Elongation in Escherichia coli: a Hydrostatic Pressure Study

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JOURNAL OF BACTERIOLOGY, July 1982, p. 516-520 0021-9193/82/070516-05$02.00/0 Vol. 151, No.

1 JOURNAL OF BACTERIOLOGY, July 1982, p /82/ $02.00/0 Vol. 151, No. 1 Effect of S12 Ribosomal Mutations on Peptide Chain Elongation in Escherichia coli: a Hydrostatic Pressure Study GERALD McMAHONt* AND JOSEPH V. LANDAU Department of Biology, Rensselaer Polytechnic Institute, Troy, New York Received 18 September 1981/Accepted 4 March 1982 Protein synthesis in Escherichia coli mutants that differ from one another in mutations which impart streptomycin resistance was investigated by the application of hydrostatic pressure. Increased pressure resistance was only observed in mutants which exhibited reduced rates of peptide chain elongation. These findings indicate that the major effect of pressure on protein synthesis in E. coli may involve the SI 2 ribosomal protein. It has been well documented that the application of high hydrostatic pressure affects a number of metabolic processes in actively growing bacterial cells (8, 21). The effect of hydrostatic pressure on any elementary process can beqt be described by the equation ko = kp e(-pvv )/RT, where ko is the reaction rate at zero pressure, kp is the rate under pressure (P), and AV* is the difference between the volume of the activated complex and the volume of the reactants (4). Landau (6) has shown that the effect of pressure on the synthesis of protein in Escherichia coli allows the measurement of an activation volume change of about 100 cm3/mol at 37 C. It has been strongly suggested that the synthesis of protein may be the process which limits the growth of E. coli and microbes in general at high pressure (10). On the other hand, the degree to which bacterial protein synthesis is sensitive to the application of pressure is independent of the environmental origin, physiological type, or taxonomic group of the organism studied (11). It has been shown that differences in protein synthesis response to high pressure among E. coli, a terrestrial pseudomonad (Pseudomonas fluorescens), and a deep-sea isolate (P. bathycetes) are due to functional differences in the small ribosomal subunit (7, 15). Moreover, it has been shown that the binding of trna to the ribosome may be the reaction which is most sensitive to the application of pressure in E. coli protein synthesis (2, 14). To investigate these findings further, we employed E. coli strains which possess rpsl (stra) mutations that confer resistance to (Str') or dependence on (Strd) the antibiotic streptomycin. It has been well documented how mutations in this locus lead to alterations in the S12 ribosomal protein and, in some cases, to pleiotropic t Present address: Cancer Research Center, Tufts University School of Medicine, Boston, MA effects chiefly by effecting a reduction in the rate of peptide chain elongation (19, 20). Moreover, it has been suggested that the S12 ribosomal protein may figure prominently in ribosometrna interactions (9, 18). Our laboratory has observed, in several cell-free translation systems, that barotolerant protein synthesis coincides with a reduction in the rate of polypeptide elongation. In this study, we used E. coli strains which exhibit differences in the rate of growth in the presence of streptomycin. Strain C600 and a Strr strain, SM11, grew at equivalent rates on minimal medium at 37 C (Table 1). However, the SM3 mutant grew 60% slower than did strains 516 SM11 and C600 at 37 C. It has been shown by Zengel et al. (20) that this pleiotropic effect may be chiefly due to a reduction in the rate of peptide chain elongation. However, it has been noted by these workers that the mutation may affect the function of the ribosome in the initial (20) and terminal (19) events of translation as well. As shown here and elsewhere (20), the Strr pleiotropic effects can be partially relieved by the addition of streptomycin. On the other hand, growth of the Str" mutant SD103 showed little deviation from that of its Strs counterpart when its growth medium was supplemented with streptomycin. Figure 1 illustrates the results of a study on the effects of pressure on polypeptide synthesis in actively growing cells at 37 C. Exponential cells synthesized polypeptides in a linear fashion for up to 7 min after the cells were mixed with labeled amino acids. Protein synthesis was measured as the incorporation of amino acids into trichloroacetic acid-precipitable polypeptides. Rates of protein synthesis during a 2-min period of 100- to 800-atm (10,133- to 81,060-kPa) pressurization were calculated as percentages of the atmospheric rates. In all strains, the inhibition of protein synthesis could be represented by a

