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

Journal: Toxins

Article Title: Pore-Forming Cardiotoxin VVA2 (Volvatoxin A2) Variant I82E/L86K Is an Atypical Duplex-Specific Nuclease

doi: 10.3390/toxins14060392

Figure Lengend Snippet: Re-VVA2 I82E/L86K cleaves dsDNA non-specifically with cleavage preference in dG-dC-rich regions. ( A ) The schematic diagram of the experimental design for mapping the cleavage sites of nuclease Re-VVA2 I82E/L86K. The cleaved products (pUC19 and pET28a, mainly in RF II) were digested by S1 nuclease and cloned into pre-linearized pBluescript II SK (+) vector by EcoRV. Colonies were selected and sequenced using universal primer M13F. The detailed protocol is stated in the Materials and Methods. ( B ) Cleavage sites of Re-VVA2 I82E/L86K on pUC19 and pET28a were marked on the related plasmid map. Specific cleavage sites are shown in . ( C ) Re-VVA2 I82E/L86K has cleavage preference in dG-dC-rich regions. The sequences around cleavage sites were aligned with WebLogo 3. The proposed cleavage sites were marked on the figure.

Article Snippet: Plasmid: pET28a, pUC19, pBluescript II SK (+) vector (Addgene, Watertown, MA, USA).

Techniques: Clone Assay, Plasmid Preparation

Journal:

Article Title: Modification of Type IV Pilus-Associated Epithelial Cell Adherence and Multicellular Behavior by the PilU Protein of Neisseria gonorrhoeae

doi: 10.1128/IAI.70.7.3891-3903.2002

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

Article Snippet: All E. coli strains and their recombinants were maintained in Luria-Bertani medium supplemented with the appropriate antibiotics. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Reference or source N. gonorrhoeae strains N400 Derived from VD300, a contains the IPTG-inducible recA6 allele b 11 GF2 (PilF − ) pilF ::m-Tn 3erm 11 GT3 pilT ::m-Tn 3erm 40 GT7 pilT ::m-Tn 3erm 40 GT17 pilT ::m-Tn 3cm at position 73 c This study GT26 pilT ::m-Tn 3erm 40 GT50 pilT ::m-Tn 3erm at position 1105 c This study GT101 pilT dud1 40 GT102 pilT Δ QSL 40 GT103 pilT ::m-Tn 3erm 40 GT104 pilT ind 40 GT105 pilT fs164 40 GTU2 pilT ::m-Tn 3cm pilU ::mTn 3erm This study GTXE1 pilT :: xylE This study GUXE2 pilU :: xylE This study GU2 pilU ::m-Tn 3erm at position 1591 c This study GU4 pilU ::m-Tn 3erm at position 1714 c This study GU5 pilU ::m-Tn 3erm at position 2102 c This study GU21 pilU ::m-Tn 3erm at position 1314 c This study GU33 pilU ::m-Tn 3erm at position 2541 c This study GU127 pilU ::m-Tn 3erm at position 1192 c This study E. coli strains BL21(DE3) F − ompT r B − m B − with prophage λ carring the T7 RNA polymerase gene 32 HB101 F − mcrB mrr hsdS20 (r B − m B − ) recA13 supE44 ara14 galK2 lacY1 proA2 rpsL20 (Sm r ) xyl5 λ − leu mtl1 GIBCO BRL Plasmids pBluescript II SK Cloning vector, Amp r 32 p6/16/11 1.1-kb Sal I fragment in pBluescript II SK This study p11/2/13 3.3-kb EagI/Xba I fragment in pBluescript II SK This study pHSS6 Vector for shuttle mutagenesis, Kan r 27 p1-49 3.3-kb Eag I/ Xba I fragment in pHSS6 This study pT7-5 T7 RNA polymerase promoter λ10, Amp r 32 pT7U pilU in pT7-5 This work pT7U (Δ Sal I) Sal I deletion subclone of pT7U This work Open in a separate window a VD300 is an MS11 derivative ( 15 ). b All remaining Gc strains are derived from N400. c Nucleotide positions correspond to those in GenBank no. {"type":"entrez-nucleotide","attrs":{"text":"S72391","term_id":"632717","term_text":"S72391"}} S72391 .

Techniques: Plasmid Preparation, Derivative Assay, Clone Assay, Mutagenesis