JOURNAL OF BACTERIOLOGY, Feb. 1993, p. 1032-10370021-9193/93/041032-06$02.00/0Copyright X 1993, American Society for Microbiology

Vol. 175, No. 4

Molecular Cloning, Sequencing, and Mapping of the Gene EncodingProtease I and Characterization of Proteinase andProteinase-Defective Escherichia coli Mutants

SHIGEYUKI ICHIHARA,l* YUKARI MATSUBARA,1 CHIE KATO,1 KAZUHITO AKASAKA,1AND SHOJI MIZUSHIMA2

Laboratory ofMicrobiology, School ofAgriculture, Nagoya University, Chikusa-ku, Nagoya 464-1,1 andInstitute ofApplied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan

Received 28 September 1992/Accepted 7 December 1992

Clones carrying the gene encoding a proteinase were isolated from Clarke and Carbon's collection, using achromogenic substrate, N-benzyloxycarbonyl-L-phenylalanine 13-naphthyl ester. The three clones isolated,pLC6-33, pLC13-1, and pLC36-46, shared the same chromosomal DNA region. A 0.9-kb Sau3AI fragmentwithin this region was found to be responsible for the overproduction of the proteinase, and the nucleotidesequence of the region was then determined. The proteinase was purified to homogeneity from the solublefraction of an overproducing strain possessing the cloned gene. N-terminal amino acid sequencing of thepurified protein revealed that the cloned gene is the structural gene for the protein, with the protein beingsynthesized in precursor form with a signal peptide. On the basis of its molecular mass (20 kDa), periplasmiclocalization, and substrate specificity, we conclude this protein to be protease I. By using the gene cloned on aplasmid, a deletion mutant was constructed in which the gene was replaced by the kanamycin resistance gene(Kmr) on the chromosome. The Kmr gene was mapped at 11.8 min, the gene order being dnaZ.adk-ushKmr_purE, which is consistent with the map position of apeA, the gene encoding protease I in Salmonellatyphimurium. Therefore, the gene was named apeA. Deletion of the apeA gene, either with or without deletionof other proteinases (protease IV and aminopeptidase N), did not have any effect on cell growth in the variousmedia tested.

There are a number of proteolytic processes in Esche-richia coli. They are involved in cellular functions such ascatabolism of abnormal proteins (8), protein secretion (26),and protein turnover during starvation (21). To understandthese processes, it is necessary to define the proteasesinvolved. More than 30 proteinase activities in E. coli havebeen reported (23). Mutants defective in any of these pro-teinases survive, except those defective in signal peptidases(5, 10, 44), methionine aminopeptidase (3), and DegP atelevated temperature (18, 37), suggesting that the role of oneproteinase can be taken over by another proteinase(s). Thismakes investigation of specific proteolytic processes diffi-cult. The proteases responsible for signal peptide digestionare one example. During the export of secretory proteinsacross the cytoplasmic membrane, the signal peptide ofprecursor proteins undergoes two successive proteolyticattacks, cleavage of the signal peptide by signal peptidaseand digestion of the cleaved signal peptide by signal peptidepeptidases. From an in vitro experiment, we have reportedthat protease IV is responsible for the activity of signalpeptide peptidase (11, 12). Although digestion of the cleavedsignal peptide was considerably slower in a protease IVdeletion mutant, it was still significant, suggesting thatanother envelope proteinase(s) also participates in signalpeptide digestion (29, 39).Of more than 30 known proteinases in E. coli, 10 have

been demonstrated to be localized in the cell surface, whichis composed of the outer membrane, periplasmic space, andcytoplasmic membrane. These include protease VII (ompT;13 min) (38) of the outer membrane; protease I (33, 34),

* Corresponding author.

protease III (ptr; 60 min) (6), and DegP (degP or htrA; 3.5min) (18, 37) of the periplasm; signal peptidase I (lep; 55 min)(42), signal peptidase II (IspA; 1 min) (14, 45), protease IV(signal peptide peptidase; sppA; 38 min) (13), alkaline phos-phatase isozyme conversion enzyme (iap; 59 min) (15), andprotease VI (35) of the cytoplasmic membrane; and proteaseV, the localization of which remains unclear (31). The genesencoding these proteinases and their map positions are givenin parentheses. The genes whose loci are known have beencloned and sequenced, and the gene products have beencharacterized. Genes coding for three proteinases, proteasesI, V, and VI, have not been cloned; hence, the properties ofthese proteinases have not been well characterized.We report the cloning, sequencing, and mapping of the

gene encoding protease I and some properties of the geneproduct. We also constructed deletion mutants and charac-terized their phenotypes.

