Bactericidal effect of ultraviolet C (UVC), direct and filtered through transparent plastic, on gram-positive cocci: an in vitro study.

The prevalence of wound infections caused by multidrug-resistant (MDR) bacteria is increasing along with concern about widespread use of antibiotics. In vitro studies have shown that ultraviolet radiation, especially UVC, is both an effective bactericidal and antifungal. However, evidence about its bactericidal effect on wounds covered with transparent dressings remains inconclusive. Transparent dressings are used to retain moisture over the wound as part of an intermittent negative pressure dressing-the Limited Access Dressing (LAD) technique. Because this dressing is designed to remain in place for a number of days, an in vitro study was conducted to explore the bactericidal effect of direct and indirect UVR through a transparent 0.15-mm thick transparent polythene sheet on Gram-positive cocci. Six bacterial strains were inoculated to sheep blood agar (SBA) plates and exposed to direct and filtered UVC (254 nm) for 5, 10, 15, 20, 25, and 30 seconds with one media serving as a control (no UVC exposure). Plates were subsequently incubated for 24 hours and bacterial growth observed. Each set of experiment was repeated three times. Direct UVC was shown to have good bactericidal effect (100% eradication of organisms inoculated) at durations ranging from a minimum of 5 seconds (methicillin-resistant, coagulase-negative Staphylococcus and Streptococcus pyogenes) to a maximum of 15 seconds (methicillin-susceptible Staphylococcus aureus and Enterococci species). No bactericidal effect was observed when UVC was filtered through a 0.15-mm thick transparent polythene sheet. The results confirm the bactericidal effect of UVC in vitro and suggest that this effect can be achieved after a very short period of time. At the same time, film dressings appear to filter UVC. This may help protect skin from exposure to UVC but also limits its utility for use with the LAD technique. In vivo studies to evaluate the shortest effective UVC treatment duration and follow-up clinical studies to ascertain treatment efficacy and effectiveness are needed.

Multiple-stress tolerance of ionizing radiation-resistant bacterial isolates obtained from various habitats: correlation between stresses.

Isolation of five ionizing radiation (IR)-resistant bacteria by screening of isolates from various habitats classified as common and stressed is reported. IR-resistant isolates exhibited varying degrees of resistance to gamma-radiation and were classified as highly and moderately radiation resistant. Resistance to ultraviolet (UV) radiation correlated well with gamma-radiation resistance, whereas a comparable desiccation resistance for all the highly and moderately radiation-resistant isolates was observed. However, salt tolerance failed to correlate with IR resistance, indicating a divergent evolution of the salt tolerance and radiation resistance. Characterization of isolates by the amplified rDNA restriction analysis profiling attested to the clustering of these isolates with their stress phenotype. 16S rRNA gene-based analysis of the isolates showed that the bacteria with similar-resistance physiologies clustered together and belonged to related genera. Hydrogen peroxide resistance and mitomycin survival patterns of the isolates indicated the roles of oxidative-stress tolerance in desiccation survival and recombination repair in higher radiation resistance, respectively.

Production of superoxide dismutase by Deinococcus radiophilus.

The production of superoxide dismutase (SOD) varied in Deinococcus radiophilus, the UV resistant bacterium, depending upon different phases of growth, UV irradiation, and superoxide treatment. A gradual increase in total SOD activity occurred up to the stationary phases. The electrophoretic resolution of the SOD in cell extracts of D. radiophilus at each growth phase revealed the occurrence of MnSOD throughout the growth phases. The SOD profiles of D. radiophilus at the exponential phase received oxidative stress by the potassium superoxide treatment or UV irradiation also revealed the occurrence of a single SOD. However, these treatments caused an increase in SOD activity. The data strongly suggest that D. radiophilus has only one species of SOD as a constitutive enzyme, which seems to be a membrane-associated protein.

RecA Protein from the extremely radioresistant bacterium Deinococcus radiodurans: expression, purification, and characterization.

