Insertion of Transposon Tn917 Derivatives into the Lactococcus lactis subsp. lactis Chromosome

Two transposition vectors, pTV32 and pLTV1, containing transposon Tn917 derivatives TV32 and LTV1, respectively, were introduced into Lactococcus lactis subsp. lactis MG1614. It was found that pTV32 and pLTV1 replicate and that TV32 and LTV1 transpose in this strain. A protocol for production of a collection of Tn917 insertions in L. lactis subsp. lactis was developed. The physical locations of TV32 on the chromosomal SmaI fragments of 62 independent transpositions were established by pulsed-field gel electrophoresis. These transpositions could be divided into at least 38 different groups that exhibited no Tn917-dominating hot spots on the L. lactis subsp. lactis chromosome. A total of 10 of the 62 transpositions resulted in strains that express β-galactosidase. This indicates that there was fusion of the promoterless lacZ of the Tn917 derivatives to a chromosomal promoter. Thus, the Tn917-derived transposons should be powerful genetic tools for studying L. lactis subsp. lactis.     

Establishment of an Arbitrary PCR for Rapid Identification of Tn917 Insertion Sites in Staphylococcus epidermidis: Characterization of Biofilm-Negative and Nonmucoid Mutants

Transposon mutagenesis with the Enterococcus faecalis transposon Tn917 is a genetic approach frequently used to identify genes related with specific phenotypes in gram-positive bacteria. We established an arbitrary PCR for the rapid and easy identification of Tn917 insertion sites in Staphylococcus epidermidis with six independent, well-characterized biofilm-negative Tn917 transposon mutants, which were clustered in the icaADBC gene locus or harbor Tn917 in the regulatory gene rsbU. For all six of these mutants, short chromosomal DNA fragments flanking both transposon ends could be amplified. All fragments were sufficient to correctly identify the Tn917 insertion sites in the published S. epidermidis genomes. By using this technique, the Tn917 insertion sites of three not-yet-characterized biofilm-negative or nonmucoid mutants were identified. In the biofilm-negative and nonmucoid mutant M12, Tn917 is inserted into a gene homologous to the regulatory gene purR of Bacillus subtilis and Staphylococcus aureus. The Tn917 insertions of the nonmucoid but biofilm-positive mutants M16 and M20 are located in genes homologous to components of the phosphoenolpyruvate-sugar phosphotransferase system (PTS) of B. subtilis, S. aureus, and Staphylococcus carnosus, indicating an influence of the PTS on the mucoid phenotype in S. epidermidis.     

A method for construction of E. coli strains with multiple DNA insertions in the chromosome

A system for construction of E. coli strains with multiple DNA insertions in the chromosome, based on elements of modules for site specific recombination of Tn1545 and phage λ, has been developed. Circular non-replicating DNA fragments containing the transposon attachment site (attTn), an excisable cassette with a selectable marker, and a gene of interest integrate randomly into the chromosome of a host E. coli strain when provided with transposon integrase, Int-Tn (the host strain was obtained by insertion of the fragment containing transposon int-Tn gene coding for Int-Tn into the chromosome). Integration of these fragments into the chromosome of int-Tn⁺ cells gives rise to a collection of antibiotic-resistant clones with single insertions at different locations in the chromosome. These insertions are transferred subsequently by P1 transduction into one strain and selected for antibiotic resistance provided by the cassette with the selectable marker. After transduction of each copy, a helper plasmid bearing phage λ xis and int genes is introduced into the cells to excise the drug resistance gene flanked with the λattL and λattR sites from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of P1 transduction/drug resistance gene excision. Each cycle adds another chromosomal copy of the foreign gene. To show the utility of the system, we constructed an E. coli strain bearing several chromosomal copies of lacZ at different locations.     

Use of Transposon-Transposase Complexes To Create Stable Insertion Mutant Strains of Francisella tularensis LVS

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 × 10⁻⁸ ± 0.87 × 10⁻⁸ per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.     

Identification of Listeria monocytogenes Genes Involved in Salt and Alkaline-pH Tolerance

The capacity of Listeria monocytogenes to tolerate salt and alkaline stresses is of particular importance, as this pathogen is often exposed to such environments during food processing and food preservation. We screened a library of Tn917-lacZ insertional mutants in order to identify genes involved in salt and/or alkaline tolerance. We isolated six mutants sensitive to salt stress and 12 mutants sensitive to salt and alkaline stresses. The position of the insertion of the transposon was located in 15 of these mutants. In six mutants the transposon was inserted in intergenic regions, and in nine mutants it was inserted in genes. Most of the genes have unknown functions, but sequence comparisons indicated that they encode putative transporters.     

