INFECTION AND IMMUNITY, OCt. 1988, p. 2702-2708 Vol. 56, No. 100019-9567/88/102702-07$02.00/0Copyright C) 1988, American Society for Microbiology

Association of Toxic Shock Toxin-1 Determinant with aHeterologous Insertion at Multiple Loci in the

Staphylococcus aureus ChromosomeMAY C. CHU,' BARRY N. KREISWIRTH,2 PETER A. PATTEE,3 RICHARD P. NOVICK,2 MARIAN E. MELISH,4

AND JOHN F. JAMES4*

Division of Vector-Borne Viral Diseases, Centers for Disease Control, Ft. Collins, Colorado 80522-20871; Department ofTropical Medicine and Medical Microbiology, University of Hawaii School of Medicine, Honolulu, Hawaii 968164;Department of Microbiology, Iowa State University, Ames, Iowa 500113; and Public Health Research Institute,

New York, New York 100162

Received 21 December 1987/Accepted 2 June 1988

Most Staphylococcus aureus strains associated with toxic shock syndrome and producing toxic shocksyndrome toxin 1 (TSST-1) require tryptophan because of a genetic defect in tryptophan biosynthesis. Theassociation between TSST-1 production and tryptophan auxotrophy was not correlated with the phage type, thecolonization site, or the disease status of the patient from whom the isolate came. Protoplast fusion andtransformation mapping located the genetic determinant of TSST-1 production (tst) very close to the trp operonin such strains and very close to tyrB in a Trp+ TSST-1+ strain. Southern blot hybridization of ClaI-restrictedchromosomal DNA with a tst-specific probe revealed a common homologous segment in all of the Trp+ strainswith tst linked to tyrB. These results confirmed that the tst determinant in Trp- strains is located at one site,whereas in Trp+ TSST-1+ strains the determinant is located elsewhere on the S. aureus chromosome. It issuggested that the TSST-1 determinant is associated with the insertion of a transposonlike segment into severalsites on the S. aureus chromosome.

Epidemiologic and laboratory data (3, 28, 31) have estab-lished Staphylococcus aureus as the cause of toxic shocksyndrome (TSS). A specific staphylococcal protein, TSStoxin-1 (TSST-1) (4), is associated with more than 90% of S.aureus isolates obtained from women with menstrual TSS (2,10) and with 62% of isolates from women with nonmenstrualTSS (29). Baboons and rabbits inoculated with TSST-1-producing strains (5, 10, 13) or purified TSST-1 (27) developsigns of illness similar to those of human TSS, which can beprevented by administration of serum with anti-TSST-1antibodies (5).

Typically, TSS strains of S. aureus are different fromstaphylococci isolated from asymptomatic carriers or fromindividuals with non-TSS staphylococcal infections. TSSstrains are predominantly lysed by group I phage types 29/52/80, are generally plasmid free, are resistant to penicillinand heavy metals, are more proteolytic, are more suscepti-ble to bacteriocins, are less hemolytic, and are less pig-mented (1, 2, 18, 20, 36, 37). Additionally, we have recentlyobserved that most TSS strains are tryptophan dependent,whereas most non-TSS strains are not (12). In a survey of154 S. aureus strains from various sources, including asymp-tomatic carriers, patients with non-TSS staphylococcal dis-ease, and patients with TSS, we found that 62 strainsproduced TSST-1 and that 44 of these were Trp- andappeared to share a common defect in the tryptophanpathway (M. C. Tom, M. E. Melish, and J. F. James,Abstr., Annu. Meet. Am. Soc. Microbiol. 1985, B215, p. 54).Among the 92 non-TSST-1-producing strains, there were 5tryptophan auxotrophs, representing several different de-fects in tryptophan biosynthesis. The remarkable correlationbetween TSST-1 production and tryptophan auxotrophy led

* Corresponding author.

us to investigate the possibility of genetic linkage between tstand trp. We have observed previously that there is consid-erable variability in blot hybridization patterns obtained withtst-specific probes and that most TSST-1- strains lack theentire element (16, 17). In this paper, we show by geneticmapping that tst is, in fact, closely linked to trp in Trp-strains and is located near tyrB in several Trp+ strains. Weshow also that the Trp- strains have in common a specificblot hybridization pattern with a tst probe that is differentfrom the pattern seen with Trp+ strains in which tst is linkedto tyrB. The results suggest that TSST-1 production isencoded by a variable, transposonlike genetic element.

