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.

SummaryIn Bacillus subtilis , the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (GapB) and the phosphoenolpyruvate carboxykinase (PckA) enzymes are necessary for efficient gluconeogenesis from Krebs cycle intermediates. gapB and pckA transcription is repressed in the presence of glucose but not via CcpA, the major transcriptional regulator for catabolite repression in B. subtilis . A B. subtilis miniTn 10 transposant library was screened for clones affected in catabolite repression of gapB . Inactivation of a previously unknown gene, yqzB (renamed ccpN for control catabolite protein of gluconeogenic genes), was found to relieve not only gapB but also pckA transcription from catabolite repression. Purified CcpN specifically bound to the gapB and pckA promoters. ccpN is co-transcribed constitutively with another unknown gene, yqfL . A yqfL deletion lowers the level of gapB and pckA transcription threefold under both glycolytic and gluconeogenic conditions and a ccpN deletion is epistatic over a yqfL deletion. YqfL is thus a positive regulator of the expression of gapB and pckA , the effect of which is not influenced by the metabolic regime of the cell but appears to be mediated by CcpN. ccpN has homologues in many Firmicutes, but not all, while yqfL homologues are widely distributed in Eubacteria and also present in some plants. In all analysed bacterial genomes, ccpN and yqfL are physically linked together or to putative gluconeogenic genes. CcpN thus orchestrates a novel CcpA-independent mechanism for catabolite repression of gluconeogenic genes highly conserved in Firmicutes and appears as a functional analogue of FruR in Enterobacteria. The physiological significance of the regulation mediated via the three B. subtilis global transcription regulators, CcpA, CggR and CcpN, is discussed.

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Antitermination by GlpP, catabolite repression via CcpA and inducer exclusion triggered by P~GlpK dephosphorylation control Bacillus subtilis glpFK expression of this metabolite in the ccpA mutant and allows the expression of the glpF ¢-lacZ fusion even when glucose is present. Similarly, despite the presence of glucose, large amounts of glycerol-3-P are formed in a glycerol-exposed strain synthesizing GlpKH230R, as this mutant GlpK is as active as P~GlpK. IntroductionGlycerol enters bacterial cells via facilitated diffusion, an energy-independent transport process catalysed by the glycerol transport facilitator GlpF (Lin, 1976;Heller et al., 1980), an integral membrane protein of the aquaporin family (Calamita et al., 1995). Intracellular glycerol is usually converted to glycerol-3-P in an ATPrequiring phosphorylation reaction catalysed by glycerol kinase (GlpK). Glycerol-3-P, the inducer of the glpFK operon, is not a substrate for GlpF and, hence, remains entrapped in the cell, where it is metabolized further. In Bacillus subtilis, glycerol-3-P activates the antiterminator GlpP (Holmberg and Rutberg, 1991). Two mechanisms seem to regulate carbon catabolite repression (CCR) of the B. subtilis glpFK operon, which both involve proteins of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS). One was expected to be mediated via the catabolite control protein A (CcpA) and its co-repressor, the PTS protein HPr phosphorylated at Ser-46 (P-Ser-HPr), as a target site for this protein complex precedes glpFK (Miwa et al., 2000). The second was thought to be based on insufficient phosphorylation and activation of GlpK by P~His-HPr in cells growing on rapidly metabolizable PTS sugars (Deutscher et al., 1993), which lowers the synthesis of glycerol-3-P (inducer exclusion).Mutant studies have revealed that, in both Grampositive and Gram-negative bacteria, the PTS regulates glycerol metabolism. Glycerol is not transported by the PTS. Nevertheless, bacteria defective in one of the general PTS components enzyme I (EI) or HPr had lost the capacity to grow on glycerol as the sole carbon source (Simoni et al., 1976;Reizer et al., 1984;Romano et al., 1990;Gonzy-Treboul et al., 1991; Beijer and Rutberg, Molecular Microbiology (2002) SummaryThe Bacillus subtilis glpFK operon encoding the glycerol transport facilitator (GlpF) and glycerol kinase (GlpK) is induced by glycerol-3-P and repressed by rapidly metabolizable sugars. Carbon catabolite repression (CCR) of glpFK is partly mediated via a catabolite response element cre preceding glpFK. This operator site is recognized by the catabolite control protein A (CcpA) in complex with one of its co-repressors, P-Ser-HPr or P-Ser-Crh. HPr is a component of the phosphoenolpyruvate:sugar phosphotransferase system (PTS), and Crh is an HPr homologue. The hprK-encoded HPr kinase phosphorylates HPr and Crh at Ser-46. But in neither ccpA nor hprK mutants was expression of a glpF ¢-lacZ fusion relieved from CCR, as a second, CcpA-independent CCR mechanism implying the terminato...