The RecA protein of Deinococcus radiodurans (RecA(Dr)) is essential for the extreme radiation resistance of this organism. The RecA(Dr) protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecA(Dr) protein and the E. coli RecA (RecA(Ec)) proteins are close functional homologues. RecA(Dr) forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecA(Ec). The RecA(Dr) protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecA(Dr) protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecA(Dr) protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecA(Dr) protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecA(Dr) protein binds much better to duplex DNA than the RecA(Ec) protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.

Synergistic cell-killing effect of a combination of hyperthermia and heavy ion beam irradiation: in expectation of a breakthrough in the treatment of refractory cancers (review).

We studied the sensitivity against heavy ion beam and hyperthermia on radioresistant procaryote, Deinococcus radiodurans, for the purpose of cancer therapy. First, we examined the decrease of the survival rate and molecular weight of DNA purified from this cell by acid heat treatment. These decreases were recognized by heating at 55 degrees C below pH 5.0. Then, we assumed that the decrease in survival of D. radiodurans in vivo and damage to its DNA in vitro by acid heating were due to the release of purine rings from the phosphodiester backbone of DNA molecules, i.e., depurination. Second, we investigated the relation between LET (linear energy transfer) and RBE (relative biological effectiveness) on D. radiodurans dry and wet cells using AVF cyclotron at the TIARA facility of JAERI-Takasaki, Japan. These cells were irradiated with carbon (12C5+) ion beam at LET of about 100 keV/microm, neon (20Ne8+) ion beam at LET of about 300 keV/microm and oxygen (16O6+) ion beam at LET of about 400 keV/microm. The peak in the figure of the relation between LET and RBE value was found to increase according to the increase of LET value from 100 keV/microm. Third, we conducted combination treatment with 4.8 kGy of alpha-particles, i.e., boron 10 neutron captured beam induced by Kyoto University Research Nuclear Reactor operated at 5 MW, and hyperthermia at 52 degrees C, which caused the synergistic killing effect on D. radiodurans wet cells. However, being dissimilar to the case of gamma-irradiation, the interval incubation at 30 degrees C in the medium between both treatments could inhibit the recovery of survival.

Promoter cloning in the radioresistant bacterium Deinococcus radiodurans.

Deinococcus radiodurans is a highly radiation-resistant bacterium that is classed in a major subbranch of the bacterial domain. Since very little is known about gene expression in this bacterium, an initial study of promoters was undertaken. In order to isolate promoters and study promoter function, a series of integrative vectors for stable chromosomal insertion in D. radiodurans were developed. These vectors are based on Escherichia coli replicons that are unable to replicate autonomously in D. radiodurans and carry homologous sequences for replacement recombination in the D. radiodurans chromosome. The resulting integration vectors were used to study expression of reporter genes fused to a number of putative promoters that were amplified from the D. radiodurans R1 genome. Further analysis of these and other putative promoters was performed by Northern hybridization and primer extension experiments. In contrast to previous reports, the -10 and -35 regions of these promoters resembled the sigma(70) consensus sequence of E. coli.

Genome of the extremely radiation-resistant bacterium Deinococcus radiodurans viewed from the perspective of comparative genomics.