MATERIALS AND METHODS

Bacteria and plasmids. The bacterial strains and plasmidsused are listed in Table 1.

Media. LB, M9, and S2 (27) media were used. For solidcultivation, these media were supplemented with 1.5% agar.When required, the following chemicals were added to themedia: amino acids (25 jxg/ml each), nucleotides (25 pg/mleach), thiamine-HCl (0.1 pug/ml), ampicillin (50 pg/ml), tet-racycline (15 pg/ml), and kanamycin (25 pg/ml).

Screening of protease I-overproducing clones in Clarke andCarbon's collection. The method of Ichihara et al. for screen-ing protease IV-overproducing clones (13) was employed,using N-benzyloxycarbonyl-L-phenylalanine P-nitrophenylester (Cbz-Phe-ONap) as the substrate instead of N-benzyl-

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TABLE 1. Bacterial strains, plasmids, and bacteriophage used

Strains, plasmids, Genotype Source or referenceand phage

StrainsJM103 A(lac-pro) thi strA supE endA sbcB hsdR/F' traD36 proAB lacIqZ15 20MH19 purE rpsL 13S121 pepN rpsL 13SI532 pepN apeA(Kmr) rpsL This studyCSH62 HfrH, thi CGSCaCK50 HfrH, thi apeA(Kmr); as CSH62, but apeA(Kmr) This studyN43 ara-14 lac-85 acrAl supE44 rpsL197 malA1 xyl-5 galK2 mtl-1 CGSCAX727 dnaZ thi lac rps CGSCCV2 tonA22 phoA8 adk-2 ompF672 fadL 701 relWl glpR2 flpD3 pit-10 spoT1 T2r CGSCCV2-4 tonA22 phoA8 ush-2 ompF672 fadL701 reLU1 glpR2 flpD3 pit-10 spoT1 T2r CGSCW4626Phe F-, purE phe thp lac-85 gal-2 mal mtl xyl-2 rpsL Laboratory stockCK67 As CV2-4, but apeA(Kmr) This studyCK82 As W4626Phe, but apeA(Kmr) This studyCK84 As W4626Phe, but adk-2 apeA(Kmr) This studyCK85 As W4626Phe, but adk-2 purE+ This studyKA36 pepNsppA(Kmr) apeA (Tcr) rpsL This study

PlasmidspLC6-33 ColElr; cloned gene apeA 4pLC13-1 ColEjr; cloned gene apeA 4pLC36-46 ColEir; cloned gene apeA 4pUC19 Apr; cloned gene, truncated lac(lacPO, Z') 20pYM008 Apr; cloned gene apeA (2.5-kb Sall fragment) This studypYM028 Apr; cloned gene apeA (0.9-kb Sau3AI fragment) This studypYM026 Apr; cloned gene apeA (1.0-kb AluI fragment) This studypYM025 As pYM026, but inserted in opposite direction This studypSI029 Apr Kmr; ori(Ts) 39pYM023 Apr ori(Ts); cloned gene apeA(KMr) This studypSI030" Apr Tcr; ori(Ts) This study

BacteriophagePlkc Laboratory stock

a CGSC, E. coli Genetic Stock Center." As pSI029, but the Kmr gene was replaced with the 1.4-kb EcoRI-AvaI fragment (tetracycline resistance gene, Tcr) of pBR322.

oxycarbonyl-L-valine 0-naphthyl ester (Cbz-Val-ONap).Briefly, individual clones from Clarke and Carbon's collec-tion (4) were conjugated with S121 in a 96-well plastic plate,and the conjugants were solubilized with 1% Triton X-100after treatment with lysozyme-EDTA. Cbz-Phe-ONap-hy-drolyzing activity was visualized by coupling released naph-thol with Fast Garnet GBC.DNA techniques. The following procedures were carried

out by standard methods described by Maniatis et al. (20):preparation of plasmid DNA, restriction endonuclease diges-tion, agarose gel electrophoresis, DNA ligation, and bacte-rial transformation. DNA sequencing was carried out by thedideoxy chain termination method (36). Southern hybridiza-tion was carried out by using a nonradioactive DNA Label-ing and Detection Kit (Boehringer Mannheim).

