# The essential histone-like protein HU plays a major role in Deinococcus radiodurans nucleoid compaction

Hong Ha Nguyen, Claire Bouthier de la Tour, Magali Toueille, Françoise

Vannier, Suzanne Sommer, Pascale Servant

► To cite this version:

Hong Ha Nguyen, Claire Bouthier de la Tour, Magali Toueille, Françoise Vannier, Suzanne Sommer, et al.. The essential histone-like protein HU plays a major role in Deinococcus radiodurans nucleoid compaction. Molecular Microbiology, 2009, 73, pp.240 - 252. 10.1111/j.1365-2958.2009.06766.x. hal-05514510

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# The essential histone-like protein HU plays a major role in Deinococcus radiodurans nucleoid compaction

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.

## Summary

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*.

## Introduction

*Deinococcus radiodurans* possesses an exceptional ability to withstand the lethal effects of DNA damaging agents, including ionizing radiation. The most striking feature of

Accepted 8 June, 2009. For correspondence. *E-mail pascale.servant@igmors.u-psud.fr; Tel. (+33) 1 69 15 58 17; Fax (+33) 1 69 15 78 08. **E-mail suzanne.sommer@igmors.u-psud.fr; Tel. (+33) 1 69 15 46 14; Fax (+33) 1 69 15 78 08.

*D. radiodurans* is its capability to reconstruct a functional genome within 2–3 h from hundreds of chromosomal fragments generated by irradiation, whereas the genome of most organisms is irreversibly shattered under the same conditions. It has been suggested that *D. radiodurans* combines many different mechanisms to efficiently repair its damaged DNA (for recent review, see Cox and Battista, 2005; Blasius *et al.*, 2008). *D. radiodurans* nucleoids adopt a condensed structure that remains unaltered after high-dose γ-irradiation, suggesting that this structure may passively contribute to radioresistance by preventing the dispersion of DNA fragments generated by irradiation (Levin-Zaidman *et al.*, 2003; Zimmerman and Battista, 2005). The tightly packed structure of the nucleoid appears to be a common trait among radioresistant bacteria (Zimmerman and Battista, 2005). Such a condensed genome may provide suitable scaffolds for DNA repair.

In prokaryotes, bacterial nucleoids are efficiently compacted by a group of nucleoid-associated proteins (NAPs), which are often referred to as histone-like proteins. In *Escherichia coli*, about a dozen distinct proteins associate with the nucleoid, although only five exist in abundant concentrations (Azam and Ishihama, 1999). These proteins include the HU protein, integration host factor (IHF) as well as H-NS, Fis and Dps (DNA-binding protein from starved cells). Based on their high intracellular abundance and DNA binding properties, these proteins are generally believed to be the most important players in the organization and compaction of bacterial chromatin (for review, see Travers and Muskhelishvili, 2005; Luijsterburg *et al.*, 2006). In exponentially growing cells, the proteins Fis and HU are the most abundant, whereas Dps is the major nucleoid component in stationary phase (Azam and Ishihama, 1999). Among these proteins, only HU encoded by the *hbs* gene and Dps are present in *D. radiodurans*.

In almost all bacteria except *Enterobacteriaceae*, HU exists as an 18 kDa homodimer. In *E. coli*, HU exists as a hetero-dimer composed of two homologous subunits (HUα and HUβ) or as homo-dimers. *E. coli* HU heterodimer is an architectural protein that binds non-specifically to double-stranded DNA and with increased affinity to distorted DNA leading to significant DNA bending, negative supercoiling and DNA compaction (Pontiggia *et al.*, 1993; Bonnefoy *et al.*, 1994; Castaing *et al.*, 1995;
Kamashev and Rouviere-Yaniv, 2000). *E. coli* strains lacking the two HU subunits have been successfully constructed, demonstrating that *E. coli* HU is not essential for viability. However, the absence of HU perturbs cell division, causing formation of anucleate cells (Huisman *et al.*, 1989) and increases sensitivity to γ and UV irradiation (Boubrik and Rouviere-Yaniv, 1995). More recently, a role for HU in DNA repair has emerged. In *E. coli*, HU preferentially binds nicks, gaps and abasic sites, harbours AP lyase activity (Kow *et al.*, 2007) and is involved in the repair of lesions that are in close proximity (Hashimoto *et al.*, 2003). Grove *et al.* have shown that the mycobacterial histone-like HU homologue (Hlp) promotes DNA end-joining in the presence of T4 DNA ligase (Mukherjee *et al.*, 2008). HU also plays regulatory roles in DNA replication and acts as a global transcriptional regulator of numerous genes in *E. coli* (Oberto *et al.*, 2009).

In bacteria, Dps is one of the critical proteins that transforms the nucleoid into a condensed state in response to environmental stresses (Kim *et al.*, 2004). Dps protects genomic DNA against oxidative stress (Martinez and Kolter, 1997), UV light (Nair and Finkel, 2004), possibly by its ability to protect DNA against damage. Moreover, Dps can reduce the intracellular level of Fe²⁺ and thus restricts the production of hydroxyl radicals presumably by suppression of Fenton chemistry (Zhao *et al.*, 2002). *D. radiodurans* encodes two proteins with homology to *E. coli* Dps: Dps1 and Dps2 which are the products of the *DR2263* and *DRB0092* genes respectively.

