ABSTRACT Deinococcus radiodurans is extremely resistant to ionizing and UV irradiation. The addition of manganese(¢º) into stationary-phase culture can trigger a new cycle of cell division ( Mn-CD effect ). Although D.radiodurans can hardly utilize sugar , but it does can incorporate glucose and fructose into the cell mass. We found that D. radiodurans can metabolize sugar by glycolysis ( EMP ) and pantose phosphate pathway ( PPP ). The activity of both pathways would be altered by the addition of manganese(¢º). The marker enzymes of the PPP ( transketolase ) and EMP pathway ( aldolase ) were also analyzed. Results show that manganese(¢º) can induce the hydrolysis of glucose by activating the EMP pathway. However , the induction effect various with different cultural conditions. In analyzing the PPP pathway , the activities of transketolase were also various with different cultural conditions.

The addition of Mn2¡Ï to stationary phase culture of Deinococcus radiodurans could induce further cell division. This type of cell division termed "Mn-induced Cell Division (Mn-CD)". The Mn-CD cells were less resistant to radiation, having smaller cell size, and forming less red-pigment. However, the activities of antioxidation enzymes such as superoxide dismutase (SOD) and catalase, were increased. No other divalent metal ions could cause Mn-CD. In this study, 2-D electrophoresis and Matrix Assisted Laser Desorption Ionization (MALDI) MASS were used to analyze the proteomic differences between Mn-CD and normal uninduced cells. The results showed that the expression of ribosomal proteins L20, L34, L35, chain 3 of the large ribosomal subunit, preprotein translocase (SecE subunit) and three hypothetical proteins (DR1423, DR1897 and one unidentified gene product), were pressed significantly in the cell culture when Mn2¡Ï were added. Since these ribosomal proteins are responsible for the synthesis of protein, it is clear that Mn2¡Ï could disturb the proteins synthesis in this bacterium. The addition of Mn2¡Ïcould also induce the expression of acetyl-CoA acetyltransferase, transcriptional regulator (TetR family), phosphinothricin acetyltransferase and three hypothetical proteins (DR0214, DR0296 and DRA0100). The functions of these hypothetical proteins were not known yet. In conclusion, it is true that Mn2¡Ï could alter the proteomic expression and some metabolic pathways in D. radiodurans.

No description available.

Abstract The highly radioresistant Deinococcus radiodurans is a heterotrophic Gram positive aerobic tetrad. The addition of 2.5 uM Mn(II) into the early stationary-phase culture would trigger a new round of cell division (Mn-CD effect) which is specific to this unique bacterium. In order to understand the reactions of this bacterium to different carbohydrates, we culture the bacterium by using complex media with various monosaccharides and disaccharides. No matter in the present of Mn(II) or not, the specific growth rate of fructose¡Vgrown cells was always higher than those of glucose-grown cells. Cell growth would be inhabited by excess Mn(II). Along with the increasing of Mn(II), the culture showed a prolonged lag phase. In chemically defined media, this bacterium grew well by using glucose, fructose, mannose, maltose or sucrose as it¡¦s sole carbon source and performed the Mn-CD growth by adding Mn(II). II

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 159-173).

Thesis (Ph. D.)--Virginia Polytechnic Institute and State University, 1992. / Vita. Abstract. Includes bibliographical references (leaf 193). Also available via the Internet.

Thesis (Ph. D.)--University of Washington, 2004. / Vita. Includes bibliographical references (leaves 151-162).