Purification of protease I. Protease I was purified tohomogeneity from E. coli JM103 carrying pYM008 as de-scribed below.

(i) Postribosomal fraction. Cells grown in 2 liters of LBmedium were harvested and then washed once with buffer A(50 mM Tris-HCl buffer [pH 7.8], 10 mM MgCl2, 200 mMKCl). The cells were suspended in 25 ml of buffer A and thenpassed through a French pressure cell (Aminco) twice at1,000 lb/in2. After removal of the envelope fraction bycentrifugation at 100,000 x g for 30 min, the supernatant wasfurther centrifuged at 200,000 x g for 3 h. The supernatantwas recovered as the postribosomal fraction.

(ii) DEAE-cellulose column chromatography. The superna-tant fraction was dialyzed against buffer B (10 mM Tris-HClbuffer [pH 7.8], 5 mM MgCl2) for 14 h at 40C. After theremoval of insoluble materials by low-speed centrifugation,the clear supernatant fraction was applied to a DE52 column(2 by 50 cm) equilibrated previously with buffer B. Thepass-through fractions, which were active against Cbz-Phe-ONap, were pooled.

(iii) Sephacryl S-300 column chromatography. The pooledfractions from step ii were concentrated with polyethyleneglycol 6,000 and then dialyzed against buffer C (25 mMsodium phosphate buffer [pH 7.1], 400 mM NaCl). After theremoval of insoluble materials by centrifugation, the super-natant was applied to a Sephacryl S-300 column which hadbeen equilibrated with buffer C. The active fractions werepooled, concentrated with polyethylene glycol, and thendialyzed against 10mM sodium phosphate buffer (pH 7.0)-50mM NaCl.

Genetic procedures. Interrupted mating and Plkc-mediatedgeneralized transduction were carried out as described byMiller (25). Mutant strains with the cloned gene deleted wereconstructed by using a temperature-sensitive replicon andhomologous recombination, as described before (39).

Scoring and selection of mutant strains. acrAl strains werescored for inability to grow on S2 medium containing meth-ylene blue (27). Temperature-sensitive dnaZ or adk2 strainswere scored for failure to grow at 420C on an LB agar plate.

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1034 ICHIHARA ET AL.

A BamHi Hindlil EcoRI Hindll tease IV, which can hydrolyze Cbz-Phe-ONap as well as0!% I I I pLC36-48 (41.8kb) Cbz-Val-ONap (13). These two clones, therefore, were not

| I | pLC13- (24.4kb) analyzed further. A more than 10-fold increase in Cbz-Phe-pLC6-33 (21.8kb) ONap-hydrolyzing activity was observed in whole-cell prep-

arations of the three transformants harboring pLC6-13,,........... - '-Cbz-Phe-ONap pLC13-1, and pLC36-46. Restriction enzyme mapping anal-

B SoaI Sim= Alul HapI SauA AluI hupI Sell Hydrolyzing Activity ysis revealed that these plasmids shared the same chromo-,,/,,,/pYMOB + somal DNA region, the 16-kb BamHI-HindIII fragment (Fig.

P ~~~/ pYMO28 + ofatvtagisCb-hOapocrwtinhs

., .,, :p, ,, / pYMO28 1A), indicating that the gene responsible for overproductionp,, | | pYM026 + of activity against Cbz-Phe-ONap occurs within this region.@@'@pYMO25 - Cbz-Phe-ONap-hydrolyzing activity in the subcellular frac-

C / pYMO23 tions of a transformant, S121/pLC6-33, was examined (TablepYM023_______- 2). The distribution of marker enzymes revealed that theKmr gone subfractionation was reasonably performed. Cbz-Phe-

