Identification of Prolipoprotein Signal Peptidase and Genomic Organization of the Zsp Gene in Escherichia coli*

The product of the lsp gene of Escherichia coli, i.e. the prolipoprotein signal peptidase, was identified by both in vivo pulse labeling experiments using a high expression XPL promoter vector and by an in vitro transcription/translation coupled system. The molecu- lar weight of prolipoprotein signal peptidase was estimated to be approximately 18,000 by its mobility on polyacrylamide gel electrophoresis and was found to be the same as that of SPase I1 purified from the wild-type cells. Analysis of SPase I1 activities in strains containing various subclones, deletion derivatives gen- erated from plasmid pMT521, and analysis of protein products in a strain harboring an ileS-lsp-fused gene indicated that ileS and lsp genes are transcribed on the same mRNA. This was further supported by the obser- vation that Tn5 insertions in the ileS gene resulted in a reduced expression of the lsp gene. In addition to an upstream promoter shared by the ileS and lsp genes, these analyses also revealed the presence of an internal promoter for the lsp gene within the coding region of the ileS gene. Escherichia coli possesses at least two distinct signal pep-tidases, the M13 procoat protein signal peptidase character-ized by Wickner and his co-workers (1, 2) and the prolipoprotein signal peptidase (SPase 11’)

' The abbreviations used are: SPase 11, prolipoprotein signal peptidase; P,,, promoter for tetracycline resistance gene in pBR322; SDS, sodium dodecyl sulfate; bp, base pair(s); kb, kilobase pairs. the functions of these genes are related to warrant a coordinate regulation.
In this paper, we describe the identification of SPase I1 molecules both by pulse labeling experiments in vivo and by employing an in vitro transcription/translation coupled system. We also present evidence that the ileS and k p genes are indeed transcribed on the same mRNA, and we report the existence of an internal promoter for the kp gene which resides in the StuI-HincII region of the ileS coding sequence.
Subcloning of the kp Gene under the XPL Promoter-The 0.8-kb RsaI-RsaI fragment of pMT521, containing the entire lsp gene and 170 bp of its 5'-and 140 bp of its 3"flanking regions, respectively, was isolated and inserted into the HpaI site of the plasmid pPLX (P-L Biochemicals), which contains the XPL promoter upstream of the HpaI site, to obtain plasmid pMT401. Strain N4830 was transformed with pMT401 and was induced for the lsp gene expression by shifting the growth temperature from 30 to 42 "C. I n Vitro Transcription/Translation Coupled System-An in vitro transcription/translation coupled system using the S-30 fraction of E. coli (Codon, Houston, T X ) was carried out according to the procedure described by Zubay (13). The supercoiled form of plasmid DNA was prepared by CsCl density gradient centrifugation and used as the template (2 pg of DNA/25 ~1 of the reaction mixture). After 40 min of incubation at 37 "C, in vitro protein synthesis was terminated by the addition of 10% trichloroacetic acid.
Construction of a n ileS-lsp Hybrid Gene-The EcoRV-XbaI fragment from pMT521 ( Fig. 1) containing the ileS-lsp genes was cloned into the M13 mplO phage vector at the SmaI-XbaI site. An oligodeoxyribonucleotide partially complementary to the junction region was synthesized using a model Sam One Automated Synthesizer from Biosearch Inc. (San Rafael, CA). This oligomer has the sequence 5'-GATTGACTCGGCGCAAACTTAC-3'; it was used to create a sequence alteration at the ileS-kp junction by the method of Zoller and Smith (14). The phage carrying the desired mutation was identified by hybridization, and the nucleotide sequence of the mutant was confirmed by direct sequence analysis using the dideoxyribonucleotide chain-termination method of Sanger et al. (15). This site-directed deletion mutagenesis removed the termination codon of ileS and the putative initiation codon of lsp. The resulting clone has the following sequence at the junction: 5'-CGTAAGTTTGCGCCGAGT-3', encoding Arg-Lys-Phe-Ala-Pro-Ser, which would direct the synthesis of an IleS-Lsp hybrid protein by replacing the fMet of the Lsp protein with Pro and linking the COOH-terminal Ala of the IleS protein to the second residue Ser of the Lsp protein. The 20-kb mutation-containing fragment (BamHI-XbaI) from the RF phage DNA was excised and cloned back into pMT521 in place of the corresponding wild-type sequence to generate the clone containing the fused gene.
