Isolation and characterization of pcp, a gene encoding a pyrrolidone carboxyl peptidase in Staphylococcus aureus

Gen6 166 (1995) 95-99 © 1995 Elsevier Science B.V. All rights reserved. 0378-1119/95/$09.50

95

GENE 09275

Isolation and characterization of pcp, a gene encoding a pyrrolidone carboxyl peptidase in Staphylococcus aureus (Enzyme; gene expression; Gram-positive bacteria; nucleotide sequence)

Joseph M. Patti ~, Amy Schneider ~, N o r m a Garza" and Jeffrey O. Boles b aAlbert B. Alkek Institute of Biosciences and Technology, Center for Extracellular Matrix Biology, Texas A&M University, Houston, TX 77030, USA; and bDepartment of Chemistry, Tennessee Technological University, Box 5055, Cookeville, TN 38505, USA. Tel. (1-615) 372-3844

Received by M.J. Benedik: 8 May 1995; Revised/Accepted: 21 July/24 July 1995; Received at publishers: 18 August 1995

SUMMARY

The pcp gene, encoding a pyrrolidone carboxyl peptidase (PYRase), was cloned from a LGTll genomic library prepared from Staphylococcus aureus FDA 574 and sequenced. The pcp gene is located 740 bp downstream from cna, a gene that encodes a collagen-binding adhesin in S. aureus. S. aureus pcp encodes a 212-amino-acid (aa) polypeptide. The pcp gene was overexpressed in Escherichia coli and the PYRase purified to homogeneity. The recombinant enzyme exhibited biological activitity, as determined using the chromogenic substrate L-pyroglutamyl-[3-napthylamide. Biochemical analysis of the PYRase using thiol-blocking chemicals suggested that the enyzme belongs to the cysteine peptidase family. Moreover, multiple sequence alignment revealed a high degree of similarity to previously described bacterial PYRases. This family of peptidases has been used to selectively remove the N-terminal pyrrolidone carboxylic acid residue found on certain blocked proteins and peptides prior to aa sequencing. However, the exact biological role of PYRases has yet to be elucidated.

INTRODUCTION

Pyrrolidone carboxyl peptidase (PCP; EC 3.4.11.8) is an enzyme that selectively cleaves L-pyroglutamic acid (PYR) from the N terminus of peptides and polypeptide chains (Doolittle and Armentrout, 1968). In addition, the Correspondence to: Dr. J.M. Patti, Albert B. Alkek Institute of Biosciences and Technology, Center for Extracellular Matrix Biology, Texas A&M University, Houston, TX 77030, USA. Tel. (1-713) 677-7556; Fax (1-713) 677-7576; e-mail: [email protected]

Abbreviations: A, absorbance (1 cm); aa, amino acid(s); bp, base pair(s); cna, gene encoding Sa collagen-binding adhesin; dNTP, deoxynucleo-

tide triphosphate; IPTG, isopropyl-13-D-thiogalactopyranoside; kb, kilobase(s) or 1000 bp; nt, nucleotide(s); ORF, open reading frame; PAGE, polyacrylamide-gel electrophoresis; PCP, pyrrolidone carboxyl peptidase; pcp, gene encoding a Sa PCP; PCR, polymerase chain reaction; PYR, L-pyroglutamic acid; Pyr-[3NA, L-pyroglutamyl-13-napthylamide; PYRase, bacterial pyrrolidone carboxyl peptidase; RBS, ribosomebinding site(s); re-, recombinant; Sa, Staphylococcus aureus; SDS, sodium dodecyl sulfate; [], denotes plasmid-carrier state. SSD1 0378-1119(95)00561-7

enzyme is capable of hydrolyzing the peptide bond of chromogenic substrates such as L-pyroglutamyl-13napthylamide (Pyr-13NA), pyroglutamyl-p-nitroanilide, and pyroglutamyl-4-methyl courinylamide (Fujiwara and Tsura, 1978). This class of enzyme commonly referred to as PYRase (Mitchell et al., 1987) is found in both Gram + and Gram- bacteria (Mulczyk and Szewczuk, 1970), plants, animal and human tissues (Szewczuk and Kwiatkowska, 1970). Although the biological role of the PYRases have yet to be elucidated, the enzymology of the protein has been studied for several years and is well established. Because of their ability to remove pyrrolidone-5-carboxylic acid residues blocking the N terminus of proteins, purified PYRases have been used in the preparation of proteins prior to sequential Edman (1950) degradation. The structural genes encoding PYRases from several bacteria including, Streptococcus pyogenes (Cleuziat et al., 1992), Bacillus subtilis (Awad~ et al., 1992), Bacillus

