Single-stranded DNA transposition is coupled to host replication.

DNA transposition has contributed significantly to evolution of eukaryotes and prokaryotes. Insertion sequences (ISs) are the simplest prokaryotic transposons and are divided into families on the basis of their organization and transposition mechanism. Here, we describe a link between transposition of IS608 and ISDra2, both members of the IS200/IS605 family, which uses obligatory single-stranded DNA intermediates, and the host replication fork. Replication direction through the IS plays a crucial role in excision: activity is maximal when the "top" IS strand is located on the lagging-strand template. Excision is stimulated upon transient inactivation of replicative helicase function or inhibition of Okazaki fragment synthesis. IS608 insertions also exhibit an orientation preference for the lagging-strand template and insertion can be specifically directed to stalled replication forks. An in silico genomic approach provides evidence that dissemination of other IS200/IS605 family members is also linked to host replication.

DdrI, a cAMP Receptor Protein Family Member, Acts as a Major Regulator for Adaptation of Deinococcus radiodurans to Various Stresses.

The DNA damage response ddrI gene encodes a transcription regulator belonging to the cAMP receptor protein (CRP) family. Cells devoid of the DdrI protein exhibit a pleiotropic phenotype, including growth defects and sensitivity to DNA-damaging agents and to oxidative stress. Here, we show that the absence of the DdrI protein also confers sensitivity to heat shock treatment, and several genes involved in heat shock response were shown to be upregulated in a DdrI-dependent manner. Interestingly, expression of the Escherichia coli CRP partially compensates for the absence of the DdrI protein. Microscopic observations of ΔddrI mutant cells revealed an increased proportion of two-tetrad and anucleated cells in the population compared to the wild-type strain, indicating that DdrI is crucial for the completion of cell division and/or chromosome segregation. We show that DdrI is also involved in the megaplasmid MP1 stability and in efficient plasmid transformation by facilitating the maintenance of the incoming plasmid in the cell. The in silico prediction of putative DdrI binding sites in the D. radiodurans genome suggests that hundreds of genes, belonging to several functional groups, may be regulated by DdrI. In addition, the DdrI protein absolutely requires cAMP for in vitro binding to specific target sequences, and it acts as a dimer. All these data underline the major role of DdrI in D. radiodurans physiology under normal and stress conditions by regulating, both directly and indirectly, a cohort of genes involved in various cellular processes, including central metabolism and specific responses to diverse harmful environments.IMPORTANCEDeinococcus radiodurans has been extensively studied to elucidate the molecular mechanisms responsible for its exceptional ability to withstand lethal effects of various DNA-damaging agents. A complex network, including efficient DNA repair, protein protection against oxidation, and diverse metabolic pathways, plays a crucial role for its radioresistance. The regulatory networks orchestrating these various pathways are still missing. Our data provide new insights into the crucial contribution of the transcription factor DdrI for the D. radiodurans ability to withstand harmful conditions, including UV radiation, mitomycin C treatment, heat shock, and oxidative stress. Finally, we highlight that DdrI is also required for accurate cell division, for maintenance of plasmid replicons, and for central metabolism processes responsible for the overall cell physiology.

ISDra2 transposition in Deinococcus radiodurans is downregulated by TnpB.

Transposable elements belonging to the recently identified IS200/IS605 family radically differ from classical insertion sequences in their transposition mechanism by strictly requiring single-stranded DNA substrates. This IS family includes elements encoding only the transposase (TnpA), and others, like ISDra2 from Deinococcus radiodurans, which contain a second gene, tnpB, dispensable for transposition and of unknown function to date. Here, we show that TnpB has an inhibitory effect on the excision and insertion steps of ISDra2 transposition. This inhibitory action of TnpB was maintained when ISDra2 transposition was induced by γ-irradiation of the host cells and required the integrity of its putative zinc finger motif. We also demonstrate the negative role of TnpB when ISDra2 transposition was monitored in a heterologous Escherichia coli host, indicating that TnpB-mediated inhibition does not involve Deinococcus-specific factors. TnpB therefore appears to play a regulatory role in ISDra2 transposition.

