The essential histone-like protein HU plays a major role inDeinococcus radiodurans nucleoid compactionmmi_6766 240..252
Hong Ha Nguyen, Claire Bouthier de la Tour,Magali Toueille, Françoise Vannier,Suzanne Sommer** and Pascale Servant*Univ. Paris-Sud 11, CNRS UMR 8621, LRC CEA 42V,Institut de Génétique et Microbiologie, Bâtiment 409,Université Paris-Sud, F-91405 Orsay Cedex, France.
SummaryThe nucleoid of radioresistant bacteria, includingD. radiodurans, adopts a highly condensed structurethat remains unaltered after exposure to high doses ofirradiation. This structure may contribute to radiore-sistance by preventing the dispersion of DNA frag-ments generated by irradiation. In this report, wefocused our study on the role of HU protein, anucleoid-associated protein referred to as a histone-like protein, in the nucleoid compaction of D. radiodu-rans. We demonstrate, using a new system allowingconditional gene expression, that HU is essential forviability in D. radiodurans. Using a tagged HU proteinand immunofluorescence microscopy, we show thatHU protein localizes all over the nucleoid and thatwhen HU is expressed from a thermosensitiveplasmid, its progressive depletion at the non-permissive temperature generates decondensation ofDNA before fractionation of the nucleoid into severalentities and subsequent cell lysis. We also tested theeffect of the absence of Dps, a protein also involved innucleoid structure. In contrast to the drastic effect ofHU depletion, no change in nucleoid morphology andcell viability was observed in dps mutants comparedwith the wild-type, reinforcing the major role of HUin nucleoid organization and DNA compaction inD. radiodurans.
IntroductionDeinococcus radiodurans possesses an exceptional abilityto withstand the lethal effects of DNA damaging agents,including ionizing radiation. The most striking feature of
D. radiodurans is its capability to reconstruct a functionalgenome within 2–3 h from hundreds of chromosomal frag-ments generated by irradiation, whereas the genome ofmost organisms is irreversibly shattered under the sameconditions. It has been suggested that D. radioduranscombines many different mechanisms to efficiently repairits damaged DNA (for recent review, see Cox and Battista,2005; Blasius et al., 2008). D. radiodurans nucleoids adopta condensed structure that remains unaltered after high-dose g-irradiation, suggesting that this structure may pas-sively contribute to radioresistance by preventing thedispersion of DNA fragments generated by irradiation(Levin-Zaidman et al., 2003; Zimmerman and Battista,2005). The tightly packed structure of the nucleoid appearsto be a common trait among radioresistant bacteria (Zim-merman and Battista, 2005). Such a condensed genomemay provide suitable scaffolds for DNA repair.
In prokaryotes, bacterial nucleoids are efficientlycompacted by a group of nucleoid-associated proteins(NAPs), which are often referred to as histone-likeproteins. In Escherichia coli, about a dozen distinct pro-teins associate with the nucleoid, although only five existin abundant concentrations (Azam and Ishihama, 1999).These proteins include the HU protein, integration hostfactor (IHF) as well as H-NS, Fis and Dps (DNA-bindingprotein from starved cells). Based on their high intracel-lular abundance and DNA binding properties, these pro-teins are generally believed to be the most importantplayers in the organization and compaction of bacterialchromatin (for review, see Travers and Muskhelishvili,2005; Luijsterburg et al., 2006). In exponentially growingcells, the proteins Fis and HU are the most abundant,whereas Dps is the major nucleoid component in station-ary phase (Azam and Ishihama, 1999). Among these pro-teins, only HU encoded by the hbs gene and Dps arepresent in D. radiodurans.
In almost all bacteria except Enterobacteriacae, HUexists as an 18 kDa homodimer. In E. coli, HU exists as ahetero-dimer composed of two homologous subunits(HUa and HUb) or as homo-dimers. E. coli HU hetero-dimer is an architectural protein that binds non-specificallyto double-stranded DNA and with increased affinity todistorted DNA leading to significant DNA bending, nega-tive supercoiling and DNA compaction (Pontiggia et al.,1993; Bonnefoy et al., 1994; Castaing et al., 1995;
Kamashev and Rouviere-Yaniv, 2000). E. coli strainslacking the two HU subunits have been successfully con-structed, demonstrating that E. coli HU is not essential forviability. However, the absence of HU perturbs cell divi-sion, causing formation of anucleate cells (Huisman et al.,1989) and increases sensitivity to g and UV irradiation(Boubrik and Rouviere-Yaniv, 1995). More recently, a rolefor HU in DNA repair has emerged. In E. coli, HU prefer-entially binds nicks, gaps and abasic sites, harbours APlyase activity (Kow et al., 2007) and is involved in therepair of lesions that are in close proximity (Hashimotoet al., 2003). Grove et al. have shown that the mycobac-terial histone-like HU homologue (Hlp) promotes DNAend-joining in the presence of T4 DNA ligase (Mukherjeeet al., 2008). HU also plays regulatory roles in DNA rep-lication and acts as a global transcriptional regulator ofnumerous genes in E. coli (Oberto et al., 2009).
