Vol. 173, No. 19
JOURNAL OF BACTERIOLOGY, OCt. 1991, p. 6289-6293
0021-9193/91/196289-05$02.00/0 Copyright © 1991, American Society for Microbiology
NOTES
Transposon Mutagenesis in Proteus mirabilist ROBERT BELAS,1,2* DEBORAH ERSKINE,1 ANID DAVID FLAHERTY1 Center of Marine Biotechnology, The University of Maryland, 600 East Lombard Street, Baltimore, Maryland 21202,1* and Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 212282 Received 21 June 1991/Accepted 24 July 1991
A technique of transposon mutagenesis involving the use of TnS on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that TnS transposed in P. mirabilis at a frequency of ca. 4.5 x 10-6 per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridization to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed. Proteus mirabilis is a motile gram-negative bacterium with the unique ability to move over agar surfaces by a locomotive process referred to as swarming motility (12). Swarming is the end result of a complex differentiation process which ultimately produces an elongated swarmer cell possessing hundreds of flagella (3, 20). Until recently (1), in-depth studies with modem techniques have not been applied to understanding the genetic regulation and sensory transduction mechanisms of swarmer cell differentiation and swarming of P. mirabilis. Earlier genetic studies of Proteus swarmer cell regulation (7) cannot unfortunately be repeated owing to the loss of strains and phage (16). Since little is known about the genetics of Proteus swarming, we wanted a means of genetic analysis which would be independent of the mutant phenotype under investigation. We chose to use transposon mutagenesis because, in addition to producing mutants with a null phenotype, this method of mutagenesis results in the insertion of a large segment of DNA encoding a selectable drug resistance marker into the target gene. Since the drug resistance marker is physically linked to the mutated gene, this target gene region can be cloned by selecting for recombinant bacteria which express the drug resistance. Furthermore, the insertion of several kilobases of transposon DNA allows precise physical mapping of the location of the mutation. Thus, the objective of this investigation was to develop transposon mutagenesis techniques for use in P. mirabilis. In addition to developing these procedures, we analyzed the resulting mutants for stability and randomness of transposon insertion through analyses of auxotrophic mutants and DNA-DNA hybridization. Bacterial strains and plasmids. The strains and plasmids used in this study are listed in Table 1. BB2000 is a spontaneously occurring rifampin-resistant mutant of PRM1 (a gift from J. Shapiro) and is used as the wild-type strain throughout this study. *
Corresponding author.
t Publication 148 from the Center of Marine Biotechnology.
Media and growth conditions. Escherichia coli and P. mirabilis strains were grown in L broth (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter of distilled water) at either 30 or 37°C. Unless otherwise noted, agar was added at a final concehtration of 15 g/liter to solidify broth media. Because P. mirabilis can swarm over many agarsolidified media (such as L agar), an agar-containing medium was prepared which prevents the phenotypic expression of swarming motility. It does not contain any metabolic poisons commonly used to prevent swarming such as P-phenethyl alcohol or p-nitrophenylglycerol, as have been used previously (20). This medium, referred to as LSW- agar, contained (per liter) 10 g of tryptone, 5 g of yeast extract, 5 ml of glycerol (Ultra Pure; Life Technologies, Inc., Gaithersburg, Md.), 0.4 g of NaCl, and 20 g of agar. Minimal salts medium for Proteus species contained 10.5 g of K2HPO4, 4.5 g of KH2PO4, 0.47 g of sodium citrate, and 1.0 g of (NH4)2SO4. After autoclave sterilization, 1 ml of 1 M MgSO4, 10 ml of 20% glycerol, and 1 ml of 1% nicotinic acid were added to 1 liter of medium prior to pouring. Amino acid requirements of the auxotrophic mutants were assessed by spreading 108 cells onto minimal medium and then placing a small amount of the solid amino acid on the plate. Growth of colonies around the point of amino acid addition was used to indicate fulfillment of the nutritional requirement. For E. coli, when appropriate, media were amended with 100 ,ug of ampicillin per ml, 40 ,ug of chloramphenicol per ml, 40 ,ug of kanamycin per ml, 100 ,g of rifampin per ml, or 30 pug of spectinomycin per ml. For antibiotic selection of Proteus mutants, the same concentrations of antibiotics were used except that chloramphenicol and kanamycin were used at 150 ,g/ml of medium. All reagents were of the highest purity available. Components of bacteriological media were purchased from Difco. Mutagenesis with TnS derivatives. Transposon mutagenesis in Proteus strains was done by means of biparental matings between an E. coli donor and a spontaneous rifampin-resistant mutant of P. mirabilis, BB2000 (Table 1). The choice of delivery system and vector was predicated on 6289
