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Transposon Mutagenesis in Proteus Mirabilist

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Transposon Mutagenesis in Proteus Mirabilist JOURNAL OF BACTERIOLOGY, OCt. 1991, p. 6289-6293 Vol. 173, No. 19 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 ofBiological 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 Media and growth conditions. Escherichia coli and P. the unique ability to move over agar surfaces by a locomo- mirabilis strains were grown in L broth (10 g of tryptone, 5 tive process referred to as swarming motility (12). Swarming g of yeast extract, and 10 g of NaCl per liter of distilled is the end result of a complex differentiation process which water) at either 30 or 37°C. Unless otherwise noted, agar was ultimately produces an elongated swarmer cell possessing added at a final concehtration of 15 g/liter to solidify broth hundreds of flagella (3, 20). Until recently (1), in-depth media. Because P. mirabilis can swarm over many agar- studies with modem techniques have not been applied to solidified media (such as L agar), an agar-containing medium understanding the genetic regulation and sensory transduc- was prepared which prevents the phenotypic expression of tion mechanisms of swarmer cell differentiation and swarm- swarming motility. It does not contain any metabolic poisons ing of P. mirabilis. Earlier genetic studies of Proteus commonly used to prevent swarming such as P-phenethyl swarmer cell regulation (7) cannot unfortunately be repeated alcohol or p-nitrophenylglycerol, as have been used previ- owing to the loss of strains and phage (16). Since little is ously (20). This medium, referred to as LSW- agar, con- known about the genetics ofProteus swarming, we wanted a tained (per liter) 10 g of tryptone, 5 g of yeast extract, 5 ml means ofgenetic analysis which would be independent ofthe of glycerol (Ultra Pure; Life Technologies, Inc., Gaithers- mutant phenotype under investigation. We chose to use burg, Md.), 0.4 g of NaCl, and 20 g of agar. Minimal salts transposon mutagenesis because, in addition to producing medium for Proteus species contained 10.5 g of K2HPO4, 4.5 mutants with a null phenotype, this method of mutagenesis g of KH2PO4, 0.47 g of sodium citrate, and 1.0 g of results in the insertion of a large segment of DNA encoding (NH4)2SO4. After autoclave sterilization, 1 ml of 1 M a selectable drug resistance marker into the target gene. MgSO4, 10 ml of 20% glycerol, and 1 ml of 1% nicotinic acid Since the drug resistance marker is physically linked to the were added to 1 liter of medium prior to pouring. Amino acid mutated gene, this target gene region can be cloned by requirements of the auxotrophic mutants were assessed by selecting for recombinant bacteria which express the drug spreading 108 cells onto minimal medium and then placing a resistance. Furthermore, the insertion of several kilobases of small amount of the solid amino acid on the plate. Growth of transposon DNA allows precise physical mapping of the colonies around the point of amino acid addition was used to location of the mutation. Thus, the objective of this investi- indicate fulfillment of the nutritional requirement. For E. gation was to develop transposon mutagenesis techniques coli, when appropriate, media were amended with 100 ,ug of for use in P. mirabilis. In addition to developing these ampicillin per ml, 40 ,ug of chloramphenicol per ml, 40 ,ug of procedures, we analyzed the resulting mutants for stability kanamycin per ml, 100 ,g of rifampin per ml, or 30 pug of and randomness of transposon insertion through analyses of spectinomycin per ml. For antibiotic selection of Proteus auxotrophic mutants and DNA-DNA hybridization. mutants, the same concentrations of antibiotics were used Bacterial strains and plasmids. The strains and plasmids except that chloramphenicol and kanamycin were used at used in this study are listed in Table 1. BB2000 is a 150 ,g/ml of medium. All reagents were of the highest purity spontaneously occurring rifampin-resistant mutant of PRM1 available. Components of bacteriological media were pur- (a gift from J. Shapiro) and is used as the