Proteus mirabilis Mutants Defective in Swarmer Cell

Vol. 173, No. 19

Proteus mirabilis Mutants Defective in Swarmer Cell Differentiationand Multicellular Behaviort

ROBERT BELAS,1,2* DEBORAH ERSKINE,' AND DAVID FLAHERTY1

Center of Marine Biotechnology, The University ofMaryland, 600 East Lombard Street, Baltimore, Maryland 21202,1*and Department ofBiological Sciences, University ofMaryland Baltimore County, Baltimore, Maryland 212282

Received 21 June 1991/Accepted 24 July 1991

Proteus mirabilis is a dimorphic bacterium which exists in liquid cultures as a 1.5- to 2.0-,um motile swimmercell possessing 6 to 10 peritrichous flagella. When swimmer cells are placed on a surface, they differentiate bya combination of events that ultimately produce a swarmer cell. Unlike the swimmer cell, the polyploidswarmer cell is 60 to 80 ,um long and possesses hundreds to thousands of surface-induced flagella. Thesefeatures, combined with multicellular behavior, allow the swarmer cells to move over a surface in a processcalled swarming. Transposon TnS was used to produce P. mirabUis mutants defective in wild-type swarmingmotility. Two general classes of mutants were found to be defective in swarming. The first class was composedof null mutants that were completely devoid of swarming motility. The majority of nonswarming mutationswere the result of defects in the synthesis of flagella or in the ability to rotate the flagella. The remainingnonswarming mutants produced flagella but were defective in surface-induced elongation. Strains in the secondgeneral class of mutants, which made up more than 65% of all defects in swarming were motile but weredefective in the control and coordination of multicellular swarming. Analysis of consolidation zones producedby such crippled mutants suggested that this pleiotropic phenotype was caused by a defect in the regulation ofmulticellular behavior. A possible mechanism controlling the cyclic process of differentiation and dedifferen-tiation involved in the swarming behavior of P. mirabilis is discussed.

Proteus mirabilis is a motile gram-negative bacterium,similar in many aspects of its physiology to other membersof the family Enterobacteriaceae, such as Escherichia coliand Salmonella typhimurium. It was originally described andnamed by Hauser in 1885 for the character in Homer'sOdyssey who "has the power of assuming different shapes inorder to escape being questioned" (quoted from reference18). P. mirabilis is considered to be an opportunistic patho-gen and is one of the principal causes of urinary infections inhospital patients with urinary catheters (32, 34). Its ability tocolonize the surfaces of catheters and the urinary tract maybe aided by the characteristic first described more than acentury ago and currently referred to as swarmer cell differ-entiation.When grown in suitable liquid media, P. mirabilis exists as

1.5- to 2.0-,um motile cells with 6 to 10 peritrichous flagella.These bacteria, called swimmer cells, display characteristicswimming and chemotactic behavior, moving toward nutri-ents and away from repellents (36). However, a dramaticchange in cell morphology takes place when cells grown inliquid are transferred to a nutrient medium solidified withagar. Shortly after encountering an agar surface, the cellsbegin to elongate. This is the first step in the production of amorphologically and biochemically differentiated cell, re-ferred to as the swarmer cell. The process of elongationtakes place with only a slight increase in cell width and is dueto an inhibition in the normal septation mechanism, althoughthe molecular mechanism of inhibition is not known. Elon-gation of the swarmer cell can give rise to cells 60 to 80 pumin length. During this process, DNA replication proceedswithout significant change in rate from that in the swimmercell (15). Not surprisingly, the rate of synthesis of certain

* Corresponding author.t Publication 154 from the Center of Marine Biotechnology.

proteins, e.g., flagellin (FliC, the product of the fliC gene),the protein subunit of the flagellar filament, is altered mark-edly in the swarmer cell (3, 5, 14). The result of this processis a very long, nonseptate, polyploid cell. The number ofchromosomes in the swarmer cell is roughly proportional tothe increase in length, such that a 40-p.m swarmer cell hasabout 20 chromosomes (6). Eventually septation and divi-sion do take place at the ends of the long swarmer cells,producing a microcolony of differentiated cells.

Concurrent with cellular elongation, changes take place inthe rate of synthesis of flagella on the swarmer cell. Althoughswimmer cells have only a few flagella, the elongatedswarmer cells are profusely covered by hundreds to thou-sands of new flagella (Fig. 1A) synthesized specifically as aconsequence of growth on the surface (4, 19, 20). The term"flagellin factories" was first used by Hoeniger (19) todescribe the tremendous synthesis of new flagella inswarmer cell differentiation. The newly synthesized surface-induced flagella are composed of the same flagellin subunitas the swimmer cell flagella, indicating that the same flagellarspecies is overproduced upon surface induction (6). Theresult of the surface-induced differentiation process is aswarmer cell, which differs from the swimmer cells byhaving the unique ability to move over solid media in atranslocation process referred to as swarming (17). How-ever, individual swarmer cells by themselves do not have theability to swarm (10). Swarming is the result of a coordi-nated, multicellular effort of groups of differentiatedswarmer cells (10). The process begins when a group ofdifferentiated swarmer cells move outwards as a mass andcontinues until the swarming mass of bacteria is reduced innumber as a result of loss of constituent cells which fallbehind on the surface or when the mass reverses direction.Swarming of P. mirabilis is cyclic. Once swarmer cells

have fully differentiated, the swarming colony moves out-ward in unison from all points along the periphery for a

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FIG. 1. Swarmer cell morphology and swarming motility of P. mirabilis. (A) Electron micrograph of a wild-type swarmer cell taken fromthe periphery of a swarming colony grown on L agar at 37°C for 6 h and negatively stained with uranyl acetate. Bar, 5 pm. (B) Swarmingcolony and resulting consolidation patterns of BB2000 grown on an L agar plate at 37°C for 16 h. Oblique lighting directed at the undersideof the agar plate was used to accent the consolidation zones, which show up as lighter concentric rings in the swarming colony.