In bacteria, Dps is one of the critical proteins that trans-forms the nucleoid into a condensed state in response toenvironmental stresses (Kim et al., 2004). Dps protectsgenomic DNA against oxidative stress (Martinez andKolter, 1997), UV light (Nair and Finkel, 2004), possibly byits ability to protect DNA against damage. Moreover, Dpscan reduce the intracellular level of Fe2+ and thus restrictsthe production of hydroxyl radicals presumably by sup-pression of Fenton chemistry (Zhao et al., 2002).D. radiodurans encodes two proteins with homology toE. coli Dps: Dps1 and Dps2 which are the products of theDR2263 and DRB0092 genes respectively.
Here, we unsuccessfully attempted to construct an hbs(DRA0065) deletion mutant in D. radiodurans, suggestingthat HU is essential for cell viability. We demonstrate theessentiality of hbs by constructing a new system allowingthe conditional expression of hbs on a temperature-sensitive replicon, whose replication is efficiently shutdown at 37°C. In parallel, we also inactivated the twodps genes and showed that their deletion did not changethe nucleoid compaction and radioresistance ofD. radiodurans. We also show that HU protein localized tothe nucleoid in D. radiodurans cells and that, when HUwas expressed from a thermosensitive plasmid, its pro-gressive depletion at the non-permissive temperaturegenerated first decondensation of DNA prior to fraction-ation of the nucleoid followed by cells lysis. Our datastrongly suggest a major role of HU in nucleoid organiza-tion and DNA compaction in D. radiodurans.
ResultsFailure to create a deletion mutant devoid of HU protein
To investigate the functions of the histone-like HU protein inD. radiodurans, we attempted to construct a deletionmutant. Gene disruption was performed by replacing the
coding sequences of hbs (DRA0065) gene with a chloram-phenicol resistance cassette using the tripartite ligationmethod (Mennecier et al., 2004). The constructs were thenintroduced into D. radiodurans by genetic transformationselecting for antibiotic resistance. This led to the replace-ment of the wild-type allele by the mutant counterpart viahomologous recombination. D. radiodurans is multige-nomic, with cells containing from 4 to 10 genome equiva-lents depending on the growth conditions (Hansen, 1978;Harsojo et al., 1981). We obtained chloramphenicol resis-tant colonies, but PCR analysis of two candidates showedthat, after two rounds of purification on chloramphenicolplates, both still contained the wild-type allele in addition tothe Dhbs::Wcat allele (Fig. 1B-1). Further purification of thecandidates did not allow recovery of cells devoid of thewild-type allele, suggesting that HU protein is essential forcell viability.
Essentiality of the hbs gene is demonstrated by a newconditional gene inactivation system
To obtain positive evidence that hbs is an essential gene,we developed a new genetic assay of gene essentialitybased on inactivation of a chromosomal gene incells containing an additional copy of this gene on atemperature-sensitive replication vector. In such a system,the wild-type allele carried by the plasmid supplies a func-tional product at the permissive temperature, while plasmidloss at the non-permissive temperature results in the decayof the gene product leading to cell death when the proteinis essential for cell viability. Small versatile shuttle plasmidsfor cloning in D. radiodurans exist but none is thermosen-sitive for replication (Meima and Lidstrom, 2000; Lecointeet al., 2004). Thus, as a first step, we constructed this typeof shuttle vector. For this purpose we mutated the repUgene of plasmid p11554, a SpcR derivative of plasmid PI8.The repU gene encodes a protein that shares similaritywith the replication initiation proteins of two differentThermus plasmids and has been shown to be essential forplasmid replication in D. radiodurans (Meima and Lid-strom, 2000). The repU gene was amplified by PCR underconditions favouring the generation of mutations andwas used to replace the wild-type counterpart inplasmid p11554 (for details, see Experimental proce-dures). Plasmid p11830 having three mutations in repUwas retained for further studies. Measurement of plasmidstability during growth in liquid medium without the selec-tive antibiotic showed that p11830 was stable at 28°C butwas rapidly lost at 37°C (Fig. 2). After 6 h at 37°C, theproportion of cells carrying p11830 was reduced to approxi-mately 0.1%, suggesting that plasmid replication wasquickly arrested after the temperature shift. To expresscloned genes, we then introduced a PSpac promoter and aterm116 transcription terminator into the thermosensitive
vector p11830 to yield p13840 (Fig. S1) and p13841 differ-ing by the orientation of the PSpac-term116 cassette.
This new genetic tool was validated using genes knownto be essential or dispensable for cell viability. As essentialgenes, we used the rpoD (DR0916) gene encoding thehousekeeping sigma factor (Kobayashi et al., 2003) andthe gyrA gene encoding a subunit of DNA gyrase, anenzyme that regulates DNA supercoiling required forbasic processes such as DNA replication, recombinationand repair (for review, see Nollmann et al., 2007). As acontrol, we also used the amyE gene (DR1472) previ-ously shown to be dispensable for cell viability (Meimaet al., 2001). The coding region of each gene was clonedunder the control of the PSpac promoter on the thermosen-sitive vector and the recombinant plasmids weretransformed into the D. radiodurans strain GY10973expressing the lacI regulatory gene. The resulting strainsare merodiploids containing plasmidic and chromosomalcopies of the tester genes. The chromosomal copies ofeach gene were subsequently inactivated using the tripar-tite ligation method to replace the coding regions with a
Fig. 2. Loss of thermosensitive plasmid p11830 at 37°C. StrainD. radiodurans GY13701 carrying plasmid p11830 was grown at28°C or 37°C. Samples were removed at 2 h intervals for plating at28°C on selective and non-selective media. Symbols: 28°C(squares), 37°C (circles).
