JOURNAL OF BACTERIOLOGY, Mar. 1971, p. 855-863 Vol. 105, No. 3Copyright © 1971 American Society for Microbiology Printed in U.S.A.

Chromosome Mapping of Pasteurellapseudotuberculosis by Interrupted Mating

WILLIAM D. LAWTON AND HAROLD B. STULL

Biological Sciences Laboratories, Department of the Army, Fort Detrick, Frederick, Maryland 21701

Received for publication 30 November 1970

Pasteurella pseudotuberculosis, containing the Escherichia coli plasmid F'lac,transferred its chromosome in an oriented manner to each of five multiply auxo-trophic strains of P. pseudotuberculosis. In a mating system containing gelatin, glu-cose, and phosphate buffer, a maximum of 0.02% of the donor cells transferred leadmarkers. The donor population was counterselected with nalidixic acid. We estab-lished the entry time of seven markers as follows: proline (11 min); arginine (14min); histidine (14 min); threonine (25 min); lysine (50 min); tyrosine (67 min); andtryptophan (77 min). However, an analysis of the inheritance of unselected markersdid not support the simplest assumption that the chromosome was transferred asOrigin ... pro ... arg his ... thr ... lys ... tyr ... trp ... . The markers commonto all five recipients, arg and his, were closely linked, but of the five other markers,each unique to a different recipient strain, only trp was linked to arg and his. Ourdata suggest that the Pasteurella chromosome is transferred in more than onelinkage group.

Gene transfer by conjugation between auxo-trophs of Pasteurella pseudotuberculosis has beendemonstrated by Lawton, Morris, and Burrows(14). Their data were obtained by mixing donor(F'lac) and recipient cells on selective plates andpermitting conjugation, transfer, and recombi-nant colony formation to occur on the plates.Alternative explanations for their results, such assyntrophy or reversion, were not substantiated byexperiments showing that: (i) replacement of thedonor by an F-lac- derivative, which would beexpected to cross-feed the recipient equally well,resulted in infertility; (ii) separation of parents bya membrane eliminated fertility; and (iii) a largenumber of recombinants showed unselecteddonor markers. The extension of the initial obser-vations to classical interrupted mating experi-ments was hindered by the apparent lac+ of genetransfer in broth, and by the difficulty of pre-venting the donor strain from remating on theselective plate.W£ have solved both of these problems and

report here our method of interrupted mating andthe resulting preliminary chromosome map of P.pseudotuberculosis.

MATERIALS AND METHODS

Organisms. All the bacterial strains used were de-rived from P. pseudotuberculosis strain 321V obtainedfrom E. Thai, Stockholm, Sweden. The initial auxo-trophs were obtained at the Microbiological ResearchEstablishment (MRE) and are described in the publica-

tion of Lawton, Morris, and Burrows (14). The donorstrain used in this study, obtained from strain MRE2027 by isolating a mutant resistant to I mg of strepto-mycin sulfate per ml, was designated YsD-20 (ade-53,str-50; F'lac). The F'lac plasmid had been transferredto P. pseudotuberculosis from Escherichia coli K-12,strain 23. 10.S. The recipient strains, derived from strainMRE 2205 (met-5, arg-8) after treatment with N-methyl-N'-nitro-N-nitrosoguanidine and subsequentisolation of spontaneous mutants resistant to 20 Aig ofnalidixic acid (nal) per ml, were designated YsD-16(met-5, arg-8, his-50, thr-50, nal-51), YsD-17 (met-5,arg-8, his-50, pro-50, nal-52), YsD-18 (met-5, arg-8,his-50, lys-50, nal-53), YsD-19 (met-5, arg-8, his-50,tyr-50, nal-54), and YsD-21 (met-S, arg-8, his-50, trp-50, nal-55).

Auxotrophic mutants were obtained by centrifugingcells growing in logarithmic phase in Difco brain heartinfusion (BHI) and suspending the sedimented cells in25 ml of 0.15 M NaCl containing 500 ,ug of N-methyl-N'-nitro-N-nitrosoguanidine per ml (Aldrich ChemicalCo., Milwaukee, Wis.). The cells were shaken at 37 Cfor 30 min, washed by centrifugation at least threetimes in BHI, resuspended in 50 ml of BHI, diluted,and plated immediately on blood agar base (BBL). Theplates were incubated at 26 C for 3 days, and thesmallest colonies were picked and tested as possibleauxotrophs.

