rRNA promoter activity in the fast-growing bacterium Vibrio natriegens

Abstract

The bacterium Vibrio natriegens can double with a generation time of less than 10 min (R. G. Eagon, J. Bacteriol. 83:736-737, 1962), a growth rate that requires an extremely high rate of protein synthesis. We show here that V. natriegens' high potential for protein synthesis results from an increase in ribosome numbers with increasing growth rate, as has been found for other bacteria. We show that V. natriegens contains a large number of rRNA operons, and its rRNA promoters are extremely strong. The V. natriegens rRNA core promoters are at least as active in vitro as Escherichia coli rRNA core promoters with either E. coli RNA polymerase (RNAP) or V. natriegens RNAP, and they are activated by UP elements, as in E. coli. In addition, the E. coli transcription factor Fis activated V. natriegens rrn P1 promoters in vitro. We conclude that the high capacity for ribosome synthesis in V. natriegens results from a high capacity for rRNA transcription, and the high capacity for rRNA transcription results, at least in part, from the same factors that contribute most to high rates of rRNA transcription in E. coli, i.e., high gene dose and strong activation by UP elements and Fis.

FIG. 1.

RNA/protein ratios as a function of growth rate in V. natriegens ATCC 14048 and E. coli VH1000. (A) RNA/protein ratios from cultures grown in media supporting different growth rates at 37°C (see Materials and Methods). (B) Curves were normalized to the same value at a growth rate of 2.0 doublings/h as described previously (14). Triangles, V. natriegens; circles, E. coli.

FIG. 2.

Southern hybridization analysis of rrn operons from V. natriegens. Lane 1, ³²P-labeled λ DNA cleaved with HindIII (control). Lane 2, E. coli chromosomal DNA cleaved with PstI plus BglII (control). Lanes 3 to 8, V. natriegens DNA cleaved with the indicated restriction enzymes. Probe RLG3133 was used for lanes 2 to 7 and is complementary to the beginning of the 16S rRNA gene, and probe RLG1492r was used for lane 8 and is complementary to the 16S rRNA gene (38).

FIG. 3.

DNA sequences from the core promoters and A+T-rich upstream sequences of V. natriegens (V.n.) rrn P1 and P2 promoters. The −35 and −10 hexamers for P1 and P2 are shown in boldface. E. coli (E.c.) rrnB and V. cholerae (V.c.) rrnA sequences are shown for comparison. The E. coli consensus sequences for the −35 and −10 hexamer (28), as well as for the UP element (16, 17), are also indicated. See Materials and Methods for GenBank accession numbers. Start sites (in boldface) have been determined experimentally only for the seven E. coli rrn promoters and for the V. natriegens rrnA promoter.

FIG. 4.

Determination of rrnA P1 and P2 transcription start sites from V. natriegens. In vitro transcription reactions and primer extension were carried out as described in Materials and Methods. The primer extension products were subjected to electrophoresis on an 8% acrylamide-8 M urea gel. DNA sequencing ladders (T, C, G, and A) are shown in the four lanes on the left and in the one lane on the right of the extension reaction and were generated with the same plasmid used for the in vitro transcription reaction. (A) Both P1 and P2 start sites are shown. (B) Gels from the same reaction shown in panel A but electrophoresed longer to improve resolution.

FIG. 5.

Transcription from plasmids containing the V. natriegens rRNA promoters in vitro. (A) UP element-dependent transcription. Lanes 1 and 2, E. coli (E.c.) RNAP. Lanes 3 to 6, V. natriegens (V.n.) RNAP. Lane 1, E. coli rrnB P1 promoter lacking (−) the UP element (pWR55; −41 to +1). Lane 2, E. coli rrnB P1 promoter including (+) the UP element (pRLG5944; −61 to +1). Lane 3, V. natriegens rrnA P1 promoter lacking upstream sequences (pRLG6098; −41 to +1). Lane 4, V. natriegens rrnA P1 promoter including upstream sequences (pRLG6099; −66 to +1). Lane 5, V. natriegens rrnA P2 promoter lacking upstream sequences (pRLG6096; −41 to +8). Lane 6, V. natriegens rrnA P2 promoter lacking upstream sequences (pRLG6097; −66 to +8). (B) Transcription with wild-type E. coli RNAP (WT; lanes 1, 3, and 5) or αΔ235 RNAP (Δ; lanes 2, 4, and 6). Lanes 1 and 2, E. coli rrnB P1 promoter (pRLG862). Lanes 3 and 4, V. natriegens rrnA P1 promoter (pRLG5102). Lanes 5 and 6, V. natriegens rrnA P2 promoter (pRLG5103). (C) Activation by Fis. Lanes 1 and 2, E. coli RNAP and E. coli rrnB P1-P2 (pRLG3858; −152 to +7). Lanes 3 and 4, V. natriegens RNAP and V. natriegens rrnA P1-P2 (pRLG5101; −248 to +8). Lanes 1 and 3, no Fis. Lanes 2 and 4, 100 nM Fis.

FIG. 6.

Characteristics of rrn P1 and P2 open complexes. (A) Open-complex half-lives. Transcription from rrn P1 and P2 promoters was measured following heparin addition. A representative gel is shown (see Materials and Methods). Lanes 1 to 8, E. coli RNAP and E. coli rrnB P1-P2 (pRLG3858). Lanes 9 to 16, V. natriegens RNAP and V. natriegens rrnA P1-P2 (pRLG5101). (B and C) Quantitation by phosphorimager analysis of lanes 1 to 8 (B) and lanes 9 to 16 (C). Solid circles, P1; open circles, P2. (D) Initiating NTP concentration dependence of V. natriegens rrnA P1 promoter in vitro. pRLG5101 was transcribed using 24 nM V. natriegens RNAP in 130 mM KCl transcription buffer (see Materials and Methods) in the presence of increasing GTP concentrations (solid circles). The −35 con promoter (22) was transcribed under the same conditions (open circles). E.c., E. coli; V.n., V. natriegens.