Escherichia coli ribosome-associated protein SRA, whose copy number increases during stationary phase

Abstract

Protein D has previously been demonstrated to be associated with Escherichia coli ribosomes by the radical-free and highly reducing method of two-dimensional polyacrylamide gel electrophoresis. In this study, we show that protein D is exclusively present in the 30S ribosomal subunit and that its gene is located at 33.6 min on the E. coli genetic map, between ompC and sfcA. The gene consists of 45 codons, coding for a protein of 5,096 Da. The copy number of protein D per ribosomal particle varied during growth and increased from 0.1 in the exponential phase to 0.4 in the stationary phase. For these reasons, protein D was named SRA (stationary-phase-induced ribosome-associated) protein and its gene was named sra. The amount of SRA protein within the cell was found to be controlled mainly at the transcriptional level: its transcription increased rapidly upon entry into the stationary phase and was partly dependent on an alternative sigma factor (sigma S). In addition, global regulators, such as factor inversion stimulation (FIS), integration host factor (IHF), cyclic AMP, and ppGpp, were found to play a role either directly or indirectly in the transcription of sra in the stationary phase.

FIG. 1

Association of SRA (protein D) with the 30S ribosomal subunit. Electropherograms from RFHR 2-D PAGE analysis of the ribosomal proteins in total cell extracts (A), crude ribosomes (B), high-salt-washed ribosomes (C), and 30S subunits (D) prepared from W3110 cells grown to the stationary phase. Protein D (SRA), S21, L32, and L33 spots are indicated.

FIG. 2

Cellular viability and copy number of SRA in the stationary phase. Cultures of W3110 cells were sampled at different times from the exponential phase through the stationary phase of growth. Optical density at 600 nm and colony-forming ability were determined, and the relative viability values obtained were normalized to that in the late exponential phase. Proteins extracted from harvested cells were analyzed by RFHR 2-D PAGE. The copy number of SRA was determined as described in Materials and Methods.

FIG. 3

Construction of phage λpF13(Psra-lacZ) and growth-phase-dependent induction from the sra promoter. (A) Construction of phage λpF13(Psra-lacZ). The sra promoter region was PCR amplified, digested, and cloned into plasmid pMS4342 digested with KpnI and SalI. The resultant sra-lacZ fusion was transferred to phage vector λpF13 as described in Materials and Methods (see the text for details). (B) Growth-phase-dependent induction from the sra promoter. MG1655 cells lysogenic for λpF13(Psra-lacZ) were grown at 37°C in EP medium for 6 days. Samples from cultures prepared in separate tubes were withdrawn at various times, and the optical density at 600 nm (OD₆₀₀) and β-galactosidase activity in Miller units were determined.

FIG. 4

Effects of global regulators on the transcription of sra. Cells of KY1461 lysogenic for λpF13(Psra-lacZ) and containing either rpoS, fis, hns, ompR, or relA and spoT mutations and cells of KY1461 wild type or containing cya or hip and him mutations and carrying pFF6(Psra-lacZ) were grown at 37°C in EP medium. Cultures in either the exponential phase (optical density at 600 nm, 0.4) or the stationary phase (after 24 h) of growth were assayed for β-galactosidase activity, expressed relative to that of wild-type cells in the stationary phase.

FIG. 5

Identification of the transcriptional start site of sra. The transcriptional start site was determined by primer extension using the oligonucleotide primer described in Materials and Methods. Sequence ladders were generated by the dideoxy method with the same primer and pKV7350 as the template. RNA was extracted from cultures of MG1655 (lane 1), MG1655Δsra::kan (lane 2), and MG1655(pKV7350) (lane 3) grown at 37°C to the stationary phase. The nucleotide sequence of the sra promoter region is indicated to the right, and an arrow indicates the transcriptional start site of the major transcript.

FIG. 6

Search for cis elements in the region upstream of the sra promoter. KY1461 cells lysogenized with various λpF13(Psra-lacZ) constructs were grown at 37°C in EP medium. Cultures in either the exponential phase (optical density at 600 nm [OD₆₀₀], 0.4) or the stationary phase (OD₆₀₀, 1.5) were subjected to a β-galactosidase assay. The negative numbers indicate the construct endpoints measured from the A of the translation initiation codon ATG. The start sites and directions of transcription of sra and osmC are indicated by arrows.

FIG. 7

Comparison of SRA and its homologs. (A) Alignment of amino acid sequences of SRA proteins from E. coli (ECO), Salmonella serovar Typhimurium (STM), Salmonella serovar Typhi (STY), Salmonella serovar Paratyphi (SPA), and K. pneumoniae (KPN). Dark boxes indicate complete sequence identity in all, and light boxes indicate identity in three or four organisms. (B) Alignment of the nucleotide sequence of the region upstream of the sra gene. Shaded boxes indicate identity in at least four organisms. The asterisk, −10 and −35, and SD show the transcriptional start site, RNA polymerase consensus sequences, and Shine-Dalgarno sequence of the E. coli sra gene, respectively.