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

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

Homologous overexpression of NpDps2 and NpDps5 increases the tolerance for oxidative stress in the multicellular cyanobacterium Nostoc punctiforme

The filamentous cyanobacterium Nostoc punctiforme has several oxidative stress-managing systems, including Dps proteins. Dps proteins belong to the ferritin superfamily and are involved in abiotic stress management in prokaryotes. Previously, we found that one of the five Dps proteins in N. punctiforme, NpDps2, was critical for H2O2 tolerance. Stress induced by high light intensities is aggravated in N. punctiforme strains deficient of either NpDps2, or the bacterioferritin-like NpDps5. Here, we have investigated the capacity of NpDps2 and NpDps5 to enhance stress tolerance by homologous overexpression of these two proteins in N. punctiforme. Both overexpression strains were found to tolerate twice as high concentrations of added H2O2 as the control strain, indicating that overexpression of either NpDps2 or NpDps5 will enhance the capacity for H2O2 tolerance. Under high light intensities, the overexpression of the two NpDps did not enhance the tolerance against general light-induced stress. However, overexpression of the heterocyst-specific NpDps5 in all cells of the filament led to a higher amount of chlorophyll-binding proteins per cell during diazotrophic growth. The OENpDps5 strain also showed an increased tolerance to ammonium-induced oxidative stress. Our results provide information of how Dps proteins may be utilised for engineering of cyanobacteria with enhanced stress tolerance

Photoautotrophic production of renewable ethylene by engineered cyanobacteria: Steering the cell metabolism towards biotechnological use

Ethylene is a volatile hydrocarbon with a massive global market in the plastic industry. The ethylene now used for commercial applications is produced exclusively from nonrenewable petroleum sources, while competitive biotechnological production systems do not yet exist. This review focuses on the currently developed photoautotrophic bioproduction strategies that enable direct solar-driven conversion of CO2 into ethylene, based on the use of genetically engineered photosynthetic cyanobacteria expressing heterologous ethylene forming enzyme (EFE) from Pseudomonas syringae. The emphasis is on the different engineering strategies to express EFE and to direct the cellular carbon flux towards the primary metabolite 2-oxoglutarate, highlighting associated metabolic constraints, and technical considerations on cultivation strategies and conditional parameters. While the research field has progressed towards more robust strains with better production profiles, and deeper understanding of the associated metabolic limitations, it is clear that there is room for significant improvement to reach industrial relevance. At the same time, existing information and the development of synthetic biology tools for engineering cyanobacteria open new possibilities for improving the prospects for the sustainable production of renewable ethylene

Transcript analysis of the extended hyp-operon in the cyanobacteria Nostoc sp. strain PCC 7120 and Nostoc punctiforme ATCC 29133

<p>Abstract</p> <p>Background</p> <p>Cyanobacteria harbor two [NiFe]-type hydrogenases consisting of a large and a small subunit, the Hup- and Hox-hydrogenase, respectively. Insertion of ligands and correct folding of nickel-iron hydrogenases require assistance of accessory maturation proteins (encoded by the <it>hyp</it>-genes). The intergenic region between the structural genes encoding the uptake hydrogenase (<it>hupSL</it>) and the accessory maturation proteins (<it>hyp </it>genes) in the cyanobacteria <it>Nostoc </it>PCC 7120 and <it>N. punctiforme </it>were analysed using molecular methods.</p> <p>Findings</p> <p>The five ORFs, located in between the uptake hydrogenase structural genes and the <it>hyp</it>-genes, can form a transcript with the <it>hyp</it>-genes. An identical genomic localization of these ORFs are found in other filamentous, N₂-fixing cyanobacterial strains. In <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120 the ORFs upstream of the <it>hyp</it>-genes showed similar transcript level profiles as <it>hupS </it>(hydrogenase structural gene), <it>nifD </it>(nitrogenase structural gene), <it>hypC </it>and <it>hypF </it>(accessory hydrogenase maturation genes) after nitrogen depletion. <it>In silico </it>analyzes showed that these ORFs in <it>N. punctiform</it>e harbor the same conserved regions as their homologues in <it>Nostoc </it>PCC 7120 and that they, like their homologues in <it>Nostoc </it>PCC 7120, can be transcribed together with the <it>hyp</it>-genes forming a larger extended <it>hyp-</it>operon. DNA binding studies showed interactions of the transcriptional regulators CalA and CalB to the promoter regions of the extended <it>hyp</it>-operon in <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120.</p> <p>Conclusions</p> <p>The five ORFs upstream of the <it>hyp</it>-genes in several filamentous N₂-fixing cyanobacteria have an identical genomic localization, in between the genes encoding the uptake hydrogenase and the maturation protein genes. In <it>N. punctiforme </it>and <it>Nostoc </it>PCC 7120 they are transcribed as one operon and may form transcripts together with the <it>hyp</it>-genes. The expression pattern of the five ORFs within the extended <it>hyp</it>-operon in both <it>Nostoc punctiforme </it>and <it>Nostoc </it>PCC 7120 is similar to the expression patterns of <it>hupS</it>, <it>nifD</it>, <it>hypF </it>and <it>hypC</it>. CalA, a known transcription factor, interacts with the promoter region between <it>hupSL </it>and the five ORFs in the extended <it>hyp</it>-operon in both <it>Nostoc </it>strains.</p

