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Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes
FEMS MicrobiologyLetters 71 (1990) 345-350 Published by Elsevier
345
FEMSLE~ 1 ~
Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes W.A. R e l f a n d K.S. Sriprakash Menzies Schoolof Health Research, Casuarina,Australia
Received29 May 1990 Accepted4 June 1990 Key words: Streptococcus pyogenes; M protein; Polymerase chain reaction; Opacity factor 1. S U M M A R Y We have amplified genomic sequences (emm) that may encode M protein from strains of Streptococcus pyogenes using the polymerase chain reaction (PER). Genomic D N A from 22 isolates representing 14 M scretypes was selected for the study. Primers which corresponded to the observed N-terminal signal sequence and the variable C-terminal sequences of emm6, emm49 and e n n X were used. PCR products using emm6 and emm49 oligonucleotides were classified into two mutually exclusive groups which corresponded to the presence or absence of serum opacity factor. These findings support the concept of limited heterogeneity in the C-terminal sequences of the M protein. 2. I N T R O D U C T I O N The M protein of S. pyogenes is a fibrillar surface protein which impedes phagocytosis by polymorphonuclear cells in the absence of typespecific antibodies [1]. Serotype specificity for more than 80 distinct M proteins is conferred by Correspondence to: R.S. Sriprakash, MenziesSchoolof Health Research, PO Box 41096, Casuarina, Austrafia 0811.
the N-tern-Anal sequence. The M protein contains a signal peptide which is conserved among all M proteins studied [2]. The membrane anchor region proximal to the C-terminus is also conserved, although variations have been observed, emm5. emm6, e m m l 2 and emm24 have very similar sequences for the membrane anchor region [3-6l while the analogous region for emm49 is divergent [7]. Earlier work using antibodies to surface-exposed domains [8] and D N A probes [9] examined differences in M proteins within the middle third of the molecule comprising the C repeat region. We have designed sets of oligonucleotide primers to amplify emm genes by PCR. Genomic D N A preparations from laboratory and field isolates of S. pyogenes yielded PCR products using either one of the two sets of primers which define emm6 and emm49, hence we report the existence of two major lineages of M protein (emm6-1ike and emm49-1ike). Interestingly, the opacity factor, the ability of some serotypes to cause serum opalescence, co-segregates with this lineage.
3. MATERIALS and METHODS 3.1. Strains and DNA extraction Table 1 lists the M type, specificities and origins of the S. pyogenes isolates selected for this study.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
346 In several instances, independent isolates of the same M type were used. G e n o m i c D N A was purified following extensive washing of the bacteria with tris-EDTA buffer [10].
3.2. Polymerase chain reaction and primers Three sets of primers (p6, p49 and p X ) were designed with either a Pstl or B a m H I restriction enzyme site at the 5' end of the oligonucleotide to facilitate cloning. All sets had the s a m e forward p r i m e r (sense strand) 5 ' - A A T C T G C A G T A T T C G CTF A G A AAA TTA AAA-3' corresponding to the N-terminal signal sequence. The reverse primers (antisense primers) c o r r e s p o n d i n g to the C-terminus differed in each case and were based on the published sequences of emm6 [4], emm49 [7] and enn X, a M-related gene [7]. T h e sequences were 5 ' - T G C G G A T C C A G C T G T T G C C A T AAC AGT AAG-3', 5'-TI'G GGA TCC TGC
T G A T C T T G A A C G G T T A G C - 3 ' and 5 ' - A A G GGA TCC TGC AGA TAC CAT CAC TGT T G C A-3' for p6, p49 a n d pX, respectively. P C R reaction mixture contained 100 ng of S. pyogenes genomic D N A with 50 ng each of the p r i m e r as described by Saiki et ai. [11]. T h e reaction cycles (25 cycles) included d e n a t u r a t i o n ( 9 4 ° C , 1 min), annealing ( 3 7 ° C , 2 w i n ) and extension (72" C, 3 rain) using a D N A thermal cycler (Perkin E l m e r / C e t u s ) . Following amplification, samples were analysed by agarose gel electrophoresis and visualised b y ethidium b r o m i d e staining.
