Modified mariner Transposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

Generating vectors for mariner genetic manipulation and delivery.mariner-based transposon insertions can be rapidly and efficiently generated in vivo using pMarA, and we used pMarA as the basis for the design of a universal delivery vector (16, 21, 29). The vector pMarA contains transposon TnYLB with a kanamycin resistance cassette internal to the transposon, the gene encoding the hyperactive C9 allele of mariner-Himar1 transposase, an E. coli origin of replication (oriC), a temperature-sensitive B. subtilis origin of replication [ori(Ts)_Bs], and an erythromycin (mls) resistance cassette external to the transposon (21). To generate a universal transposon delivery vector, pMarA was digested with PstI to excise TnYLB, and the larger backbone fragment was gel purified and religated to generate pKB176 (Fig. 1A). Once a transposon has been constructed, it can be cloned into the empty pKB176 delivery vector or into pMarA itself by the replacement of TnYLB and introduced to B. subtilis strains.

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Fig 1

Genetic maps of transposon construction and final architecture. (A) Map of the delivery vector pKB176, into which completed transposons are cloned and introduce to B. subtilis. (B to E) Maps of the intermediates in transposon assembly. All subsequent constructs were cloned into pKB157 as shown in panel E. (F to H) Generalized maps of the completed transposons for which the actual antibiotic resistance maker has been replaced by the term antibiotic^R. Arrows indicate genes. Boxes indicate the rest of the plasmid backbone and plasmid name. Circle indicates the temperature-sensitive origin of replication in B. subtilis [ori(TS)_Bs]. Triangles indicate mariner insertion sequences (IS elements) labeled L (left) and R (right) to provide an arbitrary orientation. Vertical lines indicate restriction endonuclease sites. The bent arrow represents a promoter.

Our next goal was to design a transposon consisting of two flanking mariner IS elements and an intervening polylinker for convenient genetic modification. The transposon was built in a series of four steps using a combination of PCR and molecular cloning (Fig. 1B to E). First, the kanamycin resistance (Kan^r) cassette from TnYLB was PCR amplified with primers 2077/2078 using pMarA as a template (Fig. 1B). The resulting PCR product incorporated three unique restriction sites on each end of the kanamycin resistance cassette flanked by two innermost XhoI restriction sites (Fig. 1C). The kanamycin resistance cassette shown in Fig. 1C then was used as a template for a second round of separate PCRs using primer pairs 2079/2080 and 2081/2082 to add the left and right mariner IS elements, respectively (Fig. 1D). The transposon was assembled by digesting the left arm with HindIII and EcoRI, digesting the right arm with MfeI and KpnI, and simultaneously ligating both arms into the HindIII/KpnI sites of plasmid pUC19 to generate plasmid pKB157 (Fig. 1E). Note that the EcoRI and MfeI sites have compatible overhangs which, when ligated, destroy each site in the final product (Fig. 1D and E). All subsequent genetic manipulations of the mariner transposon were generated in pKB157 and then cloned into the delivery vector pKB176 for introduction to B. subtilis.

mariner transposons with modified antibiotic cassettes.We constructed three transposons, TnKRM spec, TnKRM cat, and TnKRM kan, containing antibiotic resistance cassettes for spectinomycin, chloramphenicol, and kanamycin, respectively (Fig. 1F). To build the transposons, the genes encoding spectinomycin, chloramphenicol, and kanamycin resistance were PCR amplified from templates pAH54 (using primers 2203/2204), pAC225 (using primers 2201/2202), and pKB157 (using primers 2207/2208), respectively (18). Each of the PCR products was digested with XhoI and NheI and separately ligated into the XhoI and NheI sites of pKB157 to generate plasmids pDP383, pEP21, and pKB177. Note that pKB157 and pKB177 both contain the kanamycin resistance cassette between the mariner insertion elements, but pKB177 has compatible restriction sites that enable subsequent constructs to be built in parallel with its sibling plasmids pDP383 (carrying a spectinomycin resistance cassette) and pEP21 (carrying a chloramphenicol resistance cassette). To generate transposons for use in B. subtilis, each of the transposon-containing plasmids was used as the DNA template for PCR amplification using primers 2054/2312. The resulting PCR products were digested with HindIII and PstI and ligated into the HindIII and PstI sites of pKB176 to generate the transposon delivery vectors pDP384, pEP24, and pKB178, respectively.