(This work is a part of dissertation research completed byM.C.C. at the John A. Burns School of Medicine, Universityof Hawaii, Honolulu.)

MATERIALS AND METHODS

Nomenclature. The 1.5-kilobase genetic determinant ofTSST-1 (tst) resides on a larger unique segment of DNA 3.5kilobase pairs or more in length that may be located atdifferent chromosomal sites and is absent from most non-TSS strains (16, 17). Whereas several of the characteristicsof this element resemble those of a transposon (21), defini-tive evidence for transposition is lacking. We thereforepropose that the unique DNA segment containing tst beprovisionally designated Heterologous insertion 555 [i.e.,Q(HiS55)] and have used this nomenclature throughout thisreport. Others may wish to adopt this nomenclature forgenetic elements that consist of heterologous DNA inser-tions that have not been directly observed to transpose (21).

Bacterial strains.The 100 S. aureus strains studied in-cluded the following: (i) 21 vaginal isolates, including 3 frompatients with TSS, 8 from ill women without TSS, and 10from healthy women, which were received from A. Barbour,

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TSST-1 DETERMINANT IN S. AlUREUS CHROMOSOME

TABLE 1. Designation, characteristics, and origins of bacteria used in the genetic analysisS. aureus strain Genotype or phenotype Origin or reference

FRI 1169 fl(Hi555)402" M. S. BergdollbISP 2 8325 nov-142 pig-13 22ISP 5 8325 thy-101 thrB106 ilv-129 pig-131 22ISP 681 8325 thy-101 thrBI06 ilv-129 pig-131 tyrB282(TnS51) 23ISP 1532 W(Hi555)402 nov-142 80a-ISP2 X FRI 1169cISP 1653 8325d lys-115 trp-103 thrB106 ala-126 tmn-3106 ilv-129 pig-131 uraA141

tyrB282(TnSSI ermB321) Q1(TnS51)1035 fus-149 rl- r2- m31+ m32+S411 trp423 Q(Hj555)401" J. K. Todde

afl(Hi555)401, tst located in trp; fl(Hi555)402, tst located near tyrB.b Food Research Institute, University of Wisconsin, Madison.Bacteriophage 80a propagated on ISP 2 was used to transduce FRI 1169.

d This is a restriction-deficient, multiply marked NCTC 8325 strain derived from ISP 888 (31).eDepartment of Microbiology and Immunology, University of Colorado School of Medicine, Denver.

National Institute of Allergy and Infectious Diseases, RockyMountain Laboratories, Hamilton, Mont. (2); (ii) 10 nonva-ginal isolates from patients with TSS which were receivedfrom A. Reingold, Centers for Disease Control; Atlanta,Ga.; (iii) 20 vaginal isolates, 10 from patients with TSS and10 from healthy women, which were received from A.Chow, Department of Medicine, University of British Co-lumbia, Vancouver (11); (iv) 34 additional S. aureus strains,including 17 isolates from patients with TSS and 18 from illpatients without TSS, representing various colonizationsites, which were provided by J. Todd, Department ofPediatrics, University of Colorado School of Medicine,Denver; (v) strain FRI 1169, a vaginal isolate from a patientwith TSS, which was provided by M. S. Bergdoll, Univer-sity of Wisconsin, Madison; (vi) 15 isolates from the Depart-ment of Tropical Medicine, University of Hawaii, Honolulu,including 8 vaginal isolates from patients with confirmedTSS and 6 nonvaginal isolates from either ill patients withoutTSS or persons with asymptomatic carriage. Fifty-four pre-viously reported strains (12) were also included; overall,there were 78 vaginal isolates, 54 wound or abscess isolates,10 blood isolates, 9 nasopharyngeal isolates, and 3 throatisolates.

