GI-type T4SS-mediated horizontal transfer of the 89K pathogenicity island in epidemic Streptococcus suis serotype 2

Abstract

Pathogenicity islands (PAIs), a distinct type of genomic island (GI), play important roles in the rapid adaptation and increased virulence of pathogens. 89K is a newly identified PAI in epidemic Streptococcus suis isolates that are related to the two recent large-scale outbreaks of human infection in China. However, its mechanism of evolution and contribution to the epidemic spread of S. suis 2 remain unknown. In this study, the potential for mobilization of 89K was evaluated, and its putative transfer mechanism was investigated. We report that 89K can spontaneously excise to form an extrachromosomal circular product. The precise excision is mediated by an 89K-borne integrase through site-specific recombination, with help from an excisionase. The 89K excision intermediate acts as a substrate for lateral transfer to non-89K S. suis 2 recipients, where it reintegrates site-specifically into the target site. The conjugal transfer of 89K occurred via a GI type IV secretion system (T4SS) encoded in 89K, at a frequency of 10(-6) transconjugants per donor. This is the first demonstration of horizontal transfer of a Gram-positive PAI mediated by a GI-type T4SS. We propose that these genetic events are important in the emergence, pathogenesis and persistence of epidemic S. suis 2 strains.

Fig. 1

Genetic map of the 89K PAI in the S. suis 2 epidemic strain 05ZYH33. A. Aberrant average G+C content of 89K, which is much less than that of the chromosome. B. Gene organization of 89K. The terminal direct repeats attL and attR are indicated by red triangles. Genes predicted to likely be involved in the mobilization of 89K are shown in yellow. For more gene details, see Chen et al. (2007).

Fig. 2

Site-specific integration and excision of 89K in the chromosome of S. suis 05ZYH33. A. Schematic representation of the site-specific integration and excision of 89K. The left and right junctions (attL and attR), which are shown as red rectangles, are formed by recombination between the chromosomal attB site and the 89K attP site. The flanking genes are shown by solid arrows indicating the direction of transcription. The location and orientation of primers used for detection of integrated and excised 89K are indicated by thin arrows. B. Detection of a circular extrachromosomal form of 89K and the empty chromosomal attB site following precise excision, by PCR analysis using primer pairs which are presented upon the lanes, with 05ZYH33 genomic DNA as the template. C. Printout of the sequencing chromatogram of PCR products amplified with primer pair P2/P3, showing the attP site (boxed) formed by the joining of the two ends of 89K. D. Printout of the sequencing chromatogram of PCR products amplified with primer pair P1/P4, showing the empty chromosomal attB site (boxed) upon 89K excision.

Fig. 3

Int and Xis are required for efficient 89K excision. A. Genetic organization of the 3′ terminal region of 89K in S. suis 05ZYH33. The int gene and the upstream xis gene are shown as grey arrows (not drawn to scale). A putative ribosome binding site (RBS) upstream of xis is depicted by a small flag. The stem-loop structure represents a putative transcriptional terminator. The position of primer P5 is shown. B. PCR amplicons (indicated left) obtained from the wild-type, mutant and complemented strains (shown above each lane). The gyrA gene serves as an internal control.

Fig. 4

Functional organization of the DNA-processing region of 89K. The mobilization genes (mobA89K and mobC89K) involved in the DNA processing are marked by open arrows (not drawn to scale). The shaded regions indicate the conserved domains encoded therein, with the internal consensus motifs indicated by black rectangles. The asterisk represents the conserved motif (L/FxxxG/SxNxNQxAxxxN) within the MobC family. The 89K oriT was predicted to be positioned within the 05SSU0915 ORF, which encodes a hypothetical protein of unknown function. The direction of transfer is shown by a horizontal arrow. The lower portion of this figure shows the alignment of the 89K oriT site with other oriT regions of several mobilizable plasmids (indicated left). Arrows above the sequences represent the locations of inverted repeats, and vertical arrows show experimentally determined nick sites.

Fig. 5

Site- and strand-specific relaxation of the 89K oriT region. A. Physical map of the pMD-oriT plasmid, which contains the oriT region of 89K and serves as substrate DNA in the relaxation analysis. The putative oriT nick site (nic) is indicated by a vertical arrow. B. Equivalent substrate pMD-oriT plasmid was incubated with (+) or without (–) the proteins of interest. Reaction products were analysed by standard agarose gel electrophoresis (0.9%). OC, open circular plasmid DNA; CCC, covalently closed circular plasmid DNA. C. Schematic representation of the expected single-strand species generated by a site-specific nick (arrow) on endonuclease-linearized pMD-oriT DNA. The size of each ssDNA fragment is shown. D. pMD-oriT was linearized with either ScaI or NdeI, and the relaxation mixtures were analysed on a 0.9% alkaline agarose gel. Lanes: 1, ScaI-linearized pMD-oriT with MobA89K and MobC89K; 2, NdeI-linearized pMD-oriT with MobA89K and MobC89K; 3, ScaI-linearized pMD-oriT with MobA89KN258 and MobC89K; 4, NdeI-linearized pMD-oriT with MobA89K and MobC89K. The 1 kb DNA ladder marker is shown to the right (M).

Fig. 6

Hypothetical model for the function of the 89K GI-type T4SS. The 89K-borne integrase mediates precise excision of 89K from the chromosome with the aid of excisionase. After receiving a mating signal, the transfer initiates with the oriT nicking reaction catalysed by the MobA89K and MobC89K proteins. The cleaved strand is then unwound by the helicase and delivered to the 89K-encoded GI-type T4SS machinery. The VirB1-89K component is thought to act as a lytic transglycosylase, locally opening the peptidoglycan with contributions from some other factor(s) during the assembly of the transport channel. VirD4-89K is a coupling protein required to deliver the DNA substrate to the transport channel, and together with VirB4-89K, required to energize the substrate transport across the channel. The highly hydrophobic VirB6-89K homologue constitutes the main portion of the transport channel.