JOURNAL OF BACTERIOLOGY, Jan. 1990, p. 334-341 Vol. 172, No. 10021-9193/90/010334-08$02.00/0Copyright © 1990, American Society for Microbiology

Chemotactic Control of the Two Flagellar Systems ofVibrio parahaemolyticus

NECHEMIA SAR, LINDA McCARTER, MELVIN SIMON, AND MICHAEL SILVERMAN*The Agouron Institute, La Jolla, California 92037

Received 24 July 1989/Accepted 6 October 1989

Vibrio parahaemolyticus synthesizes two distinct flagellar organeles, the polar flagellum (Fla), which propelsthe bacterium in a liquid environment (swimming), and the lateral flagella (Laf), which are responsible formovement over surfaces (swarming). Chemotactic control of each of these flagellar systems was evaluatedseparately by analyzing the behavioral responses of strains defective in either motility system, i.e., Fla' Laf-(swimming only) or Fla- Laf+ (swarming only) mutants. Capillary assays, modified by using viscous solutionsto measure swarming motility, were used to quantitate chemotaxis by the Fla' Lafr or Fla- Laf' mutants. Thebehavior of the mutants was very similar with respect to the attractant compounds and the concentrationswhich elicited responses. The effect of chemotaxis gene defects on the operation of the two flagellar systems wasalso examined. A locus previously shown to encode functions required for chemotactic control of the polarflagellum was cloned and mutated by transposon TnS insertion in Escherichua coli, and the defects in this locus,che4 and che-5, were then transferred to the Fla' Laf or Fla- Laf+ strains of V. parahaemolyticus.Introduction of the che mutations into these strains prevented chemotaxis into capillary tubes and greatlydiminished movement of bacteria over the surface of agar media or through semisolid media. We conclude thatthe two flagellar organelles, which consist of independent motor-propeller structures, are directed by acommon chemosensory control system.

Vibrio parahaemolyticus produces two cell types, theswimmer cell and the swarmer cell, which are adapted forlocomotion in different circumstances (2, 20). The swimmercell, obtained when the bacteria are cultivated in liquidmedia, synthesizes a single polar flagellum (Fla) which issheathed by an extension of the outer membrane. Growth onsolidified agar media induces the differentiation of theswarmer cell, an elongated form which synthesizes, inaddition to the polar organeile, numerous unsheathed lateralflagella (Laf). The polar flagellum propels the bacterium in aliquid environment, and the lateral flagella are required fortranslocation over surfaces, which is called swarming. Theswarming phenotype is also observed for certain members ofthe genus Proteus. The two flagellar organelles of V. para-haemolyticus appear to be structurally distinct (15, 19). Thehelical, propeller filaments of the Fla and Laf organelles areassembled from different flagellin subunits, and no mutantlacking both flagellar structures (a Fla- Laf- phenotype) hasbeen isolated, suggesting that no motor or propeller compo-nents are common to the two appendages.Swimming of bacteria such as Escherichia coli or Salmo-

nella typhimurium relative to gradients of attractants andrepellants is directed by a complex signal transductionnetwork which controls the rotation of the flagellar filaments(7, 13). Chemotactic control of the movement of V. para-haemolyticus has not been analyzed. Is movement of theswimming (Fla) and swarming (Laf) appendages responsiveto chemotactic stimuli, and how is the flow of sensoryinformation channeled to produce a coordinated response ina cell with two propulsive systems? We report here themeasurement, by a modification of the capillary tube assay,of chemotaxis by cells with either swimming or swarmingmotility. Analysis of control of the behavior of one motilitysystem independent of that of the other was achieved byusing mutants defective in swarming (Fla' Laf) or swim-

* Corresponding author.

ming (Fla- Laf). Furthermore, the effects of particularchemotaxis gene (che) defects on the behavior of swimmerand swarmer cells was investigated. Mutations, generated inE. coli by transposon TnS insertion into a cloned chemotaxislocus, were transferred to the genomes of various strains ofV. parahaemolyticus where their influence on the chemotac-tic ability of cells with either motility system could bemeasured.

