Proc. Nati Acad. Sci. USA Vol. 80, pp. 2681-2685, May 1983 Genetics

IgAl proteases of : Cloning and characterization in K-12 (secretion/restriction maps/Southern blots/minicells/Tn5 insertions) J. BRICKER*, M. H. MULKSt, A. G. PLAUTt, E. R. MOXON*, AND A. WRIGHT* *Tufts University Schools of Medicine, Veterinary Medicine, and Dental Medicine, and fGastroenterology Unit, Department of Medicine, Tufts-New England Medical Center, Boston, Massachusetts 02111; and tEudowood Division of Pediatric Infectious Diseases, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Communicated by Phillips W. Robbins, December 27, 1982

ABSTRACT Haemophilus influenzae is one of several bacte- MATERIALS AND METHODS rial pathogens known to release IgAl proteases into the extra- cellular environment. Each H. influenzae isolate produces one of Bacterial Strains. H. influenzae strains used were serotypes at least three distinct types ofthese enzymes that differ in the spe- a, b, c, d, e, and f (American Type Culture Collection, nos. cific peptide bond they cleave in the hinge region of human IgAl. 9006, 9795, 9007, 9008, 8142, and 9833, respectively) and non- We have isolated the gene specifying type 1 IgAl protease from typable clinical isolates 18F, 19, and 30. The H. parainfluenzae a total genomic library of H. influenzae, subcloned it into plasmid strain used was isolated from the pharynx of a normal individ- vectors, and introduced these vectors into Eacherichia coli K-12. ual. The E. coli strains used were as follows: AW1061/F' lac The enzyme synthesized by E. coli was active and had the same iQ, F' lac iPA(ara-leu)7697, araD139, Alac-X74, A(brnQ-phoR), specificity as that of the H. influenzae donor. Unlike that of the donor, E. coi protease activity accumulated in the periplasm rather gaiU, galK, rpsL, tsx:: Tn5; X984, F, his, idv, nwt, purE, pdxC, than being transported extracellularly. The position of the pro- xyl, cycA, cycB, minA, minB, T3, rpsL; F-Z-AM15, Apro, Alac, tease gene in H. influenzae DNA and its direction of transcription thi, recA, (080 dlacZ, AM15); and MC1000, F-A(ara-leu)7697, was approximated by deletion mapping, Tn5 insertions, and ex- araDl39, Akac-X74, galU, galK, rpsL; KH802, hsdR-, hsdM', amination of the polypeptides synthesized by minicells. A 1-kilo- supE, lacY, galK. base probe excised from the IgAl protease gene hybridized with H. influenzae Genomic A Library. The A Charon 4 (8) ge- DNA restriction fragments of all H. influenzae serogroups but not nomic library used in these experiments was constructed in col- with DNA of a nonpathogenic H. parainfluenzae species known to laboration with Robert Deich by using DNA from an H. influ- be IgAl protease negative. enzae Rd- strain (KW20) transformed to type b capsule pro- duction by donor DNA from strain Eagan. The library was con- IgAl proteases are extracellular bacterial enzymes that cleave structed and phages were amplified in strain KH802 as de- the heavy polypeptide chain of human IgAl at sites within the scribed by Maniatis et al. (9). hinge region (1) (Fig. 1). Circumstantial evidence suggests that Specificity and Typing of IgAl Proteases. IgAl proteases these enzymes may be determinants of virulence. IgA is the were identified and typed by immunoelectrophoresis and predominant immunoglobulin involved in mucosal defense (1), NaDodSO4/polyacrylamide gel electrophoresis of IgAl digests and hydrolysis of IgAl impairs its function (2-4). In addition, as described (7). several species of human pathogens-for example, Haemophi- Localization and Quantitation of IgAl Protease Activity. E. lus influenzae, Streptococcus pneumoniae, Neisseria meningi- coli AW1061/pJB2 was grown at 370C in L broth containing tidis, and N. gonorrhoeae-elaborate these enzymes whereas tetracycline (10 pug/ml) with vigorous aeration, and 10-ml sam- nonpathogenic that are otherwise genetically related ples were removed at intervals throughout the growth cycle. to these species do not (5-6). Cells were centrifuged and the cell pellet was subjected to cold Several different IgAl proteases, characterized by their pat- osmotic shock (10), yielding a periplasmic fraction and a cell tern of IgAl hinge peptide hydrolysis, have been identified pellet which was sonicated and used as a cytoplasmic fraction. among routine clinical isolates ofH. influenzae (Fig. 1) (7). Each Each fraction was assayed for IgAl protease (11), for 13-lacta- isolate produces one or, less frequently, two IgAl proteases which mase [a periplasmic enzyme (12)], and for glucose-6-phosphate hydrolyze the hinge region at different peptide bonds. The dehydrogenase [a cytoplasmic enzyme (13)]. peptide bond cleaved by a given strain has been found to cor- Restriction Analysis and DNA Preparation. Restriction en- relate with serotype, H. influenzae serotypes a, b, d, and fyielding zymes were purchased from New England BioLabs and were type 1 hydrolysis and serotypes c and e yielding type 2 hydro- used according to the supplier's recommendations. Bacterial lysis (7). Although these enzymes are closely related in func- DNA was prepared as follows. Cells were lysed by 10 mM tion, at present there is no explanation for the differences in Tris HCl, pH 8/1 mM EDTA (TE buffer) containing 0.025 ml their substrate specificity and biochemistry. of 0.5% NaDodSO4 per ml of cells and the lysate was digested To achieve better understanding of the basis for the IgAl at 370C overnight with 1 mg of self-digested Pronase per ml. protease heterogeneity, we have initiated a series of genetic The mixture was extracted with phenol/chloroform, 1:1 (vol/ studies using H. influenzae. In this paper, we report the cloning vol) and then extracted twice with chloroform; the aqueous layer and characterization of the IgAl protease from H. influenzae was dialyzed against TE. The dialysate was treated with RNase serotype d. (100 pig/ml) for 2 hr at 37C, extracted as before, and precip- itated with ethanol. Plasmid DNA was isolated by the method The publication costs ofthis article were defrayed in part by page charge ofBirnboim and Doly (14) for large amounts or by the Cold Spring payment. This article must therefore be hereby marked "advertise- ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviation: kb, kilobase(s). 2681 Downloaded by guest on September 29, 2021 2682 Genetics: Bricker et al. Proc. Natl. Acad. Sci. USA 80 (1983)

