pgem7zf(−) , ap r , lacz + cloning vector  (Promega)

Journal: PLoS ONE

Article Title: The AprV5 Subtilase Is Required for the Optimal Processing of All Three Extracellular Serine Proteases from Dichelobacter nodosus

doi: 10.1371/journal.pone.0047932

Figure Lengend Snippet: Bacterial strains and plasmids.

Article Snippet: pGEM7Zf(−) , Ap r , lacZ + cloning vector , Promega.

Techniques: Transformation Assay, Plasmid Preparation, Clone Assay, Recombinant

Journal: Journal of Bacteriology

Article Title: Sulfide Consumption in Sulfurimonas denitrificans and Heterologous Expression of Its Three Sulfide-Quinone Reductase Homologs

doi: 10.1128/JB.01021-15

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: pGEM-T , Cloning vector with T overhangs, lacZ Ap r , Promega.

Techniques: Plasmid Preparation, Conjugation Assay, Clone Assay

Journal: Molecular Microbiology

Article Title: Two-component regulatory systems in Pseudomonas aeruginosa : an intricate network mediating fimbrial and efflux pump gene expression

doi: 10.1111/j.1365-2958.2010.07527.x

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

Article Snippet: pCR2.1 , TA cloning vector for PCR products, lacZ α ColE1 f1 ori Ap r Km r , Invitrogen.

Techniques: Mutagenesis, Plasmid Preparation, TA Cloning, Clone Assay, Expressing

Journal:

Article Title: Expression of the cpdA Gene, Encoding a 3?,5?-Cyclic AMP (cAMP) Phosphodiesterase, Is Positively Regulated by the cAMP-cAMP Receptor Protein Complex ▿ †

doi: 10.1128/JB.01350-08

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: All medium components were purchased from Difco, and chemicals and antibiotics were obtained from Sigma. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Source or reference Strains V. vulnificus ATCC 29307 Clinical isolate; virulent 14 AR ATCC 29307 but spontaneous rifampin resistance 26 HY101 AR but Δ cpdA 20 KP301 AR but Δ cya 20 KC74 ATCC 29307 but crp :: nptI 14 E. coli DH5α (φ80 lacZ ΔM15) recA1 endA1 gyrA96 relA1 thi-1 hsdR17 (r K − m K − ) supE44 deoR Δ( lacZYA-argF ) U169 Laboratory collection SM10λpir thi-1 thr leu tonA lacY supE recA ::Rp4-2-Tc::Muλ pir ; OriT of RP4; Km r 30 JM109 endA1 recA1 gyrA96 thi-1 hsdR17 (r K − m K − ) relA1 supE44 Δ( lac-proAB ) [F′ traD3 6proAB lacI q Z ΔM15] Promega Plasmids pUC19 Cloning vector; lacZ Ap r 34 pINE45 pUC19 with 1.72-kb Sau3AI fragment of V. vulnificus DNA containing the cpdA gene This study pINE45-1 pUC19 with 4.3-kb Sau3AI fragment of V. vulnificus DNA containing the cpdA gene This study pLAFR5 IncP Tc r ; derivative of pLAFR3 containing double cos cassettes 16 pHS51 pLAFR5 containing EcoRI and HindIII fragment of pINE45 This study pQE30 Expression vector; Ap r Qiagen pQE30-cpdA pQE30 with V. vulnificus cpdA This study pHK0011 Transcriptional fusion plasmid with promoterless luxAB ; Tc r 8 pSMK-cpdA-1 pHK0011 with cpdA upstream region (positions −1521 to +41 relative to cpdA IC) This study pSMK-cpdA2 pHK0011 with cpdA upstream region (positions −1178 to −1051 relative to cpdA IC) This study pSMK-cpdA3 pHK0011 with cpdA upstream region (positions −1163 to −1051 relative to cpdA IC) This study pGEM-11zf(+) General cloning vector; Ap r Promega pGEM-11zf(+)-cpdA2mt pGEM-11zf(+) with the mutagenized sequence for the putative CRP-binding site of cpdA regulatory region This study pSMK-cpdA2mt pSMK-cpdA2 but mutagenized CRP-binding site This study Open in a separate window Strains and plasmids used in this study

Techniques: Plasmid Preparation, Clone Assay, Expressing, Sequencing

Journal:

Article Title: Expression of the cpdA Gene, Encoding a 3?,5?-Cyclic AMP (cAMP) Phosphodiesterase, Is Positively Regulated by the cAMP-cAMP Receptor Protein Complex ▿ †

doi: 10.1128/JB.01350-08

Figure Lengend Snippet: Determination of the transcriptional start site of the cpdA gene. (A) Primer extension using V. vulnificus RNA and oligonucleotide primer yqiE-R (annealing to positions +130 to +150 relative to the IC of mutT). Lanes C, T, A, and G represent sequencing ladders of pINE45-1. The +1 indicates the site of transcriptional initiation. (B) Sequence of the upstream region of mutT, the first gene of the operon composed of mutT, yqiB, cpdA, and yqiA. The initiation codon for mutT is in boldface type, and the promoter and the putative −10 and −35 regions are underlined. The putative binding site for the cAMP-CRP complex is designated with a box. The primers used for the construction of the cpdA::luxAB transcription fusions are indicated with horizontal arrows.

