Sequential transposition of Tn916 among Staphylococcus aureus protoplasts

PLASMtD19, 13-20(1988)

Sequential

Transposition

of Tn976 among Staphy/ococc~s aureus Protoplasts

SUSAN CONNOLLY Department

YOST,’

of Microbiology,

JOANNE

M. JONES,’ AND P. A. PATTEE~

Iowa State

University,

Ames,

Iowa 5001 I

Received May 8, 1987; revised November 7. 1987 Transposition of the Strepfococcus faecalis conjugal tetracycline-resistance transposon Tn916 between S. aureus strains occurred when protoplasts of donor and recipient strains were regenerated together without prior fusion. Under these conditions, only Tn916 was transferred; spontaneous fusion of parental protoplasts is therefore unlikely to be responsible for Tn916 transfer. While the exact nature of this transfer remains unclear, it appears to resemble Tn916 conjugal transposition reported in S. faecalis. Evidence for sequential transpositions of Tn916 was obtained by 3-factorial transformation analyses and confirmed by DNA-DNA hybridizations. The ability of Tn916 to transpose within S. aureusand occupy diverse chromosomal sites demonstrates the value ofthis transposon in genetic studies of.% aureus. Q 1988 Academic FESS IIIC.

Development of the genetic map of Staphylococcus aureus NCTC 8325 has relied heavily on the MLS4-resistance transposon TnSSZ (Luchansky and Pattee, 1984; Pattee, 198 1; Schroeder and Pattee, 1984; Stahl and Pattee, 1983a,b). This reliance on Tn55Z has limited the genetic crosses that can be performed because crosses between two Tn551bearing (i.e., MLS-resistant) strains yields only limited information concerning the locations of the Tn55I insertions. Tn916 is a conjugal tetracycline-resistance (Tc’) transposon of Streptococcus faecalis (for review see Clewell and Gawron-Burke, 1986) that can be introduced into S. aureus by intergeneric protoplast fusion or membrane filter matings (Jones et al., 1987). The plasmid-free Tc’ S. aureus so obtained was

shown by hybridization profiles and transformation analyses to carry Tn916 inserts in various chromosomal sites. These data pointed to the potential value of Tn916 for genomic mapping in S. aureus. This report shows that Tn916 transfers between S. aureus protoplasts that have not been fused and that it transposes to diverse chromosomal regions, thereby confirming the usefulness of Tn916 in genetic studies of S. aureus. MATERIALS

AND

METHODS

Bacteria and bacteriophage. The strains of S. aureus used in this study are listed in Table 1 and were maintained as described by Stahl and Pattee (1983a). Phages 80 (Thompson and Pattee, 1977) and 80a (Novick, 1963) were maintained by propagation on ISP221 and ISP8, respectively. Lysates were stored at 4°C. Media Nutritionally complex media were enriched with 20 &ml of thymine and 5 pg/ml each of adenine, cytosine, guanine, and uracil. Regeneration agar (R W, Stahl and Pattee, 1983a) was modified to contain 1 mg/ml of sodium citrate and 1 &ml each of tetracycline and a second antibiotic selective for the recipient strain, unless otherwise

’ Present address: Molecular Therapeutics, Inc., 400 Morgan Lane, West Haven, CT 065 16. * Present address: Dental Research Unit, University of Michigan, Ann Arbor, MI 48 109-2007. ’ To whom all correspondence should be addressed. 4 Abbreviations used: Tc’, tetracycline resistance; BHI, brain-heart infusion; CDS, complete defined synthetic; SMMP, sucrose-magnesium-maleic acid-Penassay broth; SMTB, sucrose-magnesium-Tris buffer, Em’, erythromycin resistance; Tc’, tetracycline-sensitive; Nov’, novobiocin resistance; MIS, macrolide-lincosamide-stteptogrammin. 13

0147-619X/88

$3.00

Copymbt 8 1988 by Academic PITS Inc. All rights of reproduciion in any form reserved.