96 amyloliquefaciens (Yoshimoto et al., 1993) and Pseudomonas fluorescens (Gonzal6s and RobertBaudouy, 1994) have recently been cloned and sequenced. A preliminarycharacterization of the deduced aa sequences from the PYRase genes revealed a high degree of similarity among the group (Gonzal6s and Robert-Baudouy, 1994), including the conservation of CysT M (numbering based on the aa sequence from B. amyloliquefaciens; Yoshimoto et al., 1993), a residue proposed to be involved in the catalytic reaction (Yoshimoto et al., 1993). A noted absence in homology to other known m a m m a l i a n pyrrolidone carboxyl peptidases, such as the enzyme specific for the thyrotropin-releasing hormone peptide (O'Connor and O'Cuian, 1984) indicates that bacterial PYRases may belong to a new structural class of peptidases (Cleuziat, 1992). The pyrrolidonyl peptidase test has been a useful diagnostic method for the differentiation of Enterobacteriaceae (Mulczyk and Szewczuk, 1970) and staphylococci (Mulczyk and Szewczuk, 1972). Although this test was developed over two decades ago, neither the gene encoding the pyrrolidone carboxyl peptidase (Pcp) nor the enzyme in staphylococci have been previously isolated. This communication reports the cloning of pcp, the gene encoding the Sa PYRase and describes a preliminary characterization of the recombinant protein produced in Escherichia coll.

EXPERIMENTAL AND DISCUSSION

(a) Cloning of the Sa PYRase-encoding gene (pcp) We have previously reported the cloning and sequencing of cna, the gene that encodes a collagen-binding Sa adhesin in (Patti et al., 1992). Clone XCOLI contained two (EcoRI-PstI) fragments of approx. 2.9 kb and 1.7 kb that were subcloned into pUC18 and transformed into E. coli TG-1 to generate clones p C O L 1 0 and p C O L l l , respectively (Fig. l). D N A sequencing of p C O L l l

I 2.9 kb pCOLIO

EcoR I 2

]

4.6 kb XCOL1

500

1000

cnagene

1500

2000

I

2500

Pst I 3(500

t 7 kbpCOL11

3500

4000

J

EcoFt I

~_ 740nt

Fig. 1. Orientation of clones used to characterize the Sa pcp gene. Subclone pCOL11 contains 579 bp of the pcp gene, while the remaining 3' end of the gene is found in subclone pCOL10. Boxes indicate the locations and the arrows indicate the orientation of the respectivegenes. The asterisks denote the location of the stop codons within the identified genes. Standard molecular biological techniques were used to clone the pcp gene (Ausubel et al., 1989; Sambrook et al., 1989).

revealed a 579-bp ORF, suggesting that another putative gene was present within the 1.7-kb fragment. Even though an O R F of considerable size was present in p C O L l l , a stop codon could not be identified prior to the PstI cloning site. However, when the D N A sequence of pCOL10 was analyzed, a short O R F beginning from the PstI site and proceeding in the 3'-5' direction and containing a stop codon was found. Assembly of the D N A sequences from p C O L 1 0 and p C O L l l produced one intact O R F of 636 nt (Fig. 1). Sequencing of additional clones isolated by rescreening the k g t l l Sa genomic library with an internal fragment of the pcp gene confirmed the orientation of the 3' end of the gene.

(b) Sequence analysis of the pcp gene and the deduced protein The complete Sa D N A sequence of the 636-nt pep gene and flanking regions was determined (Fig. 2). Computer analysis revealed only one O R F located between nt 204 (ATG) and nt 842 (TAG) coding for a 212-aa polypeptide (23 225 Da) with a p l of 5.2. A putative RBS was found 8-11 nt upstream from the start codon. The G + C

