b pertussis type strain 18323  (ATCC)

Journal:

Article Title: Vag8, a Bordetella pertussis bvg -Regulated Protein

doi:

Figure Lengend Snippet: Bacterial strains and plasmids used in this study

Article Snippet: Alkaline phosphatase activity and chloramphenicol acetyltransferase (CAT) activity were assayed as previously described ( 13 , 23 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant feature(s) Source or reference B. pertussis strains 18323 Wild type ATCC 9797 SK8 18323::Tn phoA ; lacking 95-kDa protein 13 Tohama I Wild type Laboratory of Pertussis collection SK8 vag-8 ::pSKCAT Lacking 95-kDa protein; PhoA − This study 18323Δvag-8 18323; Kn r ; lacking 95-kDa protein This study B. bronchiseptica strains 110H 17 ; Laboratory of Pertussis collection 207 Laboratory of Pertussis collection 058 Laboratory of Pertussis collection B. parapertussis strains 500 Laboratory of Pertussis collection 482 Laboratory of Pertussis collection 23054 17 ; Laboratory of Pertussis collection 13367 Clinical isolate C. Mink (UCLA) 9807 Clinical isolate C. Mink (UCLA) E. coli strains SM10λ pir thi thr leu tonA lacY supE recA ::RP4-2-Tc::Mu λ pir R6K 16 DM1187(pCLB1) Source of colicinB 3 ; A. Weiss (University of Cincinnati) Plasmids pSS1129 Suicide vector 25 pSKCAT pJM703.1 with ′ phoA ′-CAT insert; Ap r Cm r 13 pTF470 2-kb Sal I fragment containing the fusion junction between pSKCAT and vag-8 in pUC18 This study pDA676 pMal-c2 expressing maltose binding protein–Vag8 N-terminal fusion protein This study p4A10 pLAFR2 cosmid derivative containing vag-8 13 ; this study pDA626 vag-8 in pBluescriptSK + This study pDA669 Derivative of pDA626 with a 1.3-kb Kn r cassette replacing a 0.6-kb internal fragment of vag-8 (Δvag-8) This study pDA672 4.1-kb Sst I- Kpn I fragment from pDA669 containing Δvag-8 in pUC19 This study pDA681 4.1-kb Xba I fragment from pDA672 containing Δvag-8 in pSS1129 This study Open in a separate window Bacterial strains and plasmids used in this study Standard DNA manipulation methods were used ( 15 ).

Techniques: Plasmid Preparation, Expressing, Binding Assay

Journal:

Article Title: Vag8, a Bordetella pertussis bvg -Regulated Protein

doi:

Figure Lengend Snippet: Presence of insertion element (IS) and phoA sequences in SK8 chromosomal DNA. Shown are Southern blots of BamHI-digested chromosomal DNA from SK8 probed with a probe which hybridizes to the TnphoA IS sequences and the gene encoding the kanamycin resistance determinant (A), a probe to the IS sequence (B), and a probe to the phoA sequence of TnphoA (C). The arrows indicate bands hybridizing to each probe. (D) Schematic representation of SK8 chromosomal DNA with insertion of TnphoA and IS50L containing ′phoA. The production of alkaline phosphatase activity from the latter is indicated by the arrow; the insertion which does not produce alkaline phosphatase activity is indicated by the crossed-out arrow. The probes derived from TnphoA are highlighted, as double-headed arrows, below the diagram. The letters under the probes refer to the Southern blots in panels A, B, and C.