To introduce the transposon delivery vectors to B. subtilis, each vector was concatemerized by propagation in recA⁺ E. coli TG1 and transformed into the naturally competent B. subtilis laboratory strain PY79 by mls selection at the permissive temperature of 30°C for the extrachromosomal maintenance of the replicon. Phage lysates then were made of strains harboring the desired transposon delivery vector with the generalized transducing phage SPP1, such that the vectors could be transduced to any B. subtilis strain of interest (5). For mutagenesis, cells were grown for 10 h at 25°C, serially diluted, plated onto plain LB and selective LB agar plates, and incubated overnight at 42°C. Growth at 42°C is nonpermissive for the maintenance of the delivery vector, and thus antibiotic resistance primarily arises from the chromosomal insertion of the transposon. Transposition frequencies were measured for each of the modified TnKRM series of transposons by dividing the number of CFU grown on antibiotic by the number of CFU grown in the absence of antibiotic (Table 5). The spurious integration of the transposon and delivery vehicle into the chromosome occurred at 10- to 100-fold lower frequency than transposition (Table 5).

Mutagenizing a population of B. subtilis cells with transposons is a simple and efficient method for conducting forward genetic screens. To determine the usefulness of our newly designed transposons, a forward genetic screen was designed to generate mutants defective in biofilm formation in the undomesticated B. subtilis strain 3610. Biofilms manifest as robust floating pellicles in liquid that are correlated with rough colonies of complex architecture on agar surfaces (4). To screen for biofilm-defective mutants, three strain derivatives of 3610 containing either TnKRM spec, TnKRM cat, or TnKRM kan (DS8473, DS8474, and DS8994) were mutagenized and plated on LB agar containing the corresponding antibiotic. Mutations that conferred a smooth-colony phenotype, which is correlated with biofilm defects, were isolated. SPP1 phage lysates were generated on candidate colonies, and the transposon insertions were backcrossed into 3610 to ensure inseparable linkage to the smooth-colony phenotype and the transposon insertion.

Wild-type B. subtilis 3610 forms rough colonies on complex LB medium, but biofilm formation is most robust on the defined medium MSgg (4). Each candidate mutant was toothpick inoculated on MSgg solid medium to assay colonies of complex architecture, and 10 μl of an overnight LB broth culture was inoculated into the wells of 6-well microtiter plates containing 10 ml MSgg broth to assay pellicle formation. Both plates and broth were incubated at room temperature for 3 days. Wild-type cells formed colonies of complex architecture on MSgg solid agar, but each of the candidate transposon mutants exhibited smooth-colony morphology (Fig. 2). Similarly, the wild type formed a robust floating pellicle in liquid MSgg media, but many of the smooth mutants exhibited either incomplete or shattered and sunken pellicles, which is indicative of a biofilm defect (Fig. 2).

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Fig 2

Mutants defective for biofilm formation. The pellicle column depicts microtiter wells (6-well plate) in which cells have been grown in MSgg medium for 3 days at 25°C. Scale bar is 1 cm. The colony column depicts 10× images of individual colonies grown on MSgg medium for 3 days at 25°C. Scale bar is 1 mm.

Transposon insertion sites were identified by inverse PCR, and each transposon that conferred a biofilm defect disrupted a gene that had been previously reported to be required for biofilm formation (Table 6). Mutations in the eps operon, including the epsD, epsE, epsK, epsM, and epsN genes, impaired biofilm formation, likely due to the disruption of the assembly of the extracellular polysaccharide (EPS) component of the biofilm matrix (4, 14). The mutation of galE, which encodes GalE (the UDP-glucose-4-epimerase), likely abolished the synthesis of a sugar precursor necessary for EPS synthesis (23). The mutation of remB disrupted RemB, a protein that activates extracellular matrix production (30). The mutation of slrR disrupted the transcription factor SlrR, which activates biofilm gene expression (8, 9, 20). Finally, the mutation of ymdB disrupted YmdB, a recently identified phosphoesterase shown to be required for complex colony architecture formation (12). Interestingly, although the mutation of ymdB resulted in a smooth-colony phenotype, it did not abolish or alter pellicle formation. We infer that either a secondary suppressor mutation quickly arises to restore pellicle formation to the ymdB mutant or the ymdB mutation is the first mutation identified that abolishes complex colony architecture without disrupting pellicle formation, suggesting that the two phenotypes in some cases are genetically separable. We conclude that the mutagenesis screens using the TnKRM series of transposons were successful in isolating biofilm-defective mutants.

Construction of TnHyJump, a transposon carrying an outward-oriented inducible promoter.Transposon insertions most commonly result in null loss-of-function mutations in the gene into which they have inserted. We set out to build a transposon which, when integrated, would cause the high-level expression of the adjacent gene(s) for use in artificial-expression “gene-trap” screens (33). To build transposons with an outward-oriented, strong, constitutive promoter, the P_hyspank promoter was PCR amplified using primers 1886/2271 and pDR111 as the DNA template (3). The PCR product then was digested with BamHI and SalI and ligated into the BamHI and SalI sites of pDP383, pEP21, and pKB177, resulting in pEP17, pEP23, and pEP18, respectively. To clone the TnHyJump transposons into the delivery vector, pEP17, pEP23, and pEP18 were PCR amplified using primers 2054/2312, digested with HindIII and PstI, and ligated into the HindIII and PstI sites of pKB176, generating pEP19, pEP26, and pEP20 (Fig. 1F).

