MicrobialEcology

Genetic Determinants of Swarming in Rhizobium etli

Kristien Braeken1, Ruth Daniels2, Karen Vos1, Maarten Fauvart1, Debkumari Bachaspatimayum1,Jos Vanderleyden1 and Jan Michiels1

(1) Centre of Microbial and Plant Genetics, Katholieke Universiteit Leuven, Kasteelpark Arenberg 20, B-3001, Heverlee, Belgium(2) Biomedisch Onderzoeksinstituut, Universiteit Hasselt, B-3590, Diepenbeek, Belgium

Received: 8 January 2007 / Accepted: 5 March 2007

Abstract

Swarming motility is considered to be a social phenom-enon that enables groups of bacteria to move coordinatelyatop solid surfaces. The differentiated swarmer cellpopulation is embedded in an extracellular slime layer,and the phenomenon has previously been linked withbiofilm formation and virulence. The gram-negativenitrogen-fixing soil bacterium Rhizobium etli CNPAF512was previously shown to display swarming behavior onsoft agar plates. In a search for novel genetic determi-nants of swarming, a detailed analysis of the swarmingbehavior of 700 miniTn5 mutants of R. etli wasperformed. Twenty-four mutants defective in swarmingor displaying abnormal swarming patterns were identi-fied and could be divided into three groups based ontheir swarming pattern. Fourteen mutants were com-pletely swarming deficient, five mutants showed anatypical swarming pattern with no completely smoothedge and local extrusions, and five mutants displayed anintermediate swarming phenotype. Sequence analysis ofthe targeted genes indicated that the mutants were likelyaffected in quorum-sensing, polysaccharide compositionor export, motility, and amino acid and polyaminesmetabolism. Several of the identified mutants displayed areduced symbiotic nitrogen fixation activity.

Introduction

Over 30 years ago, Henrichsen [23] identified six categoriesof bacterial surface motility based on the study of motilityin hundreds of strains from 40 bacterial species belongingto 18 different genera: swimming, swarming, gliding,twitching, sliding, and darting. Of these phenomena,

swimming in liquid medium has been studied mostly overthe past three decades, especially in Escherichia coli andSalmonella enterica serovar Typhimurium. However, thisform of motility is not restricted to liquid media, but alsooccurs when bacteria colonize medium with low agarconcentrations (0.2–0.4%). In contrast to the individualmovement of bacteria during swimming, swarming isconsidered as a bacterial group behavior associated withmigration on semisolid surfaces. In the presence ofextracellular slime (polysaccharides, peptides, surfactants,etc.), bacteria exhibit a flagella-driven movement on topof the agar (0.4–1.2%) enabling them to spread as abiofilm over the surface. In the laboratory, the concentra-tion of the agar used to solidify the medium can be criticalfor swarming likely owing to a certain level of wetnessrequired for movement. Association of cells in a grouplikely facilitates movement by increasing fluid retention.Swarming may be viewed as a specialized case ofswimming on a surface [10, 22, 53].

The swarming phenomenon has been demonstratedin a growing number of diverse bacteria including mem-bers of Aeromonas, Azospirillum, Bacillus, Burkholderia,Chromobacterium, Clostridium, Escherichia, Proteus,Pseudomonas, Salmonella, Serratia, Sinorhizobium, Vibrio,and Yersinia [10, 53]. Swarming normally requires thedifferentiation of vegetative cells into a specialized celltype called swarmer cells. Surface contact (change inviscosity), physiological parameters, and chemical signals(nutritional status) provide stimuli that trigger swarmercell differentiation. Although the pathways for signalintegration are still poorly understood, it is believed thatsignals are sensed and transmitted by two-componentregulatory systems, cytosolic regulators, or flagella lead-ing to a complex network. The differentiated swarmercells are often hyperflagellated and elongated. These cellsmove in groups or rafts organized parallel to their longaxis to maximize cell–cell contact, colonizing the availableCorrespondence to: Jan Michiels; E-mail: jan.michiels@biw.kuleuven.be

DOI: 10.1007/s00248-007-9250-1 & * Springer Science+Business Media, LLC 2007

surface. The migration front is preceded by a visible layerof slime-like extracellular material [5, 7, 10, 18]. As aresult of the embedding in an extracellular slime matrix,population densities are normally extremely high [25].