3.3. DNA hybridisation Blotting a n d hybridisation conditions have been described previously [12]. High ancI low stringency washes at 6 5 ° C were 0.1 × SSC and 2 × SSC, respectively.
Table 1 Characteristics of isolates of S. pyogenes Serotype, opacity factor (OF) and amplification of DNA with p6, p49 and pX primers are summarised. The opacity factor characteristics of IGL251 M30 and A995 M57 are unavailable. + M indicates faint multiple bands. Isolate
Type
Origin
NH182/905 N HI88/313 NHI2032 Y503S NHI1357 SS31 68/3116 728/150A/6 1GL251 B737/71/1 Y530S NH188/28 A1799 A1800 NH188/30 NHI88/32 NH188/35 A995 A1798 SS94 D335 F7/3554
M1 M1 M2 M2 M4 M13 M22 M28 M30 M49 M49 M53 M55 M55 M57 M57 M57 M57 M59 M59 M60 M75
Australia New Zealand Czechoslovakia Australia New Zealand U.K. U.K. Czechoslovakia Czechoslovakia Australia Australia Australia Australia Australia Australia Australia Australia Czechoslovakia Czechoslovakia New Zealand
Results of PCR with primers OF
p6
p49
pX
+ + + + + + ? + + ? + + + +
+ + + + + + ~ + + + -
+M +M + + + + + + +M + + +M +M +M +M +M +M +M + + + +
+ + + + + + + + + + + + + +
347 4. RESULTS
emm6, emml2 and emm24 and has limited homology to emm49 and arp4. Thus it was expected
4. !. PCR primers
that set p6 would give a PCR product in the tsolates which comained emm6-1ike genes. The reverse primers in sets p~9 and pX are 100% homologous to emm49 and 96% homologous to arp4. Therefore, using these primers, more than one PCR product may result in strains containing both emm49-1ike and arp4-1ike genes. Furthermore, the antisense primer in set pX is 100% homologous to ennX and may amplify an additional gene.
The primer corresponding to the signal peptide was 100% homologous to the published sequences of emm5, emm6, emm12, emm24 and emm49. Database analysis also revealed homology with the signal peptide regions for other M-like proteins in streptococci. These include ennX (76%), arp4 000%) and fcrA76 (81%) [7,13,14] (Table 2). The reverse primer corresponding to the C-terminal region in set p6 is 100% homologous to emm5,
Fig. 1. Agaros¢gel electrophoresisand e~hidiumbromide staining followingPCR. Results are shown for p6, p49 and pX primers. Lanes 2-12 and 15-25 are NH182/905 MI. NH188/313 M1, NH12032 M2, Y503S M2, NHl1357 M4, SS31 MI3. 68/3116 M22, 728/150A/6 M28, IGL251 M30. B737/71/1 M49, Y530S M49, NH188/28 M53. A1799 M55, A1800 M55. NH188/30 M57. NH18g/32 M57. NH188/35 M57. A995 M57. A1798 M59, SS94 M59. D335 M60 and P7/3554 M75. respectively.Lanes 1o 13, 14 and 27 show2.3 kb and 2.0 kb size markers froma Hmdlll digestof lambda. A no-DNAcontrol was includedin Lane26.