6290
J. BACTERIOL.
NOTES TABLE 1. Strains and plasmids used in this study
Strain or plasmid
Derivation
Genotype or phenotype"
Reference or source
E. coli
SM10 (Xpir) S17-1 (Xpir) P. mirabilis PRM1 BB2000 BB2030 BB2089 BB2114 BB2121 BB2131 BB2146 BB2196 BB2199
Rec- RP4-2Tc::Mu Apir Rec- RP4-2Tc::Mu Km::Tn7 Apir
C600 294
8, 18 8, 18
Wild type Rif swr-2030::TnS swr-2089::TnS swr-2114::Tn5 swr-2121::TnS swr-2131::TnS swr-2146::Tn5 swr-2196::TnS swr-2199::TnS
Spontaneous from PRM1 pUT/mini-TnS Cm x BB2000b pUT/mini-Tn5 Cm x BB2000 pUT/mini-TnS Cm x BB2000 pUT/mini-TnS Cm x BB2000 pUT/mini-TnS Cm x BB2000 pUT/mini-Tn5 Cm x BB2000 pUT/mini-TnS Cm x BB2000 pUT/mini-Tn5 Cm x BB2000
J. Shapiro, 10 This study This study This study This study This study This study This study This study This study
pGP704 pGP704
14 8 8
Cm Cm Cm Cm Cm Cm Cm Cm
SwrSwrSwrSwrSwrSwrSwrSwr-
FlaFlaFlaFlaFlaFlaFlaFla-
Plasmids
pGP704 pUT/mini-TnS Cm pUT/mini-TnS Sp
Apr Apr Cmr Apr spr
a Fla-, no flagellin produced; Swr-, absence of swarming on agar media. b p. mirabilis mutants defective in swarming motility and flagellar synthesis. Produced from conjugal mating of E. coli harboring pUT/mini-TnS Cm and BB2000. See the text for details.
the need to permit vector replication in the donor E. coli cells but not in the recipient Proteus cells. Further constraints were imposed on this choice as a result of native resistance in the wild-type Proteus strain to the antibiotics tetracycline and kanamycin, making these drugs unusable for primary genetic selection (2). P. mirabilis is, however, sensitive to ampicillin (at 100 ,ug/ml), chloramphenicol (at 150 ,ug/ml), and spectinomycin (at 100 ,ug/ml) and did not produce frequent spontaneous mutants to these drugs (2). Therefore, transposons carried on suicide vectors expressing ampicillin, chloramphenicol, and/or spectinomycin were used to mutagenize P. mirabilis. Initial efforts centered on finding a suitable transposon system for mutagenesis in P. mirabilis. Transposons mini-Mu (10, 11) and mini-TnJO (19) were explored as possible choices for mutagenesis. Neither of these transposons, however, transposed in P. mirabilis at sufficiently high frequency or with complete randomness to permit successful large-scale mutageneses. Unlike mini-Mu and mini-TnJO, transposon TnS has proven to be highly successful for mutagenesis of a wide variety of gram-negative bacteria (5). Recently, De Lorenzo et al. (8) constructed a series of TnS derivatives, referred to as mini-TnS, carried on a suicide delivery system using plasmid pUT, a derivative of pGP704 (14). This vector carries the 7F protein-dependent origin of replication from plasmid R6K (13) and is maintained only in strains producing the r protein. Plasmid pUT also carries oriT and thus can be conjugally transferred to recipient strains from donor strains expressing RP4 conjugative functions (9). Lastly, the suicide vector also provides the IS50R transposase gene, tnp, in cis and external to the transposon (8), providing additional stability to the inserted element. Donor E. coli [either SM1O(Apir) or S17-1(Xpir)] cells harboring either pUT/mini-TnS Cm or pUT/mini-TnS Sm/Sp were grown overnight at 37°C in L broth with appropriate antibiotic selection. The recipient, P. mirabilis BB2000, was incubated overnight in the presence of rifampin. Conjugal
transfer of the plasmid was achieved by spotting 100 [lI of donor cells (ca. 4.5 x 108 bacteria) and 200 ,ul of recipient bacteria (ca. 1.0 x 109 cells) onto a sterile cellulosic membrane filter (Micron Separations Inc., Westboro, Mass.; diameter, 45 mm; pore size, 0.2 ,um) placed on the surface of LSW- agar. The cells were incubated at 37°C overnight after adsorption of the culture fluid. Following incubation, the filter was removed from the agar surface and the bacteria were suspended by vortexing in 1 ml of phosphate-buffered saline (20 mM sodium phosphate [pH 7.5], 100 mM NaCl). Samples of 100 ,ul were then spread on LSW- agar containing rifampin and either chloramphenicol (for mini-Tn5 Cm) or spectinomycin (for mini-TnS Sm/Sp). These primaryselection plates were incubated at 37°C for 18 to 36 h or as required for visible colony growth. Antibiotic-resistant bacteria were then transferred to 49 colony master plates for further