- Email: [email protected]
[22] Molecular cloning of adhesion genes
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the host cells grown at 29°.s However, if required, several approaches for studying gene products whose toxic concentrations for the host cells are very low can be tested. For example, some of the pET vectors have been constructed to include the lac repressor (lacI) and a lac operator engineered between the T7 promoter and the studied gene. 23 With such plasmids, LacI downregulates both host transcription of T7 RNA polymerase, which involves the lacUV5 promoter, and T7-mediated transcription. Another alternative includes the use of plasmids expressing T7 lysozyme, which, in addition to its enzymatic activity, inhibits T7 RNA polymerase.23 However, such plasmids cannot be adapted to the two-plasmid system and require the use of strains expressing T7 RNA polymerase from a A phage stably integrated into the chromosome. The most effective solution to toxic proteins consists of introducing the gene for T7 RNA polymerase on a bacteriophage only at the time of induction. Two bacteriophages carrying T7 RNA polymerase, inducible from a lac promoter by isopropylthiogalactoside (IPTG), have been described for this purpose: mGP1-2, 2° which is an M13 derivative and requires that the host express the pili receptor, and the A derivative CE6Y
Acknowledgments I thank L. J. Bello, R. K. Taylor, and M. F. Daldal for critically reading the manuscript. This work is supported by U.S. D e p a r t m e n t of Agriculture Grant 92-37204-7901.
[22]
Molecular Cloning of Adhesion Genes By
S H E I L A I. H U L L a n d R I C H A R D A . H U L L
Introduction The molecular cloning of genes associated with bacterial adhesion requires methods different from the cloning of single genes. Adhesion of gram-negative bacteria to mammalian tissue is often promoted by complex structures called pili, or fimbriae, on the surface of the bacterium. Expression of fully functional adhesion may require expression of up to 10 genes spaced over a 10- to 15-kb region of DNA. Also, there is usually no reliable direct selection for adherent recombinants so that some sort of physical or functional screening process is required. As a consequence, successful cloning methods must result in chimeric molecules with high molecular weight inserts that represent contiguous regions of the donor chromosome. Inserts METHODSIN ENZYMOLOGY,VOL. 253
Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[22]
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must be sufficiently random such that the entire chromosome is represented in 1000-2000 recombinant molecules. In this chapter, methods are presented for preparing recombinant DNA libraries that have been used successfiJlly for cloning large bacterial operons and functional gene clusters in the 10- to 25-kb range such as adhesion organelles and carbohydrate antigens. Methods useful in screening for adhesin gene clones are also presented. Purification of High Molecular Weight DNA from Bacteria Materials
1 liter Sterile Luria (L) broth in a 2-liter flask 50 ml Overnight L broth culture of the D N A donor bacterium 25% (w/v) Sucrose in 50 mM Tris, pH 8.0, 1 mM EDTA 10 mg/ml Proteinase K in 10 mM CaC12 0.5 M EDTA, pH 8.0 10% Sodium lauryl sarcosinate (sarkosyl) 50 mg/ml Egg white lysozyme Autoclaved 10 mM Tris, pH 8.0, 1.0 mM EDTA (TE) Autoclaved 10 mM Tris, pH 7.3, 100 mM NaC1, 0.1 mM EDTA (TES) Cesium chloride solution: 23.4 g CsC1 per 20.3 ml solution in TE containing 50 p~g/ml phenylmethylsulfonyl fluoride (PMSF); the PMSF stock is prepared at 10 mg/ml in 95% (v/v) ethanol Cesium chloride solution, 868.1 mg/ml Procedure
One liter of L broth is inoculated with 50 ml overnight growth of bacteria and incubated at 37° with aeration until the OD660 is 0.2. Bacteria are harvested by centrifugation and washed once in 20 ml TE. The bacterial pellet is suspended in 20 ml of 25% sucrose solution, and 5-ml samples are dispensed into screw-cap polypropylene tubes on ice. Although the yield from one 5-ml sample is sufficient, processing of at least two tubes is recommended. One hundred microliters of lysozyme is added and incubation on ice continued for 5 rain. Proteinase K (25/xl) is added and mixed followed by 1 ml 0.5 M EDTA. Lysis is initiated on ice by adding 0.6 ml sarkosyl and gently twirling and inverting the tube to promote mixing. By keeping the sample cool, lysis is delayed, permitting more uniform mixing. After mixing is complete, the tubes are capped and incubated overnight at 50 °. The viscous sample is then transferred to a Beckman (Fullerton, CA) 60Ti ultracentrifuge tube or equivalent, by means of a funnel if heat
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sealing tubes are used. DNA samples should not be transferred using a syringe or small bore pipette. Alternately, the lysis procedure may be done directly in the ultracentrifuge tube; however, adding reagents to heat sealing tubes is difficult. Cesium chloride-PMSF mixture (20.3 ml) is added to the lysate. The density of this solution is based on the density of Escherichia coli DNA and assumes a lysate volume of 6.7 ml; the same density gradient has been used successfully for a lower %G+C organism, Proteus mirabilis. Each tube is filled to the top with CsC1 (868.1 mg/ml solution) and sealed. Prior to centrifugation, the sealed tubes are stored at room temperature overnight. The concentration of CsC1 in the gradients is near saturation; by allowing the CsC1 and lysate to equilibrate overnight, the likelihood of CsC1 precipitating during centrifugation is reduced. The tubes are centrifuged in a Beckman 60Ti rotor for 46 hr at 35,000 rpm and 20°. Shorter centrifugation times or use of vertical rotors have proved inferior. Ethidium bromide is not included in the gradient because of difficulty in subsequently removing it from purified DNA without subjecting the DNA to shear forces. After centrifugation, the DNA may appear as a cottonlike puff near the middle of the gradient. The tube is first vented with an 18-gauge needle near the top; the DNA is collected directly into boiled 1 cm diameter dialysis bags through a 12-gauge syringe needle (Hamilton, Reno, NV) by side puncture of the tube below the DNA. The 12-gauge needle should be inserted swiftly to minimize loss of sample. The flow rate through the needle can be regulated using the 18-gauge needle inserted at the top either manually with an attached rubber tube or by pumping light oil to displace the gradient. When the fraction containing DNA passes through the needle, the solution will become viscous, and positive pressure may be required to force the DNA through the 12-gauge needle. Care should be taken to collect only the viscous DNA with minimal additional CsC1 solution. The DNA samples are dialyzed versus 2 liters TES with three changes. Finally, the samples are transferred to a screw-cap plastic tube and stored at 4°. DNA concentration is about 300/xg/ml, although accurate measurement is difficult due to high viscosity of the sample. Alternate Methods
The chemical extraction method for DNA purification described by Marmur ~is still frequently used for preparing recombinant DNA libraries. It is less desirable for cloning of large inserts, however, because the product is of lower molecular weight owing to limited but unavoidable mechanical 1 j. Marmur, J. Mol. Biol. 3, 208 (1961).