348
4.2. Differential amplification with PCR primers A total of 22 independent isolates of S. pyogenes belonging to 14 distinct serotypes (M types) from several geographical locations were analysed using the three sets of primers. DNA from all isolates yielded PCR product(s) of 0.5--3.0 kb size. Nonspecific PCR products usually gave faint multiple bands. Fig. 1 shows the agarose gel analysis of the PCR products and the results are summarised in Table 1. The isolates can be divided into two groups. Firstly, an emm6-iike category which gave a PCR product with p6 primers and secondly, an emm49-like category which gave one or more products with p49 primers. The emm6-fike category contains opacity factor negative isolates whilst the emm49-like category contains opacity factor positive isolates. All emm49-fike isolates also gave PCR products with pX primers. Two emm6-1ike isolates (M53 NHI88/28 and M57 NHI88/30) also gave products with pX primers. The sizes of these pJ:oducts were different from the corresponding PCR products with p6 primers. These two isolates may have an enn X-like gene in addition to an emm6-like gene. 4.3. Partial characterisation of PCR products We cloned the PCR product obtained from the M57 isolate, A995 and the complete nucleotide sequence was determined (Manjula et al., unpubfished observations). The fully characterised emm57 PCR product was radioactively labelled and hybridised to a Southern blot of the PCR
Table2 Homologyof primersto M related genes The percent homologywas determinedfor the complete sequence of each primer. X denotes a lack of significanthomology for the full length sequence.N/A ffithe C terminal sequenceis not reported [14]. M related gene
Forward primer
Reverseprimers p6 p49
emm6 emm49
lO0~ lO0~ 100%
100~ X X
arp4 fcrA76 ennX
81~ 76~
pX
X 100~
X 96~
N/A
96% N/A
96% N/A
X
X
100~
products. The products resulting from p6 primers hybridised strongly even at high stringency, whereas the PCR products from p49 primers gave a relatively poor signal (data not shown). The differential hybridisation results are consistent with the hybridisation data of Scott et al. [9] for genomic DNA.
5. DISCUSSION Primer sets p6 and p49 products with DNA from all M types tested. The PCR product from M57 was cloned and sequenced. The derived amino acid sequence was in complete agreement with the partial peptide sequence of the purified M57 prorein (Manjula et al., unpublished observations). These data, together with the size distribution of the major PCR products and the hybridisation results suggest that the major PCR products represent emm sequences. Therefore we suggest the existence of two different (emm6-1ike and emm49-like) lineages of M protein based on the DNA sequence near the C-terminal region. Amplification with pX primers verified the emm49-like lineage as this primer was also 100~ homologous with emm49. Multiple bands using the p49 primers may represent amplification of M protein genes and M-like genes, i.e., arp and fcr. If the members of a multigene family are closely lined [7,14], a large composite band of two genes may occur following PCR. We have one instance (M57 NHI88/30) where the PCR fragment was large enough (3 kb) to contain two members of the family. However, the conditions for PCR in this study were in general not optimal for amplification of larger DNA fragments and they were not observed in all suspected cases. Bessen et al. [8] have reported the existence of two main classes of M proteins based on immunological cross reactivity with the antibodies to the C repeat region of the M6 protein. This classification correlated with MAPI (M-associated protein) or MAPII and with the absence or presence of opacity factor. Our observation on co-segregation of the lack of opacity factor with the emm6-fike gene parallels these data [8]. The two strains which may possess both emm6-fike and ennX-fike genes also
349 are devoid of opacity factor. T h u s it is unlikely t h a t the ennX-fike gene codes for this trait. B a s e d o n these results, it is t e m p t i n g to speculate t h a t t h e r e is o n l y a limited repertoire for the region p r o x i m a l to the C - t e r m i n u s o f the M p r o t e i n as d e t e r m i n e d b y D N A sequence.
ACKNOWLEDGEMENTS W e t h a n k Drs. D i a n a M a r t i n , N e w Z e a l a n d C o m m u n i c a b l e D i s e a s e C e n t r e a n d Belur M a n j u l a , Rockefeller University, N e w Y o r k for the s t r a i n s a n d opacity factor characteristics. W e are grateful to Dr. Valerie A s c h e a n d Professor J o h n M a t h e w s for helpful discussions.