MATERIALS AND METHODSStrains, media, and growth conditions. The V. parahae-

molyticus strains and various plasmids and cosmids used inthis study are listed in Table 1. The following media wereused: 2216 (28 g of marine broth 2216 [Difco Laboratories]per liter); HI (25 g of heart infusion broth [Difco], 20 g ofNaCl per liter); TM (10 g of tryptone [Difco], 20 g of NaClper liter); minimal high salt medium, HS, which containedper liter K2HPO4 (8.4 g), KH2PO4 (1.8 g), NH4Cl (2g),MgSO4 (0.2 g), NaCl (20 g), and 3 ml of sterile glycerol,added after autoclaving. Media were solidified with thefollowing agar concentrations: 2.0% agar (Difco) for non-swarming media, 1.5% agar to test swarming motility, and0.4% agar to test swimming motility. Chemotaxis mediumcontained K2HPO4 (1.4 g), KH2PO4 (0.3 g), EDTA (0.1mM), and 20 g of NaCl per liter. Chemotaxis by swarmingcells was tested in a chemotaxis medium supplemented with5% polyvinylpyrolidone (PVP) (360,000 M,; Sigma Chemi-cal Co.). The various amino acids, amino acid analogs, andmonosaccharides used were obtained from Sigma.Measurement of chemotaxis. The capillary assay of Adler

(1) was used, with some modifications to measure chemo-taxis. In brief, capillary tubes (2 ,ul; Drummond ScientificCo.) were flame sealed at one end and were filled withchemotaxis medium (with or without attractant). The openends of the capillaries were introduced into a bacterialsuspension in identical medium (without the attractant).After incubation for 50 min at room temperature (22 to 27°C),

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TABLE 1. Bacterial strains and plasmids

Strain, plasmid, or Description' Origin (references)cosmid

V. parahaemolyticusBB22 Wild type; Fla' Laf+ R. Belas (3)LM959 AflaCD2::kan L. McCarter (15)LM967 AflaC4 L. McCarter (15)RS313 laf-313: :mini-Mu lux (Tetr) R. Showalter (4)LM1017 Tets derivative of RS313 L. McCarter (15)ML191 che-J::mini-Mu lac (Tet') in LM1017 L. McCarter (15)ML204 che-2:mini-Mu lac (Tetr) in LM1017 L. McCarter (15)NS30 Tn5 insertion no. 1182 in BB22 This studyNS31 che4::TnS in BB22 This studyNS32 che-5::TnS in BB22 This studyNS33 TnS insertion no. 1182 in LM967 This studyNS34 che4::Tn5 in LM967 This studyNS35 che-S::TnS in LM967 This studyNS36 Tn5 insertion no. 1182 in LM1017 This studyNS37 che4::TnS in LM1017 This studyNS38 che-5::TnS in LM1017 This study

Plasmid or cosmidpACYC177 Apr Kmr (8)pNS1 Tetr Kmr; pACYC177 with PstI insert from ML191 This studypNS2 Tetr Kmr; pACYC177 with PstI insert from ML204 This studypLM146.29 Tetr; cosmid pLAFRII containing chemotaxis locus from V. parahaemolyticus This studypNS4 Tetr Kmr; pLM146.29 with che4::TnS mutation This studypNS5 Tetr Kmr; pLM146.29 with che-S::TnS mutation This studypLM1182 Tetr Kmr; recombinant pLAFRII with Tn5 insertion no.1182 L. McCarterpRK2013 Kmr; RK2 tra donor for conjugation G. Ditta (9)pPHlJI Gmr; for selection of segregants in gene replacement procedure R. Taylor (6)a Abbreviations: Tetr or Tets, tetracycline resistant or sensitive; Apr, ampicillin resistant; Kmr, kanamycin resistant; Gmr, gentamicin resistant.

the number of bacteria in each capillary was determined byplating appropriate dilutions of the contents of the capillar-ies. Each measurement was repeated with 2 to 4 capillaries,and each experiment was repeated at least twice. The resultswere expressed in the following ways: the actual number ofbacteria inside the capillary (the average of 2 to 4 capillar-ies), the bacterial counts normalized to the highest measure-ment (scored as 100%), and the number of bacteria in thecapillary containing attractant divided by the number ofbacteria in the control capillary (no attractant). To testchemotaxis by swimming cells, Fla' Laf- bacteria weregrown in 2216 medium to an optical density at 600 nm of 0.7to 0.9, centrifuged once at 4,000 x g for 7 min, andresuspended in chemotaxis medium to an optical density at600 nm of 0.1. To test chemotaxis by the swarming cells,Fla- Laf+ bacteria were inoculated in the center of HIswarming plates and incubated at 30°C for 10 to 12 h. Cellsfrom the periphery of the swarm were then suspended inchemotaxis medium containing 5% PVP. The cell suspensionwas then diluted 10- to 20-fold in the same medium to anoptical density at 600 nm of 0.1. Any attempt to suspendlaterally flagellated bacteria in media not containing PVPresulted in rapid loss of cell motility, as indicated by micro-scopic examination. In experiments designed to assess theeffect of viscosity on chemotaxis, bacteria (both Fla' Laf-and Fla- Laf' strains) were grown as described above andthen suspended in chemotaxis medium containing 5% PVPprior to diluting them in the appropriate chemotaxis mediumto give final PVP concentrations of 0.5 to 10%. The viscosityof 10% PVP at 22°C in this medium is 40 cP (4).