Although all of the recipient strains were derivedfrom the met- arg- MRE 2205 strain, the met- markerproved to be unsatisfactory for use in chromosomemapping. None of the five recipient strains would growwell in the absence of methionine but would producebackground growth and, after prolonged incubation,minute colonies appeared on the plates selective for

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LAWTON AND STULL

met+ recombinants that proved to be met- clones. Ourresults suggested that the met marker was closely linkedto trp, but we have postponed including the met locus inthe chromosome map until better met- auxotrophs areinvestigated.The designations for our genetic stock strains reflect

the current consensus of investigators in the field thatthe species P. pseudotuberculosis and P. pestis shouldbe distinguished from other members of the genus Pas-teurella. Anticipating the eventual renaming of thesespecies under the genus Yersinia, we have designated allour genetic stocks of P. pseudotuberculosis as Ys- andof P. pestis as Yp-. Thus YsD- has been used for allmutants derived from wild-type P. pseudotuberculosis321V. The number following the YsD- is merely an iso-lation number. Numerical genotype designations reflectan agreement between the only two laboratories cur-rently publishing on derived mutants of P. pseudotuber-culosis. T. W. Burrows and co-workers of the Micro-biological Research Establishment, Porton, U.K., usenumbers I to 49 for genotype designation, and we usenumbers 50 to 99.The designation of genotype and phenotype follows

the recommendations of Demerec et al. (9).Media. Our selective agar was made as follows: dis-

tilled water (1,000 ml); K2HPO4 (1.5 g); L-glutamicacid, Calbiochem (0.5 g); 12 N HCI (approximately 0.1ml to obtain pH 6.8); MgCl2-6H2O (1.0 g); NH4NO3(1.0 g); Difco agar (9.0 g); after autoclaving the me-dium, stock solutions were added aseptically to obtainfinal concentrations of 0.2% glucose (20% stock solu-tion, filtered), 10 ,g of nalidixic acid per ml (5 mg/mlof alkaline stock solution, filtered), and 50 gtg per ml(10 mg/ml of stock solutions) of the L-amino acids(Calbiochem) required by the recipient strain except forthe selected marker; final pH was 6.6.

BHI broth plus 0.1% lactose added aseptically (BHI+ lactose) was used to grow donor cells. Viable countswere made on Difco purple broth base supplementedwith 1.5% agar, 1.0% lactose, and 0.00125% triphenyl-tetrazolium chloride.

Mating procedure. The data to justify the matingmethods are presented in detail below. Our final matingprocedure was standardized as follows: donor cells weregrown on a shaker at 26 C in BHI + lactose until theoptical density reached 1.0 to 1.1 (650 nm, Coleman 14spectrophotometer). This took approximately 23 hr andyielded a viable count of approximately 109 cells/ml. Toachieve a standard inoculum, we added dimethylsul-foxide (final concentration 10%) to a 23-hr culture,froze it at -60 C, and used 0.07 ml of the thawed cellsto inoculate 50 ml of BHI + lactose medium in a 250-ml flask. The frozen cells have been thawed and re-frozen several times with no loss in viability. Portions (3ml) of this donor culture were exposed immediately,with agitation in a petri plate, to a single General Elec-tric 15-w ultraviolet bulb at a distance of 61 cm for 60sec (approximately 400 ergs/mm2). A 5-ml portion ofthe treated cells was added to 5 ml of BHI + lactose,and the diluted sample was shaken very gently in a 50-ml flask at 34 C for 5 hr to permit the cells to producethe F pili necessary for conjugation (2). The initial via-bility was reduced approximately 50% after ultraviolettreatment and remained at that number after the subse-quent 5-hr incubation.

Recipient cells were grown on Difco blood agar baseslants for 21 hr at 26 C. The cells were washed off thesurface into 10 ml of 1% Difco gelatin, 0.4% glucose,and 0.001 M sodium phosphate, pH 6.7. The viablecount was approximately 3 x 109 cells/ml.One milliliter of recipient cells plus 1 ml of donor

cells plus 3 ml of 0.5% gelatin, 0.2% glucose, 0.001 Msodium phosphate (pH 6.7) were placed in a 50-ml flaskand incubated statically in a water bath at 34 C. Thirtyminutes after combining recipient and donor cells, wegently added 3 ml of 0.5% gelatin, 0.2% glucose,0.1 M sodium phosphate (pH 6.7) and continued theincubation. Greater volumes were used successfully butalways in the above proportions. Periodically, we re-moved l-ml samples, blended for approximately 2 secat high speed on a Vortex mixer, and plated appropriatesamples on various selective agar plates. Samples platedat 0 min (i.e., when the donor and buffer were added tothe recipient) showed no revertants for most markersand only an average of 10 per ml or less for somemarkers. Dilutions, when necessary, were made in asolution containing 0.01% gelatin, 0.002% glucose,0.0001 M sodium phosphate (pH 6.7), and 3 gg of nali-dixic acid per ml. Plates were incubated at 26 C andcounted after 3 or 4 days.

