JOURNAL OF BACrERIOLOGY, Nov. 1994, p. 7005-70160021-9193/94/$04.00+0Copyright © 1994, American Society for Microbiology

Vol. 176, No. 22

Sequence Analysis and Molecular Characterization of GenesRequired for the Biosynthesis of Type 1 Capsular

Polysaccharide in Staphylococcus aureusWEN 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 productionfrom Staphylococcus aureus M. Subcloning experiments showed that these capsule (cap) genes are localized ina 14.6-kb region. Sequencing analysis of the 14.6-kb fragment revealed 13 open reading frames (ORFs). Usingcomplementation tests, we have mapped a collection of Cap- mutations in 10 of the 13 ORFs, indicating thatthese 10 genes are involved in capsule biosynthesis. The requirement for the remaining three ORFs in thesynthesis of the capsule was demonstrated by constructing site-specific mutations corresponding to each of thethree ORFs. Using an Escherichia coli S30 in vitro transcription-translation system, we clearly identified 7 ofthe 13 proteins predicted from the ORFs. Homology search between the predicted proteins and those in thedata 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, ahomopolymer polysaccharide consisting of N-acetylgalactosamino uronic acid, which is also one of thecomponents of the staphylococcal type 1 capsule. The homology between these sets of genes therefore suggeststhat capL, capI, and capM may be involved in the biosynthesis of amino sugar, N-acetylgalactosamino uronicacid. In addition, the search showed that CapG aligned well with the consensus sequence of a family ofacetyltransferases 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 isan important virulence factor in a mouse lethality test.

Most strains of Staphylococcus aureus produce capsularpolysaccharides. To date, 11 distinct serotypes have beenreported (19, 44). Type 1 or 2 capsule-producing strainsproduce a large amount of capsule. As a result, these strainsare mucoid, phage nontypeable, and clumping factor negativeand exhibit a halo around the cells under negative staining withIndia 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 predominantisolates, which account for about 80% in recent isolates fromall sources (2, 4, 14, 17, 19, 37, 44). These strains, which arereferred to as microencapsulated (50), produce a small amountof capsule and do not possess the characteristics typicallyassociated with type 1 or 2 strains. Microencapsulated type 5and 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 aninfected human hand laceration (41). Since the report of itsisolation, strain M has been subjected to intensive study as aprototype of encapsulated staphylococci (see reference 50 fora 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 presenceof taurine in the capsule is dependent on the presence oftaurine in the growth medium (51). The production of capsule

* Corresponding author. Mailing address: Department of Microbi-ology, Molecular Genetics and Immunology, University of KansasMedical Center, Kansas City, KS 66160. Phone: (913) 588-7014. Fax:(913) 588-7295.

in strain M has been correlated with its virulence properties inboth the in vitro phagocytic assay and the in vivo mouselethality assay (31, 36). It has been shown that the mechanismof resistance to opsonization is due to the masking of thebacterial surface-bound C3b complement by capsule, thuspreventing interaction with the receptors on phagocytic cells bythe capsule. Antibodies against the capsule were shown topromote opsonization of encapsulated organisms by depositingcomplement 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 DNAcontaining a cluster of capsule (cap) genes from the chromo-some of strain M (24). In this communication, we report thecomplete nucleotide sequence of the 14.6-kb DNA regioncontaining the cap genes and the characterization of thesegenes.

MATERIALS AND METHODS

Strains, plasmids, and growth conditions. The bacterialstrains and plasmids are listed in Table 1. Trypticase soy brothor agar (Difco Laboratories, Detroit, Mich.) was used forroutine cultivation of S. aureus strains. L broth or agar (Difco)was used for cultivation of Escherichia coli. S. aureus M hasbeen described previously (24). S. aureus RN4220, a restric-tion-deficient strain derived from strain 8325-4 (22), was usedas the recipient in electroporation. S. aureus CYL316 was usedas the recipient for the single-copy integration vector pCL83 orpCL84 (25). E. coli LE392 or HB101 was used for plasmidtransformations and preparation of plasmids. Electroporation

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TABLE 1. Bacterial strains and plasmids used in

Strain orplasmid

S.aureus strainsRN4220CYL316MCYL5022CYL5024CYL5091CYL5168CYL5176CYL5187CYL5203CYL5204CYL5206CYL5210CYL5211CYL5212CYL5213CYL5214CYL5240CYL5241CYL5242CYL5243CYL5244CYL5245CYL5246CYL5248CYL5327CYL5330CYL5346CYL5419CYL5529CYL5531CYL5542

E. coli strainsHB101

LE392

PlasmidspLI50pCL8pCL83pCL84pCL52.1

Relevant characteristics

8325-4r-RN4220(pYL112A19)Cap+M cap-23M cap-24M cap-91, MNNG mutagenesisM OM-CYL5168 cap-176CYL5168 cap-187CYL5168 cap-203CYL5168 cap-204CYL5168 cap-206CYL5168 cap-210CYL5168 cap-211CYL5168 cap-212CYL5168 cap-213CYLS168 cap-214CYL5168 cap-240CYLS168 cap-241CYL5168 cap-242CYL5168 cap-243CYL5168 cap-244CYL5168 cap-245CYL5168 cap-246CYL5168 cap-248M cap-327, spontaneous mutationM cap-330, spontaneous mutationM cap-346, 43°C treatmentM cap-419::catM cap-529::cat A(capA capB capC)M capM531M capI542

F- recA13 hsdS20 ara-14 proA2lacYl galK2 xyl-5 mtl-i supE44rpsL20 A-

F- hasR514 supE44 supF58 lacYlgalK2 galT22 metBI trpR55 X-

Shuttle cloning vectorShuttle cloning vectorSingle-copy integration vectorSingle-copy integration vectorTemperature-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 outdescribed (42). N-Methyl-N'-nitro-N-nitrosoguanidSigma Chemical Co., St. Louis, Mo.) was used foras described previously (32). Cap- mutants wer(screening nonmucoid colonies and were confirme4staining with India ink as previously described (vector pCL8 was essentially the same as pLI50, exampicillin resistance gene and the multiple cloniderived from pWSK30 (48). Plasmid pCL52.1 wasby cloning the 1.4-kb fragment containing the tsensitive origin of replication of pE194ts into thepCL72 (25) in which the HindIII site upstream oiresistance gene (Tcr, originally from pT181) has bwith 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 BamHIlinker.

Source orlikr

reference DNA manipulations. General DNA manipulations wereperformed as described by Sambrook et al. (39). Plasmid DNApurification 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,

24 Inc., Chatsworth, Calif.). Rapid small-scale plasmid DNA24 purification was done by the method of Holmes and QuigleyThis study (15). Bulk chromosomal DNA from S. aureus was purified by24 the method of Dyer and Iandolo (9). The transfer of DNA to24 nitrocellulose membranes was by the method of Southern (45).24 The conditions used for hybridization analysis have been24 described previously (26). Enzymes used in DNA manipulation24 were purchased from GIBCO-BRL (Gaithersburg, Md.) or24 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 M1324 derivatives mpl8 and mp19 (53). Overlapping deletions of the24 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 by24 the dideoxy chain-termination method of Sanger et al. (40)24 with a sequencing kit from U.S. Biochemical Corp. (Cleveland,24 Ohio).

