Rapid Reverse Purification DNA Extraction Approaches to Identify Microbial Pathogens in Wastewater.

Wastewater monitoring became a promising solution in the early detection of outbreaks. Despite the achievements in the identification of pathogens in wastewater using real-time PCR, there is still a lack of reliable rapid nucleic acid extraction protocols. Therefore, in this study, samples were subjected to alkali, proteinase K and/or bead-beating followed by reverse purification magnetic beads-based separation. Wastewater samples spiked with S. aureus, E. coli and C. parvum were used as examples for Gram-positive and -negative bacteria and protozoa, respectively. All results were compared with a spin column technology as a reference method. Proteinase K with bead beating (vortexing with 0.1 mm glass beads for three minutes) was particularly successful for bacterial DNA extraction (three- to five-fold increase). The most useful extraction protocol for protozoa was pre-treatment with proteinase K (eight-fold increase). The selected methods were sensitive as far as detecting one bacterial cell per reaction for S. aureus, ten bacterial cells for E. coli and two oocysts for C. parvum. The extraction reagents are cold chain independent and no centrifuge or other large laboratory equipment is required to perform DNA extraction. A controlled validation trial is needed to test the effectiveness at field levels.

Visible and red emissive molecular beacons for optical temperature measurements and quality control in diagnostic assays utilizing temperature-dependent amplification reactions.

Quality control requirements imposed on assays used in clinical diagnostics and point-of-care-diagnostic testing (POCT), utilizing amplification reactions performed at elevated temperatures of 35 to 95 °C are very stringent. As the temperature of a reaction vessel has a large impact on the specificity and sensitivity of the amplification reaction, simple tools for local in situ temperature sensing and monitoring are required for reaction and assay control. We describe here a platform of stem-and-loop structured DNA hairpins (molecular beacons, MBs), absorbing and emitting in the visible and red spectral region, rationally designed for precise temperature measurements in microfluidic assays for POCT, and their application for temperature measurements in a common DNA-based molecular biological assay utilizing thermophilic helicase-dependent amplification (tHDA). Spectroscopic studies of these MBs, rationally designed from DNA sequences of different thermal stabilities, chosen not to interact with the DNA probes applied in the nucleic acid amplification assay, and temperature-dependent fluorescence measurements of MB-assay mixtures revealed the suitability of these MBs for temperature measurements directly in such an assay with a temperature resolution of about 0.5 °C without interferences from assay components. Combining two spectrally distinguishable MBs provides a broader response range and an increase in temperature sensitivity up to 0.1 °C. This approach will find future application for temperature monitoring and quality control in commercialized diagnostics assays using dried reagents and microfluidic chips as well as assays read out with tube and microplate readers and PCR detection systems for temperature measurements in the range of 35 to 95 °C. Graphical abstract Molecular beacon platform for optical temperature measurements and quality control in diagnostic assays.

Archaeal transcriptional regulation of the prokaryotic KdpFABC complex mediating K(+) uptake in H. salinarum.

The genome of the halophilic archaeon Halobacterium salinarum encodes the high-affinity ATP-dependent K(+) uptake system Kdp. Previous studies have shown that the genes coding for the KdpFABC complex are arranged in a kdpFABCQ gene cluster together with an additional gene kdpQ. In bacteria, expression of the kdpFABC genes is generally regulated by the dedicated sensor kinase/response regulator pair KdpD/KdpE, which are absent in H. salinarum. Surprisingly, H. salinarum expresses the kdp genes in a manner which is strikingly similar to Escherichia coli. In this study, we show that the halobacterial kdpFABCQ genes constitute an operon and that kdpFABCQ expression is subject to a complex regulatory mechanism involving a negative transcriptional regulator and is further modulated via a so far unknown mechanism. We describe how the regulation of kdp gene expression is facilitated in H. salinarum in contrast to its bacterial counterparts. Whereas the Kdp system fulfils the same core function as an ATP-driven K(+) uptake system in both archaea and bacteria, the different mechanisms involved in the regulation of gene expression appear to have evolved separately, possibly reflecting a different physiological role of ATP-driven K(+) uptake in halophilic archaea.

On-chip analysis of respiratory viruses from nasopharyngeal samples.