Here, we unsuccessfully attempted to construct an *hbs* (*DRA0065*) deletion mutant in *D. radiodurans*, suggesting that HU is essential for cell viability. We demonstrate the essentiality of *hbs* by constructing a new system allowing the conditional expression of *hbs* on a temperature-sensitive replicon, whose replication is efficiently shut down at 37°C. In parallel, we also inactivated the two *dps* genes and showed that their deletion did not change the nucleoid compaction and radioresistance of *D. radiodurans*. We also show that HU protein localized to the nucleoid in *D. radiodurans* cells and that, when HU was expressed from a thermosensitive plasmid, its progressive depletion at the non-permissive temperature generated first decondensation of DNA prior to fractionation of the nucleoid followed by cells lysis. Our data strongly suggest a major role of HU in nucleoid organization and DNA compaction in *D. radiodurans*.

## Results

### Failure to create a deletion mutant devoid of HU protein

To investigate the functions of the histone-like HU protein in *D. radiodurans*, we attempted to construct a deletion mutant. Gene disruption was performed by replacing the

coding sequences of *hbs* (*DRA0065*) gene with a chloramphenicol resistance cassette using the tripartite ligation method (Mennecier *et al.*, 2004). The constructs were then introduced into *D. radiodurans* by genetic transformation selecting for antibiotic resistance. This led to the replacement of the wild-type allele by the mutant counterpart via homologous recombination. *D. radiodurans* is multigenic, with cells containing from 4 to 10 genome equivalents depending on the growth conditions (Hansen, 1978; Harsojo *et al.*, 1981). We obtained chloramphenicol resistant colonies, but PCR analysis of two candidates showed that, after two rounds of purification on chloramphenicol plates, both still contained the wild-type allele in addition to the Δ*hbs::Ωcat* allele (Fig. 1B-1). Further purification of the candidates did not allow recovery of cells devoid of the wild-type allele, suggesting that HU protein is essential for cell viability.

### Essentiality of the *hbs* gene is demonstrated by a new conditional gene inactivation system

To obtain positive evidence that *hbs* is an essential gene, we developed a new genetic assay of gene essentiality based on inactivation of a chromosomal gene in cells containing an additional copy of this gene on a temperature-sensitive replication vector. In such a system, the wild-type allele carried by the plasmid supplies a functional product at the permissive temperature, while plasmid loss at the non-permissive temperature results in the decay of the gene product leading to cell death when the protein is essential for cell viability. Small versatile shuttle plasmids for cloning in *D. radiodurans* exist but none is thermosensitive for replication (Meima and Lidstrom, 2000; Lecointe *et al.*, 2004). Thus, as a first step, we constructed this type of shuttle vector. For this purpose we mutated the *repU* gene of plasmid p11554, a SpcR derivative of plasmid PI8. The *repU* gene encodes a protein that shares similarity with the replication initiation proteins of two different *Thermus* plasmids and has been shown to be essential for plasmid replication in *D. radiodurans* (Meima and Lidstrom, 2000). The *repU* gene was amplified by PCR under conditions favouring the generation of mutations and was used to replace the wild-type counterpart in plasmid p11554 (for details, see *Experimental procedures*). Plasmid p11830 having three mutations in *repU* was retained for further studies. Measurement of plasmid stability during growth in liquid medium without the selective antibiotic showed that p11830 was stable at 28°C but was rapidly lost at 37°C (Fig. 2). After 6 h at 37°C, the proportion of cells carrying p11830 was reduced to approximately 0.1%, suggesting that plasmid replication was quickly arrested after the temperature shift. To express cloned genes, we then introduced a *P<sub>Spac</sub>* promoter and a *term116* transcription terminator into the thermosensitive
<figure><img src="image_2.png" /></figure>

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<figure><img src="image_5.png" /><figcaption>**Fig. 2.** Loss of thermosensitive plasmid p11830 at 37°C. Strain *D. radiodurans* GY13701 carrying plasmid p11830 was grown at 28°C or 37°C. Samples were removed at 2 h intervals for plating at 28°C on selective and non-selective media. Symbols: 28°C (squares), 37°C (circles).</figcaption></figure>

<caption>**Fig. 1.** Schematic representation and test of deletion-substitution in the *D. radiodurans* *hbs* and *dps* genes.</caption>

A. Schematic representation of the allele replacement event in *hbs* (A-1), *dps1* (A-2) and *dps2* (A-3) genes. Short arrows indicate the position of specific primers used for diagnostic PCR. Primers are described in Table S1.

B. PCR analysis of two independent candidate *hbs* mutants (B-1), two independent Δ*hbs* (*prepUTs-hbs⁺*) mutants (B-1) and the double Δ*dps1Δdps2* mutant (B-2 and B-3).

vector p11830 to yield p13840 (Fig. S1) and p13841 differing by the orientation of the *P<sub>Spac</sub>-term116* cassette.

This new genetic tool was validated using genes known to be essential or dispensable for cell viability. As essential genes, we used the *rpoD* (*DR0916*) gene encoding the housekeeping sigma factor (Kobayashi *et al.*, 2003) and the *gyrA* gene encoding a subunit of DNA gyrase, an enzyme that regulates DNA supercoiling required for basic processes such as DNA replication, recombination and repair (for review, see Nollmann *et al.*, 2007). As a control, we also used the *amyE* gene (*DR1472*) previously shown to be dispensable for cell viability (Meima *et al.*, 2001). The coding region of each gene was cloned under the control of the *P<sub>Spac</sub>* promoter on the thermosensitive vector and the recombinant plasmids were transformed into the *D. radiodurans* strain GY10973 expressing the *lacI* regulatory gene. The resulting strains are merodiploids containing plasmidic and chromosomal copies of the tester genes. The chromosomal copies of each gene were subsequently inactivated using the tripartite ligation method to replace the coding regions with a
<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Conditional viability of *D. radiodurans* cells expressing *rpoD* and *gyrA* on the thermosensitive plasmid. Strains were grown in liquid medium with spectinomycin at 28°C. The dilutions of cells were spotted on medium with or without spectinomycin at 28°C (A) or 37°C (B). Lane 1: strain GY13786 containing non-thermosensitive plasmid p11554 (prepU); lane 2: strain GY13781 containing thermosensitive plasmid p13840 (prepUTs); lane 3: strain GY13760 [*ΔamyE (prepUTs-amyE*)*] (control of nonessential gene); lane 4: strain GY13779 [*ΔgyrA (prepUTs-gyrA*)*]; lane 5: strain GY13755 [*ΔrpoD (prepUTs-rpoD*)*].</figcaption></figure>

