively with C. albicans filamentation within fungal biomasses. Comparisons between the Drosophila melanogaster infection model and the mouse model of disseminated candidiasis, suggest that metabolic CO2 sensing may be important for initial colonisation and epithelial invasion. Our results reveal the existence of a gaseous Candida signalling pathway and its molecular mechanism and provide insights into an evolutionary conserved CO2-signalling system., Author Summary Pathogenic microorganisms can produce a variety of secondary metabolites and signalling molecules which can affect the host, or provide them with a selective advantage against competing commensal organisms. We demonstrate that gaseous, metabolically generated CO2 can serve as a signalling molecule to enhance the organism's virulence during infection establishment by using the fungal pathogen Candida albicans as a model. Furthermore, we identified a CO2 receptor site within the catalytic domain of the soluble adenylyl cyclase, Cyr1p, which is critical for CO2 sensing and hence virulence of the organism. CO2 sensing is conserved in a variety of pathogenic species, and increased levels have been shown to suppress the host's immune system. Thus, CO2 sensing may represent a mechanism to enhance C. albicans virulence when the host's immune system is suppressed.

Catechol and 3-methylcatechol were produced from benzene and toluene respectively using different mutants of Pseudomonas putida. P. putida 2313 lacked the extradiol cleavage enzyme, catechol 2,3-oxygenase, allowing overproduction of 3-methylcatechol from toluene to a level of 11.5 mM (1.27 g·1-1) in glucose fed-batch culture. P. putida 6(12), a mutant of P. putida 2313, lacked both catechol-oxygenase and catechol 1,2-oxygenase, and accumulated catechol from benzene to a level of 27.5mM(3g·1-1).In both biotransformations product formation ceased within 10 hours of feeding the aromatic substrate, and this was due to product inhibition by the catechols. The primary site of catechol toxicity was inhibition of the aromatic dioxygenase. Neither cis-toluene dihydrodiol cis-1,2-dihydroxy-3-methylcyclohexa-3,5-diene), nor cis-benzene dihydrodiol (cis-l,2-dihydroxy-3-methylcyclohexa-3,5-diene) dehydrogenase was significantly inhibited by catechol overproduction whereas both ring activating dioxygenases were inhibit...

Listeria monocytogenes has previously been shown to adapt to a wide variety of environmental niches, principally those associated with low pH, and this compromises its control in food environments. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. The present study aimed to gain a further understanding of the physiological basis for the differential effects of one control strategy, namely the use of the lantibiotic nisin. Using propidium iodide (PI) to probe membrane integrity it was shown that L. monocytogenes Scott A was sensitive to nisin (8 ng mL(-)) but this was growth phase dependent with stationary phase cells (OD600=1.2) being much more resistant than exponential phase cells (OD600=0.38). We demonstrate that, using a combination of techniques including fluorescence activated cell sorting (FACS), the membrane adaptations underpinning nisin resistance are triggered much earlier (OD600

We introduce a novel technique for the visualization of data at various levels of detail. This is based on a colour-based representation of the data, where "high level" views of the data are obtained by merging colours together to obtain a summary-colour which represents a number of data-points. This is applied to the problem of visualizing biomolecular sequence data and picking out features in such data at various scales.

