Differential cellular localization of DNA gyrase and topoisomerase IB in response to DNA damage in Deinococcus radiodurans.

Topoisomerases are crucial enzymes in genome maintenance that modulate the topological changes during DNA metabolism. Deinococcus radiodurans, a Gram-positive bacterium is characterized by its resistance to many abiotic stresses including gamma radiation. Its multipartite genome encodes both type I and type II topoisomerases. Time-lapse studies using fluorescently tagged topoisomerase IB (drTopoIB-RFP) and DNA gyrase (GyrA-RFP) were performed to check the dynamics and localization with respect to DNA repair and cell division under normal and post-irradiation growth conditions. Results suggested that TopoIB and DNA gyrase are mostly found on nucleoid, highly dynamic, and show growth phase-dependent subcellular localization. The drTopoIB-RFP was also present at peripheral and septum regions but does not co-localize with the cell division protein, drFtsZ. On the other hand, DNA gyrase co-localizes with PprA a pleiotropic protein involved in radioresistance, on the nucleoid during the post-irradiation recovery (PIR). The topoIB mutant was found to be sensitive to hydroxyurea treatment, and showed more accumulation of single-stranded DNA during the PIR, compared to the wild type suggesting its role in DNA replication stress. Together, these results suggest differential localization of drTopoIB-RFP and GyrA-RFP in D. radiodurans and their interaction with PprA protein, emphasizing the functional significance and role in radioresistance.

DivIVA Phosphorylation Affects Its Dynamics and Cell Cycle in Radioresistant Deinococcus radiodurans.

DivIVA is a member of the Min family of proteins that spatially regulates septum formation at the midcell position and cell pole determination in Bacillus subtilis. Deinococcus radiodurans, a Gram-positive coccus-shaped bacterium, is characterized by its extreme resistance to DNA-damaging agents, including radiation. D. radiodurans cells exposed to gamma radiation undergo cell division arrest by as-yet-uncharacterized mechanisms. divIVA is shown to be an essential cell division gene in this bacterium, and DivIVA of D. radiodurans (drDivIVA) interacts with genome segregation proteins through its N-terminal region. Earlier, RqkA, a gamma radiation-responsive Ser/Thr quinoprotein kinase, was characterized for its role in radioresistance in D. radiodurans. Here, we showed that RqkA phosphorylates drDivIVA at the threonine 19 (T19) residue. The phospho-mimetic mutant with a mutation of T19 to E19 in DivIVA (DivIVAT19E) is found to be functionally different from the phospho-ablative mutant (DivIVAT19A) or the wild-type drDivIVA. A DivIVAT19E-red fluorescent protein (RFP) fusion expressed in the wild-type background showed the arrest in the typical dynamics of drDivIVA and the loss of its interaction with the genome segregation protein ParA2. The allelic replacement of divIVA with divIVAT19E-rfp was not tolerated unless drDivIVA was expressed episomally, while there was no phenotypic change when the wild-type allele was replaced with either divIVAT19A-rfp or divIVA-rfp. These results suggested that the phosphorylation of T19 in drDivIVA by RqkA affected its in vivo functions, which may contribute to the cell cycle arrest in this bacterium. IMPORTANCE Deinococcus radiodurans, a radioresistant bacterium, lacks LexA/RecA-mediated DNA damage response and cell cycle regulation as known in other bacteria. However, it adjusts its transcriptome and proteome upon DNA damage. In eukaryotes, the DNA damage response and cell cycle are regulated by Ser/Thr protein kinases. In D. radiodurans, we characterized a gamma radiation-responsive Ser/Thr quinoprotein kinase (RqkA) that phosphorylated DNA repair and cell division proteins in this bacterium. In previous work, the effect of S/T phosphorylation by RqkA on activity improvement of the DNA repair proteins has been demonstrated. This study reports that Ser phosphorylation by RqkA attenuates the function of a cell polarity and plane of cell division-determining protein, DivIVA, and its cellular dynamics in response to DNA damage, which might help to understand the mechanism of cell cycle regulation in this bacterium.

FtsZ phosphorylation brings about growth arrest upon DNA damage in Deinococcus radiodurans.

