Sequence Analysis and Molecular Characterization of Genes Required for the Biosynthesis of Type 1 Capsular Polysaccharide in Staphylococcus aureus


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 viru- lent 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, 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 (913) 588-7295. 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 chromo- some 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 restric- tion-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 F-recA13 hsdS20 ara-14 proA2 lacYl galK2 xyl-5 mtl-i supE44 rpsL20 A-F-hasR514 supE44 supF58 lacYl galK2 galT22 metBI trpR55 X- Shuttle cloning vector Shuttle cloning vector Single-copy integration vector Single-copy integration vector Temperature-sensitive cloning vector was carried out by the procedure of Kraemer and I Phage 52A used for transduction has been describc (24). The transduction procedure was carried out described (42). N-Methyl-N'-nitro-N-nitrosoguanid Sigma Chemical Co., St. Louis, Mo.) was used for as described previously (32). Capmutants wer( screening nonmucoid colonies and were confirme4 staining with India ink as previously described ( vector pCL8 was essentially the same as pLI50, ex ampicillin resistance gene and the multiple cloni derived from pWSK30 (48). Plasmid pCL52.1 was by cloning the 1.4-kb fragment containing the t sensitive origin of replication of pE194ts into the pCL72 (25) in which the HindIII site upstream oi resistance gene (Tcr, originally from pT181) has b with a BglII linker. Plasmid pCL7398 was co] digesting pCL7183 (see Fig. 3) with FspI (located this study reading frame [ORF] capC) and ligating it with a BamHI linker. 
Source or likr reference 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) 22 and further purified by CsCl-ethidium bromide density gradi-251 ent centrifugation or by using the Qiagen plasmid kit (Qiagen, 
Inc., Chatsworth, Calif.). Rapid small-scale plasmid DNA 24 purification was done by the method of Holmes and Quigley This study (15). Bulk chromosomal DNA from S. aureus was purified by 24 the method of Dyer and Iandolo (9). The transfer of DNA to 24 nitrocellulose membranes was by the method of Southern (45). 24 
The conditions used for hybridization analysis have been 24 
described previously (26). Enzymes used in DNA manipulation 24 
were purchased from GIBCO-BRL (Gaithersburg, Md.) or 24 New England Biolabs, Inc. (Beverly, Mass.) 
24 DNA sequence analysis. The 14.6-kb DNA fragment con- 24 taining capsule genes was subcloned into bacteriophage M13 24 derivatives mpl8 and mp19 (53). Overlapping deletions of the 24 DNA fragments for sequencing were generated by the exonu- This study clease III (New England Biolabs) deletion procedure as de- 24 scribed previously (54). DNA sequencing was carried out by 24 the dideoxy chain-termination method of the capA, capB, and capC genes were cloned into a single-copy This study integration vector, pCL83 or pCL84. The resultant plasmids This study were electroporated into strain CYL316 and then transduced This study by phage 52A into various mutants for complementation as This study described previously (24). DNA fragments containing other This study cap genes were cloned into pLI50 or pCL8. The resultant This study plasmids were electroporated into RN4220 and then trans- This study duced by phage 52A into various mutants for complementa- tion. 

