Isolation and Characterization of an Escherichia coli Clone Overproducing Prolipoprotein Signal Peptidase*

Based on the rationale that Escherichia coli cells containing increased levels of prolipoprotein signal peptidase would be highly resistant to globomycin, a specific inhibitor of the prolipoprotein signal peptidase, we have isolated a clone from the Carbon-Clarke collection, plasmid pLC3-13, which is globomycin-resistant and contains an increased level of prolipoprotein signal peptidase activity. The plasmid pMT521, a subclone of pLC3-13 in pBR322, conferred on its host cells approximately 20 times overproduction of prolipoprotein signal peptidase and an extremely high level of resistance against globomycin. The overproduced prolipoprotein signal peptidase was completely inhibited by the presence of globomycin in the in vitro assay, and the overproduced activity was found in the cell envelope fraction. Several lines of biochemical and genetic evidence suggest that the gene contained in pLC3-13 and its derivative clones is most likely the structure gene (lsp) for prolipoprotein signal peptidase.

Based on the rationale that Escherichia coli cells containing increased levels of prolipoprotein signal peptidase would be highly resistant to globomycin, a specific inhibitor of the prolipoprotein signal peptidase, we have isolated a clone from the Carbon-Clarke collection, plasmid pLC3-13, which is globomycin-resistant and contains an increased level of prolipoprotein signal peptidase activity. The plasmid pMT521, a subclone of pLC3-13 in pBR322, conferred on its host cells approximately 20 times overproduction of prolipoprotein signal peptidase and an extremely high level of resistance against globomycin. The overproduced prolipoprotein signal peptidase was completely inhibited by the presence of globomycin in the in vitro assay, and the overproduced activity was found in the cell envelope fraction. Several lines of biochemical and genetic evidence suggest that the gene contained in pLC3-13 and its derivative clones is most likely the structure gene (Zsp) for prolipoprotein signal peptidase.
Most, if not all, outer and periplasmic proteins in Escherichia coli are first synthesized as precursor forms containing NHZ-terminal signal sequences. These signal peptides are removed by unique endopeptidase(s) called signal (or the leader) peptidase(s) either during or immediately after translocation of nascent secretory proteins across the membrane.
There is a unique group of exported proteins in bacteria which are lipoproteins containing covalently-linked lipids. They include murein lipoprotein (l), the peptidoglycan-associated lipoprotein (a), the membrane-bound form of penicillinase in Bacillus species (3,4), and the so-called new lipoproteins in the E. coli cell envelope (5).
We recently described an in vitro system for the assay of enzymes which carry out the modification and processing of prolipoprotein to form the mature lipoprotein (6,7). We have shown that (i) the processing of prolipoprotein by prolipoprotein signal peptidase requires prior modification of prolipoprotein by glyceride (6) and (ii) the prolipoprotein signal peptidase is distinct from M13 procoat protein signal pepti-* This investigation was supported by United States Public Health Service Grant GM-28811 and American Heart Association Grant 81-663. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ To whom correspondence should be addressed. dase (7). Procoat protein signal peptidase has been purified and extensively characterized by Wickner and his co-workers (8)(9)(10)(11). Although this enzyme can process the M13 procoat protein as well as several other precursor forms of outer membrane and periplasmic proteins, it is unable to process prolipoprotein. The processing of prolipoproteins is specifically inhibited by a cyclic antibiotic globomycin, and the accumulation of modified prolipoproteins occurs in globomycin-treated cells in vivo (12). Furthermore, prolipoprotein signal peptidase activity is completely inhibited by globomycin in in. vitro (6). We reasoned therefore, that E. coli variants containing increased levels of prolipoprotein signal peptidase can be selected by virtue of their increased resistance to globomycin. Indeed, we have isolated one globomycin-resistant mutant of E. coli K12 (strain SM31-2B4) which was found to contain 3-4 times more prolipoprotein signal peptidase activity in the crude extracts.' We therefore employed globomycin selection in an attempt to isolate E. coli strains containing multiple copies of prolipoprotein signal peptidase gene encoded on plasmids.
In this paper, we describe the isolation of a prolipoprotein signal peptidase overproducing clone in the Carbon-Clarke collection (13), the subcloning of the prolipoprotein signal peptidase gene ( k p ) into pBR322, and preliminary characterization of E. coli strains containing the amplified k p gene.