Cloning a chemotaxis locus. Chromosomal sequenceswhich flanked the transposon insertions in Che- strainsML191 and ML204 were cloned by using the transposon-encoded drug resistance gene tet as a linked, selectablemarker (3, 15). Specifically, chromosomal DNA from the

mutants was digested with PstI, which generated fragmentscontaining the tet and lacZ genes of mini-Mu lac (Tetr) andthe chromosomal sequences at the site of insertion adjacentto those transposon genes. This DNA was ligated to PstI-digested pACYC177 DNA, and the mixture was used totransform E. coli DH5a with selection for Tetr (transposonencoded). Recombinant plasmids pNS1 with che sequencesfrom strain ML191 and pNS2 with che sequences fromML204 were recovered from transformants, and the PstIfragments with V. parahaemolyticus DNA were then puri-fied, labeled with 32P by nick translation, and used to probecolony filter blots of a library of V. parahaemolyticusfragments made previously in cosmid pLAFRII (11, 16).Probing of the cosmid library yielded four recombinants,including pLM146.29. Transformation, ligation, hybridiza-tion, DNA purification, and other general methods wereperformed by the method of Maniatis et al. (14). Restrictionendonucleases and T4 DNA ligase were obtained fromBoehringer Mannheim Biochemicals and were used accord-ing to the instructions of the supplier.Gene replacement mutagenesis. Recombinant cosmid

pLM146.29 was mutagenized with TnS by the method ofEngebrecht et al. (10). Restriction digests of cosmids withTn5 insertions were examined, and two, pNS4 and pNS5,were found to contain a TnS insertion in the 3-kilobase (kb)HindIII fragment encoding chemotaxis function. The muta-tions in pNS4 (che4::TnS) and pNS5 (che-5::TnS), as well asa TnS insertion (1182) in cosmid pLM1182, were thentransferred to the genomes of various strains of V. parahae-molyticus. The cosmids were first mobilized to V. parahae-molyticus by conjugation, which required the participationof tra donor pRK2013 in a triparental mating by the methodof Ditta et al. (9). Selection for exconjugants was uponminimal HS plates containing tetracycline (15 ,ug/ml) andkanamycin (150 ,ug/ml). Recombinants of V. parahaemolyti-

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336 SAR ET AL.

cus (i.e., Tn5 insertion crossed into chromosome) which hadsegregated the donor cosmids (pNS4, pNS5, or pLM1182)were then isolated from exconjugant cultures grown withselection for Kmr only. This was accomplished by matingsuch cultures of V. parahaemolyticus with E. coli containingpPHlJI (which encodes Gmr and is incompatible withpLAFRII-derived cosmids), with selection on minimal HSplates containing kanamycin (150 ,ug/ml) and gentamicin (50,ug/ml). Southern blot analysis was used to verify that thetransposon-generated mutation had replaced the wild-typeallele in the genome of the Kmr Gmr V. parahaemolyticus.Subsequently, plasmid pPHlJI was rapidly eliminated bygrowing the bacteria in the absence of selection for Gmr.

RESULTSAssaying chemotaxis. Analysis of the chemotactic control

of the movement of V. parahaemolyticus is complicatedbecause wild-type bacteria synthesize two kinds of flagellarappendages, Laf for swarming motility and Fla for swimmingmotility. The polar flagellum is not necessary for swarming,but it is produced constitutively and is therefore present incircumstances which induce formation of the laterally flag-ellated swarmer cell. Furthermore, semisolid agar media,which is used to study swimming movement by bacteria suchas E. coli and S. typhimurium, is not appropriate for analyz-ing the swimming of wild-type V. parahaemolyticus becausethis viscous environment induces formation of the swarmingappendages, Laf. The analysis was simplified by usingmutants capable of only one mode of motility, either swim-ming or swarming. Fla' Laf strain LM1017 (15) is notcapable of synthesis of lateral flagella as a result of muta-genesis by transposon mini-Mu lux (Tetr), and it was used toanalyze swimming motility. Fla- Laf+ strain LM959 cannotassemble a functional polar filament as a result of deletion ofa gene encoding a critical flagellin subunit (15). Strain LM959does not swim, and it was used to analyze swarming motil-ity.Chemotaxis by swimming and swarming cells was ana-