J. Lutkenhaus

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 J. J. landolo supplier's specifications. Proteins were labelled with [35S]methionine (New England Nuclear Corp., Boston, Mass.) and analyzed by sodium dodecyl sulfate-polyacrylamide gel elec- trophoresis (SDS-PAGE [11% resolving and 5% stacking]) by This study the method of Laemmli (23). 
2s 
Construction of mutants with chromosomal mutations at 25 capI and capM. The DNA insert in pCL7186 (Fig. 3) was This study recloned into a pBR322-derived vector that lacks NcoI. The 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 Iandolo (21). 
pCL52.1. This plasmid, pCL7377, was electroporated into S. ed previously aureus RN4220 and then moved by phage 52A into S. aureus as previously M. Strain M(pCL7377) was incubated at 42°C for 24 h in the ine (MNNG; presence of 3 ,ug of tetracycline per ml of medium to select mutagenesis strains with plasmid integration into the chromosome via e isolated by homologous recombination. A single colony was then incu- d by negative bated in medium without selection at 30°C for 24 to 48 h, which 24). Plasmid allowed the integrated plasmid to excise from the chromosome ,cept that the by single crossover at the duplication region created during ing site were plasmid integration. Depending on the site of crossover during s constructed excision, the mutation site on the insert of pCL7377 would temperature-either be left on the chromosome, thus generating the desired -ClaI site of mutant, or would remain on the plasmid, thus regenerating the f tetracycline wild-type strain. Because the plasmid cloned into pCL52.1 is teen replaced unstable (unpublished result), some cells would be devoid of nstructed by plasmid. These Tcs cells were isolated by replicating colonies 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 hybrid- ization (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 electropo- rated 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 South- ern blotting (not shown). 
Construction of strains CYL5529 and CYL5419. To con- struct 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 trun- cated capD, was digested with BglII and ligated with a 1.7-kb Sau3A DNA fragment containing the chloramphenicol acetyl- transferase (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- 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 con- taining tetracycline and chloramphenicol. Tcs Cmr colonies were selected, and one was designated CYL5529 and con- firmed 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 con- firmed 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 Capmutants 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 AGATCTATAT CAAAGAC ATTAATAAAATGAAATTAAAGATAC CCAATAAATAGAAC CACAG TATAAAT TATAT CAG TAT GC TTATATAATT TTTGAAATCTT 03 TAAACAAATGAAGTAATAATTGAGAAAAGTGTAGT TAAATTATT TTT CTTGAAATTAT TT GTTACATAG CATTTCGATG TAAAAT TCACTT TTT ATAAGT AA 05 ATTTAAAAAGAGTTQAAATATACAGGGGATTTATAITAATGGAAAACAAGAAAGGAAAATAGGAGGTTTATATGGAAAGTACAATTGATTTATCTGAA -3 5 -10 
SD CapA M E S T I D L S E TTATTAGGAAGAGTAAGAAAAAATATGAAATTATTAATAATATTACCACTTTTAGGTCTGCTAATTAGTGCAATAATTTCTTTTTTCTTTTTAGATGTAAAG L L G R V R K N M K L L I I L P L L G L L I S A I I S F F F L D V K TACCAAGCTTCTACTCAAATATTAGTGAATCAAAAAGGAAATGACTCACAAATTATGGCACAAGAAGTTCAAAGTAATATTCAACTAGTAAACACATATTCA Y Q A S T Q I L V N Q K G N D S Q I M A Q E V Q S N I Q L V N T Y S GAGATAGTTAAAAGCCCACGTATTCTAGATAAGGTTTCTAAAGAATTAGATGATAAATATTCACGTAGTGAGATATCAAGTATGTTAACCGTTACCAACCAA E I V K S P R I L D K V S K E L D D K Y S R S E I S S M L T V T N Q GCAGAGTCTCAAGTATTAAATATTGATGTTGAAAGTAAAAGTGGTAGTAATTCAGAAAAAATTGCTAATAAAATAGCAGAAGTATTTAGTGATGAAGTGCCT A E S Q V L N I D V E S K S G S N S E K I A N K I A E V F S D E V P GATATTATGAATGTTGATAATGTTTCTGTTTTATCTACAGCGGATAATACAGGAAAACAAGTTGCACCAAAACCTATGGTTAATTTAGTTGTTGGTTTAGTA D I M N V D N V S V L S T A D N T G K Q V A P K P M V N L V V G L V ATAGGATTGGTTATCGCATTATTAATAATTTTTATAAAAGAAGTGTTTGATAAACGTATTAAAACAGAAGAAGAAGTTGAAAATGAATTGGTGATACCTGTA I G L V I A L L I I F I K E V F D K R I K T E E E V E N E L V I P V CTGGGTTCAATCCAAAAATTTGATTAAGGAGGGTTTTATCTATGGCAAAAAAGAAAAGCACTATATCACCACTATATGTTCATGATAAACCTAAATCGACG L G S I Q K F D - SD CapB M A K K K S T I S P L Y V H D K P K S T ATAAGTGAAAAATTTAGAGGTATTCGATCAAATATCATGTTTTCTAATGCTGAAAATGAAATAAAAAGCTTGTTGATAACTTCTGAAAAATCCGCATCTGGT I S E K F R G I R S N I M F S N A E N E I K S L L I T S E K S A S G AAGAGCATACTTTCGGCAAACATTGCAGTAACATATGCACAGGCAGGTTACAAAACATTAATTATAGATGGAGATATGAGAAAGCCTACACAACATTATATC K S I L S A N I A V T Y A Q A G Y K T L I I D G D M R K P T Q H Y I TTTGATTTGCCTAATAATAGTGGGCTATCTAATTTAATTATTAATAAAACTACATATAGCGATTCAATTAAAGAAACTAGAGTAGAGAATTTAAACGTTTTA F D L P N N S G L S N L I I N K T T Y S D S I K E T R V E N L N V L ACAGCAGGACCAACACCGCCTAATCCTTCAGAATTAATTGCTTCTAGCAAATTTGCAACTATATTTAATGAACTTTTGAATCATTATGACTTCATTGTGATA T A G P T P P N P S E L I A S S K F A T I F N E L L N H Y D F I V I GACACACCTCCGATTAATACAGTAACGGATGCTCAAGTATATGCGCGAATAGTAAAAAATTGTGTGCTTGTCATAGATGCTGAAAAAAATAATAAGAGTGAA D T P P I N T V T D A Q V Y A R I V K N C V L V I D A E K N N K S E GTTAAGAAAGCGAAAGGTTTACTTACTAAAGCCGGTGGGAAAGTATTAGGCGCAGTTTTAAATAAAATGCCAATTGATAAAAATTCTAGCTACTATTACTAC V K K A K G L L T K A G G K V L G A V L N K M P I D K N S S Y Y Y Y TATGGAGAAGATTAAGAATGGTTGATATACATAATCATATTTTGGTTGATGTAGATGATGGACCAAAAAGCATTAATGAAGCAATTGAGTTGCTTAAACAA Y G E D * SD CapC M V D I H N H I L V D V D D G P K S I N E A I E L L K Q 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 3 3 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 0 7 8 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 plasmid pCL7183 was able to complement 17 mutants. Dele- overlapped by one nucleotide, whereas ORF capJ and ORF tion of 0.6 kb from the left end to the EcoRV site (at the 0.6-kb capK overlapped by eight nucleotides. No potential terminator coordinate) of the 6.2-kb insert fragment from pCL7183 was found downstream of the last ORF, although a GC-rich yielded pCL7193, which complemented six mutants (5176, inverted repeat was found 98 bp downstream of the stop codon 5187, 5206, 5210, 5212, and 5244). These six mutants were also of the last ORF (Fig. 2). 
