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Site-directed insertion and insertion–deletion mutations in the Escherichia coli chromosome simplif


Genetic Analysis: Biomolecular Engineering 15 (1999) 239 – 244 www.elsevier.com/locate/gat

Site-directed insertion and insertion–deletion mutations in the Escherichia coli chromosome simpli?ed
Brian Smith-White *
Biochemistry Department, Michigan State Uni6ersity, East Lansing, MI 48824 -1319, USA

Abstract A procedure to produce an exact chromosomal replica of an insertion or insertion – deletion mutation produced in vitro in a plasmid with a ColE 1 origin of replication is presented. This procedure uses a previously described property of recD mutations (Biek DP, Cohen SN. J. Bacteriol. 1986;167:594–603) and is limited by (1) the compatibility of the new mutation with recD; and (2) the presence of some Escherichia coli DNA ?anking the mutation. ? 1999 Published by Elsevier Science B.V. All rights reserved.
Keywords: Gene conversion; recD

The current procedures for introducing those mutations created in vitro into the Escherichia coli chromosome use particular mutations or conditions to direct nucleic acid metabolism to capture part of an unstable, and thus transient, genetic entity. The initial reports of moving mutations from an episome to the chromosome use a host that is polA- and exploit the dependence of ColE1 replicons upon PolA [1,2]. The limitations of this protocol can be circumvented with a host that is recB, recC, sbcB and linear DNA [3,4]. The physiological frailty of this genetic background [5] can be overcome by using a host that is solely recD [6] and achieve the same results. Two recent methods create a merodiploid, coerce integration of all or part of the episome into the chromosome and then recover the mutation as a consequence of resolution of the direct repeat. One method takes advantage of the consequence of two phage f1 origins in the same replicon [7].The other method utilizes a temperature-sensitive origin of replication to allow recovery of isolates with the episome in the chromosome [8]. The linear DNA protocols suffer from a compound problem. First, success depends upon two probabilistic events: (i) uptake of the transforming DNA by the competent cell; and (ii) alteration of the chromosome allele. Second, the two reasons for failure (i) insuf?cient numbers; and (ii) lethality associated with the mutation;
* Tel.: +1-517-3533247; fax: + 1-517-3539334. E-mail address: smithwhi@pilot.msu.edu (B. Smith-White)

are experimentally indistinguishable. The two merodiploid methods each depend upon a unique allele exchange vector-necessitating additional recombinant DNA manipulations. The method of [7] requires the target strain be male and suppressor-free. This requirement can be a great, sometimes insurmountable, barrier when examining the effect of combining newly isolated loci or new alleles of already known loci with the mutation that is created in vitro. The method of [8] is less constrained in that there are no requirements upon the target strain. Both of the merodiploid protocols allow the introduction of both potentially polar mutations and presumptively nonpolar mutations into the E. coli chromosome in a single step. However determining the presence of the mutation after selecting against the presence of the plasmid can be problematic. In the absence of a readily measured phenotype, determining the presence of the mutation in a strain can be expensive (in time or money or both). This report describes a method to introduce a potentially polar mutation into the E. coli chromosome which utilizes any ColE1-based replicon and a previously published property of recD [9]. Because of a dominant marker associated with the mutation, the allele can be transferred directly into any strain capable of being a recipient in general transduction. Fig. 1 schematically depicts the protocol. In this paper we show that the protocol produces an exact copy of the plasmid allele in the chromosome. Through the use of a previously described cassette which allows both positive and negative selection [10], presumptively

1050-3862/99/$ - see front matter ? 1999 Published by Elsevier Science B.V. All rights reserved. PII: S 1 0 5 0 - 3 8 6 2 ( 9 9 ) 0 0 0 3 1 - 5

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nonpolar mutations could be introduced into the E. coli chromosome by the marker exchange-eviction mutagenesis procedure [10]. Bacteria were grown in nutrient medium {1% (w/v) of tryptone, 0.5% (w/v) of yeast extract and 1% (w/v) of NaCl with the pH adjusted to 7.3 with KOH (a slight modi?cation of [11])} or NBYE medium {0.8% (w/v) DIFCO Nutrient Broth, 0.4% (w/v) yeast extract and 0.4% (w/v) NaCl with the pH adjusted to 7.4 with KOH}. Both of these media were solidi?ed with agar at 1.5% (w/v) as needed. Glycogen accumulation in bacteria was qualitatively evaluated by I2-vapor staining as previously described [12] with the modi?cation that cells were arranged as 48 groups of siblings in a grid from a commercially available multiple well dish [13] instead of streaks of colonies arising from individual cells. Within each grid there is a glycogen-positive control, MG 1655 or BW 853, and a glycogen-negative control, G6MD3. To compensate for the asd defect in