lyzed by measuring the movement of bacteria in liquidcultures into capillary tubes containing attractant. The assaymethod of Adler (1) was readily applied to the examinationof swimming cells. For example, cells of strain LM1017accumulated in capillary tubes containing 10 mM serine.However, several modifications in procedure were requiredto quantitate chemotactic movement by swarmer cells ofstrain LM959. Laterally flagellated cells, harvested eitherfrom the periphery of a colony swarming on an agar mediumor in particular circumstances (15, 17) from a liquid culture,were very poorly motile in liquid suspension and showedlittle migration into capillary tubes in a standard chemotaxisassay. Lateral flagella formation is induced by increasing theviscosity of the liquid growth medium (4), and translocationof swarmer cells occurs in viscous semisolid agar media(unpublished observations, this laboratory). These observa-tions indicated that swarmer-cell motility could be improvedin a viscous environment; therefore, the viscosity of thesolutions in the chemotaxis assay was increased by adding5% PVP, with the result that substantial migration intocapillary tubes containing 10 mM serine was observed.Viscous chemotaxis medium was also used to harvestswarmer cells, since suspension and agitation of cells inlow-viscosity solutions reduced motility, possibly becausethe lateral flagella are fragile and are easily sheared from thecells.The influence of the viscosity of the assay medium on the

migration of bacteria into capillary tubes containing 10 mM

a2a20

-

>4

100

80

60 -

40

20-

00 2 4 6 8 10

P V P CONCENTRATION (%)FIG. 1. Influence of viscosity on chemotaxis by swimming and

swarming V. parahaemolyticus. Chemotaxis was measured by thecapillary assay (see Materials and Methods). Migration of swimmingbacteria, strain LM1017, and swarming bacteria, strain LM959, intocapillary tubes containing either 10 mM serine or no attractant wasmeasured as a function of the concentration of PVP added toincrease the viscosity of the solutions in the assay. Symbols: A,LM1017 with 10 mM serine in the capillary; A, LM1017 with noattractant; 0, LM959 with 10 mM serine; and *, LM959 with noattractant.

serine was examined for both swimmer and swarmer cells(Fig. 1). Chemotaxis by swimmer cells of strain LM1017 wasbest at the lowest viscosity used, and chemotaxis byswarmer cells of strain LM959 was best at a much higherviscosity (5 to 7.5% PVP). For further assays, swimming wasanalyzed in chemotaxis medium without PVP, and swarmingwas measured in a medium containing 5% PVP. The re-sponse of V. parahaemolyticus to different concentrations ofthe attractant serine is shown in Fig. 2. Maximal migration,i.e., about 106 cells entering a capillary, was achieved withsimilar concentrations of serine (-10 to 20 mM) for both celltypes. Accumulation of swimmer and swarmer bacteria inthe capillary tubes resulted from movement in response to agradient of attractant, because elimination of the spatialgradient by including attractant at the same concentration,i.e., 10 mM serine, in the pool of bacteria as well as in thecapillary reduced accumulation to that level observed whenattractant was absent in both pool and capillary. Swimmingand swarming cells also showed chemotaxis to the samesubstances, namely, serine, a-methyl-DL-serine (a nonme-tabolizable analog), alanine, glycine, and leucine, all assayedat a concentration of 10 mM. Very little or no chemotaxiswas observed for aspartate (10 mM), oc-methyl-DL-aspartate(10 and 50 mM), glutamate (10 mM), maltose (1 mM),galactose (1 mM), glucose (1 mM), and acetate (1 mM). Theresults of chemotaxis assays, expressed as the ratio of thenumber of cells in a capillary containing a particular testeffector divided by the number of cells in a capillary withouteffector, are shown in Table 2. This ratio was found to rangefrom 9 to 61 for the attractants. With the "plug in agarmethod" (23), swimmer and swarmer cells moved awayfrom the repellant indole (1 mM) but not from nickel sulfate(10-7 to 10-4 M) (data not shown).