Article Snippet: Alkaline phosphatase activity and chloramphenicol acetyltransferase (CAT) activity were assayed as previously described ( 13 , 23 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant feature(s) Source or reference B. pertussis strains 18323 Wild type ATCC 9797 SK8 18323::Tn phoA ; lacking 95-kDa protein 13 Tohama I Wild type Laboratory of Pertussis collection SK8 vag-8 ::pSKCAT Lacking 95-kDa protein; PhoA − This study 18323Δvag-8 18323; Kn r ; lacking 95-kDa protein This study B. bronchiseptica strains 110H 17 ; Laboratory of Pertussis collection 207 Laboratory of Pertussis collection 058 Laboratory of Pertussis collection B. parapertussis strains 500 Laboratory of Pertussis collection 482 Laboratory of Pertussis collection 23054 17 ; Laboratory of Pertussis collection 13367 Clinical isolate C. Mink (UCLA) 9807 Clinical isolate C. Mink (UCLA) E. coli strains SM10λ pir thi thr leu tonA lacY supE recA ::RP4-2-Tc::Mu λ pir R6K 16 DM1187(pCLB1) Source of colicinB 3 ; A. Weiss (University of Cincinnati) Plasmids pSS1129 Suicide vector 25 pSKCAT pJM703.1 with ′ phoA ′-CAT insert; Ap r Cm r 13 pTF470 2-kb Sal I fragment containing the fusion junction between pSKCAT and vag-8 in pUC18 This study pDA676 pMal-c2 expressing maltose binding protein–Vag8 N-terminal fusion protein This study p4A10 pLAFR2 cosmid derivative containing vag-8 13 ; this study pDA626 vag-8 in pBluescriptSK + This study pDA669 Derivative of pDA626 with a 1.3-kb Kn r cassette replacing a 0.6-kb internal fragment of vag-8 (Δvag-8) This study pDA672 4.1-kb Sst I- Kpn I fragment from pDA669 containing Δvag-8 in pUC19 This study pDA681 4.1-kb Xba I fragment from pDA672 containing Δvag-8 in pSS1129 This study Open in a separate window Bacterial strains and plasmids used in this study Standard DNA manipulation methods were used ( 15 ).

Techniques: Sequencing, Activity Assay, Derivative Assay

Journal:

Article Title: Vag8, a Bordetella pertussis bvg -Regulated Protein

doi:

Figure Lengend Snippet: Insertion of pSKCAT into two chromosomal locations in SK8. (A) SmaI-digested chromosomal DNA probed with a CAT gene probe. Lane 1, SK8; lane 2, SK8::pSKCAT exconjugate PhoA+; lane 3, SK8::pSKCAT exconjugate PhoA−. (B) SmaI-digested DNA probed with an oligonucleotide derived from sequence upstream of ′phoA, which did not produce PhoA. Lane 1, SK8; lane 2, SK8::pSKCAT exconjugate PhoA+; lane 3, SK8::pSKCAT exconjugate PhoA−. (C) Schematic representation of homologous recombination between the ′phoA′ sequence of pSKCAT and either of the two chromosomal copies of ′phoA in SK8. The exconjugate resulting from the single crossover event is either PhoA+ or PhoA−. The shaded boxes represent ′phoA sequences. The area of hybridization of the CAT probe used in panel A is indicated by the double-headed arrow; the point of hybridization of the oligonucleotide probe used in panel B is indicated by the arrow.

Article Snippet: Alkaline phosphatase activity and chloramphenicol acetyltransferase (CAT) activity were assayed as previously described ( 13 , 23 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant feature(s) Source or reference B. pertussis strains 18323 Wild type ATCC 9797 SK8 18323::Tn phoA ; lacking 95-kDa protein 13 Tohama I Wild type Laboratory of Pertussis collection SK8 vag-8 ::pSKCAT Lacking 95-kDa protein; PhoA − This study 18323Δvag-8 18323; Kn r ; lacking 95-kDa protein This study B. bronchiseptica strains 110H 17 ; Laboratory of Pertussis collection 207 Laboratory of Pertussis collection 058 Laboratory of Pertussis collection B. parapertussis strains 500 Laboratory of Pertussis collection 482 Laboratory of Pertussis collection 23054 17 ; Laboratory of Pertussis collection 13367 Clinical isolate C. Mink (UCLA) 9807 Clinical isolate C. Mink (UCLA) E. coli strains SM10λ pir thi thr leu tonA lacY supE recA ::RP4-2-Tc::Mu λ pir R6K 16 DM1187(pCLB1) Source of colicinB 3 ; A. Weiss (University of Cincinnati) Plasmids pSS1129 Suicide vector 25 pSKCAT pJM703.1 with ′ phoA ′-CAT insert; Ap r Cm r 13 pTF470 2-kb Sal I fragment containing the fusion junction between pSKCAT and vag-8 in pUC18 This study pDA676 pMal-c2 expressing maltose binding protein–Vag8 N-terminal fusion protein This study p4A10 pLAFR2 cosmid derivative containing vag-8 13 ; this study pDA626 vag-8 in pBluescriptSK + This study pDA669 Derivative of pDA626 with a 1.3-kb Kn r cassette replacing a 0.6-kb internal fragment of vag-8 (Δvag-8) This study pDA672 4.1-kb Sst I- Kpn I fragment from pDA669 containing Δvag-8 in pUC19 This study pDA681 4.1-kb Xba I fragment from pDA672 containing Δvag-8 in pSS1129 This study Open in a separate window Bacterial strains and plasmids used in this study Standard DNA manipulation methods were used ( 15 ).