Assay of Prolipoprotein Signal Peptidase and Globomycin Sensitivity of E. coli Cells-Prolipoprotein signal peptidase activity was assayed as described previously, using total cell homogenate prepared by lysozyme-EDTA-Nikkol treatment (4). The globomycin sensitivity of E. coli cells harboring various plasmids was determined in microtiter plates as described previously (16).
Protein Analyses of Total Cell Extracts-Overnight cultures in L broth (1 ml) were harvested in microfuge tubes. Cell pellets were suspended in Laemmli's solubilizing buffer (17) and boiled for 5 min to solubilize total cell proteins. After centrifugation to remove peptidoglycan sacculus, aliquots of samples were applied to a SDS-polyacrylamide gel (17) for analyses of protein patterns.  (18). Plasmid pMT521 was obtained by subcloning a 4.5-kb DNA fragment of pLC3-13 into pBR322, and its physical map is shown in Fig. 1. Genetic mapping (6, 10) has indicated that the k p gene is located near dapB at 0.5-0.6 min of the E coli map. A transducing phage XdupB2 containing E. coli dupB region has been isolated (19), and several subclones of XdapB2 into pBR322 have been previously constructed (Fig. 1, Refs. 20, 21). A comparison of the physical maps of XdupB2, pGM21, and pMT521 suggests that pMT521 contains the ileS (the structural gene for isoleucyl-tRNA synthetase) as well as the k p gene. Our DNA sequence data of pMT521 have confirmed that the ileS gene is located immediately upstream of the k p gene (7).

Identification
The gene products encoded by several plasmids were examined by the in vitro transcription/translation coupled system described by Zubay (13). Plasmid pMT522 is a deletion derivative of plasmid pMT521 (see the following section). As shown in lune 3 of  been reported to be 114,000 (22), which is similar to that of the larger polypeptide detected in lunes 3 and 6. The molecular weight of the smaller band was estimated to be 18,000, which is similar to the molecular weight of the k p gene product deduced from DNA sequencing data (7). Several bands appeared in the high molecular weight range in lune 3 of Fig For the identification of the amplified ileS and k p gene products in vivo, the crude extracts of E. coli cells harboring plasmid pMT521 were analyzed by SDS-polyacrylamide gel electrophoresis. As shown in lanes 2 and 4 of Fig. 3, a polypeptide with M, of 110,000 was amplified in E. coli cells harboring plasmid pMT521. The molecular weight of this polypeptide is in good agreement with that reported for isoleucyl-tRNA synthetase. An elevated level of isoleucyl-tRNA synthetase activity was also detected in cells containing pMT521 (Table I). At the present time, the reason why a greater extent of accumulation of the IleS protein band in strain JE5505 than that in strain E609 is not clear. On the other hand, overproduction of the k p gene product was not detected by Coomassie Brilliant Blue staining of the gel in either JE5505 or E609 cells carrying plasmid pMT521. In order to obtain higher amplification of the k p gene product, we subcloned the RsaI-RsaI fragment (0.8 kb) containing the k p gene (see Fig. 6) into the HpuI site of plasmid pPLX (P-L Biochemicals) which contains the XPL promoter upstream of the HpuI site. Plasmid pMT401 was obtained and was used to transform strain N4830 which contains the temperaturesensitive allele of the X repressor gene (CIs7). After a 4-h  a Cells were grown in M9 minimal medium containing 0.4% glycerol overnight. Cells were harvested, resuspended in 10 mM Tris-HC1 buffer (pH 7.8) containing 15 mM Mg acetate, 60 mM KC1, 1 mM dithiothreitol, and 0.1 mM phenylmethylsulfonyl fluoride, and disrupted by sonication. The crude extract was centrifuged at 200,000 X g for 2 h at 4 "C, and the supernatant fraction was used for the assay of Ile-tRNA synthetase activity. The reaction mixture containing 100 mM Tris-HC1 buffer (pH 8.0), 5 mM MgC12, 2.5 mM ATP, 20 mM dithiothreitol, 10 mM KC1,500 pg of tRNA, and 20 nM [3H]isoleucine was incubated with the crude extract at 30 "C for 10 min. The tRNA fraction was collected on a Millipore filter following precipitation with 10% trichloroacetic acid.