Cloning and mutagenesis of a chemotaxis locus. Swimming

%-P -. so .50 I9 I - I

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CHEMOTAXIS OF V. PARAHAEMOLYTICUS 337

12

10

z

0 10io- 10io2 0 1

SERINE (molarity)

FIG. 2. Response of swimming and swarming strains to serine.The migration of swimming cells of strain LM1017 (A) and swarmingcells of strain LM959 (0) into capillary tubes containing differentconcentrations of the attractant serine is shown.

and swarming cells are capable of chemotaxis, and theirresponse to chemoeffectors is very similar, both with respectto the substances and to the concentrations which areeffective. Are the two flagellar organelles controlled byelements of a common chemosensory system? We sought totest this hypothesis by isolating and analyzing some of thegenes (che) which encode chemotaxis function in V. para-haemolyticus. Specifically, a region of the genome necessaryfor chemotaxis function was cloned; selectable che muta-tions were constructed in this cloned locus, and the chedefects were then transferred from recombinant E. coli intothe genomes of swimmer and swarmer strains of V. para-

TABLE 2. Capillary assays for attractants

Chemotaxisb ChemotaxisbCompound' by Fla' Laf by Fla- Laf+

strain LM1017 strain LM957

L-Serine (10 mM) 60 45L-Glycine (10 mM) 54 29L-Alanine (10 mM) 61 43L-Leucine (10 mM) 9 21a-Methyl-DL-serine (10 mM) 20 17L-Aspartate (10 mM) 2 2a-Methyl-DL-aspartate (10 mM) 1 1L-Glutamate (10 mM) 2 2D-Maltose (1 mM) 2 1D-Glucose (1 mM) 1 1D-Galactose (1 mM) 1 1D-Ribose (1 mM) 1 1Acetate (1 mM) 1 1

a Compounds to be tested were dissolved in chemotaxis buffer at theconcentration shown in parentheses.

b Chemotaxis is expressed as a ratio equal to the number of swimmer orswarmer cells which migrated into the capillary tube containing the chemo-effector compound tested divided by the number of cells which migrated intothe control capillary without effector compound. Ratios from at least twoindependent sets of capillaries were averaged and rounded to the nearestwhole number. See Materials and Methods for a detailed description of theassay.

haemolyticus for examination of chemotaxis phenotypes.Transposon mini-Mu lac (Tet') insertions in mutants ML191and ML204 were used to guide the cloning of transposon-linked che DNA (see Materials and Methods). MutantsML191 and ML204 were derived independently from theswimmer strain LM1017 (15) and had a Che- phenotype withrespect to swimming, i.e., motile, polarly flagellated cellswere produced but there was very little translocation insemisolid agar media and no migration into capillary tubescontaining attractant occurred. These mutants were not veryuseful for studying chemotaxis because they were not capa-ble of swarming, and the defects generated by mini-Mu lac(Tet') insertion could not be transferred to other strains of V.parahaemolyticus. Recombinant plasmids pNS1 and pNS2containing limited DNA sequences adjacent to the site oftransposon insertion in strains ML191 and ML204 wereconstructed. These plasmids were used as probes in South-ern blot analysis to map the location of the defects,che-l::mini-Mu lac (Tetr) and che-2::mini-Mu lac (Tetr) inChe- strains ML191 and ML204. The transposon insertionsin the mutants were located about 0.4 kb apart in the same3.0-kb HindIII fragment (data not shown).Fragments from plasmids pNS1 and pNS2 were used as

hybridization probes of a cosmid library to isolate recombi-nants with extensive DNA sequence (about 20 kb) from theche gene region (see Materials and Methods). Cosmid pLM146.29, one of four isolated, contained the 3.0-kb HindIIIche fragment and, when transferred by conjugation, restoredchemotaxis to mutants ML191 and ML204. Transposon TnSwas chosen for mutagenesis of the cloned genes on pLM146.29 because the location of the defects could be deter-mined by restriction mapping, the transposon insertionswould be relatively stable during subsequent conjugal trans-fer, and the TnS-encoded kanamycin resistance could beused to select recombinants of V. parahaemolyticus contain-ing the che mutations. Thirty-one cosmids with TnS muta-tions were analyzed, and two, pNS4 and pNS5, containedTnS insertions in the 3.0-kb HindIII fragment. Integration ofTnS interrupted the continuity of this fragment and gener-ated a 4.2-kb fragment and a 1.2-kb HindIII fragment (eachcontaining DNA from the che locus and from the arms ofTnS) for che4::TnS (pNS4) and a 3.8-kb fragment and a1.6-kb fragment for che-5::TnS (pNS5). The larger of thesepairs of fragments was detected in the Southern blot shownin Fig. 3. From these results and other restriction mapping,the TnS insertions were found to be located about 2.6 kbapart on the 3.0-kb fragment. Another cosmid, pLM1182,was chosen at random and mutagenized with TnS to producea neutral insertion mutation which could later be used toconstruct a Kmr control strain of V. parahaemolyticus. TnSinsertion mutations che4::TnS, che-S::TnS and control mu-tation no. 1182 were then transferred to the genomes ofwild-type strain BB22, swimming strain LM1017, andswarming strain LM967 (same Fla- phenotype as LM959except kanamycin sensitive). This involved conjugal transferof the mutated cosmids into V. parahaemolyticus, followedby selection for segregants in which the exogenote mutationhad recombined into the chromosome (see Materials andMethods). Replacement of the wild-type che allele with thetransposon-generated mutation was verified by Southernblot analysis. The result of examination of the gene replace-ment mutants constructed in strain BB22 is shown in Fig. 3.