Fig. 1. Schematic representation and test ofdeletion–substitution in the D. radiodurans hbsand dps genes.A. Schematic representation of the allelereplacement event in hbs (A-1), dps1 (A-2)and dps2 (A-3) genes. Short arrows indicatethe position of specific primers used fordiagnostic PCR. Primers are described inTable S1.B. PCR analysis of two independentcandidate hbs mutants (B-1), two independentDhbs (prepUTs-hbs +) mutants (B-1) and thedouble Ddps1Ddps2 mutant (B-2 and B-3).
chloramphenicol resistance cassette and the strains weretested for their viability at the non-permissive temperature.For this purpose, bacteria grown at 28°C in liquid mediumwith spectinomycin (to select the presence of the ther-mosensitive plasmid) were plated and incubated at 28°C(permissive temperature) or 37°C (non-permissive tem-perature) in the presence or absence of spectinomycin.Growth of DgyrA (prepUTs-gyrA +) bacteria (Fig. 3, lanes4) and DrpoD (prepUTs-rpoD +) bacteria (Fig. 3, lanes 5)was strictly dependent on the temperature, indicating thatrpoD and gyrA are essential genes while the normalgrowth of DamyE (prepUTs-amyE +) bacteria at 37°C onplates without spectinomycin (Fig. 3, lanes 3) showed thatthey were not affected by the loss of the prepUTs-amyE +
plasmid, as expected for a dispensable gene. DgyrA
(prepUTs-gyrA +) bacteria showed some reduced growthwhen plated at 28°C in the absence of selective pressure.It is possible that a suboptimal level of gyrase in thesebacteria caused some plasmid instability since DNAsuperhelicity is centrally involved in the partitioningmechanism of some plasmids (Miller et al., 1990).
This new genetic tool being validated, we used it to testthe essentiality of the hbs gene. The chromosomal copy ofmerodiploids carrying the prepUTs-hbs plasmid was inac-tivated by replacing the hbs coding region with a chloram-phenicol resistance cassette. The complete deletion of thechromosomal hbs allele in the CamR transformants puri-fied twice on selective plates was confirmed by PCRanalysis (Fig. 1B-1). The cells were then tested forgrowth at 37°C. As can be seen in Fig. 4, the growth of
Fig. 3. Conditional viability of D. radioduranscells expressing rpoD and gyrA on thethermosensitive plasmid. Strains were grownin liquid medium with spectinomycin at 28°C.The dilutions of cells were spotted on mediumwith or without spectinomycin at 28°C (A) or37°C (B). Lane 1: strain GY13786 containingnon-thermosensitive plasmid p11554 (prepU);lane 2: strain GY13781 containingthermosensitive plasmid p13840 (prepUTs);lane 3: strain GY13760 [DamyE(prepUTs-amyE +)] (control of nonessentialgene); lane 4: strain GY13779 [DgyrA(prepUTs-gyrA +)]; lane 5: strain GY13755[DrpoD (prepUTs-rpoD +)].
Fig. 4. HU is essential for D. radioduransviability. Strains were grown in liquid mediumwith spectinomycin at 28°C. The dilutions ofcells were spotted on medium with or withoutspectinomycin at 28°C (A) or 37°C (B). Lane1: strain GY13781 containing thermosensitiveplasmid p13840 (prepUTs); lane 2: strainGY13786 containing non-thermosensitiveplasmid p11554 (prepU); lane 3: strainGY13795 [Dhbs (prepUTs-hbs +)].
Dhbs (prepUTs-hbs +) bacteria was strictly dependent onthe temperature, confirming that hbs is essential for cellviability.
HU protein colocalizes with DNA
To visualize the HU protein by immunofluorescencemicroscopy, we fused a SPA-tag (Zeghouf et al., 2004) toits C-terminal end. The SPA-tagged hbs gene was con-structed in vitro (see Experimental procedures) and usedto replace the wild-type hbs allele to give strain GY13220.Cells expressing the HU-SPA protein had the samegrowth rate as the wild-type (data not shown), indicatingthat the tagged protein was functional. The results ofimmunofluorescence staining showed that the HU-SPAprotein localizes at the nucleoid and is uniformly distrib-uted throughout this structure (Fig. 5). This suggests thatHU might be involved in nucleoid compaction due to itsinteraction with DNA.
Depletion of HU protein generates important changesin the nucleoid structure
Next, we used the conditional gene inactivation systemto analyse the effects of HU depletion on the nucleoidstructure. For this purpose, colonies of GY13975, inwhich HU protein is expressed from the thermosensitiveplasmid, or of GY13781, a wild-type strain containingthe empty thermosensitive vector, were grown overnightat 28°C in medium supplemented with spectinomycin.The cells were diluted in antibiotic-free medium, and
incubated at 28°C or 37°C for 24 h before cellularmorphology and nucleoid structure were analysedby combining epifluorescence and deconvolutionmicroscopy.