[22]
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shear associated with this procedure. Column chromatography-based systems for purification of high molecular weight D N A are available commercially (Boehringer Mannheim), but their usefulness is limited by the finite capacity of the columns. Akhough the CsC1 procedure described here was originally designed for the enteric bacteria, it has also been used with Neisseria gonorrhoeae and should be easily adaptable to other genera. Lysozyme may be replaced with other lytic enzymes as appropriate. Partial Digestion of DNA Materials and Procedure
Partial digests are prepared by varying the digestion time while using an excess of restriction endonuclease and a constant temperature. Digestl'on times are determined empirically for each D N A batch prior to setting up preparative reactions. D N A (100 tzl) is mixed on ice with 11/xl of 0.1 mM MgCI~, 1 ~I bovine serum albumin (BSA; 10 mg/ml) and 1/.d Sau3A (10 U//zl). The undiluted, undigested D N A stock cannot be pipetted with a standard micropipette tip and is best transferred with a large bore tip. A 5-/xl sample is immediately mixed with electrophoresis sample buffer, and incubation is continued at room temperature. Additional samples are taken at 2, 4, and 6 min and at 5- to 10-min intervals thereafter. The viscosity of the digest should noticeably decrease after 10-15 rain. The samples are subsequently analyzed by electrophoresis through a 0.7% agarose gel. In the example shown in Fig. 1, the time points selected for library preparation were 2, 3, 4, and 6 rain. For preparative reactions, four fresh 100-/zl digests are set up as before; at each selected time point, one tube is transferred to ice ;and mixed with 5/xl of 0.5 M EDTA. When all partial digests are completed, the samples are heated to 70 ° for 20 rain to terminate the reaction. Alternate Methods
In place of using partial restriction endonuclease digestion to produce a representative collection of fragments, the chromosomal DNA can be mechanically sheared using sonication. The fragments are then methylated with BamHI methylase, the sheared ends repaired with T4 polymerase and deoxynucleoside triphosphates (dNTPs), ligated with BamHI linkers, and finally ligated into the BamHl site of a suitable vector. Although this procedure will likely result in a more random library, in practice the enzymatically fragmented DNA is satisfactory and easier to produce.
262
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0
1
2
3
4
5
10
15
20
FIG. 1. Partial digest of bacterial DNA with the restriction endonuclease Sau3A. DNA was digested with Sau3A at room temperature for the time intervals shown (in minutes), and samples from each time point were examined using 0.7% agarose gel electrophoresis.
Size Fractionation Materials and Procedure
Digested DNA is size fractionated using centrifugation through 5-20% NaC1 gradients. The gradients are prepared using sterile solutions in diethyl pyrocarbonate (DEPC)-treated Beckman SW41 ultracentrifuge tubes and chilled on ice prior to use. The four partial digests are pooled, and 200/A is loaded on each of two gradients. The gradients are centrifuged in a Beckman SW41 rotor for 5 hr at 35,000 rpm and 4°. Fractions (0.5 ml) are collected. If fractions are to be collected from the bottom of the gradient, it is helpful to include a 50% sucrose cushion; this will prevent larger DNA fragments from forming a pellet and plugging the collection needle. Samples (5/A) of alternate fractions are examined using electrophoresis through an 0.35% agarose gel for 18 hr at 1 V/cm. After samples for electrophoresis are taken, 1 ml of 95% ethanol is mixed with each fraction, and the DNA is allowed to precipitate overnight at - 2 0 °. The fractionated DNA can be stored indefinitely in the freezer at this step. In the example in Fig. 2, DNA in fractions 12-17 is greater than 30 kb and would be suitable for preparing cosmid libraries. DNA in fractions 9-11 is in the 20to 30-kb range and would be suitable for use with k vectors.
[221
MOLECULAR CLONING OF ADHESION GENES
5
7
9
263
11 S 13 H 15 17 19
FIG. 2. Agarose gel showing size-fractionated DNA obtained from a 5-20% NaC1 gradient. Fraction numbers from the top of the tube are indicated. Molecular weight standards are bacteriophage h DNA digested with SalI (S) (33 and 15 kb) and HindIII (H) (23.7, 9.5, 6.7, 4.3, and 2.3 kb).
Alternate Methods
The sizing step is included to remove low molecular weight DNA fragments which would otherwise ligate together randomly to form an insert sufficiently large to be packaged. This is especially important when cloning large operons because the presence of clones composed of random rearrangements will both reduce cloning efficiency and lead to potentially confusing results. The formation of randomly associated molecules can also be prevented by dephosphorylating the insert D N A prior to ligating with vector However, under such conditions, correct insert-to-vector molar ratios are difficult to calculate as the majority of insert DNA in the ligation mix, although capable of ligating to vector, is too small to be packaged. As a consequence, much of the vector is consumed in formation of unproductive molecules. D N A may also be size fractionated by preparative electrophoresis through 0.35% agarose gels. This procedure is limited by the capacity of the gels used. DNA fragments of the appropriate size for cosmid cloning represent a small fraction of the total partial digest; D N A recovered from several gels is needed to provide a sufficient amount of high quality product for a single ligation.