chloramphenicol resistance cassette and the strains were tested for their viability at the non-permissive temperature. For this purpose, bacteria grown at 28°C in liquid medium with spectinomycin (to select the presence of the thermosensitive plasmid) were plated and incubated at 28°C (permissive temperature) or 37°C (non-permissive temperature) in the presence or absence of spectinomycin. Growth of Δ*gyrA* (*prepUTs-gyrA*) bacteria (Fig. 3, lanes 4) and Δ*rpoD* (*prepUTs-rpoD*) bacteria (Fig. 3, lanes 5) was strictly dependent on the temperature, indicating that *rpoD* and *gyrA* are essential genes while the normal growth of Δ*amyE* (*prepUTs-amyE*) bacteria at 37°C on plates without spectinomycin (Fig. 3, lanes 3) showed that they were not affected by the loss of the *prepUTs-amyE* plasmid, as expected for a dispensable gene. Δ*gyrA*

(*prepUTs-gyrA*) bacteria showed some reduced growth when plated at 28°C in the absence of selective pressure. It is possible that a suboptimal level of gyrase in these bacteria caused some plasmid instability since DNA superhelicity is centrally involved in the partitioning mechanism of some plasmids (Miller *et al.*, 1990).

This new genetic tool being validated, we used it to test the essentiality of the *hbs* gene. The chromosomal copy of merodiploids carrying the *prepUTs-hbs* plasmid was inactivated by replacing the *hbs* coding region with a chloramphenicol resistance cassette. The complete deletion of the chromosomal *hbs* allele in the Cam<sup>R</sup> transformants purified twice on selective plates was confirmed by PCR analysis (Fig. 1B-1). The cells were then tested for growth at 37°C. As can be seen in Fig. 4, the growth of

<figure><img src="image_7.png" /><figcaption>**Fig. 4.** HU is essential for *D. radiodurans* viability. Strains were grown in liquid medium with spectinomycin at 28°C. The dilutions of cells were spotted on medium with or without spectinomycin at 28°C (A) or 37°C (B). Lane 1: strain GY13781 containing thermosensitive plasmid p13840 (prepUTs); lane 2: strain GY13786 containing non-thermosensitive plasmid p11554 (prepU); lane 3: strain GY13795 [*Δhbs (prepUTs-hbs*)*].</figcaption></figure>
<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Immunolocalization of the HU protein in *D. radiodurans*. *D. radiodurans* cells from strain GY13320 (*hbs::spaΩcat*) expressing the HU protein harbouring a SPA-tag (top panel) and from the wild-type strain (R1, lower panel). Cells were probed with an anti-Flag primary antibody followed by a FITC coupled secondary antibody (green) and with DAPI (blue). Cells were observed with a wide-field three-dimensional microscope. The images shown are single sections of a deconvoluted Z-series.</figcaption></figure>

Δ*hbs* (*prepUTs-hbs*⁺) bacteria was strictly dependent on the temperature, confirming that *hbs* is essential for cell viability.

### HU protein colocalizes with DNA

To visualize the HU protein by immunofluorescence microscopy, we fused a SPA-tag (Zeghouf *et al.*, 2004) to its C-terminal end. The SPA-tagged *hbs* gene was constructed *in vitro* (see *Experimental procedures*) and used to replace the wild-type *hbs* allele to give strain GY13220. Cells expressing the HU-SPA protein had the same growth rate as the wild-type (data not shown), indicating that the tagged protein was functional. The results of immunofluorescence staining showed that the HU-SPA protein localizes at the nucleoid and is uniformly distributed throughout this structure (Fig. 5). This suggests that HU might be involved in nucleoid compaction due to its interaction with DNA.

### Depletion of HU protein generates important changes in the nucleoid structure

Next, we used the conditional gene inactivation system to analyse the effects of HU depletion on the nucleoid structure. For this purpose, colonies of GY13975, in which HU protein is expressed from the thermosensitive plasmid, or of GY13781, a wild-type strain containing the empty thermosensitive vector, were grown overnight at 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 cellular morphology and nucleoid structure were analysed by combining epifluorescence and deconvolution microscopy.

While the wild-type cells containing the thermosensitive vector exhibited a normal cell morphology and nucleoid compaction at 37°C as well as at 28°C (Fig. 6), cells expressing HU from the thermosensitive plasmid were normal at 28°C but lysed after 24 h of growth at 37°C, suggesting that HU depletion caused dramatic effects. We also examined cells expressing GyrA from the thermosensitive plasmid. In this case, we did not observe cell lysis after 24 h incubation at 37°C, instead we observed anucleate cells whose proportion rose from 8% in cultures grown at 28°C to 50% in cultures grown at 37°C (Fig. 6). This is consistent with a major role of DNA gyrase in partitioning of the deinococcal chromosomes.