RESULTSCounterselection of donor. Because our donor

strain was resistant to streptomycin and no suit-able phages were available, we were unable toprevent mating on the selective plate by the use ofstreptomycin or phage. Nalidixic acid was shownby Barbour (1) to inhibit conjugational genetransfer, and we found it effective in eliminatingmating on the selective plates. Our five auxo-trophic recipient strains were isolated as sponta-neous mutants resistant to 20 ,ug of nalidixic acidper ml, but 20 Ag/ml in the selective agar yielded20% fewer recombinants than 10, 5, or 3 mg/ml,all of which yielded the same number of recombi-nants. Since 3 ,g/ml occasionally permitted anade+ revertant cell from the donor population togrow, we used 10 ,ug/ml in our selective plates.Membrane mating. We attempted initially to

study the kinetics of marker entry by permittingmating to occur on membrane filters (MilliporeCorp.) on a variety of agar media, periodicallyremoving a membrane, separating the matingpairs by blending, and scoring for recombinantcolonies on selective agar containing nalidixicacid. Our best results occurred when mating tookplace on the SD medium (gelatin, succinate, agar,and salts) used by Landman and Halle (13) formaintaining protoplasts. Membrane mating per-mitted us to establish a chromosome order forfive markers, but the technique was not exactenough to establish unequivocal entry times;therefore, we directed our effort toward a liquidmating system.

Development of a liquid mating system. Becauseof our past failures to observe chromosome

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VOL. 105, 1971 CHROMOSOME MAPPING OF PASTEURELLA BY CONJUGATION

transfer in either tryptic meat broth or in heartinfusion broth compared with the successful chro-mosome transfer observed on SD mating agar,

we started by simply removing the agar from theSD mating medium. We obtained low-frequencygene transfer (approximately 10-5 of the donorinput) and soon found that this medium could befuther simplified by removal of the succinate andmost of the salts without lowering the gene

transfer frequency. Our conjugation procedure atthat stage consisted of mixing I ml of the recip-ient culture, I ml of the donor culture, and 3 mlof 1% gelatin, 0.2% glucose, 0.001 M sodiumphosphate buffer, pH 6.7. Figure I illustrates our

initial results in studying the entry of the markerarg+. The most apparent problem appeared to bethe loss of arg+ recombinants after 90 min ofmating, correlated with and perhaps caused bythe rapidly declining pH of the system. Our at-tempt to control the pH simply by increasing themolarity of the buffer demonstrated that in-creased levels of buffer inhibited the conjugationsystem. Figure 2 shows the inhibition curves ob-tained with sodium phosphate buffer; essentiallythe same inhibition was also obtained with potas-sium phosphate or sodium malate buffer. Thelevel of buffer required to maintain the pH above6.0 was 0.05 M, and, at that level, gene transferwas essentially stopped.

Based on the hypothesis that the inhibition byhigh concentrations of buffer was preventingeffective contacts, we permitted the donor andrecipient cells to form pairs in the usual 1% gela-tin, 0.2% glucose, 0.001 M sodium phosphate (pH

0

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201

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0

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a 30C

0~~~~~~EI /° 20 -

0o.025M

00

A- 0.05 M

0 20 40 60 80 100

Time after mixing parental cultures (minutes)FIG. 2. Effect of sodium phosphate buffer, pH 6.7,

on the transfer of arg+ from YsD-20 (F'lac) to YsD-21(F-).

6.7) for 30 min and then added 3 ml of 1% gela-tin, 0.2% glucose, 0.05 M sodium phosphate (pH6.7). As shown in Fig. 3, this procedure did notinhibit chromosome transfer. On the contrary, itmaintained the pH and permitted the recovery ofan increased number of arg+ recombinants even

160

140

120F

0.05 M (c)or

.001 M(O)

buffer added

1001

80-

601

40-

20

07.0 2

16.5 0 xzE

-6.0 0f 015.5 a

E

0 20 40 60 80 100 120 140

Time after mixing parental cultures (minutes)

FIG. 1. Entry of arg+ from YsD-20 (F'lac) to YsD-21 (F-).

Time after mixing parental cultures (minutes)

FIG. 3. Effect of adding sodium phosphate buffer,pH 6.7, after 30-min contact period on the transfer ofarg+ from YsD-20 (F'lac) to YsD-21 (F-).