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PROTEUS MUTANTS DEFECTIVE IN MULTICELLULAR SWARMING 6281

TABLE 1. Strains used in this study

Strain Genotype or phenotypea Derivation Reference orsource

E. coliCSH4 trp lacZ strA thi recA FliC- Laboratory stockDH5a supE44 supF58 hsdS3 (rB mB ) Laboratory stock

dapD8 lacYl gin V44 A(gal-uvrB)47 tyrT58 gyrA29 tonA53A(thyA57)

P. mirabilisPRM1 Wild type J. ShapiroBB2000 Rif Spontaneous from PRM1 7BB2022 swr-2022::Tn5 Cm Swrcr Elo- pUT/mini-Tn5 Cm x BB2000b This studyBB2029 swr-2029::TnS Cm Swrcr pUT/mini-Tn5 Cm x BB2000 This studyBB2035 swr-2035::TnS Cm Swr- Mot- pUT/mini-TnS Cm x BB2000 This studyBB2040 swr-2040::TnS Cm Swr- Elo- pUT/mini-TnS Cm x BB2000 This studyBB2075 swr-2075::Tn5 Cm Swr- Che- pUT/mini-TnS Cm x BB2000 This studyBB2076 swr-2076::Tn5 Cm Swr- Eloc pUT/mini-TnS Cm x BB2000 This studyBB2105 swr-2105::Tn5 Cm Swrcr Eloc pUT/mini-TnS Cm x BB2000 This studyBB2128 swr-2128::Tn5 Cm Swr- Mot- pUT/mini-TnS Cm x BB2000 This studyBB2178 swr-2178::TnS Cm Swr- Che- pUT/mini-Tn5 Cm x BB2000 This studyBB2180 swr-2180::Tn5 Cm Swrcr pUT/mini-Tn$ Cm x BB2000 This studyBB2202 swr-2202::Tn5 Cm Swr Fla pUT/mini-Tn5 Cm x BB2000 This study

a Che-, produces rotating flagella but defective in chemotactic response; Eloc, swarmer cell elongation constitutive; Elo-, lacking elongation of swarmer cell;Fla-, no 36.7-kDa flagellin produced; Mot-, produces nonrotating flagella; Swr-, absence of swarming on agar media; Swrcr, non-wild-type or crippled swarmingand unusual consolidation patterns.

b p. mirabilis mutants defective in swarmer cell differentiation and swarming motility. Produced from conjugal mating of E. coli harboring pUT/mini-TnS Cmand BB2000 (7).

period of several hours and then stops (10, 12). This cessa-tion of movement is accompanied by a dedifferentiation ofthe swarmer cell back to swimmer cell morphology, in aprocess referred to as consolidation (see reference 37 for areview). The cycle of swarming and consolidation is thenrepeated several times until the agar surface is covered byconcentric rings formed by the swarming mass of bacteria(12). This cycle of events gives rise to the characteristicbull's-eye appearance of P. mirabilis colonies (Fig. 1B). Theswarmer cell requires continuous contact with the surface tomaintain the differentiated state. When removed from thesurface of an agar plate and suspended in liquid medium,cells quickly begin to septate and divide into short cells, andthe synthesis of flagella returns to the level observed inswimmer cells (21). Thus, the differentiation process isreversible both as a result of the consolidation process andas a consequence of removal of the inducing stimulus fromthe surface.Although many attempts have been made to explain the

mechanism of the swarming in Proteus species (37), includ-ing slime production (33) and chemotaxis (25, 36), theprocess of swarmer cell differentiation and multicellularswarming motility remains elusive.' However, swarmer celldifferentiation is not unique to P. mirabilis. Other bacteria,most notably Serratia (1) and Vibrio (9, 29) strains, have alsobeen reported to swarm as a result of a surface-induceddifferentiation from swimmer to swarmer cell. The regula-tion of swarmer cell differentiation is best understood for V.parahaemolyticus (29). The polar flagellum in V. parahae-molyticus serves in a sensory capacity by monitoring exter-nal conditions which cause the inhibition of flagellar rotation(9, 27). When such conditions are encountered, inhibition ofrotation of the polar flagellum sends a signal into the cellwhich triggers a chain of events ultimately producing adifferentiated swarmer cell. In addition to the control ex-erted by monitoring the inhibition of polar flagellum rotation,

a secondary signal is evidently sensed when iron becomeslimiting (28). The combination of conditions which inhibitthe rotation of the polar flagellum and limit the iron concen-tration is essential for initiation of transcription of the lafgenes (29).

Until recently (2), in-depth studies by modern techniqueshave not been applied to understanding the genetic regula-tion and sensory transduction mechanisms of swarmer celldifferentiation and multicellular swarming of P. mirabilis.Since little is known about the genetic regulation of Proteusswarming, we wanted a means of analysis which would beindependent of the mutant phenotype under investigation.The objective of this investigation was to use Tn5 mutagen-esis to develop a bank of Proteus mutants defective inwild-type swarmer cell differentiation and multicellularswarming motility. The present report is concerned with ouranalysis of the resulting mutants. By a combination ofassays, Proteus mutants defective in swarming weregrouped into two general categories. Mutants completelydefective in swarming motility were in large part due tomutations in genes affecting flagellin synthesis. The secondgroup of mutants produced non-wild-type swarming and aremost probably due to mutations in genes necessary forintercellular signaling. Analysis of the second class of mu-tants suggests that a cascade of signal molecules may berequired for the cycles of differentiation and dedifferentia-tion associated with swarmer cell motility.

MATERIALS AND METHODS

Bacterial strains. The strains used in this study are listed inTable 1. BB2000 is a spontaneously occurring rifampin-resistant mutant of PRM1 (7) and is used as the wild-typestrain throughout this study.Media and growth conditions. E. coli and P. mirabilis

strains were grown as described in the accompanying paper

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6282 BELAS ET AL.