14

YOST, JONES, AND PATTEE TABLE I DFZXZNATION,

GENOTYPE,

AND ORIGIN

Strain

OF STRAINS OF Staphylococcus

Genotype”

aureus

AND Escherichia

coli

Origin or reference

S. aweus

ISP2 ISP8 ISP15 ISP22 I ISP267 ISP823

8325 nov-142pig-131 8325-4 pig-131 8325 thy-101 lys-115 pig-131 Ps80 bla’ 8325 nov-142 ribl27pig-131

ISP983

8325 thrBlO6 uraAl41 ilv-129 nov-142 Tn4291 frpE85 Q[Chr::Tn551]5 pig131 tyrB282::Tn551 ermB321 8325 now142 pig-131 m-3490 8325-4 pig-131 recAl his-7

lSPlOO0 ISPlO63 ISPI 155 lSPl160

ISPI 199

tmn-3106

8OCR3 R[Chr::Tn916]1100 R(Chr::Tn916]1146 now142

Q[Chr::Tn551]1032 879R4 nov-142 Q[Chr::Tn916]1146 8325 thrBl06 wpE85 uraAl41 ilv-129 fus-149pig-131 nov-142 Tn4291 lyrB282::Tn551 erm321

R[Chr::Tn551]1035 r2- m31’ m32’ lrp-103

8325 rlrhrBlO6 hisGl5

Pattee and Neveln (1975) Thompson and Pattee (1977) Pattee and Neveln ( 1975) CDCb Pattee and Glatz ( 1980) Jones et al. (1987) Stahl and Pattee (1983a) ISP1038 DNA X ISP41’ ISP1061dgrown at 41°C with selection for Em’ Jones et al. (1987) Jones et al. ( 1987)

RN496’ DNA X ISP926’

ala-126 ilv-129pig-131 lys-115 nov-142

Q[Chr::Tn551]5 ISP1252 ISPl272 ISP1283 ISPI 320 ISP1321 ISP1322 ISP1323 ISPI 324 ISP1325 SAI 13

8325 thrBIO6 pig-131 R[Chr::Tn916]1101 8OCR3 nov-I42 R(Chr::Tn916]1119 8OCR3 nov-142 R[Chr::Tn9I6]1130 8325 pig-131 nov-142 R[Chr::Tn916]1103 8325 pig-131 nov-142 R[Chr::Tn916]1137 8325 pig-131 nov-142 R[Chr::Tn916)1138 8325 pig-131 nov-142 R[Chr::Tn916]1139 8325 pig-131 nov-142 R(Chr::Tn916]1140 8325 pig-131 nov-142 Q[Chr::Tn9/6]1141 8325 rl- m31’ r2- m32’pig-131

RN1855

8325 Q[Chr::Tn551]40

pig-l31

E. coli

Pattee ( 1986) Jones et al. (1987) Origin = ISP1272 ISPl252 X ISP2 fusion controP ISPI 252 X ISP2 fusion control ISP 1252 X ISP2 fusion control ISPl252 X ISP2 fusion control ISPl252 X ISP2 fusion control ISP1252 X ISP2 fusion control Iordanescu and Surdeanu (1976) Pattee and Glatz (1980) Yamamoto et al. (1987)

DHI(XXpAM620) ” 8325 and 8325-4 derivatives are in phage lytic group 111,879R4 derivatives are in lytic group II, and Ps80 and 8OCR3 are in lytic group 1. b Centers for Disease Control, Atlanta, Georgia. ‘Stahl and Pattee (1983a). ISPlOOO was constructed by transforming ISP41 with DNA from ISPlO38. ISPlO38 = 8325 ~~4141 hisGl5 nov-142 Tn4291 1a-3490 pig-131, while ISP4l = 8325 nov-142 pig-131 /us-149. Both strains are described in Stahl and Pattee (1983a). d ISPlO61 = 8325-4 pig-131 recA1 his-7 (~1258 bla1401 mer-14 repA36) (Tam and Pattee, 1986). ’ RN496 = 8325-4 pig-131 R(Chr::TnSSl]S (Pattee et (II.. 1977). lISP926 = 8325 rl- r2- m31’ m32’ lys-115 lrp-103 IhrBl06 ala-126 tmn-3106 ilv-129pig-131 hisGl5 nov-142 (Stahl and Pattee, 1983b). d Protoplasts of ISP1252 and ISP2 were regenerated together without prior fusion as described in this study.

stated. Antibiotic resistance phenotypes were selected and scored on brain-heart infusion (BHI; Difco) agar containing 1 or IO PgJrnl,

respectively, of the appropriate antibiotic. Complete defined synthetic (CDS) agar was used to select and score nutritional markers

Tn916

IN S.