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3or

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858 LAWTON A

120 min after mating commenced. The control,to which 3 ml of 1% gelatin, 0.2% glucose, and0.001 M sodium phosphate (pH 6.7) were addedafter 30 min, showed the expected decline of pHand decrease of arg+ recombinants after 75 min.

Variability in the mating system seemed to becorrelated with small variations in the method ofpreparing the donor cells. For this reason, weinvestigated several of these procedures using thetransfer of the arg+ marker from YsD-20 toYsD-21 as a measure of the best conditions toprepare the donor strain for mating. We foundthat: (i) Ultraviolet irradiation of the male in-creased the frequency of chromosome transfer(Table 1). Mitomycin C showed a similar donor-enhancing effect, but was not as efficient as wasultraviolet treatment. (ii) The donor strain pro-duced maximum recombinants if the culture wasused at a cell density of 6 x 108 to 10 x 108 perml (Table 2). A lower cell density yielded thesame donor efficiency but a lower total numberof recombinants, whereas a higher cell densitywas poorer in both efficiency and total number ofrecombinants. (iii) After ultraviolet irradiation,incubating the donor strain for 5 hr was betterthan incubating for 3, 7, or 24 hr (Table 3); the 5-hr period necessary for the production of F pili

TABLE 1. Effect of ultraviolet (UV) irradiation ofdonor cells on the transfer ofarg+ from YsD-20 (Flac)

to YsD-21 (F-)

Time of exposure of argp Recombinants as per centdonor cells to donor input (x 10-') afterUV lighta (sec) mating for 60 min

0 5920 12440 13460 147100 111120 99

a Three-milliliter portions of donor culture were ex-posed, with agitation in a petri plate, to a single Gen-eral Electric 15-w UV bulb at a distance of 61 cm. A60-sec exposure resulted in a dose of approximately 400ergs/mm2.

TABLE 2. Effect ofconcentration ofdonor cells on thetransfer ofarg+ from YsD-20 (F'lac) to YsD-21 (F-)

Concn of donor cells arg+ Recombinants afterafter growth at 26 C mating for 60 min

Viable cells/ml Optical density Per ml Per cent donor(x 10') at 650 nm (x 103) input(x 10-4)

2.3 0.440 40 1746.7 0.850 130 1947.0 0.900 146 2098.7 1.000 144 16610.7 1.070 140 13115.9 1.300 81 51

AND STULL J. BACTERIOL.

TABLE 3. Effect ofdonor expression time andtemperature on the transfer ofarg+ from YsD-20

(F'lac) to YsD-21 (F-)

argp RecombinantsTime of expres- after mating forsion after UVa Viable donor cells/ml' 60 min asexposure (hr) per cent donor

input(x 10-')

At 34 C3 3.1 x 108 875 2.8 x 108 1417 3.9 x 10 117

24 3.6 x 109 46At37C

3 1.9x108 805 2.6x 108 1187 3.3 x 108 100

24 4.2 x 108 14

a UV, ultraviolet.I Viable donor cells = 6.7 x 108/ml before UV expo-

sure and 3.1 x 108/ml immediately after 60 sec of UVexposure.

(Table 3) as well as the subsequent mating (Table4) was better at 34 C than at 37 C. Mating at 26C produced only 10% of the recombinants thatwere produced at 34 C. (iv) For mating. 0.2%glucose was better than 1.0% glucose, and 0.5%gelatin was equivalent to I or 2% and better than0.25% gelatin (Table 5). Different sources of gel-atin had a marked influence on our results. With

TABLE 4. Effect of temperature of mating on thetransfer of arg+ from YsD-20 (Flac) to YsD-21 (F-)8

arg+ Recombinants as per centdonor input (x 10-4)

Mating time (min)Mating temp Mating temp

34C 37C

60 118 8990 150 112120 182 127

a Donor cells were exposed to ultraviolet light for 60sec and expressed for 5 hr at 37 C.

TABLE 5. Effect ofgelatin and glucose concentrationon the transfer ofarg+ from YsD-20 (Flac) to YsD-21

(F-)

Per cent Per centglucose gelatin

0.2 2.00.2 1.00.2 0.50.2 0.251.0 2.01.0 1.01.0 0.51.0 0.25

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BBL gelatin, Eastman purified calf gelatin, andsome batches of Difco gelatin, we observed ap-proximately 50% of the recombinants obtainedwith the best batch of Difco gelatin. The use ofBaker gelatin produced only 5% of the recombi-nants obtained with the best batch of Difco gel-atin.

