JOURNAL OF BACTERIOLOGY, Mar. 2010, p. 1710–1718 Vol. 192, No. 60021-9193/10/$12.00 doi:10.1128/JB.01427-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

The Sinorhizobium meliloti RNA Chaperone HfqMediates Symbiosis of S. meliloti and Alfalfa�†

Lise Barra-Bily,1,2‡ Shree P. Pandey,1‡ Annie Trautwetter,2Carlos Blanco,2 and Graham C. Walker1*

Department of Biology, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139-4307,1 andOsmoregulation Chez les Bacteries, CNRS UMR 6026, Universite de Rennes I, Campus de Beaulieu,

Av. du General Leclerc, 35042 Rennes, France2

Received 30 October 2009/Accepted 7 January 2010

There exist commonalities between symbiotic Sinorhizobium meliloti and pathogenic Brucella bacteria in terms ofextensive gene synteny and the requirements for intracellular survival in their respective hosts. The RNA chaperoneHfq is essential for virulence for several bacterial groups, including Brucella; however, its role in S. meliloti has notbeen investigated. Our studies of an S. meliloti loss-of-function hfq mutant have revealed that Hfq plays a key rolein the establishment of the symbiosis between S. meliloti and its host Medicago sativa. S. meliloti Hfq is involved incontrolling the population density under a free-living state and affects the growth parameters and nodulation. Anhfq mutant poorly colonizes the infection threads that are necessary for the bacteria to invade the developing nodule.An hfq mutant is severely impaired in its ability to invade plant cells within the nodule, which leads to the formationof small, ineffective nodules unable to fix nitrogen. In culture, the hfq mutant did not accumulate transcripts of nifA,which encodes a key regulator necessary for nitrogen fixation. Hfq may be involved in regulation of several proteinsrelevant to hfq mutant phenotypes. The crucial role of Hfq in symbiosis suggests that small regulatory RNAs areimportant for its interactions with its plant host.

The alpha subgroup of proteobacteria includes memberssuch as Sinorhizobium meliloti, which establishes a symbiosiswith certain leguminous plants, as well as members, such asBrucella abortus, that are highly pathogenic to animals andhumans. Despite the very different outcomes of the chronicintracellular infections established by S. meliloti and B. abortusin their respective hosts, parallels can be drawn because in bothcases the bacteria need to survive within acidic membranecompartments for a prolonged time after endocytosis (50).Comparisons of genomic sequences between brucellae andrhizobia have revealed similarities. For example, extensivegene synteny exists between chromosome I of Brucella and thegenome of Mesorhizobium loti, while chromosome II sharesregions of gene synteny with the pSym megaplasmids of S.meliloti. In addition, the transport and metabolic capabilities ofBrucella have similarities to those of the plant symbiont (46).Genomic comparisons suggest that these two types of bacteriashare a complex evolutionary history and that Brucella evolvedfrom soil/plant-associated ancestral bacteria of the Rhizobium/Agrobacterium group (46).

The parallel between these two bacterial groups is well il-lustrated by the inner membrane protein BacA, which is crit-ical for the maintenance of the chronic intracellular infectionsthat underlie these two very diverse host-bacterial relation-ships (20, 35). S. meliloti bacA mutants lyse soon after being

endocytosed into the plant cytoplasm (20), while B. abortusbacA mutants are defective in intracellular replication in mac-rophages and are unable to establish a chronic infection inBALB/c mice (35). BacA is required for the transport of cer-tain peptides into S. meliloti (38). Furthermore, for both S.meliloti and B. abortus, loss of BacA results in a ca. 50%reduction in the very-long-chain fatty acid (27-OHC28:0 and29-OHC30:0) content of both Sinorhizobium and Brucella lipidA (15) and in increased resistance toward the glycopeptidebleomycin (20, 35). Thus, in the context of the S. meliloti-legume symbiosis, BacA is required for transition from thefree-living form of bacteria into the nonreplicating, nitrogen-fixing bacteroid form (20, 27), while in the context of B. abor-tus-host pathogenesis, BacA acts as a pathogenesis factor re-quired for the long-term survival and residence of B. abortus inits intracellular niche (35, 50).

Another factor besides BacA that is required for B. abortusto establish a chronic infection is the RNA-binding Hfq protein(48, 50). Hfq also strongly contributes to stress resistance instationary phase. A B. abortus hfq mutant shows increasedsensitivity to H2O2, shows decreased survival under acidic con-ditions, fails to replicate in cultured murine macrophages, andis rapidly cleared from the spleens and livers of experimentallyinfected BALB/c mice (48). Thus, similarly to bacA mutants, B.abortus hfq mutants are not able to establish a chronic intra-cellular infection of BALB/c mice. The role of the homologoushfq gene in S. meliloti and its contribution to symbiosis haveremained unexplored.

Hfq (HF-I), first discovered as a host factor required for Q�phage replication in Escherichia coli, is an RNA-binding pro-tein involved in regulation of RNA metabolism and is con-served in about half of all sequenced bacterial genomes (63,66). Hfq is a member of the Sm and Sm-like family of proteins

* Corresponding author. Mailing address: Department of Biology,68-633, Massachusetts Institute of Technology, 77 Massachusetts Ave.,Cambridge, MA 02139. Phone: (617) 253-6716. Fax: (617) 253-2643.E-mail: gwalker@mit.edu.

† Supplemental material for this article may be found at http://jb.asm.org/.

‡ Equal contribution.� Published ahead of print on 14 January 2010.

