M28_Spy1325 binds to salivary agglutinin, a 340-kDa protein abundantly found in human saliva. Zhang et al. [8] recently demonstrated that immunization of mice with recombinant purified

M28_Spy1325 confers protection against invasive infection. Thus, two proteins encoded by RD2 likely contribute to host-pathogen interactions. Several lines of evidence suggest that RD2 in GAS was acquired by horizontal gene transfer (HGT). First, the RD2 element is integrated into a tRNA-threonine gene and flanked by 16bp imperfect direct repeats ATTC(C/T)CGGTGGTGGCA [1, 3]. The chromosomal location of RD2 is identical in the majority MLN4924 research buy of RD2-positive strains suggesting a conserved mode of integration [1]. Second, the G+C content of RD2 (35%) is significantly lower than the average GAS genome (38%) and contains different di-nucleotide content and codon usage [1, 3].

An RD2-like Savolitinib element also has been identified in the genome of a serotype M2 GAS strain [3]. The RD2 element in this strain is virtually identical at the nucleotide level to RD2 present in M28 strains. However, the genome sequence of strain MGAS6180 (M28) and MGAS10270 (M2) are otherwise quite divergent from one another. Based on single nucleotide polymorphism (SNPs), the average SNP difference between genomes is about 137 per 1 kb (total of 14096 SNPs), while only 8 nucleotide differences are found within 37 kb RD2 region [3, 9]. The differences in SNP frequency within chromosome and RD2 region strongly

suggests that the RD2 element in these strains has had a very different evolutionary history compared to the core chromosome, and was acquired via horizontal transfer [3]. The primary goal of the experiments described herein was to test the hypothesis that the RD2 element was laterally transferable in vitro under laboratory conditions, and we found that this was the case. Moreover, we identified an RD2-like element in multiple strains of Lancefield group C and G streptococci, indicating that this genetic element is more phylogenetically widespread than previously appreciated. Methods Bacterial strains and growth Streptococcal strains of serotypes A, C, and G (Additional File 1, Table Avelestat (AZD9668) S1) were cultured routinely at 37°C in an atmosphere of 5% CO2 on Trypticase soy agar II plates containing 5% sheep blood (Becton Dickinson, Franklin Lakes, NJ) or in liquid Todd Hewitt medium supplemented with 0.5% yeast extract (THY medium). Antibiotics were used at the following concentrations: spectinomycin, 150 μg/ml; erythromycin, 1 μg/ml; and kżanamycin, 400 μg/ml. Isolation of total DNA from AG-014699 in vitro streptococci DNA was isolated from cultures grown overnight in THY medium using a modified phenol-chloroform procedure [10]. Briefly, 5 to 35 ml of overnight THY cultures were pelleted by centrifugation and suspended in TE, pH 7.5.