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A demonstration that pCU1 tra gene products are not required in the killing of Klebsiella pneumoniae
PLASMlDl7,
161-163 (1988)
SHORT A Demonstration MINNA
COMMUNICATIONS
That pCU1 tra Gene in the Killing of Klebsiella
B. ROTHEIM,*
Products Are Not Required pneumoniae
BRUCE LOVE,? V. THATTE$
AND V. N. IYER+
*Department of Microbiology and Immunology, State University ofNew York Health Science Center, Syracuse, New York 13210; tDepartment of Biochemistry, University of Rochester Medical Center, Rochester, New York 14642; and *Department of Biology and Institute qfBiochemistry, Carleton University, Ottawa, Ontario KlS 586. Canada
Received December
3, 1987; revised March 24, 1988
IncN group plasmids, including pC?U1, are able to kill Klebsiella pneumoniae when conjugatively transferred from an Escherichia coli donor. Transposon mutagenesis and deletion analysis of the known tra complementation groups were used to demonstrate that the tra gene products inactivated are not required for the Kik phenotype. Q 1988 Academic Ress, 1~. Plasmids of the N-incompatibility group, of which the conjugative plasmid pCU1 is a prototype, have been shown to kill about 90% of recipient Klebsiella cells when conjugatively transferred from an Escherichia coli donor (Rodriguez and Iyer, 198 1). This plasmid mediated killing of Klebsiella has been designated as the Kik phenotype (Thatte et al., 1985a), and previous studies have defined two noncontiguous regions, kikA and k&B (Fig. l), on the pCU1 genome that are required for optimal expression of the Kik phenotype. The requirement for simultaneous mobilization into Klebsiella and the observation that all transposon Tn5 insertions within the conjugative tra region also abolish the Kik phenotype (Thatte et al., 1985a) raise the possibility that for this phenotype some or all of the tra genes must also be transmitted into the recipient. Altematively, with such Tra- mutants, the absence of the Kik phenotype may be simply due to the failure to deliver the kikA and kikB regions into Klebsiella. In the context of these experiments, a helper plasmid is one which will not self-mobilize but which can provide pCU1 tra functions for the mobilization of other plasmids carrying born (born = basis of mobility) but deleted of tra function. We have constructed
a helper plasmid carrying the tra region of pCU1, which has been deleted of the born region and part of traH, and named it pCU80 1. This plasmid has a deletion of approximately 4 kbp, from coordinates 6.4 to 10.4 on the pCU1 map (Fig. 1). In the construction of plasmid pCU801, a Tn5 insertion derivative of pCU1, pCU29 (pCU1:: Tn5:8.2) was linearized at the XhoI sites on Tn5 (there are no XhoI sites on pCU1). The linearized plasmid was progressively shortened by treatment with Bal31 (Maniatis et al., 1982) to delete the &s-dominant sequences of born while still retaining a functional tra region. The reaction was stopped at various times, and the DNA was pooled, recircularized with T4 DNA ligase, and used to transform E. coli HB 101 (pCU800). The plasmid pCU800 is a mobilizable derivative of pCU 109 which lacks a functional tra region because of a partial deletion. It is used in these experiments to select a helper plasmid, as described below. Transformants (Sp%m’) were analyzed in a cross to HB 10 1Rf, selecting transconjugants on RfSp and RtCm (L agar plus rifampicin and spectinomycin or chloramphenicol). A potentially useful helper plasmid should not give transconjugants on RfSp (i.e.. does not self-mobilize) but should give transcon-
161
0147-619X/88 $3.00
Camrisht 0 1988 byAcademic Press, he. All rights of reproduction in any form rewrvcd
162
SHORT COMMUNICATIONS
FIG. 1. Construction of pCU80 1 and pCU 109. The heavy line in the pCU 109 map represents sequences from the vector pACYC 184.Tn5 is represented by the arrowhead, placed on the plasmid map at the point of insertion. The pCU29 region deleted in the construction of pCU80 1 is defined by the broken line. Map coordinates for pCU 109::Tn5 are based on the pCU 1 map of the tra region. The wavy line indicates that the boundary of the region is not precisely defined.
