Journal:

Article Title: Apyrase, the Product of the Virulence Plasmid-Encoded phoN2 ( apy ) Gene of Shigella flexneri , Is Necessary for Proper Unipolar IcsA Localization and for Efficient Intercellular Spread †

doi: 10.1128/JB.188.4.1620-1627.2006

Figure Lengend Snippet: Bacterial strains and plasmids a

Article Snippet: The locations of 3× FLAG-tagged OspB recombinant protein as well as of IpaB, IpaC, and apyrase proteins are indicated. na, not added. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype or characteristic(s) Source or reference Strains M90T Wild-type S. flexneri serotype 5a 32 HND115 M90T Δ phoN2 ; susceptible This work HND201 M90T Δ ospB ; susceptible This work HND215 M90T Δ ospBphoN2 ; susceptible This work HND43 M90T Δ mxiA ; susceptible This work HND53 M90T Δ virB :: aphA-3 ; Km r This work HND549 M90T ospB -3×FLAG; susceptible This work HND5311 M90T Δ mxiA ospB -3×FLAG; susceptible This work SC560 M90T Δ icsA ::Ω; Sm r 10 M15[pREP4] E. coli K-12 strain carrying plasmid pREP4 Qiagen Plasmids pGEM-T Cloning vector, Ap r Promega pBluescript SK Cloning vector, Ap r Stratagene Inc., La Jolla, CA pBAD28 Arabinose-inducible P BAD expression vector, Ap r Cm r 13 pSUB11 Template plasmid carrying a 3×FLAG epitope and Km r cassette 42 pKD46 λ Red helper plasmid; oriR101 repA101 (Ts) P-araB-gam-bet-exo Ap r 7 pKD4 Template plasmid carrying a Km r gene with FLP recognition target sequence 7 pCP20 FLP helper plasmid; pSC101 replicon (Ts) bla cat Flp (λR p ) cI 857 Ap r Cm r 7 pREP4 lacI , Km r Qiagen pQE30 Cloning vector; Ap r Qiagen pHN301 pQE30 carrying His6- phoN2 ; Ap r This work pHN28 pBAD28 carrying phoN2 ; Ap r Cm r This work pHN111 pBAD28 carrying mxiA ; Ap r Cm r This work pFB1-4 pBluescript SK carrying a mutated phoN2 gene of EIEC strain HN280 presenting the R192P amino acid substitution 36 Open in a separate window a Ap r , ampicillin resistance; Cm r , cloramphenicol resistance; Km r , kanamycin resistance.

Techniques: Plasmid Preparation, Clone Assay, Expressing, Sequencing

Journal:

Article Title: Apyrase, the Product of the Virulence Plasmid-Encoded phoN2 ( apy ) Gene of Shigella flexneri , Is Necessary for Proper Unipolar IcsA Localization and for Efficient Intercellular Spread †

doi: 10.1128/JB.188.4.1620-1627.2006

Figure Lengend Snippet: Formation of plaques on confluent monolayers of HeLa and Caco-2 cells a

Article Snippet: The locations of 3× FLAG-tagged OspB recombinant protein as well as of IpaB, IpaC, and apyrase proteins are indicated. na, not added. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype or characteristic(s) Source or reference Strains M90T Wild-type S. flexneri serotype 5a 32 HND115 M90T Δ phoN2 ; susceptible This work HND201 M90T Δ ospB ; susceptible This work HND215 M90T Δ ospBphoN2 ; susceptible This work HND43 M90T Δ mxiA ; susceptible This work HND53 M90T Δ virB :: aphA-3 ; Km r This work HND549 M90T ospB -3×FLAG; susceptible This work HND5311 M90T Δ mxiA ospB -3×FLAG; susceptible This work SC560 M90T Δ icsA ::Ω; Sm r 10 M15[pREP4] E. coli K-12 strain carrying plasmid pREP4 Qiagen Plasmids pGEM-T Cloning vector, Ap r Promega pBluescript SK Cloning vector, Ap r Stratagene Inc., La Jolla, CA pBAD28 Arabinose-inducible P BAD expression vector, Ap r Cm r 13 pSUB11 Template plasmid carrying a 3×FLAG epitope and Km r cassette 42 pKD46 λ Red helper plasmid; oriR101 repA101 (Ts) P-araB-gam-bet-exo Ap r 7 pKD4 Template plasmid carrying a Km r gene with FLP recognition target sequence 7 pCP20 FLP helper plasmid; pSC101 replicon (Ts) bla cat Flp (λR p ) cI 857 Ap r Cm r 7 pREP4 lacI , Km r Qiagen pQE30 Cloning vector; Ap r Qiagen pHN301 pQE30 carrying His6- phoN2 ; Ap r This work pHN28 pBAD28 carrying phoN2 ; Ap r Cm r This work pHN111 pBAD28 carrying mxiA ; Ap r Cm r This work pFB1-4 pBluescript SK carrying a mutated phoN2 gene of EIEC strain HN280 presenting the R192P amino acid substitution 36 Open in a separate window a Ap r , ampicillin resistance; Cm r , cloramphenicol resistance; Km r , kanamycin resistance.

