The production of the Sinorhizobium meliloti exopolysaccharide, succinoglycan, is required for the formation of infection threads inside root hairs, a critical step during the nodulation of alfalfa [Medicago sativa] by S . meliloti . Two bacterial mutations, exoR95::Tn5 and exoS96::Tn5, resulted in the overproductionof succinoglycan and a reduction in symbiosis . Systematic analysesof the symbiotic phenotypes of the two mutants demonstratedtheir reduced efficiency of root hair colonization . In addition,both the exoR95 and exoS96 mutations caused a marked reductionin the biosynthesis of flagella and consequent loss of abilityof the cells to swarm and swim . Succinoglycan overproductiondid not appear to be the cause of the suppression of flagellumbiosynthesis . Further analysis indicated that both the exoR95and exoS96 mutations affected the expression of the flagellumbiosynthesis genes . These findings suggest that both the ExoRprotein and the ExoS/ChvI two-component regulatory system are involved in the regulation of both succinoglycan and flagellum biosynthesis . These findings provide new avenues of understandingof the physiological changes S . meliloti cells go through during the early stages of symbiosis and of the signal transductionpathways that mediate such changes.

 
Sinorhizobium meliloti and its legume host, alfalfa [Medicago sativa], establish an effective nitrogen-fixing symbiosis through a series of signal exchanges that starts with the exchange of Nod [nodulation] factors and flavonoids, which results in the formation of curled alfalfa root hairs that are colonized byS . meliloti cells [13, 20, 32] . The colonized curled root hairsdevelop infection threads within the root hairs, which allowS . meliloti cells to invade the developing root nodules [14,16, 31] . A successful invasion of nodules by S . meliloti willresult in the formation of pink nitrogen-fixing nodules . The pink color is due to the presence of leghemoglobin . Nodules that are not occupied by S . meliloti and/or not capable of fixing nitrogen are most often white due to the lack of leghemoglobin [20].

The formation of infection threads inside root hairs requiresthe presence of an S . meliloti exopolysaccharide, succinoglycan[9], in addition to the Nod factor [33] . Succinoglycan is a polymer that consists of different numbers of a repeating unit consisting of one galactose and seven glucoses with three modification groups: acetyl, pyruvyl, and succinyl [17, 24] . All three modificationsmust be present in order for the S . meliloti succinoglycan tobe active in eliciting infection thread formation [9] . Surprisingly, overproduction of succinoglycan appears to reduce efficiencyof nodulation [12].

Two S . meliloti mutants, exoR95::Tn5 and exoS96::Tn5, were isolatedbased on their ability to overproduce succinoglycan [12] . TheexoR gene encodes a protein of 268 amino acids that shares nosignificant homology with any other protein in currently available databases [23] . The exoS gene encodes the membrane-bound sensorof the ExoS/ChvI two-component regulatory system, and the exoS96mutation might have resulted in the formation of a constitutivelyactive version of the sensor [10] . Two close homologs of theexoS gene, Agrobacterium tumefaciens chvG [8] and Brucella abortus bvrS [29], are essential to the pathogenicity of their respectivehosts [8, 29] . The A . tumefaciens chvG gene is involved in regulatingacid-inducible genes [18], and B . abortus bvrS is involved in regulating membrane protein expression [15] . Both the exoR95and exoS96 mutations resulted in the upregulation of the 22succinoglycan biosynthesis genes, with the exoYFQ operon asthe primary target of the regulation [23] . The exoY gene encodesa galactose transferase that carries out the first step of succinoglycanbiosynthesis, and the exoY210::Tn5 mutation completely blockssuccinoglycan biosynthesis [17, 25].

The exoR95 mutation resulted in a reduction of nodulation efficiency so that some of the plants inoculated with the exoR95 mutants were tall and green with pink nodules, while the others were short and yellow with white nodules [12] . The exoR95 mutantalso appeared to have a reduced efficiency in colonizing curledroot hairs [9] . The exoS96 mutation did not change the nodulationefficiency significantly, even though it appeared to cause areduction in the efficiency in colonizing curled root hairs[9].