analyses. Because P. mirabilis is not infected by lambda phage, the master bank of colonies was first transferred to LSW- agar which had been previously spread with bacteriophage Xvir to locate spontaneous rifampin-resistant E. coli contaminants. Such contaminants made up less than 0.05% of the total master bank and were culled from the bank at this time. From a single mating, approximately 4.5 x 103 antibiotic-resistant Proteus cells were obtained, indicating that the frequency of transposition as defined as the ratio of drug-resistant exconjugants to the initial number of recipient cells was relatively high (ca. 4.5 x 10-6 per recipient cell). Analysis of auxotrophic mutants and randomness of TnS insertions. Both mini-TnS Sm/Sp and mini-TnS Cm were equally useful for mutagenesis of P. mirabilis. Therefore, we chose mini-TnS Cm for production of the initial bank of mutants. A total of 13,052 chloramphenicol-resistant (CmD) mutants of P. mirabilis were obtained from this mutagenesis. Since transposon mutagenesis in Proteus species has not been examined in detail, we analyzed the mutants for stable insertion of the transposon and also for randomness of insertion. The bank was transferred from LSW- agar con-
NOTES
VOL. 173, 1991
taining chloramphenicol to a nonselective medium (LSWwithout antibiotic), incubated for 48 h at 37°C, and then transferred for a second time to LSW- agar. After an additional 48-h incubation (total of 96 h without selective pressure), the bank was transferred back to LSW- agar containing chloramphenicol. Of the 13,052 colonies, only 16 (0.12%) failed to grow following passage on nonselective medium, suggesting that the Tn5 element was stably inherited in these bacteria. To provide further information on insertional stability, the remaining 13,036 stable TnS-carrying mutants were screened for the presence of plasmidencoded ampicillin resistance. The pUT suicide plasmid -harbors an ampicillin resistance gene, so any ampicillinresistant colonies might be the result of a replicating plasmid in P. mirabilis. Approximately 35% of the colonies (4,542 of a bank of 13,036 mutants) were found to be resistant to ampicillin (Ap). Analysis of these Apr Cmr strains failed to find any extrachromosomal plasmid, although Southern hybridizations with plasmid pGP704 or pUT/mini-TnS Cm probes did confirm that plasmid sequences were present inserted into Proteus chromosomal DNA (data not shown). These data are interpreted to suggest that the pUT plasmid carrying mini-Tn5 Cm did act as a suicide vector and failed to replicate in P. mirabilis; however, in one-third of the mutants the failure to replicate was not concomitant with loss of plasmid DNA. Apparently, in these strains the integration of the transposon was accompanied by integration of part or all of the plasmid DNA as well. Since these strains maintained both stable integration of the mini-TnS Cm element without replication of the plasmid and were phenotypically stable as well, the integration of plasmid DNA did not hamper further analyses. The frequency of auxotrophic mutants in the bank was investigated as one criterion for estimating the randomness of mini-TnS Cm insertion in P. mirabilis. The 13,036 colonies were transferred from master plates to minimal salt medium. Approximately 2.5% (326 of 13,036 Cmr mutants) did not grow on the minimal salt medium and were judged to possess an auxotrophic defect. These auxotrophic mutants were not extensively analyzed, but preliminary results indicated that they included those that required methionine for growth on minimal salt medium, in addition to arginine auxotrophs leucine auxotrophs (data not shown). The frequency of Proteus auxotrophs produced with mini-TnS Cm agrees with data for auxotroph production in other bacteria (4) and suggests that insertion of the transposon did not occur at localized hot spots on the Proteus chromosome. The randomness of insertion was further analyzed by Southern hybridizations in which the 3.4-kb HindlIl fragment from plasmid pUT/mini-Tn5 Cm was used as a probe to locate the transposon on DNA fragments produced by digesting chromosomal DNA from mutants defective in wild-type swarming motility (Swr-) and flagellin synthesis (Fla-) with PvuII (Fig. 1). Since there is a single, asymmetric