Article Snippet: All medium components were purchased from Difco, and chemicals and antibiotics were obtained from Sigma. table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Source or reference Strains V. vulnificus ATCC 29307 Clinical isolate; virulent 14 AR ATCC 29307 but spontaneous rifampin resistance 26 HY101 AR but Δ cpdA 20 KP301 AR but Δ cya 20 KC74 ATCC 29307 but crp :: nptI 14 E. coli DH5α (φ80 lacZ ΔM15) recA1 endA1 gyrA96 relA1 thi-1 hsdR17 (r K − m K − ) supE44 deoR Δ( lacZYA-argF ) U169 Laboratory collection SM10λpir thi-1 thr leu tonA lacY supE recA ::Rp4-2-Tc::Muλ pir ; OriT of RP4; Km r 30 JM109 endA1 recA1 gyrA96 thi-1 hsdR17 (r K − m K − ) relA1 supE44 Δ( lac-proAB ) [F′ traD3 6proAB lacI q Z ΔM15] Promega Plasmids pUC19 Cloning vector; lacZ Ap r 34 pINE45 pUC19 with 1.72-kb Sau3AI fragment of V. vulnificus DNA containing the cpdA gene This study pINE45-1 pUC19 with 4.3-kb Sau3AI fragment of V. vulnificus DNA containing the cpdA gene This study pLAFR5 IncP Tc r ; derivative of pLAFR3 containing double cos cassettes 16 pHS51 pLAFR5 containing EcoRI and HindIII fragment of pINE45 This study pQE30 Expression vector; Ap r Qiagen pQE30-cpdA pQE30 with V. vulnificus cpdA This study pHK0011 Transcriptional fusion plasmid with promoterless luxAB ; Tc r 8 pSMK-cpdA-1 pHK0011 with cpdA upstream region (positions −1521 to +41 relative to cpdA IC) This study pSMK-cpdA2 pHK0011 with cpdA upstream region (positions −1178 to −1051 relative to cpdA IC) This study pSMK-cpdA3 pHK0011 with cpdA upstream region (positions −1163 to −1051 relative to cpdA IC) This study pGEM-11zf(+) General cloning vector; Ap r Promega pGEM-11zf(+)-cpdA2mt pGEM-11zf(+) with the mutagenized sequence for the putative CRP-binding site of cpdA regulatory region This study pSMK-cpdA2mt pSMK-cpdA2 but mutagenized CRP-binding site This study Open in a separate window Strains and plasmids used in this study

Techniques: Sequencing, Binding Assay

Journal:

Article Title: Assembly of Fimbrial Structures in Pseudomonas aeruginosa : Functionality and Specificity of Chaperone-Usher Machineries ▿

doi: 10.1128/JB.00093-07

Figure Lengend Snippet: Strains and plasmids used in this study a

Article Snippet: The following antibiotics at the indicated concentrations were used: for E. coli , ampicillin (50 μg/ml), gentamicin (Gm; 25 μg/ml), kanamycin (Km; 25 μg/ml), tetracycline (Tc; 15 μg/ml), and streptomycin (Sm; 50 μg/ml), and for P. aeruginosa , carbenicillin (300 μg/ml), Gm (80 μg/ml), Tc (200 μg/ml), and Sm (2 mg/ml). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Source or reference Strains E. coli TG1 supE Δ( lac-proAB ) thi hsdR Δ 5 (F′ traD36 rpoA + B + lacI q Z Δ M15 ) Lab collection TOP10F′ F′ [ lacI q Tn 10 (Tet r )] mrcA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ Δ M15 Δ lacX74 recA1 Invitrogen CC118(λ pir ) Host strain for pKNG101 replication, Δ( ara-leu ) araD Δ lacX74 galE galK phoA20 thi-1 rpsE rpoB argE (Am) recA1 Rf r (λ pir ) Lab collection P. aeruginosa PAO1 Wild type Lab collection PAO1Δ pilA Δ fliC PAO1 mutant with deletions in the pilA and fliC genes Lab collection PAO1Δ fliC PAO1 mutant with a deletion in the fliC gene Lab collection PAO1Δ pilA PAO1 mutant with a deletion in the pilA gene Lab collection PAO1Δ pilA Δ fliC Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , and cupC3 genes This study PAO1Δ pilA Δ fliC Δ cupB3 PAO1 mutant with deletions in the pilA , fliC , and cupB3 genes This study PAO1Δ pilA Δ fliC Δ cupB1 PAO1 mutant with deletions in the pilA , fliC , and cupB1 genes This study PAO1Δ pilA Δ fliC Δ cupB3 Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , cupB3 , and cupC3 genes This study PAK Wild type Lab collection PAK cupB3 ::Cb r PAK strain with an insertion in the cupB3 gene 36 PAK cupC3 ::Cb r PAK strain with an insertion in the cupC3 gene 36 Plasmids pCR2.1 TA cloning vector for PCR products, lacZ α ColE1 f1 ori Ap r Km r Invitrogen pMMB67EH Broad-host-range vector, IncQ P tac lacZ α Gm r 20 pBBR1MCS-3 Broad-host-range vector, Tc r Lab collection pKNG101 Suicide vector in P. aeruginosa sacB Sm r Lab collection pDEST42 Destination vector for gateway technology, T7 promoter, Ap r Invitrogen pBBR cupC3 cupC3 gene cloned in pBBR1MCS-3, Tc r This study pBBR cupB3 cupB3 gene cloned in pBBR1MCS-3, Tc r This study pRK2013 ColE1 ori tra + mob + Km r Lab collection pMMB rocS1 rocS1 gene cloned in pMMB67EH, Gm r 20 pDEST42- cupC1 cupC1 gene cloned in pDEST42, Ap r This study Open in a separate window a Cb, carbenicillin; Ap, ampicillin; Tet, tetracycline; Rf, rifampin.