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found in all three domains of life (45) and forms a homo-hexameric structure that binds preferentially to A/U-richRNAs (reviewed in references 6 and 67). Hfq serves as post-transcriptional regulator that binds small RNAs (sRNA) andmRNAs and facilitates their interaction (1, 22, 37, 60). Hfqmay promote duplex formation by increasing the local concen-tration of the sRNA and its target (7), changing the RNAstructure (18, 49), and accelerating strand exchange and sub-sequent annealing (2). These RNA-RNA interactions mediatemRNA turnover and/or translation (1, 23, 60).

E. coli hfq null mutants have a pleiotropic phenotype thatincludes decreased growth rate, increased cell length, and sen-sitivity to UV light (64). Since Hfq is essential for efficienttranslation of RpoS in E. coli (44) and Salmonella (3, 8, 57),some of the phenotypic properties of an hfq mutant are due toRpoS deficiency. Hfq protein also exerts effects in an RpoS-independent manner and even can bind to the poly(A) tails ofsome mRNAs, stimulating poly(A) adenylation (26, 33, 43).Salmonella hfq mutants had reduced growth, had attenuatedinfection, and showed severe defects in invasion of epithelialcells (58). Interestingly, these phenotypes in Salmonella arelargely RpoS independent (58). An RpoS homolog has notbeen identified in the alphaproteobacteria, the phylogeneticgroup to which Brucella and S. meliloti belong.

Hfq has been shown to influence the virulence of a numberof other pathogenic bacteria (e.g., references 12, 14, 32, 40, 55,and 59). The parallels between Brucella pathogenesis and S.meliloti symbiosis motivated us to construct and characterizean S. meliloti loss-of-function hfq mutant and to explore itsinvolvement in the symbiosis of S. meliloti with its host, Medi-cago sativa. Our results show that Hfq plays several importantroles in the establishment of the S. meliloti-alfalfa symbiosis aswell in aspects of the physiology of the free-living rhizobia.

MATERIALS AND METHODS

Media. Luria-Bertani (LB) was prepared as previously described (51). S.meliloti was also grown in minimal galactose aspartate salts (GAS) medium aspreviously described (24, 47). Bacto agar (Gibco) was used at 1.5% for solidmedium. Gentamicin (50 �g/ml for S. meliloti and 5 �g/ml for E. coli), kanamycin(25 �g/ml), neomycin (200 �g/ml), streptomycin (500 �g/ml), and tetracycline(10 �g/ml) were added when needed.

Nodulation assay. Nodulation of alfalfa (Medicago sativa cv. Iroquois) by S.meliloti on whole plant and nodule sections was analyzed. The alfalfa seeds weresurface sterilized as previously described (34) and germinated on 0.8% agar inwater in the dark for 62 h. One seedling was placed on top of a petri dish platefilled with 25 ml of Jensen’s agar (34). The seedlings were inoculated by 1 ml ofS. meliloti cells grown to saturation in LB, washed in sterile distilled water, andresuspended to an optical density at 600 nm (OD600) of 0.05. The plates weresealed using parafilm, wrapped in aluminum foil to maintain the root in the dark,and incubated at 25°C for a maximum of 4 weeks. Microscopy was performed byusing standard methods.

Molecular manipulation. A list of strains and plasmids used in this study aregiven in Table S1 in the supplemental material. Cloning, subcloning, and isola-tion of plasmid or genomic DNAs were performed using previously describedprocedures (51) or using kits provided by Qiagen, Inc. (Valencia, CA). Site-directed mutagenesis was performed using a QuikChange kit (Stratagene, CedarCreek, TX) and confirmed by sequencing.

Construction of an S. meliloti Rm1021 Hfq mutant. Two distinct PCRs werecarried out in order to amplify the sequences overlapping the hfq 5� and 3� ends.To make pblC60, the upstream region of hfq (including a portion of its openreading frame) was amplified from S. meliloti Rm1021 with the primers hfqL3(5�-ATAATCATGAGAATTCCTATGTGCGCCCAG-3�) and hfqin10 (5�-TAATAAGCTTGGCATGATCCTCGAGATGGCGTG-3�). The 1,306-bp PCRproduct was cloned into the pGEM-T vector (Promega, La Jolla, CA). Thedownstream region was amplified using the primers blhfqDsfwd (5�-CGGGAT

GTGAGGAACATT-3�) and blhfqR2 (5�-GAATCCATCTAGACCAAGTCAGTGG-3�). The 1,567-bp PCR product was cloned into the pGEM-T vector, givingpblB58.

The hfq::lacZ transcriptional fusion was obtained by inserting a lacZ-aacC1cassette obtained from the pAB2001 plasmid (4). This cassette was recovered bya HindIII/XhoI double digest of pAB2001 and inserted between the same sitesof pC60, giving pC67. Then, the hfq::lacZ-bearing fragment was recoveredthrough an NcoI digest and introduced in the corresponding site of pB58. Finally,the disrupted hfq gene and surrounding regions were cloned into the BamHI siteof pKmob18sacB (53), a suicide vector for S. meliloti.

Transconjugants were selected as described by Schafer et al. (53). Amongthem, only 43% had lost the neomycin resistance carried by pKmob18sacB,which reflects a double crossing over. One of the mutants was chosen for furtheranalysis, and the sequences surrounding the hfq gene were sequenced (data notshown) in order to ensure that none of the genes downstream or upstream wereaffected by a point mutation. The absence of a wild-type (WT) copy of hfq waschecked by PCR and Southern blot analysis (data not shown). The mutation wasthen purified by transduction into the parental strain Rm1021. One of thetransductants was named Smbl�500.

