THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 2, Issue of January 14, pp. 1502–1510, 2000 © 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. The Type 4 Prepilin Peptidases Comprise a Novel Family of Aspartic Acid *

(Received for publication, August 13, 1999, and in revised form, October 29, 1999)

Christian F. LaPointe‡ and Ronald K. Taylor§ From the Department of Microbiology, Dartmouth Medical School, Hanover, New Hampshire 03755

Type 4 prepilins or prepilin-like-proteins are secreted The related type 4 pilin-like proteins partially comprise the by a wide range of bacterial species and are required for main terminal branch of the general secretory pathway in a variety of functions including type 4 pilus formation, Gram-negative bacteria, also known as the type 2 secretion toxin and other secretion, gene transfer, and pathway (6). The general secretory pathway is the pathway biofilm formation. A distinctive feature of these proteins utilized by the majority of proteins that are secreted outside of is the presence of a specialized leader peptide that is Gram-negative bacteria. Such proteins include toxins such as cleaved off by a cognate membrane-bound type 4 prepi- cholera toxin of V. cholerae (7) and exotoxin A of Pseudomonas lin peptidase (TFPP) during the process of secretion. In aeruginosa (8), as well as other such as pullulanase of this report we show that the TFPPs represent a novel Klebsiella pneumoniae (9) and hemolysin of Aeromonas hy- family of bilobed aspartate proteases that is unlike any drophila (10). Type 4 pilin-like proteins are also required for other . The pairs of aspartic acids of the two TFPPs in Vibrio cholerae are found at positions natural transformation of Bacillus subtilus (11) and Strepto- 125 and 189 of TcpJ and 147 and 212 of VcpD. Corre- coccus pneumoniae (12). sponding aspartate residues are completely conserved The type 4 leader peptide is characterized by several features throughout this extensive peptidase family. that are highly conserved, yet differ from those of standard leader peptides. The N-terminal leader that is cleaved from the mature protein is highly charged and immediately precedes a Members of the family of leader peptidases known as type 4 region of approximately 20 residues that are predominantly prepilin peptidases (TFPP)1 have been identified in a vast hydrophobic and are retained within the mature protein. The number of species of Gram-negative bacteria and an increasing majority of type 4 prepilins or prepilin-like proteins are desig- number of Gram-positive bacteria. The TFPP is responsible for nated type 4a, containing leader peptides that are short (ϳ6 the cleavage and N-methylation (known collectively as process- residues). Type 4b preproteins contain a longer leader (ϳ25 ing) of the highly conserved type 4 leader peptide present at the residues). The peptide bond on the prepilin that is hydrolyzed N terminus of the families of secreted proteins known as type 4 by the TFPP lies between the charged and hydrophobic do- prepilins and type 4 prepilin-like proteins (1). Whereas the mains, immediately C-terminal to an invariant glycine. The cleavage of the type 4 leader peptide is a necessary step for new N-terminal amino acid that arises upon processing is a secretion of the mature pilin, methylation of the N terminus of N-methylated phenylalanine for type 4a proteins and is typi- the mature pilin is not required for secretion and has no known cally a N-methylated methionine for type 4b proteins. The function (2). Type 4 pilins are polymerized as the structural TFPPs themselves are integral cytoplasmic membrane proteins subunits of type 4 pili (Tfp), which are surface organelles re- with numerous transmembrane domains. The processing reac- quired for diverse activities that contribute to broad attributes tion has been postulated to occur on the cytoplasmic side of the such as genetic transfer, virulence, and environmental persist- membrane because of the membrane orientation of the prepi- ence of a wide array of Gram-negative bacterial pathogens. lin, the location of all the major nonmembrane peptidase do- Prototypical examples include microcolony formation mediated mains, and the likelihood that the cytoplasmically located S- by toxin-coregulated pilus (TCP) of Vibrio cholerae,2 bacterial adenosyl methionine would provide an available source of surface dispersal mechanisms mediated by bundle forming pi- methyl groups for the methylation step of the reaction (13). lus of enteropathogenic (3), colonization and Previous studies on the proteolytic mechanism of the TFPPs natural transformation mediated by PilE of Neisseria sp. (4), have focused on the largest cytoplasmic region of the protein and gliding motility mediated by the Tfp of Myxococcus sp. (5). designated cytoplasmic domain 1. These studies were per- formed on PilD, a prototypic TFPP from P. aeruginosa. Domain * This work was supported in part by Grant ROI AI 23096 from the 1 of the PilD peptidase contains two pairs of cysteine residues National Institutes of Health. The costs of publication of this article that are conserved among many members of the TFPP family. were defrayed in part by the payment of page charges. This article must Alteration of the 4 highly conserved cysteine residues by sub- therefore be hereby marked “advertisement” in accordance with 18 stitution of alanine or serine residues resulted in up to an U.S.C. Section 1734 solely to indicate this fact. ‡ Recipient of Predoctoral Fellowship AI F31-09635 from the Na- 80–100% reduction of peptidase activity in an in vitro assay. tional Institutes of Health. However, the processing defect varied from approximately 0% § To whom correspondence should be addressed: Dept. of Microbiol- to 80% for the various mutants in vivo, suggesting that al- ogy, Dartmouth Medical School, Hanover, NH 03755. Tel.: 603-650- though pilin cleavage was less efficient, it was not abolished 1632; Fax: 603-650-1318; E-mail: [email protected]. 1 The abbreviations used are: TFPP, type 4 prepilin peptidase; Tfp, (14). Chemical inhibitor studies supported the notion that the type 4 pili; TCP, toxin-coregulated pilus; NEM, N-ethylmaleimide; cysteine residues contributed to protease activity and PilD was PCR, polymerase chain reaction; Ab, antibody; PAGE, polyacrylamide classified as a cysteine protease, specifically as a member of the gel electrophoresis; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)carbodi- C20 family of cysteine proteases (15). The retention of partial imide hydrochloride. 2 Kirn, T. J., Lafferty, M. L., Sandoe, C. M. P., and Taylor, R. K. activity by some of the pilD mutants as well as the subsequent (2000), Mol. Microbiol., in press. discovery of TFPP’s that lack the consensus cysteine residues