Techniques: Plasmid Preparation, Mutagenesis, TA Cloning, Clone Assay

Journal:

Article Title: Assembly of Fimbrial Structures in Pseudomonas aeruginosa : Functionality and Specificity of Chaperone-Usher Machineries ▿

doi: 10.1128/JB.00093-07

Figure Lengend Snippet: Oligonucleotides used for mutation engineering and gene cloning

Article Snippet: The following antibiotics at the indicated concentrations were used: for E. coli , ampicillin (50 μg/ml), gentamicin (Gm; 25 μg/ml), kanamycin (Km; 25 μg/ml), tetracycline (Tc; 15 μg/ml), and streptomycin (Sm; 50 μg/ml), and for P. aeruginosa , carbenicillin (300 μg/ml), Gm (80 μg/ml), Tc (200 μg/ml), and Sm (2 mg/ml). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Source or reference Strains E. coli TG1 supE Δ( lac-proAB ) thi hsdR Δ 5 (F′ traD36 rpoA + B + lacI q Z Δ M15 ) Lab collection TOP10F′ F′ [ lacI q Tn 10 (Tet r )] mrcA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ Δ M15 Δ lacX74 recA1 Invitrogen CC118(λ pir ) Host strain for pKNG101 replication, Δ( ara-leu ) araD Δ lacX74 galE galK phoA20 thi-1 rpsE rpoB argE (Am) recA1 Rf r (λ pir ) Lab collection P. aeruginosa PAO1 Wild type Lab collection PAO1Δ pilA Δ fliC PAO1 mutant with deletions in the pilA and fliC genes Lab collection PAO1Δ fliC PAO1 mutant with a deletion in the fliC gene Lab collection PAO1Δ pilA PAO1 mutant with a deletion in the pilA gene Lab collection PAO1Δ pilA Δ fliC Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , and cupC3 genes This study PAO1Δ pilA Δ fliC Δ cupB3 PAO1 mutant with deletions in the pilA , fliC , and cupB3 genes This study PAO1Δ pilA Δ fliC Δ cupB1 PAO1 mutant with deletions in the pilA , fliC , and cupB1 genes This study PAO1Δ pilA Δ fliC Δ cupB3 Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , cupB3 , and cupC3 genes This study PAK Wild type Lab collection PAK cupB3 ::Cb r PAK strain with an insertion in the cupB3 gene 36 PAK cupC3 ::Cb r PAK strain with an insertion in the cupC3 gene 36 Plasmids pCR2.1 TA cloning vector for PCR products, lacZ α ColE1 f1 ori Ap r Km r Invitrogen pMMB67EH Broad-host-range vector, IncQ P tac lacZ α Gm r 20 pBBR1MCS-3 Broad-host-range vector, Tc r Lab collection pKNG101 Suicide vector in P. aeruginosa sacB Sm r Lab collection pDEST42 Destination vector for gateway technology, T7 promoter, Ap r Invitrogen pBBR cupC3 cupC3 gene cloned in pBBR1MCS-3, Tc r This study pBBR cupB3 cupB3 gene cloned in pBBR1MCS-3, Tc r This study pRK2013 ColE1 ori tra + mob + Km r Lab collection pMMB rocS1 rocS1 gene cloned in pMMB67EH, Gm r 20 pDEST42- cupC1 cupC1 gene cloned in pDEST42, Ap r This study Open in a separate window a Cb, carbenicillin; Ap, ampicillin; Tet, tetracycline; Rf, rifampin.