After we established optimal conditions formating, each of the five auxotrophic recipientswas mated with the donor strain. Figures 4

0

x

s

._06C

0co

Qa

0

CL

._

E0

+ 10

160r

1401

his + g

+ (0)

thr

Time after mixing parental cultures (minutes)FIG. 4. Interrupted mating of YsD-20 (F'lac) and

YsD-16 (F-).

through 8 show the results of a mating experi-ment with each recipient. This type of experi-ment was performed several times with eachrecipient and provided us with the entry timesshown in Table 6.Mating efficiency. We assayed the appearance

of pro+, arg+, and his+ recombinants during an

extended mating period to determine the max-imum yield of recombinants under optimalmating conditions. Approximately 3 hr aftermixing parental cultures, each of the three typesreached a plateau at approximately 2 x 105 re-combinants per 109 donor cells, indicating that a

maximum of 0.02% of the donor populationtransmitted their lead markers. Several single-colony isolates from the donor population trans-mitted markers with the same efficiency as theparent population. The number of viable donorand recipient cells per ml of mating mixture didnot change during 150 min of mating. The max-

x

0-

C.

C

h._

0

C

0

la

C

v

0.0.

C

0

+x

10r

8OF

60

401

pro+(A)/

AMAI

his + (g)orarg+ (O)

201

0 20 40 60 80

Time after mixing parental cultures (minutes)FIG. 5. Interrupted mating of YsD-20 (F'lac) ana

YsD-17 (F-).

imum number of thr+ recombinants was 60% ofthe maximum numbers of pro+, arg+, or his+recombinants. The maximum numbers of lys+,tyr+, and trp+ recombinants were not determinedbecause the slope of the recombinant curves didnot reach a plateau during a 3-hr mating period.

180

160-

II.' I or /

o 120 -rg9 (o) (X10)o. +

~0100

@ I /

X 80 1CLsoo

.C 60 -

E I

w

40 -

20or T

Time after mixing parental cultures (minutes)

FIG. 6. Interrupted mating of YsD-20 (F'lac) andYsD-18 (F-).

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LAWTON AND STULL

1400

x7 120 -

a 100 /

c 80- tyr + (X5]

his (0)

6: 0 or

60- 20 4 0 8 0 2

arg (0)

20

c

._~~.

0 20 40 60 80 100 120

Time after mixing parental cultures (minutes)

FIG. 7. Interrupted mating of YsD-20 (F'lac) and

YsD-19 (F-).

0

120

CL

10a

0C

an

-a 40C~~~~~~~~~~~E~~~ ~ ~~

w 20- ~ ~ ~ ~ ~

C0 0 4 0 8 tr20 (140

Time after mixing parental cultures (minutes)

FIG. 8. Interrupted mating of YsD-20 (F'lac) andYsD-21 (F-).

TABLE 6. Time ofentry ofmarkers from P.pseudotuberculosis (YsD-20)

Avg Range of No. of ex-Marker entry time entry times periments to

(min) (min) obtain avg

pro 10.5 7-14 15arg 13.7 13-14 13his 13.7 13-14 29thr 24.8 24-26 5lYS 50.4 48-52 5tyr 66.8 62-71 4trp 76.6 70-81 8

Attempts to isolate an Hfr type. A fluctuationtest (12) was performed by comparing 100 donorcultures started from small inocula with thestandard donor population for their ability todonate the arg+ marker. No individual cultureproduced significantly more recombinants thanthe standard donor. A second approach taken toobtain an Hfr type was to enrich Hfr cells in thedonor population by "curing" the unintegratedF'tac plasmids with acridine orange (I 1). Ourdonor strain grew in BHI (pH 7.9) plus 25 ,ug ofacridine orange per ml but did not grow in thesame medium containing 50 Ag of acridine or-

ange per ml. The curing of the F'lac plasmid was

poor (20 to 40% lac- clones compared with 5 to10% lac- clones in the control medium withoutacridine orange), and no clones with improvedchromosome donation frequency could be iso-lated from the lac+ population that was not curedby acridine orange.

Selection for linked markers. If all of our

markers were on a single linkage group, selectionfor double recombinants should follow the pat-tern established in E. coli, i.e. a constant number(approximately 50%) of recipients that inheriteda selected distal donor marker should also haveinherited a proximal donor marker regardless ofthe sampling time. Conversely, the joint inherit-ance of two markers relative to the proximalmarker should be an increasing percentage withelapsed time of mating extrapolating back to theentry time for the distal marker. Our data onlinked markers in P. pseudotuberculosis did notfit these expectations (Tables 7-10). Only themarker trp appeared to be on the same linkagegroup as arg and his. The markers pro, thr, lys,and tyr did not appear to be linked to arg or his.The results in Tables 7 to 10 were obtained bycomparing the number of colonies appearing onminimal agar selective for single or double re-

combinants. Essentially the same results wereobtained by picking several hundred colonies se-lected as single recombinants and testing themfor the inheritance of other donor markers.

