Ss-Sl2, a Novel Cell Wall Protein with PAN Modules, Is Essential for Sclerotial Development and Cellular Integrity of Sclerotinia sclerotiorum

The sclerotium is an important dormant body for many plant fungal pathogens. Here, we reported that a protein, named Ss-Sl2, is involved in sclerotial development of Sclerotinia sclerotiorum. Ss-Sl2 does not show significant homology with any protein of known function. Ss-Sl2 contains two putative PAN modules which were found in other proteins with diverse adhesion functions. Ss-Sl2 is a secreted protein, during the initial stage of sclerotial development, copious amounts of Ss-Sl2 are secreted and accumulated on the cell walls. The ability to maintain the cellular integrity of RNAi-mediated Ss-Sl2 silenced strains was reduced, but the hyphal growth and virulence of Ss-Sl2 silenced strains were not significantly different from the wild strain. Ss-Sl2 silenced strains could form interwoven hyphal masses at the initial stage of sclerotial development, but the interwoven hyphae could not consolidate and melanize. Hyphae in these interwoven bodies were thin-walled, and arranged loosely. Co-immunoprecipitation and yeast two-hybrid experiments showed that glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Woronin body major protein (Hex1) and elongation factor 1-alpha interact with Ss-Sl2. GAPDH-knockdown strains showed a similar phenotype in sclerotial development as Ss-Sl2 silenced strains. Hex1-knockdown strains showed similar impairment in maintenance of hyphal integrity as Ss-Sl2 silenced strains. The results suggested that Ss-Sl2 functions in both sclerotial development and cellular integrity of S. sclerotiorum

Population dynamics of a salmonella lytic phage and its host : implications of the host bacterial growth rate in modelling

The prevalence and impact of bacteriophages in the ecology of bacterial communities coupled with their ability to control pathogens turn essential to understand and predict the dynamics between phage and bacteria populations. To achieve this knowledge it is essential to develop mathematical models able to explain and simulate the population dynamics of phage and bacteria. We have developed an unstructured mathematical model using delay-differential equations to predict the interactions between a broad-host-range Salmonella phage and its pathogenic host. The model takes into consideration the main biological parameters that rule phage-bacteria interactions likewise the adsorption rate, latent period, burst size, bacterial growth rate, and substrate uptake rate, among others. The experimental validation of the model was performed with data from phage-interaction studies in a 5 L bioreactor. The key and innovative aspect of the model was the introduction of variations in the latent period and adsorption rate values that are considered as constants in previous developed models. By modelling the latent period as a normal distribution of values and the adsorption rate as a function of the bacterial growth rate it was possible to accurately predict the behaviour of the phage-bacteria population. The model was shown to predict simulated data with a good agreement with the experimental observations and explains how a lytic phage and its host bacteria are able to coexist.Financial support was received through the Strategic Project PEst-OE/EQB/LA0023/2013 from the FCT-Fundacao para a Ciencia e Tecnologia (http://www.fct.pt) and the projects "BioHealth - Biotechnology and Bioengineering approaches to improve health quality'', Ref. NORTE-07-0124 FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER and "Consolidating Research Expertise and Resources on Cellular and Molecular Biotechnology at CEB/IBB'', Ref. FCOMP-01-0124-FEDER-027462. Silvio B. Santos was supported by the grant SFRH/BPD/75311/2010 and Carla Carvalho was supported by the grant SFRH/BPD/79365/2011 both from the FCT-Fundacao para a Ciencia e Tecnologia (http://www.fct.pt). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Whole-cell Biosensors for Monitoring Bioremediation

Whole-cell biosensors are sensing devices that typically use biofilm-dwelling microorganisms to detect specific physical or chemical aspects of environmental samples. Microbial detection produces a signal that is transformed into user accessible data which can be as simple as colour change on a paper strip or as complex as quantitative digital display. Whole-cells can be embedded on a transducer or used separately as part of a multi-step assay format. Both unmodified and genetically modified cells have been used in this way. Metabolic reporters are used to detect toxicity that inhibits cell metabolism while catabolic reporters can be used to detect specific contaminants. Biosensors can provide data on the bioavailability of contaminants and are very relevant to monitoring bioremediation. Several commercially available sensors have been developed and some of these have been widely tested and demonstrated to be effective at measuring environmental contaminants

Agrobacterium-mediated transformation of Sclerotinia sclerotiorum

Ascospores from the phytopathogenic fungus Sclerotinia sclerotiorum were transformed to hygromycin B resistance by cocultivation 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 8105 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

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

© 2015 Elsevier B.V. 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 (-0.36V vs Ag/AgCl, i.e. -0.08V 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±19mW·m-2 (against 74±14mW·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 (0.8g·L-1 removed against the initial 1.3g·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

Comment on microbial community composition Is unaffected by anode potential

While bioelectrochemical systems (BESs) such as microbial fuel cells (MFCs) or microbial electrolysis cells (MECs) remain promising technologies, their widespread use for the sustainable production of energy from wastewaters is yet to be realized. Establishment of anode-respiring bacteria (ARB) at the anode surface is considered an important determinant of BES performance. Because ARB use the anode for respiration, it should be possible to use anode potential as a mechanism to select various ARB communities with different electron transfer properties

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

Photosynthetic biocathode enhances the power output of a sediment-type microbial fuel cell

Conventional microbial fuel cells (MFCs) consist of biological anodes and abiotic cathodes separated by a proton-exchange membrane. The abiotic cathode usually catalyses the reduction of oxygen to produce water by means of expensive catalysts such as platinum. The cathodic reaction is often limiting in MFCs and researchers are now focusing on efficient, low-cost catalysts to improve oxygen reduction at the cathode. This paper describes a photosynthetic biocathode in a sediment-type MFC constructed without a proton-exchange membrane. The carbon and stainless steel cathode did not contain any catalyst, but was covered in a biofilm composed of a complex community including microalgae and cyanobacteria. Although electroactive species were detected in the cathode biofilm, no biocatalysis of oxygen reduction was observed. Enhancement of the current output was mostly due to the production of pure oxygen near the cathode surface by the photosynthetic biofilm. Photosynthesis could produce dissolved oxygen levels approximately four times higher than oxygen levels obtained by aeration. The MFC was able to generate a maximum power density of 11 mW/m2 (projected anode area) over six months without feeding. © 2014 The Royal Society of New Zealand

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

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 -0.42V vs. Ag/AgCl. The biofilms all produced maximum power density of approximately 270mWm-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 -0.25V 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 -0.42V biofilms. This work demonstrates for the first time that closely related strains of Geobacter can have very different competitive advantages at different anode potentials. © 2013 Elsevier Ltd