Techniques: Mutagenesis

Journal:

Article Title: Assembly of Fimbrial Structures in Pseudomonas aeruginosa : Functionality and Specificity of Chaperone-Usher Machineries ▿

doi: 10.1128/JB.00093-07

Figure Lengend Snippet: Biofilm formation by P. aeruginosa upon RocS1 overproduction. Strains PAO1ΔpilAΔfliC/pMMBrocS1 and PAO1ΔpilAΔfliC/pMMB67EH were inoculated into microtiter plates. (A) After ITPG induction for 48 h at 30°C, bacterial rings that formed at the air-liquid interface were observed by crystal violet staining. (B) The amount of bacteria was quantified after extraction of crystal violet and OD570 measurements. Wells inoculated with sterile medium and with the PAO1 strain were also included as controls. ***, P < 0.001. (C) Biofilm formation at the air-liquid interface of glass slides immersed in culture medium was analyzed with the PAO1ΔpilAΔfliC/pMMBrocS1 (ΔΔ), PAO1ΔpilAΔfliCΔcupB3/pMMBrocS1 (ΔΔΔcupB3), and PAO1ΔpilAΔfliCΔcupB3/pMMBrocS1 (ΔΔΔcupC3) strains using DAPI staining and epifluorescence microscopic observation at a ×63 magnification. (D) Stacked confocal scanning laser microscopy images of biofilms of the PAO1ΔpilAΔfliC/pMMBrocS1 (ΔΔ), PAO1ΔpilAΔfliCΔcupB3/pMMBrocS1 (ΔΔΔcupB3), and PAO1ΔpilAΔfliCΔcupB3/pMMBrocS1 (ΔΔΔcupC3) strains and of the trans-complemented strains with the cupB3 (ΔΔΔcupB3 + pBBRB3) and cupC3 (ΔΔΔcupC3 + pBBRC3) genes. DAPI was used for staining, and confocal-microscopic observation was done at a ×180 magnification using z slices of 300 nm. (E) Corresponding extracted z images and their respective xy and xz planes showing biofilms formed by the PAO1ΔpilAΔfliC/pMMBrocS1 (ΔΔ), PAO1ΔpilAΔfliCΔcupB3/pMMBrocS1 (ΔΔΔcupB3), and PAO1ΔpilAΔfliCΔcupB3/pMMBrocS1 (ΔΔΔcupC3) strains and in the trans-complemented strains with the cupB3 (ΔΔΔcupB3 + pBBRB3) and cupC3 (ΔΔΔcupC3 + pBBRC3) genes.

Article Snippet: The following antibiotics at the indicated concentrations were used: for E. coli , ampicillin (50 μg/ml), gentamicin (Gm; 25 μg/ml), kanamycin (Km; 25 μg/ml), tetracycline (Tc; 15 μg/ml), and streptomycin (Sm; 50 μg/ml), and for P. aeruginosa , carbenicillin (300 μg/ml), Gm (80 μg/ml), Tc (200 μg/ml), and Sm (2 mg/ml). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Source or reference Strains E. coli TG1 supE Δ( lac-proAB ) thi hsdR Δ 5 (F′ traD36 rpoA + B + lacI q Z Δ M15 ) Lab collection TOP10F′ F′ [ lacI q Tn 10 (Tet r )] mrcA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ Δ M15 Δ lacX74 recA1 Invitrogen CC118(λ pir ) Host strain for pKNG101 replication, Δ( ara-leu ) araD Δ lacX74 galE galK phoA20 thi-1 rpsE rpoB argE (Am) recA1 Rf r (λ pir ) Lab collection P. aeruginosa PAO1 Wild type Lab collection PAO1Δ pilA Δ fliC PAO1 mutant with deletions in the pilA and fliC genes Lab collection PAO1Δ fliC PAO1 mutant with a deletion in the fliC gene Lab collection PAO1Δ pilA PAO1 mutant with a deletion in the pilA gene Lab collection PAO1Δ pilA Δ fliC Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , and cupC3 genes This study PAO1Δ pilA Δ fliC Δ cupB3 PAO1 mutant with deletions in the pilA , fliC , and cupB3 genes This study PAO1Δ pilA Δ fliC Δ cupB1 PAO1 mutant with deletions in the pilA , fliC , and cupB1 genes This study PAO1Δ pilA Δ fliC Δ cupB3 Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , cupB3 , and cupC3 genes This study PAK Wild type Lab collection PAK cupB3 ::Cb r PAK strain with an insertion in the cupB3 gene 36 PAK cupC3 ::Cb r PAK strain with an insertion in the cupC3 gene 36 Plasmids pCR2.1 TA cloning vector for PCR products, lacZ α ColE1 f1 ori Ap r Km r Invitrogen pMMB67EH Broad-host-range vector, IncQ P tac lacZ α Gm r 20 pBBR1MCS-3 Broad-host-range vector, Tc r Lab collection pKNG101 Suicide vector in P. aeruginosa sacB Sm r Lab collection pDEST42 Destination vector for gateway technology, T7 promoter, Ap r Invitrogen pBBR cupC3 cupC3 gene cloned in pBBR1MCS-3, Tc r This study pBBR cupB3 cupB3 gene cloned in pBBR1MCS-3, Tc r This study pRK2013 ColE1 ori tra + mob + Km r Lab collection pMMB rocS1 rocS1 gene cloned in pMMB67EH, Gm r 20 pDEST42- cupC1 cupC1 gene cloned in pDEST42, Ap r This study Open in a separate window a Cb, carbenicillin; Ap, ampicillin; Tet, tetracycline; Rf, rifampin.