(7). Swarming motility was observed on L agar (7), whereasLSW- agar (7) was used to inhibit swarming. For selectionof Tn5 Cm inserts, chloramphenicol was added to a finalconcentration of 150 pg/ml of medium. Swimming motility inplates was determined by using a semisolid agar mediumcontaining 10 g of tryptone, 5 g of NaCl, and 3.5 g of agar perliter. This medium is referred to as Mot agar. Swimmingmotility in liquid was assessed by microscopic examinationof cultures grown in Mot broth (Mot agar lacking agar).

Mutagenesis with Tn5 derivatives. Derivatives of transpo-son TnS carried on a suicide vector (11) were used tomutagenize P. mirabilis as described by Belas et al. (7). Abank of 13,036 chloramphenicol-resistant (Cmr) mutants wasproduced from conjugal matings of E. coli with recipient P.mirabilis (7).

Analysis of swarming and swimming motility. In all swarm-ing tests, L agar plates were used after thorough drying at42°C (45 to 60 min) to provide uniform conditions for testingswarming motility. To test swarming motility of the Proteusmutants, we replica plated colonies onto L agar. After 4 to 6h of incubation at 37°C, swarming motility was scored andpotential nonswarming mutants were transferred to a set ofsubmaster plates. The swarming analysis was repeated againuntil no colonies demonstrated active swarming during the6-h incubation. The putative nonswarming colonies werethen transferred to L agar and incubated at 37°C for 16 h.After overnight incubation, many of the putative nonswarm-ing colonies produced swarming motility on L agar, althoughnone demonstrated wild-type swarming when comparedwith BB2000. Mutants that did not show any swarmingmotility were designated as Swr- to distinguish them frommutants showing non-wild-type swarming, which were callSwrcr. Swrcr refers to the crippled nature of these mutantsdefective in wild-type swarming motility and consolidation.Swimming motility was assessed by both a semisolid agar

analysis and light-microscopic examination of the motility ofcells grown in Mot broth. As a preliminary test, the bank ofSwr- and Swrcr Proteus mutants was transferred to Motagar. Following overnight growth at 37°C, nonmotile bacte-ria were identified as those failing to migrate outwardthrough the Mot agar. This was repeated, and the resultingnonswimming (Swm-) bacteria were identified. Since Swm-mutants could be defective in either synthesis of flagella(Fla-), flagellum energetics (Mot-), or chemotaxis behavior(Che-), the motility of the Swm- bacteria was furtheranalyzed by light microscopy of individual strains grown inMot broth for 4 to 6 h at 37°C to a cell density of 107 to 108cells per ml. Strains that displayed active swimming motilityin liquid but failed to show migration through Mot agar weredesignated Che-. The remaining strains were all nonswim-ming in both liquid and semisolid media and could be eitherdefective in producing flagella (Fla-) or defective in rotatingthe flagella (Mot-). Fla- strains were separated from Mot-strains by Western immunoblot analysis with rabbit anti-flagellin antisera. In all swimming assays, in addition to thewild-type P. mirabilis, E. coli DHSa (FliC+) and CSH4(FliC-) served as positive and negative controls, respec-tively, for motility assays.

Protein analysis of flagellin and western hybridization tech-niques. Purified flagella from strain BB2000 were prepared asfollows. An overnight culture of cells was spot inoculated inthe center of each of 10 L agar plates. Following incubationat 37°C for 10 h (the time required for the swarming cells tocover the surface), cells were scraped off the agar surfacewith an L-shaped glass rod and suspended in 50 ml ofphosphate-buffered saline (PBS; 20 mM sodium phosphate

[pH 7.5], 100 mM NaCl). After vigorous vortexing for 2 min,whole cells were removed from the supernatant, whichcontained free flagella, by centrifugation in a Beckman J-21rotor at 4°C for 10 min at 10,000 rpm. Remaining whole cellswere removed by a second centrifugation under identicalconditions. Cell debris and fragmented cells were furtherremoved by centrifugation of the supernatant in a BeckmanJ-A-20.1 rotor for 20 min at 15,000 rpm and 4°C. Suspendedflagella contained within the supernatant were then pelletedby centrifugation in a Beckman SW27.1 rotor at 25,000 rpmfor 60 min at 4°C. The resulting pellet was suspended in 100to 250 Al of PBS. The crude preparation of flagella waslayered on a 25 to 75% sucrose step gradient (in PBS).Flagella were separated from membrane components andother cellular debris by centrifugation at 20,000 rpm for 3 hat 4°C in a Beckman SW27.1 rotor. A single band of >95%pure flagellin was extracted from the 30% sucrose step,extensively dialyzed against distilled water and lyophilizedto dryness. The flagella were suspended in distilled waterand used as a flagellin standard in immunoblots.To analyze the production of flagella in TnS-generated

mutants, we grew bacteria overnight in 2 ml of L brothcontaining chloramphenicol. The cells were concentrated bycentrifugation, and the pellet was suspended in ice-cold PBSto an optical density at 600 nm of 0.35. A 1-ml sample of eachculture was then pelleted in a microcentrifuge at 14,000 rpmfor 20 min at 4°C. The supernatant was removed, the pelletwas suspended in 50 ,ul of distilled water, and the sample wasstored at -20°C until needed. Wild-type whole-cell prepara-tions were treated in an identical manner.The presence of flagella on various Swr- and Swrcr

