Biochemical Properties and Roles of DprA Protein in Bacterial Natural Transformation, Virulence, and Pilin Variation.

Natural transformation enables bacteria to acquire DNA from the environment and contributes to genetic diversity, DNA repair, and nutritional requirements. DNA processing protein A (DprA) receives incoming single-stranded DNA and assists RecA loading for homology-directed natural chromosomal transformation and DNA strand annealing during plasmid transformation. The dprA gene occurs in the genomes of all known bacteria, irrespective of their natural transformation status. The DprA protein has been characterized by its molecular, cellular, biochemical, and biophysical properties in several bacteria. This review summarizes different aspects of DprA biology, collectively describing its biochemical properties, molecular interaction with DNA, and function interaction with bacterial RecA during natural transformation. Furthermore, the roles of DprA in natural transformation, bacterial virulence, and pilin variation are discussed.

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.

Characterization of DNA Processing Protein A (DprA) of the Radiation-Resistant Bacterium Deinococcus radiodurans.

Environmental DNA uptake by certain bacteria and its integration into their genome create genetic diversity and new phenotypes. DNA processing protein A (DprA) is part of a multiprotein complex and facilitates the natural transformation (NT) phenotype in most bacteria. Deinococcus radiodurans, an extremely radioresistant bacterium, is efficient in NT, and its genome encodes nearly all of the components of the natural competence complex. Here, we have characterized the DprA protein of this bacterium (DrDprA) for the known characteristics of DprA proteins of other bacteria and the mechanisms underlying the DNA-RecA interaction. DrDprA has three domains. In vitro studies showed that purified recombinant DrDprA binds to both single-strand DNA (ssDNA) and double-strand DNA (dsDNA) and is able to protect ssDNA from nucleolytic degradation. DrDprA showed a strong interaction with DrRecA and facilitated RecA-catalyzed functions in vivo. Mutational studies identified DrDprA amino acid residues crucial for oligomerization, the interaction with DrRecA, and DNA binding. Furthermore, we showed that both oligomerization and DNA binding properties of DrDprA are integral to its support of the DrRecA-catalyzed strand exchange reaction (SER) in vitro. Together, these data suggested that DrDprA is largely structurally conserved with other DprA homologs but shows some unique structure-function features like the existence of an additional C-terminal Drosophila melanogaster Miasto-like protein 1 (DML1) domain, equal affinities for ssDNA and dsDNA, and the collective roles of oligomerization and DNA binding properties in supporting DrRecA functions. IMPORTANCE Bacteria can take up extracellular DNA (eDNA) by natural transformation (NT). Many bacteria, including Deinococcus radiodurans, have constitutive competence systems and can take up eDNA throughout their growth phase. DprA (DNA processing protein A) is a transformation-specific recombination mediator protein (RMP) that plays a role in bacterial NT, and the absence of this gene significantly reduces the transformation efficiencies of both chromosomal and plasmid DNA. NT helps bacteria survive under adverse conditions and contributes to genetic diversity in bacteria. The present work describes the characterization of DprA from D. radiodurans and will add to the existing knowledge of DprA biology.

WD40 domain of RqkA regulates its kinase activity and role in extraordinary radioresistance of D. radiodurans.

RqkA, a DNA damage responsive serine/threonine kinase, is characterized for its role in DNA repair and cell division in D. radiodurans. It has a unique combination of a kinase domain at N-terminus and a WD40 type domain at C-terminus joined through a linker. WD40 domain is comprised of eight ß-propeller repeats held together via 'tryptophan-docking motifs' and forming a typical 'velcro' closure structure. RqkA mutants lacking the WD40 region (hereafter referred to as WD mutant) could not complement RqkA loss in γ radiation resistance in D. radiodurans and lacked γ radiation-mediated activation of kinase activity in vivo. WD mutants failed to phosphorylate its cognate substrate (e.g. DrRecA) in surrogate E. coli cells. Unlike wild-type enzyme, the kinase activity of its WD40 mutants was not stimulated by pyrroloquinoline quinine (PQQ) indicating the role of the WD motifs in PQQ interaction and stimulation of its kinase activity. Together, results highlighted the importance of the WD40 domain in the regulation of RqkA kinase signaling functions in vivo, and thus, the role of WD40 domain in the regulation of any STPK is first time demonstrated in bacteria.Communicated by Ramaswamy H. Sarma.