24 Complementation test. DNA fragments containing any of24 the capA, capB, and capC genes were cloned into a single-copyThis study integration vector, pCL83 or pCL84. The resultant plasmidsThis study were electroporated into strain CYL316 and then transducedThis study by phage 52A into various mutants for complementation asThis study described previously (24). DNA fragments containing otherThis study cap genes were cloned into pLI50 or pCL8. The resultantThis 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-translationsystem (Promega Corp., Madison, Wis.) according to the

J. J. landolo supplier's specifications. Proteins were labelled with [35S]me-thionine (New England Nuclear Corp., Boston, Mass.) andanalyzed 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 at25 capI and capM. The DNA insert in pCL7186 (Fig. 3) wasThis 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 about20 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. aureusas previously M. Strain M(pCL7377) was incubated at 42°C for 24 h in theine (MNNG; presence of 3 ,ug of tetracycline per ml of medium to selectmutagenesis strains with plasmid integration into the chromosome viae 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, which24). Plasmid allowed the integrated plasmid to excise from the chromosome,cept that the by single crossover at the duplication region created duringing site were plasmid integration. Depending on the site of crossover durings constructed excision, the mutation site on the insert of pCL7377 wouldtemperature- either be left on the chromosome, thus generating the desired- ClaI site of mutant, or would remain on the plasmid, thus regenerating thef tetracycline wild-type strain. Because the plasmid cloned into pCL52.1 isteen replaced unstable (unpublished result), some cells would be devoid ofnstructed by plasmid. These Tcs cells were isolated by replicating coloniesI in the open on agar plates with and without tetracycline. The desired

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GENES AFFECTING TYPE 1 CAPSULE PRODUCTION IN S. AUREUS 7007

B H

B H

cap cccapB

H

EiD~~apC~

Bm pCL7163 caPD'

s

- CL21

FIG. 1. Construction of pCL7325 and pCL7375. Solid bars, DNAfragments from strain M; hatched bars, DNA fragments containing thecat 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 pertinentrestriction 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 digestedwithAccl and religated to generate pCL7359 with a deletion of114 bp within ORF capM. Plasmid pCL7359 was electropo-rated into strain RN4220 and then transduced into strain M byphage 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 constructedas 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-kbSau3A 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 BglIIand EcoRI to release a 4.0-kb fragment containing the cat

gene. This fragment was ligated to the BamHI and EcoRI sitesof pCL7371 containing a 1.2-kb HindIII-PstI fragment (2.3- to

3.5-kb coordinates in Fig. 3; the HindIII has been replaced bya BamHI linker). The resultant plasmid, pCL7375, thus con-

tained a cat gene replacing the central 4.1-kb DNA region ofthe 8-kb DNA fragment of pCL7163. Plasmid pCL7375 waselectroporated into strain RN4220 and then transduced tostrain M by phage 52A. M(pCL7375) was incubated at 42°C inthe presence of tetracycline (3 jig/ml) and chloramphenicol (5,ug/ml) for 24 h. Cells were plated on agar plates containingchloramphenicol. Colonies were replica plated onto agarplates containing chloramphenicol and onto agar plates con-taining tetracycline and chloramphenicol. Tcs Cmr colonieswere 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 withBamHI and PstI and recloned into pCL52.1 digested with thesame enzymes. The resultant plasmid was electroporated intoRN4220 and then transduced to strain M by phage 52A. StrainCYL5419 was isolated as described for CYL5529 and con-firmed by Southern blotting (result not shown).

Virulence studies. Strains CYL5529 and CYL5419 wereincubated at 37°C for 18 h in the presence of chloramphenicol(5,ug/ml). Cultures were harvested by centrifugation, washedtwice in sterile phosphate-buffered saline (PBS), and resus-pended in PBS to 2 x 107 CFU/ml. Six mice (Charles RiverLaboratories) were injected intraperitoneally with 0.5 ml ofeach strain (i.e., 107 CFU), and lethality was assessed 24 hlater.

Nucleotide sequence accession number. The sequence re-ported here has been submitted to GenBank under accessionnumber U10927.

RESULTS

DNA sequence analysis. Previously, complementation testswith a collection of Cap- mutants derived from S. aureus Mshowed that 20 of the 22 mutants were complemented byplasmid clones containing DNA derived from a 19.4-kb DNAregion of the S. aureus M chromosome. This result suggeststhat the majority of the cap genes affecting capsule productionin S. aureus M are clustered in the 19.4-kb DNA region of thechromosome (24). To define the smallest DNA region that stillcontained this cluster of genes, we performed complementa-tion tests with plasmid subclones derived from a 20-kb DNAfragment containing the original 19.4-kb fragment. We foundthat all of the mutants previously complemented by the 19.4-kbfragment were complemented by plasmid subclones derivedfrom a shorter DNA fragment 14.6 kb in size. Plasmidscontaining inserts with additional deletions of 0.6 and 1.8 kbfrom the left and right ends of the 14.6-kb fragment, respec-tively, were unable to complement some of the mutants. Thisresult indicates that the minimum size of the DNA regioncontaining 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 shownin Fig. 2, 13 ORFs were identified. These ORFs were namedcapA through capM and could potentially encode proteins withmolecular 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 theseORFs were transcribed in one orientation from left to right.The largest spacing between the stop codon and the startcodon of two adjacent ORFs was 27 bp, indicating that theseORFs are closely linked. A potential promoter canonical tothose found in E. coli and Bacillus spp. (12, 33) was found 31bp upstream of the start site of the first ORF, capA (Fig. 2). AllORFs were preceded by potential Shine-Dalgarno sequences(43) 5 to 11 bp upstream of the start codons. GTG was the startcodon for ORF capD and ORF capF, whereas ATG was the