Genetic approaches for studying rhizosphere colonization

Most bacterial traits involved in colonization of plant roots are yet to be defined. Studies were initiated to identify genes in Pseudomonas which play significant roles in this process. The general approach is to use transposons to construct collections of insertion mutants, each of which is then screened for alterations in its interactions with the host plant. In one study a Tn5 derivative containing a constitutively expressed -galactosidase (lacZ) gene was used to generate a collection of insertion mutants which could be distinguished from the wild-type parent on X-gal plates. Each mutant was examined for its ability to colonize wheat seedlings in the presence of the wild-type parent. Mutants which gave wild-type:mutant ratio of 20:1 or greater were obtained. In a second study a Tn5 derivative which carries a promoterless lacZ gene located near one end of the transposon was constructed. Expression of the lacZ gene depends on the presence of an active promoter outside of the transposon in the correct orientation. Insertion mutants generated with this transposon were examined for changes in -galactosidase expression in the presence and absence of plant root exudate. A number of mutants which showed differential lacZ expression have been identified.     

Recombination in the Vicinity of Insertions of Transposon Tn 5 in MYXOCOCCUS XANTHUS

To test genetic recombination in the vicinity of insertions of the transposon Tn5, crosses were performed by transduction between M. xanthus strains carrying different insertions of Tn5. One member of each pair carried resistance to kanamycin (Tn5-Km); the other carried resistance to tetracycline (Tn5-Tc). The distance between each pair of Tn5 insertions was also measured by restriction mapping. The physical distance corresponding to each recombination frequency was calculated from the transductional linkage and compared with distance on the restriction map. A good correspondence between the two measures of distance was obtained for a pair of Tn5 insertions near the cglB locus and for another pair near the mgl locus. Correspondence between the two measurements of distance, the observed allelic behavior of Tn5-Km and Tn5 -Tc at the same locus and the finding of the same frequencies of recombinants in reciprocal crosses implied that recombination in the vicinity of Tn 5 was normal.     

Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis inBacillus subtilis

A total of 20Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.     

Specificity of Transposon Tn5 Insertion

Genetic mapping studies had shown that the bacterial transposon Tn5 can insert into many sites in a gene, but that some sites are preferred. To begin understanding Tn5's insertion specificity at the molecular level, we selected transpositions of Tn5 from the Escherichia coli chromosome to the plasmid pBR322 and analyzed the resultant pBR322::Tn5 plasmids by restriction endonuclease digestion and DNA sequencing. Seventy-five insertions in the tet gene were found at 28 sites including one major hotspot (with 21 insertions) and four lesser hotspots (with four to ten insertions each). All five hotspots are within the first 300 of the 1250-base pair (bp) tet gene. In contrast, 31 independent insertions in the amp gene were found in at least 27 distinct sites.—Tn5 generates 9 bp target sequence duplications when it transposes. Such transposon-induced duplications are generally taken to indicate that cleavages of complementary target DNA strands are made 9 bp apart during transposition. DNA sequence analysis indicated that GC base pairs occupy positions 1 and 9 in the duplications at each of the five hotspots examined, suggesting a GC-cutting preference during Tn5 transposition.     

A novel method for the rapid cloning in Escherichia coli of Bacillus subtilis chromosomal DNA adjacent to Tn917 insertions