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Ligation and Packaging DNA from selected size fractions is collected by centrifugation and suspended in 0.4 ml TE. The DNA concentration of the undiluted sample is determined spectrophotometrically in DEPC-treated semimicrocuvettes. The concentration is usually about 2-3 ~g/ml for fractions greater than 30 kb and 8-10/xg/ml for the 20- to 30-kb fractions. For ligation, fractions in the appropriate size range may be pooled to supply 2.5-3.5 /xg and copreeipitated with Barn HI-digested cosmid vector such as pHC79 or phage vector such as EMBL3 at a 1 : 1 molar ratio. Prior dephosphorylation of the vector is not beneficial. Dephosphorylation of the insert is also not required but may be beneficial if the insert DNA is not size fraetionated as discussed above. Ligation is done using standard conditions in a final volume of 11/xl. After ligation, the sample will become viscous. Two to three microliters of ligated DNA is then packaged in vitro into bacteriophage X transducing particles. 2 The packaged phage can be stored several years at -70 ° with minimal loss of viability. Selection of a host strain is based primarily on its susceptibility to bacteriophage k. HB101 is a suitable host for either cosmid or phage cloning of adhesin genes. However, this host expresses type 1 pili under some growth conditions, which may potentially complicate interpretation of resuits. Escherichia coli P678-54, and its recA derivative, JW369, lack genes for type 1 pili and may therefore be more useful. For experiments using phage replacement vectors, E. coli Q359 is used as a host. The transducing titer (vector antibiotic resistance phenotype) for cosmid libraries is about 103//xl phage mixture. If one assumes an average insert size of 40 kb in a cosmid clone and an E. coli genome size of 4700 kb, the frequency of any particular gene in a library would be 1 in 118 ampicillinresistant transductants. In practice, the frequency of complete adhesin gene clusters among ampicillin-resistant transductants varies between 0.1 and 1%. The titer of recombinant phage using k replacement vectors is usually about 102-103/ml. The frequency of recombinant phage containing at least part of an adhesin gene cluster is 0.1-1%. However, the frequency of complete adhesin gene clusters is lower as compared with cosmid cloning (about 0.01-0.2%) owing to the smaller size of the phage vector inserts.
Functional Screening Methods for Identifying Adhesin Clones If the adhesion phenotype of interest is a hemagglutinin, hemagglutination of suseptible erythrocytes is the most direct way to screen for recombiz L. Enquist and N. Sternberg, this series, Vol. 68, p. 281.
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nant cosmid clones. Growth conditions for maximal expression of hemagglutination in the donor strain should be established prior to screening recombinant clones. Conditions to consider include incubation temperature, growth with or without aeration, liquid versus solid media, minimal media versus L broth or other complex media, and glucose effects.
Hemagglutination Ampicillin-resistant transductants are picked and inoculated as a patch, 50-100 per plate, onto two L plates containing ampicillin and incubated overnight. Patches should be tested the following day as the hemagglutination capacity of recombinant clones is often diminished after prolonged incubation. Erythrocytes are washed and suspended at 3% (v/v) in phosphate-buffered saline (pH 7.0) containing 0.01% gelatin (BSG). Human erythrocytes should be used within 1 week of collection and washed and diluted just prior to testing hemagglutination. If the desired adhesion phenotype Jis D-mannose-resistant, 10 mM D-mannose may be included in the erythrocyte mixture. Bacteria from an individual patch are picked with a sterile toothpick and mixed with a drop of erythrocyte solution on a glass plate chilled over ice. Enough bacteria should be used so that the drop of erythrocytes becomes visibly turbid. Patches are tested in batches of 1020; afl:er each batch, the glass plate is gently rocked 5-10 times to enhance visualization of hemagglutination, which may appear immediately or only after rocking.
Gradient Enrichment The frequency of hemagglutinating clones can also be enriched, if necessary, using glycerol step gradient centrifugation. Step gradients are prepared in sterile 15-ml clear centrifuge tubes, using 3 ml of 20% glycerol in BSG for the bottom step and 3 ml of 5% (v/v) glycerol in BSG for the top step. Ampicillin-resistant recombinant bacteria are collected from overnight growth (pool 104-105 colonies), suspended gently in BSG to an OD600 of 1.5, and diluted 1 : 10 in BSG. One-half milliliter diluted bacteria is mixed with 0.5 ml 3% erythrocytes and incubated at room temperature for 20 rain with occasional gentle mixing. The bacteria-erythrocyte suspension is then layered onto the gradient and centrifuged at 600 rpm (relative centrifugual force of 77) for 12 min in a Sorvall GLC centrifuge with a swinging bucket rotor. The 5% glycerol step and sample load volume are removed with a syringe and bent 18-gauge needle and gently replaced with 3 ml sterile BSG as a wash. The BSG and remaining gradient are removed, the erythrocytes suspended in BSG, and dilutions spread on antibiotic-containing agar plates. A single gradient cycle provides approximately 10-fold enrichment. The
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maximum enrichment achieved using serial gradients with intermediate regrowth steps was 105-fold.
Advantages The hemagglutination procedure is simple and direct. It assures that at least the minimum number of genes required for the adhesion phenotype are present on the cloned molecule.
Disadvantages (1) Cloned genes, especially from genera other than Escherichia, may not be expressed or their products may not be assembled into adhesin organelles in E. coli. (2) In general, this method will not work for adhesins that are not hemagglutinins. However, if the tissue receptor for the adhesin of interest is known, latex beads coated with receptor may be substituted for erythrocytes. (3) Clones that contain only part of the adhesin gene cluster will not be detected. Antigen Screening Methods for Identifying Adhesin Clones Recombinant D N A libraries may also be screened for the presence of adhesin-associated antigens without regard to expression of functional adhesion. The methods used for screening either cosmid or phage libraries for adhesin-associated antigens do not differ significantly from general published procedures. 3,4 In the following a method is described that is useful in identifying appropriate antigens for preparation of antibody when the nature of the adhesin organelle is unknown; this method is applicable to adhesins that are not hemagglutinins, s
Materials 10 ml Minimal medium: 60 mM potassium phosphate (pH 7.4); 2 mM disodium citrate; 0.8 mM MgCI2; 15 mM NH4C1; 80/zM (NH4)SO4; 20 /xg/ml of required amino acids; 0.4% glycerol; and 0.5 mg/ml yeast extract H235SO4 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel and associated buffers 3 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 4 M. Snyder, S. Elledge, D. Sweetser, R. A. Young, and R. W. Davis, this series, Vol. 154, p. 107. s S. K. Wray, S. I. Hull, R. G. Cook, J. Barrish, and R. A. Hull, Infect. Immun. 54, 43 (1986).