jugants with high frequency on RfCm, indicating it has a functional tra region that mobilizes pCU800 in trans. Three helper plasmids were obtained, of which pCU801 had the largest deletion while still retaining a functional tra. pCU803 was deleted of born, but retained a functional traH. Representative data on self-mobilization of pCU801 and the mobilization of pCU800 by pCUl and by pCU80 1 are given in Table 1. The E. coli strains and the materials and methods used for conjugation have been described (Thatte et al., 1985a). Quantitative and qualitative determination of the Kik phenotype was also previously described (Thatte et al., 1985a). pCU109 is a plasmid constructed by cloning the HindI fragment
(27.5- 11.2) ofpCU
1, as shown in
Fig. 1, into
the HindIII site in the tetracycline gene of the plasmid vector pACYC184 (Thatte et al., 1985a). A seriesof Tn5 insertions in pCU 109 (designated pVT_) (Thatte et al., 1985b) was used in experiments to determine the role of TABLE 1 Conjugative transfer frequency (transconjugants /lOO donors) Donor C6OO(pCUl)
HnlulWWpCUsW
HBlOl(pCU801) HBlOl(pCU801/pCU800)
RfSp 1.06 x 10’ 1.2 x lo2
RfCm <10-s 1.43 x 102 <10-a
0.74 X lo-’ 0.9’ ’ 1’
163
SHORT COMMUNICATIONS
tra. The pVT series of insertions did not
contain a traA::TnS mutant, but a Tn5 insertion in traA of pCU1 (named pCU171) was available (Thatte et al., 1985b). This traA::TnS insertion was transferred to pCUIO9 by homologous recombination, and the resulting plasmid was named pCU8 12. It was shown that pCU8 12 did not complement pCU17 1, indicating that the Tn5 had been recombined into the truA of pCU109. The strains carrying these insertions are shown in Fig. 1. HBlO 1 (pCU801) was transformed with DNA derived from the pCU109::Tn5 insertions to allow the mobilization of eachderivative into Klebsiella (pCU803 was used to mobilize plasmids pVT149 and pVT 150). When the mobilization was done we observedthat in each casethere was a decrease of 70-90s in the viable cell count of the Klebsiella recipients. We also constructed a KpnI deletion derivative of pCU 109 (deleted from 5.9 to 16.2) lacking tra complementation groups C through G, which is designated pCU405. This plasmid was mobilized by pCU801 and we observeda similar decrease of 70-90s in the viable cell count of the M5a 1 recipients. Theseexperimentshave not excludeda requirement for born in the Kik phenotype. There is, however, a strong possibility that Kik expression requires the simultaneous transfer of the kik genes. This inference is based on the observations (Thatte et al., 1985a),referred to above, showing that if either of the kik regions is placed in Klebsiella, and the other region introduced by conjugation, there is no loss in Klebsiellu viability. However, this recipient, now containing the two kik regions in tram, is able to conjugatively kill a suitably marked Klebsiella recipient. An interesting aspectof the Kik phenotype is the host specificity, since the viability of E. coli is not reduced in a similar seriesof experiments. In pKMlO1, two genescalled kilA and kilB have been identified and mapped (Winans and Walker, 1985). The
expressionof thesegenesleadsto lethality in the E. coli host. A Klebsiella kik gene of pKM101 is separableby mutation from the E. coli kil genes(Winans and Walker, 1985), and maps at a position analogousto that of kikB in pCU1 (S. Winans and G. Walker, personal communication). In this paper we have shown that plasmids carrying the kikA and kikB regions,with Tn5 insertions in each of the eight tru complementation groups, are able to kill Klebsiella when mobilized by a nontransmissible helper plasmid. We can conclude, therefore, that the tru functions inactivated by the Tn5 insertions studied here are not required for the Kik phenotype. Since the deletion mutant, pCU405, is Kik+ in phenotype, we also conclude that all functions of complementation groups traC through traG are not necessary for the Kik phenotype. ACKNOWLEDGMENTS Research at Carleton University was supported by an operating research grant from the Medical Research Council of Canada and an equipment grant from the Natural Science and Engineering Research Council of Canada. M.B.R. was a recipient of an NYS/UUP Faculty Award.