Besides the differentiation into swarmer cells, wettingagents that reduce surface tension are also required forswarming in many organisms. Among the best studied arethe glycolipids or lipopeptides produced by Bacillussubtilis (surfactin), Serratia (serrawettin), Pseudomonasaeruginosa (rhamnolipids) (for an overview: [46]). A rolefor the LPS-O-antigen in improving surface wettabilityor swarming was also suggested for S. enterica serovarTyphimurium, Proteus mirabilis, and Serratia marcescens[4, 27, 58]. Similarly, the capsular polysaccharide of P.mirabilis, also called colony migration factor, plays a keyrole by enhancing medium surface fluidity [21, 43]. In B.subtilis, extracellular protease activity is also importantfor surface movement [8]. Finally, it has been shown thatP. mirabilis swarming is stimulated by peptides andamino acids, possibly generated by the three broadspecificity proteases present in this bacterium [18].

Some recent publications have used a mutantscreening approach to identify genes involved in swarm-ing in E. coli K-12 [26] and P. aeruginosa PAO1 [40] or amicroarray-based approach for genes involved in surfacemotility in S. enterica serovar Typhymurium [62].Mutations in genes belonging to various functionalcategories were identified and underscore the notionthat swarming is a complex type of motility requiring awide variety of cellular activities. In E. coli K-12, differentcomponents of the cell surface were found among theidentified genes including the lipopolysaccharide andenterobacterial common antigen. These compounds arethought to have a role in reduction of surface tension.

The involvement of quorum sensing (QS) regulationin swarming has been reported for both biosurfactantproduction and swarmer cell differentiation (reviewed byDaniels et al. [10]). Involvement of LuxIR-type systemsproducing N-acylhomoserine lactones (AHLs) in swarm-ing is mainly reported for regulation of biosurfactantproduction like serrawettin production in Serratia lique-faciens and rhamnolipid production in P. aeruginosa [14,38, 59]. Evidence also exists for a role of AHL-mediatedregulation of biosurfactant production and swarmingbehavior in S. marcescens and Burkholderia sp. [25, 31,45]. In Pantoea stewartii subsp. stewartii, mutation of theesaIR QS system was reported to affect bacterial ad-hesion, swarming, and biofilm formation [24, 29, 61].Finally, a mutation in the yenI gene, encoding the LuxI-type AHL-synthase of Yersinia enterocolitica, affects bothswimming and swarming [3].

The gram-negative nitrogen-fixing soil bacteriumRhizobium etli is the bacterial symbiotic partner of thecommon bean plant. R. etli CNPAF512 produces severalQS signal molecules via the cinIR and raiIR quorum

sensing systems [11, 47]. It was previously demonstratedthat R. etli CNPAF512 swarms and colonizes the surfaceof YEM soft agar. For swarming migration to occur, thecin QS system is required, as R. etli cinI and cinR mutantsare no longer able to move over this solid surface. Incontrast, they form a regular colony at the inoculationpoint [10]. Recent data published by our laboratoryrevealed that the CinI-made long chain AHLs have anadditional function besides high level induction of the cinand rai quorum sensing systems [9]. These moleculeswere shown to possess significant surface activity andpossibly induce liquid flows as a result of gradients insurface tension at biologically relevant concentrations,pointing to a direct role of these molecules as biosurfac-tants during swarming.

Little is known about factors involved in swarmingin rhizobia. Swarming has only been demonstrated forSinorhizobium meliloti and R. etli. In S. meliloti, a fadDmutant, altered in a gene encoding a long-chain fattyacyl-CoA ligase, displayed multicellular swarming behav-ior [55]. In this work, we initiated research toward thegenetic determinants involved in swarming in R. etliCNPAF512 by analyzing the swarming behavior of asubset of 700 mutants and investigated their symbioticperformance and a possible link with the cin QS system.

Methods

Bacterial Strains and Plasmids. The bacterial strainsand plasmids used in this work are listed in Table 1. Themutant strains isolated during the screening for mutantsaffected in swarming are represented in Table 2. R. etlistrains were cultured in complex TY-medium (0.3% Yeastextract, 0.5% Tryptone, 7 mM CaCl2) at 30-C [35], onYEM soft agar (0.75%) plates for swarming experiments[9], and TY soft agar plates (0.2%) for swimmingexperiments. Escherichia coli strains were routinely grownat 37-C in Luria–Bertani (LB) medium [49]. Agrobacteriumtumefaciens NT1 was grown in TY or in AB medium at30-C [47]. Antibiotics were supplied at the following con-centrations: ampicillin 100 mg ml

_1, gentamicin 30 mg ml_1,

kanamycin 30 mg ml_1, spectinomycin 50 mg ml

_1, nalidixicacid 30 mg ml

_1, neomycin 60 mg ml_1, or tetracycline 1 mg ml

_1

(R. etli) or 10 mg ml_1 (E. coli). For A. tumefaciens, kanamycin

was supplied at 50 mg ml_1 and carbenicillin at 100 mg ml

_1.Triparental conjugations were done as described previously[12].

