Bacterial gene transfer by natural genetic transformation in the environment.

Deinococcus radiodurans is a robust bacterium best known for its capacity to repair massive DNA damage efficiently and accurately. It is extremely resistant to many DNA-damaging agents, including ionizing radiation and UV radiation (100 to 295 nm), desiccation, and mitomycin C, which induce oxidative damage not only to DNA but also to all cellular macromolecules via the production of reactive oxygen species. The extreme resilience of D. radiodurans to oxidative stress is imparted synergistically by an efficient protection of proteins against oxidative stress and an efficient DNA repair mechanism, enhanced by functional redundancies in both systems. D. radiodurans assets for the prevention of and recovery from oxidative stress are extensively reviewed here. Radiation- and desiccation-resistant bacteria such as D. radiodurans have substantially lower protein oxidation levels than do sensitive bacteria but have similar yields of DNA double-strand breaks. These findings challenge the concept of DNA as the primary target of radiation toxicity while advancing protein damage, and the protection of proteins against oxidative damage, as a new paradigm of radiation toxicity and survival. The protection of DNA repair and other proteins against oxidative damage is imparted by enzymatic and nonenzymatic antioxidant defense systems dominated by divalent manganese complexes. Given that oxidative stress caused by the accumulation of reactive oxygen species is associated with aging and cancer, a comprehensive outlook on D. radiodurans strategies of combating oxidative stress may open new avenues for antiaging and anticancer treatments. The study of the antioxidation protection in D. radiodurans is therefore of considerable potential interest for medicine and public health.

Oxidative Stress Resistance in Deinococcus radiodurans

Deinococcus radiodurans, one of the most radioresistant organisms known to date, is able to repair efficiently hundreds of DNA double- and single-strand breaks as well as other types of DNA damages promoted by ionizing or ultraviolet radiation. We review recent discoveries concerning several aspects of radioresistance and survival under high genotoxic stress. We discuss different hypotheses and possibilities that have been suggested to contribute to radioresistance and propose that D. radiodurans combines a variety of physiological tools that are tightly coordinated. A complex network of regulatory proteins may be discovered in the near future that might allow further understanding of radioresistance.

Deinococcus radiodurans: what belongs to the survival kit?

Evaluation of the antioxidant effects of carotenoids from Deinococcus radiodurans through targeted mutagenesis, chemiluminescence, and DNA damage analyses.

In Deinococcus radiodurans, the extreme resistance to DNA–shattering treatments such as ionizing radiation or desiccation is correlated with its ability to reconstruct a functional genome from hundreds of chromosomal fragments. The rapid reconstitution of an intact genome is thought to occur through an extended synthesis-dependent strand annealing process (ESDSA) followed by DNA recombination. Here, we investigated the role of key components of the RecF pathway in ESDSA in this organism naturally devoid of RecB and RecC proteins. We demonstrate that inactivation of RecJ exonuclease results in cell lethality, indicating that this protein plays a key role in genome maintenance. Cells devoid of RecF, RecO, or RecR proteins also display greatly impaired growth and an important lethal sectoring as bacteria devoid of RecA protein. Other aspects of the phenotype of recFOR knock-out mutants paralleled that of a ΔrecA mutant: ΔrecFOR mutants are extremely radiosensitive and show a slow assembly of radiation-induced chromosomal fragments, not accompanied by DNA synthesis, and reduced DNA degradation. Cells devoid of RecQ, the major helicase implicated in repair through the RecF pathway in E. coli, are resistant to γ-irradiation and have a wild-type DNA repair capacity as also shown for cells devoid of the RecD helicase; in contrast, ΔuvrD mutants show a markedly decreased radioresistance, an increased latent period in the kinetics of DNA double-strand-break repair, and a slow rate of fragment assembly correlated with a slow rate of DNA synthesis. Combining RecQ or RecD deficiency with UvrD deficiency did not significantly accentuate the phenotype of ΔuvrD mutants. In conclusion, RecFOR proteins are essential for DNA double-strand-break repair through ESDSA whereas RecJ protein is essential for cell viability and UvrD helicase might be involved in the processing of double stranded DNA ends and/or in the DNA synthesis step of ESDSA.