Involvement of serine / threonine protein kinases in DNA damage response and cell division in bacteria.

The roles of Serine/Threonine protein kinases (STPKs) in bacterial physiology, including bacterial responses to nutritional stresses and under pathogenesis have been well documented. STPKs roles in bacterial cell cycle regulation and DNA damage response have not been much emphasized, possibly because the LexA/RecA type SOS response became the synonym to DNA damage response and cell cycle regulation in bacteria. This review summarizes current knowledge of STPKs genetics, domain organization, and their roles in DNA damage response and cell division regulation in bacteria.

MusaMPK5, a mitogen activated protein kinase is involved in regulation of cold tolerance in banana.

Mitogen activated protein kinases (MAPKs) are known to play important functions in stress responses of plants. We have functionally characterized a MAPK, MusaMPK5 from banana and demonstrated its function in cold tolerance response of banana plants. Expression of MusaMPK5 showed positive response to cold, methyl-jasmonate and salicylic acid treatment. Transgenic banana plants harbouring PMusaMPK5::GUS after exposure to cold stress (8 °C) showed strong induction of GUS in cells surrounding central vascular cylinder of corm and cortical cells of pseudostem. Transgenic banana lines overexpressing MusaMPK5 were regenerated and four different transgenic lines were confirmed for T-DNA insertions by Southern blot and PCR analysis. In an in-vitro growth assay transgenic lines gained better shoot length and fresh weight during recovery from cold stress indicating improved cold tolerance ability of transgenic lines than control plants. Leaf discs of transgenic lines bleached less and retain lower MDA content than leaf discs of control plants after cold stress (4 °C and 8 °C). Cold stress tolerance analysis using two month old plants suggested that improved cold tolerance ability of transgenic lines might be associated with increased level of proline and reduced MDA content. MusaMPK5 gets localized in cytoplasm as observed in onion epidermal cells transiently overexpressing either MusaMPK5-GFP or MusaMPK5-GUS fusion protein. MusaMPK5 is a functional kinase as it autophosphorylate itself and phosphorylate myelin basic protein (MBP) in an in vitro reaction. Purified MusaMPK5 can phosphorylate NAC042 and SNAC67 transcription factors of banana which are important regulators of stress tolerance in banana plants.

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.

drFrnE Represents a Hitherto Unknown Class of Eubacterial Cytoplasmic Disulfide Oxido-Reductases.

AIMS: Living cells employ thioredoxin and glutaredoxin disulfide oxido-reductases to protect thiol groups in intracellular proteins. FrnE protein of Deinococcus radiodurans (drFrnE) is a disulfide oxido-reductase that is induced in response to Cd2+ exposure and is involved in cadmium and radiation tolerance. The aim of this study is to probe structure, function, and cellular localization of FrnE class of proteins. RESULTS: Here, we show drFrnE as a novel cytoplasmic oxido-reductase that could be functional in eubacteria under conditions where thioredoxin/glutaredoxin systems are inhibited or absent. Crystal structure analysis of drFrnE reveals thioredoxin fold with an alpha helical insertion domain and a unique, flexible, and functionally important C-terminal tail. The C-tail harbors a novel 239-CX4C-244 motif that interacts with the active site 22-CXXC-25 motif. Crystal structures with different active site redox states, including mixed disulfide (Cys22-Cys244), are reported here. The biochemical data show that 239-CX4C-244 motif channels electrons to the active site cysteines. drFrnE is more stable in the oxidized form, compared with the reduced form, supporting its role as a disulfide reductase. Using bioinformatics analysis and fluorescence microscopy, we show cytoplasmic localization of drFrnE. We have found "true" orthologs of drFrnE in several eubacterial phyla and, interestingly, all these groups apparently lack a functional glutaredoxin system. Innovation and Conclusion: We show that drFrnE represents a new class of hitherto unknown intracellular oxido-reductases that are abundantly present in eubacteria. Unlike other well-known oxido-reductases, FrnE harbors an additional dithiol motif that acts as a conduit to channel electrons to the active site during catalytic turnover. Antioxid. Redox Signal. 28, 296-310.