Techniques: Staining, Microscopy

Journal:

Article Title: Assembly of Fimbrial Structures in Pseudomonas aeruginosa : Functionality and Specificity of Chaperone-Usher Machineries ▿

doi: 10.1128/JB.00093-07

Figure Lengend Snippet: Extracellular assembly of CupC1 and CupB1 proteins in a specific usher-dependent manner. (A) Protein samples obtained from SFs from the PAK, PAK::cupB3, and PAK::cupC3 strains transformed with pMMBrocS1. Proteins contained in the SFs were precipitated with 50% AS, separated on SDS gels, and stained with Coomassie blue. The CupB1 and the CupC1 proteins are indicated by the upper (B1) and the lower (C1) arrows, respectively. (B) Detection of the CupC1 protein in whole-cell extracts (C) (lanes 1, 3, and 5) and in SFs (lanes 2, 4, 6, 7, and 8) of PAO1ΔpilAΔfliC (ΔΔ), PAO1ΔpilAΔfliCΔcupC3 (ΔΔΔcupC3), PAO1ΔpilAΔfliCΔcupC3/pBBRcupC3 (ΔΔΔcupC3/pBBRcupC3), PAO1ΔpilAΔfliCΔcupB3 (ΔΔΔcupB3), and PAO1ΔpilAΔfliCΔcupB3ΔcupC3 (ΔΔΔcupB3ΔcupC3). All strains contained the pMMBrocS1 plasmid. (C) Coomassie blue staining of proteins contained in SFs from PAO1ΔpilAΔfliC (ΔΔ, lane 1), PAO1ΔpilAΔfliCΔcupB3 (ΔΔΔcupB3, lane 2), PAO1ΔpilAΔfliCΔcupC3 (ΔΔΔcupC3, lane 3), and PAO1ΔpilAΔfliCΔcupB3ΔcupC3 (ΔΔΔcupB3ΔcupC3, lane 4). All strains contained pMMBrocS1. The CupB1 and the CupC1 proteins are identified by the upper (B1) and the lower (C1) arrows, respectively.

Article Snippet: The following antibiotics at the indicated concentrations were used: for E. coli , ampicillin (50 μg/ml), gentamicin (Gm; 25 μg/ml), kanamycin (Km; 25 μg/ml), tetracycline (Tc; 15 μg/ml), and streptomycin (Sm; 50 μg/ml), and for P. aeruginosa , carbenicillin (300 μg/ml), Gm (80 μg/ml), Tc (200 μg/ml), and Sm (2 mg/ml). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Source or reference Strains E. coli TG1 supE Δ( lac-proAB ) thi hsdR Δ 5 (F′ traD36 rpoA + B + lacI q Z Δ M15 ) Lab collection TOP10F′ F′ [ lacI q Tn 10 (Tet r )] mrcA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ Δ M15 Δ lacX74 recA1 Invitrogen CC118(λ pir ) Host strain for pKNG101 replication, Δ( ara-leu ) araD Δ lacX74 galE galK phoA20 thi-1 rpsE rpoB argE (Am) recA1 Rf r (λ pir ) Lab collection P. aeruginosa PAO1 Wild type Lab collection PAO1Δ pilA Δ fliC PAO1 mutant with deletions in the pilA and fliC genes Lab collection PAO1Δ fliC PAO1 mutant with a deletion in the fliC gene Lab collection PAO1Δ pilA PAO1 mutant with a deletion in the pilA gene Lab collection PAO1Δ pilA Δ fliC Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , and cupC3 genes This study PAO1Δ pilA Δ fliC Δ cupB3 PAO1 mutant with deletions in the pilA , fliC , and cupB3 genes This study PAO1Δ pilA Δ fliC Δ cupB1 PAO1 mutant with deletions in the pilA , fliC , and cupB1 genes This study PAO1Δ pilA Δ fliC Δ cupB3 Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , cupB3 , and cupC3 genes This study PAK Wild type Lab collection PAK cupB3 ::Cb r PAK strain with an insertion in the cupB3 gene 36 PAK cupC3 ::Cb r PAK strain with an insertion in the cupC3 gene 36 Plasmids pCR2.1 TA cloning vector for PCR products, lacZ α ColE1 f1 ori Ap r Km r Invitrogen pMMB67EH Broad-host-range vector, IncQ P tac lacZ α Gm r 20 pBBR1MCS-3 Broad-host-range vector, Tc r Lab collection pKNG101 Suicide vector in P. aeruginosa sacB Sm r Lab collection pDEST42 Destination vector for gateway technology, T7 promoter, Ap r Invitrogen pBBR cupC3 cupC3 gene cloned in pBBR1MCS-3, Tc r This study pBBR cupB3 cupB3 gene cloned in pBBR1MCS-3, Tc r This study pRK2013 ColE1 ori tra + mob + Km r Lab collection pMMB rocS1 rocS1 gene cloned in pMMB67EH, Gm r 20 pDEST42- cupC1 cupC1 gene cloned in pDEST42, Ap r This study Open in a separate window a Cb, carbenicillin; Ap, ampicillin; Tet, tetracycline; Rf, rifampin.