Deinococcus radiodurans est l'un des organismes les plus radiorésistants connus à ce jour et de manière plus générale, présente une exceptionnelle tolérance aux agents qui endommagent son ADN. Cette bactérie est capable de reconstituer un génome intact à partir de centaines de fragments d'ADN engendrés par exposition aux radiations ionisantes. Ce phénomène de radiorésistance est la conséquence d'une association de plusieurs mécanismes et facteurs agissants de concert lorsque les cellules sont soumises à ce type de rayonnement. Parmi ces facteurs identifiés, on peut citer la présence de certaines protéines spécifiques des Deinococcaceae. Parmi elles, la protéine PprA joue un rôle important dans la radiorésistance de D. radiodurans. Cette protéine qui est fortement induite après irradiation n'a pas d'homologuechez des organismes autres que les Deinococcaceae. Pour essayer de comprendre le rôle de PprA dans la radiorésistance chez D. radiodurans, j'ai effectué une caractérisation approfondie du mutant de délétion ΔpprA. Ce mutant est très sensible aux rayons γ ainsi qu'à d'autres agents qui créent des dommages sur l'ADN comme l'acide nalidixique et la novobiocine. L'étude morphologique de ce mutant et la localisation de la protéine in vivo après irradiation suggèrent que PprA est impliquée dans un mécanisme de ségrégation des chromosomes lors de la division, après irradiation lorsque l'ADN des cellules a été réparé. L'étude du réseau d'interactants de PprA a révélé que ce cette protéine interagit in vivo avec l'ADN gyrase après irradiation et permet la stimulation de l'activité de décaténation de cette dernière sans influencer son activité de surenroulement négatif de l'ADN in vitro. Les phénotypes observés précédemment ont également suggéré une éventuelle interaction entre PprA et les protéines de la famille SMC. Chez D. radiodurans les protéines de la famille SMC sont SMC, SbcC et RecN. De façon surprenante, la délétion du gène recN dans une souche ΔpprA supprime la sensibilité aux agents endommageant l'ADN observée dans la souche simple mutante ΔpprA. Ces résultats suggèrent une interaction génétique entre les gènes pprA et recN, cependant, la nature précise du lien entre les deux protéines PprA et recN reste à établir. / Deinococcus radiodurans, one of the most radioresistant organisms known to date is able to reconstruct an intact genome from hundreds of DNA fragments generated by γ-rays. More generally, this bacterium is also tolerant to other DNA-damaging agents. This exceptional ability to overcome effects of ionizing radiations is due to a combination of several well regulated mechanisms and factors acting together when cells are exposed the radiations. Among these factors, some specific proteins of the Deinococcaceae family which are induced after irradiation can be observed. The PprA protein is one of these specific proteins and has been shown to have an important role in radioresistance in D. radiodurans. This protein is one of the most induced after γ-rays treatment. None homologous protein have been identify for the PprA protein. Characteristics of the ΔpprA mutant were investigated in order to understand the involvement of PprA in radioresistance. This mutant is very sensitive to γ-rays and other DNA damaging agents as nalidixic acid or novobiocin. Phenotypic analyses of this mutant revealed that PprA protein seems to be implicated in chromosome segregation after irradiation when DNA is repaired in cells. Moreover, the PprA protein has been shown to interact in vivo with DNA gyrase after irradiation and to stimulate in vitro the decatenation activity of DNA gyrase, without affecting its DNA negative supercoiling activity. Phenotypes previously observed also suggest a potential interaction between the PprA protein and the protein SMC family which are SMC, SbcC and RecN in D. radiodurans. Surprisingly, we found that disrupting recN gene in a ΔpprA strain abolish the sensitivity to DNA damaging agents observed in a ΔpprA strain. These results suggest that the two genes, pprA and recN interact but the accurate link between the two proteins PprA and RecN remains to be highlighted.

Deinococcus geothermalis est un organisme non-model intéressant pour les bio-productions de part sa résistance extrême et ses capacités de fermentation partant de diverses sources de carbone. Cependant les outils d’ingénierie permettant une fine maîtrise des voix métaboliques restent limités pour cet organisme. Le but de ce travail de thèse, est d’essayer de surpasser cet obstacle à travers l’observation des motifs génétiques et de leur organisation. L’analyses de ces motifs a été menée via deux approches. La première est l’étude de l’impact de la position dans le génome sur l’expression d’une cassette reportrice. Grâce à une collection de 150 souches, nous avons observé que l’expression est plus forte au niveau de l’origine de réplication que du terminus. Une autre observation concerne la présence de zone de forte expression réparties symétriquement le long du chromosome. La seconde approche est l’analyse des motifs génétiques en cas de stress grace outil GREAT:SCAN:patterns. Ces motifs sont fortement liés régulation de l’expression des gènes et sont des points intéressant pour l’ingénierie du génome. En analysant les résultats de différentes conditions de stress ainsi que les régulons décrits dans la littérature, nous avons pu observer que des stress voisins partagent les mêmes motifs et que ces motifs semblent conservés chez des organismes distants. Ces deux approches ont permis de déterminer des positions d’insertion dans le génome intéressantes pour l’ingénierie métabolique. / Deinococcus geothermalis is a non-model organism of high interest for bio-manufacturing since it shows a extreme resistance and good capacities for fermentation process on different carbon sources. However the engineering tools are limited to finely tuned metabolic pathways for bio-productions. This PhD work aims at contributing to overcome this obstacle through a whole-genome approach to the issue of understanding the genomic organization of D. geothermalis and defined interesting genomic locations. The whole-genome approach is based on the existence of genome-scale patterns that were analyzed in two different ways. A first approach consisted of studying the influence of the genome location on the expression of a reporter cassette. On a library of over 150 strains, the expression is higher near the origin of replication than near the terminus, a common observation. However, other hot spots of expression along the genome additionally appeared with a symmetric distribution about the origin of replication. The second approach consisted of analyzing the genomic patterns under stress through the in-house GREAT:SCAN:patterns software. These patterns interrelate with gene expression regulation and are an interesting key for genome engineering. Testing different stress conditions and considering the matching regulons as described in the literature, it appeared that related stresses share genomic patterns. Moreover these patterns tend to be conserved between distant organisms. These two approaches lead to define interesting genome loci for inserting genes encoding the enzymes of a pathway, with a view to metabolic engineering.