Summary A rapid and general procedure has been devised for the pBR322-mediated cloning in Escherichia coli of Bacillus subtilis chromosomal DNA extending in a specified direction from any Tn917 insertion. Derivatives of Tn917 have been constructed that contain a pBR322-derived replicon, together with a chloramphenicol-resistance (Cm^r) gene of Gram-positive origin (selectable in B. subtilis), inserted by ligation in two orientations into a SalI restriction site located near the center of the transposon. When linearized plasmid DNA carrying such derivatives was used to transform to Cm^r B. subtilis bacteria already containing a chromosomal insertion of Tn917, the pBR322 sequences efficiently became integrated into the chromosomal copy of the transposon by homologous recombination. It was then possible to clone chromosomal sequences adjacent to either transposon insertion junction into E. coli, using a selection for ampicillin-resistance, by transforming CaCl₂-treated cells with small amounts of insert-containing DNA that had been digested with various restriction enzymes and then ligated at a dilute concentration. Because pBR322 sequences may be inserted by recombination in either orientation with respect to the transposon arms, a single restriction enzyme (such as EcoRi or SphI) that has a unique recognition site in pBR322 DNA may be used to separately clone chromosomal DNA extending in either direction from the site of any transposon insertion. A family of clones generated from the region of an insertional spo mutation (spoIIH::Tn917) was used in Southern hybridization experiments to verify that cloned material isolated with this procedure accurately reflected the arrangement of sequences present in the chromosome. Strategies are discussed for taking advantage of certain properties inherent in the structure of clones generated in this way to facilitate the identification and study of promoters of insertionally mutated genes.     

Transposon Assisted Gene Insertion Technology (TAGIT): A Tool for Generating Fluorescent Fusion Proteins

We constructed a transposon (transposon assisted gene insertion technology, or TAGIT) that allows the random insertion of gfp (or other genes) into chromosomal loci without disrupting operon structure or regulation. TAGIT is a modified Tn5 transposon that uses Kan^R to select for insertions on the chromosome or plasmid, β-galactosidase to identify in-frame gene fusions, and Cre recombinase to excise the kan and lacZ genes in vivo. The resulting gfp insertions maintain target gene reading frame (to the 5′ and 3′ of gfp) and are integrated at the native chromosomal locus, thereby maintaining native expression signals. Libraries can be screened to identify GFP insertions that maintain target protein function at native expression levels, allowing more trustworthy localization studies. We here use TAGIT to generate a library of GFP insertions in the Escherichia coli lactose repressor (LacI). We identified fully functional GFP insertions and partially functional insertions that bind DNA but fail to repress the lacZ operon. Several of these latter GFP insertions localize to lacO arrays integrated in the E. coli chromosome without producing the elongated cells frequently observed when functional LacI-GFP fusions are used in chromosome tagging experiments. TAGIT thereby faciliates the isolation of fully functional insertions of fluorescent proteins into target proteins expressed from the native chromosomal locus as well as potentially useful partially functional proteins.     

Precise excision of bacterial transposon Tn5 in yeast

Summary We have demonstrated that precise excision of bacterial transposon Tn5 can occur in the yeast, Saccharomyces cerevisiae. Tn5 insertions in the yeast gene LYS2 were generated by transposon mutagenesis made in Escherichia coli by means of a ::Tn5 vector. Nine insertions of Tn5 into the structural part of the yeast LYS2 gene situated in a shuttle epsiomal plasmid were selected. All the plasmids with a Tn5 insertion were used to transform yeast strains carrying a deletion of the entire LYS2 gene or a deletion of the part of LYS2 overlapping the point of insertion.All insertions inactivated the LYS2 gene and were able to revert with low (about 10^-8) frequencies to lysine prototrophy. Restriction analysis of revertant plasmids revealed them to be indistinguishable from the original plasmid without Tn5 insertion. DNA sequencing of the regions containing the points of insertions, made for two revertants, proved that Tn5 excision was completely precise.     

Construction of a Slime Negative Transposon Mutant in Staphylococcus epidermidis Using the Enterococcus faecalis Transposon Tn917

The slime-producing Staphylococcus epidermidis strain sensu strictu CNS23 was transformed by protoplast transformation with the plasmid pTV1 which carries transposon Tn917. Using this transposon mutagenesis system we obtained the Tn917-inserted mutant CT512, which has lost the ability to produce slime. A single insertion of the trasposon Tn917 into the chromosome of CT512 could be detected by Southern hybridization. This mutant showed a significantly higher stability concerning its slime-negative phenotype compared with spontaneous slime-negative mutants of S. epidermidis strain CNS23. In slime-ELISA no slime-associated antigen could be detected in extracts of the transposon mutant. Compared to slime-positive S. epidermidis strains, CT512 lacked in accumulative growth in microtiter tube test.     