REFERENCES [1] Lancefield, R.C. (1962) J. Immunol. 89, 307-313. [2] Haanes-Fritz, E., Kraus, W., Burdett, V., Dale, J.B., Beache~-, E.H. and Cleary, P.P. (1988) Nucleic Acids Res. 16, 4667-4677.
[3] Miller, L., Gray, L., Beachey, E.H. and Kehoe, M.A. (1988) J. Biol. Chem. 263, 5668-5673. [4] Hollingshead, S.K., Fischeui, V.A. and Scott, J. (1986) J. Biol. Chem. 261, 1677-1686. [5] Robbins, J.C., Spanier, J.G., Jones, SJ., Simpson, W.J. and Cleary, P.P. (1987) J. Bacteriol. 169, 5633-5640. [6] Mouw, A.R., Beachey, E.H. and Burdett, V. (1988) J. Bacteriol. 170, 676-684. [7] Haanes, E. and Cleary, P.P. (1989) J. Bacteciol. 171, 6397-6408. [8] Bessen, D., Jones, K.F. and Fischetti, V.A. (1989) J. Exp. Med. 169, 269-283. [9] Scott, J.R., Holfingshead, S.K. and Fischetti, V.A. (1986) Infect. Immun. 52, 609-612. [10] Skjold, S.A., Quie, P.G., Fries, L.A., Bamham, M. and Cleary, P.P. (1987) J. Infect. Dis. 155,1145-1150. [11] Saiki, R.K., Scharf, S., Faloona, F., Muller, K.B., Horn, G.T., Ehrlich, H.A. and Arnheim, N. (1985) Science 230, 1350-1354. [12] Reed, K.C. and Mann. D. (1985) Nucleic Acids Res. 13, 7207-7221. [13] Frithz, E., Heden, L.-O. and Lindahl, G. (1989) Mol. Microbiol. 3, 1111-1119. [14] Heath, D.G. and Cteary, P.P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4741-4745.
345
FEMSLE~ 1 ~
Limited repertoire of the C-terminal region of the M protein in Streptococcus pyogenes W.A. R e l f a n d K.S. Sriprakash Menzies Schoolof Health Research, Casuarina,Australia
Received29 May 1990 Accepted4 June 1990 Key words: Streptococcus pyogenes; M protein; Polymerase chain reaction; Opacity factor 1. S U M M A R Y We have amplified genomic sequences (emm) that may encode M protein from strains of Streptococcus pyogenes using the polymerase chain reaction (PER). Genomic D N A from 22 isolates representing 14 M scretypes was selected for the study. Primers which corresponded to the observed N-terminal signal sequence and the variable C-terminal sequences of emm6, emm49 and e n n X were used. PCR products using emm6 and emm49 oligonucleotides were classified into two mutually exclusive groups which corresponded to the presence or absence of serum opacity factor. These findings support the concept of limited heterogeneity in the C-terminal sequences of the M protein. 2. I N T R O D U C T I O N The M protein of S. pyogenes is a fibrillar surface protein which impedes phagocytosis by polymorphonuclear cells in the absence of typespecific antibodies [1]. Serotype specificity for more than 80 distinct M proteins is conferred by Correspondence to: R.S. Sriprakash, MenziesSchoolof Health Research, PO Box 41096, Casuarina, Austrafia 0811.
the N-tern-Anal sequence. The M protein contains a signal peptide which is conserved among all M proteins studied [2]. The membrane anchor region proximal to the C-terminus is also conserved, although variations have been observed, emm5. emm6, e m m l 2 and emm24 have very similar sequences for the membrane anchor region [3-6l while the analogous region for emm49 is divergent [7]. Earlier work using antibodies to surface-exposed domains [8] and D N A probes [9] examined differences in M proteins within the middle third of the molecule comprising the C repeat region. We have designed sets of oligonucleotide primers to amplify emm genes by PCR. Genomic D N A preparations from laboratory and field isolates of S. pyogenes yielded PCR products using either one of the two sets of primers which define emm6 and emm49, hence we report the existence of two major lineages of M protein (emm6-1ike and emm49-1ike). Interestingly, the opacity factor, the ability of some serotypes to cause serum opalescence, co-segregates with this lineage.
