An improved genetically modified Escherichia coli biosensor for amperometric tetracycline measurement.

The bacterial respiratory gene, nuoA, was previously used as a reporter gene in an amperometric, whole cell biosensor for tetracycline (Tet) detection. While the nuoA-based bioassay responded sensitively to Tet, the signal declined at high Tet concentrations, probably partly due to transgene over-expression. Also, at zero concentration of Tet, the assay registered a relatively high background signal when compared to the nuoA knockout Escherichia coli strain without the biosensor transgene construct. This was probably due to incomplete repression of nuoA expression. In order to reduce gene over-expression, the sensor cells were incubated with Tet at a relatively low temperature (15 °C). Also, a low-copy number plasmid pBR322 was used to carry the transgene, instead of the high-copy number plasmid pBluescript in order to reduce over-expression and to reduce background expression. Both assays improved the biosensor response. By using a low-copy number plasmid and tetracycline resistance, the sensor was less inhibited at higher Tet concentrations; but, this did not significantly increase the linear range of the sensor. The low temperature nuoA assay could detect Tet at a range of 0.001-1 µg ml(-1). In contrast, the low-copy number nuoA assay was able to detect Tet at a range of 0.0001-1 µg ml(-1). The detection limit of Tet determined by the low-copy number nuoA assay was 0.00023 µg ml(-1), which is one order of magnitude more sensitive than in the previous nuoA assay.

Appraising freeze-drying for storage of bacteria and their ready access in a rapid toxicity assessment assay.

Direct toxicity assessment (DTA) techniques seek to measure the impact of toxic chemicals on biological materials resident in the environment. This study features the use of freeze-dried bacterial cells in combination with a rapid DTA analyser, SciTOX. The effects of three factors-cryoprotectant type, bacterial strain, and storage temperature-were tested in order to validate the shelf life of the freeze-dried cells. Three freeze-dried Gram-negative bacterial strains, Acinetobacter calcoaceticus, Escherichia coli and Pseudomonas putida, were tested by using the bacteria in the SciTox(™) DTA assay and recording their responses to two standard toxicants: 2,4-dicholorophenol and 3,5-dichlorophenol. Each freeze-dried strain of bacteria was prepared in two forms--either pre-treatment with polyethylene glycol (PEG) or with sucrose/Tween 80--prior to storing at either 4 or -20 °C for three different storage periods (1, 2 or 3 months). While the sucrose/Tween 80 pre-treated freeze-dried cells exhibited better cell viability, we concluded that PEG was a more suitable cryoprotectant for the bacteria used in the DTA assay because of EC50 parity with fresh cell and zero-time freeze-dried cell assays. The results showed that freeze-dried cells, with appropriate materials and conditions, can give reproducible DTA results for up to 3 months. The availability of a biocomponent that can be activated by simple rehydration makes the deployment of this technology much easier for an end user.

Development and applications of whole cell biosensors for ecotoxicity testing.

Whole cell biosensors are the focus of considerable and increasing interest worldwide as methods for detecting and quantifying environmental toxicity, including biochemical oxygen demand (BOD), heavy metals, antibiotics, pesticides and herbicides. This review follows the development of whole cell biosensors from attempts to utilise changes in cellular metabolism to determine BOD and general toxicity, through the exploitation of unique metabolic pathways to detect specific toxicants, to the increasingly widespread use of genetic engineering to build new, and modify existing, sensing pathways.

Reproduction without sex: conidiation in the filamentous fungus Trichoderma.

Trichoderma spp. have served as models for asexual reproduction in filamentous fungi for over 50 years. Physical stimuli, such as light exposure and mechanical injury to the mycelium, trigger conidiation; however, conidiogenesis itself is a holistic response determined by the cell's metabolic state, as influenced by the environment and endogenous biological rhythms. Key environmental parameters are the carbon and nitrogen status and the C : N ratio, the ambient pH and the level of calcium ions. Recent advances in our understanding of the molecular biology of this fungus have revealed a conserved mechanism of environmental perception through the White Collar orthologues BLR-1 and BLR-2. Also implicated in the molecular regulation are the PacC pathways and the conidial regulator VELVET. Signal transduction cascades which link environmental signals to physiological outputs have also been revealed.

Ectosymbiotic bacteria at the origin of magnetoreception in a marine protist.