Disruption of the hflX open reading frame in strain Rm1021. For disruption,a 524-bp internal fragment of the hflX open reading frame was amplified usingRm1021 genomic DNA and the oligonucleotides blhflX1 (5�-AGCTTCACCGGTCGAAGC-3�) and blhflX2 (5�-GACTGAGAAGATCGTCGACACC-3�). ThePCR amplicon was cloned into the pGEM-T vector. Subsequently, the hflX-carrying HincII-NcoI fragment was transferred between the ScaI and NcoI sitesof the pSUP102b vector (56), giving pblC51. pblC51 was transferred by triparen-tal conjugation into S. meliloti Rm1021 with selection for tetracycline resistance.One of the mutants was used as a donor strain for transduction into the Rm1021strain. The strain was named Smbl�600. The insertion was verified by PCR.

Complementing plasmids. To make the complementing plasmid, pbl100, afragment of 703 bp corresponding to the hfq gene and surrounding regions, wasamplified from S. meliloti Rm1021 with the primers Rmhfq4 (5�-ATCATGCCGTGAACG-3�) and Rmhfq6 (5�-CAGCATCTGCTTTCC-3�). The fragment wascloned into the pGEM-T vector. The open reading frame of hfq under thecontrol of its own promoter, defined using primer extension (data not shown),was cloned between PstI and SalI of pBBR1MCS-3 (31), creating the pbl100plasmid. This plasmid was transferred into S. meliloti by using triparental mat-ting. pbl100 was introduced into the strains Rm1021 and Smbl�500, creating thestrains Smbl003 and Smbl503, respectively. In parallel, the strains Smbl001 andSmbl501 were obtained by conjugation of the plasmid pBBR1MCS-3 (31) intothe strains Rm1021 and Smbl�500, respectively.

A derivative of pBBR1MCS-3 was constructed by deletion of 800 bp from thelacZ gene between the sites SspI and BamHI and then ligation of the vector onitself. The resulting plasmid was named pbl101 and introduced into the hfqmutant (Smbl�500) in order to follow the lacZ activity, in vivo, without thebackground due to the expression of lacZ from the plasmid. The strain wasnamed Smbl�502.

RNA analysis. Cultures were set up in 50-ml sterile Falcon tubes for mi-croaerobic experiments. Total RNA was isolated by TRI reagent (T 9424; Sigma,St. Louis, MO). In order to identify expression of the gene induced undermicroaerobiosis, different sets of primers were used as follows. An internalregion of nifA was amplified using the primers Sm-nifA1 (5�-CAGGAAGGTGAATTTGAGC-3�) and Sm-nifA2 (5�-CAGCGCACTGATCAACC-3�). 16SRNA was used as a control, for which the primer combination of Sm-16SL1(5�-CAATCTAGAGTCCAGAAGAGGT-3�) and Sm-16SR1 (5�-AACATCTCACGACACGAGC-3�) was used.

Phenotypic characterization of the S. meliloti hfq mutant. Growth character-istics were evaluated by analysis of growth curves. S. meliloti was grown on LBsupplemented with antibiotics when appropriate. Cells were harvested at variousdensities and resuspended in the appropriate medium (LB or GAS) to an OD600

of 1.0. Then, the bacteria were inoculated into the appropriate medium at aninitial OD600 of 0.1. Cell growth was determined by monitoring OD600.

In order to assay the effect of hfq mutation on swarming behavior, liquidcultures of S. meliloti, initiated from glycerol stocks, were grown at 30°C in LBbroth with shaking to late logarithmic phase. After incubation, cells were pelletedand washed twice in 0.85% NaCl resuspended in saline medium to obtain a cellconcentration of 1010 cells/ml. Five-microliter drops of this suspension weredeposited on the surface of air-dried (10 min) plates containing the appropriatebroth with 0.3% agar and allowed to dry for 10 min. The plates were thenincubated for 6 days at 30°C and scored for swarming by measuring the diameterof the spreading.

�-Galactosidase assays. hfq transcriptional expression was assayed by moni-toring the �-galactosidase activity expressed from a chromosomal hfq::lacZ fu-

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sion. The enzymatic activity was measured as described by Miller (42) andexpressed in Miller units.

�-Galactosidase assays in planta. �-Galactosidase activity in vivo on nodulesharvested from plants after 2, 3, or 4 weeks of culture was assayed. Nodules weretreated as described by Boivin et al. (5). The wild-type strain was used as acontrol for the efficiency of the fixation step, which inhibits the �-galactosidaseactivity of the plant. For infection thread visualization, the fixation step neverexceeded 2 h.

Microaerobiosis. Cultures were set up in 50-ml sterile Falcon tubes. A pipettediameter hole was made, under sterile conditions, into the Falcon tube lid. Asterile pipette topped with a filter (Millipore) was inserted into the lid hole. Thefilter was connected to the gas tank by using a tube. The gas tank mixture wascomposed of N2 (99.5%)-O2 (0.5%), which was bubbled into 15 to 20 ml ofmedium containing or not containing a nitrogen source.

RESULTS

Identification of the S. meliloti hfq gene. A search of the S.meliloti genome with the B. abortus Hfq sequence revealed thatthe S. meliloti gene annotated as nrfA encodes a protein 91.9%identical to the B. abortus Hfq protein (see Fig. S1 in thesupplemental material). The S. meliloti Hfq/NrfA homolog has74% similarity to NrfA (Nif regulatory factor) of Azorhizobiumcaulinodans (see Fig. S1 in the supplemental material) (30).

