PLASMID 6, 141-147(1981)

SHORT COMMUNICATIONS

Killing of Klebsiella pneumoniae Mediated by Conjugation with Bacteria Carrying Antibiotic-Resistance Plasmids of the Group N

MARGARITA RODRIGUEZ AND V. N. IYER

Department of Biology, Carleton Universify, Ottawa, Ontario KlS 5B6, Canada

Received August 25, 1980; revised December 4, 1980

Escherichia co/i K-12 strains carrying any one of a number of different plasmids of the incompatibility group N have been found to kill Klebsiella pneumoniae, MSal. A simple spot test is described that could be of value in diagnosing the presence of these plasmids. Killing is closely associated with the ability of the N+ strains to mate with M5al and all conjugation-deficient (Tra-) mutants were also unable to kill MSal (Kil-). At a low frequency, some Kil- mutants could be isolated that showed little or no transfer deficiency. Plasmids of the groups P and W which have been suspected (on other grounds) to specify conjugative systems interrelated in some manner to that specified by N plasmids, also kill MSal but to a lesser degree.

Antibiotic-resistance plasmids that are self-transmissible by bacterial conjugation (conjugative R plasmids) are usually and conveniently classified into incompatibility groups and the group N comprises one such incompatibility group (1). Studies on the genetic and molecular details of conjugation mediated by this group are in their infancy although it is known that these plasmids mediate conjugation more efficiently on solid or semisolid than in liquid media (2). Bacteriophage IKe produces plaques spe- cifically on bacteria carrying this group of plasmids (3). However, some other plasmid-specific bacteriophages (PR4, PRD 1, IKe hl, a mutant of IKe) have been described whose specificities are less narrow than IKe and encompass Enterobacteriaceae carrying conjugative plasmids of two other incompatibility groups, P and W (4 -6). This has raised the possibility that the conjuga- tive systems specified by plasmids of these three incompatibility groups may be inter- related in some way. Since plasmids of these three groups are widely dispersed and are of frequent occurrence among bacteria in na- ture, an understanding of their conjugative system(s) is likely to be rewarding. In this

paper, we show that N-group plasmids have another common property, the ability to kill Klebsiella pneumoniae. This property may be used conveniently to diagnose the pres- ence of N plasmids in a bacterial popu- lation. We describe experiments showing that killing is associated with bacterial mating. Plasmids of the groups P and W (but not those of F and I) also show this effect but to a lesser degree (Fig. 3). It is sug- gested that this property may prove useful in the analysis of the conjugative system(s) of these three groups of plasmids.

Table 1 lists the bacterial strains and plasmids used. The K. pneumoniae strain was the widely used MSal. Plasmid-medi- ated killing of this strain was studied in two ways. In the first and more quantitative procedure, a late-exponential phase Penas- say Broth (Difco) culture of the test donor was mixed with a similar culture of M5al at a donor to recipient ratio varying from 0.1 to 10 in different experiments and the mixed population was collected immediately by filtration on the surface of a Millipore filter (0.45 pm, 45 mm). The filter charged with the cells was then placed on a fresh Penas- say Agar plate with the side of the mem-

141 0147-619X/81/040141-07$02.00/0 Copyright 0 1981 by Academic Press, Inc. AU rights of reproductia, in any form reserved.

142 SHORT COMMUNICATIONS

TABLE 1

E. coli K-12 STRAINS AND THEIR PLASMIDS

Strain Conjugative group of plasmid in the strain

Phenotype source or

Chromosome specified Plasmid specified reference

CO8

C6OOT JS-3 Azi’ = J5 55-3 Nal’ C6OOT/pCU 1 C6OOT/pCU4 c6OOT/pcu8 JSlR45 JYR46 JSlR48 J5/R113 JS/Rl!%’ JSiR205 JS/pTM558 JS-3 NaWpDT200 JSIRW C6OOT/RK2 C6OOT/pRK229

J5IS-a JSlR4 1 I JS/R388 J5/R386 JS/RlOO drdl

JS/R144 drdll JSlR144 drd3 c6OOT/pcu2

No plasmid

No plasmid No plasmid No plasmid N N N N N N N N N N N P P Nonconjugative

(derived from RK2) W W W F F 1 I Unknown

Arg-His-Lac-MettMtl-Pro- Pu-Py-Thy-StrINal’

