Isolation and analysis of novel mutants of Escherichia coli prlA (secY).

Plasmid libraries of prlA mutants containing single-base-pair changes throughout the gene were generated by in vitro random mutagenesis. The prlA mutations capable of suppressing the secretion defect of LamB caused by mutations in the LamB signal peptide were selected and analyzed. Together with additional mutations generated by site-directed mutagenesis, a number of novel prlA mutations and/or suppressors were identified. These mutations provide the starting points for studying the relationship of structure and function of PrlA in its interaction with LamB and/or other component(s) in the Escherichia coli protein secretion-translocation complex.     

prlA suppression of defective export of maltose-binding protein in secB mutants of Escherichia coli.

An Escherichia coli strain containing a signal sequence mutation in the periplasmic maltose-binding protein (MBP) (malE18-1) and a point mutation in the soluble export factor SecB (secBL75Q) is completely defective in export of MBP and unable to grow on maltose (Mal- phenotype). We isolated 95 spontaneous Mal+ revertants and characterized them genetically. Three types of extragenic suppressors were identified: informational (missense) suppressors, a bypass suppressor conferring the Mal+ phenotype in the absence of MBP, and suppressors affecting the prlA gene, which encodes a component of the protein export apparatus. In this study, a novel prlA allele, designated prlA1001 and mapping in the putative second transmembrane domain of the PrlA (SecY) protein, was found. In addition, we isolated a mutation designated prlA1024 which is identical to prlA4-2, the mutation responsible for the signal sequence suppression in the prlA4 (prlA4-1 prlA4-2) double mutant (T. Sako and T. Iino, J. Bacteriol. 170:5389-5391, 1988). Comparison of the prlA1024 mutant and the prlA4 double mutant provides a possible explanation for the isolation of these prlA alleles.     

SecY variants that interfere with Escherichia coli protein export in the presence of normal secY

As an approach for studying how SecY, an integral membrane protein translocation factor of Escherichia coli, interacts with other protein molecules, we isolated a dominant negative mutation, secY-d1, of the gene carried on a plasmid. The mutant plasmid severely inhibited export of maltose-binding protein and less severely of OmpA, when introduced into sec+ cells. It inhibited growth of secY and secE mutant cells, but not of secA and secD mutant cells or wild-type cells. The mutation deletes three amino acids that should be located at the interface of cytoplasmic domain 5 and transmembrane segment 9. We also found that some SecY-PhoA fusion proteins that lacked carboxy-terminal portions of SecY but retain a region from periplasmic domain 3 to transmembrane segment 7 were inhibitory to protein export. We suggest that these SecY variants are severely defective in catalytic function of SecY, which requires cytoplasmic domain 5 and its carboxy-terminal side, but retain the ability to associate with other molecules of the protein export machinery, which requires the central portion of SecY; they probably exert the 'dominant negative' effects by competing with normal SecY for the formation of active Sec complex. These observations should provide a basis for further genetic analysis of the Sec protein complex in the membrane.     

Complementation of the protein transport defect of an Escherichia coli secY mutant (secY24) by Bacillus subtilis secY homologue

Bacillus subtilis SecY homologue shares 41.3% homology with that of E. coli and remarkably higher homologous regions (more than 80%) are present in the four cytoplasmic regions [(1990) J. Biochem. 107, 603-607]. Based on the formation of the mature form of OmpA in E. coli, we have shown that the protein transport defect of the E. coli secY mutant (secY24) is complemented by the gene product from the B. subtilis secY homologue, which is expressed under the lac promoter control. However, B. subtilis SecY could not restore growth of the E. coli mutant at nonpermissive temperature.     

secD, a new gene involved in protein export in Escherichia coli.

New mutants of Escherichia coli altered in protein export were identified in phoA-lacZ and lamB-lacZ gene fusion strains by searching for mutants that showed an altered lactose phenotype. Several mutations mapped in a new gene, secD. These mutants were, in general, cold sensitive for growth, and the mutations led to an accumulation of precursor of exported proteins. The secD gene is closely linked to tsx on the E. coli chromosome, but separable from another gene proposed to be involved in export, ssaD, which maps nearby. A plasmid carrying secD+ was identified and used to show that the mutations are recessive. The secD gene may code for a component of the cellular export machinery.     