Transfer of the F'lac plasmid. After purifica-tion of many recombinants, we observed that 20

TABLE 7. Analysis oflinked markers after matingYsD-20 x YsD-17

Percentage of single recombinants thatInterruption after inherited a second donor markermating (min)

arg his+/arg+ arg+ his+lhis+

20 58 7240 67 7660 60 7280 74 79100 69 74120 73 87

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TABLE 8. Analysis oflinked markers after matingYsD-20 x YsD-21

Percentage of single recombinants thatInterruption inherited a second donor marker

aftermating his- trp+/trp+ his, trp+/his+(min)

Expt I Expt II Expt III Expt I Expt II Expt Ill

100 21.1 26.3 36.4 0.3 0.4 0.3120 26.4 38.8 26.5 0.8 1.2 0.7140 47.8 40.3 33.7 1.8 1.5 1.3160 41.4 51.7 40.0 1.9 2.6 1.9180 40.9 48.3 41.4 2.3 2.7 2.2200 40.0 45.8 41.0 2.9 3.1 2.4

TABLE 9. A nalysis oflinked markers after matingYsD-20 x YsD-16

Percentage of single recombinants thatInterruption inherited a second donor markerafter mating

(min) arg' arg+ his+ his+thr+ /thr+ thr+ /arg+ thr+ Ithr+ thr+ /his+

80 0 0 0 0100 0.6 0.3 0.6 0.3120 0.9 0.6 0.9 0.6140 1.5 0.9 1.8 1.1160 NTa NT NT 1.6

a NT, not tested.

TABLE 10. Analysis oflinked markers after matingYsD-20 x YsD-17

Percentage of single recombinants thatInterruption inherited a second donor markerafter mating _

(min) argB arg his+ his+pro+/pro+ pro+/arg+ pro/+/pro pro+/his+

60 0 0 0 080 0.5 0.5 0.7 0.6100 1.6 1.5 1.4 1.2120 1.8 1.7 1.8 1.7140 3.0 2.7 2.1 2.2160 4.6 4.0 3.2 3.1180 4.7 4.4 3.6 3.7200 6.0 6.0 4.7 4.8

to 40% of them carried the plasmid F'lac, sug-gesting that a copy of the F'lac plasmid wastransferred from donor cells to recipient cells athigh frequency. To establish the kinetics of thistransfer, we mated YsD-20c with YsD-21 andselected for lac+nalr colonies on minimum agarwith 0.2% lactose substituted for glucose. StrainYsD-20c was a single colony isolated from theYsD-20 population and had all the propertiesdescribed above for YsD-20. The results (Fig. 9)indicated that approximately 3% of the YsD-20ccells donated the F'lac plasmid to YsD-21 withan entry time of less than 5 min.

10 sr

-i0

-o0

la

0.

-

.4-

._

.c

+v

106

10+10 4F

10 3k

101hI I I I I

0 5 10 15 20 25

Time after mixing parental cultures (minutes)

FIG. 9. Kinetics of transfer of the F'lac plasmidfrom YsD-20c to YsD-21.

DISCUSSION

Our data establish, for the first time, a set ofentry times for marker transfer in P. pseudotu-berculosis. Morris and Burrows (15) published achromosome map of P. pseudotuberculosis basedon an analysis of the frequency of unselectedmarkers that were present in recombinant colo-nies after mating on selective agar plates. Ourdata support their conclusion that the markersarg+ and his+ enter early and are closely linkedand that trp+ enters later but is on the samelinkage group as arg and his. However, only thearg-8 gene was common to all strains; our his-SOand trp-50 auxotrophs were derived independ-ently from Burrows' his-8 and trp-3 auxotrophsand may not be comparable.The development of a liquid mating system for

Pasteurella was essentially empirical. Our initialefforts to duplicate procedures well establishedfor E. coli yielded no recombinants in the Pasteu-rella system. Although P. pseudotuberculosis willgrow in most media at 37 C, BHI (the mediumthat we found to be optimum for F pili produc-tion) often caused growth inhibition at 37 C. Weavoided this problem by growing the donor cellsin BHI at 26 C and subsequently incubating themat 34 C (Table 3) for 5 hr to permit the develop-ment of F pili (2). When the donor cells were

kept at 26 C, only 10% of the maximum recombi-nants were produced.

