Molec gen. Genet. 170, 93-101 (1979) ~ by Sprmger-Verlag 1979

Transposon-Facilitated Recombination in Hbrio cholerae Steven R. Johnson* and W.R. Romig Department of Microbiologyand the MolecularBlologyInstitute, Universityof Cahforma,Los Angeles90024, USA

Summary. Improved Vibrio cholerae donors were constructed by introducing the ampicillin transposon0 Tnl, into both the conjugative plasmid, P, and the bacterial chromosome to provide "portable regions of homology." The resulting Tfr (Transposon-facilitated recombination) donors transferred genes at high frequency from origins specified by the chromosoreally inserted Tnl copies. Tnl was transposed into the chromosome from a deleted P:: Tnl vector, which was eliminated from the ceils by superinfection with a thermosensitive P::Tn9 (chloramphenicol) mutant plasmid. After eliminating the thermosensitive plasmid, the chromosomally resistant isolates were converted into donors with a P::Tnl conjugative plasmid. Tfr donors were also obtained by isolating Tnl insertion mutations in a gene for thymine biosynthesis. Chromosomal sites of Tnl relative to bacterial genes were determined by measuring gene transfer frequencies and genetic linkage. In one case, linkage of the amp gene to the chromosomal genes that defined its location was demonstrated. Chromosomal transfer by Tfr donors was reversed by isolating P::Tnl plasmids that contained Tnl inserted in the opposite orientation.

Introduction

Conjugal gene transfer in Vibrio cholerae is mediated by naturally occurring conjugative plasmid, the P factor (Bhaskaran, 1960; Parker and Romig, 1972; Johnson and Romig, 1978), but neither transduction nor transformation have been reported for this species. The V. cholerae conjugation system resembles the F*

Present address Centerfor Disease Control, Atlanta, Georgia

30333, USA For offprhzts contact W.R. Romlg

mediated system of Escherichia coli, except recombination frequencies are lower and non-selected markers are only poorly linked to selected markers (Bhaskaran, 1960; Parker et al., 1978). Datta et al. (1973) reported significant differences between the base composition of the P factor and the V. cholerae chromosome and suggested that this might bear on the failure to detect Hfr donors in this system. The apparent failure of the P factor to integrate into the chromosome (Parker et al., 1978) is perhaps the most significant practical distinction between F ÷ and P+ donors, and has impeded genetic studies with the latter bacteria. Accordingly, the goal of the present study was to develop alternative procedures for obtaining V. choIerae Hfr donors. E. coli Hfr donors are formed by recombination between the F factor and the bacterial chromosome to yield a fused, cointegrate replicon (Jacob and Wollman, 1963; Hayes, 1968; Scaife, 1968). Heteroduplex analyses strongly suggest that at least some Hfr strains are formed by reciprocal recombination between homologous sequences that are present on the F factor and the E. coli chromosome (Sharp et al., 1972; Hu et al., 1975a; Kleckner, 1977). More recently, a number of these homologous sequences have been shown to correspond to insertion sequences that are found on both replicons (Hu et al.~ 1975b: Ohtsubo and Hsu, 1978, Davidson et al., 1975). Chromosomal transfer by secondary F-prime strains is similar to that of Hfr donors, but in intermediate donors, the F-prime plasmid recombines into the bacterial chromosome through the homologous segments of bacterial DNA that they both contain (Adelberg and Burns, 1960~ Hayes, 1968; Scaife, 1967). Novel kinds of Hfr donors have been described that use translocatable elements as ""portable regions of homology'" to promote integration of conjugative plasmids into the bacterial chromosome. Zeldis et 0026-8925/79/0170/0093/$01.80

S.R. J o h n s o n and W . R Romig: V. cholerae Tfr Donors

94 Table 1. Bacterial stratus and plasmlds Plasmids

Genotype/Phenotype"

Other information

P

pSJ26 pSJ7

V. cholerae conjugative plasmid R factor, Tcs, Ap r, K m r P: : T n l , hybrid, Ap ~ P :' Tn9, hybrid, Cm r P : : T n l , hybrid, Ap r thermosensitive mutant, hts25 of pSJS, C m ~ SJ25 : : Tnl composite, Ap ~, C m r pSJ26A7 [cam :hts], AprTra -

Bhaskaran (1960); Parker and Romig (1972) Source of T n l (Datta et al., 1971) T n l in ( + ) orientation (Johnson and Romig, 1978) Tn9 (Cm) from F - C M (Kondo and Mxtsuhashi, 1966) T n l in ( - ) orientation, this paper renders host thermosensitive, this paper T n l transposed from RP4 to SJ25, this paper large deletion (80%) including Cm r of Tn9 and hts of P factor, this paper

Classical strains

Genotype/Phenotype

Other information

pur 1, leu-1 (Cm r Hts) put-l, leu-1 (Cm r) arg-1, ih,-1 trp-1 Smr (Cm ~) arg-1 ilv-1 his-1 Sp r (Ap ~) arg-1 ilv-1, hts-1 nal r