Descriptions of the S. aureus strains used for geneticmapping are presented in Table 1. Included in this table arethe two TSST-1+ strains, FRI 1169 and S411, used formapping tst; the multiply marked derivatives of S. aureusNCTC 8325; and the control strains for DNA hybridization.Working cultures were maintained on Trypticase soy agar

(BBL Microbiology Systems, Cockeysville, Md.) slantsstored at 4°C, and permanent stock cultures were kept onglass beads in Trypticase soy broth (BBL) containing 50%glycerol and stored at -70°C.

Bacteriophages. Reference typing phages and phages +11and 80a were maintained as described previously (35).

Purification of TSST-1. Crude TSST-1 was harvested from24-h shaker cultures inoculated with strain FRI 1169 inTodd-Hewitt Broth (Difco Laboratories, Detroit, Mich.).TSST-1 was purified by ion-exchange chromatography(CMC-50, Sephadex), followed by preparative isoelectricfocusing (PEGG pH 4.5 gradient; LKB Instruments, Inc.,Rockville, Md.). TSST-1 stained as a single band at pI 7.2 ona thin-layer isoelectric focusing gel and as a single band of 24kilodaltons on a 12.5% sodium dodecyl sulfate-polyacryl-amide gel electrophoresis gel by silver staining and reactedwith both TSST-1-hyperimmune rabbit serum and TSST-1-reactive monoclonal antibody AC3, described below.Anti-TSST-l serum and monoclonal antibody preparation.

Polyclonal rabbit antiserum was prepared by intramuscular

inoculation of 10 jig of purified TSST-1 per ml mixed withFreund complete adjuvant into adult New Zealand Whiterabbits. Rabbits were boosted with 10 jig of TSST-1 per mlwithout adjuvant at 2, 3, and 4 weeks after initial inoculation.The specificity of immune rabbit serum was determined bythe presence of a single precipitin line against both crude andpurified TSST-1. A mouse monoclonal antibody was pre-pared with purified TSST-1 as the immunogen by standardmethods (15). The anti-TSST-1 monoclonal antibody AC3was of isotype immunoglobulin G2a by enzyme-linked im-munosorbent assay with rabbit anti-mouse immunoglobulinsubclass sera (Organon Teknika, Malvern, Pa.). Hybridomaculture supernatants were concentrated by 50% ammoniumsulfate precipitation and purified by desorption from proteinA-Sepharose (Pharmacia, Inc., Piscataway, N.J.) with cit-rate buffer, pH 4.5 (15). The protein concentration of thepurified immunoglobulin peak was determined with theBradford assay (9) and adjusted to 2 jig/ml of protein per mlfor use in the enzyme-linked immunosorbent assay. Thespecificity of monoclonal antibody AC3 was determined byWestern blotting (immunoblotting) (38), which detected aband at 24 kilodaltons in a purified TSST-1 preparation.Western blot analysis of crude TSST-1 preparations detectedadditional bands in the 42- and 60-kDa range.TSST-1 detection. TSST-1 production by the S. aureus

strains was detected by the immunodiffusion method of Ritzet al. (30). The criterion for TSST-1 production was appear-ance of a precipitin line of identity with purified TSST-1 withrabbit hyperimmune serum.A method to detect TSST-1 production by individual

colonies was developed as a membrane-bound solid-phaseenzyme-linked immunosorbent assay at 25°C. Nitrocellulosefilters (0.45-jLm pore size; Millipore Corp., Bedford, Mass.)were placed over the growth area to adsorb for 15 min, andthe filters were washed with deionized water, soaked brieflyin 0.01 M phosphate-buffered saline (PBS; 0.01 MNa2HPO4-NaH2PO4, 0.15 M NaCl, 0.05% Tween 20, 0.01%thimersal; pH 7.5), and blocked with 3% bovine serumalbumin in PBS for 30 min with gentle shaking, followed bya second blocking solution of rabbit anti-protein A (SigmaChemical Co., St. Louis, Mo.) serum plus 100 jig of unla-beled avidin (Sigma) per ml in PBS for another 30 min. Afterbeing washed three times with water and once with PBS, thefilters were incubated in a solution containing 2 jig of proteinper ml of AC3 preblocked with protein A (Sigma) and 25 jigof biotin per ml in 1% bovine serum albumin-PBS for 2 h.Biotinylated horse anti-mouse immunoglobulin G (VecstainABC kit; Vector Laboratories, Burlingame, Calif.), alsopreblocked with protein A, was then diluted 1:200 in 1%

