Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: In vitro susceptibilities of planktonic S. mutans UA159

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: In Vitro

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Comparative killing kinetics of CLP-4. S. mutans UA159 cultures at a cell density of 6 × 105 CFU/ml were challenged with 5, 10, and 25 μg/ml CLP-4 under conditions of active growth in CDM supplemented with 0.5% (wt/vol) glucose (A) and against growth-arrested cells in CDM lacking any carbon source (B). Samples at time zero were enumerated prior to peptide treatment. Data shown are the means and standard deviations of three biological replicates from three independent experiments.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques:

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: CLP-4 prevents S. mutans biofilm formation. (A) Biofilms inoculated with 2 × 107 CFU/ml were grown for 24 h in the presence of CLP-4, chlorhexidine, or erythromycin at concentrations ranging between 0.6× and 2× their respective MICs. Biofilm formation was quantified using crystal violet staining and expressed in percentage relative to untreated control. Shown are the means and standard deviations of three biological replicates from three independent experiments. *, P < 0.05; ***, P < 0.001 compared to untreated control. (B) Corresponding growth curve kinetics showing the MIC of CLP-4 on S. mutans UA159.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Staining, Control

Journal: Antimicrobial Agents and Chemotherapy

Article Title: Antibacterial and Antibiofilm Activities of a Novel Synthetic Cyclic Lipopeptide against Cariogenic Streptococcus mutans UA159

doi: 10.1128/AAC.00776-17

Figure Lengend Snippet: Effects of CLP-4 on preformed biofilms. S. mutans UA159 biofilms were established for 24 h and then treated with increasing concentrations (1× to 10× the MIC) of CLP-4, chlorhexidine, or erythromycin. (A) Antibiofilm activities were assessed by quantifying the cell viability of treated biofilms by colony enumeration on agar plates. The means and standard deviations of three biological replicates from three independent experiments are shown. **, P < 0.01; ***, P < 0.001 compared to untreated control. (B) Biofilms treated with 10× the MICs for each antimicrobial were fluorescently labeled using the LIVE/DEAD BacLight viability stain and visualized by confocal laser scanning microscopy. Shown are the top-down three-dimensional (3D) volume rendering of biofilms at a total magnification of ×400. Bottom images represent optical planes in the xz, and vertical thin images represent yz dimensions. Membrane-compromised bacteria are stained red with propidium iodide, while intact bacteria are stained green with SYTO 9. Areas highlighted by dashed lines indicate regions of interest (ROIs) viewed at a higher magnification. Dimensions shown are 387.5 μm by 387.5 μm by 16 μm. (C) ROIs are presented at ×2,300 magnification. Dimensions shown are 68.1 μm by 68.1 μm by 16 μm.

Article Snippet: These results showed that CLP-4 is a promising agent that can effectively inhibit planktonic growth of S. mutans . table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Antimicrobial agent MIC and MBC (μg/ml) by inoculum density of: 6 × 10 5 CFU/ml 2 × 10 7 CFU/ml MIC MBC MIC MBC CLP-4 2.8 6 5 20 Erythromycin 0.016 0.6 0.062 1 Chlorhexidine dihydrochloride 1.25 3.5 1.25 5 Open in a separate window In vitro susceptibilities of planktonic S. mutans UA159 table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Streptococcus mutans strain Serotype MIC (μg/ml) MBC (μg/ml) Reference or source UA159 c 2.8 6 56 ATCC 25175 c 3.0 9 57 LM7 e 2.0 8 58 JF243 c 3.0 <5 59 568-2v-5 2.0 <5 60 764 2.0 5 C. M. Levesque 768 2.0 6 C. M. Levesque Open in a separate window S. mutans strains used in this study and their in vitro susceptibilities to CLP-4

Techniques: Control, Labeling, Staining, Confocal Laser Scanning Microscopy, Membrane, Bacteria

Journal:

Article Title: Integrated Physical and Genetic Mapping of Bacillus cereus and Other Gram-Positive Bacteria Based on IS 231 A Transposition Vectors

doi:

Figure Lengend Snippet: Bacterial strains and plasmids used

Article Snippet: All electroporations were done with a Gene Pulser II (Bio-Rad) apparatus. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Strains Bacillus cereus ATCC 14579 Type strain 38 Bacillus subtilis CU267 Host strain for pGI plasmids and first transposition assays 13 Plasmids pGIC052 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 1.4, 10 kb This study pGIC054 ori + , ori − , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 9.7 kb This study pGIC055 ori + , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 6.8 kb This study pGIC056 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 10 kb This study pGIC057 ori + , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 7.1 kb This study pGC2 ori − , Amp r , 2.9 kb 33 pHT1030 B. thuringiensis plasmid, thermosensitive replicon 24 pGC13 Fusion of pGC2 and pHT1030; ori + , ori − , Amp r , Ery r , 6.8 kb 24a pGIG010 ori − , Cm r , mini-IS 231 A Kan r , RCP 1.4, 4.4 kb 23a pGI300 ori + , suicide plasmid with Tn 10 dSp, RCP 2, 6.6 kb 28 pGI4010 ori − , Amp r , Ery r , 3.7 kb 18 pNEB193 ori − , Amp r , 2.7 kb New England Biolabs pBluescript SK ori − , Amp r , cloning vector, 3 kb Stratagene pGIC102 pBluescript with an Eco RI fragment containing the mini-IS Kan r inserted into the hot spot, 10.3 kb This study pGIC105 pBluescript with an Eco RI fragment containing the mini-IS Sp r inserted into the ade insertion site, 6.2 kb This study Open in a separate window a ori + , replicative origin for gram-positive host; ori − , replicative origin for gram-negative host; Amp r , Ery r , Kan r , and Sp r , resistance genes to ampicillin, erythromycin, kanamycin, and spectinomycin, respectively; P deg -TnpA, IS 231 A transposase gene expressed under control of the P deg promoter.

Techniques: Plasmid Preparation, Clone Assay

Journal:

Article Title: Integrated Physical and Genetic Mapping of Bacillus cereus and Other Gram-Positive Bacteria Based on IS 231 A Transposition Vectors

doi:

Figure Lengend Snippet: Transposition in B. subtilis CU267 and B. cereus ATCC 14579

Article Snippet: All electroporations were done with a Gene Pulser II (Bio-Rad) apparatus. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Strains Bacillus cereus ATCC 14579 Type strain 38 Bacillus subtilis CU267 Host strain for pGI plasmids and first transposition assays 13 Plasmids pGIC052 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 1.4, 10 kb This study pGIC054 ori + , ori − , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 9.7 kb This study pGIC055 ori + , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 6.8 kb This study pGIC056 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 10 kb This study pGIC057 ori + , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 7.1 kb This study pGC2 ori − , Amp r , 2.9 kb 33 pHT1030 B. thuringiensis plasmid, thermosensitive replicon 24 pGC13 Fusion of pGC2 and pHT1030; ori + , ori − , Amp r , Ery r , 6.8 kb 24a pGIG010 ori − , Cm r , mini-IS 231 A Kan r , RCP 1.4, 4.4 kb 23a pGI300 ori + , suicide plasmid with Tn 10 dSp, RCP 2, 6.6 kb 28 pGI4010 ori − , Amp r , Ery r , 3.7 kb 18 pNEB193 ori − , Amp r , 2.7 kb New England Biolabs pBluescript SK ori − , Amp r , cloning vector, 3 kb Stratagene pGIC102 pBluescript with an Eco RI fragment containing the mini-IS Kan r inserted into the hot spot, 10.3 kb This study pGIC105 pBluescript with an Eco RI fragment containing the mini-IS Sp r inserted into the ade insertion site, 6.2 kb This study Open in a separate window a ori + , replicative origin for gram-positive host; ori − , replicative origin for gram-negative host; Amp r , Ery r , Kan r , and Sp r , resistance genes to ampicillin, erythromycin, kanamycin, and spectinomycin, respectively; P deg -TnpA, IS 231 A transposase gene expressed under control of the P deg promoter.

Techniques: Plasmid Preparation, Incubation

Journal:

Article Title: Integrated Physical and Genetic Mapping of Bacillus cereus and Other Gram-Positive Bacteria Based on IS 231 A Transposition Vectors

doi:

Figure Lengend Snippet: PFGE profiles of total DNA from B. cereus strains restricted by NotI (A), AscI (B), SfiI (C), and I-SceI (D). Y, yeast chromosome marker; λ, lambda ladder marker (New England Biolabs); HS, hot-spot candidate with one single mini-IS Kanr insertion; TS, B. cereus ATCC 14579 type strain; A1 and A2, ade mutants; G1, gua mutant; H1 and H2, his mutants; M1 and M2, met mutants; U1, ura mutant; Aph., aphenotypic double-insertion mutant.