Classical stratus were derived from Bhaskaran's (1960) strata 162 in this laboratory

RP4 pSJ5 + pSJ8 pSJ13 pSJ25

V. cholerae strains RV34(pSJ25) RV34(pSJ8) RV94(pSJ8) RV160(pSJ5) RV107(P)

El Tor strains (developed in this study) RJ1 R J2 RJ3 RJ23 RJ24

RV79 Prototroph (pur deficient) Smr arg-2 Smr ura-3 RiP" Sm ~ tlv-1 RiP" Sm ~

R J25 RJ30 RJ40 RJ44 RJ47 RJ50 RJ57

arg-5 arg-7 arg-7 arg-7 arg-7 ura-3 arg-7

ura-2 Rif r Smr ilv-5 RiP" Sm r ilv-5 lys-1 RiP" Sm r ih,-5 his-2 RiP" Sm r ilv-5 trp-2 Rig Smr thy-2 Rig Smr ilv-5 met-4 thy-3 Rig Sm r

Received as M a k 757 (Gerdes and Romig, 1975) One-step, spontaneous, 100 gg/ml N T G from RJ2 RJ23-RJ30 were obtained by N T G from RJ1 ; selected for Rift, and scored for co-induced auxotrophic mutations; spontaneous Smr N T G from RJ30 N T G from RJ30 N T G from RJ30 Spontaneous thy from RJ23 N T G from RJ30 for met-4, spontaneous thy-3

Tnl strains (For definition of ""T n l strain" see Results section. RJ211 to RJ236

R J I ' : T n l - 1 to R J l ' : T n l - 2 6

Thy-, Tnl stratus RJ237 to RJ240

RJ1 thy::Tnl-27 to RJ1 thy:.Tnl~30

Tfr donors (For definition of Tfr Donors see Results section) RJ241-RJ270 R J271

RJ211(pSJ5) - RJ240(pSJ5) RJ234(pSJ 13)

The designations follow the recommendations of Demerec et al. (1966), Novick et al. (1976) and Campbell et al. (1977)

al. (1973) utilized this procedure to determine the relative orientation of bacteriophage Mu in the E. coli chromosome and F plasmid, and D6nari6 et al. (1977) reported RP4::Mu-mediated chromosomal transfer in several Gram negative species. R. Menzel (described in Kleckner et al., 1977) isolated Salmonella typhimurium Hfr donors by introducing the transposon, Tnl0, into an F' plasmid and bacterial chromosome. More recently Watson and Scaife (1978) described an E. coli FIfr derived from RP4 hybrid

that contained a cloned lambda att region. In the present study, Tnl (Hedges and Jacob, 1974; Heffron et al., 1975; Kleckner, 1977; Richmond and Sykes, 1972) was introduced into both the P factor and bacterial chromosome to ensure homology between the two replicons. The incompatibility of various P factor derivatives was used to isolate V. eholerae strains that contained chromosomally inserted copies of Tnl. By transferring the previously characterized P::Tnl conjugative

S.R. J o h n s o n and W.R. Romlg: V. cholerae Tfr Donors

plasmid (Johnson and Romig, 1978) into these isolates, donors were obtained whose properties resembled those of "' intermediate" E. coli donors (Adelberg and Burns, 1960; Scaife, 1967). The construction and properties of the improved mating strains, which we call " T f r " (for Transposon-facilitated recombination) donors, are described. Materials and Methods Bacterml Stratus and Plasmids Strains of V. cholerae and plasmids used In this study are listed m Table i. Strains were routinely stored at - 6 0 ° C in Brain Heart Infusion (BHI, Difco) broth containing 20% (v/v) glycerol. Media. Meat Extract Agar (MEA) and BHI broth were used as n u m e n t media (Parker et al., 1971). Liquid and solid synthetic media were Minimal M e d i u m A (Miller, 1972), appropriately supplemented as described. All liquid and synthetic media contained 50 pg/ml of adenine to satisfy an undefined pnrine requirement in RV79 and its derivatives. Antibiotics for selection and counterselection were used at the following concentratlons: streptomycin (Sm), 100 pg/ml; spectinomycin (Sp), 100 ~tg/ml; rlfamycm (RID, 30 pg/ml in complete, 15 pg/ml in synthetic m e d i u m ; Amplcillm (Ap) (or penicillin G). 300 gg/ml, and chloramphenicol, (Cm), 15 pg/ml. Qualitame Mating Procedures. The replica mating procedure of Low (1973) was used for qualitatively determining donor characteristics of large n u m b e r s of isolates. For screening purposes, semiquantitative matings were performed by mixing 0.1 ml of a donor culture with 1.0 ml of a recipient, each of which contained about 1-2 x l0 s cells/ml The mixture was incubated for 200 rain at 37 ° C, and samples were applied to small areas of selective agar plates with a calibrated loop, P Plasmzd Assay, The presence of the P plasmid and the hybrid P plasmlds was determined independently of gene transfer by testing the ability of the strain to produce lacunae on a P - indicator lawn. This procedure has been described (Parker and Romig, 1972) Mutant Isolatzon M u t a n t strains of V, cholerae were obtained by treating the cells with N-methyl-N'-nitro-N-nitrosoguanidme (NTG) by the procedures of Adelberg et al. (1965) Auxotrophic mutants were detected and characterized by the replica plating procedures described by Clowes and Hayes (1968). Mutated plasraids were transferred from the mutagen-treated cells to untreated bacteria before they were scored. For some purposes, auxotrophic mutants were isolated by selecting NTG-lnduced Rig mutants and scoring them for co-Induced auxotrophic mutations (Guerola et aI., 197 I). Other antibiotic resistant m u t a n t s were obtained as spontaneous, one-step mutations. Thy-auxotrophs were obtained by trlmethoprim selection (Stacey and Simpson, 1967; Miller, 1972). Quantitatwe Mating Procedures. Dilute lnocula of donor or recipient cultures were incubated m BHI broth at 30 o C without shaking for 12 18h. The donor was diluted 1:20 into fresh broth and shaken at 3 0 ° C at 100 rpm, and the recipient culture was diluted 1 ' 10 and shaken vigorously. After 2 h of incubation, the donor contained about 2 × l0 s and the recipient culture about 2-5 × 108 cells/ml. The bacteria were mated by adding 1 ml of donor culture to i0 ml of recipient culture and gently shaking the resulting mixture at 37 ° C for about 150 mln. After mating, samples were diluted