Polycyclic aromatic hydrocarbons (PAHs) are of great environmental concern due to their mutagenic and carcinogenic properties. Their persistence in ecosystems is due to their low water solubility which results in their binding to particulates in soils, sediments and atmosphere. Bioremediation represents the major route for the ecological recovery of PAHs contaminated sites. The aim of this work was to investigate the uptake and storage of benzo[a]pyrene by a non white-rot fungus, Fusarium solani, and to characterize, by cytological methods, the intracellular fluorescent vesicles observed when growing the fungus on synthetic medium in presence of benzo[a]pyrene. We have demonstrated that PAHs uptake into fungal hyphae was a passive phenomenon. Indeed, benzo[a]pyrène uptake and storage in Fusarium solani was not prevented at 4°C or in the presence of cytochrome oxidase inhibitor (sodium azide). The use of two cytoskeleton modulating drugs (colchicine or cytochalasin) suggested that microtubules and actine filaments were not involved in PAHs transport. Ultrastructural study showed that PAHs incorporation was not associated with specific structures. The use of Sudan III and Rhodamine B stainings showed that PAHs were accumulated in pre-existing structures corresponding to lipid vesicles. We found that Fusarium solani, was able to store into lipid vesicles and degrade efficiently a large scale of PAHs. The degradation rates were about 84, 70, 58, 34 and 40% for anthracene, pyrene, benzo[a]pyrene, benzo[ghi]perylene and coronene respectively. We also showed that the PAHs intracellular storage was not restricted to the Deuteromycotina fungus Fusarium solani, but could be generalized to numerous other fungi, even poor degraders, belonging to different genera., Les hydrocarbures polycycliques aromatiques (HPA), composés extrêmement nocifs pour l'homme et son environnement en raison des pouvoirs toxiques, mutagènes et cancérogènes reconnus pour plusieurs d'entre eux, constituent une classe de polluants particulièrement répandus dans le sol. Parmi les méthodes de réhabilitation des sols contaminés par les HPA, la bioremédiation se révèle être une des techniques les plus économiques et les plus écologiques. Isolé au laboratoire de Mycologie/Phytopathologie/Environnement de l'Université du Littoral-Côte d'Opale, à partir d'un sol artificiellement contaminé, Fusarium solani, un champignon imparfait ou Deutéromycète, est capable de dégrader efficacement une large gamme d'HPA de 2 à 7 cycles. Les taux de dégradation sont respectivement de 84, 70, 58, 34 et 40 % pour l'anthracène, le pyrène, le benzo[a]pyrène, le benzo[ghi]pérylène et le coronène. Quand Fusarium solani est cultivé sur un milieu synthétique contenant un des HPA comme substrat, des vésicules fluorescentes sont observées à l'intérieur des hyphes indiquant le prélèvement et le stockage des HPA (ou l'un de leurs métabolites) par ce champignon. Dans cette étude, nous avons démontré que ce phénomène de prélèvement et d'accumulation est passif. En effet, aucune modification n'est décelée lorsque Fusarium solani est cultive à 4°C ou en présence d'un inhibiteur de la cytochrome oxidase (l'acide de sodium). L'emploi d'inhibiteurs du cytosquelette (colchicine et cytochalasine) suggère la non implication des filaments d'actine et des microtubules dans ce transport. Des observations au microscope électronique ne montrent aucune structure intervenant spécifiquement dans l'accumulation des HPA chez Fusarium solani. En effet, grâce à l'utilisation de deux colorants : le Soudan III et la rhodamine B, nous avons démontré que les HPA s'accumulent dans des vésicules lipidiques préexistantes. D'autre part, nous avons montré que ce phénomène de prélèvement et de stockage des HPA dans les vésicules lipidiques est commun à l'ensemble des souches fongiques quelle que soit leur position taxonomique et indépendamment des taux de dégradation qu'elles présentent.

Listeria monocytogenes is a food-borne Gram-positive bacterium that is responsible for a variety of infections (worldwide) annually. The organism is able to survive a variety of environmental conditions and stresses, however, the mechanisms by which L. monocytogenes adapts to environmental change are yet to be fully elucidated. An understanding of the mechanism(s) by which L. monocytogenes survives unfavourable environmental conditions will aid in developing new food processing methods to control the organism in foodstuffs. We have utilized a proteomic approach to investigate the response of L. monocytogenes batch cultures to the transition from exponential to stationary growth phase. Proteomic analysis showed that batch cultures of L. monocytogenes perceived stress and began preparations for stationary phase much earlier (approximately A(600) = 0.75, mid-exponential) than predicted by growth characteristics alone. Global analysis of the proteome revealed that the expression levels of more than 50% of all proteins observed changed significantly over a 7-9 h period during this transition phase. We have highlighted ten proteins in particular whose expression levels appear to be important in the early onset of the stationary phase. The significance of these findings in terms of functionality and the mechanistic picture are discussed.

