JOURNAL OF BACTERIOLOGY, Nov. 1969, p. 769-785Copyright 0 1969 American Society for Microbiology

Vol. 100, No. 2Printed in U.S.A.

Isolation and Characterization of Host-IndependentBdellovibrios

RAMON J. SEIDLER' AND MORTIMER P. STARR

Department of Bacteriology, University of California, Davis, California 95616

Received for publication 24 July 1969

A reliable method has been developed for the isolation of host-independent (H-I;i.e., "saprophytic") strains of Bdellovibrio from host-dependent (H-D; i.e., "para-sitic") cultures. The technique involves growing streptomycin-resistant (Smr) H-Dcultures on streptomycin-susceptible (Sm") host cells. A lysate containing largenumbers of the Smr H-D cells and some remaining Sm8 host cells is transferred to aselection medium which contains the antibiotic. The Sm8 host cells in the lysate arekilled, and the Smr H-I strains develop in broth within 3 to 6 days. By use of thismethod, it has been possible to isolate H-I strains from 16 different H-D Bdellovibriostrains studied. The frequency of occurrence of host independence is in the range ofone H-I colony per 106 to 107 plaque-forming units of H-D bdellovibrios. The H-Icultures are nonfermentative, do not reduce nitrate, are strongly proteolytic, areoxidase-positive, and do not utilize 14 different carbon compounds as sources ofenergy for growth. Most H-I cultures are catalase-positive upon initial isolationfrom H-D lysates, but some cultures lose this enzyme upon subsequent transfersthrough host-free media. Most H-I bdellovibrios are pleomorphic, consisting ofvibrio- to spiral-shaped cells typically measuring 0.3 to 0.4,tm in width and 1 to 10Mm in length. All H-I bdellovibrios have a cytochrome a and c component (H-IA3.12 differs from the other strains in the location of the peaks of the cytochromespectrum). All are sensitive to oxytetracycline and (except for strain H-I A3.12) tothe vibriostatic pteridine 0/129; most bdellovibrios, except for H-I A3.12, are gen-erally uniformly resistant or susceptible to a given antibiotic. Bdellovibrioand Vibrio spp. have common cytochrome difference spectra and susceptibilitiesto oxytetracycline and to the vibriostatic pteridine 0/129. All H-I bdellovibrios ex-amined produce an exocellular protease which digests heat-killed host cells. Bdel-lovibrios possessing predatory and bacteriolytic properties could be reselected fromH-I bdellovibrio cultures growing in the presence of living host cells. Attempts toselect for bacteriolytic isolates from Vibrio and Spirillum spp. were unsuccessul.

Bdellovibrio bacteriovorus, the unusual bac-terium which preys upon, enters, and lyses thecells of other bacteria (4, 16-18), was at firstbelieved to be obligatorily parasitic, i.e., host-dependent (H-D). However, even in the earliestpublications on Bdellovibrio (17, 18), it was shownthat yellow colonies of "saprophytic" [i.e., host-independent (H-I)] derivatives of the H-DBdellovibrio could be found, after 5 to 6 days ofincubation, when concentrated suspensions oflysates were inoculated onto a yeast extract-peptone-agar medium. Unfortunately, this tech-nique for isolation of H-I bdellovibrios has aserious limitation: plates which receive an inocu-

1 Present address: Department of Biology, University of TexasM. D. Anderson Hospital and Tumor Institute at Houston,Houston, Tex. 77025.

lum large enough to contain the relatively rareH-I cells usually receive also a large number ofhost cells which form a nearly confluent hostlawn after 2 days of incubation. Consequently,the small colonies of the slowly growing H-Istrains cannot always be detected.

Shilo and Bruff (10) isolated H-I derivativesfrom the H-D Bdellovibrio strain A3.12 by meansof a differential filtration of the lysate through a0.45-jum membrane filter. This technique was notsuccessful for isolating H-I derivatives from theonly other H-D strain (H-D 109) tested by Bruff(B. S. Bruff, M.S. Thesis. Univ. of California,Berkeley, 1964), who reported also that only 106to 107 cells of the H-D strain A3.12 were neces-sary to initiate growth of an H-I culture.The frequency of H-I cells in H-D popula-

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tions suggests that such strains arise by mutation.The formation of plaques when a large popula-tion of H-I cells is plated on a susceptible host(17, 18) suggests the possible occurrence ofback-mutations for host dependence. However,in the absence of direct evidence that these eventsare indeed mutational, we prefer to use the non-commital terminology "H-I derivative" or "H-Istrain" rather than "H-I mutant."We describe here a reliable technique for the

isolation of H-I derivatives from H-D cultures ofbdellovibrios, together with the morphological,cultural, and biochemical characteristics of theseH-I strains. Elsewhere (8), we deal with thedeoxyribonucleic acid (DNA) characterization ofH-I (as well as of H-D) bdellovibrios.

MATERIALS AND METHODSCultures. Many of the H-D Bdellovibrio strains

used (100, 101, 109, 110, 114, 118, 120, A3.12) wereamong those isolated by Stolp and Starr (18). Addi-tional cultures were kindly supplied by M. Shilo(B, D, E), A. Guelin (Xty; reference 4), D. A. Klein(OX9-2, OX9-3), and A. J. Hansen (2484 Se-2,2484 Se-3). In addition, several Vibrio and Spirillumstrains were employed in comparative studies. Theseincluded Vibrio spp. NCTC 4711, NCTC 8042, IP5638 (21), V. metchnikovii ATCC 7708, and Spirillumgracile (2) ICPB 2853 (D-4), ICPB 2854 (D-5), andICPB 2855 (D-6). (Abbreviations used: NCTC, Na-tional Collection of Type Cultures; IP, PasteurInstitute; ATCC, American Type Culture Collection,ICPB, International Collection of PhytopathogenicBacteria.)

Media. Enriched nutrient broth (NB) contained:Difco Nutrient Broth, 8 g; Difco Casamino Acids,5 g; Difco yeast extract, 1 g, and distilled water, 1liter (17).

Dilute nutrient broth (NB/10) was prepared from1 volume of NB plus 9 volumes of distilled water.After autoclaving, the following salts (7) were addedfrom a filter-sterilized stock solution: MgC12-6H20(0.003 M Mg++, final concentration) and CaCl2-2H20(0.002 M Ca++); pH was adjusted to 7.2 with 0.1 NNaOH. This medium was used for the propagationof the H-D Bdellovibrio strains in cultures of theirhosts.

Peptone-yeast extract (PYE) broth consisted of10 g of Bacto peptone and 3 g of Difco yeast extract,added to 1 liter of distilled water [HP medium ofStolp and Petzold (17)]. Plates were prepared by theaddition of Difco agar (usually 15 g/liter) or Oxoidlonagar No. 2 (8.5 g/liter; "PYE-ion" agar). PYEbroth was used for growing H-I cultures of Bdellovi-brio. A diluted PYE broth, PYE/5, was made bymixing 1 volume of PYE broth with 4 volumes ofdistilled water.YDC agar contained: Difco yeast extract, 10 g;

glucose, 20 g; finely divided calcium carbonate, 20 g;Difco agar, 15 g; and distilled water, 1 liter.