PvuII site in the transposon (8), digestion of DNA from chloramphenicol-resistant mutants should yield two unique bands if mutagenesis was random. Preparation of plasmid and chromosomal DNA, radioactive labeling of plasmid DNA, and the conditions used for the transfer of DNA fragments to membranes and hybridizations have been described previously (4, 15, 17). Chromosomal DNA from P. mirabilis Swr- Fla- mutants was isolated and enzymatically cleaved with PvuII. The resulting DNA fragments were separated on a 1% agarose gel and transferred to Nytran membrane (Schleicher & Schuell). To identify transposon insertion sites on the chromosomal DNA, the 3.4-kb HindlIl
6291
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24
r
I.-,J
VI
1
JOURNAL OF BACTERIOLOGY, OCt. 1991, p. 6289-6293
0021-9193/91/196289-05$02.00/0 Copyright © 1991, American Society for Microbiology
NOTES
Transposon Mutagenesis in Proteus mirabilist ROBERT BELAS,1,2* DEBORAH ERSKINE,1 ANID DAVID FLAHERTY1 Center of Marine Biotechnology, The University of Maryland, 600 East Lombard Street, Baltimore, Maryland 21202,1* and Department of Biological Sciences, University of Maryland Baltimore County, Baltimore, Maryland 212282 Received 21 June 1991/Accepted 24 July 1991
A technique of transposon mutagenesis involving the use of TnS on a suicide plasmid was developed for Proteus mirabilis. Analysis of the resulting exconjugants indicated that TnS transposed in P. mirabilis at a frequency of ca. 4.5 x 10-6 per recipient cell. The resulting mutants were stable and retained the transposon-encoded antibiotic resistance when incubated for several generations under nonselective conditions. The frequency of auxotrophic mutants in the population, as well as DNA-DNA hybridization to transposon sequences, confirmed that the insertion of the transposon was random and the Proteus chromosome did not contain significant insertional hot spots of transposition. Approximately 35% of the mutants analyzed possessed plasmid-acquired ampicillin resistance, although no extrachromosomal plasmid DNA was found. In these mutants, insertion of the Tn5 element and a part or all of the plasmid had occurred. Application of this technique to the study of swarmer cell differentiation in P. mirabilis is discussed. Proteus mirabilis is a motile gram-negative bacterium with the unique ability to move over agar surfaces by a locomotive process referred to as swarming motility (12). Swarming is the end result of a complex differentiation process which ultimately produces an elongated swarmer cell possessing hundreds of flagella (3, 20). Until recently (1), in-depth studies with modem techniques have not been applied to understanding the genetic regulation and sensory transduction mechanisms of swarmer cell differentiation and swarming of P. mirabilis. Earlier genetic studies of Proteus swarmer cell regulation (7) cannot unfortunately be repeated owing to the loss of strains and phage (16). Since little is known about the genetics of Proteus swarming, we wanted a means of genetic analysis which would be independent of the mutant phenotype under investigation. We chose to use transposon mutagenesis because, in addition to producing mutants with a null phenotype, this method of mutagenesis results in the insertion of a large segment of DNA encoding a selectable drug resistance marker into the target gene. Since the drug resistance marker is physically linked to the mutated gene, this target gene region can be cloned by selecting for recombinant bacteria which express the drug resistance. Furthermore, the insertion of several kilobases of transposon DNA allows precise physical mapping of the location of the mutation. Thus, the objective of this investigation was to develop transposon mutagenesis techniques for use in P. mirabilis. In addition to developing these procedures, we analyzed the resulting mutants for stability and randomness of transposon insertion through analyses of auxotrophic mutants and DNA-DNA hybridization. Bacterial strains and plasmids. The strains and plasmids used in this study are listed in Table 1. BB2000 is a spontaneously occurring rifampin-resistant mutant of PRM1 (a gift from J. Shapiro) and is used as the wild-type strain throughout this study. *
Corresponding author.
