Association of peptidyl prolyl cis/trans isomerase Rrd1 with C terminal domain of RNA polymerase II.

The largest subunit of RNAPII extends as the conserved unstructured heptapeptide consensus repeats Y1S2P3T4S5P6S7 and their posttranslational modification, especially the phosphorylation state at Ser2, Ser5 and Ser7 of CTD recruits different transcription factors involved in transcription. In the current study, fluorescence anisotropy, pull down assay and molecular dynamics simulation studies employed to conclude that peptidyl-prolyl cis/trans-isomerase Rrd1 has strong affinity for unphosphorylated CTD rather than phosphorylated CTD for mRNA transcription. Rrd1 preferentially interacts with unphosphorylated GST-CTD in comparison to hyperphosphorylated GST-CTD in vitro. Fluorescence anisotropy revealed that recombinant Rrd1 prefers to bind unphosphorylated CTD peptide in comparison to phosphorylated CTD peptide. In computational studies, the RMSD of Rrd1-unphosphorylated CTD complex was greater than the RMSD of Rrd1-pCTD complex. During 50 ns MD simulation run Rrd1-pCTD complex get dissociated twice viz. 20 ns to 30 ns and 40 ns to 50 ns, while Rrd1-unpCTD complex remain stable throughout the process. Additionally, the Rrd1-unphosphorylated CTD complexes acquire comparatively higher number of H-bonds, water bridges and hydrophobic interactions occupancy than Rrd1-pCTD complex, concludes that the Rrd1 interacts more strongly with the unphosphorylated CTD than the pCTD.

Transcriptome Analysis to Understand Salt Stress Regulation Mechanism of Chromohalobacter salexigens ANJ207.

Soil salinity is one of the major global issues affecting soil quality and agricultural productivity. The plant growth-promoting halophilic bacteria that can thrive in regions of high salt (NaCl) concentration have the ability to promote the growth of plants in salty environments. In this study, attempts have been made to understand the salinity adaptation of plant growth-promoting moderately halophilic bacteria Chromohalobacter salexigens ANJ207 at the genetic level through transcriptome analysis. In order to identify the stress-responsive genes, the transcriptome sequencing of C. salexigens ANJ207 under different salt concentrations was carried out. Among the 8,936 transcripts obtained, 93 were upregulated while 1,149 were downregulated when the NaCl concentration was increased from 5 to 10%. At 10% NaCl concentration, genes coding for lactate dehydrogenase, catalase, and OsmC-like protein were upregulated. On the other hand, when salinity was increased from 10 to 25%, 1,954 genes were upregulated, while 1,287 were downregulated. At 25% NaCl, genes coding for PNPase, potassium transporter, aconitase, excinuclease subunit ABC, and transposase were found to be upregulated. The quantitative real-time PCR analysis showed an increase in the transcript of genes related to the biosynthesis of glycine betaine coline genes (gbcA, gbcB, and L-pro) and in the transcript of genes related to the uptake of glycine betaine (OpuAC, OpuAA, and OpuAB). The transcription of the genes involved in the biosynthesis of L-hydroxyproline (proD and proS) and one stress response proteolysis gene for periplasmic membrane stress sensing (serP) were also found to be increased. The presence of genes for various compatible solutes and their increase in expression at the high salt concentration indicated that a coordinated contribution by various compatible solutes might be responsible for salinity adaptation in ANJ207. The investigation provides new insights into the functional roles of various genes involved in salt stress tolerance and oxidative stress tolerance produced by high salt concentration in ANJ207 and further support the notion regarding the utilization of bacterium and their gene(s) in ameliorating salinity problem in agriculture.

Analysis of Biosynthetic Gene Clusters, Secretory, and Antimicrobial Peptides Reveals Environmental Suitability of Exiguobacterium profundum PHM11.

Halotolerant bacteria produce a wide range of bioactive compounds with important applications in agriculture for abiotic stress amelioration and plant growth promotion. In the present study, 17 biosynthetic gene clusters (BGCs) were identified in Exiguobacterium profundum PHM11 belonging to saccharides, desmotamide, pseudaminic acid, dipeptide aldehydes, and terpene biosynthetic pathways representing approximately one-sixth of genomes. The terpene biosynthetic pathway was conserved in Exiguobacterium spp. while the E. profundum PHM11 genome confirms the presence of the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway for the isopentenyl diphosphate (IPP) synthesis. Further, 2,877 signal peptides (SPs) were identified using the PrediSi server, out of which 592 proteins were prophesied for the secretion having a transmembrane helix (TMH). In addition, antimicrobial peptides (AMPs) were also identified using BAGEL4. The transcriptome analysis of PHM11 under salt stress reveals the differential expression of putative secretion and transporter genes having SPs and TMH. Priming of the rice, wheat and maize seeds with PHM11 under salt stress led to improvement in the root length, root diameters, surface area, number of links and forks, and shoot length. The study shows that the presence of BGCs, SPs, and secretion proteins constituting TMH and AMPs provides superior competitiveness in the environment and make E. profundum PHM11 a suitable candidate for plant growth promotion under salt stress.