induction of the cloned k p gene by shifting the culture temperature from 30 to 42 "C, SPase I1 activity was amplified by more than 100-fold over the control culture of N4830 (pPLX) which does not contain the insert (data not shown). Even under this highly induced condition, the SPase I1 polypeptide could not be detected in the crude extract by Coomassie Brilliant Blue staining of SDS-polyacrylamide gel. To identify the k p gene product in vivo by an alternative method, we labeled N4830(pMT401) cells with [35S]methionine for 15 min after a 1-hr induction of the kp gene on pMT401 under the control of XPL promoter at 42 "C. The crude membrane fraction was prepared and extracted with Tris acetate buffer (pH 4.0), Triton X-100 solution at 4 "C overnight. This extraction procedure has been reported to solubilize SPase I1 activity preferentially from the membrane fraction of E. coli (23). As  plasmid pPLX ( l a n e 2). The mobility of this band is exactly the same as that of a partially purified SPase I1 preparation from the wild-type cells (lane 6, shown by arrow) provided by Dr. Paul Ray (Wellcome Research Laboratories, Research Triangle Park, NC). When the crude membrane was extracted with Tris-HC1 buffer (pH 8.0), Triton X-100, no obvious difference in band pattern was observed between the membrane prepared from the kip-containing clone (lane 3) and that of the control (lane 4). This is likely due to the masking of the SPase I1 by the high background of other proteins extracted under this condition which co-migrated to the M, = 18,000 region of the gel.
Effects of Various Deletions in pMT521 on the Expression of the kp Gene-In order to study the genomic organization of the k p gene and its flanking sequence cloned on pMT521, we constructed several deletion derivatives from pMT521 (Fig. 5). Both the SPase I1 activities and the maximum globomycin concentrations which allowed cells harboring these plasmids to grow were determined. As shown in Table  I1 and Fig. 5, deletion of DNA flanking the single XbaI site (pMT522 and pSYC881) completely abolished the amplification of SPase I1 activity. This observation suggests that the kp gene is located around the XbaI site and is in agreement with the genetic mapping data (6) and the DNA sequence data  . Surprisingly, those plasmids with deletion extending into the promoter of tetracycline resistance gene (Pkt) in the pBR322 portion of these plasmids (pSYC887, 888, 889, and 890) lost most of the amplified expression of the Isp gene but not completely, i.e. they contained about three times higher SPase I1 activity than that of pBR322-harboring cells. This residual level of Isp gene expression appears to depend on a weak kp promoter which is located within the ileS gene (discussed below). These results indicate that the P,, located on pMT521 functions as a major promoter for the cloned k p gene, which is separated by the entire 3-kb length of ileS gene.
The deletion of the EcoRV-HpaI region (pMTlO3) still contained about 60% Isp expression as in pMT521, which is about the same level of k p expression exhibited in pMTlOl and pMTlO2. As documented in the accompanying paper (24), there is an open reading frame of 936 bp upstream of the ileS gene (designated gene x ) ; the EcoRV site in pMT521 was located at about 400 bp upstream from the 3'-end of this gene. It should be noted that the HpaI-StuI region appears to have a weak stimulation effect on the expression of the Isp gene.
