Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures.No information is available regarding the regulation of flagellar formation in B . subtilis in response to contact with a solid surface . One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates . This study describes the swarming abilityof a B . subtilis hypermotile mutant harboring a mutation inthe ifm locus that has long been known to affect the degreeof flagellation and motility in liquid media . On solid media,the mutant produces elongated and hyperflagellated cells displayinga 10-fold increase in extracellular flagellin . In contrast tothe mutant, the parental strain, as well as other laboratorystrains carrying a wild-type ifm locus, fails to activate aswarm response . Furthermore, it stops to produce flagella whentransferred from liquid to solid medium . Evidence is providedthat the absence of flagella is due to the lack of flagellingene expression . However, restoration of flagellin synthesisin cells overexpressing ^D or carrying a deletion of flgM doesnot recover the ability to assemble flagella . Thus, the ifmgene plays a determinantal role in the ability of B . subtilisto contact with solid surfaces.

 
Bacterial structures facing the cell surface play a criticalrole in establishing and maintaining interactions with the environment.In motile eubacteria, the flagellar organelle is the most complexand exposed extracytoplasmic structure and is made up of manyproteins that are sequentially assembled from the cytoplasmicmembrane outward . This highly regulated process is governed,at least in part, by the hierarchical expression of flagellargenes that are organized in classes in both the enteric bacteriaand Bacillus subtilis . While class II genes encode structuraland regulatory proteins needed for assembly of the hook-basalbody, class III genes, whose expression depends on the late-flagellar-stagesigma factor [²⁸ in the enterics and ^D in B . subtilis], encodeproteins required for the maturation of flagella and the chemosensorysystem [reviewed in references 1, 22, and 30].

The flagellum is essential for active movement of individualcells in a liquid environment [swimming] and for chemotaxisand plays an important role in interaction with surfaces asa sensor of medium viscosity [23] or as an adhesion tool [12]. Flagellum-driven motility may help pathogens to reach their target, thus contributing to bacterial virulence [32].

Bacteria may experience two different life-styles, dependingon whether they grow in liquid environments or are in contactwith solid surfaces . The transition from free to sessile growthrequires the sensing of yet unknown surface-related signalsand their processing to trigger a productive interaction withthe solid surface . This type of adaptation is particularly importantfor soil bacteria such as B . subtilis that rapidly shift fromplanktonic to sessile life, depending on microenvironmentalconditions.

Among the best-characterized responses of flagellated bacteriato contact with solid surfaces is the cooperative behavior knownas swarming motility . Swarming can be considered a strategyfor rapid spread over solid surfaces in the environment andfor active colonization of mucosal surfaces in infected hosts[2, 4] . Swarming bacteria produce highly organized communities of elongated and aseptate cells that exhibit a remarkable increase in the number of flagella in comparison to short oligoflagellated cells growing in liquid media [8, 15, 17, 19, 39] . Although it has been shown that swarming motility requires the integrity of the flagellar and chemotaxis systems in both gram-negativeand gram-positive bacteria [5, 7, 11, 14, 17, 20, 31, 39, 41], almost nothing is known about the molecular mechanisms involvedin the sensory transduction pathways of cell differentiationin response to solid clues.

Strikingly different colony patterns have been described forB . subtilis, depending on nutrient availability, agar concentration [36], and surfactin production [20] . In this study, we describethe isolation of a B . subtilis hypermotile mutant that exhibitsthe ability to swim or swarm depending on whether it is propagatedin liquid or on solid media . The hypermotile strain carriesa mutation in the ifm locus described in 1969 by Grant and Simon[13] that affects the degree of motility and level of flagellationof B . subtilis during growth in liquid media . In contrast tothe mutant, we found that the parental strain does not mounta swarming response and even stops to produce flagella whentransferred from liquid to solid medium . Evidence is also providedthat other motile B . subtilis strains carrying a wild-type ifmlocus are unable to swarm and to produce flagella over solidsurfaces.