DNA Techniques. Standard techniques were usedfor DNA manipulations [49]. Restriction endonucleases(Roche Diagnostics) and ligase (Invitrogen) were usedaccording to the manufacturer’s instructions. Total DNAfrom R. etli CNPAF512 was isolated using the Puregene\

Genomic DNA purification kit (Gentra Systems).Plasmids were isolated from E. coli using GFXi Micro

K. BRAEKEN ET AL.: SWARMING IN R. ETLI

Plasmid Prep Kit (Amersham). DNA fragments wereisolated from agarose gels using Qiaquick Gel Extractionkit (Qiagen) (94 kb) or Minelute Gel extraction Kit(Qiagen) (G4 kb). Polymerase chain reactions (PCR)were performed in a Px2 Thermal Cycler (ThermoElectron). Amplified PCR fragments were recovered fromthe PCR mix with the QIAquick PCR purification kit(Qiagen). Primers were obtained from Eurogentec. DNA

probes for Southern hybridization were labeled withdigoxigenin-dUTP using a random-primed labeling kit(Boehringer Manheim). Genomic DNA was isolated,digested with restriction enzymes, separated by electro-phoresis, and blotted to Hybond N membrane (Amersham)by standard techniques. Prehybridization and hybridizationwere carried out at 68-C. Signals were detected with achemiluminescence detection kit (Boehringer Manheim).

Table 1. Strains and plasmids used in this study

Strain or Plasmid Descriptiona Source or reference

StrainsE. coliDH5a F- (F80DlacZDM15) endA1 gyrA96 thi-1recA1 hsdR17(r�K m�K )

relA1 supE44D(lacZYA-argF)

[49]

A. tumefaciensNT1 (pJM749, pSVB33) Kmr, Cbr, AHL biosensor strain [41]NT1 (pJM749) Lacks Ti plasmid, no detectable production of AHLs [41]R.leguminosarum bv.

viciae 248Sensitive for growth-inhibition by ‘small’ [60]

R. etliCNPAF512 Nalr, wild-type [35]FAJ4013 Nalr, Spr,Nmr, rail::gusA-Sp, cinI::Km opposite orientations [11]FAJ4006 Nalr, Nmr, cinI::Km opposite orientation [11]FAJ1328 Nalr, Nmr, rail::gusA-Nm mutant, convergently oriented [47]FAJ1329 Nalr, Nmr, raiR mutant with gusA-Km reverse orientation [47]CMPG8799 to

CMPG8777Nalr, Nmr, swarming-defective mTn5 mutants identified

in this study as described in Table 2This work

CMPG8707 Swarming-defective mutant CMPG8795, cinI::W-DKm This workCMPG8708 Swarming-defective mutant CMPG8797, cinI::W-DKm This workCMPG8709 Swarming-defective mutant CMPG8789, cinI::W-DKm This workCMPG8710 Swarming-defective mutant CMPG8787, cinI::W-DKm This workCMPG8711 Swarming-defective mutant CMPG8794, cinI::W-DKm This workCMPG8712 Swarming-defective mutant CMPG8783, cinI::W-DKm This workCMPG8713 Swarming-defective mutant CMPG8786, cinI::W-DKm This workCMPG8717 Swarming-defective mutant CMPG8781, cinI::W-DKm This workCMPG8718 Swarming-defective mutant CMPG8780, cinI::W-DKm This workCMPG8720 Swarming-defective mutant CMPG8798, cinI::W-DKm This workCMPG8723 Swarming-defective mutant CMPG8796, cinI::W-DKm This workCMPG8725 Swarming-defective mutant CMPG8793, cinI::W-DKm This workCMPG8730 Swarming-defective mutant CMPG8790, cinI::W-DKm This workCMPG8731 Swarming-defective mutant CMPG8792, cinI::W-DKm This workCMPG8732 Swarming-defective mutant CMPG8782, cinI::W-DKm This workCMPG8734 Swarming-defective mutant CMPG8799, cinI::W-DKm This workCMPG8735 Swarming-defective mutant CMPG8777, cinI::W-DKm This workCMPG8736 Swarming-defective mutant CMPG8778, cinI::W-DKm This workCMPG8738 Swarming-defective mutant CMPG8784, cinI::W-DKm This workCMPG8739 Swarming-defective mutant CMPG8785, cinI::W-DKm This workCMPG8740 Swarming-defective mutant CMPG8791, cinI::W-DKm This workCMPG8742 Swarming-defective mutant CMPG8779, cinI::W-DKm This work