To determine the dynamics of the cellular alterations occurring during HU protein depletion, we analysed the cell morphologies in samples taken at different times after the shift at the non-permissive temperature (Fig. 7 and Table 1). We observed that, 10 h after the temperature shift, the cells had an increased size and enlarged nucleoids (50% of the cell population). Further incubation (t = 12 h) first resulted in the appearance of cells with fractionated nucleoids (63% of cells), showing several dense DAPI stainable bodies, and later (t = 15 h and t = 20 h) in a complete collapse of the nucleoid structure that filled the entire cellular space (45.8% and 59.8% respectively). Finally (t = 24 h), the entire cell population lysed. These data strongly suggest that HU plays a key
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** Effect of DNA gyrase or HU depletion on *D. radiodurans* nucleoid morphology. Cells in exponential growth phase cultivated at 28°C in medium supplemented with spectinomycin were harvested by centrifugation, diluted in antibiotic-free medium and incubated at 28°C (A) or 37°C (B). Cells were stained and observed with a wide-field three-dimensional microscope 24 h after inoculation. Left images: DIC (differential interference 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: Δ*gyrA* (*prepUTs-gyrA*+); 3: GY13795: Δ*hbs* (*prepUTs-hbs*+).</figcaption></figure>

role in DNA compaction and nucleoid organization in *D. radiodurans*.

*Nucleoid compaction is not changed in Δdps mutants*

In addition, we investigated the effect of the absence of Dps, also considered as major protein of nucleoids in prokaryotes. We constructed mutants deleted of *dps1* or *dps2* genes as well as the double mutant Δ*dps1Δdps2*. Homogenotes of single or double *dps* mutants were easily obtained after just one cycle of purification on selective medium, as confirmed by amplifying the targeted allele by PCR (Fig. 1B-2 and B-3). The *dps* mutants were not affected in their growth (data not shown). Since Dps is known to be abundant in stationary phase (Altuvia *et al.*, 1994), *dps* mutant cells in exponential or in stationary growth phase were analysed by combining epifluorescence and deconvolution microscopy to investigate cell morphology and nucleoid structure. Δ*dps1*, Δ*dps2* and the double mutant cells had the same morphology as the wild-type cells and contained a condensed nucleoid with a normal compact structure

(Fig. 8). Similar results were obtained when irradiated cells were examined (data not shown). When exposed to γ-irradiation, Δ*dps* mutants showed the same level of resistance as the wild-type (Fig. 9). This suggests that, contrary to HU, Dps is not essential for nucleoid compaction and cell viability.

Discussion

*Deinococcus radiodurans* nucleoid adopts a condensed structure that remains unaltered after high-dose γ-irradiation (Levin-Zaidman *et al.*, 2003; Zimmerman and Battista, 2005). Such tightly compact structure of the nucleoid, a common trait among radioresistant bacteria, may prevent the diffusion of DNA fragments generated by irradiation and contribute to the radioresistance of these species. Bacterial nucleoid associated proteins (NAPs) play a major role in DNA compaction and nucleoid organization (Travers and Muskhelishvili, 2005; Luijsterburg *et al.*, 2006). In this report, we studied the role of two major NAPs, HU and Dps proteins, on nucleoid compaction in *D. radiodurans*.
<figure><img src="image_10.png" /><figcaption>**Fig. 7.** Observation of Δhbs (*prepUTs-hbs*) cells after a temperature shift at 37°C. Cells of *D. radiodurans* strain GY13795 [Δhbs (*prepUTs-hbs*)] were treated as described in legend to Fig. 6 and were stained and observed with a wide-field three-dimensional microscope at different times during incubation at 37°C as indicated.</figcaption></figure>

The failure to obtain a homozygous *hbs* knockout mutant suggested the essentiality of this gene. To confirm this hypothesis, we developed a conditional inactivation system based on the temperature sensitive vector. Two properties of the plasmids used in this study (Meima and Lidstrom, 2000; Lecointe *et al.*, 2004) are particularly relevant for our conditional gene inactivation system: (i) their low copy number (4–8 copies per cell) in *D. radiodurans* allowing rapid loss of the *prepUTs* vector at the non-permissive temperature and (ii) their stable maintenance in the absence of selective pressure, allowing a cloned allele to efficiently complement the disrupted chromosomal counterpart at the permissive temperature. A different conditional gene inactivation system was previously described (Lecointe *et al.*, 2004). It was based on an *E. coli* plasmid containing an N-terminal fragment of an essential deinococcal gene cloned under the control of

the $P_{Spac}$ promoter to generate an insertion-duplication mutant conditionally expressing a functional protein. The two drawbacks of this system, (i) the leakage of the $P_{Spac}$ promoter and (ii) the high rate of reversion by efficient intrachromosomal recombination, are circumvented in our new conditional gene inactivation system. In this study, using our new system allowing conditional gene expression, we proved that HU is essential for cell viability in *D. radiodurans*.

We also analysed the effects of HU depletion on nucleoid structure. Severe nucleoid abnormalities were observed after loss of the plasmid encoded HU: at early times the nucleoid enlarged, then appeared as fractionated in several condensed nuclear bodies and later collapsed in a diffuse structure filling the entire cellular space. At this time, we also observed leakage of the diffuse nucleoid in the surrounding medium through cell

<table><caption>**Table 1.** Morphology of the nucleoids in cells deprived of HU.</caption><thead><tr><th></th><th>t = 0 h</th><th>t = 10 h</th><th>t = 12 h</th><th>t = 15 h</th><th>t = 20 h</th></tr></thead><tbody><tr><td>Compact nucleoid</td><td>92.4% (110/119)</td><td>27.2% (75/276)</td><td>&lt; 1.5%</td><td>&lt; 0.7%</td><td>&lt; 0.3%</td></tr><tr><td>Expanded nucleoid</td><td>&lt; 1%</td><td>26.8% (74/276)</td><td>&lt; 1.5%</td><td>&lt; 0.7%</td><td>&lt; 0.3%</td></tr><tr><td>Fractionated nucleoid</td><td>&lt; 1%</td><td>11.6% (32/276)</td><td>63.3% (50/79)</td><td>31.9% (46/144)</td><td>33.5% (101/301)</td></tr><tr><td>Collapsed nucleoid</td><td>&lt; 1%</td><td>11.2% (31/276)</td><td>12.7% (10/79)</td><td>45.8% (66/144)</td><td>59.8% (180/301)</td></tr><tr><td>Other morphologies</td><td>7.6% (9/119)</td><td>23.2% (64/276)</td><td>24% (19/79)</td><td>22% (32/144)</td><td>6.6% (20/301)</td></tr></tbody></table>

