PLASMID
25,76-80
( I99 I )
Tn5 Mutagenesis
and insertion
Replacement
in Azotobacter
vinelandii
ASLJNCIONCONTRERAS,~RAFAELMALDONAD~,ANDJOSEPCASADESUS~ Departammto
de Gen&ica, Universidad
de Sevilla, Apartado 1095, E-41080 Seville, Spain
Received September 25, 1990; revised December 3, 1990 Tn5 insertion mutants of Azotobacter vinelandii were isolated using vectors pJB4JI (IncP) and pGS9 (IncN). A procedure to replace Tn5 (Km’) by its nontransposing derivative Tn5- 13 1 (T?) was developed. For the replacement, a ColE 1 derivative harboring Tn5- 13 1 (pRZ 13 1) was conjugally mobilized by the IncN plasmid pCU 10 1 into A. vinelandii strains containing Tn5. Both plasmids are unable to be maintained in A. vinelundii, but the transient presence of pRZ I3 1 allows recombination between the incoming and the resident Tn5 elements. Genetic and physical analysis showed that insertion replacements result in lower frequencies of TnS-associated genomic rearrangements, thereby increasing the stability of TnS-containing strains. 0 1991 Academic
Press, Inc.
UW 136 is a RiT derivative of the standard, wild-type strain UW (W. J. Brill, University of Wisconsin, USA). For Tn5 mutagenesis, two vectors were used: pJB4JI (Gm’ Km’ Tra+), an IncP plasmid carrying Tn5 inserted into a Mucts62 prophage (Beringer et al., 1978), and pGS9 (Cm’ Km’ Tra+ IncN), a TnS-containing derivative of pCU 10 1 (Selvaraj and Iyer, 1983). Culture media, growth conditions, antibiotic concentrations, and genetic methods were as described elsewhere (Contreras and Casadesus (1987) and references therein). Although plasmid pJB4JI is stably maintained in A. vinelandii, it can be used for Tn5 delivery by conjugal dislodgment with the incompatible plasmid pJB3J1, a Ap’ Tc’ Km” derivative of the IncP plasmid R68.45 (Brewin et al., 1980). For dislodgment, A. vinelandii Km’ Ap’ Tc’ transconjugants were selected from matings AS9/pJB4JI x UW 136/pJB3. Over 99% of the Km’ Ap’ Tc’ colonies were GmS, indicating that efficient displacement of pJB4JI had occurred. All the TnS-carrying isolates described below were isolated in the presence of nalidixic acid; hence they derive from strain AS9. To rule out that Tn5 might preferentially transpose to plasmid pJB3JI rather than to the A. vinelandii genome, 30 independent AS9 Km’
Despite the extensive use of transposon Tn5 as a genetic tool, transposition systems with defective Tn5 derivatives have not been developed (Berg et al., 1989). As an altemative, we describe a procedure to replace Tn5 insertions by Tn5- 13 1, a nontransposing Tn5 derivative encoding tetracycline resistance (Rothstein et al., 1980b) (Fig. 1). The replacement requires a double crossover at the flanking IS50 sequences. We show that the system can be easily applied to strain construction in Azotobacter. Its use should be equally feasible in other nonenteric bacteria, provided that they can act as recipients of ColE 1 derivatives mobilized by broad-range suicide plasmids from group IncN. For Azotobacter, where genetic analysis is constrained by the extremely high DNA content (Sadoff et al., 1979; Kennedy and Toukdarian, 1987; Contreras and Casadesus, 1987), the use of strains carrying “frozen” insertions is expected to be a crucial advantage for many genetic manipulations. All the Azotobacter vinelandii mutants described in this study derive from AS9, a spontaneous Nal’ derivative of strain UW 136 (E. Santero, Universidad de Sevilla, Spain). ’ Present address: Department of Biochemistry, University of Leicester, Leicester LEl 7RH, England. * To whom correspondence should be addressed. 0147-619X/91
$3.00
Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form rrscrvcd.
76
SHORT COMMUNICATIONS
TnS-131
EcoR I
Bglll
TnlO
BglIl
77
EcoRl
BglIl
FIG. 1. Diagrams of Tn5, TnlO, and TnS- 131; maps are not drawn at scale. Tn5- 131 is a Tn5 derivative in which the central &/II fragment has been replaced by a TnlO Bg/II fragment containing the tetracycline resistance gene (Rothstein et al., 1980b); the size of the central Bg/II fragment of Tn5 is 2.7 kb.