95 and spread onto various selective media. When nutritional markers were selected, samples were diluted into half-strength BHI and plated onto appropriate synthetic media Transfer frequencies were calculated as the ratio of recombinants to input donors. W h e n recombinants were analyzed for linkage, the wild type allele of the donor was selected, and the media were supplemented for other nutritional requirements of the donor and recipient. For linkage analyses, the donor was always counterselected with antibiotics. The recombinant colonies were transferred with sterile toothpicks to gridded agar plates containing the same medium used to select them, and the master plates were rephcated to a series of supplemented media to score for the acquisition of unselected markers.

Isolation of Hybrid P Factors and Mutants. The hybrid P factor, pSJS, which contains the chloramphenicol transposon, Tn9, was obtained by procedures similar to those used to isolate pSJ5 (Johnson and Romig, 1978) V. cholerae RV107(P +) was mated to E, coli K57 (F-CM) (Kondo and Mltsuhashi, 1968), and Cm r transconjugants were obtained at a frequency of about 10 7. The resulting RV107(P +) (F-CM +) strain co-transferred C m r and lacunae-forming ability at high frequency (about 10 ~) to other P - recipients. Transconjugants from this cross, which contained pSJ8, cotransferred C m r and lacunae-forming abihty at frequencies approaching 1.0, and exhibited other characteristics of P+ donors. Strains presumed to contain the thermosensitive m u t a n t plasmid, pSJ25 (P. : Tn9 hts25, whose presence renders the host thermosensitive), were scored by purifying them on C m - M E A agar at 30 ° C. The resistant colonies were stabbed in corresponding rows along the margin of two plain M E A plates. One plate was incubated at 42 ° C and the other at 30 ° C After 18 h, the Hts phenotype was visually determined by its growth inhibition at 42 ° C. To verify that residual growth at 42 ° C consisted of bacteria which had lost the mutant plasmid, the marginal growth of each stab was streaked toward the center of the plate, and both plates were reincubated at 30 ° C. They were then replicated on C m - M E A plates and the replicates were incubated at 30 ° C. Similar procedures were used to score stares containing plasmld deletion mutants that had lost the hts and cam markers from the composite plasmid, pSJ26.

Results

Attempts to detect Hfr donors in P+ culture by conventional procedures were not successful, and therefore we attempted to directly select bacteria that contained integrated P factors using the methods described by Beckwith et al. (1966). Because P-prime plasmids were not available, attempts were made to isolate thermosensitive mutants of the hybrid P factor, pSJ8, for this purpose. Although mutants analogous to the F ' T s l l f l a c + plasmid (Cuzin and Jacob, 1964) were not identified, other kinds of thermosensitive P factor mutants were obtained, including pSJ25, whose properties are described below. When bacteria carrying pSJ25 were shifted from 30°C to 42 ° C, they produced Cm~ progeny, apparently by defectively segregating the mutant plasmid. However, further examination revealed that the hts plasmid mutation'was lethal to the cells at 42 ° C, irrespective of the host's genetic background. The lethal effects were demonstrated by measuring colony forming ability (at 30 ° C) and turbidity of a culture of