FIG. 1. Restriction mapping of the chromosomal regions of ONap-hydrolyzing activity was found in the periplasmicpLC36-48, pLC13-1, and pLC6-33 and subcloning and assaying of fraction.the overproduced protease I. (A) The chromosomal regions of the Subcloning and sequencing of the gene encoding the pro-pLC plasmids and the restriction sites are shown. (B) The indicated teinase. Subcloning of the 16-kb BamHI-HindIII regionregions were subcloned into pUC19, and then Cbz-Phe-ONap hy- revealed that the 0.9-kb Sau3AI fragment cloned on pYM028drolysis activity of the transformants was examined: +, overpro- was the minimum length DNA, which donates the enzymeducer; -, non-overproducer. ji and p indicate the position anddirectinXoftelacr e o. ( p wa activity (Fig. 1B). Cells harboring pYM026 constructed bydirection of the lac promoter on thle vector. UC) pYMu2 was liato of th .-blIfamn fpM0 noteSconstructed with a vector, pSI029, possessing a temperature-sensi- ligation of the1.0-kbAlui f ragmentof pYMot 8 intotheSeoaI

tive replicon. Part of the insertion region was replaced with Kmr, as site of pUC19, possessing the lac promoter at the end of theindicated. site, showed strong activity, whereas cells harboring

pYM025, which possesses the same fragment in the oppositedirection, did not. Thus, the direction of transcription of the

ushl and purE strains were scored for inability to grow on gene and the possible localization of its promoter wereM9 medium containing AMP as a sole carbon source and M9 determined.medium without adenine, respectively. The entire region inserted into pYM028 was sequencedOther methods. Amino acid sequencing was carried out as (Fig. 2). The sequence had only one long open reading

described previously (22). Labeling of E. coli with 35S- frame, which comprised 208 amino acid residues. A putativemethionine, preparation of cell envelope fractions, sodium promoter region was assigned to nucleotides 40 to 45,dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, TTGACA, for the -35 region, and nucleotides 63 to 68,and fluorography were carried out as described previously GAGGAT, for the -10 region. The two regions were flanked(10). For gel electrophoresis, samples containing active by 17 nucleotides. The Alu-cutting site used for construct-protease I were pretreated with 10% trichloroacetic acid at ing pYM026 was located at nucleotides 50 to 51 between the40C. putative -35 and -10 regions. This is consistent with the

Nucleotide sequence accession number. The nucleotide fact that pYM026 requires an additive promoter for synthesissequence in Fig. 3 has been deposited in the DDBJ/EMBL/ of the proteinase.GenBank DNA data bases under accession number D13180. Purification of the overproduced proteinase. The overpro-

duced proteinase was purified from the soluble cellularfraction of one of the overproducers, JM103/pYM008 (Fig.

RESULTS 1B). The three-step purification described in Materials andCloning of the gene encoding a periplasmic proteinase which Methods resulted in a single polypeptide band corresponding

hydrolyzes Cbz-Phe-ONap. Of 2,200 clones in Clarke and to a molecular mass of 20 kDa on a polyacrylamide gel (Fig.Carbon's collection (4), 5, i.e., pLC6-33, pLC7-10, pLC13-1, 3). The N-terminal 12 amino acid residues of the purifiedpLC36-46, and pLC40-13, were positive for Cbz-Phe-ONap- protein were determined. The sequence was A-D-T-L-L-I-hydrolyzing activity. We have already reported that L-G-D-S-L-S-, which was the same as that of A27-S33 of thepLC7-10 and pLC40-13 carry the sppA gene encoding pro- polypeptide chain deduced from the DNA sequence shown

TABLE 2. Localization of overproduced Cbz-Phe-ONap-hydrolyzing activity'

Activity against Alkaline Poes VPGlcoiaeStrain/plasmid Fraction Protein Cbz-Phe-ONapb phosphatasec Protease INC 13.Galactosldasec

U/mil % U/ml % U/ml % U/ml %

S121 Periplasm 5.9 14.4 99.7 20.9 88.0 0 0 0.3 4.0Membrane 22.1 0.1 0.3 0 0 21.0 71.2 0 0Cytoplasm 72.0 0 0 2.8 11.8 8.5 28.8 7.2 95.0

SI21/pLC6-33 Periplasm 7.5 208.8 90.6 20.7 85.9 0 0 1.3 20.0Membrane 23.3 1.2 0.5 0 0 19.0 71.7 0 0Cytoplasm 69.2 20.4 8.9 2.3 14.1 7.5 28.3 4.4 80.0

a The method described by Nossal and Heppel (28) was used.b Activity due to the action of protease IV, which can be estimated with Cbz-Val-ONap, has been subtracted.c Alkaline phosphatase, protease IV, and 13-galactosidase were used as marker enzymes localized in the periplasm, membranes, and cytoplasm, respectively.

