Reversible regulation of conjugation of Bacillus subtilis plasmid pLS20 by the quorum sensing peptide responsive anti-repressor RappLS20

Quorum sensing plays crucial roles in bacterial communication including in the process of conjugation, which has large economical and health-related impacts by spreading antibiotic resistance. The conjugative Bacillus subtilis plasmid pLS20 uses quorum sensing to determine when to activate the conjugation genes. The main conjugation promoter, Pc, is by default repressed by a regulator RcopLS20 involving DNA looping. A plasmid-encoded signalling peptide, Phr*pLS20, inactivates the anti-repressor of RcopLS20, named RappLS20, which belongs to the large group of RRNPP family of regulatory proteins. Here we show that DNA looping occurs through interactions between two RcopLS20 tetramers, each bound to an operator site. We determined the relative promoter strengths for all the promoters involved in synthesizing the regulatory proteins of the conjugation genes, and constructed an in vivo system uncoupling these regulatory genes to show that RappLS20 is sufficient for activating conjugation in vivo. We also show that RappLS20 actively detaches RcopLS20 from DNA by preferentially acting on the RcopLS20 molecules involved in DNA looping, resulting in sequestration but not inactivation of RcopLS20. Finally, results presented here in combination with our previous results show that activation of conjugation inhibits competence and competence development inhibits conjugation, indicating that both processes are mutually exclusive.

Ranking of Delay factors in Road Construction Project due to Improper Construction Equipment Management

Construction time overrun is considered one of the most appearing problems in construction industry. In Road construction, Equipment plays a major role, Hence there is a need for control of delay in road construction projects. This paper aims to identify the equipment delay causes in road construction projects and ranks the critical factors. The equipment delay causes were identified through pilot study and literature papers. The pilot study was done by interviewing 20 experts and the questionnaire were designed. Totally 19 causes were identified and categorized into four main groups namely 1.Maintenance related causes, 2.Equipment related causes, 3.Financial related causes and 4.Personnel related causes. The questionnaire survey received 180 responses on the opinion of Contractor, Equipment dealer, Equipment operator, Site engineer, Project manager and others. The results were analysed using Relative Important Index (RII), Ranking the equipment delay causes identifies the most risky equipment delay factors in road construction project. The most five risky factors are Lack of spare parts, Over usage of equipment leads to crashing, Inadequate equipment, Non allocation of fuel purchase staff and Inexpert or unpractised equipment operator

Origin of co-operativity in the activation of dimeric transcription factors

Cooperative behavior in the binding of ligands to a protein is often viewed as a complex phenomenon where conformational changes induced by the binding of the first ligand leads to tighter binding of subsequent ligands. We revisit the ligand-dependent activation of dimeric transcription factors and show that this process may appear cooperative even when it results from independent lig- and binding events. This effect is further accentuated through binding of the activated transcription factor to its cognate operator site on the DNA, where we demonstrate that cooperative activation is a stable fixed point. Our analysis nicely accounts for the apparent co-operativity inherent in the biological activity of many dimeric transcription factors.

Characterization of consensus operator site for Streptococcus pneumoniae copper repressor, CopY

ABSTRACTCopper is broadly toxic to bacteria. As such, bacteria have evolved specialized copper export systems (cop operons) often consisting of a DNA-binding/copper-responsive regulator (which can be a repressor or activator), a copper chaperone, and a copper exporter. For those bacteria using DNA-binding copper repressors, few studies have examined the regulation of this operon regarding the operator DNA sequence needed for repression. In Streptococcus pneumoniae (the pneumococcus), CopY is the copper repressor for the cop operon. Previously, these homologs have been characterized to bind a 10-base consensus sequence T/GACAnnTGTA. Here, we bioinformatically and empirically characterize these operator sites across species using S. pneumoniae CopY as a guide for binding. By examining the 21-base repeat operators for the pneumococcal cop operon and comparing binding of recombinant CopY to this, and the operator sites found in Enterococcus hirae, we show using biolayer interferometry that the T/GACAnnTGTA sequence is essential to binding, but it is not sufficient. We determine a more comprehensive S. pneumoniae CopY operator sequence to be RnYKACAAATGTARnY (where “R” is purine, “Y” is pyrimidine, and “K” is either G or T) binding with an affinity of 28 nM. We further propose that the cop operon operator consensus site of pneumococcal homologs be RnYKACAnnYGTARnY. This study illustrates the necessity to explore bacterial operator sites further to better understand bacterial gene regulation.

An Anticipative Control Approach and Interactive GUI to Enhance the Rendering of the Distal Robot Interaction with its Environment during Robotized Tele-Echography

Performing a robotized telemedicine act via specific networks brings forth the issue of transparency in order to enable the operator, e.g. the medical ultrasound specialist, to safely and accurately perform bilateral tele-operation tasks despite the long time delays inherent to the communication link. To counter these effects, two strategies are combined to improve, at the operator site, the rendering of the interactions between the remote robotic systems with its environment (i.e. the patient), and the control of the robot’s orientation at the operator site. The first approach is the development of a new control architecture based on an internal model providing an anticipated value of the distant environment stiffness; it is complemented with a graphic user interface (GUI) which provides the expert with the real-time relative position of the haptic probe with the robot’s end effector for better tele-operated control. These combined strategies provide the expert with an improved interactive tool for tele-diagnosis.

Vibrio cholerae LexA Coordinates CTX Prophage Gene Expression

ABSTRACT The filamentous bacteriophage CTXΦ transmits the cholera toxin genes by infecting and lysogenizing its host, Vibrio cholerae. CTXΦ genes required for virion production initiate transcription from the strong P A promoter, which is dually repressed in lysogens by the phage-encoded repressor RstR and the host-encoded SOS repressor LexA. Here we identify the neighboring divergent rstR promoter, P R, and show that RstR both positively and negatively autoregulates its own expression from this promoter. LexA is absolutely required for RstR-mediated activation of P R transcription. RstR autoactivation occurs when RstR is bound to an operator site centered 60 bp upstream of the start of transcription, and the coactivator LexA is bound to a 16-bp SOS box centered at position −23.5, within the P R spacer region. Our results indicate that LexA, when bound to its single site in the CTXΦ prophage, both represses transcription from P A and coactivates transcription from the divergent P R. We propose that LexA coordinates P A and P R prophage transcription in a gene regulatory circuit. This circuit is predicted to display transient switch behavior upon induction of CTXΦ lysogens.