96

S.R. Johnson and W.R. Romig: V. cholerae Tfr Donors

50-

~ - - 3 0 °CA

~

4 2 °C

Table 2. Superinfection ellminaUon of resident Tnl plasmid vectors by acqulmion of related Tn9 plasmid

x._._-xT

40- AB.E

-,o°

/

co

o

~_ 35z

Colomes

Selected

Frequency

Tested

Sensitive to Ap

0.41

150

104 (67%)

104

104 (100%)

a:

3o-

.io-Z u(_9

I- 25uJ

z

.~ zo-

Cross l a RV94 (pSJS) Cm* x Cm r RV160(pSJ5) Ap'

-~o-3

15-

C)

,IO- 4 ~

I o5. (

-9,-6'o-3'o

Plasmid resistance

o

6'0 9'o

+o2 o

TIME (rnin) Fig. 1. Lethal effects of pSJ25 on host cells at 42 ° C. A culture of RV34(pSJ25) was grown at 30°C for 90 mln, after which it was shifted to 42°C. Turbidity (© - o ) was monitored throughout with a Klett-Summersoncolorimeter,using a red No. 66 filter.

Viability (e - - e ) was measured by colony formation at 30°C and at to= 8.6 x 107/ml.The proportion of Cm~ colonies ( x - - x ) was determined by replica plating survivors to Cm-MEA agar at 30° C RV34(pSJ25) during growth at 30 ° C and after it was shifted to 42 ° C. The turbidity (Fig. 1) approximately doubled after the temperature shift, after which the culture almost completely lysed. Loss of viability commenced more rapidly, and the cells continued to die at a faster rate than the turbidity decreased. Most of the survivors were Cm ~, Hts + and did not produce lacunae. Thus, although the hts mutation did not result in the temperature-sensitive segregational loss of the mutant plasmid, its lethal effects at 42°C appeared to render it strongly selective f o r " cured" cells.

Procedures for Introducing Tnl into the Chromosome. Initially, pSJ5 was chosen as the vector to introduce Tnl into the chromosome, but preliminary experiments indicated that it was not efficiently eliminated by superinfection with pSJ8 (Table 2). Procedures were therefore developed to isolate deleted, transferdeficient Tnl vectors, because it seemed probable that they would be efficiently eliminated by superinfection. For this purpose a transmissible plasmid, pSJ26 (P:: Tn9, Tnl hts 25), was constructed by transposing Tnl to pSJ25 by previously described methods (Johnson and Romig, 1978). The resulting hts, Cm r, Ap r plasmid was transferred to an E1 Tor strain, RV79, which unlike classical strains (Parker et al., 1978), does not contain detectable cryptic plasmids (Johnson, S.R., unpublished observations).

Cross 2 b RV34 (pSJS) Cm r × Cm r RJ1 (pSJ7) Ap r

a Cross 1: Donor counterselected with Sp; intact Tnl resident vector b Cross 2/ Donor counterselected with nutritional requirement ( L e u ) , deletion mutant Tnl resident vector

Plasmid deletion mutants were obtained by incubating samples of RV79(pSJ26) at 42°C to select Hts+ revertants and among these, approximately 4% of the Hts +, Cm s survivors retained plasmid-determined Ap r. Of the seven Apr isolates examined by agarose gel electrophoresis (Meyers et al., 1976), five contained plasmid deletion mutants with molecular weights of about 10 × 10 6, compared to the 52 x 10 6 for pSJ5 (Johnson, S.R., Ph.D. thesis, University of California, Los Angeles, 1978). The amp gene of these five isolates was not transmissible, the cells did not produce lacunae, and they occasionally segregated Ap s progeny. When strain RJ207, which contained one of the deleted P:: Tnl plasmids, pSJ7, was superinfected with pSJ8, the resident P : : T n l deletion mutant was quantitatively eliminated from transconjugants (Table 2).

Isolation of Chromosomally Inserted TnI Strain. Bacteria containing chromosomal insertions of Tnl were isolated by superinfecting RJ207 with pSJ25 and selecting transconjugants with both Cm and Ap. The doubly resistant colonies were grown at 42°C on MEA plates to eliminate the superinfecting plasmid. Survivors which lost Cm r, but retained Apr were presumed to represent cells with chromosomally inserted copies of Tnl. Consistent with this assumption, these strains did not transfer Ap r, and did not contain plasraids as determined by agarose gel electrophoresis. In addition, when superinfected with pSJ8, these strains stably maintained both Cm r and Apr without selection, but only Cm r was transmissible. Characterization of Tnl Strains and Tfr Donors. A number of independently derived, plasmid-free strains

S.R Johnson and W.R Romig: V. cholerae Tfr Donors were isolated, that by the criteria above, c o n t a i n e d c h r o m o s o m a l l y inserted T n l t r a n s p o s o n s . F o r convenience, this type of isolate is referred to as a " T n l s t r a i n . " The T n l copy was expected to occupy a specific site in the c h r o m o s o m e of a given T n l strain b u t a m o n g the various isolates, these sites were expected to vary (Heffron et al., 1975; Kleckner, et al., 1977). All RV79 derivatives were identified with the prefix, R J, a n d a strain n u m b e r was assigned to each. The T n l insertions in the isolates were also n u m b e r e d sequentially, a n d for clarity, b o t h designations are f r e q u e n t l y used. Thus, the first T n l strain, RJ211 c o n t a i n s T n l insertion No. 1 ( T n l - 1 ) , the second, RJ212, c o n t a i n s T n l - 2 a n d so on. The term " T f r " ( T r a n s p o s o n - f a c i l i t a t e d r e c o m b i n a t i o n ) was chosen to d e n o t e our Hfr d o n o r s because this term emphasizes their similarity to c o n v e n t i o n a l Hfr d o n o r s .

