Sep 16, 1994 - 20.5% over 332 amino acids between CapI and GalE. Moderate to high ..... Arbeit, R. D., W. W. Karakawa, W. F. Vann, and J. B. Robbins. 1984.
JOURNAL
OF
BACrERIOLOGY, Nov. 1994, p. 7005-7016
Vol. 176, No. 22
0021-9193/94/$04.00+0
Copyright © 1994, American Society for Microbiology
Sequence Analysis and Molecular Characterization of Genes Required for the Biosynthesis of Type 1 Capsular Polysaccharide in Staphylococcus aureus WEN S. LIN, TIM CUNNEEN, AND CHIA Y. LEE* Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, Kansas 66160 Received 25 July 1994/Accepted 16 September 1994
We previously cloned a 19.4-kb DNA region containing a cluster of genes affecting type 1 capsule production from Staphylococcus aureus M. Subcloning experiments showed that these capsule (cap) genes are localized in a 14.6-kb region. Sequencing analysis of the 14.6-kb fragment revealed 13 open reading frames (ORFs). Using complementation tests, we have mapped a collection of Cap- mutations in 10 of the 13 ORFs, indicating that these 10 genes are involved in capsule biosynthesis. The requirement for the remaining three ORFs in the synthesis of the capsule was demonstrated by constructing site-specific mutations corresponding to each of the three ORFs. Using an Escherichia coli S30 in vitro transcription-translation system, we clearly identified 7 of the 13 proteins predicted from the ORFs. Homology search between the predicted proteins and those in the data bank showed very high homology (52.3% identity) between capL and vipA, moderate homology (29% identity) between capI and vipB, and limited homology (21.8% identity) between capM and vipC. The vipA, vipB, and vipC genes have been shown to be involved in the biosynthesis of SalmoneUla typhi Vi antigen, a homopolymer polysaccharide consisting of N-acetylgalactosamino uronic acid, which is also one of the components of the staphylococcal type 1 capsule. The homology between these sets of genes therefore suggests that capL, capI, and capM may be involved in the biosynthesis of amino sugar, N-acetylgalactosamino uronic acid. In addition, the search showed that CapG aligned well with the consensus sequence of a family of acetyltransferases from various prokaryotic organisms, suggesting that CapG may be an acetyltransferase. Using the isogenic Cap- and Cap' strains constructed in this study, we have confirmed that type 1 capsule is an important virulence factor in a mouse lethality test.
Most strains of Staphylococcus aureus produce capsular polysaccharides. To date, 11 distinct serotypes have been reported (19, 44). Type 1 or 2 capsule-producing strains produce a large amount of capsule. As a result, these strains are mucoid, phage nontypeable, and clumping factor negative and exhibit a halo around the cells under negative staining with India ink. Although these strains are antiphagocytic and virulent to mice (27, 31, 36), they are rarely encountered clinically. On the other hand, type 5 and 8 strains are the predominant isolates, which account for about 80% in recent isolates from all sources (2, 4, 14, 17, 19, 37, 44). These strains, which are referred to as microencapsulated (50), produce a small amount of capsule and do not possess the characteristics typically associated with type 1 or 2 strains. Microencapsulated type 5 and 8 strains have been shown to be antiphagocytic (18); however, this result was not supported by recent studies (1, 5, 52). S. aureus M is a type 1 capsule producer isolated from an infected human hand laceration (41). Since the report of its isolation, strain M has been subjected to intensive study as a prototype of encapsulated staphylococci (see reference 50 for a review). The capsule of strain M is composed of taurine,
in strain M has been correlated with its virulence properties in both the in vitro phagocytic assay and the in vivo mouse lethality assay (31, 36). It has been shown that the mechanism of resistance to opsonization is due to the masking of the bacterial surface-bound C3b complement by capsule, thus preventing interaction with the receptors on phagocytic cells by the capsule. Antibodies against the capsule were shown to promote opsonization of encapsulated organisms by depositing complement throughout the capsule and the bacterial surface
(47).
To study the molecular biology of capsule biosynthesis in S. aureus, our laboratory previously cloned a region of DNA containing a cluster of capsule (cap) genes from the chromosome of strain M (24). In this communication, we report the complete nucleotide sequence of the 14.6-kb DNA region containing the cap genes and the characterization of these genes. MATERIALS AND METHODS
Strains, plasmids, and growth conditions. The bacterial strains and plasmids are listed in Table 1. Trypticase soy broth or agar (Difco Laboratories, Detroit, Mich.) was used for routine cultivation of S. aureus strains. L broth or agar (Difco) was used for cultivation of Escherichia coli. S. aureus M has been described previously (24). S. aureus RN4220, a restriction-deficient strain derived from strain 8325-4 (22), was used as the recipient in electroporation. S. aureus CYL316 was used as the recipient for the single-copy integration vector pCL83 or pCL84 (25). E. coli LE392 or HB101 was used for plasmid transformations and preparation of plasmids. Electroporation
2-acetamido-2-deoxy-fucose, and 2-acetamido-2-deoxy-D-ga-
lacturonic acid in a molar ratio of 1:2:4 (29, 34). The presence of taurine in the capsule is dependent on the presence of taurine in the growth medium (51). The production of capsule * Corresponding author. Mailing address: Department of Microbiology, Molecular Genetics and Immunology, University of Kansas Medical Center, Kansas City, KS 66160. Phone: (913) 588-7014. Fax: (913) 588-7295.
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LIN ET AL.
J. BAcrERIOL.
TABLE 1. Bacterial strains and plasmids used in this study Strain or plasmid
S.aureus strains RN4220 CYL316 M CYL5022 CYL5024 CYL5091 CYL5168 CYL5176 CYL5187 CYL5203 CYL5204 CYL5206 CYL5210 CYL5211 CYL5212 CYL5213 CYL5214 CYL5240 CYL5241 CYL5242 CYL5243 CYL5244 CYL5245 CYL5246 CYL5248 CYL5327 CYL5330 CYL5346 CYL5419 CYL5529 CYL5531 CYL5542 E. coli strains HB101
LE392 Plasmids pLI50
pCL8 pCL83 pCL84 pCL52.1
Source or
Relevant characteristics
reference
22
8325-4rRN4220(pYL112A19)
251
Cap+ M cap-23 M cap-24 M cap-91, MNNG mutagenesis M OMCYL5168 cap-176 CYL5168 cap-187 CYL5168 cap-203 CYL5168 cap-204 CYL5168 cap-206 CYL5168 cap-210 CYL5168 cap-211 CYL5168 cap-212 CYL5168 cap-213 CYLS168 cap-214 CYL5168 cap-240 CYLS168 cap-241 CYL5168 cap-242 CYL5168 cap-243 CYL5168 cap-244 CYL5168 cap-245 CYL5168 cap-246 CYL5168 cap-248 M cap-327, spontaneous mutation M cap-330, spontaneous mutation M cap-346, 43°C treatment M cap-419::cat M cap-529::cat A(capA capB capC) M capM531 M capI542
24 24 This study 24 24 24 24 24
F- recA13 hsdS20 ara-14 proA2 lacYl galK2 xyl-5 mtl-i supE44 rpsL20 AF- hasR514 supE44 supF58 lacYl galK2 galT22 metBI trpR55 X-
J. Lutkenhaus
24
24 24 24 24 This study 24 24 24 24
24 24 This study This study This study This study This study This study This study
J. J. landolo
reading frame [ORF] capC) and ligating it with a BamHI linker. likr DNA manipulations. General DNA manipulations were performed as described by Sambrook et al. (39). Plasmid DNA purification was performed by the procedure of Birnboim (7) and further purified by CsCl-ethidium bromide density gradient centrifugation or by using the Qiagen plasmid kit (Qiagen,
Inc., Chatsworth, Calif.). Rapid small-scale plasmid DNA purification was done by the method of Holmes and Quigley (15). Bulk chromosomal DNA from S. aureus was purified by the method of Dyer and Iandolo (9). The transfer of DNA to nitrocellulose membranes was by the method of Southern (45). The conditions used for hybridization analysis have been described previously (26). Enzymes used in DNA manipulation were purchased from GIBCO-BRL (Gaithersburg, Md.) or New England Biolabs, Inc. (Beverly, Mass.) DNA sequence analysis. The 14.6-kb DNA fragment containing capsule genes was subcloned into bacteriophage M13 derivatives mpl8 and mp19 (53). Overlapping deletions of the DNA fragments for sequencing were generated by the exonuclease III (New England Biolabs) deletion procedure as described previously (54). DNA sequencing was carried out by the dideoxy chain-termination method of Sanger et al. (40) with a sequencing kit from U.S. Biochemical Corp. (Cleveland, Ohio). Complementation test. DNA fragments containing any of the capA, capB, and capC genes were cloned into a single-copy integration vector, pCL83 or pCL84. The resultant plasmids were electroporated into strain CYL316 and then transduced by phage 52A into various mutants for complementation as described previously (24). DNA fragments containing other cap genes were cloned into pLI50 or pCL8. The resultant plasmids were electroporated into RN4220 and then transduced by phage 52A into various mutants for complementation. In vitro protein expression. The plasmids carrying the cap genes were expressed with an in vitro transcription-translation system (Promega Corp., Madison, Wis.) according to the supplier's specifications. Proteins were labelled with [35S]methionine (New England Nuclear Corp., Boston, Mass.) and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE [11% resolving and 5% stacking]) by
Shuttle cloning vector Shuttle cloning vector
This study
Single-copy integration vector Single-copy integration vector Temperature-sensitive cloning vector
the method of Laemmli (23). Construction of mutants with chromosomal mutations at
25 This study
capI and capM. The DNA insert in pCL7186 (Fig. 3) was recloned into a pBR322-derived vector that lacks NcoI. The
2s
carried out by the procedure of Kraemer and IIandolo (21). Phage 52A used for transduction has been describced previously (24). The transduction procedure was carried out as previously described (42). N-Methyl-N'-nitro-N-nitrosoguanid ine (MNNG; Sigma Chemical Co., St. Louis, Mo.) was used for mutagenesis as described previously (32). Cap- mutants wer(e isolated by screening nonmucoid colonies and were confirme4 d by negative staining with India ink as previously described ( 24). Plasmid vector pCL8 was essentially the same as pLI50, ex,cept that the ampicillin resistance gene and the multiple cloniing site were derived from pWSK30 (48). Plasmid pCL52.1 wass constructed by cloning the 1.4-kb fragment containing the ttemperaturesensitive origin of replication of pE194ts into the ClaI site of pCL72 (25) in which the HindIII site upstream oif tetracycline resistance gene (Tcr, originally from pT181) has bteen replaced with a BglII linker. Plasmid pCL7398 was co]nstructed by digesting pCL7183 (see Fig. 3) with FspI (locatedI in the open
was
-
resultant plasmid was first digested with NcoI (located in capI) and then was digested with exonuclease Bal 31 to delete about 20 bp. The religated DNA insert was then recloned into pCL52.1. This plasmid, pCL7377, was electroporated into S. aureus RN4220 and then moved by phage 52A into S. aureus M. Strain M(pCL7377) was incubated at 42°C for 24 h in the presence of 3 ,ug of tetracycline per ml of medium to select strains with plasmid integration into the chromosome via homologous recombination. A single colony was then incubated in medium without selection at 30°C for 24 to 48 h, which allowed the integrated plasmid to excise from the chromosome by single crossover at the duplication region created during plasmid integration. Depending on the site of crossover during excision, the mutation site on the insert of pCL7377 would either be left on the chromosome, thus generating the desired mutant, or would remain on the plasmid, thus regenerating the wild-type strain. Because the plasmid cloned into pCL52.1 is unstable (unpublished result), some cells would be devoid of plasmid. These Tcs cells were isolated by replicating colonies on agar plates with and without tetracycline. The desired
GENES AFFECTING TYPE 1 CAPSULE PRODUCTION IN S. AUREUS
VOL. 176, 1994 B
H
B
cc
cap
capB E Bm
s
H
H
iD~~apC~ pCL7163
caPD'
-
CL21
FIG. 1. Construction of pCL7325 and pCL7375. Solid bars, DNA fragments from strain M; hatched bars, DNA fragments containing the cat gene of pC194; thin lines, cloning vectors. Abbreviations: B, BglII; Bm, BamHI; E, EcoRI; H, HindIII; P, PstI; S, Sau3A; 'capC, 5' truncated capC gene; capD', 3' truncated capD gene. Only pertinent restriction sites are shown.
mutant strain, CYL5542, was confirmed by Southern hybridization (not shown). To construct the mutation in capM, a 1.9-kb HindlIl (12.6-kb coordinate)-BglII (14.6-kb coordinate) fragment was recloned into pCL52.1. The resultant plasmid was digested withAccl and religated to generate pCL7359 with a deletion of 114 bp within ORF capM. Plasmid pCL7359 was electroporated into strain RN4220 and then transduced into strain M by phage 52A transduction. The same procedure for isolating a mutant with a mutation at capI described above was employed. The desired mutant strain, CYL5531, was confirmed by Southern
blotting (not shown).