Cells of GY13795 [Δhbs (*prepUTs-hbs*)] grown overnight at 28°C in medium with spectinomycin were harvested by centrifugation, diluted in antibiotic-free medium and incubated at 28°C or 37°C. Cells aliquots were removed at different times after temperature shift and observed with a wide-field three-dimensional microscope 24 h after inoculation.
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** Illustration of cell morphology in the Δ*dps1Δdps2* strain. Nucleoids stained with DAPI appear in blue and membranes stained with FM4-64 appear in red. *D. radiodurans* cells from the wild-type strain (R1, left panel) and from Δ*dps1Δdps2* strain (right panel) were observed with a wide-field three-dimensional microscope.</figcaption></figure>

A. Cells in stationary growth phase (OD<sub>650</sub> = 2).

B. Cells in exponential growth phase (OD<sub>650</sub> = 0.5). The images shown are single sections of a deconvoluted Z-series.

<figure><img src="image_12.png" /><figcaption>**Fig. 9.** Survival curve for Δ*dps1Δdps2* mutant strain to γ rays. Bacteria were exposed to γ-irradiation at doses indicated on the abscissa and dilutions were plated as described in *Experimental procedures*. Symbols: wild-type (circles), Δ*dps1Δdps2* (squares). Each value is the average of three independent experiments with standard deviations that did not exceed 10% of the mean values.</figcaption></figure>

membrane holes. Finally, the entire cell population lysed and only membrane ghosts were visible. Several reports have linked the nucleoid remodelling altered by HU with the reorganization of transcription (Dorman and Deighan, 2003; Kar et al., 2005; Oberto et al., 2009). Moreover, it has been recently shown that HU regulates the expression of as many as 8% (353 genes) of the *E. coli* genome, either cooperating with transcription regulators or binding directly to its DNA structure targets (Oberto et al., 2009). The observed dramatic effects of HU depletion in *D. radiodurans* might be caused by an alteration of the expression pattern of some genes under the control of deinococcal HU protein or by the loss of the nucleoid architecture. In *E. coli*, a transcriptional role of HU was demonstrated for the upregulation of the *proVWX* operon in hyper-osmolar environments (Manna and Gowrishankar, 1994) and recently it was shown that the HU regulon is composed of genes responding to high osmolarity (Oberto et al., 2009). Therefore, the loss of HU, through its effects on transcription of proteins needed to maintain osmolarity, may perturb the osmotic balance in the cell and provoke the lysis.
We also showed that *D. radiodurans* HU protein concentrates in the nucleoid and is uniformly distributed throughout the structure. *E. coli* HU is an architectural protein that binds non-specifically to double-stranded DNA and with increased affinity to distorted DNA to cause significant DNA bending, negative supercoiling and DNA compaction (Pontiggia *et al.*, 1993; Bonnefoy *et al.*, 1994; Castaing *et al.*, 1995; Kamashev and Rouviere-Yaniv, 2000). Ghosh and Grove (2004) have shown by *in vitro* experiments that the *D. radiodurans* HU protein is unable to bend duplex DNA, showing no preference for DNA with nicks and gaps, but does bind with high affinity to four-way junction structures. These properties of the deinococcal HU protein led the authors to propose an *in vivo* role of HU in stabilizing homologous recombination intermediates rather than to function as an architectural element. Furthermore, Minton and Daly (1995) proposed that pre-existing four-way junction structures favour pre-alignement of homologous chromosomes and stimulate DNA double strand break repair by homologous recombination or single strand annealing and serve to organize the genetic material in space. While the role of *D. radiodurans* HU in DNA repair remains an open question, our present data strongly suggest that it is a major nucleoid structural protein.

In contrast to HU essentiality, which did not allow the isolation of Δhbs mutant, *dps* mutants were easily obtained and were not affected in their growth, had normal nucleoid structure and exhibited a wild-type resistance to γ-irradiation in exponential as well as stationary growth phases. In *E. coli*, Dps protein plays an important role in cell survival during various stresses, including starvation, UV and γ-irradiation, thermal stress and oxidative stress (Nair and Finkel, 2004). Several hypotheses could be proposed to account for the radioresistance of *dps* mutant in *D. radiodurans*: (i) Dps protein from *E. coli* protects DNA from oxidative damage through physical association and/or sequestration of Fe²⁺ ions that produce hydroxyl radicals via Fenton chemistry (Zhao *et al.*, 2002). *D. radiodurans* Dps proteins might be less effective in this protective mechanism. Indeed, purified *D. radiodurans* Dps1 binds to DNA *in vitro* but does not provide protection from oxidative damage (Grove and Wilkinson, 2005; Kim *et al.*, 2006). (ii) *D. radiodurans* possesses multiple pathways to deal with reactive oxygen species (ROS) so that the mutational inactivation of one pathway might have only a small phenotypic impact. *D. radiodurans* encodes three 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 high intracellular manganese concentration that could minimize the requirement of an efficient iron homeostasis mechanism for cell survival under oxidative stress (Daly

*et al.*, 2004). Manganese ions behave as chemical quenching agent of ROS and mimic the activities of catalase and superoxide dismutase (Seib *et al.*, 2006). Indeed, a link between the cellular concentration of Mn(II) ions and protection of cellular proteins against oxidation has been recently established (Daly *et al.*, 2007). (iv) Dps is among the most abundant proteins in stationary-phase *E. coli* cells (Almiron *et al.*, 1992). In *D. radiodurans*, it was shown that Dps1 is present in the cells independently of the growth phase (Lipton *et al.*, 2002). Under these conditions, this might explain that Dps1 does not play an important role for compaction of the nucleoid in a growth-cycle specific manner. However, the growth cycle specific regulation of Dps expression in *D. radiodurans* is not yet elucidated and it is possible that Dps is expressed after prolonged nutrient starvation to play a protective role in bacteria under stress in their natural environment.

