Conjugative Transfer of the Integrative and Conjugative Element ICEBs1 from Bacillus subtilis Likely Initiates at the Donor Cell Pole

Mobile genetic elements (MGEs) are ubiquitous in bacteria. They contain genes for antibiotic resistance, symbiosis, and virulence, and their dissemination plays an important role in bacterial evolution by conferring new genes and phenotypes to their recipients. The most common MGEs are phages, plasmids, and integrative and conjugative elements (ICEs), also known as conjugative transposons. Conjugative plasmids and ICEs generally encode their own conjugation systems and are transferred from cell to cell via direct cell-cell contact (16). ICEBs1 is an ICE found integrated into the leucine tRNA gene of Bacillus subtilis strains (3, 7). When induced, ICEBs1 excises from the chromosome and can transfer to recipient cells. Gene expression and excision are induced by the SOS response or when cells are at high density surrounded by cells lacking a copy of ICEBs1. Regulation by population density is mediated by the regulator RapI and the pentapeptide PhrI (3). In this issue of the Journal of Bacteriology, Berkmen et al. (4) report that a fully functional ConE, a protein of the HerA/FtsK superfamily of ATPases, is required for ICEBs1 conjugation. A ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one additional ICEBs1 gene product was needed to target ConE to the membrane and cell poles. When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell, whereas following excision, the ICE was more often detected near a cell pole. Excision of ICEBs1 also resulted in altered subcellular positioning of the replisome.

Similarities with other ICEs.

Tn916, found in several different gram-positive cocci, is the smallest, least complex, and best-characterized ICE. The closest relative of Tn916 (similar gene organization with approximately 30% sequence identity between common genes) is ICEBs1 (8). A comparison of the gene organization of these two ICEs with that of ICE6013 and Tn5801 from Staphylococcus aureus is shown in Fig. 1.

FIG. 1.

Comparison of the gene organization of ICEBs1 with other MGEs. The direction of transcription for each predicted ORF is shown with large arrows. oriT_ICEBs1 is indicated by a vertical line within the nicK coding region, oriT_Tn916 by a vertical line between the orf20 and orf21 coding region (15). The boxed region in ICE6013 indicates a Tn552 insertion. Colored arrows indicate that the ICEBs1, Tn916, or Tn5801 ORF matches the corresponding ICE6013 ORF in pairwise BLASTP comparisons (modified with permission from reference 21).

Putative functions of proteins encoded by ICEBs1.

For several proteins likely involved in conjugative transfer of ICEBs1, putative functions have been assigned. Lee and Grossman identified the ICEBs1-encoded relaxase, YdcR, renamed NicK (16). NicK is homologous to the pT181 family of plasmid relaxases (16) and shows similarity to the predicted relaxase Orf12, encoded by ICE6013 (Fig. 1) (21). The Grossman group found that transfer of ICEBs1 requires nicK and identified a cis-acting oriT that is also necessary for transfer (16). Expression of nicK results in nicking of ICEBs1 between a GC-rich inverted repeat in oriT. NicK was the only protein needed for nicking. The oriT nic-site is located within the nicK open reading frame (16). Fukushima and coworkers identified and characterized the cell wall hydrolase, YddH, renamed CwlT (11). CwlT has two hydrolase domains, a dl-endopeptidase domain at its C terminus and an N-acetylmuramidase domain at its N terminus (11). Both activities might help locally open the B. subtilis peptidoglycan to facilitate conjugative transfer of ICEBs1 to recipient cells. YdcQ shows similarities to FtsK-like proteins, and it is predicted to act as the coupling protein in the ICEBs1 transport apparatus. It could serve to link processed donor DNA to the mating apparatus. YddE, renamed ConE (4), is most similar to Orf8 of ICE6013 (Fig. 1). It shows a VirB4 domain across its C-terminal half (21). VirB4 proteins are thought to deliver energy for substrate transport in the conjugative DNA transfer process by NTP-hydrolysis. ICEBs1 appears to encode homologues of all the type IV secretion system key players found in the transfer region of the conjugative plasmids pIP501/pRE25 and pSK41/pGO1 from gram-positive organisms (1, 14).

Regulation of conjugative transfer of ICEBs1: similarities with bacteriophages.