Transfer Properties of Tfr Donors. The T n l strains were converted into d o n o r s with the P : : T n l plasmid, pSJ5, a n d their P+ state was c o n f i r m e d by l a c u n a e assays (Parker a n d Romig, 1972). These strains, which n o w possessed h o m o l o g o u s T n l sequences in their sex factors a n d c h r o m o s o m e s , were screened for transfer abilities, a n d the m o r e efficient d o n o r s were e x a m i n e d q u a n t i t a t i v e l y by the b r o t h m a t i n g procedures described in Methods. T o avoid the possible complications of i n t e r s t r a i n crosses, the recipients were all derived f r o m strain RV79. F o r controls, p a r e n t a l RV79 strains (which lack the T n l insertion) were converted into d o n o r s with b o t h the P factor a n d with pSJ5. The c o n t r o l d o n o r s transferred all m a r k e r s at about the same, low frequencies (Table 3) that were previously reported for the c o r r e s p o n d i n g classical d o n o r s (Parker et al., 1978). However, Tfr d o n o r s transferred various genes at significantly different fre-

97 1

"1'41~ ¢

"~l~ lys

II

~a

(~

abc

i v lyS

abc

~r

ilv ly s

Fig. 2. Tnl lnsert~on at two chromosomal sites The Tnl element is represented by shaded rectangles, and the letters are included for orientation. The arrow on the P::Tnl plasmid indicates ~ts direction of transfer Chromosomal genes are ilr, lys, and arg. In Panel I. arg is transferred at high frequency, but not &, and lys. In Panel II, dv and lys and arg are transferred at high frequency

quencies, p r e s u m a b l y as a result of their h o m o l o g o u s T n l sequences.

PositionOTg Tnl Insertions Re&tire to Chromosomal Markers. As illustrated in Fig. 2, both the origin and direction of transfer by Tfr d o n o r s can be inferred by m e a s u r i n g gene transfer frequencies. Based on these a s s u m p t i o n s , the d a t a in T a b l e 3 suggested that T n l in RJ253 (strain T n l - 1 3 ) was relatively distant f r o m ilv, arg, and lys, because this d o n o r transferred all three markers only slightly m o r e frequently t h a n the controls. The same r e a s o n i n g suggested that T n l was closer to ih,, arg, a n d lys in RV241 (strain T n l - 1 ) , because it transferred these three genes m o r e freq u e n t l y t h a n the controls, or RJ253. Likewise, T n l appeared to be near ih, in RJ258 (strain T n l - 1 8 ) , a n d near arg in RJ264 (strain T n l - 2 4 ) . However, the greatly reduced transfer of arg by RJ258 a n d of ih, by RJ264 indicated that T n l in b o t h strains was located between these two m a r k e r s a n d that it was oriented differently in them. The position of @s relative

Table 3, Recombination frequencies" for selected markers with Tfr and control donors Recipient

Selected marker c

Tfr donors b

Tnl site RJ3 RJ25 RJ30 RJ25 RJ23 RJ24 RJ30 RJ40 u c

arg-2 arg-5 arg-7 ura-2

ura-3 dim dr-5 /ys-1

Control donors

RJ241 (Tnl-1)

RJ253 (Tnl-13)

RJ264 (Tnl-24)

2,6 X 1 0 - 3 2,5x 10-3 2.8xi0 3 8Ax 10-3 5 5x 10-5 2.6 x 10-3 2.2x 10 3 2.6x 10-3

3.7 x i0 -'~ -2.1x10 -4 3.4 x 10-4 4.8x 10 4

3 1 x 10-3 91x10 3 2 0x 10-3 8.2x 10-s 1 6x10-5 6.4x 10-5

Frequencies are presented as recombmants per input donor The sex factor in Tfr donors is pSJ5 Donors were counterselected with Sm

RJ258 (Tnl-18)

RJ 1(P +) (None)

53x10 -5 4.4x 10~5 5.1 x 10.5

2.8x 10-5 3.3x 10-s 3.0x 10-5 1.6 x 10 5 5.5x 10 6

RJI(pSJ5) (Plasmld only)

7 . 7 × 10 _3

4.0x 10-3 1.Ox 10-5

I A x 10

5

m

4.4x 10-s 4.0x 10-s 2.1 xl0 -5

98

S.R. J o h n s o n and W.R. Romlg: V. cholerae Tfr Donors

Table 4. Segregation of selected and unselected markers in RJ30 recombinants with Tfr and control donors Linkage scored a