PlasmidspFAJ1458 Tcr, railI-gusA fusion in pFAJ1703 E. Luyten, pers. communicationpFAJ4013 Smr, Spr, EcoRI fragment (cinR cinI orf123 orf140) in

pPZP200 derivative[11]

pFAJ4014 Tcr, cinI-gusA fusion in pFAJ1703 [11]pRK2013 Kmr, ColE1 replicon with RK2 transfer genes [15]pRK2073 Kmr, Spr, pRK2013::Tn7 [15]pCMPG8799 Gmr, pFAJ4006 containing cinI::W-DKm (opposite orientation)

with an internal PstI fragment, encoding Km resistance,deleted from the pHP45-W-Km cassette

This work

aNalr, nalidixic acid resistance, Kmr, kanamycin resistance, Nmr, neomycin resistance, Spr, spectinomycin resistance, Apr, ampicillin resistance, Tcr,tetracycline resistance, Cbr, carbenicillin resistance, Gmr, gentamycin resistance, Smr, streptomycin resistance.

K. BRAEKEN ET AL.: SWARMING IN R. ETLI

Tab

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K. BRAEKEN ET AL.: SWARMING IN R. ETLI

Motility Assays and Sequence Determination.

Swarming assays were performed on YEM soft agar plates(0.75%) (containing 0.4 g/l MgSO4

. 7H2O) as described inDaniels et al. [9]. For swimming, TY plates containing 0.2%agar were spot inoculated with bacteria previously grownon YEM agar plates. Each strain was tested in tenfold inthree independent experiments. The plates were incubatedat 30-C in a closed container before swimming behaviorwas checked. Images of representative surface motilityplates were captured using a digital camera (Sony).

For the selected mutants, total DNA was digestedovernight with the XhoI enzyme, leaving the transposonintact, religated, and transformed into E. coli. For eachmutant, plasmid DNA of at least two independentcolonies was submitted for sequencing. The DNAsequence of the genomic region flanking the 50 end ofthe gusA gene was determined using the gusA primerRHI-628 (50-CTCCTTAGCTAGTCAGGTACCG-30),whereas primer AZO-497 (50-GTCTGACGCTCAGTGGAAC-30) was used for the region 30 of the transposon.Southern hybridization using a gusA probe was per-formed to confirm insertion of the transposon in a singlelocus. The putative open reading frame (ORF)s locatedon the flanking sequences were identified by BlastXsearches [2].

Determination of Quorum Sensing Super-regulation.

To determine if AHL production was affected in the mutantstrains, the A. tumefaciens tra reporter system was used. Thereporter strain was grown overnight and diluted 40-foldinto TY soft medium (0.75%). The mutants were spotinoculated in the middle of the agar plates on the surfaceand incubated for 3 days. The A. tumefaciens strain isdeficient in its own AHLs, but exogenously produced AHLscan induce a traG-lacZ fusion, observable by a colorreaction in the presence of the substrate X-gal [41]. AHLproduction was monitored by the examination of thediameter of the blue halo observed.

Expression Analysis. To investigate a possibleregulation of the identified genes by AHLs, all selectedtransposon mutants were additionally mutated in the cinIgene. Inactivation of cinI was carried out with pCMPG8799.cinI mutants were selected by screening for loss ofbacteriostatic activity toward the sensitive strain Rhizobiumleguminosarum bv. viciae 248 and the loss of activation ofthe A. tumefaciens tra reporter system [11]. Mutants wereconfirmed by PCR analysis. Quorum sensing signalmolecules were isolated from cultures of wild-type R. etlior A. tumefaciens NT1 carrying the cinIR locus on pFAJ4013as described by Daniels et al. [9]. Overnight cultures of themutants were diluted 40-fold in fresh medium containingthe dissolved QS signal molecules extract (finalconcentration 1�) or a control. These cultures wereincubated in microtiter dishes sealed with a breathable

membrane (Greiner Bio-One) for 24 or 40 h after whichexpression was analyzed. Quantitative analysis of GusAactivity was carried out in microtiter dishes with p-nitrophenyl-b-D-glucuronide (pNPG) as the substrate bythe method of Miller. GusA activity was examined inVERSAmax microplate reader (Molecular Devices) [9].

Growth Analysis. Overnight cultures of R. etlistrains were washed twice in 10 mM MgSO4, brought toan OD600 of 0.5, and subsequently diluted 6,000-fold inTY-medium. Three hundred microliters of the cultures,five repetitions for each condition, were introduced inthe wells of sterile Honeycomb plates. The OD600 valuewas recorded every hour in a Bioscreen C MicrobiologyWorkstation (Labsystems Oy, Zellik, Belgium) [37].

Plant Experiments. Seeds of Phaseolus vulgaris cv.Limburgse vroege were sterilized and germinated aspreviously described [36] and plants were inoculatedwith 100 ml of an overnight bacterial culture andincubated as described by Dombrecht et al. [13]. Foreach R. etli strain, at least ten plants were inoculated.Nitrogenase activity was determined by measuringacetylene reduction activity (ARA) of nodulated rootsin a closed vessel 3 weeks after inoculation [13].

