John R. Battista

Deinococcus radiodurans — the consummate survivor

Relatively little is known about the biochemical basis of the capacity of Deinococcus radiodurans to endure the genetic insult that results from exposure to ionizing radiation and can include hundreds of DNA double-strand breaks. However, recent reports indicate that this species compensates for extensive DNA damage through adaptations that allow cells to avoid the potentially detrimental effects of DNA strand breaks. It seems that D. radiodurans uses mechanisms that limit DNA degradation and that restrict the diffusion of DNA fragments that are produced following irradiation, to preserve genetic integrity. These mechanisms also increase the efficiency of the DNA-repair proteins.

Global analysis of the Deinococcus radiodurans proteome by using accurate mass tags

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organisms dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

Extensive Diversity of Ionizing-Radiation-Resistant Bacteria Recovered from Sonoran Desert Soil and Description of Nine New Species of the Genus Deinococcus Obtained from a Single Soil Sample

ABSTRACT The ionizing-radiation-resistant fractions of two soil bacterial communities were investigated by exposing an arid soil from the Sonoran Desert and a nonarid soil from a Louisiana forest to various doses of ionizing radiation using a 60Co source. The numbers of surviving bacteria decreased as the dose of gamma radiation to which the soils were exposed increased. Bacterial isolates surviving doses of 30 kGy were recovered from the Sonoran Desert soil, while no isolates were recovered from the nonarid forest soil after exposure to doses greater than 13 kGy. The phylogenetic diversities of the surviving culturable bacteria were compared for the two soils using 16S rRNA gene sequence analysis. In addition to a bacterial population that was more resistant to higher doses of ionizing radiation, the diversity of the isolates was greater in the arid soil. The taxonomic diversity of the isolates recovered was found to decrease as the level of ionizing-radiation exposure increased. Bacterial isolates of the genera Deinococcus, Geodermatophilus, and Hymenobacter were still recovered from the arid soil after exposure to doses of 17 to 30 kGy. The recovery of large numbers of extremely ionizing-radiation-resistant bacteria from an arid soil and not from a nonarid soil provides further ecological support for the hypothesis that the ionizing-radiation resistance phenotype is a consequence of the evolution of other DNA repair systems that protect cells against commonly encountered environmental stressors, such as desiccation. The diverse group of bacterial strains isolated from the arid soil sample included 60 Deinococcus strains, the characterization of which revealed nine novel species of this genus.

Global Analysis of Deinococcus Radiodurans Proteome by Csing Accurate Mass Tags

Understanding biological systems and the roles of their constituents is facilitated by the ability to make quantitative, sensitive, and comprehensive measurements of how their proteome changes, e.g., in response to environmental perturbations. To this end, we have developed a high-throughput methodology to characterize an organisms dynamic proteome based on the combination of global enzymatic digestion, high-resolution liquid chromatographic separations, and analysis by Fourier transform ion cyclotron resonance mass spectrometry. The peptides produced serve as accurate mass tags for the proteins and have been used to identify with high confidence >61% of the predicted proteome for the ionizing radiation-resistant bacterium Deinococcus radiodurans. This fraction represents the broadest proteome coverage for any organism to date and includes 715 proteins previously annotated as either hypothetical or conserved hypothetical.

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.

Characterization and radiation resistance of new isolates of Rubrobacter radiotolerans and Rubrobacter xylanophilus

Abstract In this study we characterized new strains of the slightly thermophilic species Rubrobacter radiotolerans and the thermophilic species Rubrobacter xylanophilus, both of which were previously represented only by the type strains isolated, respectively, from Japan and the United Kingdom. The new isolates were recovered from two hot springs in central Portugal after gamma irradiation of water and biofilm samples. We assessed biochemical characteristics, performed DNA–DNA hybridization, and carried out 16S rDNA sequence analysis to demonstrate that the new Rubrobacter isolates belong to the species R. radiotolerans and R. xylanophilus. We also show for the first time that the strains of R. xylanophilus and other strains of R. radiotolerans are extremely gamma radiation resistant.

