JOURNAL

OF

BACTERIOLOGY, Jan. 1991, p. 487-494

Vol. 173,

0021-9193/91/020487-08$02.00/0 Copyright C) 1991, American Society for Microbiology

No. 2

Molecular Analysis of Lipoteichoic Acid from Streptococcus agalactiae Department

JOHN J. MAURER AND STEPHEN J. MATTINGLY* University of Texas Health Science Center at San Antonio, San Antonio, Texas 78284

of Microbiology,

Received 5 July 1990/Accepted 24 October 1990

A method for the analysis of lipoteichoic acid (LTA) by polyacrylamide gel electrophoresis (PAGE) is described. Purified LTA from Streptococcus agalactiae tended to smear in the upper two-thirds of a 30 to 40% linear polyacrylamide gel, while the chemically deacylated form (cdLTA) migrated as a ladder of discrete bands, reminiscent of lipopolysaccharides. The deacylated polymer appeared to separate in this system on the basis of size, as evident from results obtained from PAGE analysis of cdLTA subjected to limited acid hydrolysis and LTA that had been fractionated by gel filtration. A survey of cdLTA from other streptococci revealed similarities in molecular weight ranges. The polymer from Enterococcus hirae was of a higher molecular weight. This procedure was used to examine the effect of penicillin and chloroamphenicol on the synthesis, turnover, and heterogeneity of LTA in S. agalactiae. Penicillin appeared to enhance LTA synthesis while causing the release of this polymer into the supernatant fluid. In contrast, chloramphenicol inhibited the synthesis of this molecule and resulted in its depletion from the cell surface. Penicillin did not alter the heterogeneity of this polymer, but chloramphenicol caused an apparent shift to a lower-molecular-weight form of the LTA, as determined by PAGE. This shift in the heterogeneity of LTA did not appear to be due to increased carbohydrate substitution, since chloramphenicol did not alter the electrophoretic migration profile of LTA from E. hirae. From a pulse-chase study, it was determined that LTA was released as a consequence of deacylation.

Lipoteichoic acid (LTA) is an amphiphilic molecule found in most gram-positive bacteria (12). This molecule is a glycerol-phosphate (GP) polymer that extends through the peptidoglycan layer of the cell wall and is bound to the cell surface through a hydrophobic interaction between its glycolipid anchor and the cytoplasmic membrane (12). Various functions have been attributed to this polymer; they include scavenging divalent cations (15), regulating autolysin activity (9), and mediating adherence of a number of grampositive bacteria to eucaryotic cells (2, 6, 8, 23, 31, 33). In this last role, it is believed to play a major role in the pathogenesis of several species of streptococci, including Streptococcus pyogenes, the cause of streptococcal pharyngitis (2), and S. agalactiae, one of the leading causes of neonatal sepsis and meningitis in the United States. A recent study demonstrated differences among clinical isolates of S. agalactiae in terms of the levels of LTA synthesized (30) and the relative binding activity of these strains to human fetal lung epithelial cells (31). The chain length of LTA in S. agalactiae (32) and other gram-positive bacteria has previously been defined as the total number of GP units per terminal GP unit or fatty acids present in the glycolipid (12). In this report, an alternative method for analysis of polymer length by polyacrylamide gel electrophoresis (PAGE) is described which allows visualization of the LTA GP backbone. The synthesis, turnover, and heterogeneity of the GP backbone of LTA of S. agalactiae were examined by this method.

