14 research outputs found

    The 5′ recombination sites in SD repeat regions of different variants.

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    A<p>: The DNA before slash is homologous sequence located at 5′ of two recombination sites of the mutant.</p>B<p>: The nucleotides at 5′ of two recombination sites are not totally homologous in this mutant.</p

    Instability of SD repeats shown by restriction enzyme analyses.

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    <p>(A) pNZ3004-ClfA.SAR.rRWM.C9 plasmid map showed locations of the restriction enzymes and sizes of different segments. (B) After 3 rounds of propagation, instability of constructs pNZ3004-ClfA.SAR.rRWM.C9.C1 was detected by restriction enzyme analyses. Lane 1: Uncut plasmid; Lane 2: Digested by <i>Bam</i>HI; Lane 3: Digested by <i>Bam</i>HI/<i>Sal</i>I; Lane 4: Digested by <i>Pst</i>I; Lane 5: Digested by <i>Sal</i>I; Lane 6: Digested by <i>Eco</i>RI; Lane 7: <i>Bam</i>HI restriction enzyme activity control. Plasmid PF01 digested by <i>Bam</i>HI; Lane 8: ultraRanger 1 kb DNA ladder (Kb). Arrows A and B indicated the incomplete digestion by <i>Bam</i>HI and the failed double digestion by <i>Bam</i>HI/<i>Sal</i>I, respectively. Arrow C showed the mixture of the shorter plasmid DNA. Arrow D revealed the rearrangement in the region between two <i>Pst</i>I sites. (C) The rearranged variants from the transformants of pNZ3004-ClfA.SAR.rRWM.C9.C7 digested by <i>Pst</i>I. Lane 1: pNZ3004-ClfA.SAR.rRWM.C9.C7.C1 (deletion in SD repeats between SAR and rRWM). Lane 2: pNZ3004-ClfA.SAR.rRWM.C9.C7.C2 (deletion in SD repeats between SAR and rRWM). Lane 3: pNZ3004-ClfA.SAR.rRWM.C9.C7.C3 (deletion in SD repeats between SAR and rRWM). Lane 4: pNZ3004-ClfA.SAR.rRWM.C9.C7.C4 (deletion in SD repeats between SAR and rRWM). Lane 5: pNZ3004-ClfA.SAR.rRWM.C9.C7.C5 (deletion in SD repeats between SAR and rRWM). Lane 6: pNZ3004-ClfA.SAR.rRWM.C9.C7.C6 (deletion in SD repeats between SAR and rRWM). Lane 7: pNZ3004-ClfA.SAR.rRWM.C9.C7.C7 (expansion in SD repeats between SAR and rRWM). Lane 8: pNZ3004-ClfA.SAR.rRWM.C9.C7.C8 (deletion in SD repeats between SAR and rRWM). Lane 9: pNZ3004-ClfA.SAR.rRWM.C9.C7.C9 (no rearrangement). Lane10: pNZ3004-ClfA.SAR.rRWM.C9.C7.C10 (deletion in SD repeats between SAR and rRWM). (D) The rearranged variants from the transformants of pNZ3004-ClfA.SAR.rRWM.C9.C7 double-digested by <i>Bam</i>HI and <i>Sal</i>I. Lane 1: pNZ3004-ClfA.SAR.rRWM.C9.C7.C1 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 2: pNZ3004-ClfA.SAR.rRWM.C9.C7.C2 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 3: pNZ3004-ClfA.SAR.rRWM.C9.C7.C3 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 4: pNZ3004-ClfA.SAR.rRWM.C9.C7.C4 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 5: pNZ3004-ClfA.SAR.rRWM.C9.C7.C5 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 6: pNZ3004-ClfA.SAR.rRWM.C9.C7.C6 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 7: pNZ3004-ClfA.SAR.rRWM.C9.C7.C7 (SAR segment increase, may have expansion in SD repeats of SAR). Lane 8: pNZ3004-ClfA.SAR.rRWM.C9.C7.C8 (deletion in SD repeats of SAR). Lane 9: pNZ3004-ClfA.SAR.rRWM.C9.C7.C9 (no rearrangement). Lane10: pNZ3004-ClfA.SAR.rRWM.C9.C7.C10 (rearrangement occurred between SAR and rRWM, lost <i>Bam</i>HI site). Lane 11: ultraRanger 1 kb DNA ladder (Kb).</p

    The definition of perfect and imperfect SD repeats.

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    <p>The TRs sequence “SDSDSD” was defined as a perfect consensus and an imperfect consensus was defined by the repeat contains 1–3 residues which did not follow the consensus “SDSDSD” sequence. Both perfect consensus and imperfect consensus repeats were counted as SD repeats.</p

    DNA sequence characterization of <i>clfA</i> SD repeats of <i>S. aureus</i> Smith Cp and the locations of rearranged sites from 9 variants of pNZ3004-ClfA.rRWM and 1 variant of pNZ3004-ClfA.SAR.rRWM.C9.