Directed Evolution of Ionizing Radiation Resistance in Escherichia coli

We have generated extreme ionizing radiation resistance in a relatively sensitive bacterial species, Escherichia coli, by directed evolution. Four populations of Escherichia coli K-12 were derived independently from strain MG1655, with each specifically adapted to survive exposure to high doses of ionizing radiation. D(37) values for strains isolated from two of the populations approached that exhibited by Deinococcus radiodurans. Complete genomic sequencing was carried out on nine purified strains derived from these populations. Clear mutational patterns were observed that both pointed to key underlying mechanisms and guided further characterization of the strains. In these evolved populations, passive genomic protection is not in evidence. Instead, enhanced recombinational DNA repair makes a prominent but probably not exclusive contribution to genome reconstitution. Multiple genes, multiple alleles of some genes, multiple mechanisms, and multiple evolutionary pathways all play a role in the evolutionary acquisition of extreme radiation resistance. Several mutations in the recA gene and a deletion of the e14 prophage both demonstrably contribute to and partially explain the new phenotype. Mutations in additional components of the bacterial recombinational repair system and the replication restart primosome are also prominent, as are mutations in genes involved in cell division, protein turnover, and glutamate transport. At least some evolutionary pathways to extreme radiation resistance are constrained by the temporally ordered appearance of specific alleles.

Description of four novel psychrophilic, ionizing radiation-sensitive Deinococcus species from alpine environments.

Five psychrophilic bacterial strains were isolated from soil samples collected above the treeline of alpine environments. Phylogenetic analysis based on 16S rRNA gene sequences indicated that these organisms represent four novel species of the genus Deinococcus; levels of sequence similarity to the type strains of recognized Deinococcus species were in the range 89.3-94.7 %. Strains PO-04-20-132T, PO-04-20-144, PO-04-19-125T, ME-04-01-32T and ME-04-04-52T grew aerobically, with optimum growth at 10 degrees C and at pH 6-9. The major respiratory menaquinone was MK-8. The fatty acid profiles of strains PO-04-20-132T, PO-04-20-144, PO-04-19-125T and ME-04-01-32T were dominated by 16 : 1omega7c, 17 : 0 iso and 15 : 1omega6c, whereas 16 : 1omega7c, 17 : 0 cyclo and 16 : 0 predominated in strain ME-04-04-52T. The DNA G+C contents of strains PO-04-20-132T, PO-04-19-125T, ME-04-01-32T and ME-04-04-52T were 63.2, 63.1, 65.9 and 62.6 mol%, respectively. Strains PO-04-20-132T, PO-04-19-125T, ME-04-01-32T and ME-04-04-52T had gamma radiation D10 (dose required to reduce the bacterial population by 10-fold) values of < or =4 kGy. These four strains showed sensitivity to UV radiation and extended desiccation as compared with Deinococcus radiodurans. On the basis of the phylogenetic analyses, and chemotaxonomic and phenotypic data, it is proposed that strains PO-04-20-132T (=LMG 24019T=NRRL B-41950T; Deinococcus radiomollis sp. nov.), PO-04-19-125T (=LMG 24282T=NRRL B-41949T; Deinococcus claudionis sp. nov.), ME-04-01-32T (=LMG 24022T=NRRL B-41947T; Deinococcus altitudinis sp. nov.) and ME-04-04-52T (=LMG 24283T=NRRL B-41948T; Deinococcus alpinitundrae sp. nov.) represent the type strains of four novel species of the genus Deinococcus.

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.

An aroA Mutant of Edwardsiella ictaluri Is Safe and Efficacious as a Live, Attenuated Vaccine

Abstract The aroA gene of Edwardsiella ictaluri was cloned and sequenced, and the sequence data were used to construct a deletion–insertion mutation in the aroA gene. The mutated gene was transferred into a virulent, wild-type E. ictaluri strain by conjugation and allelic exchange. Putative aroA mutants were confirmed phenotypically by demonstrating a need for supplementation with aromatic metabolites to support growth in minimal media. The genetic construction was evaluated by using the polymerase chain reaction to amplify appropriate regions of the aroA deletion–insertion, and DNA sequencing of the amplified products confirmed the predicted construction. A selected mutant, LSU-E1, was passed 30 times in nonselective media with no reversion to the wild-type following screening of 1.6 × 1011 colony-forming units. The mutant was demonstrated via injection to be attenuated more than 5 logs10 compared with the wild-type E. ictaluri strain, and it was avirulent by immersion and oral routes. Tissue persistence...