Construction of strains CYL5529 and CYL5419. To construct strain CYL5529, plasmid pCL7375 was first constructed as outlined in Fig. 1. Plasmid pCL7163, which contained an 8-kb PstI-EcoRI fragment carrying capA-capC and the truncated capD, was digested with BglII and ligated with a 1.7-kb Sau3A DNA fragment containing the chloramphenicol acetyltransferase (cat) gene of pLI50 originally derived from pC194 (16). A BglII site between the cat gene and the capA gene was regenerated. This plasmid, pCL7325, was digested with BglII and EcoRI to release a 4.0-kb fragment containing the cat gene. This fragment was ligated to the BamHI and EcoRI sites of pCL7371 containing a 1.2-kb HindIII-PstI fragment (2.3- to 3.5-kb coordinates in Fig. 3; the HindIII has been replaced by a BamHI linker). The resultant plasmid, pCL7375, thus con-
7007
tained a cat gene replacing the central 4.1-kb DNA region of the 8-kb DNA fragment of pCL7163. Plasmid pCL7375 was electroporated into strain RN4220 and then transduced to strain M by phage 52A. M(pCL7375) was incubated at 42°C in the presence of tetracycline (3 jig/ml) and chloramphenicol (5 ,ug/ml) for 24 h. Cells were plated on agar plates containing chloramphenicol. Colonies were replica plated onto agar plates containing chloramphenicol and onto agar plates containing tetracycline and chloramphenicol. Tcs Cmr colonies were selected, and one was designated CYL5529 and confirmed by Southern hybridization (not shown). To construct strain CYL5419, the 7.9-kb insert of the plasmid pCL7325 described above (Fig. 1) was digested with BamHI and PstI and recloned into pCL52.1 digested with the same enzymes. The resultant plasmid was electroporated into RN4220 and then transduced to strain M by phage 52A. Strain CYL5419 was isolated as described for CYL5529 and confirmed by Southern blotting (result not shown). Virulence studies. Strains CYL5529 and CYL5419 were incubated at 37°C for 18 h in the presence of chloramphenicol (5 ,ug/ml). Cultures were harvested by centrifugation, washed twice in sterile phosphate-buffered saline (PBS), and resuspended in PBS to 2 x 107 CFU/ml. Six mice (Charles River Laboratories) were injected intraperitoneally with 0.5 ml of each strain (i.e., 107 CFU), and lethality was assessed 24 h later. Nucleotide sequence accession number. The sequence reported here has been submitted to GenBank under accession number U10927. RESULTS DNA sequence analysis. Previously, complementation tests with a collection of Cap- mutants derived from S. aureus M showed that 20 of the 22 mutants were complemented by plasmid clones containing DNA derived from a 19.4-kb DNA region of the S. aureus M chromosome. This result suggests that the majority of the cap genes affecting capsule production in S. aureus M are clustered in the 19.4-kb DNA region of the chromosome (24). To define the smallest DNA region that still contained this cluster of genes, we performed complementation tests with plasmid subclones derived from a 20-kb DNA fragment containing the original 19.4-kb fragment. We found that all of the mutants previously complemented by the 19.4-kb fragment were complemented by plasmid subclones derived from a shorter DNA fragment 14.6 kb in size. Plasmids containing inserts with additional deletions of 0.6 and 1.8 kb from the left and right ends of the 14.6-kb fragment, respectively, were unable to complement some of the mutants. This result indicates that the minimum size of the DNA region containing this cluster of cap genes is about 14.6 kb. The nucleotide sequence of the 14.6-kb cap region was then determined as described in Materials and Methods. As shown in Fig. 2, 13 ORFs were identified. These ORFs were named capA through capM and could potentially encode proteins with molecular masses of 24.5, 25.2, 29.4, 67.5, 50.7, 45.0, 19.4, 41.4, 37.9, 44.5, 52.4, 47.2, and 43.1 kDa, respectively. All of these ORFs were transcribed in one orientation from left to right. The largest spacing between the stop codon and the start codon of two adjacent ORFs was 27 bp, indicating that these ORFs are closely linked. A potential promoter canonical to those found in E. coli and Bacillus spp. (12, 33) was found 31 bp upstream of the start site of the first ORF, capA (Fig. 2). All ORFs were preceded by potential Shine-Dalgarno sequences (43) 5 to 11 bp upstream of the start codons. GTG was the start codon for ORF capD and ORF capF, whereas ATG was the
7008
LIN ET AL. 1 1 03 2 05 1 306 10 408 44 510 78 612 112 714 146 816 180 918 214 1019 21 1121 55 1223 89 1325 123
1427 157 1529 191 1631 225
J. BACTERIOL.
AGATCTATAT CAAAGAC ATTAATAAAATGAAATTAAAGATAC CCAATAAATAGAAC CACAG TATAAAT TATAT CAG TAT GC TTATATAATT TTT GAAATCT T T AAACAAATGAAGTAATAATTGAGAAAAGTGTAGT TAAATTATT TTT CTTGAAATTAT TT GTTACATAG CATTT CGATG TAAAAT TCACT T TTT ATAAGT AA
ATTTAAAAAGAGTTQAAATATACAGGGGATTTATAITAATGGAAAACAAGAAAGGAAAATAGGAGGTTTATATGGAAAGTACAATTGATTTATCTGAA -3 5 L
L
G
R
2344
233 2446 3 2548 37 2650 71 2752 105 2854 139 2956 173 3068 207 3170 241 3272 275 3374 319 3476 343 3578 377 3680 411 3782 445 3884 478 3976 513 4078
V
R
K
N
M
K
L
L
I
I
SD L
L
P
G
L
L
L
CapA M
S
I
A
I
S
E
S
I
I
T
F
F
D
L
L
F
S
E
V
D
K
TACCAAGCTTCTACTCAAATATTAGTGAATCAAAAAGGAAATGACTCACAAATTATGGCACAAGAAGTTCAAAGTAATATTCAACTAGTAAACACATATTCA Y
Q
S
A
T
Q
I
L
V
Q
N
K
G
D
N
Q
S
I
M
Q
A
E
Q
V
S
N
Q
I
L
V
N
Y
T
S
GAGATAGTTAAAAGCCCACGTATTCTAGATAAGGTTTCTAAAGAATTAGATGATAAATATTCACGTAGTGAGATATCAAGTATGTTAACCGTTACCAACCAA E
I
K
V
S
P
R
I
L
D
K
S
V
E
K
L
D
K
D
Y
S
S
R
E
I
S
S
L
M
T
V
N
T
Q
GCAGAGTCTCAAGTATTAAATATTGATGTTGAAAGTAAAAGTGGTAGTAATTCAGAAAAAATTGCTAATAAAATAGCAGAAGTATTTAGTGATGAAGTGCCT A
E
S
Q
V
L
N
I
D
V
S
E
S
K
G
S
S
N
K
E
I
A
N
I
K
A
V
E
S
F
D
V
E
P
GATATTATGAATGTTGATAATGTTTCTGTTTTATCTACAGCGGATAATACAGGAAAACAAGTTGCACCAAAACCTATGGTTAATTTAGTTGTTGGTTTAGTA D
I
M
N
V
D
N
V
S
V
L
S
T
A
N
D
T
G
Q
K
A
V
P
K
P
M
V
L
N
V
G
V
L
V
ATAGGATTGGTTATCGCATTATTAATAATTTTTATAAAAGAAGTGTTTGATAAACGTATTAAAACAGAAGAAGAAGTTGAAAATGAATTGGTGATACCTGTA I
G
V
L
I
A
L
L
I
F
I
I
K
V
E
F
D
K
R
I
K
T
E
E
E
V
E
E
N
V
L
I
P
V
CTGGGTTCAATCCAAAAATTTGATTAAGGAGGGTTTTATCTATGGCAAAAAAGAAAAGCACTATATCACCACTATATGTTCATGATAAACCTAAATCGACG L
G
S
I
Q
K
F
SD
-
D
CapB M
A
K
S
K
K
I
T
S
L
P
Y
V
H
K
D
P
K
S
T
ATAAGTGAAAAATTTAGAGGTATTCGATCAAATATCATGTTTTCTAATGCTGAAAATGAAATAAAAAGCTTGTTGATAACTTCTGAAAAATCCGCATCTGGT I
S
K
E
F
R
G
I
S
R
I
N
F
M
S
N
A
N
E
I
E
S
K
L
I
L
S
T
E
S
K
G
S
A
AAGAGCATACTTTCGGCAAACATTGCAGTAACATATGCACAGGCAGGTTACAAAACATTAATTATAGATGGAGATATGAGAAAGCCTACACAACATTATATC K
S
L
I
S
A
N
I
V
A
T
Q
A
Y
A
G
Y
K
T
L
I
I
G
D
D
M
K
R
P
T
Q
Y
H
I
TTTGATTTGCCTAATAATAGTGGGCTATCTAATTTAATTATTAATAAAACTACATATAGCGATTCAATTAAAGAAACTAGAGTAGAGAATTTAAACGTTTTA F
D
P
L
N
N
S
G
S
L
N
I
L
I
N
K
T
T
S
Y
D
S
I
K
E
T
R
E
V
N
L
N
L
V
ACAGCAGGACCAACACCGCCTAATCCTTCAGAATTAATTGCTTCTAGCAAATTTGCAACTATATTTAATGAACTTTTGAATCATTATGACTTCATTGTGATA T
A
G
P
T
P
P
N
S
P
L
E
I
S
A
S
K
F
A
T
I
F
N
L
E
L
N
H
Y
D
F
V
I
I
GACACACCTCCGATTAATACAGTAACGGATGCTCAAGTATATGCGCGAATAGTAAAAAATTGTGTGCTTGTCATAGATGCTGAAAAAAATAATAAGAGTGAA D
T
P
P
I
N
T
V
T
D
Q
A
V
Y
R
A
I
K
V
C
N
V
L
V
I
D
A
K
E
N
N
K
S
E
GTTAAGAAAGCGAAAGGTTTACTTACTAAAGCCGGTGGGAAAGTATTAGGCGCAGTTTTAAATAAAATGCCAATTGATAAAAATTCTAGCTACTATTACTAC V
K
K
A
K
G
L
L
T
K
A
G
G
K
V
G
L
A
V
L
N
K
M
P
I
K
D
S
S
N
Y
Y
Y
Y
TATGGAGAAGATTAAGAATGGTTGATATACATAATCATATTTTGGTTGATGTAGATGATGGACCAAAAAGCATTAATGAAGCAATTGAGTTGCTTAAACAA Y
G
E
D
*
CapC M
SD