In *E. coli*, about a dozen distinct proteins (Azam and Ishihama, 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 subtle phenotypes, indicating that some of the roles of one NAP can be likely fulfilled by another. Therefore, these NAPs may functionally compensate for one another. In contrast, *D. radiodurans* lacks HNS, IHF and FIS, cells devoid of Dps contain a condensed nucleoid whereas cells lacking *hbs* are not viable. Thus, we propose that the essential protein HU is responsible for most of the compaction of the *D. radiodurans* nucleoid. Whether other *Deinococcus*-specific nucleoid associated proteins cooperate with HU in determining the very compact structure of the nucleoid in this radioresistant bacterium remains to be determined.

## Experimental procedures

### Bacterial strains, cultures, media and transformation

Bacterial strains are listed in Table 2. The *Escherichia coli* strain DH5α was used as the general cloning host and strain SCS110 was used to propagate plasmids prior to introduction into *D. radiodurans* via transformation (Meima *et al.*, 2001). All *D. radiodurans* strains were derivatives of strain R1 (ATCC13939). *D. radiodurans* was grown in TGY2X (1% tryptone, 0.2% dextrose, 0.6% yeast extract) at 30°C with aeration or on TGY1X plates solidified with 1.5% agar. *E. coli* strains were grown in Luria-Bertani (LB) broth (Gibco Laboratories). When necessary, media were supplemented with the appropriate antibiotics used at the following final concentrations: chloramphenicol 3.5 µg ml⁻¹ for *D. radiodurans*; kanamycin 6 µg ml⁻¹ for *D. radiodurans*; tetracycline 2.5 µg ml⁻¹ for *D. radiodurans*; spectinomycin 40 µg ml⁻¹ for *E. coli* and 75 µg ml⁻¹ for *D. radiodurans*. Transformation of *D. radiodurans* with PCR products, genomic DNA or plasmids was performed as previously described (Bonacossa de Almeida *et al.*, 2002).
<table><caption>**Table 2.** Bacterial strains.</caption><thead><tr><th>Bacterial strains</th><th>Genotype</th><th>Reference</th></tr></thead><tbody><tr><td><strong>E. coli</strong></td><td></td><td></td></tr><tr><td>DH5α</td><td>supE44 ΔlacU(φ80lacZΔM15) hsdR17 recA1 endA1 gyrA96 thi-1 relA1</td><td>Stock laboratory</td></tr><tr><td>SCS110</td><td>endA dam dcm supE44 Δ(lac-proAB) (F' traD36 proAB lacIq ZΔM15)</td><td>Stock laboratory</td></tr><tr><td><strong>D. radiodurans</strong></td><td></td><td></td></tr><tr><td>R1</td><td>ATCC13939</td><td>Stock laboratory</td></tr><tr><td>GY10973</td><td>amyE Ω PtufA:lacI-kan</td><td>(Lecointe et al., 2004)</td></tr><tr><td>GY13308</td><td>Δdps1Ωkan</td><td>This work</td></tr><tr><td>GY13313</td><td>Δdps2Ωtet</td><td>This work</td></tr><tr><td>GY13314</td><td>Δdps1Ωkan Δdps2Ωtet</td><td>This work</td></tr><tr><td>GY13320</td><td>hbs::spaΩcat</td><td>This work</td></tr><tr><td>GY13701</td><td>R1/p11830</td><td>This work</td></tr><tr><td>GY13751</td><td>GY10973/p13844 (prepUTs::rpoD)</td><td>This work</td></tr><tr><td>GY13755</td><td>As GY13751 but ΔrpoDΩcat</td><td>This work</td></tr><tr><td>GY13760</td><td>As GY13771 but amyEΩPtufA:lacI-kan</td><td>This work</td></tr><tr><td>GY13767</td><td>GY10973/p13856 (prepUTs::gyrA)</td><td>This work</td></tr><tr><td>GY13771</td><td>R1/p13849 (prepUTs::amyE)</td><td>This work</td></tr><tr><td>GY13779</td><td>As GY13767 but ΔgyrAΩcat</td><td>This work</td></tr><tr><td>GY13781</td><td>GY10973/p13840</td><td>This work</td></tr><tr><td>GY13786</td><td>GY10973/p11554</td><td>This work</td></tr><tr><td>GY13789</td><td>GY10973/p13863 (prepUTs::hbs)</td><td>This work</td></tr><tr><td>GY13792</td><td>Non-homogenotized ΔhbsΩcat</td><td>This work</td></tr><tr><td>GY13795</td><td>As GY13789 but ΔhbsΩcat</td><td>This work</td></tr></tbody></table>