Thr-LeuThi-Thy- Pro-Met-Azi’ Pro-Met-Nal’ As for C6OOT As for C6OOT As for C6OOT As for J5 As for J5 As for J5 As for J5 As for J5 As for J5 As for J5 As for J5 As for J5 As for C600T As for C6OOT

As for J5 As for J5 As for J5 As for J5 As for J5 As for J5 As for J5 As for C6OOT

- -

Ap’Sm’Sp’ AprSmrSprTcr AP’ Ap’SuTc’ Ap’Sm’Su’Tc’ Ap’Sm’Su’Tc’ Tc’ Ap’Sp’Tcr Ap%’ AprKmrSmr Sm’SurTpr Ap’KmlTc’ Ap’Sm’ Ap’Tc’

Cm’Km’Sm’Su’ SuTp’ SVTP’ Tc’ CmrSmrSuTcrdrd 1 SmrSuTcrdrdl 1 Kmlc’drd3 SmTc’

This laboratory

(8) (7) (I) (9) (9) (9) (1) (1) (1)

(10) (2)

(11) (12) (13)

(1) (14) (15)

(1) (16) (16)

(1) (1) (1) (1) (9)

Note. Pro, proline; Thi, thiamine; Thr, threonine; Thy, thymine; Ap, ampicillin; Cm, chloramphenicol; Pn, penicillin; Km, kanamycin; Sm, streptomycin; Sp, spectinomycin; Su, sulfonamide; Tc, tetracycline; Tp, trimethoprim; drd, plasmid mutation that derepresses conjugal transfer; r and s, resistance and sensitivity; -, nutritional dependency.

brane carrying the cells facing up and in- cubated at 37°C. After varying intervals, the cells were washed off the filter and col- lected in sterile 0.85% saline and immedi- ately diluted extensively to prevent further matings. The viable counts of M5al were then determined by plating on glucose mini- mal agar (7) on which donors were counter- selected. In a few experiments, a nalidixic acid-resistant spontaneous mutant of M5al was used as the recipient, nalidixic acid (50 pg/ml) being used for counterselecting the donors. Two types of controls were used: the recipient alone or mixtures of the recipient and the appropriate R- donor. Under these experimental conditions and in different experiments, about 80% of the

M5al cells lost colony-forming ability when mated with N+ cells (see Fig. 1). Further- more about 50% of the surviving colonies on minimal agar were found to have an ab- normal morphology (Fig. 2). Killing could be detected at a donor to recipient ratio of as low as 0.1.

In the second procedure adapted for screening purposes, 10H to lo9 M5al cells were spread on a minimal agar plate and air-dried. Drops of the test “donor” were then placed on the surface of the medium using a multipoint inoculating device and in- cubated at 37°C when the inoculated areas remained relatively clear if the donor strain carried an appropriate plasmid (Fig. 3). These procedures were used with all the

SHORT COMMUNICATIOPjS 143

strains listed in Table 1. Escherichia co/i K-12 strains tested (J5 and C600, Table 1) were not susceptible to killing.

Evidence Indicating That Killing Requires Mating

As indicated earlier, bacteria carrying an N-group plasmid mate more efficiently on solid than in liquid media. Four kinds of experiments indicated that killing was not mediated by a cell-free substance and that it could be detected only under conditions permitting efficient conjugation: (a) When the N+-strain C6OO/pCUl was mixed and incubated with M5al in a liquid culture even at a very high donor:recipient ratio (100: l), killing or abnormal colony-formation by M5al was not detected. (Under these con- ditions, and even between E. cofi strains, mating was relatively inefficient.) (b) Cell- free culture filtrates did not have a killing effect. (c) If membrane filter matings were conducted under conditions where donor and recipient cells were brought into con- tact by the apposition of filter surfaces charged with M5al and N+ cells, killing could be observed. Under similar conditions with a third uncharged filter sandwiched between the two charged surfaces, killing was not detected. (d) The production of a cell-bound antibiotic-like substance by the E. coli strain was excluded by nega- tive results of tests similar to those used for colicins ( 17,18).

Conjugative Plasmids That Are Able to Mediate Killing

Table 2 shows the results of experiments in which a number of E. co/i strains carry- ing different plasmids were tested for their ability to kill MSal and for their conjugal transfer proficiency. From these results it can be seen that with one exception (plasmid pTM558) all the N plasmids tested killed M5al. The inability to detect killing with the strain carrying pTM558 is correlated with the relatively poor conjugative ability

100 1

80.

