Tracing the serendipitous genesis of radiation resistance.

Free-living organisms frequently encounter unfavorable abiotic environmental factors. Those who adapt and cope with sudden changes in the external environment survive. Desiccation is one of the most common and frequently encountered stresses in nature. On the contrary, ionizing radiations are limited to high local concentrations of naturally occurring radioactive materials and related anthropogenic activities. Yet, resistance to high doses of ionizing radiation is evident across the tree of life. The evolution of desiccation resistance has been linked to the evolution of ionizing radiation resistance, although, evidence to support the idea that the evolution of desiccation tolerance is a necessary precursor to ionizing radiation resistance is lacking. Moreover, the presence of radioresistance in hyperthermophiles suggests multiple paths lead to radiation resistance. In this minireview, we focus on the molecular aspects of damage dynamics and damage response pathways comprising protective and restorative functions with a definitive survival advantage, to explore the serendipitous genesis of ionizing radiation resistance.

An in vivo Interaction Network of DNA-Repair Proteins: A Snapshot at Double Strand Break Repair in Deinococcus radiodurans.

An extremophile Deinococcus radiodurans survives massive DNA damage by efficiently mending hundreds of double strand breaks through homology-dependent DNA repair pathways. Although DNA repair proteins that contribute to its impressive DNA repair capacity are fairly known, interactions among them or with proteins related to other relevant pathways remain unexplored. Here, we report in vivo cross-linking of the interactomes of key DNA repair proteins DdrA, DdrB, RecA, and Ssb (baits) in D. radiodurans cells recovering from gamma irradiation. The protein-protein interactions were systematically investigated through co-immunoprecipitation experiments coupled to mass spectrometry. From a total of 399 proteins co-eluted with the baits, we recovered interactions among diverse biological pathways such as DNA repair, transcription, translation, chromosome partitioning, cell division, antioxidation, protein folding/turnover, metabolism, cell wall architecture, membrane transporters, and uncharacterized proteins. Among these, about 80 proteins were relevant to the DNA damage resistance of the organism based on integration of data on inducible expression following DNA damage, radiation sensitive phenotype of deletion mutant, etc. Further, we cloned ORFs of 23 interactors in heterologous E. coli and expressed corresponding proteins with N-terminal His-tag, which were used for pull-down assays. A total of 95 interactions were assayed, in which we confirmed 25 previously unknown binary interactions between the proteins associated with radiation resistance, and 2 known interactions between DdrB and Ssb or DR_1245. Among these, five interactions were positive even under non-stress conditions. The confirmed interactions cover a wide range of biological processes such as DNA repair, negative regulation of cell division, chromosome partitioning, membrane anchorage, etc., and their functional relevance is discussed from the perspective of DNA repair. Overall, the study substantially advances our understanding on the cross-talk between different homology-dependent DNA repair pathways and other relevant biological processes that essentially contribute to the extraordinary DNA damage repair capability of D. radiodurans. The data sets generated and analyzed in this study have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD021822.

Membrane Vesicles of Group B Streptococcus Disrupt Feto-Maternal Barrier Leading to Preterm Birth.

Infection of the genitourinary tract with Group B Streptococcus (GBS), an opportunistic gram positive pathogen, is associated with premature rupture of amniotic membrane and preterm birth. In this work, we demonstrate that GBS produces membrane vesicles (MVs) in a serotype independent manner. These MVs are loaded with virulence factors including extracellular matrix degrading proteases and pore forming toxins. Mice chorio-decidual membranes challenged with MVs ex vivo resulted in extensive collagen degradation leading to loss of stiffness and mechanical weakening. MVs when instilled vaginally are capable of anterograde transport in mouse reproductive tract. Intra-amniotic injections of GBS MVs in mice led to upregulation of pro-inflammatory cytokines and inflammation mimicking features of chorio-amnionitis; it also led to apoptosis in the chorio-decidual tissue. Instillation of MVs in the amniotic sac also resulted in intrauterine fetal death and preterm delivery. Our findings suggest that GBS MVs can independently orchestrate events at the feto-maternal interface causing chorio-amnionitis and membrane damage leading to preterm birth or fetal death.

Significant alterations of the novel 15 gene signature identified from macrophage-tumor interactions in breast cancer.