2 VOL. 151, 1982 NOTES 517 TABLE 1. Activation volume changes (AVt) and P50 values of protein synthesis in strains grown in the absence or presence of streptomycin (Str) Growth rate (doublings/h) AV V (cm3/mo) c'mlofptensh of th protein P50 (atm) with': Straina Phenotype with: Temp ( C) esis wi No Str Str No Str Str No Str Str C600 Strs 1.05 d ± 7 (7) 360 SMi1 Strr ± 2 (2) NDf 458 ND SM3 Strr ± 15 (6) 58 ± 2 (4) SD103 Strd ± 3 (2) C600 Strs ± 11 (2) 420 SM3 Strr 0.50 ND ± 5 (3) ND 458 ND a The strains were grown on a modified glucose minimal medium (18), and, in some cases, streptomycin sulfate was added to a final concentration of 200 Fig/ml. Strains C600, SM3, and SM11 were obtained from John Yates, University of Wisconsin, Madison. The parental strain, C600, is a K-12 derivative with a mutation in the lacy locus (1). Strains SM3 and SM11 are spontaneous Strr mutants isolated by selection at 37 C in the presence of 200 p.g of streptomycin sulfate per ml (20). Strains SS7 and SD103 were obtained from Charles Hurwitz, Veterans Administration Hospital, Albany, N.Y. Strain SD103 is a spontaneous Strc mutant derived from a 1Bgalactosidase strain K-12 parent. b AV* values were calculated from the second phase of the pressure curve in the equation AVt = RT ln [(k1lk2) (P2 - Pj)], where k, and k2 are the rates of protein synthesis at pressures P1 and P2, respectively. c Multiply atmosphere values by to get kilopascal values. d No growth. Number of determinations. f ND, Not determined. pressure profile which is characteristic of the E. coli protein-synthesizing process at 37 C (12). Activation volume changes (AV*) were calculated from the slope of the second phase of the profile in the equation AV* = RTln[(kl/k2) (P2 - P1)], where k1 and k2 are the reaction rates at pressures P1 and P2, respectively (Table 2). Although the Strf mutation may decrease the AV* of protein synthesis, the most apparent effect of the mutation is that the pressure necessary to inhibit protein synthesis is increased. We expressed this phenomenon as an increase in the pressure necessary to inhibit protein synthesis by 50% at a given temperature (P50 value) (Table 1). It became apparent that strain SM3 could synthesize protein at a pressure which would normally arrest polyribosome function in its Strr counterpart. Furthermore, protein synthesis in strain SM3 is as resistant to pressure as protein synthesis in P.fluorescens or P. bathycetes (12). All of these findings are in agreement with the initial observations of Pope et al. (12) that protein synthesis in a StrT E. coli mutant is more barotolerant at 680 atm (68,901 kpa) than is its Strs parent. In addition, we have been able to show this effect by using a rpsl22 mutant strain (DEV2) which contains Gorini's stra2 allele (3; data not shown). When protein syntheses in strains C600 and SM3 were tested at 25 C, there was little difference in response to the application of pressure, suggesting that the role of the S12 mutation during pressurization is minimized at the lower temperature. This finding is reflected in the observation that the growth rates of the mutant and parental strains are similar at 25 C, suggesting a suppression of the pleiotropic effect by growth at the lower temperature (Table 1). In addition, we measured the effect of streptomycin on the responses of the mutant and parental strains to pressure at 37 C (Table 1). Since it has been demonstrated that the addition of streptomycin partially relieves pleiotropic effects in some Strr strains (20), we predicted that the growth of these strains in the presence of streptomycin would also enhance the inhibition of protein synthesis by pressure. For the most part, this prediction was borne out in vivo and through the use of the in vitro peptide chain elongation assay described below. Since it has been shown by Schwarz and Landau (13) that the major effect of pressure on protein synthesis is the reduction of the polypeptide chain elongation rate, we expected that our findings with whole cells would be mimicked in vitro. We isolated initiation-free polysomes from strains C600 and SM3 (16), incubated the polysomes with saturating amounts of E. coli "S100" extract, and measured the rate of ribosomal runoff as the incorporation of amino acids into trichloroacetic acid-precipitable polypeptides. E. coli MRE600 can be used as a source of supernatant factors since the response of protein synthesis to pressure in this strain is similar to that in the parental strain, C600. Moreover, it has been shown that the source of factors does not influence the pressure response (12). Figure 2 illustrates the results of these experiments in