Point-of-care (PoC) testing followed by personalized efficient therapy of infectious diseases may result in a considerable reduction of associated health care costs. Lab-on-a-chip (LoC) systems represent a potentially high efficient class of PoC tools. Here, we present a LoC system for automated pathogen analysis of respiratory viruses from nasopharyngeal specimens. The device prepares total nucleic acids from extracted swab samples using magnetic silica beads. After reverse transcription the co-purified viral RNA is amplified in accordance with the QIAplex multiplex PCR technology. Hybridized to corresponding QIAGEN LiquiChip beads and labelled with streptavidin R-phycoerythrin, the amplified target sequences are finally detected using a QIAGEN LiquiChip200 workstation. All chemicals needed are either stored freeze-dried on the disposable chip or are provided in liquid form in a reagent cartridge for up to 24 runs. Magnetic stir bars for mixing as well as turning valves with metering structures are integrated into the injection-moulded disposable chip. The core of the controlling instrument is a rotating heating bar construction providing fixed temperatures for fast cycling. PCR times of about half an hour (for 30 cycles) could be achieved for 120 µl reactions, making this system the fastest currently available high-volume PCR chip. The functionality of the system was shown by comparing automatically processed nasopharyngeal samples to ones processed manually according to the QIAGEN "ResPlex™ II Panel v2.0" respiratory virus detection kit. A prototype of the present instrument revealed slightly weaker signal intensities with a similar sensitivity in comparison to the commercially available kit and automated nucleic acid preparation devices, even without protocol optimization.

Transcriptional control by two leucine-responsive regulatory proteins in Halobacterium salinarum R1.

BACKGROUND: Archaea combine bacterial-as well as eukaryotic-like features to regulate cellular processes. Halobacterium salinarum R1 encodes eight leucine-responsive regulatory protein (Lrp)-homologues. The function of two of them, Irp (OE3923F) and lrpA1 (OE2621R), were analyzed by gene deletion and overexpression, including genome scale impacts using microarrays. RESULTS: It was shown that Lrp affects the transcription of multiple target genes, including those encoding enzymes involved in amino acid synthesis, central metabolism, transport processes and other regulators of transcription. In contrast, LrpA1 regulates transcription in a more specific manner. The aspB3 gene, coding for an aspartate transaminase, was repressed by LrpA1 in the presence of L-aspartate. Analytical DNA-affinity chromatography was adapted to high salt, and demonstrated binding of LrpA1 to its own promoter, as well as L-aspartate dependent binding to the aspB3 promoter. CONCLUSION: The gene expression profiles of two archaeal Lrp-homologues report in detail their role in H. salinarum R1. LrpA1 and Lrp show similar functions to those already described in bacteria, but in addition they play a key role in regulatory networks, such as controlling the transcription of other regulators. In a more detailed analysis ligand dependent binding of LrpA1 was demonstrated to its target gene aspB3.

Structural and biochemical characterization of a halophilic archaeal alkaline phosphatase.

Phosphate is an essential component of all cells that must be taken up from the environment. Prokaryotes commonly secrete alkaline phosphatases (APs) to recruit phosphate from organic compounds by hydrolysis. In this study, the AP from Halobacterium salinarum, an archaeon that lives in a saturated salt environment, has been functionally and structurally characterized. The core fold and the active-site architecture of the H. salinarum enzyme are similar to other AP structures. These generally form dimers composed of dominant beta-sheet structures sandwiched by alpha-helices and have well-accessible active sites. The surface of the enzyme is predicted to be highly negatively charged, like other proteins of extreme halophiles. In addition to the conserved core, most APs contain a crown domain that strongly varies within species. In the H. salinarum AP, the crown domain is made of an acyl-carrier-protein-like fold. Different from other APs, it is not involved in dimer formation. We compare the archaeal AP with its bacterial and eukaryotic counterparts, and we focus on the role of crown domains in enhancing protein stability, regulating enzyme function, and guiding phosphoesters into the active-site funnel.

Regulation of phosphate uptake via Pst transporters in Halobacterium salinarum R1.

The genome of the archaeon Halobacterium salinarum contains two copies of the pst (phosphate-specific transport) operon, the genes of which are related to well-studied bacterial homologues. Both operons (pst1 and pst2) were shown to be polycistronic and, when under P(i)-limited conditions, transcription initiated 1 bp upstream of the translational starts. Under P(i) saturation, the pst1 operon utilized an additional transcription start site 59 bp upstream of the first one. The leaderless pst1 transcript was found to be more efficiently translated than the leadered transcript. Promoter strengths differed significantly between the two operons and when P(i) levels changed. The basal pst1 promoter activity in P(i)-saturated conditions was minimal while the pst2 promoter was active. In contrast, phosphate limitation induced the pst1 operon threefold more than the pst2 operon. We identified basic and phosphate-dependent cis-acting elements in both promoters. Phosphate-uptake assays conducted with several Pst1 and Pst2 mutant strains revealed differences in the substrate affinities between the two transporters and also suggested that the P(i)-binding proteins PstS1 and PstS2 can interact with either of the two permease subunits of the transporters. The tactic behaviour of wild type and pst-deletion strains showed that the Pst1 transporter plays an important role for phosphate-directed chemotaxis.