1732 29 1834 63 1936 97 2038 131 2140 165 2242 199
-10
TTATTAGGAAGAGTAAGAAAAAATATGAAATTATTAATAATATTACCACTTTTAGGTCTGCTAATTAGTGCAATAATTTCTTTTTTCTTTTTAGATGTAAAG
V
D
I
N
H
I
H
L
V
D
V
D
G
D
P
S
K
N
I
E
A
E
I
L
Q
K
L
GCCCAAAGTGAAAATGTAACAGATATAGTTGCAACACCTCACCATCTTCATAAGAGATATAGTAATGATATTGAAAAAGTGAAAATAAAATTAAATGAATTA A
Q
S
E
N
V
T
D
I
V
A
T
P
H
H
L
H
K
R
Y
S
N
D
I
E
K
V
K
I
K
L
N
E
L
AAAAATAACTCAGAAATAAAAAAATTAGGACTTAATTTATATGTTGGACAAGAGATTCGTATAACGGATCAAATTATAGAAGGTATAAAAAATAAAGAGATT K
N
N
S
E
I
K
K
L
G
L
N
L
Y
V
G
Q
E
I
R
I
T
D
Q
I
I
E
G
I
K
N
K
E
I
GAAGGAATTAATGAATCTAGGTATTTGTTAATAGAATTTCCTAGCAATGAGATTCCTTATTATACGAATCAATTATTCTATGAGTTACAAACAATGGGATAT E
G
I
N
E
S
R
Y
L
L
I
E
F
P
S
N
E
I
P
Y
Y
T
N
Q
L
F
Y
E
L
Q
T
M
G
Y
ATTCCAATCATTGCGCATCCAGAACGGAATAAAGCGATAGTTCAAAATTTAGATTTACTATTTGAATTAATAAATGGTGGTGCATTAAGTCAGATTACAGCT I
P
I
I
A
H
P
E
R
N
K
A
I
V
Q
N
L
D
L
L
F
E
L
I
N
G
G
A
L
S
Q
I
T
A
TCTTCATTATTGGGAGATTTTGGGAATAACATAAGAAAATTATCATTAAAAATGATAGATAGTAATTTAGCACATTTTATTGCATCAGATGCACATAGTATA S
S
L
L
G
D
F
G
N
N
I
R
K
L
S
L
K
M
I
D
S
N
L
A
H
F
I
A
S
D
A
H
S
I
ACAAATCGACCTTTTATGCTAAAACAGTTGTTTAATGATAGAAAGTTAAAAGCTTATTATGAGGAATTAGAAAGTTATTTAAAAAACGGGAAGTTAGTTTTA T
N
R
P
F
M
L
K
Q
L
F
N
D
R
K
L
K
A
Y
Y
E
E
L
E
S
Y
L
K
N
G
K
L
V
L
ACAAATGAAAGAATTTCCAAACAGATACCAACTCAAGATTATAAGCAGAAAAAATGGTTTGGTCTTTTATAGAAAAAGTGAGTGAGGGGATTAAAAGTGACA T N E R I S K Q I P T Q D Y K Q K K W F G L L * SD CapD M T AGTATTTCTGCGAAATTGCGATTTTTAATATTAATTATTATAGATTCGTTTATTGTTACATTTTCAGTATTTTTAGGATATGCAATATTAGAACCGTATTTT S
I
S
A
K
L
R
F
L
I
L
I
I
I
D
S
F
I
V
T
F
S
V
F
L
G
Y
A
I
L
E
P
Y
F
AAAGGATATTCAATAGATTTATTAGTATTATCATCTGTTATATTGTTGGTATCACATCATATATTCGCATATGTATTTAATTTATATCATCGAGCGTGGGAG K
G
Y
S
I
D
L
L
V
L
S
S
V
I
L
L
V
S
H
H
I
F
A
Y
V
F
N
L
Y
H
R
A
W
E
TATGCGAGTGTAAGTGAATTGATGTCAGTTTTAAAAGCGGTTACAAGTTCAATAGTGGTAACACTTTTATTAGTTTCATTACTTATAAGTGAAAGTCCATTT Y
A
S
V
S
E
L
M
S
V
L
K
A
V
T
S
S
I
V
V
T
L
L
L
V
S
L
L
I
S
E
S
P
F
CTAAGGTTATATTTTATAACGTGGATGATGCATTTGCTATTGATTGGTGGCTCTAGATTGTTTTGGCGGGTTTATAGAAGGTATTTTATTGATAACGCTGTC L
R
L
Y
F
I
T
W
M
M
H
L
L
L
I
G
G
S
R
L
F
W
R
V
Y
R
R
Y
F
I
D
N
A
V
GAAAAAAAAGCCACATTAGTAGTAGGGGCTGGACAAGGCGGATCTGTTTTAATTCGTGAAATGTTAAGAAGCCAAGATATGCGTATGCAACCAGTTTTAGCT E K K A T L V V G A G Q G G S V L I R E M L R S Q D M R M Q P V L A GTTGATGACGATAAAAACAAACAAAAAATGACTATTACTGAACGTGTTAAAGTTCAAGGTTATGTTGAAGATATACCGGAACTAGTAAAAAAATTTAGAATA V D D D K N K Q K M T I T E R V K V Q G Y V E D I P E L V K K F R I AAAAAGATTATTATTGCTATACCGACATTAAGCCAAAAAAGGTTAAATGAAATTAATAAAATATGTAATATTGAGGGCGTTGAATTATTTAAAATGCCTAAT K
K
I
I
I
A
I
P
T
L
S
Q
K
R
L
N
E
I
N
K
I
C
N
I
E
G
V
E
L
F
K
M
P
N
ATAGAAGATGTGTTATCTGGAGAATTAGAAGTCAATAATTTGAAAAAAGTAGAAGTAGAAGATTTACTTGGAAGAGATCCAGTTGAACTAGACATGGCATTA I
E
D
V
L
S
G
E
L
E
V
N
N
L
K
K
V
E
V
E
D
L
L
G
R
D
P
V
E
L
D
M
A
L
ATTTCAAGAGAATTAACGAATAAAACAATATTGGTAACTGGAGCTGGAGGATCAATAGGTTCAGAAATTTGCAGACAAGTAAGTAAATTTGATCCTCAAAAA I S R E L T N K T I L V T G A G G S I G S E I C R Q V S K F D P Q K ATTATTTTGTTAGGACATGGAGAGAACAGTATTTATTCAATTCATCAAGAATTAAGTAAAACTTATGGAAATAGAATTGAATTTGTTCCTGTTATAGCTGAT I
I
L
L
G
H
G
E
N
S
I
Y
S
I
H
Q
E
L
S
K
T
Y
G
N
R
I
E
F
V
P
V
I
A
D
GTACAAAATAAAACACGTATTTTAGAAGTCATGAATGAATTTAAGCCATATGCTGTTTATCATGCTGCAGCACATAAGCATGTACCATTAATGGAATACAAC V Q N K T R I L E V M N E F K P Y A V Y H A A A H K H V P L M E Y N CCTCATGAAGCTATTAGAAATAATATTTTAGGTACTAAAAATGTAGCTGAGTCTGCAAAAGAAGGAGAAGTAAGTAAATTTGTAATGATTTCAACAGATAAA P
H
E
A
I
R
N
N
I
L
G
T
K
N
V
A
E
S
A
K
E
G
E
V
S
K
F
V
M
I
S
T
D
K
GCTGTAAATCCATCTAATGTAATGGGAGCAACAAAACGCATTGCTGAAATGGTAATACAAAGTTTAAATGAAGATAATTCTAAGACAAGTTTTGTAGCAGTA A
V
N
P
S
N
V
M
G
A
T
K
R
I
A
E
M
V
I
Q
S
L
N
E
D
N
S
K
T
S
F
V
A
V
AGATTTGGAAATGTGCTTGGATCAAGAGGATCGGTCATACCTCTATTTAAAAATCAAATTGAATCAGGTGGTCCGGTTACAGTAACTCACCCTGAAATGACA R
F
G
N
V
L
G
S
R
G
S
V
I
P
L
F
K
N
Q
I
E
S
G
G
P
V
T
V
T
H
P
E
M
T
CGATATTTCATGACTATACCAGAAGCATCAAGACTTGTATTACAGGCCGGGGCATTGGCACAAGGTGGCGAAGTATTTGTGCTAGACATGGGTAAACCAGTT R
Y
F
M
T
I
P
E
A
S
R
L
V
L
Q
A
G
A
L
A
Q
G
G
E
V
F
V
L
D
M
G
K
P
V
AAAATAGTTGATTTAGCTAAGAATTTAATACGGTTGAGCGGTAAAAAAGAAGAAGATATTGGTATTGAATTTTCAGGGATTAGACCAGGAGAAAAATTATAT K
I
V
D
L
A
K
N
L
I
R
L
S
G
K
K
E
E
D
I
G
I
E
F
S
G
I
R
P
G
E
K
L
Y
GAAGAATTATTAAATAAAAATGAAATTCATCCGCAGCAGGTATATGAAAAAATTTATAGAGGCAAAGTTGACCACTATATTAAAACGGAAGTTGATTTGATT
FIG. 2. Nucleotide sequence of the 14.6-kb region containing cap genes and the predicted amino acid sequences of the CapA to CapM proteins. The potential -35 and -10 sequences and Shine-Dalgarno (SD) sequences are underlined. Termination codons are designated by asterisks. The inverted repeat downstream of capM is indicated by inverted arrows.
start codon for all other ORFs. ORF capG and ORF capH overlapped by one nucleotide, whereas ORF capJ and ORF capK overlapped by eight nucleotides. No potential terminator was found downstream of the last ORF, although a GC-rich inverted repeat was found 98 bp downstream of the stop codon of the last ORF (Fig. 2). Genetic complementation. To determine whether each of the ORFs is involved in the synthesis of the capsule and to study the organization of the genes, capsule mutants previously described (24) and those newly identified in this study (Table 1) were tested by complementation tests with various subclones derived from the 14.6-kb region. As shown in Fig. 3,
plasmid pCL7183 was able to complement 17 mutants. Deletion of 0.6 kb from the left end to the EcoRV site (at the 0.6-kb coordinate) of the 6.2-kb insert fragment from pCL7183 yielded pCL7193, which complemented six mutants (5176, 5187, 5206, 5210, 5212, and 5244). These six mutants were also complemented by pCL5201 with a 2.3-kb insert that contains only intact capE, suggesting that the mutations in these six mutants mapped in capE. Deletion from the right end of the 6.2-kb insert fragment of pCL7183 to the PstI site (at the 3.5-kb coordinate) resulted in pWL7315 with a 3.5-kb insert containing intact capA, capB, and capC and truncated capD. This plasmid lost its ability to complement nine mutants (5023,
VOL. 176, 1994 547 418 0 581 4282 6 4384 40 4486 74 4588 108 4690 142 4792 176 4894 210 4996 244 5098
278 5200 312 5302 346 5404 380 5506 414 5609 7
5711 41 5813 75 5913 109 6017 143 6119 '177 6221 211 6323 245 6425 279 6527 313 6629 347 6731 381 6834 15 6936 49
7038 83 7140 117 7242 151
7346 13 7448 47 7550 81 7652 115 7754 149 7856 183 7958 217 8060 251
GENES AFFECTING TYPE 1 CAPSULE PRODUCTION IN S. AUREUS E
E
L
L
N
K
N
E
I
H
P
Q
Q
V
Y
E
K
I
R
Y
G
K
V
H
D
Y
I
K
T
V
E
L
D
7009
I
GTGGAAGATTTAATAAATAATTTCTCAAAAGAGAAGCTCTTAAAGATAGCAAATAGATAAATATAAAATGTATATTGGAGTTTTAAAATGAAACTAAAGTAC V E D L I N N F S K E K L L K I A N R * SD CapE M K L K Y AAAGTAATTTTAATTATAAATTTTGTTACAGTATTATTTAGTATATTTACATTCATCGGTTATTTAAATAACTTGATAGGTTTTAGAGTTGTTACAATATCA K V I L I I N F V T V L F S I F T F I G Y L N N L I G F R V V T I S TTATGTATAACTATAGCTATGACCGTGTACTTACTTTACAAAAAAGAAAAGGTTTTTTATTAGTTTATTTAATATATTTATTTTTAACTAATTTTGGTGTA L C I T I A M T V Y L L Y K K R K G F L L V Y L I Y L F L T N F G V TTTGTAACAAATATATTTTTAGCCAATCCTTTAGTGGAGTATCACGGTGATTTATCTTGGTATTATATAAATACATCTAATTTGTTTAGCATTGCTACTTTT F V T N I F L A N P L V E Y H G D L S W Y Y I N T S N L F S I A T F GCGATATTAACTTTTACAATTTTAAGTAATTTTATTAGTGTGTTTAGCAAAATAAATCCAAGTAGAAAGTTTGATATTAAAAGTAAAGGAAATAATTTATTC A I L T F T I L S N F I S V F S K I N P S R K F D I K S K G N N L F TACTACACAGGAATATTATTTATCATTGGATTTACTATACAATTTCTTTTTTATATTATAACTGGTCGATTAGCAATTAATACTTATGGAGATTATGTAAGT Y Y T G I L F I I G F T I Q F L F Y I I T G R L A I N T Y G D Y V S AGCATACAAGAATTACCAATGTATACGTACGGTATATTCTTTTTTTCAATTGGGATTGCATTTGCATTTTCTAATGTAAAAAAAACACATATTAAATATCTA S I Q E L P M Y T Y G I F F F S I G I A F A F S N V K K T H I K Y L GTAATTATATTAACTCCCCAAGTGTTATTTTTTTTAATAACCGGAAATAGGGGAGAAGTATTTTATCCAATTCTATCTGCACTTGGAGTGTTAATAGTAAGG V I I L T P Q V L F F L I T G N R G E V F Y P I L S A L G V L I V R AATTATAAAATTAAATGGTGGATGATAATTACAATTGTTTTTACATTATTTTTTGTAATACCATTTATTAAAGTCTTTAGAAATATGGATAGTAGCTCAATT N Y K I K W W M I I T I V F T L F F V I P F I K V F R N M D S S S I GAAAAGGTTGATATAAATTGGTTTTCTTCACTTGTTGAAATAGGCTACACACTGCGCCCTTTAGGTTATGTAACTAGGTGGATTGACGGTGGGGAAAGCATA E K V D I N W F S S L V E I G Y T L R P L G Y V T R W I D G G E S I GTTTATGGCAAAAGTTACTTAGCACCTATTCAAAATATATTTTCATATATTATACCAGGGTTACAACCTGTAAACTATGAAATGGTTGGTTACGGTTTTAGG V