Relative distance b Donor. RJ241 (Tn 1-1)

arg ilv arg r/f ih' arg ih, rtf

2.1 > 100 2.3 100

RJ253 RJ264 RJ258 RJ1 (pSJ5) (Tn 1-13) (Tn 1-24) (Tn 1-18) (control) 1.1 3.1 4.3 2.6

> 100 > 100 8 21.0

20.8 > 100 3.5

4.1 34.5 4.0 11.4

a Selected marker is given first b Relative distance is 1/Linkage frequency (Parker et al., 1978), values greater than 100 represent unlinked genes

to arg, ih,, and the Tnl insertions is presented separately. Linkage frequencies were determined because genes that were transferred at about the same high frequencies by a given Tfr donor should not be separated by the Tnl site, and should remain linked regardless of which was selected. Conversely, if two genes were separated by the Tnl insertion, they should be transferred at widely different frequencies and their linkage should be biased by selection. That is, recombinants selected for "proximal'' markers should rarely contain unselected distal markers, whereas the rare recombinants selected for "distal" markers should more often contain unselected proximal markers. The placement of Tnl relative to the linked arg, ill,, and nfmarkers by these considerations is presented below for the Tnl strains previously discussed. As shown in Table 4, rif, arg and ilv are linked when they are transferred to RJ30 by either the P+ control or by RJ253. Because ih, and arg are transferred at approximately equal frequencies by RJ253, and they remain linked to r/f and to each other, the placement of Tnl outside the region containing these markers was indicated (i.e., they are not separated by Tnl in this strain). Similarly the linkage of arg and ih, was consistent with their elevated transfer when transferred by RJ241. However, recombinants selected for ih, did not contain the unselected donor rlf allele. (This is shown in Table 4 by a relative distance of greater than 100 between them.) These results indicated that Tnl was inserted between r/f and ih, in this Tnl strain, and that ih, was the proximal marker. Analogous results were obtained with RJ258, but whereas this donor efficiently transferred ih', the selected ih ,+ recombinants did not contain the unselected donor arg allele. Therefore, Tnl was placed between ill' and arg, with ih, as the proximal marker. The data for RJ264 also suggested that its Tnl insertion

rff Tn1-13 RJ253

ys

~Iv Tn1-1 RJ241

TnM8 PJ258

]rg Tnl-24 RJ264

/

Fig. 3. Placement of T n l insertions relative to bacterial chromosomal markers The arrows indicate direction of transfer by various Tfr donors containing the specified T n l insertions and the conjugatire plasmld, pSJ5

site was between arg and ih,. However, recombinants selected for the efficiently transferred arg marker from RJ264 did not contain the unselected donor ih, allele, suggesting that its Tnl insertion was in the opposite orientation from that of RJ258. As indicated by its enhanced transfer from strains RJ241 and RJ253, lys was closely linked to arg and ill, (Table 3). However, transfer of the lys marker was not enhanced by either RJ258, which appeared to transfer ill, as a proximal marker, or by RJ264, which transferred arg at high frequency. These results suggested: (i) that lys was between arg and ilv; (ii) that it was between the Tnl insertion sites in both strains; and (iii) that lys is distal to the transfer origin of both donors. The location of the Tnl insertions in this group of Tfr donors and their apparent direction of transfer with pSJ5 are presented in Fig. 3. Isolation of "Directed Tnl Insertion Mutants. " None of the Tnl strains contained auxotrophic or other detectable mutations (Kleckner, 1977; Kleckner et al., 1977). Procedures were therefore devised to select specifically for Tnl insertions into the thymidylate synthetase gene (Miller, 1972). RJI(pSJ7) was superinfected with pSJ25 as before to eliminate the Tnl vector. The culture was then plated onto Minimal A agar which contained Ap and Cm to select for Tnl insertions, and which also contained trimethoprim and thymine to select for thymine auxotrophs (Miller, 1972). After incubation, the surviving colonies were verified as thymine auxotrophs, and their Tnl chromosomal insertions were confirmed by the procedures described previously for other Tnl strains. Thymine-requiring Tnl strains occurred at a frequency (about 10-10) which was compatible with reported frequencies for the occurrence of "directed transpositions" into bacterial genes (Beckwith et al., 1966). Also consistent with insertion mutations, the reversion frequencies of the thymine-requiring Tnl strains (with one exception) were not enhanced with NTG under conditions that enhanced reversion of control thy mutants over 1,000-fold (Starlinger and Saedler, 1972). The presumptive insertion mutants were converted into donors with pSJ5 and screened for transfer ability. These donors enhanced transfer of a group of genes that was not transferred efficiently by the previous set of Tfr donors (Table 5), indicating that their Tnl insertions were located in another region of the chromosome.

