In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of Cl- ions

Abstract

In vitro protein synthesis in Vibrio costicola [poly(U)-directed incorporation of phenylalanine] was studied.The extent of protein synthesis was limited by the number of ribosomes present. Density gradient centrifugation experiments suggested that, after runoff of ribosomes from the artificial messenger, the 50S subunit was unable to attach to the 30S-messenger complex. As shown previously (M. Kamekura and D. J. Kushner, J. Bacteriol. 160:385-390, 1984), Cl-ions inhibited protein synthesis; indeed, the highest rate of synthesis took place in the lowest attainable Cl-concentration (37 mM). The inhibitory effects were partly reversed by glutamate and betaine, both of which are concentrated within cells of V. costicola. The strongest reversal was seen when both glutamate and betaine were present. Cl-ions car! prevent binding of ribosomes to poly(U) and displace ribosomes already bound to this artificial messenger. The effects of Cl-ions on binding were also reversed by glutamate and betaine. Cl-ions did not affect accuracy of translation; they were shown previously (Kamekura and Kushner, J. Bacteriol. 160:385-390, 1984) not to affect phenylalanyl-tRNA synthetase. It was also found that washing ribosomes with inhibitory NaCl concentrations did not interfere with their ability to carry out protein synthesis later in optimal (low) salt concentrations. On the contrary, these ribosomes were more active than before they were washed. We conclude that the main site of action of Cl-in the system studied is on the binding of ribosomes to the mRNA.Vibrio costicola is a moderate halophile which grows in media containing a wide range (0.4 to 3.5 M) of NaCl concentrations, with its optimum growth occurring at a concentration of around 1 M NaCl (7). Although its cellassociated concentration of Na+ and K+ ions is generally equal to the external Na+ concentration (15), in vitro protein synthesis [poly(U)-dependent phenylalanine incorporation] by cellular extracts of this bacterium, after growth in the presence of 1 M NaCl, was found to have maximal activity in 0.1 to 0.2 M concentrations of NaCl, KCl, or NH4C1 and no, or very little, activity in 0.6 M concentrations of these salts. Amino acid incorporation dependent on "endogenous" mRNA had similar responses to these salts (19).The inhibitory effects were shown to be due mainly to the C1-ions, which are normally excluded from the cells: V. costicola grown in the presence of 1.0 M NaCl contains only 0.2 M C1-(6). The effects of higher concentrations of these ions were partly reversed by a number of organic substances, including glycine betaine (betaine) and glutamate, which are accumulated within the cells.The site of action of C1-has not yet been identified. It is not phenylalanyl-tRNA synthetase, which is barely inhibited by as much as 0.9 M NaCl (6). In this paper, we report further studies of the in vitro protein-synthesizing system of V. costicola, with special reference to the site of action of Cl-ions.

Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola

“…These results confirm the toxic nature of the Cl-ions on in vitro protein synthesis by V. costicola, effects previously observed with poly(U) as artificial mRNA (3,9,38). The greater sensitivity of the R17-directed system than the endogenous system to added Cl-ions suggests that these ions are more toxic to initiation than to elongation.…”

“…Therefore, it seems that betaine can afford some protection against the toxic action of Cl-ions, as was previously seen with the poly(U) system (3,9). Although glutamate also affords protection against Cl-ions in the poly(U) system (3,9), further addition of 0.2 M sodium or potassium glutamate was inhibitory in the systems studied now, especially the R17 RNA-directed system (Fig. 4A and B).…”

“…Both systems are more physiological than the poly(U)-directed system studied previously (3,9), but there are important differences between them. In the R17-directed system, every ribosome that becomes involved in protein synthesis must first go through normal initiation.…”

“…Polyuridylic acid [poly(U)]-directed incorporation of phenylalanine by cell extracts of the moderate halophile Vibrio costicola is inhibited by Cl-ions (9), which prevent ribosome binding to the artificial mRNA (3).…”

Comparative Toxicities of Salts on Microbial Processes in Soil

“…A lower toxicity of SO 4 2Ϫ ions than Cl Ϫ ions to soil microorganisms has been suggested previously (28), e.g., cultured rhizobial strains were found to be less affected by SO 4 2Ϫ salts than the corresponding Cl Ϫ salts (50). Chloride ions inside cells have been shown to inhibit protein synthesis by preventing the binding of ribosomes to mRNA (51,52). SO 4 2Ϫ ions, on the other hand, can be metabolized by many bacteria and fungi and have no ion-specific toxicity to cellular processes.…”