The exoR95 and exoS96 mutants have been linked to the reduction of cellular motility, based on the findings that the two mutants formed smaller colonies on swarming plates [35] . Two S . melilotimutations have been isolated that increase motility and thesizes of colonies on swarming plates and reduce succinoglycan production [35] . These findings raised the possibility thatthe regulation of succinoglycan production and cellular motility could be coupled . The coupling of the regulation of bacterial exopolysaccharide production and the regulation of cell motilityhas been found in Ralstonia solanacearum [5], Vibrio cholerae[1, 34], and Salmonella enterica [7], which presumably increases the ability of the cells to interact with their prospective host cells.

S . meliloti cellular motility is supported by two to eight peritrichoussemirigid flagella which allow the bacterium to move effectivelyin viscous liquid [6] . These flagella rotate in one directionat different speeds, which allows the bacterial cells to alterdirection [27] . The S . meliloti cellular motility requires thefunction of the chemotaxis, flagellum, and motility genes, whichare tightly regulated in a hierarchical order from class I toclass III [30] . Class I genes comprise the visN and visR genes[30] . Class II genes, which comprise flagellar assembly andmotor genes, are controlled by the class I genes [30] . Class III genes include flagellin and chemotaxis genes [30] . Any lossor change of gene function in the hierarchy could result in the loss of cellular motility, which would reduce nodulation efficiency [2-4, 6].

To further understand the roles of the ExoR protein and the ExoS/ChvI two-component signal transduction system in the S. meliloti-alfalfa symbiosis, systematic analysis of the symbiotic properties and cell motility of both mutants was conducted.Global gene expression profiles were analyzed by using oligonucleotide microarrays and confirmed by reverse transcription-PCR [RT-PCR]. These results link the regulation of succinoglycan biosynthesisto the expression of the flagellum biosynthesis genes.

 
Strains and bacterial media. The S . meliloti strains used in this study are shown in Table1 . S . meliloti Rm1021 [Str^r] was used as the wild-type strain [17], and MT616[pRK600] [Cm^r] was used as a helper for conjugation[17] . The S . meliloti phage used for general transduction was M12 [19] . The S . meliloti succinoglycan-deficient exoY210::Tn5[Neo^r] mutant was used as a control [17] . Two S . meliloti succinoglycan overproduction mutants, exoR95::Tn5 [Neo^r] and exoS96::Tn5 [Neo^r],were used to study the function of the exoR and exoS genes.To facilitate the construction of double mutants, the transposonTn5 in the exoR95::Tn5 and exoS96::Tn5 mutants was replaced with the transposon Tn5-233 to generate exoR395::Tn5-233 [Gm^r]and exoS396::Tn5-233 [Gm^r] mutants, so that the exoR395::Tn5-233and exoS396::Tn5-233 mutants were the same as the exoR95::Tn5 and exoS96::Tn5 mutants with the exception of the antibiotic markers [12] . The exoR95 and exoR395 mutants have been usedinterchangeably in some of the experiments described in thispaper . The same is true for the exoS96 and exoS396 mutants.

 
Luria-Bertani [LB] medium was used for the growth of Escherichia coli strains, and LB supplemented with 2.5 mM MgSO₄ and 2.5 mM CaCl₂ [LB/MC] was used for all S . meliloti strains [17]. TY medium was used for growth of S . meliloti strains for microarray analysis . Agar [1.5%] was added to make solid medium . Antibiotics were used at the indicated concentrations: chloramphenicol [Cm], 10 µg/ml; gentamicin [Gm], 15 µg/ml; kanamycin,25 µg/ml; neomycin [Neo], 200 µg/ml; streptomycin[Str], 500 µg/ml.

Nodulation efficiency. To characterize the functions of ExoR and ExoS/ChvI proteins,exoR95 and exoS96 mutants and other strains were analyzed fortheir ability to establish nitrogen-fixing symbiosis with alfalfaby determining their nodulation efficiency, which was measuredby the color and the number of nodules on roots of alfalfa plantsinoculated with these strains . The symbiotic ability of thebacterial strains can also be measured by their ability to colonizecurled alfalfa root hairs [root hair colonization efficiency],to invade alfalfa roots through root hairs [root hair invasionefficiency], and to invade root nodules [nodule invasion efficiency].