1502 This paper is available on line at http://www.jbc.org Protease Active Site of TFPP 1503

have brought this mechanism of protease activity into question. TcpA Prepilin—The method for membrane preparation was adapted For example, the XpsO TFPP of Xanthomonas campestris was from a previously described cell fractionation protocol (25). A 100-ml characterized and shown to lack all the domain 1 cysteine overnight culture was placed on ice for 20 min, and the cells were then collected by centrifugation at 5000 ϫ g for 10 min. The cell pellet was residues, yet the xpsO gene fully complements a pilD deletion resuspended in 1 ml of 200 mM Tris, pH 8.0, to which 1 ml of 50 mM Tris, for prepilin processing (16). Some newer members of the rap- ␮ ␮ pH 8.0, 1 M sucrose, 2 ml of H2O, 20 lof0.5M EDTA, and 20 lof idly growing family lack the entire domain 1 region. Analysis of lysozyme (10 mg/ml) were added. The cell suspension was allowed to ␮ a current TFPP alignment also reveals a complete lack of any incubate on ice for an additional 30 min. 100 lof1M MgSO4 was conserved histidine residue, a necessary component of the cys- added, and the suspension was centrifuged at 5000 ϫ g for 10 min. The ϫ teine protease catalytic mechanism (15). These observations cell pellet is resuspended in 5 ml of 50 mM Tris, pH 8.0, sonicated 3 15 s at 50% duty, and centrifuged at 5000 ϫ g for 10 min. The super- have led to a revised classification of PilD, and therefore the natant was then centrifuged at 230,000 ϫ g (60,000 rpm in a TLA 100.3 complete family of TFPPs, to the U12 category of proteases rotor) for 15 min. The resulting pellet, which contained the total mem- with unknown catalytic mechanism (17). brane fraction, was resuspended in 200 ␮lof50mM Tris, pH 8.0. In the study reported here, an approach similar to the com- TcpA Prepilin Quantitation—Membrane preparations were prepared prehensive mutational strategy employed to identify the signal from 42 °C grown overnight cultures of E. coli K38, which does not peptidase I active site (18) was used to determine the active site express TcpA, and MK90, which overexpresses TcpA prepilin at ele- vated temperature. 20 ␮l of a membrane preparation from K38 and residues of TcpJ, the TFPP responsible for processing the TcpA from MK90 were subjected to electrophoresis on a 12.5% SDS-PAGE gel type 4 prepilin of V. cholerae. A protein sequence alignment of alongside a standard of 3 ␮g of , followed by staining with the TFPP family revealed several highly conserved potential Coomassie Blue. The stained gel was dried using the Novex gel drying protease active site residues in addition to the two cysteine system. The dried gel was scanned by a Molecular Dynamics Personal pairs that exist in this protease family. These include serine, Densitometer SI, and densitometry analysis was performed using Im- aspartic acid, lysine, and glutamic acid residues, most of which ageQuant version 1.2 software. The intensities (measured in pixels) of the 3-␮g trypsin band, the TcpA prepilin band (MK90), the correspond- are located in cytoplasmic domains of TFPPs. Mutations in tcpJ ing location of the TcpA prepilin band in the TcpA-negative control lane corresponding to these positions, as well as a deletion of the (K38), and an approximately 28-kDa band from both K38 and MK90 entire region encoding cytoplasmic domain 1, were constructed. were measured while adjusting to a single background value. The mass In vivo activity and in vitro protease assays indicate that only of the TcpA prepilin present in the MK90 lane was determined by first Asp125 and Asp189, which are completely conserved throughout subtracting the intensity of the corresponding area in the K38 TcpA the entire TFPP family, are absolutely required for protease prepilin-lacking lane. This corrected for the staining intensity of minor protein bands that co-migrate with TcpA prepilin. By comparing the activity in TcpJ. Chemical protease inhibition studies are con- intensity values of the 28-kDa bands from the TcpA-positive and TcpA- sistent with this finding. The mutational analysis was ex- negative lanes, a further adjustment was made to the TcpA prepilin tended to a second TFPP of V. cholerae, VcpD, which is required value to account for any loading difference between the lanes. The for toxin secretion (19). Taken together, the results lead to the trypsin band intensity was divided by 3 ␮g to determine an intensity/␮g conclusion that members of the TFPP family are bilobed aspar- value, which was applied to the final TcpA prepilin band value to ␮ tic acid proteases and due to their complete insensitivity to determine the mass of TcpA prepilin/ l in the membrane preparation. The TcpA prepilin membrane preparation was diluted with 50 mM Tris, pepstatin should be regarded as a novel family of non-- pH 8.0, buffer to a concentration of 0.2 ␮g/␮l. like acid proteases. These findings allow the rational develop- In Vitro Cleavage Assay—The in vitro TcpJ cleavage assay was ment of inhibitors with practical therapeutic value. designed, based on the assay developed for PilD of P. aeruginosa (26), except that pre-TcpA was provided in a membrane preparation rather MATERIALS AND METHODS than as purified protein. TcpJ derivatives were provided in membrane Bacterial Strains, Plasmids, Media, and Antibiotics—E. coli strains preparations of the TcpJ-expressing strain JM109 (pCL9) (wild-type used were K38 (20) and JM109 (21); Amber-Lys, Amber-Leu, Amber- and mutant alleles). The 100-␮l processing reaction was prepared by Gln, Amber-Ser, and Amber-Tyr are chromosomal amber suppressor combining a 50-␮l substrate fraction with a 50-␮l enzyme fraction (an strains from Promega’s INTERCHANGETM in vivo Amber suppression equivalent of 1 unit of wild-type activity) and incubated at 37 °C for 1 h. mutagenesis system. Strain JM290 is JM109 carrying pTrc99A/EpsI The substrate fraction was prepared by combining 5 ␮l of the TcpA (6)His (19). Strain MK90 is K38 carrying p3Z-A, which provides tcpA prepilin-containing membrane preparation (1 ␮g of TcpA prepilin), 10 under the control of a T7 promoter, and pGP1–2, which provides the ␮l 0.5% (w/v) cardiolipin, 20 ␮lof5ϫ assay buffer (125 mM triethanol- heat-inducible T7 RNA polymerase that drives transcription of the tcpA amine HCl, pH 7.5, 2.5% v/v Triton X-100), and brought up to the total