Ap’ Tc’ isolates (carrying pJB3JI and Tn5) were used as donors to transfer pJB3JI to E. coli HB 10 1. Selection was carried out on MacConkey galactose plates supplemented with tetracycline. Km’ colonies were not found among the E. coli Tc’ transconjugants, indicating that Tn5 does not transpose at high frequency to plasmid pJB3JI. Thus, A. vinelandii Km’ transconjugants were presumed to carry mostly chromosomal Tn5 insertions. Tn5 mutagenesis with vector pGS9 was achieved in matings E. coli HB 10 l/pGS9 x A. vinelandii AS9, under conditions similar to those used by other authors (Joerger et al., 1986; Toukdarian and Kennedy, 1986). A. vinelandii Km’ Rif transconjugants appeared at a frequency of about 1Oe6per recipient bacterium. Rifampicin (but not nalidixic acid) exerted efficient counterselection of the E. coli donor. Over 95% of the Km’ Rif transconjugants were Cm’, indicating that the vector was not stably maintained in A.
vinelandii. To be classified as TnS-induced mutants, Km’ isolates were required to passthe reversion and linkage tests described elsewhere
(Contreras and Casadesus, 1987), to prove that the kanamycin resistance and the mutation were genetically associated. In addition, genomic DNAs from Km’ isolates generated by pJB4JI were tested for Southern hybridization against a Mu probe, plasmid pGP1 (Giphart-Gassler and van de Putte, 1979). Isolates carrying Mu sequenceswere not further analyzed. TnS-induced mutants generated by either procedure are listed, together with their characteristics, in Table 1. All mutants were stable enough to fulfill the requirements of strain construction and preservation. However, secondary transposition of Tn5 was often detected: (I) 14-99s of the prototrophic revertants of Tn5 insertion mutants were Km’. (II) Complete ( 100%)linkage was rarely found. Stable heterozygotes, like the ade+/ ade: : TnlOHH 104 isolates described by Contreras and Casadesus( 1987), were not found; thus every stable Km’ prototrophic revertant or transformant was likely generated by a secondary Tn5 transposition. Moreover, physical analysis indicated that additional rearrangements, not detectable in genetic experiments, also occurred in TnS-containing
78
SHORT COMMUNICATIONS TABLE 1 GENETICANALYSISOFTN~~NSER~~NMUTANTSINA. vinelandii
Strain No.
Phenotype
Frequency of reversion
Km’ revertants
Linkage (direct)”
Linkage (reverse)b
ASlOl’ ASl02’,’ AS1 1l’,’ ASI 12’,’ ASI 13’ ASI 14’ ASI 15d AS1 16d ASI 17d AS191d
Adecyscyscyssee below8 NifMetMtlNifMet-
1o-s 1o-9
25,76-80
( I99 I )
Tn5 Mutagenesis
and insertion
Replacement
in Azotobacter
vinelandii
ASLJNCIONCONTRERAS,~RAFAELMALDONAD~,ANDJOSEPCASADESUS~ Departammto
de Gen&ica, Universidad
de Sevilla, Apartado 1095, E-41080 Seville, Spain
Received September 25, 1990; revised December 3, 1990 Tn5 insertion mutants of Azotobacter vinelandii were isolated using vectors pJB4JI (IncP) and pGS9 (IncN). A procedure to replace Tn5 (Km’) by its nontransposing derivative Tn5- 13 1 (T?) was developed. For the replacement, a ColE 1 derivative harboring Tn5- 13 1 (pRZ 13 1) was conjugally mobilized by the IncN plasmid pCU 10 1 into A. vinelandii strains containing Tn5. Both plasmids are unable to be maintained in A. vinelundii, but the transient presence of pRZ I3 1 allows recombination between the incoming and the resident Tn5 elements. Genetic and physical analysis showed that insertion replacements result in lower frequencies of TnS-associated genomic rearrangements, thereby increasing the stability of TnS-containing strains. 0 1991 Academic
Press, Inc.