 
Bacterial strains and media. Table 1 lists the B . subtilis strains used in this study . Formapping purposes, a set of B . subtilis mutants constructed byseveral laboratories [38] was used . The strains were grown at 37°C in either tryptone-NaCl [1% tryptone, 0.5% NaCl [TrB]], nutrient broth, Schaeffer sporulation medium, or Luria-Bertanibroth [LB] . The media were routinely solidified with 1.5% agarunless otherwise noted . Surfactin production was assayed onblood agar plates [Columbia agar with sheep blood; Oxoid] asdescribed by Nakano et al . [29] . Escherichia coli DH5 supE44lacU169 [80 lacZM15] hsdR17 recA1 endA1 gyrA96 thi-1 relA1 wasused as the host for construction of recombinant plasmids . E.coli cells were grown in LB broth . When necessary, antibioticswere used at the following concentrations: 100 µg of ampicillin/ml;1 µg of erythromycin/ml; 2.5 µg of kanamycin/ml;and 5 µg of chloramphenicol/ml.

 
Genetic techniques. B . subtilis strains were transformed with chromosomal or plasmidDNA by using the procedure of Kunst and Rapoport [21] . Transductionmapping with the PBS1 phage was performed according to Hochet al . [18]. E . coli transformation was performed followingstandard protocols [37].

Motility assays. For swimming motility, strains were propagated in gelatin-agar[motility plate] having the following composition: 1% BactoPeptone, 8% Bacto Gelatin, 1% Bacto Agar, 0.5% NaCl, and 25µg of the appropriate growth requirement/ml [Table 1].Phenotypic assays for swarming were initiated by spotting 2µl of an overnight culture at the center of tryptone-NaClplates containing 1.5% agar [TrA] . The plates were analyzedafter growth for up to 24 to 48 h of incubation at 37°C. Bacteria were Gram stained for microscopy to evaluate the presence of elongated swarm cells [39] . Flagellum staining was performedas described by Harshey and Matsuyama [16].

Construction of mutant strains. To construct strain PB5250 with a deletion of the flagellin-encodinggene [hag], a sequence of 717 bp was replaced with the kanamycinresistance determinant . First, a 330-bp DNA fragment correspondingto the region upstream of the hag coding sequence [from nucleotide375 to nucleotide 45 with respect to the initiation of transcription]was amplified by PCR by using the primers HagB [5'-CGGGATCCCATTATTG TGAATCGCAAG-3'] and HagE [5'-CGGAATTCAAAAAAATCCTCACTTTTTTTGTGAGGAT-3']. The BamHI [HabB] and EcoRI [HagE] recognition sequences are in bold . The template was chromosomal DNA of B . subtilis PB168. The amplified fragment was purified by acrylamide gel electrophoresis, electroeluted, and restricted with EcoRI and BamHI . After purification,the DNA fragment was ligated into the EcoRI and BamHI sitesof plasmid pJM114 [33], thus generating pTCH1 . The ligated plasmidwas used to transform E . coli DH5 . A second DNA fragment of180 bp, extending from codon 224 to codon 283 of the hag codingsequence, was obtained by PCR by using primers HagC [5'-CCATCGATCAACCAAGTTTCTTCTCAACGT-3']and HagK [5'-GGGGTACCTGAGAAAGAATGTTGTTCTTTG-3'] . The ClaI [HagC] and KpnI [HagK] sites are in bold . The amplified DNA was restricted with ClaI and KpnI, treated as above, and cloned into pTCH1, the pJM114 derivative digested with ClaI and KpnI . The final plasmid, named pTCH2, contained two DNA fragments derived from PB168 flanking the kanamycin resistance determinant . The plasmidwas verified by sequencing . The plasmid DNA [pTCH2] was linearizedby BamHI digestion and used to transform competent cells ofPB168 . After selection for kanamycin resistance, one transformantwas characterized by PCR and named PB5250 . To construct strainPB5332, we followed the marker congression procedure using DNAfrom the surfactin producer strain PB1927 as described by Nakanoet al . [29] . Selection was for Trp⁺ transformants, followedby screening for surfactin production on blood agar plates.

ß-Galactosidase activity assay. To measure ß-galactosidase activity, overnight culturesin sporulation medium were diluted in fresh medium and samples[1.0 ml] were taken at 30-min intervals for reading of opticaldensity at 525 nm [OD₅₂₅] and determination of ß-galactosidaseactivity, which is expressed in modified Miller units [34].