on p3Z-A (22). Strain CL318 is JM109 carrying pCL9. V. cholerae volume with H2O. The enzyme fraction was prepared by combining a strains are derivatives of classical Ogawa O395 Smr. Strain J71K-1 is standardized volume of TcpJ-containing membrane preparation and r ␮ O395 tcpJ::kan (22). Strain JM315 is O395 vcpD1 (19). Strain CL381 is H2O to bring the volume to 50 l. The cleavage reaction was stopped by O395 vcpD1, tcpJ::kanr. Plasmid pCL9, a derivative of pUC18 (21), was the addition of 100 ␮lof2ϫ protein sample buffer. The volume of TcpJ constructed by the cloning of a 980-bp EcoRI/NsiI fragment containing preparation for all TcpJ derivatives was standardized to be equivalent tcpJ into the EcoRI and PstI sites in the polylinker of pUC18. Plasmid to the volume containing an amount of wild-type TcpJ sufficient to pCL10 was constructed by the additional cloning of a 2.0-kilobase pair cleave 50% of 1 ␮g of TcpA prepilin in 1 h, which is defined as 1 unit of HindIII fragment that contains tcpA into the HindIII site of the pCL9 TcpJ peptidase activity. The formation of mature TcpA from precursor polylinker. Plasmid pCL11 was constructed by the PCR amplification of was monitored by Western immunoblot analysis. tcpJ with primer pair J-ATG NcoI(5Ј- AGACCATGGAATACGTT- Chemical Protease Inhibitors—Chemical inhibitors were tested for TACTTGATCCTATTTTCGATTG) and J-Histag (5Ј-CCCCTGCAGCT- their ability to prevent the cleavage of TcpA from the prepilin to mature CAATGATGATGATGATGATGCATTAAACGGATTG) and the cloning form in the in vitro cleavage assay. Various concentrations of inhibitors of the resulting fragment into the NcoI and PstI sites of the pKK233–2 were added to both the enzyme (1 unit of TcpJ) and substrate fraction based expression vector, pTrc99A (Amersham Pharmacia Biotech). so that, when combined, the inhibitor concentration would remain Primer J-Histag 3Ј codes for 6 His residues, which are added to the C constant. The inhibitor was incubated in the enzyme fraction at room terminus of the TcpJ protein. Plasmid p3Z-A was constructed by the temperature for 30 min prior to the combination of the two fractions. cloning of the 1-kilobase pair DraI tcpA fragment into the SmaI site of The 100-␮l reaction was allowed to proceed for1hat37°Cbefore 100 the Promega expression vector, pGEM3Z (22). Plasmid pJM294 is a ␮lof2ϫ SDS protein sample buffer was added to stop the reaction. The pACYC184 derivative containing vcpD (19). Plasmid pRTG7H3 is a specific concentrations of each inhibitor are listed in Table I. The tcpA-expressing plasmid (23). Plasmid pGP1–2 carries the thermoin- 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride ducible T7 polymerase gene (24). (EDAC)/glycinamide inhibition protocol is a two-step chemical reaction E. coli strains were grown in LB at 37 °C. V. Cholerae strains were based on the procedure developed for the selective modification of grown in LB, pH 6.5, at 30 °C to induce expression of TCP. Antibiotics carboxyl side groups of aspartic and glutamic acids in proteins (27). were added to the media when needed at the following concentrations: Takahashi et al. (28) first suggested the use of this procedure as a 100 ␮g/ml ampicillin, 45 ␮g/ml kanamycin, 34 ␮g/ml chloramphenicol. method to specifically inhibit acid proteases. Membranes containing the Preparation of Membranes Containing TcpJ Prepilin Peptidase or equivalent of 6 units of TcpJ activity are incubated in 80 ␮lof25mM 1504 Protease Active Site of TFPP

FIG.1.Percentage of conserved ho- mology of potential protease active site residues from 27 TFPP homologs. All potential protease active site residues (Cys, Ser, Asp, Lys, His, Glu) in TcpJ were analyzed for their percentage of ho- mology in a protein sequence alignment of the TFPP family. Potential protease ac- tive site residues that are the major con- served amino acids at a position in the peptidase family alignment but are not conserved in TcpJ are enclosed in paren- theses and are designated with respect to the relative position in the TcpJ protein sequence. When a residue in TcpJ and a conserved residue not found in TcpJ exist at the same relative position, then both are indicated by a sectored bar with the TcpJ portion represented by the lower portion of the bar. The remaining portion of the bar represents the conserved resi- due not in TcpJ. Predicted membrane to- pology is indicated by C (cytoplasmic), P (periplasmic), or M (transmembrane) be- low each residue. Gray bars indicate the conserved residues that have been altered during this study. Alignment was done in DNASTAR MegAlign, using the Clustal method of alignment.

potassium biphthalate, pH 4.0, and 100 mM EDAC for 30 min at room The total sample was boiled for 5 min, and 50 ␮l was subjected to temperature. 20 ␮lof1M glycinamide was added to the mixture, and electrophoresis on a 12.5% SDS-PAGE gel. For both in vivo and in vitro incubation at room temperature was continued for an additional 30 min processing, the proteins were electroblotted to nitrocellulose and probed before the reaction was stopped by the addition of 150 ␮l of 200 mM Tris with anti-TcpA peptide 6 antiserum. The secondary Ab, an anti-rabbit buffer, pH 8.0. The mixture was spot dialyzed against 50 mM Tris IgG conjugated to horseradish peroxidase, was used to detect TcpA by buffer, pH 8.0, for 1 h and then centrifuged at 230,000 ϫ g (60,000 rpm chemiluminescence using ECL reagents (Amersham Pharmacia Bio- in TLA 100.3 rotor) for 15 min. The resulting membrane pellet, which tech), followed by autoradiography. contains the chemically modified TcpJ was resuspended in 60 ␮lof50 For detection of TcpJ, 20 ␮l of a membrane preparation from a mM Tris, pH 8.0. 20 ␮l of the membrane suspension, which contains 2 TcpJ-expressing strain was combined with 20 ␮lof2ϫ SDS-PAGE units of TcpJ, was used in the in vitro cleavage assay to determine the sample buffer and incubated at 37 °C for 15 min. 20 ␮l of the sample peptidase activity. Activity of the modified TcpJ was compared with an was then subjected to electrophoresis on a 12.5% SDS-PAGE gel. Pro- equivalent amount of TcpJ, which had been subjected to a mock modi- teins were transferred to nitrocellulose and probed with anti-TcpJ fication procedure that did not include EDAC or glycinamide. peptide J3–1 antiserum or, in the case of TcpJ (6)His and TcpJ Mutagenesis of tcpJ—The TcpJ S46A, C76A, D189A, and K191A (6)His⌬35–81, with TetraHis Ab (Qiagen). Detection with secondary alterations were constructed in pCL10 by the QuikchangeTM (Strat- antibody was carried out as described for TcpA. agene) method of mutagenesis, which utilizes inverse PCR primed by RESULTS divergent overlapping primers containing the desired complementary nucleotide changes. The S18A, C48A, C51A, S65A, C73A, S81A, E88A, A Limited Set of Potential Peptidase Active Site Residues Are D125A, D125E, D125N, D125Amb, S172A, D183N, D189E, D189N, Conserved among TFPPs—In order to characterize the bio- ⌬ D189Amb, S212A/S213A, and 35–81 were constructed in pCL10, and chemical mechanism of the cleavage reaction catalyzed by TcpJ D147A and D212A were constructed in pJM294 using other methods of and other TFPPs, we sought to identify the critical active site inverse PCR primed by nonoverlapping divergent primers. Both prim- ers contained an engineered common restriction site that allowed for amino acids. First, a limited set of candidate active site resi- the restriction of the PCR product by the appropriate enzyme followed dues were identified for targeted changes. Since only the amino by the ligation at the complementary DNA overhangs. In these cases, acids cysteine, serine, aspartic acid, histidine, lysine, or glu- only one primer carried the desired mutation that was incorporated into tamic acid have been identified as critical active site residues in the amplified DNA. In some cases the engineering of restriction sites proteases, an alignment of 27 TFPPs was used to quantitate into the primers was accompanied by the incorporation of silent muta- the conservation of these residues (Fig. 1). Since the active site tions. In other primers, seamless cloning and mutagenesis was per- formed by incorporating the restriction enzyme site EarIorSapIatthe is thought to be located on the cytoplasmic face of the cellular 5Ј end of each primer in the pair. These enzymes cleave just outside membrane, the analysis was further refined to focus on those their recognition site, which makes possible a primer design that en- residues located within the three major theoretical cytoplasmic sures that the recognition site is cleaved off the end of the PCR product, domains of TcpJ. Twelve conserved positions were identified, while complementary gene sequence overhangs remain to promote an- including eight in cytoplasmic domain 1, one in domain 2, and nealing prior to ligation. three in domain 3 as indicated by asterisks (Fig. 2A). Of these Immunoblot Analyses—In vivo TcpA processing was monitored in E. 125 189 coli and V. cholerae strains grown to mid-log phase except where indi- residues, only Asp and Asp are present in every single cated. 0.5 ml of cell culture was centrifuged and resuspended in 100 ␮l TFPP examined. The positions of all potentially catalytic resi- of LB. 100 ␮lof2ϫ SDS-PAGE sample buffer was added to the sample. dues with respect to the membrane topology model of TcpJ are Protease Active Site of TFPP 1505