Techniques: Transformation Assay, Staining, Plasmid Preparation

Journal:

Article Title: Assembly of Fimbrial Structures in Pseudomonas aeruginosa : Functionality and Specificity of Chaperone-Usher Machineries ▿

doi: 10.1128/JB.00093-07

Figure Lengend Snippet: Images of CupB and CupC appendages at the P. aeruginosa cell surface. Long fimbriae decorated with gold particles coupled to specific antibodies directed against CupC1 were peritrichously distributed at the cell surface of the PAO1ΔpilAΔfliC strain (A), in which RocS1 was overproduced (pMMBrocS1) (inset in upper left-hand corner, ×20,000 magnification of central boxed area). The CupC1-labeled fimbriae were absent in the mutant PAO1ΔpilAΔfliCΔcupC3/pMMBrocS1 (magnification, ×50,000) (B) but recovered when the cupC3 mutation was trans-complemented with the cupC3 gene (magnification, ×20,000) (C). The unlabeled CupB fimbriae were visualized (arrowheads) in the cupC3 mutant (B) and absent in the PAO1ΔpilAΔfliCΔcupB3ΔcupC3 mutant (D) (magnification, ×50,000) but recovered when the cupB3 mutation was trans-complemented with the cupB3 gene (magnification, ×20,000) (E).

Article Snippet: The following antibiotics at the indicated concentrations were used: for E. coli , ampicillin (50 μg/ml), gentamicin (Gm; 25 μg/ml), kanamycin (Km; 25 μg/ml), tetracycline (Tc; 15 μg/ml), and streptomycin (Sm; 50 μg/ml), and for P. aeruginosa , carbenicillin (300 μg/ml), Gm (80 μg/ml), Tc (200 μg/ml), and Sm (2 mg/ml). table ft1 table-wrap mode="anchored" t5 TABLE 1. caption a7 Strain or plasmid Relevant characteristic(s) Source or reference Strains E. coli TG1 supE Δ( lac-proAB ) thi hsdR Δ 5 (F′ traD36 rpoA + B + lacI q Z Δ M15 ) Lab collection TOP10F′ F′ [ lacI q Tn 10 (Tet r )] mrcA Δ( mrr-hsdRMS-mcrBC ) φ80 lacZ Δ M15 Δ lacX74 recA1 Invitrogen CC118(λ pir ) Host strain for pKNG101 replication, Δ( ara-leu ) araD Δ lacX74 galE galK phoA20 thi-1 rpsE rpoB argE (Am) recA1 Rf r (λ pir ) Lab collection P. aeruginosa PAO1 Wild type Lab collection PAO1Δ pilA Δ fliC PAO1 mutant with deletions in the pilA and fliC genes Lab collection PAO1Δ fliC PAO1 mutant with a deletion in the fliC gene Lab collection PAO1Δ pilA PAO1 mutant with a deletion in the pilA gene Lab collection PAO1Δ pilA Δ fliC Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , and cupC3 genes This study PAO1Δ pilA Δ fliC Δ cupB3 PAO1 mutant with deletions in the pilA , fliC , and cupB3 genes This study PAO1Δ pilA Δ fliC Δ cupB1 PAO1 mutant with deletions in the pilA , fliC , and cupB1 genes This study PAO1Δ pilA Δ fliC Δ cupB3 Δ cupC3 PAO1 mutant with deletions in the pilA , fliC , cupB3 , and cupC3 genes This study PAK Wild type Lab collection PAK cupB3 ::Cb r PAK strain with an insertion in the cupB3 gene 36 PAK cupC3 ::Cb r PAK strain with an insertion in the cupC3 gene 36 Plasmids pCR2.1 TA cloning vector for PCR products, lacZ α ColE1 f1 ori Ap r Km r Invitrogen pMMB67EH Broad-host-range vector, IncQ P tac lacZ α Gm r 20 pBBR1MCS-3 Broad-host-range vector, Tc r Lab collection pKNG101 Suicide vector in P. aeruginosa sacB Sm r Lab collection pDEST42 Destination vector for gateway technology, T7 promoter, Ap r Invitrogen pBBR cupC3 cupC3 gene cloned in pBBR1MCS-3, Tc r This study pBBR cupB3 cupB3 gene cloned in pBBR1MCS-3, Tc r This study pRK2013 ColE1 ori tra + mob + Km r Lab collection pMMB rocS1 rocS1 gene cloned in pMMB67EH, Gm r 20 pDEST42- cupC1 cupC1 gene cloned in pDEST42, Ap r This study Open in a separate window a Cb, carbenicillin; Ap, ampicillin; Tet, tetracycline; Rf, rifampin.