Article Snippet: All electroporations were done with a Gene Pulser II (Bio-Rad) apparatus. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Strains Bacillus cereus ATCC 14579 Type strain 38 Bacillus subtilis CU267 Host strain for pGI plasmids and first transposition assays 13 Plasmids pGIC052 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 1.4, 10 kb This study pGIC054 ori + , ori − , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 9.7 kb This study pGIC055 ori + , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 6.8 kb This study pGIC056 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 10 kb This study pGIC057 ori + , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 7.1 kb This study pGC2 ori − , Amp r , 2.9 kb 33 pHT1030 B. thuringiensis plasmid, thermosensitive replicon 24 pGC13 Fusion of pGC2 and pHT1030; ori + , ori − , Amp r , Ery r , 6.8 kb 24a pGIG010 ori − , Cm r , mini-IS 231 A Kan r , RCP 1.4, 4.4 kb 23a pGI300 ori + , suicide plasmid with Tn 10 dSp, RCP 2, 6.6 kb 28 pGI4010 ori − , Amp r , Ery r , 3.7 kb 18 pNEB193 ori − , Amp r , 2.7 kb New England Biolabs pBluescript SK ori − , Amp r , cloning vector, 3 kb Stratagene pGIC102 pBluescript with an Eco RI fragment containing the mini-IS Kan r inserted into the hot spot, 10.3 kb This study pGIC105 pBluescript with an Eco RI fragment containing the mini-IS Sp r inserted into the ade insertion site, 6.2 kb This study Open in a separate window a ori + , replicative origin for gram-positive host; ori − , replicative origin for gram-negative host; Amp r , Ery r , Kan r , and Sp r , resistance genes to ampicillin, erythromycin, kanamycin, and spectinomycin, respectively; P deg -TnpA, IS 231 A transposase gene expressed under control of the P deg promoter.

Techniques: Marker, Mutagenesis

Journal:

Article Title: Integrated Physical and Genetic Mapping of Bacillus cereus and Other Gram-Positive Bacteria Based on IS 231 A Transposition Vectors

doi:

Figure Lengend Snippet: B. cereus chromosomal map. Based on the position of the dnaA gene and by comparison of the locations of other genes with those of B. subtilis, the restriction map recently published by Carlson et al. (7) has been reoriented to place the potential replicative origin site at the 0°/360° point. Several relevant loci have been retained; their positions were arbitrarily set in the middle of the chromosomal fragment to which they hybridize (7). plc, phospholipase C; inA, inhibitor A; pdh, pyruvate dehydrogenase. Nx and Ax correspond to NotI and AscI restriction fragments, respectively. The hot-spot insertion site as well as eight other insertions generating auxotrophies are indicated. Aph represents an aphenotypic secondary transposition event.

Article Snippet: All electroporations were done with a Gene Pulser II (Bio-Rad) apparatus. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Strains Bacillus cereus ATCC 14579 Type strain 38 Bacillus subtilis CU267 Host strain for pGI plasmids and first transposition assays 13 Plasmids pGIC052 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 1.4, 10 kb This study pGIC054 ori + , ori − , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 9.7 kb This study pGIC055 ori + , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 6.8 kb This study pGIC056 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 10 kb This study pGIC057 ori + , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 7.1 kb This study pGC2 ori − , Amp r , 2.9 kb 33 pHT1030 B. thuringiensis plasmid, thermosensitive replicon 24 pGC13 Fusion of pGC2 and pHT1030; ori + , ori − , Amp r , Ery r , 6.8 kb 24a pGIG010 ori − , Cm r , mini-IS 231 A Kan r , RCP 1.4, 4.4 kb 23a pGI300 ori + , suicide plasmid with Tn 10 dSp, RCP 2, 6.6 kb 28 pGI4010 ori − , Amp r , Ery r , 3.7 kb 18 pNEB193 ori − , Amp r , 2.7 kb New England Biolabs pBluescript SK ori − , Amp r , cloning vector, 3 kb Stratagene pGIC102 pBluescript with an Eco RI fragment containing the mini-IS Kan r inserted into the hot spot, 10.3 kb This study pGIC105 pBluescript with an Eco RI fragment containing the mini-IS Sp r inserted into the ade insertion site, 6.2 kb This study Open in a separate window a ori + , replicative origin for gram-positive host; ori − , replicative origin for gram-negative host; Amp r , Ery r , Kan r , and Sp r , resistance genes to ampicillin, erythromycin, kanamycin, and spectinomycin, respectively; P deg -TnpA, IS 231 A transposase gene expressed under control of the P deg promoter.

Techniques:

Journal:

Article Title: Integrated Physical and Genetic Mapping of Bacillus cereus and Other Gram-Positive Bacteria Based on IS 231 A Transposition Vectors

doi:

Figure Lengend Snippet: IS231A target DNA sequence of the hot-spot insertion site (A) and Ade− insertion site (B) and representation of their respective EcoRI fragments cloned in pGIC102 (A) and pGIC105 (B). The 11 bp boxed on the sequence represent the duplicated target site (DR). IRL and IRR refer to IR left and right, respectively, by reference to the transposase gene orientation in IS231A; small black arrows indicate the cleavage generated by the transposase on each strand of the DNA. The size of the EcoRI fragment cloned in pGIC102 (A) is 7.3 kb, including 1.9 kb for the mini-IS Kanr. This fragment is 3.2 kb in the case of pGIC105 (B), from which 1.6 kb pertains to the mini-IS Spr. As for B. subtilis, the putative B. cereus purL and purF genes share a 25-bp overlap.