Y
G
K
S
Y
L
A
P
I
Q
N
I
F
S
Y
I
I
P
G
L
Q
P
N
V
Y
E
M
G
V
G
Y
F
R
TATAGACTACCTGGTATGGGTTTTAATGTAATAGCAGAAGCGTATTACAATGGTGCAATTGTTGGAGTATTGATTGTAATGGTGTTATTAGTGCTTTTACTT Y
R
L
P
G
M
G
F
N
V
I
A
E
A
Y
Y
N
G
A
I
V
G
V
L
I
V
V
M
L
L
V
L
L
L
TGGAAATTTACAAATTTTAAATCTTTTGAAATGCTGTCAATGGGTACTGCAATTGTAAGTGTATTAATCAATAATATAAGAAACGCATTTTCATTCGTACCT
W
K
F
T
N
F
K
S
F
E
M
L
S
M
G
T
A
I
S
V
V
L
I
N
N
I
R
T
K SD
A
D
N
A
S
F
V
F
P
GCATACATATTAATTATCATTGTAATAGTGATTATATTGCTGTTTATAGATAGTTATTTAAAAAAAACGAADGCCGATTAAAGTGGTTAAAAATTTTAAT Y I L I I *
A
I
V
I
V
I
I
L
L
F
I
D
S
Y
L
K
K
E
CapF M
V
K
N
F
N
TATATGTTTGTTGCTAATATACTGTCTGCATTGTGCAAATTTTTAATTTTACTAGTAATAGTTAGATTAGGAACACCTGAGGATGTAGGACGTTATAATTAT Y
M
F
V
A
N
I
L
S
A
L
C
K
F
L
I
L
L
V
I
V
R
L
G
T
P
E
D
V
G
R
E
Y
Y
N
Y
S
A
GCTTTAGTTATAACTGCTCCAATTTTTTTATTTATATCCCTGAAAATAAGGTCTGTAATTGTCACGAATGATAAATATAGTCCAAATGAATATATATCAGCA
A
L
V
I
T
A
P
I
F
L
F
I
S
L
K
I
R
S
V
I
V
T
N
K
D
S
Y
N
P
I
ATTTTATCATTAAATATTATTACTTTAATATTTGTTGCAATTTTTGTTTATGTATTAGGAAATGGTGATTTAACTACTATATTAATAGTATCGTTAATAAAA I
L
S
L
N
I
I
T
L
I
F
V
A
I
F
V
Y
V
L
G
N
G
D
T
L
T
I
L
S
V
I
L
K
I
TTATTTGAAAATATAAAAGAAGTACCTTATGGAATATATCAAAAAAATGAGAGTCTCAAATTACTCGGAATCTCAATGGGTATTTATAATATACTAAGTTTA L
F
E
N
I
K
E
V
P
Y
G
I
Y
Q
K
N
E
S
L
K
L
L
G
S
I
M
G
I
N
Y
S
L
I
L
ATTTTGTTTTATATTATATATTCTTTTTCACACAACTTGAATATGGCACTTTTATTTTTAGTAATATCCTGTATATTCTCATTTGCTATTATCGATAGATGG I L F Y I I Y S F S H N L N M A L L F L V I S C I F S F A I I D R W TATCTAAGTAAGTATTATAATATAAAACTACACTATAATAACAACATTGCAAAGTTTAAGGAGATTTTTATTCTTACAATACCTCTTGCTTTTTCAAGTGCG Y L S K Y Y N I K L H Y N N N I A K F K E I F I L T I P L A F S S A TTAGGATCATTAAATACAGGAATTCCTAGAATAGTATTAGAAAATCTATTTGGAAAATATACATTAGGTATATTTTCTACAATTGCGTATGTACTGGTAATC L G S L N T G I P R I V L E N L F G K Y T L G I F S T I A Y V L V I GGTGGGTTATTCGCAAATTCAATCAGTCAAGTTTTTTTACCCAAATTAAGAAAATTATATAAAGATGAAAATTGAATTTGAAAAGTTAACTAGAAAA G G L F A N S I S Q V F L P K L R .K L Y K D E K K I E F E K L T R K ATGGTGTTTATTGGAATTTTTATTGGTATGTGCTCGGTAATATTGAGTTTGTTTTTAGGTGAGGCCTTGTTATCATTGTTATTTGGTAAAGAATATGGTGAA M V F I G I F I G M C S V I L S L F L G E A L L S L L F .G K E Y G E AATAATATAATATTAATCATTCTTTCTTTTGGTTTGCTTTTTATACTCAGTGGTATTTTTTTGGGGACAACTATAATAGCCACTGGAAAGTATAATGTGAAT N N I I L I I L S F G L L F I L S G I F L G T T I I A T G K Y N V N TACAAAATTTCTCTAATACTATTGTTTTGTATTTTGATATTTAGTTTCTTATTAATACCAAAATATTCTTTATTAGGTGCTGCTTTAACTATTACTATTTCA Y
K
I
S
L
I
L
L
F
C
I
L
I
F
S
F
L
L
CAATTCGTTGCTTTAATAAGCTATTACTATTTTTACAAAAGGATATTTTGAG Q
F
V
A
L
I
S
Y
Y
Y
F
Y
K
R
I
SD
F
I
P
K
Y
S
L
L
G
A
A
L
I
T
T
S
I
GAAAATATGAAAAAATTATTTTTAAAGTTAATGAAAAGGAATCTATCT CapG M K K L F L K L M .K R N L S
GAAGATAAGATAAGGAAATTGGGTGTACAAGTTGGAAATGATTGTAGGTTTTTAAGTGTTGATAGATCAACATTTGGATCTGAGCCTTACCTGATTCAAATA E D K I R K L G V Q V G N D C R F L S V D R S T F G S E P Y L. I Q I GGAAATCATGTAACAATAACTAGTGGTGTAAAATTTGCTACCCATGATGGTGGGGTATGGATTTTTAGAAAAAAATATCCTGAGATAGATAATTTTCATAGA G
N
H
V
T
I
T
S
G
V
K
F
A
T
H
D
G
G
V
W
I
F
R
K
K
Y
P
E
I
D
N
F
H
R
N
V
V
V
G
A
G
S
V
V
T
ATATTTATCGGTAATAATGTTTTCATAGGGATTAATTCAATAATTTTGCCAGGAGTAACAATAGGAAATAATGTTGTAGTAGGTGCTGGGAGTGTGGTAACG I
F
I
G
N
N
V
F
I
G
I
N
S
I
I
L
P
G
V
T
I
G
N
AAAGATGTACCTGATAATGTAATTATTGGTGGTAATCCTGCCAAAAAAATTAAAAGTATAGAGGCCTATGAAACTAAAATATTAGAAAATGCGGATTACACA K D V P D N V I I G G N P A K K I K S I E A Y E T K I L E N A D Y T AAAAAACTTAATTATAATGAAAAGAAGATATATTTATTAAATAAATTCAAAGAGAATAGGTATAATTAATGATTAAAGTTATGCATATATTTAGCAGAATGAAT K
K
L
N
Y
N
E
K
K
I
Y
L
L
N
K
F
K
E
N
R Y SD
N
*
CapH M
I
K
V
M
H
I
F
S
R
M
N
CGTGGCGGTGCAGAATTAAGAACTATGGATACAATGAAACTATTAAATAGGGAGTTTGAGTTTCATGTATGTGCTACATCAGGAAAAAGGGGCGAATTAGAT R G G A E L R T M D T M K L L N R E F E F H V C A T S G K R G E L D GATGAATTAGAATCTATGGGTATTACGATACATTATTTAGATATTAAAAAATTTAGTTTTCCTTTCAAGTTCATAAAGTTATTGAAAAAGAAAAATATAGAT D E L E S M G I T I H Y L D I K K F S F P F K F I K L L K K K N I D GTTGTACATAGTCACATACTTTTTATGAGTGGATTGATTCAATTACTTTCTTTTTCAGCAAATGTAAGAAATAGAATAACACACTTTAGAACTTCCAAAGAT V V H S H I L F M S G L I Q L L S F S A N V R N R I T H F R T S K D AGTAAAGAACAATATAATAAAATAAGAAAAGCTAGAAATAAAGTACTGAAAGCCATTATAGAAATCTTTAGTACGAAAATCTTATATGTAAGTAATATAGCA S K E Q Y N K I R K A R N K V L K A I I E I F S T K I L Y V S N I A AATAGGAATTTAATCTCAATGAAACTTTTTCCGAAAAAACATAAAACTATTTACAATGGATTTGAAATAAGTAATATAAACAAAAATTTTAAAAAAGAGGAA N R N L I S M K L F P K K H K T I Y N G F E I S N I N K N F K K E E AATAGTTTTATTTATGTCGGTAGGTTCATTCATACTAAGAACCAATTATTTTTGTTAGATGTAATAGAAATTTTAAAAAAAGAATTTAATACTAACATTGAA N S F I Y V G R F I H T K N Q L F L L D V I E I L K K E F N T N I E ATTACTTTTGTTGGCAATATTCAAACTGATTATGGAAAAAAATTCTTGAGTATAGCAAATGAAAGAGGTTTAAATAAAAATATTAAGGTTATTGGAGAAGTG I T F V G N I Q T D Y G K K F L S I A N E R G L N K N I K V I G E V AATAACCCATTAGATTATTTAAAAACGAGTGAATATTTTTTGTTTCCTAGTGAATTAGAGGGATTACCGGGAGCATTGATAGAAGCGCATCATCACAATTGT N
N
P
L
D
Y
L
K
T
S
E
Y
F
L
F
P
S
E
L
E
G
L
P
G
A
L
I
E
A
H
H
H
N
C
FIG. 2-Continued.
5203, 5214, 5226, 5242, 5243, 5245, 5246, and 5248) in addition to the six mutations mapped in capE but was able to complement 5213 and 5346. These results indicate that the mutations in those nine mutants that were not complemented by either pWL7315 or pCL5201 mapped in capD. Deletion up to near the Hindlll site in ORF capC (at the 2.3-kb coordinate) by Bal 31 nuclease resulted in pWL7309 with a 2.3-kb insert that still complemented 5213 and 5346. Further deletion up to the HindIII site at the 1.1-kb coordinate resulted in plasmid pCL7407, which did not complement 5346 but complemented 5213. Because pWL7309 contains intact capA and capB, whereas pCL7407 contains only intact capA, these results indicate that the mutation in 5346 mapped in capB and the mutation in 5213 mapped in capA. The complementation
results presented above indicate that we have mapped 17 mutations into four complementation groups (represented by ORF capA, capB, capD, and capE) on the 6.2-kb BglII-EcoRI insert fragment of pCL7183. The fact that the six mutants complemented by pCL7193 were also complemented by pCL5201 (contains only intact capE) indicates that the capE gene is transcribed independently from capA, capB, and capD. On the other hand, mutations mapped in capA, capB, and capD were not complemented by pCL7193, which carries a fragment with a short deletion from the left side of the 6.2-kb fragment of pCL7183. These results suggest that capA, capB, and capD, as well as capC, are transcribed as a transcriptional unit from left to right. Subcloning of the right portion of the 14.6-kb region re-
