By simultaneously amplifying more than one locus in the same reaction, multiplex PCR is becoming a rapid and convenient screening assay in both the clinical and the research laboratory. While numerous papers and manuals discuss in detail conditions influencing the quality of PCR in general, relatively little has been published about the important experimental factors and the common difficulties frequently encountered with multiplex PCR. We have examined various conditions of the multiplex PCR, using a large number of primer pairs. Especially important for a successful multiplex PCR assay are the relative concentrations of the primers at the various loci, the concentration of the PCR buffer, the cycling temperatures and the balance between the magnesium chloride and deoxynucleotide concentrations. Based on our experience, we propose a protocol for developing a multiplex PCR assay and suggest ways to overcome commonly encountered problems.

Multiplex PCR: Critical Parameters and Step-by-Step Protocol

http://link.springer.com/content/pdf/10.1007/BF02927128.pdf

Aquatic microbiology: F. A. Skinner andJ. M. Shewan Academic Press, London 1978; 370 pp.

Cyanobacteria are key organisms in the global ecosystem, useful models for studying metabolic and physiological processes conserved in photosynthetic organisms, and potential renewable platforms for production of chemicals. Characterizing cyanobacterial metabolism and physiology is key to understanding their role in the environment and unlocking their potential for biotechnology applications. Many aspects of cyanobacterial biology differ from heterotrophic bacteria. For example, most cyanobacteria incorporate a series of internal thylakoid membranes where both oxygenic photosynthesis and respiration occur, while CO2 fixation takes place in specialized compartments termed carboxysomes. In this review, we provide a comprehensive summary of our knowledge on cyanobacterial physiology and the pathways in Synechocystis sp. PCC 6803 (Synechocystis) involved in biosynthesis of sugar-based metabolites, amino acids, nucleotides, lipids, cofactors, vitamins, isoprenoids, pigments and cell wall components, in addition to the proteins involved in metabolite transport. While some pathways are conserved between model cyanobacteria, such as Synechocystis, and model heterotrophic bacteria like Escherichia coli, many enzymes and/or pathways involved in the biosynthesis of key metabolites in cyanobacteria have not been completely characterized. These include pathways required for biosynthesis of chorismate and membrane lipids, nucleotides, several amino acids, vitamins and cofactors, and isoprenoids such as plastoquinone, carotenoids, and tocopherols. Moreover, our understanding of photorespiration, lipopolysaccharide assembly and transport, and degradation of lipids, sucrose, most vitamins and amino acids, and haem, is incomplete. We discuss tools that may aid our understanding of cyanobacterial metabolism, notably CyanoSource, a barcoded library of targeted Synechocystis mutants, which will significantly accelerate characterization of individual proteins.

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Current knowledge and recent advances in understanding metabolism of the model cyanobacterium Synechocystis sp. PCC 6803.

Functional Proteomic Discovery of Slr0110 as a Central Regulator of Carbohydrate Metabolism in Synechocystis Species PCC6803

Metabolic flux of the oxidative pentose phosphate pathway under low light conditions in Synechocystis sp. PCC 6803.

Previous studies have shown that mutation of cyanobacterialzwf genes leads to markedly impaired dark viability, however, it is not entirely clear whether this phenotype is due to a polar effect on other genes in the transcriptional unit, in particularopcA. In the cyanobacteriumSynechocystis sp. PCC 6803 thezwf andopcA genes are widely separated in the genome and can be mutated without polar effects on each other. Thezwf gene ofSynechocystis sp. PCC 6803 was deleted and no glucose-6-phosphate dehydrogenase activity was detectable in the mutant. Growth of the mutant was similar to the wild-type in the light, but also viability was unimpaired in the dark. The result of this study indicates that previously reported reduced dark viability in cyanobacterialzwf mutants may actually have been a result of impairedopcA transcription.

A Δzwf (glucose-6-phosphate dehydrogenase) mutant of the cyanobacteriumSynechocystis sp. PCC 6803 exhibits unimpaired dark viability

The genes opcA and zwf are located close to each other in most of cyanobacterial strains and the mutations in the opcA gene were reported to lose most of G6PDH activity. One of the reasons suggested for this loss was the polar effect of the mutation on expression of the zwf gene in the same operon and the other was absence of the OpcA polypeptide necessary for the catalytic activity of G6PDH. Synechocystis sp. PCC 6803 exhibits a gene organisation in which opcA and zwf are far away from each other and is ideal for analysis of an opcA mutation alone. In this study, an opcA single mutant and an opcA-zwf double mutant were constructed and effects of the opcA mutation on G6PDH activity and dark viability were then investigated. Contrary to the previous observations, no negative effect of the mutation on G6PDH activity was found under the optimal substrate concentrations. However, when one of the substrates, G6P or NADP, was reduced gradually, G6PDH activity in the mutant cells decreased faster than the wild types. Our results indicated that an opcA mutation did not affect G6PDH activity severely when zwf and opcA were in different operons. Similarly, dark viability of the opcA single and the zwf-opcA double mutants did not exhibit meaningful difference from the wild type.