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Although our marker entry times are based on

matings with five different recipients, each of thefive accepted their common markers, arg-8 andhis-50, from the donor at the same entry time,making it possible to compare all recipients forthe entry of their unique markers.

Since each marker had a specific entry time,we assumed that the F'iac plasmid mobilized thechromosome of P. pseudotuberculosis at a spe-

cific origin and was responsible for its transfer ina specific direction. In our system, approximately0.02% of the male cells donated lead markers.Based on the negative results of a fluctuation testand the failure of acridine orange to enrich forHfr cells, chromosome transfer did not appear toresult from the presence of a few Hfr cells in our

male population. The behavior of F'iac in Pasteu-rella may be analogous to the occurrence of type11 strains of E. coli (8). F+ donor strains of type11 do not give rise to stable Hfr derivatives as dotype I F+ donor strains, although both types givenearly identical recombination frequencies whenmated with the same F- parent. Also, acridineorange was less effective in curing F from type 11strains than it was in type I strains, and acridineorange was quite ineffective in curing F'iac fromPasteurella. The basis for the difference betweentype I and type II F+ strains in E. coli is stillunknown, so that the anology between type 11 F+strains of E. coli and F'iac in P. pseudotuber-culosis, although interesting, does not provide in-sight into the behavior of the F factor in Pasteu-rella.The donor-enhancing effect shown by ultravi-

olet irradiation or by mitomycin C treatmentsuggested that F'iac in P. pseudotuberculosis mayfunction in the same manner that Evenchik et al.(10) proposed for F or F' factors in E. coli. Theysuggested a mechanism whereby the excision ofsingle-stranded fragments of the bacterial chro-mosome, during the repair of ultraviolet damage,facilitates pairing with homologous regions of thecomplementary sex factor. An alternate explana-tion of the ultraviolet effect (suggested by RoyCurtiss I1l) is the possibility that "snakes" are

formed after ultraviolet exposure, resulting inmore donor surface to mate. However, our donorstrain of P. pseudotuberculosis showed no dis-cernible "snake" formation after ultraviolet ex-

posure and subsequent 5-hr expression period. Ofcourse, it is possible that only cells able to donateformed snakes and that these cells were too rare

to observe microscopically.It is puzzling that the number of recombinants

for lead markers reached a plateau as late as 3 hrafter the entry time. Since pairing of donor andrecipient cells appeared to be inhibited by thepresence of 0.05 M buffer (Fig. 2 and 3), the addi-

tion of 0.1 M sodium phosphate buffer 30 minafter mixing donor and recipient cells should havelimited all new pair formation at least to the ini-tial 30-min period. Donor cells can pair rapidlywith recipient cells, as measured by the initiationof F'iac plasmid transfer in less than 5 min aftermixing parental cultures (Fig. 9). We envision theE. coil F'lac plasmid mobilizing the chromosomeof P. pseudotuberculosis in a relatively inefficientand asynchronous manner, i.e., the frequency ofchromosome transfer may depend on the stage inthe vegetative chromosome replication cycle, asnoted in E. coli K-12 by Curtiss et al. (6). Sincethe growth of our donor strain was poor underthe conditions used for F pili expression (Table3) and for mating, the chromosome replicationcycle may have been slowed. If linearization ofthe chromosome, preparatory to transfer, oc-curred between completion of one round of repli-cation and initiation of another (7), we mightexpect that chromosome mobilization under poorgrowth conditions could extend over a 3-hr pe-riod.Our data do not prove that the E. coil F'iac

plasmid can integrate into the chromosome of P.pseudotuberculosis, as integration may not benecessary for chromosome transfer. Clowes andMoody (5) showed that donor strains of E. colipossessing a mutation conferring inability tocarry out genetic recombination (rec-; see refer-ence 4) were deficient in chromosome transfermobilized by F, F'iac, or Col I factors, butnormal chromosome transfer occurred whenmobilized by the Col I factor. Perhaps F'iac inPasteurella is analogous to Col I in rec- E. coli,and the F'iac plasmid is necessary only to providea means to conjugate.Our data on linked markers did not fit our ini-

tial presumption that the Pasteurella chromo-some was a single linkage group as follows:

Origin.. pro.. arg his ...... lys..ystyr.. trp.(Entry time-min) .. .(I 1) (14) (14) (25) (50) (67) (77)

This situation predicts that thr+, lys+, ortrp+ recombinants should inherit the markersarg+ or his+ at a frequency close to 50%. Infact, only trp+ recombinants seemed linked to argor his (Tables 7-10). Even the pro+ marker,which had an entry time only 3 min before thearg his loci, was not linked as expected to eitherthe arg+ or his+ markers. The only linkage be-tween arg+ or his+ and pro+, thr+, lys+, ortyr+ was observed later than 80 min after matingand at a low but increasing frequency through thelatest time sample at 200 min (Tables 7-10). Thesedata suggest that P. pseudotuberculosis donates