H. infIuenZe-2 N.meningitidlis-2 Hmeningitidis-/ CHO,S fnQUIS H.influenzwe-/ I S~~pnwmcvWoe~~N gonorrhoeae :CHO: - CA6.~~~~~~~~~~~~~~~~~SrTh-r SrP-CsId--S I -Pro-Ser-Thr-Pro-Pro -Thr P S Pro Ser -ProPr- o - Pro Thr-Pro Ser - Pro-Ser- CY 225 'w 230 ' ' 235 ' , ! , 240 FIG. 1. Hinge region of human IgAl heavy chain, showing the location of the peptide bonds cleaved by the various microbial IgAl proteases. Harbor procedure (15) for small amounts. A DNA was isolated sulting mixture. DNA was isolated from an agarose Eel after from purified virions as reported (15). Enzyme-cut DNA was electrophoresis and then nick-translated by using [a-3 P]dATP fractionated on agarose submarine type gels with an SB3 buffer (15). The probe extends 0.25 kb to the left and 0.75 kb to the (16) and SeaKem ME agarose purchased from Marine Colloids, right of the Pst I site shown in Fig. 2c. Rockland, ME. When necessary, DNA was isolated from agar- Minicells and Protein Labeling. The minicell-forming strain ose gels by the glass powder technique (17). X984 was transformed with various plasmids (see Results) and Ligation and Transformation of Plasmid DNA. Ligation of the minicells (109 in 0.2 ml) isolated from cultures of these strains fragments was carried out by using T4 DNA ligase (New En- were labeled for 60 min with 100 ,Ci (1 Ci = 3.7 X 101' Bq) gland BioLabs). The plasmids that were used as vectors for the of [3S]methionine (22). The 3S-labeled products were ana- H. influenzae DNA were pUR222 (18) and pBR322. For trans- lyzed on NaDodSO4/polyacrylamide gels (23), and labeled bands formation, ligated DNA was added to E. coli cells that had been were detected by fluorography. pretreated with calcium chloride (15). Transposition of Tn5. Tn5 insertions in pJB2 were selected as described by Felton et al. (19). RESULTS Transfer and Hybridization of DNA with an IgAl Protease- Identification of the IgAl Protease Gene in a A Library. A Specific Probe. Chromosomal DNA was isolated from Hae- genomic library of H. influenzae DNA carried on a A Charon mophilus strains, restricted with EcoRI, and run on a 1% agar- 4 was screened for the IgAl protease gene by using the overlay ose gel as described above. DNA was transferred to a nitro- technique of Gilbert and Plaut (24). Of 3,000 plaques examined cellulose filter by the Southern procedure (20) and hybridized in this way, 2 were found to be enzyme positive, a frequency to probe DNA (21). The probe was prepared by isolating the compatible with that expected from the average size of the ge- 6.9-kilobase (kb) EcoRI H. influenzae DNA fragment from pJB2 nomic fragments in the A Charon 4 library. There was little (see Fig. 2c), digesting this fragment with Hinfl, and purifying background in the assay, indicating that neither E. coli nor phage the single Pst I-sensitive segment (1 kb in size) from the re- A Charon 4 produced detectable amounts ofproteases that cleave