Tn917 Targets the Region Where DNA Replication Terminates in Bacillus subtilis,Highlighting a Difference in Chromosome Processing in the Firmicutes

The bacterial transposon Tn917 inserts preferentially in the terminus region of some members of the Firmicutes. To determine what molecular process was being targeted by the element, we analyzed Tn917 target site selection in Bacillus subtilis. We find that Tn917 insertions accumulate around the central terminators, terI and terII, in wild-type cells with or without the SPβ lysogen. Highly focused targeting around terI and terII requires the trans-acting termination protein RTP, but it is unaffected in strains compromised in dimer resolution or chromosome translocation. This work indicates that Tn917 is sensitive to differences in DNA replication termination between the Firmicutes.Certain transposons are known to target features associated with DNA metabolism, and these elements have the potential to offer greater insight into these host processes (6). The bacterial transposon Tn917 was originally isolated from Enterococcus faecalis and has been used as an insertion mutagen in this and other gram-positive bacteria (5, 28, 32). However, Tn917 has been shown to have an extreme regional preference for insertion into the terminus region in E. faecalis, and the molecular mechanism responsible for this bias is unknown (10). Multiple processing events associated with chromosome duplication occur in the terminus region, a portion of the chromosome we define here as equidistant from the origin of DNA replication in circular genomes. In some bacteria, a system exists that actively terminates DNA replication forks at specific sites within the terminus region, called ter sites, through the use of a trans-acting protein called Tus in Escherichia coli and RTP in Bacillus subtilis (8, 9, 18). Another processing event that occurs in the terminus region involves the resolution of dimer chromosomes at a site called dif (2). The resolution of dimer chromosome is usually catalyzed by two tyrosine recombinases called XerC and XerD in E. coli and RipX and CodV in B. subtilis.To better understand processing events in bacterial chromosomes, we investigated Tn917 target site selection in the model low-G+C gram-positive bacterium, B. subtilis, where replication and recombination are well studied. We were specifically interested in knowing if Tn917 targeted DNA replication termination or dimer resolution as suggested previously (10). There are multiple examples where elements have been suggested to recognize these molecular processes as targets for transposition in E. coli (20, 23, 29). In the case of Tn917, it is of special interest to understand targeting because this behavior differs even between closely related species; Tn917 transposition preferentially occurs in the terminus region of Enterococcus faecalis and Streptococcus equi, but this behavior is not found in Listeria monocytogenes and Streptococcus suis (see references 10 and 28 and see below).Tn917 insertions were collected in B. subtilis using plasmid pTV1-OK in the strain CU1065 (W168 trpC2 SPβ⁻) background using a procedure that prevented the isolation and sequencing of the same transposition event (11). The temperature-sensitive plasmid pTV1-OK imparts resistance to kanamycin and contains the erythromycin resistance-encoding transposon Tn917 (11). To generate transposants, single colonies of purified pTV1-OK transformants (12) were used to inoculate individual test tubes containing LB liquid medium with kanamycin (10 μg/ml) plus erythromycin at a sublethal concentration (1 ng/ml), a level previously shown to induce the erm and transposase genes of Tn917 (30). Cultures were incubated at 30°C overnight and then plated on LB medium containing erythromycin (1 μg/ml) and incubated at 42°C overnight. The latter incubation step was repeated to ensure loss of the transposon delivery plasmid. We determined the position and orientation of individual transposition events in the chromosome using arbitrary PCR analysis and the sequence of the B. subtilis 168 chromosome version {"type":"entrez-nucleotide","attrs":{"text":"AL009126.1","term_id":"30407132","term_text":"AL009126.1"}}AL009126.1 as described previously (10, 11, 14). This analysis indicated that transposition events occur preferentially around the first ter sites encountered by DNA replication forks in the chromosome, terI and terII (Fig. ​(Fig.1A);1A); 30% (25/82) of the transposition events occurred within 15 kb of these sites even though this region comprises less than 1% of the chromosome (Fig. ​(Fig.1C).1C). Previously it was shown that Tn917 transposition showed a strong bias for the gltA gene in screens for auxotrophs, a gene known to be close to the region where DNA replication terminated (25, 31, 32) and now known to be ∼3.6 kb from terI-terII (14). However, our work here is the first indication that transposition actually occurred around the central terI-terII terminators.Open in a separate windowFIG. 