Preparation and analysis of environmental DNA: optimisation of techniques for phylogenetic analysis of ATAD sludge

peer-reviewedNon-culture based analysis of microbial populations via phylogenetic analysis is becoming an increasingly important tool in the determination of microbial communities and community flux in a large number of environmental niches. The results obtained are however only as good as the preparative techniques used to isolate the original templates and the PCR processing techniques used to amplify these templates. In thermal niches such as autothermal thermophilic aerobic digestion systems (ATAD) there are many features that limit the optimal recovery of microbial diversity. Such features include lysis and release of products from the microbial population such as nucleases and proteases which effect both template recovery and polymerase functionality, inhibitory substances from the environmental source and even the nature of the primers used to amplify the resultant templates. In optimisation of the extraction and amplification of environmental DNA from an ATAD system we have identified a number of issues that affect the recovery of diversity which may have widespread applicability to the recovery of diversity by molecular techniques from other environmental niches. Methods of optimisation and analysis of diversity recovery will be discussed.HE

A robust procedure for the measurement of Serum Magnesium on the Hitachi 704 using Calmagite

peer-reviewedA robust procedure for measurement of magnesium in serum is described using calmagite in CAPS buffer at pH 11.5. Interference from other magnesium reagents was reduced and evaluated for chromophoric interference. The procedure was not effected by bilirubin or hemoglobin in serum samples

The Potential of the Photoautotroph Synechocystis for Metal Bioremediation

The photoautotrophic cyanobacterium Synechocystis PCC6803 has received much attention as a model photosynthetic cell factory for the production of a range of important biotech products. The biomass remaining from this activity may then have further utility in processes such as metal bioremediation. In addition Synechocystis being an inhabitant of many natural aquatic environments is seen as an environmentally friendly alternative to using chemical precipitation methodologies for metal remediation. Synechocystis produces a range of extracellular polysaccharide substances (EPS) that can undergo modification as a function of culture age and growth nutrients which have been implicated in metal biosorption. Many studies have demonstrated that high levels of charged groups present in EPS are important in forming polymeric matrices with metallic ions allowing their biosorption. Genetic studies has revealed genes involved in such metal binding indicating that EPS can be modified for potential enhancement of binding or modification of the types of metals bound. The utility of metal binding to live and dead biomass of Synechocystis has been demonstrated for a range of metals including Cr(VI), Cd(II), Cu(II), Pb(II), Sb, Ni(II), Mn(II), Mn(IV), As(III), As(V), Cs and Hg. The potential of using Synechocystis as a biosorption platform is discussed

New and emerging SXT/R391 integrative conjugative elements as vehicles for stable mobile element transfer and spread of antibiotic resistance in both human and animals.

peer-reviewedThe integrative conjugative elements, ICE s SXT and R391 are the prototypes of a group of gram negative integrative elements known as the SXT/R391 group. R391 was identified in a clinical isolate of Providencia in the late 1960 s in South Africa, while SXT was initially isolated in 1992 in a clinical isolate of Vibrio cholerae O139 and variants have since been isolated in pandemic strains throughout the world. Subsequent sequencing of both elements demonstrated a high degree of structural similarity leading to the group being classified as the SXT/R391 group. The SXT/R391 ICE elements are characterised as integrating into a specific chromosomal site within gram ve hosts, being extremely stable and promiscuous and possessing a number of element hotspots for integration of heterologous DNA including increasingly, antibiotic resistance determinants. This makes such ICE s highly adapted for antibiotic spread. New evidence emerging indicates that SXT/R391-like ICE s are increasingly being identified worldwide particularly in Asia not only from Vibrio species, where they have been found widely in human clinical isolates, but from other gram -ve associated infections of domestic animals and fish. Evidence of more such elements may emerge in the future as a new trapping vector pIceCap has been developed to capture them in a circular form, aiding characterisation. The types of the novel ICE s now emerging, their comparison with prototype elements and the antibiotic resistances associated with them are important given their promiscuous nature and stability. PUBLISHEDPeer reviewe