Enumeration of plaques. Plaque counts on hostlawns on double-layer NB/10 plates were used to

enumerate the H-D cells. For the bottom layer, 1%Difco agar was used, and the top layer (2.5 ml) con-tained 0.8% Difco agar and 0.5 ml of an overnightNB culture of host bacteria. For further details, seeSeidler and Starr (7).

Maintenance of stock cultures. Stock cultures ofthe various H-D Bdellovibrio strains were maintainedin NB/10 cultures of the host bacteria at 4 C. Nearlyall cultures remained viable for up to 4 to 6 monthsin this condition. H-D A3.12 lost its viability withinseveral weeks at 4 C; it was stored at room tempera-ture in an NB/10 lysate and transferred every 3 to 5weeks. All H-I cultures were maintained at roomtemperature on PYE agar plates, and transferred atapproximately 4-week intervals. Cultures of the hostbacteria were maintained on YDC agar slants, whichwere stored at 4 C and transferred every 5 to 6 months.All Bdellovibrio cultures have been lyophilized (13)and are stored in the International Collection ofPhytopathogenic Bacteria maintained in this Depart-ment.

Electron microscopy. To obtain actively growingcultures of H-I bdellovibrios for morphologicalstudies, H-I cells were inoculated into 25 ml of PYEbroth from PYE-ion agar plates, and were incubatedat 28 C; after 2 to 3 days, an actively growing turbidculture had developed. A 15 to 20% (v/v) inoculumwas transferred to fresh broth and incubated over-night. The cells were fixed in 2% Formalin (finalconcentration) for 30 min, washed twice, and allowedto resuspend slowly in 2% Formalin. Most prepara-tions were stained with 0.5% uranyl acetate; somewere stained with 0.5% phosphotungstic acid (pH7.2). Specimens were examined in an RCA-EMUelectron microscope.

Cytochrome difference spectra. Cultures were grownfor 2 to 3 days in PYE broth, shaken at 28 to 30 C.The cells were washed twice and resuspended in cold0.03 M phosphate buffer (pH 7.2) at a density of 3,100to 3,600 Klett units (filter 42). Cytochrome differ-ence spectra of several H-I bdellovibrios and of fourstrains of Vibrio spp. were determined by the tech-nique of Stanier, Palleroni, and Doudoroff (15). Thedifference spectra of one H-D culture (H-D 110)was determined and compared with that of its H-Iderivative; this parasite was propagated on Erwiniaamylovora (ICPB EAl1). Absorption peaks weredetermined for Pseudomonas maltophilia (ICPB2648-67) as a reference culture, and the values werecompared with those of a previous determination(15).

Protocol for the isolation of H-I bdellovibrios. Inaddition to trying the previously described proce-dures (10, 17), we used an original method for theisolation of H-I strains, which involves three phases.Phase I was the selection of streptomycin-resistent(Smr) mutants of the H-D cultures. H-D cells werepropagated on an Smr host in NB/10 at 30 C for24 hr. Streptomycin was added to 500 Ag/ml, and theculture was incubated for an additional 12 hr. Then,4 to 6 ml was transferred to 25 ml of NB/10 contain-ing streptomycin (500 ,ug/ml) and an Smr host. Whenthe H-D bdellovibrios became predominant (3 to 4days), a 10-ml sample of the lysate was washed free

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from streptomycin by centrifugation. The Smr H-Dcells were separated from the Smr host cells by differ-ential filtration through a 0.45-jAm membrane filter(Millipore Corp., Bedford, Mass.). In phase II, thefiltered H-D Smr culture was inoculated into NB/10containing a suitable Sm9 host. When the H-D cellsreached about 109 plaque-forming units (PFU)/ml,5 to 10 ml of the lysate was transferred to the selectionmedia, which contained 500 ,ug of streptomycin/ml(phase III). The selection media employed were NB/10 (which contained approximately 109 autoclavedEscherichia coli B cells per ml) and PYE broth. Thecultures were incubated for 3 to 6 days at 28 C, duringwhich time growth of the H-I cultures became ap-parent. Cultures were transferned at least twice throughPYE broth, with the use of large inocula (about 15to 25%, v/v). The H-I isolates were then transferredto PYE agar plates.

Diagnostic tests. Bacteriological diagnostic testswere performed in the manner recommended in theManual of Microbiological Methods (13).

For carbon-utilization experiments, cultures weregrown to the stationary phase in PYE broth (1.5 to2 days, 28 C). A 10% (v/v) inoculum was transferredinto a complex, growth-limiting medium consistingof 0.2% Difco yeast extract, 0.2% Bacto peptone,and 0.2% of the carbon source being tested. Theamount of growth in the control broth (withoutadded carbon source) was compared with the growthin the experimental flasks and in a medium containingan additional 0.2% Bacto peptone, by means of aKlett colorimeter (filter 42), after 2 days of incuba-tion at 28 C.The test for gelatin liquefaction was carried out in

12% Difco gelatin made up in PYE broth. Nitratereduction was tested in static tube cultures of PYEbroth (4 ml) supplemented with 0.1% KNO3. Samplesof the cultures were tested for nitrite and ammoniaproduction after 3, 5, and 7 days of incubation. Forindole production, cultures were grown in 0.3% Difcoyeast extract plus 1.0% Difco Proteose Peptone No. 3,instead of PYE broth, because of the higher trypto-phan content in the former medium (Difco Manual,9th ed.); all H-I cultures grew well in this medium.

Antibiotic and antimetabolite susceptibility wasdetermined with Bacto-sensitivity disks, mediumconcentration (Difco Laboratories). Tests for suscep-tibility to the vibriostatic pteridine 0/129 (9) wereaccomplished by streaking duplicate plates of PYEagar with the culture under investigation and sprink-ling one plate with crystals of the vibriostatic material.Inhibition of growth by the antibiotics and anti-metabolites was scored after 5 to 6 days of incubationat 30 C on PYE agar.The oxidase test was performed in the manner

described by Stanier, Palleroni, and Doudoroff (15),with the use of a 1% (w/v) aqueous solution of N, N'-dimethyl-p-phenylenediamine.

Assay of exocellular protease. For the assay ofprotease, the substrate consisted of an autoclavedsuspension of E. coli B cells in 0.001 M tris(hydroxy-methyl)aminomethane buffer (pH 7.2). The E. coliB culture was prepared by growing it to the stationaryphase in NB, washing the cells with distilled water,

and concentrating them 10-fold in the buffer. The cellsuspension was autoclaved (15 lb) for 7 min. For thedetection of protease, actively growing cultures ofthe H-I bdellovibrios were propagated in PYE brothovernight at 30 C. Culture samples were centrifugedto remove the cells, and sufficient autoclaved E. coliB cells were added to the spent culture medium togive approximately 200 Klett units of turbidity (filter42). These turbid mixtures were incubated in a waterbath at 37 C in Klett tubes, and readings were re-corded periodically. In addition to using PYE broth,some H-I cultures were also grown in PYE/5 brothcontaining approximately 109 autoclaved E. coli Bcells/ml.

