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1.
FIG. 1.

FIG. 1. From: Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease Spirochete Borrelia burgdorferi .

Shuttle vector transformations. Schematic representation of the various DNA sources, strains and methods used to assess the contributions of bbe02 and bbq67 to the restriction-modification (R-M) systems of B. burgdorferi.

Ryan O. M. Rego, et al. J Bacteriol. 2011 March;193(5):1161-1171.
2.
FIG. 2.

FIG. 2. From: Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease Spirochete Borrelia burgdorferi .

Transformation of B. burgdorferi with shuttle vector DNA from E. coli. Transformation frequencies of B. burgdorferi strains used in this study with shuttle vector DNAs isolated from E. coli (dam+ dcm+ hsd) were calculated by determining the proportion of viable bacteria after electroporation that contained the shuttle vector. The shuttle vectors used in each transformation are identified below the bars. Recipient B. burgdorferi strain genotypes are distinguished by the different bars identified to the right of the graph (+, intact gene; Δ, gene deletion; −, missing plasmid). An asterisk denotes the limit of detection when no transformants were obtained from a defined number of viable bacteria. The percentage of transformants retaining lp25 when initially present in the recipient clone is indicated in parentheses above the bars.

Ryan O. M. Rego, et al. J Bacteriol. 2011 March;193(5):1161-1171.
3.
FIG. 5.

FIG. 5. From: Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease Spirochete Borrelia burgdorferi .

Adenine methylation of DNA by B. burgdorferi clones harboring bbe02 or bbq67. (A) RsaI digestion products of total plasmid DNA prepared from B. burgdorferi and E. coli (dam+ dcm+ hsd) carrying the shuttle vector pKFSS1, separated by gel electrophoresis, and stained with GelRed. (B) Southwestern blot analysis of plasmid DNA from B. burgdorferi and E. coli. The gel shown in panel A was transferred to a membrane and incubated with a polyclonal rabbit antiserum that recognizes methylated adenine (N6-A). The pertinent bbe02 and bbq67 genotypes of B. burgdorferi that served as sources of plasmid DNA are identified beneath the lanes (+, intact gene; Δ, gene deletion; −, missing plasmid). Lane 1, A3; lane 2, A3ΔBBE02; lane 3, A3-68; lane 4, A3-68ΔBBE02; lane 5, E. coli (Ec) (dam+ dcm+ hsd).

Ryan O. M. Rego, et al. J Bacteriol. 2011 March;193(5):1161-1171.
4.
FIG. 3.

FIG. 3. From: Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease Spirochete Borrelia burgdorferi .

Influence of shuttle vector DNA methylation by E. coli on transformation of B. burgdorferi. B. burgdorferi clones were transformed with pBSV2 shuttle vector DNA prepared from E. coli containing or lacking Dam, Dcm, and Hsd methyltransferases. Recipient B. burgdorferi strain genotypes are indicated below the bars (+, intact gene; Δ, gene deletion; −, missing plasmid). Strain genotypes for the E. coli DNA donors are distinguished by the different bars identified to the right of the graph. Transformation frequency was calculated by determining the proportion of viable bacteria after electroporation that contained the shuttle vector. An asterisk denotes the limit of detection when no transformants were obtained from a defined number of viable bacteria. The absence of lp25 in all recovered transformants is indicated (zero), although lp25 was present in >90% of viable bacteria after electroporation. Transformations were performed twice for recipients containing both bbe02 and bbq67 or only bbq67 and once for all other recipients, in parallel with pBSV2 DNA from all three E. coli sources.

Ryan O. M. Rego, et al. J Bacteriol. 2011 March;193(5):1161-1171.
5.
FIG. 4.

FIG. 4. From: Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease Spirochete Borrelia burgdorferi .

