Extracytoplasmic function sigma factors regulate expression of the Bacillus subtilis yabE gene via a cis-acting antisense RNA - PubMed (original) (raw)

Extracytoplasmic function sigma factors regulate expression of the Bacillus subtilis yabE gene via a cis-acting antisense RNA

Warawan Eiamphungporn et al. J Bacteriol. 2009 Feb.

Abstract

Bacillus subtilis yabE encodes a predicted resuscitation-promoting factor/stationary-phase survival (Rpf/Sps) family autolysin. Here, we demonstrate that yabE is negatively regulated by a cis-acting antisense RNA which, in turn, is regulated by two extracytoplasmic function sigma factors: sigma(X) and sigma(M).

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Figures

FIG. 1.

(A) Organization of yabE and the downstream PECF element. Promoter sites are indicated by bent arrows with a subscript to indicate the relevant holoenzyme(s). A putative transcription terminator downstream of yabE is indicated by an oval. RnmV is an RNase M5/primase-related protein involved in the maturation of the 5S rRNA (6). (B) Alignment of PECF elements from various bacilli. The intergenic region between yabE and rnmV of B. subtilis (Bsu) was aligned with the corresponding sequences from B. amyloliquefaciens (Bam) and B. licheniformis (Bli) by using the ClustalW2 program (

http://www.ebi.ac.uk/Tools/clustalw2/index.html

). The putative −10 and −35 regions are indicated. The run of seven A residues near the end of the sequence shown corresponds to the run of U residues in the yabE transcription terminator.

FIG. 2.

(A) Growth phase regulation of PECF. The β-galactosidase activity of the PECF_-lacZ_ fusion in the wild type (CU1065) was assayed during growth in LB medium, with samples taken every 1 h for the measurement of the optical density at 600 nm (OD600) and the β-galactosidase activity. Solid squares represent growth at an optical density at 600 nm, while solid triangles represent β-galactosidase activity. (B) Mapping of the transcriptional start sites of yabE and ECF-directed antisense transcripts by the rapid amplification of cDNA 5′ ends. The putative −10 and −35 regions are underlined. The transcription start sites are in bold, and the translation start site is italicized. PA is the promoter corresponding to σA. Met indicates the start codon (methionine).

FIG. 3.

(A) PECF-directed antisense expression is dependent on σX and σM. The β-galactosidase activity of the PECF-lacZ fusion was assayed in various ECF deletion strains indicated by the relevant gene. Cultures were grown in LB medium, and samples were taken when cells reached late log phase. Data are expressed as averages ± standard deviations of results for triplicate samples. wt, wild type. (B) Regulation of yabE by an antisense RNA. Total RNA (15-μg) samples prepared from late-log-phase cultures of various deletion strains after growth in LB medium were analyzed by Northern blotting. RNA was separated, blotted, and hybridized with [γ-32P]ATP-labeled strand-specific probes for the yabE and the antisense RNA transcripts (indicated with arrowheads). The level of 23S rRNA is shown underneath as a loading control (2 μg of RNA was loaded). nt, nucleotides.

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Extracytoplasmic function sigma factors regulate expression of the Bacillus subtilis yabE gene via a cis-acting antisense RNA - PubMed (original) (raw)