Suppression of growth and protein secretion defects in Escherichia coli secA mutants by decreasing protein synthesis

Abstract

We devised a new selection for conditionally lethal suppressors of secA mutants. This selection allows the isolation of both temperature-sensitive and cold-sensitive suppressor mutations, whereas previous studies were limited to nonlethal or cold-sensitive suppressor mutations. Two temperature-sensitive suppressor mutations lie in genes required for protein synthesis: asnS, the gene for the asparaginyl-tRNA synthetase, and divE, which encodes the tRNAser. A previously characterized mutation in alaS, the gene for the alanyl-tRNA synthetase, suppresses the growth and secretion defects of a secA mutant. Although the primary effects of these suppressor mutations are different, it is likely that they cause suppression of secA mutations by altering the rate of protein synthesis, since the protein synthesis inhibitors, chloramphenicol and tetracycline, also suppress secA mutations. Chloramphenicol also suppresses the growth defect of certain other sec mutants. We postulate that the impaired secretory capacity of sec mutants can be offset by decreasing the rate of elongation of secreted proteins or by decreasing the total amount of secreted proteins per cell. The results indicate that our initial goal to identify cellular secretory components as suppressors of secA mutations might be difficult to achieve because of a high frequency of nonspecific suppressors that alter protein synthesis. Unexpectedly, the suppressor approach provides a direct genetic selection for mutants in protein synthesis.

Trigger Factor Retards Protein Export in Escherichia coli

“…To examine the relationship between translation rate and protein export in different genetic backgrounds, we pulse-labeled MC4100 and HDB55 and monitored OmpA export in the presence of varying concentrations of the peptidyltransferase inhibitor chloramphenicol. Sublethal concentrations of this drug have been shown to suppress export defects caused by mutations in the translocation machinery (38). If disruption of tig reduces the SecB requirement by slowing translation, then OmpA should be exported in HDB55, which contains a threshold concentration of chloramphenicol.…”

SecA Is Required for the Insertion of Inner Membrane Proteins Targeted by the Escherichia coli Signal Recognition Particle

“…In these experiments we used a strain that contains a secA am mutation and a temperature-sensitive amber suppressor. At low temperature full-length SecA protein is synthesized, but at high temperature only a small unstable fragment of the protein is produced (16 (28). Selective media contained 100 g/ml ampicillin or 40 g/ml chloramphenicol.…”

ssaD1, a suppressor of secA51(Ts) that renders growth of Escherichia coli cold sensitive, is an early amber mutation in the transcription factor gene nusB

“…These suppressor mutations have been located to a surprisingly large number of genes, most of them involved in protein synthesis (9,14,18,20). It has been suggested that most of these suppressors operate indirectly by reducing the rate of protein synthesis, thereby bringing the processes of protein synthesis and secretion back into balance (9,20).…”

“…It has been suggested that most of these suppressors operate indirectly by reducing the rate of protein synthesis, thereby bringing the processes of protein synthesis and secretion back into balance (9,20). This proposal was also supported by the observation that even low levels of protein synthesis inhibitors such as chloramphenicol suppressed both temperature-sensitive and cold-sensitive sec mutants and resulted in increased rates of protein export under normally nonpermissive conditions (9). One suppressor of the secY24(Ts) mutation ssyB63(Cs) has been shown recently to be an insertional disruption of nusB, a gene important in transcription antitermination and essential for the growth of E. coli at low temperatures (5,12,22).…”

“…These suppressor mutations have been located to a surprisingly large number of genes, most of them involved in protein synthesis (9,14,18,20). It has been suggested that most of these suppressors operate indirectly by reducing the rate of protein synthesis, thereby bringing the processes of protein synthesis and secretion back into balance (9,20). This proposal was also supported by the observation that even low levels of protein synthesis inhibitors such as chloramphenicol suppressed both temperature-sensitive and cold-sensitive sec mutants and resulted in increased rates of protein export under normally nonpermissive conditions (9).…”