G6MD3, sterile 5 mg/ml diaminopimelic acid was added after autoclaving to bring the concentration to 50 mg/ml in all media. Ampicillin was used at 100 mg/ml; kanamycin was used at 50 mg/ml; chloramphenicol was used at 50 mg/ml. Table 1 presents the bacterial strains, and their genotypes, employed in this study. All allele numbers are on record with the Coli Genetic Stock Center. MG 1655 (BJW 235) is from Mark Guyer [14]. DH5 (BJW 312) was purchased from BRL. BW 853 (BJW 466) is from Bernard Weiss. G6MD3 (BJW 524) is from Maxine Schwartz [15]. All have been maintained in the lab. Transformation of bacteria follows the protocol of [16] with the use of sterile 1.5 ml conical centrifuge tubes instead of Falcon 2059 tubes. P1 CMcir100 [17] was the gift from S. Adhya via N. Rao. Preparation of lysogens and induction of these lysogens to produce transducing stocks are described elsewhere [17]. Selection of transductants is performed in the presence of 10 mM citrate

Fig. 1. A schematic of the protocol to exchange a plasmid allele into the chromosome.

B. Smith-White / Genetic Analysis: Biomolecular Engineering 15 (1999) 239–244 Table 1 Bacterial strainsa Strain number BJW 235 BJW 312 BJW BJW BJW BJW BJW BJW 466 467 468 469 470 524 Source MG 1655 DH5 BW 853 This work This work This work This work G6MD3 Genotype

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supE44, gyrA96, recA1, relA1, endA1, hsdS17, thi-1, deoR recD::mini Tn10 glg-200::npt, recD::mini Tn 10 glg-201::npt, recD::mini Tn10 glg-200::npt glg-201::npt F+, D(asd-glpD)

a glg-200::npt is a 0.6 kbp deletion from the Cla 1 site (250 bp 5% of the translation-initiation codon of glgC) to the BstE2 site (360 bp 3% of the translation-initiation codon of glgC) with a 1.5 kbp DNA fragment encoding neomycin phosphotransferase from Tn5 inserted such that the direction of transcription of the insert and the ?anking DNA is identical. glg-201::npt is the same as glg-200::npt except the inserted DNA fragment is in the opposite orientation.

to eliminate secondary reinfection of growing cells by the progeny of the P1 virions in the transducing stock. The plasmids utilized or created during this work are described in the text. The plasmid mnemonic ‘pBJ’ is on record with the Plasmid Reference Center [18]. Recombinant DNA procedures followed the instructions of the enzyme vendors with one exception. Blunt ligation was performed as described elsewhere [19]. Plasmids were maintained and propagated in the DH5 background. Chemical amounts of plasmid DNA were puri?ed using the QIAGEN maxi kit from 500 ml of overnight stationary phase cells grown in NBYE supplemented with 100 mg/ml ampicillin, 30 mM potassium phosphate (pH 7.2), 0.5% (v/v) glycerol and 0.01% (v/v) antifoam A. The dominant marker tag for the alleles was the neomycin phosphotransferaseresistance (npt) portion of Tn5 [20] which had been captured in pKC7 [21]. The tag was prepared by digesting pKC7 with Sal 1 and Hind 3, treating the bulk digestion products with Klenow fragment to ?ll-in the 5% overhang at the end of each DNA fragment and isolating the 1.5 kbp fragment from agarose following electrophoretic resolution of the three fragments. The mutations were created using restriction sites in pOP 12 [22]. The allele construction was performed upon subcloned portions of pOP 12 to facilitate cleavage at a particular site of multiple sites present in the entirety of pOP 12. pglgC is described previously [23]. pBJ 302 is the 3.7 kbp Hpa 1 fragment of pOP 12 which contains all of glgC, all of glgA, and a 5% portion of glgP. pBJ 303 is the 3.7 kbp EcoR 5 fragment of pOP 12 which contains a 3% portion of asd, all of glgB and a 5% portion of glgX The identity of pBJ 302 and pBJ 303 was con?rmed by (1) correlating restriction maps of the resulting plasmids with that expected from the nucleic acid sequence; and (2) show-