The Effect of Iron Limitation on the Transcriptome and Proteome of Pseudomonas fluorescens Pf-5

One of the most important micronutrients for bacterial growth is iron, whose bioavailability in soil is limited. Consequently, rhizospheric bacteria such as Pseudomonas fluorescens employ a range of mechanisms to acquire or compete for iron. We investigated the transcriptomic and proteomic effects of iron limitation on P. fluorescens Pf-5 by employing microarray and iTRAQ techniques, respectively. Analysis of this data revealed that genes encoding functions related to iron homeostasis, including pyoverdine and enantio-pyochelin biosynthesis, a number of TonB-dependent receptor systems, as well as some inner-membrane transporters, were significantly up-regulated in response to iron limitation. Transcription of a ribosomal protein L36-encoding gene was also highly up-regulated during iron limitation. Certain genes or proteins involved in biosynthesis of secondary metabolites such as 2,4-diacetylphloroglucinol (DAPG), orfamide A and pyrrolnitrin, as well as a chitinase, were over-expressed under iron-limited conditions. In contrast, we observed that expression of genes involved in hydrogen cyanide production and flagellar biosynthesis were down-regulated in an iron-depleted culture medium. Phenotypic tests revealed that Pf-5 had reduced swarming motility on semi-solid agar in response to iron limitation. Comparison of the transcriptomic data with the proteomic data suggested that iron acquisition is regulated at both the transcriptional and post-transcriptional levels

Doing synthetic biology with photosynthetic microorganisms

The use of photosynthetic microbes as synthetic biology hosts for the sustainable production of commodity chemicals and even fuels has received increasing attention over the last decade. The number of studies published, tools implemented, and resources made available for microalgae have increased beyond expectations during the last few years. However, the tools available for genetic engineering in these organisms still lag those available for the more commonly used heterotrophic host organisms. In this mini-review, we provide an overview of the photosynthetic microbes most commonly used in synthetic biology studies, namely cyanobacteria, chlorophytes, eustigmatophytes and diatoms. We provide basic information on the techniques and tools available for each model group of organisms, we outline the state-of-the-art, and we list the synthetic biology tools that have been successfully used. We specifically focus on the latest CRISPR developments, as we believe that precision editing and advanced genetic engineering tools will be pivotal to the advancement of the field. Finally, we discuss the relative strengths and weaknesses of each group of organisms and examine the challenges that need to be overcome to achieve their synthetic biology potential.Peer reviewe

Det ansvarsfulla företaget. En språkvetenskaplig studie av årsredovisningsgenrens expansion och utveckling under åren 1981-2001

Den här studien syftar till att åskådliggöra hur årsredovisningsgenren har expanderat och utvecklats under åren 1981-2001. Uppsatsen innehåller dels en kvantitativ undersökning av vilka textavsnitt som tillkommit i årsredovisningarna under undersökningsperioden, dels en djupare analys av dispositionen och textmönstret i de texter som behandlar olika aspekter av begreppet ”Det ansvarsfulla företaget”. Undersökningsmaterialet består av årsredovisningar från de svenska storföretagen Hennes och Mauritz, Trelleborg och Volvo. Undersökningen visar att både de obligatoriska avsnitten, som årsredovisningen enligt lag måste innehålla, och de kompletterande ökar i omfattning under den aktuella tidsperioden, men att de kompletterande avsnitten dominerar storleksmässigt. Utmärkande för de analyserade årsredovisningstexterna är att samtliga har en övergripande, tematisk disposition och att styckenas innehåll är specifikt och additivt disponerat. Urvalet för textanalysen och den kvantitativa undersökningen synliggör hur begreppet ”Det ansvarsfulla företaget” får sitt genomslag i mitten av 1990-talet och att det med tiden kommer att omfatta alltfler ämnesområden. Beskrivningarna av hur företagen engagerar sig i humanitära och miljörelaterade frågor är ett återkommande drag i årsredovisningarnas textmönster och de visar att företagen försöker möta omvärldens krav på att de skall ta ett större samhälligt ansvar. Det är tydligt att årsredovisningarna har fått en bredare publik genom åren och att deras texter inte längre bara är avsedda för företagens aktieägare. Begreppet ”Det ansvarsfulla företaget” omfattar med tiden även företagens personalpolitik. Årsredovisningarna presenterar inte längre enbart ett ekonomiskt perspektiv på de frågor som rör personalen, utan uttrycker en ambition att se till varje anställds individuella behov och utveckling. I slutet av undersökningsperioden är ”Det ansvarsfulla företaget” ett etablerat begrepp inom årsredovisningsgenren och i vissa avseenden har nu fokus flyttats från företagens affärsverksamhet till deras sociala engagemang