BACKGROUND: Tumor microenvironment is composed of a largely altered extracellular matrix with different cell types. The complex interplay between macrophages and tumor cells through several soluble factors and signaling is an important factor in breast cancer progression. METHODS: We have extended our earlier studies on monocyte and macrophage conditioned medium (MϕCM) and have carried out proteomic analysis to identify its constituents as well as validation. The 8-gene signature identified through macrophage-breast cancer cell interactions was queried in cBioportal for bioinformatic analyses. RESULTS: Proteomic analysis (MALDI-TOF and LC-MS/MS) revealed integrin and matrix metalloproteinases in MϕCM which activated TGF-ß1, IL-6, TGF- ßRII and EGFR as well as its downstream STAT and SMAD signaling in breast cancer cells. Neutralization of pro-inflammatory cytokines (TNF-α. Il-1ß, IL-6) abrogated the MϕCM induced migration but invasion to lesser extent. The 8- gene signature identified by macrophage-tumor interactions (TNF-α, IL-1ß, IL-6, MMP1, MMP9, TGF-ß1, TGF-ßRII, EGFR) significantly co-occurred with TP53 mutation, WTAPP1 deletion and SLC12A5 amplification along with differential expression of PSAT1 and ESR1 at the mRNA level and TPD52and PRKCD at the protein level in TCGA (cBioportal). Together these genes form a novel 15 gene signature which is altered in 63.6% of TCGA (1105 samples) data and was associated with high risk and poor survival (p<0.05) in many breast cancer datasets (SurvExpress). CONCLUSIONS: These results highlight the importance of macrophage signaling in breast cancer and the prognostic role of the15-gene signature. GENERAL SIGNIFICANCE: Our study may facilitate novel prognostic markers based on tumor-macrophage interaction.

Developmental proteome dynamics of silk glands in the 5th instar larval stage of Bombyx mori L (CSR2×CSR4).

UNLABELLED: Bivoltine breed of Bombyx mori (B. mori), CSR2×CSR4 is an Indian high yielding silkworm strain. Silk gland proteome of this strain was not studied till now. Methods of improving silk production by chemical approaches have reached saturation and transgenic methods are needed in further to boost silk production. An understanding of proteomic changes during silk gland development helps in designing experiments to enhance silk production by transgenic approaches. The present study reports comprehensive developmental proteomic analysis of CSR2×CSR4, 5th instar whole silk glands. Eighty six unique protein IDs were obtained from the analysis of one hundred and twenty protein spots. Among the identified proteins, majority of the proteins were involved in metabolism (41%) followed by proteins involved in protein homeostasis (30%). Sixty percent of the identified proteins showed dynamic nature by expression analysis from day 1, day 3, day 5 and day 7 gels. In comparison to the published data till now on silk gland proteomics this study reports identification of 20 new proteins from the silk glands for the first time. SIGNIFICANCE: The paper reports for the first time proteomic analysis of high yielding silkworm strain of India. The study analyzes whole silk glands to understand the tissue in total during 5th instar development. Lowering fibroin content made us to identify a large number of new proteins which were not reported till now in the silk gland proteome. Proteins which are involved in silk synthesis and release were found to be developmentally regulated. The study identified alanine, serine and glycine tRNA ligases for the first time and also showed their up-regulation on day 7 of 5th instar larval stage. The amino acid repeat of fibroin protein is enriched with the three amino acids, glycine, serine and alanine. The identified proteins could be studied further to understand their functional role in-depth.

Proteome dynamics during post-desiccation recovery reveal convergence of desiccation and gamma radiation stress response pathways in Deinococcus radiodurans.