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1 AGATCTATATCAAAGACATTAATAAAATGAAATTAAAGATACCCAATAAATAGAACCACAGTATAAATTATATCAGTATGCTTATATAATTTTTGAAATCTT1 03 TAAACAAATGAAGTAATAATTGAGAAAAGTGTAGTTAAATTATTTTTCTTGAAATTATTTGTTACATAGCATTTCGATGTAAAATTCACTTTTTATAAGTAA2 05 ATTTAAAAAGAGTTQAAATATACAGGGGATTTATAITAATGGAAAACAAGAAAGGAAAATAGGAGGTTTATATGGAAAGTACAATTGATTTATCTGAA1 -3 5 -10 SD CapA M E S T I D L S E306 TTATTAGGAAGAGTAAGAAAAAATATGAAATTATTAATAATATTACCACTTTTAGGTCTGCTAATTAGTGCAATAATTTCTTTTTTCTTTTTAGATGTAAAG10 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 K408 TACCAAGCTTCTACTCAAATATTAGTGAATCAAAAAGGAAATGACTCACAAATTATGGCACAAGAAGTTCAAAGTAATATTCAACTAGTAAACACATATTCA44 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 S510 GAGATAGTTAAAAGCCCACGTATTCTAGATAAGGTTTCTAAAGAATTAGATGATAAATATTCACGTAGTGAGATATCAAGTATGTTAACCGTTACCAACCAA78 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 Q612 GCAGAGTCTCAAGTATTAAATATTGATGTTGAAAGTAAAAGTGGTAGTAATTCAGAAAAAATTGCTAATAAAATAGCAGAAGTATTTAGTGATGAAGTGCCT112 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 P714 GATATTATGAATGTTGATAATGTTTCTGTTTTATCTACAGCGGATAATACAGGAAAACAAGTTGCACCAAAACCTATGGTTAATTTAGTTGTTGGTTTAGTA146 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 V816 ATAGGATTGGTTATCGCATTATTAATAATTTTTATAAAAGAAGTGTTTGATAAACGTATTAAAACAGAAGAAGAAGTTGAAAATGAATTGGTGATACCTGTA180 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 V918 CTGGGTTCAATCCAAAAATTTGATTAAGGAGGGTTTTATCTATGGCAAAAAAGAAAAGCACTATATCACCACTATATGTTCATGATAAACCTAAATCGACG214 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 T1019 ATAAGTGAAAAATTTAGAGGTATTCGATCAAATATCATGTTTTCTAATGCTGAAAATGAAATAAAAAGCTTGTTGATAACTTCTGAAAAATCCGCATCTGGT21 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 G1121 AAGAGCATACTTTCGGCAAACATTGCAGTAACATATGCACAGGCAGGTTACAAAACATTAATTATAGATGGAGATATGAGAAAGCCTACACAACATTATATC55 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 I1223 TTTGATTTGCCTAATAATAGTGGGCTATCTAATTTAATTATTAATAAAACTACATATAGCGATTCAATTAAAGAAACTAGAGTAGAGAATTTAAACGTTTTA89 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 L1325 ACAGCAGGACCAACACCGCCTAATCCTTCAGAATTAATTGCTTCTAGCAAATTTGCAACTATATTTAATGAACTTTTGAATCATTATGACTTCATTGTGATA123 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 I1427 GACACACCTCCGATTAATACAGTAACGGATGCTCAAGTATATGCGCGAATAGTAAAAAATTGTGTGCTTGTCATAGATGCTGAAAAAAATAATAAGAGTGAA157 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 E1529 GTTAAGAAAGCGAAAGGTTTACTTACTAAAGCCGGTGGGAAAGTATTAGGCGCAGTTTTAAATAAAATGCCAATTGATAAAAATTCTAGCTACTATTACTAC191 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 Y1631 TATGGAGAAGATTAAGAATGGTTGATATACATAATCATATTTTGGTTGATGTAGATGATGGACCAAAAAGCATTAATGAAGCAATTGAGTTGCTTAAACAA225 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 Q1732 GCCCAAAGTGAAAATGTAACAGATATAGTTGCAACACCTCACCATCTTCATAAGAGATATAGTAATGATATTGAAAAAGTGAAAATAAAATTAAATGAATTA29 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 L1834 AAAAATAACTCAGAAATAAAAAAATTAGGACTTAATTTATATGTTGGACAAGAGATTCGTATAACGGATCAAATTATAGAAGGTATAAAAAATAAAGAGATT63 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 I1936 GAAGGAATTAATGAATCTAGGTATTTGTTAATAGAATTTCCTAGCAATGAGATTCCTTATTATACGAATCAATTATTCTATGAGTTACAAACAATGGGATAT97 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 Y2038 ATTCCAATCATTGCGCATCCAGAACGGAATAAAGCGATAGTTCAAAATTTAGATTTACTATTTGAATTAATAAATGGTGGTGCATTAAGTCAGATTACAGCT131 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 A2140 TCTTCATTATTGGGAGATTTTGGGAATAACATAAGAAAATTATCATTAAAAATGATAGATAGTAATTTAGCACATTTTATTGCATCAGATGCACATAGTATA165 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 I2242 ACAAATCGACCTTTTATGCTAAAACAGTTGTTTAATGATAGAAAGTTAAAAGCTTATTATGAGGAATTAGAAAGTTATTTAAAAAACGGGAAGTTAGTTTTA199 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 L2344 ACAAATGAAAGAATTTCCAAACAGATACCAACTCAAGATTATAAGCAGAAAAAATGGTTTGGTCTTTTATAGAAAAAGTGAGTGAGGGGATTAAAAGTGACA2 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 T2446 AGTATTTCTGCGAAATTGCGATTTTTAATATTAATTATTATAGATTCGTTTATTGTTACATTTTCAGTATTTTTAGGATATGCAATATTAGAACCGTATTTT3 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 F2548 AAAGGATATTCAATAGATTTATTAGTATTATCATCTGTTATATTGTTGGTATCACATCATATATTCGCATATGTATTTAATTTATATCATCGAGCGTGGGAG37 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 E2650 TATGCGAGTGTAAGTGAATTGATGTCAGTTTTAAAAGCGGTTACAAGTTCAATAGTGGTAACACTTTTATTAGTTTCATTACTTATAAGTGAAAGTCCATTT71 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 F2752 CTAAGGTTATATTTTATAACGTGGATGATGCATTTGCTATTGATTGGTGGCTCTAGATTGTTTTGGCGGGTTTATAGAAGGTATTTTATTGATAACGCTGTC105 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 V2854 GAAAAAAAAGCCACATTAGTAGTAGGGGCTGGACAAGGCGGATCTGTTTTAATTCGTGAAATGTTAAGAAGCCAAGATATGCGTATGCAACCAGTTTTAGCT139 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 A2956 GTTGATGACGATAAAAACAAACAAAAAATGACTATTACTGAACGTGTTAAAGTTCAAGGTTATGTTGAAGATATACCGGAACTAGTAAAAAAATTTAGAATA173 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 I3068 AAAAAGATTATTATTGCTATACCGACATTAAGCCAAAAAAGGTTAAATGAAATTAATAAAATATGTAATATTGAGGGCGTTGAATTATTTAAAATGCCTAAT207 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 N3170 ATAGAAGATGTGTTATCTGGAGAATTAGAAGTCAATAATTTGAAAAAAGTAGAAGTAGAAGATTTACTTGGAAGAGATCCAGTTGAACTAGACATGGCATTA241 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 L3272 ATTTCAAGAGAATTAACGAATAAAACAATATTGGTAACTGGAGCTGGAGGATCAATAGGTTCAGAAATTTGCAGACAAGTAAGTAAATTTGATCCTCAAAAA275 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 K3374 ATTATTTTGTTAGGACATGGAGAGAACAGTATTTATTCAATTCATCAAGAATTAAGTAAAACTTATGGAAATAGAATTGAATTTGTTCCTGTTATAGCTGAT319 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 D3476 GTACAAAATAAAACACGTATTTTAGAAGTCATGAATGAATTTAAGCCATATGCTGTTTATCATGCTGCAGCACATAAGCATGTACCATTAATGGAATACAAC343 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 N3578 CCTCATGAAGCTATTAGAAATAATATTTTAGGTACTAAAAATGTAGCTGAGTCTGCAAAAGAAGGAGAAGTAAGTAAATTTGTAATGATTTCAACAGATAAA377 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 K3680 GCTGTAAATCCATCTAATGTAATGGGAGCAACAAAACGCATTGCTGAAATGGTAATACAAAGTTTAAATGAAGATAATTCTAAGACAAGTTTTGTAGCAGTA411 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 V3782 AGATTTGGAAATGTGCTTGGATCAAGAGGATCGGTCATACCTCTATTTAAAAATCAAATTGAATCAGGTGGTCCGGTTACAGTAACTCACCCTGAAATGACA445 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 T3884 CGATATTTCATGACTATACCAGAAGCATCAAGACTTGTATTACAGGCCGGGGCATTGGCACAAGGTGGCGAAGTATTTGTGCTAGACATGGGTAAACCAGTT478 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 V3976 AAAATAGTTGATTTAGCTAAGAATTTAATACGGTTGAGCGGTAAAAAAGAAGAAGATATTGGTATTGAATTTTCAGGGATTAGACCAGGAGAAAAATTATAT513 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 Y4 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. Theinverted 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-kbcapK overlapped by eight nucleotides. No potential terminator coordinate) of the 6.2-kb insert fragment from pCL7183was 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 alsoof 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 sixthe ORFs is involved in the synthesis of the capsule and to mutants mapped in capE. Deletion from the right end of thestudy the organization of the genes, capsule mutants previously 6.2-kb insert fragment of pCL7183 to the PstI site (at the 3.5-kbdescribed (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 sub- ing intact capA, capB, and capC and truncated capD. Thisclones derived from the 14.6-kb region. As shown in Fig. 3, plasmid lost its ability to complement nine mutants (5023,

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VOL. 176, 1994 GENES AFFECTING TYPE 1 CAPSULE PRODUCTION IN S. AUREUS 7009