Article Snippet: All electroporations were done with a Gene Pulser II (Bio-Rad) apparatus. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Strains Bacillus cereus ATCC 14579 Type strain 38 Bacillus subtilis CU267 Host strain for pGI plasmids and first transposition assays 13 Plasmids pGIC052 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 1.4, 10 kb This study pGIC054 ori + , ori − , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 9.7 kb This study pGIC055 ori + , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 6.8 kb This study pGIC056 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 10 kb This study pGIC057 ori + , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 7.1 kb This study pGC2 ori − , Amp r , 2.9 kb 33 pHT1030 B. thuringiensis plasmid, thermosensitive replicon 24 pGC13 Fusion of pGC2 and pHT1030; ori + , ori − , Amp r , Ery r , 6.8 kb 24a pGIG010 ori − , Cm r , mini-IS 231 A Kan r , RCP 1.4, 4.4 kb 23a pGI300 ori + , suicide plasmid with Tn 10 dSp, RCP 2, 6.6 kb 28 pGI4010 ori − , Amp r , Ery r , 3.7 kb 18 pNEB193 ori − , Amp r , 2.7 kb New England Biolabs pBluescript SK ori − , Amp r , cloning vector, 3 kb Stratagene pGIC102 pBluescript with an Eco RI fragment containing the mini-IS Kan r inserted into the hot spot, 10.3 kb This study pGIC105 pBluescript with an Eco RI fragment containing the mini-IS Sp r inserted into the ade insertion site, 6.2 kb This study Open in a separate window a ori + , replicative origin for gram-positive host; ori − , replicative origin for gram-negative host; Amp r , Ery r , Kan r , and Sp r , resistance genes to ampicillin, erythromycin, kanamycin, and spectinomycin, respectively; P deg -TnpA, IS 231 A transposase gene expressed under control of the P deg promoter.

Techniques: Sequencing, Clone Assay, Generated

Journal:

Article Title: Integrated Physical and Genetic Mapping of Bacillus cereus and Other Gram-Positive Bacteria Based on IS 231 A Transposition Vectors

doi:

Figure Lengend Snippet: Hybridization of the total DNA of B. cereus auxotrophs restricted by EcoRI with a probe corresponding to the Spr gene. HS, hot-spot candidate with one single mini-IS Kanr insertion (negative control); A1, A2, A3, and A4, ade mutants; C1, cys mutant; G1, gua mutant; H1 and H2, his mutants; M1 and M2, met mutants; U1, ura mutant.

Article Snippet: All electroporations were done with a Gene Pulser II (Bio-Rad) apparatus. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Description a Reference or source Strains Bacillus cereus ATCC 14579 Type strain 38 Bacillus subtilis CU267 Host strain for pGI plasmids and first transposition assays 13 Plasmids pGIC052 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 1.4, 10 kb This study pGIC054 ori + , ori − , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 9.7 kb This study pGIC055 ori + , Ery r , mini-IS 231 A Sp r , P deg -TnpA, RCP 2, 6.8 kb This study pGIC056 ori + , ori − , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 10 kb This study pGIC057 ori + , Ery r , mini-IS 231 A Kan r , P deg -TnpA, RCP 2, 7.1 kb This study pGC2 ori − , Amp r , 2.9 kb 33 pHT1030 B. thuringiensis plasmid, thermosensitive replicon 24 pGC13 Fusion of pGC2 and pHT1030; ori + , ori − , Amp r , Ery r , 6.8 kb 24a pGIG010 ori − , Cm r , mini-IS 231 A Kan r , RCP 1.4, 4.4 kb 23a pGI300 ori + , suicide plasmid with Tn 10 dSp, RCP 2, 6.6 kb 28 pGI4010 ori − , Amp r , Ery r , 3.7 kb 18 pNEB193 ori − , Amp r , 2.7 kb New England Biolabs pBluescript SK ori − , Amp r , cloning vector, 3 kb Stratagene pGIC102 pBluescript with an Eco RI fragment containing the mini-IS Kan r inserted into the hot spot, 10.3 kb This study pGIC105 pBluescript with an Eco RI fragment containing the mini-IS Sp r inserted into the ade insertion site, 6.2 kb This study Open in a separate window a ori + , replicative origin for gram-positive host; ori − , replicative origin for gram-negative host; Amp r , Ery r , Kan r , and Sp r , resistance genes to ampicillin, erythromycin, kanamycin, and spectinomycin, respectively; P deg -TnpA, IS 231 A transposase gene expressed under control of the P deg promoter.

Techniques: Hybridization, Negative Control, Mutagenesis