Figure 1.
S. aureus extra-chromosomal DNA isolated from VISA, VRSA, MRSA, VISE and MSSA strains.
Extra-chromosomal DNA from S. aureus strains was separated by electrophoresis on a 0.7% agarose gel and stained with ethidium bromide. Numbers on top designate NRS or VRS strain designations from the NARSA repository (http://www.narsa.net/content/staphLinks.jsp): NRS numbers 18, 19, 21, 24, 26, 27, 28, 29, and 35 are VISA strains; VRS 2 and 3a are VRSA strains; NRS 70, 108, 119, 120, 123, 271 and 408 are MRSA strains; 53 is a VISE; and 104, 109, 145, 149, and 161 are MSSA strains. Separate 0.7% agarose gels profiling the extra-chromosomal isolations were combined; numbers next to the black dashes on the left next indicate the position of molecular weight standards in kb.
Figure 2.
Identification and classification of contig sequences that contain phage genes and other genetic elements from S. aureus extra-chromosomal DNA.
BLAST nucleotide sequence analysis identified the sequence identity of contigs generated from S. aureus extra-chromosomal DNA. Contigs were labeled based on homology to published nucleotide sequences: bacteriophage (black), plasmid (green), transposon/insertion sequences (IS) (blue), or not in GenBank database (n/a; purple). Contigs were labeled transposon/(IS) if a transposon or insertion element was unassociated or associated with plasmid or chromosome. Numbers above rectangular bars represent the total contigs within an individual sequencing sample. Y-axis lists total contigs identified as mobile genetic elements (MGE) based on BLAST homology identifications.
Figure 3.
Strategy for genome sequencing of φBU01 from VISA strain NRS19 and completion of a genetic map resulting in a single circular contig.
(A) Initial 454 sequencing of an unknown ExPФ in VISA strain NRS19 was assembled relative to S. aureus bacteriophage ФNM3 (NC_008617.1) template sequence. Alignment was generated using an algorithm with default settings in MacVector Version 12.6 [30], [31]. The preliminary alignments of 454 sequencing contigs (orange arrows, Table S3 in File S1, File S2) were used as a reference to generate PCR amplifications (black bars) to be used for primer walking and to determine unknown sequences. Numbers inside orange arrows designate specific contig numbers. Primers used to generate the ExPФ sequences (numbers within black bars designate PCR product) through primer walking (Sanger) are listed in Table S2 in File S1. (B) Physical map of VISA ΦBU01. Genes in orange indicate toxins encoded on the prophage, blue indicates genes associated with DNA replication and processing, and green indicates structural, miscellaneous, and hypothetical (blank arrows) genes. A list of the genes and descriptions are in Table S6 in File S1.
Figure 4.
VISA extra-chromosomal DNA contains circular ExPФ's.
Extra-chromosomal DNA of VISA strains NRS19 (ΦBU01, A and B) and NRS26 (C and D) was treated with or without Plasmid-Safe linear DNase treatment (DNase-linear, +/−), separated on a 0.7% agarose gel (B and D), and visualized with SYBR Safe DNA stain. DNA bands separated on the gel were transferred to a nylon membrane and analyzed by Southern blot using probes based on bacteriophage genes (A and C). Probes were generated by PCR amplification of genes located in bacteriophage phage DNA using primers sets bdu211/bdu212 for NRS19 (ΦBU01) and bdu215/bdu216 for NRS26 (Table S2 in File S1). MRSA strain NRS70 (70, A and C) was used as a negative control in the Southern blot.
Figure 5.
VISA ExPΦ's localize in and outside of the chromosome.
S. aureus culture samples were taken at 6, 8 and 24 hours following 1∶100 dilution of overnight cultures. Samples were concentrated to an adjusted OD600 of 5.0 to 10. Chromosomal DNA was prepared inside 0.75% agarose beads and cut with SmaI restriction digestion enzyme. DNA bands separated by PFGE (left in each set) were transferred to a nylon membrane (right in each set) and analyzed by Southern blot. (A and C) Biotin incorporated labeled probe specific to 16S DNA of S aureus DNA encoding 16S ribosomal RNA. (B) Biotin labeled probe specific to an ExPΦ found in NRS19 (ΦBU01) was used for a Southern blot probe on NRS19 cultures. (D) Biotin labeled probe specific to an ExPΦ found in NRS26 was used for a Southern blot probe on NRS26 cultures. Black arrows indicate location of bands on the Southern blots.