While the wild-type cells containing the thermosensitivevector exhibited a normal cell morphology and nucleoidcompaction at 37°C as well as at 28°C (Fig. 6), cellsexpressing HU from the thermosensitive plasmid werenormal at 28°C but lysed after 24 h of growth at 37°C,suggesting that HU depletion caused dramatic effects. Wealso examined cells expressing GyrA from the thermosen-sitive plasmid. In this case, we did not observe cell lysisafter 24 h incubation at 37°C, instead we observedanucleate cells whose proportion rose from 8% in culturesgrown at 28°C to 50% in cultures grown at 37°C (Fig. 6).This is consistent with a major role of DNA gyrase inpartitioning of the deinococcal chromosomes.
To determine the dynamics of the cellular alterationsoccurring during HU protein depletion, we analysed thecell morphologies in samples taken at different times afterthe shift at the non-permissive temperature (Fig. 7 andTable 1). We observed that, 10 h after the temperatureshift, the cells had an increased size and enlarged nucle-oids (50% of the cell population). Further incubation(t = 12 h) first resulted in the appearance of cells withfractionated nucleoids (63% of cells), showing severaldense DAPI stainable bodies, and later (t = 15 h andt = 20 h) in a complete collapse of the nucleoid structurethat filled the entire cellular space (45.8% and 59.8%respectively). Finally (t = 24 h), the entire cell populationlysed. These data strongly suggest that HU plays a key
Fig. 5. Immunolocalization of the HU protein in D. radiodurans. D. radiodurans cells from strain GY13320 (hbs::spaWcat) expressing the HUprotein harbouring a SPA-tag (top panel) and from the wild-type strain (R1, lower panel). Cells were probed with an anti-Flag primary antibodyfollowed by a FITC coupled secondary antibody (green) and with DAPI (blue). Cells were observed with a wide-field three-dimensionalmicroscope. The images shown are single sections of a deconvoluted Z-series.
role in DNA compaction and nucleoid organization inD. radiodurans.
Nucleoid compaction is not changed in Ddps mutants
In addition, we investigated the effect of the absence ofDps, also considered as major protein of nucleoids inprokaryotes. We constructed mutants deleted of dps1 ordps2 genes as well as the double mutant Ddps1Ddps2.Homogenotes of single or double dps mutants wereeasily obtained after just one cycle of purification onselective medium, as confirmed by amplifying the tar-geted allele by PCR (Fig. 1B-2 and B-3). The dpsmutants were not affected in their growth (data notshown). Since Dps is known to be abundant in station-ary phase (Altuvia et al., 1994), dps mutant cells inexponential or in stationary growth phase were analysedby combining epifluorescence and deconvolution micros-copy to investigate cell morphology and nucleoid struc-ture. Ddps1, Ddps2 and the double mutant cells had thesame morphology as the wild-type cells and contained acondensed nucleoid with a normal compact structure
(Fig. 8). Similar results were obtained when irradiatedcells were examined (data not shown). When exposedto g-irradiation, Ddps mutants showed the same level ofresistance as the wild-type (Fig. 9). This suggests that,contrary to HU, Dps is not essential for nucleoid com-paction and cell viability.
DiscussionDeinococcus radiodurans nucleoid adopts a condensedstructure that remains unaltered after high-doseg-irradiation (Levin-Zaidman et al., 2003; Zimmerman andBattista, 2005). Such tightly compact structure of thenucleoid, a common trait among radioresistant bacteria,may prevent the diffusion of DNA fragments generated byirradiation and contribute to the radioresistance of thesespecies. Bacterial nucleoid associated proteins (NAPs)play a major role in DNA compaction and nucleoid orga-nization (Travers and Muskhelishvili, 2005; Luijsterburget al., 2006). In this report, we studied the role of twomajor NAPs, HU and Dps proteins, on nucleoid compac-tion in D. radiodurans.
Fig. 6. Effect of DNA gyrase or HU depletion on D. radiodurans nucleoid morphology. Cells in exponential growth phase cultivated at 28°C inmedium supplemented with spectinomycin were harvested by centrifugation, diluted in antibiotic-free medium and incubated at 28°C (A) or37°C (B). Cells were stained and observed with a wide-field three-dimensional microscope 24 h after inoculation. Left images: DIC (differentialinterference contrast) signals; right images: nucleoids stained with DAPI appear in blue and membranes stained with FM4-64 appear in red.The images shown are single sections of a deconvoluted Z-series. 1: GY13781: wt (prepUTs); 2: GY13779: DgyrA (prepUTs-gyrA +);3: GY13795: Dhbs (prepUTs-hbs +).
The failure to obtain a homozygous hbs knockoutmutant suggested the essentiality of this gene. To confirmthis hypothesis, we developed a conditional inactivationsystem based on the temperature sensitive vector. Twoproperties of the plasmids used in this study (Meima andLidstrom, 2000; Lecointe et al., 2004) are particularly rel-evant for our conditional gene inactivation system: (i) theirlow copy number (4–8 copies per cell) in D. radioduransallowing rapid loss of the prepUTs vector at the non-permissive temperature and (ii) their stable maintenancein the absence of selective pressure, allowing a clonedallele to efficiently complement the disrupted chromo-somal counterpart at the permissive temperature. A differ-ent conditional gene inactivation system was previouslydescribed (Lecointe et al., 2004). It was based on anE. coli plasmid containing an N-terminal fragment of anessential deinococcal gene cloned under the control of
the PSpac promoter to generate an insertion-duplicationmutant conditionally expressing a functional protein. Thetwo drawbacks of this system, (i) the leakage of the PSpac
promoter and (ii) the high rate of reversion by efficientintrachromosomal recombination, are circumvented in ournew conditional gene inactivation system. In this study,using our new system allowing conditional gene expres-sion, we proved that HU is essential for cell viability inD. radiodurans.
