[[The]] role of the bacterial ribosome in barotolerance

Abstract

Examination of the pressure response of the protein synthesizing systems utilizing all the possible 30s and 50s ribosomal subunit combinations from E. coli, P. fluorescens and P. bathycetes indicated that the salt-mediated barotolerant characteristic associated with P. bathycetes ribosomes lies within the functional capabilities of the 3Os subunit. Furthermore, the concentration of selected ions utilized in the process of separation and isolation of P. bathycetes ribosomal subunits was a critical factor. Utilization of isolation media containing either ImM Mg++, ImM Mg++ plus 150mM Na+, or lmM Mg++ plus 150mM K+ resulted in particles being obtained which were either respectively unable to synthesize polypeptide, capabLe of polypeptide synthesis which was barosenstive at both low and high salts, or capable of synthesis which was barosensitive in low salts and barotolerant in high salts. The inability to synthesize polypeptide has been shown to be due to an in ability to form messenger RNA-ribosomecampLexes , Furthermore riboosome isolated in 1mM Mg++ salts display altered sedimentation profiles, i.e. 30s particles appear as 26s particles. This change in s value may be due to the loss of specific ribosomal proteins during the isolation proceedure. The loss of the barotolerant characteristic may be due to specific ion-macromolecular interactions whLch cause irreversible conformational changes in the 30s ribosomal subunit.

The effects of high hydrostatic pressures on protein synthesis by whole cells and cell-free preparations of Escherichia coli, Pseudomonaa flourescens, and Pseudomonas bathycetes were determined. Actively growing cells of P. bathycetes and P. fluorescens were less sensitive than were E. coli cells. Protein synthesis by cell-free preparations of E. coli and P. fluorescens showed the same extent of inhibition as their respective whole cell preparations, whereas cell-free preparations of P. bathycetes showed a marked increase in pressure sensitivity over whole cells. Protein synthesis by hybrid protein synthesizing cell-free preparations (the ribosomes from one organism and the 5-100 supernatant fraction from another) demonstrated that response to high pressure is dependent on the source of ribosomes employed. A hybrid system containing E. coli ribosomes and P. fluorescens 5-100 shows the same sensitivity to pressure as a homologous E. coli system, whereas a hybrid containing P. fluorescens ribosomes and E. coli S-100 shows the greater pressure tolerance characteristic of the P. fluorescens homologous system. Experiments utilizing protein synthesizing systems employing various combinations of 308 and 50s ribosomal subunits from E. coli and P. fluorescens demonstrated the barotolerant nature of protein synthesis in P. fluorescens to be associated with the 308 ribosomal subunit.

The degree of barotolerance exhibited by P. fluorescens and P. bathycetes in vitro polyphenylalanine synthesizing systems can be modified by altering theeoncentrat1ons of specific ions in the reaction mixture. Rybrid protein synthesizing systems utilizing all the posstble S-100 supernatant fluid and ribosome combinations from E. coli, P. fluorencens and P. bathycetes were tested for barotolerance under conditions of low (l6mM Mg++,OmM Na+) and high (60mM: Mg++, 150mM Na+) ion concentrations. The results reveal that barotolerant protein synthesis is a characteristic determined by the origin of the ribosome. Systems utilizing E. coli ribosomes are barosensitive at both low and high ion concentrations. P. fluorescens barotolerant under both conditions, and P. bathycetes ribosomes barosensitive at low and barotolerant at high ion concentrations. Therefore, certain concentrations of specific ions will increase harotolerance, but only if the ribosomes are capab1e of functioning at high pressure.