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2704 CHU ET AL.

bovine serum albumin-PBS and reacted with the washedfilters for 1 h. This was followed by incubation with anavidin-peroxidase complex for an additional hour. A sub-strate solution of 3 mg of 4-chloro-1-naphthol (Sigma) per mlwith 0.004% hydrogen peroxide was used to visualize posi-tive TSST-1 reactions.

S. aureus auxotyping. A suspension of 108 CFU/ml wasmade in buffered salts (2.48 g of Na2HPO4, 0.4 g of KH2PO4,5 ml of a salt solution containing 1% MgSO4, 1% MnCl2,0.32% sodium citrate, and 0.32% FeSO4 per liter of deio-nized water) and washed twice by centrifugation at 350 x g(12,000 rpm) for 10 min. The chemically defined medium ofEmmett and Kloos (14) was adapted to micromethods. In a96-well sterile, flat-bottom polystyrene microtiter plate(Costar, Cambridge, Mass.), 5 RI of washed cells was addedto each of various wells with 200 ,ul of medium lacking one ofthe 18 amino acids. Control wells included those withoutniacin, without thiamine, without glucose, with completemedium, and with only buffered salts and phenol red. After24 h of incubation at 37°C, the wells were examined forgrowth by visual turbidity, and a change in color from red toyellow was recorded as acid production. Auxotype profilesfor each strain were repeated blindly and independently onthree to five different occasions.Tryptophan biosynthetic block. Tryptophan-requiring

strains were characterized for response to tryptophan syn-thesis pathway intermediates, L-tryptophan, and tryptophananalogs (22, 26). Semidefined medium consisted of 20 g oftryptophan-free Casamino Acids (Difco), 20 g of ,-glycero-phosphate, 2 g of D-glucose, and 15 g of Bacto-Agar (Difco)dissolved in 1 liter of buffered salts and autoclaved. Tenmicrograms each of niacin and thiamine per liter was addedto the autoclaved medium. The surface of semidefinedmedium was heavily streaked with a washed suspension of108 CFU of the test strain per ml. Filter disks soaked with 10,ul of one of the prepared stocks (200 ,ug of each substrateand tryptophan analog per ml) was then placed on theinoculated agar surface. Plates were incubated overnight at37°C. Response to the exogenous substrate was noted asgrowth around the filter disk.

Chemicals. Tryptophan pathway intermediates includedshikimic acid, chorismic acid, anthranilic acid, and indole.Tryptophan analogs included 7-azotryptophan; 4-, 5-, and6-methyltryptophan; and 5-fluorotryptophan (SFT). All ofthe above-listed reagents were from Sigma.

Protoplast fusions and transformations. Protoplast fusionsand transformations were performed as described previously(33, 34). Trypticase soy broth was supplemented with thy-mine (20 ,ug/ml) and adenine, guanine, cytosine, and uracil (5,ug of each per ml). The composition of complete definedsynthetic agar (25) was modified by omitting the appropriateamino acids, purines, and pyrimidines as needed to scoreand select nutritional markers (33). Antibiotic resistancephenotypes were selected and scored on brain heart infusion(Difco) agar containing 0.1% (wt/vol) sodium citrate and theappropriate concentration of the antibiotic (33).DNA hybridization with the TSST-1 probe. For hybridiza-

tion analysis by Southern blotting, whole-cell DNA wasisolated from each strain by a modification of the method ofMekalanos (23). The procedure was adapted for S. aureus bysubstituting lysostaphin (Sigma) (final concentration, 500 ,g/ml) for lysozyme. DNA preparations were digested to com-pletion with the restriction enzyme ClaI, electrophoresed ina 0.9% agarose gel, transferred to nitrocellulose, and thenhybridized with a radiolabeled 297-base-pair BamHI-HindIItst-specific DNA fragment (8). Hybridization and washes in

0.1x SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodiumcitrate) were performed at 65°C.