1.Distribution of Tn917 insertions in Bacillus subtilis wild-type strain CU1065 and the rtp::cat strain. Tn917 transposition events were mapped on the B. subtilis CU1065 chromosome in cells that were wild type (A) or had an rtp deletion allele (rtp::cat) (B). (C and D) The total number of insertions collected and mapped and the number that occurred in the 200-kb region centered around the terI-terII sites (terI, 2,017,644; terII, 2,017,520) is indicated and the distribution graphed across this 200-kb region in 10-kb bins. The numbering of the graph is centered on terI-terII as the zero position. The location of Tn917 insertions (arrows) and a putative dif site are indicated in kilobase pairs on the B. subtilis strain 168 genome. The orientation of individual insertion events is indicated by placement of the arrow inside (right to left) or outside (left to right) the circle. Triangles indicate the positions of the nine ter sites in the B. subtilis chromosome; the flat side of the triangle indicates the side where approaching replication forks are actively terminated. Positions on the B. subtilis chromosome are according to the B. subtilis 168 chromosome sequence version {"type":"entrez-nucleotide","attrs":{"text":"AL009126.1","term_id":"30407132","term_text":"AL009126.1"}}AL009126.1 (14).To further confirm the Tn917 preference for the region around the terI-terII sites, we analyzed targeting in B. subtilis strain JH642 containing the lysogen SPβ. In this strain the terI-terII sites move 134 kb relative to oriC from the position found in B. subtilis strain CU1065 (15). We found that Tn917 insertions continued to have a preference for the terI-terII sites in the lysogen strain where 17% (4/23) of the insertions still occurred within 15 kb of these sites even though this region comprises less than 1% of the chromosome (data not shown). This supported the idea that targeting is not dependent on the relative position in the chromosome but is instead a sequence or process directly associated with this particular region of the chromosome.To determine if targeting required active termination of DNA replication, we examined Tn917 transposition in an Δrtp strain. Active termination is not an essential process in bacteria, and the only phenotype associated with an Δrtp allele is dependent on the inactivation of other systems (17). We created a CU1065 Δrtp::cat strain using long-flanking-homology PCR analysis (19). The oligonucleotide primers JEP158 (5′-GGGTAACTAGCCTCGCCGGTCCACGATATTAAAGACTGATAGTCC-3′) and JEP159 (5′-CCGGCATCAGCAAATTTGGCGG-3′) were used to amplify the region 5′ to the deletion; JEP137 (5′-AATGCTTCGGCCAGCTTCTTCAGG-3′) and JEP138 (5′-CTTGATAATAAGGGTAACTATTGCCTTTAATAGAAACAAACACC-3′) were used to amplify the region 3′ to the deletion. The primers and plasmids used for amplification of the antibiotic resistance cassettes have been described previously (3). Deletion of the rtp gene in the B. subtilis chromosome was confirmed by PCR analysis.We found that there was still a general preference for Tn917 transposition across the terminus region in the Δrtp background (e.g., in both the rtp⁺ and Δrtp backgrounds about 40% of the insertions occurred within a 200-kb window centered around the terI-terII sites) (Fig. 1C and D). However, the extreme preference for the region around the terI-terII sites was lost in the Δrtp background, and only 6% (5/83) of the transposition events occurred within 15 kb of the terI-terII sites (Fig. ​(Fig.1B).1B). Our data indicate that within the terminus region, while insertions occur at a greater-than-expected frequency within 15 kb of the central terI-terII sites in the wild-type background (P < 0.001; χ² statistic), this was not true in the Δrtp strain (P = 0.22; χ² statistic). These data are consistent with a model where Tn917 targets the region where DNA replication terminates in the chromosome. While there is no requirement for active termination of DNA replication, the RTP-mediated process likely focuses Tn917 insertions around the central terI-terII sites.In E. faecalis, Tn917 insertions occur with a strong grouping where 65% of the insertions occurred in a 200-kb region (1,450 to 1,650 kb) centered around the predicted natural position of replication termination in this organism as indicated by the skew of the chromosome (10, 13, 16). This position also correlates with the dif site used to resolve dimer chromosomes in E. faecalis (1,550,523 bp), and 23% of the insertions occurred within 15 kb of the predicted dif site (a region which constitutes ∼1% of the chromosome) (13, 16). While Tn917 did not appear to target the dif site in B. subtilis (Fig. ​(Fig.1C),1C), we wanted to decisively rule out any role of dimer resolution in Tn917 targeting. The RipX and CodV proteins are involved in dif recombination in B. subtilis, but the ripX gene product is known to play the essential role in this process. Therefore, we monitored transposition in an otherwise isogenic ΔripX B. subtilis strain which is deficient in chromosome dimer resolution (26). A CU1065 ΔripX::cat strain was constructed using transformation with chromosomal DNA from strain PAL422 (ΔripX::cat) (27). The distribution of Tn917 insertions indicates that there is still a preference for transposition in the terminus region (Fig. ​(Fig.2A).2A). In addition, Tn917 insertions still preferentially occurred around terI-terII within the terminus region in the ΔripX strain; 22% (17/77) of the insertions occurred within 15 kb of terI-terII sites in the ΔripX background (Fig. 2A and C). Similar to the result found with the wild-type strain, we found that insertions occur at a greater-than-expected frequency within 15 kb of the central terI-terII sites within the region shown in Fig. ​Fig.2C2C in the ΔripX background (P < 0.001; χ² statistic). This confirms that dimer resolution is not responsible for the attraction of Tn917 insertion for the terminus region in B. subtilis.Open in a separate windowFIG. 