Autogenous Control Is Not Sufficient To Ensure Steady-State Growth ...

Vol. 172, No. 1

JOURNAL OF BACTERIOLOGY, Jan. 1990, p. 305-309

0021-9193/90/010305-05$02.00/0 Copyright © 1990, American Society for Microbiology

Autogenous Control Is Not Sufficient To Ensure Steady-State Growth Rate-Dependent Regulation of the S10 Ribosomal Protein Operon of Escherichia coli LASSE LINDAHL AND JANICE M. ZENGEL

Department of Biology, The University of Rochester, Rochester, New York 14627 Received 19 July 1989/Accepted 4 October 1989

The regulation of the S10 ribosomal protein operon of Escherichia coli was studied by using a lambda prophage containing the beginning of the S10 operon (including the promoter, leader, and first one and one-half structural genes) fused to lacZ. The synthesis of the lacZ fusion protein encoded by the phage showed the expected inhibition during oversynthesis of ribosomal protein L4, the autogenous regulatory protein of the S10 operon. Moreover, the fusion gene responded to a nutritional shift-up in the same way that genuine ribosomal protein genes did. However, the gene did not exhibit the expected growth rate-dependent regulation during steady-state growth. Thus, the genetic information carried on the prophage is sufficient for L4-mediated autogenous control and a normal nutritional shift-up response but is not sufficient for steady-state growth rate-dependent control. These results suggest that, at least for the 11-gene S10 ribosomal protein operon, additional regulatory processes are required to coordinate the synthesis of ribosomal proteins with cell growth rate and, furthermore, that sequences downstream of the proximal one and one-half genes of the operon are involved in this control.

In the 11-gene S10 r-protein operon, the regulatory protein is L4, encoded by the third gene of the operon (24, 27). The S10 operon is distinct from other investigated r-protein operons in that L4 not only regulates translation but also modulates transcription of the operon via an L4-stimulated transcription terminator (attenuator) in the leader (9, 17, 26). We have shown that L4-mediated attenuation of transcription plays a major role in the increased expression of the S10 operon observed immediately after a nutritional shift-up (10, 25). In contrast, attenuation control cannot account for

In bacteria the synthesis of ribosomes is regulated in response to the environment of the cells. Two aspects of growth rate-dependent regulation have been observed. During steady-state (or balanced) growth, the rate of ribosome formation is correlated with the medium-determined growth rate, such that rapidly growing cells devote a larger fraction of their energy and mass to ribosomes than do slowly growing cells (7, 19). Furthermore, immediately after a nutritional shift-up from one medium to another supporting faster growth, the synthesis of ribosomal components is rapidly accelerated, going through several oscillations before the final postshift steady-state rate is attained (6, 12, 19, 26). The mechanisms for regulating ribosome synthesis have long been subject to scrutiny. Autogenous control in Escherichia coli is the best-understood mechanism affecting ribosomal protein (r-protein) synthesis (for reviews, see references 14 and 18). Autogenous control is accomplished by key r-proteins which, in addition to being constituents of the ribosome, also work as repressors of their own operons. Thus, when the regulatory protein from a given operon accumulates in excess of its available binding site on nascent rRNA, the free protein inhibits the expression of its own mRNA, usually at the level of translation. If r-protein synthesis were regulated exclusively by the autogenous control mechanism, then the regulation of ribosome synthesis could be accomplished by regulating only rRNA. For example, the growth medium-dependent control of ribosome synthesis might reflect a direct control of rRNA synthesis and an indirect effect, via autogenous control, on r-protein synthesis. This simple model predicts that the autogenous control mechanism is both necessary and sufficient for correlating r-protein synthesis with the growth rate of the cell. In fact, Cole and Nomura (3) have shown that autogenous control of the Lll-Li operon is required for the steady-state growth medium-dependent regulation of that operon. However, their study could not determine if autogenous control is sufficient for this control.