complemented by pCL5201 with a 2.3-kb insert that contains Genetic complementation. To determine whether each of only intact capE, suggesting that the mutations in these six the ORFs is involved in the synthesis of the capsule and to mutants mapped in capE. Deletion from the right end of the study the organization of the genes, capsule mutants previously 6.2-kb insert fragment of pCL7183 to the PstI site (at the 3.5-kb described (24) and those newly identified in this study (Table coordinate) resulted in pWL7315 with a 3.5-kb insert contain-1) were tested by complementation tests with various subing intact capA, capB, and capC and truncated capD. This clones derived from the 14.6-kb region. As shown in Fig. 3, plasmid lost its ability to complement nine mutants (5023 complemented 5213 and 5346. Further deletion up to the On the other hand, mutations mapped in capA, capB, and HindIII site at the 1.1-kb coordinate resulted in plasmid capD were not complemented by pCL7193, which carries a pCL7407, which did not complement 5346 but complemented fragment with a short deletion from the left side of the 6.2-kb 5213. Because pWL7309 contains intact capA and capB, fragment of pCL7183. These results suggest that capA, capB, whereas pCL7407 contains only intact capA, these results and capD, as well as capC, are transcribed as a transcriptional indicate that the mutation in 5346 mapped in capB and the unit from left to right. mutation in 5213 mapped in capA. The complementation Subcloning of the right portion of the 14.6-kb region re- 
, E E L L N K N E I H P Q Q V Y E K I Y R G K V D H Y I K T E V D L I 0 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 V N Y E M V G Y G 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 M V 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 V S V L I N N I R N A F S F V P GCATACATATTAATTATCATTGTAATAGTGATTATATTGCTGTTTATAGATAGTTATTTAAAAAAAACGAADGCCGATTAAAGTGGTTAAAAATTTTAAT A Y I L I I I V I V I I L L F I D S Y L K K E T K A D * SD 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 Y N Y GCTTTAGTTATAACTGCTCCAATTTTTTTATTTATATCCCTGAAAATAAGGTCTGTAATTGTCACGAATGATAAATATAGTCCAAATGAATATATATCAGCA A L V I T A P I F L F I S L K I R S V I V T N D K Y S P N E Y I S A ATTTTATCATTAAATATTATTACTTTAATATTTGTTGCAATTTTTGTTTATGTATTAGGAAATGGTGATTTAACTACTATATTAATAGTATCGTTAATAAAA I L S L N I I T L I F V A I F V Y V L G N G D L T T I L I V S L I K TTATTTGAAAATATAAAAGAAGTACCTTATGGAATATATCAAAAAAATGAGAGTCTCAAATTACTCGGAATCTCAATGGGTATTTATAATATACTAAGTTTA L F E N I K E V P Y G I Y Q K N E S L K L L G I S M G I Y N I L S 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 '177 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 I P K Y S L L G A A L T I T I S CAATTCGTTGCTTTAATAAGCTATTACTATTTTTACAAAAGGATATTTTGAG GAAAATATGAAAAAATTATTTTTAAAGTTAATGAAAAGGAATCTATCT Q F V A L I S Y Y Y F Y K R I F -SD 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 ATATTTATCGGTAATAATGTTTTCATAGGGATTAATTCAATAATTTTGCCAGGAGTAACAATAGGAAATAATGTTGTAGTAGGTGCTGGGAGTGTGGTAACG I F I G N N V F I G I N S I I L P G V T I G N N V V V G A G S V V T 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 N * SD 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.
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 19 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 3 66 AAATTTGT____TGTTTAGAAATATAAGAACGGTTTGGCCACTCAAACTATACAG 53 K T L SD 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 Y S V S L K K D R L K S I G K E N F T F N K GGATAAATAGCATACAATTTTGTACACGATGATATTGTCCACGTTAAAATT V K L E N Y D D L S K V F V D E Q P E V V V N L A A Q A G V N Y S I GGACAGAGAATATTAATTGTTAGAATTGAGATGCTTATTCAATATTTCTAC E N P R T Y I D S N I V G F M N I L E C S R H F N I Q N L I Y A S S ATCGTAGTCATCTTACATACCTGAATTGTACGTATTTTCGACAAATAAGGT 27 5 5 V Y G A N T S K P F S T S D N I D H P L S L Y A A T K K S N E L AGCCTCTTGCTTTCATACAACGATAATTCCGAAGTCTGGAACGTTGGTTTA M A H T Y S H L Y N L P T T G L R F F T V Y G P N G R P D M A L F K TTCAACACTATACACAAAGCAATAGAAAGTAAATTCTCTGTAATTGACATG 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 I S AGGCTTGTTAAGAAACCAGCGTCCCCTAATAAAGAATGGTCAGGGGCCGACCCTGACCCTGGTTCCTCATATGCACCATATAAAGTCTACAATATTGGGAAC R L V K K P A S P N K E W S G A D P D P G S S Y A P Y K V Y N I G N ACGCATAATAGATTTGGCAAAATATAGAAAGTGAAATCTGTTCACGAAGAC 63 N S P V R L M E F V E A I E N K L G K E A R K N Y M D L Q P G D V P GACTTCATTGTATGTAAAATATCACAAATCATAGTGGAAAATGTATGATAA K T Y A N V D D L F R D I D F K P E T T I Q D G V N K F V D N Y L K 3 86 TATTAAAGLAALL TTATTCATCCTGAAGTTCATGAATTAGTATTATATGTC 31 Y Y K K SD CapJ M Q L L F V H D F P V E K Y K D N Y Y S I G F 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 K S N 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 I S I K AAGGTGGTTATGGACATTTAGTTTGACCATTTAAATATACTAAGTGATGTG K S 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 V E V V C 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 A V K N A S I GGTTTTATAAAATGGACATTCTTAGGAGGTAAGATCACTCACGAAAATATA