Mutualistic symbioses are often a source of evolutionary innovation and drivers of biological diversification1. Widely distributed in the microbial world, particularly in anoxic settings2,3, they often rely on metabolic exchanges and syntrophy2,4. Here, we report a mutualistic symbiosis observed in marine anoxic sediments between excavate protists (Symbiontida, Euglenozoa)5 and ectosymbiotic Deltaproteobacteria biomineralizing ferrimagnetic nanoparticles. Light and electron microscopy observations as well as genomic data support a multi-layered mutualism based on collective magnetotactic motility with division of labour and interspecies hydrogen-transfer-based syntrophy6. The guided motility of the consortia along the geomagnetic field is allowed by the magnetic moment of the non-motile ectosymbiotic bacteria combined with the protist motor activity, which is a unique example of eukaryotic magnetoreception7 acquired by symbiosis. The nearly complete deltaproteobacterial genome assembled from a single consortium contains a full magnetosome gene set8, but shows signs of reduction, with the probable loss of flagellar genes. Based on the metabolic gene content, the ectosymbiotic bacteria are anaerobic sulfate-reducing chemolithoautotrophs that likely reduce sulfate with hydrogen produced by hydrogenosome-like organelles6 underlying the plasma membrane of the protist. In addition to being necessary hydrogen sinks, ectosymbionts may provide organics to the protist by diffusion and predation, as shown by magnetosome-containing digestive vacuoles. Phylogenetic analyses of 16S and 18S ribosomal RNA genes from magnetotactic consortia in marine sediments across the Northern and Southern hemispheres indicate a host-ectosymbiont specificity and co-evolution. This suggests a historical acquisition of magnetoreception by a euglenozoan ancestor from Deltaproteobacteria followed by subsequent diversification. It also supports the cosmopolitan nature of this type of symbiosis in marine anoxic sediments.

Approaches to functional genomics in filamentous fungi.

The study of gene function in filamentous fungi is a field of research that has made great advances in very recent years. A number of transformation and gene manipulation strategies have been developed and applied to a diverse and rapidly expanding list of economically important filamentous fungi and oomycetes. With the significant number of fungal genomes now sequenced or being sequenced, functional genomics promises to uncover a great deal of new information in coming years. This review discusses recent advances that have been made in examining gene function in filamentous fungi and describes the advantages and limitations of the different approaches.

Agrobacterium-mediated transformation of Sclerotinia sclerotiorum.

Ascospores from the phytopathogenic fungus Sclerotinia sclerotiorum were transformed to hygromycin B resistance by co-cultivation with Agrobacterium tumefaciens. Transformed spores germinated and grew on PDA supplemented with 100 ug/ml hygromycin B. The presence of mitotically stable hph gene integration at random sites in the genome was confirmed by PCR and Southern blot analysis. A transformation frequency of 8 x 10(-5) was achieved in five separate experiments. This study is the first report of success co-cultivating A. tumefaciens with S. sclerotiorum. This report of a reproducible Agrobacterium-mediated transformation method should allow the development of T-DNA tagging as a system for insertional mutagenesis in S. sclerotiorum and provide a simple and reliable method for genetic manipulation.

Genome-scale investigation of phenotypically distinct but nearly clonal Trichoderma strains.

Biological control agents (BCA) are beneficial organisms that are applied to protect plants from pests. Many fungi of the genus Trichoderma are successful BCAs but the underlying mechanisms are not yet fully understood. Trichoderma cf. atroviride strain LU132 is a remarkably effective BCA compared to T. cf. atroviride strain LU140 but these strains were found to be highly similar at the DNA sequence level. This unusual combination of phenotypic variability and high DNA sequence similarity between separately isolated strains prompted us to undertake a genome comparison study in order to identify DNA polymorphisms. We further investigated if the polymorphisms had functional effects on the phenotypes. The two strains were clearly identified as individuals, exhibiting different growth rates, conidiation and metabolism. Superior pathogen control demonstrated by LU132 depended on its faster growth, which is a prerequisite for successful distribution and competition. Genome sequencing identified only one non-synonymous single nucleotide polymorphism (SNP) between the strains. Based on this SNP, we successfully designed and validated an RFLP protocol that can be used to differentiate LU132 from LU140 and other Trichoderma strains. This SNP changed the amino acid sequence of SERF, encoded by the previously undescribed single copy gene "small EDRK-rich factor" (serf). A deletion of serf in the two strains did not lead to identical phenotypes, suggesting that, in addition to the single functional SNP between the nearly clonal Trichoderma cf. atroviride strains, other non-genomic factors contribute to their phenotypic variation. This finding is significant as it shows that genomics is an extremely useful but not exhaustive tool for the study of biocontrol complexity and for strain typing.

Maintenance of Geobacter-dominated biofilms in microbial fuel cells treating synthetic wastewater.