The assay of globomycin resistance of E. coli cells provides a very sensitive measurement of a small difference in Isp expression, because this assay is based on cumulative cell growth. With increasing length in deletion between HpaI and StuI (compare SPase I1 activities between pMT103 and pMT104 or globomycin sensitivities among pSYC887, pSYC888, pSYC889, and pSYC890), cells harboring these smaller plasmids had become increasingly more sensitive towards globomycin. mRNA for ikS and Isp Genes Are on the Same Tramcript- We constructed the plasmid pMT521-7 which contains a deletion between the EcoRI and EcoRV sites (the P,, region) of pMT521 (Fig. 6). Cells harboring the plasmid contained only 15% of SPase I1 activity as compared to that in pMT521, indicating that P, was functioning as a major promoter for the cloned Isp gene in pMT521. This result is consistent with the DNA sequencing data (7)   Pt,, and the k p gene in pMT521 significantly reduced k p expression.
Detection of an Internal Promoter for the k p Gene within the ileS Gene-The plasmid pSYC890 is deleted for P,, together with about 5/6 of the iles gene (Fig. 5). The SPase I1 activity in E. coli cells harboring this plasmid was still about three times higher than that found in the control cells (Table   11). In addition, we have reported previously that E. coli cells harboring plasmid pSYC931 which contains the StuI-AccI fragment of pMT521 at the SmaI site of plasmid pUC8 overproduce SPase I1 activity (11,667 units/mg protein) in the absence of isopropyl-P-D-thiogalactopyranoside as compared to 25,000 units/mg protein in its presence (7). The latter observation could be explained either by the existence of an internal promoter in the StuI-AccI fragment, or by an incomplete repression of the lac-operator. On the other hand, when the same fragment (StuI-AccI) was inserted into the same site of plasmid pUC9 so that the direction of tanscription is opposite to that of the lac promoter in the vector, the resulting plasmid overproduced SPase I1 activity to similar extents, 10,681 or 10,352 units/mg protein with or without isopropyl-@-D-thiogalactopyranoside induction, respectively. These results strongly suggest the existence of an internal promoter within the COOH-terminal '/6 of the iles gene.
In order to ascertain this promoter activity for the k p gene expression, we subcloned various lengths of DNA fragments flanking the k p gene into the pBR322 vector devoid of the upstream promoter of the tetracycline resistance gene (Fig.  6). Initially, the EcoRI-StuI region was removed from pMT521 (see Fig. 6) which generated pMT521-20. At the junction of EcoRI-StuI site of pMT521-20, a new EcoRI site was generated. We replaced the EcoRI-XbaI fragment with the HincII-XbaI and ScaI-XbaI fragments containing the k p gene, and obtained pMT521-18 and -16, respectively. In these constructions, the P, , in pBR322 had been removed, and the expression of downstream gene should depend entirely on the activity of the promoter located within the inserted fragment. Plasmid pMT521-20 confers about two times higher SPase I1 activity on the host cells than the control experiment (Fig.  7). On the other hand, cells harboring pMT521-18 and pMT521-16 did not exhibit enhanced SPase I1 activities, indicating that there exists an internal promoter for the kp gene and that it resides between the StuI and Hind1 region located about 400 to 200 bp upstream from the ileS stop codon.
Construction and Expression of an ileS-kp Fusion Gene-The DNA sequencing data have demonstrated that the stop codon of the ileS gene overlaps with the initiation codon of the k p gene. The junction between the ileS and the kp genes in pMT521 was changed from CTGATG to GCCG, which created a fusion gene of ileS-kp (see "Experimental Procedures"). The amino acid sequence encoded by the junction region of this hybrid gene was changed from (1leS)-Ala-Stop-Met-Ser-(Lsp) to (1leS)-Ala-Pro-Ser-(Lsp). Cells harboring this plasmid, pSYC1009, accumulated this hybrid protein in the membrane fraction (Fig. 8, lane 5), in contrast to accumulation of the native IleS protein in the soluble fraction of extracts prepared from cells harboring the pMT521 plasmid (Fig. 8, lane 2). MM294(pSYC1009) cells exhibited 25% of SPase I1 activity as compared to MM294(pMT521) cells and showed increased resistance to globomycin (data not shown).
These data further indicate that the ileS and k p genes are cotranscribed into a single polycistronic mRNA.