Phenotypes of mutants. The nine strains constructed bycrossing three TnS insertion mutations (che4: :TnS,che-5::TnS, and control insertion no. 1182) into three V.parahaemolyticus strains (wild-type BB22, swarmer LM967,

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eu..m

4.2 kb-.

3.8kl).3.~O_k_

.

FIG. 3. Southern blot analysis of DNA from recombinantcosmids and strains of V. parahaemolyticus containing transposonTnS insertions in a chemotaxis locus. 32P-labeled cosmid pLM146.29was used to probe HindIII-digested DNA from the following: lane 1,pLM146.29 (parent cosmid); lane 2, pNS4 (che-4::TnS); lane 3,pNS5 (che-5::TnS); lane 4, strain BB22 (wild type); lane 5, strainNS31 (che-4::TnS in BB22); lane 6, strain NS32 (che-5::Tn5 inBB22); lane 7, strain NS30 (control TnS insertion no. 1182 in BB22).Sizes of particular HindIII fragments are shown at left in kilobases.

and swimmer LM1017) are listed in Table 1. Introduction ofthe three mutations did not affect production of Fla or Lafappendages as judged by Western blot (immunoblot) mea-surements with antisera specific for the subunits of the Flaand Laf filaments (data not shown). Bacterial suspensionswere also examined under a microscope, and the mutationsdid not cause paralysis of the flagellar motors, i.e., animmotile or Mot- phenotype. The movement of the variousstrains through semisolid agar medium or over the surface ofsolidified agar medium is shown in Fig. 4. Control strains,those with neutral TnS insertion no. 1182, are shown in thetop row. The presence of control TnS insertion no. 1182 didnot alter the ability of V. parahaemolyticus to swim orswarm. Strain NS30 (wild-type BB22 with insertion no.1182), which produces both Fla and Laf appendages, movedthrough the semisolid medium and swarmed over the surfaceof the solidified medium. Strain NS33 (swarmer strainLM967 with insertion no. 1182) moved through the semisolidmedium and over the solidified medium. Strain NS36 (swim-mer strain LM1017 with insertion no. 1182) was capable ofmovement through the semisolid medium but could notswarm over the surface of the solidified medium. Note thatthe swarmer bacteria (i.e., strain NS33) could translocate,"swim," in the semisolid medium, but the swimmer bacteria(i.e., NS36) could not "swarm" over the surface of thesolidified medium. Apparently, there are environments inwhich both Fla and Laf flagella function well, such as asemisolid agar medium and a medium containing an inter-mediate concentration of PVP (see Fig. 1).

Introduction of the che defects influenced the movementof V. parahaemolyticus through and over agar media (Fig.4A and B). Movement of strain NS31 (che4::TnS in BB22)and strain NS32 (che-5::TnS in BB22) through the semisolid

medium was impaired, and swarming over the solidifiedmedium did not occur. Similarly strain NS34 (che4::TnS inLM967) and NS35 (che-5::TnS in LM967) showed relativelyslow progress through the semisolid environment and almostno swarming over the surface of the solidified medium.Strains NS37 (che4::TnS in LM1017) and NS38 (che-5::TnSin LM1017) no longer had the ability of their parent to swimthrough the semisolid medium. Impairment caused by chemutations was more severe for translocation on the solidifiedagar medium than on semisolid agar, where more growth ata distance from the site of inoculation was observed. Thedefect in motility was also more extreme with the che4: :Tn5mutation than with the che-5::TnS mutation. These muta-tions appeared to differ in their effect on the pattern ofmovement of individual swarmer and swimmer cells.Swarmer and swimmer cells with a che+ composition,examined microscopically in a high- or low-viscosity me-dium, respectively, exhibited a pattern of behavior consist-ing of smooth runs interrupted by occasional tumbles orreversals. This pattern was similar to the alternation ofswimming modes described for bacteria such as E. coli (5).The behavior of strains with che4::TnS consisted mostly ofsmooth runs, while strains with che-S::TnS showed an in-creased frequency of reversals. Migration of mutants intocapillary tubes containing 10 mM serine was also measured,and all the strains with che defects were unresponsive, i.e.,approximately the same numbers of cells were found intubes with and without attractant.