FIG.2.Primary sequence alignment of the three major cytoplasmic domains of the TFPP family and topology relative to TcpJ. A, partial protein sequence alignment of 27 TFPP homologs. The first region shown corresponds approximately to the large cytoplasmic domain 1. The following two regions correspond precisely to the predicted residues, which constitute cytoplasmic domains 2 and 3. Asterisks designate positions in these regions for which mutations were constructed for this study. The alignment was done in DNASTAR MegAlign, using the Clustal method for alignment. B, membrane topology model of TcpJ. The predicted topology of TcpJ was deduced by comparing the hydrophobicity profile of TcpJ with the ␤-lactamase fusion data of Erwinia carotovora OutO (36) and the alkaline phosphatase fusion data of P. aeruginosa PilD (37). Cytoplasmic domains 1, 2, and 3 are indicated. All potential protease active site residues (Cys, Ser, Asp, Lys, His, Glu) have been designated. Gray shaded residues indicate each position that has been altered during this study. Active site residues are additionally indicated with a star. indicated in Fig. 2B. 16 residues including the 7 serines, 4 (S171) in Fig. 1, which, in combination, are approximately 65% cysteines, 3 aspartic acids, 1 lysine, and 1 glutamic acid indi- conserved. Asp183 and Glu183 (Fig. 1, (E183)) in combination cated as shaded residues in Fig. 1 and 2B were selected for are conserved in nearly 80% of the 27 TFPPs. Aspartic acid and mutagenesis. 13 of the 16 potential active site residues tar- glutamic acid can function equivalently as the active site of an geted for mutagenesis are all of the residues conserved at acid protease (29). Mutagenesis of Ser212 was required due to greater than the arbitrary level of 50% in TcpJ. The three the construction of the S212A/S213A double mutant, which selected residues that did not meet this criterion are Ser172, was necessary so the analysis of the phenotype of the S213A Asp183, and Ser212. Ser172 of TcpJ may be functionally equiva- would not be ambiguous due to the remaining serine in the lent to residues Ser168 and Ser171 indicated as (S168) and adjacent position. 1506 Protease Active Site of TFPP

activity. The four mutants D125A, D125N, D189A, and D189N completely lacked all peptidase activity, as would be expected when an active site residue of a protease is converted to a nonfunctional substitute. The D125E and D189E mutants re- tain the carboxylic acid moiety at the R-group terminus and both retain partial peptidase activity. Processing by S212A/ S213A double mutant is not shown because it was later deter- mined that the mutant TcpJ is unstable. In order to test the peptidase activity expressed by the tcpJ mutant constructs in V. cholerae, they were introduced into V. cholerae strain CL381, an O395 derivative with an insertional disruption in tcpJ and an in-frame deletion of vcpD, which render it completely devoid of TcpA processing activity. The resultant strains were grown under TCP-inducing conditions (LB, pH 6.5, 30 °C) and total cell protein extracts were exam- ined by Western immunoblot analysis of TcpA (Fig. 3B). The CL381 double mutant was utilized for this analysis, because disruption of tcpJ alone does not lead to a complete loss of TcpA processing, as evidenced by the detection of some processed TcpA in the J71K-1 extract. The CL381 extract shows that the residual processing is due to VcpD, a second TFPP present in V. cholerae. Similar to the results seen in JM109, tcpJ mutants S18A, S46A, C51A, S65A, C73A, C76A, S81L, E88A, S172A, D183N, and K191A exhibited peptidase activity identical to wild-type TcpJ while C48A possessed an intermediate pepti- dase activity. D125A, D125N, D189A, and D189N all com- pletely lacked peptidase activity, whereas the D125E and D189E mutants restored peptidase activity, providing further support for the assignment of Asp125 and Asp189 as the active site residues. To further extend the number of different amino acid substi- tutions analyzed at the 125 and 189 positions of TcpJ, amber (TAG) codons were individually engineered at each correspond- ing position on the pCL10 plasmid and the resultant constructs were introduced into Amber-Lys, Amber-Leu, Amber-Gln, Am- ber-Ser, and Amber-Tyr suppressor strains of E. coli. The