mutants was determined by Western blotting (immunoblot-ting) with antisera directed against the 36.7-kDa flagellinprotein of P. mirabilis. Immunoblotting was done by stan-dard methods with only minor modifications (16). The pro-tein concentration of purified preparations of flagella andwhole cells was determined by using the bicinchoninic acid(BCA) protein assay reagent (Pierce Chemical). For whole-cell preparations, 3 ,ug of protein was loaded per lane,whereas for purified flagellin, 0.8 ,ug was analyzed. Briefly,proteins in sample buffer were separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) ona 12% polyacrylamide resolving gel according to the proce-dure of Laemmeli (23). Electrophoretic transfer of proteins(35) to Immobilon-P membranes (Millipore) was done at 4°Cfor 1 h at 100 V in a buffer consisting of 0.096 M glycine,0.125 M Tris base, and 20% methanol. Western blots wereblocked with saturant (5% nonfat dry milk in 0.9% NaCl-20mM Tris [pH 7.5]). The immobilized proteins were thenreacted for 2 to 12 h with antiserum directed against thepurified 36.7-kDa flagellin protein. Antiserum 497 (anti-flagellin antiserum) was used throughout this study at adilution of 1:1,000. At this concentration a small amount ofbackground hybridization to three unidentified nonflagellinproteins was observed. This background did not hamper theanalysis of flagellin and was subtracted from the analyses.Unbound anti-flagellin immunoglobulin G (IgG) was washedaway, and goat anti-rabbit IgG conjugated to alkaline phos-phatase (Sigma Chemical) was then reacted with the immo-bilized proteins for 2 h at room temperature. The secondaryantibody was removed by washing in 0.9% NaCl-20 mM Tris(pH 7.5). To visualize the bound IgG conjugate, we reactedthe blot with 5-bromo-4-chloro-3-indolylphosphate and NitroBlue Tetrazolium substrate (BCIP-NBT substrate) until theprotein bands were suitably dark (usually 30 min). Thereaction was stopped by rinsing the blot in PBS containing 20

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mM EDTA. Once dried, blots were photographed and storedat room temperature. A silver-staining procedure (16) wasused to visualize proteins from whole-cell extracts whencomparisons between immunoblots and total whole-cell pro-tein samples were required.Antibody production. Polyclonal antisera were prepared

against the purified and denatured Proteus 36.7-kDa flagellinprotein. Purified flagellin was separated by SDS-PAGE asdescribed above. Proteins were visualized by Coomassieblue staining by standard procedures (13). The 36.7-kDaflagellin protein was excised from the stained gel, minced tohomogeneity with Freund's incomplete adjuvant, and sub-cutaneously administered to New Zealand White rabbits.Initially, 200 ,ug of purified protein was injected. Boostershots of an additional 50 ,ug of flagellin were given every 2weeks. Antibody titer was tested 1 week following anantigen boost and empirically determined by using immuno-blots to purified flagellin. Administration of the antigencontinued until the titer peaked, usually 45 to 60 days afterinitial injection. At the time of maximum titer, blood waswithdrawn from the ear artery and collected in a glass tube.Following clotting, the serum was removed and stored at-20°C until needed.Swarmer cell elongation analysis. The ability of P. mirabilis

Swr- and Swrcr mutants to elongate as a result of surfaceinduction was analyzed as follows. An overnight brothculture of P. mirabilis cells grown in L broth plus chloram-phenicol was inoculated on fresh L agar containing chloram-phenicol. After incubation at 37°C for 6 h, a loopful of cellsfrom the edge of the colony was removed aseptically, placedin 1 ml of PBS, and vortexed briefly. A sample was removedand immediately examined by phase-contrast light micros-copy. Wild-type P. mirabilis served as a control for elonga-tion comparison. A strain was considered to be Elo+ (sur-face-induced cell elongation) if more than 10% of thepopulation had an average length of at least 20 ,um (10swimmer cell lengths). To test for constitutive elongation(Eloc) mutants, a 16-h broth culture of P. mirabilis wasinoculated in fresh L broth containing chloramphenicol. Theculture was incubated with shaking for 6 h at 37°C, afterwhich a sample was removed for light-microscopic analysis.A strain was designated Eloc if, in this noninducing environ-ment, at least 10% of the cells were 20 ,um or longer. BB2000served as a control and typically had less than 0.5% swarmercells in an uninduced population.

Materials and reagents. All reagents were of the highestpurity available. Components of bacteriological media werepurchased from Difco. The indicators BCIP and NBT, usedto assay alkaline phosphatase, were purchased from Boehr-inger Mannheim Biochemicals and Sigma Chemical Co.,respectively.

RESULTS

Analysis of mutants defective in swarmer cell differentiationand swarming motility. Our interest in P. mirabilis concernsthe genetic regulation of swarmer cell differentiation and themechanisms of sensory transduction controlling multicellu-lar swarming behavior. To accomplish these goals, weundertook an analysis of mutants defective in wild-typeswarming. As described in the accompanying paper (7), aderivative of TnS was used to produce a bank of 13,036 P.mirabilis Cmr mutants. Initially, the bank of mutants wasmaintained on agar plates which prevented swarming motil-ity (LSW- agar). To detect strains which were defective inthe swarming phenotype, we replica plated the bank of

mutants onto L agar medium, which permitted swarmingmotility after incubation at 37°C for 4 to 6 h. Wild-typeswarming-proficient colonies, designated Swr+ (Fig. 1B),were distinguished from those completely lacking swarming(Swr-, Fig. 2A) and those showing abnormal or crippledmulticellular swarming behavior (Swrcr [cr for crippled]; Fig.2B to D) by examining the zone immediately around thereplicated colony. When strong backlighting was used, a thinfilm of swarming bacteria could be observed to move out-ward from the point of initial inoculation in Swr+ strains,whereas Swr- and Swrcr strains showed no bacteria past thepoint of inoculation after 6 h of growth at 37°C. Of the 13,036original Cmr mutants, 70 were found to be completelydefective in swarming motility (Swr-) and 142 producednon-wild-type swarming. This latter category containedpleiotropic Swrcr mutants, demonstrating a wide variety ofswarming behaviors including changes in the rate of swarm-ing motility and variations in consolidation pattern formationfrom the wild type. Although it was possible to distinguishwild-type swarming from Swr- or Swrcr mutants at 6 h orearlier, the distinction between Swr- and Swrcr was not seenuntil these mutants had been grown for 16 to 24 h at 37°C.After such incubations, Swr- colonies were observed toshow no growth past the point of inoculation (Fig. 2A),whereas Swrcr strains translocated outward from the inocu-lation point (Fig. 2B to D). However, although this group ofSwrcr mutants did manifest swarming motility, their move-ment and multicellular behavior was not wild type. Thegroup of 142 Swrcr mutants included strains which showedchanges in the spatial organization of consolidation zones(Fig. 2B and C), as well as mutants whose consolidationzones were indeterminate or ill defined (Fig. 2D).