The bacterium Deinococcus radiodurans shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. D. radiodurans is best known for its extreme resistance to ionizing radiation; not only can it grow continuously in the presence of chronic radiation (6 kilorads/h), but also it can survive acute exposures to gamma radiation exceeding 1,500 kilorads without dying or undergoing induced mutation. These characteristics were the impetus for sequencing the genome of D. radiodurans and the ongoing development of its use for bioremediation of radioactive wastes. Although it is known that these multiple resistance phenotypes stem from efficient DNA repair processes, the mechanisms underlying these extraordinary repair capabilities remain poorly understood. In this work we present an extensive comparative sequence analysis of the Deinococcus genome. Deinococcus is the first representative with a completely sequenced genome from a distinct bacterial lineage of extremophiles, the Thermus-Deinococcus group. Phylogenetic tree analysis, combined with the identification of several synapomorphies between Thermus and Deinococcus, supports the hypothesis that it is an ancient group with no clear affinities to any of the other known bacterial lineages. Distinctive features of the Deinococcus genome as well as features shared with other free-living bacteria were revealed by comparison of its proteome to the collection of clusters of orthologous groups of proteins. Analysis of paralogs in Deinococcus has revealed several unique protein families. In addition, specific expansions of several other families including phosphatases, proteases, acyltransferases, and Nudix family pyrophosphohydrolases were detected. Genes that potentially affect DNA repair and recombination and stress responses were investigated in detail. Some proteins appear to have been horizontally transferred from eukaryotes and are not present in other bacteria. For example, three proteins homologous to plant desiccation resistance proteins were identified, and these are particularly interesting because of the correlation between desiccation and radiation resistance. Compared to other bacteria, the D. radiodurans genome is enriched in repetitive sequences, namely, IS-like transposons and small intergenic repeats. In combination, these observations suggest that several different biological mechanisms contribute to the multiple DNA repair-dependent phenotypes of this organism.

Physiologic determinants of radiation resistance in Deinococcus radiodurans.

Immense volumes of radioactive wastes, which were generated during nuclear weapons production, were disposed of directly in the ground during the Cold War, a period when national security priorities often surmounted concerns over the environment. The bacterium Deinococcus radiodurans is the most radiation-resistant organism known and is currently being engineered for remediation of the toxic metal and organic components of these environmental wastes. Understanding the biotic potential of D. radiodurans and its global physiological integrity in nutritionally restricted radioactive environments is important in development of this organism for in situ bioremediation. We have previously shown that D. radiodurans can grow on rich medium in the presence of continuous radiation (6,000 rads/h) without lethality. In this study we developed a chemically defined minimal medium that can be used to analyze growth of this organism in the presence and in the absence of continuous radiation; whereas cell growth was not affected in the absence of radiation, cells did not grow and were killed in the presence of continuous radiation. Under nutrient-limiting conditions, DNA repair was found to be limited by the metabolic capabilities of D. radiodurans and not by any nutritionally induced defect in genetic repair. The results of our growth studies and analysis of the complete D. radiodurans genomic sequence support the hypothesis that there are several defects in D. radiodurans global metabolic regulation that limit carbon, nitrogen, and DNA metabolism. We identified key nutritional constituents that restore growth of D. radiodurans in nutritionally limiting radioactive environments.

Engineering radiation-resistant bacteria for environmental biotechnology.

Seventy million cubic meters of ground and three trillion liters of groundwater have been contaminated by leaking radioactive waste generated in the United States during the Cold War. A cleanup technology is being developed based on the radiation-resistant bacterium Deinococcus radiodurans, which is being engineered to express bioremediating functions.

Ro ribonucleoproteins contribute to the resistance of Deinococcus radiodurans to ultraviolet irradiation.

The genome of the radiation-resistant eubacterium Deinococcus radiodurans contains an ortholog of an RNA-binding protein known as the Ro 60-kD autoantigen. This protein, which was previously identified only in higher eukaryotes, is normally bound to small RNAs known as Y RNAs. We show that the Ro protein ortholog Rsr contributes to the resistance of D. radiodurans to UV irradiation. Rsr binds several small RNAs, encoded upstream of rsr, that accumulate following UV irradiation. One of these RNAs resembles a Y RNA. These results suggest that Ro RNPs could similarly contribute to the recovery of higher cells following UV irradiation.

Production of two different catalase-peroxidases by Deinococcus radiophilus.