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BSG (phosphate-buffered saline, pH 7.0, with 0.01% gelatin) Target tissue to which bacteria adheres dispersed as single cells or groups of two or three cells
Procedure Bacteria are grown overnight in 10 ml minimal medium containing 50 i~Ci/ml H235SO4 (or [14C]glucose and additional unlabeled SO4 if the adhesin is thought not to be a protein). Other growth conditions may be used to enhance the adhesion phenotype. The bacteria are washed once with 1 ml BSG and suspended in 1 ml BSG at a density of 1-2 × 10l° cells/ml. Outer membrane material is sheared from the bacterial surface by multiple passage through a 26 gauge needle. Whole bacteria are removed by centrifugation for 10 min at 10,000 × g, 4°. Target cells are washed in BSG, and pellets containing 2 × l0 s cells are suspended in BSG containing radiolabeled outer membrane material. After incubation at 37° for 1 hr with continuous mixing, the reaction mix is centrifuged, and both the pellet and supernatant are retained. The cell pellet is washed 3 times with BSG and suspended in 1 ml BSG. The radioactivity of the cell and original supernatant fractions is determined, and an equal number of counts per minute (cpm) from each are dialyzed versus distilled water and freezedried. Samples are then examined using SDS-polyacrylamide gel electrophoresis and autoradiography. Adhesin antigens will be those enriched in the cell fraction as compared to the supernatant. This method may also be used in development of purification strategies for adhesin molecules. Presence of the adhesin in fractions obtained at different purification steps can be monitored by including a sample of the fraction in the reaction mix and measuring its capacity to competitively reduce binding of radiolabeled adhesin to tissue.
Advantages (1) The antigen screening method does not require assembly of a functional adhesin organelle. Adhesin genes from other genera of bacteria that may be cloned into E. coli but not assembled and clones that contain only part of an adhesin operon can be identified. (2) This method does not require that the adhesin-associated molecules be proteins, only that they be antigenic. (3) The method can be used with both phage and cosmid libraries.
Disad vantages (1), The adhesin molecule must be purified in advance for production of antibody; alternatively, a homogenic mutant derivative of the donor
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strain that does not express the adhesin antigen must be available to adsorb irrelevant antibodies. (2) Clones that express cross-reactive antigens may be inadvertently selected. DNA Hybridization Screening Method for Identifying Adhesin Gene Clones Recombinant DNA libraries may also be screened for the presence of adhesin-associated gene sequences without regard to the expression of any cloned gene. For screening cosmid libraries, the colony blot method described by Maas 6 is both sensitive and convenient. Filters for hybridization are prepared directly from the transduced colonies, and hybridizing colonies can be recovered from growth remaining after blotting. Materials
Whatman (Clifton, N J) No. 3 paper and autoclaved Whatman 541 paper 0.5 M NaOH, 1.5 M NaC1 1 M Tris (pH 7.0), 2 M NaC1 Procedure
Sterile Whatman 541 paper is placed on agar plates containing 100200 colonies. The filters are gently massaged to remove any bubbles. The orientation of the filter is marked with a pencil. The Whatman 541 papers are placed bacteria side up in a glass petri dish on three layers of Whatman No. 3 paper saturated with NaOH/NaC1 solution. Papers are steamed for 4 min in a covered beaker of boiling water. The Whatman 541 paper is removed and immersed in 300-500 ml of 1 M Tris (pH 7.0), 2 M NaC1 for 4 min. Air dry. Prior to hybridization, the filters are washed once briefly in hybridization solution and incubated with hybridization solution containing the selected probe and competitor DNA. No filter baking or prehybridization step is required. For screening phage libraries, standard plaque hybridization methods are appropriate. 3 Advantages
(1) No gene expression is required so adhesin genes that are neither functional nor expressed in E. coli can be cloned. (2) The procedure can be used to screen phage libraries so adhesin genes producing products that inhibit growth of E. coli can be cloned. 6R. Maas, Plasmid 10, 296 (1983).
MOLECULAR ANALYSISOF S. pyogenes ADHESION
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Disadvantages The D N A hybridization method is useful only when D N A or amino acid sequence information is available for the gene of interest. Unless the adhesin genes share D N A sequence similarity with previously cloned genes wherein the previous clone can be used as a hybridization probe, it is necessary to design synthetic oligonucleotide probes based on the amino acid sequence of an adhesin-associated protein. As a consequence of the degeneracy of the triplet code, such probes are usually ambiguous at one or mare positions and have reduced specificity. Isolation of incorrect crossreactive clones is possible and even likely, leading to considerable wasted effort. Alternately, converging oligonucleotides designed on the basis of the amino acid sequence from different regions of the protein can be used as polymerase chain reaction (PCR) primers to produce a D N A probe. For this method, the D N A sequence of any PCR product should be determined to confirm its appropriateness prior to using it as a probe. The deduced amino acid sequence predicted from the PCR product should match the actual amino acid sequence of the target protein.
Acknowledgments This work was supported by U.S. Public Health Service Grants A I 21009 and A I 18462.
[23] M o l e c u l a r
By E M A N U E L
Analysis of Streptococcus pyogenes Adhesion
HANSKI, GEORGE FOGG, AVIVA I S R A E L BURSTEIN,
and
Tovi,
NOBUH1KO O K A D A ,
MICHAEL CAPARON
Introduction
Streptococcus pyogenes (the group A streptococcus) is one of the most versatile human pathogens as regards the number of different tissues it can infect and the wide range of different diseases it can cause. Streptococcus pyogenes can cause disease through three basic pathogenic mechanisms. Multiplication on the surface of or within a tissue results in destruction of host cells and is accompanied by an intense inflammatory response. These types of diseases can range from the more mild and self-limiting infections of the throat (e.g., pharyngitis, commonly known as "strep throat") and the skin (e.g., impetigo) to infections which involve increasingly deeper METHODSIN ENZYMOLOGY.VOL.253
Copyright © 1995by AcademicPress. Inc. All rightsof reproductionin any formreserved.