RESULTS

Detection of TSST-1. Of the 154 strains, 62 producedTSST-1 by immunodiffusion against polyclonal immune rab-bit serum. TSST-1 monoclonal antibody AC3 confirmed all62 strains as TSST-1 positive. Of the 62 TSST-1+ strains, 45were isolated from patients with TSS, whereas only 17 (16%)of 109 non-TSS isolates produced TSST-1, a figure consis-tent with previously determined frequencies of TSST-1production by non-TSS-associated S. aureus (10).

Nutritional requirements. The spectrum of amino acidrequirements of the 154 S. aureus strains was generally inagreement with the results obtained by Emmett and Kloos(14). Eighty-nine percent of the strains were arginine auxo-trophs, 82% required valine, 68% were proline requiring, and32% required tryptophan. Except for tryptophan require-ment, there was no strong link between any single aminoacid requirement and TSST-1 production. Among the 48Trp- strains, 44 were TSST-1 producers (90%); conversely,44 (71%) of 62 TSST-1 producers were Trp-, whereas only 5(5%) of 92 non-TSST-1 producers were Trp- (P < 0.001; chisquare).

Relationship of tryptophan auxotrophy to colonization site.Since many of the previously reported TSST-1-producingstrains were vaginal isolates from patients with TSS (2, 3),the next step was to determine whether the observed tryp-tophan requirement was associated with the site of infectionrather than with TSST-1 production. Thirty-six (81%) of the44 TSST-1+ vaginal isolates were tryptophan auxotrophs,whereas 2 (6%) of 34 TSST-1- vaginal isolates requiredtryptophan (P < 0.001; Fisher exact test). Tryptophanauxotrophs represented 5 (42%) of 12 TSST-1+ wound orabscess isolates and 2 (50%) of 4 TSST-1+ blood isolatescompared with 2 (5%) of 42 and 1 (16%) of 6, respectively,TSST-1- strains from comparable sources (P < 0.001;Fisher exact test). Tryptophan requirement and TSST-1production thus covaried, irrespective of the isolation site.Tryptophan genetic block characterization. All 44 Trp-

TSST-1+ strains grew and produced TSST-1 in response toexogenous indole and tryptophan. All strains grew withoutdetectable TSST-1 in response to 5FT, an analog that may beincorporated into proteins in place of tryptophan (32), anddid not respond to anthranilic acid or earlier intermediates(4-, 5-, and 6-methyltryptophan). Two of five Trp- TSST-1-strains responded to these compounds, whereas two re-sponded only to indole and the other responded to anthra-nilic acid as well. Thus, all of the Trp- TSST-1- strainsappeared to have defects at different points in the tryptophanbiosynthetic pathway (26). Growth of Trp+ TSST-1+ strainswas not affected by addition of the same compounds. Sincethe results suggested that tst was directly related to trypto-phan auxotrophy, mapping studies were next performed totest for genetic linkage of tst and trp.

Compatible strains for genetic exchange. Most TSST-1+strains of S. aureus are in lytic group I, whereas a minorityeither exhibit a combination of group I and III typingreactions or are in group III. Because of this diversity andbecause the lytic group of a strain is a good preliminaryindicator of how well that strain will exchange geneticmaterial with another, the phage typing patterns of the 62TSST-1+ strains available in this study were determined.Fourteen strains exhibited sensitivity to one or more phagesin lytic group III and therefore were likely candidates to use

INFECT. IMMUN.

TSST-1 DETERMINANT IN S. AUREUS CHROMOSOME 2705

TABLE 2. Analysis of tht-101, trB282::Tn55I, and 1stdeterminants by transformation of ISP 681 (Thy- Tyr- Em'

TSST-1-) with DNA from ISP 1532 (Thy' Tyr' Ems TSST-l+)"

Class Phenotype'} X of totalrecombinants'

A Thy- Tyr' TSST-1- 44B Thy- Tyr' TSST-1+ 50C Thy' Tyr' TSST-1+ )

D Thy' Tyr' TSST-1 4

"The transformation frequency was 314 Tyr' transformants per 109 CFU,and the reversion frequency was four Tyr' colonies per 109 CFU. We used 2.0x 109 CFU and excess DNA in the transformation suspension. All Tyr'colonies were erythromycin sensitive, and all Tyr colonies were erythromy-cin resistant.

