The transcription termination factor Rho is essential and autoregulated in Caulobacter crescentus

Abstract

The impossibility of obtaining a rho null mutant and sensitivity to bicyclomycin have indicated that rho is essential for the viability of Caulobacter crescentus. Transcription gene fusions of sequences with serial deletions of the rho 5' untranslated region (5'-UTR) with a lacZ reporter gene indicated that rho is autoregulated at the level of attenuation of transcription in the 5'-UTR.

FIG. 1.

(A) Immunoblot of C. crescentus cell extracts with polyclonal anti-Rho antiserum. Lane 1, extract from strain SP3710; lane 2, extract from strain NA1000; lane 3, extract from SP3710 containing the pMR20-Rho plasmid. (B) Analysis of the sensitivity of C. crescentus NA1000 cells to bicyclomycin. Growth was determined by measuring the optical density at 600 nm after 48 h at 30°C of cultures in peptone-yeast extract medium containing increasing concentrations of bicyclomycin: 0, 50, 75, 100, 200, and 300 μg/ml.

FIG. 2.

(A) Determination of the transcription start site of the rho gene. Primer extension analysis was carried out with 50 μg total RNA from exponential-phase cells (P.E.). Primer Rho1 was 5′-end labeled with ³²P, extended with reverse transcriptase to determine the transcription start site (arrowhead), and also used in the sequencing reaction (shown on the left). (B) Nucleotide sequence of the rho regulatory region. The bent arrow indicates the transcription start site and was designated position +1, and the −35/−10 sequences are boxed. C-rich sequences are shaded, the ribosome-binding site is double underlined, and the PstI restriction sites are indicated. The arrows indicate the positions of each oligonucleotide used for primer extension, transcriptional fusions, and RNA gel mobility shift assays.

FIG. 3.

Analysis of expression driven by rho regulatory regions. (A) Map (not to scale) of the region surrounding the rho gene in C. crescentus. Boxes indicate open reading frames, and the arrows above them show the direction of transcription. Coordinates in nucleotides are relative to the first nucleotide of the rho transcription start site (bent arrow), determined by primer extension analysis (+1). The promoter is indicated as P1. The stem and loop indicate a putative Rho-independent transcription terminator. Below the map, each transcriptional fusion construct is indicated, where coordinates indicate the extent of the regulatory rho region cloned in front of the reporter lacZ gene. (B) β-Galactosidase activity of each construct in the NA1000 and SP3710 strains. The results are in Miller units (21) and are the average of results of at least three independent assays.

FIG. 4.

Analysis of expression driven by rho regulatory regions in the presence of bicyclomycin. Expression was determined from NA1000 cells harboring the respective constructs described in Fig. 3 after incubation at 30°C for 3 h in the presence or absence of 100 μg/ml bicyclomycin. The β-galactosidase (β-gal) activities are expressed in Miller units (21) and are the averages of results of at least three independent assays.

FIG. 5.

RNA electrophoretic mobility shift assay of rho mRNA. The indicated amount of purified Rho was incubated with 70 ng of ³²P-labeled RNA fragment for 20 min, and the complexes were resolved in a 5% polyacrylamide gel. Lane 1, RNA probe; lanes 2 to 5, increasing amounts of Rho (0.5, 1, 3, and 5 μg, respectively) added to the RNA probe; lane 6, the probe incubated with 3 μg of Rho and 0.7 μg of the same nonradioactive RNA competitor; lane 7, the probe incubated with 3 μg of Rho and 1 μg of tRNA; lane 8, the probe incubated with bovine serum albumin (5 μg).