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VOL. 105, 1971 CHROMOSOME MAPPING OF PASTEURELLA BY CONJUGATION

its chromosome in more than one linkage group.Since we presently have no strain that is auxo-trophic for more than one of the markers pro,thr, lys, or tyr, we have not determined thepossible linkage of these markers to each other.Although we realize that these four markers arenot necessarily on the same linkage group, wehave hypothesized that the chromosome of P.pseudotuberculosis may have two (or more)linkage groups, each of which has an equalprobability of being transferred to a single re-cipient, but that no recipient receives both linkagegroups concurrently. Thus, a recipient cell mightreceive either origin A.. arg his ... trp ... or

(14) (14) (77)origin B ... pro ... .thr .. . lys . . . tyr. . . We

(I 1) (25) (50) (67)can account for the low frequency, late linkageseen in Tables 7 to 10 by assuming that, after onelinkage group had been transferred, the same malecell may then donate its second linkage group.This process can be envisioned as separate chro-mosomes or as a single chromosome with two sexfactor attachment sites, each with an approxi-mately equal probability of being the origin of thechromosome to be donated. Our hypothesis is verysimilar to the description by Clark (3) of a doublemale strain of E. coli. We are currently gatheringadditional evidence to clarify the linkage relation-ships on the Pasteurella chromosome.

LITERATURE CITED

1. Barbour, S. D. 1967. Effect of nalidixic acid on conjugationtransfer and expression of episomal lac genes in Escher-ichia coli K12. J. Mol. Biol. 28:373-376.

2. Brinton, C. C., Jr., P. Gemski, and J. Carnahan. 1964. Anew type of bacterial pilus genetically controlled by thefertility factor of E. coli K- 12 and its role in chromosometransfer. Proc. Nat. Acad. Sci. U.S.A. 52:776-783.

3. Clark, A. J. 1963. Genetic analysis of a "double male"strain of Escherichia coli K- 12. Genetics 48:105-120.

4. Clark, A. J., and A. D. Margulies. 1965. Isolation andcharacterization of recombination-deficient mutants ofEscherichia coli K-12. Proc. Nat. Acad. Sci. U.S.A. 53:45 1-459.

5. Clowes, R. C., and E. E. M. Moody. 1966. Chromosomaltransfer from "recombination-deficient" strains of Esch-erichia coli K- 12. Genetics 53:717-726.

6. Curtiss, R., D. R. Stallions, J. A. Mays, and J. Renshaw.1967. Mechanism of chromosome mobilization andtransfer by F+ donors of Escherichia coli K-12. Genetics56:553-554.

7. Curtiss, R. 1969. Bacterial conjugation. Annu. Rev. Micro-biol. 23:69-136.

8. Curtiss, R., and J. Renshaw. 1969. F+ strains of Escher-ichia coli K-12 defective in Hfr formation. Genetics 63:7--26.

9. Demerec, M., E. A. Adelberg, A. J. Clark, and P. E. Hart-man, 1966. A proposal for a uniform nomenclature inbacterial genetics. Genetics 54:61-76.

10. Evenchik, Z., K. A. Stacey, and W. Hayes. 1969. Ultravi-olet induction of chromosome transfer by autonomoussex factors in Escherichia coli. J. Gen. Microbiol. 56:1 -14.

11. Hirota, Y. 1960. The effect of acridine dyes on mating typefactors in Escherichia coli. Proc. Nat. Acad. Sci. U.S.A.46:57-64.

12. Jacob, F., and E. L. Wollman. 1956. Recombinaisong6n6tique et mutants de fertilit6 chez Escherichia coli. C.R. H. Acad. Sci. Ser. D 242:303-306.

13. Landman, 0. E., and S. Halle. 1963. Enzymically andphysically induced inheritance changes in Bacillus sub-tilis. J. Mol. Biol. 7:721-738.

14. Lawton, W. D., B. C. Morris, and T. W. Burrows. 1968.Gene transfer in strains of Pasteurelia pseudotubercu-losis. J. Gen. Microbiol. 52:25-34.

15. Morris, B. C., and T. W. Burrows. 1968. Gene transferstudies with Pasteurelia pseudotuberculosis. Interna-tional Symposium on Pseudotuberculosis, Paris, 1967.Symp. Ser. Immunobiol. Stand. 9:275-284.

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