DISCUSSION

Bacteria such as E. coli are capable of directing theirswimming movement in relation to gradients of chemicals intheir environment, and knowledge about the genes, geneproducts, and biochemistry involved in chemotaxis hasreached a high level of refinement (7, 13). The role ofchemotaxis in surface translocation phenomena such asswarming has not been studied extensively. Investigators ofProteus mirabilis concluded that chemotaxis was not in-volved in swarming (25), but it is not clear whether thisfinding has a bearing on the behavior of V. parahaemolyti-cus. The similarity between the swarming of Proteus spp.and Vibrio spp. could be superficial, since the flagellarsystems of the two bacteria are considerably different.Experiments with Vibrio alginolyticus suggested that nega-tive chemotaxis was directing bacteria away from regions ofhigh culture density (24). Development of quantitative as-says and genetic methods would be very useful in analyzingbehavioral control of swarming of bacteria like V. parahae-molyticus, but several problems have been encountered.Swarming cells of V. parahaemolyticus synthesize both Flaand Laf organelles; therefore, it is difficult to assess thecontribution which each type of flagella makes to the move-ment of the cell. We were able to analyze control of theswimming and swarming appendages separately by usingswimming only (Fla' Laf-) and swarming only (Fla- Laf+)mutants. The capillary assay makes possible accurate quan-titation of a chemotactic response, but swarming cellsshowed very poor migration into capillary tubes in thestandard protocol. There are several explanations for theproblem encountered with the standard assay. Cells at theperiphery of a swarming colony do not move independentlybeyond the group in an "adventurous" mode and appear torequire cell-cell contact or some "social" interaction, sug-gesting a similarity to the movement of Myxococcus xanthus(12). Possibly, this interaction could not be reproduced in the

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FIG. 4. Swimming and swarming of chemotaxis mutants. Mutants with transposon TnS insertions were inoculated into a semisolid (0.4%agar) tryptone medium plate (A) and onto a solid (1.5% agar) heart infusion medium plate (B) to test swimming and swarming motility,respectively. Plates were photographed after a 12-h incubation at 30°C. The strains in plates A and B are as follows: in left column, NS30 (top),NS31 (middle), NS32 (bottom); in central column, NS33 (top), NS34 (middle), NS35 (bottom); and in right column, NS36 (top), NS37(middle), NS38 (bottom).

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340 SAR ET AL. J. BACTERIOL.

liquid environment of the capillary assay. Alternatively,swarmer cells could dedifferentiate during the assay becausethe conditions which induce differentiation are absent. Otherexplanations include loss of fragile Laf structures duringharvesting and a failure of Laf to generate propulsive force inthe liquid environment of the assay.Knowledge of the conditions which induce differentiation

led us to try increasing the viscosity of the assay medium.This gave a substantial improvement in the migration ofswarming bacteria into the capillary tubes. Increasing vis-cosity was not actually necessary for induction of Lafformation, since constitutive production of lateral flagellahad already been accomplished by genetic manipulation (15).However, a viscous medium was required in harvesting toprevent loss of flagella from cells and to improve propulsionby the lateral flagella. Cell-cell contact could be necessaryfor movement in a swarming colony, but it is apparently notimportant for movement in a viscous fluid, since cellsdispersed in solution did migrate into capillary tubes. Fromexamination of the effect of viscosity on chemotaxis, it isclear that the Fla and Laf organelles function best in differentphysical environments: Fla is most effective in a low-viscosity liquid and Laf operates best on surfaces and also inviscous environments, perhaps including mucous films andslime layers. Despite these differences, the responses of theFla and Laf appendages are "tuned" to the same spectrumof stimuli. This group of chemoeffector compounds, whichincludes serine, resembles the group which in E. coli elicitsresponses mediated by the Tsr (taxis to serine and repel-lants) receptor-signal transducer protein (21, 22). The corre-spondence of responses of the swimmer and swarmer cellssuggests several possibilities for channeling information tothe flagella of V. parahaemolyticus. There could be twoparallel but similar systems for independent sensory controlof the two flagellar organelles, or there could be one commonpathway for processing the sensory information which di-rects the movement of both organelles.

