Downloaded by guest on September 23, 2021 Proc. Natl. Acad. Sci. USA Vol. 90, pp. 3511-3515, April 1993 Biochemistry Heterogeneity of the principal cr factor in Escherichia coli: The rpoS gene product, r38, is a second principal cr factor of RNA polymerase in stationary-phase Escherichia coli KAN TANAKA*, YUKO TAKAYANAGI*, NOBUYUKI FUJITAt, AKIRA ISHIHAMAt, AND HIDEO TAKAHASHI** *Institute of Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan; and tDepartment of Molecular Genetics, National Institute of Genetics, Mishima, Shizuoka 411, Japan Communicated by R. Haselkorn, January 19, 1993 ABSTRACT The rpoS gene of Escherichia coli encodes a recognized by Ea70 and Eo-38 holoenzymes, but other pro- putative RNA polymerase a factor that is considered to be the moters are transcribed by only one of the two holoenzymes. central regulator of gene expression in stationary phase. The In addition, we examined the expression ofor38 protein in vivo gene product (ar3) was overproduced using the cloned rpoS by Western analysis. gene and purified to homogeneity. Reconstituted RNA poly- merase holoenzyme (Ea38) was found to recognize in vitro a MATERIALS AND METHODS number of typical a70-type promoters, including the lacUV5 and thp promoters. Some, however, were recognized exclu- Bacterial Strains. E. coli strains DH1 (13), XLlblue (14), sively or preferentially by Ear70, whereas at least one, fic, was MC4100 (4), and BL21(DE3) (15) were used in this study. favored by E&rm. Thus E. coli promoters can be classified into Plasmid pTZ19R was obtained from Pharmacia. pET3b, three groups: the first group is recognized by Ea70 and Ea,38 pLysS, and pLysE plasmids are as described (15). but the second and third groups are recognized substantially by Cloning of the rpoS Gene and Determination of the Nucle- either Ea70 or Eam alone. In contrast to other minor afactors, otide Sequence. Purification of DNA from E. coli DH1 and am shares a set of amino acid sequences common among the cloning ofthe 2.3-kb Kpn I fragment containing the rpoS gene principal a factors of eubacteria and is therefore a member of were performed as described (8). The nucleotide sequence of the RpoD-related protein family. The intracellular level of a38 the 1.6-kb Kpn I-Hpa I fragment was determined on both was demonstrated to increase in vivo upon entry into stationary strands using modified T7 DNA polymerase (Sequenase; phase. These results together indicate that am is a second United States Biochemical) as described by the supplier. principal a factor in stationary-phase E. coli. Overproduction and Purification of the rpoS Gene Product. DNA primers for the polymerase chain reaction (PCR), The promoter selectivity of RNA polymerase holoenzyme, 5'-ATCATATGAGTCAGAATACGCTGAAA-3' and 5'- controlled by replacing cr factors, is the primary mechanism TTGGATCCTTATTGTGCACAGAAAAAACCAGC-3', for the global switching ofgene expression in eubacteria. The were synthesized with an Applied Biosystems model 392 principal Cr factor or70 (the rpoD gene product) in exponen- DNA synthesizer. PCR was carried out for 30 cycles as the described (16). Each cycle was at 95°C for 1.5 min, 40°C for tially growing Escherichia coli potentiates transcription 2 min, and 72°C for 4 min. PCR products were digested with of genes controlled by the consensus promoters consisting of Nde I and BamHI and cloned into the corresponding region two hexanucleotide sequences, TTGACA and TATAAT (1, ofexpression plasmid pET3b. The nucleotide sequence ofthe 2). Other minor species of alternative cr factors recognize amplified product was determined and verified to be identical different sets of promoter sequences, each being associated with that of the cloned rpoS sequence (this study). The with a limited number of genes that are mostly expressed in outlines of expression of rpoS and purification of or38 were response to various stress conditions (1, 2). The rpoS (katF) performed as described (15, 17). The expressing strain was gene of E. coli was first identified as a positive regulator of inoculated in 1 liter of LB medium. When the OD660 of the the katE gene encoding hydroperoxidase II (HPII) (3). Later, culture reached 0.7, isopropyl ,3-D-thiogalactoside was added several other genes that are, for the most part, expressed in at the final concentration of 0.5 mM. Cells were collected by the stationary growth phase were shown to be regulated by centrifugation after 3 hr of further cultivation. The overex- the rpoS allele (4, 5). Thus the rpoS gene was assumed to be pressed protein was recovered as inclusion bodies and sol- the central regulator of gene expression in the stationary ubilized in TGED buffer (10 