DNA recognition and the precleavage state during single-stranded DNA transposition in D. radiodurans.

Bacterial insertion sequences (ISs) from the IS200/IS605 family encode the smallest known DNA transposases and mobilize through single-stranded DNA transposition. Transposition by one particular family member, ISDra2 from Deinococcus radiodurans, is dramatically stimulated upon massive γ irradiation. We have determined the crystal structures of four ISDra2 transposase/IS end complexes; combined with in vivo activity assays and fluorescence anisotropy binding measurements, these have revealed the molecular basis of strand discrimination and transposase action. The structures also show that previously established structural rules of target site recognition that allow different specific sequences to be targeted are only partially conserved among family members. Furthermore, we have captured a fully assembled active site including the scissile phosphate bound by a divalent metal ion cofactor (Cd²(+)) that supports DNA cleavage. Finally, the observed active site rearrangements when the transposase binds a metal ion in which it is inactive provide a clear rationale for metal ion specificity.

Irradiation-induced Deinococcus radiodurans genome fragmentation triggers transposition of a single resident insertion sequence.

Stress-induced transposition is an attractive notion since it is potentially important in creating diversity to facilitate adaptation of the host to severe environmental conditions. One common major stress is radiation-induced DNA damage. Deinococcus radiodurans has an exceptional ability to withstand the lethal effects of DNA-damaging agents (ionizing radiation, UV light, and desiccation). High radiation levels result in genome fragmentation and reassembly in a process which generates significant amounts of single-stranded DNA. This capacity of D. radiodurans to withstand irradiation raises important questions concerning its response to radiation-induced mutagenic lesions. A recent study analyzed the mutational profile in the thyA gene following irradiation. The majority of thyA mutants resulted from transposition of one particular Insertion Sequence (IS), ISDra2, of the many different ISs in the D. radiodurans genome. ISDra2 is a member of a newly recognised class of ISs, the IS200/IS605 family of insertion sequences.

The Role of ISCR1-Borne POUT Promoters in the Expression of Antibiotic Resistance Genes.

The ISCR1 (Insertion sequence Common Region) element is the most widespread member of the ISCR family, and is frequently present within γ-proteobacteria that occur in clinical settings. ISCR1 is always associated with the 3'Conserved Segment (3'CS) of class 1 integrons. ISCR1 contains outward-oriented promoters POUT, that may contribute to the expression of downstream genes. In ISCR1, there are two POUT promoters named PCR1-1 and PCR1-2. We performed an in silico analysis of all publically available ISCR1 sequences and identified numerous downstream genes that mainly encode antibiotic resistance genes and that are oriented in the same direction as the POUT promoters. Here, we showed that both PCR1-1 and PCR1-2 significantly increase the expression of the downstream genes bla CTX-M-9 and dfrA19. Our data highlight the role of ISCR1 in the expression of antibiotic resistance genes, which may explain why ISCR1 is so frequent in clinical settings.

Thioredoxin can influence gene expression by affecting gyrase activity.

The expression of many genes of facultatively photosynthetic bacteria of the genus Rhodobacter is controlled by the oxygen tension. Among these are the genes of the puf and puc operons, which encode proteins of the photosynthetic apparatus. Previous results revealed that thioredoxins are involved in the regulated expression of these operons, but it remained unsolved as to the mechanisms by which thioredoxins affect puf and puc expression. Here we show that reduced TrxA of Rhodobacter capsulatus and Rhodobacter sphaeroides and oxidized TrxC of R.capsulatus interact with DNA gyrase and alter its DNA supercoiling activity. While TrxA enhances supercoiling, TrxC exerts a negative effect on this activity. Furthermore, inhibition of gyrase activity strongly reduces puf and puc expression. Our results reveal a new signaling pathway by which oxygen can affect the expression of bacterial genes.

