Concerted changes in gene expression and cell physiology of the cyanobacterium Synechocystis sp. strain PCC 6803 during transitions between nitrogen and light-limited growth.

Physiological adaptation and genome-wide expression profiles of the cyanobacterium Synechocystis sp. strain PCC 6803 in response to gradual transitions between nitrogen-limited and light-limited growth conditions were measured in continuous cultures. Transitions induced changes in pigment composition, light absorption coefficient, photosynthetic electron transport, and specific growth rate. Physiological changes were accompanied by reproducible changes in the expression of several hundred open reading frames, genes with functions in photosynthesis and respiration, carbon and nitrogen assimilation, protein synthesis, phosphorus metabolism, and overall regulation of cell function and proliferation. Cluster analysis of the nearly 1,600 regulated open reading frames identified eight clusters, each showing a different temporal response during the transitions. Two large clusters mirrored each other. One cluster included genes involved in photosynthesis, which were up-regulated during light-limited growth but down-regulated during nitrogen-limited growth. Conversely, genes in the other cluster were down-regulated during light-limited growth but up-regulated during nitrogen-limited growth; this cluster included several genes involved in nitrogen uptake and assimilation. These results demonstrate complementary regulation of gene expression for two major metabolic activities of cyanobacteria. Comparison with batch-culture experiments revealed interesting differences in gene expression between batch and continuous culture and illustrates that continuous-culture experiments can pick up subtle changes in cell physiology and gene expression.

Reliable detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis by using multiplex qPCR including internal controls for nucleic acid extraction and amplification.

BACKGROUND: Several pathogens could seriously affect public health if not recognized timely. To reduce the impact of such highly pathogenic micro-organisms, rapid and accurate diagnostic tools are needed for their detection in various samples, including environmental samples. RESULTS: Multiplex real-time PCRs were designed for rapid and reliable detection of three major pathogens that have the potential to cause high morbidity and mortality in humans: B. anthracis, F. tularensis and Y. pestis. The developed assays detect three pathogen-specific targets, including at least one chromosomal target, and one target from B. thuringiensis which is used as an internal control for nucleic acid extraction from refractory spores as well as successful DNA amplification. Validation of the PCRs showed a high analytical sensitivity, specificity and coverage of diverse pathogen strains. CONCLUSIONS: The multiplex qPCR assays that were developed allow the rapid detection of 3 pathogen-specific targets simultaneously, without compromising sensitivity. The application of B. thuringiensis spores as internal controls further reduces false negative results. This ensures highly reliable detection, while template consumption and laboratory effort are kept at a minimum.

Transcript profiling indicates that the absence of PsbO affects the coordination of C and N metabolism in Synechocystis sp. PCC 6803.

Transcript profiling of nitrate-grown Synechocystis sp. PCC 6803 PsbO-free mutant cells in comparison to wild-type (WT) detected substantial deviations. Because we had previously observed phenotypical differences between Synechocystis sp. PCC 6803 WT and its corresponding PsbO-free mutant when cultivated with l-arginine as sole N source and a light intensity of 200 mumol photons m(-2) s(-1), we also performed transcript profiling for both strains grown either with nitrate or with l-arginine as sole N source. We observed a total number of 520 differentially regulated transcripts in Synechocystis WT because of a shift from nitrate- to l-arginine-containing BG11 medium, while we detected only 13 differentially regulated transcripts for the PsbO-free mutant. Thus, the PsbO-free Synechocystis mutant had already undergone a preconditioning process for growth with l-arginine in comparison to WT. While Synechocystis WT suffered from growth with l-arginine at a light intensity of 200 mumol photons m(-2) s(-1), the PsbO-free mutant developed only a minor stress phenotype. In summary, our results suggest that the absence of PsbO in Synechocystis affects the coordination of photosynthesis/respiration and l-arginine metabolism through complex probably redox-mediated regulatory pathways. In addition, we show that a comparison of the transcriptomes of nitrate-grown Synechococcus elongatus PCC 7942 WT cells and its corresponding PsbO-free mutant cells resulted in only a few differentially regulated transcripts between both strains. The absence of the manganese/calcium-stabilizing PsbO protein of PSII with an assigned regulatory function for photosynthetic water oxidation causes bigger changes in the transcriptome of the permissive photoheterotrophically growing Synechocystis sp. PCC 6803 than in the transcriptome of the obligate photoautotrophically growing S. elongatus PCC 7942.

A simple remedy against artifactual double bands in denaturing gradient gel electrophoresis.

Denaturant gradient gel electrophoresis (DGGE) is a widely used method for mutation analysis and for studies of microbial diversity. Particular combinations of target gene fragments and primers may give rise to erroneous DGGE profiles. We report on a very straightforward means to eliminate the artifactual 'double bands' that can be encountered in several applications.

Development and comparison of two assay formats for parallel detection of four biothreat pathogens by using suspension microarrays.

Microarrays provide a powerful analytical tool for the simultaneous detection of multiple pathogens. We developed diagnostic suspension microarrays for sensitive and specific detection of the biothreat pathogens Bacillus anthracis, Yersinia pestis, Francisella tularensis and Coxiella burnetii. Two assay chemistries for amplification and labeling were developed, one method using direct hybridization and the other using target-specific primer extension, combined with hybridization to universal arrays. Asymmetric PCR products for both assay chemistries were produced by using a multiplex asymmetric PCR amplifying 16 DNA signatures (16-plex). The performances of both assay chemistries were compared and their advantages and disadvantages are discussed. The developed microarrays detected multiple signature sequences and an internal control which made it possible to confidently identify the targeted pathogens and assess their virulence potential. The microarrays were highly specific and detected various strains of the targeted pathogens. Detection limits for the different pathogen signatures were similar or slightly higher compared to real-time PCR. Probit analysis showed that even a few genomic copies could be detected with 95% confidence. The microarrays detected DNA from different pathogens mixed in different ratios and from spiked or naturally contaminated samples. The assays that were developed have a potential for application in surveillance and diagnostics.