Analysis of the frequency of acquiring Swr- and Swrcr inthe population suggests that the regulation of swarmer celldifferentiation and multicellular swarming motility must in-volve a complex set of genes. Although these experimentsdid not specifically address the question of how many genesare involved in swarming, our data show that approximately1.6% (212 Swr- and Swrcr mutants + 13,036 total mutants)of all Cmr mutants were defective in some aspect of swarm-ing. If the chromosome size and coding capacity of P.mirabilis is roughly equivalent to that of E. coli, the datasuggest that roughly 60 genes ([212 Swr- and Swrcr mutants+ 13,036 total mutants] x 4,000 genes per chromosome) mayplay a role in the process of swarming motility. If only Swr-mutants are considered in this type of analysis, the numberof genes involved in swarming and swarmer cell differentia-tion drops to around 20. These estimates compare favorablyto the 40-gene estimate reported by others (2). Since thedevelopment of swarmer cells and multicellular swarmingbehavior is a complex phenotype, most probably involvingbetween 20 and 60 genes, we analyzed the group of 212 Swr-and Swrcr mutants for other characteristics associated withswarmer cell differentiation and multicellular swarming mo-tility in an attempt to further define the nature of eachmutation.Swarmer cell differentiation and swarming motility in P.

mirabilis are the end results of at least four separate phe-nomena (37). First, the cells must sense the cue from thesurface and respond accordingly by initiating transcription ofthe set of genes whose expression ultimately produces thedifferentiated cell. Second, surface-induced transcription ofgenes regulating cell division must produce an elongatedcell. Third, the cell must synthesize vastly increasedamounts of flagellin for the new surface-induced flagella andmust express the appropriate genes whose products will

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FIG. 2. Colony morphology of P. mirabilis mutants defective in wild-type swarming motility. A loopful of an overnight culture grown inL broth was inoculated as a 1.5- to 2-cm line at the center of an L agar plate, and the cells were incubated for 16 h at 37°C. (A) BB2202 (Swr-Swm- Fla-) is an example of a mutant totally devoid of swarming motility. (B to D) Strains BB2180, BB2022, and BB2029, respectively, shownormal swimming and flagellar synthesis (Swm' Fla'), but are crippled in wild-type swarming motility (Swrcl) because of loss of spatial ortemporal control of consolidation (BB2180 and BB2022) or as a result of uncoordinated multicellular migration (BB2029), resulting in anindeterminate consolidation pattern.

rotate the flagella. Fourth, multicellular interactions andsignaling must occur between swarmer cells to allow them tomove out on the agar surface in groups and to consolidate(dedifferentiate) in unison. If any one of these events isdefective, the end result will be either complete loss ofswarming motility or abnormal swarming behavior. Thus, atleast four possible phenotypic categories should be observedin our analysis of Proteus Swr- and Swrcr mutants.

As mentioned above, one of the principal defects leadingto a Swr- strain is the failure to synthesize flagella. P.mirabilis produces only one type of flagellum, whose expres-sion is surface induced such that cells become hyperflagel-lated when swarmer cell differentiation occurs (4, 6). There-fore, Fla- Proteus mutants would be expected to not only bedefective in swarming motility, but also to have defects intheir swimming motility. To test for the presence and func-

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FIG. 3. Western blot analysis of flagellin synthesis in P. mirabilis swarming mutants. Overnight broth cultures or cells suspended fromplates were prepared as described in Materials and Methods. Except where noted, all preparations were from cells grown in L broth. Lanes:1, purified flagellin; 2, BB2000 (wild type, broth); 3, BB2000 (wild type, agar);'4, BB2035 (Swr- Swm- Fla'); 5, BB2128 (Swr- Swm- Fla');6, BB2075 (Swr- Swm+ Che- Fla'); 7, BB2178 (Swr- Swm+ Che- Fla'); 8, BB2040 (Swr- Swm+ Elo- Fla'); 9, BB2076 (Swr- Swm+ ElocFla'); 10, BB2105 (Swrcr Swm+ Eloc Fla+); 11, BB2029 (Swrcr Swm+ Fla'); 12, BB2202 (Swr- Swm- Fla-). (A) Silver stain of whole-cellpreparations and (B) Western blot with rabbit polyclonal antiserum 497 which is directed against the P. mirabilis 36.7-kDa flagellin (FliC)subunit. The blot was subsequently developed with goat anti-rabbit IgG-alkaline phosphatase secondary antiserum and NBT-BCIP substrate.The arrow indicates the presence of the 36.7-kDa FliC subunit. Molecular size markers in kilodaltons are indicated on the left.

tioning of flagella in the Swr- and Swrcr strains, we analyzedswimming motility, chemotactic behavior, and the ability tosynthesize flagellin. Overnight broth cultures of Swr- andSwrcr mutants were inoculated into the center of a plate ofsemisolid Mot agar. The bacteria were incubated at 37°C for

TABLE 2. Phenotypes of P. mirabilis Swr- and Swrcr mutants

Phenotypea No.mof Commentsmutants Cmet

Swr- Swm- Fla- 47 Nonswarming; do notsynthesize flagellin

Swr- Swm- Mot- 2 Nonswarming; paralyzedflagella

Swr- Swm+ Che- 6 Nonswarming; defective inchemotaxis

Swr- Swm+ Elo- 7 Nonswarming; defect inelongation

Swr- Swm+ Eloc 4 Nonswarming; constitutiveelongation

Swr- Swm+ Elo+ ??? 4 Nonswarming; unknownmutation

Swrcr Swm- 0 Crippled swarming; do notswim

Swrcr Swm+ Che- 13 Crippled swarming; defect inchemotaxis

Swrcr Swm+ Elo- 26 Crippled swarming; defect inelongation

Swrcr Swm+ Eloc 10 Crippled swarming; constitutiveelongation

Swrcr Swm+ ??? 93 Crippled swarming; unknownmutation

a ???, undefined mutants, defective in a currently unknown locus. For otherdefinitions, see Table 1, footnote a.