547 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 I418 0 GTGGAAGATTTAATAAATAATTTCTCAAAAGAGAAGCTCTTAAAGATAGCAAATAGATAAATATAAAATGTATATTGGAGTTTTAAAATGAAACTAAAGTAC581 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 Y4282 AAAGTAATTTTAATTATAAATTTTGTTACAGTATTATTTAGTATATTTACATTCATCGGTTATTTAAATAACTTGATAGGTTTTAGAGTTGTTACAATATCA6 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 S4384 TTATGTATAACTATAGCTATGACCGTGTACTTACTTTACAAAAAAGAAAAGGTTTTTTATTAGTTTATTTAATATATTTATTTTTAACTAATTTTGGTGTA40 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 V4486 TTTGTAACAAATATATTTTTAGCCAATCCTTTAGTGGAGTATCACGGTGATTTATCTTGGTATTATATAAATACATCTAATTTGTTTAGCATTGCTACTTTT74 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 F4588 GCGATATTAACTTTTACAATTTTAAGTAATTTTATTAGTGTGTTTAGCAAAATAAATCCAAGTAGAAAGTTTGATATTAAAAGTAAAGGAAATAATTTATTC108 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 F4690 TACTACACAGGAATATTATTTATCATTGGATTTACTATACAATTTCTTTTTTATATTATAACTGGTCGATTAGCAATTAATACTTATGGAGATTATGTAAGT142 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 S4792 AGCATACAAGAATTACCAATGTATACGTACGGTATATTCTTTTTTTCAATTGGGATTGCATTTGCATTTTCTAATGTAAAAAAAACACATATTAAATATCTA176 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 L4894 GTAATTATATTAACTCCCCAAGTGTTATTTTTTTTAATAACCGGAAATAGGGGAGAAGTATTTTATCCAATTCTATCTGCACTTGGAGTGTTAATAGTAAGG210 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 R4996 AATTATAAAATTAAATGGTGGATGATAATTACAATTGTTTTTACATTATTTTTTGTAATACCATTTATTAAAGTCTTTAGAAATATGGATAGTAGCTCAATT244 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 I5098 GAAAAGGTTGATATAAATTGGTTTTCTTCACTTGTTGAAATAGGCTACACACTGCGCCCTTTAGGTTATGTAACTAGGTGGATTGACGGTGGGGAAAGCATA278 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 I5200 GTTTATGGCAAAAGTTACTTAGCACCTATTCAAAATATATTTTCATATATTATACCAGGGTTACAACCTGTAAACTATGAAATGGTTGGTTACGGTTTTAGG312 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 R5302 TATAGACTACCTGGTATGGGTTTTAATGTAATAGCAGAAGCGTATTACAATGGTGCAATTGTTGGAGTATTGATTGTAATGGTGTTATTAGTGCTTTTACTT346 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 L5404 TGGAAATTTACAAATTTTAAATCTTTTGAAATGCTGTCAATGGGTACTGCAATTGTAAGTGTATTAATCAATAATATAAGAAACGCATTTTCATTCGTACCT380 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 P5506 GCATACATATTAATTATCATTGTAATAGTGATTATATTGCTGTTTATAGATAGTTATTTAAAAAAAACGAADGCCGATTAAAGTGGTTAAAAATTTTAAT414 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 N5609 TATATGTTTGTTGCTAATATACTGTCTGCATTGTGCAAATTTTTAATTTTACTAGTAATAGTTAGATTAGGAACACCTGAGGATGTAGGACGTTATAATTAT7 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 Y5711 GCTTTAGTTATAACTGCTCCAATTTTTTTATTTATATCCCTGAAAATAAGGTCTGTAATTGTCACGAATGATAAATATAGTCCAAATGAATATATATCAGCA41 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 A5813 ATTTTATCATTAAATATTATTACTTTAATATTTGTTGCAATTTTTGTTTATGTATTAGGAAATGGTGATTTAACTACTATATTAATAGTATCGTTAATAAAA75 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 K5913 TTATTTGAAAATATAAAAGAAGTACCTTATGGAATATATCAAAAAAATGAGAGTCTCAAATTACTCGGAATCTCAATGGGTATTTATAATATACTAAGTTTA109 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 L6017 ATTTTGTTTTATATTATATATTCTTTTTCACACAACTTGAATATGGCACTTTTATTTTTAGTAATATCCTGTATATTCTCATTTGCTATTATCGATAGATGG143 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 W6119 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 A6221 TTAGGATCATTAAATACAGGAATTCCTAGAATAGTATTAGAAAATCTATTTGGAAAATATACATTAGGTATATTTTCTACAATTGCGTATGTACTGGTAATC211 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 I6323 GGTGGGTTATTCGCAAATTCAATCAGTCAAGTTTTTTTACCCAAATTAAGAAAATTATATAAAGATGAAAATTGAATTTGAAAAGTTAACTAGAAAA245 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 K6425 ATGGTGTTTATTGGAATTTTTATTGGTATGTGCTCGGTAATATTGAGTTTGTTTTTAGGTGAGGCCTTGTTATCATTGTTATTTGGTAAAGAATATGGTGAA279 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 E6527 AATAATATAATATTAATCATTCTTTCTTTTGGTTTGCTTTTTATACTCAGTGGTATTTTTTTGGGGACAACTATAATAGCCACTGGAAAGTATAATGTGAAT313 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 N6629 TACAAAATTTCTCTAATACTATTGTTTTGTATTTTGATATTTAGTTTCTTATTAATACCAAAATATTCTTTATTAGGTGCTGCTTTAACTATTACTATTTCA347 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 S6731 CAATTCGTTGCTTTAATAAGCTATTACTATTTTTACAAAAGGATATTTTGAG GAAAATATGAAAAAATTATTTTTAAAGTTAATGAAAAGGAATCTATCT381 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 S6834 GAAGATAAGATAAGGAAATTGGGTGTACAAGTTGGAAATGATTGTAGGTTTTTAAGTGTTGATAGATCAACATTTGGATCTGAGCCTTACCTGATTCAAATA15 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 I6936 GGAAATCATGTAACAATAACTAGTGGTGTAAAATTTGCTACCCATGATGGTGGGGTATGGATTTTTAGAAAAAAATATCCTGAGATAGATAATTTTCATAGA49 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 R7038 ATATTTATCGGTAATAATGTTTTCATAGGGATTAATTCAATAATTTTGCCAGGAGTAACAATAGGAAATAATGTTGTAGTAGGTGCTGGGAGTGTGGTAACG83 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 T7140 AAAGATGTACCTGATAATGTAATTATTGGTGGTAATCCTGCCAAAAAAATTAAAAGTATAGAGGCCTATGAAACTAAAATATTAGAAAATGCGGATTACACA117 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 T7242 AAAAAACTTAATTATAATGAAAAGAAGATATATTTATTAAATAAATTCAAAGAGAATAGGTATAATTAATGATTAAAGTTATGCATATATTTAGCAGAATGAAT151 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 N7346 CGTGGCGGTGCAGAATTAAGAACTATGGATACAATGAAACTATTAAATAGGGAGTTTGAGTTTCATGTATGTGCTACATCAGGAAAAAGGGGCGAATTAGAT13 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 D7448 GATGAATTAGAATCTATGGGTATTACGATACATTATTTAGATATTAAAAAATTTAGTTTTCCTTTCAAGTTCATAAAGTTATTGAAAAAGAAAAATATAGAT47 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 D7550 GTTGTACATAGTCACATACTTTTTATGAGTGGATTGATTCAATTACTTTCTTTTTCAGCAAATGTAAGAAATAGAATAACACACTTTAGAACTTCCAAAGAT81 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 D7652 AGTAAAGAACAATATAATAAAATAAGAAAAGCTAGAAATAAAGTACTGAAAGCCATTATAGAAATCTTTAGTACGAAAATCTTATATGTAAGTAATATAGCA115 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 A7754 AATAGGAATTTAATCTCAATGAAACTTTTTCCGAAAAAACATAAAACTATTTACAATGGATTTGAAATAAGTAATATAAACAAAAATTTTAAAAAAGAGGAA149 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 E7856 AATAGTTTTATTTATGTCGGTAGGTTCATTCATACTAAGAACCAATTATTTTTGTTAGATGTAATAGAAATTTTAAAAAAAGAATTTAATACTAACATTGAA183 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 E7958 ATTACTTTTGTTGGCAATATTCAAACTGATTATGGAAAAAAATTCTTGAGTATAGCAAATGAAAGAGGTTTAAATAAAAATATTAAGGTTATTGGAGAAGTG217 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 V8060 AATAACCCATTAGATTATTTAAAAACGAGTGAATATTTTTTGTTTCCTAGTGAATTAGAGGGATTACCGGGAGCATTGATAGAAGCGCATCATCACAATTGT251 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 results presented above indicate that we have mapped 17to the six mutations mapped in capE but was able to comple- mutations into four complementation groups (represented byment 5213 and 5346. These results indicate that the mutations ORF capA, capB, capD, and capE) on the 6.2-kb BglII-EcoRIin those nine mutants that were not complemented by either insert fragment of pCL7183. The fact that the six mutantspWL7315 or pCL5201 mapped in capD. Deletion up to near complemented by pCL7193 were also complemented bythe Hindlll site in ORF capC (at the 2.3-kb coordinate) by Bal pCL5201 (contains only intact capE) indicates that the capE31 nuclease resulted in pWL7309 with a 2.3-kb insert that still gene is transcribed independently from capA, capB, and capD.complemented 5213 and 5346. Further deletion up to the On the other hand, mutations mapped in capA, capB, andHindIII site at the 1.1-kb coordinate resulted in plasmid capD were not complemented by pCL7193, which carries apCL7407, which did not complement 5346 but complemented fragment with a short deletion from the left side of the 6.2-kb5213. 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 transcriptionalindicate 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-