The polymerization/depolymerization dynamics of FtsZ play a pivotal role in cell division in the majority of the bacteria. Deinococcus radiodurans, a radiation-resistant bacterium, shows an arrest of growth in response to DNA damage with no change in the level of FtsZ. This bacterium does not deploy LexA/RecA type of DNA damage response and cell cycle regulation, and its genome does not encode SulA homologues of Escherichia coli, which attenuate FtsZ functions in response to DNA damage in other bacteria. A radiation-responsive Ser/Thr quinoprotein kinase (RqkA), characterized for its role in radiation resistance in this bacterium, could phosphorylate several cognate proteins, including FtsZ (drFtsZ) at Serine 235 (S235) and Serine 335 (S335) residues. Here, we reported the detailed characterization of S235 and S335 phosphorylation effects in the regulation of drFtsZ functions and demonstrated that the phospho-mimetic replacements of these residues in drFtsZ had grossly affected its functions that could result in cell cycle arrest in response to DNA damage in D. radiodurans. Interestingly, the phospho-ablative replacements were found to be nearly similar to drFtsZ, whereas the phospho-mimetic mutant lost the wild-type protein's signature characteristics, including its dynamics under normal conditions. The kinetics of post-bleaching recovery for drFtsZ and phospho-mimetic mutant were nearly similar at 2 h post-irradiation recovery but were found to be different under normal conditions. These results highlighted the role of S/T phosphorylation in the regulation of drFtsZ functions and cell cycle arrest in response to DNA damage, which is demonstrated for the first time, in any bacteria.

LC-MS/MS method for the simultaneous quantification of pyriproxyfen and bifenthrin and their dissipation kinetics under field conditions in chili and brinjal.

Bifenthrin, a synthetic pyrethroid, and pyriproxyfen, a plant growth regulator, are used extensively in agriculture for controlling the different insect pests. The present study was undertaken to examine the dissipation behavior of a formulation with a combination of pyriproxyfen and bifenthrin on chili and brinjal under field conditions at four different locations. Dissipation study of combination of pyriproxyfen and bifenthrin revealed swift degradation in both crops with a half-life of 2.5-2.6 and 2.0-2.1 days in brinjal and chili, respectively. Also, a simple method for simultaneous quantification of pyriproxyfen and bifenthrin was developed and validated using modified QuEChERS (quick, easy, cheap, effective, rugged, and safe) technique on liquid chromatography with tandem mass spectrometry (LC-MS/MS). Recovery of the method was found to be under an acceptable range of 90.0%-93.5% and 88.7%-94.3% in chili and 92.4%-96.6% and 97.4%-100.9% in brinjal for pyriproxyfen and bifenthrin, respectively. At harvest time, the terminal residues of bifenthrin and pyriproxyfen were below the maximum residue limits set by European Union in chili and brinjal, respectively, suggesting that the use of this pesticide formulation is safe and does not impose harmful effects on human health. PRACTICAL APPLICATION: In this paper, dissipation behavior of a pesticide formulation with a combination of pyriproxyfen and bifenthrin was undertaken under field conditions at four different locations on chili and brinjal in India. The simultaneous quantification of pyriproxyfen and bifenthrin using LC-MS/MS technique has been validated incorporating modified QuEChERS extraction method with limit of detection at 0.005 µg/g and limit of quantification at 0.01 µg/g, which is well below the EU-MRLs (European Union legislation Maximum Residue Level) of pyriproxyfen and bifenthrin in both chili and brinjal. Furthermore, dissipation kinetics of a formulation undertaken under field conditions at four different locations on chili and brinjal suggested that the terminal residues of both bifenthrin and pyriproxyfen were below the maximum residue limits set by European Union in chili and brinjal, respectively, at the time of harvest and that the use of this pesticide formulation is safe.

Guanine quadruplexes and their roles in molecular processes.

The role of guanine quadruplexes (G4) in fundamental biological processes like DNA replication, transcription, translation and telomere maintenance is recognized. G4 structure dynamics is regulated by G4 structure binding proteins and is thought to be crucial for the maintenance of genome integrity in both prokaryotic and eukaryotic cells. Growing research over the last decade has expanded the existing knowledge of the functional diversity of G4 (DNA and RNA) structures across the working models. The control of G4 structure dynamics using G4 binding drugs has been suggested as the putative targets in the control of cancer and bacterial pathogenesis. This review has brought forth the collections of recent information that indicate G4 (mostly G4 DNA) roles in microbial pathogenesis, DNA damaging stress response in bacteria and mammalian cells. Studies in mitochondrial gene function regulation by G4s have also been underscored. Finally, the interdependence of G4s and epigenetic modifications and their speculated medical implications through G4 interacting proteins has been discussed.

DivIVA Regulates Its Expression and the Orientation of New Septum Growth in Deinococcus radiodurans.