mM Tris-HCl, pH 8.0/5% phase (4). From the nucleotide sequence ofthe rpoS gene, the glycerol/0.1 mM EDTA/0.1 mM dithiothreitol) containing 6 gene product has been believed to be an RNA polymerase cr M guanidine hydrochloride. r38 was renatured by gradual factor (6, 7). However, a, activity has not been detected dilution with 60 volumes of TGED buffer and absorbed to previously for the rpoS gene product. DE-52 resin (Whatman). The resin was packed into a column, In this study, we determined the nucleotide sequence ofthe and the protein was eluted by TGED buffer containing 0.2 M rpoS gene§ and found that the gene product is structurally NaCl and precipitated with ammonium sulfate. The fraction similar to or70 and the principal or factors from divergent was redissolved in TGED buffer and loaded onto a Sephacryl eubacterial species (7-12). To assess the functional similarity S-300 gel filtration column (Pharmacia). The peak fraction between the rpoS gene product (or38) and cr70, we reconsti- was further purified by high-speed liquid chromatography tuted Eor38 holoenzyme using a highly purified rpoS gene (Waters, model 510) on a protein pack G-DEAE column product and examined its promoter selectivity in in vitro (Waters, 8.2 mm x 75 mm). Elution was with alinear gradient transcription assays using various E. coli promoters. The of0-1.0 M NaCl in the same buffer. cr38 was precipitated with results indicate that a number of E. coli promoters are Abbreviation: E, RNA polymerase core enzyme. The publication costs of this article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" §The sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. D13548). 3511 3512 Biochemistry: Tanaka et al. Proc. Natl. Acad. Sci. USA 90 (1993) ammonium sulfate, dialyzed against the storage buffer lated to be 37,956. Thus this protein was henceforth referred [TGED plus 0.5 M NaCl with 50% (wt/vol) glycerol], and to as 738. stored at -20°C. The total yield was about 8 mg from 2.7 g 038 shares a common structure with the principal o,factors of E. coli cells (wet weight). The quantity of protein was of divergent eubacteria and is a member of the RpoD-related estimated using a protein assay kit (Bio-Rad) with bovine protein family (7-12). The similarity includes the RpoD box serum albumin as a standard. sequence and the sequences in the DNA binding regions In Vitro Transcription. The purification of RNA polymer- (7-12), which may indicate that a38 is functionally similar to ase core enzyme (E) and o70 subunit have been described (J70. A comparative study of a factors categorized a38 into (18). The core enzyme was mixed with equimolar amounts of group 2, which includes cr factors that are not essential for either a70 or ov38, and the mixtures were incubated for 10 min exponential growth but are similar to the principal of factors at 37°C. Single-round transcription reactions were performed (29). with 3 pmol of reconstituted holoenzyme under standard Overproduction of the rpoS Gene Product. To produce the conditions (19). The templates in each reaction were as rpoS gene product (o,38), a DNA fragment containing the rpoS follows: (a) a 205-bp EcoRI fragment from pKB252 carrying coding region was amplified by PCR and inserted into an the lacUV5 promoter (20); (b) a 512-bp HindIII fragment overexpression plasmid (pET3b) under the control of a T7 trp promoter (20); (c) a 210-bp promoter (15). The nucleotide sequence ofthe PCR-amplified from pWT101 carrying the product proved to be identical to that of the rpoS gene. The Hinfl fragment from pJLO-2 carrying the rpU promoter (20); resultant plasmid was named pETF and found to be the most (d) a 391-bp Sal I-Hinfl fragment from pSP261 carrying the productive when the host BL21(DE3) strain harbored the rpsApl promoter (21); (e) a 0.8-kb EcoRI-HindIII fragment pLysS plasmid (15). After isopropyl f3-D-thiogalactoside in- from pRP1 carrying the rrnEpl promoter (22); (f) a 459-bp duction, o-38 accumulated as inclusion bodies in overexpress- Nru I-ApaLI fragment from plasmid ColEl carrying the ing E. coli cells. Thus, a38 was recovered as inclusion bodies, RNA I and RNA II promoters (23); (g) a 287-bp BamHI-Kpn solubilized in a guanidine solution, and purified to near I fragment from p48 plasmid carrying the alaS promoter (19); homogeneity by a combination ofcolumn chromatographies. (h) a 320-bp Hpa II fragment from pSY343 carrying the leuX The purity of (38 was carefully examined by SDS/PAGE and promoter (19); (i) a 950-bp Bgl II-Sma I fragment from by Western analysis using monospecific ao70 antiserum. Anal- pTUB2 carrying the tufB promoter (24); (j) a 485-bp BamHI ysis revealed no trace contamination of o,70.