G A G T G G C A A C A A C G q ' F ? A A C A A A ~ T G A T ] ' T A C A T C - A A C A T G C I T A G T A q ' F A A q ' F A 60 AATACC~CACGCITTGAAAATCCGATFFATAAAGG'Vlqq'?CAA~'I'I' Fi'±' 120 q'?ATGCGCATGCATAACCTGATATG'F I'I'ITAATTATGCGCATGTTATACTAAAq'FAAATT 180 q'FAATACTGCGGGG T G T C E T A A A A ~ Z C A C A q q ' I T A G T A A C A G G G T T C G C G C C ~ C A 240 M H I L V T G F A P F D 12 A T C A A G A T A T T A A T C C T I L q ' I ~ G G A A G C T G T G A C T C A A C T A G A A A A T A ~ T A T I ~ G C A C A C 300 N Q D I N P S W E A V T Q L E N I I G T 32 ATACAATCGAT~.AAq'F~kAACTACC~CCqLT F I"Y~AC~kAAGTAGATACFAq'TAT~JkATA 360 H T I D K L K L P T S F K K V D T I I N 52 AAACGq'FC43CAq~:T~TCAq'FATGAEX]q*YGTACTAGCTATAC~ACAAC~EI3GTAGAA 420 K T L A S N H Y D V V L A I G Q A G G R 72 A T G C C A T T A C C C CAGAACGTGTCGCCAq'FAATATEB3ATGATGCAC GTA~qL'CAGATAATG 480 N A I T P E R V A I N I D D A R I P D N 92 A T G A T E ~ A A C C T A T T G A T C A A G C C A T T C A q ' F F A G A C G G T G CGC CACGq'FA'I"t"i"I"PCAA 540 D D F Q P I D Q A I H L D G A P R Y F S 112 A T I T A C C A G T T A A A G C A A T G A C T C A A A G T G T T A T T A A C C A A G G A ~ C T C g G A G C A C q ' I T 600 N L P V K A M T Q S V I N Q G L P G A L 132 C A A A T A G C G C A G G T A C G T I~:GTATGTAAT~:ACGTACTTTATCACTTAGGq*FAqq~TACAAG 660 S N S A G T F V C N H V L Y H L G Y L Q 152 ATAAGCAq'FAC CCTCAC CTI~CGATTCGGATTTAq'I~ATGTG CCATATATAC<~AGAGCAAG 72(] D K H Y P H L R F G F I H V P Y I P E Q 172 TCGT]~GGTAAATCCGATACACCATCTATCC C A T T A G A A C A G A T A G T T G CAGG T T T G A C T G 780 V V G K S D T P S M P L E Q I V A G L T 192 C A G C C A E ' F G A A G C T A T E ~ G A T C A C G A q l I 3 A T ~ A C G T A T A G C T C T A G G C A C A A C G G A A T 840 A A I E A I S D H D D L R I A L G T T E 212 AG~CTATAAATGCAC T A A C A A A A T A C A T T C C T T A A A T G A C T A A C A A ~ 2"TAATAGGGT 900 AATACTTACGGAAGTATG'I' I'I"I'ATTTATGGG GGAGGAATTAATAAT(3ACTACAAAAACGG 960 TATTTGAqlDTCATT~3ATAq]3(;G TT?AG GATAqTPAGTAAATGTGTATQATACq~IZZG AAAG 1020 q~YGAAA 1026

Fig. 2. Nucleotide sequence of the pep gene, including upstream and downstream sequence information and deduced aa sequence of the Sa pyrrolidone carboxyl peptidase (PYRase), The putative RBS is underlined, the start codon is shown in bold-face type, and the stop codon is indicated by an asterisk. Methods: Alkali-denatured plasmid DNA containing the pep gene was sequenced by the dideoxy-chain termination method (Sanger et al., 1977), using [-~-3SS]dATP (Amersham, Arlington Heights, IL, USA) and Sequenase Version 2.0 (US Biochemical,Cleveland, OH, USA). Forward and reverse universal primers, as well as custom made oligodeoxynucleotides (Gene Technologies Laboratory, Institute of Developmental and Molecular Biology, Texas A&M University)were used as sequencing primers. The reported nt sequence has been deposited in the GenBank database under accession No. U19770.

97 content of pcp, calculated as 35%, correlates well with that of the staphylococcal g e n o m e (Kloos and Schleifer, 1986). The deduced aa sequence from the 636-nt O R F was c o m p a r e d with protein sequences in the National Center for Biotechnology I n f o r m a t i o n databases using a B L A S T P search (Altschul et al., 1990). Significant similarities were detected with bacterial PYRases from S. pyogenes (Cleuziat et al., 1992), B. subtilis (Awad6 et al., 1992), B. amyloliquefaciens (Yoshimoto et al., 1993) and P.fluorescens (Gonzal6s and R o b e r t - B a u d o u y , 1994). The Sa P Y R a s e exhibited the highest degree of similarity to the PYRase from S. pyogenes (50% identity) (Cleuziat et al., 1992). Biochemical characterization of the published PYRases suggest that they function as cysteine peptidases and therefore m a y possess a putative catalytic triad of Cys and His with Asp, Glu, Asn, or Gln (Rawlings and Barret, 1993). The sequence alignment revealed the conservation of residues Cys TM and His 165 within the Sa P Y R a s e (numbering based on Fig. 2). The potential third residue involved in enzyme catalysis could be Glu 78, Asp 9~, or Asp z°3. Site-directed mutagenesis of the B. amyloliquefaciens P Y R a s e has shown that Cys 144 (Cys TM of B. amyloliquefaciens P Y R a s e is c o m p a r a b l e to Cys 141 of Sa PYRase) is critical for full enzymatic activity (Yoshimoto et al., 1993); however, the other aa involved in catalysis have not been experimentally determined.

