Article Figures & Data
Figures
- FIG. 1.
Map of pNS2Trc used for gene expression and gene knockdown in Bartonella. Ptrc, trc hybrid promoter; MCS, multiple-cloning site; Kan, kanamycin resistance gene; Rep, gene required for plasmid replication; 6xHis, His6 tag fusion; LacO, lac operator; rrnB TT, T1 and T2 transcription terminators derived from the rrnB rRNA operon.
- FIG. 2.
Invasion of HMEC-1 cells by B. henselae strains harboring plasmids with various promoters transcribing ompR as-RNA. Infected cells were treated with gentamicin (200 μg/ml for 1 hour) to kill extracellular bacteria, and the lysate was plated to determine the number of intracellular bacteria as CFU per well for each promoter transcribing plasmid-borne ompR. Wild-type B. henselae Houston-1 with and without (“no promoter”) the promoterless plasmid (pNS2) served as controls. B. henselae (ompROC) overexpressing ompR from the T5 promoter in pNS2T5 is also shown. P < 0.05 compared to Houston-1.
Tables
- TABLE 1.
Descriptions of the plasmids used in this study and the β-galactosidase activities resulting from transcription of lacZ directionally cloned immediately downstream of the vector-specific promoter
Plasmid Size (bp) Promoter GenBank accession no. Μean β-galactosidase activity ± SD (Miller units) pNS2Trc 3,109 Ptrc EU600162 36,531 ± 3,326 pNS2T5 3,000 PT5 EU600163 1,204 ± 162.5 pNS2Ch 2,917 PCh EU600164 1,166 ± 161.4 pNS2Kan 2,823 Pkan EU600165 687 ± 70.1 pNs2Amp 2,905 Pamp EU600169 394 ± 67.2 pNS2GroE 2,877 PgroE EU600166 273 ± 56.0 pNS2 2,680 None NAa 153 ± 43.4 ↵ a NA, not applicable.
Supplemental material
Files in this Data Supplement:
- Supplemental file 1
-
Bacterial strain and plasmid data (Table S1), PCR primers used for amplification (Table S2), and immunoblotting for detection
of beta-galactosidase produced by transcribing lacZ from different promoters (Fig. S1).
Word file, 67K. - Supplemental file 2
-
Supplemental materials and methods (bacterial strains, plasmids, and oligonucleotides; recombinant DNA methods; vector construction;
promoter replacement; lacZ expression; and Western blotting).
Word file, 70K.
- Supplemental file 1
-
Bacterial strain and plasmid data (Table S1), PCR primers used for amplification (Table S2), and immunoblotting for detection
of beta-galactosidase produced by transcribing lacZ from different promoters (Fig. S1).