Proteome analysis of Saccharomyces cerevisiae after methyl methane sulfonate (MMS) treatment.

The treatment of methyl methane sulfonate (MMS) increases sensitivity to the DNA damage which, further leads to the cell death followed by a cell cycle delay. Delay in the cell cycle is because of the change in global transcription regulation which results into proteome change. There are several microarray studies on the transcriptome changes after MMS treatment, but very few studies are reported related to proteome change. The proteome analysis in this report identified subgroups of proteins, belonging to known cell cycle regulators, metabolic pathways and protein folding. About 53 proteins were identified by MS/MS and found that 36 of them were induced, 10 were repressed and few of them showed insignificant change. Our results indicated the change in the interactome as well as phosphorylation status of carboxy terminal domain (CTD) of RNA Polymerase II (RNAP-II) after MMS treatment. The RNAP-II complex was affinity purified and ~1640 peptides were identified using nano LC/MS corresponding to 27 interacting proteins along with the twelve RNAP-II subunit. These identified proteins participated in the repair of the damage, changes the function of the main energetic pathways and the carbon flux in various end products. The main metabolic enzymes in the glycolysis, pyruvate phosphate and amino acid biosynthesis pathways showed significant change. Our results indicate that DNA damage is somehow related to these pathways and is co-regulated simultaneously.

Pan-genome analysis of Exiguobacterium reveals species delineation and genomic similarity with Exiguobacterium profundum PHM 11.

The stint of the bacterial species is convoluting, but the new algorithms to calculate genome-to-genome distance (GGD) and DNA-DNA hybridization (DDH) for comparative genome analysis have rejuvenated the exploration of species and sub-species characterization. The present study reports the first whole genome sequence of Exiguobacterium profundum PHM11. PHM11 genome consist of ~ 2.92 Mb comprising 48 contigs, 47.93% G + C content. Functional annotations revealed a total of 3033 protein coding genes and 33 non-protein coding genes. Out of these, only 2316 could be characterized and others reported as hypothetical proteins. The comparative analysis of predicted proteome of PHM11 with five other Exiguobacterium sp. identified 3806 clusters, out of which the PHM11 shared a total of 2723 clusters having 1664 common clusters, 131 singletons and 928 distributed between five species. The pan-genome analysis of 70 different genomic sequences of Exigubacterium strains devoid of a species taxon was done on the basis of GGD and the DDH which identified eight genomes analogous to the PHM11 at species level and may be characterized as E. profundum. The ANI value and phylogenetic tree analysis also support the same. The results regarding pan-genome analysis provide a convincing insight for delineation of these eight strains to species.

pH and alcohol induced structural transition in Ntf2 a nuclear transport factor of Saccharomyces cerevisiae.

Ntf2 is a nuclear envelope protein, which play a pivotal role in nucleocytoplasmic transport and mediates the nuclear import of RanGDP. It interacts with various nucleoporins along with Ran-GDP and part of a multicomponent system that assembles at the nuclear pore complex (NCP) during nuclear import. Here, we have described the biophysical characterization of Ntf2 from Saccharomyces cerevisiae. Recombinant Ntf2 showed increment in the ß-sheet content as well as decrement in the α-helix content from pH-7.0 to pH-4.0. A subsequent decrease in the pH led to increment in the α-helical content along with decrement in ß-sheet content. Intrinsic fluorescence studies demonstrated the unfolding of the protein below physiological pH. Ntf2 showed stabilization as well as phenomenal phase transition (ß sheet to α helix) by increase in alcohol concentration from 10% to 70%. Further increase in alcohol concentration (90%) resulted in residual secondary structure in Ntf2 protein. Presence of ammonium sulfate also stabilizes the secondary structure of Ntf2 protein. The structural characterization reveals the flexibility and the stability of Ntf2 at various conditions. These structural alterations in Ntf2 protein probably occurs in the course of nucleocytoplasmic transport when it interacts with other proteins moving towards its final destination.

An insight into structural plasticity and conformational transitions of transcriptional co-activator Sus1.