Construction and growth characteristics of the S. meliloti hfqmutant. In order to investigate the physiological roles of hfqand to follow its expression both in the free-living stage and inplanta, we disrupted the hfq gene by a partial gene deletionthat also created a transcriptional fusion to a promoterlesslacZ gene derived from the vector pAB2001 (4). The result-ing hfq::lacZ mutation was crossed into the Rm1021 genomeby a double-recombination event enabled by the use of thepKmob18sacB vector (53). One clone, designated Smbl499,was randomly chosen from the transformants for furtheranalysis. PCR and Southern blotting were performed toconfirm the partial deletion (of 60 nucleotides [nt]) of hfqand insertion of the lacZ-Gm cassette derived from thepAB2001 vector (data not shown). The hfq mutation wastransduced into the parental strain Rm1021 to ensure theabsence of unlinked mutations, and the resulting strain wasnamed Smbl�500.

It was immediately evident that the loss of Hfq functionaffected the growth of S. meliloti, as the hfq mutant was delayed1 to 2 days in forming colonies on LB agar in comparison to thehfq� parental strain Rm1021. When grown in LB, the hfqmutant (Smbl�500) exhibited a doubling time of 5 h, comparedto 4 h for the hfq� parent. The maximum cell density obtainedwith the hfq mutant was �60% relative to the level for the WTwhen grown in LB (Fig. 1B). A similar effect was observed inminimal medium.

hfq transcription is growth phase regulated. Smbl�500 car-rying the transcriptional fusion of hfq with the promoterlesslacZ gene was grown in minimal medium, and samples wereharvested at different time points and assayed for �-galactosi-dase activity (42). Hfq was expressed during all the stages ofthe growth (Fig. 1C). As a control for the background levels of�-galactosidase activity, the wild-type strain was grown simul-taneously and under the same conditions. Its background levelnever exceeded 30 Miller units (data not shown). Relative tothe levels seen in exponential phase, we observed a two- tothreefold increase in �-galactosidase activity that began as thecells entered stationary phase (Fig. 1C). The same pattern of

induction was observed in strain Smbl503 (complemented hfqmutant) (data not shown). Further experiments will be requiredto elucidate whether S. meliloti Hfq auto-controls its own expres-sion at a translational level, as described for E. coli (65). Thegreater expression level of hfq during stationary phase suggeststhat Hfq plays a role in the stationary phase adaptation and mayaccount, at least in part, for the lower cell density reached by themutant than by the parental strain. It has been suggested that,during symbiosis, S. meliloti is in a physiological state closer tostationary phase than to exponential phase (28), so it seems likelythat an hfq mutation might have a substantial impact on its abilityto interact with its host alfalfa.

Hfq is required to establish the symbiosis between S. melilotiand alfalfa. To investigate the role of Hfq in symbiosis, 2-day-old alfalfa seedlings were inoculated with the hfq mutant(Smbl�500) or the WT strain (Rm1021). After 4 weeks ofincubation, plants inoculated with the hfq mutant reached only

FIG. 1. Characterization of hfq in S. meliloti. (A) Organization ofthe hfq locus on chromosome of S. meliloti. (B) Growth of wild-type(circles) and hfq mutant (squares) strains in LB (closed symbols) orGAS (open symbols) medium. (C) Transcriptional activity of the hfqgene. Results are shown for a time course experiment performedfollowing the growth of the mutant by measuring the optical density ofthe culture at different times of the growth (open circles; expressed inOD600). Simultaneously, samples were used to assay for �-galactosi-dase activity resulting from the expression of the hfq::lacZ fusion.Results are expressed in Miller units, function of time, and OD600 ofsample (filled squares). The error bars correspond to the variationobtained between 4 repeats of independent dilution of the sample.

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2.2 � 0.8 cm (Fig. 2A). The yellow-to-pale-green color of theleaves indicates the lack of nitrogen fixation. In contrast, theplants inoculated with the hfq� parent strain Rm1021 reacheda height of 8.1 � 2 cm and exhibited dark green leaves (Fig.2A). The analysis of the surrounding region of hfq in thegenome of S. meliloti Rm1021 (16) revealed that, as in B.abortus (46), the gene immediately downstream of S. melilotihfq is hflX, which encodes a putative GTP-binding protein (Fig.1A). To test whether the impaired growth of plants was dueonly to a lack of Hfq and not to a polar effect on hflX, weperformed a complementation experiment. A 700-bp DNAfragment carrying hfq� under the control of its own promoterwas cloned into the plasmid pBBR1-MCS3, creating pbl100.This plasmid was then introduced by triparental mating intostrain Rm1021 and the hfq mutant strain Smbl�500. Seedlingswere then inoculated with the strains carrying the hfq� plasmidpbl100 and/or the control vector pbl101. At 4 weeks postin-oculation, the plants inoculated with the hfq� (complemented)strain reached a height of 7 � 1.4 cm (Fig. 2A), showing nosignificant differences from the wild-type strain carrying thesame plasmid. To confirm directly that the downstream hflXgene was not required for the symbiosis, an hflX mutant,Smbl�600, was constructed. This mutation had no impact onthe symbiosis (data not shown), showing that the function ofhflX is not essential for establishment of an effective symbiosis.Therefore, the symbiosis defect of the hfq mutant can be at-tributed to the mutation of hfq.