t Publication 148 from the Center of Marine Biotechnology.
Media and growth conditions. Escherichia coli and P. mirabilis strains were grown in L broth (10 g of tryptone, 5 g of yeast extract, and 10 g of NaCl per liter of distilled water) at either 30 or 37°C. Unless otherwise noted, agar was added at a final concehtration of 15 g/liter to solidify broth media. Because P. mirabilis can swarm over many agarsolidified media (such as L agar), an agar-containing medium was prepared which prevents the phenotypic expression of swarming motility. It does not contain any metabolic poisons commonly used to prevent swarming such as P-phenethyl alcohol or p-nitrophenylglycerol, as have been used previously (20). This medium, referred to as LSW- agar, contained (per liter) 10 g of tryptone, 5 g of yeast extract, 5 ml of glycerol (Ultra Pure; Life Technologies, Inc., Gaithersburg, Md.), 0.4 g of NaCl, and 20 g of agar. Minimal salts medium for Proteus species contained 10.5 g of K2HPO4, 4.5 g of KH2PO4, 0.47 g of sodium citrate, and 1.0 g of (NH4)2SO4. After autoclave sterilization, 1 ml of 1 M MgSO4, 10 ml of 20% glycerol, and 1 ml of 1% nicotinic acid were added to 1 liter of medium prior to pouring. Amino acid requirements of the auxotrophic mutants were assessed by spreading 108 cells onto minimal medium and then placing a small amount of the solid amino acid on the plate. Growth of colonies around the point of amino acid addition was used to indicate fulfillment of the nutritional requirement. For E. coli, when appropriate, media were amended with 100 ,ug of ampicillin per ml, 40 ,ug of chloramphenicol per ml, 40 ,ug of kanamycin per ml, 100 ,g of rifampin per ml, or 30 pug of spectinomycin per ml. For antibiotic selection of Proteus mutants, the same concentrations of antibiotics were used except that chloramphenicol and kanamycin were used at 150 ,g/ml of medium. All reagents were of the highest purity available. Components of bacteriological media were purchased from Difco. Mutagenesis with TnS derivatives. Transposon mutagenesis in Proteus strains was done by means of biparental matings between an E. coli donor and a spontaneous rifampin-resistant mutant of P. mirabilis, BB2000 (Table 1). The choice of delivery system and vector was predicated on 6289
6290
J. BACTERIOL.
NOTES TABLE 1. Strains and plasmids used in this study
Strain or plasmid
Derivation
Genotype or phenotype"
Reference or source
E. coli
SM10 (Xpir) S17-1 (Xpir) P. mirabilis PRM1 BB2000 BB2030 BB2089 BB2114 BB2121 BB2131 BB2146 BB2196 BB2199
Rec- RP4-2Tc::Mu Apir Rec- RP4-2Tc::Mu Km::Tn7 Apir
C600 294
8, 18 8, 18
Wild type Rif swr-2030::TnS swr-2089::TnS swr-2114::Tn5 swr-2121::TnS swr-2131::TnS swr-2146::Tn5 swr-2196::TnS swr-2199::TnS
Spontaneous from PRM1 pUT/mini-TnS Cm x BB2000b pUT/mini-Tn5 Cm x BB2000 pUT/mini-TnS Cm x BB2000 pUT/mini-TnS Cm x BB2000 pUT/mini-TnS Cm x BB2000 pUT/mini-Tn5 Cm x BB2000 pUT/mini-TnS Cm x BB2000 pUT/mini-Tn5 Cm x BB2000
J. Shapiro, 10 This study This study This study This study This study This study This study This study This study
pGP704 pGP704
14 8 8
Cm Cm Cm Cm Cm Cm Cm Cm
SwrSwrSwrSwrSwrSwrSwrSwr-
FlaFlaFlaFlaFlaFlaFlaFla-
Plasmids
pGP704 pUT/mini-TnS Cm pUT/mini-TnS Sp
Apr Apr Cmr Apr spr
a Fla-, no flagellin produced; Swr-, absence of swarming on agar media. b p. mirabilis mutants defective in swarming motility and flagellar synthesis. Produced from conjugal mating of E. coli harboring pUT/mini-TnS Cm and BB2000. See the text for details.