In rod-shaped Gram-negative bacteria, FtsZ localization at midcell position is regulated by the gradient of MinCDE complex across the poles. In round-shaped bacteria, which lack predefined poles, the next plane of cell division is perpendicular to the previous plane, and determination of the FtsZ assembly site is still intriguing. Deinococcus radiodurans, a coccus bacterium, is characterized by its extraordinary resistance to DNA damage. DivIVA, a putative component of the Min system in this bacterium, interacts with cognate cell division and genome segregation proteins. Here, we report that deletion of a chromosomal copy of DivIVA was possible only when the wild-type copy of DivIVA was expressed in trans on a plasmid. However, deletion of the C-terminal domain (CTD) of DivIVA (CTD mutant) was possible but produced distinguishable phenotypes, like smaller cells, slower growth, and tilted septum orientation, in D. radiodurans. In trans expression of DivIVA in the CTD mutant could restore these features of the wild type. Interestingly, the overexpression of DivIVA led to delayed separation of tetrads from an octet state in both trans-complemented divIVA-mutant and wild-type cells. The CTD mutant showed upregulation of the yggS-divIVAN operon. Both the wild type and CTD mutant formed FtsZ foci; however, unlike wild type, the position of foci in the mutant cells was found to be away from conjectural midcell position in cocci. Notably, DivIVA-red fluorescent protein (DivIVA-RFP) localizes to the septum during cell division at the new division site. These results suggested that DivIVA is an essential protein in D. radiodurans, and its C-terminal domain plays an important role in the regulation of its expression and orientation of new septal growth in this bacterium. IMPORTANCE In rod-shaped Gram-negative bacteria, the midcell position for binary fission is relatively easy to model. In cocci that do not have predefined poles, the plane of next cell division is shown to be perpendicular to the previous plane. However, the molecular basis of perpendicularity is not known in cocci. The DivIVA protein of Deinococcus radiodurans, a coccus bacterium, physically interacts with the septum and establishes macromolecular interactions with genome segregation proteins through its N-terminal domain and with MinC through the C-terminal domain. Here, we have brought forth some evidence to suggest that DivIVA is essential for growth and plays an important role in cell polarity determination, and its C-terminal domain plays a crucial role in the growth of new septa in the correct orientation as well as in the regulation of DivIVA expression.

Molecular insights into replication initiation in a multipartite genome harboring bacterium Deinococcus radiodurans.

Deinococcus radiodurans harbors a multipartite ploid genome system consisting of two chromosomes and two plasmids present in multiple copies. How these discrete genome elements are maintained and inherited is not well understood. PprA, a pleiotropic protein involved in radioresistance, has been characterized for its roles in DNA repair, genome segregation, and cell division in this bacterium. Here, we show that PprA regulates ploidy of chromosome I and II and inhibits the activity of drDnaA, the initiator protein in D. radiodurans. We found that pprA deletion resulted in an increased genomic content and ploidy of both the chromosomal elements. Expression of PprA in trans rescued the phenotypes of the pprA mutant. To understand the molecular mechanism underlying these phenotypes, we characterized drDnaA and drDnaB. As expected for an initiator protein, recombinant drDnaA showed sequence-specific interactions with the putative oriC sequence in chromosome I (oriCI). Both drDnaA and drDnaB showed ATPase activity, also typical of initiator proteins, but only drDnaB exhibited 5'→3' dsDNA helicase activity in vitro. drDnaA and drDnaB showed homotypic and heterotypic interactions with each other, which were perturbed by PprA. Interestingly, PprA has inhibited the ATPase activity of drDnaA but showed no effect on the activity of drDnaB. Regulation of chromosome copy number and inhibition of the initiator protein functions by PprA strongly suggest that it plays a role as a checkpoint regulator of the DNA replication initiation in D. radiodurans perhaps through its interaction with the replication initiation machinery.

Molecular interactions and their predictive roles in cell pole determination in bacteria.

Bacterial cell cycle is divided into well-coordinated phases; chromosome duplication and segregation, cell elongation, septum formation, and cytokinesis. The temporal separation of these phases depends upon the growth rates and doubling time in different bacteria. The entire process of cell division starts with the assembly of divisome complex at mid-cell position followed by constriction of the cell wall and septum formation. In the mapping of mid-cell position for septum formation, the gradient of oscillating Min proteins across the poles plays a pivotal role in several bacteria genus. The cues in the cell that defines the poles and plane of cell division are not fully characterized in cocci. Recent studies have shed some lights on molecular interactions at the poles and the underlying mechanisms involved in pole determination in non-cocci. In this review, we have brought forth recent findings on these aspects together, which would suggest a model to explain the mechanisms of pole determination in rod shaped bacteria and could be extrapolated as a working model in cocci.