Bacteria of the radiation resistant genus 𝘋𝘦𝘪𝘯𝘰𝘤𝘰𝘤𝘤𝘶𝘴 have a high resistance to the lethal and mutagenic effects of many DNA damaging agents, however, the mechanisms involved in the response of these bacteria to oxidative stress are poorly understood. To investigate antioxidant enzyme responses in 𝘋𝘦𝘪𝘯𝘰𝘤𝘰𝘤𝘤𝘶𝘴 sp., the types of catalase and superoxide dismutase (SOD) produced by these bacteria were identified by visualization of the enzyme activities on non-denaturing polyacrylamide gels (PAGE) and the growth inhibition of selected strains by various concentrations of hydrogen peroxide and paraquat (a superoxide generating agent) was tested. 𝘋𝘦𝘪𝘯𝘰𝘤𝘰𝘤𝘤𝘶𝘴 sp. were found to be more resistant to hydrogen peroxide and more sensitive to paraquat than 𝘌𝘴𝘤𝘩𝘦𝘳𝘪𝘤𝘩𝘪𝘢 𝘤𝘰𝘭𝘪 K12. They possess relatively high levels of catalase and exhibit similar electrophoretic patterns on catalase zymograms compared to 𝘌. 𝘤𝘰𝘭𝘪, but all the tested strains produce only one SOD except 𝘋𝘦𝘪𝘯𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘳𝘢𝘥𝘪𝘰𝘱𝘩𝘪𝘭𝘶𝘴 which produces two. The two catalases of 𝘋𝘦𝘪𝘯𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘳𝘢𝘥𝘪𝘰𝘥𝘶𝘳𝘢𝘯𝘴 were found to be regulated independently. Cultures of 𝘋. 𝘳𝘢𝘥𝘪𝘰𝘥𝘶𝘳𝘢𝘯𝘴, when pretreated with sublethal levels of hydrogen peroxide, became relatively resistant to the lethal effects of H₂O₂ and exhibited higher levels of catalase than untreated control cultures. The pretreated cells were also relatively resistant to UV-and γ-ray-mediated lethality. These results suggest that 𝘋𝘦𝘪𝘯𝘰𝘤𝘰𝘤𝘤𝘶𝘴 sp. possess inducible defense mechanisms against the deleterious effects of oxidants and ionizing and UV radiation. The resistance to the lethal effects of hydrogen peroxide and UV radiation can not be induced by pretreatment with sublethal levels of hydrogen peroxide in 𝘋. 𝘳𝘢𝘥𝘪𝘰𝘥𝘶𝘳𝘢𝘯𝘴 rec30 mutant. Effects of manganese and magnesium on the growth and catalase activity of 𝘋. 𝘳𝘢𝘥𝘪𝘰𝘥𝘶𝘳𝘢𝘯𝘴 were determined. Homology between 𝘌. 𝘤𝘰𝘭𝘪 catalase and SOD genes and those of 𝘋𝘦𝘪𝘯𝘰𝘤𝘰𝘤𝘤𝘶𝘴 sp. was tested by Southern blot analysis using previously cloned genes from 𝘌. 𝘤𝘰𝘭𝘪. / Thesis / Master of Science (MS)