growth rate-dependent control during steady-state growth (25). However, since our earlier experiments did not monitor L4-mediated regulation of translation, we could not exclude the possibility that this form of autogenous control is responsible for regulating expression of the S10 operon during steady-state growth. We have reinvestigated the role of autogenous control in the growth medium-dependent regulation of the S10 operon, using a system which could detect L4 regulation of either transcription or translation. Our results suggest that the combined L4-mediated autogenous controls of transcription and translation are not sufficient to obtain the steady-state growth medium-dependent regulation of the S10 operon. Thus, regulation of the S10 operon during balanced growth appears to involve additional processes which are not necessary for a nutritional shift-up response. MATERIAL AND METHODS Strains. E. coli K-12 LL306 is A(pro-lac) recA nalA supE thi. Bacteriophages XLF2 and XLF22 were constructed from the plasmids pLF2 and pLF22 (10), respectively, by transferring a HindIII-NaeI fragment carrying the beginning of the S10 operon fused to the lacZ gene to DNA from phage XD69 (21) cleaved with the same enzymes. The ligated DNA was packaged into lambda particles in vitro (by using a commercial packaging extract from Promega) and used to infect LL306. Recombinant phages carrying the L3'-lacZ'

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LINDAHL AND ZENGEL

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FIG. 1. Construction of lambda phages containing the beginning of the S10 operon fused to lacZ. The structure of the HindIII-NaeI fragment from plasmid pLF2 is shown at the top. This fragment was transferred to XD69 as shown at the bottom, and the resulting phage was called XLF2. The region of the S10 operon deleted in plasmid pLF22 is indicated by the solid bar below the pLF2 map. The deletion removes most of the S10 leader (except the proximal 17 bases), as well as the first 64 bases of the S10 structural gene (10). Phage XLF22 carrying this deletion was constructed in a way analogous to the construction of XLF2. kb, Kilobases.

fusion gene were identified as blue plaques on TB plates (22) containing X-gal (5-bromo-4-chloro-3-indolyl-,-D-galactopyranoside). Purified stocks of the recombinant phages were used to lysogenize LL306, using a multiplicity of infection of about 0.1 to ensure single-copy lysogeny. Lysogens were identified as blue colonies on AB-glucose-Casamino Acids plates containing X-gal. Where indicated, the lac repressor gene was introduced into the lysogenic LL306 derivatives by cross-streaking with a leucine-requiring donor strain carrying F' lacIq lacZ::TnS pro' (obtained from Niels Fiil) and selecting for prototrophy and kanamycin resistance. The plasmid pLL127 (17) carries an L4 gene under control of the lacUVS promoter and was transformed into the F' lacIq strains by standard procedures. Media and growth conditions. Cultures were grown in LB (20) or AB minimal medium (2) supplemented with 0.4% glycerol, with 0.2% glucose, or with 0.2% glucose and 40 ,ug of each of 19 amino acids (not including methionine) per ml. Growth of the cultures was monitored spectrophotometrically at 450 nm. Cultures were labeled or assayed for enzyme activity at an A450 of 0.3 to 0.8 (5 x 107 to 1 x 108 cells per ml). L4 oversynthesis from plasmid pLL127 was induced by adding isopropyl thiogalactoside to 1 mM. A nutritional shift-up was induced in cells growing in minimal glycerol medium by adding glucose to 0.2% and 19 amino acids (not including methionine) to 20 ,ug/ml. Accumulation and synthesis of fusion protein. The differential accumulation of L3'-lacZ' fusion protein was measured as specific 0-galactosidase activity determined in cells permeabilized with sodium dodecyl sulfate and chloroform (20). The differential rate of fusion protein synthesis was measured as described previously (25). Briefly, portions of the culture were grown with [35S]methionine for 1 min and then with an excess of nonradioactive methionine for 2 min. The labeled cells were then lysed in sodium dodecyl sulfate sample buffer. Portions of the lysates were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and an autoradiogram of the gel was made. The amount of radioactivity in the fusion bands was then determined and corrected for the background found in the position of the fusion bands in lanes of the gel loaded with labeled extracts from a strain, grown under the same conditions, that does not contain the fusion gene. Finally, the differential rate of fusion protein synthesis was calculated as the ratio between the radioactivity in the fusion band and the total amount of trichloroacetic acid-precipitable radioactivity loaded in each lane of the gel.