Nodulation efficiency assays were carried out as previously described [17] . Alfalfa [M . sativa cv . Iroquois] seeds weresurface sterilized and germinated in the dark . The bacterialcells [0.1 ml] were plated on the surface of solid plant growthmedium, Jensen's agar [22], inside a square petri dish . Foreach bacterial strain, 0.1 ml of cell suspension was placedon top of solid plant growth medium, Jensen's agar . Ten 2-day-oldalfalfa seedlings were spread out evenly across the plate . Astack of 10 plates was wrapped with aluminum foil and placed in a plant growth chamber on one side of the petri dishes so that the shoots of the seedlings were pointing up . To determinethe effect of the size of the inoculum on nodulation efficiency,plants were inoculated with decreasing amounts of inoculum.One milliliter of cell suspensions at three different cell concentrations,10⁷, 10⁵, and 10³ cells/ml, was used to inoculate a set of 10 plants in one square petri dish . Ten plants were used for each strain at each cell concentration to minimize the influenceof the individual alfalfa plant on the results . The alfalfaplants were checked for plant height, the numbers of nodules,and the percentage of nodules that were pink each week . Thegrowth of the plants was documented using a Kodak digital camera.

The nodule invasion efficiency was measured as the percentageof the nodules that were pink . The formation of pink nodulesis the result of bacterial colonization of the plant cells insidethe nodule, which elicits the production of leghemoglobin [11].

Root hair invasion efficiency assays were carried out as previously described [9] . Briefly, alfalfa seedlings growing on microscopeslides covered with a layer of solid plant growth medium wereinoculated with bacterial cell suspension and covered with a single layer of dialysis membrane . These slide assemblies were placed inside a 50-ml culture tube with beveled bottom . Culturetubes were filled with liquid Jensen's medium to cover the lowerpart of the slide to support growth . Plants were examined dailyusing an Olympus AX70TRF fluorescence microscope to score thenumbers of colonized curled root hairs, root hairs with initiatedinfection threads, and extended infection threads . The combinednumber of all three events represents the total number of eventsof colonization of curled root hairs, because initiated infectionthreads and extended infection threads are the result of colonizationof curled root hairs . The percentage of colonization of curledroot hairs that further developed into extended root hairs representsthe root hair invasion efficiency.

Cell motility and flagellum staining. Cell motility was examined using swarming plates and phase-contrastmicroscopy as described previously [35] . Briefly, bacterial strains were inoculated onto LB/MC soft agar medium and incubatedfor 4 days to determine colony size . Bacterial cells from mid-logphase were mixed with an equal volume of 0.4% high-viscositycarboxymethyl cellulose and observed under a phase-contrastmicroscope to determine their motility.

Bacterial flagella were stained using a method similar to that used in prior work [21] . Slides were rinsed with 95% ethyl alcohol,dried with lintless paper tissue, and passed through a flame.A loopful of bacterial cell suspension was transferred to one end of the clean slide, and the slide was tilted to allow the drop of cell suspension to run to the other end . Cell suspensions were air dried . The slide was flooded with the flagellum mordantfor 10 min, rinsed gently with distilled water, and floodedwith Ziehl's carbol fuchsin for 5 min . The slide was rinsedoff gently and air dried . Bacterial flagella were examined andphotographed under a phase-contrast microscope.

Whole-genome analysis of gene expression . [i] Printing and layout of Sm6kOligo microarrays. Each array contained 6.223 70-mer oligonucleotides and threePCR fragments printed in three replicates, except for a setof 12 control genes [sma1118, smb21183, smb21295, smc00323,smc00335, smc00363, smc00646, smc01106, smc02857, smc03859, smc03979, and smc04040], which were printed in 51 replicates. The 70-mer oligonucleotide set was designed and synthesizedby QIAGEN [Hildesheim, Germany] based on the three NCBI refseqs: NC_003047 [chromosome], with the database file NC_003047.ffn; NC_003037 [pSymA], with the database file NC_003037.ffn; and NC_003078 [pSymB], with the database file NC_003078.ffn [updatedin March 2002; ftp://ftp.ncbi.nih.gov/genomes/Bacteria/Sinorhizobium_meliloti/]. As further controls, three alien DNA PCR fragments [spot reportalien PCR product 1, Stratagene 252551; spot report alien PCRproduct 2, Stratagene 252552; spot report alien PCR product3, Stratagene 252553 [Stratagene, La Jolla, Calif.]], four 70-meroligonucleotides directed against transgenes [gusA, lacZ, nptII, and aacC1], and two 70-mer stringency control oligonucleotides directed against smc02725 and smc03990 [80% identity] were spotted in three replicates . As negative controls, 12 alien 70-mer oligonucleotideswere spotted in 48 replicates.

