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Applied and Environmental Microbiology, June 2008, p. 3419-3425, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.00476-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

In Vitro Mutagenesis of Bacillus subtilis by Using a Modified Tn7 Transposon with an Outward-Facing Inducible Promoter{triangledown}

Christophe Bordi,{dagger} Bronwyn G. Butcher, Qiaojuan Shi, Anna-Barbara Hachmann, Joseph E. Peters, and John D. Helmann*

Department of Microbiology, Cornell University, Ithaca, New York 14853-8101

Received 27 February 2008/ Accepted 4 April 2008


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ABSTRACT
 
A Tn7 donor plasmid, pTn7SX, was constructed for use with the model gram-positive bacterium Bacillus subtilis. This new mini-Tn7, mTn7SX, contains a spectinomycin resistance cassette and an outward-facing, xylose-inducible promoter, thereby allowing for the regulated expression of genes downstream of the transposon. We demonstrate that mTn7SX inserts are obtained at a high frequency and occur randomly throughout the B. subtilis genome. The utility of this system was demonstrated by the selection of mutants with increased resistance to the antibiotic fosfomycin or duramycin.


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INTRODUCTION
 
Transposons are mobile genetic elements that can move from one site to another in the genome with the aid of a recombinase called a transposase. They have been used as tools for genetic analysis in many biological systems, and engineered transposons have been developed that incorporate a variety of useful features. Numerous transposon delivery systems have been developed for Escherichia coli and other gram-negative bacteria, and several are commercially available. However, in many cases these incorporate selectable markers that are not conducive to their use in gram-positive bacteria.

Transposons have played a major role in genetic analysis in Bacillus subtilis, which serves as a model gram-positive bacterium. The first to be developed was Tn917, a member of the Tn3 family of replicative insertion elements. Tn917 was adapted by the incorporation of a promoterless lacZ gene, and the resulting Tn917lac transposon was used to generate large numbers of reporter fusions (51). Analysis of these fusions played a major role in early molecular genetic studies of sporulation and other post-exponential-phase processes in B. subtilis. Despite their wide use, Tn917 has significant shortfalls. For example, Tn917 has a preference for certain genes (40, 44, 49, 52), an observation likely explained by its preferential insertion near the terminus region in several low-G+C, gram-positive bacteria (18).

More recently, the Tn10 and mariner transposons were adapted for in vivo transposition in this organism. Tn10, a transposon isolated from E. coli, was adapted for B. subtilis by fusion of the transposase gene to expression signals appropriate for this bacterium (36). In these systems, the transposon and the specific transposase are cloned on a plasmid with a temperature-sensitive origin of replication for gram-positive hosts. Transposition events are selected by growing the strain containing this plasmid at nonpermissive temperatures (at which the plasmid will not replicate) and selecting for the antibiotic resistance marker found on the transposon.

We and others have successfully used a mini-Tn10 delivery plasmid, pIC333, in many genetic screens. Tn10 has not been analyzed for any "regional biases," but it is known to have a strong preference for a 6-bp target sequence (20). Therefore, while Tn10 insertion events are distributed throughout the genome, the number of potential insertion sites is reduced. In our studies, we have often found only one or two sites of insertion per gene, and in other cases, we failed to recover insertions in expected targets (5, 16). Moreover, the pIC333 plasmid is unstable in both E. coli and B. subtilis (T. Msadek, personal communication) and care needs to be taken when isolating and working with this plasmid. Recently, an improved and stabilized mini-Tn10 system was constructed for use in B. anthracis and was shown to be fully functional in B. subtilis (50).

In an attempt to develop a more efficient and random system, the mariner transposable element Himar1 was adapted for in vivo transposition in B. subtilis (26). The TnYLB-1 element (consisting of Himar-recognized inverted terminal repeats flanking a Kanr cassette) and the Himar1 transposase (modified for expression in B. subtilis) were cloned on a delivery vector containing a temperature-sensitive origin of replication. Transposon systems using the mariner element have been applied to a number of species and did not display regional hot spots. Insertions are highly random, since they occur in the small target sequence TA (2, 8, 26, 41, 47).