Another indicator of normal symbiosis is the presence ofleghemoglobin in the elongated nodules, which makes the nod-ule pink. Whereas the plants inoculated with the hfq� parentexhibited more than 95% elongated pink nodules (Fig. 2B),more than 50 plants inoculated with the hfq mutant in distinctexperiments exhibited fewer than 10% pink nodules, very fewof which were elongated (Fig. 2B). Representative pink nod-ules were crushed and the bacteria recovered and reinoculated,but we did not observe more pink nodules on plants inoculatedwith these recovered hfq mutants than on plants inoculatedwith the parental hfq mutant.

hfq missense mutants that altered residues in the Sm1 do-

main that is highly conserved in bacterial Hfq proteins wereconstructed (see Fig. S1 in the supplemental material). Plas-mids carrying mutations altering Gly35 (pbl100-G35V), Ser39(pbl100-S39T), or Phe40 (pbl100-F40W) failed to complementthe hfq mutant phenotype. No significant differences were ob-served between the plants inoculated with the hfq mutant andthose inoculated with the derivatives carrying the mutant plas-mids (data not shown). These residues form the part of �2strand in the Hfq structure (36, 54). The �2 and �4 strandscomprise the “A site,” responsible for the specificity of Hfq foradenosine nucleotides of the poly(A) RNA (36). This Glyresidue, present in most of the described Hfq proteins andlocated in the middle of �2, is critical for maintenance of thehighly distorted Sm fold (54). The Gly35 residue has beensuggested to be involved in binding with ATP (61). The failureof the G35V mutant to complement and the high degree ofconservation of Gly35 indicate that this residue is importantfor Hfq function. The Phe40 residue, which corresponds to thePhe39 residue of E. coli Hfq, has been shown to be involved inthe affinity of Hfq toward the DsrA sRNA (62). If such smallRNAs are involved in the symbiosis, it would not be surprisingthat this mutation alters the ability of hfq to complement thehfq null mutation.

hfq is expressed in planta and is required for efficient inva-sion through infection threads. Expression of hfq in planta wasassessed using the transcriptional fusion hfq::lacZ in the Smbl502strain, lacking Hfq and carrying the control plasmid pbl101, aswell as in the complemented strain Smbl503. �-Galactosidaseexpression was visualized, as previously described (5), in sectionsor in whole nodules harvested at 3 to 4 weeks postinoculation.�-Galactosidase activity, which correlates with hfq expression, wasobserved at different stages of the infection process. Three stagesof the infection process include colonization of the curled roothair, invasion though infection threads, and finally release into thecytoplasm of plant cells in the infection zone of the nodule. Sig-nificant differences in the pattern of hfq expression were observedbetween the mutant and the complemented strains; plants inoc-ulated with the hfq mutant (Smbl502) exhibited a high frequencyof infection thread abortion.

After 4 weeks of incubation, the majority of the nodules inplants inoculated with the hfq mutant (Smbl502) failed to showany substantial staining of plant cells in the interior of thenodule (59.7% � 10.4%), except at the surfaces of the nodulesin the remnants of colonized curled root hairs and aborted roothairs (Fig. 3A). Furthermore, only 1.7% � 1.5% of the noduleselicited by the hfq mutant were fully colonized (Fig. 3B1, leftnodule). The remaining nodules (38.6%) exhibited staining ofonly a few invaded cells in the interior of the nodule (Fig. 3B1,right nodule). Even if some cells were invaded by the hfqmutant, these nodules did not elongate as they normally dowhen inoculated with a WT strain; the nodules did not differ-entiate, and leghemoglobin seemed to be lacking, as they ap-peared white. They did not enable nitrogen fixation, as theplant remained small and chlorotic. In contrast, for the plantsinoculated with complemented strain (Smbl502), only 13.6% �5.7% of the nodules failed to exhibit substantial staining and72% � 7% of the nodules inoculated with the complementedstrain exhibited colonization of the whole nodule (Fig. 3B2).These results further indicate that Hfq is expressed in plantaand is required for an efficient colonization of the infection

FIG. 2. Evidence of the symbiosis defects of an hfq mutant.(A) Live alfalfa plants, 4 weeks after inoculation with the hfq mutant(panel 1), the hfq mutant carrying pbl101 (panel 2), the complementedhfq mutant (panel 3), and the WT strain (panel 4). (B) Enlargedimages of nodules elicited by a plant inoculated with Rm1021, the hfq�

parent (panel 1) and the hfq mutant (panel 2).

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threads and invasion into the developing nodule. The highfrequency of infection thread abortion seen with the hfq mu-tant evidently reduces the frequency with which the bacteriaare able to invade plant cells in the infection zone of thenodule.

The severe deficiency of the hfq mutant in invading noduleswas also observed in a competition experiment. When plantswere inoculated with a mixture of the hfq mutant (Smbl�500)and its hfq� parent (Rm1021) in a ratio of 1,000:1, no hfqmutant bacteria could be recovered from the nodules.

Hfq is required for efficient release and differentiation of thebacteria inside the plant cell. To gain further insights into the

nature of the symbiotic deficiency of the hfq mutant, ultrastruc-tural analysis was performed on 4-week-old nodules by usingelectron microscopy. Nodules from plants inoculated with thewild-type strain showed the presence of bacteroids in most ofthe cells (Fig. 4A). In contrast, for plants inoculated with thehfq mutant, most of the plant cells within white nodules lackedbacteria or bacteroids and instead were filled with the starchgranules that accumulate prior to the uptake of the bacteria.At a much lower frequency than in the wild type, we also sawinfected plant cells that contained some bacteria or bacteroid-like cells, as shown in a representative section (Fig. 4B); wealso noted occasional dividing cells (Fig. 4C). These observa-tions are consistent with our other results indicating that thehfq mutant is much less efficient than wild type at invadingplant cells and establishing the chronic intracellular infectionthat underlies the symbiosis.