(Pattee and Neveln, 1975). The sucrose-based buffers SMMP [sucrose-magnesium-maleic acid-Penassay broth (antibiotic medium 3; Difco)] and SMTB (sucrose-magnesium-Tris buffer) have been described (Chang and Cohen, 1979; Stahl and Pattee, 1983a). Transductions and transformations. The preparation of transducing lysates and the transduction procedure have been described (Schroeder and Pattee, 1984). DNA used for transformation was prepared as described by Pattee and Neveln (1975). Transformations were performed as previously described (Luchansky and Pattee, 1984; Stahl and Pattee, 1983b). Map distances between two markers, A and B, are expressed as 1 minus the estimated cotransfer frequency (C), where C = frequency of cotransformation of two markers, A and B (C = AB/A or C = AB/B) (Pattee and Neveln, 1975). Transfer of Tn916 between S, aureus protoplasts.S. aureus protoplasts were prepared according to Stahl and Pattee (1983a). Transfer of Tn916 was accomplished by mixing aliquots of protoplasts of the Tn916containing strain and a recipient carrying a counterselectable antibiotic resistance determinant in a 1: 1 ratio. After stationary incubation at 32°C for 18 h in SMMP (Chang and Cohen, 1979) containing 0.6 mg/ml sodium citrate, 0.2-ml samples were spread to R agar plates containing 1 mg/ml sodium citrate and 1 gg each of tetracycline and the antibiotic (erythromycin or novobiocin) selective for the recipient strain. Protoplasts were allowed to regenerate for 7 days at 35°C in a humidified incubator. Phage-typing patterns and complete phenotypes of the resulting recombinants were then determined to identify those cells which had acquired only Tn916 from the donor strain. Purification of chromosomal DNA. Chromosomal DNA was purified on CsCl-ethidium bromide gradients and then digested with restriction endonucleases as recommended by the supplier (Bethesda Research Laboratories). After agarose electrophoresis in Tris-borate-EDTA running buffer (Meyers et al., 1976) the DNA fragments

15

auwus

were stained with ethidium sualized by transillumination.

bromide

and vi-

Preparation and detection of biotinylated DNA. A DNA detection system based on biotin-streptavidin (BluGene; Bethesda Research Laboratories) was used to identify chromosomal DNA fragments containing insertions of Tn916 (Jones et al., 1987). pAM620 (Yamamoto et al., 1987) biotinylated with photoactivatable biotin (Clontech Laboratories) served as the probe in these studies. RESULTS

Protoplast-mediated transfer of Tn916 between S. aureus strains. Transfer of Tn916 between strains of S. aureus was first observed when protoplasts were mixed in SMTB in the absence of the fusion-inducing agent polyethylene glycol and then regenerated on nonselective regeneration agar. Usually between 1 and 50 recombinants/ 1O9 colony-forming units between multiply marked parental strains were observed regardless of the selective markers used (Stahl and Pattee, 1983a). In contrast, when protoplasts of ISP823 (carrying Tn916) and ISP983 were mixed and then regenerated without prior fusion, several thousand Tc’ colonies that otherwise exhibited the ISP983 phenotype were obtained. Acquisition of the Tc’ phenotype was not inhibited by DNase in the protoplast suspensions and in the regeneration medium, and Tc’ ISP983 colonies were not recovered when intact cells of these strains were used or when ISP983 protoplasts were incubated without ISP823. Selection for uracil- or threonine-independent ISP983 colonies did not yield recombinants, suggesting that the transfer was restricted to Tn916. Tn916 transfer between nonfused ISP823 and ISP983 protoplasts was observed under various conditions (Table 2). Tn916 transfer was reduced when nonfused protoplasts were regenerated on R agar containing twice the normal concentration of sodium citrate. Because this higher level of sodium citrate did not impair protoplast regeneration (data not shown), these results suggest that transduc-