Topoisomerase IB interacts with genome segregation proteins and is involved in multipartite genome maintenance in Deinococcus radiodurans.

Deinococcus radiodurans, an extremophile, resistant to many abiotic stresses including ionizing radiation, has 2 type I topoisomerases (drTopo IA and drTopo IB) and one type II topoisomerase (DNA gyrase). The role of drTopo IB in guanine quadruplex DNA (G4 DNA) metabolism was demonstrated earlier in vitro. Here, we report that D. radiodurans cells lacking drTopo IB (ΔtopoIB) show sensitivity to G4 DNA binding drug (NMM) under normal growth conditions. The activity of G4 motif containing promoters like mutL and recQ was reduced in the presence of NMM in mutant cells. In mutant, the percentage of anucleate cells was more while the copy number of genome elements were less as compared to wild type. Protein-protein interaction studies showed that drTopo IB interacts with genome segregation and DNA replication initiation (DnaA) proteins. The typical patterns of cellular localization of GFP-PprA were affected in the mutant cells. Microscopic examination of D. radiodurans cells expressing drTopo IB-RFP showed its localization on nucleoid forming a streak parallel to the old division septum and perpendicular to newly formed septum. These results together suggest the role of drTopo IB in genome maintenance in this bacterium.

Guanine Quadruplex DNA Regulates Gamma Radiation Response of Genome Functions in the Radioresistant Bacterium Deinococcus radiodurans.

Guanine quadruplex (G4) DNA/RNA are secondary structures that regulate the various cellular processes in both eukaryotes and bacteria. Deinococcus radiodurans, a Gram-positive bacterium known for its extraordinary radioresistance, shows a genomewide occurrence of putative G4 DNA-forming motifs in its GC-rich genome. N-Methyl mesoporphyrin (NMM), a G4 DNA structure-stabilizing drug, did not affect bacterial growth under normal conditions but inhibited the postirradiation recovery of gamma-irradiated cells. Transcriptome sequencing analysis of cells treated with both radiation and NMM showed repression of gamma radiation-responsive gene expression, which was observed in the absence of NMM. Notably, this effect of NMM on the expression of housekeeping genes involved in other cellular processes was not observed. Stabilization of G4 DNA structures mapped at the upstream of recA and in the encoding region of DR_2199 had negatively affected promoter activity in vivo, DNA synthesis in vitro and protein translation in Escherichia coli host. These results suggested that G4 DNA plays an important role in DNA damage response and in the regulation of expression of the DNA repair proteins required for radioresistance in D. radioduransIMPORTANCEDeinococcus radiodurans can recover from extensive DNA damage caused by many genotoxic agents. It lacks LexA/RecA-mediated canonical SOS response. Therefore, the molecular mechanisms underlying the regulation of DNA damage response would be worth investigating in this bacterium. D. radiodurans genome is GC-rich and contains numerous islands of putative guanine quadruplex (G4) DNA structure-forming motifs. Here, we showed that in vivo stabilization of G4 DNA structures can impair DNA damage response processes in D. radiodurans Essential cellular processes such as transcription, DNA synthesis, and protein translation, which are also an integral part of the double-strand DNA break repair pathway, are affected by the arrest of G4 DNA structure dynamics. Thus, the role of DNA secondary structures in DNA damage response and radioresistance is demonstrated.

N-terminal domain of DivIVA contributes to its dimerization and interaction with genome segregation proteins in a radioresistant bacterium Deinococcus radiodurans.

Unlike in rod-shaped bacteria, cell polarity is not well defined in cocci and possibly gets marked during molecular events around cytokinesis. DivIVA is a member of Min system that is involved in spatial regulation of septum formation in bacteria. Recently, we showed that DivIVA of Deinococcus radiodurans (drDivIVA) interacts with proteins involved in cell division and genome segregation (segrosome). To map drDivIVA domain (s) that interact with these proteins, the N-terminal (DivIVA-N), C-terminal (DivIVA-C) and a middle (DivIVA-M) region/section of drDivIVA were generated. Circular Dichroism (CD) studies suggested that all three variants of drDivIVA fold properly, but they appeared different under transmission electron microscopy (TEM). Full length drDivIVA showed bundles under TEM whereas variants did not. Both full length drDivIVA and N-terminal domain showed repeats of heptad motifs, a characteristic of alpha-helical coiled-coil proteins. DivIVA-N showed dimerization and interaction with segrosome while DivIVA-M interacted with MinC, a cell division regulatory protein. Further, the C-terminal region seems to be crucial for the structural and functional integrity of drDivIVA. These results suggested that drDivIVA dimerizes through its N-terminal domain while both segrosome and MinC interact through different regions of this protein.