*

MATERIALS AND METHODS Bacterial strains, culture media, and growth conditions. S. agalactiae 110, a clinical group B streptococcal isolate, has been well characterized in terms of LTA (30), type-specific antigen (40), neuraminidase (26), and virulence in an animal model (10). Other bacteria examined in this study included S. pyogenes 147; a strain of group A streptococcus isolated from a patient with pharyngitis; S. mutans GS-5, a wellcharacterized serotype c isolate; and Enterococcus hirae ATCC 9790 (formerly S. faecalis). Organisms were cultured on 5% sheep blood agar plates (BBL Microbiology Systems, Cockeysville, Md.) at 37°C for 16 to 24 h before each experiment. S. mutans was incubated in an anaerobic chamber containing 5% CO2. A chemically defined medium, FMC, was prepared as described previously (38) and was always used within 24 h of preparation. Cultivation of S. pyogenes required the addition of 2% (vol/vol) Todd-Hewitt broth (Difco Laboratories, Detroit, Mich.) to the chemically defined medium. Growth was monitored spectrophotometrically (Junior model 35; The Perkin-Elmer Corp., Oak Brooks, Ill.) as described previously (27), with 1 adjusted optical density unit (AOD) being equivalent to 0.45 p.g of cells (dry weight) per ml. A starter culture was prepared by inoculating two or three colonies from a 24-h blood agar plate into 10 ml of FMC and allowing the culture to reach the mid-exponential phase of growth (approximately 170 p,g of cells [dry weight] per ml). The initial cell density of a 100-ml culture was adjusted to 13 to 15 p.g of cells (dry weight) per ml (30 to 35 AOD units) with the appropriate amount of inoculum from the starter culture. LTA was specifically radiolabeled with [3H]glycerol (0.5 ,uCi/ml) in the presence of unlabeled glycerol (15 ,ug/ml) as described previously (30). The culture pH was maintained at 7.0 with periodic addition of 2.5 N NaOH

Corresponding author. 487

488

MAURER AND MATTINGLY

once cells reached the mid-exponential phase of growth. Stationary phase was defined as the time at which the cell density failed to increase exponentially over a 30- to 60-min interval. In studies on the effect of chloramphenicol (CAM) on LTA synthesis in S. agalactiae, cells were grown in the presence of [3H]glycerol (0.5 ,uCi/ml) and carrier glycerol (15 jig/ml) with an initial inoculum of 10 ,ug of cells (dry weight) per ml (25 AOD). A 1-liter culture was grown to 500 AOD (0.23 mg of cells per ml) and split into two 500-ml cultures, one of which was treated with CAM (10 ,ug/ml). Cells were harvested once the untreated culture reached stationary phase. Studies on the effect of antibiotics on the de novo synthesis and turnover of LTA involved growing 1-liter cultures to mid-exponential phase (500 AOD) in FMC containing unlabeled glycerol (15 ,ug/ml) and then dividing the culture into equal portions. Along with the addition of [3H]glycerol (0.5 pRCi/ml) to all cultures, penicillin G (PEN) (5 ,ug/ml) or CAM (10 ,ug/ml) was added, and the cultures were incubated at 37°C until the control (untreated) reached stationary phase. Turnover of LTA was examined by labeling a 500-ml culture with [3H]glycerol (0.5 ,uCilml; 15 ,ug of carrier glycerol per ml), starting with an initial cell density of 10 ,ug of cells per ml. One the culture reached exponential phase (500 AOD), the cells were washed three times with sterile phosphate-buffered saline (PBS) and resuspended in FMC containing glycerol (15 ,ug/ml) but no radiolabel. Aliquots (100 ml) were obtained periodically, and the LTA and deacylated LTA (dLTA) from cells and supernatant fluids were purified and analyzed as described below. Extraction, purification, quantitation, and biochemical analysis of LTA. The extraction and purification of LTA has been detailed in previous work (25, 30). The LTA was concentrated for analysis by lyophilization. Total organic phosphate was determined by the method of Chen et al. (7). Purified LTA was chemically deacylated by mild alkali treatment with NH40H as described previously (31) to obtain chemically deacylated LTA (cdLTA). PAGE of cdLTA. The protocol for PAGE analysis of cdLTA was adapted from the procedure of Pelkonen et al. (34). PAGE was performed on a gel (10 by 12 cm) with a 30 to 40% polyacrylamide gradient in Tris-borate (lx TBE is 0.089 M Tris base, 0.089 M boric acid, and 0.002 M EDTA [pH 8.3]) gel buffer. Other buffers examined in this study included Tris-acetate (TAE) and Tris-phosphate (TPE) (24). Gels were preelectrophoresed at 10 V/cm for 1 h at 4°C. Samples were weighted with 1/10 volume of 1 M sucrose with 1Ox TBE and loaded at 10 RI per well. Material was applied to each well in terms of micrograms of total organic phosphate present in each sample. Dyes included xylene cyanol, bromophenol blue, and phenol red and were run in separate lanes from the cdLTA samples. Samples were electrophoresed at 20 V/cm for 12 h at 4°C. Electrophoresis was discontinued once the phenol red dye front reached the bottom of the gel. Gels were stained by the alcian blue-silver stain technique of Min and Cowman (28). Alcian blue and silver staining reagents were both purchased from Bio-Rad (Richmond, Calif.). Limited acid hydrolysis and separation of cdLTA by PAGE. cdLTA was subjected to limited acid hydrolysis by treating 2 ,ug (total organic phosphate) of sample with 2 N HCI at 80°C for 0, 1, 5, or 10 min. Samples were immediately placed on ice after each time period. Acid was removed from the sample with nitrogen gas, and the acid-hydrolyzed cdLTA was dissolved in 10 RI of double-distilled H20. Electrophoresis was performed at 35 V/cm until the phenol red migrated