## DNA manipulations

Plasmid DNA was extracted from *E. coli* using the QIAprep spin miniprep kit (Qiagen). Chromosomal DNA of *D. radiodurans* was isolated as previously described (Mennecier et al., 2006). Plasmid DNA of *D. radiodurans* was extracted from exponentially growing cells. Two millilitres of cultures was harvested by centrifugation (12 000 r.p.m. at room temperature). Pellets were resuspended in 300 µl of lysis buffer (glucose 20%, 25 mM Tris HCl pH 8, EDTA 10 mM) and disrupted with a fastprep desintegrator (Savant; Bio101) using 0.1 g of glass beads (500 µm). After centrifugation for 3 min at 12 000 r.p.m., 250 µl of buffer P2 (1% SDS, 200 mM NaOH) was added to the supernatant and mixed, and 350 µl of buffer P3 (Na acetate 3M, pH 5.2) was added and mixed immediately. After centrifugation for 10 min at 12 000 r.p.m., the supernatants were applied to the Qiaprep spin miniprep column and purification was performed as recommended by Qiagen. Amplification of plasmid or genomic DNA by PCR was performed with DyNAzyme EXT DNA polymerase (Finnzyme) or Extensor Hi-Fidelity PCR enzyme Mix (ABgene). Oligonucleotides used are listed in Table S1.

Mutations in the *repU* gene were randomly generated by PCR amplification using p11554 as template and oligonucleotides PS310-PS311. The PCRs contained the proofreading-defective Taq polymerase (Invitrogen) and unbalanced dNTPs pools, either 0.05 mM dGTP, 0.2 mM dTTP, 0.2 mM dCTP and 0.2 mM dATP or 0.05 mM dTTP, 0.2 mM dGTP, 0.2 mM dCTP and 0.2 mM dATP.

## Construction of thermosensitive plasmids

Plasmids are described in Table 3. To construct the thermosensitive plasmids, we first made a derivative of the vector p11554 (shuttle vector that contains a spectinomycin resistance gene) deleted of the *repU* gene. For this purpose, p11554 was cleaved by PpuMI/AclI to remove a fragment containing the *repU* gene and the backbone was circularized using the annealed product of two oligonucleotides (PS308-PS309). The resulting plasmid p11829 can only replicate in *E. coli*. The mutated *repU* gene was generated by PCR mutagenesis using p11554 as template and PS310-PS311 oligonucleotides, and introduced between the PpuMI and AclI sites of plasmid p11829 to reconstitute the original p11554 plasmid with random mutations in the *repU* gene. The ligation mixture was directly used to transform *D. radiodurans* selecting for SpcR transformants at 28°C. A total of 10 000 SpcR colonies were screened by replica plating for defective growth on spectinomycin plates at 37°C. The plasmids of 10 thermosensitive candidates (p11830 to p11839) were purified from the deinococcal cells, introduced into the *E. coli* DH5α host, and the *repU* gene was sequenced. Each plasmid (p11830 to p11839) carried at least one mutation leading to an amino acid substitution in the RepU protein (see Table S2). Plasmid p11830, which harbours three amino acid changes in RepU (C71S, E130V and V290A), was retained for further experiments. To express cloned genes, the IPTG-regulated P<sub>Spac</sub> promoter and a transcription terminator Term116 were introduced in p11830. For this purpose, the P<sub>Spac</sub>-term116 cassette was amplified by PCR from plasmid p11559 (Mennecier et al., 2004), using the primer pair HH70/HH71 and cloned into p11830 between the BamHI and BstZ171 sites or between the BglII and Stul sites to give plasmids p13840 (Fig. S1) and p13841, respectively, which differ in the orientation of the cassette.

To construct plasmid p13844, the *rpoD* gene was amplified by PCR using the primer pair (HH39/HH45) and the product was cloned into plasmid p13840 between the Ndel/XhoI sites. Plasmid p13849 containing *amyE* (DR1472) gene was constructed in a similar way using the primer pairs HH44/HH55. The *gyrA* gene was cloned into plasmid p13841
<table><caption>**Table 3. Plasmids.**</caption><thead><tr><th>Plasmids</th><th>Description</th><th>Reference</th></tr></thead><tbody><tr><td>pGTC101</td><td>Source of chloramphenicol cassette in <i>D. radiodurans</i></td><td>(Earl et al., 2002)</td></tr><tr><td>p11086</td><td>Source of kanamycin cassette in <i>D. radiodurans</i></td><td>Laboratory stock</td></tr><tr><td>p11615</td><td>Source of tetracyclin cassette in <i>D. radiodurans</i></td><td>Laboratory stock</td></tr><tr><td>p11554</td><td>Shuttle vector <i>E. coli</i>/D. radiodurans, Spc<sup>R</sup></td><td>Laboratory stock</td></tr><tr><td>p11559</td><td>Source of P<sub>Spac-term116</sub> cassette</td><td>Laboratory stock</td></tr><tr><td>p11829</td><td>Non-replicative plasmid in <i>D. radiodurans</i> (Δ<sub>repU</sub>), Spc<sup>R</sup></td><td>This work</td></tr><tr><td>p11830 to p11839</td><td>Vector thermosensitive for replication in <i>D. radiodurans</i>, Spc<sup>R</sup>, prepUTs</td><td>This work</td></tr><tr><td>p12723</td><td>Source of <i>spa-tag</i> chloramphenicol cassette</td><td>This work</td></tr><tr><td>p13840</td><td>p11830 P<sub>Spac-term 116</sub><sup>a</sup></td><td>This work</td></tr><tr><td>p13841</td><td>p11830 P<sub>Spac-term 116</sub><sup>a</sup></td><td>This work</td></tr><tr><td>p13844</td><td>p13840: prepUTs::rpoD</td><td>This work</td></tr><tr><td>p13849</td><td>p13840: prepUTs::amyE</td><td>This work</td></tr><tr><td>p13856</td><td>p13841: prepUTs::gyrA</td><td>This work</td></tr><tr><td>p13862</td><td>p13841: prepUTs::hbs</td><td>This work</td></tr></tbody></table>

a. The two plasmids p13840 and p13841 differ by the orientation of the P<sub>Spac-term116</sub> cassette.

between the Ndel/Xhol sites in a similar way using the primers HH32/HH33 to obtain plasmid p13856. The *hbs* gene was amplified by PCR using the oligonucleotides *huNdel/ huDral* and cloned into plasmid p13841 between the Ndel/ Dral sites to yield plasmid p13863. All constructions were verified by DNA sequencing.