2.The distribution of Tn917 insertions in B. subtilis CU1065 ripX::cat and spoIIIE::spc strains. Tn917 transposition events were mapped on the B. subtilis CU1065 chromosome from ripX (ripX::cat) (A) or spoIIIE (ΔspoIIIE::spc) (B) strains. (C and D) The total number of insertions and the number that occurred in the 200-kb region centered on the terI-terII sites are indicated, and the distribution is graphed across 10-kb bins. The designations are as described in legend to Fig. ​Fig.11.We also determined if the DNA translocation protein SpoIIIE played any role in targeting Tn917 insertions to the terminus region. SpoIIIE monitors signals in the bacterial chromosome that convene in the terminus region. In E. coli, the chromosome dimer resolution proteins require a partner protein, FtsK, for completing recombination (2, 4). The FtsK and SpoIIIE proteins are both able to monitor DNA sequences in the chromosome and translocate DNA in one direction relative to the chromosomal dif site (reviewed in reference 1). While the role of the SpoIIIE protein in actively growing cells is unclear, we were still interested in this gene product because Tn917 inserts at a region in the chromosome where the sequences recognized by SpoIIIE converge. A CU1065 spoIIIE::spc strain was constructed using transformation with chromosomal DNA from strain KPL708 (spoIIIE::spc) (24). After examining the distribution of insertions, we found that there was still a significant bias for Tn917 insertion within the terminus region where 20% (16/77) of the insertions fell within 15 kb of the terI-terII sites in the ΔspoIIIE background (P < 0.001; χ² statistic) (Fig. 2B and D). Our results indicate that neither the B. subtilis dimer resolution system nor the translocation function of SpoIIIE is responsible for attracting Tn917 events to the terminus region of the chromosome. B. subtilis has two poorly understood proteins with homology to DNA translocases like SpoIIIE and FtsK called YtpT and YtpS (26). The YtpT protein is not required for dif recombination as monitored in a plasmid-based assay, and any role for the YtpT and YtpS proteins in Tn917 targeting was not investigated here. The result with the ΔripX and ΔspoIIIE strains also indicates that a direct interaction with the RipX and SpoIIIE proteins does not direct Tn917 transposition into the terminus region.In summary, we can now confirm that Tn917 transposition events are attracted to the place where DNA replication forks are expected to terminate through the action of the RTP protein in the B. subtilis chromosome in a process that is unaffected by the dimer resolution and DNA translocation systems (Fig. ​(Fig.11 and ​and2).2). Other transposons have been shown to target features of DNA replication termination in bacteria (23, 29). In Tn7 target site selection, at least two molecular signals are used to recognize active DNA replication, a gapped DNA structure and an interaction with the β-clamp processivity factor (21). Presumably these features become more available when DNA replication terminates. Interestingly, it was also noted that the Tn917 transposase has a sequence that resembles a protein motif used by a variety of proteins to interact with the processivity factor (21). It seems possible that in both Tn7 and Tn917 transposition, the β-clamp processivity factor may provide part of the signal for identifying insertion sites and that the β-clamp may become available when replication is terminated by either passive or active means. Further research will be needed to confirm that a sequence within the Tn917 transposase interacts with the processivity factor and if this interaction is important for recognizing replication termination and perhaps other replication targets (e.g., DNA repair signals and the replication of mobile DNA elements, as in the case of Tn7 [22]).Tn917 appears to be sensitive to an unknown difference in either how DNA replication is terminated or how termination events are processed within the Firmicutes. While Tn917 insertions are focused around the predicted point of replication termination in E. faecalis, Tn917 targeting did not show this bias in Listeria monocytogenes (10). A similar discrepancy was found within the closely related Streptococcus equi and Streptococcus suis species. Tn917 insertions did not occur with any obvious bias in the S. suis genome; however, in S. equi, 60% of the Tn917 insertions occurred in a 15-kb region (28). By comparing the genes found in this region of the chromosome with the DNA sequence from S. equi, we can report that Tn917 also targets the terminus region in S. equi (personal observation), a region where dimer chromosomes are likely to be resolved via an unconventional system (dif_SL) found in the Streptococcus and Lactococcus genera (16). This is a region where replication termination is likely to occur in S. equi based on the skew of the genome (16).In E. faecalis and in S. equi, the grouping of Tn917 insertions was surprisingly strong despite the absence of an active replication termination system (23% and 60% of the insertions, respectively, fell in a region around dif that made up about 1% of the chromosome [10, 28]); in B. subtilis, insertions were found only to focus tightly in a very small region in the presence of active termination via RTP (30% of the insertions in a region around terI-terII that comprised about 1% of the chromosome) (compare Fig. 1C and D). This could suggest that an accessory termination system may be acting in E. faecalis and S. equi to actively terminate DNA replication. While dimer resolution systems have been suggested to be capable of terminating DNA replication (13), work in E. coli using 2-D electrophoresis suggests that only a very small percentage of DNA replication forks actually stall or slow around the dif site (7). It is formally possible that dimer resolution systems may show an altered ability to stall or slow replication forks in different species of bacteria.Our results firmly establish that Tn917 is capable of recognizing features of replication termination when selecting where to insert in the chromosome. Further research will be needed to determine if accessory proteins in some members of the Firmicutes alter the ability of Tn917 to preferentially target where DNA replication terminates. Alternatively, processing of replication forks following replication termination may differ in some fundamental way within the Firmicutes.     