We also analysed the effects of HU depletion on nucle-oid structure. Severe nucleoid abnormalities wereobserved after loss of the plasmid encoded HU: at earlytimes the nucleoid enlarged, then appeared as fraction-ated in several condensed nuclear bodies and later col-lapsed in a diffuse structure filling the entire cellularspace. At this time, we also observed leakage of thediffuse nucleoid in the surrounding medium through cell
Fig. 7. Observation of Dhbs (prepUTs-hbs +) cells after a temperature shift at 37°C. Cells of D. radiodurans strain GY13795 [Dhbs(prepUTs-hbs +)] were treated as described in legend to Fig. 6 and were stained and observed with a wide-field three-dimensional microscopeat different times during incubation at 37°C as indicated.
Table 1. Morphology of the nucleoids in cells deprived of HU.
Cells of GY13795 [Dhbs (prepUTs-hbs +)] grown overnight at 28°C in medium with spectinomycin were harvested by centrifugation, diluted inantibiotic-free medium and incubated at 28°C or 37°C. Cells aliquots were removed at different times after temperature shift and observed witha wide-field three-dimensional microscope 24 h after inoculation.
membrane holes. Finally, the entire cell population lysedand only membrane ghosts were visible. Several reportshave linked the nucleoid remodelling altered by HU withthe reorganization of transcription (Dorman and Deighan,2003; Kar et al., 2005; Oberto et al., 2009). Moreover, ithas been recently shown that HU regulates the expres-sion of as many as 8% (353 genes) of the E. coli genome,either cooperating with transcription regulators or bindingdirectly to its DNA structure targets (Oberto et al., 2009).The observed dramatic effects of HU depletion inD. radiodurans might be caused by an alteration of theexpression pattern of some genes under the control ofdeinococcal HU protein or by the loss of the nucleoidarchitecture. In E. coli, a transcriptional role of HU wasdemonstrated for the upregulation of the proVWX operonin hyper-osmolar environments (Manna and Gowrishan-kar, 1994) and recently it was shown that the HU regulonis composed of genes responding to high osmolarity(Oberto et al., 2009). Therefore, the loss of HU, throughits effects on transcription of proteins needed to maintainosmolarity, may perturb the osmotic balance in the celland provoke the lysis.
Fig. 8. Illustration of cell morphology in theDdps1Ddps2 strain. Nucleoids stained withDAPI appear in blue and membranes stainedwith FM4-64 appear in red. D. radioduranscells from the wild-type strain (R1, left panel)and from Ddps1Ddps2 strain (right panel)were observed with a wide-fieldthree-dimensional microscope.A. Cells in stationary growth phase(OD650 = 2).B. Cells in exponential growth phase(OD650 = 0.5). The images shown are singlesections of a deconvoluted Z-series.
Fig. 9. Survival curve for Ddps1Ddps2 mutant strain to g rays.Bacteria were exposed to g-irradiation at doses indicated on theabscissa and dilutions were plated as described in Experimentalprocedures. Symbols: wild-type (circles), Ddps1Ddps2 (squares).Each value is the average of three independent experiments withstandard deviations that did not exceed 10% of the mean values.
We also showed that D. radiodurans HU protein concen-trates in the nucleoid and is uniformly distributed through-out the structure. E. coli HU is an architectural protein thatbinds non-specifically to double-stranded DNA and withincreased affinity to distorted DNA to cause significantDNA bending, negative supercoiling and DNA compaction(Pontiggia et al., 1993; Bonnefoy et al., 1994; Castainget al., 1995; Kamashev and Rouviere-Yaniv, 2000). Ghoshand Grove (2004) have shown by in vitro experiments thatthe D. radiodurans HU protein is unable to bend duplexDNA, showing no preference for DNA with nicks and gaps,but does bind with high affinity to four-way junction struc-tures. These properties of the deinococcal HU protein ledthe authors to propose an in vivo role of HU in stabilizinghomologous recombination intermediates rather than tofunction as an architectural element. Furthermore, Mintonand Daly (1995) proposed that pre-existing four-way junc-tion structures favour pre-alignement of homologous chro-mosomes and stimulate DNAdouble strand break repair byhomologous recombination or single strand annealing andserve to organize the genetic material in space. While therole of D. radiodurans HU in DNA repair remains an openquestion, our present data strongly suggest that it is amajor nucleoid structural protein.