RNA isolation and RT-PCR. Total RNA was purified from B . subtilis cultures grown in TrBor TrA for 6 h . Cells were harvested from plates by washingthe surface of agar plates with cold diethylpyrocarbonate-treatedwater or collected from liquid cultures by centrifugation at4,000 x g for 15 min . After being washed with diethylpyrocarbonate-treated water, about 1 x 10⁸ bacterial cells were resuspended in 450µl of RLT buffer [RNeasy Mini kit; QIAGEN] containing0.35 g of glass beads [diameter 0.1 mm] and vortexed for 15min to break the cells . Samples were centrifuged for 2 min at10,000 x g, and the aqueous phase was removed . Two hundred microlitersof absolute ethanol was added, and the mixture was applied toan RNeasy Mini Spin column [QIAGEN] . After being digested with40 Kunitz units of RNase-free DNase [QIAGEN] for 20 h, totalRNA was eluted from the column according to the instructionsof the manufacturer . An aliquot of the RNA was examined on agarosegel to ensure its integrity . Reverse transcription [RT]-PCRswere performed in one-step reactions . Up to 1 µg of RNAwas mixed with 0.8 µM [each] primer in AMV/Tfl buffer[50 mM Tris HCl [pH 8.3], 50 mM KCl, 10 mM MgCl₂, 10 mM dithiothreitol,0.5 mM spermidine] containing 1.0 mM MgSO4, 0.1 mM deoxynucleosidetriphosphate, 25 U Tfl polymerase [Promega], and 3.75 U of AMVreverse transcriptase [Promega] in a final volume of 25 µl.Reactions were incubated at 48°C for 60 min . PCR amplification was as follows: 30 cycles at 94°C for 1 min, 58°C for1 min, and 72°C for 1 min for the hag gene; 30 cycles at94°C for 1 min, 55°C for 1 min, and 72°C for 1 minfor fliM, flgM, sigD, and cheW . The following primers were used:HAGU1 [5'-CACAATATTGCAGCGCTTAA-3'] and HAGL1 [5'-TAATAATTGAAGTACGTTTTG-3'] for hag, BSFLIMU [5'-CTCCCAAAATGAAATAGATG-3'] and BSFLIML [5'-TTCTCCATCTTGTTCACCTCT-3']for fliM, FLGMF1 [5'-AATCAATTTGGAACACA-3'] and FLGMR3 [5'-ATTTGCGTCTACTTTGTA-3']for flgM, BSSIGDU [5'-GAATTATGAAGATCAGGTG-3'] and BSSIGDL [5'-TTGTATCACTTTTTCCAGCAG-3']for sigD, and CHEWF [5'-GGTAAATGGCAAAGAATATG-3'] and CHEWR [5'-AGCTTGATCGGGCACAG-3']for cheW . Contamination by DNA was checked by performing reactions without the addition of the AMV reverse transcriptase . Positive controls were obtained by using genomic DNA as the template.

Protein samples, gel electrophoresis, and immunoblot analysis. Bacterial cells were grown in TrB or TrA for 8 h at 37°C.Cells were harvested from the plates by washing the surfacesof agar plates with cold water and were normalized with respectto the OD₆₀₀ of liquid cultures . Cell suspensions were vortexedand centrifuged at 5,000 x g for 15 min at 4°C . Flagellarfilaments were collected from the supernatants by high-speedcentrifugation at 100,000 x g for 1 h and suspended in proteinsample buffer containing ß-mercaptoethanol [37] . Bacterialpellets were lysed in the same buffer by heating at 95°Cfor 5 min . Protein samples were heated at 95°C for 10 minand subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis[SDS-PAGE] . Gels were either silver stained [6] or electrotransferred to nitrocellulose and probed with rabbit antibodies to B . subtilis flagellin followed by incubation with a secondary antibody conjugated with horseradish peroxidase . The peroxidase activity was visualized by diaminobenzidine colorimetric reaction in accordance with standard procedures [37].

 
Isolation of a B . subtilis hypermotile mutant. A spontaneous hypermotile mutant was selected by spotting thestrain PB1831 [trpC2 pheA1] in the middle of a motility plate. Following overnight incubation at 37°C, the rim of the growthhalo was picked and transferred onto nutrient agar, and singlecolonies were tested for increased motility, essentially asdescribed by Pooley and Karamata [35] . One isolate, referredto as PB5249, was retained and used for subsequent analysis.

The mutation responsible for the hypermotile phenotype was mapped by transformation crosses . B . subtilis strains with antibiotic resistance markers present in different positions on the chromosome[38] were used as DNA donors . Competent cells of the mutantwere transformed, selected for antibiotic resistance, and screenedfor motility . The mutation was mapped between hag and uvrA, at approximately 310° on the B . subtilis chromosome . In particular, 80% cotransformation with a resistance marker presentin the gene yvjA was observed . A more detailed mapping was not attempted . This map position agrees with the previously reported location of an ifm mutation [13, 35]; thus, the PB5249 mutantwas tentatively named ifmP.