Integration of a transposable DNA sequence which mediates ampicillin resistance into Clo DF13 plasmid DNA: determination of the site and orientation of TnA insertions.

The isolation and characterization of Clo DF13 plasmids containing a transposable DNA sequence (TnA) that specifies for ampicillin resistance is described. The particular transposon is derived from the R plasmid pRI30, and is designated Tn901. In order to determine the site and orientation of Tn901 insertions into the Clo DF13 genome, we made use of restriction endonucleases and heteroduplex mapping. For this purpose, Clo DF13 plasmid DNA and DNA of Clo DF13::Tn901 plasmids were digested with endonucleases HincII, PstI, BamH-I, SalI, and HpaI or with a combination of two of these enzymes. By analysis of the resulting fragmentation patterns, the physical maps of Clo DF13 DNA and Tn901 DNA could be derived. Furthermore, the site and orientation of Tn901 insertions into the Clo DF13 genome could be determined by this approach. The data obtained were verified by heteroduplex mapping. Analysis of 33 independently isolated Clo DF13 recombinant plasmids showed that insertion of Tn901 had occurred at 31 different sites. No preference with respect to the orientation of Tn901 was observed. Insertion of Tn901 into a segment of about 20% of the Clo DF13 genome resulted in the loss of cloacin production, indicating that the structural gene coding for cloacin is located in this area. The sites of Tn901 insertions within Clo DF13 were more or less scattered; however, no Tn901 insertion sites were found in two distinct areas comprising 11 and 17%, respectively, of the Clo DF13 genome. Transposition of Tn901 DNA to the copy mutant Clo DF13-rep3 showed that the β-lactamase activity and the minimal inhibitory concentration of ampicillin were correlated to the number of plasmid copies per cell.     

