Jan 15, 1991 - DANIEL GENTRY,' HUA XIAO,1t RICHARD BURGESS,2 AND MICHAEL CASHEL'* ..... Neidhardt, F. C., P. L. Bloch, and D. F. Smith. 1974.
JOURNAL OF BACTERIOLOGY, June 1991, p. 3901-3903
Vol. 173, No. 12
0021-9193/91/123901-03$02.00/0 Copyright © 1991, American Society for Microbiology
The Omega Subunit of Escherichia coli K-12 RNA Polymerase Is Not Required for Stringent RNA Control In Vivo DANIEL GENTRY,' HUA XIAO,1t RICHARD BURGESS,2 AND MICHAEL CASHEL'* Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, Bethesda, Maryland 20892,1 and McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 537062
Received 15 January 1991/Accepted 28 March 1991
Igarashi et al. (K. Igarashi, N. Fujita, and A. Ishihama, Nucleic Acids Res. 17:8755-8765, 1989) reported that the omega (w) subunit of Escherichia coli RNA polymerase was required for stringent control as judged by in vitro transcription assays in the presence and absence of guanosine 3',5'-bispyrophosphate (ppGpp). This conclusion predicts that a deletion of the gene (designated rpoZ or spoS) should show a relaxed RNA control phenotype in vivo. However, we find that wild-type stringent control of stable RNA accumulation is unaffected by a spoS null allele that abolishes cellular production of the X protein. We conclude that protein is not necessary for the operation of the stringent RNA control response. w
The bacterial stringent response to amino acid, or aminoacyl tRNA, limitation is a pleiotropic global response whose hallmark is a decreased rate of stable RNA accumulation; mutant strains in which stable RNA accumulation persists under these conditions are said to have "relaxed" RNA control. There are several genetic lesions (relA, relB, and reiC) that can result in a relaxed RNA control phenotype. The occurrence of the stringent response is correlated with an increase in the cellular concentration of guanosine 3',5'-bispyrophosphate (ppGpp). Despite the existence of strong correlative evidence that stable RNA synthesis is inhibited during the stringent response, the in vitro evidence that ppGpp inhibits stable RNA synthesis at the level of transcription initiation by a direct interaction with RNA polymerase is not yet conclusive (for a review, see reference
oped by Kajitani and Ishihama (9) and measuring the effect of ppGpp on transcription in the presence or absence of These studies concluded that is required for stringent control in vitro (8). A prediction of this conclusion is that a strain lacking protein should show relaxed RNA control. The RNA control properties of a strain bearing a deletioninsertion allele of spoS (rpoZ) are compared here with those of otherwise isogenic but relaxed and stringent strains. A null allele of relA was used in the relaxed strain (10). The spoS3 deletion-insertion mutation. The construction of a null allele for the gene encoding has been described (14). Except for the first two codons and the last A of the TAA stop codon, all of the spoS gene on a plasmid was deleted and replaced by a chloramphenicol resistance cassette, giving the AspoS3::cat allele, which is designated spoS3. spoS3 was recombined into the chromosome by way of a X intermediate, and the location of the insertion was verified by P1 transduction (14). Unlike insertion mutations reported previously (6, 13), the spoS3 mutation appears to exhibit only weak polar effects on the expression of spoT as judged by the half-life of ppGpp after isoleucine starvation and supplementation; whereas the spoT+ strain MG1655 has a half-life of 45 s and a spoS: :kan insertion strain has a half-life of 24 min, the AspoS3: :cat mutant has a half-life of 3 min (4). The spoS3 mutation also complements the growth defect of severe spoT mutants (14). Thus, the only phenotypes attributable to the null mutation can be explained as secondary effects of a slight decrease in spoT expression; no known C-specific phenotype has yet been found. Measurements of RNA accumulation. Figure 1 shows the effects of serine hydroxamate addition (200 ,ug/ml) on rates of accumulation of alkali-labile, trichloroacetic acid (TCA)precipitable 32P activity for three strains growing with uniform labeling with H332P04. The center panel shows data for the spoS3 strain CF2790, which is otherwise identical to the wild-type strain CF1943 (alias W3110) (left panel). The right panel shows the behavior of strain CF1944, which differs from CF1943 only by the presence of a relA deletion. All three cultures were grown in microtiter dishes in a Wellwarm incubator-shaker at 32°C in wells containing 150 ,ul of MOPS C.