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8162 ATTAACTGAAAAGAATTAGAACAATTAGCCTATGATGATATCAACTGCTAC285 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 T8264 ACAAGTATCAGAAGAAAGTTAGTCATTTTAACAAGCAAAGATAAAATAAGG319 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 S8366 AAATTTGT____TGTTTAGAAATATAAGAACGGTTTGGCCACTCAAACTATACAG353 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 G8468 CTAGTTGTTGTGAAAGTATTCGTCTGAGGAAATAACAAGAAAATTATTATA25 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 K8570 GGATAAATAGCATACAATTTTGTACACGATGATATTGTCCACGTTAAAATT59 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 I8672 GGACAGAGAATATTAATTGTTAGAATTGAGATGCTTATTCAATATTTCTAC93 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 S8774 ATCGTAGTCATCTTACATACCTGAATTGTACGTATTTTCGACAAATAAGGT127 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 L8876 AGCCTCTTGCTTTCATACAACGATAATTCCGAAGTCTGGAACGTTGGTTTA161 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 K8978 TTCAACACTATACACAAAGCAATAGAAAGTAAATTCTCTGTAATTGACATG195 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 S9080 AGGCTTGTTAAGAAACCAGCGTCCCCTAATAAAGAATGGTCAGGGGCCGACCCTGACCCTGGTTCCTCATATGCACCATATAAAGTCTACAATATTGGGAAC229 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 N9182 ACGCATAATAGATTTGGCAAAATATAGAAAGTGAAATCTGTTCACGAAGAC263 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 P9284 GACTTCATTGTATGTAAAATATCACAAATCATAGTGGAAAATGTATGATAA297 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 K9386 TATTAAAGLAALL TTATTCATCCTGAAGTTCATGAATTAGTATTATATGTC331 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 F9489 TTAAATTGACAATACATTGTAAGTAAATAGGGAATTGTAGGAATTATATGA24 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 N9591 GAAAATATTAACAAAACAAACCTATTTATTAAAAAAAATGACCTCATCACA58 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 K9693 AAGGTGGTTATGGACATTTAGTTTGACCATTTAAATATACTAAGTGATGTG92 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 C9795 GACTTAGAATGTCTGTATTTGAATTACTACAGATTTCAAATCGGAATCACT126 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 I9897 GGTTTTATAAAATGGACATTCTTAGGAGGTAAGATCACTCACGAAAATATA160 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 K9999 ATTGTTGGAAATAATGATATGTTCTTTGTAAAGCTAGACAAATATATATGT194 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 V10101 AATGAACGTTATAACATTCAATTAGAATTTGTGGGTCATGGGCCCAGTAAGAAATTTATGGAAATGGCTAAAAAATACAATGTTGAAAATAATGTTATATTT228 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 F10203 AAAGGTAAAATTTATGACAAGACAGCGTTAAATAATTGGTTTAGAAATTTAGATTTGTATATTCAACCAAGTTTAACAGAGGGACATTGTCGAGCAATTGTG262 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 V10305 GACAAGATGGGCAATGGCATCGTGATCGTGGTAAAATTTTTACAAGTTGTA296 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 K10407 TACAATATAAACATTTTACAACAAGAAGTTGAAAAAATTTAGTCACGAATT330 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 I10509 CATGAGGAAGATCTATTAAATAAAGTTTTTGGAQzAAAAAAGTATAAAAATA364 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-1 SD CapK M L N Y I Y N H10612 TACAATTCAATTAGTTATAGGAATTCTAAAAAAATACTATTAGATAGGCTG9 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 K10714 GAGTGTAATGATTTATTTGAACTTCAAAATCAAAGATTTGAAGAATTTTATAATTATATCAAAAAAAATAGTGAATTTTATTCTGAAATAATTAAAAAT43 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 N10816 ATTACGAAAACCGTCATTACATGCGATAGAGTAATGAAAGTAAATATCAAA77 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 K10918 AAAAATAAAAGGATGGTCATGAAGAGTTTAATAGAAGTTTTGAATGTACTA111 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 D11020 TTTAAACAAGGTTTAGTTAAGGTGGAGGTGAATGACATACAAAGAGATTGG145 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 R11122 TTAAACCAACATAGTTTCTTAGTAGTAATTAAATCTAAATAAAATCGCGAC179 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 T11224 TTGGATGGCTATACTACAGTTATTCATAGGATTGCGAGATATATTTTAGATAACAATATAGAATTAAGTTTTACACCAATTGCTATTTTTCCTAATGCAGAA213 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 E11326 ACTTTAACTGATTTAATGAGGGATGATATTGAAAAGCCTTTTAATTGTCCAGTGCGTAATCAATATGCTTCTTCTGAAGGAGCACCTTTTATTACAGAAAA247 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 N11428 AAACAAGCCCAACTACAAATTAATCTACCTACTCCACTCTTTCACTCTAAACAAATTCATCCTAATATCTATCAATTAATACTCACACCTTTTTATACTACT281 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 T11530 ATCCATTGGTTAACGGTCGAATAAATACTCGAATTACAAGTTAATAAGATT315 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 I11632 GTGACAGTTTAATTGGAAGATGACATTATTTACGATAATGTAATAGTTGAC349 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 S11734 CATGAAATAATAACTATTTTTGATATAGTCGTAATAATTAGATAATTATGA383 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 K11836 TCGTTGAAAATATCATTATTTATAAACTACCTGGAAAAGTTCATAACTAAA417 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 K11938 aAAGAAGAAAAATCGATGTTGTAGAGTACGACGACTTGATACTAGACGTT1 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 F12040 GTTATATAGATAGATAAAATAGTGAAAGATAAAATATAAACCATTGAAATC31 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 S12142 AATGCAGAAGATTTGAAAAAGGCTGATTTTATTATTATAGCTGTGCCAACACCAATCGATAAGCATAATAAACCTGATTTATTACCATTATTAAAACCAGT65 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 thetants, 5024, 5241, 5330, 5327, 5204, and 5211. Sequential mutations of.the six mutants were mapped in five ORFs: 5024deletion from the right side of pWL7327 resulted in six in capG, 5241 in capH, 5330 in capJ, 5327 in capK, and 5204plasmids (Fig. 3), pCL7134, pCL7187, pCL7186, pCL7191, and 5211 in capL. We also deleted pWL7327 from the left sidepWL7316, 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 ofthe 12.6-kb coordinate), capK (HindIll site at the 11.4-kb the six mutants complemented by pWL7327. Thus, it is likelycoordinate), 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 to8.3-kb coordinate), respectively. These plasmids were used to right. However, because the deletion in pCL7165 also deletedcomplement the six mutants complemented by pWL7327. caDG, capH, and capI, it is possible that these ORFs could beBecause the end points of these deletions are located in transcribed independently from the downstream genes.