Geobacter-dominated biofilms can be selected under stringent conditions that limit the growth of competing bacteria. However, in many practical applications, such stringent conditions cannot be maintained and the efficacy and stability of these artificial biofilms may be challenged. In this work, biofilms were selected on low-potential anodes (-0.36 V vs Ag/AgCl, i.e. -0.08 V vs SHE) in minimal acetate or ethanol media. Selection conditions were then relaxed by transferring the biofilms to synthetic wastewater supplemented with soil as a source of competing bacteria. We tracked community succession and functional changes in these biofilms. The Geobacter-dominated biofilms showed stability in their community composition and electrochemical properties, with Geobacter sp. being still electrically active after six weeks in synthetic wastewater with power densities of 100±19 mW·m(-2) (against 74±14 mW·m(-2) at week 0) for all treatments. After six weeks, the ethanol-selected biofilms, despite their high taxon richness and their efficiency at removing the chemical oxygen demand (0.8 g·L(-1) removed against the initial 1.3 g·L(-1) injected), were the least stable in terms of community structure. These findings have important implications for environmental microbial fuel cells based on Geobacter-dominated biofilms and suggest that they could be stable in challenging environments.

Influence of inoculum and anode surface properties on the selection of Geobacter-dominated biofilms.

This study evaluated the impact of inoculum source and anode surface modification (carboxylate -COO(-) and sulfonamide -SO2NH2 groups) on the microbial composition of anode-respiring biofilms. These two factors have not previously been considered in detail. Three different inoculum sources were investigated, a dry aerobic soil, brackish estuarine mud and freshwater sediment. The biofilms were selected using a poised anode (-0.36 V vs Ag/AgCl) and acetate as the electron donor in a three-electrode configuration microbial fuel cell (MFC). Population profiling and cloning showed that all biofilms selected were dominated by Geobacter sp., although their electrochemical properties varied depending on the source inoculum and electrode surface modification. These findings suggest that Geobacter sp. are widespread in soils, even those that do not provide a continuously anaerobic environment, and are better at growing in the MFC conditions than other bacteria.

Influence of anode potentials on selection of Geobacter strains in microbial electrolysis cells.

Through their ability to directly transfer electrons to electrodes, Geobacter sp. are key organisms for microbial fuel cell technology. This study presents a simple method to reproducibly select Geobacter-dominated anode biofilms from a mixed inoculum of bacteria using graphite electrodes initially poised at -0.25, -0.36 and -0.42 V vs. Ag/AgCl. The biofilms all produced maximum power density of approximately 270 m Wm(-2) (projected anode surface area). Analysis of 16S rRNA genes and intergenic spacer (ITS) sequences found that the biofilm communities were all dominated by bacteria closely related to Geobacter psychrophilus. Anodes initially poised at -0.25 V reproducibly selected biofilms that were dominated by a strain of G. psychrophilus that was genetically distinct from the strain that dominated the -0.36 and -0.42 V biofilms. This work demonstrates for the first time that closely related strains of Geobacter can have very different competitive advantages at different anode potentials.

Comparison of three genetically modified Escherichia coli biosensor strains for amperometric tetracycline measurement.

Three separate genetic strategies, based upon the induced expression of three different genes (lacZ, selA and nuoA) were tested to provide the SciTox assay with sensitive and specific detection of the antibiotic tetracycline (Tet). All three strategies relied on gene induction from the Tn10 tetA promoter. Both lacZ and nuoA biosensors responded specifically and sensitively to sub-inhibitory concentrations of Tet. However, the selA-based assay was not sensitive enough to detect Tet in the SciTox assay. The detection limits for Tet of the lacZ and nuoA biosensor strains were 0.11 µg ml(-1) and 0.0026 µg ml(-1), respectively, and their linear ranges were 0.1-1 µg ml(-1) and 0-0.01 µg ml(-1), respectively. While lacZ has previously been used as a reporter gene in an amperometric bioassay, nuoA is a novel and more sensitive reporter gene. This is the first report in which a respiratory gene was used as a reporter gene in an amperometric biosensor. The results indicate that this approach can produce a highly sensitive detection system. In order to test whether the new system could be used to detect other chemicals, the nuoA gene was re-engineered to be driven by the copper-inducible copA promoter. Using this strain, the SciTox assay was found to be able to specifically detect copper and silver ions.

Functional stability of a hybrid anaerobic digester/microbial fuel cell system treating municipal wastewater.