J. BACTERIOL.

FIG. 1. PAGE of native and cdLTA from S. agalactiae. Gel electrophoresis was performed on a 30 to 40% polyacrylamide gel gradient as described in Materials and Methods. Lane 1, Dye markers; lane 2, native LTA (5 ,ug of organic phosphate); lane 3, native LTA (10 ,ug of organic phosphate); lane 4, cdLTA (1 p,g of organic phosphate).

one-third of the way through the gel. The gel was stained as described above. Determination of the molecular weight range of cdLTA. Lipoteichoic acid was fractionated on the basis of size by column chromatography with G-100 Sephadex (Sigma, St. Louis, Mo.). The sample was dissolved in a deoxycholate buffer (0.25% deoxycholate, 0.2 M NaCl, 1 mM EDTA, 0.02% NaN3, 10 mM Tris [pH 8.0]; originally described for dissociating aggregates of purified lipopolysaccharides [35]). One milliliter of sample (107 dpm) was applied to a column previously equilibrated with deoxycholate buffer. Eluent was collected from the column in 0.8-ml fractions with a Gilson microfractionator (Gilson, Stafford, Tex.). The elution profile for the LTA was determined by liquid scintillation. Fractions contained within this peak were chemically deacylated as previously described (30). A chloroformmethanol (2:1) extraction was included to ensure removal of fatty acid residues and detergent. Samples were analyzed for total organic phosphate by the procedure of Chen et al. (7). Fractionated cdLTA (0.25 ,ug) was electrophoresed until the phenol red dye front migrated two-thirds of the way through the gel. The polyacrylamide gel was stained by the procedures outlined above. RESULTS PAGE of LTA. By using a modified version of the PAGE technique of Pelkonen et al. (34) for analyzing negatively charged polysaccharides, LTA was observed to migrate as a smear in the upper two-thirds of the gel (Fig. 1, lanes 2 and 3). At least 5 ,ug of sample was required for detection. Although electrophoretic methods involving SDS-PAGE are commonly used to separate molecules on the basis of size, this detergent appeared to interfere with staining (data not shown). This is attributed to the recognized ability of this amphipathic molecule to form micelles (12). To test this hypothesis further, the polymer was chemically deacylated by mild alkali treatment. Electrophoresis was initially performed at 20 V/cm with a conventional 10% native polyacrylamide gel, and electrophoresis was discontinued once the dye front (bromophenol blue) reached the bottom of the gel. Only the acylated form of the polymer was evident. How-