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GAAACTGTTGGGAAGGTAATTACTCCAGACACAATCGTTGTATATGAATCTACAGTTTATCCTGGCGCAACAGAAGAAGAATGTGTACCTGTATTGGAAAAAE 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 KTATTCTGGACTCGTTTGTGGTAAAGATTTTTTTGTTGGTTACTCACCTGAAAGAATTAATCCTGGGGATAAGGTACATACTTTTGAAACCATTACTAAGGTTY 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 VGTATCTGGCCAAACGCTTGAAGTATTAGAAATAGTTGCAGACGTATATAGTTCAGTAGTCACAGCAGGAGTTCATAAAGCATCTTCTATTAAAGTAGCAGAAV 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 EGCAGCAAAAGTCATTGAAAACACACAACGTGATGTAAATATTGCCCTAATGAATGAATTGGCAATTATTTTTGATAAATTAGATATAGATAC TAACGAGGTAA 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 VTTAAAAGCTTCAGGAACAAAATGGAACTTCTTGAATTTTAAACCAGGATTAGTAGGGGGACATTGCATTGGTGTGGACCCATATTATTTAACACATAAAGCTL 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 ACAGGAAGTTGGGCACCATCCTGAAGTGATTTTAGCAGGTAGAAGAATAAATGATAATATGGCTAAATATATTGCTTCTAACGTTATTAAAGAGTTATTGAAGQ 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 KCAAGGATTAGAAGTACAAGGAGCAACAGTTAATGTGCTAGGTCTTACATTTAAAGAGAATTGTCCGGATTTAAGAAATACAAAGGTTATTCATATTATTGAAQ 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 EGAACTGAAAGAGTATGGATTAAACGTAACAGTGAATGATGTTGAAGCGGATAAAAATGAAGCTAAAAAGTTCTTTGGTTTAGATTTGATAGATACAAAAGAAE 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 ETTAAAAATGGTGGATGTAGTGTTATTTGCAGTGCCACATAAAGACTATATGGAAAATAAAAAGGATTATATCAATTTAGTTAAAGATTGTGGCATAGTGTTTL 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 FGACATTAAAGGCATAATCAATAGTGATGAACTTAATGTAAGTCAACGATTATGGAGATTATAAGTGTAAAATTACATTGGAGGTTTTGTATGAAAAATCAAD I K G I I N S D E L N V S Q R L W R L * SD CapM M K N QAAAATATTTCATTTAGTTACTGTTTCTAAGAGTATTCCACTTATGAGAGGACAAATAGAATTTTTAAGAAAAAAATATGGATGTTCACATTGTTTCGAGTK 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 SGATGGTAAGGAATTAAAGCAGTATGATAATGAAATAGC TCATGTTATACCTATGAAAAGAGATATAGCATTATTCAGTGATTTAAAGTCATTATTAAAAATGD 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 MATATTACTATTTCACAAAGAAAAACCATTTATTGTTAATTCTGGTACTCCAAAAGCAGGATTAATAGGAACAATAGCTGCGTTTATTACCCAAAGAC CTATTI 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 IAGAATATATACTGTGAGAGGTTTAAGGCTTGAAACAGTTAAAGGATTCAAATATTTTGTATTGTATTTGATGGAAAAGATAGCAATGTTTTGTGCAACTGATR 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 DATAATAGCAATTTCTGAAAGTTTAAAGCATAAAATTATTACATCTAATTTGGCTAAGGAAAATAAAATTACTGTTTTGGGATTTGGTAGTTCTAATGGTATAI 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 ICAATTTGAAAAATTCCAATTAGATAACAATAAATTAGAAGAAAAATACCATAAATTATTAAATGATAATTTTGTTATTGGCTATGTAGGAAGAATTGTAAAAQ 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 KGATAAAGGTATACATGAATTAATTCAGTCATTTAAAATTATTGTAAGTAAAGGATATAATGTCAAATTGCTTGTTATTGGTAGTTTAGAGACAGAAAATTCTD 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 SATTGATGAATCTGACTATTTATTTTTAACTCAAAATCCTAATGTAGTACTAATCAAGCATGTTTCAGATCCAATTTCATTTTATAATAATATGAATGTATTTI 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 FGTTTTTCCAACTCATAGAGAAGGGTTTGGAAATGTTAGTATAGAGGCTCAAGCACTTGAAGTGC CAGTAATTACTACTAATGTTACAGGCGCTATTGATACTV 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 TGTAGTAAATGGAGAAACTGGATTTATTGTTGAAAAAGGTGACTTTAAAGCAATCGCTGAAAAAATTGAAAAATTAATTAATGACGAGAGTTTAAGAGAAACTV 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 TATTGGTCATAATGGAAGAAAGAGAGTGGAAAATAAATTTTCAAGC CAAATTATATGGGAAGAATTGGAAAGTATGTACAATACTTTTCTAAAAGAAAGTGAGI 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 EGGAAAGTAATGAAAAGAATATTTGATATATTTAGTTCATTATATGCAATTATTATATTTTTGCGCTGCTCTTTCTAGTGTCTATTGCAATTAAAATAGAGTCG K *

TAAAGGTCCGATlYGTATTTAAACAGGACCGACCAGGCGTAAAGAACAAATTATTTAAAATTTATAAATTTAGATCT 14563

FIG. 2-Continued.

Because the EcoRI site (6.2-kb coordinate) that divides theinsert fragments cloned in pCL7183 and pWL7327 is located inORF capF, it is possible that the mutations of the twopreviously isolated mutants, 5025 and 5027 (24), which were

not complemented by either pCL7183 or pWL7327, mapped incapF. To test this possibility, the 6.2-kb PstI-KpnI (3.5- to9.7-kb coordinates) fragment in the middle portion of the14.6-kb fragment was cloned into a plasmid vector. Theresultant plasmid, pCL7164, which contains five intact ORFs,capE to capI, was able to complement mutations mapped incapE, capG, and capH as expected but was not able tocomplement 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 additionalmutants generated by MNNG mutagenesis. We found one

mutant, 5091, complemented by pCL7164 but not by pCL7183or pWL7327 (Fig. 3), indicating that the mutation in 5091mapped in capF. Most of the other MNNG-induced mutationsmapped in capE, and no additional mutation was mapped incapC, capI, or capM.

Mutagenesis. Because no mutation was mapped in theORFs capC, capI, and capM, we were unable to conclude thatthese ORFs are involved in capsule biosynthesis. Therefore, weattempted to construct mutants with chromosomal mutationsmapped in these three ORFs to determine whether they are

involved in biosynthesis of type 1 capsule. Strains CYL5542and CYL5531, which have mutations corresponding to capIand capM, respectively were constructed as described in Ma-terials and Methods. These site-specific mutants were allCap-, indicating that both ORF capI and capM are involved inthe synthesis of capsule. To confirm that the mutations instrains CYL5542 and CYL5531 are indeed located in ORFs

capI and capM, respectively, and to show that the mutation incapI is not polar, we performed complementation tests with theplasmids described in Fig. 3. The results showed that strainCYL5542 was complemented by pCL7191, which containsintact capG through capI but not by pWL7316 in which most ofthe ORF capI was deleted, whereas strain CYL5531 was

complemented by pWL7327 containing capH through capMbut 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 themutation 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 incapC. However, we were unable to obtain the desired mutationin capC despite several independent attempts. To determine ifORF capC is involved in capsule biosynthesis, we sought a

different approach. We constructed a deletion mutation ofstrain M, CYL5529, in which the DNA fragment between theHindIII site (2.3-kb coordinate within capC) and 1.8 kbupstream of the BglII site (at the 0-kb coordinate) of thegenome was deleted and replaced with a 1.7-kb DNA fragmentcontaining the cat gene of pC194 by allelic replacement (seeMaterials and Methods). Thus, strain CYL5529 is a nullmutant with deletion of ORFs capA and capB and most ofcapC. We then constructed plasmid pCL7398 containing intactcapA, capB, and capD and a BamHI linker insertion at the FspIsite (2.0-kb coordinate) within ORF capC by using the single-copy integration vector pCL84, as described in Materials andMethods. The plasmid pCL7398 was then transferred toCYL5529. The resultant strain, CYL5529(pCL7398), which isgenotypically equivalent to the wild-type strain M with a capCmutation, was Cap-, implying that ORF capC is involved incapsule biosynthesis. However, it is possible that CYL5529could have a mutation at other cap genes, which would give riseto the Cap- phenotype regardless of whether capC is essential

VOL. 176, 1994

12244991234613312448167125502011265223512754269128563031295833713060371131624051 3 26451 3 3 663913468731357010713 6721411377417513876209139782431408027714182311142843451438637914488

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7012 LIN ET AL.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 kbI I I I I I I I, , | I

BHEvHcH FH P Hc A C E Hc Ev N A C KHcBEVHHcC H Hc A B

I\L / \1 I \11 'll I/ / I/ I 1 I11 I-- ~ ~+>--I +, +,>->> >30 .- A.