6.9 kb -4.-- 5.7 kb -*1

rr - E 0 0 0 00-.- u C U w x w

o I I IL- a ME-m-1C%4 ... tH54) "in 5 X JB1 A ,, H. influenzoe insert

-I I

Q 0 _x 0_a u Th > z g w w~~~_w0. iS c aco 0 VIa. b I ..I11 LI pJB1 Pstl deletion pUR 222 Qi a

a. @_ li -c. 0 _;: -,,ES S- > 0 00 w w~ .ii c I I II pJB2

pBR322 1) J) 21 kb (1) I --deleted in pJB4 3.9kb

direction of transciption

FIG. 2. Restriction maps of H. influenzae DNA coding for IgAl protease in AJB1 and in subclones pJB1 and pJB2. H. influenzae DNA is rep- resented by an unbroken line. The orientations of the vectors are indicated by a few restriction sites; other vector sites are not shown. The plasmids pJB1 and pJB2 are illustrated as though they had been cut once withEcoRI. Homologous regions in theH. influenzae insert are connected by broken lines. (a) AJB1. Only those restriction sites necessary to delineate the extent of the insert and show its orientation are indicated. (b) pJB1. DNA deletedby complete digestion of pJB1 withPst I is indicated by an arrow below the map. (c) pJB2. Region deleted from pJB2 to give pJB4 is indicated by a line below the map. Tn5 insertions are represented by a circle connected to the map by a line. The number within the circle identifies the specific insert. Plasmids with Tn5 inserts nos. 2, 3, and 4 specify production of active IgA protease; the plasmid with Tn5 insert no. 1 does not. The probable