While these in vivo systems have proven successful, transposition can be biased by cell processes. Many transposons are inhibited from transposition into actively transcribed regions (9, 11, 13, 29). Some elements, like Tn916, show biases for noncoding DNA (32). Presumably, the in vivo architecture of the chromosome and DNA-binding proteins could also inhibit transposition into some regions. Therefore, in vitro transposition systems have been developed for use in E. coli and several are commercially available (e.g., the EZ-Tn5 and HyperMu systems from Epicenter and the GPS-1 genome-priming system from New England BioLabs). These systems use purified transposase and a transposon donor to mutagenize a DNA target of choice (plasmid, cosmid, or chromosomal DNA).

Tn7 transposition requires the TnsA and TnsB proteins (forming the transposase), which recognize and cleave the left and right ends of the Tn7 transposon (34). TnsC is a AAA protein (21) that regulates the transposase, usually by interaction with one of two target site selecting proteins, TnsD or TnsE. Using a mutant TnsC protein (46) that no longer requires TnsD or TnsE results in random integration into any DNA target (1, 4, 10, 19, 24). Transposons, like Tn7, engineered for very low target selectivity have been used in many organisms (1, 4, 10, 19, 24). In addition to randomness, Tn7 also displays "target site immunity" in vivo and in vitro, which greatly inhibits transposition proximal to a copy of the Tn7 element in a DNA (12, 45, 46). By preventing in vitro transposition events from occurring close to one another, the Tn7 system minimizes the generation of linked insertions that would otherwise be introduced simultaneously into the recipient strain.

Here we report a Tn7-based in vitro system adapted for use in B. subtilis by the introduction of a spectinomycin (SPEC) resistance cassette. In addition, we have altered Tn7 to contain an outward-facing, xylose-inducible promoter (PxylA). PxylA is routinely used for the regulated expression of proteins (e.g., with the integrational plasmids pXT [14] and pSWEET [3]) and is induced by xylose and repressed by glucose under the control of the chromosomally encoded XylR repressor. The resulting mTn7SX transposon inserts itself randomly into the genome and serves as a useful tool for both the insertional disruption of genes and the regulated expression of downstream genes.


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MATERIALS AND METHODS
 
Bacterial strains and culture media.
All of the B. subtilis and E. coli strains and oligonucleotide primers used in this study are listed in Table 1. Bacteria were grown in liquid Luria-Bertani medium (LB) at 37°C with vigorous shaking or on solid medium containing 1.5% Bacto agar (Difco). Screening for auxotrophs and pJPM122::mTn7SX insertions was performed on solid minimal medium (MM) (17) supplemented with 10 µM FeSO4 (note that this medium contains tryptophan as the strains used are tryptophan auxotrophs). Sporulation proficiency was assessed on Difco sporulation medium (22). Antibiotics were added to the growth medium at the following concentrations: SPEC, 100 µg/ml; neomycin (NEO), 8 µg/ml; kanamycin (KAN), 10 µg/ml; tetracycline, 20 µg/ml; chloramphenicol (CM), 10 µg/ml. Where indicated, 2% xylose or 40 µg/ml X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside) was used. E. coli cells were made competent by treatment with ice-cold 0.1 M CaCl2 (33). Transformation of B. subtilis was carried out as described previously (22).