RESULTS AND DISCUSSION Our experiments were done by using the fusion gene operon shown in Fig. 1. This operon consists of the beginning of the S10 operon, including the promoter, the entire S10 gene, and the proximal part of the second gene, coding for L3, fused to a partial lacZ gene. We have previously shown that, when carried on a plasmid (pLF2), this fusion gene operon is regulated like the intact chromosomal S10 operon during the autogenous response. That is, the synthesis of the L3'-lacZ' fusion protein is inhibited 10- to 20-fold by excess L4 (10). As a control, we also used a derivative of this fusion operon which lacks most of the leader and the proximal part of the S10 gene. This deletion, carried by plasmid pLF22, eliminates the L4-mediated regulation of fusion protein synthesis, because the target(s) for both attenuation and translation control by L4 has been removed

(10; Fig. 1). To avoid growth medium-induced alterations of plasmid

copy number, we constructed single-copy versions of the fusion operons carried on plasmids pLF2 and pLF22 by transferring the fusion operons to the phage vector XD69 (Fig. 1; 21). E. coli K-12 LL306 was then lysogenized with

the resulting recombinant phages. We first analyzed the L4-mediated autogenous control of the L3'-lacZ' fusion genes on the two A prophages to verify that the regulation of the fusion gene had not been altered by being placed in a new context. To determine the effect of L4 on the recombinant prophages, we introduced into the lysogenic strains an F' episome carrying a lac repressor gene and then transformed them with the plasmid pLF127, which carries an L4 gene under the control of the lacUVS promoter and operator. We then pulse-labeled cultures with [35S]methionine before and 10 min after inducing L4 oversynthesis from pLL127 by addition of isopropyl thiogalactoside. Gel electrophoretic analysis of extracts from the pulselabeled cells showed that, as expected, the differential synthesis of L3'-lacZ' fusion protein from XLF2 was drastically decreased when L4 was oversynthesized (Fig. 2). Also as expected, fusion protein synthesis from the leader deletion derivative on XLF22 did not respond to excess L4 (Fig. 2). We quantitated the effect of L4 on fusion protein synthesis from XLF2 by measuring the amount of radioactivity in the fusion protein bands before and after L4 induction. Oversynthesis of L4 led to a 20-fold decrease in fusion protein synthesis (data not shown). Thus, the L4 effect on the fusion genes on the prophages appears to be identical to

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GROWTH RATE CONTROL OF S10 r-PROTEIN OPERON

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regulation of the L3'-lacZ' fusion gene by the transfer from the plasmid to the phage vector, we analyzed the synthesis of fusion protein after a nutritional shift-up. The rate of L3'-lacZ' fusion protein synthesis was determined at different times after the shift by pulse-labeling portions of the culture and analyzing extracts by gel electrophoresis as described above. Protein synthesis from the L3'-lacZ' fusion gene carried by XLF2 exhibited a temporary pronounced increase after the shift (Fig. 3a). Quantitation of this effect showed that the syntheTo confirm that the

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protein increased about threefold 2 to 3 mmn (Fig. 3b). These results are consistent with our previous results with the plasmid-borne fusion gene (10) and indicate that the XLF2 prophage is subject to normal nutritional shift-up regulation. A much weaker response was observed for the fusion gene on the XLF22 prophage (Fig. 3), consistent with our conclusion from previous experiments using plasmid pLF22 (10) that L4-mediated autogenous regulation contributes strongly to the shift-up control. (The diminished response from the XLF22 fusion gene is probably due to regulation of initiation of transcription at the S10 promoter [10].) Having established that the XLF2 prophage contains the genetic information sufficient for L4-mediated autogenous control and nutritional shift-up response, we examined the effect of the growth medium on expression of the L3'-lacZ' fusion gene during steady-state growth. If the L4-mediated autogenous control by itself were sufficient to ensure steadystate growth rate control, the differential rate of synthesis of the L3'-lacZ' fusion protein from XLF2, like synthesis of the genuine L3 r-protein, should increase more than twofold as sis of the fusion

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2 3 4 5 Minutes after shift Minutes after shift FIG. 3. Effect of a nutritional shift-up on lacZ fusion protein synthesis. Cells carrying XLF2 or XLF22 were grown in minimal glycerol medium. Three samples were removed and pulse-labeled with [35S]methionine (0-min samples), and the cells were then subjected to a nutritional shift-up. At the indicated times after the shift-up, samples were removed and pulse-labeled with [35S]methionine. (a) Electrophoretic analysis of total protein extracts after a nutritional shift-up. An autoradiogram of the gel is shown. The positions of the L3'-lacZ' fusion protein and the P and P' subunits of RNA polymerase are indicated. The control lane contained labeled protein from cells containing no prophage and therefore synthesizing no fusion protein. (b) Quantitation of fusion protein synthesis after a nutritional shift-up. The differential synthesis rates were calculated as described in Materials and Methods. The postshift values were normalized to the average of the three preshift values. 0 1