Deinococcus radiodurans is inherently resistant to both ionizing radiation and desiccation. Fifteen months of desiccation was found to be the LD50 dose for D. radiodurans. Desiccated cells of D. radiodurans entered 6h of growth arrest during post-desiccation recovery (PDR). Proteome dynamics during PDR were mapped by resolving cellular proteins by 2-dimensional gel electrophoresis coupled with mass spectrometry. At least 41 proteins, represented by 51 spots on proteome profiles, were differentially expressed throughout PDR. High upregulation in expression was observed for DNA repair proteins involved in single strand annealing (DdrA and DdrB), nucleotide excision repair (UvrA and UvrB), homologous recombination (RecA) and other vital proteins that contribute to DNA replication, recombination and repair (Ssb, GyrA and GyrB). Expression of CRP/FNR family transcriptional regulator (Crp) remained high throughout PDR. Other pathways such as cellular detoxification, protein homeostasis and metabolism displayed both, moderately induced and repressed proteins. Functional relevance of proteomic modulations to surviving desiccation stress is discussed in detail. Comparison of our data with the published literature revealed convergence of radiation and desiccation stress responses of D. radiodurans. This is the first report that substantiates the hypothesis that the radiation stress resistance of D. radiodurans is incidental to its desiccation stress resistance.

Putative DNA modification methylase DR_C0020 of Deinococcus radiodurans is an atypical SAM dependent C-5 cytosine DNA methylase.

BACKGROUND: Control of cellular processes by epigenetic modification of cytosine in DNA is widespread among living organisms, but, is hitherto unknown in the extremely radioresistant microbe D. radiodurans. METHODS: C-5 methyl cytosines (m5C) were detected by immuno-blotting with m5C-specific antibody. Site of cytosine methylation by DR_C0020 encoded protein was investigated by bisulfite sequencing. The DR_C0020 knockout mutant (Δdcm), constructed by site directed mutagenesis, was assessed for effect on growth, radiation resistance and proteome. Proteins were identified by mass spectrometry. RESULTS: Methylated cytosines were detected in the D. radiodurans genome. The DR_C0020 encoded protein (Dcm, NCBI accession: WP_034351354.1), whose amino acid sequence resembles m4C methylases, was shown to be the lone SAM-dependent C-5 cytosine methyltransferase. Purified Dcm protein was found to methylate CpN sequence with a preference for methylation of two consecutive cytosines. The Δdcm strain completely lost m5C modification from its genome, had no effect on growth but became radiation sensitive. The Δdcm cells exhibited minor alterations in the abundance of several proteins involved primarily in protein homeostasis, oxidative stress defense, metabolism, etc. CONCLUSION: DR_C0020 encoded SAM-dependent methyltransferase Dcm is solely responsible for C-5cytosine methylation at CpN sites in the genome of D. radiodurans and regulates protein homeostasis under normal growth conditions. The protein is an unusual case of an amino methyltransferase that has evolved to producing m5C. GENERAL SIGNIFICANCE: Although, dispensable under optimal growth conditions, the presence of m5C may be important for recognition of parent strand and, thus, could contribute to the extraordinary DNA repair in D. radiodurans.

Surface (S)-layer proteins of Deinococcus radiodurans and their utility as vehicles for surface localization of functional proteins.

The radiation resistant bacterium, Deinococcus radiodurans contains two major surface (S)-layer proteins, Hpi and SlpA. The Hpi protein was shown to (a) undergo specific in vivo cleavage, and (b) closely associate with the SlpA protein. Using a non-specific acid phosphatase from Salmonella enterica serovar Typhi, PhoN as a reporter, the Surface Layer Homology (SLH) domain of SlpA was shown to bind deinococcal peptidoglycan-containing cell wall sacculi. The association of SlpA with Hpi on one side and peptidoglycan on the other, localizes this protein in the 'interstitial' layer of the deinoccocal cell wall. Gene chimeras of hpi-phoN and slh-phoN were constructed to test efficacy of S-layer proteins, as vehicles for cell surface localization in D. radiodurans. The Hpi-PhoN protein localized exclusively in the membrane fraction, and displayed cell-based phosphatase activity in vivo. The SLH-PhoN, which localized to both cytosolic and membrane fractions, displayed in vitro activity but no cell-based in vivo activity. Hpi, therefore, emerged as an efficient surface localizing protein and can be exploited for suitable applications of this superbug.

Involvement of phosphoesterases in tributyl phosphate degradation in Sphingobium sp. strain RSMS.