Identification of new genes contributing to the extreme radioresistance of Deinococcus radiodurans using a Tn5-based transposon mutant library.

Here, we have developed an extremely efficient in vivo Tn5-based mutagenesis procedure to construct a Deinococcus radiodurans insertion mutant library subsequently screened for sensitivity to genotoxic agents such as γ and UV radiations or mitomycin C. The genes inactivated in radiosensitive mutants belong to various functional categories, including DNA repair functions, stress responses, signal transduction, membrane transport, several metabolic pathways, and genes of unknown function. Interestingly, preliminary characterization of previously undescribed radiosensitive mutants suggests the contribution of cyclic di-AMP signaling in the recovery of D. radiodurans cells from genotoxic stresses, probably by modulating several pathways involved in the overall cell response. Our analyses also point out a new transcriptional regulator belonging to the GntR family, encoded by DR0265, and a predicted RNase belonging to the newly described Y family, both contributing to the extreme radioresistance of D. radiodurans. Altogether, this work has revealed new cell responses involved either directly or indirectly in repair of various cell damage and confirmed that D. radiodurans extreme radiation resistance is determined by a multiplicity of pathways acting as a complex network.

Expression of the trxA gene for thioredoxin 1 in Rhodobacter sphaeroides during oxidative stress.

Expression of the thioredoxin ( trxA) gene of Rhodobacter sphaeroides is regulated by oxidative stress at the transcriptional and post-transcriptional levels. All oxidative stress agents tested resulted in a moderate or strong increase of trxA mRNA levels, which was not due to increased mRNA stability. While the kinetics of increased trxA mRNA and of sodB mRNA, encoding superoxide dismutase, were similar after addition of tert-butyl hydroperoxide ( t-BOOH) or hydrogen peroxide (H(2)O(2)), different kinetics were observed after addition of diamide or paraquat, indicating the involvement of different stress responses in the regulation of these genes. The level of TrxA did not increase to the same extent as trxA mRNA levels. Furthermore, the addition of H(2)O(2) or t-BOOH led to increased turnover of the protein. Apparently, increased txA transcription compensated, at least in part, for the reduced stability of the protein. A strain expressing lower levels of thioredoxin 1 showed decreased resistance to diamide and H(2)O(2) but increased resistance to paraquat and t-BOOH compared to the wild-type. These data implicate the involvement of various systems in the response to different types of oxidative stress and the participation of thioredoxin 1 in the defense against oxidative stress caused by diamide or H(2)O(2).

Thioredoxin is essential for Rhodobacter sphaeroides growth by aerobic and anaerobic respiration.

To investigate the biological role of thioredoxin in the facultative photosynthetic bacterium Rhodobacter sphaeroides, attempts were made to construct a thioredoxin-deficient mutant by site-specific mutagenesis, using the Tn903 kanamycin resistance gene for selection. In situ and Southern hybridization analyses have demonstrated that the TrxA- mutation is lethal for R. sphaeroides growth under anaerobic conditions with DMSO as terminal electron acceptor and under aerobic conditions. In addition, the DNA region upstream of the trxA initiation codon is essential for aerobic growth of R. sphaeroides. An ORF of unknown function was identified in this region and is suggested to encode a product essential for aerobic metabolism of R. sphaeroides. The mechanism of thioredoxin action was also analysed by using the procedure for gene replacement to introduce a Cys33 to Ser mutation into the trxA chromosomal copy. The strain carrying this mutation produced a thioredoxin impaired in its protein-disulfide reductase activity and was also not viable. These data suggest that the physiological function of R. sphaeroides thioredoxin is redox-dependent. Thioredoxin purified from R. sphaeroides was shown to have a glutathione-disulfide oxidoreductase activity typical of glutaredoxins. This unexpected finding suggests that R. sphaeroides thioredoxin, in contrast to Escherichia coli thioredoxin, has the potential to act in GSH-dependent processes. Thus, the fundamental role of R. sphaeroides thioredoxin in cell growth probably originates from the multiple functions it can serve in vivo.