The production of two kinds of catalase-peroxidase, viz. catalase-2 and catalase-3 of Deinococcus radiophilus varied depending upon growth phases and oxidative stress. A gradual increase in total catalase activity occurred during exponential and stationary phase. Electrophoretic resolution of these catalases in Deinococcal cell extracts revealed the uniform occurrence of catalase-2 and the appearance of catalase-3 only during the late exponential and stationary phase. A substantial increase in total catalase was observed in either hydrogen peroxide- or UV-treated cells. Monitoring of D. radiophilus catalase activity in the oxidative stressed and non-treated cells by gel electrophoresis followed by densitometry revealed the several-fold increase in catalase-3, which is above the constant level of catalase-2. The occurrence of catalase-3 and catalase-2 revealed by fractionation of sucrose-shocked cells suggests that catalase-3 is a cytosolic inducible enzyme whereas catalase-2 is the membrane-associated constitutive enzyme.

Radiation resistance: the fragments that remain.

The complete genome sequence of the bacterium, Deinococcus radiodurans R1 has been released. This achievement will greatly aid efforts to study this organism, but analysis of the sequence reveals little that helps explain the extreme ionizing radiation resistance of this species.

Induction of a futile Embden-Meyerhof-Parnas pathway in Deinococcus radiodurans by Mn: possible role of the pentose phosphate pathway in cell survival.

Statistical models were used to predict the effects of tryptone, glucose, yeast extract (TGY) and Mn on biomass formation of the highly radioresistant bacterium Deinococcus radiodurans. Results suggested that glucose had marginal effect on biomass buildup, but Mn was a significant factor for biomass formation. Mn also facilitated glucose interactions with other nutrient components. These predictions were verified by in vivo and in vitro experiments. TGY-grown cells metabolized glucose solely by the pentose phosphate pathway (PPP). Although only a fraction of glucose from the medium was transported into the cells, glucose was incorporated into the DNA efficiently after cells were exposed to UV light. The presence of glucose also enhanced the radioresistance of the culture. Mn could induce an Embden-Meyerhof-Parnas (EMP) pathway in D. radiodurans. The EMP pathway and the PPP of the Mn-treated cells oxidized glucose simultaneously at a 6:1 ratio. Although glucose was hydrolyzed rapidly by the Mn-treated cells, most glucose was released as CO(2). Mn-treated cultures retained less glucose per cell than cells grown without Mn, and still less glucose was incorporated into the DNA after cells were exposed to UV light. Mn-treated cells were also more sensitive to UV light. Results suggested that metabolites of glucose generated from the PPP enhanced the survival of D. radiodurans. Induction of the EMP pathway by Mn may deplete metabolites for DNA repair and may induce oxidative stress for the cell, leading to reduction of radioresistance.

Effect of the space environment on the induction of DNA-repair related proteins and recovery from radiation damage.

Recovery of bacterial cells from radiation damage and the effects of microgravity were examined in an STS-79 Shuttle/Mir Mission-4 experiment using the extremely radioresistant bacterium Deinococcus radiodurans. The cells were irradiated with gamma rays before the space flight and incubated on board the Space-Shuttle. The survival of the wild type cells incubated in space increased compared with the ground controls, suggesting that the recovery of this bacterium from radiation damage was enhanced under microgravity. No difference was observed for the survival of radiosensitive mutant rec30 cells whether incubated in space or on the ground. The amount of DNA-repair related RecA protein induced under microgravity was similar to those of ground controls, however, induction of PprA protein, the product of a newly found gene related to the DNA repair mechanism of D. radiodurans, was enhanced under microgravity compared with ground controls.

Identification and disruption analysis of the recN gene in the extremely radioresistant bacterium Deinococcus radiodurans.