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  		TABLE 1. Strains and primers used in this study

Construction of pTn7SX.
The xylA promoter (PxylA) was amplified from pXT (14) with Pfu polymerase (Stratagene) and primers 2514 (containing a BssHII site; Table 1) and 2516 (SpeI) and purified through the QIAquick spin column (Qiagen). This PxylA PCR product was digested sequentially with BssHII and SpeI (New England BioLabs) and cloned into pGPS2.1aph3± (a derivative of pGPS2.1 [catalog no. N7121S from New England BioLabs], where the cat gene has been replaced with the aph3 [Kanr] gene; J. C. Huguet and J. Peters, unpublished data) digested with the same enzymes. After transformation into E. coli BW23474 (pir+), constructs with PxylA in the correct orientation were identified by PCR with 1.1x Thermo-Start PCR Master Mix (ABgene) and primers 2517 (complementary to the Tn7 right region) and 2516. The aph3 gene was then replaced with the spec gene as follows. The spec gene was amplified from pXT with 1.1x Thermo-Start PCR Master Mix (ABgene) and primers 2511 (containing a BssHII site) and 2510 (NotI), and the product was purified through the QIAquick spin column (Qiagen). This spec PCR product and the plasmid were then sequentially digested with BssHII and NotI, ligated with T4 ligase (Invitrogen), and transformed into E. coli BW23474, and clones were selected on LB plus SPEC. Correct clones were confirmed by PCR with primers 2510 and 2516. The resulting plasmid, pTn7SX, was purified with the QIAprep miniprep kit (Qiagen) and eluted with 50 µl sterile Milli-Q water. Sequence information for this plasmid is available at http://www.micro.cornell.edu/cals/micro/research/labs/helmann-lab/supplements.cfm.

Purification of Tns proteins.
TnsA and TnsB were purified as previously described (31, 42). The TnsCA225V mutation was introduced into TnsC expression vector pRS550 (a kind gift from Nancy Craig), a pCYB1 (New England BioLabs)-based vector, for the expression of a TnsC-intein-chitin-binding domain fusion protein by replacing the SacI-NheI fragment of pRS550 with a 904-bp SacI-NheI fragment from plasmid pCW15 which carried the TnsABCA225V mutation (46), creating pCYB-TnsC*.

TnsC* was purified with a chitin column as recommended by the manufacturer (New England BioLabs), but the buffer contained 25 mM HEPES (pH 7.5), 1 M NaCl, 1 mM ATP, 10 mM MgCl2, 0.1 mM EDTA, 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), and 10% glycerol. TnsC* was separated from the intein-chitin-binding domain tag by intein-mediated self-cleavage and stored in 25 mM HEPES (pH 7.5)-1 M NaCl-2.5 mM dithiothreitol-1 mM ATP-10 mM MgCl2-0.1 mM EDTA-10 mM CHAPS-10% glycerol at –80°C. The TnsABC* proteins allowed 10 to 15% conversion of a target plasmid to simple insert products in a 40-min reaction (data not shown).

In vitro transposition.
Chromosomal DNA was isolated from a 5-ml overnight culture of B. subtilis CU1065 with the GFX Genomic Blood DNA purification kit (Amersham Biosciences) as described by the manufacturer, with the following changes. Cells were resuspended in 200 µl lysozyme buffer, the optional RNase A treatment was included, and the DNA was eluted in 50 µl sterile Milli-Q water. The in vitro transposition reactions were performed essentially as previously described (4), with the indicated modifications. The 100-µl in vitro reaction mixture contained (final concentrations) 26 mM HEPES, 4.4 mM Tris (pH 7.6), 50 µg/ml bovine serum albumin, 2 mM ATP (pH 7.0), 2.1 mM dithiothreitol, 0.05 mM EDTA, 0.1 mM MgCl2, 0.1 mM CHAPS, 17.3 mM NaCl, 16.5 mM KCl, 15 mM magnesium acetate, 1.33% glycerol, 39 ng TnsA (12.5 nM), 25 ng TnsB (3 nM), and 50 ng TnsCA225V (8 nM). The reaction mixtures contained 100 ng mini-Tn7-containing donor DNA and 600 ng target chromosomal DNA. All of the reaction mixture components except TnsA, TnsB, magnesium acetate, and the donor DNA were incubated for 20 min at 30°C. The final reaction mixture components were then added, and the reaction was allowed to proceed for an additional 45 min at 30°C. The reaction was stopped by phenol-chloroform extraction and ethanol precipitation, and the DNA was resuspended in 15 µl H2O.