FIG.3.Peptidase activity exhibited by tcpJ wild-type and mu- strains were grown to mid-log phase, and whole cell protein tant strains. A, Western immunoblot analysis to detect cleavage levels extracts were examined for cleavage of TcpA by Western im- of TcpA in E. coli JM109 strains containing pCL10- and pCL11-based munoblot analysis (Fig. 3C). No amino acid substitution at mutations in tcpJ. The TcpJ-negative control lane is an extract from either position 125 or 189 exhibited any peptidase activity. The JM109 carrying only the tcpA-encoding plasmid pRTG7H3. All muta- tions are present on pCL10 except WT6His and ⌬35–81, which are on protein levels and membrane localization of the altered TcpJ pCL11. PP denotes prepilin TcpA (uncleaved), and MP denotes mature proteins were all comparable to wild-type TcpJ. pilin (cleaved). B, Western immunoblot analysis to detect cleavage TcpJ peptidase activity was also be measured by an in vitro r levels of TcpA in V. cholerae O395. J71K-1 is an O395 tcpJ::kan assay modeled after the in vitro peptidase assay devised for mutant, which retains some residual TcpA cleavage activity due to a second TFPP, VcpD. CL381 is a tcpJ::kanr, ⌬vcpD double mutant that PilD of P. aeruginosa (26). TcpA-containing membranes from lacks both TFPP activities present in V. cholerae. All remaining strains MK90 and TcpJ-containing membranes from CL318 were com- are CL381 containing pCL10- and pCL11-based mutations as in A. C, bined in the in vitro assay. A series of assays with an increasing Western immunoblot analysis to detect TcpJ D125am and D189am cleavage activity in E. coli amber-suppressor strains and a non-sup- volume of TcpJ-containing membranes was used to determine pressor control strain, JM109. The level of TcpJ detected by Western the volume of membrane preparation corresponding to 1 unit of immunoblot analysis of TcpJ in membrane fractions is shown below the TcpJ activity, which is the amount required to cleave 50% of 1 cleavage reaction for each strain. The TcpJ-negative control is an ex- ␮g of TcpA prepilin into the mature form in 1 h (Fig. 4A). tract from JM109 (pRTG7H3). The wild-type extract is from JM109 (pCL10). The tcpJ amber (TAG) mutations are present on pCL10. The peptidase activity of proteins encoded by a subset of the tcpJ mutants was determined in the in vitro cleavage assay. Asp125 and Asp189 Are the Sole Residues Required for Pepti- TcpJ proteins with alterations S46A, C51A, S65A, C73A, and dase Activity—Alteration of the targeted residues was per- C76A were all capable of cleaving TcpA prepilin in the in vitro formed by site-directed mutagenesis of the 16 selected positions assay. C48A, the double mutant S212A/S213A, and the Ala, in tcpJ present on plasmid pCL10, which also carries tcpA Glu, and Asn changes at positions 125 and 189 abolished all encoding the TcpA type 4 prepilin that is the cognate substrate peptidase activity (Fig. 4B). These results are consistent with of TcpJ. Mutant and wild-type constructs were introduced into the in vivo analysis except for the C48A, D125E, and D189E E. coli JM109, and total cell protein extracts from midlog proteins, which exhibited intermediate peptidase activity in cultures were analyzed for TcpJ protease activity by Western vivo had no peptidase activity in vitro. This is not surprising immunoblot detection of precursor and mature forms of TcpA since studies on PilD of P. aeruginosa have shown that the in (Fig. 3A). TcpJ proteins with alterations S18A, S46A, C51A, vitro assay is a more stringent test of peptidase activity than S65A, C73A, C76A, S81L, E88A, S172A, D183N, and K191A the in vivo conditions (14). The double mutant S212A/S213A exhibited peptidase activity identical to wild-type TcpJ, has no peptidase activity due to the fact that the enzyme is whereas C48A displayed an intermediate level of peptidase unstable as shown by the lack of any TcpJ present in TcpJ Protease Active Site of TFPP 1507

FIG.4.In vitro assay for peptidase ac- tivity of wild-type and altered TcpJ proteins. A, the in vitro cleavage assay with increasing amounts of TcpJ containing membrane fraction. 1 unit cleaves 50% of 1 ␮g of TcpA prepilin (PP) to the mature pilin form (MP)in1hat37°C.B, the activity of 15 mutants of TcpJ in the in vitro peptidase assay. The TcpJ(6)His and TcpJ⌬35–81 de- rivatives are in pCL11 constructions. All other mutations are in pCL9. A Western im- munoblot analysis showing levels of TcpJ in the membrane fractions is shown below each reaction lane. TcpJ(6)His and TcpJ⌬35–81 TcpJ levels were detected by Tetra-His Ab.

immunoblot. All other TcpJ derivatives were detected at levels approximately equal to wild-type except for C48A and C51A, which gave faint or undetectable TcpJ signals. This is likely due to the anti-TcpJ antiserum, which is directed against a peptide that encompasses Cys48 and Cys51. It is likely that C48A and C51A mutants alter the epitope in TcpJ sufficiently so that the protein is not detected by the antiserum. Cytoplasmic Domain 1 Is Not Required for TcpJ Peptidase Activity—In order to definitively determine whether the cys- teine residues or any portion of cytoplasmic domain 1 is re- quired for TFPP cleavage activity, an internal in-frame dele- tion corresponding to positions 35–81 of TcpJ was constructed. Since our TcpJ antibody is directed against a peptide within the region removed by the deletion, the construction was made in the 6-His epitope-tagged version of tcpJ contained on plas- mid pCL11, yielding plasmid pCL11⌬35–81. TcpJ (6)His and the deletion derivative expressed from pCL11 and pCL11⌬35–81 were both able to cleave TcpA to equivalent extents in E. coli (Fig. 3A), V. cholerae (Fig. 3B), or in vitro (Fig. 4B). Interestingly, TcpJ (6)His⌬35–81 was found to process TcpA efficiently enough to restore piliation to the tcpJ mutant strain, J71K-1 (Fig. 5). VcpD Requires a Pair of Aspartic Acid Residues Correspond- ing to Those Necessary for TcpJ Activity—In order to determine the extent to which the TcpJ requirement for the aspartic acid residue pair can be generalized to other members of the TFPP family, the corresponding changes of D147A and D212A were made in VcpD. The VcpD TFPP is responsible for processing FIG.5.The complementation of V. cholerae O395 tcpJ mutant by TcpJ(6)His⌬35–81 for TcpA cleavage and TCP biogenesis. A, type 4 prepilins such as those involved in mannose-sensitive Transmission electron microscopy of V. cholerae strains to detect for pilus formation and processes the extracellular protein secre- their ability to secrete and assemble TCP. Strains: 1, O395; 2, J71K-1; tion type 4 prepilin-like proteins required for the secretion of 3, J71K-1 pCL11⌬35–81; 4, J71K-1 pCL11. Arrows indicate lateral toxin and other enzymes by V. cholerae. VcpD can also cleave bundles of TCP. Magnification is 50,000-fold. B, Western immunoblot analysis to detect levels of precursor (PP) and mature (MP) forms of TcpA prepilin, although the cognate TFPP for TcpA is TcpJ TcpA produced by the same V. cholerae strains as in A. (19). Wild-type, D147A, and D212A forms of VcpD expressed from plasmid pJM294 derivatives were introduced into E. coli ogous result was seen with the cleavage of the type 4 prepilin- JM109 carrying the tcpA-expressing plasmid pRTH3G7. like protein substrate of VcpD, EpsI (Fig. 6B). These experi- Strains were grown to midlog phase, whole cell protein extracts ments demonstrate that alanine substitutions at Asp147 and were made prepared, and the extracts were examined by SDS- Asp212, the aspartic acid residues in VcpD that correspond to PAGE and Western immunoblot analysis with anti-TcpA anti- protease active site Asp125 and Asp189 of TcpJ, completely serum (Fig. 6A). Wild-type VcpD cleaved TcpA prepilin com- eliminate peptidase activity for multiple substrates. pletely under these conditions, whereas the D147A and D212A Protease Inhibitor Studies of TcpJ Activity Support the Es- mutant derivatives were totally defective. Protein levels of sential Role of the Aspartic Acid Residues for Protease Activi- VcpD in the four strains were determined by Western immu- ty—Utilizing the TcpJ in vitro cleavage assay, a number of noblot analysis using the anti-TcpJ antiserum, which cross- chemical protease inhibitors were examined for their ability to reacts with VcpD. The D147A and D212A forms of VcpD are block the peptidase activity of TcpJ (Table I). Two or more present at levels similar to those in wild-type VcpD. An anal- inhibitors specific to each class of protease family were tested 1508 Protease Active Site of TFPP