Studies of protein-protein interactions in Fanconi anemia pathway to unravel the DNA interstrand crosslink repair mechanism.

Fanconi anemia (FA), a cancer predisposition syndrome exhibits hallmark feature of radial chromosome formation, and hypersensitivity to DNA crosslinking agents. A set of FA pathway proteins mainly FANCI, FANCD2 and BRCA2 are expressed to repair the covalent crosslink between the dsDNA. However, FA, BRCA pathways play an important role in DNA ICL repair as well as in homologous recombination repair, but the presumptive role of FA-BRCA proteins has not clearly explored particularly in context to function associated protein-protein interactions (PPIs). Here, in-vivo, in-vitro and in-silico studies have been performed for functionally relevant domains of FANCI, FANCD2 and BRCA2. To our conclusion, FANCI ARM repeat interacts with FANCD2 CUE domain and BRCA2 C-terminal region. Interestingly, FANCD2 CUE domain also interacts strongly with BRCA2 C-terminal region. Interactions between BRCA2 CTR and functionally relevant mutations Ser222Ala (cell cycle checkpoint mutant) and Leu231Arg (DNA ICL repair mutant) present in FANCD2 CUE domain have been analysed. To our finding, these mutations abrogate the binding between FANCD2 CUE domain and BRCA2 CTR. Furthermore, (1) different domain of FANCI, FANCD2 and BRCA2 are playing important role in PPIs, (2) mutations cause the impairment in the PPIs which in turn may disrupt the DNA ICL repair mechanism.

Structural and biophysical properties of h-FANCI ARM repeat protein.

Fanconi anemia complementation groups - I (FANCI) protein facilitates DNA ICL (Inter-Cross-link) repair and plays a crucial role in genomic integrity. FANCI is a 1328 amino acids protein which contains armadillo (ARM) repeats and EDGE motif at the C-terminus. ARM repeats are functionally diverse and evolutionarily conserved domain that plays a pivotal role in protein-protein and protein-DNA interactions. Considering the importance of ARM repeats, we have explored comprehensive in silico and in vitro approach to examine folding pattern. Size exclusion chromatography, dynamic light scattering (DLS) and glutaraldehyde crosslinking studies suggest that FANCI ARM repeat exist as monomer as well as in oligomeric forms. Circular dichroism (CD) and fluorescence spectroscopy results demonstrate that protein has predominantly α- helices and well-folded tertiary structure. DNA binding was analysed using electrophoretic mobility shift assay by autoradiography. Temperature-dependent CD, Fluorescence spectroscopy and DLS studies concluded that protein unfolds and start forming oligomer from 30°C. The existence of stable portion within FANCI ARM repeat was examined using limited proteolysis and mass spectrometry. The normal mode analysis, molecular dynamics and principal component analysis demonstrated that helix-turn-helix (HTH) motif present in ARM repeat is highly dynamic and has anti-correlated motion. Furthermore, FANCI ARM repeat has HTH structural motif which binds to double-stranded DNA.

Phosphorylation of Deinococcus radiodurans RecA Regulates Its Activity and May Contribute to Radioresistance.

Deinococcus radiodurans has a remarkable capacity to survive exposure to extreme levels of radiation that cause hundreds of DNA double strand breaks (DSBs). DSB repair in this bacterium depends on its recombinase A protein (DrRecA). DrRecA plays a pivotal role in both extended synthesis-dependent strand annealing and slow crossover events of DSB repair during the organism's recovery from DNA damage. The mechanisms that control DrRecA activity during the D. radiodurans response to γ radiation exposure are unknown. Here, we show that DrRecA undergoes phosphorylation at Tyr-77 and Thr-318 by a DNA damage-responsive serine threonine/tyrosine protein kinase (RqkA). Phosphorylation modifies the activity of DrRecA in several ways, including increasing its affinity for dsDNA and its preference for dATP over ATP. Strand exchange reactions catalyzed by phosphorylated versus unphosphorylated DrRecA also differ. In silico analysis of DrRecA structure support the idea that phosphorylation can modulate crucial functions of this protein. Collectively, our findings suggest that phosphorylation of DrRecA enables the recombinase to selectively use abundant dsDNA substrate present during post-irradiation recovery for efficient DSB repair, thereby promoting the extraordinary radioresistance of D. radiodurans.