' See Table 1 for genotypes. Thy-, Thymine requirement; Tyr, tyrosinerequirement; TSST-1+, TSST-1 production.

' We analyzed 832 recombinants for unselected markers.

as transformation donors with multiply marked derivativesof NCTC 8325, which is a lytic group III strain. When these14 strains were infected with phage 80O propagated on strainISP 2, only 4 (including FRI 1169, a Trp+ TSST-1+ strain,and S411, a Trp- TSST-1' strain) yielded novobiocin-resistant (Novr) transductants at frequencies reflecting theabsence of restriction barriers between them and ISP 2.These two strains were then used in the mapping experi-ments described below.

Genetic mapping. A protoplast fusion between ISP1532and ISP1653 was performed to map the tst locus in ISP 1532.ISP 1532 is a novobiocin-resistant (Novr) derivative of FRI1169 (Trp+ TSST-1+), and ISP 1653 is a multiply markedderivative of strain 8325 carrying chromosomal markers forfusidic acid resistance (Fusr) and tetracycline resistance(Tetr). In this cross, Novr Tetr and Novr Fusr recombinantswere selected, 1,120 of which were scored for the distribu-tion and coinheritance of unselected markers, including Trpand TSST-1. The results indicated that the tst determinant ofISP 1532 was probably in the thrB-trp-thy-t'yrB region of thechromosome. Several transformation experiments were thenperformed to define more precisely the chromosomal locusof the tst determinant of ISP 1532. Table 2 summarizes theresults of one such experiment, in which ISP 681 wastransformed with ISP 1532 DNA with selection for Tyr'.These results showed that the tst marker of ISP 1532 wasimmediately adjacent to tyrB in the gene order thy-tyrB-tst(Fig. 1).

S411 is representative of Trp- TSST-1+ isolates. To testfor linkage of tst and trp, we transformed ISP 5 with DNAisolated from S411, selecting for Thy'. The trp operon isknown to be approximately midway between thy and thirB(25). Among 436 Thy' transformants, none were Trp-

TABLE 3. Analysis of tliiBI06, ist, and tliv-101 determinants bytransformation of ISP 5 (Thr- TSST-1- Trp+ Thy-) with DNA

from S411 (Thr' TSST-l Trp- Thy')"

Class Phenotype'> %7c of totalrecombinants'

A Thr- TSST-1- Trp+ Thy' 90B Thr- TSST-1+ Trp- Thy' 0C Thr+ TSST-1+ Trp- Thy' 9D Thr+ TSST-1 Trp+ Thy' 1

"The transformation frequency was 436 Thy' transformants per 109 CFU.and the reversion frequency was two Thy' colonies per 109 CFU. We used 6.6x 109 CFU and excess DNA in the transformation suspension.

" See Table 1 for genotypes. Thr-, Threonine requirements; TSST-1+,TSST-1 production; Trp-, tryptophan requirement; Thy, thymine require-ment.

We analyzed 630 recombinants for unselected markers.

TSST-1+, indicating extremely close linkage of the twomarkers. thv and tihrB showed 9% cotransformation (Table3), a figure that is unexpectedly high relative to the repro-ducible value of 2% in previous crosses between TSST-1-strains (25). This result can be attributed to suppression ofrecombination as a consequence of the tst element betweenthe thy and thrB markers. The results of the two crosses,plus several others in which the tst marker in these two wasmapped with respect to tihrA and the Tn55I insertions M8and fl1, are summarized in Fig. 1, suggesting two separatesites for tst in nutritionally different strains. On the basis ofthese data, we have assigned to the trp-linked tst gene theinsertion number fQ401 and to the tyrB-linked tst gene theinsertion number Q1402.