A B C24.5 25.2 29.4

D67.5

E F G50.7 45.0 19.4

H41.4 37.9

J44.5

K52.4

L M47.2 43.1 kd

+ +

+ +- pWL,

+ +

- pCL7407+

pCL7183+ +_

pCL7193_ pWL7315

7309

- pCL5201

_ + + + +

-

_- - + +

_ + +

- + +

_ _ _ + + pA

- pCL7185_ _ - + -

pCL7164

pWL7327+ + +

+ + _

+ _ _

- pCL7186+ -

pCL7191

VL7316

5091 5024 5241 5330 5327 52045211

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 fragmentscloned into plasmid vectors. The inserts in plasmids pCL7183, pCL7193, pWL7315, pWL7309, and pCL7407 were cloned into single-copyintegration 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 topCL7398, was used to complement CYL5529. The resultantstrain, CYL5529(pCL7183), was Cap', suggesting that CYL5529 does not contain an additional mutation outside ORFscapA 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 capAaffects the expression of the downstream capD gene. To testthis possibility, the plasmid pCL7398 was transferred intostrain 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 thatcould also result in the Cap- phenotype seen in CYL5529(pCL7398). This concern was ruled out in experiments inwhich we showed that pCL7398 complemented both 5213 (acapA mutant) and 5346 (a capB mutant).

Analysis of gene products. To confirm the ORFs predicted

from the sequence data, plasmid subclones containing thecapsule genes were expressed in an in vitro transcription-translation system with E. coli S30 extract (55). When plasmidpCL7152 containing five complete ORFs (capA to capE) was

expressed, five proteins with approximate molecular masses of24.5, 25.2, 29.4, 67.5, and 50.7 kDa were detected (comparelane 2 with lane 1 of Fig. 4), which corresponded to thepredicted molecular masses of ORFs capA to capE, respec-tively. When plasmid pCL7164 containing the complete ORFscapE to capI was expressed, although several other minorbands were also detected, no protein bands corresponding tothese ORFs could be clearly demonstrated. By expressingplasmid 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 withlane 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-

0

pCL7134

pCL7187

5213 5346 517651875206521052125244

502352035214522652425243524552465248

pCL7165

.

I-

J. BACTERIOL.

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GENES AFFECTING TYPE 1 CAPSULE PRODUCTION IN S. AUREUS 7013

kb 0 1 2 3 4 5 6 7 8 9 10 i1 12 13 14

=glcRVPilacR Noo Kpnl BgIII EcoRVC9111I_-" \I cli I / / / I~~~~~~~~~~cla

-430io1 >- -4- -30-A 9 C D E F 0 H J K L M

kd 24.5 25.2 29.4 67.5 50.7 45.0 19.4 41.4 37.9 4.5 52.4 47.2 43.1

pCL7152

pCL714

pWL7327

FIG. 4. Autoradiogram of E. coli S30 in vitro transcription trans-lation of the gene products. [35S]methionine-labelled proteins wereseparated by SDS-PAGE as described in Materials and Methods. Lane1, 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 predictedCap proteins.

PROT) protein databases by using the algorithm of Lipmanand Pearson (30) in the Genetics Computer Group computerprograms. The most significant homology was between CapLand VipA, which showed 52.3% identity and 89.4% similaritywith a 423-amino-acid overlap (Fig. 5). CapL also showed24.0% identity to AlgD with a 416-amino-acid overlap. VipA isa protein involved in the biosynthesis of Vi polysaccharide inSalmonella typhi (11), and AlgD is GDP-mannose dehydroge-nase, which catalyzes oxidation of GDP-mannose to GDP-mannuronic acid required for alginate biosynthesis by mucoidPseudomonas aeruginosa (38). A high degree of homology wasalso found between the CapI protein and an unidentifiedreading frame (ORF2) adjacent to the chain-length determi-nant, which confers a modal distribution of chain length on the0 antigen component of lipopolysaccharide (LPS) of E. coli(6). The homology showed 57.7% identity and 89.2% similarityin a 333-amino-acid overlap. In addition, the CapI proteinshowed limited homology to VipB protein, another enzymeinvolved in the Vi antigen synthesis of S. typhi (11), and showedlimited homology to various epimerases from prokaryotic andeukaryotic origins, including GalE protein (UDP-N-acetylglu-

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 179I11111:111 :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:::1:111:11111:11:::::111: 1: 1 1:1 ::

VipA MIKKGINVEGSSVLILGFTFKENCPDIRNTRIIDVVKELGKYSCKVDIFDPWVDAEEVRR 364

CapL FFGLDLIDTKELKMVDWLFAVPHKDYME-NKKDYINLVKDCGIVFDIKGIINSDELNV 417:1: :: 11 1:::.: : 11 :::1:1 :: ::: :1

VipA EYGIIPVSEVKSSHYDAIIVAVGHQQFKQMGSEDIRGFGKDKHVLYDLKYVLPAEQSDV 423

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

cosamine epimerase) of E. coli (28). There were identities of29.0% over 335 amino acids between Capl and VipB and20.5% 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 conservedregion of about 50 amino acids of various members of theNodL-LacA-CysE acetyltransferase family recently identified(8). Twenty-two of the 29 amino acids between residues 86 and132 of CapG were identical to the consensus of the conservedregion of the acetyltransferases, and 5 of the mismatches wereconserved substitutions (Fig. 6). Thus, CapG could be anacetyltransferase required for the synthesis of type 1 capsularpolysaccharide.The deduced amino acid sequence of CapM showed limited

homology to RfpB protein of Shigella dysenteriae (10, 20) with24.1% identity over 370 amino acids. RfpB has been implicatedas a galactosyl transferase that adds a galactose moiety to thecore region in LPS biosynthesis (10). CapM protein also

Tn2424Cat

NodL

LacA

CysE

At cat

Sa vat

CapG

Consensus

113-164 IGSDVWIGSEAMIMPGIKIGHGAVIGSRALVAKDVEPYTIVGGNPAKSIRKR

134 -185 IGRHAWIGGGAIILPGVTIGDHAVIGAGSVVTRDVPAGSTAMGNPARVKAGG

134-185 IGNNVWIGSHVVINPGVTIGDNSVIGAGSIVTKDIPPNVVAAGVPCRVIRET

195-246 IREGVMIGAGAKILGNIEVGRGAKIGAGSVVLQPVPPHTTAAGVPARIVGKP

112-163 IGNDVWIGSEAIIMPGITVGDGAVIGTRALVTKDVEPYAIVGGNPAKTIRKR

121-172 IGNDVWIGRDVTIMPGVKIGDGAIIAAEAVVTKNVAPYSIVGGNPLKFIRKR

86-137 IGNNVFIGINSIILPGVTIGNNVVVGAGSVVTKDVPDNVIIGGNPAKKIKSI

IG- -VWIG ---- I-PGV-IG--AVIGA- - -VTKDV-P----- GNPA- -I

FIG. 6. Alignment of CapG with Tn2424Cat (Cat protein ofTn2424 [35]), NodL (the probable acetyltransferase of Rhizobiumleguminosarum), 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, andCysE is taken from Parent and Roy (35).