Influence of putative restriction-modification systems in B. burgdorferi on the transformation efficiency of pKFSS1 shuttle vector DNA prepared from B. burgdorferi. Three different B. burgdorferi clones, containing or lacking the bbe02 and bbq67 loci, were transformed by electroporation with B. burgdorferi plasmid DNA prepared from three different B. burdorferi clones containing the shuttle vector pKFSS1. The transformation efficiency for pKFSS1 was calculated for the fraction of total B. burgdorferi plasmid DNA that it represents. The pertinent bbe02 and bbq67 genotypes of both donor and recipient B. burgdorferi are distinguished by shading beneath the bars (plasmid source) and shading of the bars (plasmid recipient) identified to the right of the graph (+, intact gene; Δ, gene deletion; −, missing plasmid). The transformation efficiency of in vitro-methylated (M.SssI) pKFSS1 DNA from E. coli (dam+ dcm+ hsd) is included for comparison. Transformations for all donor-recipient combinations were carried out at least twice except for the following, which was carried out once: donor A3Δbbe02/recipient A3. Plasmids lp25 and lp56 were retained in all transformants when initially present in the recipient. The transformation efficiency of pKFSS1 from clone A3 into all recipients is also presented in Table .

Ryan O. M. Rego, et al. J Bacteriol. 2011 March;193(5):1161-1171.
6.
FIG. 7.

FIG. 7. From: Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease Spirochete Borrelia burgdorferi .

Differences between shuttle vectors and sequences surrounding cleaved versus protected RsaI sites. (A) Schematic diagram depicting the differences between the three shuttle vectors used in this study. ▴, protected RsaI restriction sites on pBSV2G; Δ, unprotected RsaI restriction site on pBSV2G; IR1, inverted repeat 1; ColE1, E. coli origin of replication; MCS-LacZ, alpha-peptide and multiple-cloning site (MCS) of pUC18; Orf1, Orf2, and Orf3, plasmid replication genes from cp9 of B. burgdorferi; Zeo, zeocin resistance marker; Kan, kanamycin resistance gene; Spec, spectinomycin-streptomycin resistance gene (aadA); Gent, gentamicin resistance gene (aacC1). (B) Alignment of nucleotide sequences surrounding the protected and unprotected RsaI sites (unique and shared) on shuttle vectors pBSV2G, pKFSS1, and pBSV2. Nucleotides that are identical to those in the top line are indicated by periods. The top line shows the protected RsaI site in the MCS shared by all three shuttle vectors. The RsaI restriction site, GTAC, is shown in bold type and shaded. The underlined sequence, TCGA, indicates the conservation of this sequence upstream of the RsaI cleavage site in both protected sequences.

Ryan O. M. Rego, et al. J Bacteriol. 2011 March;193(5):1161-1171.
7.
FIG. 6.

FIG. 6. From: Defining the Plasmid-Borne Restriction-Modification Systems of the Lyme Disease Spirochete Borrelia burgdorferi .

Protection of an RsaI site on pKFSS1 by BBQ67. (A) RsaI digestion products of total plasmid DNA from B. burgdorferi and E. coli transformants carrying the shuttle vector pKFSS1, separated by gel electrophoresis, and stained with GelRed. The migration positions of size standards (in base pairs) are indicated to the right of the gel. (B) Southern blot analysis of RsaI-digested plasmid DNA from B. burgdorferi and E. coli. The gel shown in panel A was transferred to a membrane and hybridized with labeled pKFSS1 fragments. The pertinent bbe02 and bbq67 genotypes of B. burgdorferi that served as sources of plasmid DNA are identified beneath the gels (+, intact gene; Δ, gene deletion; −, missing plasmid). Lanes 1, A3; lanes 2, A3ΔBBE02; lanes 3, A3-68; lanes 4, A3-68ΔBBE02; lanes 5, E. coli (dam+ dcm+ hsd); lane 6, 1-kb Plus DNA ladder (Invitrogen). The numbering of pKFSS1 RsaI fragments from E. coli corresponds with the numbering in the schematic diagram in panel C. The arrow identifies the pKFSS1 fragment that results when cleavage of a particular RsaI site is blocked. (C) Schematic diagram of the shuttle vector pKFSS1. The RsaI sites are indicated, and fragments are numbered for comparison with the restriction profiles in panels A and B. The RsaI site indicated in bold type is protected from cleavage on plasmid DNA from B. burgdorferi carrying bbq67.

Ryan O. M. Rego, et al. J Bacteriol. 2011 March;193(5):1161-1171.

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