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    <p>The number at two sides represents the nucleotide number. The perfect consensus repeats are marked in red and the imperfect consensus repeats are marked in black. The repeat right after 15 central perfect consensus repeats has only 12 nucleotides, as this arrangement makes the whole repeats maximally conform the consensus. Each rearranged site in 9 variants of pNZ3004-ClfA.rRWM was marked by the name of variants in parentheses in green, with the region between them looped out during the rearrangement. For 1 variant of pNZ3004-ClfA.SAR.rRWM.C9, 5 SD repeats were added between the two arrangement sites and were not shown in the figure, marked by the name of the variant in parentheses in blue.</p

    Primers used in this study.

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    <p>Primers used in this study.</p

    SD repeat variations in the surface proteins of different bacteria strains.

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    A<p>:The detected proteins available in the Reference, Swissprot and Non-redundant protein sequences database (accessed on August 3<sup>rd</sup>, 2011). The number in the parentheses is the number of strains which contain same protein.</p>B<p>:The variation site in the SD repeats, the letters represent amino acids single-letter codes (SLC).</p>C<p>:The distribution and number of variation sites in the proteins. N, C, M and W represent the mutations in the N terminus, C terminus, middle, or whole sequences of the SD repeats, respectively.</p

    A schematic diagram of <i>S. aureus</i> ClfA organization and the insert fragments in the constructs.

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    <p>S: the signal sequence; A: the binding domain; R: the SD repeat region; W: the wall-spanning region; M: the membrane-spanning region. ClfA.RWM: the segment RWM of ClfA from <i>S. aureus</i> Smith Cp which was cloned into pNZ3004. ClfA.SA.rRWM: the inserted portion of pNZ3004-ClfA.SA.rRWM after cloning the segment SA of ClfA from <i>S. aureus</i> Smith Cp cloned into pNZ3004-ClfA.rRWM. ClfA.SAR.rRWM: the inserted portion of pNZ3004-ClfA.SAR.rRWM after cloning the segment SAR of ClfA from <i>S. aureus</i> Smith Cp into pNZ3004-ClfA.rRWM.</p

    Plasmids and their relevant features in this study.

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    A<p>: The copy numbers of the SD repeats were same for the following plasmids: pNZ3004-ClfA.SAR.rRWM.C9.C1 and pNZ3004-ClfA.SAR.rRWM.C9.C3 to pNZ3004-ClfA.SAR.rRWM.C9.C10.</p

    Image_1_Arginine Catabolic Mobile Elements in Livestock-Associated Methicillin-Resistant Staphylococcal Isolates From Bovine Mastitic Milk in China.TIF

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    <p>The arginine catabolic mobile element (ACME) facilitates colonization of staphylococci on skin and mucous membranes by improving their tolerances to polyamines and acidic conditions. ACME is inserted in tandem with the SCCmec element and Staphylococcus epidermidis has been proposed to be a reservoir of ACME for other staphylococci. In this study, we investigated the existence of ACME in 146 staphylococcal isolates from mastitic milk and found 21 of them carried ACME. Almost half of the investigated S. epidermidis isolates contained the element. The whole genome of a S. epidermidis strain Y24 with ACME was further sequenced and the ACME-SCCmec composite island was assembled. This composite island is 81.3 kb long and consisted of 77 ORFs including a methicillin resistance gene mecA, a type II’ ACME gene cluster, a virulence gene pls and eight heavy metal tolerance genes. Wide existence of ACME in livestock-associated staphylococci from this study and a potential risk of spreading ACME among different staphylococcal species warrant close monitoring and further studies.</p

    Table_1_Arginine Catabolic Mobile Elements in Livestock-Associated Methicillin-Resistant Staphylococcal Isolates From Bovine Mastitic Milk in China.DOCX

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    <p>The arginine catabolic mobile element (ACME) facilitates colonization of staphylococci on skin and mucous membranes by improving their tolerances to polyamines and acidic conditions. ACME is inserted in tandem with the SCCmec element and Staphylococcus epidermidis has been proposed to be a reservoir of ACME for other staphylococci. In this study, we investigated the existence of ACME in 146 staphylococcal isolates from mastitic milk and found 21 of them carried ACME. Almost half of the investigated S. epidermidis isolates contained the element. The whole genome of a S. epidermidis strain Y24 with ACME was further sequenced and the ACME-SCCmec composite island was assembled. This composite island is 81.3 kb long and consisted of 77 ORFs including a methicillin resistance gene mecA, a type II’ ACME gene cluster, a virulence gene pls and eight heavy metal tolerance genes. Wide existence of ACME in livestock-associated staphylococci from this study and a potential risk of spreading ACME among different staphylococcal species warrant close monitoring and further studies.</p
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