S.R Johnson and W R Romlg K_mr,_c_ m_r, H_Ls_,Lcn_+ -

RP4

~l~.ht s

99

V. cholerae Tfr Donors

KrnSCrnr,Ap r Hts, Lcn~ Transfer to .................. ~hts I] RJ1 (~10-4 ) ~ I Cm,Ap Ap I

Table 5. Recombination frequencies a for selected markers with T n l t h y - insertion m u t a n t s as donors

Tc

Cm

/~ Crnm~ 4Z°C Hts,Tra •~

pSJ 7 TransposeAp

Ap

~. L'~ .

.

.

.

.

.

Selected b marker

Tfr donors c RJ267

RJ268

RJ269

RJ270

RJ50 RJ47

ura-3 arg-7 H~,-5 trp-2 hzs-2 met-4

3 2 x l 0 -3 2.8x10 -s 2.5x I0 - 6 6.4x 10 4 high high

3x10 2 7 . 6 × 1 0 -5 1.3x 10 -5 5.6x 10 -3 6 8 × i0- 3 3 5 x 10-3

2.7x10-3 low low high high high

2.3x10-3 low low high high high

RJ44 RJ57

pSJ7 [ .h~ ]

\Ap J i _ _

Reclplent

.

CmS,Apr(Tra-I,Lcn Fig. 4. Constructmn of deleted P: T n l vector, pSJ7. Upper left. RP4 transferred to RV34(pSJ25), transpose T n l to pSJ25 to form pSJ26 Upper right Transfer pSJ26 to RJ1, verify transfer by resistance pattern and by subsequent transfer abdlty Lower right: Select P ' : T n l deletion mutants by incubating RJI(pSJ26) at 42 ° C, verify retention of non-transferrable Ap r. Lower left T n l transposed from pSJ7 to bacterial chromosome

a Recombination frequencies are recomblnants per input donor ; "h~gh" and " l o w " represent semNuantitative results of screening experiments b Donors counterselected with Sm Sex factor, pSJ5

Table 6. Linkage of the amp gene of T n l in RJ234 (Tnl-24) to the arg locus Selected marker a

_A£r_(T_ra-_),Lcn- _ _ pSJ7

Superinfect pSJ25

l __T___J

@hts

ll." arg arg lys

I (Ap) 420C

Tfr Donor

d b c

Tnl Strata

~____

Transfer pSJ5 ~~ P A p ~

Apr (Tra+) Lcn +

Frequency b

Apr colomes 30 ° C (Cm ~)

Hts colonies c 42 ° C

Ap ~ colonies 42 ° C (Cm ~)

7x10 5 7x10 -s 1 x 10- 7

0/16 6/16 1/4

16/16 16/16 4/4

0/16 4/16 1/4

Ap'~_ ~

Cm

......

A_(_(!r_a l),_qm_r, _H_ts, C.,cn+ ~tS I

Cross' RJ234(pSJ25) x RJ40

D o n o r counterselected with Sm Frequency is recombmants per input donor Hts colomes are thermosensltive at 42 ° C

L A pA~ L .............. Ap r (Yra-) Cm s,Hls +

Lcn-

Fig. 5. Isolation of " T n l strains" and Tfr donors. Upper left: RJ1 containing T n l chromosomally inserted and pSJ7 deletion mutant. Upper right: Superinfect with pSJ25 to ehmmate pSJ7, select for Apr and Cm r transconjugants. Lower right: Eliminate pSJ25 by 42 ° C incubation, verify non-transferrable Apr to ldenufy "'Tnl strains." Lower left: Transfer hybrid conjugative plasmid, pSJ5 to T n l strain to form Tfr donor, verify by lacunae production AbbreHauons. Transfer deficient ( T r a ) . lacunae production (kcn+), others as previously defined. Locations of the amplcfllin transposon, T n l , the chloramphenicol transposon, Tn9, transfer origins and other plasmld markers are for illustrative purposes and the pIasmMs are not drawn to scale

Tnl insertion sites in additional Tfr donors and their locations relative to other V. cholerae genes have since been determined. Results of these mapping experiments will be presented separately.

Linkage of amp to Chromosomal Genes. To test the reliability of our Tnl placements, genetic linkage was

measured between the amp gene and the chromosomal gene(s) that defined its location. These experiments were not feasible with Tfr donors, because most recombinants for chromosomal markers also contain the P :: Tnl plasmid. Therefore, strain RJ234(Tnl-24), whose Tnl transposon was placed between arg and lys, was converted into a donor with the mutant plasmid, pSJ25, that does not contain the homologous Tnl insertion. To circumvent the possibility that Tnl in the bacterial chromosome might be transposed to this plasmid, it was subsequently eliminated from the recombinants. RJ234(pSJ25) was crossed to RJ40 and recombinants were selected for the arg, ilv, and arg lys donor alleles. The ih' locus was used as an outside marker because it was only moderately linked to arg in P-mediated crosses, The recombinants were scored for Apr before and after they were incubated at 42 ° C. Apr was only rarely plasmid-borne in these recombinants (Table 6), and the donor amp and arg markers were linked. These results support the interpretation that Tnl is chromosomally inserted in this strain, and are consistent with its previously determined location.