DNA Gyrase of Deinococcus radiodurans is characterized as Type II bacterial topoisomerase and its activity is differentially regulated by PprA in vitro.

The multipartite genome of Deinococcus radiodurans forms toroidal structure. It encodes topoisomerase IB and both the subunits of DNA gyrase (DrGyr) while lacks other bacterial topoisomerases. Recently, PprA a pleiotropic protein involved in radiation resistance in D. radiodurans has been suggested for having roles in cell division and genome maintenance. In vivo interaction of PprA with topoisomerases has also been shown. DrGyr constituted from recombinant gyrase A and gyrase B subunits showed decatenation, relaxation and supercoiling activities. Wild type PprA stimulated DNA relaxation activity while inhibited supercoiling activity of DrGyr. Lysine133 to glutamic acid (K133E) and tryptophane183 to arginine (W183R) replacements resulted loss of DNA binding activity in PprA and that showed very little effect on DrGyr activities in vitro. Interestingly, wild type PprA and its K133E derivative continued interacting with GyrA in vivo while W183R, which formed relatively short oligomers did not interact with GyrA. The size of nucleoid in PprA mutant (1.9564 ± 0.324 µm) was significantly bigger than the wild type (1.6437 ± 0.345 µm). Thus, we showed that DrGyr confers all three activities of bacterial type IIA family DNA topoisomerases, which are differentially regulated by PprA, highlighting the significant role of PprA in DrGyr activity regulation and genome maintenance in D. radiodurans.

Structure-function study of deinococcal serine/threonine protein kinase implicates its kinase activity and DNA repair protein phosphorylation roles in radioresistance of Deinococcus radiodurans.

The DR2518 (RqkA) a eukaryotic type serine/threonine protein kinase in Deinococcus radiodurans was characterized for its role in bacterial response to oxidative stress and DNA damage. The K42A, S162A, T169A and S171A mutation in RqkA differentially affected its kinase activity and functional complementation for γ radiation resistance in Δdr2518 mutant. For example, K42A mutant was completely inactive and showed no complementation while S171A, T169A and T169A/S171A mutants were less active and complemented proportionally to different levels as compared to wild type. Amongst, different DNA binding proteins that purified RqkA could phosphorylate, PprA a DNA repair protein, phosphorylation had improved its affinity to DNA by 4 fold and could enhance its supportive role in intermolecular ligation by T4 DNA ligase. RqkA phosphorylates PprA at threonine 72 (T72), serine 112 (S112) and threonine 144 (T144) in vitro with the majority of it goes to T72 site. Unlike wild type PprA and single mutants of T72, S112 and T144 residues, the T72AS112A double and T72AS112AT144A triple mutant derivatives of PprA did not phosphorylate in vivo and also failed to complement PprA loss in D. radiodurans. Deletion of rqkA in pprA::cat background enhanced radiosensitivity of pprA mutant, which became nearly similar to ΔrqkA resistance to γ radiation. These results suggested that K42 of RqkA is essential for catalytic functions and the kinase activity of RqkA as well as phosphorylation of PprA have roles in γ radiation resistance of D. radiodurans.

DR1769, a protein with N-terminal beta propeller repeats and a low-complexity hydrophilic tail, plays a role in desiccation tolerance of Deinococcus radiodurans.