4 to 6 h, and the zone of swimming migration was measuredand compared with that of the wild type. Since lack ofmigration through Mot agar can be attributed either to adefect in flagellar synthesis (Fla-), or in the ability to rotatethe flagellum (Mot-) or to a defect in chemotactic sensing(Che-) of the attractant gradient established by the growingcells (24), we conducted tests to distinguish among the threepossibilities. Strains that failed to migrate in Mot agar weregrown for 4 to 5 h at 37°C in Mot broth and then examined forswimming motility by using light microscopy. If motile cellswere observed by light microscopy but motility was not seenon Mot agar, the strain was considered Che-. If no motilitywas seen in either case, the cells were considered either Fla-or Mot-. To distinguish between these two possibilities, wedetermined the presence of flagellin in whole-cell prepara-tions by using a Western analysis with rabbit polyclonalantisera elicited against the purified 36.7-kDa flagellin pro-tein of BB2000 (Fig. 3). The presence of a flagellin band wasused to confirm the existence of flagella. It is presumed forthe purposes of this assay that the only detectable flagellinpresent in the cell exists in the assembled flagellar filament,since a pool of unassembled flagellin monomers has not beenfound to exist in P. mirabilis (6). Strains possessing para-lyzed flagella (Fla') were designated as Mot-.The results of the motility, chemotaxis, and flagellin

testing are shown in Table 2. Not surprisingly, the loss ofswimming motility was consistent with the loss of swarmingmotility. The predominant defect (47 of 70) in Swr- mutantswas due to a failure to synthesize flagellin. The frequency ofSwr- Fla- mutants in this analysis is comparable to thefrequency of P. mirabilis Swr- Fla- mutants reportedelsewhere (2). These results support the idea that the sameflagella used for swimming motility are overexpressed duringswarmer cell differentiation in P. mirabilis. As seen in Fig. 3,Western analysis with polyclonal antiserum directed againstthe 36.7-kDa flagellin protein indicated that the amount of

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flagellin synthesized per cell was dependent on the type ofswarming defect. For example, Mot- strains BB2035 andBB2128 (Fig. 3, lanes 4 and 5) appeared to produce lessflagellin than did the wild type (lane 2) or Che- mutants(BB2075 and 2178 [lanes 6 and 7]), which produce slightlymore flagellin than the wild type. The cause of this variationin flagellar synthesis and its effect on multicellular swarmingmotility is not fully understood, but possible explanationsare discussed below.Loss of swimming motility was not observed in Swrcr

mutants. Since Swrcr mutants are strains that are defective inmulticellular behavior but display swarming motility, it is notsurprising that they have rotating flagella. Interestingly, 13 ofthe 142 Swrcr mutants (and 6 of 70 Swr- mutants) weredefective in chemotaxis behavior in semisolid and liquidmedia. The interpretation of this result is that a functioningchemotaxis sensory system is necessary for swarmer celldifferentiation and/or multicellular swarming motility. Thisagrees with the evidence on the involvement of chemotaxisin V. parahaemolyticus swarming motility (31), but is con-tradictory to the evidence accumulated for P. mirabilis (36).Possible explanations for this contradiction are discussedbelow.The second general defect that can give rise to a Swr- or

Swrcr mutant pertains to the set of genes controlling swarmercell elongation. Because elongation appears to be intimatelyinvolved in the process of swarming and differentiation, weexamined the 212 Swr- and Swrcr mutants to determinewhether defects in surface-induced elongation (Elo-) werepresent. Two criteria were used to assess swarmer cellelongation: (i) the percentage of the induced populationwhose length equaled or exceeded 20 ,um, and (ii) theaverage length of the swarmer cell population. BB2000grown on agar was used as a control and typically showed>80% swarmer cells at 40-,um average length under inducingconditions. As shown in Table 2, Elo- mutants accountedfor 10% (7 of 70) of the Swr- mutants and 18% (26 the 142)Swrcr mutants. Some of the Swrcr Elo- mutants (15 of 26)produced tightly arranged consolidation zones with limitedswarming motility, such as demonstrated by BB2022 (Fig.2C). The remaining Swrcr Elo- mutants in this group,although producing unusual, non-wild-type consolidationzones, were pleiotropic, showing a range of swarming ratesand consolidation patterns. The Swr- Elo- mutants did notswarm on agar, although swimming motility in this groupappeared to be wild type.An interesting category of swarming-defective mutants

appeared while we were examining the strains for swimmingmotility. Some mutants from both Swr- and Swrcr groupsconstitutively expressed the elongated swarmer cell (Eloc).Both groups had wild-type swimming motility and wereChe+. Flagellin synthesis in these cells was unusual. It wasanticipated that a constitutive swarmer cell would havegreatly increased flagellin synthesis, typical of surface-in-duced BB2000; however, the Western analysis (Fig. 3, lanes9 and 10) indicated that flagellin synthesis in broth-grownSwr- Eloc and Swrcr Eloc mutants was slightly lower or justequal to that seen in broth-grown wild-type cells (lane 2).Flagellin synthesis on Swr- Eloc and Swrcr Eloc mutantsgrown under inducing conditions was wild type, and the cellsappeared to have no other defects. One possible explanationfor this is that these Eloc mutants are constitutive only forelongation and not the entire pathway leading to the differ-entiated swarmer cell. Thus, Eloc mutants are partiallydefective in swarmer cell differentiation.Many of the Swrcr mutants (more than 65%) and a few of