100

S.R. Johnson and W.R. Romig: V. cholerae Tfr Donors

Table 7. Gene transfer frequenciesa from same origin by different P" Tnl plasmids

Recipient

RJ40

a b

Selected marker ilv-5 lys-1 arg-7

Tfr Donorb RJ234(pSJ5)

RJ234(pSJ13)

8.3x10 5 6.4x 10-s 1.2x 10-2

1.3x10 2 9.5x 10-a 2.1 x 10 s

Recombinationfrequenciesare recombinants/lnput donor Donor counterselectedwith Sm

Reversed Chromosomal Transfer in Tfr Donors. According to the model in Fig. 2, the orientation of Tnl with respect to the P factor origin of transfer, directly determines the direction of gene transfer in a given Tfr donor. Because Tnl transposons can insert themselves into D N A sequences in either of two orientations (Heffron et al., 1975), it seemed likely that other P : : T n l plasmids could be obtained which could transfer chromosomal genes from the same origin, but in the opposite direction. Nine independently isolated P : : T n l plasmids were transferred to RJ234 and the resulting donors were screened by crossing them to R J40. Transfer properties of 7 of these donors were indistinguishable from control pSJ5 donors, but 2 of them apparently reversed the polarity of chromosome transfer. Transfer frequencies of the Tfr donors containing the two exceptional plasmids, pSJ13 (or pSJ14) were essentially reversed compared to those of RJ234(pSJ5) (Table 7). Chromosomal transfer in both directions has also been demonstrated in the other Tnl strains, and this capability greatly increases the utility of Tfr donors.

Discussion

More efficient V. cholerae donors have been developed by separately introducing the Tnl transposon into the P conjugative plasmid and the bacterial chromosome to create regions of accurate homology in each. In contrast to P+ donors, the improved donors initiated chromosomal transfer from a specific origin (the Tnl insertion site), and high frequency gene transfer was polarized with respect to the origin. The Tnl transposons appear to be stable in these strains, judged by their unaltered donor activities and continued resistance to Ap after repeated transfer without selection. The Transposon-facilitated recombination (Tfr) donors are similar in construction and behavior to those described by Zeldis et al. (1973), Kleckner et al. (1977), and Watson and Scaife (1978). Tfr donors are more closely similar to F' "interme-

diate d o n o r s " (Adelberg and Burns, 1960; Scaife, 1967), than to conventional Hfr strains. These similarities include their moderately high, polarized chromosomal transfer and their efficient transfer of the hybrid plasmid. These properties suggest that our Tfr donors are likewise a mixed population, consisting mainly of P+ donors and relatively fewer Hfr donors. The small fraction of Hfr's in Tfr populations probably account for the observed high frequency, polarized gene transfer. However, most of the cells can presumably transfer chromosomal genes at low frequency by normal P factor activity. If this is so, at least a portion of the low frequency recombinants that received markers distal to the origin (Table 4), may have received them this way. Although the linkage relationships of markers transferred at high frequency were probably not affected by this activity, data indicating linkage between selected terminal and unselected proximal markers are questionable. It is not clear why thermosensitive replication mutants of pSJ8 (or pSJ5) were not obtained among the 40,000 mutagenized clones examined. Neither do we understand the nature of the hts mutation that was repeatedly (25 independent isolates) identified. The behavior of RV34(pSJ25) after it was shifted to 42 ° C (Fig. 1) suggested prophage induction (Gerdes and Romig, 1975); however phage-like structures were not detected by electron microscopy. The hts mutation may be related to the growth-inhibiting property that causes lacunae, but this possibility has not been established. The E1 Tot biotype is the principal etiological agent of the current cholera pandemic (Gallut, 1974). It was felt that genetic studies with these strains might be potentially more useful than similar studies with classical strains. Although suitable conditions for time of entry mapping with these donors have not been established, these strains appear to provide the flexibility required for an effective mating system. It is apparent that the general principles utilized here (Kleckner et al., 1977; also see Bukhari et al., 1977), should be adaptable to other bacteria, perhaps including classical V. cholerae biotypes. Other transposons may be more advantageous than Tnl for these purposes, but because ampicillin is not therapeutically useful against V. cholerae (Northrup, 1969), the T n l transposon was preferred for developing the E1 Tor mating system. Acknowledgments. This investigation was supported by the United States-Japan Cooperative Medical Science Program administered by the National Institute of Allergy and Infectious Diseases. and by a grant from the AcademicSenate, Universityof California, Los Angeles. We would like to thank Pat Zamenhof, R. Martinez and G Wilcox for reading.the manuscript.

S.R. Johnson and W.R Romlg' V. cholerae Tfr Donors

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Communicated

by H.W. Boyer

Received September 30, 1978