wild-type strain chased from Difco. throughout this study. Mutagenesis with TnS derivatives. Transposon mutagene- sis in Proteus strains was done by means of biparental matings between an E. coli donor and a spontaneous ri- * Corresponding author. fampin-resistant mutant of P. mirabilis, BB2000 (Table 1). t Publication 148 from the Center of Marine Biotechnology. The choice of delivery system and vector was predicated on 6289 6290 NOTES J. BACTERIOL. TABLE 1. Strains and plasmids used in this study Strain or plasmid Genotype or phenotype" Derivation Reference or source E. coli SM10 (Xpir) Rec- RP4-2Tc::Mu Apir C600 8, 18 S17-1 (Xpir) Rec- RP4-2Tc::Mu Km::Tn7 Apir 294 8, 18 P. mirabilis PRM1 Wild type J. Shapiro, 10 BB2000 Rif Spontaneous from PRM1 This study BB2030 swr-2030::TnS Cm Swr- Fla- pUT/mini-TnS Cm x BB2000b This study BB2089 swr-2089::TnS Cm Swr- Fla- pUT/mini-Tn5 Cm x BB2000 This study BB2114 swr-2114::Tn5 Cm Swr- Fla- pUT/mini-TnS Cm x BB2000 This study BB2121 swr-2121::TnS Cm Swr- Fla- pUT/mini-TnS Cm x BB2000 This study BB2131 swr-2131::TnS Cm Swr- Fla- pUT/mini-TnS Cm x BB2000 This study BB2146 swr-2146::Tn5 Cm Swr- Fla- pUT/mini-Tn5 Cm x BB2000 This study BB2196 swr-2196::TnS Cm Swr- Fla- pUT/mini-TnS Cm x BB2000 This study BB2199 swr-2199::TnS Cm Swr- Fla- pUT/mini-Tn5 Cm x BB2000 This study Plasmids pGP704 Apr 14 pUT/mini-TnS Cm Apr Cmr pGP704 8 pUT/mini-TnS Sp Apr spr pGP704 8 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 ofE. coli harboring pUT/mini-TnS Cm and BB2000. See the text for details. the need to permit vector replication in the donor E. coli transfer of the plasmid was achieved by spotting 100 [lI of cells but not in the recipient Proteus cells. Further con- donor cells (ca. 4.5 x 108 bacteria) and 200 ,ul of recipient straints were imposed on this choice as a result of native bacteria (ca. 1.0 x 109 cells) onto a sterile cellulosic mem- resistance in the wild-type Proteus strain to the antibiotics brane filter (Micron Separations Inc., Westboro, Mass.; tetracycline and kanamycin, making these drugs unusable diameter, 45 mm; pore size, 0.2 ,um) placed on the surface of for primary genetic selection (2). P. mirabilis is, however, LSW- agar. The cells were incubated at 37°C overnight after sensitive to ampicillin (at 100 ,ug/ml), chloramphenicol (at adsorption of the culture fluid. Following incubation, the 150 ,ug/ml), and spectinomycin (at 100 ,ug/ml) and did not filter was removed from the agar surface and the bacteria produce frequent spontaneous mutants to these drugs (2). were suspended by vortexing in 1 ml of phosphate-buffered Therefore, transposons carried on suicide vectors express- saline (20 mM sodium phosphate [pH 7.5], 100 mM NaCl). ing ampicillin, chloramphenicol, and/or spectinomycin were Samples of 100 ,ul were then spread on LSW- agar contain- used to mutagenize P. mirabilis. ing rifampin and either chloramphenicol (for mini-Tn5 Cm) Initial efforts centered on finding a suitable transposon or spectinomycin (for mini-TnS Sm/Sp). These primary- system for mutagenesis in P. mirabilis. Transposons selection plates were incubated at 37°C for 18 to 36 h or as mini-Mu (10, 11) and mini-TnJO (19) were explored as required for visible colony growth. Antibiotic-resistant bac- possible choices for mutagenesis. Neither of these trans- teria were then transferred to 49 colony master plates for posons, however, transposed in P. mirabilis at sufficiently further analyses. Because P. mirabilis is not infected by high frequency or with complete randomness to permit lambda phage, the master bank of colonies was first trans- successful large-scale mutageneses. Unlike mini-Mu and ferred to LSW- agar which had been previously spread with mini-TnJO, transposon TnS has proven to be highly success- bacteriophage Xvir to locate spontaneous rifampin-resistant ful for mutagenesis of a wide variety of gram-negative E. coli contaminants. Such contaminants made up less than bacteria (5). Recently, De Lorenzo et al. (8) constructed a 0.05% of the total master bank and were culled from the series of TnS derivatives, referred to as mini-TnS, carried on bank at this time.
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