7010
J. BACTERIOL.
LIN ET AL. 8162 285 8264 3 19 8 3 66
3 53 8468 25 8570 59 8672 93 8774 1 27 8876 161 8978 195 9080 2 29 9182 2 63 9284 297 9 3 86 3 31 9489 24 9591 58 9693 92 9795 126 9897 160 9999 1 94 10101 228 10203 262 10305 2 96 10407 3 30 1 0 509 3 64 1 10612 9 10714 43 10816 77 10918 111
11020 145 11122 17 9 11224 2 13 11326 247 11428 281 11530 3 15 11632 3 49 11734 3 83 11836 417 1 19 3 8 1 12040 31 12142 65
ATTAACTGAAAAGAATTAGAACAATTAGCCTATGATGATATCAACTGCTAC I V I S S N I K S N S E V N Q Y F K D S S F E L E L I P K T N A S T ACAAGTATCAGAAGAAAGTTAGTCATTTTAACAAGCAAAGATAAAATAAGG I K K L I S R K K H I S F N D S N V F D I N M T T Q E L K E I Y M S AAATTTGT____TGTTTAGAAATATAAGAACGGTTTGGCCACTCAAACTATACAG SD K T L CapI M K I L I T G T A G F I G S N L A K K L I K Q G CTAGTTGTTGTGAAAGTATTCGTCTGAGGAAATAACAAGAAAATTATTATA N
Y
V
I
G
V
D
S
I
N
D
Y
S
Y
S
V
K
K
L
D
R
L
S
K
G
I
K
N
E
F
T
K
N
F
GGATAAATAGCATACAATTTTGTACACGATGATATTGTCCACGTTAAAATT K
V
L
E
N
Y
D
D
L
S
K
V
F
V
D
Q
E
P
V
E
V
N
V
A
L
Q
A
G
A
N
V
Y
I
S
GGACAGAGAATATTAATTGTTAGAATTGAGATGCTTATTCAATATTTCTAC N
E
P
R
T
Y
I
D
S
N
I
V
G
F
N
M
I
S
C
E
L
H
R
F
N
Q
I
N
I
L
A
Y
S
S
ATCGTAGTCATCTTACATACCTGAATTGTACGTATTTTCGACAAATAAGGT 5
5
V
Y
G
A
N
T
S
K
P
F
S
S
T
D
I
N
H
D
S
L
P
L
Y
A
A
K
T
S
K
N
L
E
AGCCTCTTGCTTTCATACAACGATAATTCCGAAGTCTGGAACGTTGGTTTA A
M
H
T
Y
S
H
L
Y
N
L
P
G
T
T
L
R
F
F
V
T
G
Y
N
P
P
R
G
D
M
A
K
F
L
TTCAACACTATACACAAAGCAATAGAAAGTAAATTCTCTGTAATTGACATG S I F T K A I V N D Q A I D V Y N H G N M M R D F T Y V D D I V E A AGGCTTGTTAAGAAACCAGCGTCCCCTAATAAAGAATGGTCAGGGGCCGACCCTGACCCTGGTTCCTCATATGCACCATATAAAGTCTACAATATTGGGAAC L
R
V
K
K
P
A
S
P
N
K
E
S
W
G
A
D
D
P
S
G
P
S
A
Y
Y
P
K
Y
V
N
N
G
I
ACGCATAATAGATTTGGCAAAATATAGAAAGTGAAATCTGTTCACGAAGAC S
N
P
V
R
L
M
E
F
V
E
A
I
N
E
K
G
L
E
K
K
R
A
N
Y
M
P
Q
L
D
G
P
V
D
GACTTCATTGTATGTAAAATATCACAAATCATAGTGGAAAATGTATGATAA K
T
Y
A
N
V
D
D
L
Y
K
D
I
K
F
D
Q
CapJ M
SD
K
R
P
E
T
Q
I
T
V
G
D
N
K
F
V
N
D
K
L
Y
TTATTCATCCTGAAGTTCATGAATTAGTATTATATGTC
TATTAAAGLAALL Y
F
L
L
V
F
D
H
P
F
E
V
Y
K
K
D
Y
N
S
Y
F
G
I
TTAAATTGACAATACATTGTAAGTAAATAGGGAATTGTAGGAATTATATGA 5
H
K
I
N
N
N
Y
L
T
I
F
D
K
M
L
I
N
S
R
V
K
N
V
D
N
C
E
I
I
N
N
S
K
GAAAATATTAACAAAACAAACCTATTTATTAAAAAAAATGACCTCATCACA G
E
K
V
N
F
K
T
I
D
S
Y
K
S
P
K
S
L
I
F
K
H
K
K
I
F
S
A
L
T
S
I
K
I
AAGGTGGTTATGGACATTTAGTTTGACCATTTAAATATACTAAGTGATGTG S
K
D
C
V
L
I
R
V
P
5
V
L
C
F
I
A
A
L
I
C
K
K
I
N
K
P
Y
M
E
V
C
V
V
GACTTAGAATGTCTGTATTTGAATTACTACAGATTTCAAATCGGAATCACT A
A
F
D
A
Y
N
F
H
G
S
I
F
G
K
I
L
S
L
P
M
E
Y
L
Q
K
N
V
N
A
V
N
K
I
S
A
GGTTTTATAAAATGGACATTCTTAGGAGGTAAGATCACTCACGAAAATATA I
A
Y
V
T
K
K
Y
L
S
N
K
Y
P
C
N
G
K
E
F
K
G
I
S
Q
S
V
F
K
E
K
N
ATTGTTGGAAATAATGATATGTTCTTTGTAAAGCTAGACAAATATATATGT L
N
D
I
G
N
K
I
K
I
C
L
I
G
S
T
F
V
D
Y
K
G
H
N
V
A
I
K
S
N
S
I
V
L
AATGAACGTTATAACATTCAATTAGAATTTGTGGGTCATGGGCCCAGTAAGAAATTTATGGAAATGGCTAAAAAATACAATGTTGAAAATAATGTTATATTT N
E
G
G
K
Y
N
I
E
L
E
F
V
G
D
C
P
5
K
K
F
M
S
M
A
K
K
Y
N
V
E
N
N
F
I
V
AAAGGTAAAATTTATGACAAGACAGCGTTAAATAATTGGTTTAGAAATTTAGATTTGTATATTCAACCAAGTTTAACAGAGGGACATTGTCGAGCAATTGTG K
I
Y
D
K
T
A
L
N
N
N
F
N
N
L
D
L
Y
I
Q
P
K
C SD
5
L
T
S
G
H
A
N
C
V
I
GACAAGATGGGCAATGGCATCGTGATCGTGGTAAAATTTTTACAAGTTGTA E C N C V P T L A S N A G C N S D S V N K E Y L F K P K D V V K A I TACAATATAAACATTTTACAACAAGAAGTTGAAAAAATTTAGTCACGAATT L T K L I N R S I L S K Q Y R E E N V L E N K K N I S G Y N L S N I CATGAGGAAGATCTATTAAATAAAGTTTTTGGAQzAAAAAAGTATAAAAATA Q
I
E
N
E
K
A
L
L
N
Y
K
K
I
I
N
D
F
Y
L
A
I
N
K
N
CapK M
AL N
I
Y
H
N
Y
TACAATTCAATTAGTTATAGGAATTCTAAAAAAATACTATTAGATAGGCTG 5 P I1 F Q N L M V S IK G K I F M K Q R Y T K H Y YESE IK R L K GAGTGTAATGATTTATTTGAACTTCAAAATCAAAGATTTGAAGAATTTTATAATTATATCAAAAAAAATAGTGAATTTTATTCTGAAATAATTAAAAAT E C N D L F E L Q N Q R F E K F Y N Y I K K N S E F Y S E I I K K N ATTACGAAAACCGTCATTACATGCGATAGAGTAATGAAAGTAAATATCAAA I I T K K N L S C K K I T V A N I N Q L P E I T K D D I R K N V D K AAAAATAAAAGGATGGTCATGAAGAGTTTAATAGAAGTTTTGAATGTACTA R Y L D I A K T D S N A Y N N F Y K S V K C T G N C S T G N K L I K TTTAAACAAGGTTTAGTTAAGGTGGAGGTGAATGACATACAAAGAGATTGG V F N R K K K R V P I K K I V G G R V S Y N C K K G V Y Q F K E Q H TTAAACCAACATAGTTTCTTAGTAGTAATTAAATCTAAATAAAATCGCGAC P E T K F K K L N I Y Y N L K A D C E Y N A Y H I S Q L N K P L N Q TTGGATGGCTATACTACAGTTATTCATAGGATTGCGAGATATATTTTAGATAACAATATAGAATTAAGTTTTACACCAATTGCTATTTTTCCTAATGCAGAA L D C Y T T V I H R I A R Y I L D N N I S L S F T P I A I F P N A E ACTTTAACTGATTTAATGAGGGATGATATTGAAAAGCCTTTTAATTGTCCAGTGCGTAATCAATATGCTTCTTCTGAAGGAGCACCTTTTATTACAGAAAA T
L
T
C
L
N
R
C
C
I
E
K
A
F
N
C
P
V
R
N
0
Y
A
S
S
S
C
A
P
F
I
T
E
N
AAACAAGCCCAACTACAAATTAATCTACCTACTCCACTCTTTCACTCTAAACAAATTCATCCTAATATCTATCAATTAATACTCACACCTTTTTATACTACT K
E
C
R
F
E
L
C
T
S
I
N
V
A
T
C
V
F
E
C
K
0
I
N
C
N
I
Y
S
L
I
V
T
C
F
Y
T
T
ATCCATTGGTTAACGGTCGAATAAATACTCGAATTACAAGTTAATAAGATT I I K I K R I T T P L L R Y K I C C S V S L E N E L P V N Y Q 0 K C GTGACAGTTTAATTGGAAGATGACATTATTTACGATAATGTAATAGTTGAC E S C R N N C F L Q 5 R E K C I V T N V N L S T A I R F V S N C V I CATGAAATAATAACTATTTTTGATATAGTCGTAATAATTAGATAATTATGA I K K L K Y S L K Q C N I I V Y L V I S N C A C K N N I F V Q N C I TCGTTGAAAATATCATTATTTATAAACTACCTGGAAAAGTTCATAACTAAA F
N
T
N
F
H
F
S
F
V
N
K
I
P
5
T
P
C
C
K
K
R
F
A
I
N
N
I
K
aAAGAAGAAAAATCGATGTTGTAGAGTACGACGACTTGATACTAGACGTT N I A V V C L C Y V C L P V A V T F C N K H K V I C F N SC CapLNM
R
GTTATATAGATAGATAAAATAGTGAAAGATAAAATATAAACCATTGAAATC C I K E L K N N Y C R T N E V T E N K L K N T N I E Y T S I N E S R AATGCAGAAGATTTGAAAAAGGCTGATTTTATTATTATAGCTGTGCCAACACCAATCGATAAGCATAATAAACCTGATTTATTACCATTATTAAAACCAGT N
A
E
C
L
K
K
A
C
F
I
I
I
A
V
P
T
P
I
C
K
N
N
K
P
C
L
L
P
L
L
K
A
S
FIG. 2-Continued.
an 8.4-kb EcoRI-Bglll insert coordinates), which complemented six mutants, 5024, 5241, 5330, 5327, 5204, and 5211. Sequential deletion from the right side of pWL7327 resulted in six plasmids (Fig. 3), pCL7134, pCL7187, pCL7186, pCL7191, pWL7316, and pCL7185, with deletion end points located in ORFs capM (at the 14.0-kb coordinate), capL (HPiindIll site at the 12.6-kb coordinate), capK (HindIll site at the 11.4-kb coordinate), cap.! (Clal site at the 9.5-kb coordinate), capI (Ncol at the 8.9-kb coordinate), and capH (EcoRV at the 8.3-kb coordinate), respectively. These plasmids were used to
suited in plasmid pWL7327 with
(6.2-
to 14.6-kb
complement the six mutants complemented by pWL7327. Because the end points of these deletions are located in
different ORFs, we were able to map each mutation in this region to a specific ORF. The results in Fig. 3 showed that the mutations of.the six mutants were mapped in five ORFs: 5024 in capG, 5241 in capH, 5330 in capJ, 5327 in capK, and 5204 and 5211 in capL. We also deleted pWL7327 from the left side of the 8.4-kb insert to the Kpnl site (at the 9.7-kb coordinate). The resultant plasmid pCL7165 failed to complement any of the six mutants complemented by pWL7327. Thus, it is likely that the five complementation groups defined by the six mutants were transcribed as a transcriptional unit from left to right. However, because the deletion in pCL7165 also deleted caDG, capH, and capI, it is possible that these ORFs could be transcribed independently from the downstream genes.