UW 136 is a RiT derivative of the standard, wild-type strain UW (W. J. Brill, University of Wisconsin, USA). For Tn5 mutagenesis, two vectors were used: pJB4JI (Gm’ Km’ Tra+), an IncP plasmid carrying Tn5 inserted into a Mucts62 prophage (Beringer et al., 1978), and pGS9 (Cm’ Km’ Tra+ IncN), a TnS-containing derivative of pCU 10 1 (Selvaraj and Iyer, 1983). Culture media, growth conditions, antibiotic concentrations, and genetic methods were as described elsewhere (Contreras and Casadesus (1987) and references therein). Although plasmid pJB4JI is stably maintained in A. vinelandii, it can be used for Tn5 delivery by conjugal dislodgment with the incompatible plasmid pJB3J1, a Ap’ Tc’ Km” derivative of the IncP plasmid R68.45 (Brewin et al., 1980). For dislodgment, A. vinelandii Km’ Ap’ Tc’ transconjugants were selected from matings AS9/pJB4JI x UW 136/pJB3. Over 99% of the Km’ Ap’ Tc’ colonies were GmS, indicating that efficient displacement of pJB4JI had occurred. All the TnS-carrying isolates described below were isolated in the presence of nalidixic acid; hence they derive from strain AS9. To rule out that Tn5 might preferentially transpose to plasmid pJB3JI rather than to the A. vinelandii genome, 30 independent AS9 Km’
Despite the extensive use of transposon Tn5 as a genetic tool, transposition systems with defective Tn5 derivatives have not been developed (Berg et al., 1989). As an altemative, we describe a procedure to replace Tn5 insertions by Tn5- 13 1, a nontransposing Tn5 derivative encoding tetracycline resistance (Rothstein et al., 1980b) (Fig. 1). The replacement requires a double crossover at the flanking IS50 sequences. We show that the system can be easily applied to strain construction in Azotobacter. Its use should be equally feasible in other nonenteric bacteria, provided that they can act as recipients of ColE 1 derivatives mobilized by broad-range suicide plasmids from group IncN. For Azotobacter, where genetic analysis is constrained by the extremely high DNA content (Sadoff et al., 1979; Kennedy and Toukdarian, 1987; Contreras and Casadesus, 1987), the use of strains carrying “frozen” insertions is expected to be a crucial advantage for many genetic manipulations. All the Azotobacter vinelandii mutants described in this study derive from AS9, a spontaneous Nal’ derivative of strain UW 136 (E. Santero, Universidad de Sevilla, Spain). ’ Present address: Department of Biochemistry, University of Leicester, Leicester LEl 7RH, England. * To whom correspondence should be addressed. 0147-619X/91
$3.00
Copyright 0 1991 by Academic Press. Inc. All rights of reproduction in any form rrscrvcd.
76
SHORT COMMUNICATIONS
TnS-131
EcoR I
Bglll
TnlO
BglIl
77
EcoRl
BglIl
FIG. 1. Diagrams of Tn5, TnlO, and TnS- 131; maps are not drawn at scale. Tn5- 131 is a Tn5 derivative in which the central &/II fragment has been replaced by a TnlO Bg/II fragment containing the tetracycline resistance gene (Rothstein et al., 1980b); the size of the central Bg/II fragment of Tn5 is 2.7 kb.
Ap’ Tc’ isolates (carrying pJB3JI and Tn5) were used as donors to transfer pJB3JI to E. coli HB 10 1. Selection was carried out on MacConkey galactose plates supplemented with tetracycline. Km’ colonies were not found among the E. coli Tc’ transconjugants, indicating that Tn5 does not transpose at high frequency to plasmid pJB3JI. Thus, A. vinelandii Km’ transconjugants were presumed to carry mostly chromosomal Tn5 insertions. Tn5 mutagenesis with vector pGS9 was achieved in matings E. coli HB 10 l/pGS9 x A. vinelandii AS9, under conditions similar to those used by other authors (Joerger et al., 1986; Toukdarian and Kennedy, 1986). A. vinelandii Km’ Rif transconjugants appeared at a frequency of about 1Oe6per recipient bacterium. Rifampicin (but not nalidixic acid) exerted efficient counterselection of the E. coli donor. Over 95% of the Km’ Rif transconjugants were Cm’, indicating that the vector was not stably maintained in A.
vinelandii. To be classified as TnS-induced mutants, Km’ isolates were required to passthe reversion and linkage tests described elsewhere
(Contreras and Casadesus, 1987), to prove that the kanamycin resistance and the mutation were genetically associated. In addition, genomic DNAs from Km’ isolates generated by pJB4JI were tested for Southern hybridization against a Mu probe, plasmid pGP1 (Giphart-Gassler and van de Putte, 1979). Isolates carrying Mu sequenceswere not further analyzed. TnS-induced mutants generated by either procedure are listed, together with their characteristics, in Table 1. All mutants were stable enough to fulfill the requirements of strain construction and preservation. However, secondary transposition of Tn5 was often detected: (I) 14-99s of the prototrophic revertants of Tn5 insertion mutants were Km’. (II) Complete ( 100%)linkage was rarely found. Stable heterozygotes, like the ade+/ ade: : TnlOHH 104 isolates described by Contreras and Casadesus( 1987), were not found; thus every stable Km’ prototrophic revertant or transformant was likely generated by a secondary Tn5 transposition. Moreover, physical analysis indicated that additional rearrangements, not detectable in genetic experiments, also occurred in TnS-containing
78
SHORT COMMUNICATIONS TABLE 1 GENETICANALYSISOFTN~~NSER~~NMUTANTSINA. vinelandii
Strain No.
Phenotype
Frequency of reversion
Km’ revertants
Linkage (direct)”
Linkage (reverse)b
ASlOl’ ASl02’,’ AS1 1l’,’ ASI 12’,’ ASI 13’ ASI 14’ ASI 15d AS1 16d ASI 17d AS191d
Adecyscyscyssee below8 NifMetMtlNifMet-
1o-s 1o-9