the Swr- mutants (4 of 70) were wild type for swimming(Fig. 3, lane 11) and elongation phenotypes. Although ouranalysis does not provide definite answers about the identityof these mutants, many of the Swrcr mutants in this categoryproduced very unusual swarming zones. As shown in Fig.2D, this atypical swarming migration was unlike wild-typeswarming (Fig. 1A) and was very different from the tightconsolidation zones seen in other Swrcr mutants (Fig. 2C).The ruffled or frilled appearance of these indeterminatemutants suggests that the cells in this swarming colony aredefective either in synthesizing or in sensing the intercellularsignals which coordinate the processes of swarmer celldifferentiation and consolidation (or dedifferentiation).

DISCUSSION

It is intuitively evident that swarmer cell differentiationand swarming motility are the result of complex sensorytransduction and global control mechanisms. Since alterna-tive genetic methods, such as those for E. coli, are notcurrently available for use in P. mirabilis, we have devel-oped transposon mutagenesis techniques and applied themto our long-term study of the genetic regulatory mechanismscontrolling swarmer cell differentiation and multicellularswarming motility in this organism. More than 1.5% of allchloramphenicol-resistant mutants were in some way defec-tive in the swarming phenotype. Since this phenotype iscomplex, involving recognition of a surface, elaboration of amultiflagellated, elongated, polyploid swarmer cell, and in-tercellular communication necessary for swarming motility,it is not unreasonable to expect this frequency of Swr- andSwrcr mutants. Our data indicate that upward of 60 genesmight be involved in some aspect of swarming in P. mira-bilis. Other reports of P. mirabilis mutants defective inswarming (2) agree closely with our own estimate of 60genes, in this case placing the value at around 40 genes.Furthermore, for another swarming bacterium, V. parahae-molyticus, estimates of genes essential for its swarmingbehavior fall in the range of 25 to 50 genes (8), furthersupporting the data for P. mirabilis. Thus, approximately 40to 60 genes are involved in P. mirabilis swarming behavior,suggesting that this system is as complex as other globalregulatory systems (30).

Genetic analysis of Proteus strains showing non-wild-typeswarming resulted in the identification of two general classesof mutants. Bacteria in the first class of swarming mutantsmanifested no outward swarming motility when placed on asubstrate (L agar) known to induce swarmer cell differenti-ation and swarming motility in the wild type. This phenotypeis referred to as Swr-. P. mirabilis synthesizes only one typeof flagellin and flagella, although the synthesis is surfaceinduced (Fig. 3, lanes 2 and 3). This statement is supportedby the following observations. (i) In the present analysis, allFla- mutants were also Swr-. (ii) Only one flagellin bandwas observed by PAGE and Western hybridization. (iii) Nullmutants (such as BB2202 [Fig. 3, lane 12]), which arecompletely defective in flagellin synthesis, are defective forboth swimming and swarming. (iv) The Proteus fliC (flagel-lin) gene has recently been cloned (6) in our laboratory.Hybridization of cloned fliC sequences to Proteus chromo-somal DNA does not indicate the presence of multiple genes(6). Because the same flagellin is used for swimming andswarming motility, it is not unusual to find that all mutantsdefective for swimming motility (Swm-) were also Swr-.Thus, the flagellar genes which encode the constituent partsof the "swarming" flagellar structure are the same genes

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encoding the "swarming" flagella in P. mirabilis. This issimilar to the case in S. marcescens (1), but is in contrast toswarmer cell differentiation in V. parahaemolyticus, inwhich the constitutively synthesized polar flagellum genesare apparently unique and not shared in the production of thesurface-induced lateral flagella, whose synthesis is encodedby a different set of genes (29).Western blots with rabbit polyclonal antisera directed

against the 36.7-kDa flagellin protein from P. mirabilis wereused to detect the presence of flagellin in whole cells grownunder noninducing (liquid) conditions. These analyses wereused principally to confirm that cells were synthesizingflagella and to qualitatively measure the amount of flagellinsynthesized in uninduced cultures. P. mirabilis flagellin is agood indicator of swarmer cell induction, since its synthesisis surface induced. For example, lanes 2 and 3 in Fig. 3 showthe difference in the amount of flagellin produced by wild-type cells grown under noninducing conditions (lane 2,broth-grown cells) and inducing conditions (lane 3, agar-grown cells). Densitometric measurements of the stained36.7-kDa protein indicate that there is at least a 40- to 50-foldincrease in flagellin synthesis in cells grown on agar com-pared with that in cells grown in liquid medium (Fig. 3).Silver stains of whole-cell preparations also reveal that manyother proteins are differentially regulated as a consequenceof surface induction (lanes 2 and 3). The identity of theseother surface-induced proteins is currently not known, butthey may be essential for the formation of cell walls orflagellar components in the differentiated cell. This evidencecoincides with that in other reports showing similar differ-ences in enzymatic activity and protein composition whenswimmer and swarmer cells are compared (3, 5, 14).Immunoblots also demonstrated that although many of the