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PROTEASE I IN E. COLI 1035

GATCCCGACTCGCCCACCAGTGCGATGGTCTCGCCACGTTTGACAACCAGCTCAACTCCG 60-35 Alo

GTGAGGATGGAGAGTTCATGCTCCCCCTGACCGACGGACTTCTTAAGATGATGAACTTCA 120-10 SD M M N F N

ACAATGTTTTCCGCTGGCATTTGCCCTTCCTGTTCCTGGTCCTGTTAACCTTCCGTGCCG 180N V F R W H L P F L F L V L L T F R A A

CCGCAGCGGACACGTTATTGATTCTGGGTGATAGCCTGAGCGCCGGGTATCGAATGTCTG 240A^,A D T L L I L G D S L S A G Y R M S A

CCAGCGCGGCCTGGCCTGCCTTGTTGAATGATAAGTGGCAGAGTAAAACGTCGGTAGTTA 300S A A W P A L L N D K W Q S K T S V V N

ATGCCAGCATCAGCGGCGACACCTCGCAACAAGGACTGGCGCGCCTTCCGGCTCTGCTGA 360A S I S G D T S Q Q G L A R L P A L L K

AACAGCATCAGCCGCGTTGGGTGCTGGTTGAACTGGGCGGCAATGACGGTTTGCGTGGTT 420Q H Q P R W V L V E L G G N D G L R G F

TTCAGCCACAGCAAACCGAGCAAACGCTGCGCCAGATTTTGCAGGATGTCAAAGCCGCCA 480Q P Q Q T E Q T L R Q I L Q D V K A A N

ACGCTGAACCATTGTTAATGCAAATACGTCTGCCTGCAAACTATGGTCGCCGTTATAATG 540A E P L L M Q I R L P A N Y G R R Y N E

AAGCCTTTAGCGCCATTTACCCCAAACTCGCCAAAGAGTTTGATGTTCCGCTGCTGCCCT 600A F S A I Y P K L A K E F D V P L L P F

TTTTTATGGAAGAGGTCTACCTCAAGCCACAATGGATGCAGGATGACGGTATTCATCCCA 660F M E E V Y L K P Q W M Q D D G I H P N

ACCGCGACGCCCAGCCGTTTATTGCCGACTGGATGGCGAAGCAGTTGCAGCCTTTAGTAA 720R D A Q P F I A D W M A K Q L Q P L V N

ATCATGACTCATAAAGCAACGGAGATCCTGACAGGTAAAGTTATGCAAAAATCGGTCTTA 780H D S *

ATTACCGGATGTTCCAGTGGAATTGGCCTGGAAAGCGCGCTCGAATTAAAACGCCAGGGT 840

TTTCATGTGCTGGCAGGTTGCCGGAAACCGGATGATGTTGAGCGCATGAACAGCATGGGA 900

TTTACCGGCGTGGTTGATC 919

FIG. 2. Nucleotide sequence encompassing the 0.9-kb Sau3AIfragment. The amino acid sequence of an open reading framededuced from the nucleotide sequence is also shown. A possiblepromoter sequence, -35 and -10, and a possible ribosome-bindingsequence, SD, are indicated. A indicates the cleavage site of thesignal peptide. The AluI site used for the construction of pYM026and pYM025 is also shown.

in Fig. 2. The sequence was preceded by an N-terminalsequence possessing typical features of the signal peptide.The results indicate that the polypeptide deduced from thenucleotide sequence represents this proteinase, which issynthesized as a precursor possessing a signal peptide com-posed of 26 amino acid residues.The following properties support the view that the purified

proteinase is protease I described in references 33 and 17. (i)

94K-

67K-_

43K-_

FIG. 3. Purification of the overproduced proteinase. The over-produced proteinase was purified from the postnibosomal fraction(A) through successive chromatographies on a DEAE-cellulosecolumn (B) and a Sephacryl S-300 column (C). Each fraction wasanalyzed by SDS-polyacrylamide gel electrophoresis.