Bacteriolysis of autoclaved and living host cells.H-I cultures were grown to the stationary phase in50 ml of PYE broth and 5- to 7-ml amounts weretransferred to fresh broth at 30 C about 24 hr beforethe experiments. Host bacteria were grown on nu-trient agar slants for 24 hr at 30 C, washed, and sus-pended (approximately IO9cells/ml) in sterile distilledwater; these preparations served as the living host cellsuspension. For the autoclaved host cell suspension,P. putida A3.12 (ICPB 2484) was grown to the sta-tionary phase in PYE broth at 30 C, washed, resus-pended five-times, concentrated in distilled water, andautoclaved (15 lb, 10 min). Amounts of 0.5 ml ofliving host cell suspensions were inoculated ontodouble-layer NB/10 plates, and 1 ml was inoculatedinto 25 ml of NB/10. About 2.5 ml of the autoclavedhost cell suspension was placed into 25 ml of NB/10.The experimental protocol involved inoculation ofsamples of the 24-hr H-I cultures into the followingmedia containing host cells: 1-ml samples diluted100, 10-1, and 10-s inoculated onto NB/10 agar platescontaining living host cells; 2-ml samples into 25 mlof NB/10 containing living host cells; and 3-ml sam-ples of H-I cultures inoculated into NB/10 containingautoclaved cells of P. putida (approximately 109/ml).All cultures were incubated at 30 C. Cultures in brothwere observed after 1 and 3 days of incubation; theNB/10 plates were observed after 4 days. The hoststrains used in this study were P. putida A3.12 (ICPB2484), E. coli B (ICPB 2262), Enterobacter aerogenes(ICPB 2001), Proteus mirabilis (ICPB 2575), andSerratia marcescens (ICPB 2031).

RESULTS

Isolation ofhost-independent bdellovibrio strains.Three isolation techniques were used in our at-tempts to obtain H-I cultures of Bdellovibrio.However, only one method proved to be con-sistently reliable in our hands. The disadvantagesand limitations of the other techniques willfirst be presented.The initial attempts to isolate H-I strains of

Bdellovibrio followed in principle the method ofStolp and Petzold (17). H-D cultures were grownwith their hosts in NB/10 for 2 days at 28 C.Samples of the lysate were removed, concentratedby centrifugation, and plated onto PYE agar or

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NB agar. Normally, after overnight incubation,the plates of these rich media were covered withhost colonies. After 5 to 6 days of incubation, itwas futile to attempt to locate any H-I coloniesin the heavy growth of the host bacteria. Stolp(personal communication) has suggested that useof older lysates, which have fewer residual hostcells, might have resulted in greater success inisolating H-I strains by this method. However, inolder lysates there is a rapid decline in H-DBdellovibrio titer (18, 19).

Further trials employed the differential filtra-tration procedure of Shilo and Bruff (10). Ourfirst successful isolation by this technique was ofan H-I strain from H-D A3.12. Broth inoculatedwith filtrates of H-D 100, H-D 101, H-D 109,and H-D 118 did not show growth after 5 to 7days of incubation. Isolation attempts were re-

peated several times with the latter strains andwere eventually successful for isolating H-Istrains from H-D 109 and H-D 118.The successful technique we originated involves

the propagation of Smr H-D bdellovibrios onSm5 host cells. Upon inoculation of large num-bers of the H-D cells into the selection media(see Materials and Methods), growth of the H-Istrain was apparent within 3 to 6 days. H-Istrains were isolated by this method from everyone of the H-D cultures listed in Materials andMethods.Frequency of host independence. The use of

Smr H-D bdellovibrios also makes it possible todetermine the frequency with which H-I coloniesappear on agar media. Thus, by inoculation oflarge numbers of Smr H-D Bdellovibrio cells onagar plates containing streptomycin, only SmrH-I cells would form colonies, whereas the growthof the Sm8 host .cells, and consequently that ofH-D cells, would be inhibited. The H-D titercould be determined in terms of PFU on double-layer NB/10 plates. The results of one suchenumeration of H-I derivatives is shown in Table1.

After 7 days of incubation at 30 C, the PYEagar plates were examined. Those plates withgrowth contained hundreds of yellow colonieswhich were quite heterogeneous in size, rangingfrom 1 to 2 mm down to barely perceptible. Theplates were transferred to room temperature andincubated for an additional 6 days. At this time,many colonies were large (2 to 3 mm), but theplates still contained many colonies that weresmall and difficult to count. The colony countsgiven in Table 1 are thus only estimates and rep-

resent counts accurate to the nearest 100 colonies(about 5 to 15% error). The frequency of oc-currence of host independence (106 to 107 PFU/H-I colony) appears rather uniform in the cul-

tures tested; it is in the range of a single muta-tional event. Our results are comparable to thevalue reported for Bdellovibrio strain A3.12 byBruff (M.S. Thesis, Univ. of California, Berkeley,1964). A lower frequency (5 X 108 PFU/H-Icolony) has been reported for H-D 321 (17); wedo not know whether this is due to strain differ-ences or perhaps to an insufficient detection ofH-I 321 colonies on plates contaminated withhost cell colonies.

Diagnostic tests. A number of bacteriologicaldiagnostic tests were made with 16 H-I cultures;E. coli B was used as a control for each test. Allofthese H-I Bdellovibrio strains were oxidase-posi-tive, liquefied gelatin, produced ammonia fromPYE broth, and could grow on an autoclavedE. coli cell suspension in PYE/5 broth. None ofthe cultures produced indole nor reduced nitrate;growth did not occur at 12 and 42 C. Most H-Icultures (12 of 16) were catalase-positive uponprimary isolation. However, after transfer onhost-free media for 7 months, subsequent testsfor catalase were negative in five strains (H-I101, H-I 110, H-I 114, H-I 118, and H-I 120).Four strains were catalase-negative upon primaryisolation (H-I 100, H-I 109, H-I A3.12, and H-IOX9-2). Cultures H-I OX9-3, H-I 2484 Se-2,H-I 2484 Se-3, H-I B, H-I D, H-I E, and H-I Xtyhad been transferred for 3 to 4 months on host-free media and, at that time, remained catalase-positive.

Antibiotic susceptibility tests. A relativelyuniform response to 20 antibiotics and antime-tabolites was observed among the 11 H-I Bdel-lovibrio strains subjected to these trials. Thebdellovibrios were generally susceptible toAltafur, kanamycin (three strains resistant),methenamine mandelate (one strain resistant),neomycin, novobiocin, polymyxin B (two strainsresistant), oxytetracycline, and vibriostat 0/129(one strain resistant); they were not discerniblysusceptible to colistin (one strain susceptible),sulfisomadine sulfisoxazole (one strain suscepti-ble), isoniazid, sulfadimethoxine mystatin, olean-domycin (one strain susceptible), penicillin(three strains susceptible), sulfadiazine, sulfa-methoxy-pyradazine, triple sulfa, and vancomy-cin. It is noteworthy (Table 4) that the unusualstrain H-I A3.12 was uniquely susceptible tocolistin, sulfisoxazole, and oleandomycin, andwas the only Bdellovibrio strain not susceptibleto the vibriostatic pteridine 0/129. In seven cases,at least two of the Vibrio cultures showed re-actions different from those of the bdellovibrios.Carbon utflization by H-I bdellovibrios. Nine

strains of H-I bdellovibrios were tested for theutilization of 14 carbon sources, which includedcarbohydrates (deoxyribose, ribose, xylose,

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HOST-INDEPENDENT BDELLOVIBRIOS

galactose, glucose, sucrose), acids (formate,acetate, pyruvate, citrate, asparate, glutamate,a-ketoglutarate), and glycerol. Except that gluta-mate and a-ketoglutarate appeared stimulatoryfor three cultures (H-I 101, H-I 120, and H-I 2484Se-2), there was no other significant increment ingrowth with any carbon compound over that ob-tained in the carbon-limiting control culture, and itmust be concluded that none of these substancesserved as a major source of carbon or energy,or of both. Of possible metabolic significance isthe uniform strong inhibition of growth in allcultures by citrate.