PCR fragments [200 ng/µl] and oligonucleotides [40 µM]in 1.5 M betaine, 3x SSC [1x SSC is 0.15 M sodium chloride plus0.015 M sodium citrate] [11a] were printed onto Creative Chip3D slides [Eppendorf, Hamburg, Germany] using the MicroGridII 610 spotter [BioRobotics, Cambridge, United Kingdom] equippedwith 48 SMP3 stealth pins [TeleChem International, Sunnyvale,Calif.] . DNA was cross-linked to the surface by incubation ofthe slides for 1 h at 80°C . Processing of the slides includedthe following washes: 0.1% Triton X-100 for 5 min at 20°C;0.05% HCl, two times for 2 min at 20°C; 0.1 M KCl for 10 min at 20°C; H₂O for 1 min at 20°C; 0.05% HCl-25% ethylene glycol for 15 min at 50°C; and H₂O for 1 min at 20°C. Slides were dried by centrifugation [3 min, 185 x g, 20°C].

[ii] RNA purification and synthesis of labeled cDNA. RNA was isolated as described previously [31] . Cy3- and Cy5-labeled cDNA was prepared according to the method of deRisi et al . [12] from 10 to 15 µg of total RNA [http://www.microarrays.org/protocols.html]. For each microarray experiment, five slide hybridizations were performed using the labeled cDNA synthesized from three independent RNA preparations obtained from three independent bacterial cultures.

Hybridizations, image acquisition, and data analysis. For each comparison, hybridizations accounting for at leasttwo biological and up to two technical replicates were conducted. Hybridizations, image acquisition, and data analysis were performed as described previously [31] . Shortly, mean signal and mean local background intensities were obtained for each spot ofthe microarray images using the ImaGene 5.0 software for spotdetection, image segmentation, and signal quantification [BiodiscoveryInc., Los Angeles, Calif.] . The log₂ value of the ratio of intensities was calculated for each spot by using the formula M_i = log₂[R_i/G_i].R_i = I_ch1i – Bg_ch1i, and G_i = I_ch2i – Bg_ch2i, whereI_ch1i or I_ch2i is the intensity of a spot in channel 1 or channel2 and Bg_ch1i or Bg_ch2i is the background intensity of a spotin channel 1 or channel 2, respectively . A normalization methodbased on local regression that accounts for intensity and spatialdependence in dye biases was applied [37] . Normalization andt-statistics were carried out using the EMMA 1.1 microarraydata analysis software developed at the Bioinformatics ResourceFacility [Center of Biotechnology, Bielefeld University; www.genetik.uni-bielefeld.de/EMMA/][14] . Genes were regarded as differentially expressed if P was 0.05 and M was 1.00 or [–1.00] [at least a twofold difference].

Detection of gene expression using RT-PCR. Total RNA was collected from the cells of S . meliloti Rm1021and exoR95 and exoS96 mutants by using an RNA extraction kit[RNAex reagent; Huashun Biotechnology Co., Ltd., Shanghai].Briefly, bacterial cells were collected, mixed with RNAex reagent,and then extracted with chloroform . Total RNA was precipitatedwith isopropanol, washed with ethanol, dried, resuspended, andstored at –70°C . The detection of gene expressionwas carried out using an RT-PCR kit [TaKaRa Biotechnology, Dalian,China] following the instructions from the manufacturer . Theset of primers for targeted genes [visR, visN, and flaA] anda set of primers for the control gene [rpsF] were used in thesame RT-PCR . The RT-PCR products were resolved on agarose gelto determine whether a targeted gene was expressed.

 
Succinoglycan overproduction reduces nodulation efficiency of the exoR95 mutant but not of the exoS96 mutant. To systematically and quantitatively determine the nodulationefficiency of exoR95 and exoS96 mutants, alfalfa plants growing inside petri dishes were inoculated with one of the two mutantsor the wild-type strain Rm1021 as control . To better characterizethe difference in nodulation efficiency, plants were inoculatedwith decreasing amounts of bacterial cells.