The purified proteinase hydrolyzed Cbz-Phe-ONap andN-benzyloxycarbonyl-L-phenylalanine p-nitrophenyl ester,but not Cbz-Val-ONap or N-benzyloxycarbonyl-L-valinep-nitrophenyl ester; (ii) it was localized in the periplasm; (iii)although it exhibited a chymotrypsin-like substrate specific-ity, its activity was not inhibited by tosylphenylalaninechloromethyl ketone; (iv) it passed through a DEAE-cellu-lose column; and (v) its molecular mass was 20 kDa.Hereafter, the enzyme will be called protease I.

Stability of the purified protease I against SDS and heating.The activity of protease I was stable in the presence of 1%Triton X-100, which was in the reaction mixture. We exam-ined its stability against a strong ionic detergent, SDS, andheating. No enzyme activity was lost on treatment with 1%SDS at 370C for 30 min; in fact, it increased by 13%.Furthermore, about 50% of the activity remained afterheating at 100'C for 10 min. The combination of treatmentwith 1% SDS and 5 min of heating completely inactivated theenzyme, however.

Construction of a mutant strain with the gene encodingprotease I deleted. Plasmid pYM023, which possesses atemperature-sensitive replication origin and a 3.0-kb Sallfragment in which the 1.6-kb HpaI region is replaced by anintact kanamycin resistance gene (Kmr) (Fig. 1C), wasconstructed. As described previously (39), two successivehomologous recombinations at 420C should result in replace-ment of the Sal fragment of the chromosomal DNA with theKmr gene-possessing Sal fragment of the plasmid. Such arecombinant, SI532, selected for kanamycin resistance andampicillin sensitivity, possessed no plasmid and little activ-ity against Cbz-Phe-ONap, suggesting that the strain is amutant with the gene encoding protease I deleted. Southernblot analysis with the 3.0-kb Sall fragment and the Kmr generevealed that the replacement took place correctly on thechromosome.

Fine mapping of the gene encoding protease I, using thedeletion mutant. The Kmr region on the chromosome ofSI532 was transferred to CSH62 (Hfr; clockwise from 98.5min) by Plkc-mediated generalized transduction to constructCK50. The chromosomal locus of the Kmr gene of CK50 wasinvestigated by the interrupted-mating technique, usingJE1011 as the recipient. The Kmr gene was introduced to theconjugants in the order lac-Kmr-gal, suggesting that the Kmrgene is located between 8 and 17 min on the chromosome.The gene encoding protease I of Salmonella typhimurium

has been mapped at 12 min as apeA (24). Five genes,acrA-dnaZ-adk-ush-purE, have been mapped in this orderaround 12 min on the E. coli chromosome (la). Results ofcotransduction experiments with these mutants suggest thatthe Kmr gene is located around the ush andpurE genes. Finemapping of the Kmr gene was then carried out by a three-factor transduction cross with the adk, ush, andpurE genes.The results summarized in Table 3 indicate that the Kmrgene is located between the ush andpurE genes. When Wu'sequation (43) was used, the map position of the Kmr genewas calculated to be 11.8 min on the basis of the data shownin Table 3 and the map positions of the adk, ush, andpurEgenes (la). This, in turn, indicates that the gene encodingprotease I is located at this map position and suggests thatprotease I of E. coli and that of S. typhimurium are theproducts of corresponding genes. The E. coli gene, there-fore, was named apeA also.

Characterization of apeA mutants. The effects of the apeAdeletion on cell growth were examined in various mediacontaining peptone, albumin, or one of the following pep-tides as a sole carbon source: Gly-Gly-Gly, Gly-Gly, Phe-

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1036 ICHIHARA ET AL.

TABLE 3. Three-factor transduction crosses

Donor and Recipient and Selected Unselected markermarker' marker marker Class No. (%)

CK67 W4626phe purE' ush+ KmS 93 (60)ush purE ,sh+Kmr 49 (32)Kmr Ush Kmr 13 (8.4)

ush KmS 0 (0)Kmr ush+ purE 67 (46)

ush purE 61 (41)ush+ purE+ 10 (7)ush purE+ 9 (6)

CK82 CK85 adk+ KmspurE+ 104 (74)Kmr adk KmrpurE+ 27 (19)purE KmrpurE 10 (7.1)

Kms purE 0 (0)Kmr adkpurE+ 119 (74)

adk+ purE+ 16 (10)adkpurE 23 (14)adk+ purE 2 (1.3)

CK84 W4626phe purE+ adk+ Kms 99 (63)adk purE adk+ Kmr 60 (38)Kmr adk Kmr 1 (0.6)

adk KmS 0 (0)Kmr adk+ purE 121 (77)

adkpurE 7 (4.4)adk+ purE+ 27 (17)adkpurE' 3 (1.9)

a The KMr gene was the replacement for the structural gene for protease I.