MOLECULAR BIOLOGY OF ADHESINS
[22]
the host cells grown at 29°.s However, if required, several approaches for studying gene products whose toxic concentrations for the host cells are very low can be tested. For example, some of the pET vectors have been constructed to include the lac repressor (lacI) and a lac operator engineered between the T7 promoter and the studied gene. 23 With such plasmids, LacI downregulates both host transcription of T7 RNA polymerase, which involves the lacUV5 promoter, and T7-mediated transcription. Another alternative includes the use of plasmids expressing T7 lysozyme, which, in addition to its enzymatic activity, inhibits T7 RNA polymerase.23 However, such plasmids cannot be adapted to the two-plasmid system and require the use of strains expressing T7 RNA polymerase from a A phage stably integrated into the chromosome. The most effective solution to toxic proteins consists of introducing the gene for T7 RNA polymerase on a bacteriophage only at the time of induction. Two bacteriophages carrying T7 RNA polymerase, inducible from a lac promoter by isopropylthiogalactoside (IPTG), have been described for this purpose: mGP1-2, 2° which is an M13 derivative and requires that the host express the pili receptor, and the A derivative CE6Y
Acknowledgments I thank L. J. Bello, R. K. Taylor, and M. F. Daldal for critically reading the manuscript. This work is supported by U.S. D e p a r t m e n t of Agriculture Grant 92-37204-7901.
[22]
Molecular Cloning of Adhesion Genes By
S H E I L A I. H U L L a n d R I C H A R D A . H U L L
Introduction The molecular cloning of genes associated with bacterial adhesion requires methods different from the cloning of single genes. Adhesion of gram-negative bacteria to mammalian tissue is often promoted by complex structures called pili, or fimbriae, on the surface of the bacterium. Expression of fully functional adhesion may require expression of up to 10 genes spaced over a 10- to 15-kb region of DNA. Also, there is usually no reliable direct selection for adherent recombinants so that some sort of physical or functional screening process is required. As a consequence, successful cloning methods must result in chimeric molecules with high molecular weight inserts that represent contiguous regions of the donor chromosome. Inserts METHODSIN ENZYMOLOGY,VOL. 253
Copyright© 1995by AcademicPress,Inc. All rightsof reproductionin any formreserved.
[22]
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must be sufficiently random such that the entire chromosome is represented in 1000-2000 recombinant molecules. In this chapter, methods are presented for preparing recombinant DNA libraries that have been used successfiJlly for cloning large bacterial operons and functional gene clusters in the 10- to 25-kb range such as adhesion organelles and carbohydrate antigens. Methods useful in screening for adhesin gene clones are also presented. Purification of High Molecular Weight DNA from Bacteria Materials
1 liter Sterile Luria (L) broth in a 2-liter flask 50 ml Overnight L broth culture of the D N A donor bacterium 25% (w/v) Sucrose in 50 mM Tris, pH 8.0, 1 mM EDTA 10 mg/ml Proteinase K in 10 mM CaC12 0.5 M EDTA, pH 8.0 10% Sodium lauryl sarcosinate (sarkosyl) 50 mg/ml Egg white lysozyme Autoclaved 10 mM Tris, pH 8.0, 1.0 mM EDTA (TE) Autoclaved 10 mM Tris, pH 7.3, 100 mM NaC1, 0.1 mM EDTA (TES) Cesium chloride solution: 23.4 g CsC1 per 20.3 ml solution in TE containing 50 p~g/ml phenylmethylsulfonyl fluoride (PMSF); the PMSF stock is prepared at 10 mg/ml in 95% (v/v) ethanol Cesium chloride solution, 868.1 mg/ml Procedure
One liter of L broth is inoculated with 50 ml overnight growth of bacteria and incubated at 37° with aeration until the OD660 is 0.2. Bacteria are harvested by centrifugation and washed once in 20 ml TE. The bacterial pellet is suspended in 20 ml of 25% sucrose solution, and 5-ml samples are dispensed into screw-cap polypropylene tubes on ice. Although the yield from one 5-ml sample is sufficient, processing of at least two tubes is recommended. One hundred microliters of lysozyme is added and incubation on ice continued for 5 rain. Proteinase K (25/xl) is added and mixed followed by 1 ml 0.5 M EDTA. Lysis is initiated on ice by adding 0.6 ml sarkosyl and gently twirling and inverting the tube to promote mixing. By keeping the sample cool, lysis is delayed, permitting more uniform mixing. After mixing is complete, the tubes are capped and incubated overnight at 50 °. The viscous sample is then transferred to a Beckman (Fullerton, CA) 60Ti ultracentrifuge tube or equivalent, by means of a funnel if heat
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sealing tubes are used. DNA samples should not be transferred using a syringe or small bore pipette. Alternately, the lysis procedure may be done directly in the ultracentrifuge tube; however, adding reagents to heat sealing tubes is difficult. Cesium chloride-PMSF mixture (20.3 ml) is added to the lysate. The density of this solution is based on the density of Escherichia coli DNA and assumes a lysate volume of 6.7 ml; the same density gradient has been used successfully for a lower %G+C organism, Proteus mirabilis. Each tube is filled to the top with CsC1 (868.1 mg/ml solution) and sealed. Prior to centrifugation, the sealed tubes are stored at room temperature overnight. The concentration of CsC1 in the gradients is near saturation; by allowing the CsC1 and lysate to equilibrate overnight, the likelihood of CsC1 precipitating during centrifugation is reduced. The tubes are centrifuged in a Beckman 60Ti rotor for 46 hr at 35,000 rpm and 20°. Shorter centrifugation times or use of vertical rotors have proved inferior. Ethidium bromide is not included in the gradient because of difficulty in subsequently removing it from purified DNA without subjecting the DNA to shear forces. After centrifugation, the DNA may appear as a cottonlike puff near the middle of the gradient. The tube is first vented with an 18-gauge needle near the top; the DNA is collected directly into boiled 1 cm diameter dialysis bags through a 12-gauge syringe needle (Hamilton, Reno, NV) by side puncture of the tube below the DNA. The 12-gauge needle should be inserted swiftly to minimize loss of sample. The flow rate through the needle can be regulated using the 18-gauge needle inserted at the top either manually with an attached rubber tube or by pumping light oil to displace the gradient. When the fraction containing DNA passes through the needle, the solution will become viscous, and positive pressure may be required to force the DNA through the 12-gauge needle. Care should be taken to collect only the viscous DNA with minimal additional CsC1 solution. The DNA samples are dialyzed versus 2 liters TES with three changes. Finally, the samples are transferred to a screw-cap plastic tube and stored at 4°. DNA concentration is about 300/xg/ml, although accurate measurement is difficult due to high viscosity of the sample. Alternate Methods
The chemical extraction method for DNA purification described by Marmur ~is still frequently used for preparing recombinant DNA libraries. It is less desirable for cloning of large inserts, however, because the product is of lower molecular weight owing to limited but unavoidable mechanical 1 j. Marmur, J. Mol. Biol. 3, 208 (1961).