the need to permit vector replication in the donor E. coli cells but not in the recipient Proteus cells. Further constraints were imposed on this choice as a result of native resistance in the wild-type Proteus strain to the antibiotics tetracycline and kanamycin, making these drugs unusable for primary genetic selection (2). P. mirabilis is, however, sensitive to ampicillin (at 100 ,ug/ml), chloramphenicol (at 150 ,ug/ml), and spectinomycin (at 100 ,ug/ml) and did not produce frequent spontaneous mutants to these drugs (2). Therefore, transposons carried on suicide vectors expressing ampicillin, chloramphenicol, and/or spectinomycin were used to mutagenize P. mirabilis. Initial efforts centered on finding a suitable transposon system for mutagenesis in P. mirabilis. Transposons mini-Mu (10, 11) and mini-TnJO (19) were explored as possible choices for mutagenesis. Neither of these transposons, however, transposed in P. mirabilis at sufficiently high frequency or with complete randomness to permit successful large-scale mutageneses. Unlike mini-Mu and mini-TnJO, transposon TnS has proven to be highly successful for mutagenesis of a wide variety of gram-negative bacteria (5). Recently, De Lorenzo et al. (8) constructed a series of TnS derivatives, referred to as mini-TnS, carried on a suicide delivery system using plasmid pUT, a derivative of pGP704 (14). This vector carries the 7F protein-dependent origin of replication from plasmid R6K (13) and is maintained only in strains producing the r protein. Plasmid pUT also carries oriT and thus can be conjugally transferred to recipient strains from donor strains expressing RP4 conjugative functions (9). Lastly, the suicide vector also provides the IS50R transposase gene, tnp, in cis and external to the transposon (8), providing additional stability to the inserted element. Donor E. coli [either SM1O(Apir) or S17-1(Xpir)] cells harboring either pUT/mini-TnS Cm or pUT/mini-TnS Sm/Sp were grown overnight at 37°C in L broth with appropriate antibiotic selection. The recipient, P. mirabilis BB2000, was incubated overnight in the presence of rifampin. Conjugal
transfer of the plasmid was achieved by spotting 100 [lI of donor cells (ca. 4.5 x 108 bacteria) and 200 ,ul of recipient bacteria (ca. 1.0 x 109 cells) onto a sterile cellulosic membrane filter (Micron Separations Inc., Westboro, Mass.; diameter, 45 mm; pore size, 0.2 ,um) placed on the surface of LSW- agar. The cells were incubated at 37°C overnight after adsorption of the culture fluid. Following incubation, the filter was removed from the agar surface and the bacteria were suspended by vortexing in 1 ml of phosphate-buffered saline (20 mM sodium phosphate [pH 7.5], 100 mM NaCl). Samples of 100 ,ul were then spread on LSW- agar containing rifampin and either chloramphenicol (for mini-Tn5 Cm) or spectinomycin (for mini-TnS Sm/Sp). These primaryselection plates were incubated at 37°C for 18 to 36 h or as required for visible colony growth. Antibiotic-resistant bacteria were then transferred to 49 colony master plates for further analyses. Because P. mirabilis is not infected by lambda phage, the master bank of colonies was first transferred to LSW- agar which had been previously spread with bacteriophage Xvir to locate spontaneous rifampin-resistant E. coli contaminants. Such contaminants made up less than 0.05% of the total master bank and were culled from the bank at this time. From a single mating, approximately 4.5 x 103 antibiotic-resistant Proteus cells were obtained, indicating that the frequency of transposition as defined as the ratio of drug-resistant exconjugants to the initial number of recipient cells was relatively high (ca. 4.5 x 10-6 per recipient cell). Analysis of auxotrophic mutants and randomness of TnS insertions. Both mini-TnS Sm/Sp and mini-TnS Cm were equally useful for mutagenesis of P. mirabilis. Therefore, we chose mini-TnS Cm for production of the initial bank of mutants. A total of 13,052 chloramphenicol-resistant (CmD) mutants of P. mirabilis were obtained from this mutagenesis. Since transposon mutagenesis in Proteus species has not been examined in detail, we analyzed the mutants for stable insertion of the transposon and also for randomness of insertion. The bank was transferred from LSW- agar con-
