Presence and expression of hydrogenase specific C-terminal endopeptidases in cyanobacteria

BACKGROUND: Hydrogenases catalyze the simplest of all chemical reactions: the reduction of protons to molecular hydrogen or vice versa. Cyanobacteria can express an uptake, a bidirectional or both NiFe-hydrogenases. Maturation of those depends on accessory proteins encoded by hyp-genes. The last maturation step involves the cleavage of a ca. 30 amino acid long peptide from the large subunit by a C-terminal endopeptidase. Until know, nothing is known about the maturation of cyanobacterial NiFe-hydrogenases. The availability of three complete cyanobacterial genome sequences from strains with either only the uptake (Nostoc punctiforme ATCC 29133/PCC 73102), only the bidirectional (Synechocystis PCC 6803) or both NiFe-hydrogenases (Anabaena PCC 7120) prompted us to mine these genomes for hydrogenase maturation related genes. In this communication we focus on the presence and the expression of the NiFe-hydrogenases and the corresponding C-terminal endopeptidases, in the three strains mentioned above. RESULTS: We identified genes encoding putative cyanobacterial hydrogenase specific C-terminal endopeptidases in all analyzed cyanobacterial genomes. The genes are not part of any known hydrogenase related gene cluster. The derived amino acid sequences show only low similarity (28–41%) to the well-analyzed hydrogenase specific C-terminal endopeptidase HybD from Escherichia coli, the crystal structure of which is known. However, computational secondary and tertiary structure modeling revealed the presence of conserved structural patterns around the highly conserved active site. Gene expression analysis shows that the endopeptidase encoding genes are expressed under both nitrogen-fixing and non-nitrogen-fixing conditions. CONCLUSION: Anabaena PCC 7120 possesses two NiFe-hydrogenases and two hydrogenase specific C-terminal endopeptidases but only one set of hyp-genes. Thus, in contrast to the Hyp-proteins, the C-terminal endopeptidases are the only known hydrogenase maturation factors that are specific. Therefore, in accordance with previous nomenclature, we propose the gene names hoxW and hupW for the bidirectional and uptake hydrogenase processing endopeptidases, respectively. Due to their constitutive expression we expect that, at least in cyanobacteria, the endopeptidases take over multiple functions

Webtag: a new web tool providing tags/anchors for RT-PCR experiments with prokaryotes

<p>Abstract</p> <p>Background</p> <p>Webtag is a tool providing oligonucleotide sequences (usually called tags or anchors) that are absent from a specified genome. These tags/anchors can be appended to gene specific primers for reverse transcriptase polymerase chain reaction experiments, circumventing genomic DNA contamination.</p> <p>Results</p> <p>The use of a relational database, in conjunction with a series of scripts written in PHP and Perl, allows the user to rapidly obtain tags that are: 1) suitable for a specific organism, and 2) compatible with other oligonucleotides to be used in the experimental procedures.</p> <p>Conclusion</p> <p>This new web tool allows scientists to easily and rapidly obtain suitable tags for RT-PCR experiments, and is available at <url>http://www.egs.uu.se/software/webtag/</url>.</p

Generation of non-genomic oligonucleotide tag sequences for RNA template-specific PCR

BACKGROUND: In order to overcome genomic DNA contamination in transcriptional studies, reverse template-specific polymerase chain reaction, a modification of reverse transcriptase polymerase chain reaction, is used. The possibility of using tags whose sequences are not found in the genome further improves reverse specific polymerase chain reaction experiments. Given the absence of software available to produce genome suitable tags, a simple tool to fulfill such need was developed. RESULTS: The program was developed in Perl, with separate use of the basic local alignment search tool, making the tool platform independent (known to run on Windows XP and Linux). In order to test the performance of the generated tags, several molecular experiments were performed. The results show that Tagenerator is capable of generating tags with good priming properties, which will deliberately not result in PCR amplification of genomic DNA. CONCLUSION: The program Tagenerator is capable of generating tag sequences that combine genome absence with good priming properties for RT-PCR based experiments, circumventing the effects of genomic DNA contamination in an RNA sample

Diversity and transcription of proteases involved in the maturation of hydrogenases in Nostoc punctiforme ATCC 29133 and Nostoc sp. strain PCC 7120