tivity of TFPPs to commercially available protease inhibitors. In addition, none of the consensus protease active site motifs present in the vast majority of other protease families are obvious in this peptidase family. For example, the typical cat- alytic residues of serine proteases are the three amino acids serine, aspartic acid, and histidine, but several families of serine proteases such as the signal peptidase I family utilize only serine and lysine as the catalytic residues (30). The most conserved histidine residue in the TFPP family, His30, is only present in 30% of the members (Fig. 1, (H30)), thus ruling out involvement of the S/D/H motif in the catalytic mechanism. The numerous highly conserved serines as well as Lys191, conserved in nearly 80% of the TFPP family members, raised the possi- bility that the proteolytic activity might involve a Ser/Lys cat- alytic mechanism. A further possibility was that the TFPP could be a cysteine protease. This potential mechanism was suggested by the pilD mutational analysis of the 4 highly conserved cysteines in the peptidase family. Mutations at the 4 cysteines failed to completely eliminate peptidase activity in vivo, but in an in vitro assay these altered proteins were nearly completely deficient of peptidase activity (14). However, since all cysteine proteases require both a cysteine and a histidine as the catalytic dyad (15), the lack of a highly conserved histidine FIG.6.Mutations that alter the predicted protease active site aspartic acid residues in VcpD eliminate peptidase activity. A, in the TFPP family excludes any known cysteine catalytic Western immunoblot analysis using anti-TcpA antisera to detect cleav- mechanism. The possibility of an aspartic acid protease mech- age levels of TcpA in JM109 by VcpD mutants. All strains are JM109 anism is strongly suggested for the TFPP family due to the and carry the tcpA-expressing plasmid, pRTG7H3. The VcpD negative presence of two completely conserved aspartic acid residues strain contains no other plasmid. The wild-type and mutant alleles of vcpD are expressed from pJM294 derivatives. A Western immunoblot corresponding to TcpJ positions 125 and 189 in domains 2 and analysis showing levels of VcpD for each strain is also shown. B, 3, respectively (Fig. 2B). However, Asp125 and Asp189 do not Western immunoblot analysis using Tetra-His Ab to detect cleavage exist in the motif D(T/S)G, which is common to the majority of levels of EpsI(6)His in JM109 by VcpD derivatives. All strains are aspartic acid proteases (31). In addition, the pH optimum, JM109 and carry the EpsI(6)His-expressing plasmid, pJM290 and vcpD derivatives as in A. which has been determined to be near neutral for TcpJ (data not shown) and PilD of P. aeruginosa (26), is in contrast to the highly acidic pH optimum of the majority of aspartic acid for their ability to prevent TcpJ peptidase activity. The major- proteases (32). The final protease family comprises the metal- ity of the inhibitors had little or no effect on the peptidase loproteases, for which over half contain the active site motif, activity, as measured by the comparison of cleaved TcpA pro- HEXXH (32). The TFPP family lacks any homology to the duced in the in vitro assay in the presence or absence of inhib- HEXXH metalloprotease motif. In light of this sequence align- itor. All inhibitors were tested at the maximum suggested ment analysis, an aspartic acid protease mechanism was concentration except for the EDAC/glycinamide inhibition pro- deemed to be the most probable correct assignment for TFPPs. tocol, for which it was not required. Of all the common protease However, all potential protease active site residues conserved inhibitors used, only NEM inhibited cleavage greater than at greater than 50% were targeted for mutagenesis in order to 50%. This agrees with the strong inhibitory effect of NEM on determine the active site with certainty. PilD of P. aeruginosa (14). In contrast, a combination of EDAC and glycinamide, which is known to modify aspartic acid resi- The results from the mutational analysis of TcpJ at the 16 dues and is the only method known to inhibit the non-pepsin- selected positions clearly demonstrate that only the two aspar- type acid proteinases such as proteinase A from Aspergillus tic acids corresponding to positions 125 and 189 of TcpJ are (28), completely inhibited TcpJ peptidase activity. A 20–100- essential for protease activity. Substitution of the amino acids fold excess of the maximum suggested concentration of pepsta- alanine, asparagine, leucine, serine, tyrosine, glutamine, and tin only exhibited a minor inhibitory effect on peptidase activ- lysine into positions 125 and 189 of TcpJ produced a stable and ity, thus ruling out a pepsin-like active site in TcpJ. properly localized enzyme that lacked any protease activity. The cause for this loss of protease activity of the mutants at DISCUSSION positions 125 and 189 was determined to be the loss of the The action of a family of leader peptidases known as the carboxylic acid group of the aspartic acid. The carboxylic acid TFPPs is required for extracellular protein secretion, genetic functional groups of the active site aspartic acids directly par- exchange, and organelle formation in a wide array of bacterial ticipate in the general acid-base catalysis that causes the hy- systems. Their involvement in the processing of prepilins to drolysis (cleavage) of the peptide bond (32). When the carbox- mature forms is a prerequisite to type 4 pilin secretion and ylic acid functional group was restored at positions 125 or 189 assembly of the pilus structure. Other secreted proteins that by the substitution of glutamic acid, the protease activity was have type 4 leader peptides, but are not pilin subunits, are also restored. The additional one carbon in length of the glu- referred to as type 4 prepilin-like proteins. Such proteins are tamic acid R-group was less tolerated at position 189 than 125, components of general secretory pathways involved in protein suggesting that the location in space of the R-group carboxylic secretion and pilus biogenesis, the natural transformation acid at position 189 is critical to protease activity. It can be pathways, and conjugal plasmid DNA transfer pathways. inferred from the constraint at position 189 that the carboxylic Despite the probable ubiquitous presence of TFPPs among acid group of Asp189 directly participates in the initial chemical bacterial species, identification of the protease active site has reduction of the prepilin peptide bond while Asp125 contributes been elusive. This may have been due in part to the insensi- to the hydrolysis through interaction with a water molecule. Protease Active Site of TFPP 1509