Identification of σs-dependent genes associated with the stationary-phase acid-resistance phenotype of Shigella flexneri

Shigella flexneri grown to stationary phase has the ability to survive for several hours at pH 2.5. This acid resistance, which may contribute to the low infective dose associated with shigellosis, is dependent upon the expression of the stationary-phase-specific sigma factor σ^s. Using random TnphoA and TnlacZ mutagenesis we isolated five acid-sensitive mutants of S. flexneri, which had lost their ability to survive at pH 2.5 for 2 h in vitro. Each transposon insertion with flanking S. flexneri DNA was cloned and sequenced. Database searches indicated that two TnlacZ mutants had an insertion within the hdeA gene, which is the first gene in the hdeAB operon. Acid resistance was restored in one of these mutants by a plasmid carrying the entire hdeAB operon. Further sequence analysis from the remaining TnlacZ and two TnphoA mutants demonstrated that they all had insertions within a previously unidentified open reading frame (ORF), which is directly downstream from the gadB gene. This putative ORF encodes a protein that has homology to a number of inner membrane amino acid antiporters. A 1.8 kb polymerase chain reaction (PCR) product containing this gene was cloned, which was able to restore acid resistance in each mutant. These fusions were induced during entry into late exponential phase and were positively regulated by RpoS. We confirmed that the expression of the acid-resistance phenotype in acidified minimal media was dependent upon the supplementation of glutamic acid and that this glutamate-dependent system was RpoS regulated. Southern hybridization revealed that both the gadC and hdeAB loci are absent in Salmonella. An rpoS deletion mutant of S. flexneri was also constructed to confirm the important role played by this gene in acid resistance. This rpoS ^? derivative was extremely acid sensitive. Two-dimensional gel electrophoresis of this mutant revealed that it no longer expressed 27 proteins in late log phase that were present in its isogenic parent. These data indicate that the expression of acid resistance in S. flexneri may be multifactorial and involve proteins located at different subcellular locations.     

Tellurium and Selenium Resistance in Rhizobia and Its Potential Use for Direct Isolation of Rhizobium meliloti from Soil

Forty-eight Rhizobium and Bradyrhizobium strains were screened for resistance to tellurite, selenite, and selenate. High levels of resistance to the metals were observed only in Rhizobium meliloti and Rhizobium fredii strains; the MICs were 2 to 8 mM for Te(IV), >200 mM for Se(VI), and 50 to 100 mM for Se(IV). Incorporation of Se and Te into growth media permitted us to directly isolate R. meliloti strains from soil. Mutant strains of rhizobia having decreased levels of Se and Te resistance were constructed by Tn5 mutagenesis and were found to have transposon insertions in DNA fragments of different sizes. Genomic DNAs from Te^r rhizobium strains failed to hybridize with Te^r determinants from plasmids RP4, pHH1508a, and pMER610.     

Transfer of Tn5385, a Composite, Multiresistance Chromosomal Element from Enterococcus faecalis

Tn5385 is a ca. 65-kb element integrated into the chromosomes of clinical Enterococcus faecalis strains CH19 and CH116. It confers resistance to erythromycin, gentamicin, mercuric chloride, streptomycin, tetracycline-minocycline, and penicillin via β-lactamase production. Tn5385 is a composite structure containing regions previously found in staphylococcal and enterococcal plasmids. Several transposons and transposon-like elements within Tn5385 have been identified, including conjugative transposon Tn5381, composite transposon Tn5384, and elements indistinguishable from staphylococcal transposons Tn4001 and Tn552. The divergent regions of Tn5385 are linked by a series of insertion sequence (IS) elements (IS256, IS257, and IS1216) of staphylococcal and enterococcal origin. The ends of Tn5385 consist of directly repeated copies of enterococcal IS1216. Within the chromosomes of strains CH19 and CH116, Tn5385 has interrupted an open reading frame with substantial homology to previously described alkyl hydrogen peroxide reductase genes. Segments of this open reading frame in both CH19 and CH116 have been deleted, but the amount of deleted DNA differs for the two insertions. Transfer of Tn5385 from both donors into E. faecalis recipients occurs at a low frequency. Two types of transconjugants have been identified. In one type, the target alkyl hydrogen peroxide reductase open reading frame has been deleted, and sequences flanking Tn5385 in the respective donors are carried over to the transconjugants. These data suggest that the mechanism of Tn5385 insertion into the recipient chromosome in these transconjugants was recombination across flanking regions in the donors and homologous sequences in the recipients. The second type of transconjugant appears to have resulted from excision of Tn5385 from the CH19 chromosome by recombination across the terminal IS1216 elements and insertion into the recipient chromosome by recombination across Tn5381 (within Tn5385) and a previously transferred Tn5381 copy in the recipient chromosome. These data confirm that Tn5385 is a composite structure with genetic material from diverse genera and suggest that it is a functional transposon. They also suggest that chromosomal recombination is a mechanism of genetic exchange in enterococci.