Hfq is required for efficient expression of nifA and nfeB. Thehfq mutant (Smbl�500) has a Fix� phenotype since the plantsinoculated were clearly deficient in fixing nitrogen. The prox-imal cause of the Fix� phenotype results from the combinationof the deficiency of the hfq mutant in invading the root hairsand its apparent inability to differentiate into bacteroids afterendocytosis. However, we were interested in whether the roleof Hfq in symbiosis might extend to regulation of nitrogenfixation as well. In an attempt to address this issue, we inves-tigated whether loss of hfq function might be affecting nifAexpression. The nifA gene is an activator of nitrogen fixation inplanta, so that loss of nifA expression results in the formationof small, white nodules (21). In the free-living state, the S.meliloti nifA gene can be induced under microaerobic condi-tions in nitrogen-free medium (9). After the parental strainRm1021 and the hfq mutant (Smbl�500) were grown in GASminimal medium or nitrogen-free GS minimal medium undermicroaerobic conditions, total RNAs were extracted and sub-jected to reverse transcriptase PCR (RT-PCR). nifA expres-sion was observed only in the wild-type strain after a 4-hincubation in nitrogen-free medium and microaerobiosis. Incontrast, no nifA amplification product was observed in the hfqmutant (Fig. 5), suggesting that a deficiency in nifA expressionin an hfq mutant may also contribute to the Fix� phenotype.

NifA controls the expression of nfeB (nodule formation ef-ficiency), which, in S. meliloti, is expressed in nitrogen fixationzone III (17). nfeB expression in the hfq mutant was therefore

FIG. 3. Defects of an hfq mutant during infection and nodulation.(A) Optical microscopy images showing a nodule colonized by an hfqmutant strain expressing �-galactosidase (panel 1). Pictures focusingon curled root hair colonized by the hfq mutant revealed by their�-galactosidase activity (panel 2) and abortive infection threads (pan-els 3 and 4). (B) Optic microscopy on hand sections of nodules inoc-ulated with the hfq mutant (panel 1; Smbl502) and its complementedderivative (panel 2; Smbl503). The B1 panel, left nodule, representsrare nodules elicited after inoculation by the hfq mutant, which arecolonized but fail to elongate; panel 1, the right nodule represents themajority of nodules observed after inoculation by the hfq mutant.�-Galactosidase activity expressed by the hfq::lacZ fusion was revealedand corresponds to the blue staining.

FIG. 4. Ultrastructural evidence of hfq mutant invasion deficiency and the lack of differentiation observed using electron microscopy. (A) WTcells differentiated into bacteroids (scale bar, 1 �m). (B) hfq mutant (Smbl�500)-inoculated plants exhibit nodules with cells invaded (I) or notinvaded (NI) by bacteria (scale bar, 10 �m). Noninvaded cells contain large granules of starch (S). Panel C shows a dividing cell failing todifferentiate into a bacteroid (designated by an arrow) (scale bar, 1.5 �m).

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analyzed. Plasmids carrying nfeB translational fusions withuidA (17) were transferred in the Rm1021 and hfq strains(Smbl�500) by triparental mating. The resulting strains wereinoculated on 2-day-old alfalfa seedlings and incubated. At 4weeks postinoculation, the nodules were stained for �-glucu-ronidase activity. In the wild-type background, nfeB is specifi-cally expressed in the nitrogen fixation zone (Fig. 6A). Incontrast to what was found for Rm1021, in the hfq mutant(Smbl�500), nfeB was not expressed in most nodules, while inrare nodules, aberrant nfe expression was observed (Fig. 6B).

Mutating Hfq reduces swarming efficiency without affectingsuccinoglycan synthesis. We evaluated the physiological con-sequences of loss of hfq function on two processes associatedwith bacterial adaptation to extra- and intracellular niches,succinoglycan production, and swarming efficiency. Efficientsynthesis of succinoglycan is required for the initiation andextension of the infection threads from the colonized curledroot hairs (10, 29, 34). To test whether loss of Hfq functionaffects succinoglycan biosynthesis, the hfq mutant and its wild-type parent were plated on LB plates containing Calcofluor,which allows this exopolysaccharide to be visualized under UVlight. No significant differences were observed between thedifferent strains (data not shown), an observation suggestingthat the role of Hfq in infection thread invasion is due to itsregulation of a process or processes other than succinoglycanbiosynthesis. We also noted that the hfq mutants induced thecurling of root hairs as efficiently as the wild-type strain. Since,both root hair curling and nodule formation require Nod fac-tors, our observations suggest that loss of hfq function haslittle, if any, effect on Nod factor synthesis.

To assay its ability to swarm, the hfq mutant and its wild-typeparent were spotted at the surface of a swarming plate. Thespreading was measured (Fig. 7A) every day during 6 days. Re-sults show that the hfq mutant is retarded in swarming (Fig. 7A).Interestingly, a nifA mutation reduces the ability of S. meliloti toswarm (21). Further experiments will be needed to decipherwhether the swarming retardation phenotype in a nifA mutant is

controlled or not by Hfq. We noted an additional phenotypewhere bacterial cultures were maintained in GAS medium for 1month. The hfq mutant cells aggregated, whereas the wild-typecells did not (Fig. 7B). The aggregation was not associated withloss of viability. Taken together, our results suggest that hfq mu-tants may be at a disadvantage in adapting to new environments,including the various environments that the bacterium encountersin the course of establishing symbiosis.