### Deletion of genes in *D. radiodurans* and construction of strain GY13320 (*hbs::spaΩcat*)

The *dps1*, *dps2*, *rpoD*, *hbs*, *gyrA* disruption mutants were constructed by the tripartite ligation method (Mennecier *et al.*, 2004). The *hbs* gene was fused with a *spa* tag (sequential peptide affinity) using a fragment containing a cassette with the *spa* and the gene conferring resistance to chloramphenicol (Zeghouf *et al.*, 2004) by the tripartite ligation method. The mutated alleles constructed *in vitro* were then used to transform *D. radiodurans* to replace their wild-type counterpart by homologous recombination. The genetic structure and the purity of the mutants were checked by PCR using primers described in Table S1.

### Treatment of *D. radiodurans* with gamma irradiation

Exponential or stationary phase cultures, grown in TGY2X (supplemented with spectinomycin when necessary), were concentrated to an A<sub>650</sub> = 30 in TGY2X and irradiated on ice with a <sup>137</sup>Cs irradiation system (Institut Curie, Orsay, France) at a dose rate of 44.7 Gy min<sup>-1</sup>. Following irradiation, diluted samples were plated on TGY plates. Colonies were counted after 3–4 days incubations at 30°C.

### Assay of plasmid thermosensitivity

Plasmid containing bacteria growing exponentially at 28°C in TGY2X 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 selective and non-selective TGY agar and incubated 3–4 days at 28°C.

The frequency of the plasmid-containing bacteria was measured as the percentage of antibiotic resistant colonies over the total viable count.

### Assay of genes essentiality

The essentiality of genes was evaluated in a growth experiment, in which the strains grown at 28°C in liquid medium with spectinomycin were serially diluted, plated on TGY agar and incubated at 28°C or 37°C in the presence or the absence of spectinomycin.

### Fluorescence microscopy

Culture aliquots (1 ml) were removed and the corresponding cells were fixed using toluene at 3% final concentration. Cell membranes were stained with N-(3-triethylammonium-umpropyl)-4-(6-(4-(diethylamino)phenyl)hexatrienyl) pyridinium dibromide (FM 4–64) at 0.01 µg µl<sup>-1</sup>, and the nucleoid with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) at 2 µg ml<sup>-1</sup>. FM 4–64 stains the lipid membranes with red fluorescence (excitation/emission ~515/640 nm) and DAPI stains the nucleoid with blue fluorescence (excitation/emission ~350/470 nm). The stained cells were observed using a Leica DM RXA microscope. Images were captured with a CDD camera 5 MHz Micromax 1300Y (Roper Instruments). The final reconstructed images were obtained by deconvoluting Z-series with the Metamorph software (Universal Imaging). Before fluorescence microscopy, for depletion of HU, the cells were grown overnight at 28°C in medium supplemented with spectinomycin. The cells were then harvested by centrifugation, washed, resuspended, diluted in antibiotic-free medium 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::spaΩcat*) and wild-type strains were grown exponentially in TGY2X (OD<sub>650</sub>:0.25).
Aliquots of 0.4 ml of the exponential phase culture were taken and fixed by addition of 1/10 volume of 37% paraformaldehyde in the culture medium and incubation for 2 h at 4°C. The cell pellet was subsequently washed once in 1× PBS. In order to permeabilize the cell envelope, the cells were treated with 2 mg ml⁻¹ lysozyme for 30 min at 37°C followed by incubation with 0.1% Triton X-100 in PBS for 5 min at room temperature. Finally, the cells were washed in PBS and resuspended in 25 µl of PBS. A 3 µl aliquot was applied to a poly L-lysine pretreated slide spot, allowed to air dry and fixed by incubating in 4% PFA for 20 min at 37°C. Cells were then blocked in 2% BSA in PBS-T (0.05% Tween 20 in PBS) and incubated for 2 h at 37°C with a monoclonal mouse anti-Flag antibody (Sigma-Aldrich) diluted 1/700 in blocking solution. After 20 min washing in PBS-T, the cells were incubated for 1 h at 37°C with a FITC conjugated goat anti-mouse antibody (Jackson Immunoresearch Laboratories) diluted 1/250 in blocking solution and washed for 20 min in PBS-T. Cells were finally stained with DAPI 10 µg ml⁻¹ for 10 min at room temperature. After a final wash in PBS-T slides were mounted using fluoromount G as a mounting medium (Fluoprobes). Image acquisition and treatment were performed as described above.

## Acknowledgements

We thank Adriana Bailone for valuable discussions and critical reading of the manuscript. We thank Esma Bentchikou for her expert assistance in microscopy, M. DuBow for help with English. We thank the Institut Curie for the use of the ¹³⁷Cs irradiation system, V. Favaudon for help with gamma-irradiation. The authors are grateful to M. Prigent for technical help and advice concerning immunofluorescent labelling and picture acquisition. This work was supported by the Centre National de la Recherche Scientifique, the University Paris-Sud 11, the Commissariat à l'Energie Atomique (CEA LRC42V), Electricité de France (RB2007-11), and the Agence Nationale de la Recherche (ANR-07-BLAN-0106). This work was carried out in compliance with the current laws governing genetic experimentation in France.

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