Figure 1.
Transcript levels of lacZ transcribed from various rrn promoter regions.
The pACYC plasmids were constructed so that they contained various rrn operon promoter regions fused upstream a promoterless lacZ gene, as described in Materials and Methods. The short promoter region of rrn contained only the P1 sequence of the rrn promoter (S = short), the second segment contained the P1, P2 and nut -like sequences (boxA, boxB and boxC) (M = medium length) and a longer segment contained the P1, P2, nut like sequences and tL region of the rrn operon (L = long). All the promoter regions contained the FIS elements and other elements required for initiation of transcription. The pACYC plasmids were transformed into the lacZ and ybeY, lacZ deletion strains. (A) a schematic description of the three promoter regions which were fused to lacZ. The rrn sequence, the FIS elements, P1, P2, nut -like sequences and tL region are marked. (B) Cultures of ΔlacZ (“wild type ybeY”) and ΔlacZΔybeY (“ΔybeY”) carrying the pACYC plasmids were grown in LB at 37°C and harvested at O.D600 = 0.45. RNA was extracted from these samples and analyzed by qRTPCR.
Figure 2.
Stability and levels of RNA expressed from various rrn promoter regions in ybeY deletion mutant.
(A) Stability of lacZ gene transcripts was determined by measuring its residual levels following addition of Rifampicin (200 µg/ml). Cultures and growth conditions were as described in Figure 1. All the bacteria were deleted for the chromosomal lacZ gene and transformed with pACYC plasmids carrying various rrn promoter sequences fused upstream to a promoterless lacZ. The plasmids carried the “S” region, “M” region, or “L” region. Rifampicin was added at time 0 and the cultures were harvested at 0, 3, 6 min. RNA was extracted and analyzed by qRTPCR. Bacteria with wild type ybeY gene and plasmid with “S” region (filled circles), with “M” region (open circles) and with “L” region (filled triangles). ΔybeY mutant carrying a plasmid with “S” region (open triangles), with “M” region (filled squares) and with “L” region (opened squares). (B) Levels of RNA encoded from the region prior to the antitermination sequences. Cultures of wild type and ΔybeY mutant were grown in LB at 37°C and harvested at O.D600 = 0.45. RNA was extracted from these samples and the levels of the transcripts encoded by the region before the antitermination sequences were quantified by qRTPCR from the primers described above.
Figure 3.
Effect of plasmids expressing transcriptional antitermination factors on growth of ybeY deletion mutant.
Over-expression of NusA, NusB, NusG and NusE, was obtained by introducing multi-copy plasmids containing these genes cloned downstream to a lacZ promoter. These plasmids were transformed into the ybeY deletion mutant (A) and into the wild type strain (B). Cultures were grown overnight in LB medium at 30°C. They were then diluted to A600 of 0.04 in 2 ml wells (in a 24 well plate) containing 1 ml of LB medium supplemented with 0.1 mM IPTG for inducing the lacZ promoter. The cultures were transferred to 42°C and turbidity was measured at 600 nm for 12 hours. (A) wild type (filled circles), ybeY deletion mutant (open circles), ΔybeY mutant carrying pCA24N nusA (filled triangles), ΔybeY mutant carrying pCA24N nusB (open triangles), ΔybeY mutant carrying pCA24N nusE (filled squares), ΔybeY mutant carrying pCA24N nusG (opened squares) and ΔybeY mutant carrying pCA24N empty vector (filled diamonds). (B) wild type (filled circles), wild type carrying pCA24N nusA (open circles), wild type carrying pCA24N nusB (filled triangles), wild type carrying pCA24N nusE (open triangles) and wild type carrying pCA24N nusG (filled squares). (C) Growth of ΔybeY mutant carrying pCA24N encoding Nus genes on LB agar plates, supplemented with 0.1 mM IPTG, overnight at 42°C.
Table 1.
Primers used for plasmids construction.
Table 2.
Primers used for quantifying the level of transcripts in Quantitative PCR.