DNA bands separated by PFGE (left in each set) were transferred to a nylon membrane (right in each set) and analyzed by Southern blot. Chromosomal DNA was prepared inside 0.75% agarose beads and cut with SmaI restriction digestion enzyme. Samples were concentrated to an adjusted OD 600 of 5.0 to 10. aureus culture samples were taken at 6, 8 and 24 hours following 1∶100 dilution of overnight cultures. VISA ExPΦ's localize in and outside of the chromosome. MRSA strain NRS70 (70, A and C) was used as a negative control in the Southern blot. 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). 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). VISA extra-chromosomal DNA contains circular ExPФ's.Įxtra-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. A list of the genes and descriptions are in Table S6 in File S1. 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. 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. Numbers inside orange arrows designate specific contig numbers. 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. Alignment was generated using an algorithm with default settings in MacVector Version 12.6. aureus bacteriophage ФNM3 (NC_008617.1) template sequence. (A) Initial 454 sequencing of an unknown ExPФ in VISA strain NRS19 was assembled relative to S. Strategy for genome sequencing of φBU01 from VISA strain NRS19 and completion of a genetic map resulting in a single circular contig. Y-axis lists total contigs identified as mobile genetic elements (MGE) based on BLAST homology identifications. Numbers above rectangular bars represent the total contigs within an individual sequencing sample. Contigs were labeled transposon/(IS) if a transposon or insertion element was unassociated or associated with plasmid or chromosome. 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). aureus extra-chromosomal DNA.īLAST nucleotide sequence analysis identified the sequence identity of contigs generated from S. Identification and classification of contig sequences that contain phage genes and other genetic elements from S. 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. Numbers on top designate NRS or VRS strain designations from the NARSA repository ( ): 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. aureus strains was separated by electrophoresis on a 0.7% agarose gel and stained with ethidium bromide. aureus extra-chromosomal DNA isolated from VISA, VRSA, MRSA, VISE and MSSA strains.Įxtra-chromosomal DNA from S.
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