REFERENCES MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. ( 1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. RODRIGUEZ,M., AND IYER, V. N. (1981). Killing of Ktebsieiia pneumoniae mediated by conjugation with bacteria carrying antibiotic-resistance plasmids of the Group N. Plasmid6, 141-147. THATTE, V., GILL, S., AND IYER, V. N. (1985a). Regions on plasmid pCU 1 required for the killing of Klebsiella pneumoniae. J. Bacterial. 163, 1296-1299. THA~E, V., BRADLEY,D. E., AND IYER, V. N. (1985b). N conjugative transfer system of plasmid pCU 1. J. Bacterial. 163, 1229- 1236, WINANS, S. C., AND WALKER, G. C. (1985). Identification of pKM lOl-encoded loci specifying lethal gene products. J. Bacterial. 161,4 17-424. Communicated by Barry Polisky
161-163 (1988)
SHORT A Demonstration MINNA
COMMUNICATIONS
That pCU1 tra Gene in the Killing of Klebsiella
B. ROTHEIM,*
Products Are Not Required pneumoniae
BRUCE LOVE,? V. THATTE$
AND V. N. IYER+
*Department of Microbiology and Immunology, State University ofNew York Health Science Center, Syracuse, New York 13210; tDepartment of Biochemistry, University of Rochester Medical Center, Rochester, New York 14642; and *Department of Biology and Institute qfBiochemistry, Carleton University, Ottawa, Ontario KlS 586. Canada
Received December
3, 1987; revised March 24, 1988
IncN group plasmids, including pC?U1, are able to kill Klebsiella pneumoniae when conjugatively transferred from an Escherichia coli donor. Transposon mutagenesis and deletion analysis of the known tra complementation groups were used to demonstrate that the tra gene products inactivated are not required for the Kik phenotype. Q 1988 Academic Ress, 1~. Plasmids of the N-incompatibility group, of which the conjugative plasmid pCU1 is a prototype, have been shown to kill about 90% of recipient Klebsiella cells when conjugatively transferred from an Escherichia coli donor (Rodriguez and Iyer, 198 1). This plasmid mediated killing of Klebsiella has been designated as the Kik phenotype (Thatte et al., 1985a), and previous studies have defined two noncontiguous regions, kikA and k&B (Fig. l), on the pCU1 genome that are required for optimal expression of the Kik phenotype. The requirement for simultaneous mobilization into Klebsiella and the observation that all transposon Tn5 insertions within the conjugative tra region also abolish the Kik phenotype (Thatte et al., 1985a) raise the possibility that for this phenotype some or all of the tra genes must also be transmitted into the recipient. Altematively, with such Tra- mutants, the absence of the Kik phenotype may be simply due to the failure to deliver the kikA and kikB regions into Klebsiella. In the context of these experiments, a helper plasmid is one which will not self-mobilize but which can provide pCU1 tra functions for the mobilization of other plasmids carrying born (born = basis of mobility) but deleted of tra function. We have constructed
a helper plasmid carrying the tra region of pCU1, which has been deleted of the born region and part of traH, and named it pCU80 1. This plasmid has a deletion of approximately 4 kbp, from coordinates 6.4 to 10.4 on the pCU1 map (Fig. 1). In the construction of plasmid pCU801, a Tn5 insertion derivative of pCU1, pCU29 (pCU1:: Tn5:8.2) was linearized at the XhoI sites on Tn5 (there are no XhoI sites on pCU1). The linearized plasmid was progressively shortened by treatment with Bal31 (Maniatis et al., 1982) to delete the &s-dominant sequences of born while still retaining a functional tra region. The reaction was stopped at various times, and the DNA was pooled, recircularized with T4 DNA ligase, and used to transform E. coli HB 101 (pCU800). The plasmid pCU800 is a mobilizable derivative of pCU 109 which lacks a functional tra region because of a partial deletion. It is used in these experiments to select a helper plasmid, as described below. Transformants (Sp%m’) were analyzed in a cross to HB 10 1Rf, selecting transconjugants on RfSp and RtCm (L agar plus rifampicin and spectinomycin or chloramphenicol). A potentially useful helper plasmid should not give transconjugants on RfSp (i.e.. does not self-mobilize) but should give transcon-
161
0147-619X/88 $3.00
Camrisht 0 1988 byAcademic Press, he. All rights of reproduction in any form rewrvcd
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SHORT COMMUNICATIONS
FIG. 1. Construction of pCU80 1 and pCU 109. The heavy line in the pCU 109 map represents sequences from the vector pACYC 184.Tn5 is represented by the arrowhead, placed on the plasmid map at the point of insertion. The pCU29 region deleted in the construction of pCU80 1 is defined by the broken line. Map coordinates for pCU 109::Tn5 are based on the pCU 1 map of the tra region. The wavy line indicates that the boundary of the region is not precisely defined.