3). Highly purified RNA polymerase from Escherichia coli consists of a, , 13', and subunits as well as a peptide of Mr 10,105 called w (2). The function of the protein is unknown; the spoS (alias rpoZ) gene encoding can be insertionally inactivated or deleted without a known phenotype. Nevertheless, binds stoichiometrically to RNA polymerase, cross-links specifically to the 1' subunit, and is immunologically conserved among bacteria (4). A single spoS (rpoZ) gene is located at about 82 min on the E. coli chromosome, just upstream from and in the same operon as spoT (5, 6, 13). The product of the spoT gene is the principal enzyme responsible for the degradation of ppGpp and also is implicated as an alternative source of ppGpp synthesis that exists in the addition to the reaction catalyzed by the relA gene product (14). It is a reasonable conjecture that might be a participant in some aspect of regulation mediated by ppGpp because spoS and spoT are in the same operon. Igarashi et al. (8) have tested whether X is required for stringent control by using a "mixed-template" in vitro transcription assay develu
* Corresponding author. t Present address: Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada.
CF 1 943 10
CF 1 944
20 40 60
20 40 60 80
20 40 60 80
time (minutes) FIG. 1. RNA accumulation after serine hydroxamate addition. Cells were grown in the presence of 32Pi to achieve uniform labeling. At the point indicated by the arrow, serine hydroxamate was added (200 ,ug/ml) to one culture aliquot. Samples were processed to measure alkali-labile, TCA-precipitable radioactivity as described in the text. Left panel, strain CF1943 (alias W3110); center panel, CF2790 (CF1943 spoS3); right panel, CF1944 (CF1943 relA251). Closed squares, no serine hydroxamate; open squares, serine hydroxamate.
complete medium (11) but containing 0.4 mM phosphate (approximately 2 pXCi of 32P per ml added two generations before the first sampling) and supplemented with 40 ,ug of each of the 19 amino acids (omitting serine) per ml. Growth was monitored with a microtiter plate reader with a 595-nm filter; the increase in turbidity was in parallel with rates of RNA accumulation in cultures not treated with serine hydroxamate (data not shown). At the time points indicated in Fig. 1, 5-pul samples were removed and directly spotted in duplicate on each of two Whatman 3M filters. Pilot experiments indicated that cell growth ceases when culture aliquots are spotted in this manner. One of the filters was then soaked three times in 10% TCA with slow agitation for 30 min for each wash and finally for 15 min in ethanol before drying. The duplicate filter was soaked in 0.33 M NaOH for 3 h at 42°C to hydrolyze RNA and then treated with TCA in the same manner as for the other filter. Total counts per minute per spot were then quantitated on an Ambis Radioanalytical Imaging System. For each point plotted, the counts on the filter treated with NaOH (predominately DNA) were averaged and subtracted from the average counts of the corresponding sample treated only with TCA. The proportion of alkali-resistant counts never exceeded 10% of the total counts. The alkali-sensitive counts obtained in this manner largely represent labelled RNA. Figure 1 shows that serine hydroxamate addition results in markedly reduced rates of RNA accumulation in both the wild-type strain and the omega-deleted strain. Nearly normal rates of accumulation of RNA in the standard relaxed strain CF1944 persist after serine hydroxamate addition. Western blots (immunoblots) show that the spoS3 strain in fact is deficient in omega protein (4); Southern blots of DNA from spoS3 strains verify that the wild-type copy of the gene encoding omega is absent (6). Conclusions. We take the results shown in Fig. 1 as a clear indication that is not a necessary component for operation of the stringent RNA control response in vivo. Thus, the results obtained with the in vitro mixed-template transcription assay that implicated w protein as a participant in the w