The GAP repair procedure was performed as described for the GPS-M Mutagenesis System (New England BioLabs catalog no. E7101S) with the following modifications. The reaction mixture was incubated for 30 min at 37°C, and the ligation was performed at 16°C for 24 h. This reaction mixture was directly transformed into the B. subtilis strain of choice without further precipitation or digestion. The number of transposants in the library was estimated by plating 1% of the reaction mixture on LB-SPEC plates, and the remaining transformation can then be plated on the selection media of choice. Alternatively, the transposon library may be stored by plating the reaction mixture in 100-µl aliquots on LB-SPEC-xylose plates, allowing growth overnight, and then washing these colonies off the plates with LB and storing them at –80°C in 15% glycerol. This library may be used to inoculate a culture either to prepare chromosomal DNA for transformation or to plate directly on a selective medium.

We used pTn7SX as the transposon donor with the GPS-M Mutagenesis System from New England BioLabs (catalog no. E7101S), with the following modifications. A 120-ng sample of target chromosomal DNA was used in each reaction mixture, and instead of heat inactivation, the reaction was stopped by phenol-chloroform extraction, followed by ethanol precipitation. Five reaction mixtures were pooled and resuspended in a total of 15 µl of H2O, and GAP repair was performed as described above.

Arbitrary PCR to map insertions.
The positions of the mTn7SX insertions were mapped by arbitrary PCR as described previously (35). All PCRs were performed with the 1.1x Thermo-Start PCR Master Mix (ABgene). A first-round PCR to amplify the junction between the transposon and the genome was performed with arbitrary primer 2509 (or 2505) and a primer specific to the transposon (2501). Arbitrary primer 2509 ends in ATGCA, a sequence that has a probability of being found every 1 kb throughout the B. subtilis genome, while 2505 has a different 3' sequence (GATCA). This PCR can be performed on 1 µl of purified chromosomal DNA, or the colony of interest may be resuspended in the PCR mixture. Reaction conditions were 94°C for 15 min; 6 cycles of 94°C for 30 s, 30°C for 45 s, and 72°C for 3 min; 30 cycles of 94°C for 30 s, 42°C for 45 s, and 72°C for 3 min; and a final extension at 72°C for 6 min. The PCR product was purified with the QIAquick spin column (Qiagen) eluted in 30 µl of distilled H2O.

To further amplify the desired product, a second-round PCR was performed with primers 2508 (matches the 5' tail of the random primer) and 2502 (complementary to PxylA within mTn7SX and downstream of primer 2501). As a template, a 1:5 dilution of the first-round PCR product was used. The reaction conditions were 94°C for 15 min; 35 cycles of 94°C for 30 s, 55°C for 40 s, and 72°C for 3 min; and a final extension at 72°C for 6 min. After purification of the PCR product, the mTn7SX::genome junction site was determined by sequencing with the 2502 primer at the Cornell University Life Sciences Core Laboratories Center.

mTn7SX transposition into pJPM122.
pJPM122 (43) was purified with the QIAprep miniprep kit (Qiagen). In vitro transposition was performed as described above, except that 400 ng of the target, pJPM122, and 100 ng of the donor, pTn7SX, were used. After gap repair, the plasmid was linearized with ScaI (New England BioLabs) and the entire reaction mixture was used to transform B. subtilis ZB703A. pJPM122 carries lacZ and pBR322 sequences that allow recombination through a double-crossover event into the SPβc2{Delta}2::Tn917::pSK10{Delta}6 prophage of B. subtilis ZB703A (43). Transformants containing mTn7SX were selected on MM containing SPEC. Thirty colonies were patched onto MM-SPEC-X-Gal with or without xylose, MM-SPEC-CM with or without xylose, and MM-SPEC with or without NEO. Insertion of mTn7SX in an orientation in which the PxylA promoter is driving the expression of cat and/or lacZ was identified by xylose-dependent growth on CM and/or blue color on the X-Gal plates, while insertion within the neo gene resulted in no growth on the NEO-containing plates.

