Fig 1.
Gene organization of seven rrn operons in the E. coli K-12 genome.
Seven rRNA operons exist in more than 2,000 E. coli strains so far sequenced [39]. Two K-12 strains, MG1655 [7] and W3110 [8], are widely used as the model E. coli strains. Strain W3110 carries an inversion between rrnD and rrnE operons [A], but the direction of transcription for all seven rrn operons are the same with that of DNA replication in both strains. The order of rRNA organization in seven rrn operons are the same, but each rrn operon contains one to three different tRNA genes [B]. Details are described in text.
Fig 2.
Promoter-region sequences of seven rrn operons used for construction of the promoter assay vectors.
For construction of the promoter assay vectors of rrn operons, a total of approximately 500 bp-long sequence upstream from 5’ terminus of 16S rRNA gene, as indicated above each DNA lane, were PCR-amplified using specific set of primers (for primer sequences see S1 Table) and inserted into pGRS vector, a modified form of pGRP [28], containing an SD sequence at the junction of GRF-coding sequence. Open box and closed boxes on each probe represent the relative location of UP element and predicted Fis-binding sites, respectively (see Fig 8). Triangles downstream of the UP element indicate two promoters, upstream P1 and downstream P2. The number of Fis sites on the rrnE promoter was suggested to be more than those hitherto identified (see Fig 3). The whole length used for the construction of pGRS vector is described in parenthesis at right-side end of each lane.
Fig 3.
Gel shift assay of Fis binding to promoter DNA probes of seven rrn operons.
Gel shift assay of Fis-binding sites was performed under the standard procedure as described in Materials and Methods using each of seven rrn promoter probes as shown in Fig 2 and increasing amounts of purified Fis protein. With the rrnE and rrnG promoters, Fis exhibited high-level of cooperative binding, forming multiple ladders.
Fig 4.
Map of the two-fluorescent reporter vector for the promoter assay of seven rrn operons.
For determination of the promoter strength and regulation of seven rrn operons, approximately 500 bp-long DNA fragments (indicated by blue; for details see Fig 2) covering each of the rrn promoters, as shown in Fig 2, were PCR-amplified and inserted into pGRS vector (the modified version of pGRP vector with an inserted SD sequence (indicated by yellow).
Table 1.
Bacteria and plasmids used in this study.
Fig 5.
Growth phase-dependent variation of the promoter activity of seven rrn operons: LB and M9-Glc-CA growth.
Promoter activity of seven rrn operons was determined using a series of pGRS vector, two fluorescent reporter assay vectors (for the plasmid map see Fig 4), which were transformed into E. coli W3110 type-A. Transformants were grown at 37°C in LB (red) and M9+glucose+casamino acids (green). The growth curve (dotted lines) was determined by measuring turbidity the promoter activity (straight lines) was determined by measuring the fluorescent intensity and is shown as the relative values with the reference of lacUV5-directed activity. Purple symbols and lines represent the lacUV5 promoter-directed GFP level that was set at 1.0.
Fig 6.
Growth phase-dependent variation of the promoter activity of seven rrn operons: LB and M9-Glc growth.
Promoter activity of seven rrn operons was determined for the culture grown in LB (red) and M9-glucose (orange) media. Growth curve and promoter activity were measured as in Fig 5, and are shown in red (LB) and orange (M9-Glc).
Fig 7.
Growth phase-dependent variation of the promoter activity of seven rrn operons: LB and M9-Gly growth.
Promoter activity of seven rrn operons was determined for the culture grown in LB (red) and M9-glycerol (blue) media. Growth curve and promoter activity were measured as in Fig 5, and are shown in red (LB) and blue (M9-Gly).
Fig 8.
Growth rate-dependent variation of the promoter activity of seven rrn operons.
The promoter activity of seven rrn operons of E. coli was separately determined as described in Fig 5. [A] The ratio between the cell growth rate in four different culture media and the highest level of promoter activity in the middle of exponential growth phase is plotted for each of seven rrn promoters. [B] The highest level of promoter activity in four different cultures is shown for each of seven rrn operon promoters. The activity of rrnE promoter was the highest under all the culture conditions employed.
Fig 9.
Promoter activity of seven rrn operons in mutants lacking the fis or hns genes.
Promoter activity of seven rrn operons was determined using the double-fluorescent protein reporter system as in Fig 5. [A] The promoter assay of seven rrn operons was performed using wild-type and a mutant lacking the fis gene. The ratio of maximum promoter activity between the fis mutant and wild-type is shown for each of seven rrn promoters. The maximum reduction in the absence of Fis was observed for the rrnE promoter. [B] The promoter assay of seven rrn operons was carried out for wild-type and a mutant lacking the hns gene. The ratio of maximum promoter activity between the hns mutant and wild-type is shown for each of seven rrn promoters. The maximum activation in the absence of H-NS was observed for the rrnB promoter that carries a strong binding site for H-NS [37]. The assay was repeated at least twice and the fluctuation level was less than 10%.
Fig 10.
Sequence difference between seven rrn operons.
[A] Upper panel shows the 500 bp-long sequences upstream of the start site of mature 16S rRNA from all seven rrn operons. The sequences conserved between seven rrn operons are shown in black but the positions that differ from the conserved sequences are shown in white. Red bars indicated transcription start sites of upstream P1 and downstream P2 promoters. Marked variation was identified in the promoter regions, in particular, upstream from P1 promoter. [B] The positions of sequence variation within 16S and 23S rRNA are shown along the gene organization of rrn operons. A total of 33 and 77 differences were identified in 16S rRNA and 23S rRNA, respectively. [C] The number of bases that are different from the conserved sequence are shown for 16S and 23S rRNA for each rrn operon.