Convenient broad-host-range unstable vectors for studying stabilization cassettes in diverse bacteria

Background: Low-copy-number vectors of potential wide application in biotechnology need to encode stabilization modules ensuring their stable inheritance. The efficiency of stabilization may vary depending on the plasmid host so a thorough analysis of stabilization functions is required before use. Results: To facilitate such analysis highly unstable, mobilizable, broad-host-range (BHR) vectors based on RK2 replicon were constructed. The vectors are suitable for testing of various stabilization functions, including plasmid and chromosomal partitioning cassettes encoding ParB homologues capable of spreading on DNA. The xylE or lacZ reporter systems facilitate easy monitoring of plasmid segregation. Conclusion: The range of BHR vectors with different reporter cassettes and alternative mobilization systems expands their application in diverse bacterial species

Mutation in HvCBP20 (Cap binding protein 20) adapts barley to drought stress at phenotypic and transcriptomic levels

This work was supported by the European Regional Development Fund through the Innovative Economy for Poland 2007–2013, project WND-POIG.01.03.01-00-101/08 POLAPGEN-BD “Biotechnological tools for breeding cereals with increased resistance to drought,” task 22; National Science Centre, Poland, project SONATA 2015/19/D/NZ9/03573 “Translational genomics approach to identify the mechanisms of CBP20 signalosome in Arabidopsis and barley under drought stress.”CBP20 (Cap-Binding Protein 20) encodes a small subunit of the cap-binding complex (CBC), which is involved in the conserved cell processes related to RNA metabolism in plants and, simultaneously, engaged in the signaling network of drought response, which is dependent on ABA. Here, we report the enhanced tolerance to drought stress of barley mutant in the HvCBP20 gene manifested at the morphological, physiological, and transcriptomic levels. Physiological analyses revealed differences between the hvcbp20.ab mutant and its WT in response to a water deficiency. The mutant exhibited a higher relative water content (RWC), a lower stomatal conductance and changed epidermal pattern compared to the WT after drought stress. Transcriptome analysis using the Agilent Barley Microarray integrated with observed phenotypic traits allowed to conclude that the hvcbp20.ab mutant exhibited better fitness to stress conditions by its much more efficient and earlier activation of stress-preventing mechanisms. The network hubs involved in the adjustment of hvcbp20.ab mutant to the drought conditions were proposed. These results enabled to make a significant progress in understanding the role of CBP20 in the drought stress response.European Regional Development Fund; National Science Centre, Polan

MobC of conjugative RA3 plasmid from IncU group autoregulates the expression of bicistronic mobC-nic operon and stimulates conjugative transfer

The IncU conjugative transfer module represents highly efficient promiscuous system widespread among conjugative plasmids of different incompatibility groups. Despite its frequent occurrence the mechanisms of relaxosome formation/action are far from understood. Here we analyzed the putative transfer auxiliary protein MobC of the conjugative plasmid RA3 from the IncU incompatibility group. MobC is a protein of 176 amino acids encoded in the bicistronic operon mobC-nic adjacent to oriT. MobC is homologous to prokaryotic transcription factors of the ribbon-helix-helix (RHH) superfamily. Conserved LxxugxNlNQiaxxLn motif clusters MobC with the clade of conjugative transfer auxilliary proteins of MobP relaxases. MobC forms dimers and tetramers in solution and autoregulates the expression of mobCp by binding to an imperfect palindromic sequence (OM) located between putative -35 and -10 motifs of the promoter. Medium-copy number test plasmid containing the oriT-mobCp region is mobilized with a high frequency by the RA3 conjugative system. The mutations introduced into OM that abolished MobC binding in vitro decreased 2-3 fold the frequency of mobilization of the test plasmids. The deletion of OM within the RA3 conjugative module had no effect on transfer if the mobC-nic operon was expressed from the heterologous promoter. If only nic was expressed from the heterologous promoter (no mobC) the conjugative transfer frequency of such plasmid was 1000-fold lower. The MobC is an auxiliary transfer protein of dual function. It autoregulates the expression of mobC-nic operon while its presence significantly stimulates transfer efficiency

1st FPGA Developers' Forum (FDF) meeting

Large FPGA firmware designs, such as the ones used in the trigger systems of HEP experiments, typically contain many hundreds of configuration/status registers and memories. Managing the required HDL code and software for these can become challenging. We therefore developed a dedicated tool, called HardwareCompiler, which parses an XML description of the registers and memories and generates the required HDL code as well as C++ access functions used to configure and monitor the modules. The tool has been successfully applied in several generations of FPGA-based modules developed for the ATLAS Central Trigger system, greatly simplifying their development and testing. We present the capabilities of the HardwareCompiler with examples of generated VHDL register packages and address decoders as well as low-level C++ software. The latter is also used to generate wrappers for Python, which simplifies the development of scripts for configuring and testing the hardware

Global Transcriptional Regulation of Backbone Genes in Broad-Host-Range Plasmid RA3 from the IncU Group Involves Segregation Protein KorB (ParB Family).