kDa

(c) Expression of the pcp gene and purification of the re-protein

Fig. 3. Expression and purification of the recombinant Sa PYRase. Lanes 1, Mark 12 molecular mass standards (Novex, San Diego, CA, USA); 2, whole cell lysate from E. coli[pQE-700] prior to IPTG induction; 3, whole cell lysate from E. coli[pQE-700] after IPTG induction; and 4, purified PYRase following metal chelate chromatography. Methods:Samples were analyzed by 0.1% SDS-12% PAGE and stained with Coomassie brilliant blue R-250. A saturated overnight culture of E. coil JM101[pQE-700] was diluted 1:50 in LB supplemented with ampicillin and allowed to grow until the culture reached a n A60o of 0.6 0.7. IPTG (final concentration 0.2 raM) was added to the cells and growth continued for another 4 h at 37°C. The bacteria were collected by centrifugation and the bacterial pellets were resuspended in PBS (10 mM phosphate/0.14 M NaC1, pH 7.4). The cells were lysed by passage through a French press twice at 20000 Ib/in2. The bacterial lysate was centrifuged at 102 000 × g for 10 rain to remove bacterial debris. The supernatant containing soluble proteins was filtered through a 0.45 ~tm membrane (Coming; Corning, NY, USA) and retained for further purification. The re-Sa His6-PYRase protein was purified by immobilized metal-chelate-affinity chromatography. A column containing iminodiacetic acid Sepharose 6B Fast Flow (Sigma, St. Louis, MO, USA), connected to a FPLC system, was charged with 150 mM Ni 2+ and equilibrated with buffer A (5 mM imidazole/0.5 M NaCI/20 mM Tris, pH 7.9). After equilibration, the bacterial supernatant was applied to the column and the column was washed with 10 bed volumes of buffer A. Subsequently, the column was eluted with buffer B (200 mM imidazole/0.5 M NaCI/20 mM Tris, pH 7.9). The eluate was monitored for protein by the absorbance at 280 nm and peak fractions were analyzed by SDS-PAGE, as above.

An expression plasmid pQE-700, containing the P C R amplified pep gene, was constructed using the E. coli vector p Q E - 3 0 (Qiagen, Catsworth, CA, USA). To construct the plasmid, the pep gene was amplified from Sa F D A 574 genomic D N A ( M a r m u r , 1961) by P C R together with flanking oligodeoxynucleotides, p y r l (5'GGTACCGGATCCATGCACATTTTAGTAACAGG G T T C ) and pyr6 ( 5 ' - G G T A C C G T C G A C T T T A A G G A A T G T A T T T T G T T A ) . After amplification, the P C R p r o d u c t was treated with proteinase K, phenol chloroform extracted, and ethanol precipitated as described (Crowe et al., 1991). The P C R p r o d u c t was digested with the B a m H I + S a l I , then purified by agarose gel electrophoresis (Elu-Quik; Schleicher&Schuell, Keene, N H , USA) and ligated to the vector p Q E - 3 0 previously linearized by digestion with the same enzymes. The re-protein p r o d u c e d from this vector contains a N-terminal His 6 tag. The His6-PYRase protein was purified by immobilized metal-chelate-affinity c h r o m a t o g r a phy. S D S - P A G E analysis was used to m o n i t o r the purification of the enzyme (Fig. 3) and under reducing conditions the purified protein was detected as a single 30-kDa band. The additional aa from the N-terminal linker and the acidic nature of the protein appear to cause

1

2

3

4

2OO 116 97 66 SS 36 31

21

14 6

the protein to run slightly larger than predicted by the nt sequence. W h e n the purified PYRase was run on a non-denaturing polyacrylamide gel, a single 46-kDa band

98 was detected, suggesting the presence of a dimer (data not shown). Gel-filtration analysis of the P. fluorescens PYRase expressed in E. coli suggested that the active form of the enzyme was a dimer (Gonzal6s and RobertBaudouy, 1994). However, PYRases from S. pyogenes and B. subtilus (Awad6 et al., 1992) were determined to be tetramers when examined by non-denaturing polyacrylamide gels and gel filtration chromatography, respectively. The re-Sa PYRase was purified 6,4-fold from the cell free extract, with a 98% yield. Overall, approx. 45 mg of the PYRase were purified from 1 liter of cells (295 mg total protein from soluble cell free extract). The results of each step in the enzyme purification scheme are shown in Table I.