At the end of the fifth week, alfalfa plants that were not inoculated with any S . meliloti cells had already turned yellow and had started dying from lack of nitrogen . The alfalfa plants inoculated with all three concentrations, 10⁷, 10⁵, and 10³ cells/ml, ofthe wild-type strain Rm1021 were tall and green [Fig. 1] . Theefficiency of the nodule invasion by the wild-type cells decreased30% when the concentration of cells in the inoculum decreasedto 10³ cells/ml.

 
The plants inoculated with the exoR95 mutant were mostly yellow with a few light green leaves . Some of the plants were slightly greener than the others, which corresponded to a higher percentageof nodules that were pink on those plants . The nodule invasion efficiency was dramatically lower for the exoR95 mutant than for wild-type strain Rm1021, which suggested that the exoR95 mutant was unable to invade the nodules that it elicited onthe alfalfa plants [Fig . 1].

The plants similarly inoculated with the exoS96 mutant were mostly green and tall, like those inoculated with the wild-type cells . The nodule invasion efficiency of the exoS96 mutant was not reduced but was higher than that of the wild-type cells[Fig. 1] . The average number of pink nodules per plant was about the same for the plants inoculated with the wild-type cellsor the exoS96 mutant, which is consistent with the notion that alfalfa plants can regulate the number of pink nodules basedon their needs [11].

These results suggest that even though both the exoR95 and exoS96 mutations result in the overproduction of succinoglycan, only the exoR95 mutation causes a reduction in nodule invasion efficiency and, subsequently, a reduction in nodulation efficiency . The exoS96 mutation does not significantly alter the nodule invasion efficiency or nodulation efficiency.

Root hair invasion efficiency of the exoR95 and exoS96 mutants. Root hair invasion is the key step in nodulation, and it can be quantitatively measured using fluorescently labeled S . meliloti cells as described in detail in Materials and Methods . Succinoglycan plays an important role in the initiation of infection thread formation during root hair invasion, and succinoglycan overproduction appears to interfere with root hair invasion [9] . A large systematicanalysis of the impact of succinoglycan overproduction on roothair invasion was needed to understand the role of the exoR and exoS/ChvI genes in symbiosis.

Root hair invasion can be divided into three stages: colonization of curled root hairs, initiation of infection thread formation,and extension of infection threads to the base of the root hairs.Root hair invasion can be blocked at the initiation of infectionthread formation or the development of infection threads beforethey reach the base of the root hairs . On 12 alfalfa plantsinoculated with wild-type strain Rm1021, the average numberof total root hair invasion events per plant was 3.3 [Fig . 2A],and they were all in the form of long extended infection threadsinside root hairs . This suggested that every root hair thatwas colonized by the bacterial cells developed long and extendedinfection threads . Root hair invasion by the wild-type strainwas 100% efficient . On the 12 alfalfa plants inoculated withthe exoY210 mutant, the average number of total root hair invasionevents per plant was 0.67 . None of them developed into infectionthreads, and so the root hair invasion efficiency of this mutantwas 0% [Fig . 2B].

 
On the 12 alfalfa plants inoculated with the exoR95 mutant, the average number of root hair invasion events per plant was0.08 [Fig . 2A] . Only one root hair containing an extended infection thread was found . No other forms of root hair invasion events were found, so that the one single curled root hair that was colonized by the exoR95 mutant developed an infection thread inside the colonized root hair . Similar results have been foundin different sets of experiments [data not shown], suggestingthat the root hair invasion by the exoR95 mutant was extremely efficient [Fig . 2B], but the number of root hairs that were colonized by the exoR95 mutant dropped dramatically compared to that of the wild-type strain . These findings suggest that nodulation of alfalfa by the exoR95 mutant might be blocked before or at the step of colonizing curled alfalfa root hairs.

On the 12 alfalfa plants inoculated with the exoS96 mutant, the average number of root hair invasion events per plant was0.42 [Fig . 2A], which represented five root hairs with extended infection threads . No colonized root hairs or initiated infection threads were found, so that all of the curled colonized root hairs developed infection threads . The root hair invasion bythe exoS96 mutant can be considered 100% efficient [Fig . 2B], but similar to that of the exoR95 mutant, the number of total root hair invasion events was dramatically lower for the exoS96 mutant . These findings suggest that nodulation of alfalfa by the exoS96 mutant is most likely blocked before or at the step of colonizing curled alfalfa root hairs.