A tri- and dibutyl phosphate (TBP/DBP) non-degrading spontaneous mutant, Sphingobium SS22, was derived from the Sphingobium sp. strain RSMS (wild type). Unlike the wild type strain, Sphingobium SS22 could not grow in a minimal medium supplemented with TBP or DBP as the sole source of carbon or phosphorous. Sphingobium SS22 also did not form any of the intermediates or end products of TBP or DBP degradation, namely DBP, butanol or inorganic phosphate. Proteomic analysis revealed the absence of three prominent proteins in Sphingobium SS22 as compared to wild type. These proteins were identified by MALDI mass spectrometry, and they showed similarities to phosphohydrolase- and exopolyphosphatase-like proteins from other bacteria, which belong to the class of phosphoesterases. Cellular proteins of Sphingobium SS22 showed none or negligible phosphodiesterase (PDE) and phosphomonoesterase (PME) activities at pH 7 and displayed approximately five- and approximately twofold less DBP and monobutyl phosphate (MBP) degradation activity, respectively, in comparison to the wild type strain. In-gel zymographic analysis revealed two PDE and PME activity bands in the wild type strain, one of which was absent in the Sphingobium SS22 mutant. The corresponding proteins from the wild type strain could degrade DBP and MBP. The results demonstrate the involvement of phosphoesterase enzymes in the TBP degradation pathway elucidated earlier.

Depletion of reduction potential and key energy generation metabolic enzymes underlies tellurite toxicity in Deinococcus radiodurans.

Oxidative stress resistant Deinococcus radiodurans surprisingly exhibited moderate sensitivity to tellurite induced oxidative stress (LD50 = 40 µM tellurite, 40 min exposure). The organism reduced 70% of 40 µM potassium tellurite within 5 h. Tellurite exposure significantly modulated cellular redox status. The level of ROS and protein carbonyl contents increased while the cellular reduction potential substantially decreased following tellurite exposure. Cellular thiols levels initially increased (within 30 min) of tellurite exposure but decreased at later time points. At proteome level, tellurite resistance proteins (TerB and TerD), tellurite reducing enzymes (pyruvate dehydrogense subunits E1 and E3), ROS detoxification enzymes (superoxide dismutase and thioredoxin reductase), and protein folding chaperones (DnaK, EF-Ts, and PPIase) displayed increased abundance in tellurite-stressed cells. However, remarkably decreased levels of key metabolic enzymes (aconitase, transketolase, 3-hydroxy acyl-CoA dehydrogenase, acyl-CoA dehydrogenase, electron transfer flavoprotein alpha, and beta) involved in carbon and energy metabolism were observed upon tellurite stress. The results demonstrate that depletion of reduction potential in intensive tellurite reduction with impaired energy metabolism lead to tellurite toxicity in D. radiodurans.

Methyl viologen responsive proteome dynamics of Anabaena sp. strain PCC7120.

A proteomic approach was employed to elucidate the response of an agriculturally important microbe, Anabaena sp. strain PCC7120, to methyl viologen (MV). Exposure to 2 µM MV caused 50% lethality (LD50 ) within 6 h and modified the cellular levels of several proteins. About 31 proteins increased in abundance and 24 proteins decreased in abundance, while 55 proteins showed only a minor change in abundance. Of these, 103 proteins were identified by MS. Levels of proteins involved in ROS detoxification and chaperoning activities were enhanced but that of crucial proteins involved in light and dark reactions of photosynthesis declined or constitutive. The abundance of proteins involved in carbon and energy biogenesis were altered. The study elaborated the oxidative stress defense mechanism deployed by Anabaena, identified carbon metabolism and energy biogenesis as possible major targets of MV sensitivity, and suggested potential biotechnological interventions for improved stress tolerance in Anabaena 7120.

Gamma radiation-induced proteome of Deinococcus radiodurans primarily targets DNA repair and oxidative stress alleviation.