Metabolic engineering of the model photoautotrophic cyanobacterium synechocystis for ethanol production: optimization strategies and challenges

peer-reviewedPhotoautotrophic ethanol production using model cyanobacteria is an attractive technology that offers potential for sustainable ethanol production as a biofuel. Model strains of Synechocystis PCC6803 have been metabolically engineered to convert central metabolic intermediates such as pyruvate to acetaldehyde via cloned heterologous pyruvate decarboxylase and from acetaldehyde to ethanol via cloned homologous or heterologous alcohol dehydrogenase. While the technology is now proven, strategies are required to increase the ethanol levels through metabolic and genetic engineering and in addition, production and process strategies are required to make the process sustainable. Here we discuss both genetic and molecular strategies in combination with do wnstream strategies that are being applied while also discussing challenges to future application

Molecular analysis of bacterial community DNA in sludge undergoing autothermal thermophilic aerobic digestion (ATAD): pitfalls and improved methodology to enhance diversity recovery

peer-reviewedMolecular analysis of the bacterial community structure associated with sludge processed by autothermal thermophilic aerobic digestion (ATAD), was performed using a number of extraction and amplification procedures which differed in yield, integrity, ability to amplify extracted templates and specificity in recovering species present. Interference to PCR and qPCR amplification was observed due to chelation, nuclease activity and the presence of thermolabile components derived from the ATAD sludge. Addition of selected adjuvant restored the ability to amplify community DNA, derived from the thermophilic sludge, via a number of primer sets of ecological importance and various DNA polymerases. Resolution of community profiles by molecular techniques was also influenced by the ATAD sludge extraction procedure as demonstrated by PCR-DGGE profiling and comparison of taxonomic affiliations of the most predominant members within 16S rRNA gene libraries constructed from ATAD DNA extracted by different methods. Several modifications have been shown to be necessary to optimize the molecular analysis of the ATAD thermal niche which may have general applicability to diversity recovery from similar environments

Genotypic and phenotypic diversity of Ralstonia pickettii and Ralstonia insidiosa isolates from clinical and environmental sources including High-purity Water. Diversity in Ralstonia pickettii

<p>Abstract</p> <p>Background</p> <p><it>Ralstonia pickettii </it>is a nosocomial infectious agent and a significant industrial contaminant. It has been found in many different environments including clinical situations, soil and industrial High Purity Water. This study compares the phenotypic and genotypic diversity of a selection of strains of <it>Ralstonia </it>collected from a variety of sources.</p> <p>Results</p> <p><it>Ralstonia </it>isolates (fifty-nine) from clinical, industrial and environmental origins were compared genotypically using i) Species-specific-PCR, ii) PCR and sequencing of the 16<it>S-</it>23<it>S </it>rRNA Interspatial region (ISR) iii) the <it>fliC </it>gene genes, iv) RAPD and BOX-PCR and v) phenotypically using biochemical testing. The species specific-PCR identified fifteen out of fifty-nine designated <it>R. pickettii </it>isolates as actually being the closely related species <it>R. insidiosa</it>. PCR-ribotyping of the 16<it>S-</it>23<it>S </it>rRNA ISR indicated few major differences between the isolates. Analysis of all isolates demonstrated different banding patterns for both the RAPD and BOX primers however these were found not to vary significantly.</p> <p>Conclusions</p> <p><it>R. pickettii </it>species isolated from wide geographic and environmental sources appear to be reasonably homogenous based on genotypic and phenotypic characteristics. <it>R. insidiosa </it>can at present only be distinguished from <it>R. pickettii </it>using species specific PCR. <it>R. pickettii </it>and <it>R. insidiosa </it>isolates do not differ significantly phenotypically or genotypically based on environmental or geographical origin.</p