GENES AFFECTING TYPE 1 CAPSULE PRODUCTION IN S. AUREUS
VOL. 176, 1994 12244 99 12346 133 12448 167 12550 201 12652 235 127 54 269 12856 303 12958 337 13060 371 13162 405 1 3 2 64 5 1 3 3 66 39 13468 73 13570 107 13 672 141 13774 175 13876 209 13978 243 14080 277 14182 311 14 2 84 345 14 3 86 379 14488
7011
GAAACTGTTGGGAAGGTAATTACTCCAGACACAATCGTTGTATATGAATCTACAGTTTATCCTGGCGCAACAGAAGAAGAATGTGTACCTGTATTGGAAAAA E
T
V
G
K
V
I
T
P
D
T
I
V
V
Y
E
S
T
V
Y
P
G
A
T
E
E
E
C
V
P
V
L
E
K
TATTCTGGACTCGTTTGTGGTAAAGATTTTTTTGTTGGTTACTCACCTGAAAGAATTAATCCTGGGGATAAGGTACATACTTTTGAAACCATTACTAAGGTT Y
S
G
L
V
C
G
K
D
F
F
V
G
Y
S
P
E
R
I
N
P
G
D
K
V
H
T
F
E
T
I
T
K
V
GTATCTGGCCAAACGCTTGAAGTATTAGAAATAGTTGCAGACGTATATAGTTCAGTAGTCACAGCAGGAGTTCATAAAGCATCTTCTATTAAAGTAGCAGAA V
S
G
Q
T
L
E
V
L
E
I
V
A
D
V
Y
S
S
V
V
T
A
G
V
H
K
A
S
S
I
K
V
A
E
GCAGCAAAAGTCATTGAAAACACACAACGTGATGTAAATATTGCCCTAATGAATGAATTGGCAATTATTTTTGATAAATTAGATATAGATAC TAACGAGGTA A
A
K
V
I
E
N
T
Q
R
D
V
N
I
A
L
M
N
E
L
A
I
I
F
D
K
L
D
I
D
T
N
E
V
TTAAAAGCTTCAGGAACAAAATGGAACTTCTTGAATTTTAAACCAGGATTAGTAGGGGGACATTGCATTGGTGTGGACCCATATTATTTAACACATAAAGCT L K A S G T K W N F L N F K P G L V G G H C I G V D P Y Y L T H K A C AGGAAG TTGGGC AC CATC CTGAAGTGATT TTAGCAGGTAGAAGAATAAATGATAATATGGCTAAATATATTGC TTCTAACGT TATTAAAGAGT TAT TGAAG Q E V G H H P E V I L A G R R I N D N M A K Y I A S N V I K E L L K
CAAGGATTAGAAGTACAAGGAGCAACAGTTAATGTGCTAGGTCTTACATTTAAAGAGAATTGTCCGGATTTAAGAAATACAAAGGTTATTCATATTATTGAA Q
G
L
L
K
M
E
V
Q
G
A
T
V
N
V
L
G
L
T
F
K
E
N
C
P
D
L
R
N
T
K
V
I
H
I
I
E
GAACTGAAAGAGTATGGATTAAACGTAACAGTGAATGATGTTGAAGCGGATAAAAATGAAGCTAAAAAGTTCTTTGGTTTAGATTTGATAGATACAAAAGAA E L K E Y G L N V T V N D V E A D K N E A K K F F G L D L I D T K E TTAAAAATGGTGGATGTAGTGTTATTTGCAGTGCCACATAAAGACTATATGGAAAATAAAAAGGATTATATCAATTTAGTTAAAGATTGTGGCATAGTGTTT V
D
V
V
L
F
A
V
P
H
K
D
Y
M
E
N
K
K
D
Y
I
L
N
V
K
D
C
G
I
V
F
GACATTAAAGGCATAATCAATAGTGATGAACTTAATGTAAGTCAACGATTATGGAGATTATAAGTGTAAAATTACATTGGAGGTTTTGTATGAAAAATCAA D
I
K
G
I
I
N
S
D
E
L
N
V
S
Q
R
L
W
R
L
CapM M
SD
*
K
N
Q
AAAATATTTCATTTAGTTACTGTTTCTAAGAGTATTCCACTTATGAGAGGACAAATAGAATTTTTAAGAAAAAAATATGGATGTTCACATTGTTTCGAGT K
I
F
H
L
V
T
V
S
K
S
I
P
L
M
R
G
Q
I
E
F
L
R
K
K
N
M
D
V
H
I
V
S
S
GATGGTAAGGAATTAAAGCAGTATGATAATGAAATAGC TCATGTTATACCTATGAAAAGAGATATAGCATTATTCAGTGATTTAAAGTCATTATTAAAAATG D G K E L K Q Y D N E I A H V I P M K R D I A L F S D L K S L L K M ATATTAC TATTTC ACAAAGAAAAACCATTTATTGTTAATTCTGGTACTCCAAAAGCAGGATTAATAGGAACAATAGCTGCGTTTATTACCCAAAGAC CTATT I L L F H K E K P F I V N S G T P K A G L I G T I A A F I T Q R P I AGAATATATAC TGTGAGAGGTTTAAGGCTTGAAACAGTTAAAGGATTCAAATATTTTGTATTGTATTTGATGGAAAAGATAGCAATGTTTTGTGCAACTGAT R I Y T V R G L R L E T V K G F K Y F V L Y L M E K I A M F C A T D ATAATAGCAATTT CTGAAAGTTTAAAGCATAAAATTATTACATCTAATTTGGCTAAGGAAAATAAAATTACTGTTTTGGGATTTGGTAGTTCTAATGGTATA I I A I S E S L K H K I I T S N L A K E N K I T V L G F G S S N G I
CAATTTGAAAAATTCCAATTAGATAACAATAAATTAGAAGAAAAATACCATAAATTATTAAATGATAATTTTGTTATTGGCTATGTAGGAAGAATTGTAAAA Q
F
E
K
F
Q
L
D
N
N
K
L
E
E
K
Y
H
K
L
L
N
D
N
F
V
I
G
Y
V
G
R
I
V
K
GATAAAGGTATACATGAATTAATTCAGTCATTTAAAATTATTGTAAGTAAAGGATATAATGTCAAATTGCTTGTTATTGGTAGTTTAGAGACAGAAAATTCT D
K
G
I
H
E
L
I
Q
S
F
K
I
I
V
S
K
G
Y
N
V
K
L
L
V
I
G
S
L
E
T
E
N
S
ATTGATGAATCTGACTATTTATTTTTAACTCAAAATCCTAATGTAGTACTAATCAAGCATGTTTCAGATCCAATTTCATTTTATAATAATATGAATGTATTT
N N M N V F I D E S D Y L GTTTTTCCAACTCATAGAGAAGGGTTTGGAAATGTTAGTATAGAGGCTCAAGCACTTGAAGTGC CAGTAATTACTACTAATGTTACAGGCGC TATTGATACT V F P T H R E G F G N V S I E A Q A L E V P V I T T N V T G A I D T F
L
T
Q
N
P
N
V
V
L
I
K
H
V
S
D
P
I
S
F
Y
GTAGTAAATGGAGAAACTGGATTTATTGTTGAAAAAGGTGACTTTAAAGCAATCGCTGAAAAAATTGAAAAATTAATTAATGACGAGAGTTTAAGAGAAACT V
V
N
G
E
T
G
F
I
V
E
K
G
D
F
K
A
I
A
E
K
I
E
K
L
I
N
D
E
S
L
R
E
T
ATTGGTCATAATGGAAGAAAGAGAGTGGAAAATAAATTTTCAAGC CAAATTATATGGGAAGAATTGGAAAGTATGTACAATACTTTTCTAAAAGAAAGTGAG I
G
H
N
G
R
K
R
V
E
N
K
F
S
S
Q
I
I
W
E
E
L
E
S
M
Y
N
T
F
L
K
E
S
E
GGAAAGTAATGAAAAGAATATTTGATATATTTAGTTCATTATATGCAATTATTATATTTTTGCGCTGCTCTTTCTAGTGTCTATTGCAATTAAAATAGAGTC G
K
*
TAAAGGTCCGATlYGTATTTAAACAGGACCGACCAGGCGTAAAGAACAAATTATTTAAAATTTATAAATTTAGATCT
14563
FIG. 2-Continued.
Because the EcoRI site (6.2-kb coordinate) that divides the insert fragments cloned in pCL7183 and pWL7327 is located in ORF capF, it is possible that the mutations of the two previously isolated mutants, 5025 and 5027 (24), which were not complemented by either pCL7183 or pWL7327, mapped in capF. To test this possibility, the 6.2-kb PstI-KpnI (3.5- to 9.7-kb coordinates) fragment in the middle portion of the 14.6-kb fragment was cloned into a plasmid vector. The resultant plasmid, pCL7164, which contains five intact ORFs, capE to capI, was able to complement mutations mapped in capE, capG, and capH as expected but was not able to complement mutant 5025 or mutant 5027. The complementation results presented above showed that 9 of the 13 ORFs were represented by at least one mutant, whereas no mutation was mapped in capC, capF, capI, or capM. To search for additional mutants that may have mutations mapped in these ORFs, we screened 25 additional mutants generated by MNNG mutagenesis. We found one mutant, 5091, complemented by pCL7164 but not by pCL7183 or pWL7327 (Fig. 3), indicating that the mutation in 5091 mapped in capF. Most of the other MNNG-induced mutations mapped in capE, and no additional mutation was mapped in capC, capI, or capM. Mutagenesis. Because no mutation was mapped in the ORFs capC, capI, and capM, we were unable to conclude that these ORFs are involved in capsule biosynthesis. Therefore, we attempted to construct mutants with chromosomal mutations mapped in these three ORFs to determine whether they are involved in biosynthesis of type 1 capsule. Strains CYL5542 and CYL5531, which have mutations corresponding to capI and capM, respectively were constructed as described in Materials and Methods. These site-specific mutants were all Cap-, indicating that both ORF capI and capM are involved in the synthesis of capsule. To confirm that the mutations in strains CYL5542 and CYL5531 are indeed located in ORFs
capI and capM, respectively, and to show that the mutation in capI is not polar, we performed complementation tests with the plasmids described in Fig. 3. The results showed that strain CYL5542 was complemented by pCL7191, which contains intact capG through capI but not by pWL7316 in which most of the ORF capI was deleted, whereas strain CYL5531 was complemented by pWL7327 containing capH through capM but not by pCL7134 containing intact capH through capL. These results indicate that the mutation sites of these sitespecific mutants are mapped at the expected genes and that the mutation in capI is not polar. The same strategy used to construct the site-specific mutations in capI and capM was used to construct the mutation in capC. However, we were unable to obtain the desired mutation in capC despite several independent attempts. To determine if ORF capC is involved in capsule biosynthesis, we sought a different approach. We constructed a deletion mutation of strain M, CYL5529, in which the DNA fragment between the HindIII site (2.3-kb coordinate within capC) and 1.8 kb upstream of the BglII site (at the 0-kb coordinate) of the genome was deleted and replaced with a 1.7-kb DNA fragment containing the cat gene of pC194 by allelic replacement (see Materials and Methods). Thus, strain CYL5529 is a null mutant with deletion of ORFs capA and capB and most of capC. We then constructed plasmid pCL7398 containing intact capA, capB, and capD and a BamHI linker insertion at the FspI site (2.0-kb coordinate) within ORF capC by using the singlecopy integration vector pCL84, as described in Materials and Methods. The plasmid pCL7398 was then transferred to CYL5529. The resultant strain, CYL5529(pCL7398), which is genotypically equivalent to the wild-type strain M with a capC mutation, was Cap-, implying that ORF capC is involved in capsule biosynthesis. However, it is possible that CYL5529 could have a mutation at other cap genes, which would give rise to the Cap- phenotype regardless of whether capC is essential
7012
J. BACTERIOL.
LIN ET AL. 0
1
2
3
4
5
6
7
8
I
I
I
I
,
I
,
I
|
I
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P Hc
BHEvHcH FH
I\L / \1 I
A
~ ~ +> --I
A B C 24.5 25.2 29.4
D 67.5
E 50.7
I
Ev N A C KHcBEVHHcC H Hc A
C E Hc F 45.0
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'll I/ /
\11
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kb
14
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J 37.9 44.5
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K 52.4
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A.
.-
L 47.2
M 43.1
B
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kd
pCL7183 +
+
+
+_
pCL7193 _ pWL7315 +
+
+
-
pWL,7309
+
pCL7407
+
- pCL5201
pCL7164 _
+
+
+
+
pWL7327 -
+
+
+
pCL7134 -
_-
+
+
+
+
_
+
+
+
_
_
pCL7187 _
.