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showed limited homology to VipC protein, a gene productinvolved in the synthesis of Vi antigen of S. typhi (11) with21.8% identity over a 275-amino-acid overlap. No other ORF-encoded proteins predicted from the sequence analysis showedsignificant homology to the reported proteins in the databanks.

Virulence studies. Previous studies have shown type 1 cap-

sule is a virulence factor (27, 31). However, these studiesemployed mutant strains that were spontaneously arisen,chemically induced, or transposon mutagenized. To unequiv-ocally assess whether type 1 capsule is a virulence factor, weconstructed two isogenic strains, CYL5419 with a Cap' phe-notype and CYL5529 with a Cap- phenotype, derived fromstrain M as described in Materials and Methods. Two groups ofsix mice were injected with the isogenic strains. All six miceinjected with 107 CFU of CYL5419 died within 24 h, while allsix mice injected with 107 CFU of CYL5529 lived even after 72h. This result reconfirmed the findings of previous studies thattype 1 capsule is an important virulence factor in S. aureus.

DISCUSSION

We have previously cloned the genes affecting capsulesynthesis from S. aureus M in a 19.4-kb chromosomal DNAfragment. In this report, we further localized the genes to a

14.6-kb DNA fragment. Sequencing of this 14.6-kb fragmentshowed that there were 13 ORFs. Genetic experiments withdeleted fragments to complement various Cap- mutantsshowed that only 10 of these ORFs were represented by atleast one mutant despite the large number of mutants thatwere 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 themutations occurred at hot spots. The fact that the mutationsresulting in the Cap- phenotype were mapped in 10 of the 13ORFs suggests that these genes are involved in type 1 capsulebiosynthesis. To determine whether the remaining three ORFsare involved in the synthesis of capsule in strain M, we

attempted to construct mutants with nonpolar chromosomalmutations 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 thesame approach to construct mutants with mutations in capCfailed. One possibility for the failure may be due to the factthat 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 totransform 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 samefragment was cloned into a single-copy vector (24). Recogniz-ing this fact, we deliberately cloned the DNA fragment forreplacement without the promoter upstream of the capA, yetthis approach continued to fail, indicating that there may be a

weak promoter from the multiple-copy vector that allowsenough 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 othercap genes was not affected. We then constructed plasmidpCL7398 by cloning a DNA fragment containing capA to capEgenes with a nonpolar mutation in capC. This plasmid was

constructed with a single-copy plasmid vector that integrates atthe geh locus of the staphylococcal chromosome (26) to avoidthe high-gene-dosage effect of multiple-copy vectors. Because

deletion in CYL5529 did not affect the expression of othercapsule genes, transferring the pCL7398 into CYL5529 gener-ated a strain equivalent to the M strain with a specific mutationat capC. By using such an approach, we have shown that ORFcapC is involved in capsule production.

In the in vitro transcription-translation system, we were ableto clearly identify protein bands corresponding to seven pre-dicted ORFs (capA to -E and capJ to -K; Fig. 4). This resultlends additional support to the argument that these predictedORFs are likely to be the functional genes. The reason thatprotein bands corresponding to other ORFs were not detectedcould be due to the fact that these gene products were notefficiently expressed in the E. coli system or, alternatively, itcould be due to the fact that the bands were not resolved in theelectrophoretic system we employed. It is interesting thatCapE was readily detected, although weakly expressed inpCL7152, but no protein band corresponding to CapE wasdetected when pCL7164 was expressed, even though ourgenetic data suggested that CapE was expressed frompCL7164. This discrepancy could be due to the fact that thepromoter further upstream of capE, which is not present inpCL7164, may be required for full expression of CapE in E.coli, whereas in S. aureus, CapE may be readily expressed fromits own promoter.The genetic complementation data suggest that the cap

genes are organized into at least two operonic structures: oneoperon consists of capA through capD, with a likely transcrip-tion start upstream of capA; the other consists of at least capJ,capK, and capL but may also include capG, capH, capI, andcapM, with possible transcription initiated from upstream ofcapG. In addition, the data also indicate that there is anothertranscription start upstream of capE. Indeed, by using theGenetics Computer Group program, we have found a potentialpromoter with close homology to the consensus promotersequences found in E. coli and Bacillus spp. just upstream ofcapA (Fig. 2). Potential promoter sequences were also foundupstream of capE and capG, although they were less homolo-gous to the canonical sequences. Although these genetic datasuggest multiple transcription units, sequencing data showedthat all 13 ORFs are closely linked and that there is no obviousinverted repeat at any of the intercistronic noncoding regions,suggesting that all of these genes may be transcribed in a singletranscription unit. Thus, taking both the genetic complemen-tation results and the sequence data together, it is likely thatthese cap genes are transcribed in a long operon with addi-tional internal promoters. Because the more distal a gene is theless efficiently it is transcribed, the internal promoters wouldensure that the downstream genes are produced in adequateamounts. We are currently employing Northern (RNA) blot-ting to study the gene transcription and Si mapping todetermine the transcription start sites.

Recently, Hashimoto et al. (11) reported the nucleotidesequence of the ViaB region encoding Vi antigen in S. typhiand showed that three genes, vipA, vipB, and vipC, wereinvolved in biosynthesis of the Vi polysaccharide. Vi polysac-charide is a homopolymer of N-acetylgalactosamino uronicacid (13), which is one of the monomeric components of type1 capsule in S. aureus M (34). In fact, antiserum raised againstVi antigen cross-reacts with strain M capsule (19). The veryhigh degree of conservation between CapL and VipA, moder-ate homology between CapI and VipB, and limited homologybetween CapM and VipC imply that they are probably in-volved in the same biosynthetic pathway leading to the synthe-sis of N-acetylgalactosaminouronic acid. Because Salmonellaspp. and Staphylococcus spp. are two very evolutionarily diver-gent organisms, the similarity, especially between CapL and

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GENES AFFECTING TYPE 1 CAPSULE PRODUCTION IN S. AUREUS 7015

a Loop fi

LDHM 22-52 I - CAISILMKDLAD--EVALV1ADHE 192-223 CA L- LSVIMGCKAAGAA---RtIGVDG3P 3-33 G F- R RNVGRAALKNPR--IEVVAVNfVIPA 8-35 I I L- LPLAVEFGKSR-----QVVGFfMDHM 2-33 S QPLSLLLKNSPL--VSRLTLYDMDHE 2-24 A QALALLLKTQLPS-GSELSLYOVIPB 17-46 SGLLEELLFLN----QTVIGLRGALE 2-31 GS SKTCVQLLQNG----HD IILICAPL 4-32 IA L- LPVAVTFGNKH-----KVIGFDCAPI 2-31 IL TA E SHLAKKLIKQG-----HYrVIGVD

FIG. 7. Alignment of CapL and CapI with proteins containing theNAD-binding domain. Abbreviations: LDMH, spiny dogfish lactatedehydrogenase; ADHE, horse liver alcohol dehydrogenase; G3P,Bacillus stearothermophilus glyceraldehyde phosphate dehydrogenase;MDHM, porcine heart malate dehydrogenase; MDHE, E. coli malatedehydrogenase; 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). ThisNAD-binding domain, which consists of about 30 amino acidswith a three-dimensional structure of Pa,, is highly conservedamong NAD-dependent dehydrogenases (49). The deducedamino acid sequences of CapL and CapI also aligned well withthis NAD-binding domain, suggesting that CapL and CapI are

likely to have an NAD-binding domain (Fig. 7). On the basis ofthe 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-dependentenzymes involved in the synthesis of N-acetylgalactosamino-uronic acid.

ACKNOWLEDGMENTS

This work was supported in part by grant A128373 from the NationalInstitute of Allergy and Infectious Diseases.We thank Michael Black for isolating some of the Cap- mutants and

for participating in the initial sequencing work.

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