If the reduction of colonization of the curled root hairs werethe result of succinoglycan overproduction, blocking succinoglycanshould allow these two mutants to become as efficient as theexoY210 mutant in colonizing curled root hairs . To test thishypothesis, the exoR395exoY210 and exoS396exoY210 double mutantswere constructed by transducing the exoY210::Tn5 mutation into the exoR395::Tn5-233 or exoS396::Tn5-233 mutants . Both doublemutants, exoR395exoY210 and exoS396exoY210, were even less efficientin colonizing curled root hairs . On the sets of 12 alfalfa plantsinoculated with either one of the two double mutants, no curledroot hair was found to be colonized . These findings raise thepossibility that overproduction of succinoglycan might not bethe only factor contributing to decreased efficiency in colonizingcurled alfalfa root hairs.

Effects of the exoR95 and exoS96 mutations on cell motility. The loss of cell motility has been shown to reduce the nodulationefficiency of S . meliloti [3], which could account for the lowernodulation efficiency of the exoR95 and exoS96 mutants . Thesefindings and previous reports of the reduced cellular motilityby the exoR95 and exoS96 mutations [35] made it essential to test the motility of these two mutants.

To examine the link between cellular motility and succinoglycan production, both wild-type strain Rm1021 and its succinoglycan-deficient mutant, exoY210, were tested on swarming plates as controls. Both Rm1021and the exoY210 mutant formed large diffuse colonies [Fig . 3], suggesting that blocking succinoglycan productiondoes not affect cellular motility . When the exoR95 and exoS96mutants were similarly examined on swarming plates, they formedsmaller and smooth colonies, suggesting that both the exoR95and exoS96 mutations decreased cellular motility . If overproductionof succinoglycan was the reason for the decrease in motility,blocking succinoglycan production by these two mutants shouldrestore their cellular motility . Two double mutants, exoR395exoY210and exoS396exoY210, were similarly tested on swarming plates,and they both formed small tightly packed colonies that weresimilar to those formed by single exoR95 and exoS96 mutants.These findings suggested that the suppression of cellular motilityby the exoR95 and exoS96 mutation was not just the result ofoverproduction of succinoglycan.

 
To directly confirm that decreased cellular motility was relatedto the exoR95 and exoS96 mutations, bacterial cells of wild-type strain Rm1021 and the exoR95, exoS96, exoY210, exoR395exoY210,and exoS396exoY210 mutants were observed directly under a phase-contrastmicroscope . Bacterial cells of Rm1021 and the exoY210 mutantwere motile, while the exoR95, exoS96, exoR395exoY210, and exoS396exoY210 mutant cells were nonmotile . These results were consistent withthe findings of the swarming plate experiments and again suggestedthe overproduction of succinoglycan was not responsible forthe decreased motility of the exoR95 and exoS96 mutants.

The exoR95 and exoS96 mutations result in the loss of flagella. The production of flagella and the speed of flagellum rotationare highly regulated in S . meliloti [30] . The loss of cell motilitycould be the result of the loss of flagella or interferencein flagellum rotation . To determine whether the exoR95 and exoS96mutations resulted in the loss of flagella, cells of the wild-typestrain and the exoR95, exoS96, exoY210, exoR395exoY210, and exoS396exoY210 mutants were stained for flagella and examined under a phase-contrast microscope . The cells of wild-type Rm1021and the exoY210 mutant possessed flagella [Fig . 4] . The flagellaof Rm1021 cells were examined in detail by using transmissionelectron microscopy, and the findings were in agreement withprevious reports [data not shown] . The cells of the exoR95, exoS96, exoR395exoY210, and exoS396exoY210 mutants did not haveflagella . The fact that the exoY210 mutant produced flagellaand that neither the exoR395exoY210 nor exoS396exoY210 doublemutants produced flagella suggested that succinoglycan overproductionwas not responsible for blocking flagellum biosynthesis . Thesefindings also raised the possibility that ExoR and ExoS mayplay some roles in regulating flagellum biosynthesis in additionto regulating succinoglycan biosynthesis.