A I Y V T K K Y L S N K Y P C N G K E F K G I S N V Q S V E K F N K ATTGTTGGAAATAATGATATGTTCTTTGTAAAGCTAGACAAATATATATGT 94 N L 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 I S N L V AATGAACGTTATAACATTCAATTAGAATTTGTGGGTCATGGGCCCAGTAAGAAATTTATGGAAATGGCTAAAAAATACAATGTTGAAAATAATGTTATATTT N E G 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 V I F AAAGGTAAAATTTATGACAAGACAGCGTTAAATAATTGGTTTAGAAATTTAGATTTGTATATTCAACCAAGTTTAACAGAGGGACATTGTCGAGCAATTGTG K G K I Y D K T A L N N N F N N L D L Y I Q P 5 L T S G H C N A I V GACAAGATGGGCAATGGCATCGTGATCGTGGTAAAATTTTTACAAGTTGTA 96 E A I 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 TACAATATAAACATTTTACAACAAGAAGTTGAAAAAATTTAGTCACGAATT 30 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 0 509 CATGAGGAAGATCTATTAAATAAAGTTTTTGGAQzAAAAAAGTATAAAAATA 64 Q I E N E K A L L N Y K K I I N D F Y L A K C I N K N A- SD CapK M L N Y I Y N H 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 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 I I T K K AAAAATAAAAGGATGGTCATGAAGAGTTTAATAGAAGTTTTGAATGTACTA K N K L I K N C T G C S T G K S N V F Y T N A Y D N S R K I A Y L D TTTAAACAAGGTTTAGTTAAGGTGGAGGTGAATGACATACAAAGAGATTGG Y F K E Q H C V Y K G N K R V S V G G R K I V P I K Q K K K V F N R TTAAACCAACATAGTTTCTTAGTAGTAATTAAATCTAAATAAAATCGCGAC 9 Y N K P L N Q L N I S A Y H A D C E N L K Y Y I K K L N K F Q P E T TTGGATGGCTATACTACAGTTATTCATAGGATTGCGAGATATATTTTAGATAACAATATAGAATTAAGTTTTACACCAATTGCTATTTTTCCTAATGCAGAA 13 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 E L 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 15 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 I K I K R I I 11632 GTGACAGTTTAATTGGAAGATGACATTATTTACGATAATGTAATAGTTGAC 49 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 E S 11734 CATGAAATAATAACTATTTTTGATATAGTCGTAATAATTAGATAATTATGA 3 83 Q F V Q N C I C N I I V Y L V I S N C A C K N N I I K K L K Y S L K 11836 TCGTTGAAAATATCATTATTTATAAACTACCTGGAAAAGTTCATAACTAAA F R F C T 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 1 19 3 8 aAAGAAGAAAAATCGATGTTGTAGAGTACGACGACTTGATACTAGACGTT 1 SC CapLNM N R 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 12040 GTTATATAGATAGATAAAATAGTGAAAGATAAAATATAAACCATTGAAATC 31 C I N E S R 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 12142 AATGCAGAAGATTTGAAAAAGGCTGATTTTATTATTATAGCTGTGCCAACACCAATCGATAAGCATAATAAACCTGATTTATTACCATTATTAAAACCAGT 65 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.
suited in plasmid pWL7327 with an 8.4-kb EcoRI-Bglll insert different ORFs, we were able to map each mutation in this (6.2-to 14.6-kb coordinates), which complemented six mu-region to a specific ORF. The results in Fig. 3 showed that the tants, 5024, 5241, 5330, 5327, 5204, and 5211. Sequential mutations of.the six mutants were mapped in five ORFs: 5024 deletion from the right side of pWL7327 resulted in six in capG, 5241 in capH, 5330 in capJ, 5327 in capK, and 5204 plasmids (Fig. 3), pCL7134, pCL7187, pCL7186, pCL7191, and 5211 in capL. We also deleted pWL7327 from the left side pWL7316, and pCL7185, with deletion end points located in of the 8.4-kb insert to the Kpnl site (at the 9.7-kb coordinate). 
ORFs capM (at the 14.0-kb coordinate), capL (HPiindIll site at The resultant plasmid pCL7165 failed to complement any of the 12.6-kb coordinate), capK (HindIll site at the 11.4-kb the six mutants complemented by pWL7327. Thus, it is likely coordinate), cap.! (Clal site at the 9.5-kb coordinate), capI that the five complementation groups defined by the six (Ncol at the 8.9-kb coordinate), and capH (EcoRV at the mutants were transcribed as a transcriptional unit from left to 8.3-kb coordinate), respectively. These plasmids were used to right. However, because the deletion in pCL7165 also deleted complement the six mutants complemented by pWL7327. caDG, capH, and capI, it is possible that these ORFs could be Because the end points of these deletions are located in transcribed independently from the downstream genes. 
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 CAGGAAG TTGGGCACCATC CTGAAGTGATT TTAGCAGGTAGAAGAATAAATGATAATATGGCTAAATATATTGC TTCTAACGTTATTAAAGAGT TATTGAAG 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 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 L K M V D V V L F A V P H K D Y M E N K K D Y I N L 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 * SD CapM M 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 I D E S D Y L F L T Q N P N V V L I K H V S D P I S F Y N N M N V F 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 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 muta- tions 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 Ma- terials 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 site- specific 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 muta- tions 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 single- copy 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 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 de- scribed 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 transcription- translation 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, respec- tively. 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 Gen- Bank and European Molecular Biology Laboratory (SWISS- [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 dehydroge- nase, which catalyzes oxidation of GDP-mannose to GDP- mannuronic 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 determi- nant, 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 