Tests for fermentative capabilities of H-I bdel-lovibrios. Three cultures (H-I A3.12, H-I 100,and H-I 109) were tested for possible fermenta-tive abilities in the growth-limiting complex me-dium (0.2% Difco yeast extract plus 0.2% Bactopeptone) supplemented with 1% glucose, sucrose,or fructose. Cultures were incubated in test tubes(16 x 200 mm) containing 5 ml of medium for 8days at 30 C. Samples were removed after 3 and8 days of incubation, and the pH was determined.No detectable acid was produced. There stillremained the possibility that the H-I cultureswere capable of utilizing amino acids or peptidesanaerobically. For the determination of such a

trait, cultures were inoculated into tubes of BBLThioglycollate broth supplemented with 0.1%Oxoid lonagar. The presence of oxygen in themedium was detected by resazurin in the BBLbroth. After 4 to 6 days of incubation at 28 C,growth of the eight H-I cultures was detectableas a narrow turbid region within and at the bot-tom depth of oxygen penetration (but not belowit); none of the cultures tested formed a surfacepellicle. These observations indicate that H-Ibdellovibrios can grow at full atmospheric oxygentension (shaken broth cultures and on agarplates) but, when confronted with a "choice,"they appear to prefer an oxygen tension some-what less than atmospheric.Cytochrome difference spectra. The cytochrome

difference spectra are recorded in Table 2 and inFig. 1. The peaks at 522 to 524 nm indicate thepresence of a cytochrome c in all of the Bdellovi-brio cultures examined (12). On the basis of theabsorption peaks corresponding to a cytochromea and the soret region, two patterns occurredamong the bdellovibrios: the pattern of H-IA3.12 (607 nm, and 417 nm) and the pattern com-mon to all other H-I strains (596 to 600 nm and421 to 423 nm). Some cultures showed peaks at486 to 488 nm; it has not been determined whatmaterial absorbs in this range, but, according toSmith (12), it is not a cytochrome component.The cytochrome absorption spectra of three ofthe Vibrio strains examined are essentially the

TABLE 1. Enumeration of, andfrequency ofalterationto, host independence in bdellovibriosa

H-D PFU per ml H-I coloniesBdellovibrio on NB/10 per ml on Frequency

strain double-layer PYE + (PFU/H-Iplates 0.85% Difco colony)

Xty...... 42 X 109 8,000 5 X 108OX9-2. 27 X 109 8,000 3 X 106OX9-3. 37 X 109 12,000 3 X 106B...... 42 X 109 8,000 5 X 106

a Smr H-D cultures were propagated on E. coliB Sms in NB/10 at 34 C for 14 hr. Samples of thelysates were centrifuged and concentrated 20-foldin NB/10. Samples were diluted and plated intriplicate for enumeration of plaques (PFU),while 0.05, 0.1, and 0.2 ml were plated from theconcentrated lysates onto duplicate plates of PYEagar for enumeration of H-I colonies.

same as those of the bdellovibrios; however, thespectrum of V. metchnikovii differed significantlyfrom those of the bdellovibrios and from those ofthe other three strains of Vibrio spp. examined.The spectrum of Bdellovibrio H-D 110 is the sameas that of its H-I derivative.Morphology of H-I isolates. Most H-I Bdello-

vibrio strains we have examined show a strongdegree of pleomorphism, as described earlier(17). Within the same culture, the populationcontains rather uniform vibrio-shaped cells toquite long spiral- or bizarre-shaped cells, with manygradations between. This pleomorphism is par-ticularly well illustrated in cultures of H-I 101,H-I 110, H-I 114, and H-I 118 (Fig. 2 and 4-6).The cells are relatively uniform in thickness,ranging from 0.3 to 0.4 MAm. Cell length is quitevariable; it ranges from 1 to 2 Am and commonlyextends up to 6 to 10 ,m in the same culture.The long spiral-shaped cells do not always con-sist of chains of individual vibrios that have notseparated. No sign of cross-wall formation wasobserved in most of the long cells (Fig. 2, 4, and6).

In agreement with previous reports (17, 18),essentially all H-I cells became nonmotile whengrown repeatedly on nutrient medium unsupple-mented with living host cells. From Fig. 7, it canbe seen that most H-I A3.12 cells were not flagel-lated at the time these observations were made,although they had been flagellated some monthsearlier upon primary isolation from H-D A3.12(see illustrations in reference 6). All flagellatedcells have a single ensheathed polar flagellumcomparable in size and structure to those pre-viously reported by us in H-D 109 and H-DA3.12 (6). Most flagellated H-I strains illus-trated here have a flagellum with decreasing

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TABLE 2. Absorption peaks (nanometers) of cytochrome differenlce spectra of H-I Bdellovibrioand Pseudomonias culturesa

a-bands ,-bands

Organism Cytochrome CoYteCytochrome cyto- Cytochrome t

a b C b c

Bdellovibrio strains H-I 100, H-I 101, H-I 109,H-I 110, H-I 114, H-I 118, H-I 120, H-I 2484Se-2, H-I 2484 Se-3, H-I Xty, H-I OX9-2, H-IOX9-3, H-I B, H-I D, H-I E.................. 596-600 553-554 522-524 421-423

Bdellovibrio strain H-IA3.12. 607 554 523 417Vibrio spp. NCTC 4711, NCTC 4715, IP 5638b... 595 552 522 424V. metchnikovii................................ NOC 561 530 428-429P. maltophilia ........ 629, 595 559 530 430P. maltophiliad................................ 628, 597 558 530 430

a See Materials and Methods for preparation of cells and determination of cytochrome differencespectra.bNCTC: National Collection of Type Cultures; IP: Pasteur Institute.c Could not be observed.d Data from Stanier, Palleroni, and Doudoroff (15).

424

VIBRIO sp.

422

H-I10952417

cn ~~H-I 110 IIROs.41z H

AODwO.04590~

WAVELENGTH (nm)FIG. 1. Tracintgs of the cytochrome differeince spectra of three H-1 bdellovibrios and a Vibrio sp.

amplitude and wavelength (Fig. 3-7). However,the flagella of the bizzare-shaped cells of H-IBdellovibrio 110 (Fig. 5) have lost this morpho-logical regularity. With respect to the singleflagellum, these cultures differ from the micro-graphs presented previously of one H-I strain(Sp 19) originating from H-D 321 (17). Unfor-

tunately, H-I strain Sp 19 was not available at thetime our comparative studies were made, and wehave not yet been able to isolate another H-Iderivative from H-D 321; hence, this puzzlingpoint regarding the multiflagellar condition ofSp 19 could not be rechecked. Shilo (personalcommunication) has informed us that he also has

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HOST-INDEPENDENT BDELLOVIBRIOS

t~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.................... ..FIG. 2. H-l Bdellovibrio 101 stained with 0.5% uranyl acetate. Cells illustrate the typical pleomorphism ob-

served in most cultures. Cultures grown and treated as described in Materials and Methods.

at times observed more than one flagellum onsome bdellovibrio cells.