Phosphorylation of FtsZ and FtsA by a DNA Damage-Responsive Ser/Thr Protein Kinase Affects Their Functional Interactions in Deinococcus radiodurans.

Deinococcus radiodurans, a highly radioresistant bacterium, does not show LexA-dependent regulation of recA expression in response to DNA damage. On the other hand, phosphorylation of DNA repair proteins such as PprA and RecA by a DNA damage-responsive Ser/Thr protein kinase (STPK) (RqkA) could improve their DNA metabolic activities as well as their roles in the radioresistance of D. radiodurans Here we report RqkA-mediated phosphorylation of cell division proteins FtsZ and FtsA in vitro and in surrogate Escherichia coli bacteria expressing RqkA. Mass spectrometric analysis mapped serine 235 and serine 335 in FtsZ and threonine 272, serine 370, and serine 386 in FtsA as potential phosphorylation sites. Although the levels of FtsZ did not change during postirradiation recovery (PIR), phosphorylation of both FtsZ and FtsA showed a kinetic change during PIR. However, in an rqkA mutant of D. radiodurans, though FtsZ underwent phosphorylation, no kinetic change in phosphorylation was observed. Further, RqkA adversely affected FtsA interaction with FtsZ, and phosphorylated FtsZ showed higher GTPase activity than unphosphorylated FtsZ. These results suggest that both FtsZ and FtsA are phosphoproteins in D. radiodurans The increased phosphorylation of FtsZ in response to radiation damage in the wild-type strain but not in an rqkA mutant seems to be regulating the functional interaction of FtsZ with FtsA. For the first time, we demonstrate the role of a DNA damage-responsive STPK (RqkA) in the regulation of functional interaction of cell division proteins in this bacterium.IMPORTANCE The LexA/RecA-type SOS response is the only characterized mechanism of DNA damage response in bacteria. It regulates cell cycle by attenuating the functions of cell division protein FtsZ and inducing the expression of DNA repair proteins. There are bacteria, including Deinococcus radiodurans, that do not show this classical SOS response. D. radiodurans is known for its extraordinary resistance to gamma radiation, and a DNA damage-responsive Ser/Thr protein kinase (RqkA) has been characterized for its role in radioresistance. RqkA phosphorylates a large number of proteins in solution. The phosphorylation of RecA and PprA by RqkA enhanced their activities. FtsZ phosphorylation is inducible by gamma radiation in wild-type D. radiodurans but not in an rqkA mutant. Phosphorylation affected the interaction of FtsZ and FtsA in this bacterium. This study, therefore, brought forth some findings that might lead to the discovery of a new mechanism regulating the bacterial cell cycle in response to DNA damage.

Interdependence of bacterial cell division and genome segregation and its potential in drug development.

Cell division and genome segregation are mutually interdependent processes, which are tightly linked with bacterial multiplication. Mechanisms underlying cell division and the cellular machinery involved are largely conserved across bacteria. Segregation of genome elements on the other hand, follows different pathways depending upon its type and the functional components encoded on these elements. Small molecules, that are known to inhibit cell division and/or resolution of intertwined circular chromosome and maintenace of DNA topology have earlier been tested as antibacterial agents. The utility of such drugs in controlling bacterial infections has witnessed only partial success, possibly due to functional redundancy associated with targeted components. However, in due course, literature has grown with newer information. This review has brought forth some recent findings on bacterial cell division with special emphasis on crosstalk between cell division and genome segregation that could be explored as novel targets in drug development.

Posterior atlantoaxial dislocation without odontoid fracture.

We report a case of posterior atlantoaxial dislocation without a fracture of the odontoid in a 35-year-old woman. There have been nine reported cases of similar injury in the English literature. The integrity of the transverse ligament following posterior atlantoaxial dislocations has not been well documented in these reports. In the present case, MRI revealed an intact transverse ligament, which probably contributed to the stability of the C1-C2 complex following closed reduction.