In contrast to HU essentiality, which did not allow theisolation of Dhbs mutant, dps mutants were easilyobtained and were not affected in their growth, hadnormal nucleoid structure and exhibited a wild-type resis-tance to g-irradiation in exponential as well as stationarygrowth phases. In E. coli, Dps protein plays an importantrole in cell survival during various stresses, including star-vation, UV and g-irradiation, thermal stress and oxidativestress (Nair and Finkel, 2004). Several hypotheses couldbe proposed to account for the radioresistance of dpsmutant in D. radiodurans: (i) Dps protein from E. coli pro-tects DNA from oxidative damage through physical asso-ciation and/or sequestration of Fe2+ ions that producehydroxyl radicals via Fenton chemistry (Zhao et al., 2002).D. radiodurans Dps proteins might be less effective in thisprotective mechanism. Indeed, purified D. radioduransDps1 binds to DNA in vitro but does not provide protectionfrom oxidative damage (Grove and Wilkinson, 2005; Kimet al., 2006). (ii) D. radiodurans possesses multiple path-ways to deal with reactive oxygen species (ROS) so thatthe mutational inactivation of one pathway might haveonly a small phenotypic impact. D. radiodurans encodesthree predicted superoxide dismutases (DR1279,DR1546 and DRA0202) and three predicted catalases(DR1998, DRA0259 and DRA0146) (Omelchenko et al.,2005), which can protect biomolecules from ROS-mediated damage. (iii) D. radiodurans has a very highintracellular manganese concentration that could mini-mize the requirement of an efficient iron homeostasismechanism for cell survival under oxidative stress (Daly
et al., 2004). Manganese ions behave as chemicalquenching agent of ROS and mimic the activities of cata-lase and superoxide dismutase (Seib et al., 2006).Indeed, a link between the cellular concentration of Mn(II)ions and protection of cellular proteins against oxidationhas been recently established (Daly et al., 2007). (iv) Dpsis among the most abundant proteins in stationary-phaseE. coli cells (Almiron et al., 1992). In D. radiodurans, itwas shown that Dps1 is present in the cells independentlyof the growth phase (Lipton et al., 2002). Under theseconditions, this might explain that Dps1 does not play animportant role for compaction of the nucleoid in a growth-cycle specific manner. However, the growth cycle specificregulation of Dps expression in D. radiodurans is not yetelucidated and it is possible that Dps is expressed afterprolonged nutrient starvation to play a protective role inbacteria under stress in their natural environment.
In E. coli, about a dozen distinct proteins (Azam andIshihama, 1999; Zimmerman, 2006), including HU, IHF,HNS, FIS and Dps, associate with the nucleoid. In vitro,these NAPs clearly have the ability to compact DNA.E. coli cells deficient in one of these NAPs display subtlephenotypes, indicating that some of the roles of one NAPcan be likely fulfilled by another. Therefore, these NAPsmay functionally compensate for one another. In con-trast, D. radiodurans lacks HNS, IHF and FIS, cellsdevoid of Dps contain a condensed nucleoid whereascells lacking hbs are not viable. Thus, we propose thatthe essential protein HU is responsible for most of thecompaction of the D. radiodurans nucleoid. Whetherother Deinococcus-specific nucleoid associated proteinscooperate with HU in determining the very compactstructure of the nucleoid in this radioresistant bacteriumremains to be determined.
Experimental proceduresBacterial strains, cultures, media and transformation
Bacterial strains are listed in Table 2. The Escherichia colistrain DH5a was used as the general cloning host and strainSCS110 was used to propagate plasmids prior to introductioninto D. radiodurans via transformation (Meima et al., 2001).AllD. radiodurans strains were derivatives of strain R1(ATCC13939). D. radiodurans was grown in TGY2X (1% tryp-tone, 0.2% dextrose, 0.6% yeast extract) at 30°C with aerationor on TGY1X plates solidified with 1.5% agar. E. coli strainswere grown in Luria–Bertani (LB) broth (Gibco Laboratories).When necessary, media were supplemented with the appro-priate antibiotics used at the following final concentrations:chloramphenicol 3.5 mg ml-1 for D. radiodurans; kanamycin6 mg ml-1 for D. radiodurans; tetracycline 2.5 mg ml-1 for D. ra-diodurans; spectinomycin 40 mg ml-1 for E. coli and 75 mg ml-1
for D. radiodurans. Transformation of D. radiodurans withPCR products, genomic DNA or plasmids was performed aspreviously described (Bonacossa de Almeida et al., 2002).
Plasmid DNA was extracted from E. coli using the QIAprepspin miniprep kit (Qiagen). Chromosomal DNA ofD. radiodurans was isolated as previously described (Men-necier et al., 2006). Plasmid DNA of D. radiodurans wasextracted from exponentially growing cells. Two millilitres ofcultures was harvested by centrifugation (12 000 r.p.m. atroom temperature). Pellets were resuspended in 300 ml oflysis buffer (glucose 20%, 25 mM Tris HCl pH 8, EDTA10 mM) and disrupted with a fastprep desintegrator (Savant;Bio101) using 0.1 g of glass beads (500 mm). After centrifu-gation for 3 min at 12 000 r.p.m., 250 ml of buffer P2 (1%SDS, 200 mM NaOH) was added to the supernatant andmixed, and 350 ml of buffer P3 (Na acetate 3M, pH 5.2) wasadded and mixed immediately. After centrifugation for 10 minat 12 000 r.p.m., the supernatants were applied to theQiaprep spin miniprep column and purification was per-formed as recommended by Qiagen. Amplification of plasmidor genomic DNA by PCR was performed with DyNAzymeEXT DNA polymerase (Finnzyme) or Extensor Hi-FidelityPCR enzyme Mix (ABgene). Oligonucleotides used are listedin Table S1.