[22]
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shear associated with this procedure. Column chromatography-based systems for purification of high molecular weight D N A are available commercially (Boehringer Mannheim), but their usefulness is limited by the finite capacity of the columns. Akhough the CsC1 procedure described here was originally designed for the enteric bacteria, it has also been used with Neisseria gonorrhoeae and should be easily adaptable to other genera. Lysozyme may be replaced with other lytic enzymes as appropriate. Partial Digestion of DNA Materials and Procedure
Partial digests are prepared by varying the digestion time while using an excess of restriction endonuclease and a constant temperature. Digestl'on times are determined empirically for each D N A batch prior to setting up preparative reactions. D N A (100 tzl) is mixed on ice with 11/xl of 0.1 mM MgCI~, 1 ~I bovine serum albumin (BSA; 10 mg/ml) and 1/.d Sau3A (10 U//zl). The undiluted, undigested D N A stock cannot be pipetted with a standard micropipette tip and is best transferred with a large bore tip. A 5-/xl sample is immediately mixed with electrophoresis sample buffer, and incubation is continued at room temperature. Additional samples are taken at 2, 4, and 6 min and at 5- to 10-min intervals thereafter. The viscosity of the digest should noticeably decrease after 10-15 rain. The samples are subsequently analyzed by electrophoresis through a 0.7% agarose gel. In the example shown in Fig. 1, the time points selected for library preparation were 2, 3, 4, and 6 rain. For preparative reactions, four fresh 100-/zl digests are set up as before; at each selected time point, one tube is transferred to ice ;and mixed with 5/xl of 0.5 M EDTA. When all partial digests are completed, the samples are heated to 70 ° for 20 rain to terminate the reaction. Alternate Methods
In place of using partial restriction endonuclease digestion to produce a representative collection of fragments, the chromosomal DNA can be mechanically sheared using sonication. The fragments are then methylated with BamHI methylase, the sheared ends repaired with T4 polymerase and deoxynucleoside triphosphates (dNTPs), ligated with BamHI linkers, and finally ligated into the BamHl site of a suitable vector. Although this procedure will likely result in a more random library, in practice the enzymatically fragmented DNA is satisfactory and easier to produce.
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1
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FIG. 1. Partial digest of bacterial DNA with the restriction endonuclease Sau3A. DNA was digested with Sau3A at room temperature for the time intervals shown (in minutes), and samples from each time point were examined using 0.7% agarose gel electrophoresis.
Size Fractionation Materials and Procedure
Digested DNA is size fractionated using centrifugation through 5-20% NaC1 gradients. The gradients are prepared using sterile solutions in diethyl pyrocarbonate (DEPC)-treated Beckman SW41 ultracentrifuge tubes and chilled on ice prior to use. The four partial digests are pooled, and 200/A is loaded on each of two gradients. The gradients are centrifuged in a Beckman SW41 rotor for 5 hr at 35,000 rpm and 4°. Fractions (0.5 ml) are collected. If fractions are to be collected from the bottom of the gradient, it is helpful to include a 50% sucrose cushion; this will prevent larger DNA fragments from forming a pellet and plugging the collection needle. Samples (5/A) of alternate fractions are examined using electrophoresis through an 0.35% agarose gel for 18 hr at 1 V/cm. After samples for electrophoresis are taken, 1 ml of 95% ethanol is mixed with each fraction, and the DNA is allowed to precipitate overnight at - 2 0 °. The fractionated DNA can be stored indefinitely in the freezer at this step. In the example in Fig. 2, DNA in fractions 12-17 is greater than 30 kb and would be suitable for preparing cosmid libraries. DNA in fractions 9-11 is in the 20to 30-kb range and would be suitable for use with k vectors.
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11 S 13 H 15 17 19
FIG. 2. Agarose gel showing size-fractionated DNA obtained from a 5-20% NaC1 gradient. Fraction numbers from the top of the tube are indicated. Molecular weight standards are bacteriophage h DNA digested with SalI (S) (33 and 15 kb) and HindIII (H) (23.7, 9.5, 6.7, 4.3, and 2.3 kb).
Alternate Methods
The sizing step is included to remove low molecular weight DNA fragments which would otherwise ligate together randomly to form an insert sufficiently large to be packaged. This is especially important when cloning large operons because the presence of clones composed of random rearrangements will both reduce cloning efficiency and lead to potentially confusing results. The formation of randomly associated molecules can also be prevented by dephosphorylating the insert D N A prior to ligating with vector However, under such conditions, correct insert-to-vector molar ratios are difficult to calculate as the majority of insert DNA in the ligation mix, although capable of ligating to vector, is too small to be packaged. As a consequence, much of the vector is consumed in formation of unproductive molecules. D N A may also be size fractionated by preparative electrophoresis through 0.35% agarose gels. This procedure is limited by the capacity of the gels used. DNA fragments of the appropriate size for cosmid cloning represent a small fraction of the total partial digest; D N A recovered from several gels is needed to provide a sufficient amount of high quality product for a single ligation.
