Pitfalls using tributyrin agar screening to detect lipolytic activity in metagenomic studies

The metagenomics approach is an efficient method for obtaining novel biocatalysts and useful genes from uncultured microorganisms within diverse environments. In this study, we constructed a metagenomic library using a South African deep mine biofilm sample. The library was screened forlipolytic activity using LB Tributyrin (TLB). Although we were able to identify 3 diverse esterase enzymes, we found that 70% of the obtained sequence data revealed the presence of enzymes and genes completely unrelated to that of lipolytic enzymes thereby highlighting the limitation of screening with TLB

A deep gold mine metagenome as a source of novel esterases

New sources of enzymes for biotechnological applications are continually being sought for. While diverse microbial ecosysyems have been demonstrated in the deep subsurfaces, deep mines provide easy access to these specialist communities. Therefore, the aim of this study was to assess a deep mine biofilm as a source of novel esterase enzymes. Biofilm was collected from the Beatrix Mine in South Africa, at a depth of 808 m. Assessment of the diversity revealed a group of previously uncultured bacteria and archaea. A metagenome library was screened for esterolytic activity, producing two esterolytic clones: a phospholipase patatin protein and an isochorismatase family protein. The isochorismatase family protein contained the catalytic Asp and Cys but not the Arg, which is considered as important for catalysis. The patatin showed 55% similarity to its closest relative; the patatin family protein from Plesiocystis pacifica. The expressed patatin displayed a preference for the C6 ester and was maximally active at pH 8 and 30°C. This study reported that screening of a relatively small metagenome library from the deep mine biofilm provided two esterolytic clones, which differed from their known counterparts. This indicates that the deep mine ecosystems contain an untapped resource of novel and potentially useful enzymes which may have applications in chemical syntheses.Key words: Metagenome library, functional screening, lipolytic activity, patatin, isochorismatas

Bacterial diversity of biofilm samples from deep mines in South Africa

The Au, Pt and diamond mines of South Africa provide access to microorganism bearing fluids emanating from fractures at depths ranging from 0.7 to 3.2 km. Due to the unique characteristic of mine environment as demonstrated by extreme pH, pressure, temperatureand/or salinity, it is anticipated that it could hold the promise for novel gene sequences and hence gene products of industrial and pharmaceutical importance. To provide insight into themicrobial diversity of mines in South Africa, biofilm samples were collected from Goldfield and diamond mines and their bacterial diversity determined using molecular approaches. 16S rRNA genes were amplified from DNA extracted from these samples using polymerase chain reaction with universal bacterial primers 27F (5’- AGA GTT TGA TCM TGG CTC AG-3’) and 1492R (5’- GGT TAC CTT GTT ACG ACT T-3’). Metagenomic clone libraries were constructed and restriction fragment length polymorphism (RFLP) analysis of >100 derivedclones resulted in four major restriction patterns from which 40 clones were chosen for sequencing. More than half (53%) of the sequences were affiliated with the bacterial phylum Proteobacteria, forty-one percent (41%) of the sequences with yet uncultured bacteria andthe phyla Firmicutes and Planctomycetes were accounted for by 4% and 2% of the sequences respectively. DGGE analysis of PCR-amplified 16S rRNA genes showed characteristic fingerprints for each sample. The differences in community structure observed account for the uniqueness of each of the mines with respect to its microbial diversity

Using current molecular techniques for rapid differentiation of Salmonella Typhi and Salmonella Typhimurium

Typhoid fever is responsible for the deaths of many people annually. However, conventional and timeconsuming detection methods for Salmonella Typhi still dominate. By using a molecular basedapproach, it was possible to identify Salmonella Typhi by amplifying two specific genes (viaB and tyv) and by using RFLP analysis on the 16S rRNA gene as a first step for identification. We were also able to identify Salmonella Typhi using multiple gene targets in a single multiplex PCR reaction. Here we show that, as opposed to conventional methods, molecular based approaches are more rapid and should thus be used for any initial detection of Salmonella Typhi

Chemical speciation of environmentally significant metals with inorganic ligands. Part 4: The Cd2+ + OH–, Cl–, CO32–, SO42–, and PO43– systems (IUPAC Technical Report)

The numerical modeling of Cd-II speciation amongst the environmental inorganic ligands Cl-, OH-, CO32-, SO42-, and PO43- requires reliable values for the relevant stability (formation) constants. This paper compiles and provides a critical review of these constants and related thermodynamic data. It recommends values of log(10) beta(p,q,r) valid at I-m = 0 mol kg(-1) and 25 degrees C (298.15 K), along with the equations and empirical reaction ion interaction coefficients, Delta epsilon, required to calculate log(10)beta(p),(,q,r) values at higher ionic strengths using the Bronsted-Guggenheim-Scatchard specific ion interaction theory (SIT). Values for the corresponding reaction enthalpies, Delta H-r, are reported where available. Unfortunately, with the exception of the Cd-II-chlorido system and (at low ionic strengths) the Cd-II-sulfato system, the equilibrium reactions for the title systems are relatively poorly characterized. In weakly acidic fresh water systems (-log(10){[H+]/c degrees} < 6), in the absence of organic ligands (e. g., humic substances), Cd-II speciation is dominated by Cd2+(aq), with CdSO4(aq) as a minor species. In this respect, Cd-II is similar to Cu-II [2007PBa] and Pb-II [2009PBa]. However, in weakly alkaline fresh water solutions, 7.5 < -log(10) {[H+]/c degrees} < 8.6, the speciation of Cd-II is still dominated by Cd2+(aq), whereas for Cu-II [2007PBa] and Pb-II [2009PBa] the carbonato-species MCO3(aq) dominates. In weakly acidic saline systems (-log(10) {[H+]/c degrees} < 6; -log(10) {[Cl-]/c degrees} < 2.0) the speciation is dominated by CdCln(2-n)+ complexes, (n = 1-3), with Cd2+(aq) as a minor species. This is qualitatively similar to the situation for Cu-II and Pb-II. However, in weakly alkaline saline solutions, including seawater, the chlorido-complexes still dominate the speciation of Cd-II because of the relatively low stability of CdCO3(aq). In contrast, the speciation of Cu-II [2007PBa] and Pb-II [2009PBa] in seawater is dominated by the respective species MCO3(aq). There is scope for additional high-quality measurements in the Cd2+ + H+ + CO32- system as the large uncertainties in the stability constants for the Cd2+-carbonato complexes significantly affect the speciation calculations