TABLE I The effect of chemical inhibitors on the in vitro TcpJ cleavage assay The effect of chemical protease inhibitors on the peptidase activity of TcpJ was determined by incubating the inhibitor with an amount of membrane preparation known to contain 1 unit of activity of TcpJ for 30 min at room temperature. The TcpJ/inhibitor mixture is then tested for peptidase activity in the in vitro processing assay (see “Materials and Methods”). Amounts of cleaved TcpA prepilin were quantitated and applied to the formula: % cleavage ϭ (amount of cleaved TcpA in inhibition assay/amount of cleaved TcpA in no inhibitor control assay) ϫ 100. The inhibition by EDAC/glycinamide is a two-step protocol conducted in acidic conditions (see “Materials and Methods”).

Chemical(s) Specificity Suggested concentration Concentration Cleavage

% No inhibitor 100 E-64 Cysteine proteases (1.4–28 ␮M)a (1–10 ␮M)b 28 ␮M 96 Calpain inhibitor I Cysteine proteases 45 ␮Ma 45 ␮M 87 NEM Cysteine proteases 1 mMc 1mM 15 Aprotinin Serine proteases (0.01–0.3 ␮M)a 0.3 ␮M 100 3,4-DCI Serine proteases (5–200 ␮M)a (5–100 ␮M)b 100 ␮M 65 200 ␮M 54 Pefabloc SC Serine proteases (0.4–4 mM)a 2mM 62 4mM 50 Leupeptin Ser and Cys proteases 1␮Ma (1–10 ␮M)b 10 ␮M 85 PMSF Ser and Cys proteases (0.1–1 mM)a 3mMc 1mM 89 3mM 53 Phosphoramidon Metallopeptidases (0.007–0.6 mM)a 0.6 mM 89 EDTA-Na Metalloproteases (0.5–1.3 mM)a 1.3 mM 100 Bestatin Amino peptidase(metallo-) 130 ␮Ma 130 ␮M 81 Pepstatin Aspartic proteases 1 ␮Ma (1–5 ␮M)b 1 ␮M 95 10 ␮M 92 100 ␮M 74 EDAC/glycinamide Acid protease 0.1 M/1.0 Md 0.1 M/0.2 M 2 a Roche Molecular Biochemicals. b Prolysis, a protease and protease inhibitor Web server (T. Moreau). c Ref. 14. d K. Takahashi, personal communication.