3. MATERIALS and METHODS 3.1. Strains and DNA extraction Table 1 lists the M type, specificities and origins of the S. pyogenes isolates selected for this study.
0378-1097/90/$03.50 © 1990 Federation of European MicrobiologicalSocieties
346 In several instances, independent isolates of the same M type were used. G e n o m i c D N A was purified following extensive washing of the bacteria with tris-EDTA buffer [10].
3.2. Polymerase chain reaction and primers Three sets of primers (p6, p49 and p X ) were designed with either a Pstl or B a m H I restriction enzyme site at the 5' end of the oligonucleotide to facilitate cloning. All sets had the s a m e forward p r i m e r (sense strand) 5 ' - A A T C T G C A G T A T T C G CTF A G A AAA TTA AAA-3' corresponding to the N-terminal signal sequence. The reverse primers (antisense primers) c o r r e s p o n d i n g to the C-terminus differed in each case and were based on the published sequences of emm6 [4], emm49 [7] and enn X, a M-related gene [7]. T h e sequences were 5 ' - T G C G G A T C C A G C T G T T G C C A T AAC AGT AAG-3', 5'-TI'G GGA TCC TGC
T G A T C T T G A A C G G T T A G C - 3 ' and 5 ' - A A G GGA TCC TGC AGA TAC CAT CAC TGT T G C A-3' for p6, p49 a n d pX, respectively. P C R reaction mixture contained 100 ng of S. pyogenes genomic D N A with 50 ng each of the p r i m e r as described by Saiki et ai. [11]. T h e reaction cycles (25 cycles) included d e n a t u r a t i o n ( 9 4 ° C , 1 min), annealing ( 3 7 ° C , 2 w i n ) and extension (72" C, 3 rain) using a D N A thermal cycler (Perkin E l m e r / C e t u s ) . Following amplification, samples were analysed by agarose gel electrophoresis and visualised b y ethidium b r o m i d e staining.
3.3. DNA hybridisation Blotting a n d hybridisation conditions have been described previously [12]. High ancI low stringency washes at 6 5 ° C were 0.1 × SSC and 2 × SSC, respectively.
Table 1 Characteristics of isolates of S. pyogenes Serotype, opacity factor (OF) and amplification of DNA with p6, p49 and pX primers are summarised. The opacity factor characteristics of IGL251 M30 and A995 M57 are unavailable. + M indicates faint multiple bands. Isolate
Type
Origin
NH182/905 N HI88/313 NHI2032 Y503S NHI1357 SS31 68/3116 728/150A/6 1GL251 B737/71/1 Y530S NH188/28 A1799 A1800 NH188/30 NHI88/32 NH188/35 A995 A1798 SS94 D335 F7/3554
M1 M1 M2 M2 M4 M13 M22 M28 M30 M49 M49 M53 M55 M55 M57 M57 M57 M57 M59 M59 M60 M75
Australia New Zealand Czechoslovakia Australia New Zealand U.K. U.K. Czechoslovakia Czechoslovakia Australia Australia Australia Australia Australia Australia Australia Australia Czechoslovakia Czechoslovakia New Zealand
Results of PCR with primers OF
p6
p49
pX
+ + + + + + ? + + ? + + + +
+ + + + + + ~ + + + -
+M +M + + + + + + +M + + +M +M +M +M +M +M +M + + + +
+ + + + + + + + + + + + + +
347 4. RESULTS
emm6, emml2 and emm24 and has limited homology to emm49 and arp4. Thus it was expected
4. !. PCR primers
that set p6 would give a PCR product in the tsolates which comained emm6-1ike genes. The reverse primers in sets p~9 and pX are 100% homologous to emm49 and 96% homologous to arp4. Therefore, using these primers, more than one PCR product may result in strains containing both emm49-1ike and arp4-1ike genes. Furthermore, the antisense primer in set pX is 100% homologous to ennX and may amplify an additional gene.