A thermophilic anaerobic digester (AD) was combined with a microbial fuel cell (MFC) to evaluate whether either component had increased stability when operated in combination as a hybrid system, perturbed by the addition of acetic acid. The MFC and the anaerobic digester were able to operate effectively together. The MFC was more susceptible to high acetic acid load than the AD. The hybrid system did not have increased resilience compared to the solitary systems in the conditions tested. However, the low pH had a relatively delayed effect on the MFC compared to the AD, allowing the hybrid system to have a more stable energy output. Also, at very low pH, when operating as a hybrid, the AD component was able to recover pH to normal levels when the MFC component failed. These results demonstrate that there are synergies that can be gained from this hybrid system.

Isolate-specific conidiation in Trichoderma in response to different nitrogen sources.

A characteristic feature of Trichoderma is the production of concentric rings of conidia in response to alternating light/dark conditions and a single ring of conidia in response to a single burst of light. In this study, conidiation was investigated in four biocontrol isolates (T. hamatum, T. atroviride, T. asperellum, T. virens) and one isolate from the mushroom pathogen species, T. pleuroticola. All five isolates produced concentric conidial rings under alternating light/dark conditions on potato-dextrose agar (PDA), however, in response to a 15min burst of blue light, only T. asperellum and T. virens produced a clearly defined conidial ring. Both T. pleuroticola and T. hamatum photoconidiated in a disk-like fashion and T. atroviride produced a broken ring with a partially filled in appearance. In the presence of primary nitrogen, T. asperellum and T. pleuroticola conidiated in a disk, whereas, when grown in the presence of secondary nitrogen, a ring of conidia was produced. Primary nitrogen promoted photoconidiation and competency to conidiate in response to light appeared dependent on the nitrogen catabolite repression state of the cell. Mycelial injury was also investigated in the same five isolates of Trichoderma on PDA and under different nitrogen statuses. For the first time, we report that conidiation in response to injury is differentially regulated in different isolates/species of Trichoderma.

Ambient pH intrinsically influences Trichoderma conidiation and colony morphology.

Conidiation in Trichoderma has been demonstrated to be favoured by a low ambient pH and more recently PacC (Pac1) mediated pH-regulation has been implicated in the control of conidiation. In this study, ambient pH effects on conidiation were investigated in three isolates (Trichoderma hamatum, Trichoderma atroviride and Trichoderma pleuroticola) exposed to a single blue-light burst or to mycelial injury. Disks of conidiation were observed for T. atroviride in response to a single light exposure, which clearly demonstrates that all cells are potentially competent for photoconidiation. Previous studies have suggested T. hamatum does not conidiate in response to mycelial injury, however, in this study a clear injury response was observed from pH 2.8 to 3.2. T. pleuroticola displayed three distinct pH-dependent colony morphologies from pH 2.8 to 5.2. Conidiation was strictly low pH-dependent on buffered media and observed at all pH values on unbuffered media. The dependence of the conidial phenotype on the buffering state of the medium rather than the pH per se, was unexpected as it has been suggested that conidiation is PacC regulated. Conversely, excretion of an anthraquinone was strictly pH-dependent regardless of the buffering state. These studies highlight the complexity of ambient pH effects on Trichoderma spp. and demonstrate a need to widen the scope of research to multiple species.

Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola.

Trichoderma species conidiate in response to blue light, however, unlike in the blue-light model fungus Neurospora crassa, conidiation in Trichoderma spp. has been considered to be non-circadian. In this study we uncovered evidence for circadian conidiation in Trichoderma pleuroticola and identified orthologues of the key N. crassa clock components, wc-1 (blr-1) and frq.

Models of phage growth and their applicability to phage therapy.

Phage therapy is complicated by the self-replicating nature of phage. It is difficult to extrapolate from in vitro phage growth data to in vivo expectations, difficult to interpret in vivo data and difficult to generalize from one in vivo situation to another. Various generic models of phage growth have been used as the theoretical basis for understanding the kinetics of phage therapy. Here, we have experimentally tested the efficacy of such simple models to predict, qualitatively and quantitatively, the growth of phage and the phage proliferation threshold in vitro. Naturally occurring, antibiotic-resistant bacteria were used to measure the growth of phage in vivo. In homogenous, in vitro environments, the models were predictive of T4 phage growth on Escherichia coli RR1. However, the models were not able to predict growth of T4 phage or K1-5 phage in the more complex environment of the rat's digestive tract. To explore fully the kinetics of phage therapy, more complex models need to be devised. We suggest that it may be necessary to consider and model the interactions between phage growth parameters and bacterial growth parameters.