Identification of a new gene (secA) and gene product involved in the secretion of envelope proteins in Escherichia coli

Bacteroides fragilis TMP10, which is clindamycin-erythromycin resistant (Clnr) and tetracycline resistant (Tetr), contains several plasmids and is capable of transferring drug resistance markers to suitable recipients. We were able to separate a 14.6-kilobase self-transmissible Clnr plasmid, pBFTM10, from the other plasmids of TMP10 in a tetracycline-sensitive recipient strain, B. fragilis TM4000. All Clnr transconjugants acquired an unaltered pBFTM10 and became plasmid donor strains. Transfer is proposed to occur by conjugation since it required to cell-to-cell contact of filter matings and was insensitive to DNase, but sensitive to chloroform treatment of donor cells. The efficiency of transfer of pBFTM10 in a Tets background (TM4003) was not affected by pretreatment of donor cells with clindamycin. A spontaneously occurring Clns derivative, pBFTM10 delta 1, suffered a deletion of DNA, which included a 4.4-kilobase EcoRI fragment. A complex interaction between the autonomous plasmid pBFTM10 and a tetracycline transfer element also present in strain TMP10 was observed since pretreatment of this donor with tetracycline or clindamycin resulted in a marked increase in transfer of both tetracycline and clindamycin resistance.     

A signal sequence is not required for protein export in prlA mutants of Escherichia coli.

The prlA/secY gene, which codes for an integral membrane protein component of the Escherichia coli protein export machinery, is the locus of the strongest suppressors of signal sequence mutations. We demonstrate that two exported proteins of E.coli, maltose-binding protein and alkaline phosphatase, each lacking its entire signal sequence, are exported to the periplasm in several prlA mutants. The export efficiency can be substantial; in a strain carrying the prlA4 allele, 30% of signal-sequenceless alkaline phosphatase is exported to the periplasm. Other components of the E.coli export machinery, including SecA, are required for this export. SecB is required for the export of signal-sequenceless alkaline phosphatase even though the normal export of alkaline phosphatase does not require this chaperonin. Our findings indicate that signal sequences confer speed and efficiency upon the export process, but that they are not always essential for export. Entry into the export pathway may involve components that so overlap in function that the absence of a signal sequence can be compensated for, or there may exist one or more means of entry that do not require signal sequences at all.     

Yeast gene which suppresses the defect in protein export of a secY mutant of E. coli

To find factors participating in protein translocation in yeast, we screened a yeast genomic library for genes which, when introduced into Escherichia coli, suppressed secY24, a temperature sensitive mutation of an essential integral membrane protein (SecY) required for protein export. We isolated and characterized a gene (YSY6) which improved the translocation of the OmpA protein in mutant strain IQ85(secY24). It could also suppress another mutant [rplO215(Am)], in which the level of expression of the SecY protein is decreased at high-temperature. The YSY6 gene encodes a small amphiphilic peptide consisting of 65 amino acids, which can be expressed in E. coli cells.     

Identification of the heat-inducible protein C15.4 as the groES gene product in Escherichia coli

The product of the Escherichia coli morphogenetic gene groES (mopB) was identified as the heat-inducible protein C15.4 by two-dimensional gel analysis of the products of wild-type and mutant alleles carried on the bacterial chromosome, on a hybrid plasmid, and on a transducing phage.     

Characterization of an amber mutation in the structural gene for ribosomal protein L15, which impairs the expression of the protein export gene, secY, in Escherichia coli

[This corrects the article on p. 2323 in vol. 3.].     

Characterization of an amber mutation in the structural gene for ribosomal protein L15, which impairs the expression of the protein export gene, secY, in Escherichia coli

We have previously described a temperature-sensitive mutant, ts215, which is defective in protein secretion. Complementation studies indicated that the mutation was located at the distal part of the spc ribosomal protein operon and the gene secY is required for efficient protein secretion. We now report a more complete genetic and biochemical analysis of the ts215 mutant. These studies revealed that the ts215 mutant has an amber mutation in the gene rp10 for ribosomal protein L15, which is located upstream and adjacent to secY. The amber mutation exerts a polar effect on secY causing a defect in protein secretion. These conclusions were supported by the following observations. The mutant strain carries a phi 80 prophage containing a temperature-sensitive suppressor, supFts6. The strain contains decreased amounts of L15 and is suppressible by a temperature-independent nonsense suppressor. In addition, L15 contains an extra tyrosine residue when suppressed by supF. DNA sequence analysis revealed the presence of a single base change in rp10 resulting in an amber codon at the 38th codon of L15. The mutant phenotype is complemented by a plasmid carrying only the secY gene under lac promoter control. The mutant cells complemented by secY can grow and synthesize proteins at normal rates and abundances at 42 degrees C, despite the fact that their ribosomes contain barely detectable levels of L15. These results indicate that ribosomal protein L15 is dispensable for protein synthesis and cell growth. In contrast, the decreased level of expression of the secY gene leads to defective protein secretion and defective cell growth.     