 
The exoR and exoS96 mutations suppress the expression of the flagellin genes. To confirm that the expression of S . meliloti flagellin biosynthesisgenes is regulated by the ExoR protein or the ExoS/ChvI two-componentregulatory system, the expression of all S . meliloti genes inthe exoR95 and exoS96 mutant backgrounds was analyzed . TotalRNA was isolated from free-living exoR95 and exoS96 mutantsas well as from the wild-type strain, Rm1021 . The total RNAwas reverse transcribed into fluorescently labeled cDNA andhybridized to Sm6kOligo microarrays containing probes for allpredicted protein-coding genes of S . meliloti Rm1021 . The exoR95mutation resulted in downregulation of the expression of 160genes and putative open reading frames and in upregulation of136 genes and putative open reading frames in the range of two-to eightfold . The exoS96 mutation caused downregulation of 129genes and putative open reading frames and upregulation of 131genes and putative open reading frames in the range of two-to eightfold . Both exoR95 and exoS96 mutations downregulatedthe expression of all five S . meliloti flagellum biosynthesisand regulatory genes 2.7- to 5.5-fold [Table 2] . The expressionof the fla genes was downregulated further in the exoR95 mutantbackground.

 
The effects of the exoR395 and exoS396 mutations, which were the same as the effects of the exoR95 and exoS96 mutations, on the expression of the flagellin gene were further analyzed using RT-PCR [Fig . 5] . Expression of the rpsF gene, which encodesthe S6 protein for the 30S ribosome subunit, was used as aninternal control . Expression of the rpsF gene was detected inthe wild type and the exoR395 and exoS396 mutants . Expressionof the flaA gene, which encodes a flagellin subunit, was detectedin the wild type but not in either of the mutants . Expressionof the visN and visR genes, which serve as the primary regulatorsof flagellum biosynthesis, was detected in the wild type andthe exoR395 and exoS396 mutants.

 
Together, these findings suggest that both ExoR and ExoS govern flagellum synthesis by regulating the expression of the fla genes, but they are not involved in regulating the expressionthe visN and visR genes, which play key roles in regulating S . meliloti flagellum biosynthesis.

 
The presence of succinoglycan is essential to successful roothair invasion and nodulation of alfalfa by S . meliloti [9, 17],and so it was puzzling that succinoglycan overproduction appearedto change nodulation efficiency of the exoR95 mutant but notof the exoS96 mutant [12] . This suggests that there might beother factors related to the overproduction of succinoglycan.Since both the ExoR and ExoS proteins are involved in signaltransduction, both of them could be involved in other symbioticprocesses in addition to regulating succinoglycan production.

The systematic analysis of nodulation efficiency of the mutants showed that the exoR95 mutant was less efficient in nodule invasion than its wild-type parent, while the exoS96 mutant was as efficient as its wild-type parent . To determine at which point the exoR95 mutant becomes less efficient in nodule invasion, the efficiencies of colonizing curled root hair and root hair invasion were compared for the exoR95 and exoS96 mutants and other strains.

The efficiencies of root hair invasions of the two mutants were not affected, but the efficiencies of colonizing curled roothairs by these mutants were reduced dramatically . This loweredefficiency of colonizing curled root hairs could be compensatedby a longer time of interaction between the mutants and alfalfa,which could explain why the drop in the efficiency of colonizingroot hairs showed little impact on nodulation efficiency bythe exoS96 mutant . These findings and the report of the exoR95and exoS96 mutants showing reduced motility on swarming plates[35], as well as another report of the isolation of an exopolysaccharide-overproducing mutant with decreased motility [26], brought the cellular motilityof these two mutants into focus.

Our analysis showed that neither the exoR95 nor exoS96 mutants were able to swarm or swim . When stained directly for flagella, neither exoR95 nor exoS96 mutants showed flagella, while the wild-type cells were flagellated . This reduction in flagellum production could be the result of energy stress on the cellsor direct involvement of the ExoR and ExoS proteins in regulating flagellum biosynthesis . Further analyses showed that blocking succinoglycan production in the exoR395 and exoS396 mutant backgroundsdid not restore flagellum production . These findings are consistentwith the possibility that the ExoR protein and ExoS/ChvI two-componentregulatory system are involved in the regulation of flagellumbiosynthesis directly or indirectly.