Selection of fosfomycin- and duramycin-resistant mutants.
Fosfomycin-resistant (Fosr) mutants were selected by plating B. subtilis CU1065 transformed with the gap-repaired in vitro transposition reaction mixture on solid LB plates containing SPEC, 2% xylose, and 30 µg/ml fosfomycin. MICs for these resistant mutants and wild-type B. subtilis were determined by growth (optical density at 600 nm) after incubation overnight at 37°C in microtiter plates with LB containing 0, 10, 20, 30, 40, 50, 60, 70, 80, 100, 150, and 200 µg/ml fosfomycin and either 2% xylose or glucose. Insertions within the glpTQ operon were screened by PCR with primers 2461 and 2462 and 1.1x Thermo-Start PCR Master Mix (ABgene).

Duramycin-resistant (Durr) mutants were selected by plating the transformed B. subtilis CU1065 cells on solid LB containing SPEC and xylose. After incubation for 5 h at 37°C, these plates were overlaid with 5 ml of soft LB agar (0.7%) containing SPEC, xylose, and 6 µg/ml duramycin and incubated at 37°C overnight. Clones were checked for insertion of mTn7SX into the pssA operon by PCR with primers 2369 and 1916. MICs were determined as described above, with the following concentrations of duramycin: 0, 1, 1.5, 2, 5, 20, 30, 40, 50, 75, 100, 150, and 200 µg/ml.

Allelic replacement by long-flanking homology PCR.
The mTn7SX insertions in 10 transposants were replaced with a kan cassette by long-flanking homology PCR as previously described (30). Briefly, regions up- and downstream of the mTn7SX insertion site were amplified and joined with a kan cassette by PCR. The corresponding transposants were transformed with the joined PCR product, and transformants were selected on LB-KAN and tested for SPEC sensitivity. Kanr Specs colonies indicated that mTn7SX had been replaced with the kan cassette by double recombination and that no other mTn7SX insertion was present in the genome. Primers used for creation of the joined PCR products are available upon request.


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RESULTS AND DISCUSSION
 
Construction of pTn7SX.
We constructed a Tn7 transposon donor plasmid, pTn7SX, that contains a SPEC resistance cassette and an outward-facing, xylose-inducible promoter, PxylA (Fig. 1). In addition to screening for insertions that disrupt genes, this transposon also allows for upregulation of genes not expressed under the conditions of the experiment or of cryptic genes. B. subtilis PxylA and the spec gene were amplified from pXT and cloned into pGPS2.1aph3± (a derivative of pGPS2.1 in which the cat gene was replaced with aph3) to create pTn7SX (see Materials and Methods).


Figure 1
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  		FIG. 1. Physical map of the pTn7SX transposon donor plasmid. This plasmid is derived from the pGPS2.1 donor plasmid from New England BioLabs (catalog no. N7121S) and contains the origin of replication from the transmissible plasmid R6K (which depends on the {Pi} replication initiation protein, the product of the pir gene) and a tetracycline resistance gene. The transposon, flanked by the Tn7 left and right ends (Tn7L and Tn7R, respectively), contains a gram-positive SPEC resistance cassette and the outward-facing xylA promoter (PxylA).

We tested this new transposon by performing in vitro transposition into a plasmid carrying a NEO resistance gene and a promoterless cat-lacZ operon (pJPM122). After in vitro transposition, gap repair, and linearization, the plasmid was transformed into B. subtilis ZB703A (to allow for integration of the plasmid into the SPβ prophage carried in this strain). Thirty SPEC-resistant transformants were selected and screened for xylose-inducible upregulation of the cat and/or lacZ genes or disruption of the neo gene. Seven clones contained mTn7SX insertions that resulted in the expression of β-galactosidase in the presence of xylose, indicating that the transposon-encoded PxylA promoter was inserted upstream of the lacZ gene. Two of these insertions also exhibited xylose-inducible CM resistance due to insertion of the transposon upstream of the cat gene (thereby activating the cat-lacZ operon fusion). Another 17 clones were NEO sensitive as a result of the insertion of Tn7 into the neo gene. The remaining six clones presumably have Tn7 insertions either in the opposite orientation within the cat or lacZ gene or elsewhere in the plasmid. These data confirmed that the transposon is active and that the outward-facing PxylA promoter is functional and suggest random insertion throughout the target plasmid DNA.