The P_hyspank promoter may be controlled by the addition of IPTG to the culture medium when the lacI gene encoding the LacI repressor protein is present in the genome. To control the expression of the P_hyspank promoter on the TnHyJump transposons, a vector was constructed in which the lacI gene of E. coli and a tetracycline resistance cassette was integrated between the arms of the B. subtilis lacA gene. The insertion of lacI at lacA serves a dual purpose: first, it disrupts the lacA gene that encodes an endogenous LacZ homolog in B. subtilis which, when misregulated, can confound blue/white β-galactosidase screens (11), and second, the construct expresses the LacI repressor protein to regulate the IPTG-inducible expression of the P_hyspank promoter in trans. To build a lacA::tet integration vector, the region containing the 5′ end of the lacA gene and adjacent upstream DNA was PCR amplified with primers 2224/2225 using 3610 chromosomal DNA as a template, digested with BglII and PstI, and cloned into the BglII/PstI sites of pDG1515 (13) carrying the tetracycline resistance gene to generate pKB169. The region containing the 3′ end of the lacA gene and adjacent downstream DNA was PCR amplified with primers 2226/2227 using 3610 chromosomal DNA as a template, digested with HindIII and KpnI, and cloned into the HindIII/KpnI sites of pKB169 to generate pKB170. Finally, the lacI gene was PCR amplified using primer pair 2209/2211 with plasmid pDR111 as a template, digested with SalI and BamHI, and cloned into the BamHI/SalI sites of pKB170 to generate pKB175. Thus, when pKB175 is integrated into the chromosome, lacA will be disrupted by the insertion of the lacI gene and a tetracycline resistance marker.

To demonstrate proof of principle with the TnHyJump transposons, B. subtilis strains DS7996, DS7997, and DS8137 were generated containing separate TnHyJump transposon delivery vectors, a promoterless lacZ gene encoding β-galactosidase integrated at the amyE locus, and the lacI gene integrated into the lacA locus. The promoterless lacZ in the chromosome was included in an attempt to isolate and identify TnHyJump insertions that inserted upstream of, and in the same orientation as, lacZ and thereby direct the transcription of the lacZ gene to turn colonies a blue color on media containing X-gal and the appropriate antibiotic selection. The inserted lacI gene enabled the control of the P_hyspank promoter such that blue colony color, if any, should be dependent on the presence of IPTG.

Cells mutagenized with the TnHyJump transposon were plated on LB agar plates containing the antibiotic appropriate for the TnHyJump used, the chromogenic β-galactosidase substrate X-gal, and 1 mM IPTG, and they were grown overnight at 42°C, the nonpermissive temperature for the transposon delivery vector. Blue colonies were found at a low frequency (Fig. 3A). The rare blue colonies were isolated and restreaked onto selective media containing X-gal with and without the inducer IPTG. In all cases, colonies had a more intense blue color in the presence of IPTG, and colonies had a less intense blue color in the absence of IPTG (Fig. 4). None of the IPTG-dependent blue colonies contained transposon insertions upstream of the promoterless lacZ gene that was integrated at the amyE locus. Rather, each transposon was inserted within the large 25-kb yes putative operon, such that the P_hyspank promoter was oriented to express the downstream yes genes. The most distal insertion that resulted in blue colony color in the presence of X-gal was located within yesX, suggesting that β-galactosidase activity originated from one of the three colinear genes expressed downstream, yesY, yesZ, and yetA. Of these three genes, the most likely candidate to encode β-galactosidase activity is yesZ, encoding YesZ, a putative β-galactosidase (26). We infer that yesZ encodes a cryptic β-galactosidase, the native regulation and expression of which is unknown. We speculate that insertions were favored in front of yesZ rather than in front of the artificially integrated lacZ gene, because yesZ appears to be preceded by 25 kb of genes with which it may be cotranscribed and is thus a much larger target. We conclude that the TnHyJump transposons insert efficiently and express downstream genes when the P_hyspank promoter is activated.

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Fig 3

Primary plates for blue/white β-galactosidase screens. (A) A petri plate with colonies of cells mutagenized with TnHyJump. (B) A petri plate with colonies of cells mutagenized with TnLacJump. Both plates contain antibiotic and the chromogenic LacZ substrate X-gal. Black carets point to representative blue colonies.

