Transposon Mutagenesis in Proteus Mirabilist

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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 mutagenesis 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. From a single mating, approximately 4.5 x a suicide delivery system using plasmid pUT, a derivative of 103 antibiotic-resistant Proteus cells were obtained, indicat- pGP704 (14). This vector carries the 7F protein-dependent ing that the frequency of transposition as defined as the ratio origin ofreplication from plasmid R6K (13) and is maintained of drug-resistant exconjugants to the initial number of recip- only in strains producing the r protein. Plasmid pUT also ient cells was relatively high (ca. 4.5 x 10-6 per recipient carries oriT and thus can be conjugally transferred to recip- cell). ient strains from donor strains expressing RP4 conjugative Analysis of auxotrophic mutants and randomness of TnS functions (9). Lastly, the suicide vector also provides the insertions. Both mini-TnS Sm/Sp and mini-TnS Cm were IS50R transposase gene, tnp, in cis and external to the equally useful for mutagenesis of P. mirabilis. Therefore, we transposon (8), providing additional stability to the inserted chose mini-TnS Cm for production of the initial bank of element. mutants. A total of 13,052 chloramphenicol-resistant (CmD) Donor E. coli [either SM1O(Apir) or S17-1(Xpir)] cells mutants of P. mirabilis were obtained from this mutagenesis. harboring either pUT/mini-TnS Cm or pUT/mini-TnS Sm/Sp Since transposon mutagenesis in Proteus species has not were grown overnight at 37°C in L broth with appropriate been examined in detail, we analyzed the mutants for stable antibiotic selection. The recipient, P. mirabilis BB2000, was insertion of the transposon and also for randomness of incubated overnight in the presence of rifampin. Conjugal insertion. The bank was transferred from LSW- agar con- VOL. 173, 1991 NOTES 6291

taining chloramphenicol to a nonselective medium (LSW- %-4 without antibiotic), incubated for 48 h at 37°C, and then transferred for a second time to LSW- agar. After an 24 r additional 48-h incubation (total of 96 h without selective I.-,J 1<- pressure), the bank was transferred back to LSW- agar VI r. containing chloramphenicol. Of the 13,052 colonies, only 16 r._ o o o -1- r-i P- \C \c (0.12%) failed to grow following passage on nonselective o e oc "-4 (% e# j-t CN -- W- "- I-~ 1- 7- medium, suggesting that the Tn5 element was stably inher- cq C\) ( (NJCM "llC\)CIQe l ited in these bacteria. To provide further information on m n *N v insertional stability, the remaining 13,036 stable TnS-carry- "GR . = P ing mutants were screened for the presence of plasmid- U- encoded ampicillin resistance. The pUT suicide plasmid 21.2 -harbors an ampicillin resistance gene, so any ampicillin- resistant colonies might be the result of a replicating plasmid Un in P. mirabilis. Approximately 35% of the colonies (4,542 of qw a bank of 13,036 mutants) were found to be resistant to 5.2_ ampicillin (Ap). Analysis of these Apr Cmr strains failed to .wl find any extrachromosomal plasmid, although Southern hy- 3.5 a.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~*- bridizations with plasmid pGP704 or pUT/mini-TnS Cm probes did confirm that plasmid sequences were present inserted into Proteus chromosomal DNA (data not shown). 2.0 These data are interpreted to suggest that the pUT plasmid carrying mini-Tn5 Cm did act as a suicide vector and failed 1 -4 O- to replicate in P. mirabilis; however, in one-third of the mutants the failure to replicate was not concomitant with 0.9 - loss of plasmid DNA. Apparently, in these strains the integration of the transposon was accompanied by integra- FIG. 1. Southern hybridization to chromosomal DNA from eight tion of part or all of the plasmid DNA as well. Since these Swr- Fla- mutants of P. mirabilis. Chromosomal DNA was pre- strains maintained both stable integration of the mini-TnS pared from each Swr- Fla- strain, as well as BB2000 (wild type), as Cm element without replication of the plasmid and were described in the text, and digested with PvuII. The resulting DNA phenotypically stable as well, the fragments were separated on a 1% agarose gel in 1x TAE (15) and integration of plasmid transferred to a charged nylon membrane (Nytran). DNA fragments DNA did not hamper further analyses. containing TnS insertions were identified by using the 3.4-kb HindlIl The frequency of auxotrophic mutants in the bank was fragment containing the chloramphenicol resistance gene from plas- investigated as one criterion for estimating the randomness mid pUT/mini-TnS Cm labeled with [Ot-32P]dCTP as a probe. A of mini-TnS Cm insertion in P. mirabilis. The 13,036 colonies single asymmetric PvuII site exists in the 3.4-kb HindlIl fragment, were transferred from master plates to minimal salt medium. resulting in two DNA fragments; this indicates a single insertion of Approximately 2.5% (326 of 13,036 Cmr mutants) did not the transposon. Size markers (in kilobases) are indicated to the left grow on the minimal salt medium and were judged to possess of the autoradiograph. an auxotrophic defect. These auxotrophic mutants were not extensively analyzed, but preliminary results indicated that they included those that required methionine for growth on fragment containing the chloramphenicol resistance gene minimal salt medium, in addition to arginine auxotrophs from plasmid pUT/mini-TnS Cm was labeled with leucine auxotrophs (data not shown). The frequency of [a-32P]dCTP (New England Nuclear) and used as a probe for Proteus auxotrophs produced with mini-TnS Cm agrees with the transposon. DNA fragments hybridizing to the TnS data for auxotroph production in other bacteria (4) and probe were