Techniques:

Journal:

Article Title: Apyrase, the Product of the Virulence Plasmid-Encoded phoN2 ( apy ) Gene of Shigella flexneri , Is Necessary for Proper Unipolar IcsA Localization and for Efficient Intercellular Spread †

doi: 10.1128/JB.188.4.1620-1627.2006

Figure Lengend Snippet: phoN2 mutant strain HND115 forms plaques of approximately wild-type size on Caco-2 cell monolayers when complemented with plasmid pFB1-4. pFB1-4 (Table ​(Table1)1) contains a randomly generated mutant of the apy gene, the orthologue of phoN2 in the EIEC strain HN280, encoding a recombinant apyrase presenting the R192P substitution which inactivates its dNTP-hydrolyzing activity. (A) Western blot analysis with anti-apyrase antibodies of whole-cell extracts of M90T, HND115 (phoN2), and HND115/pFB1-4 (pBluescript SKapyR192P). (B) Plaques formed by M90T, HND115 (phoN2), and HND115/pFB1-4 (pBluescript SKapyR192P). Dishes were photographed after 48 h of infection.

Article Snippet: The locations of 3× FLAG-tagged OspB recombinant protein as well as of IpaB, IpaC, and apyrase proteins are indicated. na, not added. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant genotype or characteristic(s) Source or reference Strains M90T Wild-type S. flexneri serotype 5a 32 HND115 M90T Δ phoN2 ; susceptible This work HND201 M90T Δ ospB ; susceptible This work HND215 M90T Δ ospBphoN2 ; susceptible This work HND43 M90T Δ mxiA ; susceptible This work HND53 M90T Δ virB :: aphA-3 ; Km r This work HND549 M90T ospB -3×FLAG; susceptible This work HND5311 M90T Δ mxiA ospB -3×FLAG; susceptible This work SC560 M90T Δ icsA ::Ω; Sm r 10 M15[pREP4] E. coli K-12 strain carrying plasmid pREP4 Qiagen Plasmids pGEM-T Cloning vector, Ap r Promega pBluescript SK Cloning vector, Ap r Stratagene Inc., La Jolla, CA pBAD28 Arabinose-inducible P BAD expression vector, Ap r Cm r 13 pSUB11 Template plasmid carrying a 3×FLAG epitope and Km r cassette 42 pKD46 λ Red helper plasmid; oriR101 repA101 (Ts) P-araB-gam-bet-exo Ap r 7 pKD4 Template plasmid carrying a Km r gene with FLP recognition target sequence 7 pCP20 FLP helper plasmid; pSC101 replicon (Ts) bla cat Flp (λR p ) cI 857 Ap r Cm r 7 pREP4 lacI , Km r Qiagen pQE30 Cloning vector; Ap r Qiagen pHN301 pQE30 carrying His6- phoN2 ; Ap r This work pHN28 pBAD28 carrying phoN2 ; Ap r Cm r This work pHN111 pBAD28 carrying mxiA ; Ap r Cm r This work pFB1-4 pBluescript SK carrying a mutated phoN2 gene of EIEC strain HN280 presenting the R192P amino acid substitution 36 Open in a separate window a Ap r , ampicillin resistance; Cm r , cloramphenicol resistance; Km r , kanamycin resistance.

Techniques: Mutagenesis, Plasmid Preparation, Generated, Recombinant, Activity Assay, Western Blot, Infection