The extraordinary radioresistance of Deinococcus radiodurans primarily originates from its efficient DNA repair ability. The kinetics of proteomic changes induced by a 6-kGy dose of gamma irradiation was mapped during the post-irradiation growth arrest phase by two-dimensional protein electrophoresis coupled with mass spectrometry. The results revealed that at least 37 proteins displayed either enhanced or de novo expression in the first 1 h of post-irradiation recovery. All of the radiation-responsive proteins were identified, and they belonged to the major functional categories of DNA repair, oxidative stress alleviation, and protein translation/folding. The dynamics of radiation-responsive protein levels throughout the growth arrest phase demonstrated (i) sequential up-regulation and processing of DNA repair proteins such as single-stranded DNA-binding protein (Ssb), DNA damage response protein A (DdrA), DNA damage response protein B (DdrB), pleiotropic protein promoting DNA repair (PprA), and recombinase A (RecA) substantiating stepwise genome restitution by different DNA repair pathways and (ii) concurrent early up-regulation of proteins involved in both DNA repair and oxidative stress alleviation. Among DNA repair proteins, Ssb was found to be the first and most abundant radiation-induced protein only to be followed by alternate Ssb, DdrB, indicating aggressive protection of single strand DNA fragments as the first line of defense by D. radiodurans, thereby preserving genetic information following radiation stress. The implications of both qualitative or quantitative and sequential or co-induction of radiation-responsive proteins for envisaged DNA repair mechanism in D. radiodurans are discussed.

Application of Machine Learning in Predicting Hepatic Metastasis or Primary Site in Gastroenteropancreatic Neuroendocrine Tumors.

Gastroenteropancreatic neuroendocrine tumors (GEP-NETs) account for 80% of gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs). GEP-NETs are well-differentiated tumors, highly heterogeneous in biology and origin, and are often diagnosed at the metastatic stage. Diagnosis is commonly through clinical symptoms, histopathology, and PET-CT imaging, while molecular markers for metastasis and the primary site are unknown. Here, we report the identification of multi-gene signatures for hepatic metastasis and primary sites through analyses on RNA-SEQ datasets of pancreatic and small intestinal NETs tissue samples. Relevant gene features, identified from the normalized RNA-SEQ data using the mRMRe algorithm, were used to develop seven Machine Learning models (LDA, RF, CART, k-NN, SVM, XGBOOST, GBM). Two multi-gene random forest (RF) models classified primary and metastatic samples with 100% accuracy in training and test cohorts and >90% accuracy in an independent validation cohort. Similarly, three multi-gene RF models identified the pancreas or small intestine as the primary site with 100% accuracy in training and test cohorts, and >95% accuracy in an independent cohort. Multi-label models for concurrent prediction of hepatic metastasis and primary site returned >98.42% and >87.42% accuracies on training and test cohorts, respectively. A robust molecular signature to predict liver metastasis or the primary site for GEP-NETs is reported for the first time and could complement the clinical management of GEP-NETs.

Deinococcus lineage and Rad52 family-related protein DR0041 is involved in DNA protection and compaction.

DR0041 ORF encodes an uncharacterized Deinococcus lineage protein. We earlier reported presence of DR0041 protein in DNA repair complexes of Ssb and RecA in Deinococcus radiodurans. Here, we systematically examined the role of DR0041 in DNA metabolism using various experimental methodologies including electrophoretic mobility assays, nuclease assays, strand exchange assays and transmission electron microscopy. Interaction between DR0041 and the C-terminal acidic tail of Ssb was assessed through co-expression and in vivo cross-linking studies. A knockout mutant was constructed to understand importance of DR0041 ORF for various physiological processes. Results highlight binding of DR0041 protein to single-stranded and double-stranded DNA, interaction with Ssb-coated single-stranded DNA without interference with RecA-mediated strand exchange, protection of DNA from exonucleases, and compaction of high molecular weight DNA molecules into tightly condensed forms. Bridging and compaction of sheared DNA by DR0041 protein might have implications in the preservation of damaged DNA templates to maintain genome integrity upon exposure to gamma irradiation. Our results suggest that DR0041 protein is dispensable for growth under standard growth conditions and following gamma irradiation but contributes to protection of DNA during transformation. We discuss the role of DR0041 protein from the perspective of protection of broken DNA templates and functional redundancy.

The radiophiles of Deinococcaceae family: Resourceful microbes for innovative biotechnological applications.

Extremophiles are nature's tiny warriors as they call inhospitable environments their home. They possess special factor(s) that offer an edge over other life forms susceptible to harsh conditions. One such family of extremophiles under discussion here is Deinococcaceae. The microbes belonging to Deinococcaceae are primarily radiophiles, the world's most radiation resistant bacteria, in addition to having resistance to high temperature, metals, cold etc. in specific species. Gamma rays have always been known to be lethal to living cells as it damages DNA, the blueprint of life. But, Deinococci sustain extremely high doses of gamma radiation, about 3000 times more than the dose humans succumb to. This review brings forth the utility of these special factors of Deinococcaceae for a broad range of biotechnological applications.