Transposition into B. subtilis chromosomal DNA with mTn7SX.
In vitro transposition was carried out with chromosomal DNA isolated from B. subtilis CU1065. After gap repair, this mutagenized chromosomal DNA was used to transform B. subtilis CU1065 and strains carrying transposon insertions were selected on LB agar containing SPEC. We obtained an average of ~57,000 clones per transformation with one in vitro transposition reaction. The B. subtilis genome is about 4,215 kb in size. Thus, this corresponds statistically to one insertion for every 73 bp (or about 13 insertions per gene). Five hundred colonies were selected for further characterization. Of these, ~2.6% were sporulation deficient (as monitored on Difco sporulation medium) and ~1.8% were auxotrophs (did not grow on MM). The chromosomal DNA from 30 SPEC-resistant colonies was purified, and the positions of the mTn7SX insertions were mapped by arbitrary PCR and DNA sequencing (see Materials and Methods). These insertions were found throughout the genome (Fig. 2), further highlighting the random nature of transposition. Ten clones were selected, and the transposon was replaced with a kan gene by transformation with a PCR product containing homology to the regions flanking the transposon insertion and an intervening kan cassette. The resulting transformants were all KAN resistant and SPEC sensitive, indicating that there was not a second transposon within these genomes. Taken together, these data show that mutagenesis with this transposon occurs with high efficiency and the resulting insertion events are highly distributed.


Figure 2
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  		FIG. 2. (A) Random insertion of mTn7SX across the B. subtilis chromosome. The insertion sites of 30 transposants were determined by arbitrary PCR and DNA sequencing, and their locations are shown as gray circles. (B) Insertions resulting in fosfomycin resistance. The positions of 10 insertions within the glpTQ genes and 2 upstream of the fosB gene were determined. Each insertion was at a different position within these genes and is indicated by a vertical arrow. (C) Insertions resulting in duramycin resistance. The positions of 10 insertions within the pssA-ybfM-psd operon were determined and are indicated by black arrows. Open arrows represent the open reading frames, and the –10 and –35 promoter regions are shown as gray boxes.

While the above reactions were performed with proteins purified in our laboratory (see Materials and Methods), this mTn7SX donor plasmid can also be used with the commercially available GPS-M Mutagenesis System (New England BioLabs). In comparative experiments with the same amount of target chromosomal and donor plasmid DNAs, we found that the efficiency of transposition was similar (we obtained a bank size of 56,800 transposants with the commercial kit).

Isolation of fosfomycin-resistant mutants.
We used mTn7SX to select for resistance to fosfomycin, an antibiotic produced by Streptomyces species and used in the treatment of lower urinary tract infections (37). This antibiotic is also effective against methicillin-resistant and vancomycin-resistant strains of Staphylococcus aureus.

After transformation of CU1065 with the in vitro transposition reaction mixture, these cells were plated on LB plates containing SPEC, xylose, and 30 µg/ml fosfomycin. Thirty-seven fosfomycin-resistant colonies were isolated and patched to plates containing xylose and 50 µg/ml fosfomycin. All but two of these isolates were resistant to this higher concentration of fosfomycin. The position of the insertion was mapped in 10 of the clones able to grow on the higher fosfomycin concentration, and in each case the transposon was inserted at a different position within the glpTQ operon (Fig. 2B). We also mapped the insertions in the two clones that grew only on plates containing 30 µg/ml fosfomycin. In both cases, the transposon was inserted upstream of fosB with PxylA driving the expression of this gene (Fig. 2B). The remaining 25 clones also grew at the higher fosfomycin concentration, and PCR with primers specific to regions up- and downstream of glpTQ confirmed that all insertions were within this operon. Since some transposons were oriented such that they place the glpQ gene under xylose control, we tested the MICs for these strains in liquid media with and without 2% xylose (Table 2). In these insertions, fosfomycin resistance was not dependent on xylose, indicating that resistance is caused by disruption of either gene. On the other hand, the fosB promoter insertions were sensitive to fosfomycin in the absence of xylose. In this case, resistance is provided by overexpression of the fosB gene.


