A combination of bioinformatics and functional analyses revealed a novel member of the GI-type T4SS family that plays a key role in the conjugal transfer of 89K. Throughout the past half century, investigations of T4SSs have largely focused on defining the mechanisms of action of several model systems of Gramnegative bacteria, such as the F (IncF), R388 (IncW), RP4 (IncP) and pKM101 (IncN) plasmid conjugation systems and the A. tumefaciens VirB/VirD4 system. However, little is known about the architectures or mechanisms of T4SSs in Gram-positives (Alvarez-Martinez and Christie, 2009) . Although T4SS-mediated mobilization of genetic elements has been characterized in some species of Gram-positive bacteria, such elements are currently limited to conjugative plasmids (e.g. E. faecalis pCF10, S. agalactiae pIP501 and C. perfringens pCW3) and transposons (e.g. E. faecalis Tn916, B. subtilis ICEBs1 and S. agalactiae TnGBS2). Indeed, transfer of Gram-positive GIs (especially PAIs) by T4SSs has not yet been reported. Our findings expand the repertoire of Gram-positive ICEs that can be mobilized by T4SS, and this is the first characterization of horizontal transfer of a Gram-positive PAI by a GI-type T4SS. In contrast, GI-type T4SSs have been identified in various Gram-negative GIs such as pKLC102, PAPI-1, SPI-7 and the Pseudomonas clc element, as well as others derived from a wide variety of Gram-negative plant and animal pathogens, including Erwinia carotovora (Chen et al., 2008) , Xylella fastidiosa (Zaini et al., 2008) , Photorhabdus luminescens (Held et al., 2007) and Yersinia pseudotuberculosis (Collyn et al., 2006) . The complete set of 24 genes thought to encode the putative GI-type T4SS in H. influenzae ICEHin1056 has been fully or largely identified in the GIs of the above-mentioned Gram-negative pathogens, suggesting that GI-type T4SSs are highly conserved among these species (Juhas et al., 2008) . Comparative genomic and functional analysis suggested that 89K encodes a GI-type T4SS at its 5′ terminus (05SSU0961-05SSU0982), differing in gene content and organization with the Gramnegative counterparts (Fig. S2 ). This indicates that there may be an evolutionary divergence of GI-type T4SSs between Gram-negative and Gram-positive bacteria, perhaps due to the differences in cell wall structure.