Figure 7 illustrates the effects of cultural con-ditions and staining with phosphotungstic acidon the cellular morphology of H-I A3.12. Theactively growing cells in the young culture(upper micrographs in Fig. 7) consist of ratheruniform plump vibrios, measuring about 0.4to 0.5 ,um in thickness and about 1 to 1.5 ,um inlength. The two lower micrographs of Fig. 7 areof cells from a 2-day PYE broth culture into whichthe cells had been inoculated from a PYE agarplate. Unlike the young (overnight) culture fromwhich the cells shown at the top of Fig. 7 had

been taken (see Materials and Methods), theculture shown in the lower micrographs of Fig. 7had not been transferred and grown overnight infresh broth before examination. The cells, fixedin Formalin and stained with 0.5% phosphotung-stic acid, are quite variable in length (3 to 10 Am)and consist of vibrio- or spiral-shaped cells. Ofparticular interest are the blebs or extrusions oc-curring around the surface of the cells; this ispossibly an artifact of the staining procedure andmay not represent the true morphological stateof the living cells. All H-I isolates show a strongtendency to spheroplast formation in older cul-tures. A representative illustration of spheroplast

775VOL. 100, 1969

SEIDLER AND STARR~~~~~~~~~~... ........ .

. ..........

aeR..._..... X_

FIG. 3. H-I Bdellovibrio 109 stained with 0.5% uranyl acetate. Cells in the upper micrographs are morpho-logically uniform. The two lower micrographs illustrate spheroplastformation (48-hr culture) typical ofall cultures.

776 J. BACTERIOL.

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HOST-INDEPENDENT BDELLOVIBRIOS

FIG. 4. H-I Bdellovibrio 110 stained with 0.5% uranyl acetate. Some cells are long and highly pleomorphic;others are typically vibrio-shaped. All flagellated cells contain a single polar flagellum. The flagella of the largecells have lost all regularity in appearance.

formation is shown for a 48-hr culture of H-I109 (lower micrographs, Fig. 3). The upper mi-crographs of Fig. 3 show cells taken from activelygrowing H-I 109 cultures; like those of young H-IA3.12 in the upper micrographs of Fig. 7, thesecells are rather uniform and typically vibrio-shaped.

Production of exocellular protease by H-Ibdellovibrios. The presence of an exocellularproteolytic enzyme in cultures of H-D and H-Istrains of Bdellovibrio has been reported (10,18). This enzyme could dissolve heat-killed bac-teria. The protease of Bdellovibrio A3.12 was be-lieved by Shilo and Bruff (10) to play an im-portant role in bringing about a nonspecific

dissolution of host cell components after inva-sion by the parasite. However, these workersstated that two other Bdellovibrio strains pro-duced little, if any, protease. Because of the en-zyme's potential importance in the Bdellovibriolife-cycle, we tested 12 H-I strains for the pro-duction of this enzyme (Table 3). All 12 of thecultures tested showed some proteolytic activityin PYE broth. The proteolytic activities of all ofthe strains in PYE broth (with the exception ofH-I A3.12) were similar, and produced a relativedecrease in turbidity ranging from 11% (H-I2484 Se-3) to 27% (H-I D). Even though H-IA3.12 had been transferred on host-free mediafor over 1 year, it still had the highest proteolytic

777VOL. 100, 1969

0

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SEIDLER AND STARR

FIG. 5. H-I Bdellovibrio 114, 0.5% uranyl acetate stain. Cells of this culture consist of individual vibrio- to

spiral-shaped cells. Some of the spirals have undergone division, but the newly formed cells have not yet separated.Some of these spirals are reminiscent of the intracellular growth phases ofH-D bdellovibrios.

activity (80% decrease in turbidity). Figure 8illustrates the kinetics of the dissolution of auto-claved host cells by the protease produced bythree representative H-I cultures. The kinetics ofturbidity decrease were usually the same forcultures grown in PYE or PYE/5 supplementedwith autoclaved host cells (cf. H-I A3.12 and

H-I 118). However, in two cases [H-I 2484Se-2 and H-I E (see also Table 3)], significantlymore proteolytic activity was obtained whencultures were grown in the medium supplementedwith autoclaved host cells.

Observations of H-I strains growing on auto-claved and living hosts. A semi-quantitative study

J. BACTEPIOL.778

HOST-INDEPENDENT BDELLOVIBRIOS

FIG. 6. H-I Bdellovibrio 118 stained with 0.5% uranyl acetate. Cells of this culture represenlt the extreme inthe formation of long spiral-shaped cells. The cell in the lower micrograph is nearly 10 pAm in length.

has been undertaken to check the ability of 5

H-I isolates to grow on living or autoclaved hostcells under a variety of conditions. Growth onliving and autoclaved host cells would provide ameans of measuring some of the physiologicalcapabilities of these cultures. Furthermore, thesetrials might provide a convenient way of screening

other bacteria as possible free-living, but un-recognized, relatives of H-D bdellovibrios.

After 1 day of incubation in NB/10 broth withliving host cells, an examination of cultures H-I109, H-I 110, H-I 114, and H-I 118 showed that afew Bdellovibrio-like bacteria (i.e.< looking likeH-D bdellovibrios: small, vibrio-shaped, rapidly

VOL. 100, 1969 779

411Z

SEIDLER AND STARR

.._-

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FIG. 7. H-I Bdellovibrio A3.12 stained with 0.5% uranyl acetate or 0.5% phosphotungstic acid (two lowermicrographs). The cells in the upper micrographs were grown and prepared as described in Materials and Methods.The cells in the lower micrographs were not subcultured in fresh medium before examination. The blebs surroundingthe cells are believed to be artifacts of the staining procedure.

motile) were present. With the exception of somecells in the H-I 110 culture, none of the Bdello-vibrio-like cells were observed actually attachedto the living host cells at this point. In H-I 110,fewer host cells remained, and many Bdello-vibrio-like cells were observed attached to and in-vading host cells. After an additional 2 days ofincubation, examination of the cultures of H-I109, H-I 114, and H-I 118 revealed that manysmall, actively motile bacteria were now present.

Spheroplast formation of many host cells wasobserved. H-I A3.12 was the most virulent of thecultures examined; after 24-hr incubation, essen-tially all host cells were replaced by an activelymotile population of Bdellovibrio-like cells.The H-I cultures inoculated into NB/10 con-

taining autoclaved P. putida showed, after 1day of incubation, an increase in the number ofsmall Bdellovibrio-like cells. Most of the auto-claved cells had been digested, and much debris

780 J. BACTERIOL.

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HOST-INDEPENDENT BDELLOVIBRIOS

remained which probably consisted of fragmentsof the autoclaved cells. Unlike the case of growthwith living host cells, most of the Bdellovibrio-likecells in the various cultures grown with auto-claved P. putida were not motile, with the excep-

tion of H-I A3.12. After 72-hr incubation, allbroth cultures became yellow from the growthof the yellow-pigmented Bdellovibrio-like cells.