Techniques: Labeling, Mutagenesis

Journal:

Article Title: Molecular Characterization and Substrate Preference of a Polycyclic Aromatic Hydrocarbon Dioxygenase from Cycloclasticus sp. Strain A5

doi: 10.1128/AEM.69.11.6688-6697.2003

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

Article Snippet: 24 E. coli JM109 recA1 relA1 thi-1 Δ( lac-proAB ) gyrA96 hsdR17 endA1 supE44 (r k − m k − ) [F7 traD36 proAB lacI r Z ΔM15] 60 E. coli XL1-Blue hsdR17 supE44 recA1 endA1 gyrA46 thi-1 relA1 lac /F′ [ proAB + lacI q lacZ ΔM15::Tn 10 (Tet r )] Stratagene E. coli XL1-Blue MRA Δ( mcrA ) 183 Δ( mcrCB-hsdSMR-mrr ) 173 endA1 supE44 thi-1 recA96 gyrA1 relA1 lac Stratagene Plasmids Lorist6 Km r : 5.28-kb cosmid vector 15 pBluescript II SK+ lacZ Ap r : 2.96-kb cloning vector Stratagene pH1a Km r Lorist6::11-kb Sau 3A1 fragment from A5 containing phnA1A2A3A4 This study pH1b Km r Lorist6::11-kb Sau 3A1 fragment from A5 containing phnA1A2A3A4 This study pPhnA Ap r : pBluescript:: ring-hydroxylating dioxygenase gene phnA1A2A3A4 from A5 This study pPhnC Ap r : pBluescript:: extradiol dioxygenase gene phnC from A5 This study Open in a separate window Bacterial strains and plasmids used in this study

Techniques: Plasmid Preparation, Clone Assay

Journal:

Article Title: Redefining the Role of psr in ?-Lactam Resistance and Cell Autolysis of Enterococcus hirae

doi: 10.1128/JB.185.20.5925-5935.2003

Figure Lengend Snippet: Strains and plasmids used in this study

Article Snippet: pUC18 , Ap r lacZ E. coli cloning vector , Amersham Pharmacia Biotech.

Techniques: Plasmid Preparation, Mutagenesis, Derivative Assay, Clone Assay, TA Cloning, Expressing

Journal:

Article Title: Analysis of dofA , a fruA -Dependent Developmental Gene, and Its Homologue, dofB , in Myxococcus xanthus

doi: 10.1128/JB.184.24.6803-6810.2002

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

Article Snippet: pGEM-T easy , Ap r lacZ cloning vector , Promega.

Techniques: Plasmid Preparation, Clone Assay

Journal:

Article Title: Genetic and Functional Analysis of the tbc Operons for Catabolism of Alkyl- and Chloroaromatic Compounds in Burkholderia sp. Strain JS150