RNA biogenesis and mRNA transport are an intricate process for every eukaryotic cell. SAGA, a transcriptional coactivator and TREX-2 are the two major complexes participate in this process. Sus1 is a transcription export factor and part of both the SAGA and the TREX-2 complex. The competitive exchange of Sus1 molecule between SAGA and TREX-2 complex modulates their function which is credited to structural plasticity of Sus1. Here, we portray the biophysical characterization of Sus1 from S. cerevisiae. The recombinant Sus1 is a α-helical structure which is stable at various pH conditions. We reported the α-helix to ß-sheet transition at the low pH as well as at high pH. Sus1 showed 50% reduction in the fluorescence intensity at pH-2 as compared to native protein. The fluorescence studies demonstrated the unfolding of tertiary structure of the protein with variation in pH as compared to neutral pH. The same results were obtained in the ANS binding and acrylamide quenching studies. Similarly, the secondary structure of the Sus1 was found to be stable till 55% alcohol concentration while tertiary structure was stable up to 20% alcohol concentration. Further increase in the alcohol concentration destabilizes the secondary as well as tertiary structure. The 300 mM concentration of ammonium sulfate also stabilizes the secondary structure of the protein. The structural characterization of this protein is expected to unfold the process of the transportation of the mRNA with cooperation of different proteins.

Construction of strains to identify novel factors for regulation of centromeric cohesion protection (CCP) and sister kinetochore mono-orientation (SKM).

BACKGROUND: Meiosis-I is a unique type of chromosome segregation where each chromosome aligns and segregates from its homolog. The mechanism of meiosis I homolog separation in different eukaryotes depends on their centromere and kinetochore architecture which in turn relies mainly on two processes, first on a specialized four protein complex known as monopolin and second, the centromeric cohesion protection (CCP). However, in mammals the complex has not been identified. Furthermore, in budding yeast, there could be additional factors in this process which includes some meiosis specific and some non meiosis specific factors. RESULT: We constructed two strains. In the first strain we expressed Mam1 and Cdc5 which leads to sister kinetochore monoorientation (SKM) and in the second case we expressed Rec8 and Spo13 which enhanced CCP even in mitosis. The expression of these proteins in mitotically dividing cells caused co-orientation of the chromosomes, which lead to the cell death followed by miss-segregation of chromosomes. Then we utilized these strains to screen the cDNA libraries from yeast and mammals to identify the novel factors which participate in CCP and SKM. Finally, SGY4119 strain expressing Spo13 and Rec8 was transformed with pRS316 gal cDNA library and transformants were screened for lethality on galactose. We screened ~ 105 transformants colonies. Out of these ~ 3000 colonies were able to survive on galactose plate which was narrow down to 6 on the basis of desired phenotype. CONCLUSION: So far, meiosis specific kinetochore proteins have been identified only in two yeasts. Recently, in mammals a meiosis specific kinetochore protein (MEIKIN) has been identified with similar function. Till now a single protein in mammals and four proteins monopolin complex in budding yeast has been identified to coorient the centromere. Many more novel factors have to be identified yet. That is why we wished to device genetic screen using a functional genomics approach. Since the list of proteins already identified in yeast is not exhaustive as the circumstantial evidence suggests, we wish to use the same yeast strains to identify additional novel yeast proteins that may be involved in the execution of meiosis.

Draft genome sequence of a cold-adapted phosphorous-solubilizing Pseudomonas koreensis P2 isolated from Sela Lake, India.

The draft genome sequence of a cold-adapted phosphorus-solubilizing strain Pseudomonas koreensis P2 isolated from the Sela Lake contains 6,436,246 bp with G + C content of 59.8%. The genome sequence includes 5743 protein coding genes, 68 non-protein coding genes, 1007 putative proteins, 5 rRNA genes, 64 tRNAs and two prophage regions in 40 contigs. Besides these, genes involved in phosphate solubilization, siderophore production, iron uptake, heat shock and cold shock tolerance, multidrug resistance and glycine-betaine production were also identified.

Draft Genome Sequence of Halotolerant Bacterium Chromohalobacter salexigens ANJ207, Isolated from Salt Crystal Deposits in Pipelines.

Chromohalobacter salexigens ANJ207 was isolated from a salt crystal and is known to tolerate up to 30% NaCl concentration. Here, we report the de novo draft assembly of C. salexigens ANJ207. The genome was determined to have 3.66 Mb represented in 13 scaffolds, with a total of 3,406 genes predicted.

An interplay between Shugoshin and Spo13 for centromeric cohesin protection and sister kinetochore mono-orientation during meiosis I in Saccharomyces cerevisiae.