Techniques: Derivative Assay, Sequencing, Homologous Recombination, Hybridization

Journal:

Article Title: Vag8, a Bordetella pertussis bvg -Regulated Protein

doi:

Figure Lengend Snippet: Immunoblots of the maltose binding protein–Vag8 fusion and reactivity of antiserum raised to this fusion protein. (A) Fusion protein (5 μg) was subjected to SDS-PAGE, transferred to nitrocellulose, and probed with monoclonal antibody P8E7. (B) Fusion protein and whole-cell lysates from Tohama I, 18323, and SK8 were subjected to SDS-PAGE, transferred to nitrocellulose, and probed with antiserum raised in mice to the maltose binding protein–Vag8 fusion. Lane 1, Tohama I; lane 2, 18323; lane 3, SK8; lane 4, 5 μg of maltose binding protein fusion to an N-terminal fragment of Vag8. The large arrow indicates Vag8; the smaller arrows indicate the 103-kDa fusion protein.

Article Snippet: Alkaline phosphatase activity and chloramphenicol acetyltransferase (CAT) activity were assayed as previously described ( 13 , 23 ). table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant feature(s) Source or reference B. pertussis strains 18323 Wild type ATCC 9797 SK8 18323::Tn phoA ; lacking 95-kDa protein 13 Tohama I Wild type Laboratory of Pertussis collection SK8 vag-8 ::pSKCAT Lacking 95-kDa protein; PhoA − This study 18323Δvag-8 18323; Kn r ; lacking 95-kDa protein This study B. bronchiseptica strains 110H 17 ; Laboratory of Pertussis collection 207 Laboratory of Pertussis collection 058 Laboratory of Pertussis collection B. parapertussis strains 500 Laboratory of Pertussis collection 482 Laboratory of Pertussis collection 23054 17 ; Laboratory of Pertussis collection 13367 Clinical isolate C. Mink (UCLA) 9807 Clinical isolate C. Mink (UCLA) E. coli strains SM10λ pir thi thr leu tonA lacY supE recA ::RP4-2-Tc::Mu λ pir R6K 16 DM1187(pCLB1) Source of colicinB 3 ; A. Weiss (University of Cincinnati) Plasmids pSS1129 Suicide vector 25 pSKCAT pJM703.1 with ′ phoA ′-CAT insert; Ap r Cm r 13 pTF470 2-kb Sal I fragment containing the fusion junction between pSKCAT and vag-8 in pUC18 This study pDA676 pMal-c2 expressing maltose binding protein–Vag8 N-terminal fusion protein This study p4A10 pLAFR2 cosmid derivative containing vag-8 13 ; this study pDA626 vag-8 in pBluescriptSK + This study pDA669 Derivative of pDA626 with a 1.3-kb Kn r cassette replacing a 0.6-kb internal fragment of vag-8 (Δvag-8) This study pDA672 4.1-kb Sst I- Kpn I fragment from pDA669 containing Δvag-8 in pUC19 This study pDA681 4.1-kb Xba I fragment from pDA672 containing Δvag-8 in pSS1129 This study Open in a separate window Bacterial strains and plasmids used in this study Standard DNA manipulation methods were used ( 15 ).

Techniques: Western Blot, Binding Assay, SDS Page