The Deinococcus radiodurans genome encodes five putative quinoproteins. Among these, the Δdr2518 and Δdr1769 mutants became sensitive to gamma radiation. DR2518 with beta propeller repeats in the C-terminal domain was characterized as a radiation-responsive serine/threonine protein kinase in this bacterium. DR1769 contains beta propeller repeats at the N terminus, while its C-terminal domain is a proline-rich disordered structure and constitutes a low-complexity hydrophilic region with aliphatic-proline dipeptide motifs. The Δdr1769 mutant showed nearly a 3-log cycle sensitivity to desiccation at 5% humidity compared to that of the wild type. Interestingly, the gamma radiation and mitomycin C (MMC) resistance in mutant cells also dropped by ∼1-log cycle at 10 kGy and ∼1.5-fold, respectively, compared to those in wild-type cells. But there was no effect of UV (254 nm) exposure up to 800 J · m(-2). These cells showed defective DNA double-strand break repair, and the average size of the nucleoid in desiccated wild-type and Δdr1769 cells was reduced by approximately 2-fold compared to that of respective controls. However, the nucleoid in wild-type cells returned to a size almost similar to that of the untreated control, which did not happen in mutant cells, at least up to 24 h postdesiccation. These results suggest that DR1769 plays an important role in desiccation and radiation resistance of D. radiodurans, possibly by protecting genome integrity under extreme conditions.

Characterization of the role of the RadS/RadR two-component system in the radiation resistance of Deinococcus radiodurans.

Deinococcus radiodurans shows extraordinary tolerance to DNA damage, and exhibits differential gene expression and protein recycling. A putative response regulator, the DRB0091 (RadR) ORF, was identified from a pool of DNA-binding proteins induced in response to gamma radiation in this bacterium. radR is located upstream of drB0090, which encodes a putative sensor histidine kinase (RadS) on the megaplasmid. Deletion of these genes both individually and together resulted in hypersensitivity to DNA-damaging agents and a delayed or altered double-strand break repair. A ΔradRradS double mutant and a ΔradR single mutant showed nearly identical responses to gamma radiation and UVC. Wild-type RadR and RadS complemented the corresponding mutant strains, but also exhibited significant cross-complementation, albeit at lower doses of gamma radiation. The radS transcript was not detected in the ΔradR mutant, suggesting the existence of a radRS operon. Recombinant RadS was autophosphorylated and could catalyse the transfer of γ phosphate from ATP to RadR in vitro. These results indicated the functional interaction of RadS and RadR, and suggested a role for the RadS/RadR two-component system in the radiation resistance of this bacterium.

Survival of phosphate-solubilizing bacteria against DNA damaging agents.

Phosphate-solubilizing bacteria (PSBs) were isolated from different plant rhizosphere soils of various agroecological regions of India. These isolates showed synthesis of pyrroloquinoline quinone (PQQ), production of gluconic acid, and release of phosphorus from insoluble tricalcium phosphate. The bacterial isolates synthesizing PQQ also showed higher tolerance to ultraviolet C radiation and mitomycin C as compared to Escherichia coli but were less tolerant than Deinococcus radiodurans. Unlike E. coli, PSB isolates showed higher tolerance to DNA damage when grown in the absence of inorganic phosphate. Higher tolerance to ultraviolet C radiation and oxidative stress in these PSBs grown under PQQ synthesis inducible conditions, namely phosphate starvation, might suggest the possible additional role of this redox cofactor in the survival of these isolates under extreme abiotic stress conditions.

ATP-type DNA ligase requires other proteins for its activity in vitro and its operon components for radiation resistance in Deinococcus radiodurans in vivo.

A multiprotein DNA processing complex isolated from Deinococcus radiodurans contains the DNA repair protein PprA, an ATP-type DNA repair ligase (LigB) encoded by the drB0100 gene, and protein kinase activity. An ATP-dependent DNA end-joining activity was detected in the complex. To elucidate the function of the drB0100 gene, we generated the deletion mutant for the DR_B0100 ORF. The mutant exhibited a nearly 2-log cycle reduction in growth rate when exposed to a 10,000 Gray dose of γ-radiation, and a significant loss in mitomycin C and methylmethane sulphonate tolerance as compared with wild type. Functional complementation of these phenotypes required the wild-type copy of drB0100 along with other genes such as drb0099 and drb0098, organized downstream in the operon. The in vitro DNA ligase activity of LigB was stimulated severalfold by PprA in the presence of the recombinant DRB0098 protein. However, this activity did not improve when PprA was substituted with purified DRB0099 protein or when DRB0098 protein was substituted with the DRB0099 protein in the presence of PprA in solution. These results suggest that PprA and DRB0098 protein are required for LigB function. Furthermore, they also suggest that the LigB operon components contribute to radiation resistance and double-strand break (DSB) repair in D. radiodurans.

