Mismatch repair ensures fidelity of replication and recombination in the radioresistant organism Deinococcus radiodurans.

We have characterized the mismatch repair system (MMR) of the highly radiation-resistant type strain of Deinococcus radiodurans, ATCC 13939. We show that the MMR system is functional in this organism, where it participates in ensuring the fidelity of DNA replication and recombination. The system relies on the activity of two key proteins, MutS1 and MutL, which constitute a conserved core involved in mismatch recognition. Inactivation of MutS1 or MutL resulted in a seven-fold increase in the frequency of spontaneous RifR mutagenesis and a ten-fold increase in the efficiency of integration of a donor point-mutation marker during bacterial transformation. Inactivation of the mismatch repair-associated UvrD helicase increased the level of spontaneous mutagenesis, but had no effect on marker integration--suggesting that binding of MutS1 and MutL proteins to a mismatched heteroduplex suffices to inhibit recombination between non identical (homeologous) DNAs. In contrast, inactivation of MutS2, encoded by the second mutS -related gene present in D. radiodurans, had no effect on mutagenesis or recombination. Cells devoid of MutS1 or MutL proteins were as resistant to gamma-rays, mitomycin C and UV-irradiation as wild-type bacteria, suggesting that the mismatch repair system is not essential for the reconstitution of a functional genome after DNA damage.

Negative regulation of the heat shock response in Streptomyces.

All organisms respond to a sudden increase in temperature by inducing the synthesis of a set of proteins called heat shock proteins (HSPs). Although the induction of HSPs is a universal response, a diversity of mechanisms control HSP synthesis in different organisms. In Streptomyces, the synthesis of major HSPs, such as the widespread molecular chaperones DnaK, ClpB, GroEL and HSP18, is negatively controlled at the transcriptional level by at least three different repressors. The control of groE gene expression involves an inverted repeat (called the CIRCE element) that is highly conserved among eubacteria, and the HrcA repressor. The dnaK operon and clpB belong to the HspR /HAIR regulon. The HspR repressor-HAIR operator system is used in some bacteria but is not widespread. In particular, it has not been found in gram-positive bacteria with low G+C content. Transcription of hsp18, which encodes a small HSP, is regulated by the RheA repressor. This repressor, which has intrinsic thermosensor activity, has to date been identified only in Streptomyces.

The RheA repressor is the thermosensor of the HSP18 heat shock response in Streptomyces albus.

Microorganisms have mechanisms to sense their environment and rapidly adapt to survive changes in conditions. In Streptomyces albus, various transcriptional repressors mediate the induction of heat shock genes. The RheA repressor regulates the synthesis of HSP18, a small heat shock protein, which plays a role in thermotolerance. The RheA protein was purified to determine how it responds rapidly to temperature. Gel retardation assays and footprinting experiments identified the specific target of RheA as an inverted repeat (TGTCATC 5N GATGACA) located in Phsp18, PrheA which is the common promoter region of the divergon. Gel retardation assays detected RheA-complexes formed with the hsp18-rheA promoters. The complexes did not form at higher temperature. In vitro transcription experiments showed that RheA is an autoregulatory protein and that its activity is inhibited by high temperature. The temperature-induced derepression by RheA is reversible. Dichroism circular spectroscopy revealed a reversible change of RheA conformation in relation with the temperature that could represent a transition between an active and an inactive form. Our experiments demonstrate that RheA acts as a cellular thermometer in hsp18 regulation.

Production of Cry11A and Cry11Ba toxins in Bacillus sphaericus confers toxicity towards Aedes aegypti and resistant Culex populations.

Cry11A from Bacillus thuringiensis subsp. israelensis and Cry11Ba from Bacillus thuringiensis subsp. jegathesan were introduced, separately and in combination, into the chromosome of Bacillus sphaericus 2297 by in vivo recombination. Two loci on the B. sphaericus chromosome were chosen as target sites for recombination: the binary toxin locus and the gene encoding the 36-kDa protease that may be responsible for the cleavage of the Mtx protein. Disruption of the protease gene did not increase the larvicidal activity of the recombinant strain against Aedes aegypti and Culex pipiens. Synthesis of the Cry11A and Cry11Ba toxins made the recombinant strains toxic to A. aegypti larvae to which the parental strain was not toxic. The strain containing Cry11Ba was more toxic than strains containing the added Cry11A or both Cry11A and Cry11Ba. The production of the two toxins together with the binary toxin did not significantly increase the toxicity of the recombinant strain to susceptible C. pipiens larvae. However, the production of Cry11A and/or Cry11Ba partially overcame the resistance of C. pipiens SPHAE and Culex quinquefasciatus GeoR to B. sphaericus strain 2297.

Disruption of hspR, the repressor gene of the dnaK operon in Streptomyces albus G.

hspR is the distal gene of the Streptomyces albus dnaK operon. It encodes a protein similar to GlnR, the repressor of the Bacillus subtilis glutamine synthetase gene. Transcriptional analysis showed that disruption of hspR led to constitutive high-level expression of the dnaK operon, SDS-PAGE analysis revealed over-production and accumulation of the chaperone DnaK at low temperature HSP94, a heat-inducible protein cross-reacting with anti-CipB antibodies, was also shown to be constitutively overexpressed at low temperature in the hspR mutant. Those features were lost when the mutant was complemented in trans by an intact copy of hspR. The hspR mutant was impaired in its growth on solid rich medium: colonies grow slowly at 30 degrees C. However, formation of aerial mycelium and sporulation was not prevented. In liquid culture growth curves of the mutant and the wild type were similar. The kinetics of groEL gene induction were not modified by the hspR null mutation, indicating that HspR was not directly involved in the control of groEL transcription. Thus, in contrast with B. subtilis and other Gram-positive bacteria, transcription of Streptomyces dnaK and groEL operons is not controlled by the same regulator.