Chemotactic control of swimming and swarming was alsoanalyzed by measuring the effect of specific mutations in achemotaxis gene locus. Che- mutants ML191 and ML204,isolated previously from a collection of transposon mini-Mulac (Tetr) insertions in Fla+ Laf strain LM1017, were aparticularly valuable resource because the transposon inser-tions in these mutants linked a selectable marker to a locusrequired for chemotaxis. Consequently, this locus, whichwas known initially to be required only for chemotacticcontrol of the Fla system, could be cloned. Then it wasmutated with TnS in E. coli, and the defects, che-4::TnS andche-5::TnS, were transferred to the wild-type, swimmer-only, and swarmer-only strains of V. parahaemolyticus. Theche-4 and che-5 mutations prevented swimming through asemisolid medium and swimming into capillary tubes, e.g.,strains NS37 and NS38. These defects also preventedswarming movement over the surface of a solidified mediumor through a viscous medium into capillaries, e.g., strainsNS34 and NS35. It is evident that swimming and swarmingcells need che-encoded functions to move into capillarytubes or to translocate through or over the surface of variousmedia, and swimming and swarming cells probably requirethe same che-encoded functions because the mutant pheno-types were caused by the same mutations. Transposoninsertions usually have a polar influence and greatly reduceexpression of genes downstream in an operon; therefore,there is the alternative interpretation that separate che genesfor each flagellar system exist but that these occupy the sametranscriptional units. Alternatively, expression of separate

che genes for each flagellar system could be controlled bycommon regulatory genes. We think that the simplest inter-pretation of the various evidence is that the two flagellarorganelles are controlled by a single chemosensory circuit.This arrangement would appear to be advantageous becausecoordination of the movement of different propulsive organ-elles could be achieved by coupling both to one chemosen-sory or decision-making apparatus.

Transfer of che-4::TnS and che-5::TnS into wild-typestrain BB22 resulted in impairment of its swimming andswarming movement. We have observed that no mutantswith a Fla- Laf phenotype were recovered from librariescontaining more than 40,000 strains generated by transposonmini-Mu lac (Tetr) or mini-Mu lux (Tetr) insertion into BB22(15). This observation was interpreted to mean that nocommon component was necessary for the function of bothFla and Laf appendages. The phenotypes observed for theche mutations would appear to be inconsistent with ourassertion about common flagellar components. However,the Che- phenotype (slow movement through semisolid agarmedium) was not detected, because in searching the collec-tions of strains with transposon insertions for mutants withmotility defects, only strains incapable of any translocationthrough semisolid media were sought. We now concludefrom the results of this study and from previous observationsthat there are two separate, structurally distinct propulsiveorganelles, Fla and Laf, in V. parahaemolyticus and that theoperation of these organelles is coupled to a single chemo-taxis control system.The two flagellar organelles are also related to each other

in another way. The polar flagellum functions as a sensorcontrolling swarmer-cell differentiation. Specifically, theperformance of the polar appendage is coupled to the tran-scription of the swarmer cell genes, laf, such that when thefunction of Fla is perturbed, either physically by constraintof its movement or genetically by introduction of mutationsintofla (or che) genes, lafgenes are induced (15). We wonderif there is some connection between the behavioral controlanalyzed here and the mechanism of transcriptional controlof laf. Although chemotaxis gene products have not beendemonstrated to regulate gene expression, the amino acidsequences of chemotaxis proteins of E. coli show homolo-gies to the sequences of signaling proteins that coupleenvironmental stimuli to gene transcription (18). Furtherwork is needed to define the individual che genes and theirfunction. Antibody raised to the CheA protein of E. coli alsobinds in Western blots to a protein from V. parahaemolyti-cus, and this protein is absent in the mutant of V. parahae-molyticus containing the che-4::TnS mutation (unpublishedresults). It should, therefore, be possible to relate specificcomponents of V. parahaemolyticus to those in well-studiedmodel organisms.

ACKNOWLEDGMENTS

We thank Doug Bartlett, Nachum Kaplan, Richard Showalter,and Miriam Wright for helpful advice and assistance and LeticiaWolpert for preparing the manuscript.

This research was supported by a contract (N00014-87-K-0322)from The Office of Naval Research. N.S. was a recipient of aFulbright Postdoctoral Fellowship.

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