-
+
-
+
+
+
_
_
_
+
_
_
-
+
-
pCL7186
-
pCL7191 pAVL7316
pCL7185 -
I-
pCL7165
5213 5346
5204 5327 5091 5024 5241 5330 5176 5023 5211 5187 5203 5206 5214 5210 5226 5212 5242 5244 5243 5245 5246 5248 FIG. 3. Mapping of the mutants by complementation tests. ORFs are indicated by arrows. Capital letters under arrows represent the corresponding genes. The predicted molecular mass of each predicted polypeptide is shown under each gene. Solid bars represent DNA fragments cloned into plasmid vectors. The inserts in plasmids pCL7183, pCL7193, pWL7315, pWL7309, and pCL7407 were cloned into single-copy integration vector pCL83 or pCL84. The others are cloned into either pLI50 or its derivative, pCL8. A + indicates the Cap' phenotype, and a - indicates the Cap- phenotype. The mutants used for the tests are shown at the bottom of the figure. A, AccI; B, BglII; C, ClaI; E, EcoRI; Ev,
EcoRV; F, FspI; H, HindIII; Hc, HincII; K, KpnI; P, PstI; N, NcoI.
for capsule biosynthesis. To rule out this possibility, pCL7183, which carries the wild-type DNA fragment equivalent to pCL7398, was used to complement CYL5529. The resultant strain, CYL5529(pCL7183), was Cap', suggesting that CYL 5529 does not contain an additional mutation outside ORFs capA to capC. Because capA, capB, capC, and capD were shown to be coexpressed by the complementation tests described above (Fig. 3), it is possible that the mutation at capA affects the expression of the downstream capD gene. To test this possibility, the plasmid pCL7398 was transferred into strain CYL5246 (a capD mutant). The resultant strain was Cap', indicating that the mutation in pCL7398 is not polar. It is also possible that during in vitro construction of pCL7398, a mutation could be introduced into capA or capB that could also result in the Cap- phenotype seen in CYL5529 (pCL7398). This concern was ruled out in experiments in which we showed that pCL7398 complemented both 5213 (a capA mutant) and 5346 (a capB mutant). Analysis of gene products. To confirm the ORFs predicted
from the sequence data, plasmid subclones containing the capsule genes were expressed in an in vitro transcriptiontranslation system with E. coli S30 extract (55). When plasmid pCL7152 containing five complete ORFs (capA to capE) was expressed, five proteins with approximate molecular masses of 24.5, 25.2, 29.4, 67.5, and 50.7 kDa were detected (compare lane 2 with lane 1 of Fig. 4), which corresponded to the predicted molecular masses of ORFs capA to capE, respectively. When plasmid pCL7164 containing the complete ORFs capE to capI was expressed, although several other minor bands were also detected, no protein bands corresponding to these ORFs could be clearly demonstrated. By expressing plasmid pWL7327 containing complete ORFs capG to capM, we detected two prominent bands that could be identified as CapJ (44.5 kDa) and CapK (52.4 kDa) (compare lane 4 with lane 5). Homology search. The deduced amino acid sequences of CapA to CapM were compared with sequences in the GenBank and European Molecular Biology Laboratory (SWISS-
VOL. 176, 1994
GENES AFFECTING TYPE 1 CAPSULE PRODUCTION IN S. AUREUS
7013
CapL MNRNIAWGLGYVGLPVAVTFGNKHKVIGFDINESRIKELKNNYDRTNEVTENKLKNTN- 59 : :1::111111:1 1: :1:11:1 11 1111 1 11: 1::: VipA DEVKIAIIGLGYVGLPLAVEFGKSRQWGFDVNKKRILELKNGVDVNLETTEEELREARY 64 CapL IEYTSNAEDLKKADFIIIAVPTPIDKHNKPDLLPLLKASETVGKVITPDTIWYESTVYP 119 :11:
:1
:1 11:11111:
::111 11:1111111 1:
1111111111
VipA LKFTSEIEKIKECNFYIITVPTPINTYKQPDLTPLIKASETVGTVLNRGDIVVYESTVYP 124
CapL GATEEECVPVLEKYSGLVCGKDFFVGYSPERINPGDKVHTFETITKVVSGQTLEVLEIVA 179 I 11111:1 11 :1:1111111111
1:11: 11 1:: 1::
VipA GCTEEECVPILARMSGMTFNQDFYVGYSPERINPGDKKHRLTNIKKITSGSTAQIAELID 184
CapL DVYSSVVTAGVHKASSIKVAEAAKVIENTQRDVNIALMNELAIIFDKLDIDTNEVLKASG 239 :11 ::1 11111111111111111::1:1:: VipA EVYQQIISAGTYKAESIKVAEAAKVIENTQRDLNIALVNELAIIFNRLNIDTEAVLRAAG 244 CapL TKWNFLNFKPGLVGGHCIGVDPYYLTHKAQEVGHHPEVILAGRRINDNMAKYIASNVIKE 299 VipA SKWNFLPFRPGLVGGHCIGVDPYYLTHKSQGIGYYPEIILAGRRLNDNMGNYVSEQLIKA 304
CapL LLKQGLEVQGATVNVLGLTFKENCPDLRNTKVIHIIEELKEYGLNVTVNDVEADKNEAKK 359 ::11:::1:1 11:11111:11:::::111:
1:
1 1:1 ::
VipA MIKKGINVEGSSVLILGFTFKENCPDIRNTRIIDVVKELGKYSCKVDIFDPWVDAEEVRR 364 CapL FFGLDLIDTKELKMVDWLFAVPHKDYME-NKKDYINLVKDCGIVFDIKGIINSDELNV 417 :: 11 1:::.:
:1: kb 0
3
2
1
5
4
6
7
8
9
la cR Noo =gl cRV Pi
Kpnl BgIII
cli -4 30io1
_-" I
A
9
C
kd 24.5 25.2 29.4
\I
D
E
F
67.5
50.7
45.0
I /
0
H
19.4 41.4
37.9
i1
10
/
12
13
14
EcoRV C9111 /
I~~~~~~~~~~cla
: 11
:::1:1 :: ::: :1
VipA EYGIIPVSEVKSSHYDAIIVAVGHQQFKQMGSEDIRGFGKDKHVLYDLKYVLPAEQSDV 423
FIG. 5. Comparison of the deduced protein sequences of CapL and VipA. A line indicates identical residues, and two dots indicate similar residues.
>- -4- -30-
J
K
L
M
4.5
52.4
47.2
43.1
pCL7152 pCL714 pWL7327
FIG. 4. Autoradiogram of E. coli S30 in vitro transcription translation of the gene products. [35S]methionine-labelled proteins were separated by SDS-PAGE as described in Materials and Methods. Lane 1, plasmid vector pLI50; lane 2, pCL7152 (pLI50 replicon); lane 3, pCL7164 (pLI50 replicon); lane 4, pWL7327 (pCL8 replicon); lane 5, plasmid vector pCL8. Letters indicate the corresponding predicted
Cap proteins.
PROT) protein databases by using the algorithm of Lipman and Pearson (30) in the Genetics Computer Group computer programs. The most significant homology was between CapL and VipA, which showed 52.3% identity and 89.4% similarity with a 423-amino-acid overlap (Fig. 5). CapL also showed 24.0% identity to AlgD with a 416-amino-acid overlap. VipA is a protein involved in the biosynthesis of Vi polysaccharide in Salmonella typhi (11), and AlgD is GDP-mannose dehydrogenase, which catalyzes oxidation of GDP-mannose to GDPmannuronic acid required for alginate biosynthesis by mucoid Pseudomonas aeruginosa (38). A high degree of homology was also found between the CapI protein and an unidentified reading frame (ORF2) adjacent to the chain-length determinant, which confers a modal distribution of chain length on the 0 antigen component of lipopolysaccharide (LPS) of E. coli (6). The homology showed 57.7% identity and 89.2% similarity in a 333-amino-acid overlap. In addition, the CapI protein showed limited homology to VipB protein, another enzyme involved in the Vi antigen synthesis of S. typhi (11), and showed limited homology to various epimerases from prokaryotic and eukaryotic origins, including GalE protein (UDP-N-acetylglu-
cosamine epimerase) of E. coli (28). There were identities of 29.0% over 335 amino acids between Capl and VipB and 20.5% over 332 amino acids between CapI and GalE. Moderate to high levels of homology were found between the deduced amino acid sequence of CapG and the conserved region of about 50 amino acids of various members of the NodL-LacA-CysE acetyltransferase family recently identified (8). Twenty-two of the 29 amino acids between residues 86 and 132 of CapG were identical to the consensus of the conserved region of the acetyltransferases, and 5 of the mismatches were conserved substitutions (Fig. 6). Thus, CapG could be an acetyltransferase required for the synthesis of type 1 capsular
polysaccharide. The deduced amino acid sequence of CapM showed limited homology to RfpB protein of Shigella dysenteriae (10, 20) with 24.1% identity over 370 amino acids. RfpB has been implicated as a galactosyl transferase that adds a galactose moiety to the core region in LPS biosynthesis (10). CapM protein also
Tn2424Cat
113-164
NodL
134 -185
LacA
134-185
CysE
195-246
At cat
112-163
Sa vat
121-172
CapG
86-137
Consensus
IGSDVWIGSEAMIMPGIKIGHGAVIGSRALVAKDVEPYTIVGGNPAKSIRKR IGRHAWIGGGAIILPGVTIGDHAVIGAGSVVTRDVPAGSTAMGNPARVKAGG IGNNVWIGSHVVINPGVTIGDNSVIGAGSIVTKDIPPNVVAAGVPCRVIRET IREGVMIGAGAKILGNIEVGRGAKIGAGSVVLQPVPPHTTAAGVPARIVGKP IGNDVWIGSEAIIMPGITVGDGAVIGTRALVTKDVEPYAIVGGNPAKTIRKR IGNDVWIGRDVTIMPGVKIGDGAIIAAEAVVTKNVAPYSIVGGNPLKFIRKR IGNNVFIGINSIILPGVTIGNNVVVGAGSVVTKDVPDNVIIGGNPAKKIKSI IG- -VWIG ---- I-PGV-IG--AVIGA- - -VTKDV-P----- GNPA- -I
FIG. 6. Alignment of CapG with Tn2424Cat (Cat protein of Tn2424 [35]), NodL (the probable acetyltransferase of Rhizobium leguminosarum), LacA (thiogalactoside acetyltransferase of E. coli), CysE (serine acetyltransferase of E. coli), AtCat (Cat protein of A. tumefaciens [46]), and Sa Vat (virginiamycin-like antibiotic acetyltransferase of S. aureus [3]). The alignment of Tn2424Cat, NodL, LacA, and CysE is taken from Parent and Roy (35).
7014
LIN ET AL.
showed limited homology to VipC protein, a gene product involved in the synthesis of Vi antigen of S. typhi (11) with 21.8% identity over a 275-amino-acid overlap. No other ORFencoded proteins predicted from the sequence analysis showed significant homology to the reported proteins in the data banks. Virulence studies. Previous studies have shown type 1 capsule is a virulence factor (27, 31). However, these studies employed mutant strains that were spontaneously arisen, chemically induced, or transposon mutagenized. To unequivocally assess whether type 1 capsule is a virulence factor, we constructed two isogenic strains, CYL5419 with a Cap' phenotype and CYL5529 with a Cap- phenotype, derived from strain M as described in Materials and Methods. Two groups of six mice were injected with the isogenic strains. All six mice injected with 107 CFU of CYL5419 died within 24 h, while all six mice injected with 107 CFU of CYL5529 lived even after 72 h. This result reconfirmed the findings of previous studies that type 1 capsule is an important virulence factor in S. aureus.