16

YOST, JONES, AND PATTEE TABLE 2

used and about lo-* when the incubation was 18 h. To enhance the recovery of Tc’ recombinants, protoplasts were incubated for I8 h in SMMP containing sodium citrate before spreading on R agar plates. No. of Tc’ Em’ The spectrum of transfer-proficient 5’. Conditions of protoplast recombinants uurcus was examined. Transfer between proincubation before recovered’ on plating” toplasts of ISP823 or ISP 1272 (8OCR3 derivStandard Modified atives containing primary insertions of Buffer Time R agar R agar Tn916) and ISP983 or ISPll60 resulted in low but reproducible frequencies (data not SMTB 1 min 2 I shown). ISPl I55 and ISP1283 also served as SMTB + citrate 4h 2 0 SMTB 18h 1 0 donors in transfer to ISP I 199, but at a highly SMTB + citrate 18h I 0 variable frequency. This variability was not SMMP 4h 75 5 examined further. No Tn916 transfer was SMMP + citrate 4h 250 2 detected from ISP823 or ISP1272 to the reSMMP 18 h 50 10 combination-deficient strain ISP1063. We SMMP + citrate 18h 5 15 attribute this observation to the low viability ’ Protoplasts of ISP823 and ISP983 were incubated in of ISP1063, not to a role of Rec. Transfer of either SMTB or SMMP buffer with or without 0.6 Tc’ was specific for Tn916 since Tc’ ISP983 mg/ml sodium citrate for different time intervals before spreading onto standard or modified R agar. Both plat- colonies were not recovered when ISPlOOO ing media contained 1 &ml each of tetracycline and or ISP267 carrying the readily transducible erythromycin; standard R agar contained 0.5 mg/ml so- tet-3490 and tmn-3106 markers (Asheshov, dium citrate, while modified R agar contained I.0 1975) were used as donors. mg/ml sodium citrate. Transposition of Tn916 within S. aureus. ’ Recombinants are expressed as the numbers of Tc’ To determine whether transfer involved Em’ recombinants recovered per 0.2-ml sample containing an average of 4 X lo9 protoplasts. For each ex- transposition or homologous recombination, Tc’ recipients were screened to identify those periment, protoplasts of strains ISP823 and ISP983 were individually plated; in no instance were Tc’ Em’ colonies that carried Tn916 at a site different from recovered on the regeneration medium. that of the donor. Three levels of transposition are distinguished in this study. Primary inserts originated from intergeneric crosses tion was responsible for a significant portion between S. fhecalis carrying Tn916 and Tc” of the recombinants. ISP983 carries phage S. aureus (Jones et al., 1987). First-round $1 1, a serological group B transducing transposition constitutes the initial transposition of Tn916 between two S. aureus phage, and ISP823 may carry a similar strains. Second-round transpositions refer to phage. Phage capable of mediating generalized transduction in S. aureus usually are in Tn916 insertions obtained using the product serological group B, and their adsorption is of a first-round transposition as donor. All such transpositions were rare and somewhat Ca2+ dependent and thus inhibited by citrate difficult to detect because of the accompanyions (Dowel1 and Rosenblum, 1962). Addition of 0.6 mg/ml of sodium citrate to the ing transfer of Tn916 from donor to recipiincubation buffers did not affect Tn916 ent without transposition. Location of‘first-round transposition intransfer in any consistent way (Table 2). Nonetheless, except where noted, sodium ci- serts. The estimated frequency of first-round transfer was very low, ca. lo-* to 10e9, using trate was routinely added to incubation buffers (0.6 mg/ml) and R agar (1 mgjml) to Tn916 donors derived from either strain reduce transduction. The observed fre- ISP823 or strain SAI 13. Using ISP823, quency of Tc’ recombinants was about 10e9 which carries two copies of Tn916, one of if a 4-h incubation period before plating was which is in segment 8 (Fig. I), and lSP983 as EFFE~X OFPROTOPLASTINCUBATIONTIMEAND INCUBATIONCONDITIONSONTHETRANSFEROF T~~I~BE~EENISP~~~ANDISW~~

