Article Figures & Data
Figures
- Fig 1
Map of the inducible expression vector pKIX. The multiple-cloning site (MCS) and the inserted kdp promoter (Pkdp) with the basal transcription initiation elements (TATA box [TATA], transcription factor B recognition element [BRE], and initiation region [INR]) are shown in detail. The transcription start site in Pkdp is denoted by an arrow. Unique restriction sites suitable for the cloning of target genes within the MCS are highlighted in light gray.
- Fig 2
(a) Sequence of Pkdp between the TATA box and the transcription start site (arrow). Exchanged nucleotides (ex) are denoted. (b) β-Galactosidase activities of transcriptional fusions of bgaH to a truncated −42-bp Pkdp (−42 Pkdp) and to the −42-bp Pkdp with the nucleotide exchanges (−42 Pkdp ex) shown in panel a. Measurements were done in triplicate with H. salinarum R1 ΔkdpFABC cultures grown under inducing and noninducing conditions (3 and 100 mM initial K+, respectively).
- Fig 3
Growth of H. salinarum R1 ΔkdpFABC transformed with pKIX_bgaH in medium supplemented with 3 mM, 5 mM, or 100 mM KCl. Another 3 mM KCl culture was supplied with 100 mM KCl in the late exponential growth phase (arrow) directly upon sampling for subsequent real-time RT-PCR analyses (see Fig. 5). Samples were collected at the times indicated by arrows in the early exponential (early exp.), the late exponential (late exp.), the early stationary (early stat.), and the late stationary (late stat.) growth phases. In the case of the 3 mM KCl culture supplied with 100 mM KCl, an additional sample was taken 1.5 h following KCl addition.
- Fig 4
Normalized transcript levels of bgaH expressed from pKIX_bgaH in H. salinarum R1 ΔkdpFABC with various initial K+ concentrations in the medium (3 mM, 5 mM, and 100 mM KCl). Samples were taken from cultures as described in the legend to Fig. 3. All samples were assayed in triplicate by real-time RT-PCR analysis.
- Fig 5
Normalized transcript levels of kdpFABCQ expressed in H. salinarum R1 ΔkdpFABC from pKIX_kdp with various initial K+ concentrations in the medium (3 mM, 5 mM, and 100 mM KCl). Sampling was done as described in the legend to Fig. 3. All samples were assayed in triplicate by real-time RT-PCR analysis.
- Fig 6
(a) Sequence of Pkdp between the TATA box and the transcription start site (arrow). Mutations introduced in the regions of bp −17 to −20 (−17_−20) and bp −8 to −11 (−8_−11) are denoted. (b) β-Galactosidase activities of H. salinarum R1 ΔkdpFABC encoding Pkdp::bgaH fusions with a truncated 42-bp Pkdp (−42). The bars labeled −17_−20 and −8_−11 indicate the effects of the exchanges shown in panel a. Samples were taken from cultures grown under inducing and noninducing conditions (3 and 100 mM KCl, respectively) and analyzed in triplicate. (c) Normalized expression levels of kdpFABCQ expressed in H. salinarum R1 ΔkdpFABCQ from the full-length kdp promoter (−206) and from the promoter comprising both sets of mutations (−206 ex) depicted in panel a. Measurements were performed in triplicate by real-time RT-PCR analysis.
Additional Files
Supplemental material
Files in this Data Supplement:
- Supplemental file 1 -
Detailed description of the construction of pKIX (Text S1).
PDF file, 14K. - Supplemental file 2 -
List of plasmids (Table S1).
PDF file, 52K. - Supplemental file 3 -
List of primers (Table S2).
PDF file, 24K. - Supplemental file 4 -
Construction of the K+-dependent inducible expression vector pKIX (Fig. S1).
PDF file, 34K. - Supplemental file 5 -
Sequence information of plasmid pKIX (Text S2).
PDF file, 51K. - Supplemental file 6 -
β-Galactosidase activities of H. salinarum ΔkdpFABC transformed with a plasmid expressing bgaH under the control of Pkdp (Fig. S2).
PDF file, 58K.
- Supplemental file 1 -
Detailed description of the construction of pKIX (Text S1).