Mutations in the repU gene were randomly generated byPCR amplification using p11554 as template and oligonucle-otides PS310-PS311. The PCRs contained the proofreading-defective Taq polymerase (Invitrogen) and unbalanceddNTPs pools, either 0.05 mM dGTP, 0.2 mM dTTP, 0.2 mMdCTP and 0.2 mM dATP or 0.05 mM dTTP, 0.2 mM dGTP, 0.2mMdCTP and 0.2 mM dATP.
Construction of thermosensitive plasmids
Plasmids are described in Table 3. To construct the ther-mosensitive plasmids, we first made a derivative of the vectorp11554 (shuttle vector that contains a spectinomycin resis-
tance gene) deleted of the repU gene. For this purpose,p11554 was cleaved by PpuMI/AclI to remove a fragmentcontaining the repU gene and the backbone was circularizedusing the annealed product of two oligonucleotides (PS308-PS309). The resulting plasmid p11829 can only replicate inE. coli. The mutated repU gene was generated by PCRmutagenesis using p11554 as template and PS310-PS311oligonucleotides, and introduced between the PpuMI and AclIsites of plasmid p11829 to reconstitute the original p11554plasmid with random mutations in the repU gene. The ligationmixture was directly used to transform D. radiodurans select-ing for SpcR transformants at 28°C. A total of 10 000 SpcR
colonies were screened by replica plating for defectivegrowth on spectinomycin plates at 37°C. The plasmids of 10thermosensitive candidates (p11830 to p11839) were purifiedfrom the deinococcal cells, introduced into the E. coli DH5ahost, and the repU gene was sequenced. Each plasmid(p11830 to p11839) carried at least one mutation leadingto an amino acid substitution in the RepU protein (seeTable S2). Plasmid p11830, which harbours three amino acidchanges in RepU (C71S, E130V and V290A), was retainedfor further experiments. To express cloned genes, the IPTG-regulated PSpac promoter and a transcription terminatorTerm116 were introduced in p11830. For this purpose, thePSpac-term116 cassette was amplified by PCR from plasmidp11559 (Mennecier et al., 2004), using the primer pair HH70/HH71 and cloned into p11830 between the BamHI andBstZ171 sites or between the BglII and StuI sites to giveplasmids p13840 (Fig. S1) and p13841, respectively, whichdiffer in the orientation of the cassette.
To construct plasmid p13844, the rpoD gene was amplifiedby PCR using the primer pair (HH39/HH45) and the productwas cloned into plasmid p13840 between the NdeI/XhoIsites. Plasmid p13849 containing amyE (DR1472) gene wasconstructed in a similar way using the primer pairs HH44/HH55. The gyrA gene was cloned into plasmid p13841
Table 2. Bacterial strains.
Bacterial strains Genotype Reference
E. coliDH5a supE44 DlacU(f80lacZDM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1 Stock laboratorySCS110 endA dam dcm supE44 D(lac-proAB) (F!traD36 proAB lacI qZDM15) Stock laboratory
D. radioduransR1 ATCC13939 Stock laboratoryGY10973 amyE W PtufA:lacI-kan (Lecointe et al., 2004)GY13308 Ddps1Wkan This workGY13313 Ddps2Wtet This workGY13314 Ddps1Wkan Ddps2Wtet This workGY13320 hbs::spaWcat This workGY13701 R1/p11830 This workGY13751 GY10973/p13844 (prepUTs::rpoD) This workGY13755 As GY13751 but DrpoDWcat This workGY13760 As GY13771 but amyEWPtufA:lacI-kan This workGY13767 GY10973/p13856 (prepUTs::gyrA) This workGY13771 R1/p13849 (prepUTs::amyE) This workGY13779 As GY13767 but DgyrAWcat This workGY13781 GY10973/p13840 This workGY13786 GY10973/p11554 This workGY13789 GY10973/p13863 (prepUTs::hbs) This workGY13792 Non-homogenotized DhbsWcat This workGY13795 As GY13789 but DhbsWcat This work
between the NdeI/XhoI sites in a similar way using theprimers HH32/HH33 to obtain plasmid p13856. The hbs genewas amplified by PCR using the oligonucleotides huNdeI/huDraI and cloned into plasmid p13841 between the NdeI/DraI sites to yield plasmid p13863. All constructions wereverified by DNA sequencing.
Deletion of genes in D. radiodurans and constructionof strain GY13320 (hbs::spaWcat)
The dps1, dps2, rpoD, hbs, gyrA disruption mutants wereconstructed by the tripartite ligation method (Mennecier et al.,2004). The hbs gene was fused with a spa tag (sequentialpeptide affinity) using a fragment containing a cassette withthe spa and the gene conferring resistance to chlorampheni-col (Zeghouf et al., 2004) by the tripartite ligation method. Themutated alleles constructed in vitro were then used to trans-form D. radiodurans to replace their wild-type counterpart byhomologous recombination. The genetic structure and thepurity of the mutants were checked by PCR using primersdescribed in Table S1.
Treatment of D. radiodurans with gamma irradiation
Exponential or stationary phase cultures, grown in TGY2X(supplemented with spectinomycin when necessary), wereconcentrated to an A650 = 30 in TGY2X and irradiated on icewith a 137Cs irradiation system (Institut Curie, Orsay, France)at a dose rate of 44.7 Gy min-1. Following irradiation, dilutedsamples were plated on TGY plates. Colonies were countedafter 3–4 days incubations at 30°C.