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Ligation and Packaging DNA from selected size fractions is collected by centrifugation and suspended in 0.4 ml TE. The DNA concentration of the undiluted sample is determined spectrophotometrically in DEPC-treated semimicrocuvettes. The concentration is usually about 2-3 ~g/ml for fractions greater than 30 kb and 8-10/xg/ml for the 20- to 30-kb fractions. For ligation, fractions in the appropriate size range may be pooled to supply 2.5-3.5 /xg and copreeipitated with Barn HI-digested cosmid vector such as pHC79 or phage vector such as EMBL3 at a 1 : 1 molar ratio. Prior dephosphorylation of the vector is not beneficial. Dephosphorylation of the insert is also not required but may be beneficial if the insert DNA is not size fraetionated as discussed above. Ligation is done using standard conditions in a final volume of 11/xl. After ligation, the sample will become viscous. Two to three microliters of ligated DNA is then packaged in vitro into bacteriophage X transducing particles. 2 The packaged phage can be stored several years at -70 ° with minimal loss of viability. Selection of a host strain is based primarily on its susceptibility to bacteriophage k. HB101 is a suitable host for either cosmid or phage cloning of adhesin genes. However, this host expresses type 1 pili under some growth conditions, which may potentially complicate interpretation of resuits. Escherichia coli P678-54, and its recA derivative, JW369, lack genes for type 1 pili and may therefore be more useful. For experiments using phage replacement vectors, E. coli Q359 is used as a host. The transducing titer (vector antibiotic resistance phenotype) for cosmid libraries is about 103//xl phage mixture. If one assumes an average insert size of 40 kb in a cosmid clone and an E. coli genome size of 4700 kb, the frequency of any particular gene in a library would be 1 in 118 ampicillinresistant transductants. In practice, the frequency of complete adhesin gene clusters among ampicillin-resistant transductants varies between 0.1 and 1%. The titer of recombinant phage using k replacement vectors is usually about 102-103/ml. The frequency of recombinant phage containing at least part of an adhesin gene cluster is 0.1-1%. However, the frequency of complete adhesin gene clusters is lower as compared with cosmid cloning (about 0.01-0.2%) owing to the smaller size of the phage vector inserts.
Functional Screening Methods for Identifying Adhesin Clones If the adhesion phenotype of interest is a hemagglutinin, hemagglutination of suseptible erythrocytes is the most direct way to screen for recombiz L. Enquist and N. Sternberg, this series, Vol. 68, p. 281.
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nant cosmid clones. Growth conditions for maximal expression of hemagglutination in the donor strain should be established prior to screening recombinant clones. Conditions to consider include incubation temperature, growth with or without aeration, liquid versus solid media, minimal media versus L broth or other complex media, and glucose effects.
Hemagglutination Ampicillin-resistant transductants are picked and inoculated as a patch, 50-100 per plate, onto two L plates containing ampicillin and incubated overnight. Patches should be tested the following day as the hemagglutination capacity of recombinant clones is often diminished after prolonged incubation. Erythrocytes are washed and suspended at 3% (v/v) in phosphate-buffered saline (pH 7.0) containing 0.01% gelatin (BSG). Human erythrocytes should be used within 1 week of collection and washed and diluted just prior to testing hemagglutination. If the desired adhesion phenotype Jis D-mannose-resistant, 10 mM D-mannose may be included in the erythrocyte mixture. Bacteria from an individual patch are picked with a sterile toothpick and mixed with a drop of erythrocyte solution on a glass plate chilled over ice. Enough bacteria should be used so that the drop of erythrocytes becomes visibly turbid. Patches are tested in batches of 1020; afl:er each batch, the glass plate is gently rocked 5-10 times to enhance visualization of hemagglutination, which may appear immediately or only after rocking.
Gradient Enrichment The frequency of hemagglutinating clones can also be enriched, if necessary, using glycerol step gradient centrifugation. Step gradients are prepared in sterile 15-ml clear centrifuge tubes, using 3 ml of 20% glycerol in BSG for the bottom step and 3 ml of 5% (v/v) glycerol in BSG for the top step. Ampicillin-resistant recombinant bacteria are collected from overnight growth (pool 104-105 colonies), suspended gently in BSG to an OD600 of 1.5, and diluted 1 : 10 in BSG. One-half milliliter diluted bacteria is mixed with 0.5 ml 3% erythrocytes and incubated at room temperature for 20 rain with occasional gentle mixing. The bacteria-erythrocyte suspension is then layered onto the gradient and centrifuged at 600 rpm (relative centrifugual force of 77) for 12 min in a Sorvall GLC centrifuge with a swinging bucket rotor. The 5% glycerol step and sample load volume are removed with a syringe and bent 18-gauge needle and gently replaced with 3 ml sterile BSG as a wash. The BSG and remaining gradient are removed, the erythrocytes suspended in BSG, and dilutions spread on antibiotic-containing agar plates. A single gradient cycle provides approximately 10-fold enrichment. The
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maximum enrichment achieved using serial gradients with intermediate regrowth steps was 105-fold.
Advantages The hemagglutination procedure is simple and direct. It assures that at least the minimum number of genes required for the adhesion phenotype are present on the cloned molecule.
Disadvantages (1) Cloned genes, especially from genera other than Escherichia, may not be expressed or their products may not be assembled into adhesin organelles in E. coli. (2) In general, this method will not work for adhesins that are not hemagglutinins. However, if the tissue receptor for the adhesin of interest is known, latex beads coated with receptor may be substituted for erythrocytes. (3) Clones that contain only part of the adhesin gene cluster will not be detected. Antigen Screening Methods for Identifying Adhesin Clones Recombinant D N A libraries may also be screened for the presence of adhesin-associated antigens without regard to expression of functional adhesion. The methods used for screening either cosmid or phage libraries for adhesin-associated antigens do not differ significantly from general published procedures. 3,4 In the following a method is described that is useful in identifying appropriate antigens for preparation of antibody when the nature of the adhesin organelle is unknown; this method is applicable to adhesins that are not hemagglutinins, s
Materials 10 ml Minimal medium: 60 mM potassium phosphate (pH 7.4); 2 mM disodium citrate; 0.8 mM MgCI2; 15 mM NH4C1; 80/zM (NH4)SO4; 20 /xg/ml of required amino acids; 0.4% glycerol; and 0.5 mg/ml yeast extract H235SO4 12.5% sodium dodecyl sulfate (SDS)-polyacrylamide gel and associated buffers 3 j. Sambrook, E. F. Fritsch, and T. Maniatis, "Molecular Cloning: A Laboratory Manual," 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989. 4 M. Snyder, S. Elledge, D. Sweetser, R. A. Young, and R. W. Davis, this series, Vol. 154, p. 107. s S. K. Wray, S. I. Hull, R. G. Cook, J. Barrish, and R. A. Hull, Infect. Immun. 54, 43 (1986).