DISCUSSION

Our study reveals that the RNA chaperone Hfq, which playsdiverse regulatory roles in many free-living bacteria (25, 66) andis needed for the virulence of numerous pathogens (e.g., refer-ences 12, 14, 32, 40, 55, and 59), is critically required for S. melilotito establish a nitrogen-fixing symbiosis with its host plant alfalfa.This study was motivated in part by the phylogenetic relationshipsbetween the rhizobiaceae and the brucellae (46), both of whichestablish chronic intracellular infections within acidic membranecompartments in their respective hosts (50). Furthermore, ourfinding that the S. meliloti BacA protein is important for thechronic intracellular infection phase of symbiosis had previouslyled us to discover that B. abortus BacA is specifically required forthe chronic phase of pathogenesis in the BALB/c mouse model(35). Since, like BacA, B. abortus Hfq is specifically required forthe chronic phase of pathogenesis, we were curious to testwhether Hfq might play an important role in the interactions of S.meliloti with its plant host.

Hfq affects at least three aspects of the symbiosis: (i) theability of the rhizobia to invade the developing nodule throughhost-derived infection threads, (ii) the capability of the rhizo-bia to invade plant cells within the nodule and establish thechronic intracellular infection that underlies the symbiosis, and(iii) the ability of the bacteria to express various functionsnecessary for nitrogen fixation. Our study shows that the roleof Hfq goes far beyond the regulation of nitrogen fixation thathad been previously observed in A. caulinodans (30) andRhodobacter capsulatus (13) and reveals additional parallelsbetween pathogenesis and symbiosis. As discussed below andin more detail in our accompanying paper (3a), Hfq also playsimportant roles in regulating numerous responses of free-livingS. meliloti as well.

In the S. meliloti strain Rm1021-alfalfa symbiosis, infectionthread initiation, elongation, and extension require that therhizobia be able to synthesize the exopolysaccharide succino-glycan. Mutants, such as the exoY strain, that are unable to

FIG. 6. Optical microscopy images of hand sections of nodulesfrom plants inoculated with the wild-type strain (A) or the hfq mutant(B) containing the plasmid pGus2 carrying the nfeB::uidA fusion(SmblK7 or SmblK8, respectively). Expression of this fusion is revealedby the blue staining in nodule zone III in the wild-type strain and indifferent zones of a nodule in the hfq mutant (SmblK8), which, al-though colonized, fails to elongate (arrows).

FIG. 5. Analysis of nifA gene expression using RT-PCR. RNA wereextracted from the wild-type strain Rm1021 (lanes 1 and 2) and the hfqmutant Smbl�500 (lanes 3 and 4) before (�; lanes 1 and 3) or after (�;lanes 2 and 4) 4 h of incubation in nitrogen-free medium and mi-croaerobiosis. An equal amount of RNA was used to perform theRT-PCR, and an equal volume of the amplification product wasloaded onto the gel. The nifA gene is expressed only 4 h after incuba-tion in WT strains (upper panel). The lower panel shows RT-PCRamplification of 16S RNA, used as a control. MW, molecular weightmarker.

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synthesize succinoglycan form small white Fix� nodules (29,34), because most of the colonized curled root hairs fail toinitiate infection threads and those infection threads that areinitiated then abort (10). The hfq mutant is able to synthesizesuccinoglycan, which allows the bacteria to initiate infectionsthreads and stimulate their elongation. Nevertheless, a largeproportion of the infection threads do abort before the releaseof the bacteria within the plant cells in the infection zone of thenodule. The infection thread abortion phenotype of the hfqmutant may be due to the fact that, while in the infectionthread, S. meliloti is being subjected to a prolonged oxidativeburst released by its host (52). Like the oxidative burst gener-ated by mammalian cells in response to pathogens, this oxida-tive stress consists of at least superoxide and hydrogen perox-ide, both of which are deleterious to cell survival. Theprolonged burst is a chronic stress for S. meliloti, with bothsuperoxide and hydrogen peroxide being readily detectable innodules several weeks after the initial infection (52). Our ac-companying study shows that Hfq function is important for S.meliloti to survive superoxide and hydrogen peroxide. The hfqmutant’s ability to spread inside the infection thread may alsobe impaired by its increased doubling time and possibly by itsdecreased ability to swarm.

Despite its reduced ability to invade developing nodulesthrough infection threads, the S. meliloti hfq mutant is endo-cytosed into the cytoplasm of plant cells in a significant fractionof the alfalfa nodules. Normally, the rhizobia stop dividingafter endocytosis and then differentiate into bacteroids, a pro-cess that includes rounds of endoreduplication (19, 29, 41).However, in our ultrastructural studies of the nodules infectedby the hfq rhizobia, many fewer bacteroid-like cells could be

observed than with the wild type, and in some cases, we evenobserved what appear to be dividing bacteria. Thus, Hfq mayalso affect symbiosis by regulating the differentiation of thebacteria into bacteroids.