17

Tn916 IN S. arcre

nants were transformed with RN 1855 DNA with selection for Em’ (=R[Chr: : Tn552]40, an insert linked to the Q[Chr::Tn916]1101 site); seven recombinants no longer carrying Tn916 at the Q 110 I site were identified. These isolates represent transpositions of Tn916 to new chromosomal sites. The other 28 recombinants were judged to retain Tn916 at the Ql 101 site based on the Tc” phenotype of most Em’ transformants. These probably resulted from transduction of Tn916 from ISP1252 to ISP2. Two of the seven recombinants representing secondround transposition events carried Tn916 in the fl[Chr::Tn551]1 I-tyrl? region, but at different sites (Q[Chr: :Tn916] 1 139 and Q[Chr::Tn916]1140 in Table 3). Hybridization studies. To obtain molecular evidence for the sequential transposition of Tn916 in S. aureus, chromosomal DNAs from ISP8, ISP823, ISP1252, and ISP1320 through ISP1325 were digested with HindIII, which cuts Tn916 once (Gawron-Burke and Clewell. 1984) and analyzed in Southern ( 1975) blots using biotinylated pAM620 probe DNA (Fig. 2). ISP823, the parent of Q[Chr: : Tn916] 110 1, contains two copies of Tn916 (Jones et al., 1987) and either copy of Tn916 could have served as the source of R[Chr: : Tn916] 110 1. Figure 2 shows that subsequent transfer of Tn916 from ISP1252 FIG. 1. Abbreviated chromosome map of (containing R[Chr : : Tn916] 110 1) also inStaphylococcus aureus NCTC 8325 showing the location volved transposition, as evidenced by unique of the major markers used for mapping and the other DNA junction fragmarkers relevant to mapping chromosomal inserts of Tn916-chromosomal Tn916 in the present study. thy, thymine requirement; ments in strains ISP1320 through ISP1325. Ql I, Q[Chr: : TnSSI] 1 I ; tyrB, L-tyrosine requirement; The hybridization data suggest that R[CHr: : att&lZ, integration site for prophage d12; Q34, fi[Tn916] 1 I37 and R[Chr: :Tn916] 1138 (Fig. Chr: :Tn551]34; rib, riboflavin requirement: tmn,tetracycline-minocycline resistance; purB. adenine + gua- 2B, lanes E and F) although independently isolated, may occupy the same site. Siminine requirement; ilv, L-isoleucine + L-valine requirelarly, U[Chr::Tn916]1 140 and t2[Chr:: ment; pig, absence of golden-yellow pigment; uraA and uraB. uracil requirements; his, L-histidine requirement; Tn916]1141 (Fig. 2B, lanes H and I) may nov. novobiccin resistance; Tn4291. methicillin resis- also be at one site.

recipient, two insertions resulting from firstround transposition were identified by the loss of linkage (about 50% cotransformation) between Tn916 and h-129. One (Q[Chr:: Tn91611101) was in segment 15 and the other (Q[Chr::Tn916] 1102) was in segment 1 (Table 3). Location of second-round transposition inserts. Protoplasts of ISP1252, carrying Q[Chr::Tn916]1101 adjacent to Q[Chr:: Tn.551]40, were regenerated with protoplasts of ISP2 using SMMP (devoid of citrate) and R agar (reduced citrate), conditions that allow some transduction (Table 2). Among 484 Tc’ Nov’ recombinants recovered, 60% were Thr- and thus presumably arose from transduction of nov-242 into ISP1252 instead of Tn916 transfer into ISP2. Thirtyfive of the prototrophic Tc’ (ISP2) recombi-

tance (formerly met-4916; Trees and Iandolo, 1988); tet, tetracycline resistance;fus, fusidic acid resistance; purC, purine requirement; MO, Q[Chr::Tn551]40; thrB, Lthreonine requirement; trp. L-tryptophan require.ment; lys, L-lysine requirement. The map is divided into 18 segments, the ends of each of which are defined by a pair of cotransformable markers except where the circle is shown interrupted; the latter reflect gaps that exceed the capacity of currently available transforming DNA (redrawn from Pattee (I 987)).

DISCUSSION

Jones et al. (1987) demonstrated the transfer of Tn916 from S. faecalis carrying pADI::Tn916 and pPDS::Tn916 to S. aureus by intergeneric protoplast fusions. The Tc’ S. aureus so obtained were plasmid

18

YOST,

JONES,

AND

TABLE

PATTEE

3

SUMMARY OF CHROMOSOMAL LOCI OCCUPIED BY PRIMARY, FIRST-ROUND, AND SECOND-ROUND TRANSPOSITIONSOF Tn916 Chromosomal location” Insertion Primary QllOO First-round RI 101 RI 102 Second-round RI 103 Rll39 n1140

Origin pADI::Tn916

Segment

8

Rl 100 or RI 146 in ISP823 RI 100 or RI 146 in ISP832

15

RI 101 in BP1252 RI 101 in BP1252 RI 101 in BP1252

18

1

Marker order

~~I1OO-pig-I3I-ilv-129

uruB232-R

110

Map distance(s)*

0.06

I -R40

from pig-131;