518 NOTES Id I-. 100 100 200 300 400 500 600 700 800 PRESSURE (ATM) FIG. 1. Effect of pressure on protein synthesis in E. coli C600 (0), SM11 (A), SM3 (V), and SD103 (0). A portion (2.

3 518 NOTES Id I PRESSURE (ATM) FIG. 1. Effect of pressure on protein synthesis in E. coli C600 (0), SM11 (A), SM3 (V), and SD103 (0). A portion (2.0 ml) of exponential culture, grown at 25 or 37 C, was rapidly mixed with 2,uCi of 14C-labeled L-amino acids and 50,uM each (final concentration) 20 amino acids. The reaction mixture was incubated at the desired temperature. After 1 min of incubation, a 1.0-ml volume was drawn into a tuberculin syringe which was then capped with a serum stopper and placed in a reaction vessel which had been filled with water and equilibrated to the incubation temperature. The reaction vessel was capped and brought to the desired pressure with a hydraulic hand pump. The pressure was attained in 7 s and maintained for 2 min; the reaction vessel was then depressurized and disassembled, and the contents of the syringe were emptied into a vial at the reaction temperature. Samples were processed as previously described (7), trichloroacetic acid precipitates were collected onto filters, and radioactivity was determined by scintillation counting. Atmospheric incorporation was linear for the duration of the experiment, and pressurized samples showed linear kinetics after pressure release. Rates under pressure were extrapolated over the period of pressurization and expressed as a percentage of the atmospheric rate (5). J. BACTERIOL. the absence or presence of streptomycin. As in whole cells, SM3 polysome function was more resistant to pressure than was its strain C600 (Strs) counterpart. Moreover, the in vitro atmospheric chain elongation rate found in this study paralleled the in vivo 3-galactosidase chain elongation rate found by Zengel et al. (20). One unexplained finding, however, is that the addition of streptomycin, as an inhibitor of StrP peptide chain elongation, resulted in the lowering of the activation volume change both in vivo and in vitro. Because the inhibitory action of streptomycin is antagonized by magnesium (17) and because the magnesium ion concentration greatly influences the activation volume change of protein synthesis (7), the reduction in the AVt which accompanies the streptomycin-induced inhibition may be related to ribosome rigidity. Our evidence suggests that changes in the rate of peptide chain elongation due to modifications in the S12 ribosomal protein dramatically alter the effect of pressure on the rate of ribosomal runoff in vitro and the rate of protein synthesis in vivo. This conclusion is partially based upon evidence which suggests that the pleiotropic effects we observed in the SM3 mutant were a result of a mutation in the structural gene for the S12 ribosomal protein (20). Since previous studies implicate the trna binding reaction (14) as the reaction most inhibited by the application of pressure, a mutation in the rpsl locus could very well influence a pressure-reactive site on the small ribosomal subunit, resulting in the effects presented here. Although there is a correlation between the atmospheric rate of polypeptide chain elongation and the P50 value, it would be premature to say that only modifications at the S12 genetic locus can lead to changes in the pressure response. In fact, it has been shown by Pope et al. (12) that the synthesis of protein in E. coli may be more barotolerant at 15 and 25 C than at 37 C, implicating a more general, temperaturedependent ribosomal reaction. We suggest, however, that lowering the reaction temperature influences the ribosomal site in a way that is analogous to the genetic modifications we used. Although the addition of streptomycin, a known effector of chain elongation, influences the inhibitory effect of pressure, the addition of low concentrations of fusidic acid, another inhibitor of chain elongation, did not lead to changes in pressure response, although it did dramatically reduce the growth rate (data not shown). These findings suggest that modifications in the amino acid sequence of the S12 ribosomal protein and the modulation of the resulting ribosomal structure by the binding of streptomycin may have a unique capacity to influence the pressure-reactive site of the ribosome. It appears that the rpsl locus mutation and streptomycin both serve to specifically alter the chain extension rate enough to influence a ribosome site crucial to the inhibitory effect of pressure. We have also found that the application of hydrostatic pressure restricts streptomycin-induced misreading of a polyuridylate template (G. McMahon and J. V. Landau, manuscript in preparation). Such a finding