Screening of Globomycin-resistant Clones among the Carbon Collection-Individual clones of the Carbon collection (generous gift of H. Tabor, National Institutes of Health) were grown in 50 p1 of L broth containing colicin E l in microtiter plates at 37 "C overnight. Approximately lo6 cells of overnight culture were inoculated into 50 ~1 of L broth containing 50 pg/ml of globomycin and incubated overnight at 37 "C. Globomycin-resistant clones were further screened by the in vitro prolipoprotein signal peptidase assay described below.
In Vitro Prolipoprotein Signal Peptidase Assay-E. coli cells grown in 300 pl of L broth containing colicin E l were harvested in a microfuge and suspended in 30 pl of lysis buffer (0.1% lysozyme, 10 mM EDTA, 20% sucrose, 50 mM Tris-HC1 buffer (pH 8.0), and 10 pg each of DNase and RNase/ml). After a 15-min incubation at 37 "C, 270 p1 of 50 mM Tris-HC1 buffer (pH 7.4) containing 0.25% Nikkol was added and the tube was mixed by brief vortexing and bath sonication. The crude homogenate (7 pl) was then added to 3 pl of a reaction mixture containing glyceride-modified prolipoprotein (20,000 cpm) (prepared according to Ref. 7), 0.25% Nikkol, 50 mM Tris-HCl buffer (pH 7.4) and 0.25% 0-mercaptoethanol and the incubation was continued for 60 min at 37 "C. Termination of enzyme reaction, gel electrophoresis, and calculation of prolipoprotein signal peptidase activity were described previously (7).
Plasmid Preparation and Subcloning of lsp Gene-Plasmid DNA purified by a NaOH-sodium dodecyl sulfate rapid method (14) was utilized for plasmid screening, restriction endonuclease analysis, and transformation. The procedures described by Maniatis

RESULTS
Strains Carrying pLC3-13 Ouerproduce Prolipoprotein Signal Peptidase-Since globomycin is a specific inhibitor of prolipoprotein signal peptidase, we assumed that overproduction of prolipoprotein signal peptidase would overcome the effect of globomycin and that E. coli strains with an amplified Isp gene would be globmycin-resistant. Among the approximately 2200 clones of the Carbon collection, the clone carrying plasmid pLC3-13 was found to be globomycin-resistant (Fig. 1A, lane I). This was confirmed by the in uitro prolipoprotein signal peptidase assay which revealed that this clone, JA200 (pLC3-13), indeed contained 4-5 times higher prolipoprotein signal peptidase activity (Fig. 1B, lane 1 ) as compared with the parental strain (Fig. lB, lane 4). When strain JA200 was cured of the pLC3-13 plasmid, both globomycin sensitivity and prolipoprotein signal peptidase activity were restored to normal levels. (Fig. l,A and B, lane 3). Other E. coli strains transformed by plasmid pLC3-13 became globomycin-resistant and contained increased levels of prolipoprotein signal peptidase (Fig. 1, A and B, lanes 2 and 5).
Subcloning of Prolipoprotein Signal Peptidase Gene into Plasmid pBR322"The restriction map of pLC3-13 is shown in Fig. 2. Plasmid pLC3-13 DNA was digested by restriction endonuclease EcoRV yielding two fragments (10.4 and 9.0 kb'). EcoRV-digested pLC3-13 DNA was ligated with pBR322 DNA which had been digested with the same enzyme, and the ligated DNA was used to transform strain SM31. SM31 was used as the host for transformation with subcloned plasmids because of the high efficiency of transformation in this strain. Amp'Tet" transformants were selected and further screened for resistance to globomycin (50 pglml). One of these clones (containing plasmid pMT503) was found to have an insertion of the larger fragment (10.4 kb) of pLC3-13 into the EcoRV site of pBR322 (Fig. 2). The E. coli strain containing pMT503 showed the expected phenotypes, Amp', Tet*, and Gmb' and contained increased levels of prolipoprotein signal peptidase activity as determined by the in uitro assay. The plasmid pMT503 DNA was totally digested by NruI and religated by T4 DNA ligase. Although the religation at the NruI site was incomplete, about half of the AmpTet"Gbm' transformants obtained by transformation of strain SM31 with the religated DNA were found to contain plasmid DNA of a smaller size (8.5 kb) than that of pMT503. This smaller plasmid, designated pMT521, resulted from the deletion of 6 kb of the ColEl region and 0.8 kb of pBR322 DNA from pMT503 (Fig. 2). The strains carrying pMT521 contained approximately 20 times higher prolipoprotein signal peptidase activity than the parental strains (data not shown). The prolipoprotein signal peptidase activity overproduced in strains carrying pMT521 was completely inhibited by globomycin in the in uitro assay (Fig. 3). Furthermore, all prolipo-* The abbreviations used are: kb, kilobases; PLP, prolipoprotein; LP, lipoprotein; Amp', ampicillin-resistant; Tet", tetracycline-sensitive; Gbm', globomycin-resistant. protein signal peptidase activity in JE5505 (pMT521) was found in the cell envelope fraction (Fig. 4) which corresponds to the subcellular localization of prolipoprotein signal peptidase in wild type cells (16).
