Tn 917 Targets the Region Where DNA Replication Terminates in Bacillus subtilis , Highlighting a Difference in Chromosome Processing in the Firmicutes

Abstract

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 SP␤ 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-GϩC 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 SP␤ Ϫ ) background using a procedure that prevented the isolation and sequencing of the s...

The Tn 3 -family of Replicative Transposons

“…Consistent with this, several Tn3-family transposons preferentially insert within or close to the replication origin of target plasmids (161,174). On the other hand, Tn917 transposition into the chromosome of Enterococcus faecalis and other Firmicutes showed a strong preference for the replication termination region (172,175,176). This region is subject to extensive processing, including numerous replication forks collapse and repair events (177)), which may provide the required transposition signal.…”

TnFLX: a third-generation mariner-based transposon system forBacillus subtilis

“…Bacillus subtilis is a model genetic organism and a number of transposon systems have been developed to aid genetic analyses. The earliest transposon system developed for B. subtilis was Tn917 which generated random insertions but exhibited a strong bias toward insertions near the chromosome’s terminus (9–11). The next system used transposon Tn 10 that improved insertion diversity and provided an easier method for insertion site identification but was still prone to insertions at particular locations or “hotspots” (12–16).…”

A mariner transposon vector adapted for mutagenesis in oral streptococci

“…An extreme regional preference for Tn917 mutagenesis has also been shown in Enterococcus faecalis and Bacillus subtilis (Shi et al. ). On the contrary, the mariner transposon system has been used in many bacterial species without any apparent insertion bias in the chromosome (Rubin et al.…”

“…; Shi et al. ). Moreover, transposon mutagenesis with ISS1 may be problematic as some Streptococcus species contains endogenous ISS1 elements that can recombine with the inserted ISS1, resulting in chromosomal deletions (Thibessard et al.…”