We isolated a radiosensitive mutant strain, KR4128, from a wild-type strain of Deinococcus radiodurans, which is known as a extremely radioresistant bacterium. The gene that restore the defect of the mutant in DNA repair was cloned, and it turned out to be the homolog of the recN gene of Escherichia coli. The recN gene encoded a protein of 58 kDa, and, in its N-terminal region, a potential ATP binding domain was conserved as expected for a prokaryotic RecN protein. An analysis of sequence of the mutant recN gene revealed a G:C to T:A transversion near the 3' end of the coding region. This alteration causes an ochre mutation, and results in the truncation of 47 amino acids from the C-terminal region of the RecN protein. The null mutant of recN gene was constructed by insertional mutagenesis, and it showed substantial sensitivities to various types of DNA damaging agents, indicating that a single defect in the recN gene can directly affect the DNA damage resistant phenotype in D. radiodurans. The recN locus of KR4128 was also disrupted and the disruptant indicated the sensitivity that was indistinguishable from its progenitor. The result indicate that the transversion in the recN gene of KR4128 cells causes a complete loss of function of the RecN protein and thus the C-terminal region of the RecN protein includes domain essential to its function.

Genome sequence of the radioresistant bacterium Deinococcus radiodurans R1.

The complete genome sequence of the radiation-resistant bacterium Deinococcus radiodurans R1 is composed of two chromosomes (2,648,638 and 412,348 base pairs), a megaplasmid (177,466 base pairs), and a small plasmid (45,704 base pairs), yielding a total genome of 3,284, 156 base pairs. Multiple components distributed on the chromosomes and megaplasmid that contribute to the ability of D. radiodurans to survive under conditions of starvation, oxidative stress, and high amounts of DNA damage were identified. Deinococcus radiodurans represents an organism in which all systems for DNA repair, DNA damage export, desiccation and starvation recovery, and genetic redundancy are present in one cell.

Why is Deinococcus radiodurans so resistant to ionizing radiation?

When exponential-phase cultures of Deinococcus radiodurans are exposed to a 5000-Gray dose of gamma radiation, individual cells suffer massive DNA damage. Despite this insult to their genetic integrity, these cells survive without loss of viability or evidence of mutation, repairing the damage by as-yet-poorly-understood mechanisms.

Whole-genome shotgun optical mapping of Deinococcus radiodurans.

A whole-genome restriction map of Deinococcus radiodurans, a radiation-resistant bacterium able to survive up to 15,000 grays of ionizing radiation, was constructed without using DNA libraries, the polymerase chain reaction, or electrophoresis. Very large, randomly sheared, genomic DNA fragments were used to construct maps from individual DNA molecules that were assembled into two circular overlapping maps (2.6 and 0.415 megabases), without gaps. A third smaller chromosome (176 kilobases) was identified and characterized. Aberrant nonlinear DNA structures that may define chromosome structure and organization, as well as intermediates in DNA repair, were directly visualized by optical mapping techniques after gamma irradiation.

Damage to DNA purified from the radioresistant prokaryote, Deinococcus radiodurans, by acid heating.

Using a highly radioresistant bacterium, Deinococcus radiodurans, the mechanism of degradation of the purified DNA molecules by heating was examined under acidic conditions. Setting the treatment temperature at 55ûC with a duration of 0 to 20 min and adjusting the pH of the cell suspension to 3, 5 or 7, cell viabilities after the treatment were compared. The survival rate decreased in proportion to the reduction of pH. DNA purified from D. radiodurans was then damaged by irradiation with gamma-rays at 0.22 kGy or 1 kGy. It was considered that the radioresistance of D. radiodurans was due to its high repair capability, rather than any specificity of DNA structure. Purified D. radiodurans DNA was resistant to heating up to 90ûC at neutral pH. However, marked DNA damage occurred when it was heated at pH values below 5.0. Then, DNA labeled with [3H]adenine was examined. Treatment at lower pH and higher temperature resulted in release of more adenine base, i.e., the purine ring, from the DNA molecules. Therefore, we assumed that the decrease in survival of D. radiodurans in vivo and damage to its DNA in vitro by acid heating were due to the release of adenine and guanine from the DNA, i.e., depurination.