Demonstration that the complete loss of protease activity for all all TFPPs share the aspartic acid active site. The result with amino acid substitutions at positions 125 and 189 was not VcpD was of further importance because its cognate targets are simply due to a conformational change was also addressed by within the type 4a subgroup of type 4 prepilins or prepilin-like the asparagine and serine substitutions at these positions. proteins, which have a short signal peptide of typically 5–8 Asparagine and serine have small polar R-groups similar to the amino acids. In contrast, the cognate substrate of TcpJ is TcpA, R-group of aspartic acid and thus would likely maintain the which is a type 4b prepilin with a 25-amino acid leader peptide. wild-type conformation of the enzyme. The chemical protease inhibitor studies served to confirm the Only one of the altered forms of TcpJ, the double mutant mutational analysis that determined that TcpJ is not a cysteine S212A/S213A form, was unstable and could not be assessed for protease, , metalloprotease, or a typical activity. The replacement of both hydrophilic serine acid protease. The protocol using EDAC and glycinamide residues with hydrophobic alanine residues occurs at a loop strongly implicates the dependence on aspartic acids for activ- region between transmembrane domains and likely prevented ity of the enzyme. Inhibition by the EDAC/glycinamide protocol the proper integration of the mutant peptidase into the mem- also suggests that the active site aspartic acids are accessible to brane, resulting in its degradation. In lieu of a cleavage activity chemical inhibitors and not shielded by the protein conforma- result of the S212A/S213A mutant, other evidence indicates a tion of the cytoplasmic domains. The only protease inhibitor protease active site serine does not exist at position 212 or 213. other than EDAC/glycinamide that inhibited at greater than Ser212 and Ser213 are both located on the periplasmic side on 50% was NEM, which acts by modifying thiol groups of the the inner membrane and could not participate in a protease cysteine R-groups of cysteine proteases. The partial inhibition reaction occurring on the cytoplasmic side on the membrane. of TcpJ could occur due to the modification of Cys48, which Additionally, the only possible catalytic partners are Lys191, when altered by mutation to an alanine, caused a comparable which is not required for protease activity or Lys180, which if loss of protease activity. This would suggest that, when Cys48 is considered functionally equivalent to Lys182 (Fig. 1, (K182)) not in its natural form, it can lead to a conformational change would only be found in approximately 60% of peptidases. in the enzyme that partially prevents the functioning of the Conclusive evidence that no amino acid residue between protease active site. position 35 and 81 is required individually, or in combination, The TFPPs differ from the majority of aspartic acid proteases for peptidase activity or for TCP biogenesis was provided by the in that the active site aspartic acids are not found in the internal deletion in TcpJ that removed the majority of cytoplas- D(T/S)G motif, the optimum pH for in vitro activity is near mic domain 1. This eliminates the possibility that two or more neutral as opposed to pH 2–4, and peptidase activity is not of the four conserved cysteines act collectively in some manner inhibited by pepstatin. Other aspartic acid proteases exist that as a protease active site cysteine. The deletion also demon- possess these unusual traits individually. Signal peptidase II, strates that domain 1 is not in any way required for substrate an aspartic acid protease that cleaves prolipoproteins, does not recognition, binding, orientation, or any function that leads to possess the D(T/S)G motif at the active site (33). The human the assembly of the pilin into a pilus structure. aspartic acid protease has a pH optimum range of 5.5–7.5 Evidence for conservation of the protease active site among (34). Pseudomonas sp. 101 carboxyl proteinase is a bacterial multiple members of the TFPP family was provided by muta- aspartic acid protease that is insensitive to pepstatin (35). tions that altered either of the two aspartic acids of VcpD Aspartic acid proteases, which are insensitive to pepstatin, are corresponding to the active site residues in TcpJ. These alter- often referred to as non-pepsin-like acid proteases (28). The ations abolished VcpD activity, thus strongly suggesting that majority of aspartic acid proteases like pepsin and human 1510 Protease Active Site of TFPP immunodeficiency virus type 1 protease are bilobed molecules 8. Strom, M. S., Nunn, D., and Lory, S. (1991) J. Bacteriol. 173, 1175–1180 9. Pugsley, A. P., and Reyss, I. (1990) Mol. Microbiol. 4, 365–379 in which the active site cleft is located between the lobes with 10. Pepe, C. M., Eklund, M. W., and Strom, M. S. (1996) Mol. Microbiol. 19, an active site aspartic acid contributed from each lobe. In this 857–869 respect, the TFPPs display a remarkable similarity. The active 11. Dubnau, D. (1997) Gene (Amst.) 192, 191–198 12. Pestova, E. V., and Morrison, D. A. (1998) J. Bacteriol. 180, 2701–2710 site can be envisioned as a cleft between two lobes correspond- 13. Strom, M. S., and Lory, S. (1987) J. Bacteriol. 169, 3181–3188 ing to domains 2 and 3 with one active site aspartic acid residue 14. Strom, M. S., Bergman, P., and Lory, S. (1993) J. Biol. Chem. 268, 15788–15794 in each lobe. 15. Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 461–486 The comprehensive mutational analysis and chemical inhib- 16. Hu, N. T., Lee, P. F., and Chen, C. (1995) Mol. Microbiol. 18, 769–777 itor studies that led to the discovery of the active site of TcpJ 17. Rawlings, N. D., and Barrett, A. J. (1999) Nucleic Acids Res. 27, 325–331 18. Tschantz, W. R., Sung, M., Delgado-Partin, V. M., and Dalbey, R. E. (1993) are ultimately most significant in that they allow for the pre- J. Biol. Chem. 268, 27349–27354 diction of the protease active site of the entire TFPP family. 19. Marsh, J. W., and Taylor R. K. (1998) Mol. Microbiol. 29, 1481–1492 The identity of the active site, as well as the ability to specifi- 20. Russel, M., and Model, P. (1984) J. Bacteriol. 159, 1034–1039 21. Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene (Amst.) 33, 103–119 cally target the active site aspartic acids by a chemical protocol, 22. Kaufman, M. R., Seyer, J. M., and Taylor, R. K. (1991) Genes Dev. 5, provide the foundation for the development of specific protease 1834–1846 inhibitors of all members of the TFPP family. Such inhibitors 23. Shaw, C. E., and Taylor, R. K. (1990) Infect. Immun. 58, 3042–3049 24. Tabor, S., and Richardson, C. C. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, would have substantial antimicrobial effects on a wide range of 1074–1078 pathogenic bacteria. 25. Pfau, J. D., and Taylor, R. K. (1998) J. Bacteriol. 180, 4724–4733 26. Nunn, D. N., and Lory, S. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 3281–3285 Acknowledgments—We thank J. Marsh for strains and plasmids, 27. Hoare, D. G., and Koshland, D. E., Jr. (1966) J. Am. Chem. Soc. 88, 2057–2058 28. Takahashi, K., Tanokura, M., Inoue, H., Kojima, M., Muto, Y., Yamasaki, M., Ripple Electron Microscope Facility at Dartmouth College for electron Makabe, O., Kimura, T., Takizawa, T., Hamaya, T., Suzuki, E., and Miyano, microscopy, Dartmouth College Molecular Biology Core Facility for H. (1991) Structure and Function of the Aspartic Proteinases, pp. 203–211, oligonucleotide synthesis and DNA sequencing, and D. Pippen for help- Plenum, New York ful discussions. 29. Takahashi, K., Kagami, N., Huang, X. P., Kojima, M., and Inoue, H. (1998) Adv. in Exp. Med. Biol. 436, 275–282 REFERENCES 30. Rawlings, N. D., and Barrett, A. J. (1994) Methods Enzymol. 244, 19–61 1. Strom, M. S., Nunn, D. N., and Lory, S. (1993) Proc. Natl. Acad. Sci. U. S. A. 31. Rawlings, N. D., and Barrett, A. J. (1995) Methods Enzymol. 248, 105–120 90, 2404–2408 32. Rao, M. B., Tanksale, A. M., Ghatge, M. S., and Deshpande, V. V. (1998) 2. Pepe, J. C., and Lory, S. (1998) J. Biol. Chem. 273, 19120–19129 Microbiol. Mol. Biol. Rev. 62, 597–635 3. Bieber, D., Ramer, S. W., Wu, C. Y., Murray, W. J., Tobe, T., Fernandez, R., 33. Sankaran, K., and Wu, H. C. (1995) Methods Enzymol. 248, 169–180 and Schoolnik, G. K. (1998) Science 280, 2114–2118 34. Inagami, T. (1981) Biochemical Regulation of Blood Pressure, pp. 39–73, John 4. Tonjum, T., and Koomey, M. (1997) Gene (Amst.) 192, 155–163 Wiley & Sons, Inc., New York 5. Wu, S. S., and Kaiser, D. (1995) Mol. Microbiol. 18, 547–558 35. Oda, K., Takahashi, S., Ito, M., and Dunn, B. M. (1998) Adv. Exp. Med. Biol. 6. Pugsley, A. P. (1993) Microbiol. Rev. 57, 50–108 436, 349–353 7. Sandkvist, M., Michel, L. O., Hough, L. P., Morales, V. M., Bagdasarian, M., 36. Reeves, P. J., Douglas, P., and Salmond, G. P. (1994) Mol. Microbiol. 12, Koomey, M., DiRita, V. J., and Bagdasarian, M. (1997) J. Bacteriol. 179, 445–457 6994–7003 37. Lory, S., and Strom, M. S. (1997) Gene (Amst.) 192, 117–121