Phosphate-dependent behavior of the archaeon Halobacterium salinarum strain R1.

Phosphate is essential for life on earth, since it is an integral part of important biomolecules. The mechanisms applied by bacteria and eukarya to combat phosphate limitation are fairly well understood. However, it is not known how archaea sense phosphate limitation or which genes are regulated upon limitation. We conducted a microarray analysis to explore the phosphate-dependent gene expression of Halobacterium salinarum strain R1. We identified a set of 17 genes whose transcript levels increased up to several hundredfold upon phosphate limitation. Analysis of deletion mutants showed that this set of genes, the PHO stimulon, is very likely independent of signaling via two-component systems. Our experiments further indicate that PHO stimulon induction might be dependent on the intracellular phosphate concentration, which turned out to be subject to substantial changes. Finally, the study revealed that H. salinarum exhibits a phosphate-directed chemotaxis, which is induced by phosphate starvation.

Microarray analysis in the archaeon Halobacterium salinarum strain R1.

BACKGROUND: Phototrophy of the extremely halophilic archaeon Halobacterium salinarum was explored for decades. The research was mainly focused on the expression of bacteriorhodopsin and its functional properties. In contrast, less is known about genome wide transcriptional changes and their impact on the physiological adaptation to phototrophy. The tool of choice to record transcriptional profiles is the DNA microarray technique. However, the technique is still rarely used for transcriptome analysis in archaea. METHODOLOGY/PRINCIPAL FINDINGS: We developed a whole-genome DNA microarray based on our sequence data of the Hbt. salinarum strain R1 genome. The potential of our tool is exemplified by the comparison of cells growing under aerobic and phototrophic conditions, respectively. We processed the raw fluorescence data by several stringent filtering steps and a subsequent MAANOVA analysis. The study revealed a lot of transcriptional differences between the two cell states. We found that the transcriptional changes were relatively weak, though significant. Finally, the DNA microarray data were independently verified by a real-time PCR analysis. CONCLUSION/SIGNIFICANCE: This is the first DNA microarray analysis of Hbt. salinarum cells that were actually grown under phototrophic conditions. By comparing the transcriptomics data with current knowledge we could show that our DNA microarray tool is well applicable for transcriptome analysis in the extremely halophilic archaeon Hbt. salinarum. The reliability of our tool is based on both the high-quality array of DNA probes and the stringent data handling including MAANOVA analysis. Among the regulated genes more than 50% had unknown functions. This underlines the fact that haloarchaeal phototrophy is still far away from being completely understood. Hence, the data recorded in this study will be subject to future systems biology analysis.

The genome of the square archaeon Haloquadratum walsbyi : life at the limits of water activity.

BACKGROUND: The square halophilic archaeon Haloquadratum walsbyi dominates NaCl-saturated and MgCl2 enriched aquatic ecosystems, which imposes a serious desiccation stress, caused by the extremely low water activity. The genome sequence was analyzed and physiological and physical experiments were carried out in order to reveal how H. walsbyi has specialized into its narrow and hostile ecological niche and found ways to cope with the desiccation stress. RESULTS: A rich repertoire of proteins involved in phosphate metabolism, phototrophic growth and extracellular protective polymers, including the largest archaeal protein (9159 amino acids), a homolog to eukaryotic mucins, are amongst the most outstanding features. A relatively low GC content (47.9%), 15-20% less than in other halophilic archaea, and one of the lowest coding densities (76.5%) known for prokaryotes might be an indication for the specialization in its unique environment CONCLUSION: Although no direct genetic indication was found that can explain how this peculiar organism retains its square shape, the genome revealed several unique adaptive traits that allow this organism to thrive in its specific and extreme niche.

Regulation of the Bacillus subtilis heat shock gene htpG is under positive control.

The heat shock genes of Bacillus subtilis are assigned to four classes on the basis of their regulation mechanisms. While classes I and III are negatively controlled by two different transcriptional repressors, class II is regulated by the alternative sigma factor sigma(B). All heat shock genes with unidentified regulatory mechanisms, among them htpG, constitute class IV. Here, we show that expression of htpG is under positive control. We identified a DNA sequence (GAAAAGG) located downstream of the sigma(A)-dependent promoter of htpG. The heat inducibility of the promoter could be destroyed by inversion, nucleotide replacements, or removal of this DNA sequence. Fusion of this sequence to the vegetative lepA promoter conferred heat inducibility. Furthermore, we were able to show that the heat induction factor is dependent on the absolute temperature rather than the temperature increment and that nonnative proteins within the cytoplasm fail to induce htpG.