Electron Microscopy. A formvar (0.7%) carbon-coated grid (50 mesh, Cu-OD-841005, VECO\) was placedon the surface of a swarmer plate for 10 s; the grid was placedon a drop of phosphotungsten acid (0.25%, pH 7) for 30 s forstaining, and excess liquid was drained. Samples wereexamined with a Philips\ EM 208 S transmission electronmicroscope at 80 kV. Images were digitized using the SIS\

image analysis system.

Results

Identification of Mutants with an Altered Swarming

Behavior. Swarming in rhizobia is a relativelyrecently described phenomenon [9, 10, 55]. Besides thecinIR system, little is known about other genes involvedin R. etli and the contribution of this form of motility tothe symbiotic relationship with the host plant. Toidentify a first set of genes that are involved in theswarming behavior of R. etli CNPAF512, a group of 700mutants were analyzed with respect to their swarmingbehavior. The 700 mutants in this library were previouslyselected in a screening of a mTn5gusA-oriV library inwild-type R. etli CNPAF512 for genes potentially involvedin the symbiotic process (M. Moris, unpublished).Moreover, as these mutants were isolated based ondifferential GusA activity, the orientation of the gusAgene in these mutants enables us to investigate if thedefect in swarming is related to quorum sensing-mediated gene regulation.

K. BRAEKEN ET AL.: SWARMING IN R. ETLI

First, the mutants were grown overnight in microtiterdishes containing TY-medium. After washing the cultureswith liquid YEM medium, 1.2 ml was spot inoculated on aswarm plate. After 3 days incubation, swarming behaviorwas monitored. The wild type and quorum sensingmutants, FAJ1328 and FAJ1329 (swarming positive),and FAJ4006, FAJ4013 (swarming negative) were includ-ed as controls for swarming conditions in every round ofthe screening.

Mutants, 183, showing no swarming behavior or aswarming behavior different from the wild type (e.g.,swarming diameter, presence of extrusions, etc.) wereselected. These mutants were retested after growth inmicrotiter dishes. From this analysis, 60 strains were se-lected for detailed analysis, and overnight cultures in 5 mlTY-medium were brought to equal optical densities andused to inoculate swarmer plates in triplicate. The latterstep of the screening was repeated independently threetimes.

Finally, 24 mutants consistently showing no or anatypical (e.g., diameter, extrusions) swarming pattern inall repeats of the experiment were subjected to sequenceanalysis. The results are summarized in Table 2. Uponsequence analysis, mutants carrying the transposon inypch00541 (CMPG8799) and ctaD (CMPG8790) werefound to be present twice in the selection. The 22 differentmutants were classified in three groups, based on theshape of the colony edge. Twelve mutants were identifiedthat were completely swarming deficient (smooth edge;group 1), five mutants showed an atypical swarming

pattern (no completely smooth edge with local extrusions;group 2), and five mutants displayed an intermediateswarming phenotype (less pronounced scalloping of thecolony edge compared to the wild type; group 3).Representative pictures of each group are shown in Fig. 1.

Swimming Behavior of Swarm Mutants. Genes notonly involved in swarming but also important forswimming behavior were identified by performing swim-ming tests. Most mutants displayed a wild-type swim-ming pattern (Fig. 2A and B). As expected, mutantsknocked-out in components of the flagellar system(CMPG8787, CMPG8786 see Fig. 2D, CMPG8785) werealso defective in swimming, whereas CMPG8796 dis-played a reduced swimming behavior as shown by thesmaller swimming diameter. As the latter strain alsodisplays a growth delay, we cannot exclude that themotility phenotype of this mutant is due to a moregeneral defect. Therefore, to exclude that the swarmingbehavior is due to a growth defect, Bioscreen C curves ofall mutants were obtained for growth (data not shown).Most strains displayed a wild-type growth pattern, threemutants displayed a small growth retardation (indicatedas T in Table 2), while three mutants (CMPG8796,CMPG8790, CMPG8777) were more significantly retard-ed. As this could also affect the outcome of the swarmingassay, (e.g., by a defect in the energy supply as can beexpected for CMPG8790 mutated in cytochrome Coxidase subunit 1), these three strains will not be furtherdiscussed.

Figure 1. Swarming behavior ofselected mutants. Representativesof the different groups are shownincluding the wild type (A) and amutant designated as swarmingpositive (B). C–G representdifferent mutants classified asswarming negative in group 1(C: CMPG8799; D: CMPG8788;E: CMPG8797; F: CMPG8791;G: CMPG8796). Three mutantsshowing local extrusions (classi-fied in group 2) are represented inH to J (H: CMPG8787;I: CMPG8783: J: CMPG8786).Finally, CMPG8777 (K) andCMPG8781 (L) represent mutantsclassified in group 3.