pMT503 DNA was totally digested by BanHI and religated by T4 DNA ligase resulting in the construction of plasmid pMT522 which contains further deletion of 2 kb of E. coli DNA (Fig. 2). The deletion of the 2-kb E. coli DNA fragment from pMT503 resulted in the loss of both the globomycinresistance phenotype and increased levels of prolipoprotein signal peptidase. It is clear, therefore, that this 2-kb region must contain the promoter and/or part of the prolipoprotein signal peptidase structural gene.   pMT52I"Plasmid pMT521 was used to transform E. coli strains E609, E610 ( n l p A), JE5505 (lpo), and SM31-2B4 (a spontaneous globomycin-resistant mutant of strain SM31).' All transformants were extremely resistant to globomycin (>300 pg/ml) as compared to their parental strains regardless of the particular genetic background of these strains ( Table   I).
The murein lipoprotein structural gene is deleted in strain JE5505 (17). Accordingly, this strain is globomycin-resistant (120 pg/ml) since the major cause of globomycin-mediated lethality is the accumulation of modified murein prolipoprotein in treated cells (12). However, while the murein lipoprotein is the most abundant lipoprotein in E. coli and is therefore the primary target of globomycin action, other relatively minor lipoprotein species also accumulate as their glyceridemodified precursor forms in globomycin-treated cells (5). The fact that JE5505 (pMT521) is much more resistant to globomycin than JE5505 strongly suggests that these secondary target(s) of globomycin action are responsible for the eventual globomycin-mediated lethality of JE5505. Furthermore, it provides further support that prolipoprotein signal peptidase is responsible for the processing of all these prolipoproteins. The same explanation can be applied to the results obtained with strains E610 (mlpA) (18) and E610 (pMT521).
Strain SM31-2B4 was selected by globomycin treatment of SM31, and it presumably contained a spontaneous chromosomal mutation resulting in an increase level of prolipoprotein signal peptidase activity.' Because of the apparent overproduction of prolipoprotein signal peptidase in mutant SM31-2B4 as compared to strain SM31, it is globomycin-resistant (120 pglml). Introduction of pMT521 into SM31-2B4 resulted in further overproduction of prolipoprotein signal peptidase and increased resistance to globomycin (>300 pglml).

DISCUSSION
We have identified among the Carbon-Clarke collection of E. coli genomic fragments a plasmid pLC3-13 which confers upon its host cell increased levels of prolipoprotein signal peptidase activity and a concomitant increase in globomycin resistance. Several lines of evidence suggest that the gene contained in pLC3-13 and its derivative clones is most likely the structural gene for prolipoprotein signal peptidase. The evidence supporting this conclusion is as follows. 1) The increased prolipoprotein signal peptidase activity in strains containing pMT521 is sensitive to globomycin in uitro (Fig.  3). 2) The gene product is a membrane-bound enzyme (Fig.  4). 3) The prolipoprotein signal peptidase activity in strains containing the cloned gene has the same mobility in nondenaturing gel as the enzyme present in the parental strain (data not shown). These properties are the same as observed for the prolipoprotein signal peptidase present in strains not harboring the plasmid (6,16). 4) The crude extract of strains containing pMT521 does not contain any activity which would inactivate globomycin irreversibly (data not shown). 5) Genetic evidence indicates that the E. coli gene(s) in pMT521 is located approximately a t 0.5 min of the E. coli map." This is nearly the same location as the gene altered in the ts signal peptidase mutant reported recently ( 1 9~~ 6) Strains harboring an F'-plasmid (F'101) containing E. coli genes between thr and leu loci were found to contain 1.7 times prolipoprotein signal peptidase activity, as compared to the strains cured for the F'101 plasmid. The apparent gene dosage-prolipoprotein signal peptidase activity relationship observed among strains containing F'-plasmid, ColEl-plasmid, and pBR322 derivatives with the lsp gene strongly suggests that the cloned gene is indeed the structural gene for prolipoprotein signal peptidase.
The availability of this plasmid will now permit a detailed study of the genetic organization of the lsp gene. In addition, the subcloning of the lsp gene into a runaway plasmid would facilitate the biochemical and physiological studies of this unique enzyme.