<p>Abstract</p> <p>Background</p> <p>The last step in the maturation process of the large subunit of [NiFe]-hydrogenases is a proteolytic cleavage of the C-terminal by a hydrogenase specific protease. Contrary to other accessory proteins these hydrogenase proteases are believed to be specific whereby one type of hydrogenases specific protease only cleaves one type of hydrogenase. In cyanobacteria this is achieved by the gene product of either <it>hupW </it>or <it>hoxW</it>, specific for the uptake or the bidirectional hydrogenase respectively. The filamentous cyanobacteria <it>Nostoc punctiforme </it>ATCC 29133 and <it>Nostoc </it>sp strain PCC 7120 may contain a single uptake hydrogenase or both an uptake and a bidirectional hydrogenase respectively.</p> <p>Results</p> <p>In order to examine these proteases in cyanobacteria, transcriptional analyses were performed of <it>hupW </it>in <it>Nostoc punctiforme </it>ATCC 29133 and <it>hupW </it>and <it>hoxW </it>in <it>Nostoc </it>sp. strain PCC 7120. These studies revealed numerous transcriptional start points together with putative binding sites for NtcA (<it>hupW</it>) and LexA (<it>hoxW</it>). In order to investigate the diversity and specificity among hydrogeanse specific proteases we constructed a phylogenetic tree which revealed several subgroups that showed a striking resemblance to the subgroups previously described for [NiFe]-hydrogenases. Additionally the proteases specificity was also addressed by amino acid sequence analysis and protein-protein docking experiments with 3D-models derived from bioinformatic studies. These studies revealed a so called "HOXBOX"; an amino acid sequence specific for protease of Hox-type which might be involved in docking with the large subunit of the hydrogenase.</p> <p>Conclusion</p> <p>Our findings suggest that the hydrogenase specific proteases are under similar regulatory control as the hydrogenases they cleave. The result from the phylogenetic study also indicates that the hydrogenase and the protease have co-evolved since ancient time and suggests that at least one major horizontal gene transfer has occurred. This co-evolution could be the result of a close interaction between the protease and the large subunit of the [NiFe]-hydrogenases, a theory supported by protein-protein docking experiments performed with 3D-models. Finally we present data that may explain the specificity seen among hydrogenase specific proteases, the so called "HOXBOX"; an amino acid sequence specific for proteases of Hox-type. This opens the door for more detailed studies of the specificity found among hydrogenase specific proteases and the structural properties behind it.</p

Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120

<p>Abstract</p> <p>Background</p> <p>The maturation of hydrogenases into active enzymes is a complex process and e.g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by <it>hypABCDEF </it>and a hydrogenase specific protease, encoded either by <it>hupW </it>or <it>hoxW</it>. The N₂-fixing cyanobacterium <it>Nostoc </it>sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of <it>hyp</it>-genes in <it>Nostoc </it>sp. strain PCC 7120.</p> <p>Results</p> <p>RT-PCRs demonstrated that the six <it>hyp</it>-genes together with one ORF may be transcribed as a single operon. Transcriptional start points (TSPs) were identified 280 bp upstream from <it>hypF </it>and 445 bp upstream of <it>hypC</it>, respectively, demonstrating the existence of several transcripts. In addition, five upstream ORFs located in between <it>hupSL</it>, encoding the small and large subunits of the uptake hydrogenase, and the <it>hyp</it>-operon, and two downstream ORFs from the <it>hyp</it>-genes were shown to be part of the same transcript unit. A third TSP was identified 45 bp upstream of asr0689, the first of five ORFs in this operon. The ORFs are annotated as encoding unknown proteins, with the exception of alr0692 which is identified as a NifU-like protein. Orthologues of the four ORFs asr0689-alr0692, with a highly conserved genomic arrangement positioned between <it>hupSL</it>, and the <it>hyp </it>genes are found in several other N₂-fixing cyanobacteria, but are absent in non N₂-fixing cyanobacteria with only the bidirectional hydrogenase. Short conserved sequences were found in six intergenic regions of the extended <it>hyp</it>-operon, appearing between 11 and 79 times in the genome.</p> <p>Conclusion</p> <p>This study demonstrated that five ORFs upstream of the <it>hyp</it>-gene cluster are co-transcribed with the <it>hyp</it>-genes, and identified three TSPs in the extended <it>hyp</it>-gene cluster in <it>Nostoc </it>sp. strain PCC 7120. This may indicate a function related to the assembly of a functional uptake hydrogenase, hypothetically in the assembly of the small subunit of the enzyme.</p

Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria

<p>Abstract</p> <p>Background</p> <p>The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll <it>a </it>and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of <it>Nostoc punctiforme </it>ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.</p> <p>Results</p> <p>With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution.</p> <p>Conclusion</p> <p>It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.</p

Characterization of the hupSL promoter activity in Nostoc punctiforme ATCC 29133

<p>Abstract</p> <p>Background</p> <p>In cyanobacteria three enzymes are directly involved in the hydrogen metabolism; a nitrogenase that produces molecular hydrogen, H₂, as a by-product of nitrogen fixation, an uptake hydrogenase that recaptures H₂and oxidize it, and a bidirectional hydrogenase that can both oxidize and produce H₂.<it>Nostoc punctiforme </it>ATCC 29133 is a filamentous dinitrogen fixing cyanobacterium containing a nitrogenase and an uptake hydrogenase but no bidirectional hydrogenase. Generally, little is known about the transcriptional regulation of the cyanobacterial uptake hydrogenases. In this study gel shift assays showed that NtcA has a specific affinity to a region of the <it>hupSL </it>promoter containing a predicted NtcA binding site. The predicted NtcA binding site is centred at 258.5 bp upstream the transcription start point (tsp). To further investigate the <it>hupSL </it>promoter, truncated versions of the <it>hupSL </it>promoter were fused to either <it>gfp </it>or <it>luxAB</it>, encoding the reporter proteins Green Fluorescent Protein and Luciferase, respectively.</p> <p>Results</p> <p>Interestingly, all <it>hupsSL </it>promoter deletion constructs showed heterocyst specific expression. Unexpectedly the shortest promoter fragment, a fragment covering 57 bp upstream and 258 bp downstream the tsp, exhibited the highest promoter activity. Deletion of the NtcA binding site neither affected the expression to any larger extent nor the heterocyst specificity.</p> <p>Conclusion</p> <p>Obtained data suggest that the <it>hupSL </it>promoter in <it>N. punctiforme </it>is not strictly dependent on the upstream NtcA cis element and that the shortest promoter fragment (-57 to tsp) is enough for a high and heterocyst specific expression of <it>hupSL</it>. This is highly interesting because it indicates that the information that determines heterocyst specific gene expression might be confined to this short sequence or in the downstream untranslated leader sequence.</p

Dextran 500 Improves Recovery of Inflammatory Markers: An In Vitro Microdialysis Study.

Cerebral microdialysis (CMD) is used in severe traumatic brain injury (TBI) in order to recover metabolites in brain extracellular fluid (ECF). To recover larger proteins and avoid fluid loss, albumin supplemented perfusion fluid (PF) has been utilized, but because of regulatory changes in the European Union, this is no longer practicable. The aim with this study was to see whether fluid, absolute (AR), and relative (RR) recovery for the novel carrier, Dextran 500, was better than conventional PF for a range of cytokines and chemokines. An in vitro setup mimicking conditions observed in the neurocritical care of TBI patients was used, utilizing 100-kDa molecular-weight cut-off CMD catheters inserted through a triple-lumen bolt cranial access device into an external solution with diluted cytokine standards in known concentrations for 48 h (divided into 6-h epochs). Samples were run on a 39-plex Luminex (Luminex Corporation, Austin, TX) assay to assess cytokine concentrations. We found that fluid recovery was inadequate in 50% of epochs with conventional PF, whereas Dextran PF overcame this limitation. The AR was higher in the Dextran PF samples for a majority of cytokines, and RR was significantly increased for macrophage colony-stimulating factor and transforming growth factor-alpha. In summary, Dextran PF improved fluid and cytokine recovery as compared to conventional PF and is a suitable alternative to albumin supplemented PF for protein microdialysis.The work was supported by funding for SGC and KLHC from the National Institute for Health Research Biomedical Research Centre, Cambridge (Neuroscience Theme; Brain Injury and Repair Theme). PJH is funded by a National Institute for Health Research (NIHR) Professorship, Academy of Medical Sciences/Health Foundation Senior Surgical Scientist Fellowship and the National Institute for Health Research Biomedical Research Centre, Cambridge. EPT has received salary support from Swedish Society for Medical Research. AH is supported by the Royal College of Surgeons of England and the National Institute for Health Research Biomedical Research Centre, Cambridge. The study consumables were purchased through the NIHR Research Professorship (Peter Hutchinson) and the Luminex 200 analyser was purchased with Medical Research Council (MRC) funding (G0600986 ID79068)