jugants with high frequency on RfCm, indicating it has a functional tra region that mobilizes pCU800 in trans. Three helper plasmids were obtained, of which pCU801 had the largest deletion while still retaining a functional tra. pCU803 was deleted of born, but retained a functional traH. Representative data on self-mobilization of pCU801 and the mobilization of pCU800 by pCUl and by pCU80 1 are given in Table 1. The E. coli strains and the materials and methods used for conjugation have been described (Thatte et al., 1985a). Quantitative and qualitative determination of the Kik phenotype was also previously described (Thatte et al., 1985a). pCU109 is a plasmid constructed by cloning the HindI fragment
(27.5- 11.2) ofpCU
1, as shown in
Fig. 1, into
the HindIII site in the tetracycline gene of the plasmid vector pACYC184 (Thatte et al., 1985a). A seriesof Tn5 insertions in pCU 109 (designated pVT_) (Thatte et al., 1985b) was used in experiments to determine the role of TABLE 1 Conjugative transfer frequency (transconjugants /lOO donors) Donor C6OO(pCUl)
HnlulWWpCUsW
HBlOl(pCU801) HBlOl(pCU801/pCU800)
RfSp 1.06 x 10’ 1.2 x lo2
RfCm <10-s 1.43 x 102 <10-a
0.74 X lo-’ 0.9’ ’ 1’
163
SHORT COMMUNICATIONS
tra. The pVT series of insertions did not
contain a traA::TnS mutant, but a Tn5 insertion in traA of pCU1 (named pCU171) was available (Thatte et al., 1985b). This traA::TnS insertion was transferred to pCUIO9 by homologous recombination, and the resulting plasmid was named pCU8 12. It was shown that pCU8 12 did not complement pCU17 1, indicating that the Tn5 had been recombined into the truA of pCU109. The strains carrying these insertions are shown in Fig. 1. HBlO 1 (pCU801) was transformed with DNA derived from the pCU109::Tn5 insertions to allow the mobilization of eachderivative into Klebsiella (pCU803 was used to mobilize plasmids pVT149 and pVT 150). When the mobilization was done we observedthat in each casethere was a decrease of 70-90s in the viable cell count of the Klebsiella recipients. We also constructed a KpnI deletion derivative of pCU 109 (deleted from 5.9 to 16.2) lacking tra complementation groups C through G, which is designated pCU405. This plasmid was mobilized by pCU801 and we observeda similar decrease of 70-90s in the viable cell count of the M5a 1 recipients. Theseexperimentshave not excludeda requirement for born in the Kik phenotype. There is, however, a strong possibility that Kik expression requires the simultaneous transfer of the kik genes. This inference is based on the observations (Thatte et al., 1985a),referred to above, showing that if either of the kik regions is placed in Klebsiella, and the other region introduced by conjugation, there is no loss in Klebsiellu viability. However, this recipient, now containing the two kik regions in tram, is able to conjugatively kill a suitably marked Klebsiella recipient. An interesting aspectof the Kik phenotype is the host specificity, since the viability of E. coli is not reduced in a similar seriesof experiments. In pKMlO1, two genescalled kilA and kilB have been identified and mapped (Winans and Walker, 1985). The
expressionof thesegenesleadsto lethality in the E. coli host. A Klebsiella kik gene of pKM101 is separableby mutation from the E. coli kil genes(Winans and Walker, 1985), and maps at a position analogousto that of kikB in pCU1 (S. Winans and G. Walker, personal communication). In this paper we have shown that plasmids carrying the kikA and kikB regions,with Tn5 insertions in each of the eight tru complementation groups, are able to kill Klebsiella when mobilized by a nontransmissible helper plasmid. We can conclude, therefore, that the tru functions inactivated by the Tn5 insertions studied here are not required for the Kik phenotype. Since the deletion mutant, pCU405, is Kik+ in phenotype, we also conclude that all functions of complementation groups traC through traG are not necessary for the Kik phenotype. ACKNOWLEDGMENTS Research at Carleton University was supported by an operating research grant from the Medical Research Council of Canada and an equipment grant from the Natural Science and Engineering Research Council of Canada. M.B.R. was a recipient of an NYS/UUP Faculty Award.
REFERENCES MANIATIS, T., FRITSCH, E. F., AND SAMBROOK, J. ( 1982). “Molecular Cloning: A Laboratory Manual.” Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. RODRIGUEZ,M., AND IYER, V. N. (1981). Killing of Ktebsieiia pneumoniae mediated by conjugation with bacteria carrying antibiotic-resistance plasmids of the Group N. Plasmid6, 141-147. THATTE, V., GILL, S., AND IYER, V. N. (1985a). Regions on plasmid pCU 1 required for the killing of Klebsiella pneumoniae. J. Bacterial. 163, 1296-1299. THA~E, V., BRADLEY,D. E., AND IYER, V. N. (1985b). N conjugative transfer system of plasmid pCU 1. J. Bacterial. 163, 1229- 1236, WINANS, S. C., AND WALKER, G. C. (1985). Identification of pKM lOl-encoded loci specifying lethal gene products. J. Bacterial. 161,4 17-424. Communicated by Barry Polisky