Observations of H-I cells grown with livinghost cells on NB/10 plates were made after 4 daysof incubation. Plates inoculated directly with 1ml of each of the H-I cultures we have studiedshowed confluent lysis; Stolp (personal communi-cation) has isolated some H-I strains which donot form plaques under these conditions. Wetmounts prepared from the agar surfaces did showsome differences in the number of host cells re-

maining. In this respect, H-I 118 appeared the

1.0

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z

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TIME (hrs)FIG 8. Kinetics of the dissolution of autoclaved

E. coli B cells by H-I Bdellovibrio culture supernatantfluids tested at 37 C. Symbols: 0, P YE broth control;A and A, H-I Bdellovibrio 2484 Se-2; 0 and 0,

H-IBdellovibrio A3.12; 0 and *, H-I Bdellovibrio 118.Solid symbols indicate supernatant fluids from culturesgrown in PYE/5 medium which contained about 109cells of autoclaved E. coli B/ml; open symbols are

PYE supernatant fluids.

TABLE 3. Production of exocellular protease byselected H-I Bdellovibrio culturesa

H-I Bdellovibrio strain PYE PYE/S +E. coli B

2484 Se-2 ...................... 0.80 0.51OX9-2.. 0.77D....................... 0.73 0.74E. 0.84 0.60A3.12 . 0.20 0.25Xty.0.742484Se-30..89 0.82OX9-3....................... 0.86B. 0.82101....................... 0.84 0.84118....................... 0.76 0.77120....................... 0.80 0.82

a Actively growing cultures were propagatedovernight at 30 C in PYE broth or PYE/5 brothcontaining approximately 109 cells/ml of auto-claved E. coli. The cells were removed by centrifu-gation, and the proteolytic activity of the super-natant fluids was determined as described inMaterials and Methods. The results are expressedas the fraction of the original turbidity (200 Klettunits) remaining after 2-hr incubation at 37 C.

least virulent, while examination of H-I A3.12plates revealed very few host cells remaining. Atthe 10-3 dilution of H-I A3.12, there was plaqueformation and a nearly confluent lysis of thelawns of living host cells. No plaques were formedby any of the remaining H-I cultures at this dilu-tion, although microscopic examination showed afew small, vibrio-shaped cells.These observations provide additional evidence

that these H-I isolates are indeed descendants ofH-D Bdellovibrio and are not contaminants.Inoculation of these isolates with living host cellsindicated that there was a reselection for cul-tures possessing predatory and bacteriolyticabilities seemingly identical to those of the H-Dcultures. This comparative study indicates,however, that there is a significant difference be-tween the degree of virulence of H-I A3.12 andthat of the other H-I Bdellovibrio strains. Al-though all cultures were able to revert and attackliving host cells, as well as to digest autoclavedcells, H-I A3.12 was always the most virulentstrain. In addition, H-I A3.12 was the onlyBdellovibrio culture to form isolated plaques at a10-3 dilution on lawns on NB/10 plates. Theseobservations point to the generally unusual be-havior of H-I A3.12 compared to other H-I deriv-atives (Table 4). The H-I isolates other than A3.12were able to effect complete lysis of host lawnsonly when plated directly onto NB/10 plates.The absence of plaques at 10-1 and 10-3 dilutions

781VOL. 100, 1969

SEIDLER AND STARR

TABLE 4. Comparison of the properties of H-1 Bdellovibrio strain A3.12 and other H-IBdellovibrio strains

Property H-I A3.12 Other H-I bdellovibrios

Antibiotic susceptibilitya0/129 ................................._ +Colistin................................ +Sulfisoxazole ........................... +Oleandomycin.......................... +

Cytochrome absorption peaks............. 607, 554, 523, 417 nm 596-600, 553-554, 522-524,421-423 nm

GC content of DNA........ 42.7% (8) 50-51%b (8)Isolated plaques (at 10 dilution) on hostlawns................................ Yes No

Relative proteolytic activity . High Low

a The plus sign indicates susceptibility to the antibiotic; the minus sign indicates resistance to theantibiotic.

I See reference 8 for an exception.

of the other H-I strains is possibly due to thedilution of certain factors present in the PYEbroth cultures. One such factor playing a role inplaque formation may be the exocellular pro-tease formed in small amounts by most H-I cul-tures, but produced in much larger amounts byH-I A3.12.Because it has been possible to reselect clones

with predatory and bacteriolytic properties fromH-I cultures of Bdellovibrio, an attempt was madeto select such clones from other bacteria of knownsystematic position. In this manner, it might bepossible to identify free-living relatives of H-DBdellovibrio which are masquerading under othernames. Four Vibrio spp. and three isolates ofS. gracile (2) were examined for bacteriolyticactivity. The experimental protocol was identicalto that employed for the H-I Bdellovibrio cul-tures, as described in the previous section. Allattempts to detect bacteriolytic and predatoryactivity in broth preparations of these mixturesby turbidimetric measurements and by micro-scopic observations were unsuccessful. Thus,on the basis of these tests, neither the Vibrio spp.nor the S. gracile strains would appear to be soclosely related to Bdellovibrio that H-D variantsare detectable.

DISCUSSION

Of the publications concerning B. bacteriovorus,not many (8, 10, 17, 18; R. J. Seidler and M. P.Starr, Bacteriol. Proc., p. 23, 1968) have placedemphasis on an examination of H-I Bdellovibriocultures. This deficiency is not due to a lack ofinterest in this aspect of Bdellovibrio research,but might be attributed to the difficulty en-countered previously in isolating H-I strains.The methods of Stolp and Petzold (17) and

Shilo and Bruff (10) for the isolation of H-IBdellovibrio gave limited success in our hands.The H-I strains which we have studied were iso-lated by an original technique in which largenumbers of Smr H-D cells (approximately 5 X109 to 10 X 109) are placed into a medium whichcan support the growth of any Smr H-I cellswhich may be present and at the same time pre-vent the growth of Sms host cells carried overwith the inoculum. The chore of isolating spon-taneous Smr mutants of the H-D bdellovibrios iscounterbalanced by the reliability of this isola-tion technique. Using this method, we were ableto isolate H-I strains from every one of the 16different H-D strains we have used, and we mayconclude that the parasitism of Bdellovibrio is notobligatory.

Enumeration of the frequency of occurrence ofH-I derivatives indicates that the parameter is inthe range of a single mutational event (Table 1).The number of H-I colonies appearing on platesis influenced by the composition of the mediumand type of agar, though only casual attentionwas paid to this point in the present study.The results of the bacteriological diagnostic

tests reveal a high degree of uniformity among theH-I Bdellovibrio isolates. All cultures liquefygelatin, produce ammonia from peptone (PYEbroth), are oxidase-positive, and grow in thetemperature range of 23 to 37 C (none of theH-I cultures grow at the two temperature ex-tremes of 12 and 42 C); under the conditionstested, none of the cultures reduced nitrate norproduced indole. All cultures except H-I E pro-duce a small amount of a yellow, water-insolublepigment (18) on PYE agar. The amount of pig-ment produced appears to decrease with succes-sive transfers on the host-free medium.