Genome-wide lone strand adenine methylation in Deinococcus radiodurans R1: Regulation of gene expression through DR0643-dependent adenine methylation.

DNA methylation is a covalent modification of adenine or cytosine in the genome of an organism and is found in diverse microbes including the radiation resistant bacterium Deinococcus radiodurans R1. Although earlier findings have confirmed repression or de-repression of certain genes in adenine methyltransferase (DR_0643/Dam1DR) deficient D. radiodurans mutant however, the overall regulatory aspects of Dam1DR-mediated adenine methylation remain mostly unexplored. In the present study, we compared the genome-wide methylome and the corresponding transcriptome of D. radiodurans WT and Δdam1 mutant to explore the correlation between methylation and gene expression. In D. radiodurans, deletion of DR_0643 ORF (Δdam1) led to hypomethylation of 512 genes resulting in differential expression of 168 genes (99 genes are upregulated and 69 genes are downregulated). The modification patterns deduced for Dam1DR (DR_0643) and Dam2DR (DR_2267) were non-palindromic and atypical. Moreover, we observed methylation at opportunistic sites that show adenine methylation only in D. radiodurans Δdam1 and not in D. radiodurans WT. Correlation between the methylome and transcriptome suggests that hypomethylation at Dam1DR specific sites had both negative as well as a positive effects on gene expression. Pathways such as amino acid metabolism, transport, oxidative phosphorylation, quorum sensing, signal transduction, two-component system, glycolysis/gluconeogenesis, TCA cycle, glyoxylate and dicarboxylate metabolism were modulated by Dam1DR-mediated adenine methylation in D. radiodurans. Processes such as DNA repair, recombination, ATPase and transmembrane transporter activity were enriched when Dam1DR mutant was subjected to radiation stress. We further evaluated the molecular interactions and mode of binding between Dam1DR protein and S-adenosyl methionine using molecular docking followed by MD simulation. To get a better insight into the methylation mechanism, the Dam1DR-SAM complex was also docked with a DNA molecule to elucidate DNA-Dam1DR structural interaction during methyl-group transfer reaction. In summary, our work presents comprehensive and integrative approaches to investigate both functional and structural aspects of DNA adenine methyltransferase (Dam1DR) in D. radiodurans biology.

Proteomic Perspective of Cadmium Tolerance in Providencia rettgeri Strain KDM3 and Its In-situ Bioremediation Potential in Rice Ecosystem.

In this study, a multi-metal-tolerant natural bacterial isolate Providencia rettgeri strain KDM3 from an industrial effluent in Mumbai, India, showed high cadmium (Cd) tolerance. Providencia rettgeri grew in the presence of more than 100 ppm (880 µM) Cd (LD50 = 100 ppm) and accumulated Cd intracellularly. Following Cd exposure, a comparative proteome analysis revealed molecular mechanisms underlying Cd tolerance. Among a total of 69 differentially expressed proteins (DEPs) in Cd-exposed cells, de novo induction of ahpCF operon proteins and L-cysteine/L-cystine shuttle protein FliY was observed, while Dps and superoxide dismutase proteins were overexpressed, indicating upregulation of a robust oxidative stress defense. ENTRA1, a membrane transporter showing homology to heavy metal transporter, was also induced de novo. In addition, the protein disaggregation chaperone ClpB, trigger factor, and protease HslU were also overexpressed. Notably, 46 proteins from the major functional category of energy metabolism were found to be downregulated. Furthermore, the addition of P. rettgeri to Cd-spiked soil resulted in a significant reduction in the Cd content [roots (11%), shoot (50%), and grains (46%)] of the rice plants. Cd bioaccumulation of P. rettgeri improved plant growth and grain yield. We conclude that P. rettgeri, a highly Cd-tolerant bacterium, is an ideal candidate for in-situ bioremediation of Cd-contaminated agricultural soils.

Repression of the glucose-inducible outer-membrane protein OprB during utilization of aromatic compounds and organic acids in Pseudomonas putida CSV86.

