Evidence for an accumulation of messenger RNA specific for extracellular protease and its relevance to the mechanism of enzyme secretion in bacteria

Washed cells of Bacillus amyloliquefaciens rapidly synthesize and secrete a neutral protease into the extracellular medium. In a medium containing low concentrations of amino acids the production occurs almost linearly but in high concentrations of amino acids there is a rapid phase (phase 1) of production for 30 minutes, followed by a levelling for 50 minutes after which synthesis resumes linearly (phase 2). The inhibitors of RNA synthesis, rifampicin and actinomycin D, inhibit phase 2 synthesis promptly but do not inhibit phase 1 production. This latter production is nevertheless sensitive to inhibitors of protein synthesis and labelling studies have confirmed that the protease production involves de novo synthesis of the enzyme. The evidence indicates that there is present in harvested cells an accumulated pool of mRNA for protease, capable of supporting synthesis of the enzyme for 80 minutes. It is proposed that mRNA for protease migrates from the gene to specific translational-extrasion sites in the membrane and that the accumulation represents a queuing of messenger molecules for these sites. The biphasic time-course in the presence of high concentrations of amino acids is attributed to amino-acid repression of protease mRNA formation, phase 1 production representing translation and exhaustion of the accumulated pool. The protein synthesis inhibitors, pactamycin and fusidic acid, at low concentrations completely inhibit protease production without affecting general intracellular protein synthesis. This is interpreted as supporting the concept that protease synthesis occurs on ribosomes located at the periphery of the cell.