The primer corresponding to the signal peptide was 100% homologous to the published sequences of emm5, emm6, emm12, emm24 and emm49. Database analysis also revealed homology with the signal peptide regions for other M-like proteins in streptococci. These include ennX (76%), arp4 000%) and fcrA76 (81%) [7,13,14] (Table 2). The reverse primer corresponding to the C-terminal region in set p6 is 100% homologous to emm5,
Fig. 1. Agaros¢gel electrophoresisand e~hidiumbromide staining followingPCR. Results are shown for p6, p49 and pX primers. Lanes 2-12 and 15-25 are NH182/905 MI. NH188/313 M1, NH12032 M2, Y503S M2, NHl1357 M4, SS31 MI3. 68/3116 M22, 728/150A/6 M28, IGL251 M30. B737/71/1 M49, Y530S M49, NH188/28 M53. A1799 M55, A1800 M55. NH188/30 M57. NH18g/32 M57. NH188/35 M57. A995 M57. A1798 M59, SS94 M59. D335 M60 and P7/3554 M75. respectively.Lanes 1o 13, 14 and 27 show2.3 kb and 2.0 kb size markers froma Hmdlll digestof lambda. A no-DNAcontrol was includedin Lane26.
348
4.2. Differential amplification with PCR primers A total of 22 independent isolates of S. pyogenes belonging to 14 distinct serotypes (M types) from several geographical locations were analysed using the three sets of primers. DNA from all isolates yielded PCR product(s) of 0.5--3.0 kb size. Nonspecific PCR products usually gave faint multiple bands. Fig. 1 shows the agarose gel analysis of the PCR products and the results are summarised in Table 1. The isolates can be divided into two groups. Firstly, an emm6-iike category which gave a PCR product with p6 primers and secondly, an emm49-like category which gave one or more products with p49 primers. The emm6-fike category contains opacity factor negative isolates whilst the emm49-like category contains opacity factor positive isolates. All emm49-fike isolates also gave PCR products with pX primers. Two emm6-1ike isolates (M53 NHI88/28 and M57 NHI88/30) also gave products with pX primers. The sizes of these pJ:oducts were different from the corresponding PCR products with p6 primers. These two isolates may have an enn X-like gene in addition to an emm6-like gene. 4.3. Partial characterisation of PCR products We cloned the PCR product obtained from the M57 isolate, A995 and the complete nucleotide sequence was determined (Manjula et al., unpubfished observations). The fully characterised emm57 PCR product was radioactively labelled and hybridised to a Southern blot of the PCR
Table2 Homologyof primersto M related genes The percent homologywas determinedfor the complete sequence of each primer. X denotes a lack of significanthomology for the full length sequence.N/A ffithe C terminal sequenceis not reported [14]. M related gene
Forward primer
Reverseprimers p6 p49
emm6 emm49
lO0~ lO0~ 100%
100~ X X
arp4 fcrA76 ennX
81~ 76~
pX
X 100~
X 96~
N/A
96% N/A
96% N/A
X
X
100~
products. The products resulting from p6 primers hybridised strongly even at high stringency, whereas the PCR products from p49 primers gave a relatively poor signal (data not shown). The differential hybridisation results are consistent with the hybridisation data of Scott et al. [9] for genomic DNA.