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489

45

FIG. 2. PAGE of cdLTA subjected to limited acid hydrolysis. Samples (2 ,ug of organic phosphate) were hydrolyzed with 2 N HCI at 80°C for 0, 1, 5, or 10 min. Material was prepared and separated by gel electrophoresis as described in the text. Lane 1, Dye markers; lane 2, cdLTA (0-min hydrolysis); lane 3, cdLTA (1-min hydrolysis); lane 4, cdLTA (5-min hydrolysis); lane 5, cdLTA (10-min hydrolysis).

discontinuing electrophoresis once the dye front had migrated halfway through the gel revealed that cdLTA had migrated to the bottom one-third of the gel. Since cdLTA migrated ahead of bromophenol blue, a lower-molecular-weight dye, phenol red, was included. With a 30 to 40% linear polyacrylamide gadient gel, the cdLTA formed a ladder of discrete bands (Fig. 1, lane 4) in the bottom one-third of the gel, reminiscent of lipopolysaccharide (13). Less sample (1 Rg) was required for visualization with the alcian blue-silver stain than for the acylated form of the polymer. Separation by gel electrophoresis was similar for native dLTA and cdLTA (data not shown), indicating that the migration of cdLTA was not attributable to partial hydrolysis resulting from the mild alkali treatment. In examining other buffer systems, it was found that the electrophoretic profile of this polymer was not dependent on the buffer used when TBE was compared with a Tris-acetate or a Tris-phosphate gel buffer (data not shown). The presence of a broad range of bands suggested that cdLTA may represent a heterogeneous population of molecules that vary in length. Determination of the relative molecular weight of cdLTA separated by gel electrophoresis. The heterogeneiety exhibited in the electrophoretic migration of this polymer could represent LTA at various stages in polymerization from the lower- to the higher-molecular-weight form. The fastermigrating material could either represent the smaller cdLTA or a more negatively charged form of the molecule unrelated to size. To distinguish between these possibilities, cdLTA was subjected to limited acid hydrolysis (2 N HCl at 80°C for 1 to 10 min). A shift in the electrophoretic migration pattern of cdLTA was readily apparent. Acid hydrolysis for 1 min (Fig. 2, lane 3) resulted in the disappearance of bands in the upper region of the gel and an increase in the staining intensity in preexisting bands present in the lower portion of the gel (Fig. 2, compare lanes 2 and 3) along with the appearance of new bands that migrated ahead of the last band present in the control (Fig. 2, lane 2). Further hydrolysis resulted in an increase in the staining intensity of the "new" bands present in the lower region (Fig. 2, lanes 3 and 4) until only one band could be detected after a 10-min hydrolysis (Fig. 2, lane 5). ever,

FIG. 3. PAGE analysis of LTA fractionated by gel filtration. Lipoteichoic acid was fractionated on the basis of size by column chromatography with G-100 Sephadex according to the procedure

outlined in Materials and Methods. The fractions representing the high and low molecular weight ranges of the LTA peak were loaded in descending order from left to right into the gel lanes. Fractions contained within the LTA peak were chemically deacylated. Fractionated cdLTA (0.25 jig) was electrophoresed until the phenol red dye front migrated two-thirds of the way through the gel. Gel electrophoresis was performed as described in the text.

The results from this limited hydrolysis suggest that the more slowly migrating bands represent the higher-molecular-

weight cdLTA. This was supported by evidence obtained from the electrophoretic profile of LTA that had been fractionated according to size by Sephadex G-100 column chromatography. Fractions eluting within the LTA peak (Kav = 0.67) were chemically deacylated and applied to a 30 to 40% polyacrylamide gradient gel in descending order from left to right, with the left lane representing the initial fraction from the LTA peak. As shown in Fig. 3, the cdLTA representing the entire size range of fractionated LTA migrated as a diagonal. The diagonal would only be seen if a descending order of high- to low-molecular-weight molecules were followed in separation of cdLTA by gel electrophoresis. This approach allowed an estimation of the molecular weight range of LTA. Since LTA is a linear polymer of repeating a-1,3 GP units, dextrans were chosen as appropriate molecular weight standards for sizing the fractions from Sephadex G-100 column. Based on dextran standards, the size range of cdLTA was estimated at 4,000 to 8,000 Da, which is within the size range of LTA determined in previous reports of the chain length of the polymer (12). Survey of cdLTA from gram-positive bacteria by PAGE. The relative PAGE migration patterns of cdLTA from S. pyogenes, S. mutans, and E. hirae in addition to S. agalactiae were examined. The electrophoretic migration of cdLTA from the various organisms revealed that these polymers were also quite heterogeneous (Fig. 4). The cdLTAs appeared to be similar among the streptococci, although there were some differences in staining intensities, particularly in the higher-molecular-weight region. The enterococcus clearly produced a higher-molecular-weight form of this molecule. Also, the lower-molecular-weight bands found in the other organisms were not evident in E. hirae (Fig. 4, lane 4). Requirement of protein or cell wall synthesis for the production of LTA in S. agalactiae. Both CAM and PEN have been found to affect the adherence of pathogens to the host,