K. BRAEKEN ET AL.: SWARMING IN R. ETLI

Symbiotic Phenotype of Swarm Mutants. Theswarmer mutants were evaluated for symbiotic nitrogenfixation capacity by measuring ARA values of inoculatedplants. Eleven and nine strains displayed reduced orwild-type nitrogen fixation levels, respectively. MutantCMPG8794 was Nod-. A summary of the results ispresented in Table 2.

R. etli Swarmer Cells are Hyperflagellated. Toobtain a more detailed view on the morphological differ-entiation associated with the swarming phenomenon in R.etli, we analyzed transmission electron micrographs of cells,directly taken from the edge of a wild-type R. etli-swarmingcolony. In R. etli, swarming coincides with hyperflagellationas found in other bacteria (Fig. 3). The hyperflagellation asobtained during swarming was not observed in bacterialcultures grown in broth (data not shown).

Relationship with Quorum Sensing. As cin mutantsare no longer capable of swarming, and this phenotype canbe restored by complementation, we determined if any ofthe mutants identified here is impaired in AHL production.Therefore, mutants were spot inoculated on TY soft agarplates containing the A. tumefaciens reporter strain. After 3days of incubation at 30-C, the diameter of the blue halo,representing diffused AHL molecules produced by the

mutant that activate the reporter, was compared with thatobserved for the wild-type strain. All strains, except mutantCMPG8788 (cinI), produced a blue halo indistinguishablefrom that of the wild type in different repeats of theexperiment (data not shown). This indicates that theswarming phenotype is not caused by reduced productionor secretion of AHL molecules and that no super-regulatorsof the R. etli QS systems were present, at least not operatingin the conditions tested.

To determine whether any of the genes is regulatedby cinI, the selected mutants were mutagenized in cinI byusing pCMPG8799. Expression tests were carried out todetermine the expression of the gusA fusions in thepresence of QS signal molecule extract obtained fromwild-type R. etli CNPAF512, from A. tumefaciens NT1carrying cinIR on a plasmid and from A. tumefaciens NT1negative control. Expression of none of the genesidentified was found to be significantly affected bymutation of cinI (data not shown).

Discussion

It was previously shown that R. etli CNPAF512 swarmsand promotes surface colonization of YEM soft agar plates[10]. To better understand the mechanism of swarming inR. etli, a first subset of mTn5gusA-oriV mutants of R. etliwas analyzed for their swarming phenotype. Twenty-twodifferent mutants showed no or an altered swarmingbehavior. These are summarized in Table 2. The effect ofthe transposon insertion is due to inactivation of the genementioned in the table or to a possible polar effect. Asthese 700 mutants were previously isolated based ondifferential expression patterns in micro-aerobic condi-tions or in the presence of nodule extract, the number ofmutants identified here is probably not representative for

Figure 2. Swimming behavior. Swimming behavior observed on0.2% agar plates. Most mutants showed a wild-type swimmingbehavior (as shown in A and B), reaching the edge of the platesafter approximately 3 days. Mutant CMPG8796 displayed astrongly reduced diameter of swimming after 3 days, while themutants in genes involved in synthesis of flagella like CMPG8786(D) displayed only growth at the inoculation point.

Figure 3. Microscopic observation of swarmer cells. Electronmicrographic picture of wild-type R. etli, isolated from the edge ofa swarming colony and colored with phosphotungsten acid(0.25%, pH 7). The differentiated swarmer cells are hyperflagel-lated (bar, 1,000 nm).

K. BRAEKEN ET AL.: SWARMING IN R. ETLI

the percentage of genes involved in swarming. Among thehitherto identified genes, genes encoding elements of theflagellar system and modifying the EPS matrix surround-ing the bacteria were identified. Overall, mutants isolatedhere could be affected in the swarmer cell differentiation,in surface translocation, or both. Detailed microscopicobservation of the bacteria will enable us to distinguishbetween these possibilities. As some of the mutantsisolated also display a growth defect in rich medium, thismight affect the outcome and timing of swarmer celldifferentiation or surface translocation, leading to theobserved phenotypes. Finally, as most mutants identifiedin this screening still await further confirmation, wecannot rule out at this stage that some of the observedeffects are due to secondary site mutations.