direction of synthesis of IgA protease is indicated by an arrow. The restriction enzymes Acc I, BamHI, Bcl I, Hae II, HinclI, HindIII, Hpa I, Nde I, Sac I, Sal I, Sst I, Stu I, and TthlllI do not cut the H. influenzae insert in pJB2. Downloaded by guest on September 29, 2021 Genetics: Bricker et al. Proc. Natl. Acad. Sci. USA 80 (1983) 2683 IgAl. Also, sonicates of E. coli cells used in this study showed no evidence of IgAl protease activity. One ofthe two IgAl-positive phages, ACh4: KW20:JB1 (AJB1), was purified and its DNA was characterized by using restriction enzyme analysis. A map of the restriction sites of AJB1 relevant to our analysis of the IgAl protease gene is shown in Fig. 2a. .0 *3-! *-H Characterization of the Protease Produced by AJB1. Im- munoelectrophoretic analysis demonstrated that an ammonium sulfate fraction from a AJB1 lysate ofE. coli KH802 hydrolyzed human IgAl to yield intact Faba and Fca fragments (Fig. 3). NaDodSO4/polyacrylamide gel electrophoresis of IgAl cleaved I Fc with the AJB1 enzyme showed that the cleavage products were _i -- t I Fd identical to those of the H. influenzae donor (Fig. 4). to_. w-** -L Cloning of the IgAl Protease Gene in E. coli Plasmids. EcoRI fragments from partial and complete digests of AJB1 were li- gated separately into the cloning vectors pUR222 and pBR322. Transformants were screened for IgAl protease production by using the "MI-labeled IgAl/agar overlay method (24). Plasmids that contained the 6.9-kb EcoRI fragment of AJB1 alone (pJB2) 1 2 3 4 5 6 or the 6.9-kb fragment plus the 5.7-kb fragment (pJBl) spec- ified IgAl protease. Restriction maps of these plasmids and their FIG. 4. Autoradiograph ofIgAl protease digests of 125I-labeled hu- relationship to AJB1 are shown in Fig. 2 b and c. Plasmids con- man IgAl examined by NaDodS04/polyacrylamide gel electrophore- taining the 5.7-kb fragment or the 1.3-kb fragment alone were sis. Samples were reduced with mercaptoethanol and electrophoresed also identified, and cells carrying these were IgAl under reducing conditions. The lanes show human IgAl incubated with: plasmids 1, buffer; 2, H. influenzae type 2 protease; 3, H. influenzae type 1 pro- protease negative. The protease produced by cells carrying tease; 4, concentrated lysate of E. coli KH802 infected with AJB1; 5, plasmid pJB2 had the IgAl cleavage specificity of the original sonicatedE. coli AW1061/F'lac iQ/pJB2; 6, buffer. H, heavy chain; Fca, H. influenzae enzyme (Fig. 4). Thus we conclude that the 6.9- Fca fragment; Fda, heavy chain component of the Faba fragment; L, kb EcoRI fragment of DNA carries the structural gene for IgAl light chain. protease. This fragment is flanked on both sides by H. influ- enzae DNA in AJB1, suggesting that it represents the corre- resulted in loss of its ability to specify IgAl protease. Tn5 in- sponding fragment from the H. influenzae chromosome. sertions located 2.2 kb to the left of the Pst I site, Tn5 no. 2, Analysis of Subclones of Plasmids pJBl and pJB2. We used and 1.2 kb to the right of the Pst I site, Tn5 no. 4, are IgAl both deletion analysis and transposon mutagenesis to localize protease positive. Thus, the coding sequence specifying IgAl the IgAl protease encoding sequence within the 6.9-kb EcoRI protease activity lies between Tn5 insertions nos. 2 and 4. As fragment of DNA. The Pst I-generated deletion of pJBl DNA, will be shown below, Tn5 insertion no. 2 probably lies within indicated in Fig. 2b, eliminated the ability of the plasmid to the polypeptide encoding sequence near the 3' end ofthe gene. specify IgAl- protease. In contrast, deletion of DNA from the Localization of IgAl Protease in E. coli. Plasmid encoded opposite end of the 6.9-kb fragment, generating plasmid pJB4 IgAl protease activity was found primarily in the periplasm of (Fig. 2c), had no effect on IgAl protease production. Insertion E. coli cells (Table 1), in contrast to H. influenzae in which more of Tn5 near the Pst I site of plasmid pJB2, Tn5 no. 1 in Fig. 2, than 98% of IgAl protease is extracellular. The total amount of IgAl protease produced by the plasmid-containing E. coli cells was 0.05 unit (11) ofIgAl protease per 109 colony-forming units whereas H. influenzae produced 16.5 units/109 colony-forming units, or 300 times more active enzyme. Growth of AJB1 in E. coli/pJB2 cells produced a lysate containing 0.56 unit/109 cells, Aw or 10 times as much enzyme as the E. coli/pJB2 cells alone. U This may be attributed to the greater number of phage copies than of plasmid copies per cell, or it may be due to increased transcription from the APL promoter. Identification of the Gene Product. The IgAl-protease gene product was identified by comparing polypeptides specified by the IgAl protease-positive plasmids pJB2 and pJB4 with those specified by plasmids pJB2: :Tn5 nos. 1 and 2. The plasmids were introduced by transformation into the minicell-forming