490

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MAURER AND MATTINGLY

FIG. 4. Survey in PAGE profiles of cdLTAs from streptococci and an enterococcus. Conditions for electrophoresis and staining of gel are described in Materials and Methods. Lane 1, Dye markers; lane 2, S. pyogenes cdLTA; lane 3, S. agalactiae cdLTA; lane 4, E. hirae cdLTA; lane 5, S. mutans cdLTA.

depending on the organism and the nature of the adhesin (29). The ability to alter the binding properties of the organism is probably due to inhibition of additional synthesis or turnover of preexisting adhesins by these antibiotics. The effects of these two antibiotics were examined to determine their effect on LTA synthesis and/or turnover in S. agalac-

tiae. Inhibition of protein synthesis with CAM resulted in a reduction in LTA (Fig. 5). With the decrease in LTA, there was a corresponding increase in the natural dLTA extracted from whole cells. When protein synthesis was inhibited by depriving cells of the essential amino acid leucine (27), a similar change in the elution profile of [3H]glycerol-labeled teichoic acid was found (data not shown). These results could be attributed to either inhibition of additional LTA synthesis or enhanced turnover of this polymer. To address this question, de novo synthesis of LTA in the presence and absence of antibiotics was examined (see Materials and Methods). Antibiotics and [3H]glycerol were added to the culture once cells reached the mid-exponential phase of growth. In previous studies, >99% of the [3H]glycerol incorporated was associated with dLTA and LTA from whole cells, suggesting direct incorporation into LTA (30). This is in contrast to the results of Kessler and Shockman (20), who found that preequilibration of the radiolabel was necessary, since [3H]glycerol was incorporated into phosphatidylglycerol as well as cellular and extracellular LTA in E. hirae. The present study therefore measured the effect of antibiotics on the incorporation of [3H]glycerol into LTA (synthesis). Examination of total teichoic acid (dLTA and LTA) present in both the supernatant fluid and whole cells revealed that CAM treatment resulted in a one-third reduction

-a

a-)

E rS

0 E X

C0

CM

a

I

a..

0Ne 30

40

Fractions FIG. 5. Effect of CAM on LTA synthesis of symptomatic isolate 110. A 1-liter culture was grown to 500 AOD (see inset) in the presence of [3H]glycerol. The culture was split into two 500-ml cultures, one of which was treated with CAM (10 ,ug/ml). Cells were grown to stationary phase, and LTA was extracted from the whole cells and purified by hydrophobic liquid chromatography with Octyl-Sepharose and a 0 to 60%

1-propanol gradient.