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  		TABLE 2. Fosfomycin-resistant mutants used in this study

GlpT is an ABC transporter that actively transports glycerol-3-phosphate to the cytoplasm (27), while the glpQ gene encodes a glycerophosphodiester phosphodiesterase that processes the glycerophophodiesters produced from membrane phospholipids into glycerol-3-phosphate and their respective alcohols (25). Fosfomycin enters the cell via the GlpT transporter, where it inactivates MurA, a cytoplasmic enzyme involved in the first step of peptidoglycan biosynthesis (27). It has long been known that mutations in glpT of E. coli result in increased fosfomycin resistance (28). In B. subtilis, the glpT locus was originally mapped by selection for Fosr mutants (28). No Fosr mutations have been mapped to glpQ. However, a glpQ mutant of Enterococcus faecalis was recently found to be more resistant to the class IIa bacteriocin divercin V41 (6). These authors propose that this mutation results in a change in the fatty acid and phospholipid composition of the membrane, therefore preventing the activity of this membrane-active antibiotic. This would not explain the involvement of GlpQ in resistance to fosfomycin, which acts on a target located in the cytoplasm. In B. subtilis, expression of the glp regulon is induced by the antiterminator GlpP in the presence of glycerol-3-phosphate (38). We speculate that disruption of glpQ results in lower levels of glpT expression, due to reduced levels of glycerol-3-phosphate, and therefore less transport of fosfomycin into the cells.

FosB encodes an enzyme that catalyzes the addition of L-cysteine to fosfomycin, rendering it inactive (37). In B. subtilis, the extracytoplasmic function {sigma} factor {sigma}W controls the expression of fosB (7). The transposon mutants isolated in this screen contained insertions in the fosB promoter region, thereby uncoupling the expression of fosB from {sigma}W and instead placing this gene under xylose control.

These data highlight the versatility of mTn7SX: in addition to mutations disrupting genes, we also isolated mutations that caused increased resistance due to the upregulation of a resistance gene. This second class of mutations would not be easily detected with conventional transposon screens.

Isolation of duramycin-resistant mutants.
We also used mTn7SX to isolate mutants with increased resistance to the lantibiotic duramycin. Duramycin is a 10-amino-acid tetracyclic peptide produced by Streptoverticillium cinnamoneum that binds exclusively to ethanolamine phospholipids (23). Duramycin-resistant B. subtilis mutants have little or no phosphatidylethanolamine (PE) and cardiolipin, but the mutant loci were not determined (15).

Our first attempts to isolate Durr mutants by plating the B. subtilis transformants directly on duramycin-containing LB plates failed. Since duramycin resistance appears to be conferred by a change in membrane composition, we hypothesized that our method did not allow for sufficient growth in the absence of selection for the effects of mTn7SX insertions to manifest themselves as changes in the membrane lipid composition. Therefore, we plated the transformed cells on LB agar containing SPEC (for selection of the mutants) and xylose (to allow for upregulation from the PxylA promoter) and incubated these plates for 5 h to allow time for potential alterations in membrane composition to become manifest. These plates were then overlaid with 0.8% soft LB agar containing SPEC, xylose, and 6 µg/ml duramycin and incubated overnight. We obtained 60 clones resistant to duramycin upon restreaking on plates containing 10 µg/ml duramycin.