5. DISCUSSION Primer sets p6 and p49 products with DNA from all M types tested. The PCR product from M57 was cloned and sequenced. The derived amino acid sequence was in complete agreement with the partial peptide sequence of the purified M57 prorein (Manjula et al., unpublished observations). These data, together with the size distribution of the major PCR products and the hybridisation results suggest that the major PCR products represent emm sequences. Therefore we suggest the existence of two different (emm6-1ike and emm49-like) lineages of M protein based on the DNA sequence near the C-terminal region. Amplification with pX primers verified the emm49-like lineage as this primer was also 100~ homologous with emm49. Multiple bands using the p49 primers may represent amplification of M protein genes and M-like genes, i.e., arp and fcr. If the members of a multigene family are closely lined [7,14], a large composite band of two genes may occur following PCR. We have one instance (M57 NHI88/30) where the PCR fragment was large enough (3 kb) to contain two members of the family. However, the conditions for PCR in this study were in general not optimal for amplification of larger DNA fragments and they were not observed in all suspected cases. Bessen et al. [8] have reported the existence of two main classes of M proteins based on immunological cross reactivity with the antibodies to the C repeat region of the M6 protein. This classification correlated with MAPI (M-associated protein) or MAPII and with the absence or presence of opacity factor. Our observation on co-segregation of the lack of opacity factor with the emm6-fike gene parallels these data [8]. The two strains which may possess both emm6-fike and ennX-fike genes also
349 are devoid of opacity factor. T h u s it is unlikely t h a t the ennX-fike gene codes for this trait. B a s e d o n these results, it is t e m p t i n g to speculate t h a t t h e r e is o n l y a limited repertoire for the region p r o x i m a l to the C - t e r m i n u s o f the M p r o t e i n as d e t e r m i n e d b y D N A sequence.
ACKNOWLEDGEMENTS W e t h a n k Drs. D i a n a M a r t i n , N e w Z e a l a n d C o m m u n i c a b l e D i s e a s e C e n t r e a n d Belur M a n j u l a , Rockefeller University, N e w Y o r k for the s t r a i n s a n d opacity factor characteristics. W e are grateful to Dr. Valerie A s c h e a n d Professor J o h n M a t h e w s for helpful discussions.
REFERENCES [1] Lancefield, R.C. (1962) J. Immunol. 89, 307-313. [2] Haanes-Fritz, E., Kraus, W., Burdett, V., Dale, J.B., Beache~-, E.H. and Cleary, P.P. (1988) Nucleic Acids Res. 16, 4667-4677.
[3] Miller, L., Gray, L., Beachey, E.H. and Kehoe, M.A. (1988) J. Biol. Chem. 263, 5668-5673. [4] Hollingshead, S.K., Fischeui, V.A. and Scott, J. (1986) J. Biol. Chem. 261, 1677-1686. [5] Robbins, J.C., Spanier, J.G., Jones, SJ., Simpson, W.J. and Cleary, P.P. (1987) J. Bacteriol. 169, 5633-5640. [6] Mouw, A.R., Beachey, E.H. and Burdett, V. (1988) J. Bacteriol. 170, 676-684. [7] Haanes, E. and Cleary, P.P. (1989) J. Bacteciol. 171, 6397-6408. [8] Bessen, D., Jones, K.F. and Fischetti, V.A. (1989) J. Exp. Med. 169, 269-283. [9] Scott, J.R., Holfingshead, S.K. and Fischetti, V.A. (1986) Infect. Immun. 52, 609-612. [10] Skjold, S.A., Quie, P.G., Fries, L.A., Bamham, M. and Cleary, P.P. (1987) J. Infect. Dis. 155,1145-1150. [11] Saiki, R.K., Scharf, S., Faloona, F., Muller, K.B., Horn, G.T., Ehrlich, H.A. and Arnheim, N. (1985) Science 230, 1350-1354. [12] Reed, K.C. and Mann. D. (1985) Nucleic Acids Res. 13, 7207-7221. [13] Frithz, E., Heden, L.-O. and Lindahl, G. (1989) Mol. Microbiol. 3, 1111-1119. [14] Heath, D.G. and Cteary, P.P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4741-4745.