Swr- and Swrcr mutants produced flagellin, the synthesis ofthis protein was lower than that in the wild type. The reasonfor this lowered production is not clear from our data, butmay reflect the nature of the mutation. For instance, bothMot- strains examined in Fig 3 (lanes 4 and 5) showeddecreased flagellin synthesis, whereas both Che- strainsproduced wild-type amounts of flagellin. This correlation isnot very tight, however, when extended to other pheno-types, particularly in Elo- and Eloc mutants (lanes 8 to 10).In this case, Swr- Elo- and Swr- Eloc strains produceddecreased level of flagellin (compared with the wild type),but a Swrcr Eloc mutant appeared to synthesize normalamounts of flagellin. One possible explanation for this resultis that the transposon has inserted in genes at different pointsin the regulatory hierarchy controlling swarmer cell differ-entiation. Such a possibility is currently being explored.Some of the Swr- (as well as Swrc) mutants were

defective in chemotaxis behavior. These mutants possessedfunctioning flagella, but had lost the ability to detect thepresence of attractants or repellents in the surroundingenvironment. Sar et al. (31) have shown that in V. parahae-molyticus, the same chemotaxis system which controls therotational bias of the polar flagellum functions to regulate thedirection of rotation of the lateral flagella. This conclusionsupports the work presented here and indicates that muta-tions in che genes give rise to defects in swarming behaviorin P. mirabilis. However, Williams et al. (36), in an attemptto substantiate the hypothesis that chemotaxis is the drivingforce in Proteus swarming (25), failed to find any evidence tosuggest that chemotactic behavior was necessary for thesignal initiating swarming motility. The current data cannotresolve the obvious contradiction between the earlier workwith P. mirabilis; however, the fact that both Swr- and

Swrcr phenotypes shared defects in chemotaxis behaviorsuggests that the role of chemotaxis in Proteus swarmingmay have to be reevaluated. Moreover, our data implicatechemotaxis behavior not in the initial onset of swarmer celldifferentiation, but in the latter stages of swarming motility,specifically the stages involving multicellular coordination ofthe consolidation process.

Although most of the Swr- mutants were also Swm-, theremaining 30% of Swr- strains were wild type in their abilityto synthesize and rotate their flagella (Fla' Mot') and inchemotaxis (Che+). These Swr- Swm+ mutants fell into atleast three distinct groups. Some of the mutants weredefective in wild-type swarmer cell elongation (Table 2,Elo- and Eloc groups). Unlike the wild-type cells, which donot septate properly when grown on a surface, Elo- mutantsseptated normally, and did not elongate, when grown underinducing conditions. This may be due to a defect in a globalsurface induction response or in a specific gene responsiblefor regulating septation. In contrast, Eloc mutants wereconstitutive for swarmer cell elongation and demonstratedinhibited septum formation at all times. Although suchdefects in P. mirabilis have not been analyzed, possiblecandidates for such mutations might be analogs of theftsZAQ genes of E. coli. In E. coli, these genes regulatenormal septation and, when mutated (26), can cause aphenotype similar to that of the elongated Proteus swarmercell. The E. coliftsZAQ genes are not known to be regulatedby growth on surfaces, so if Proteus analogs offtsZAQ areinvolved in swarmer cell elongation, they must be under adifferent control mechanism from their E. coli counterparts.The majority of swarming mutants isolated in this study

were not completely defective in swarmer cell differentiationand swarming motility (null mutants [Fig. 2A]); rather, theydisplayed pleiotropic phenotypes, suggesting that for many,the mutation might be in a gene affecting multicellularbehavior. In this report we have opted to designate mutantsthat show non-wild-type swarming as Swrcr mutants todistinguish them from mutants which fail to swarm at all(Swr-). These Swrcr mutants failed to produce wild-typeconsolidation zones (Fig. 2B to D). Mutants in this broadgroup fell into phenotypic classes that produced eithernarrow or broad consolidation zones, nonuniform consoli-dation zones, or indeterminate consolidation zones. The firstphenotypic class appeared to have a defect in the spatial ortemporal control of the multicellular behavior presumed tocontrol the sequence of events involving differentiation anddedifferentiation. It may be that in such mutants the signalsregulating swarming behavior are either synthesized morerapidly than in the wild type, giving rise to faster consolida-tion and narrower zones (Fig. 2B to C), or synthesized moreslowly, resulting in broad zones due to a diminished consol-idation process. The class of Swrcr mutants which showsnonuniform consolidation zones is depicted in Fig. 2B.These mutants form swarming colonies that do not have theequal spacing between these zones typical of the wild type.The last class of Swrcr mutants found in P. mirabilis mani-fests no discrete pattern of consolidation zones (Fig. 2D).Only a few such mutants (9 of 142) were isolated; however,they are perhaps the most interesting of all our mutantsbecause of the presumed nature of the defect. The swarmingpattern produced by these indeterminate mutants suggeststhat the swarming colony fails to either produce or sense thepresence of the signals needed for consolidation. The resultis a swarming colony with many ruffles or frills that areproduced as a result of uncoordinated differentiation anddedifferentiation cycles. We hypothesize that the mutations

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producing this phenotype are localized in genetic loci whichencode proteins necessary for the production or recognitionof putative signal molecules whose primary activity is tocoordinate each cycle of differentiation and dedifferentia-tion. Such Proteus mutants have been observed by others(2), implying that this phenotype is real and not the result ofa double mutation or some spurious event. Genetic analysisin Myxococcus xanthus has defined at least four signalmolecules (csgA, csgB, csgC, and csgD) that apparentlycontrol the multicellular differentiation leading to fruiting-body formation (22). Although merely speculative at thisjuncture, it is nevertheless plausible that Proteus swarmnercell differentiation and swarming motility share some of thefeatures of the sensory transduction pathways observed inthe myxobacteria. The use of transposon insertions to pro-duce mutations in swr genes will facilitate our efforts tounderstand the nature of these defects, since a selectablemarker (the chloramphenicol resistance gene) has now beenphysically linked to the Proteus gene. We are currently usingthis approach to clone the flanking Proteus DNA into E. colifor further analyses aimed at understanding cell-to-cell com-munication in P. mirabilis.

ACKNOWLEDGMENTS

This research was supported by Public Health Service grantAI-27107 from the National Institute of Allergy and InfectiousDiseases of the National Institutes of Health.We thank Roger Chien for his contribution to some of the

experiments described in this paper; Philip Rutledge for assistancewith the electron microscope; and Paul Lovett, Harold Schreier,and James Shapiro for useful discussions.

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Proteus mirabilis Mutants Defective in Swarmer Cell