Engineering of a promoter repressed by a light-regulated transcription factor in escherichia coli

Light-regulated gene expression systems allow controlling gene expression in space and time with high accuracy. Contrary to previous synthetic light sensors that incorporate two-component systems which require localization at the plasma membrane, soluble one-component repression systems provide several advantageous characteristics. Firstly, they are soluble and able to diffuse across the cytoplasm. Secondly, they are smaller and of lower complexity, enabling less taxing expression and optimization of fewer parts. Thirdly, repression through steric hindrance is a widespread regulation mechanism that does not require specific interaction with host factors, potentially enabling implementation in different organisms. Herein, we present the design of the synthetic promoter PEL that in combination with the light-regulated dimer EL222 constitutes a one-component repression system. Inspired by previously engineered synthetic promoters and the Escherichia coli lacZYA promoter, we designed PEL with two EL222 operators positioned to hinder RNA polymerase binding when EL222 is bound. PEL is repressed by EL222 under conditions of white light with a light-regulated repression ratio of five. Further, alternating conditions of darkness and light in cycles as short as one hour showed that repression is reversible. The design of the PEL-EL222 system herein presented could aid the design and implementation of analogous one-component optogenetic repression systems. Finally, we compare the PEL-EL222 system with similar systems and suggest general improvements that could optimize and extend the functionality of EL222-based as well as other one-component repression systems

Assessment of Platelet Function in Traumatic Brain Injury-A Retrospective Observational Study in the Neuro-Critical Care Setting.

BACKGROUND: Despite seemingly functional coagulation, hemorrhagic lesion progression is a common and devastating condition following traumatic brain injury (TBI), stressing the need for new diagnostic techniques. Multiple electrode aggregometry (MEA) measures platelet function and could aid in coagulopathy assessment following TBI. The aims of this study were to evaluate MEA temporal dynamics, influence of concomitant therapy, and its capabilities to predict lesion progression and clinical outcome in a TBI cohort. MATERIAL AND METHODS: Adult TBI patients in a neurointensive care unit that underwent MEA sampling were retrospectively included. MEA was sampled if the patient was treated with antiplatelet therapy, bled heavily during surgery, or had abnormal baseline coagulation values. We assessed platelet activation pathways involving the arachidonic acid receptor (ASPI), P2Y12 receptor, and thrombin receptor (TRAP). ASPI was the primary focus of analysis. If several samples were obtained, they were included. Retrospective data were extracted from hospital charts. Outcome variables were radiologic hemorrhagic progression and Glasgow Outcome Scale assessed prospectively at 12 months posttrauma. MEA levels were compared between patients on antiplatelet therapy. Linear mixed effect models and uni-/multivariable regression models were used to study longitudinal dynamics, hemorrhagic progression and outcome, respectively. RESULTS: In total, 178 patients were included (48% unfavorable outcome). ASPI levels increased from initially low values in a time-dependent fashion (p < 0.001). Patients on cyclooxygenase inhibitors demonstrated low ASPI levels (p < 0.001), while platelet transfusion increased them (p < 0.001). The first ASPI (p = 0.039) and TRAP (p = 0.009) were significant predictors of outcome, but not lesion progression, in univariate analyses. In multivariable analysis, MEA values were not independently correlated with outcome. CONCLUSION: A general longitudinal trend of MEA is identified in this TBI cohort, even in patients without known antiplatelet therapies. Values appear also affected by platelet inhibitory treatment and by platelet transfusions. While significant in univariate models to predict outcome, MEA values did not independently correlate to outcome or lesion progression in multivariable analyses. Further prospective studies to monitor coagulation in TBI patients are warranted, in particular the interpretation of pathological MEA values in patients without antiplatelet therapies