Characterisation of an opcA Mutant of the Unicellular Cyanobacterium Synechocystis sp. PCC 6803.

[Abstract] Glucose-6-phosphate dehydrogenase (G6PDH) (EC 1.1.1.49) is the first enzyme of the oxidative pentose phosphate cycle and catalyses the conversion of glucose-6-phosphate (G6P) to 6phosphoglucono-δ-lactone and transfers one electron to NADP+ producing one NADPH. Conversion of G6P to 6-phosphoglucono-δ-lactone is proportional to the production of NADPH. The increase in NADPH concentration results in an increase in absorbance at 340 nm. To assay G6PDH activity, therefore, production of NADPH is determined by measuring increase in absorbance at 340 nm spectrophotometrically. This increase rate is then converted to unit of activity and specific activity of G6PDH. In this procedure, a generalized method is given for bacterial G6PDH assays emphasizing on a cyanobacterium Synechocystis sp. PCC6803 (Schaeffer and Stanier, 1978; Karakaya et al., 2008, 2012) and a heterotrophic bacterium E.coli (Hylemon and Phibbs, 1972; Barnel et al., 1990).

/pdf/measurement-of-glucose-6-phosphate-dehydrogenase-activity-in-tnf7chupny.pdf

Measurement of Glucose-6-phosphate Dehydrogenase Activity in Bacterial Cell-free Extracts

Bacterial contamination levels of representative 4 natural springs were detected seasonally through indicator bacterial contents in a rural area of Ordu, Turkiye. Total coliform bacteria, enterococi, Escherichia coli and Clostridium perfringens spores were used as the indicator organisms. According to the bacterial parameters, none of the detected springs comply with the standards implemented by the state health authorities. Contamination level of the springs increases with human and domestic animal activities in spring and summer. Using PCR and duplex PCR, samples collected from the most densely contaminated spring, Soguksu, were analyzed for three pathogenic bacterial groups. After eight-hour enrichment of the samples, positive results were found only for Salmonella spp. In conclusion, this investigation revealed that the natural springs used for drinking water supply in the research area are exposed to bacterial contaminations and the level of contamination increased with settlement density.

/pdf/an-investigation-on-the-bacterial-contents-of-natural-5ciidf3xlz.pdf

An Investigation on the Bacterial Contents of Natural Springs in a Rural Area of the Middle Blacksea Region in Turkey

The aim of this study was to express the zwf gene of Synechococcus sp. PCC 7942 in zwf mutant Escherichia coli DF214 cells and to analyse glucose-6-phosphate dehydrogenase (G6PDH) activity. Initially, mutant cells were transformed with plasmid pNUT1 containing a Synechococcus sp. PCC 7942 zwf gene with a 1 kb upstream region that is expected to contain promoter elements. Transformant DF214 cells were not complemented by this fragment in a glucose minimal medium, nor did they exhibit statistically meaningful G6PDH activity. Therefore, the zwf gene was cloned in the lac operon to express the Zwf as a fusion protein; this yielded the construct pSG162. The pSG162 transformant E. coli DF214 cells were complemented in a glucose minimal medium, indicating that cyanobacterial Zwf protein fused with the part of LacZ′ polypeptide, enabling the cells to utilize glucose via the oxidative pentose phosphate pathway. Compared with wild-type E. coli cells, approximately ten times more G6PDH activity was measured in transformant cells. This indicated that the Synechococcus sp. PCC 7942 zwf gene was expressed under the control of the E. coli lac promoter as a fusion protein and the zwf product was converted into an active G6PDH form. Analyses was also carried out to determine whether dithiothreitol (DTT) was an in vitro reducing agent affected the enzyme activity, as was previously reported for this cyanobacterial strain. The results showed no variation in enzyme activity in the reduced assay conditions. Therefore, the zwf mutant E. coli strain DF214 was found to provide a rapid system for analysis of cyanobacterial G6PDH enzymes, but not for the redox state analysis of this enzyme.

Kübra Özkul

Papers

A Δzwf (glucose-6-phosphate dehydrogenase) mutant of the cyanobacteriumSynechocystis sp. PCC 6803 exhibits unimpaired dark viability

Characterisation of an opcA Mutant of the Unicellular Cyanobacterium Synechocystis sp. PCC 6803.

Measurement of Glucose-6-phosphate Dehydrogenase Activity in Bacterial Cell-free Extracts

An Investigation on the Bacterial Contents of Natural Springs in a Rural Area of the Middle Blacksea Region in Turkey

Analysis of glucose-6-phosphate dehydrogenase of the cyanobacterium Synechococcus sp. PCC 7942 in the zwf mutant Escherichia coli DF214 cells