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 acetyltrans- ferase of S. aureus [3]). The alignment of Tn2424Cat, NodL, LacA, and CysE is taken from Parent and Roy (35). 
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 ORF- encoded 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 cap- sule is a virulence factor (27, 31). However, these studies employed mutant strains that were spontaneously arisen, chemically induced, or transposon mutagenized. To unequiv- ocally assess whether type 1 capsule is a virulence factor, we constructed two isogenic strains, CYL5419 with a Cap' phe- notype 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. Cap- mutants with mutations mapped in capI and capM were successfully obtained, implying that these two genes are in- volved 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). Recogniz- ing 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 ap- proach and constructed a null mutant, CYL5529, which pro- duced 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 deletion in CYL5529 did not affect the expression of other capsule genes, transferring the pCL7398 into CYL5529 gener- ated 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 pre- dicted 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 complemen- tation results and the sequence data together, it is likely that these cap genes are transcribed in a long operon with addi- tional 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) blot- ting 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 polysac- charide 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, moder- ate homology between CapI and VipB, and limited homology between CapM and VipC imply that they are probably in- volved in the same biosynthetic pathway leading to the synthesis of N-acetylgalactosaminouronic acid. Because Salmonella spp. and Staphylococcus spp. are two very evolutionarily diver- gent organisms, the similarity, especially between CapL and 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 align- ment 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 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-acetylgalactosamino- uronic acid. 