The KorB protein of the broad-host-range conjugative plasmid RA3 from the IncU group belongs to the ParB family of plasmid and chromosomal segregation proteins. As a partitioning DNA-binding factor, KorB specifically recognizes a 16-bp palindrome which is an essential motif in the centromere-like sequenceparSRA3, forms a segrosome, and together with its partner IncC (ParA family) participates in active DNA segregation ensuring stable plasmid maintenance. Here we show that by binding to this palindromic sequence, KorB also acts as a repressor for the adjacentmobCpromoter driving expression of themobC-nicoperon, which is involved in DNA processing during conjugation. Three other promoters, one buried in the conjugative transfer module and two divergent promoters located at the border between the replication and stability regions, are regulated by KorB binding to additional KorB operators (OBs). KorB acts as a repressor at a distance, binding to OBs separated from their cognate promoters by between 46 and 1,317 nucleotides. This repressor activity is facilitated by KorB spreading along DNA, since a polymerization-deficient KorB variant with its dimerization and DNA-binding abilities intact is inactive in transcriptional repression. KorB may act as a global regulator of RA3 plasmid functions inEscherichia coli, since its overexpression intransnegatively interferes with mini-RA3 replication and stable maintenance of RA3

High-throughput sequencing identification of novel and conserved miRNAs in the Brassica oleracea leaves.

Conclusively, for the first time, the large set of miRNAs was identified in mature cabbage leaves. Potential targets designation for these miRNAs may suggest their essential role in many plants primary biological processes. Presented study not only supplements the knowledge about B. oleracea miRNAs, but additionally it may be used in other research concerning the improvement of the cabbage cultivation

Genomic and Functional Characterization of the Modular Broad-Host-Range RA3 Plasmid, the Archetype of the IncU Group▿ †

IncU plasmids are a distinctive group of mobile elements with highly conserved backbone functions and variable antibiotic resistance gene cassettes. The IncU archetype is conjugative plasmid RA3, whose sequence (45,909 bp) shows it to be a mosaic, modular replicon with a class I integron different from that of other IncU replicons. Functional analysis demonstrated that RA3 possesses a broad host range and can efficiently self-transfer, replicate, and be maintained stably in alpha-, beta-, and gammaproteobacteria. RA3 contains 50 open reading frames clustered in distinct functional modules. The replication module encompasses the repA and repB genes embedded in long repetitive sequences. RepA, which is homologous to antitoxin proteins from alpha- and gammaproteobacteria, contains a Cro/cI-type DNA-binding domain present in the XRE family of transcriptional regulators. The repA promoter is repressed by RepA and RepB. The minireplicon encompasses repB and the downstream repetitive sequence r1/r2. RepB shows up to 80% similarity to putative replication initiation proteins from environmental plasmids of beta- and gammaproteobacteria, as well as similarity to replication proteins from alphaproteobacteria and Firmicutes. Stable maintenance functions of RA3 are most like those of IncP-1 broad-host-range plasmids and comprise the active partitioning apparatus formed by IncC (ParA) and KorB (ParB), the antirestriction protein KlcA, and accessory stability components KfrA and KfrC. The RA3 origin of transfer was localized experimentally between the maintenance and conjugative-transfer operons. The putative conjugative-transfer module is highly similar in organization and in its products to transfer regions of certain broad-host-range environmental plasmids

Down-regulation of CBP80 gene expression as a strategy to engineer a drought-tolerant potato

Developing new strategies for crop plants to respond to drought is crucial for their innovative breeding. The down-regulation of nuclear cap-binding proteins in Arabidopsis renders plants drought tolerant. The CBP80 gene in the potato cultivar Desiree was silenced using artificial microRNAs. Transgenic plants displayed a higher tolerance to drought, ABA-hypersensitive stomatal closing, an increase in leaf stomata and trichome density, and compact cuticle structures with a lower number of microchannels. These findings were correlated with a higher tolerance to water stress. The level of miR159 was decreased, and the levels of its target mRNAs MYB33 and MYB101 increased in the transgenic plants subjected to drought. Similar trends were observed in an Arabidopsis cbp80 mutant. The evolutionary conservation of CBP80, a gene that plays a role in the response to drought, suggests that it is a candidate for genetic manipulations that aim to obtain improved water-deficit tolerance of crop plants

Novel broad-host-range vehicles for cloning and shuffling of gene cassettes

Novel vectors for cloning and shuffling of gene cassettes based on minireplicon of broadhost- range RA3 plasmid from IncU incompatibility group were constructed. A series of minireplicon variants were prepared with copy number ranging from low (1-2 copies per chromosome), medium (10-15 copies per chromosome) to high copy number (80-90 copies per chromosome). The new cloning vectors are relatively small in size (4.5-5.4 kb) and carry various resistance determinants: kanamycin (KmR), tetracycline (TcR) or chloramphenicol (CmR). The vectors were engineered to facilitate cloning and shuffling of the functional modules with or without transcriptional terminators. Using the described strategy, a bank of functional modules, ready for exchange, has been initiated