..... I '.' Table 1. Localization of IgAl protease activity in E. coli AW1061/pJB2 % of total enzyme activity Cellular IgAl (-) (+) fraction protease f-Lactamase Dehydrogenase* FIG. 3. Immunoelectrophoresis of human serum IgAl and its IgAl Cytoplasmic 21.2 0.3 83.1 proteasecleavage products: 1 and3, intactIgAl; 2, IgAl incubated with Periplasmic 78.8 99.7 16.9 the concentrated lysate ofE. coli KH802 infected with AJB1; and 4, IgAl Extracellular 0 0 0 incubated with type 1 IgAl protease prepared from the H. influenzae bk/Rd- strain used to construct the genomic library. * Glucose-6-phosphate dehydrogenase. Downloaded by guest on September 29, 2021 2684 Genetics: Bricker et al. Proc. Natl. Acad. Sci. USA 80 (1983)

strain X984. The 35S-labeled polypeptides produced in mini- A B C D E F G H J cells prepared from each strain were analyzed by polyacryl- amide gel electrophoresis. Several polypeptides were specified by each of the plasmids but only one polypeptide, Mr 137,000, appeared to be unique to pJB2 and pJB4 (Fig. 5). The unique polypeptides of Mr 47,000 and 125,000 specified by pJB2:: Tn5 nos. 1 and 2, respectively, may be premature termination prod- ucts from the IgAl protease gene caused by insertion of Tn5. 2380- If this is the case, then Tn5 insertion no. 1 is closer to the be- 9 65- ginning of the gene for the Mr 137,000 protein than is Tn5 in- 6.57- sertion no. 2, suggesting that transcription of the gene occurs 4 31 - in the direction indicated in Fig. 2c. Given the protease phe- notypes of the various plasmids, it is likely that the Mr 137,000 polypeptide is the IgAl protease. It should be noted that cells FIG. 6. RestrictionandSouthern blot analysis ofH. influenzaeDNA. containing plasmid pJB2:: Tn5 no. 2 retained IgAl protease ac- The probe used was a 2P-labeled 1-kb segment fromthe IgAl encoding tivity. region of H. influenzae DNA. The DNA from various H. influenzae strains was digestedwithEcoRl priorto electrophoresis in a 1.0% agar- A B C D ose gel. Lanes: A-F, DNA fromH. influenzae serotypes a, b, c, d, e, and f, respectively; G, DNA from H. influenzae strain 18F, which produces more than one IgAl protease; H and I, DNA fromH. influenzae strains 19 and 30, which produce no IgAl protease; J, DNA from H. parain- fluenzae, whichproduces no enzyme. The sizes of thevariousbands are given in the text; the numbers indicate size in kb. --137 -125 IgAl-Protease Genetic Variability in Haemophilus. It has been reported that there are at least five different IgAl-pro- tease types produced by various strains of H. influenzae; this includes some strains that produce two types of enzyme at once (7, 27). Moreover, nonpathogenic H. parainfluenzae strains do not produce an IgAl protease (6). In order to examine this di- versity, DNA from various strains ofH. influenzae was digested 58 with EcoRI and the resulting fragments were separated by 54 CY) agarose gel electrophoresis and transferred to a cellulose nitrate -- filter by the method of Southern (20). The filter was hybridized t0-a. 'N95349 0 to a 1-kb probe isolated from a DNA fragment located at the --47 Pst I site ofthe 6.9-kb H. influenzae EcoRI segment. The EcoRI fragments ofserotypes a, b, d, and fwere heterogeneous in size L- and had bands of 7.9, 9.7, 6.9, and 6.4 kb, respectively (Fig. 6). Serotypes c and e, both of which make type two enzymes, had a common band of3.4 kb. Strain 18F, which produced more than one enzyme, had only one band, of 5.0 kb. Strains 19 and 30, which produced little or no enzyme, still retained homology to the probe and had bands of 18.5 and 5.3 kb. The protease- negative H. parainfluenzae had no homology to the probe un- der the conditions used. DISCUSSION We have identified and cloned a segment of DNA from H. in- fluenzae that codes for IgAl protease. This DNA is present as a 6.9-kb EcoRI fragment and is probably contained within a sin- gle gene of approximately 3.5 kb. The gene is expressed by E. coli K-12 when cloned in either A Charon 4 or in a plasmid, thus FIG. 5. Proteins synthesized in E. coli minicells containing IgAl making it probable that its own promoter is present and func- protease encoding plasmids. Minicells containing various plasmids tional in E. coli. having H. influenzae DNA inserts were incubated in the presence of The IgAl protease arising from the cloning of the DNA into [35S]methionine, and the resulting labeled proteins were fractionated E. coli K-12 had the same substrate specificity as that of the H. by NaDodSO4/polyacrylamide gel electrophoresis. Lanes A-D con- tained proteins specified by plasmids pJB2, pJB4, pJB2: :Tn5 no. 2, and influenzae donor but differed in other characteristics. In con- pJB2: :Tn5 no. 1, respectively. The Mr 137,000 band seen in lanesA and trast to H. influenzae, in which enzyme is secreted into the growth B is the product of the IgAl-protease gene. Shorter fragments of this medium, the IgAl protease encoded by the cloned DNA ac- polypeptide due to Tn5 insertions within the gene are seen at the Mr cumulated in the periplasm ofE. coli. Similar observations have 125,000 position in lane C and Mr 47,000 position in lane D. The bands, been made in the case of two other secreted bacterial proteins, in lanes C and D, of Mr 58,000, 54,000,53,000, and 49,000 are Tn5-spec- toxin and of E. ified polypeptides (25). The bands at Mr 28,000 and 31,000 in all lanes (28) plasmid-specified hemolysin coli are l3-lactamase and its precursor. The band at Mr 37,000 in all lanes (29). The fact that IgAl protease activity is found in the peri- is coded for by the tet gene (26). These proteins, as well as ,B-galacto- plasm of E. coli suggests that it might be synthesized as a pre- sidase with Mr 116,000 (not shown) were used as size standards for the protein with a signal sequence at its amino terminus. calculations of the molecular weights of the IgA proteases. Another characteristic of the IgAl protease gene cloned into Downloaded by guest on September 29, 2021 Genetics: Bricker et al. Proc. Natl. Acad. Sci. USA 80 (1983) 2685