I

  in the open on agar plates with and without tetracycline.The desired 


FIG. 3 .

 3 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 


FIG. 4 .

 4 FIG. 4. Autoradiogram of E. coli S30 in vitro transcription trans- lation of the gene products. [35S]methionine-labelled proteins were 




  FIG. 5. Comparison of the deduced protein sequences of CapL and VipA. A line indicates identical residues, and two dots indicate similar residues. 




  5203, 5214, 5226, 5242, 5243, 5245, 5246, and 5248) in addition results presented above indicate that we have mapped 17 to the six mutations mapped in capE but was able to comple- mutations into four complementation groups (represented by ment 5213 and 5346. These results indicate that the mutations ORF capA, capB, capD, and capE) on the 6.2-kb BglII-EcoRI in those nine mutants that were not complemented by either insert fragment of pCL7183. The fact that the six mutants pWL7315 or pCL5201 mapped in capD. Deletion up to near complemented by pCL7193 were also complemented by the Hindlll site in ORF capC (at the 2.3-kb coordinate) by Bal pCL5201 (contains only intact capE) indicates that the capE 31 nuclease resulted in pWL7309 with a 2.3-kb insert that still gene is transcribed independently from capA, capB, and capD. 



J. BAcrERIOL. 

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.
Wen S Lin, AND Tim Cunneen, Chia Y Lee
Sequence Analysis and Molecular Characterization of Genes Required for the Biosynthesis of Type 1 Capsular Polysaccharide in Staphylococcus aureus