ing that the plasmids conferred increased enzyme activity speci?c for the subcloned fragment, respectively, ADPglucose pyrophosphorylase and glycogen synthase for pBJ 302 and branching enzyme for pBJ 303 (data not presented). Recovery of plasmid borne alleles was achieved by examining partially puri?ed nucleic acid for various anticipated fragments resulting from digestion with restriction endonucleases. The allele was characterized by the restriction map which the allele confers to the piece of E. coli DNA following puri?cation of chemical amounts of the plasmid carrying the allele. This protocol involved three steps once the chimeric plasmid with a desired mutation is produced. The procedure is schematically depicted in Fig. 1. First, the merodiploid condition was achieved in BJW 466. Second, the population was allowed to proliferate for a determined number of generations in the absence of any selection. This allows those cells that fail to partition the plasmid the opportunity to grow. Some of these plasmid-free cells will have changed the chromosomal allele to the plasmid allele prior to losing the plasmid. Third, the population was treated to selectively remove those cells which lack the dominant marker associated with the allele. There were three possible groups of cells in the population surviving this protocol: 1. those having retained the episome but not converting the chromosomal allele 2. those having retained the episome after either homogenizing both alleles to the allele initially present only on the plasmid or exchanging the plasmid and the chromosomal alleles 3. those having lost the episome after converting the chromosomal allele. Those members of the third group were identi?ed by screening siblings of individual cells for the presence or absence of the dominant marker associated with the vector portion of the chimeric plasmid. Distinguishing between the ?rst and second group requires a readily measured phenotype associated with the mutation in the chromosome. As depicted in Fig. 1, two serial passages at 1:30 each time yields the three classes in roughly equal numbers (data not shown). pglgC was completely digested with Cla 1 and BstE 2. The location of these sites relative to the translationinitiation codon of glgC is in the footnote in Table 1 and graphically presented in Fig. 2. Treatment of the digestion products with Klenow fragment produced blunt ended fragments. The 3.6 kbp fragment was puri?ed from agarose following electrophoretic resolution of the digestion products. Following blunt ligation to introduce the DNA fragment encoding kanamycinresistance, both orientations of this dominant marker were recovered: pBJ 311 and pBJ 312. Fig. 2A shows the results of analytical digestion of puri?ed material. Fig. 2B is a cartoon of the structure of the starting

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material and the two products. Digestion with BamH 1 and Bgl 2 simultaneously or Pst 1 utilize a site in the vector and an asymmetrically positioned site in the DNA encoding kanamycin-resistance. Digestion with Pst 1 also shows that there is a single copy of the inserted DNA because none of the possible fragments anticipated from either direct duplication or inverted duplication are observed. Digestion with EcoR 1 or Hind 3 con?rms that there is a single copy of the inserted DNA. The mutation, when present in the chromosome, is anticipated to render a cell unable to deposit glycogen. Following allele exchange with three 100-fold passages without selection, 24 of 24 kanamycin resistant colonies arising from an orientation of the inserted dominant marker had lost the episome (ampicillin-sensitive) and were unable to deposit glycogen.

One from each orientation was saved, BJW 467 and BJW 468. There are at least two possible regions of the chromosome where gene disruption might adversely affect glycogen deposition — the structural genes for the anabolic enzymes at 75 min and glgQ (over 2 min removed from 75 min) [24]. The other locus known to in?uence glycogen deposition, csrA, can be ruled out as a target since previous work has shown that disruption of csrA leads to hyperdeposition rather hypodeposition [25]. The location of the mutation in the chromosome was ascertained by transductional linkage to known loci. Transducing stocks for both alleles were prepared as described above. P1 (467) and P1 (468) were used to transduce G6MD3 to either kanamycin-resistance or diaminopimelic acid prototrophy. For both alleles

Fig. 2. (A) Photograph of fragments resulting from analytic digestions of the plasmids pBJ 311 and pBJ 312 after electrophoretic resolution in agarose. The DNA is rendered visible by ethidium bromide staining. The two progeny plasmids ?ank the parental plasmid in each of four digestions. (B) The cartoon schematic of the structures of the three different plasmids. The position of restriction endonuclease sites used either in construction or diagnostic characterization of the two alleles are indicated by a vertical line that is labeled.

B. Smith-White / Genetic Analysis: Biomolecular Engineering 15 (1999) 239–244

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Fig. 3. (A) Autoradiogram of the blot of E. coli genomic DNA fragments probed with glgC material. (B) Autoradiogram of the blot of E. coli genomic DNA fragments probed with the Tn5 material from pKC7 [21].