Characterization of a DNA damage-inducible membrane protein kinase from Deinococcus radiodurans and its role in bacterial radioresistance and DNA strand break repair.

Deinococcus radiodurans mutant lacking pyrroloquinoline-quinone (PQQ) synthesis shows sensitivity to γ-rays and impairment of DNA double strand break repair. The genome of this bacterium encodes five putative proteins having multiple PQQ binding motifs. The deletion mutants of corresponding genes were generated, and their response to DNA damage was monitored. Only the Δdr2518 mutant exhibited higher sensitivity to DNA damage. Survival of these cells decreased by 3-log cycle both at 6 kGy γ-rays and 1200 Jm(-2) UV (254 nm) radiation, and 2.5-log cycle upon 14 days desiccation at 5% humidity. The Δdr2518 mutant showed complete inhibition of DSB repair until 24 h PIR and disappearance of a few phosphoproteins. The Δdr2518pqqE:cat double mutant showed γ-ray sensitivity similar to Δdr2518 indicating functional interaction of these genes in D. radiodurans. DR2518 contains a eukaryotic type Ser/Thr kinase domain and structural topology suggesting stress responsive transmembrane protein. Its autokinase activity in solution was stimulated by nearly threefold with PQQ and twofold with linear DNA, but not with circular plasmid DNA. More than 15-fold increase in dr2518 transcription and several-fold enhanced in vivo phosphorylation of DR2518 were observed in response to γ irradiation. These results suggest that DR2518 as a DNA damage-responsive protein kinase plays an important role in radiation resistance and DNA strand break repair in D. radiodurans.

Increased synthesis of signaling molecules coincides with reversible inhibition of nucleolytic activity during postirradiation recovery of Deinococcus radiodurans.

Deinococcus radiodurans tolerates extensive DNA damage and exhibits differential expression of various genes associated with the growth of the organism and DNA repair. In cells treated with gamma radiation, the levels of cyclic AMP (cAMP) and ATP increased rapidly by differentially regulating adenylyl cyclase (AC) and 2'3' cAMP phosphodiesterase. The levels of cAMP, ATP, AC and protein kinases were high when phosphodiesterase activity was low. These cells exhibited in vivo inhibition of nucleolytic function by reversible protein phosphorylation and contained the comparatively higher levels of total phosphoproteins. We suggest that Deinococcus, a prokaryote, uses DNA damage-induced signaling mechanism as evidenced by gamma radiation-induced synthesis of secondary messengers and signaling enzymes.

Involvement of a protein kinase activity inducer in DNA double strand break repair and radioresistance of Deinococcus radiodurans.

Transgenic bacteria producing pyrroloquinoline quinone, a known cofactor for dehydrogenases and an inducer of a periplasmic protein kinase activity, show resistance to both oxidative stress and protection from nonoxidative effects of radiation and DNA-damaging agents. Deinococcus radiodurans R1 encodes an active pyrroloquinoline quinone synthase, and constitutive synthesis of pyrroloquinoline quinone occurred in wild-type bacteria. Disruption of a genomic copy of pqqE resulted in cells that lacked this cofactor. The mutant showed a nearly 3-log decrease in gamma radiation resistance and a 2-log decrease in mitomycin C tolerance compared to wild-type cells. The mutant cells did not show sensitivity to UVC radiation. Expression of pyrroloquinoline quinone synthase in trans showed that there was functional complementation of gamma resistance and mitomycin C tolerance in the pqqE mutant. The sensitivity to gamma radiation was due to impairment or slow kinetics of DNA double strand break repair. Low levels of (32)P incorporation were observed in total soluble proteins of mutant cells compared to the wild type. The results suggest that pyrroloquinoline quinone has a regulatory role as a cofactor for dehydrogenases and an inducer of selected protein kinase activity in radiation resistance and DNA strand break repair in a radioresistant bacterium.