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A Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radiodurans

Summary Deinococcus radiodurans R1 recovering from acute dose of γ radiation shows a biphasic mechanism of DNA double-strand break repair. The possible involvement of microsequence homology-dependent, or nonhomologous end joining type mechanisms during initial period followed by RecA-dependent homologous recombination pathways has been suggested for the reconstruction of complete genomes in this microbe. We have exploited the known roles of exonuclease I in DNA recombination to elucidate the nature of recombination involved in DNA doublestrand break repair during post-irradiation recovery of D. radiodurans . Transgenic Deinococcus cells expressing exonuclease I functions of Escherichia coli showed significant reduction in γ radiation radioresistance, while the resistance to far-UV and hydrogen peroxide remained unaffected. The overexpression of E. coli exonuclease I in Deinococcus inhibited DNA double-strand break repair. Such cells exhibited normal post-irradiation expression kinetics of RecA, PprA and single-stranded DNA-binding proteins but lacked the divalent cation manganese [(Mn(II)]-dependent protection from γ γ γ radiation. The results strongly suggest that 3 ′′ ( ρ ) 5 ′′ single-stranded DNA ends constitute an important component in recombination pathway involved in DNA doublestrand break repair and that absence of sbcB from deinococcal genome may significantly aid its extreme radioresistance phenotype.

/pdf/an-exonuclease-i-sensitive-dna-repair-pathway-in-deinococcus-1r6rrjezov.pdf

An exonuclease I‐sensitive DNA repair pathway in Deinococcus radiodurans: a major determinant of radiation resistance

We report here the construction of a homozygous recA460::cam insertion mutant of Synechocystis sp. PCC 6803 that may be useful for plant molecular genetics by providing a plant like host free of interference from homologous recombination. The homozygous recA460::cam mutant is highly sensitive to UVC under both photoreactivating and nonphotoreactivating conditions compared to the wild type (WT). The liquid culture of the mutant growing in ~800 lx accumulates nonviable cells to the tune of 86% as estimated by colony counts on plates incubated at the same temperature and light intensity. The generation time of recA mutant in standard light intensity (2,500 lx) increases to 50 h compared to 28 h in lower light intensity (~800 lx) that was used for selection, thus explaining the earlier failures to obtain a homozygous recA mutant. The WT, in contrast, grows at faster rate (23 h generation time) in standard light intensity compared to that at ~800 lx (26 h). The Synechocystis RecA protein supports homologous recombination during conjugation in recA— mutant of Escherichia coli, but not the SOS response as measured by UV sensitivity. It is suggested that using this homozygous recA460::cam mutant, investigations can now be extended to dissect the network of DNA repair pathways involved in housekeeping activities that may be more active in cyanobacteria than in heterotrophs. Using this mutant for the first time we provide a genetic evidence of a mechanism independent of RecA that causes enhanced UVC resistance on light to dark transition.

A homozygous recA mutant of Synechocystis PCC6803: construction strategy and characteristics eliciting a novel RecA independent UVC resistance in dark

Certain species of bacteria can become competent to take up high molecular weight DNA from the surrounding medium. DNA homologous to resident chromosomal DNA is transported, processed and recombined with the resident DNA. There are some variations in steps leading to transformation between Gram-positive bacteria likebiplococcus pneumoniae and Gram-negative bacteria represented byHaemophilus influenzae but the integration is by single-strand displacement in both cases. Plasmid (RSF0885) transformation is low inHaemophilus influenzae but this is increased significantly if (homologous) chromosomal DNA is spliced to plasmid DNA. InHaemophilus influenzae, rec1 function is required for peak transformation with chimeric plasmids. Chimeric plasmid fixed presumably extrachromosomally undergoes frequent recombination between homologous segments contained in resident chromosome and the plasmid.

http://repository.ias.ac.in/35214/1/35214.pdf

Genetic transformation in bacteria

Haemophilus influenzae UvrA: overexpression, purification, and in cell complementation

A new, high-efficiency, DNA-cloning vector pJ1-8 was derived in two steps from the chimeric plasmid pD7 consisting of RSF 0885(amp
 r) andHaemophilus influenzae chromosomal DNA. pJl-8 has only oneEcoRI site and a molecular weight of only 2.5 × 106. No detectableamp
 r transformation was obtained with pJl-8 DNA. However,amp
 r transformation increases markedly ifHaemophilus influenzae chromosomal DNA segments are spliced into it, providing a very facile assay for detecting inserts.

/pdf/a-new-dna-cloning-vector-for-haemophilus-influenzae-rd-23ecqi5opk.pdf

Vasudha P. Joshi

Papers

An exonuclease I‐sensitive DNA repair pathway in Deinococcus radiodurans: a major determinant of radiation resistance

A homozygous recA mutant of Synechocystis PCC6803: construction strategy and characteristics eliciting a novel RecA independent UVC resistance in dark

Genetic transformation in bacteria

Haemophilus influenzae UvrA: overexpression, purification, and in cell complementation

A new DNA-cloning vector for Haemophilus influenzae Rd.