VOL. 151, 1982 NOTES 519 TABLE 2. Activation volume changes (AVW) and P50 values for polysomal runoff at 37 C in the absence or presence of streptomycin (Str) Rate of runoff (cpm/min per mg of P.

4 VOL. 151, 1982 NOTES 519 TABLE 2. Activation volume changes (AVW) and P50 values for polysomal runoff at 37 C in the absence or presence of streptomycin (Str) Rate of runoff (cpm/min per mg of P. (atm) with': AV* (cm3/mol) of polysomal Source of ribosomes) with: runoff in vitro with: polysomes No NoNoStr Str" Stra ~~~~~Str Str No Str Str Strain C ± 5 (2) 914 ± 15 (2) ± 5 (2)` 50 1 (2) Strain SM ± 12 (3) 1738 ± 10 (2) ± 8 (3) 79 ± 5 (2) a Streptomycin was added to reaction mixtures containing polysomes from strain C600 or SM3 to a final concentration of 1 or 10 FM, respectively. b Multiply atmosphere values by to get kilopascal values. c Number of determinations. would not be unexpected if hydrostatic pressure alters the kinetics of trna-ribosome interaction in such a way that the ribosome is able to eject noncognate trnas more efficiently (9). These observations support the contention of other studies that the binding of trna to the ribosome may be the reaction in E. coli which is most sensitive to pressure. (This work is taken in part from a thesis submitted by G.M. in partial fulfillment of the requirement for the Ph.D. in the Department of Biology, Rensselaer Polytechnic Institute.) A Poo o PRESSURE (ATM ) FIG. 2. B o 50060o 700 S0 PRESSURE (ATM) Effect of streptomycin on the response of polypeptide extension to pressure. A portion (1.0 ml) of the reaction mixture containing 1 mg of E. coli C600 (0) or SM3 (0) polysomes was incubated at 37 C in the presence of 50 mm Tris-hydrochloride (ph 7.6)-60 mm NH4Cl-8 mm Mg (acetate)2-1 mm dithiothreitol- 1 mm Tris-ATP-20 F.M Tris-GTP-5 mm potassium phosphoenolpyruvate-1 pci of '4C-labeled L-amino acids-30 pg of pyruvate kinase-1 mg of "S100" extract. Initiation-free polysomes were prepared by the gel filtration method of Tai et al. (16). Portions (1.0 ml) of the reaction mixtures were tested at the pressures indicated, and samples were processed as previously described (7). Streptomycin sulfate was either omitted from (B) or added to the reaction mixtures at a concentration of 10 pug/ml (A). Incorporation of amino acids into trichloroacetic acid-precipitable polypeptides was ribosome dependent, and pressurized samples resumed atmospheric rates of incorporation upon the release of pressure. We thank John Yates and Charles Hurwitz for kindly donating the bacterial strains for this study and Daniel Pope, P.-C. Tai, and Robert Broeze for helpful suggestions and discussions. LITERATURE CITED 1. Appleyard, R. K Segregation of new lysogenic types during growth of a doubly lysogenic strain derived from Escherichia coli K12. Genetics 39: Arnold, R. M., and L. J. Albright Hydrostatic pressure effects on the translation stages of protein synthesis in a cell-free system from Escherichia coli. Biochim. Biophys. Acta 238: Bjare, U., and L. Gorini Drug dependence reversed by a ribosomal ambiguity mutation, ram, in Escherichia coli. J. Mol. Biol. 57: Johnson, F. H., H. Eyring, and B. J. Stover Hydrostatic pressure molecular and volume changes, p In F. H. Johnson, H. Eyring, and B. J. Stover (ed.), The theory of rate processes in biology and medicine. John Wiley & Sons, Inc., New York. 5. Landau, J. V Protein and nucleic acid synthesis in Escherichia coli: pressure and temperature effects. Science 153: Landau, J. V Induction, transcription, and translation in Escherichia coli: a hydrostatic pressure study. Biochim. Biophys. Acta 149: Landau, J. V., W. P. Smith, and D. H. Pope Role of the 30S ribosomal subunit, initiation factors, and specific ion concentration in barotolerant protein synthesis in Pseudomonas bathycetes. J. Bacteriol. 130: Marquis, R. E High-pressure microbial physiology. Adv. Microbiol. Physiol. 14: Ninio, J A semiquantitative treatment of missense and nonsense suppression in the stra and ram ribosomal mutants of Escherichia coli. J. Mol. Biol. 84: Pope, D. H., and L. R. Berger Inhibition of metabolism by hydrostatic pressure: what limits microbial growth? Arch. Mikrobiol. 93: Pope, D. H., W. P. Smith, M. A. Orgrinc, and J. V. Landau Protein synthesis at 680 atm: is it related to environmental origin, physiological type, or taxonomic group? Appl. Environ. Microbiol. 31: Pope, D. H., W. P. Smith, R. W. Swartz, and J. V. Landau Role of bacterial ribosomes in barotolerance. J. Bacteriol. 121: Schwarz, J. R., and J. V. Landau Hydrostatic pressure effects in Escherichia coli: site of inhibition of protein synthesis. J. Bacteriol. 109: Schwarz, J. R., and J. V. Landau Inhibition of cellfree protein synthesis by hydrostatic pressure. J. Bacteriol. 112: Smith, W. P., D. H. Pope, and J. V. Landau Role of bacterial ribosome subunits in barotolerance. J. Bacteriol. 124: Tai, P.-C., B. J. Walace, E. L. Herzog, and B. D. Davis Properties of initiation-free polysomes ofescherichia coli. Biochemistry 12:

520 NOTES J. BACTERIOL. 17. Wallace, B. J., P.-C. Tal, E. L. Herzog, and B. D. Davis. 1973. Partial inhibition of polysomal ribosomes of Escherichia coli by streptomycin. Proc. Natl. Acad. Sci. U.S.A. 70:1234-1237.

5 520 NOTES J. BACTERIOL. 17. Wallace, B. J., P.-C. Tal, E. L. Herzog, and B. D. Davis Partial inhibition of polysomal ribosomes of Escherichia coli by streptomycin. Proc. Natl. Acad. Sci. U.S.A. 70: Yates, J. L Role of ribosomal protein S12 in discrimination of aminoacyl-trna. J. Biol. Chem. 254: Yates, J. L., W. R. Gette, M. E. Furth, and M. Nomura Effects of ribosomal mutations on the read-through of a chain termination signal: studies on the synthesis of bacteriophage 0 gene protein in vitro. Proc. Natl. Acad. Sci. U.S.A. 74: Zengel, J. M., R. Young, P. P. Dennis, and M. Nomura Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli. J. Bacteriol. 129: Zimmerman, A. M. (ed.) High pressure effects on cellular processes. Academic Press, Inc., New York.

IN VIVO AND IN VITRO CROSS-RESISTANCE OF KANAMYCIN-RESISTANT MUTANTS OF E. COLI TO OTHER

1527 IN VIVO AND IN VITRO CROSS-RESISTANCE OF KANAMYCIN-RESISTANT MUTANTS OF E. COLI TO OTHER AMINOGLYCOSIDE ANTIBIOTICS EUNG CHIL CHOI, TOSHIO NISHIMURA, YOKO TANAKA and NOBUO TANAKA Institute of Applied