E. coli is that the enzymatic activity ofthe gene product is about tutes of Health. M.H.M. is supported by a Research Fellowship awarded 1/300th that of the donor strain. This low enzyme activity may by The Medical Foundation, Inc., of Boston. be due to decreased transcription of the gene- or to a defect; in 1. Kornfeld, S. J. & Plaut, A. G.. (1981) Rev. Infect. Dis. 3, 521-534. processing or secretion. The carboxyl terminus of the protein 2. Plaut, A. G;, Gilbert, J. V. & Wistar, R. (1977) Infect. Immun. 17, does not appear to be necessary for enzymatic activity because 130-135. a M, 125,000 polypeptide resulting from a Tn5 insertion was 3. Mulks, M. H., Plaut, A. G. & Lamm, M. (1980) in Genetics and active; further analysis of such truncated peptides may define Immunobiology of Pathogenic Neisseria, eds. Normark, S. & functional domains within the IgAl protease. Danielsson, D. (Univ. of Umea, Umea, Sweden), pp. 217-221. 4. Van Epps, D. E., Plaut, A. G., Bernier, G. M. & Williams, R. The substrate specificity of IgAl proteases from individual C., Jr. (1980) Inflammation 4, 137-144. H. influenzae isolates varies (7, 27): types 1, 2, and 3 IgAl pro- 5. Mulks, M. H. & Plaut, A. G. (1978) N. Engl. J. Med. 299, 973- teases each cleaves a distinct peptide bond in the human IgAl 976. hinge region. Furthermore, these enzyme types are strongly 6. Mulks, M. H., Kornfeld, S. J. & Plaut, A. G. (1980)J. Infect. Dis. correlated to serotype (capsular antigen). Not unexpectedly, 141, 450-456. Southern'blot analysis using a type 1 IgAl protease-specific DNA 7. Mulks, M. H., Kornfeld, S. J., Frangione, B. & Plaut, A. G. (1982) probe shows a J. Infect. Dis. 146, 266-274. variability at the genomic level. EcoRI-digested 8. Blattner, F., Williams, B., Blechl, A., Dennison-Thompson, K., chromosomal DNA from H. influenzae serotypes a, b, d, and Faber, H., Furlong, L., Grunwald, D., Keifer, D., Moore, D., f, which produce type 1 IgAl proteases, possess regions of ho- Shumm, J., Sheldon, E. & Smithies, 0. (1977) Science 196, 161- mology to the probe, although the sizes of the hybridizing EcoRI 169. fragments are different. H. influenzae serotypes c and e each 9. Maniatis, T., Hardison, R., Lacy, E., Lauer, J., O'Connell, C., produce IgAl type 2 proteases and each has a 3.4-kb EcoRI Quon, D., Sim, G. & Efstratiadis, A. (1978) Cell 15, 687-701. fragment 10. Neu, H. C. & Heppel, L. A. (1965)J. Biol. Chem. 240, 3685-3692. homologous with the probe. Because H. influenzae 11. Plaut, A. G., Gilbert, J. V. & Heller, I. (1978) Adv. Exp. Med. Biol. serotypes a, b, d, and fsecrete IgAl proteases ofthe same spec- 107, 495-498. ificity, it was reasonable that each would have genomic ho- 12. Jack, G. W. & Richmond, M. H. (1970)J. Gen. Microbiol. 61, 43- mology; however, in the case of serotypes c and e, further stud- 61. ies will be needed to determine whether the probe actually 13. Morse, S. A., Stein, S. & Hines, J. (1974)J. Bacteriol. 