782 J. BACTERIOL.

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Most of the H-I cultures (12 of 16) producedcatalase upon initial isolation of the H-I strains.After 7 months of growth on host-free media, fiveof these initially catalase-positive cultures nolonger gave a positive reaction for catalase. Withthe exception of the lactic acid bacteria, Shigelladysenteriae, and a few other isolated cases, theabsence of catalase is an unusual characteristicfor aerobic bacteria (14).The majority of the Bdellovibrio cultures were

susceptible to 9 of the 20 antibiotics and anti-metabolites tested (including penicillin beyond10,000 units/ml) and, except for one culture(H-I A3.12), were quite uniformly resistant or

susceptible to a given antibiotic. The antibioticsusceptibility results show that there are fourcases in which H-I A3.12 has a different reactionfrom most other H-I bdellovibrios. In addition,H-I A3.12 is the strain most susceptible to penicil-lin. These results probably indicate differences inseveral metabolically sensitive sites between H-IA3.12 and the other Bdellovibrio cultures ex-amined (see Table 4). Shewan, Hodgkiss, andListon (9) published a method for the dif-ferentiation of Vibrio spp. from other relatedbacteria based on antibiotic susceptibility. Of thenearly 200 cultures examined, all Vibrio cultureswere susceptible to oxytetracycline and to thevibriostatic pteridine 0/129. All of the bdello-vibrios tested in this study are sensitive to oxy-tetracycline and 0/129, except that H-I A3.12 isnot sensitive to 0/129. This probably is indica-tive of some degree of metabolic similaritiesbetween these two groups of bacteria.

All nine H-I Bdellovibrio cultures examinedshow a general inability to utilize any of the 14carbon compounds tested. These results are whatmight be expected of an organism that (duringparasitic growth) is suspected of utilizing hostcell cytoplasm (7). The observations on gelatinliquefaction and ammonia production from pep-tones suggest that Bdellovibrio has become meta-bolically specialized and is limited only to theutilization of complex mixtures of proteins, pep-tides, and amino acids. The failure to utilizedeoxyribose and ribose may indicate that theH-D Bdellovibrio does not use host cell nucleicacids as a source of carbon and energy.Our observations on carbon utilization are

compatible with the results of a study on energy-producing systems recently reported by Simpsonand Robinson (11), who concluded that thesingle strain of H-D Bdellovibrio which theystudied probably has only a limited ability toutilize free sugars. Their report indicates that theenzymes of the tricarboxylic acid cycle as well asglutamic dehydrogenase are present. This mightaccount for our observations on growth stimula-

tion of some H-I Bdellovibrio cultures by a-ketoglutarate and glutamate. Simpson and Robin-son (11) mentioned that their Bdellovibrio strain(6-S-5) is an obligate parasite, although theyreported that it has the necessary mechanismsfor obtaining energy to permit host-independentexistence. However, these investigators did notelaborate on the efforts they made to isolate H-Iderivatives from strain 6-S-5. Our experiencein the isolation and cultivation of numerous H-Istrains gives us cause for reservation about thealleged obligatory nature of parasitism in bdel-lovibrios.

All tests for the fermentation of carbohydratesby H-I Bdellovibrio were negative. The absence ofgrowth below the zone of oxygen diffusion inthioglycolate broth precludes an anaerobic dis-similation of amino acids or peptides. Other re-ports indicate that H-D Bdellovibrio strains alsocannot grow in a completely anaerobic environ-ment (1, 11, 18).The cytochrome difference spectra of 16 cul-

tures of H-I bdellovibrios indicate the presenceof a cytochrome c component. Fifteen cultureshave absorption peaks at 596 to 600 mm corre-sponding to a cytochrome a, and at 421 to 423nm for the soret region. H-I A3.12 differs fromthe remainder of the cultures with respect tothese components, and shows peaks at 607 nmand 417 nm, respectively (Table 2, Fig. 1; seealso Table 4). There is a similarity in the cyto-chrome difference spectra of three of four strainsof Vibrio spp. examined and the 15 Bdellovibriostrains.For some time, a search has been made for

bacteria of known systematic position that mightbe free-living relatives of H-D bdellovibrios. Re-peated attempts to select for bacteriolytic isolatesfrom several Vibrio spp. and from three strains ofS. gracile were unsuccessful. As of this time, theonly known free-living relatives of H-D Bdello-vibrio are the H-I bdellovibrios. Table 5 com-pares some of the properties of the H-I de-rivatives with those of H-D Bdellovibrio strains.The morphological studies of H-I strains indi-

cate that most cultures form spiral- and bizarre-shaped cells (17). These spiral forms are reminis-cent of the intracellular spirillar stage of the H-Dcultures (16; D. Abram and M. Shilo, Bacteriol.Proc., p. 22, 1967). A recent report by Burger,Drews, and Ladwig (1) illustrates that a cultureof H-D Bdellovibrio would grow (i.e., elongateinto spiral-shaped cells) in cell-free host extracts,but would not undergo cell division into vibrios.The ability of H-I Bdellovibrio cultures to undergocellular multiplication, rather than only cellgrowth, in the absence of living host cells, maythus represent the major obstacle in the successful

783VOL. 100, 1969

TABLE 5. Comparison of some properties of H-I and H-D bdellovibrios

Property H-I bdellovibrios H-D bdellovibrios

Morphology Vibrios, spirals, and other forms Alternations of vibrio and spiral(pleomorphic), 0.3-0.4 ,m in shapes (7, 16), 0.3-0.4 jam inwidth; spheroplasts in older width; spheroplasts in oldercultures; single (but see 17) and cultures; single and ensheathedensheathed polar flagellum polar flagellum

Motility Loses motility in host-free media Actively motile during growth onhost cells

Growth on:PYE Yes NoAutoclaved host cells Yes NoLiving host cells Upon massive inoculation Yes

Plaques on living host cells Confluent lysis upon massive Isolated plaques on susceptibleinoculation; isolated plaques host bacteriaformed only by H-I A3.12

Protease formed Yes Yes (three strains investigated;see 10)

Cytochrome difference spectra Same peaks for H-D Bdellovibrio and its H-I derivativeGC content Same GC contents for H-D Bdellovibrio and its H-I derivative (8)DNA compositional hetero- Same for the H-D Bdellovibrio and its H-I derivative (8)

geneityRelation to oxygen No growth in anaerobic zone of No plaques under anaerobic con-

thioglycolate broth ditions (1, 11 18)

isolation of H-I strains. The ability of H-I bdel-lovibrios to undergo division in the absence ofliving host cells could conceivably result from analteration in the control over cellular divisionmechanisms. This might account for the extrememorphological heterogeneity in cell lengths andshapes observed in most H-I cultures.Although, as noted above, not all H-I cultures

have flagella, all flagellated cells examined withan electron microscope show a single (but seereference 17), ensheathed, polar flagellum (Fig.3-7). The flagella are comparable in size andstructure to those previously reported by us forH-D Bdellovibrio 109 and H-D and H-I Bdelio-vibrio A3.12 (6) and by others (3) in Vibrio.