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Fig 4

TnHyJump transposons inserted in the yes operon that induce blue colony color. Genetic map of the yes operon. Arrows indicate genes. Gray triangles indicate transposon insertions. Bent arrows indicate promoters and the direction of transcription. The yesZ gene is indicated in blue, as it is predicted to encode YesZ, a putative β-galactosidase. Pictures are images of colonies grown on LB containing X-gal and either the presence or absence of IPTG.

Construction of TnLacJump, a transposon carrying a promoterless lacZ gene.Transposons are useful for inserting other genes into the genome besides antibiotic markers. For instance, a promoterless lacZ gene may be integrated within a transposon to generate randomly inserted transcriptional reporter fusions (6, 7, 17, 24). To begin building transposons with a promoterless lacZ gene, the lacZ gene was PCR amplified using primers 1888/1889 and the lacZ plasmid pDG268 as the DNA template (2). The PCR product then was digested with BamHI and XhoI and ligated into the BamHI and XhoI sites of pDP383, pEP21, and pKB177, resulting in pEP1, pEP22, and pEP3, respectively. To insert the TnLacJump derivatives into the delivery vector, pEP1, pEP22, and pEP3 were used as templates for PCR using primers 2054/2312 to generate a product containing the IS elements, the lacZ gene, and the antibiotic resistance cassette. The PCR product was digested with HinDIII and PstI and ligated into the HinDIII and PstI sites of the delivery vector pKB176, generating pEP4, pEP25, and pEP6 (Fig. 1H).

To demonstrate proof of principle with the TnLacJump transposons, strains containing a TnLacJump delivery vector (DS7487, DS7133, and DS8274) were mutagenized by plating on LB agar plates containing X-gal and the appropriate antibiotic at the nonpermissive temperature of 42°C. The rationale of the screen was that the promoterless lacZ in the transposon should generate blue colonies on X-gal only if the TnLacJump transposon had inserted itself within a transcriptionally active region and in the same orientation as an upstream endogenous promoter. Blue colonies of various color intensity were common (Fig. 3B). The identification of the transposon insertion site revealed that all transposons conferring a blue colony color were colinear within the gene in which they were inserted (Fig. 5 and Table 6). Two insertions require additional comment. One transposon, TnΩ8725, was inserted within 1 of the 10 23S rRNA genes in the B. subtilis genome, and due to the high level of sequence identity between the homologous genes, we were unable to identify which specific 23S rRNA gene was disrupted. Nonetheless, the 23S rRNA insertion exhibited the most intense blue colony color of all of the insertions, which is consistent with integration within one of the highly expressed rRNA operons. Another transposon, TnΩ8329, was integrated in an intergenic region upstream of, and colinear with, the gene yvrM. We infer that the insertion lies downstream of an active promoter, and we note that the lacZ insertion appears to disrupt a small, previously unannotated open reading frame colinear with yvrM. We conclude that the TnLacJump transposons are an efficient method for generating insertional lacZ reporters in B. subtilis.

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Fig 5

TnLacJump transposons insertion sites and orientation. Colony indicates a colony of the indicated strain grown on LB agar containing the chromogenic LacZ substrate X-gal. Blue color indicates that β-galactosidase is being expressed, and therefore lacZ in the transposon is being transcribed. Transposon insertion site indicates the location of the transposon insertion and the surrounding genetic neighborhood. Open arrows indicate genes. The gray triangle indicates the location of TnLacJump insertion. The blue arrow indicates the orientation of the lacZ gene. The position of the insertion in the chromosome, given in degrees is indicated at the right.

White colonies also were obtained from the TnLacJump transposon mutagenesis (Fig. 3B). Two of the white colonies contained transposon insertions TnΩ7169 and TnΩ8606 that were oriented in the opposite direction from the disrupted gene and thus were not transcribed by an upstream promoter. One of the white colonies contained the transposon insertion TnΩ8359, in which the lacZ was inserted within, and colinear with, the gene (yeeA) that was disrupted. We infer that there may be a promoter upstream of yeeA that directs yeeA transcription but that the putative yeeA promoter was not expressed under the conditions of the screen. We conclude that TnLacJump insertions only generate functional reporters when inserted colinearly with an upstream promoter and only under conditions in which the upstream promoter is active.

In sum, we have designed a series of three transposons that include not only different antibiotic markers but also either a strong outward-oriented promoter for overexpression screens or a promoterless lacZ gene for the random generation of transcriptional reporter fusions. Furthermore, we demonstrate that all nine of the transposons mobilize at high frequency, and we demonstrate proof of principle for insertional mutagenesis, the inducible expression of a cryptic β-galactosidase YesZ, and the successful generation of random functional transcriptional reporters. Finally, we note that the base transposon in pKB157 can be modified to suit the needs of bacteria besides B. subtilis, which, when delivered by in vitro mutagenesis (1) or by a species-specific in vivo strategy, should be useful for generating any transposon of interest in any model organism (15).