The results of the ARA measurements reveal thatthere is no direct link between the absolute nitrogenfixation capacity and swarming capacity. This raises thequestion about the possible role of swarming in thebacteria–plant interaction (reviewed by [10, 44]). Ingeneral, the phenomenon of swarming is often linkedwith biofilm formation, although the relationship be-tween these phenomena appears to be rather dual. Recentresearch in P. aeruginosa revealed that early in biofilmformation, the extent of swarming, which depends on theconditions used, determines the structure of the biofilmformed. Moreover, the nutritional environment wasfound to influence the functions required for swarmingincluding quorum sensing-mediated regulation [54].Interestingly, attachment of bacteria to plants proceedsthrough a similar mechanism as observed for initiationof biofilm formation and the direct observation ofbacteria adhered to plant cells often reveals multicellularassemblies variably described as microcolonies, aggre-gates, or cell clusters. These resemble each other in thefact that they all consist of bacteria embedded in a matrixon a surface [44].

The main role of surface motility in P. aeruginosabiofilm formation was proposed to be after the initialcontact is established, when cells move over the surface,where they finally aggregate and form microcolonies [39,42]. In these early steps of biofilm formation, bacteriaundergo a variety of metabolic changes after attachmentincluding differential expression of proteins involved inamino acid metabolism, membrane proteins involved intransport processes, and proteins necessary for theproduction of extracellular polymers and organellesinvolved in motility such as flagella and pili [51].Metabolic differentiation in swarmer cells and genesspecifically induced by surface growth are also reportedfor Salmonella [28, 63]. However, in other bacteria suchas B. cepacia, no direct link between swarming andbiofilm formation was observed, leading to the sugges-tion that swarming per se is not required for biofilmformation [25]. Moreover, a growing number of bio-

surfactants, important for surface translocation, arereported to be involved in biofilm dispersion, suggestingalso a possible role for swarming in the dispersal ofbiofilms (reviewed by [6, 10]).

Evidence that surface movement is important duringthe colonization process, especially of distal parts of theroots, is provided by research on P. fluorescens F113during the colonization of the alfalfa rhizosphere [50].During the colonization process, P. fluorescens F113undergoes phenotypic variation resulting in three phasevariants, C, F, and S, with C representing the wild-typebehavior. The F and S variants could swim faster andswarmed in conditions that did not allow swarming inthe wild type. These variants preferentially colonizeddistal parts of the roots, which are not easily reached bythe wild-type strain. The observation that P. fluorescensmutants in site-specific recombinases, responsible for thegeneration of subpopulations, are seriously affected incompetitive rhizosphere colonization [33], demonstratesthe importance of the generation of these specializedswarming subpopulations in colonization. As plantinoculation is carried out here with pure cultures directlyinoculated on the developing root, we do not haveinformation about the effect of mutants defective inswarming on the colonization of roots especially whenthey have to compete with wild-type bacteria. Carefulmicroscopic observation of the distribution pattern ofbacteria of mixed cultures and competition experimentsmight help to extend our understanding of the impor-tance of swarming during symbiosis in R. etli.

The identification of a cinI mutant (CMPG8788) inthe screening confirms previous observations that thecinI system is required for R. etli CNPAF512 swarming.As swarming is a flagellum-driven movement, theobservation that three of the mutants identified herecarry the transposon in genes important for accurateflagellum synthesis is not surprising. Motility, in general,is affected in these mutants, which is further confirmedby their defect in swimming behavior. CMPG8787 carriesthe transposon in the flbT gene, most similar to S.meliloti flbT (SMc03051) (85% amino acid identity).Study of the function of this gene has mainly beencarried out for the corresponding gene in Caulobactercrescentus. In the latter bacterium, it was shown that theflbT gene product functions as a negative regulator offlagellin expression in the absence of flagellum assembly[32]. CMPG8785 carries the insertion in flgE, probablyencoding the structural protein of the hook that connectsthe basal body of the flagellum with the filament. Ingeneral, the hook is important in bacterial flagellabecause it allows the synchronous rotation of severalfilaments driven by their motors in a bundle formedbehind the cell (swimming) [48]. As this gene is predictedto be co-transcribed with flbT and other genes, theobserved phenotypes of these two mutants can be due

K. BRAEKEN ET AL.: SWARMING IN R. ETLI

to polar effects on downstream genes in the operon.Finally, in mutant CMPG8786, the transposon is insertedin flaCch1, the first of four genes encoding putativeflagellin subunits. The gene cluster flaA–flaB–flaD–flaC(Smc03037–Smc03040) is found at the correspondingposition in the chemotaxis, flagellar, and motility regionof the S. meliloti genome [56]. Generally, the flagellarfilament consists of an assembly of about 20,000 flagellinsubunits, whose molecular mass typically ranges from 40to 60 kDa [30]. Mutational analyses revealed that in S.meliloti, flagellin A is the principal, absolutely essentialsubunit but that in addition, at least one of the secondaryflagellin species is needed for assembling a functionalfilament [52]. No information on the importance ofdifferent potential fla genes in R. etli CNPAF512 isavailable, but in the R. etli CFN42 genome, additionalgenes encoding putative flagellin subunits have beenannotated, and it would be interesting to test whetherthese play more specific roles in the adaptation of flagellatoward different forms of motility like swimming andswarming.