Two gamma-and UV-radiation-tolerant, Gram-negative, rod-shaped bacterial strains, VCD115 T and VCD117, were isolated from a mixture of sand samples collected in the Sahara Desert in Morocco and Tunisia, after exposure of the sand to 15 kGy gamma radiation. Phylogenetic analysis based on 16S rRNA gene sequences and DNA-DNA hybridizations showed that VCD115 T and VCD117 are members of a novel species belonging to the genus Deinococcus, with Deinococcus grandis as its closest relative. The DNA G+C contents of VCD115 T and VCD117 are 59?8 and 60?6 mol%, respectively. The major fatty acids (straight-chain 15 : 1, 16 : 1, 17 : 1 and 16 : 0), polar lipids (dominated by phosphoglycolipids and glycolipids) and quinone type support the affiliation to the genus Deinococcus. The strains did not grow on rich medium such as trypticase soy broth (TSB), but did grow as whitish colonies on tenfold-diluted TSB. The genotypic and phenotypic properties allowed differentiation of VCD115 T and VCD117 from recognized Deinococcus species. Strains VCD115 T and VCD117 are therefore identified as representing a novel species, for which the name Deinococcus deserti sp. nov. is proposed, with the type strain VCD115 T (=DSM 17065 T =LMG 22923 T ).Various bacterial species have the capacity to survive under conditions that are commonly considered as extreme, for example in environments experiencing high pressure or high salt concentrations. In our laboratory, we are studying bacteria that live in the upper sand layers of deserts, where they are exposed to cycles of high and low temperatures, and to cycles of desiccation and hydration. De-and rehydration may cause DNA damage in these bacteria, and in order to survive they probably possess efficient DNA-repair mechanisms. Ionizing radiation causes similar types of DNA damage including double-strand breaks, which are the most deleterious to the organism (Mattimore & Battista, 1996). Bacteria belonging to the genus Deinococcus, in particular the well-studied Deinococcus radiodurans, have the distinctive feature of being the most radiation-tolerant of vegetative cells. D. radiodurans can withstand doses of radiation a thousand times higher than a human can. It can survive doses of radiation that do not exist naturally on Earth. Therefore, it is likely that this radiation tolerance is related to the bacterial response to natural non-radioactive DNA-damaging conditions such as desiccation (Makarova et al., 2001). At the time of writing, eight recognized species belong to the genus Deinococcus (Ferreira et al., 1997;Rainey et al., 1997;Suresh et al., 2004). Three other species have been described very recently, 'Deinococcus frigens', 'Deinococcus saxicola' and 'Deinococcus marmoris ' (Hirsch et al., 2004). Only D. radiodurans R 1 T has been studied extensively. Its genome has been sequenced (White et al., 1999), and analyses of the transcriptome (Liu et al., 2003;Tanaka et al., 2004) . For all other tests, bacterial strains were cultivated at 30 u C in TSB/10 or on agar plates containing the same m...

Deinococcus radiodurans is known for its extreme radioresistance. Comparative genomics identified a radiation-desiccation response (RDR) regulon comprising genes that are highly induced after DNA damage and containing a conserved motif (RDRM) upstream of their coding region. We demonstrated that the RDRM sequence is involved in cis-regulation of the RDR gene ddrB in vivo. Using a transposon mutagenesis approach, we showed that, in addition to ddrO encoding a predicted RDR repressor and irrE encoding a positive regulator recently shown to cleave DdrO in Deinococcus deserti, two genes encoding α-keto-glutarate dehydrogenase subunits are involved in ddrB regulation. In wild-type cells, the DdrO cell concentration decreased transiently in an IrrE-dependent manner at early times after irradiation. Using a conditional gene inactivation system, we showed that DdrO depletion enhanced expression of three RDR proteins, consistent with the hypothesis that DdrO acts as a repressor of the RDR regulon. DdrO-depleted cells loose viability and showed morphological changes evocative of an apoptotic-like response, including membrane blebbing, defects in cell division and DNA fragmentation. We propose that DNA repair and apoptotic-like death might be two responses mediated by the same regulators, IrrE and DdrO, but differently activated depending on the persistence of IrrE-dependent DdrO cleavage.

The nucleoid of radioresistant bacteria, including D. radiodurans, adopts a highly condensed structure that remains unaltered after exposure to high doses of irradiation. This structure may contribute to radioresistance by preventing the dispersion of DNA fragments generated by irradiation. In this report, we focused our study on the role of HU protein, a nucleoid-associated protein referred to as a histone-like protein, in the nucleoid compaction of D. radiodurans. We demonstrate, using a new system allowing conditional gene expression, that HU is essential for viability in D. radiodurans. Using a tagged HU protein and immunofluorescence microscopy, we show that HU protein localizes all over the nucleoid and that when HU is expressed from a thermosensitive plasmid, its progressive depletion at the non-permissive temperature generates decondensation of DNA before fractionation of the nucleoid into several entities and subsequent cell lysis. We also tested the effect of the absence of Dps, a protein also involved in nucleoid structure. In contrast to the drastic effect of HU depletion, no change in nucleoid morphology and cell viability was observed in dps mutants compared with the wild-type, reinforcing the major role of HU in nucleoid organization and DNA compaction in D. radiodurans.

A transcriptome comparison of a wild-type Bacillus subtilis strain growing under glycolytic or gluconeogenic conditions was performed. In particular, it revealed that the ywkA gene, one of the four paralogues putatively encoding a malic enzyme, was more transcribed during gluconeogenesis. Using a lacZ reporter fusion to the ywkA promoter, it was shown that ywkA was specifically induced by external malate and not subject to glucose catabolite repression. Northern analysis confirmed this expression pattern and demonstrated that ywkA is cotranscribed with the downstream ywkB gene. The ywkA gene product was purified and biochemical studies demonstrated its malic enzyme activity, which was 10-fold higher with NAD than with NADP (k cat/K m 102 and 10 s−1 mM−1, respectively). However, physiological tests with single and multiple mutant strains affected in ywkA and/or in ywkA paralogues showed that ywkA does not contribute to efficient utilization of malate for growth. Transposon mutagenesis allowed the identification of the uncharacterized YufL/YufM two-component system as being responsible for the control of ywkA expression. Genetic analysis and in vitro studies with purified YufM protein showed that YufM binds just upstream of ywkA promoter and activates ywkA transcription in response to the presence of malate in the extracellular medium, transmitted by YufL. ywkA and yufL/yufM could thus be renamed maeA for malic enzyme and malK/malR for malate kinase sensor/malate response regulator, respectively.