All H-I cultures examined produced some exo-cellular protease (Table 3). H-I A3.12 had thehighest proteolytic activity, even after beingtransferred on host-free media for over 1 year.With respect to proteolytic activity, the culturestested form three groups: those producing arelatively small amount of protease activity whengrown in the presence or absence of heat-killedbacteria (most cultures); two cultures (H-I 2484Se-2 and H-I E) which produced more proteolyticactivity in the presence of heat-killed host cellsthan in their absence; and H-I A3.12, which pro-duced large amounts of protease, both in thepresence and absence of heat-killed bacteria (Ta-ble 3, Fig. 8).As a result of the observations reported here

and elsewhere (8); it is now possible to add to the

definition of the genus Bdellovibrio furthermorphological, physiological, and biochemicalcharacteristics. These bacteria are vibrio- to spiral-shaped during host-independent and host-de-pendent growth; flagellated cells have a single,ensheathed, polar flagellum (H-I 321 may havemore than one flagellum; 17). They do not utilizeany carbohydrate as an energy source; they arenonfermentative; they do not grow anaerobicallywith nitrate; they do not produce indole; theyare strongly proteolytic; they are oxidase-positive;they all have cytochromes a and c; they are sensi-tive to oxytetracycline and to the vibriostaticpteridine 0/129 (except that H-I A2.12 is notsensitive to 0/129). The molar percent guanine-plus-cytosine (GC) content of the DNA is ap-proximately 43 to 51% (most strains 50 to 51%);the DNA appears quite homogeneous both inoptical melting behavior and in equilibriumdistribution in an isopycnic gradient (8).On the basis of gross morphological similarities

as well as certain physiological features, Bdello-vibrio appears to be related to members of thegenera Spirillum or Vibrio. Veron (20, 21) hasdiscussed the taxonomic position of Vibrio andrelated bacteria and has given defining character-istics for the families Spirillaceae and Vibrio-naceae. Because bdellovibrios are not fermenta-tive, do not use carbohydrates as an energysource, and are oxidase-positive, it might appearthat these bacteria are physiologically related tomembers of the family Spirillaceae as described

784 SEIDLER AND STARR J. BACTERIOL.

HOST-INDEPENDENT BDELLOVIBRIOS

by Wron. We feel that there is not a close taxo-nomic relationship between Bdellovibrio andCampylobacter (family Spirillaceae); the lattergroup has been described as being nonproteoly-tic, able to reduce nitrate, and as having amuch lower GC content than Bdellovibrio [29 to35% GC (5, 20)]. However, Bdellovibrio does showsome properties in common with members of thegenus Vibrio. The mutual similarities involve keyantibiotic sensitivities (oxytetracycline and O/129), cytochrome content and absorption peaks,as well as similarities in GC contents (44 to 50%for Vibrio; 20). Taking all this into account, weremain in agreement with an earlier proposal(18) regarding the establishment of a separategenus Bdellovibrio, not only on the original basis(predatory and parasitic nature), but also byvirtue of the distinctive biochemical and physio-logical properties of the bdellovibrios. It is likelythat more than one species exists in this genus(Table 4); however, we are not yet prepared torecommend a nomenclatural change.

ACKNOWLEDGMENTS

This investigation was supported by Public Health Servicetraining grant GM-01041 from the National Institute of GeneralMedical Sciences and research grant AI-08426 from the NationalInstitute of Allergy and Infectious Diseases. A Fellowship fromthe John Simon Guggenheim Memorial Foundation (to M.P.S.)is gratefully acknowledged.We thank Diana C. Davis for her advice on the determination

of cytochrome difference spectra. We are grateful to M. Shilo,D. A. Klein, A. Guelin, and A. J. Hansen, who kindly providedBdellovibrio strains for this study. We express appreciation toH. Stolp, J. C. C. Huang, and M. Shilo for their critical readingsof the manuscript and much helpful advice, and to Karen Williamsfor technical assistance.

LITERATURE CITED

1. Burger, A., G. Drews, and R. Ladwig. 1968. Wirtskreis andInfektionscyclus eines neu isolierten Bdellovibrio bacterio-vorus-Stammes. Arch. Mikrobiol. 61:261-279.

2. Canale-Parola, E., S. L. Rosenthal, and D. G. Kupfer. 1966.Morphological and physiological characteristics of Spirillumgracile sp. n. Antonie van Leeuwenhoek J. Microbiol.Gerol. 32:113-124.

3. Saluert, A., D. Kerridge, and R. W. Home. 1963. The finestructure and mode of attachment of the sheathed flagellumof Vibrio metchnikovil. J. Cell Biol. 18:327-336.

4. Lepine, P., A. Gudlin, J. Sisman, and D. Lamblin. 1967.

Etude au microscope electronique de la lyse de Salmonellapar Bdellovibrio bacteriovorus. Compt. Rend. Acad. Sci.Paris 264:2957-2960.

5. Sebald, M., and M. Veron. 1963. Teneur en bases de l'ADNet classification des Vibrions. Ann. Inst. Pasteur 105:897-910.

6. Seidler, R. J., and M. P. Starr. 1968. Structure of the flagellumof Bdellovibrio bacteriovorus. J. Bacteriol. 95:1952-1955.

7. Seidler, R. J., and M. P. Starr. 1969. Factors affecting theintracellular parasitic growth of Bdellovibrio bacteriovorusdeveloping within Escherichia coll. J. Bacteriol. 97:912-923.

8. Seidler, R. J., M. P. Starr, and M. Mandel. 1969. Deoxyribo-nucleic acid characterization of bdellovibrios. J. Bacteriol.100:786-790.

9. Shewan, J. M., W. Hodgkiss, and J. Liston. 1954. A methodfor the rapid differentiation of certain non-pathogenic,asporogenous bacilli. Nature (London) 173:208-209.

10. Shilo, M., and B. Bruff. 1965. Lysis of gram-negative bacteriaby host-independent ectoparasitic Bdellovibrio bacteriovorusisolates. J. Gen. Microbiol. 40:317-328.

11. Simpson, F. J., and J. Robinson. 1968. Some energy-produc-ing systems in Bdellovibrio bacteriovorus, strain 6-5-S. Can.J. Biochem. 46:865-873.

12. Smith, L. 1961. Cytochrome systems in aerobic electrontransport, p. 365-396. In I. C. Gunsalus and R. Y. Stanier(ed.), The bacteria, vol. 2. Academic Press Inc., New York.

13. Society of American Bacteriologists. 1957. Manual of micro-biological methods. McGraw-Hill Book Co., Inc., NewYork.

14. Stanier, R. Y., M. Doudoroff, and E. Adelberg. 1964. Themicrobial world. Prentice-Hall, Inc., Englewood Cliffs,N.J.

15. Stanier, R. Y., N. J. Palleroni, and M. Doudoroff. 1966. Theaerobic pseudomonads: a taxonomic study. J. Gen. Micro-biol. 43:159-271.

16. Starr, M. P., and N. L. Baigent. 1966. Parasitic interaction ofBdellovibrio bacteriovorus vith other bacteria. J. Bacteriol.91:2006-2017.

17. Stolp, H., and H. Petzold. 1962. Untersuchungen Uber einenobligat parasitischen Mikroorganismus mit lytischerAktivitat fur Pseudomonas-bacterien. Phytopathol. Z.45:364-390.

18. Stolp, H., and M. P. Starr. 1963. Bdellovibrio bacterlovorus,gen. et sp. n., a predatory, ectoparasitic, and bacteriolyticmicroorganism. Antonie van Leeuwenhoek J. Microbiol.Serol. 29:217-248.

19. Varon, M., and M. Shilo. 1968. Interaction of Bdellovibriobacterlovorus and host bacteria. I. Kinetic studies of attach-ment and invasion of Escherichia coli B by Bdellovibriobacterlovorus. J. Bacteriol. 95:744-753.

20. Vron, M. 1965. La position taxonomique des Vibrio et decertaines bact6ries comparables. Compt. Rend. Acad.Sci. Paris. 261:5243-5246.

21. Veron, M. 1966. Taxonomique numerique des vibrions et decertains bacteries comparables. II. Corr6lation entre lessimilitudes ph6n6tiques et la composition en bases deI'ADN. Ann. Inst. Pasteur 111:671-709.

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