The increased motility of PB5249 might be explained by an increased number of flagella . This possibility was supported by the observation that the mutant produced more flagellar antigen than the parental strain . In fact, the presence of a flagellum-specific antiserum completely inhibited motility of the parental strain when includedin motility agar at a final dilution of 1:10,000 . This dilutionwas ineffective with the mutant strain, which required a 10-foldhigher concentration of antiserum [data not shown] . A higheramount of flagellin was detected by gel electrophoresis andimmunoblotting in supernatants from broth cultures of the mutantthan from the parental strain; moreover, flagellin accumulationduring growth started 1 h earlier in the mutant than in theparental strain [data not shown] . The molecular mass of theflagellin monomer from the mutant was the same as the monomerfrom the parent; when mixed in a one-to-one ratio and analyzedby MALDI-TOF mass spectrometry, a single peak was found, withan estimated mass of 32,540 Da [calculated mass, 32,472 Da].

The increased level of flagellin in the ifmP mutant could have different explanations, such as higher transcription rate, increased stability of the protein, or faster assembly of the flagellin monomers . We measured the level of expression of the flagellingene by using a hag-lacZ transcriptional fusion, integrated ectopically into the chromosomes of PB1831 and PB5249, thus generating the strains PB5308 and PB5309, respectively . Thekinetics of ß-galactosidase synthesis was similarin the two strains [Fig. 1], but the peak value in PB5309 [2,500U] was about three times the value obtained with the strainPB5308 [850 U] . Therefore, the observed difference between PB1831and PB5249 in the level of flagellin detected by immunoblottingwas consistent with an increased level of hag gene transcription.

 
ifm and the surface contact response. On TrA plates, the strains PB1831 and PB5249 produced morphologicallydistinct colonies . The parental strain produced extremely roughcolonies [Fig. 2A], while the colonies produced by PB5249 were less rough and often exhibited a layered, terraced appearanceupon aging [Fig . 3A] . Microscopic inspection of the colonies showed that the parental strain formed long cell chains preferentially aligned along the major axis and curled [Fig . 2B] . The filamentswere long, up to 50 µm, and septa were always present [Fig . 2C] . It appeared that the whole community was constitutedby short [2 to 3 µm], rod-shaped cells that were indistinguishablefrom those growing in liquid media [Fig. 2D] . More strikingwas the complete absence of cells bearing flagellar filamentsat every stage of colony development . Flagella were never viewedat agar concentrations ranging from 2 to 0.5% [Fig . 2H]; however,the ability of PB1831 to produce flagella in liquid medium [Fig. 2E] was retained at agar concentrations lower than or equal to 0.3% [Fig . 2F and G] . Of great interest was the observationthat this behavior was not peculiar to PB1831 but was sharedby all the B . subtilis strains carrying a wild-type ifm locus,such as the reference strain 168 [PB168] and two of its oldestderivatives, PB25 and PB1424 [Table 1] . Cells taken from theifmP mutant colonies [Fig . 3B] were longer [8 to 16 µmlong] [Fig . 3C] than cells grown in liquid medium [Fig . 3D]and were without septa . Moreover, an increased number of flagellawas seen on elongated cells [Fig . 3E and F] in comparison with that for the oligoflagellated cells grown in liquid media [Fig. 3G] . Flagella of the hyperflagellated cells appeared to be veryfragile, since large amounts of flagellar filaments were very often observed detached from cells in stained preparations [Fig. 3F] . The finding that the strain PB5249 produces elongated andhyperflagellated cells when propagated over the surface of solidmedia demonstrates that B . subtilis is provided with the abilityto undergo swarming differentiation . Swarming by PB5249 occurredat a wide range of temperatures [20 to 37°C] and on differentsolid media [TrA, nutrient, and LB agar] . Macroscopically, B.subtilis swarm colonies did not exhibit regularly layered consolidationphases alternated with swarming migration [Fig. 3A], as hasalready been reported for other Bacillus species [11, 39].

