Topoisomerase IV (topo IV), an essential factor during chromosome segregation, resolves the catenated chromosomes at the end of each replication cycle. How the decatenating activity of the topo IV is regulated during the early stages of the chromosome cycle despite being in continuous association with the chromosome remains poorly understood. Here we report a novel cell cycle-regulated protein in Caulobacter crescentus, NstA (negative switch for topo IV decatenation activity), that inhibits the decatenation activity of the topo IV during early stages of the cell cycle. We demonstrate that in C. crescentus, NstA acts by binding to the ParC DNA-binding subunit of topo IV. Most importantly, we uncover a dynamic oscillation of the intracellular redox state during the cell cycle, which correlates with and controls NstA activity. Thus, we propose that predetermined dynamic intracellular redox fluctuations may act as a global regulatory switch to control cellular development and cell cycle progression and may help retain pathogens in a suitable cell cycle state when encountering redox stress from the host immune response.

Cell fate determination in the asymmetric bacterium Caulobacter crescentus (Caulobacter) is triggered by the localization of the developmental regulator SpmX to the old (stalked) cell pole during the G1→S transition. Although SpmX is required to localize and activate the cell fate-determining kinase DivJ at the stalked pole in Caulobacter, in cousins such as Asticcacaulis, SpmX directs organelle (stalk) positioning and possibly other functions. We define the conserved σ 54 -dependent transcriptional activator TacA as a global regulator in Caulobacter whose activation by phosphorylation is indirectly down-regulated by SpmX. Using a combination of forward genetics and cytological screening, we uncover a previously uncharacterized and polarized component (SpmY) of the TacA phosphorylation control system, and we show that SpmY function and localization are conserved. Thus, SpmX organizes a site-specific, ancestral, and multifunctional regulatory hub integrating the in-phase oscillation of two global transcriptional regulators, CtrA (the master cell cycle transcriptional regulator A) and TacA, that perform important cell cycle functions.cell cycle | Caulobacter crescentus | σ 54 | SpmX | α-proteobacteria

Sophisticated signaling mechanisms allow bacterial cells to cope with environmental and intracellular challenges. Activation of specific pathways ameliorates these challenges and thereby warrants integrity. Here, we demonstrate the pliability of the CckA-CtrA two-component signaling system in the freshwater bacterium Caulobacter crescentus. Our forward genetic screen to analyze suppressor mutations that can negate the chromosome segregation block induced by the topoisomerase IV inhibitor, NstA, yielded various point mutations in the cell cycle histidine kinase, CckA. Notably, we identified a point mutation in the PAS-B domain of CckA, which resulted in increased levels of phosphorylated CtrA (CtrA~P), the master cell cycle regulator. Surprisingly, this increase in CtrA~P levels did not translate into a genome-wide increase in the DNA occupancy of CtrA, but specifically enriched its affinity for the chromosomal origin of replication, Cori, and for a very small sub-set of CtrA regulated promoters. We show that through this enhanced binding of CtrA to the Cori, cells are able to overcome the toxic defects rendered by stable NstA through a possible slow down in the chromosome replication cycle. Taken together, our work opens up an unexplored and intriguing aspect of the CckA-CtrA signal transduction pathway. The distinctive DNA binding nature of CtrA and its regulation by CckA might also be crucial for pathogenesis because of the highly conserved nature of the CckA-CtrA pathway in alphaproteobacteria.

21Sophisticated signaling mechanisms allow bacterial cells to cope with environmental 22 and intracellular challenges. Activation of specific pathways facilitates the cells to 23 overcome cellular damage and thereby warrant integrity. Here we demonstrate the 24 pliability of the CckA-CtrA two component signaling system in the freshwater bacterium, 25 Caulobacter crescentus. Our forward genetic screen to analyse suppressor mutations 26 that can negate the chromosome segregation block induced by the topoisomerase IV 27 inhibitor, NstA, yielded various point mutations in the cell cycle histidine kinase, CckA. 28Notably, we identified a point mutation in the PAS-B domain of CckA, which resulted in 29 increased levels of phosphorylated CtrA (CtrA~P), the master cell cycle regulator.30 Surprisingly, this increase in CtrA~P levels did not translate into a genome-wide 31 increase in the DNA occupancy of CtrA, but specifically enriched its affinity to the 32 chromosomal origin of replication, C ori , and a very small sub-set of CtrA regulated 33 promoters. We show that through this enhanced binding of CtrA to the C ori , cells are 34 able to overcome the toxic defects rendered by stable NstA through a possible slow 35 down in the chromosome cycle. Taken together, our work opens up an unexplored and 36 intriguing aspect of the CckA-CtrA signal transduction pathway. The distinctive DNA 37 binding nature of CtrA and its regulation by CckA might also be crucial for pathogenesis 38 because of the highly conserved nature of CckA-CtrA pathway in alphaproteobacteria.39 40 41 42 2 48 crescentus (henceforth Caulobacter), has emerged as a powerful model organism for 49 studying the complex signaling mechanisms that control cell cycle and development in 50 response to environmental cues. During its cell cycle, Caulobacter undergoes 51 asymmetric division to produce progenies with distinct developmental fates. One of the 52 daughter cells, the swarmer cell, acquires a dispersal fate wherein its motility is assisted 53 by the polar flagellum and a tuft of pili (1,2). In contrast, the stalked daughter cell 54 acquires a sedentary fate and is in an S-phase-like state capable of replicating its 55 chromosome and proliferating by cytokinesis ( Figure 1A) (3,4). The G1-like swarmer cell 56 has to terminally differentiate into a stalked cell to enter into the proliferative phase. This 57 G1 to S-like transition is marked by the shedding of the flagellum, retraction of the pili, 58 and production of a stalk at the same cell pole ( Figure 1A). 59In the swarmer cells, the master transcriptional regulator, CtrA, inhibits the DNA 60 replication. The Caulobacter origin of replication, C ori , is bound by CtrA, which prevents 61 replisome formation in the swarmer cells (5). Concurrent with the swarmer to stalked 62 cell transition, CtrA is degraded by proteolysis and thus facilitating the binding of DnaA, 63 the replication initiator, to the C ori triggering chromosome replication.(6). Apart from 64 blocking DNA replication initiation, CtrA also...

This work focusses on analysing mechanical properties and microscopic assessment into Inconel-718 plates in welded and unwelded conditions. Welding was performed by tungsten inert gas welding technique. Two mechanical tests such as tensile test and hardness were performed on both the types of plates to compare the properties of welded joint and unwelded plate. Although Inconel 718 possesses good weldability, the strength, ductility, and hardness of welded joint were reported lesser than these of Unwelded plate. The microstructural images revealed that metal carbides present in Inconel plate had reduced after welding. The ultimate tensile stress and elongation before breaking of welded joint were 16% and 72% lower than Unwelded plate. The fractography analysis of the ruptured part revealed that Unwelded plate possessed higher ductility than welded plate.