visualized after autoradiography. As seen in Fig. suggests that insertion of the transposon did not occur at 1, for each of the eight Swr- Fla- strains, the 3.4-kb HindIll localized hot spots on the Proteus chromosome. fragment hybridized to two unique DNA fragments. This is The randomness of insertion was further analyzed by expected if there is a single transposon insertion at a unique Southern hybridizations in which the 3.4-kb HindlIl frag- site on the chromosome in each of these strains. Although ment from plasmid pUT/mini-Tn5 Cm was used as a probe to the site of transposition in five of the eight strains examined locate the transposon on DNA fragments produced by in Fig. 1 is different, comparison of DNA fragment sizes digesting chromosomal DNA from mutants defective in from three of the mutants (BB2030, BB2196, and BB2199) wild-type swarming motility (Swr-) and flagellin synthesis suggests that the insertion points are identical. Further (Fla-) with PvuII (Fig. 1). Since there is a single, asymmet- analysis of these chromosomes digested with HinclI con- ric PvuII site in the transposon (8), digestion of DNA from firmed that the transposon had inserted at unique sites within chloramphenicol-resistant mutants should yield two unique a 0.1-kb region on the Proteus genome (data not shown). bands if mutagenesis was random. Preparation of plasmid These data suggest that the mutations in BB2030, BB2196, and chromosomal DNA, radioactive labeling of plasmid and BB2199 may be in the same gene. DNA, and the conditions used for the transfer of DNA Significance. Very little is known about the frequency and fragments to membranes and hybridizations have been de- stability of transposition of TnS or other transposons in scribed previously (4, 15, 17). Chromosomal DNA from P. Proteus species. A derivative of mini-Mu (10) has been mirabilis Swr- Fla- mutants was isolated and enzymatically previously used to mutate and clone genes from P. mirabilis. cleaved with PvuII. The resulting DNA fragments were In that study, mutations in different Proteus genes, e.g., met, separated on a 1% agarose gel and transferred to Nytran trp, crp, and proC, were isolated at different frequencies. membrane (Schleicher & Schuell). To identify transposon The difference in obtaining the various mutants was believed insertion sites on the chromosomal DNA, the 3.4-kb HindlIl to reflect the DNA insertion specificity (hot spots of inser- 6292 NOTES J. BACTERIOL. tion) of phage Mu (10, 11). In the present study, we initially observed; therefore, transposition was not limited to a few attempted to use mini-Mu as well as mini-TnJO (19) trans- sites. To provide further evidence of the randomness of posons in P. mirabilis. We found that both transposons were mutagenesis, the DNA from several strains possessing iden- unsuitable for use in P. mirabilis, principally because these tical phenotypes was isolated and hybridized to a chloram- elements appeared to have a low frequency of transposition phenicol resistance gene probe (Fig. 1). Analysis of the in P. mirabilis and also because of insertional hot spots on genomic fragments hybridizing to this probe suggests that the Proteus chromosome (data not shown). In contrast to the transposon had inserted at different loci in these pheno- mini-Mu and mini-TnJO, the current study proves that mini- typically similar strains. We did not perform linkage analysis TnS derivatives are very successful in mutagenizing P. on the entire bank of mutants generated from the present mirabilis. The estimated frequency of transposition, al- study, so there is no way of knowing from our data how though not directly measured in our work, was sufficiently closely related the Tn5 inserts are in these mutants or what high (ca. 4.5 x 10-6 per recipient cell) to allow for acquisi- the percentage of siblings is in the population. However, tion of a bank of more than 13,000 mutants used in the solely on the basis of size, the DNA from three of these present study. Several factors probably influence the effi- Swr- Fla- mutants produced similar hybridization patterns ciency of TnS mutagenesis in P. mirabilis. These could (Fig. 1, BB2030, BB2196, and BB2199), suggesting that the include the proportion of Proteus bacteria receptive to transposon inserted within close proximity, but not at an conjugal mating, the presence of enzymes that restrict or identical site, in these genomes. modify incoming foreign DNA introduced into the recipient The development of transposon mutagenesis techniques bacteria, the capability of the vector transferring TnS to for use in P. mirabilis provides new ways to explore the "suicide" in the Proteus host, and the intrinsic activity of regulation of swarming motility and swarmer cell differenti- ISSOR-encoded transposase in P. mirabilis. The minimal ation. We have used this technique to construct a set of effective concentration of chloramphenicol (150 pg/ml) for mutants defective in swarming (3) and are currently analyz- the P. mirabilis strain used in this study was more than 300% ing a bank of Swr- mutants in P. mirabilis produced by of that used in E. coli, so the ability to express the antibiotic transposition of Tn5-lacZJ. Analysis of the transcriptional resistance phenotype encoded by the transposon may be regulation of swr genes will be the subject of future reports. strain dependent. The utility of transposon mutagenesis for a given Proteus strain will depend on the presence of barriers This research was supported by Public Health Service grant such as those mentioned above. AI-27107 from the National Institute of Allergy and Infectious The insertion of TnS in Proteus chromosomal DNA ap- Diseases of the National Institutes of Health. 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