(d) Enzymology of the recombinant Sa PYRase The specific activity of the enzyme was initially found to be 69 gmol/min per mg PYRase, which is similar to the activities reported for other PYRases (Sullivan et al., 1977; Awad6 et al., 1992; Yoshimoto et al., 1993; Gonzal6s and Robert-Baudouy, 1994). However, after 12 h dialysis against buffer C (50mM Tris.OH, p H 7 . 2 / l m M E D T A / l m M 2-mercaptoethanol/150mM KC1) the specific activity of the enzyme increased to 74 gmol/min per mg PYRase. It is possible that the enzyme preparation contained Ni z+ that had leached of the charged iminodiacetic acid column during the affinity purification resulting in a slight inhibition of PYRase activity. Previous studies have indicated that divalent cations such as Zn 2+ and Co 2+ can cause a reduction in the enzymatic activity of bacterial PYRases (Szewczuk and Mulczyk, 1969; Awad6 et al., 1992). To further investigate this possibility, the purified Sa PYRase was titrated with 1-100mM Ni 2+ (as NiC12) and tested for enzymatic activity. The Sa PYRase exhibited a loss of approx. 5% of its activity when compared to the untitrated sample and this compares favorably with the activity enhancement (7%) found after the initial dialysis following affinity purification. Titration with 1 200 mM imidazole had no effect on activity. Taken together these data suggest

that Ni 2+ can cause a reduction in the enzymatic activity of the PYRase and that dialysis of the affinity purified protein i n a buffer containing a divalent chelator may assist in the recovery of enzyme activity. The pH dependence of PYRase activity was determined by titration of the enzyme into appropriate buffers containing HCI over the pH range 6.5-8.5. The maximal activity was found to occur at pH 7.8. Additionally the turnover number, kcat, of the recombinant Sa PYRase was determined to be 60 s -~. This value compares favorably with the PYRase isolated from P. fluorescens (Gonzal6s and Robert-Baudouy, 1994), but is twice as active as the cloned PYRase from B. subtilus (Yoshimoto et al., 1993). The enzymatic activity of most bacterial PYRases are sensitive to thiol-blocking agents. To determine whether the Sa PYRase was also sensitive to these agents, the enzymatic activity was studied in the presence of the various concentrations (1, 10 and 100 mM) of iodoacetamide, N-ethylmaleimide and p-chloromercuribenzoate. At the lowest concentration (1 mM), the enzymatic activity of the PYRase was completely inhibited by each of the inhibitors after a 10 rain incubation at 30°C. These data suggest that the activity of the Sa PYRase is similar to other previously isolated bacterial PYRases and suggests that an active site thiol may be directly involved in enzymatic catalysis.

(e) Conclusions (1) The Sa pcp gene encoding a pyrrolidone carboxyl peptidase (PYRase) has been cloned, sequenced and overexpressed in E. coll. (2) The deduced aa sequence is similar to other PYRases cloned from both Gram + and Gram- bacteria. (3) The re-Sa PYRase protein has been purified to homogeneity and exhibits enzymatic activity (74 pmol/min per mg, kea t 60 s -1) as determined by the ability to cleave Pyr-[3NA. Identification of the active site residues is currently underway.

TABLE I Purification of the recombinant Sa PYRase overexpressed from the pcp gene in E. col? Purification step

Total protein (mg)

Total units (u)

Specific activity a (u/mg)

Percent recovery

Purification (fold)

Soluble cell-free extract Metal-chelate-affinity chromatography Dialysis

295 45 45

3393 3105 3330

11.5 69 74

100 92 98

1 6 6.4

a The enzymatic activity of the purified Sa PYRase was quantitated using Pyr-~NA (Sigma) as a model substrate as described by Lee et al. (1971). Following a 3-min incubation (30°C) of the PYRase diluted in 900 pl of 50 m M phosphate buffer (pH 7.0), 100 gl of 10 m M Pyr-[3NA in methanol was added, and the release of 13NA was assayed on a spectrophotometer (Milton Roy 501) at 340 nm. One unit (u) of PYRase activity is defined as 1 gmol of Pyr-[3NA liberated per min at 30°C.

99 ACKNOWLEDGEMENTS

This work was supported by a grant from the Arthritis Foundation. We thank Peggy Buford for excellent technical assistance.

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