doi: 10.1128/AEM.67.10.4805-4816.2001

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

Article Snippet: Cultures maintained in liquid or solid media were incubated at 37°C. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristic(s) a Source or reference Strains E. coli JM109 recA1 relA1 thi-1 Δ( lac-proAB ) gyrA96 hsdR17 endA1 supE44 (r K − m K + ) [F′ traD36 proAB lacI q Z Δ M15 ] Promega E. coli BL21 F − dcm ompT hsdR (r B − m B + ) gal Stratagene Burkholderia sp. strain JS150 Tol + Ben + Chlben + Crl + Phl + 15 P. aeruginosa PAO1c Prototroph 20 Plasmids pBluescript II KS + (SK + ) lacZ Ap r ; 2.96-kb cloning vector Stratagene pRO1727 Tc r Cb r ; 3.77-kb Pseudomonas cloning vector 8 pHYK2000 Cb r ; pRO1727::14.3-kb Hin dIII- Bam HI fragment from JS150 containing tbc1 and tbc2 This study pHYK2001 Cb r ; pRO1727::11.3-kb Hin dIII- Bam HI fragment of pHYK2000, with a 3-kb Hin dIII deletion This study pHYK2002 Cb r ; pRO1727::10.1-kb Hin dIII- Bam HI fragment of pHYK2000, with a 4.2-kb internal Not I deletion This study pHYK2003 Cb r ; pRO1727::8.9-kb Hin dIII- Bam HI fragment of pHYK2000, with a 5.4-kb internal Xho I deletion This study pHYK2004 Cb r ; pRO1727::10.5-kb Hin dIII- Xho I fragment of pHYK2000, with a 3.8-kb deletion fusing an internal Xho I site with a vector Sal I site This study pHYK2005 Cb r ; pRO1727::9.2-kb Bam HI- Xho I fragment of pHYK2000 ligated to Bam HI- and Xho I-digested pRO1727 This study pJCM1000 Ap r ; pBluescript::5.4-kb Xho I fragment of pHYK2000 This study pJCM1001 Ap r ; pBluescript::6.2-kb Hin dIII- Not I fragment of pHYK2000 This study Open in a separate window a Abbreviations: Tol, toluene; Ben, benzene; Chlben, chlorobenzene; Crl, cresol; Phl, phenol; Ap, ampicillin; Tc, tetracycline; Cb, carbenicillin.

Techniques: Plasmid Preparation, Clone Assay

Journal:

Article Title: Genetic and Functional Analysis of the tbc Operons for Catabolism of Alkyl- and Chloroaromatic Compounds in Burkholderia sp. Strain JS150

doi: 10.1128/AEM.67.10.4805-4816.2001

Figure Lengend Snippet: Physical and functional map of pHYK2000. (A) Restriction map of the 14.3-kb BamHI-HindIII DNA fragment from Burkholderia sp. strain JS150 cloned into vector pRO1727 as pHYK2000. The XhoI-HindIII fragment used as a probe is indicated. (B) Southern hybridization results for DNA digested with XhoI and HindIII. Lanes: C, control (cloned) DNA; Ch, chromosomal DNA; P, plasmid DNA. (C) Construction of deletion mutants of pHYK2000 and assays for substrates oxidized by each construct. For functional mapping of pHYK2000, HindIII, NotI, XhoI, BamHI-XhoI, and HindIII-XhoI deletions were constructed, and the constructs were used to assay for oxidation (+, weak; ++, intermediate; +++, strong) of toluene (Tol), benzene (Ben), and chlorobenzene (Chlb).

Article Snippet: Cultures maintained in liquid or solid media were incubated at 37°C. table ft1 table-wrap mode="anchored" t5 TABLE 1 caption a7 Strain or plasmid Relevant characteristic(s) a Source or reference Strains E. coli JM109 recA1 relA1 thi-1 Δ( lac-proAB ) gyrA96 hsdR17 endA1 supE44 (r K − m K + ) [F′ traD36 proAB lacI q Z Δ M15 ] Promega E. coli BL21 F − dcm ompT hsdR (r B − m B + ) gal Stratagene Burkholderia sp. strain JS150 Tol + Ben + Chlben + Crl + Phl + 15 P. aeruginosa PAO1c Prototroph 20 Plasmids pBluescript II KS + (SK + ) lacZ Ap r ; 2.96-kb cloning vector Stratagene pRO1727 Tc r Cb r ; 3.77-kb Pseudomonas cloning vector 8 pHYK2000 Cb r ; pRO1727::14.3-kb Hin dIII- Bam HI fragment from JS150 containing tbc1 and tbc2 This study pHYK2001 Cb r ; pRO1727::11.3-kb Hin dIII- Bam HI fragment of pHYK2000, with a 3-kb Hin dIII deletion This study pHYK2002 Cb r ; pRO1727::10.1-kb Hin dIII- Bam HI fragment of pHYK2000, with a 4.2-kb internal Not I deletion This study pHYK2003 Cb r ; pRO1727::8.9-kb Hin dIII- Bam HI fragment of pHYK2000, with a 5.4-kb internal Xho I deletion This study pHYK2004 Cb r ; pRO1727::10.5-kb Hin dIII- Xho I fragment of pHYK2000, with a 3.8-kb deletion fusing an internal Xho I site with a vector Sal I site This study pHYK2005 Cb r ; pRO1727::9.2-kb Bam HI- Xho I fragment of pHYK2000 ligated to Bam HI- and Xho I-digested pRO1727 This study pJCM1000 Ap r ; pBluescript::5.4-kb Xho I fragment of pHYK2000 This study pJCM1001 Ap r ; pBluescript::6.2-kb Hin dIII- Not I fragment of pHYK2000 This study Open in a separate window a Abbreviations: Tol, toluene; Ben, benzene; Chlben, chlorobenzene; Crl, cresol; Phl, phenol; Ap, ampicillin; Tc, tetracycline; Cb, carbenicillin.

Techniques: Functional Assay, Clone Assay, Plasmid Preparation, Hybridization, Construct