Meiosis is a specialized cell division process by which haploid gametes are produced from a diploid mother cell. Reductional chromosome segregation during meiosis I (MI) is achieved by two unique and conserved events: centromeric cohesin protection (CCP) and sister kinetochore mono-orientation (SKM). In Saccharomyces cerevisiae, a meiosis-specific protein Spo13 plays a role in both these centromere-specific events. Despite genome-wide association of Spo13, we failed to detect its function in global processes such as cohesin loading, cohesion establishment and homologs pairing. While Shugoshin (Sgo1) and protein phosphatase 2A (PP2ARts1) play a central role in CCP, it is not fully understood whether Spo13 functions in the process through a Sgo1- PP2ARts1-dependent or -independent mechanism. To delineate this and to find the relative contribution of each of these proteins in CCP and SKM, we meticulously observed the sister chromatid segregation pattern in the wild type, sgo1Δ, rts1Δ and spo13Δ single mutants and in their respective double mutants. We found that Spo13 protects centromeric cohesin through a Sgo1- PP2ARts1-independent mechanism. To our surprise, we observed a hitherto unknown role of Sgo1 in SKM. Further investigation revealed that Sgo1-mediated recruitment of aurora kinase Ipl1 to the centromere facilitates monopolin loading at the kinetochore during MI. Hence, this study uncovers the role of Sgo1 in SKM and demonstartes how the regulators (Sgo1, PP2ARts1, Spo13) work in a coordinated manner to achieve faithful chromosome segregation during meiosis, the failure of which leads to aneuploidy and birth defects.

The mRNA capping enzyme of Saccharomyces cerevisiae has dual specificity to interact with CTD of RNA Polymerase II.

RNA Polymerase II (RNAPII) uniquely possesses an extended carboxy terminal domain (CTD) on its largest subunit, Rpb1, comprising a repetitive Tyr1Ser2Pro3Thr4 Ser5Pro6Ser7 motif with potential phosphorylation sites. The phosphorylation of the CTD serves as a signal for the binding of various transcription regulators for mRNA biogenesis including the mRNA capping complex. In eukaryotes, the 5 prime capping of the nascent transcript is the first detectable mRNA processing event, and is crucial for the productive transcript elongation. The binding of capping enzyme, RNA guanylyltransferases to the transcribing RNAPII is known to be primarily facilitated by the CTD, phosphorylated at Ser5 (Ser5P). Here we report that the Saccharomyces cerevesiae RNA guanylyltransferase (Ceg1) has dual specificity and interacts not only with Ser5P but also with Ser7P of the CTD. The Ser7 of CTD is essential for the unconditional growth and efficient priming of the mRNA capping complex. The Arg159 and Arg185 of Ceg1 are the key residues that interact with the Ser5P, while the Lys175 with Ser7P of CTD. These interactions appear to be in a specific pattern of Ser5PSer7PSer5P in a tri-heptad CTD (YSPTSPPS YSPTSPSP YSPTSPPS) and provide molecular insights into the Ceg1-CTD interaction for mRNA transcription.

The prophage-encoded hyaluronate lyase has broad substrate specificity and is regulated by the N-terminal domain.

Streptococcus equi is the causative agent of the highly contagious disease "strangles" in equines and zoonotic meningitis in human. Spreading of infection in host tissues is thought to be facilitated by the bacterial gene encoded extracellular hyaluronate lyase (HL), which degrades hyaluronan (HA), chondroitin 6-sulfate, and dermatan sulfate of the extracellular matrix). The clinical strain S. equi 4047 however, lacks a functional extracellular HL. The prophages of S. equi and other streptococci encode intracellular HLs which are reported to partially degrade HA and do not cleave any other glycosaminoglycans. The phage HLs are thus thought to play a role limited to the penetration of streptococcal HA capsules, facilitating bacterial lysogenization and not in the bacterial pathogenesis. Here we systematically looked into the structure-function relationship of S. equi 4047 phage HL. Although HA is the preferred substrate, this HL has weak activity toward chondroitin 6-sulfate and dermatan sulfate and can completely degrade all of them. Even though the catalytic triple-stranded ß-helix domain of phage HL is functionally independent, its catalytic efficiency and specificity is influenced by the N-terminal domain. The phage HL also interacts with human transmembrane glycoprotein CD44. The above results suggest that the streptococci can use phage HLs to degrade glycosaminoglycans of the extracellular matrix for spreading virulence factors and toxins while utilizing the disaccharides as a nutrient source for proliferation at the site of infection.