CMPG8799 is mutated in a gene encoding a probablepolysaccharide export system protein. This protein containsthe characteristics of the Poly_export Pfam family (PF02563)of periplasmic proteins involved in polysaccharide biosyn-thesis and/or export. The gene is most similar to S. melilotiSMc01794. As (secreted) polysaccharides are a componentof the extracellular slime, important for the physicalmovement of the cells, and possibly also acting as a sinkfor the accumulation of signals involved in swarming andQS, the observed effect on swarming is not unexpected.However, no information is available at the moment aboutthe precise polysaccharide(s) synthesized and/or secreted bythe system involving this gene in R. etli CNPAF512.

In CMPG8783, the transposon is inserted in a genedesignated plyA1 and encoding a polysaccharidase gene.These genes have been mainly studied in R. leguminosarum.Two similar glycanases, PlyA and PlyB, capable ofdegrading EPS and carboxymethyl cellulose, were de-scribed in R. leguminosarum. These genes are secreted viathe PrsDE-encoded type I secretion complex that is alsorequired for secretion of NodO and several other proteins[16, 17]. The plyA gene is located immediately upstreamof prsDE and does not greatly affect EPS processing in theconditions tested unless the protein is overexpressed. PlyBis encoded elsewhere on the chromosome, and the plyBmutant is severely affected in the degradation of EPS,forms sticky colonies, and cultures were twice as viscouscompared to the wild type [16]. In R. etli CFN42, threegenes are designated plyA based on their similarity withthe plyA gene of R. leguminosarum. Of these genes, plyA3is also located upstream of the prsDE genes. In the mutantidentified here, the transposon is located in plyA1. Giventhat this mutant also forms very sticky colonies on plate,the gene interrupted here might correspond to R.

leguminosarum plyB. The fact that a mutation of thisgene increases the stickiness of the matrix in which thebacteria are embedded explains the observed effect onswarming.

Finally, a number of genes involved in diversemetabolic pathways were identified. A number of thesegenes are related to or positioned in operons containinggenes possibly involved in the metabolism of arginineand derivatives (including polyamines) or transport ofthese compounds (e.g., CMPG8781 and CMPG8791). InP. mirabilis, it was recently shown that the polyamineputrescine functions indeed as an extracellular signal forswarming, and mutants in speB (encoding SpeB, aprotein involved in the conversion of agmatine toputrescine) display reduced swarming differentiationand migration [57]. The mechanism for the response toputrescine is currently unknown. However, as putrescinehas been shown to form a complex with galacturonicacid in P. mirabilis LPS, this might contribute to efficientcell translocation [57]. Little is known about the role ofdifferent polyamines in rhizobia, although it was previous-ly demonstrated that homospermidine is the polyaminepresent in highest concentration in all rhizobial strainstested [19]. In the related bacterium, A. tumefaciens,mutation of the att locus, in which most genes display thehighest similarity with proteins involved in the transportof spermidine and putrescine, resulted in no or stronglyreduced attachment to carrot suspension culture cells.This could be reversed by addition of conditionedmedium [34]. Finally, peptides and glutamine have alsobeen identified as being involved in P. mirabilis swarmercell differentiation [1]. However, the positive effect ofglutamine on P. mirabilis swarming has only been studiedon minimal media.

In contrast to other bacteria, no obvious genesinvolved in biosurfactant production were identified inthis screening. This is in agreement with recent researchdemonstrating that long chain AHLs, produced by CinI,possess significant surfactant activity. This fact mayaccount for the swarming phenotype upon mutation ofthe cin system [9].

Acknowledgments

KB was aspirant of the Fund for Scientific Research-Flanders and is recipient of a postdoctoral fellowshipfrom the Research Council of the K. U. Leuven (PDM/06/196). KV and MF are recipients of a fellowship fromthe IWT-Flanders. This work was supported by grantsfrom the Research Council of the K. U. Leuven (GOA/2003/09 and IDO/05/012) and from the Fund forScientific Research-Flanders (G.0637.06 and G.0287.04).We thank Prof. I. Lambrichts (Hasselt University) forhelp with the TEM protocol and C. Heusdens fortechnical assistance.

K. BRAEKEN ET AL.: SWARMING IN R. ETLI

Kristien Braeken, Ruth Daniels, Karen Vos contrib-uted equally to this article.

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K. BRAEKEN ET AL.: SWARMING IN R. ETLI