Pseudomonas putida CSV86 shows preferential utilization of aromatic compounds over glucose. Protein analysis and [¹4C]glucose-binding studies of the outer membrane fraction of cells grown on different carbon sources revealed a 40 kDa protein that was transcriptionally induced by glucose and repressed by aromatics and succinate. Based on 2D gel electrophoresis and liquid chromatography-tandem mass spectrometry analysis, the 40 kDa protein closely resembled the porin B of P. putida KT2440 and carbohydrate-selective porin OprB of various Pseudomonas strains. The purified native protein (i) was estimated to be a homotrimer of 125 kDa with a subunit molecular mass of 40 kDa, (ii) displayed heat modifiability of electrophoretic mobility, (iii) showed channel conductance of 166 pS in 1 M KCl, (iv) permeated various sugars (mono-, di- and tri-saccharides), organic acids, amino acids and aromatic compounds, and (v) harboured a glucose-specific and saturable binding site with a dissociation constant of 1.3 µM. These results identify the glucose-inducible outer-membrane protein of P. putida CSV86 as a carbohydrate-selective protein OprB. Besides modulation of intracellular glucose-metabolizing enzymes and specific glucose-binding periplasmic space protein, the repression of OprB by aromatics and organic acids, even in the presence of glucose, also contributes significantly to the strain's ability to utilize aromatics and organic acids over glucose.

New insights into the activation of Radiation Desiccation Response regulon in Deinococcus radiodurans.

The highly radiation-resistant bacterium Deinococcus radiodurans responds to gamma radiation or desiccation through the coordinated expression of genes belonging to Radiation and Desiccation Resistance/Response (RDR) regulon. RDR regulon is operated through cis-acting sequence RDRM (Radiation Desiccation Response Motif), trans-acting repressor DdrO and protease IrrE (also called PprI). The present study evaluated whether RDR regulon controls the response of D. radiodurans to various other DNA damaging stressors, to which it is resistant, such as UV rays, mitomycin C (MMC), methyl methanesulfonate (MMS), ethidium bromide (EtBr), etc. Activation of 3 RDR regulon genes (ddrB, gyrB and DR1143) was studied by tagging their promoter sequences with a highly sensitive GFP reporter. Here we demonstrated that all the DNA damaging stressors elicited activation of RDR regulon of D. radiodurans in a dose-dependent and RDRM-/IrrE-dependent manner. However, ROS-mediated indirect effects [induced by hydrogen peroxide (H2O2), methyl viologen (MV), heavy metal/metalloid (zinc or tellurite), etc.] did not activate RDR regulon. We also showed that level of activation was inversely proportional to cellular abundance of repressor DdrO. Our data strongly suggests that direct DNA damage activates RDR regulon in D. radiodurans.

N6-methyladenine and epigenetic immunity of Deinococcus radiodurans.

DNA methylation is ubiquitously found in all three domains of life. This epigenetic modification on adenine or cytosine residues serves to regulate gene expression or to defend against invading DNA in bacteria. Here, we report the significance of N6-methyladenine (6mA) to epigenetic immunity in Deinococcus radiodurans. Putative protein encoded by DR_2267 ORF (Dam2DR) contributed 35% of genomic 6mA in D. radiodurans but did not influence gene expression or radiation resistance. Dam2DR was characterized to be a functional S-adenosyl methionine (SAM)-dependent N6-adenine DNA methyltransferase (MTase) but with no endonuclease activity. Adenine methylation from Dam2DR or Dam1DR (N6-adenine MTase encoded by DR_0643) improved DNA uptake during natural transformation. To the contrary, methylation from Escherichia coli N6-adenine MTase (DamEC that methylates adenine in GATC sequence) on donor plasmid drastically reduced DNA uptake in D. radiodurans, even in presence of Dam2DR or Dam1DR methylated adenines. With these results, we conclude that self-type N6-adenine methylation on donor DNA had a protective effect in absence of additional foreign methylation, a separate methylation-dependent Restriction Modification (R-M) system effectively identifies and limits uptake of G6mATC sequence containing donor DNA. This is the first report demonstrating presence of epigenetic immunity in D. radiodurans.