. .

I 0 IO 20 3c ,

MINUTES OF MATING

FIG. 1. Loss of viability of Klebsieh pneumoniae, MSal as a function of mating with the strain C6OOT/ pCU1 carrying a conjugative N-group antibiotic- resistance plasmid. A donor to recipient ratio of 10 was used in this experiment.

of this strain (about lOO-fold lower than for strains carrying the other N plasmids). N-Group plasmids are transferred stably to MSal at a frequency that is less than to E. cofi K-12 (Table 2). This is a result that would be expected if potential transcon- jugants were killed. Plasmids of the groups P and W were transferred to M5al as ef- ficiently as were the N-group plasmids. Strains carrying a P or W group plasmid also killed M5al (although the transfer- deficient pRK229 did not mediate killing) but to a degree significantly less than with strains carrying an N plasmid. We have also tested E. cofi strains carrying com- patible plasmids from the N, P, and W groups in every combination. Such strains did not show a degree of killing greater than those carrying a plasmid with a greater killing ability.

Plasmids of two other major conjugative groups (F and I) did not mediate any de- tectable killing (Table 2). Thus, this prop- erty is characteristic of conjugative N-group plasmids and is shared to some degree by conjugative P- and W-group plasmids. The N group of plasmids has a higher efficiency of killing.

144 SHORT COMMUNICATIONS

SHORT COMMUNICATIONS

FIG. 3. Illustration of the results of the spot test used for the detection of the killing phenotype. Top and from left to right: Control JS; phenotype of a strain carrying an N-group plasmid @CU 1); phenotype of a strain carrying a W-group plasmid (S-a). Bottom: detection of a Kil- mutant of pCU1 (top row, third from left).

The Ability of an N Plasmid (pCU1) to agarose gel electrophoresis patterns to that Promote Killing Is Not Altered in Those observed previously for pCU1 (9). Thus, Recipients That Survive Killing none of the genetic or physical properties

As indicated in Table 2, 90% of the ex- of pCU1 appear to have altered during its

posed cells of MSal failed to form colonies. maintenance in the surviving cells of M5al.

When the colonies surviving from mating with a pCU1 donor were tested, they were Isolation and Relevant Properties of

usually found to carry the antibiotic-resist- Plasmid Mutants Selected for Deficiency

ance markers of the donor plasmid. These in Killing or in Conjugal Transfer

markers could be transferred by conjugation Supercoiled DNA was isolated from to an appropriate E. coli strain which strain C6OO/pCUl using procedures de- then acquired the ability to kill M5al. The scribed by Clewell and Helinski (19) and plasmid-positive M5al surviving cells could Humphreys et al. (20). This DNA was then also be shown to kill a nalidixic acid- subjected to hydroxylamine mutagenesis resistant mutant of MSal. Plasmid DNA iso- in vitro (21) and used to transform strain lated from these primary and secondary C600 selecting for Sp’ transformants. These E. coli strains and digested with the restric- transformants were then regrown and tested tion endonuclease Hpa I, showed similar separately for the presence of their original

146 SHORT COMMUNICATIONS

TABLE 2

TESTS OF THE ABILITY OF E. coli K-12 STRAINS CARRYING DIFFERENT CONJUGATIVE PLASMIDS TO KILL K. pneumoniae MSal

Donor strain of plasmid

c6Oo/pcu 1 c6Oo/pcu4 C6OO/pCU8 JSlR205 C08lRl99 JSlpTM558 JSlR48 JSlRP4 C6OO/RK2 C6OOlpRK229 JS/S-a J5lR386 JS/RlOO-1 JS/R64- 11 J5/R144-3 c6Oo/pcu2

Conjugative group(s)

N N N N N N N P P P( Tra-) W F F I I ?