Heat induction of hsp18 gene expression in Streptomyces albus G: transcriptional and posttranscriptional regulation.

In Streptomyces albus G, HSP18, a protein belonging to the small heat shock protein family, could be detected only at high temperature. The nucleotide sequence of the DNA region upstream from hsp18 contains an open reading frame (orfY) which is in the opposite orientation and 150 bp upstream. This open reading frame encodes a basic protein of 225 amino acids showing no significant similarity to any proteins found in data banks. Disruption of this gene in the S. albus chromosome generated mutants that synthesized hsp18 RNA at 30 degrees C, suggesting that orfY plays either a direct or indirect role in the transcriptional regulation of the hsp18 gene. In addition, thermally induced expression of the hsp18 gene is subject to posttranscriptional regulation. In the orfY mutant, although hsp18 RNA was synthesized at a high level at 30 degrees C, the HSP18 protein could not be detected except after heat shock. Synthesis of the HSP18 protein in the orfY mutant was also heat inducible when transcription was inhibited by rifampin. Furthermore, when wild-type cultures of S. albus were shifted from high temperature to 30 degrees C, synthesis of the gene product could no longer be detected, even though large amounts of hsp18 RNA were present.

Characterization of Streptomyces albus 18-kilodalton heat shock-responsive protein.

In Streptomyces albus during the heat shock response, a small heat shock protein of 18 kDa is dramatically induced. This protein was purified, and internal sequences revealed that S. albus HSP18 showed a marked homology with proteins belonging to the family of small heat shock proteins. The corresponding gene was isolated and sequenced. DNA sequence analysis confirmed that the hsp18 gene product is an analog of the 18-kDa antigen of Mycobacterium leprae. No hsp18 mRNA could be detected at 30 degrees C, but transcription of this gene was strongly induced following heat shock. The transcription initiation site was determined by nuclease S1 protection. A typical streptomycete vegetative promoter sequence was identified upstream from the initiation site. Disruption mutagenesis of hsp18 showed that HSP18 is not essential for growth in the 30 to 42 degrees C temperature range. However, HSP18 is involved in thermotolerance at extreme temperatures.

Post-transcriptional regulation of the groEL1 gene of Streptomyces albus.

Thermally induced expression of the heat-shock gene groEL is subject to post-transcriptional regulation in Streptomyces albus. When S. albus cells were shifted from 30 degrees C to 41 degrees C, synthesis of three GroEL-like proteins was induced from two genes transcribed from associated promoters P1 and P2. Surprisingly, analyses of transcriptional fusions of these promoters with various reporter genes indicated constitutive expression independent of heat shock. In contrast, neo expression was thermally inducible as a GroEL1-APH translational fusion protein. Furthermore, expression of the groEL1-neo gene was heat inducible even after the groEL1 promoter region was replaced by a heterologous non-heat-inducible promoter such as the Escherichia coli lac promoter. Finally, synthesis of GroE proteins, as well as the GroEL-APH fusion protein, was heat inducible when their transcription was inhibited by rifampicin. Post-transcriptional regulatory signals needed for heat-induced GroEL1 synthesis were mapped within of the groEL1 structural gene.

Use of new Escherichia coli/Streptomyces conjugative vectors to probe the functions of the two groEL-like genes of Streptomyces albus G by gene disruption.

Streptomyces albus G contains two groEL-like genes encoding three related proteins [Guglielmi et al., J. Bacteriol. 173 (1991) 7374-7381; Mazodier et al., J. Bacteriol. 173 (1991) 7382-7386]. Two proteins, HSP58 and HSP18, are synthesized from a single start codon site in groEL1. HSP18 may be a processed form of HSP58 or the result of early termination after frameshifting. The third protein, HSP56 is encoded by groEL2. In order to determine the physiological roles of these different proteins, both groEL genes were mutagenized by using a new approach for obtaining insertions in the streptomycete chromosome. Escherichia coli plasmids containing fragments homologous to groEL1 or groEL2 are unable to replicate in Streptomyces. They were introduced into S. albus by conjugation with E. coli. We then screened for mutants in which groEL1 or groEL2 had been disrupted due to recombination events (single or double crossover) at specific sites. Using this approach, the functionally indispensable domain of HSP58 was localized to within 249 amino acids of the N-terminus. HSP58 was not detected in the mutant generated by the most upstream insertion into the groEL1 coding sequence. However, HSP18 was synthesized in this mutant after heat shock. This groEL1 mutant was not impaired in growth in the 30-41 degrees C temperature range and SDS-PAGE analysis showed its overall pattern of gene expression to be indistinguishable from the parental strain. The inability to generate strains containing groEL2 disruptions strongly suggests that HSP56 is indispensable for growth.(ABSTRACT TRUNCATED AT 250 WORDS)

[Action of radiation of the head on the capacity for lymphoblastic transformation in the adult rabbit]. / Action d'une irradiation céphalique sur la capacité de transformation lymphoblastique chez le lapin adulte.

The temporary immunological depression previously demonstrated after high level head irradiation of adult rabbits seems to be due to decrease in lymphoblastic transformation capacity connected with encephalon injury.