Proper maintenance of genomic content is a major task for all organisms. A variety of cellular processes are devoted to reliably replicating and segregating completed genomes prior to cell division. Different mechanisms have evolved to increase the chances of survival by monitoring and responding to the status of genome integrity. When replication does not work properly, these mechanisms are activated (12, 13). Their role is to repair the DNA replication defect and to delay subsequent cell cycle events (e.g., see references 6 and 10). One of the best-known mechanisms that detect DNA damage and replication arrest is the bacterial SOS response (13). There are two regulatory components to this response: RecA and LexA. Goranov and coworkers measured the global transcriptional response to DNA damage and perturbations in replication on mRNA levels of B. subtilis by using whole-genome DNA microarrays. They found that many genes from lysogenic phages and from ICEBs1 required recA but not lexA for transcriptional induction (13). These results are consistent with reports from Auchtung et al. (3) and McVeigh and Yasbin (17) that showed that the induction of SPβ and ICEBs1 is recA dependent. It is thought that this regulation allows the MGE a better chance of survival by enabling it to leave if the host cell is being damaged (13).

ICEBs1 is normally propagated by the host cell through chromosomal replication and cell division. When the host cell undergoes DNA damage or cells harboring the element are surrounded by cells lacking ICEBs1, it can excise from the chromosome and transfer to recipient cells (2). Most of the genes in ICEBs1 are located downstream from xis, the gene for the excisionase (Fig. 1). Transcription of most of these genes appears to be coregulated (3). Intercellular peptide signaling also regulates transcription of xis through yddJ, as well as excision and transfer of the element (3). This regulation is mediated by the rapI-phrI signaling cassette present in ICEBs1; it occurs independently of the global DNA damage response. RapI stimulates expression of xis through yddJ, and this stimulatory activity is antagonized by the secreted signaling peptide PhrI. PhrI is a pentapeptide that is reimported by the oligopeptide permease (3).

ICEBs1 also encodes a repressor, ImmR. The Grossman group found that ImmR represses transcription of the promoter that initiates expression of xis (2). This repression is responsible for preventing excision of ICEBs1 and expression of most of its genes (xis through yddJ). These properties of ImmR are similar to those of other repressor proteins from many MGEs, including coliphage lambda (9, 18, 20). Additionally, ImmR functions as an immunity repressor. This means that when it is expressed in a potential recipient, it greatly decreases the frequency of acquisition of the element via conjugation. Thus, the acquisition of additional copies of ICEBs1 by cells already containing the element is limited. ImmR-mediated immunity appears to occur by limiting expression or activity of the integrase Int (2).

Bose and coworkers detected that an ICEBs1-encoded antirepressor ImmA controls horizontal transfer of ICEBs1 by proteolysis of the repressor ImmR (5). ImmR is rapidly degraded in vivo when antagonized by ImmA. Repressor and antirepressor interacted directly in a yeast two-hybrid assay. Purified ImmA caused in vitro cleavage of purified ImmR, likely by site-specific cleavage, resulting in ImmR degradation and derepression of ICEBs1 in vivo (5). Homologues of immA and immR are found in many other MGEs; e.g., an immA homologue was found in the B. subtilis phage Φ105 (5), and immA and immR homologues were detected in a putative MGE in the genome of the vancomycin-resistant Enterococcus faecalis strain V583 (19).

Localization of conjugative transfer proteins in gram-positive bacteria.

Localization of the putative ATPase ConE, a VirB4 homologue encoded by ICEBs1, to the B. subtilis cell pole by the Grossman laboratory (4) presents a milestone in deciphering the conjugative transfer mechanism of MGEs in gram-positive bacteria. Only two conjugative transfer proteins from gram-positive bacteria have been localized so far: the VirB6-like protein TcpH and the putative ATPase TcpF, encoded by the Clostridium perfringens plasmid pCW3. By using immunofluorescence microscopy, the Julian Rood laboratory showed them to be located at both poles of C. perfringens donor cells (22). This indicates that the mating-pair formation complex also appears to be located at the cell poles (22). Attempts to localize the conjugative transfer proteins required for pIP501-mediated conjugation in Enterococcus are in progress (K. Arends and E. Grohmann, unpublished data).

Perspectives.

The elucidation of the regulation of the ICEBs1 conjugative transfer and the mechanism by which the excised MGE is transferred to recipient cells could provide us with valuable tools to simplify the genetic analysis of gram-positive bacteria. Additionally, together with studies on conjugative plasmids from gram-positive bacteria, such as pIP501 and pCW3, the work on ICEBs1 will aid in understanding the mechanisms of conjugative DNA transfer in gram-positive bacteria.