DISCUSSION
Recently, a second signal peptidase, prolipoprotein signal peptidase, was found.in E. coli and extensively studied (3)(4)(5)(6)(7)(8)(9)(10)(11). Since the amount of this enzyme in the wild-type cells is extremely low and the activity is relatively unstable, the identification of the SPase I1 protein has been very difficult. This difficulty has been overcome by extensive purification of this enzyme with the use of stabilizing agents such as sulfhydryl compounds and glycerol (23) and by the cloning of the k p gene (4) into a high expression vector such as the XPL promoter vector, as reported in this paper. By both in vivo and in vitro studies using pMT401 and pMT521, respectively, we have identified a 18,000-Da polypeptide as the subunit for SPase 11. The mobility of overproduced in vivo product in cells harboring pMT401 is the same as that of partially purified SPase I1 from the wild-type cells and also coincides with the predicted molecular weight of SPase I1 based on DNA sequence data.
SPase I1 is an essential gene, and the regulation of its expression poses an interesting problem. The first unexpected finding comes from the DNA sequencing data which indicate t- an overlap between the initiation codon of the k p gene and the stop codon of the upstream ileS gene. This observation suggests the possibility that ileS and kp genes may share a common promoter on the chromosome. In this paper, we have clearly demonstrated that the Pet of pBR322 functions as a common promoter for the expression of the cloned ileS and lsp genes in pMT521 by analysis of the deletion derivatives (Fig. 6) and Tn5 insertion mutations (Fig. 5) of pMT521. Further evidence showing that the native promoter for the ileS-kp transcriptional unit is located upstream of ileS is presented in the accompanying paper (24). In addition to this promoter common for ileS and kp, we identified a weak internal promoter for the kp gene located 200-400 bp upstream of the ileS stop codon. We propose, therefore, that the transcription for the k p gene may be initiated at least from two sites in vivo, one upstream of the gene x and the ileS gene (24) and the other from the distal portion of the ileS structural gene. Ribosomes translating the large polycistronic mRNA coding for x, ileS, and k p gene products may continue through the ileS-kp junction to the end of kp or beyond. In this case, it might not need a good Shine/Dalgarno sequence (25) for an efficient initiation of translation of the k p coding sequence. After completing the translation of the ileS coding sequence, ribosomes can immediately initiate the translation of the adjacent k p sequence at its AUG codon. On the other hand, for the smaller monocistronic mRNA initiated within the distal portion of the ileS gene, a Shine/Dalgarno sequence would be required for translation initiation because the k p coding sequence is preceded by a nontranslated sequence. At -13 to -6 bp from the initiation codon of k p is found the GTAAGTTT sequence which is complementary to the 3'-end of the 16 S rRNA of E. coli (25); this may serve as the Shine/ Dalgarno sequence for the kp gene. The DNA region within the ileS structure gene, i.e. the HpaI-StuI region, appears to be required for the maximal expression of the kp gene (Table  11), suggesting the possible existence of other weak promoter(s) within the ileS gene. The physiological significance of coordinated expression of ileS and kp genes by forming a single transcript and the presence of internal promoter(s) of kp in the ileS gene remain obscure.

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The synthesis of a hybrid protein of the expected molecular weight encoded by the ileS-kp fused gene provides further evidence for the cotranscriptional expression of the ileS and k p genes. In addition, it supports the conclusion based on the nucleotide sequence of the kp gene that SPase I1 polypeptide does not contain a functional signal peptide at its NHp terminus. Otherwise, one might have expected the removal of the IleS portion of the hybrid protein along with the putative signal peptide during insertion of the hybrid protein into the inner membrane. The hybrid protein is found in the membrane fraction of the cell extract as the native SPase 11.
Apparently, the localization of the hybrid protein, which contains an extra 111,000-Da polypeptide amino-terminal to SPase 11, is determined entirely by the sequence in the SPase I1 portion at the COOH terminus of the hybrid protein.
Furthermore, this result indicates that the abilities of the SPase I1 domains in the hybrid protein to assemble into the membrane and to function enzymatically as SPase I1 have not been severely affected by the sequence alteration at its NH, terminus.