Assay of plasmid thermosensitivity
Plasmid containing bacteria growing exponentially at 28°C inTGY2X supplemented with spectinomycin were diluted 100-fold in antibiotic-free medium and incubated at 28°C or 37°C.At various times, samples were removed, plated on selectiveand non-selective TGY agar and incubated 3–4 days at 28°C.
The frequency of the plasmid-containing bacteria was mea-sured as the percentage of antibiotic resistant colonies overthe total viable count.
Assay of genes essentiality
The essentiality of genes was evaluated in a growth experi-ment, in which the strains grown at 28°C in liquid mediumwith spectinomycin were serially diluted, plated on TGY agarand incubated at 28°C or 37°C in the presence or theabsence of spectinomycin.
Fluorescence microscopy
Culture aliquots (1 ml) were removed and the correspondingcells were fixed using toluene at 3% final concentration. Cellmembranes were stained with N-(3-triethylammonium-umpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl)pyridinium dibromide (FM 4–64) at 0.01 mg ml-1, and the nucle-oid with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI)at 2 mg ml-1. FM 4–64 stains the lipid membranes with redfluorescence (excitation/emission ~515/640 nm) and DAPIstains the nucleoid with blue fluorescence (excitation/emission ~350/470 nm). The stained cells were observedusing a Leica DM RXA microscope. Images were capturedwith a CDD camera 5 MHz Micromax 1300Y (Roper Instru-ments). The final reconstructed images were obtained bydeconvoluting Z-series with the Metamorph software (Univer-sal Imaging). Before fluorescence microscopy, for depletion ofHU, the cells were grown overnight at 28°C in medium supple-mented with spectinomycin. The cells were then harvested bycentrifugation, washed, resuspended, diluted in antibiotic-freemedium and incubated at 28°C or 37°C. Culture aliquots (1 ml)were removed at different times after the shift at the non-permissive temperature and treated as previously described.
Immunofluorescence labelling and microscopy
Deinococcus radiodurans GY13320 (hbs::spaWcat) and wild-type strains were grown exponentially in TGY2X (OD650:0.25).
Table 3. Plasmids.
Plasmids Description Reference
pGTC101 Source of chloramphenicol cassette in D. radiodurans (Earl et al., 2002)p11086 Source of kanamycin cassette in D. radiodurans Laboratory stockp11615 Source of tetracyclin cassette in D. radiodurans Laboratory stockp11554 Shuttle vector E. coli/D. radiodurans, SpcR Laboratory stockp11559 Source of PSpac-term116 cassette Laboratory stockp11829 Non-replicative plasmid in D. radiodurans (DrepU), SpcR This workp11830 to p11839 Vector thermosensitive for replication in D. radiodurans, SpcR, prepUTs This workp12723 Source of spa-tag chloramphenicol cassette This workp13840 p11830 PSpac-term 116a This workp13841 p11830 PSpac-term 116a This workp13844 p13840: prepUTs::rpoD This workp13849 p13840: prepUTs::amyE This workp13856 p13841: prepUTs::gyrA This workp13862 p13841: prepUTs::hbs This work
a. The two plasmids p13840 and p13841 differ by the orientation of the PSpac-term116 cassette.
Aliquots of 0.4 ml of the exponential phase culture were takenand fixed by addition of 1/10 volume of 37% paraformalde-hyde in the culture medium and incubation for 2 h at 4°C. Thecell pellet was subsequently washed once in 1¥ PBS. In orderto permeabilize the cell envelope, the cells were treated with2 mg ml-1 lysozyme for 30 min at 37°C followed by incubationwith 0.1% Triton X-100 in PBS for 5 min at room temperature.Finally, the cells were washed in PBS and resuspended in25 ml of PBS. A 3 ml aliquot was applied to a poly L-lysinepretreated slide spot, allowed to air dry and fixed by incubat-ing in 4% PFA for 20 min at 37°C. Cells were then blocked in2% BSA in PBS-T (0.05% Tween 20 in PBS) and incubatedfor 2 h at 37°C with a monoclonal mouse anti-Flag antibody(Sigma-Aldrich) diluted 1/700 in blocking solution. After20 min washing in PBS-T, the cells were incubated for 1 h at37°C with a FITC conjugated goat anti-mouse antibody(Jackson Immunoresearch Laboratories) diluted 1/250 inblocking solution and washed for 20 min in PBS-T. Cells werefinally stained with DAPI 10 mg ml-1 for 10 min at roomtemperature. After a final wash in PBS-T slides were mountedusing fluoromount G as a mounting medium (Fluoprobes).Image acquisition and treatment were performed asdescribed above.
AcknowledgementsWe thank Adriana Bailone for valuable discussions and criti-cal reading of the manuscript. We thank Esma Bentchikou forher expert assistance in microscopy, M. DuBow for help withEnglish. We thank the Institut Curie for the use of the 137Csirradiation system, V. Favaudon for help with gamma-irradiation. The authors are grateful to M. Prigent for technicalhelp and advice concerning immunofluorescent labelling andpicture acquisition. This work was supported by the CentreNational de la Recherche Scientifique, the University Paris-Sud 11, the Commissariat à l’Energie Atomique (CEALRC42V), Electricité de France (RB2007-11), and the AgenceNationale de la Recherche (ANR-07-BLAN-0106). This workwas carried out in compliance with the current laws governinggenetic experimentation in France.
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