Because the hfq mutant is so inefficient in establishing achronic intracellular infection, we were unable to examinewhether Hfq regulates functions required for nitrogen fixationin the context of the nodule, a possibility suggested by theearlier observations that Hfq regulates nitrogen fixation in A.caulinodans (30) and R. capsulatus (13). However, by takingadvantage of the fact that S. meliloti genes specifically involvedin nitrogen fixation can be induced in free-living bacteria undermicroaerophilic conditions in nitrogen-free medium, we wereable to show that Hfq also regulates the expression of nifA andnfeB. While nfeB is probably under the control of nifA, nifA isessential for several cellular processes and nitrogen fixation.nifA mutants fail to fix nitrogen and elicit small, ineffectivewhite nodules (21). The interior of these nodules is reminiscentof a hypersensitive response characteristic of noncompatiblehost-pathogen interaction and is likely due to alteration inexpression of several genes in signal transduction, cellular me-tabolism, growth, and development (21). As a consequence,disruption of nifA influences multiple cellular processes, suchas growth, swarming, and extracellular protein production(21). hfq mutants not only failed to express nifA but showedphenotypes strikingly similar to those of nifA mutants, such aslower levels of swarming behavior, alteration in protein accu-mulation (as shown in the second study), and reduced ability tonodulate on the host. Thus, it seems likely that Hfq is impor-tant not only for infection thread invasion and for differenti-

FIG. 7. hfq regulates physiological responses to changes in environment. (A) The S. meliloti hfq mutant swarms less efficiently than the wild-typestrain. (Aa) Five-day colonies from the hfq mutant Smbl�500 (panel 1) and the WT (panel 2) strains were plated on a swarming plate (0.3% agar)and incubated at 30°C. The white bar represents 0.5 cm. (Ab) Results of swarming for the WT (gray bars) and the hfq mutant (white bars) strains.Colony progression was measured every day. The bars represent the means of results from 3 experiments. (B) When hfq mutant cultures (panel1) in GAS medium were maintained for 1 month, aggregates were observed. Similar aggregates were not observed in WT cells (panel 2).

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ation into bacteroids but for the induction of nitrogen-fixationfunctions as well.

Hfq has been shown to serve as an RNA chaperone thatinteracts with the sRNAs at single-stranded AU-rich regionsand helps the sRNAs to pair with their target mRNAs (22,66). Although Hfq can exert regulatory roles that are inde-pendent of sRNAs, the multiple crucial roles of Hfq inestablishing S. meliloti symbiosis with its legume hoststrongly suggests the involvement of sRNAs in regulatingmultiple aspects of symbiosis. The requirement for a func-tional Sm1 domain, previously shown to be needed for RNAinteraction of Hfq, is also consistent with the idea thatregulatory S. meliloti sRNAs play an important role in itsinteractions with its plant host. Such a regulatory strategywould allow the bacteria to react quickly and at lower en-ergetic cost to the stresses and sudden variations in theirenvironments that they experience during the establishmentof the symbiosis (39); as recently reviewed by Waters andStorz (69), sRNA-mediated regulation has several advan-tages compared to protein-regulated regulation duringstress responses. Recent comparative genome analysis andprediction studies have suggested the existence of a numberof sRNAs in S. meliloti, and some have been experimentallyverified (11, 65, 68), but their regulatory roles in bacterialphysiology and symbiosis has not yet been investigated. Ex-pression of several of these predicted sRNAs in S. melilotihas been confirmed by Northern blots and microarray anal-ysis. The expression patterns of these sRNAs show differ-ential expression with respect to the stage of growth (e.g.,early, exponential, or stationary). Furthermore, expressionof several of the sRNAs is regulated by environmentalstresses, such as heat, salinity, H2O2, alteration in pH, nu-trients, and surfactants, etc. (68). The phenotypes of the S.meliloti hfq mutant suggest the involvement of multiplesRNAs during both the free-living and the symbiotic life-styles of the bacteria.

The accompanying paper describes a proteomic analysis thatidentified 55 proteins whose levels are significantly altered inthe free-living S. meliloti hfq mutant, most of which are in-volved in cell metabolism or stress resistance (3a). In entericbacteria, Hfq has been shown to regulate the alternate sigmafactors RpoS, which is involved in stationary-phase adaptation,and RpoE, which controls the envelope stress response. Al-though no RpoS or RpoS-like protein has been found in any ofthe sequenced alphaproteobacteria, this study shows that lossof Hfq function reduces the expression of the alternate sigmafactors RpoE1, RpoE2, RpoE3, and RpoE4 (3a). Thus, someof the phenotypes of the hfq mutants are an indirect conse-quence of Hfq modulating a key regulatory protein, such as theRpoE sigma factor.

In conclusion, we have shown that Hfq plays crucial rolesin mediating symbiosis by helping the bacteria adapt toextra- and intracellular niches during its interaction with thehost plant cells. The involvement of sRNAs in symbioticinteractions in an Hfq-dependent manner certainly warrantsfurther investigation. Parallel studies for identification ofsRNAs in B. abortus would enable better understanding ofthese key regulatory elements during intracellular survival,virulence, and symbiosis.

ACKNOWLEDGMENTS

We thank S. Georgeault, C. Monnier, M. Uguet, and M. C. Savaryfor technical assistance.

This work was supported by National Institutes of Health grantGM31010 (to G.C.W.), MIT Center for Environmental Health Sci-ences NIEHS P30 ES002109, the Centre National de la RechercheScientifique, and the Ministere de la Recherche et de l’EducationNationale. L.B.-B. was supported by Region Bretagne. G.C.W. is anAmerican Cancer Society Research Professor.

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