Phe, or Gly-Phe. No significant effect of the deletion wasobserved, however. The results were coincident with thoseobtained by Kowit et al. (17), although they used a lesswell-defined mutant. We further constructed a triple mutant,KA36, in which the following three genes, encoding protei-nases, are defective: pepN (7, 13, 19), ape4(Tcr), andsppA(Kmr) (1). This strain also did not exhibit any differencein cell growth in the media described above. Pulse-chaseexperiments with KA36 and its wild-type strain, MH19, didnot show any difference in the decay pattern of envelopeproteins or that of whole-cell proteins.

DISCUSSION

In this study, we cloned and sequenced the gene encodingprotease I, purified the gene product, and determined themap position of the gene on the E. coli chromosome.Protease I was computer searched (National BiomedicalResearch Foundation Protein Data Base) for homologieswith all registered proteins. No significant homologies wereobserved, however (data not shown).Pacaud and Uriel (34) first reported protease I as a soluble

protease of E. coli which hydrolyzed a chromogenic sub-strate, N-acetyl-Phe-ONap. Because of the low water solu-bility of the substrate, they used a reaction mixture contain-ing 20% dioxane, an organic solvent. In our study, wefurther showed that protease I was quite resistant to deter-gents such as Triton X-100 and SDS. In addition, the enzymeremained active after treatment at 100'C. These resultsindicate that protease I is an unusually stable enzyme, atleast in terms of its activity against Cbz-Phe-ONap. How-ever, because the real substrate in the cells is unknown, thephysiological or enzymological significance of the stabilityremains unknown.

In Clarke and Carbon's collection, two genes, sppA andapeA, were found to be responsible for the overproductionof Cbz-Phe-ONap-hydrolyzing activity. Genes encodingother proteinases, which possibly hydrolyze Cbz-Phe-ONap, were not cloned. Two such proteinases have beenreported (32). These proteinases may have been inactivatedunder the assay conditions employed. The gene encodingprotease V could have been cloned in the present work,since this proteinase hydrolyzes N-benzyloxycarbonyl-L-phenylalaninep-nitrophenyl ester under the conditions used(13, 31). We found, however, that this proteinase barelyhydrolyzed Cbz-Phe-ONap (data not shown).Sequence analysis of the N-terminal region of protease I

revealed that the cleavage of the signal peptide took placebetween the third and fourth alanine residues of four alanineresidues in a row. The cleavage site for signal peptidase I is,in most cases, Ala-X. The specific cleavage at one siteamong the four Ala-X sites suggests that other factors alsoplay a role in determining the cleavage site. The distancefrom the hydrophobic region (five to six amino acid residues)and the size of amino acid residues around the site (-3, -1recognition) have been discussed in relation to cleavagespecificity (40). As judged from these discussions, cleavageat Ala-Asp would be favorable. The N-terminal amino acidresidues of periplasmic proteins are, in most cases, charged(2, 9, 16, 30), whereas those of outer membrane proteins arehydrophobic (41). On the basis of this, cleavage at Ala-Aspwould also be favorable for protease I, a periplasmic en-zyme. Factors determining the cleavage specificity in pro-tease I should be studied further.The physiological importance of protease I has been

questioned, since a spontaneous mutation on protease I didnot have a significant effect on the cell physiology, includingcell growth (17). In the present work, we showed that adeletion mutation on this proteinase did not affect cellgrowth either. We further showed that a mutant strain,KA36, defective in three proteinases, including protease I,grew as well as the wild-type strain on various media. It isprobable that the functions of these proteinases can becompensated for by other proteinases. Alternatively, indi-vidual proteinases, including protease I, have importantfunctions under certain circumstances, such as in the intes-tine or in natural environments.

ACKNOWLEDGMENTS

We thank T. Mizuno for valuable suggestions.This work was supported by a grant from the Ministry of

Education, Science and Culture of Japan.

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