Identification of the purC gene product of Escherichia coli.

The purC region of the Escherichia coli chromosome was isolated from in vivo-derived lambda transducing bacteriophages and cloned in high-copy-number plasmids. The product of the purC gene, phosphoribosylaminoimidazolesuccinocarboxamide synthetase, was identified as a protein with an Mr of ca. 27,000. The level of the protein is increased by more than 60-fold in strains carrying the gene on a high-copy-number plasmid. Purine addition represses the enzyme level in both plasmid- and non-plasmid-containing strains.     

Identification of the Escherichia coli recN gene product as a major SOS protein.

The recA+ lexA+-dependent induction of four Escherichia coli SOS proteins was readily observed by two-dimensional gel analysis. In addition to the 38-kilodalton (kDa) RecA protein, which was induced in the greatest amounts and was readily identified, three other proteins of 115, 62, and 12 kDa were seen. The 115-kDa protein is the product of the uvrA gene, which is required for nucleotide excision repair and has previously been shown to be induced in the SOS response. The 62-kDa protein, which was induced to high intracellular levels, is the product of recN, a gene required for recBC-independent recombination. The recA and recN genes were partially derepressed in a recBC sbcB genetic background, a phenomenon which might account for the recombination proficiency of such strains. The 12-kDa protein has yet to be identified.     

Mutation that suppresses the protein export defect of the secY mutation and causes cold-sensitive growth of Escherichia coli.

A cold-sensitive mutant was isolated among temperature-resistant revertants of the secY24 mutant defective in secretion of envelope proteins across the cytoplasmic membrane at 42 degrees C. A single mutation, designated ssyA3, is responsible both for the extragenic suppression of secY and for the cold-sensitive growth. In contrast to the parental secY24 mutant, the suppressed cells do not accumulate precursors of envelope proteins at any temperatures. The cells containing the ssyA3 mutation, whether in combination with secY24 or not, show an optimal growth at 42 degrees C and a very poor growth at 30 degrees C. At the low temperature, protein synthesis is generally slowed down, probably at the step of chain elongation. The gene ssyA was mapped at a new locus between hisS and glyA on the chromosome. It is possible that the product of this gene interacts both with the protein secretion system and the protein synthesizing system.     

Expression of the pspA gene stimulates efficient protein export in Escherichia coli

Expression of several mutant forms of outer membrane protein PhoE of Escherichia coli, which are disturbed in normal biogenesis, resulted in high expression of a 26kDa protein. This 26kDa protein fractionated as a peripherally bound inner membrane protein. It appeared to be identical to a previously identified protein (PspA = phage shock protein A) of unknown function that is induced upon infection of E. coli with filamentous phages. PspA was not expressed upon synthesis of mutant PhoE proteins in a secB mutant, nor upon expression of a PhoE mutant that lacks the signal sequence, suggesting that entrance into the export pathway of prePhoE is essential for induction. PspA synthesis was also induced under other conditions that are known to block the export apparatus, i.e. in secA, secD and secF mutants when grown at their non-permissive temperature or upon induction of the synthesis of MalE-LacZ or LamB-LacZ hybrid proteins. The inducing conditions for PspA synthesis suggested a rote for this protein in export. In vivo pulse-chase experiments showed that the translocation of (mutant) prePhoE and of the precursors of other exported proteins was retarded in a pspA mutant strain. Also, in in vitro translocation assays, a role for PspA in protein transport could be demonstrated.     

A defined mutation in the protein export gene within the spc ribosomal protein operon of Escherichia coli: isolation and characterization of a new temperature-sensitive secY mutant.

We describe the properties of a temperature-sensitive mutant, ts24, of Escherichia coli. The mutant has a conditional defect in export of periplasmic and outer membrane proteins. At 42 degrees C, precursor forms of these proteins accumulate within the cell where they are protected from digestion by externally added trypsin. The accumulated precursors are secreted and processed very slowly at 42 degrees C. The mutation is complemented by expression of the wild-type secY (or prlA) gene, which has been cloned into a plasmid vector from the promoter-distal part of the spc ribosomal protein operon. The mutant has a single base change in the middle of the secY gene, which would result in the replacement of a glycine residue by aspartic acid in the protein product. These results demonstrate that the gene secY (prlA) is essential for protein translocation across the E. coli cytoplasmic membrane.     

Escherichia coli protein X is the recA gene product.

Escherichia coli protein X is known to be made in large amounts following DNA damage or inhibition of DNA replication. We have shown that it is identical to the recA gene product by partial proteolytic digestion of the radiochemically pure proteins and analysis by electrophoresis on polyacrylamide-sodium dodecyl sulfate gels.