Since the pssA-ybfM-psd operon is required for the synthesis of PE and cardiolipin, we checked whether any of these mutations were at this locus and found that 53 of the 60 clones gave a PCR product consistent with insertion of mTn7SX within this region. We prepared chromosomal DNA from 10 of the 53 putative pssA-ybfM-psd::mTn7SX clones and used this to transform B. subtilis CU1065. Transformants were selected on SPEC, and in all cases subsequent testing revealed that these transformants were resistant to more than 200 µg/ml duramycin. This confirmed that the resistance phenotype was linked to disruption of the pssA operon by mTn7SX. Mapping of the mTn7SX junctions indicated that each insertion was unique, and they were found regularly spaced throughout all three genes of the operon (Fig. 2C). Analysis of chromosomal DNA from the other seven isolates indicated that the Durr phenotype was not linked to the mTn7SX insertion, suggesting that a second mutation (presumably within the pssA-ybfM-psd operon) was responsible for the resistance phenotype.

Phosphatidylserine (PS) synthase (encoded by pssA) catalyzes the formation of PS, the first step in PE synthesis. PS is rapidly converted to PE by PS decarboxylase (encoded by psd). Therefore, the disruption of these genes results in a lack of PE in the membrane and insensitivity to duramycin (our data; L. Salzberg and J. D. Helmann, unpublished data). The function of YbfM is unknown; however, we have shown that nonpolar mutations within its gene do not result in duramycin resistance (Salzberg and Helmann, unpublished). Therefore, the insertions that we isolated within this gene are predicted to be polar on the downstream psd gene. Since we obtained multiple unique insertions across the pssA-ybfM-psd operon, we suspect that we have saturated the screen, indicating that there are no other genes within the B. subtilis genome that, when disrupted or overexpressed, result in duramycin resistance.

Conclusion.
The development of mTn7SX enables in vitro transposition in the gram-positive bacterium B. subtilis. The addition of the outward-facing, xylose-inducible promoter further improves the system by allowing the identification of "cryptic" or unexpressed genes or simply by ensuring the expression of genes downstream of the insertion. This is analogous to the GeneHunter transposon, based on the EZ-Tn5 system, in which the transposon contains an outward-facing, inducible pTAC promoter (39). Moreover, the observation that some transposants selected in the presence of xylose are unable to grow in medium lacking xylose (data not shown) suggests that this system also generates conditional lethal mutations by insertion of the PxylA promoter upstream of essential genes. The mTn7SX element has several advantages over in vivo transposon mutagenesis systems. Firstly, there is no need for high-temperature curing of the plasmid, which increases the chance of isolating siblings resulting from outgrowth of the same insertion event. We have not identified any siblings in our experiments. Secondly, mTn7SX displays no obvious insertional bias or requirement for a target sequence, as highlighted by the random distribution of insertions within the glpTQ and pssA-ybfM-psd operons and across the genome (Fig. 2). Lastly, Tn7-mediated immunity greatly diminishes the probability of multiple insertions per DNA fragment. This results in libraries of transformants containing a single insertion event per chromosome.


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ACKNOWLEDGMENTS
 
We thank Nancy Craig (Johns Hopkins Medical School/HHMI) for sharing reagents.

This work was supported by a Cornell Innovations Grant (to J.E.P.) and NIH grants GM069508 (to J.E.P.) and GM047446 (to J.D.H.).


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FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, Wing Hall, Cornell University, Ithaca, NY 14853-8101. Phone: (607) 255-6570. Fax: (607) 255-3904. E-mail: jdh9{at}cornell.edu Back

{triangledown} Published ahead of print on 11 April 2008. Back

{dagger} Present address: Département de Biologie, Faculté des Sciences de Luminy, Université de la Méditerranée, 13402 Marseille Cedex 20, France. Back


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Applied and Environmental Microbiology, June 2008, p. 3419-3425, Vol. 74, No. 11
0099-2240/08/$08.00+0     doi:10.1128/AEM.00476-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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