To examine the possible involvement of the ExoR protein andthe ExoS/ChvI two-component regulatory system in regulatingexpression of the flagellum biosynthesis genes, expression ofthe entire genome of the exoR95 and exoS96 mutants was examinedusing DNA microarray analysis technology . While the expressionof numerous genes was changed by either the exoR95 mutationor the exoS96 mutation [the complete set of data will be publishedin the future], the expression of all five flagellum biosynthesisgenes was consistently repressed by both mutations . These findingssuggest that both the ExoR protein and the ExoS/ChvI two-componentsignal transduction system play key roles in regulating bothsuccinoglycan production and flagellum biosynthesis, directlyor indirectly.

S . meliloti fla genes are at the bottom of the chemotaxis regulatory hierarchy, and their expression is regulated by the VisR and VisN proteins at the first level and by the FliM protein andthe protein encoded by open reading frame 38 at the second level[30] . The regulatory mechanism[s] of the VisR and VisN proteinsis not clear . It has been suggested that they might form heterodimers[30] . Our findings that both exoR95 and exoS96 mutations downregulated the fla genes but not the visR and visN genes provided the firstnew set of links between succinoglycan and flagellum productionthrough the ExoR protein and the ExoS/ChvI two-component regulatorysystem . The ExoR protein and the ExoS/ChvI system could regulateboth succinoglycan and flagellum production via signal transductionpathways . Suppressor mutations have been isolated that alterthe swarming and succinoglycan production phenotypes of theexoR95 mutant [data not shown], making it more likely that thereare genes downstream from the ExoR protein and ExoS/ChvI system that are involved in regulating both succinoglycan and flagellum production . Alternatively, the expression of the genes regulated by the ExoR protein and ExoS/ChvI system could lead to physiological or structure changes that are sensed by the VisR and VisN proteins in regulating flagellum production . Efforts are currently under way to identify the genes that encode proteins that are downstreamof ExoR or ExoS/ChvI and are involved in regulating succinoglycanand flagellum production . Other studies are being carried outto further study the clear differences in nodulation invasionefficiency between the exoR95 and exoS96 mutants.

All together, these findings suggested that flagellum biosynthesis and succinoglycan production might be coordinated at the levelof gene expression . The multiple peritrichous flagella couldincrease the ability of S . meliloti cells to move towards alfalfaroots to bring themselves closer to the root hair surface [6], although flagella themselves might not be involved in attaching to the surfaces of root hairs [28] . The flagellum production might be downregulated after the attachment . Once attached to and colonizing alfalfa roots, the expression of succinoglycan biosynthesis was induced [H.-P . Cheng, unpublished results],and succinoglycan elicits the formation of infection threadsinside curled root hairs [9] . Such coordinated production of flagella and succinoglycan could be necessary to ensure efficient nodulation.

The closely coordinated biosynthesis of flagella and exopolysaccharide has been found in many other bacterium-host interactions, since flagella play an important role in bacterial pathogenicity [36]. The V . cholerae epsD and epsE genes appear to coordinate the biosynthesis of flagella and exopolysaccharide during biofilm formation [1, 34] . The S . enterica igaA gene regulates the productionof capsule polysaccharide and cell motility [7] . The R . solanacearumphcA gene regulates polysaccharide production, endoglucanaseactivity, and motility [5] . The studies of the coordinated biosynthesis of S . meliloti flagella and succinoglycan biosynthesis will contribute to the general understanding of the interactions between bacteria and their prospective hosts.

 
We thank Eva Schulte-Berndt for technical assistance.

This work was supported by grants from NIH [5S06GM08225] and PSC-CUNY [617320030 and 632140032], from Bundesministerium für Forschung und Technologie, Germany [national grants 0311752and 031U213D] and the "Bioinformatik Initiative" by Deutsche Forschungsgemeinschaft [BIZ 7], and from The National High Technology [863] International Research Program [2001AA214211] and theNational Key Program for Basic Research of China [2001CB108901].

 
* Corresponding author . Mailing address: Biological Sciences Department, Lehman College, The City University of New York, 250 Bedford Park Blvd., West, Bronx, NY 10468 . Phone: [718] 960-7190 . Fax: [718] 960-8236 . E-mail: haiping@lehman.cuny.edu.

Present address: Greenwich High School, Greenwich, CT 06830.

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