120, 702- hybridizes to the type 2 protease gene or whether it hybridizes 714. to a 14. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 7, 1513-1523. region unrelated to IgAl protease synthesis. 15. Berman, M. L., Enquist, L. W. & Silhavy, T. J. (1981) Advanced Use of the DNA probe to examine a strain of H. influenzae Bacterial Genetics (Cold Spring-Harbor Laboratory, Cold Spring that expresses two IgAl protease types showed a single ho- Harbor, NY). mologous EcoRl fragment. This result may indicate that only 16. Haywood, G. & Smith, M. (1972) J. Mol Biol 63, 383-395. one enzyme with two specificities is synthesized, or alterna- 17. Vogelstein, B. & Gillespie, D. (1979) Proc. Natl Acad. Sci. USA tively the probe may hybridize to only one of two separate genes 76, 615-619. specifying IgAl proteases, or a gene duplication event may have 18. Ruther, U., Koenen, M., Otto, K. & Muller-Hill, B. (1981) Nu- yielded adjacent, distinct genes that are present on the same cleic Acids Res. 9, 4087-4098. 19. Felton, J., Michaelis, S. & Wright, A. (1980)J. Bacteriol 142, 221- EcoRI fragment. Two H. influenzae strains that do not produce 228. IgAl protease showed homology to the probe, suggesting that 20. Southern, E. M. (1975)J. Mol Biol 98, 503-517. the gene in these strains may be defective or that other genes 21. Barnett, T., Pachl, C., Gergen, J. & Wensink, P: (1980) Cell 21, are needed for full expression of protease activity. Finally, H. 729-738. parainfluenzae, which is known to be IgAl protease negative, 22. Levy, S. B. (1974)J. Bacteriol 120, 1451-1463. possessed no homology with the probe. 23. Dharmalingam, K. & Goldberg, E. (1979) Virology 96, 393-403. Cloning of an IgAl protease gene of H. should 24. Gilbert, J. V. & Plaut, A. G. (1983) Immunol Methods 57, in press. influenzae 25. Rothstein, S. & Reznikoff, W (1981) Cell 23, 191-199. permit further studies of the molecular actions of this enzyme 26. Sancar, A., Hack, A. & Rupp, W. D. (1979) J. Bacteriol 137, 692- and its potential biological role in the pathogenesis of local and 693. disseminated infections caused by H. influenzae. Cloning of a 27. Insel, R., Allen, P. & Berkowitz, I. (1982) in Seminars in Infec- gene specifying IgAl protease from , as tious Disease, eds. Weinstein, L. & Fields, B. (Stratton, New York), reported by Koomey et al. (30), will allow similar studies of the Vol. 4, pp. 225-231. role of this enzyme in gonococcal disease. 28. Pearson, G. & Mekalanos, J. (1982) Proc. Natl Acad. Sci. USA 79, 2976-2980. This work was supported by Grants GM15837, AI-14648, and NS12554 29. Goebel, W. & Hedgpeth, J. (1982)J. Bacteriol 151, 1290-1298. from the National Institutes of Health. E.R. M. is a recipient of Re- 30. Koomey, J. M., Gill, R. E. & Falkow, S. (1982) Proc. Nati Acad. search Career Development Award AI-00300 from the National Insti- Sci. USA 79, 7881-7885. Downloaded by guest on September 29, 2021