kanamycin-resistance and the inability to deposit glycogen were linked 100%. For both alleles diaminopimelic acid prototrophy and the inability to deposit glycogen were linked 100%. The lack of recombination between asd and this mutation suggest greater linkage than previously reported in mapping of the glg loci [26,27]. However, this is exactly the expected result when both the selected marker and the scored marker are within the endpoints of a deletion in the recipient chromosome. The same transducing stocks were used to transduce BJW 235 to kanamycin-resistance. The tight linkage between kanamycin-resistance and glycogen deposition seen with G6MD3 as the recipient was con?rmed with 100% linkage between the two phenotypes. Also this data in BJW 235 rules out the possibility that tight linkage between the selected marker and glycogen deposition in G6MD3 was an inadvertent consequence of the recipient being a deletion for this region. One strain from each orientation was saved, BJW 469 and BJW 470. The molecular details of the consequences of the allele exchange were ascertained by elucidating the re-

striction map using hybridization techniques. Genomic DNA was prepared from BJW 235, BJW 469 and BJW 470 by a previously published procedure [28]. The new allele is expected to remove a BamH 1 site, have an EcoR 1 fragment with a 1.5? 0.6 kbp= +0.9 kbp alteration of mobility and have 2 Pst 1 sites and 1 Sph 1 site asymmetrically introduced by the insertion–deletion event. Fig. 3A shows the result of probing with the wild-type glgC nucleic acid. The BamH 1 site is removed and the EcoR 1 fragment is appropriately affected. The Pst 1 and Sph 1 digestions show that glg-200::npt and glg-201::npt are different. Fig. 3B shows the results of probing with the DNA fragment encoding kanamycin-resistance. Only the strains with the mutant alleles have hybridizing material. The EcoR 1 and Pst 1 digestions show that there is a single copy of this DNA in the genome; con?rming the transduction data and the plasmid structure data. The Sph 1 digestion shows that the genomic alleles have the dominant marker in opposite orientations. These data taken together lead to the conclusion that the genomic allele has been changed to an exact replica of the plasmid allele.

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The process which changes the original chromosomal allele to the episomal allele appears to absolutely require E. coli DNA to be ?anking the mutation. pBJ 302 has two Pst 1 sites. One is in the multicloning site of the vector pT7T3-U, less than 30 nucleotides from one end of the E. coli DNA. The other is 1 kbp 5% of the translation-initiation codon of the glgA portion of the E. coli DNA. pBJ 302 was digested to completion with Pst 1. The resulting 3% overhang was removed by treatment of the bulk digestion products with Klenow fragment. The 5.8 kbp fragment was puri?ed from agarose following electrophoretic resolution of the digestion products. The DNA fragment encoding kanamycin-resistance was introduced by blunt ligation resulting in pBJ 320. This plasmid has 2.8 kbp of E. coli DNA adjacent to a 1.5 kbp fragment of DNA encoding kanamycin-resistance (data not shown). Since the mutation removes over half of the coding region of the structural gene for glycogen synthase, a cell with this mutation in the chromosome was anticipated to be unable to deposit glycogen. Following the allele exchange protocol, scoring 24 of the resulting isolates for the presence or absence of the plasmid and ability to deposit glycogen showed that all strains retained the episome and remained able to deposit glycogen. This can not be due to unanticipated lethality of the mutation. A simple insertion at 400 bp 5% of the Pst 1 site (and within the E. coli material deleted in pBJ 320) is viable and eliminates glycogen synthase activity concomitantly with glycogen deposition (data not presented). We conclude that the absence of DNA homologous to E. coli DNA on one side of the episomal allele is an insurmountable barrier to the process of converting the genomic allele. pBJ 303 was digested to completion with Nru 1. The 6 kbp fragment was puri?ed from agarose following electrophoretic resolution of the ?ve fragments. pBJ 323 was recovered following blunt ligation with the DNA fragment encoding kanamycin-resistance. This plasmid has 2.0 kbp of E. coli DNA replaced by two copies of the 1.5 kbp piece of DNA encoding kanamycin-resistance (data not presented). The insertion is ?anked by 50 bp and 1.7 kbp of E. coli DNA. Examination of the kanamycin-resistant colonies following the allele exchange protocol showed that 0/8 were ampicillin-resistant and able to deposit glycogen, 4/8 were ampicillin-resistant and unable to deposit glycogen, 0/8 were ampicillin-sensitive and able to deposit glycogen and 4/8 were ampicillin-sensitive and unable to deposit glycogen. We conclude that 50 nucleotides is suf?cient to satisfy the requirement for one of the ?anking E. coli DNA elements in the process to change the genomic allele to the episomal allele.
.

Acknowledgements I would like to acknowledge ?nancial support from the Department of Energy grant DE-FG02-93ER20121 to Jack Preiss and encouragement by Jack Preiss to complete this work. I would like to thank one reviewer of a previously submitted manuscript for cogent, useful comments which were instrumental in preparing this manuscript.

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