 
To estimate the extent of surface-induced hyperflagellationin strain PB5249, we measured the differences in the amountof extracellular flagellin in equivalent numbers of cells takenfrom TrB and TrA by SDS-PAGE and immunoblotting with a B . subtilisantiflagellin antiserum . As shown in Fig . 3H, cells grown onthe solid medium exhibited an almost 10-fold increase in flagellin compared to those grown in liquid, and the flagellin monomers appeared to have the same molecular weight [Fig . 3I] . To confirmthe identity of flagellin subunits in swim and swarm cells, a mutant with a deletion of the hag gene [PB5250] was constructed by gene replacement with a kanamycin resistance determinant.As expected, the cells failed to swim in liquid media as wellas to swarm on solid surfaces, further supporting the findingthat the same type of flagellar subunit is employed for theassembly of flagella in swim and swarm cells.

Surfactin is a B . subtilis lipopeptide antimicrobial surfactant whose activity has recently been related to the ability of an undomesticated B . subtilis strain to swarm [20] . Many B . subtilislaboratory strains derived from 168, including PB1831, are defectivein surfactin biosynthesis due to a frameshift mutation in thesfp gene [28] . Nevertheless, we tested PB5249 for the abilityto produce surfactin . No hemolytic activity was observed onblood agar plates, indicating that the strain is not a surfactinproducer . To evaluate whether the swarming behavior of PB5249was affected by the ability to produce surfactin, we constructedstrain PB5332, a surfactin-producing derivative of PB5249 . Theeffect of surfactin production on swarming was limited to aslight increase in the size of colonies [Fig. 4] that did notshow alternate cycles of swarming migration and consolidation,as already noted for strain PB5249.

 
Transcriptional analysis of flagellar genes in PB1831. The observation that PB1831 failed to undergo surface-inducedswarming differentiation and, what is more, failed to produceflagella upon growth on a solid surface induced us to get furtherinsights on the molecular events involved in the response ofPB1831 to the solid surface . To this end, the transcriptionof genes required for flagella formation was analyzed . First,we checked for transcripts of the hag class III gene by RT-PCR,and expression of hag was never detected when PB1831 was grownon solid media [Fig. 5, lane 3] . This result was in accordancewith the observation that PB5308 produced white colonies onplates containing X-gal [data not shown].

 
Since the hag gene is transcribed from a ^D-dependent promoter[25], we analyzed the expression of the fla/che class II operonthat includes the ^D-encoding gene [sigD] by RT-PCRs performedat three different positions along the approximately 26-kb operon.Transcriptional scanning showed that the operon was expressedall over its length and included sigD [Fig . 5, lanes 5, 7, 9].Thus, the lack of hag expression in solid media could not beinterpreted simply as the consequence of a missed transcriptionof the late-flagellar-stage sigma factor . To understand whetherthe level of sigD expression played a key role in hag transcription in strain PB1831 grown on solid surfaces, PB1831 was transformedwith the plasmid pSigD, which carries a copy of the sigD geneunder control of the IPTG [isopropyl-ß-D-thiogalactopyranoside]-inducible P_spac promoter [3] . PB1831 harboring pSigD restored the abilityto produce flagellin when grown on solid agar plates in thepresence of the inducer [Fig . 6; lane 2] . This result demonstratesthat the level of intracellular ^D in PB1831 propagated on solidsurfaces is not sufficiently high to promote hag expression.

 
The activity of ^D is intracellularly balanced by the anti-sigmafactor FlgM [10] . Therefore, experiments were performed to evaluateif on solid media [i] the flgM was transcribed in PB1831 and[ii] flagellin was synthesized in a PB1831 derivative carryinga deletion in flgM [PB5148] . A positive signal was obtainedby RT-PCR for the flgM gene in cells of strain PB1831 [Fig.5, lane 11], and flagellin synthesis was restored in strainPB5148, as demonstrated by immunoblot analysis [Fig . 6, lane 4] . These results show that intracellular FlgM can potentiallybe responsible for ^D inactivity and, consequently, for missedhag expression in strain PB1831 grown on solid media . However,cells overexpressing the transcription factor ^D and cells lackingFlgM recovered only part of their biosynthetic potential, sincethey did not acquire the ability to produce flagella when grownon solid media . The lack of flagella in PB1831 suggests thatthe ifmP locus regulates the complex process of flagella formationin response to external stimuli coming from liquid or solid environments at a not-yet-defined level.