Conjugal transfer frequency (% of donors) to

E. coli K-12 K. pneumoniae M5al

30 0.44 25 0.4 8 NT

23 0.57 26 0.38

0.22 0.003 10 0.2 31 17 20 14 0 0

10 0.1 1.9 4 x 10-d 3.5 1 x 10-d

16 0.1 0.23 3.7 x 10-S 0.1 10-S

Fraction (%) of MSal cells killed

upon mating

90 96 93 63 88 0

93 20 12 0

20 0 0 0 0 0

Note. In addition to the N-, P-, and W-group plasmids shown here, other N-group plasmids (R45, R46, R113, pDT200) or W-group plasmids (R411, R388) tested have been found to kill MSal in the spot test. Tra, transfer deficient; NT, not tested as MSal is resistant to ampicillin.

antibiotic-resistance markers and abilities to either kill K. pneumoniae M5Al (Fig. 3) or to transfer the plasmid by conjugation to an appropriate E. coli K-12 strain. 1900 trans- formants were tested using the “spot- mating” or “spot-killing” tests described. Of these, 132 showed no evidence of either killing or transfer (Kil- Tra-), 4 showed no evidence of killing but did show some evi- dence of transfer (Kil- Tra+ or Kil- Tra’), and none were Trap Kil+. The absence of the last phenotype and the high coincidence between the mutational impairment of con- jugation proficiency and of the killing ability confirms the association between these two properties. The four rare mutants that ap- peared to be Kil- but Tra+ or Tra’ are the subject of further studies which may help explain the mechanism of killing.

ACKNOWLEDGMENTS

This study was supported by a grant from the Medical Research Council of Canada. M.R. was the

recipient of a Gran Mariscal de Ayacucho scholar- ship from the government of Venezuela. We thank D. Bouanchand, N. Datta, E. Lederberg, D. Helinski, S. Jorgensen, C. Monti-Bragadin, and D. Taylor for several strains and plasmids used in this study. For gifts of antibiotics, we are grateful to Bristol Meyers of Canada, Montreal.

REFERENCES

1. DATTA, N., In “Microbiology 1974” (D. Schles- singer, ed.), pp. 9-15. Amer. Sot. Microbial., Washington, D. C., 1975.

2. DENNISON, S., AND BAUMBERG, S., Mol. Gen. Gene?. 138, 323-331 (1975).

3. KHATOON, H., IYER, R. V., AND IYER, V. N., Virology 48, 145- 155 (1972).

4. BRADLEY, D. E., AND COHEN, D. R., J. Gen. Micro&ok 98, 619-623 (1977).

5. OLSEN, R. H., SIAK, J., AND GRAY, R., J. Viral. 14, 688-699 (1974).

6. GRANT, R. B., AND JACKOWSKI, B. J., In “Plas- mids and Transposons” (C. Studdard and K. R. Rozee, eds.), pp. 343-349. Academic Press, New York, 1980.

7. DAVIS, B. D., AND MINGIOLI, E. S., J. Bacterial. 60, 17-28 (1950).

8. APPLEYARD, R. K., Genetics 39,440-452 (1954).

SHORT COMMUNICATIONS 147

9. KONARSKA-KOZLOWSKA, M., AND IYER, V. N., In “Plasmids and Transposons” (C. Studdard and K. R. Rozee, eds.), pp. 361-369. Academic Press, New York, 1980.

10. SCAVIZZI, M. R., Ann. Microbial. (Inst. Pasteur) 124B, 153-159 (1974).

II. MATTHEW, M., AND HEDGES, R. W., J. Bacterial. 125, 713-717 (1976).

12. BABUDRI, N., AND MONTI-BRAGADIN, C., Mol. Gen. Genet. 155, 287-290 (1977).

13. TAYLOR, D. E., KEYSTONE, J. S., AND DEVLIN, H. R., Lance? 1, 426 (1980).

14. INGRAM, L. C., RICHMOND, M. H., AND SYKES, R. B., Antimicrob. Ag. Chemother. 3, 279-288 (1973).

15. HELINSKI, D. R., In “Genetic Engineering for

Nitrogen Fixation” (A. Hollaender, ed.), pp. 19-49. Plenum, New York, 1977.

16. DATTA, N., AND HEDGES, R. W., J. Gen. Micro- biol. 72, 349-355 (1972).

17. CLOWES, R. C., AND HAYES, W., “Experiments in Microbial Genetics.” Blackwell, Oxford, 1968.

18. FREDERICQ, P., C. R. Sot. Biol. 144, 730-734 (1950).

19. CLEWELL, D. B., AND HELINSKI, D. R., Proc. Nat. Acad. Sci. USA 62, 1166-1171 (1%9).

20. HUMPHREYS, G. D., WILLSHAW, G. A., AND ANDERSON, E. S., Biochem. Biophys. Acta 383,457-463 (1975).

21. HASHIMOTO, T., AND SEKIGUCHI, M., J. Bac- teriol. 127, 1561-1563 (1972).