A bacterial consortium comprising four different species was isolated from an Indonesian agricultural soil using a mixture of aniline and 4-chloroaniline (4CA) as principal carbon sources. The four species were identified as Chryseobacterium indologenes SB1, Comamonas testosteroni SB2, Pseudomonas corrugata SB4 and Stenotrophomonas maltophilia SB5. Growth studies on aniline and 4CA as single and mixed substrates demonstrated that the bacteria preferred to grow on and utilize aniline rather than 4CA, although both compounds were eventually depleted from the culture supernatant. However, despite 100 % disappearance of the parent substrates, the degradation of 4CA was always characterized by incomplete dechlorination and 4-chlorocatechol accumulation. This result suggests that further degradation of 4-chlorocatechol may be the rate-limiting step in the metabolism of 4CA by the bacterial consortium. HPLC-UV analysis showed that 4-chlorocatechol was further degraded via an ortho-cleavage pathway by the bacterial consortium. This hypothesis was supported by the results from enzyme assays of the crude cell extract of the consortium revealing catechol 1,2-dioxygenase activity which converted catechol and 4-chlorocatechol to cis,cis-muconic acid and 3-chloro-cis,cis-muconic acid respectively. However, the enzyme had a much higher conversion rate for catechol [156 U (g protein)−1] than for 4-chlorocatechol [17·2 U (g protein)−1], indicating preference for non-chlorinated substrates. Members of the bacterial consortium were also characterized individually. All isolates were able to assimilate aniline. P. corrugata SB4 was able to grow on 4CA solely, while S. maltophilia SB5 was able to grow on 4-chlorocatechol. These results suggest that the degradation of 4CA in the presence of aniline by the bacterial consortium was a result of interspecies interactions.

Degradation of 4-chloroaniline in the presence of aniline by a microbial community in a laboratory-scale biofilm reactor was evaluated. The starter inoculum was isolated and reconstructed from a percolating column enrichment of Indonesian agricultural soil. The capacity to mineralise and detoxify 4-chloroaniline in the presence of aniline was demonstrated by the biofilm reactor when operated at high hydraulic retention time (HRT; 0.87 h). At low HRT (0.23 h and 0.39 h) 4-chlorocatechol accumulated in the effluent, accompanied by a decrease in dechlorination and detoxification. When returned to high HRT (2.14 h), the accumulation of 4-chlorocatechol stopped and the extent of dechlorination and detoxification increased. Bacteria other than the original inoculum appeared in the reactor when the operating mode was switched from closed cycle to open cycle. One of these bacteria, identified as Pseudomonas putida R1 by partial 16S rDNA sequencing, subsequently dominated the reactor at every HRT imposed. PCR-based single-strand conformational polymorphism of 16 s rDNA and traditional cultivation procedures indicated that the bacterial composition in the reactor shifted in response to applied HRT. The relationship between the bacterial abundance and the degradation capacity of the reactor is discussed.

Two oil-degrading bacteria identified as Pseudomonas aeruginosa and Micrococcus luteus were isolated from crude-oil-polluted soils in Nigeria. The organisms were grown on n-hexadecane and sodium succinate and then examined for the presence of hydrocarbon inclusions. Inclusion bodies were found in n-hexadecane-grown cells and were absent in succinate-grown cells. Formation of hydrocarbon inclusion bodies appears to be a general phenomenon among hydrocarbon utilizers.

There is considerable interest in the development of biological processes for PCB degradation. However, a consideration must be made for the effects of the accumulation of toxic intermediates on process design, and on release to the environment. Chlorobenzoates are the most noted intermediates of aerobic PCB degradation.

(1994). The Bioremediation of Polychlorinated Biphenyls (PCBs): Problems and Perspectives. Biotechnology and Genetic Engineering Reviews: Vol. 12, No. 1, pp. 139-188.