stringent response (8) should be reevaluated. One possible explanation of the in vitro results could be that the omega preparations employed were contaminated with small amounts of an active factor mistakenly assumed to be omega. Another, perhaps more baroque, possibility is that the highly purified components of the in vitro system have led to removal of a component that shares the activity of omega and that this additional gene product persists in cells deleted for the gene encoding omega. The mixed-template transcription assay was proposed as a means of circumventing the difficulties frequently encountered in reproducibly obtaining ppGpp inhibition of transcription initiation from promoters judged to show negative stringent control in vivo (9). The findings reported here suggest that the validity of the in vitro transcription assay might be viewed with skepticism. This skepticism is reinforced by a report that the mixed-template transcription assay system also led to the apparently erroneous conclusion that a class of RNA polymerase rpoB mutants was unresponsive to ppGpp (7). These mutants, obtained by Nene and Glass (12), were originally thought to possess a ppGpp resistance phenotype in vivo (12) but were later shown by Baracchini et al. (1) to be instead defective in ppGpp synthesis. The basis for this ppGpp synthesis defect remains puzzling. ACKNOWLEDGMENT A portion of this work was performed while D.G. held a National Research Council-Laboratory of Molecular Genetics Research Associateship. REFERENCES 1. Baracchini, E., R. Glass, and H. Bremer. 1988. Studies in vivo on Escherichia coli RNA polymerase mutants altered in the stringent response. Mol. Gen. Genet. 213:379-387. 2. Burgess, R. R. 1969. Separation and characterization of the subunits of RNA polymerase. J. Biol. Chem. 244:2168-2176. 3. Cashel, M., and K. E. Rudd. 1987. The stringent response, p. 1410-1438. In F. C. Neidhardt, J. L. Ingraham, K. B. Low, B.
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Magasanik, M. Schaechter, and H. E. Umbarger (ed.), Escherichia coli and Salmonella typhimurium: cellular and molecular biology. American Society for Microbiology, Washington, D.C. Gentry, D. R. 1990. Ph.D. dissertation. University of Wisconsin, Madison. Gentry, D. R., and R. R. Burgess. 1986. The cloning and sequence of the gene encoding the omega subunit of Escherichia coli RNA polymerase. Gene 48:33-40. Gentry, D. R., and R. R. Burgess. 1989. rpoZ, encoding the omega subunit of Escherichia coli RNA polymerase, is in the same operon as spoT. J. Bacteriol. 171:1271-1277. Glass, R., S. Jones, and A. Ishihama. 1986. Genetic studies on the P subunit of Escherichia coli RNA polymerase VII. RNA polymerase is a target for ppGpp. Mol. Gen. Genet. 203:265268. Igarashi, K., N. Fujita, and A. Ishihama. 1989. Promoter selectivity of Escherichia coli RNA polymerase: omega factor is responsible for the ppGpp sensitivity. Nucleic Acids Res.
17:8755-8765. 9. Kajitani, M., and A. Ishihama. 1984. Promoter selectivity of Escherichia coli RNA polymerase. J. Biol. Chem. 259:19511957. 10. Metzger, S., G. Schreiber, E. Aizenman, M. Cashel, and G. Glaser. 1989. Characterization of relAl and new relA null alleles in Escherichia coli. J. Biol. Chem. 264:21146-21152. 11. Neidhardt, F. C., P. L. Bloch, and D. F. Smith. 1974. Culture medium for enterobacteria. J. Bacteriol. 119:736-747. 12. Nene, V., and R. E. Glass. 1982. Relaxed mutants of Escherichia coli RNA polymerase. FEBS Lett. 153:307-310. 13. Sarubbi, E., K. E. Rudd, H. Xiao, K. Ikehara, M. Kalman, and M. Cashel. 1989. Characterization of the spoT gene of Escherichia coli. J. Biol. Chem. 264:15074-15082. 14. Xiao, H., M. Kalman, K. Ikehara, S. Zemel, G. Glaser, and M. Cashel. 1991. Residual guanosine 3',5'-bispyrophosphate (ppGpp) synthetic activity of relA null mutants can be eliminated by spoT null mutations. J. Biol. Chem. 266:5980-5990.