Blot hybridization analysis. A series of Trp- and Trp+toxin-producing strains were then analyzed by Southern blothybridization with a 297-base-pair tst-specific fragment ofthe cloned Hi555 element as a 32P-labeled probe. If Hi555were inserted into the same site in the trp region in thetryptophan auxotrophs and at another site adjacent to thetyrB marker in ISP 1526, different hybridization patternswith a tst probe would be expected for chromosomal digestsof these strains. The results (Fig. 2) fulfilled this prediction.Lanes A to E contained ClaI-digested chromosomal DNAfrom a series of epidemiologically unrelated Trp- TSST-1+strains, including S411 (lane D). The Trp+ TSST-1+ strainFRI 1169, in which tst mapped near thrB, exhibited adifferent hybridization pattern (lane G). The FRI 1169 hy-bridization pattern was identical to that of RN 4282 (lane F),the Trp+ TSST-1+ strain from which the TSST-1 genesegment was cloned (17).

FIG. 1. Summary of the linkage relationships between S2(Hi555)401 and II(Hi555)402 of ISP 1532 and S411 and the thrB-trp-thy-tyrBregion of the chromosome of S. auireius NCTC 8325. Relative map distances are taken from data obtained from the transformations shownin Tables 2 and 3, together with additional transformations between the Q(HiS55)401 determinant of ISP 1532 and the thrA,£i(chr::Tn551)8, and f1(chr::Tn551)11 markers (details not shown) and with DNA-blot patterns.

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2706 CHU ET AL.

82Cs El e h2 H3 C. 42 61 C. B.. H2 at H2 Sl

-A L _....1121

42 Bi

D--AROBCDFC:

A B C D E F G A B C D E F G

... ..

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1-...

lS~~~~~~~~~sa~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.FIG. 2. Diagram of DNA probe and results of DNA hybridiza-

tion probing of chromosomal ClaI fragments obtained from TSST-1+ S. aureus strains. Left panel, ethidium bromide-stained agarosegel patterns of DNA digested with ClaI. Right panel, autoradiogramof a Southern blot of the same gel hybridized with DNA probediagrammed above. Lanes: A, Harrisburg; B, RN4485; C, 587; D,ISP 1637; E, ISP 1645; F, RN4282; G, FRI 1169.

DISCUSSIONTSST-1 production by S. aureus is frequently associated

with a specific genetic lesion somewhere between anthrani-late and indol in the tryptophan pathway. TSST-1 productionand tryptophan auxotrophy do not appear to be associatedwith any phage group, although we have recently observed astrong association with chromosomal bla, cad, and asamarkers in these strains. The profile of amino acid require-ments of the TSST-1+ strains was otherwise similar to thoseof the TSST-1- strains and human skin S. aureus isolates(14).The tryptophan block common to all of the 44 Trp-

TSST-1+ strains is between anthranilate and indol, and weassume that they are the same three steps between these twointermediates in S. aureus as in Escherichia coli, namely,N-5'-phosphoribosylanthranilic acid and 1(o-carboxyphenyl-amino)-1-deoxyribulose-5-phosphate. Thus, it is not certainthat all of the strains are blocked at precisely the same point,an uncertainty that can be resolved by biochemical studiesor more detailed mapping and hybridization studies.Another question raised by our results is that of whether

the Trp- phenotype is due to insertional inactivation of oneof the trp genes by the transposonlike tst element. Prelimi-nary results suggest that this is not the case. We obtainedTrp+ reversions of two strains and found that TSST-1 wasstill produced; in one case, we showed also that the blottingpattern with a tst probe was unchanged. Additionally, werecently found two strains (Trp+ and TSST-1+) that had thesame tst blotting pattern as that seen with the Trp- strains,

suggesting that tst is in the same location in both sets ofstrains. The possibility remains, however, that tst is insertedinto a larger mobile element that is in turn inserted into thetrp gene and that this larger element has moved elsewhere inTrp+ revertants. This possibility of a larger element would,incidentally, be consistent with the crossover suppression inthe tst region observed in crosses of Trp- TSST-1+ strainswith Trp+ TSST-1- strains.Our finding that 5FT restored growth but not TSST-1