 
Bacterial flagella are organelles of the cell envelope instrumental to interaction with the environment . Flagellum-driven motilityis one of the most impressive features in the cellular physiologyof eubacteria and allows active bacterial movement in liquidas well as over solid surfaces . In the environment, the abilityto swim enables individual cells to rapidly respond to changesin nutrient availability, moving toward attractants or awayfrom repellents through a signal transduction chemotactic network.In contrast to swimming, the movement of flagellated bacteriaover solid surfaces is a striking multicellular behavior, whichenables bacterial cells to move collectively in a coordinatedfashion referred to as swarming motility [17] . Swarming, whichis not triggered by starvation but by contact with solid surfaces,closely depends on the ability of swimmer cells to undergo asurface-induced differentiation process leading to the productionof elongated and hyperflagellated swarm cells [8, 15, 17, 19,39] . Swarming over solid surfaces is a way for bacteria to disperseand to colonize the environment that favors the establishmentof commensal, symbiotic, or pathogenic associations with plantsand animals to reach optimal colonization niches [27, 32].

B . subtilis is a flagellated soil bacterium ubiquitously distributed in the environment . It possesses a remarkable metabolic and physiological versatility that facilitates its propagation ina wide range of growth conditions, including liquid and solidsubstrates . As with other flagellated bacteria, the abilityto alternate swimming with swarming motility in response toa surface stimulus may be of evident adaptive value for thisorganism . In this report, we show that swarming is not a widespreadbehavior of B . subtilis laboratory strains but can be observedonly in a strain exhibiting a hypermotile phenotype due to amutation in the ifm locus.

The hypermotile ifmP mutant responds to contact with a solid surface similarly to other Bacillus species [11, 20, 39] . Theswarm colonies produced by the ifmP mutant only occasionallyhad a terraced appearance and never exhibited the consolidationphases that are peculiar to Proteus mirabilis [5, 17]. B . subtilisswarm cells are 3 to 5 times longer and almost 10 times moreflagellated than the swimmer cells . The ifmP mutant was unableto produce surfactin, which has been shown to play a role inB . subtilis swarming [20] . In this study, we provide evidencethat surfactin facilitates bacterial migration over solid surfacesbut is not essential for swarming differentiation in an ifmPbackground.

The same motile organelle appears to be required for swimmingand swarming movements in B . subtilis . Indeed, a flagellin monomer of the same molecular weight is assembled by B . subtilis grown in liquid or on solid media, and disruption of the hag gene abolishes surface translocation . This phenomenon is similarto that found in some members of the Enterobacteriaceae [15, 41] but differs from that observed in Vibrio parahaemolyticus,which produces distinct flagellar organelles for swimming andswarming motility [24].

Integrity in the expression of flagellar and chemotaxis genesis an essential requirement for swimming and swarming motilityin several gram-negative and gram-positive bacteria [5, 7, 11,17, 20, 31, 39, 41] . That a flagellar locus can influence the ability to generate flagella only upon contact with a solidsurface has never been described before, however . Indeed, theparental strain and all the strains carrying a wild-type ifmlocus not only failed to activate a swarm response upon contactwith the surface but, remarkably, completely stopped synthesizingflagellin and assembling flagellar filaments . The lack of haggene expression can be explained at the regulatory level bya functional deficiency of the transcription factor ^D . Thisinterpretation is supported by the results obtained by overproductionof ^D in the presence of the plasmid pSigD or by deletion ofthe flgM gene encoding the anti-sigma factor FlgM . In both experimental systems, cells grown on solid surfaces succeeded in expressing flagellin; nevertheless, the ability to assemble flagellar filaments was not restored.

Thus, in strains having a wild-type ifm background, some of the steps required for the integration of signals derived from contact with a solid surface are missing or not functioningproperly . The ifmP mutation, therefore, can be regarded as again of a mutated function, restoring a response lost in theprocess of domestication of B . subtilis.

The presence of a mutant ifm gene appears to be necessary to correctly perceive the solid-surface signal in order to process and integrate it into the different regulatory pathways thataffect flagellar assembly and functions.

 
This work was supported by Fondo d'Ateneo per la Ricerca of Università di Pisa and Università degli Studidi Pavia, Italy.

 
* Corresponding author . Mailing address: Dipartimento di Genetica e Microbiologia, Via Abbiategrasso 207, 27100 Pavia, Italy . Phone: [39] 0382 505548 . Fax: [39] 0382 528496 . E-mail: galizzi@ipvgen.unipv.it.

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