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BSG (phosphate-buffered saline, pH 7.0, with 0.01% gelatin) Target tissue to which bacteria adheres dispersed as single cells or groups of two or three cells
Procedure Bacteria are grown overnight in 10 ml minimal medium containing 50 i~Ci/ml H235SO4 (or [14C]glucose and additional unlabeled SO4 if the adhesin is thought not to be a protein). Other growth conditions may be used to enhance the adhesion phenotype. The bacteria are washed once with 1 ml BSG and suspended in 1 ml BSG at a density of 1-2 × 10l° cells/ml. Outer membrane material is sheared from the bacterial surface by multiple passage through a 26 gauge needle. Whole bacteria are removed by centrifugation for 10 min at 10,000 × g, 4°. Target cells are washed in BSG, and pellets containing 2 × l0 s cells are suspended in BSG containing radiolabeled outer membrane material. After incubation at 37° for 1 hr with continuous mixing, the reaction mix is centrifuged, and both the pellet and supernatant are retained. The cell pellet is washed 3 times with BSG and suspended in 1 ml BSG. The radioactivity of the cell and original supernatant fractions is determined, and an equal number of counts per minute (cpm) from each are dialyzed versus distilled water and freezedried. Samples are then examined using SDS-polyacrylamide gel electrophoresis and autoradiography. Adhesin antigens will be those enriched in the cell fraction as compared to the supernatant. This method may also be used in development of purification strategies for adhesin molecules. Presence of the adhesin in fractions obtained at different purification steps can be monitored by including a sample of the fraction in the reaction mix and measuring its capacity to competitively reduce binding of radiolabeled adhesin to tissue.
Advantages (1) The antigen screening method does not require assembly of a functional adhesin organelle. Adhesin genes from other genera of bacteria that may be cloned into E. coli but not assembled and clones that contain only part of an adhesin operon can be identified. (2) This method does not require that the adhesin-associated molecules be proteins, only that they be antigenic. (3) The method can be used with both phage and cosmid libraries.
Disad vantages (1), The adhesin molecule must be purified in advance for production of antibody; alternatively, a homogenic mutant derivative of the donor
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strain that does not express the adhesin antigen must be available to adsorb irrelevant antibodies. (2) Clones that express cross-reactive antigens may be inadvertently selected. DNA Hybridization Screening Method for Identifying Adhesin Gene Clones Recombinant DNA libraries may also be screened for the presence of adhesin-associated gene sequences without regard to the expression of any cloned gene. For screening cosmid libraries, the colony blot method described by Maas 6 is both sensitive and convenient. Filters for hybridization are prepared directly from the transduced colonies, and hybridizing colonies can be recovered from growth remaining after blotting. Materials
Whatman (Clifton, N J) No. 3 paper and autoclaved Whatman 541 paper 0.5 M NaOH, 1.5 M NaC1 1 M Tris (pH 7.0), 2 M NaC1 Procedure
Sterile Whatman 541 paper is placed on agar plates containing 100200 colonies. The filters are gently massaged to remove any bubbles. The orientation of the filter is marked with a pencil. The Whatman 541 papers are placed bacteria side up in a glass petri dish on three layers of Whatman No. 3 paper saturated with NaOH/NaC1 solution. Papers are steamed for 4 min in a covered beaker of boiling water. The Whatman 541 paper is removed and immersed in 300-500 ml of 1 M Tris (pH 7.0), 2 M NaC1 for 4 min. Air dry. Prior to hybridization, the filters are washed once briefly in hybridization solution and incubated with hybridization solution containing the selected probe and competitor DNA. No filter baking or prehybridization step is required. For screening phage libraries, standard plaque hybridization methods are appropriate. 3 Advantages
(1) No gene expression is required so adhesin genes that are neither functional nor expressed in E. coli can be cloned. (2) The procedure can be used to screen phage libraries so adhesin genes producing products that inhibit growth of E. coli can be cloned. 6R. Maas, Plasmid 10, 296 (1983).
MOLECULAR ANALYSISOF S. pyogenes ADHESION
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Disadvantages The D N A hybridization method is useful only when D N A or amino acid sequence information is available for the gene of interest. Unless the adhesin genes share D N A sequence similarity with previously cloned genes wherein the previous clone can be used as a hybridization probe, it is necessary to design synthetic oligonucleotide probes based on the amino acid sequence of an adhesin-associated protein. As a consequence of the degeneracy of the triplet code, such probes are usually ambiguous at one or mare positions and have reduced specificity. Isolation of incorrect crossreactive clones is possible and even likely, leading to considerable wasted effort. Alternately, converging oligonucleotides designed on the basis of the amino acid sequence from different regions of the protein can be used as polymerase chain reaction (PCR) primers to produce a D N A probe. For this method, the D N A sequence of any PCR product should be determined to confirm its appropriateness prior to using it as a probe. The deduced amino acid sequence predicted from the PCR product should match the actual amino acid sequence of the target protein.
Acknowledgments This work was supported by U.S. Public Health Service Grants A I 21009 and A I 18462.
[23] M o l e c u l a r
By E M A N U E L
Analysis of Streptococcus pyogenes Adhesion
HANSKI, GEORGE FOGG, AVIVA I S R A E L BURSTEIN,
and
Tovi,
NOBUH1KO O K A D A ,
MICHAEL CAPARON
Introduction
Streptococcus pyogenes (the group A streptococcus) is one of the most versatile human pathogens as regards the number of different tissues it can infect and the wide range of different diseases it can cause. Streptococcus pyogenes can cause disease through three basic pathogenic mechanisms. Multiplication on the surface of or within a tissue results in destruction of host cells and is accompanied by an intense inflammatory response. These types of diseases can range from the more mild and self-limiting infections of the throat (e.g., pharyngitis, commonly known as "strep throat") and the skin (e.g., impetigo) to infections which involve increasingly deeper METHODSIN ENZYMOLOGY.VOL.253
Copyright © 1995by AcademicPress. Inc. All rightsof reproductionin any formreserved.