Lateral gene transfer of streptococcal ICE element RD2 (region of difference 2) encoding secreted proteins

Abstract

Background: The genome of serotype M28 group A Streptococcus (GAS) strain MGAS6180 contains a novel genetic element named Region of Difference 2 (RD2) that encodes seven putative secreted extracellular proteins. RD2 is present in all serotype M28 strains and strains of several other GAS serotypes associated with female urogenital infections. We show here that the GAS RD2 element is present in strain MGAS6180 both as an integrative chromosomal form and a circular extrachromosomal element. RD2-like regions were identified in publicly available genome sequences of strains representing three of the five major group B streptococcal serotypes causing human disease. Ten RD2-encoded proteins have significant similarity to proteins involved in conjugative transfer of Streptococcus thermophilus integrative chromosomal elements (ICEs).

Results: We transferred RD2 from GAS strain MGAS6180 (serotype M28) to serotype M1 and M4 GAS strains by filter mating. The copy number of the RD2 element was rapidly and significantly increased following treatment of strain MGAS6180 with mitomycin C, a DNA damaging agent. Using a PCR-based method, we also identified RD2-like regions in multiple group C and G strains of Streptococcus dysgalactiae subsp.equisimilis cultured from invasive human infections.

Conclusions: Taken together, the data indicate that the RD2 element has disseminated by lateral gene transfer to genetically diverse strains of human-pathogenic streptococci.

Figure 1

Comparison of the organization of RD2 ORFs in diverse GAS and GBS strains. Slanted red lines indicate discontinuity between fragments homologous to RD2 element present in MGAS6180. RD2 open reading frames are marked as white rectangles, open reading frames of GBS are color coded according to their homology to RD2 on the protein level. Numbers within each rectangle represent ORF designation number in particular GBS strain

Figure 2

RD2 encodes homologues of conjugative transfer genes present in the ICESt1 and ICESt3 elements of S. thermophilus.

Figure 3

Detection of RD2 transfer from donor strain MGAS6180 (emm28) to recipient strain MGAS10750 (emm4). Amplicons 1-12 generated by PCR tiling across the RD2 element. A. transconjugant; * denote amplicons encompassing deleted M28_Spy1325-1326 region that is replaced by spectinomycin resistance cassette; B. control with chromosomal DNA isolated from strain MGAS6180. M - 1 kb ladder (Invitrogen)

Figure 4

PCR screen detects multiple or circular copy of RD2. A. Primer combinations used for detection of seven potential arrangements of RD2. Thick black arrows represent RD2 element; thin gray line represents the chromosome. Red, purple, blue, and green arrows represent locations of primer #1, #2, #3, and #4, respectively. Potential arrangement variants are: arrangement as determined by genome sequencing [1], at least two head-to-tail copies of RD2, tail-to-tail, single copy arranged in reverse orientation than the integrative copy determined by genome sequencing, head-to-tail arrangement of copies in reverse orientation than the integrative copy determined by genome sequencing, head-to-head, and circular form. B. PCR screen detects product amplified with primer pairs #1+#2, #3+#4, and #2+#3, corresponding to arrangement variant (head-to-tail) or (circular form), and #1+#4 detecting chromosomal integration site lacking RD2. C. Primers #2+#3 detect arrangement variant 2 or 7 in multiple RD2 positive strains [1]. Serotype M1 strain MGAS5005 (lacks RD2) was used as a negative control of amplification.

Figure 5

Mitomycin C treatment results in amplification of RD2. A Rapid decrease in O.D. of a liquid culture of strain MGAS6180 after mitomycin C addition. The decreased O.D. is likely due to prophage induction followed by lytic cycle phage release. Smaller drop in OD is observed after treatment with hydrogen peroxide. B. The RD2 element is present in 6-9 copies per chromosome in the absence of inducer. C. The RD2 element is not induced by oxidative stress. Bars in each group represent the RD2 copy number after 1 h, 2 h, 3 h, and 16 h after treatment with hydrogen peroxide. D. RD2 is induced by DNA damage. Bars in each group represent the increase in copy number at 1 h, 2 h, 3 h, and 16 h after treatment with mitomycin C. The statistical significance of the increase in RD2 copy number was determined by t-test, *** on the graph denotes p value below 0.001.