0.43 from h-129

0.02 from WO

Ll I I-R I 102~cyrB282

0.01 from sll I; 0.92 from tyrE282

1ys-11541103-thy-I0I

0.45

2

tyrB2X2-R

0.52 from 12I 1; 0.60 from fyrB282

I

(11 I-CL1IOZ-ryrB282

I 139~R34

from thy-101

0.58 from Ql I; 0.33 from fyrB282

0 Refer to Fig. I for segment number. R 1 I, Q34, and Q40 are chromosomal insertions of TnSSI. * Map distances are 1 - the estimated cotransformation frequency.

free and carried Tn916 at diverse chromosomal loci. These experiments did not test, however, whether Tn916 could transpose in the absence of S. faecalis. The present study provides genetic and physical evidence that Tn916 is indeed capable of sequential transpositions in S. aureus. The transfer and transposition of Tn916 must occur at some point during the incubation and regeneration of parental protoplasts, although neither the exact time nor the mechanism of transfer is known. Transfer might involve protoplasts or regenerating intermediates in the formation of walled cells. It probably does not occur between fully regenerated walled cells, because no Tn916 transposition was found when walled cells were spread together on regeneration agar. Gasson (1980) observed the transfer of pAMP1 between nonfused protoplasts of lactic streptococci. This plasmid is conjugally active in the lactic streptococci by membrane filter mating, and protoplast formation may have facilitated PAM/~ 1 transfer in these strains.

The absence of recombinants involving other markers even in cells that had undergone Tn916 transposition implicates an active role for this conjugative transposon in transfer and argues against spontaneous fusion. This conclusion is reinforced by the stability of Tn916 at given chromosomal sites when protoplasts are fused and then recombinants analyzed after regeneration (Pattee, 1986), the transfer of only Tn916, and transfer frequencies equivalent to those for Tn916 conjugal transposition in S. faecalis (Franke and Clewell, 198 1) when transduction was stringently inhibited. Some Tn916 transfer can be attributed to transduction followed by homologous recombination or possibly to Tn916 conjugal transfer from one strain to another in the absence of transposition. The failure to recover Tc’ recombinants when using a recombination-deficient recipient (ISP1063, derived from RN98 1) is attributed to the very low transfer frequency of Tn916 in S. aureus, combined with a low incidence of viable cells (less than 10%;

Tn916 IN S. aureus

19

B

A ABCDBFGHIJKL

ABCDBFGHIJKL

FIG. 2. Hybridization analysis of Tn916 chromosomal insertions in various Staphylococcus aureus using biotinylated pAM620 to probe HindUI-digested chromosomal DNA. Lane A, biotinylated HindIII-digested X-DNA; B, ISP823 containing the primary insertions G 1100 and Q1146; C, ISPl252 containing the first-round insertion 52110 1; D, ISPI 320 containing the second-round insertion Q 1103; E, ISP132 1 containing the second-round insertion Ql137; F, ISP1322 containing the second-round insertion 01138; G, ISP1323 containing the second-round insertion G 1139; H, ISP1324 containing the second-round insertion Q 1140; I, ISPl325 containing the second-round insertion tl 114 1; J, ISPI devoid of Tn916 insertions; K, pAM620 digested with HindIII; L, biotinylated HindIII-digested X-DNA. (A) Ethidium bromide-stained 0.7% agarose gel. (B) Hybridization analysis of the DNAs shown in (A) after transfer to a nylon membrane and hybridization with biotinylated pAM620.

Wyman et al., 1974), rather than to an inability of this element to transpose to or reside in Ret- hosts. In S. fuecalis, Tn916 is known to transpose to a recombination-deficient recipient (Franke and Clewell, 198 1). The results of this study indicate that Tn916 is a functional transposon in S. aureus and has considerable potential as a genetic tool in this bacterium. The Tn916 insertions identified in this study were, however, generated by an inefficient method, and a more efficient delivery system for the transposon is needed. With the development of such a delivery system, this transposon can

be used to full advantage in genetic studies of S. aureus. ACKNOWLEDGMENTS The technical assistance of Robyn L. Hottman is gratefully acknowledged. This investigation was sup ported by National Science Foundation Grants PCM-8 110058, PCM-83 1068 1, and DMB-8705408.

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by Douglas

E. Berg