production in Trp- strains raises the possibility of a meta-bolic relationship between tryptophan metabolism andTSST-1 synthesis. Since the culture supernatants of Trp-TSST-1+ strains grown in the presence of 5FT did not reactwith either hyperimmune serum or monoclonal antibodyAC3 (unpublished data), it is unlikely that inactive TSST-1containing 5FT in place of tryptophan is produced in thepresence of the analog.These results appear to be similar to reports of inhibition

of a- and P-hemolysin production in S. aureus by 5FT atconcentrations that did not significantly inhibit growth (32)and to a report that serine protease is not produced in mediarelatively deficient in certain amino acids (7). Although 5FTcan fulfill a growth requirement for tryptophan, suggestingthat it can be incorporated into proteins, it causes a partialinhibition of growth in Trp+ strains that is reversed bytryptophan (19), suggesting that growth in the presence of5FT is metabolically equivalent to a relative deficiency of thenatural amino acid. This interpretation is supported by arecent observation that when Trp- TSST-1+ strains aregrown in limiting tryptophan they fail to produce nucleaseand a-hemolysin (R. P. Novick, unpublished data).

Overall, these results suggest that production of exopro-teins, which starts at the end of the exponential phase andcontinues into the stationary phase in laboratory cultures, isvery sensitive to a limitation in the availability of tryptophanand probably other amino acids as well. This phenomenon iscurrently under investigation.Although the genetic evidence presented here suggests

that tst is carried by a mobile, possibly transposonlikeelement, we never directly observed any instability nor wasany event detected in the laboratory that might suggestmovement of the element to a new location. Trp+ revertantsoccur, as noted, and they do so at a frequency of lo-7 to10-8 per plated CFU, a frequency typical of point mutations.Additionally, because in several cases different cultures ofthe same S. aureus strain were obtained from differentlaboratories, we were able to examine the stability of theauxotrophic and DNA blot hybridization profile of a partic-ular strain. For example, the Harrisburg strain (Trp- TSST-1+) was a part of three collections, those of the University ofHawaii (S244), the National Institute of Allergy and Infec-tious Diseases (Harrisburg), and the Public Health ResearchInstitute (RN3984). These three cultures had indistinguish-able auxotrophic and blot profiles (unpublished data). Inaddition, an isolate of FRI 1169 maintained at the PublicHealth Research Institute retained the same profile as FRI1169 during multiple independent passages of the two cul-tures. It is also noteworthy that strains A to E (Fig. 2) hadthe same blot hybridization pattern and had remarkablysimilar ClaI chromosomal fingerprints, despite being epide-miologically unrelated, and the same can be said for F and G,suggesting that the strains within each set are clonallyrelated.Our finding that tst can occupy more than one chromo-

somal site is consistent with observations that the prophage-linked gene entA is a variable gene that may be located at

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TSST-1 DETERMINANT IN S. AUREUS CHROMOSOME

more than one site (6) and may reflect a generality withrespect to genes encoding toxins and possibly other exopro-teins in S. aureus. A number of these genes have beencloned; blot hybridization analysis in conjunction withpulsed-field gel electrophoresis, which can rapidly and un-equivocally establish genomic locations, is in progress.

ACKNOWLEDGMENTS

We thank A. Barbour, M. S. Bergdoll, A. Chow, A. Reingold, andJ. Todd for providing the bacterial strains. We thank S. Hirata, S.Murata, and K. Froegner for production of purified TSST-1 andrabbit anti-TSST-1 sera. We acknowledge A. A. Benedict and theMonoclonal Service Center, Department of Microbiology, Univer-sity of Hawaii, Honolulu, for producing the anti-TSST-1-secretinghybridoma cell line.M.C.C. was partially funded by the Sidney Gaines Scholarship,

University of Hawaii, Honolulu, in part by grant PCM-8310681 fromthe National Science Foundation to P.A.P., and by a grant fromInternational Playtex, Inc., to J.F.J. Travel funding for M.C.C. wasprovided by the American Society for Microbiology President'sFellowship Award.

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