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the growth rate increases from 0.5 to 2 doublings per h (5). On the other hand, the fusion gene on XLF22 should not show this growth rate response. Cells carrying the L3'-lacZ' prophages were grown exponentially in LB medium or in minimal medium supplemented with glycerol, glucose, or glucose plus 19 amino acids (not including methionine). We measured the expression of the L3'-lacZ' fusion gene in two ways, by determining the specific activity of accumulated ,B-galactosidase enzyme and, for all but the LB medium cultures, by pulse-labeling with [35S]methionine and analyzing extracts by gel electrophoresis. (An autoradiogram of a typical gel is shown in Fig. 4.) For each experiment, both the P-galactosidase activities and the differential synthesis rates in the various growth media were normalized to the corresponding value determined for the glycerol culture in the same experiment. The results of these experiments are summarized in Fig. 5, which shows the normalized P-galactosidase accumulation or fusion protein differential synthesis rate plotted as a function of the medium-determined growth rate. Clearly, protein synthesis from the L3'-lacZ' fusion gene carried on

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XLF2 did not exhibit the expected increase with increasing growth rate. Rather, the synthesis of the fusion protein decreased about twofold as the growth rate increased from 0.5 to 2.0 doublings. Furthermore, we observed little difference in the growth rate dependence of the fusion protein synthesis whether the fusion protein was being expressed from the L4-sensitive XLF2 prophage or from the L4insensitive XLF22 prophage. This unexpected response cannot result from a strain-specific defect in steady-state growth rate-dependent control of ribosome synthesis, since control experiments showed that the accumulation of stable RNA I c

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GROWTH RATE CONTROL OF S10 r-PROTEIN OPERON

VOL. 172, 1990

ing XLF2 or XLF22. Gel electrophoresis of extracts from pulse-labeled cells showed that cAMP had no detectable effect on the synthesis of the L3'-lacZ' fusion protein from either XLF2 or XLF22 (data not shown). Thus, the proposed effect of cAMP on the transcription of lacZ cannot account for the reduced expression of the L3'-lacZ' fusion protein at high growth rates. The results of these steady-state growth rate studies were somewhat unexpected. The L3'-lacZ' fusion gene on XLF2 is under normal L4-mediated autogenous control and exhibits a nutritional shift-up response similar to that characteristic of r-protein synthesis. Yet, this gene does not exhibit the steady-state growth rate stimulation characteristic of genuine r-protein genes, including all the genes of the S10 operon (5). Therefore, either L4-mediated autogenous control is not at all involved in growth medium-dependent control of the S10 operon or, if it is necessary, it is certainly not sufficient for this control. Furthermore, the regulatory mechanisms which accomplish a nutritional shift-up response are not sufficient to achieve steady-state growth rate-dependent control of the S10 operon. These results support our earlier proposal (25) that growth rate regulation of ribosome synthesis depends on an interaction between several different molecular mechanisms. It is not clear what additional processes might contribute to the steady-state control of ribosome synthesis. One possibility is the regulation of r-protein mRNA stability. In fact, the half-life of r-protein mRNA appears to increase with the growth rate, since the differential accumulation of r-protein mRNA increases with the growth rate (8) but the differential rate of r-protein mRNA synthesis is constant (11). Perhaps there is a mechanism which actively regulates the r-protein mRNA stability as a function of the growth rate. Alternatively, the turnover rate of the r-protein mRNA could be modified simply as a consequence of altered rates of the frequency of translation due to autogenous control (4, 23). Whatever the processes that coordinate ribosome synthesis with the growth rate might be, it is evident that the S10 operon DNA cloned on XLF2, including 500 bases upstream of the promoter, all of the S10 leader, and the first one and one-half structural genes, does not contain the signals sufficient to ensure this control. What additional sequences are required is not at all clear. One speculation is that the transcription terminator at the end of the S10 operon is involved, since there is evidence that hairpins at the 3' ends of mRNA molecules can affect their stability (1). ACKNOWLEDGMENTS We thank Sankar Adhya for phage XD69, Niels Fiil for the F' lacpl episome, and Richard Archer for technical assistance. This work was supported by a research grant and a Research Career Development Award to L.L. from the National Institute of Allergy and Infectious Diseases.

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