DISCUSSION We have previously cloned the genes affecting capsule synthesis from S. aureus M in a 19.4-kb chromosomal DNA fragment. In this report, we further localized the genes to a 14.6-kb DNA fragment. Sequencing of this 14.6-kb fragment showed that there were 13 ORFs. Genetic experiments with deleted fragments to complement various Cap- mutants showed that only 10 of these ORFs were represented by at least one mutant despite the large number of mutants that were screened. No mutations were mapped in ORFs capC, capI, and capM, and more than half of the mutations were mapped in either ORF capD or ORF capE, indicating that the mutations occurred at hot spots. The fact that the mutations resulting in the Cap- phenotype were mapped in 10 of the 13 ORFs suggests that these genes are involved in type 1 capsule biosynthesis. To determine whether the remaining three ORFs are involved in the synthesis of capsule in strain M, we attempted to construct mutants with nonpolar chromosomal mutations mapped within each of these three ORFs. Capmutants with mutations mapped in capI and capM were successfully obtained, implying that these two genes are involved in capsule production. However, our attempts to use the same approach to construct mutants with mutations in capC failed. One possibility for the failure may be due to the fact that the presence of a high dosage of one or more of the genes (capA, capB, and capC) is lethal to the staphylococcal cells. Evidence for this came from the fact that we were unable to transform the DNA fragment containing intact capA, capB, and capC cloned in a multiple-copy vector into S. aureus cells, whereas transformation was readily established when the same fragment was cloned into a single-copy vector (24). Recognizing this fact, we deliberately cloned the DNA fragment for replacement without the promoter upstream of the capA, yet this approach continued to fail, indicating that there may be a weak promoter from the multiple-copy vector that allows enough gene expression. To circumvent this problem, we employed a different approach and constructed a null mutant, CYL5529, which produced no capA, capB, capC, or capD; yet expression of other cap genes was not affected. We then constructed plasmid pCL7398 by cloning a DNA fragment containing capA to capE genes with a nonpolar mutation in capC. This plasmid was constructed with a single-copy plasmid vector that integrates at the geh locus of the staphylococcal chromosome (26) to avoid the high-gene-dosage effect of multiple-copy vectors. Because
J. BAcrERIOL.
deletion in CYL5529 did not affect the expression of other capsule genes, transferring the pCL7398 into CYL5529 generated a strain equivalent to the M strain with a specific mutation at capC. By using such an approach, we have shown that ORF capC is involved in capsule production. In the in vitro transcription-translation system, we were able to clearly identify protein bands corresponding to seven predicted ORFs (capA to -E and capJ to -K; Fig. 4). This result lends additional support to the argument that these predicted ORFs are likely to be the functional genes. The reason that protein bands corresponding to other ORFs were not detected could be due to the fact that these gene products were not efficiently expressed in the E. coli system or, alternatively, it could be due to the fact that the bands were not resolved in the electrophoretic system we employed. It is interesting that CapE was readily detected, although weakly expressed in pCL7152, but no protein band corresponding to CapE was detected when pCL7164 was expressed, even though our genetic data suggested that CapE was expressed from pCL7164. This discrepancy could be due to the fact that the promoter further upstream of capE, which is not present in pCL7164, may be required for full expression of CapE in E. coli, whereas in S. aureus, CapE may be readily expressed from its own promoter. The genetic complementation data suggest that the cap genes are organized into at least two operonic structures: one operon consists of capA through capD, with a likely transcription start upstream of capA; the other consists of at least capJ, capK, and capL but may also include capG, capH, capI, and capM, with possible transcription initiated from upstream of capG. In addition, the data also indicate that there is another transcription start upstream of capE. Indeed, by using the Genetics Computer Group program, we have found a potential promoter with close homology to the consensus promoter sequences found in E. coli and Bacillus spp. just upstream of capA (Fig. 2). Potential promoter sequences were also found upstream of capE and capG, although they were less homologous to the canonical sequences. Although these genetic data suggest multiple transcription units, sequencing data showed that all 13 ORFs are closely linked and that there is no obvious inverted repeat at any of the intercistronic noncoding regions, suggesting that all of these genes may be transcribed in a single transcription unit. Thus, taking both the genetic complementation results and the sequence data together, it is likely that these cap genes are transcribed in a long operon with additional internal promoters. Because the more distal a gene is the less efficiently it is transcribed, the internal promoters would ensure that the downstream genes are produced in adequate amounts. We are currently employing Northern (RNA) blotting to study the gene transcription and Si mapping to determine the transcription start sites. Recently, Hashimoto et al. (11) reported the nucleotide sequence of the ViaB region encoding Vi antigen in S. typhi and showed that three genes, vipA, vipB, and vipC, were involved in biosynthesis of the Vi polysaccharide. Vi polysaccharide is a homopolymer of N-acetylgalactosamino uronic acid (13), which is one of the monomeric components of type 1 capsule in S. aureus M (34). In fact, antiserum raised against Vi antigen cross-reacts with strain M capsule (19). The very high degree of conservation between CapL and VipA, moderate homology between CapI and VipB, and limited homology between CapM and VipC imply that they are probably involved in the same biosynthetic pathway leading to the synthesis of N-acetylgalactosaminouronic acid. Because Salmonella spp. and Staphylococcus spp. are two very evolutionarily divergent organisms, the similarity, especially between CapL and
GENES AFFECTING TYPE 1 CAPSULE PRODUCTION IN S. AUREUS
VOL. 176, 1994 a
LDHM ADHE G3P VIPA MDHM MDHE VIPB GALE
22-52 192-223 3-33 8-35 2-33 2-24 17-46
2-31 CAPL 4-32 CAPI 2-31
I G
I
I
LFLS
fi
CAISILMKDLAD--EVALV1
-
CA
Loop
LSVIMGCKAAGAA---RtIGVD R
RNVGRAALKNPR--IEVVAVNf
LPLAVEFGKSR-----QVVGFf QPLSLLLKNSPL--VSRLTLYD
QALALLLKTQLPS-GSELSLYO
A
SGLLEELLFLN----QTVIGLR GS
SKTCVQLLQNG---- HD IILI
LPVAVTFGNKH-----KVIGFD TA E SHLAKKLIKQG-----HYrVIGVD FIG. 7. Alignment of CapL and CapI with proteins containing the NAD-binding domain. Abbreviations: LDMH, spiny dogfish lactate dehydrogenase; ADHE, horse liver alcohol dehydrogenase; G3P, Bacillus stearothermophilus glyceraldehyde phosphate dehydrogenase; MDHM, porcine heart malate dehydrogenase; MDHE, E. coli malate dehydrogenase; GALE, E. coli UDP-glucose epimerase. The alignment of the proteins LDHM, ADHE, G3P, VIPA, MDHM, MDHE, VIPB, and GALE is taken from Fig. 7 of Hashimoto et al. (11).
VipA,
across two
IA IL
L-
distantly related bacterial
genera
is intrigu-
ing.
The VipA and VipB proteins have been shown to possess a putative NAD-binding domain by homology search (11). This NAD-binding domain, which consists of about 30 amino acids with a three-dimensional structure of Pa,, is highly conserved among NAD-dependent dehydrogenases (49). The deduced amino acid sequences of CapL and CapI also aligned well with this NAD-binding domain, suggesting that CapL and CapI are likely to have an NAD-binding domain (Fig. 7). On the basis of the identification of an NAD-binding domain and sequence homology to VipA and VipB protein sequences, we propose that CapL and CapI may be the NAD- or NADP-dependent enzymes involved in the synthesis of N-acetylgalactosaminouronic acid.
ACKNOWLEDGMENTS
This work was supported in part by grant A128373 from the National Institute of Allergy and Infectious Diseases. We thank Michael Black for isolating some of the Cap- mutants and for participating in the initial sequencing work. REFERENCES
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Staphylococcus aureus mutants altered in tion. Infect. Immun. 59:1008-1014.
type 5
capsule produc-
2. Albus, A., J.-M. Fournier, C. Wolz, A. Boutonnier, M. Ranke, N. H0iby, H. Hochkeppel, and G. Doring. 1988. Staphylococcus aureus capsular types and antibody response to lung infection in patients with cystic fibrosis. J. Clin. Microbiol. 26:2505-2509. 3. Allignet, J., V. Loncle, C. Simenel, M. Delepierre, and N. El Solh. 1993. Sequence of a staphylococcal gene, vat, encoding an acetyltransferase inactivating the A-type compounds of virginiamycinlike antibiotics. Gene 130:91-98. 4. Arbeit, R. D., W. W. Karakawa, W. F. Vann, and J. B. Robbins. 1984. Predominance of two newly described capsular polysaccharide types among clinical isolates of Staphylococcus aureus. Diagn. Microbiol. Infect. Dis. 2:85-91. 5. Baddour, L. M., C. Lowrance, A. Albus, J. H. Lowrance, S. K. Anderson, and J. C. Lee. 1992. Staphylococcus aureus microcapsule expression attenuates bacterial virulence in a rat model of experimental endocarditis. J. Infect. Dis. 165:749-753. 6. Bastin, D. A., G. Stevenson, P. K. Brown, A. Haase, and P. R. Reeves. 1993. Repeat unit polysaccharides of bacteria: a model for polymerization resembling that of ribosomes and fatty acid synthetase, with a novel mechanism for determining chain length. Mol. Microbiol. 7:725-734. 7. Birnboim, H. C. 1983. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 100:243-255.
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8. Downie, J. A. 1989. The nodL gene from Rhizobium leguminosarum is homologous to the acetyl transferases encoded by lacA and cysE. Mol. Microbiol. 3:1649-1651. 9. Dyer, D. W., and J. J. landolo. 1983. Rapid isolation of DNA from Staphylococcus aureus. Appl. Environ. Microbiol. 46:283-285. 10. Gohmann, S., P. A. Manning, C.-A. Alpert, M. J. Walkers, and K. N. Timmis. 1994. Lipopolysaccharide 0-antigen biosynthesis in Shigella dysenteriae serotype 1: analysis of the plasmid-carried rfp determinant. Microb. Pathog. 16:53-64. 11. Hashimoto, Y., N. Li, H. Yokoyama, and T. Ezaki. 1993. Complete nucleotide sequence and molecular characterization of ViaB region encoding Vi antigen in Salmonella typhi. J. Bacteriol. 175: 4456-4465. 12. Hawley, D. K., and W. R. McClure. 1983. Compilation and analysis of Eschenichia coli promoter DNA sequence. Nucleic Acids Res. 11:2237-2255. 13. Heyns, K., G. Kiessling, W. Lindenberg, H. Paulsen, and M. E. Webster. 1959. D-Galaktosaminurosaure (2-Amino-2-Deoxy-DGalakturonsaure) als Baustein des Vi-Antigen. Chem. Ber. 92: 2435-2437. 14. Hochkeppel, H. K., D. G. Braun, W. Vischer, A. Imm, S. Sutter, U. Staeubli, R. Guggenheim, E. L. Kaplan, A. Boutonnier, and J. M. Fournier. 1987. Serotyping and electron microscopy studies of Staphylococcus aureus clinical isolates with monoclonal antibodies to capsular polysaccharide types 5 and 8. J. Clin. Microbiol. 25: 526-530. 15. Holmes, D. S., and M. Quigley. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114:193197. 16. Horinouchi, S., and B. Weisblum. 1982. Nucleotide sequence and functional map of pC194, a plasmid that specifies inducible chloramphenicol resistance. J. Bacteriol. 150:815-825. 17. Karakawa, W. W., J. M. Fournier, W. F. Vann, R. Arbeit, R. S. Schneerson, and J. B. Robbins. 1985. Method for the serological typing of the capsular polysaccharides of Staphylococcus aureus. J. Clin. Microbiol. 22:445-447. 18. Karakawa, W. W., A. Sutton, R. Schneerson, A. Karpas, and W. F. Vann. 1988. Capsular antibodies induce type-specific phagocytosis of capsulated Staphylococcus aureus by human polymorphonuclear leukocytes. Infect. Immun. 56:1090-1095. 19. Karakawa, W. W., and W. F. Vann. 1982. Capsular polysaccharides of S. aureus. Semin. Infect. Dis. 4:285-293. 20. Klena, J. D., R. S. Ashford II, and C. A. Schnaitman. 1992. Role
of Escherichia coli K-12 rfa genes and the rfp gene of Shigella dysenteriae 1 in generation of lipopolysaccharide core heterogeneity and attachment of 0 antigen. J. Bacteriol. 174:7297-7307. 21. Kraemer, G. R., and J. J. Iandolo. 1990. High-frequency transformation of Staphylococcus aureus by electroporation. Curr. Microbiol. 21:373-376. 22. Kreiswirth, B. N., S. Lofdahl, M. J. Betley, M. O'Reilly, P. M. Shlievert, M. S. Bergdoll, and R. P. Novick. 1983. The toxic shock syndrome exotoxin structural gene is not detectably transmitted by a prophage. Nature (London) 305:709-712. 23. Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227:
680-685. 24. Lee, C. Y. 1992. Cloning of genes affecting capsule expression in Staphylococcus aureus strain M. Mol. Microbiol. 6:1515-1522. 25. Lee, C. Y., S. L. Buranen, and Z. H. Ye. 1991. Construction of single-copy integration vectors for Staphylococcus aureus. Gene 103:101-105. 26. Lee, C. Y., and J. J. landolo. 1986. Integration of staphylococcal phage L54a occurs by site-specific recombination: structural analysis of the attachment sites. Proc. Natl. Acad. Sci. USA 83:54745478. 27. Lee, J. C., M. J. Betley, C. A. Hopkins, N. E. Perez, and G. B. Pier. 1987. Virulence studies, in mice, of transposon-induced mutants of Staphylococcus aureus differing in capsule size. J. Infect. Dis. 156:741-750. 28. Lemaire, H. G., and B. Muller-Hill. 1986. Nucleotide sequences of the galE gene and the galT gene of Escherichia coli. Nucleic Acids Res. 14:7705-7711. 29. Liau, D. F., and J. H. Hash. 1977. Structural analysis of the surface
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45. 46.
47.
48. 49.
50. 51.
52.
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