NOTES
VOL. 173, 1991
taining chloramphenicol to a nonselective medium (LSWwithout antibiotic), incubated for 48 h at 37°C, and then transferred for a second time to LSW- agar. After an additional 48-h incubation (total of 96 h without selective pressure), the bank was transferred back to LSW- agar containing chloramphenicol. Of the 13,052 colonies, only 16 (0.12%) failed to grow following passage on nonselective medium, suggesting that the Tn5 element was stably inherited in these bacteria. To provide further information on insertional stability, the remaining 13,036 stable TnS-carrying mutants were screened for the presence of plasmidencoded ampicillin resistance. The pUT suicide plasmid -harbors an ampicillin resistance gene, so any ampicillinresistant colonies might be the result of a replicating plasmid in P. mirabilis. Approximately 35% of the colonies (4,542 of a bank of 13,036 mutants) were found to be resistant to ampicillin (Ap). Analysis of these Apr Cmr strains failed to find any extrachromosomal plasmid, although Southern hybridizations with plasmid pGP704 or pUT/mini-TnS Cm probes did confirm that plasmid sequences were present inserted into Proteus chromosomal DNA (data not shown). These data are interpreted to suggest that the pUT plasmid carrying mini-Tn5 Cm did act as a suicide vector and failed to replicate in P. mirabilis; however, in one-third of the mutants the failure to replicate was not concomitant with loss of plasmid DNA. Apparently, in these strains the integration of the transposon was accompanied by integration of part or all of the plasmid DNA as well. Since these strains maintained both stable integration of the mini-TnS Cm element without replication of the plasmid and were phenotypically stable as well, the integration of plasmid DNA did not hamper further analyses. The frequency of auxotrophic mutants in the bank was investigated as one criterion for estimating the randomness of mini-TnS Cm insertion in P. mirabilis. The 13,036 colonies were transferred from master plates to minimal salt medium. Approximately 2.5% (326 of 13,036 Cmr mutants) did not grow on the minimal salt medium and were judged to possess an auxotrophic defect. These auxotrophic mutants were not extensively analyzed, but preliminary results indicated that they included those that required methionine for growth on minimal salt medium, in addition to arginine auxotrophs leucine auxotrophs (data not shown). The frequency of Proteus auxotrophs produced with mini-TnS Cm agrees with data for auxotroph production in other bacteria (4) and suggests that insertion of the transposon did not occur at localized hot spots on the Proteus chromosome. The randomness of insertion was further analyzed by Southern hybridizations in which the 3.4-kb HindlIl fragment from plasmid pUT/mini-Tn5 Cm was used as a probe to locate the transposon on DNA fragments produced by digesting chromosomal DNA from mutants defective in wild-type swarming motility (Swr-) and flagellin synthesis (Fla-) with PvuII (Fig. 1). Since there is a single, asymmetric PvuII site in the transposon (8), digestion of DNA from chloramphenicol-resistant mutants should yield two unique bands if mutagenesis was random. Preparation of plasmid and chromosomal DNA, radioactive labeling of plasmid DNA, and the conditions used for the transfer of DNA fragments to membranes and hybridizations have been described previously (4, 15, 17). Chromosomal DNA from P. mirabilis Swr- Fla- mutants was isolated and enzymatically cleaved with PvuII. The resulting DNA fragments were separated on a 1% agarose gel and transferred to Nytran membrane (Schleicher & Schuell). To identify transposon insertion sites on the chromosomal DNA, the 3.4-kb HindlIl
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