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MOLECULAR ANALYSIS OF LIPOTEICHOIC ACID

TABLE 1. Effect of CAM and PEN on cell surface and extracellular teichoic acid synthesis in S. agalactiae Treatment and

fraction None Whole cell

Supernatant

CAM Whole cell Supernatant PEN Whole cell

Supernatant

Teichoic acid

Label incorporation (104 cellsdpm/mg [dry wt])of

dLTA LTA dLTA LTA

0.31 0.11 2.61 0.01

dLTA LTA dLTA LTA

0.44 0.09 0.32 0.01

dLTA LTA dLTA LTA

1.14 0.41 2.81 0.56

491

1 2 3 4 5

Total teichoic acida (% of control) 100

28.0

162.0

a Compared with the sum of the total cellular and extracellular LTA and dLTA present in the untreated control.

in incorporation of [3H]glycerol compared with that in the untreated control (Table 1). However, inhibition of protein synthesis did not result in the release of LTA into the medium. The relatively low amount of LTA present in CAM-treated cells compared with the total amount of dLTA may reflect the reduction in de novo synthesis and the normal turnover of this amphiphile (see below). It was necessary to also examine the effect of PEN on LTA synthesis, since this beta-lactam has been well recognized as affecting more than peptidoglycan synthesis (3, 4, 37). It has been demonstrated previously that PEN caused secretion of this polymer into the environment in S. agalactiae (25). Unlike CAM, PEN did not result in the reduction of [3H]glycerol incorporation into LTA. In fact, a 1.5-fold increase was detected in total teichoic acid synthesized compared with that in the untreated culture (Table 1). Gel electrophoresis of cdLTA extracted from S. agalactiae grown under various conditions. A comparison was made of the electrophoretic migration pattern of cdLTA taken from exponential- and stationary-phase cells as well as cdLTA from CAM- or PEN-treated cells. No apparent differences in the electrophoretic patterns of cdLTA from stationary-phase (Fig. 6, lane 2) or exponential-phase (Fig. 6, lane 3) cells were detected. However, CAM-treated cells exhibited a shift in the distribution of cdLTA, with the majority of the bands migrating in the lower-molecular-weight range, as evident from the increased staining intensity (Fig. 6, lane 4). PEN treatment did not have a significant effect on the distribution of cdLTA (Fig. 6, lane 5) compared with that in the untreated culture (Fig. 6, lane 2). The shift in the migration of cdLTA could be attributed to either inhibition of the synthesis of components necessary for polymerization of the larger form of LTA as cells make the transition into stationary phase or increased glycosylation of the polymer as a consequence of the inhibition of protein synthesis. The latter possibility has been demonstrated in E. hirae, in which inhibition of protein synthesis by either CAM or amino acid deprivation resulted in an increase in glucosyl substitution in this polymer (21). This resulted in a shift in the migration of the LTA in an agarose gel. Removal of carbohydrates with glucosidase from LTA extracted from CAM-treated cell

FIG. 6. Effect of growth phase and the antibiotics CAM and PEN on the heterogeneity of cdLTA from S. agalactiae 110. Strain 110 was grown to mid-exponential phase in a chemically defined medium. Cells were either harvested at this point (exponential), allowed to continue into stationary phase, or treated with antibiotics (CAM, 10 jig/ml; PEN, 5 ,ug/ml) for 1 h at 37°C. LTA was extracted and purified as described in Materials and Methods. Gel electrophoresis was performed as described in the text. Lane 1, Dye markers; lane 2, cdLTA from stationary-phase cells (1 ,ug of organic phosphate); lane 3, cdLTA from mid-exponential-phase cells (1 p.g of organic phosphate); lane 4, cdLTA from CAM-treated cells (1 ,ug of organic phosphate); lane 5, cdLTA from PEN-treated cells (1 pg of

organic phosphate).

resulted in a change in the electrophoretic migration profile to that observed for LTA extracted from untreated cells (21). To distinguish between these two possibilities, a comparison was made between the cdLTAs from S. agalactiae and E. hirae following treatment with CAM. As shown in Fig. 7, inhibition of protein synthesis with CAM resulted in a shift in the distribution of cdLTA towards the lower-molecular-

FIG. 7. Comparison of the electrophoretic migration profiles of cdLTA from S. agalactiae and E. hirae treated with CAM. One liter of cells were grown to mid-exponential phase in a chemically defined medium and divided into two 500-ml portions. CAM (10 ,ug/ml) was added to one of the 500-ml cultures. The cultures were incubated for 1 h at 37°C. LTA was extracted and purified as stated in Materials and Methods. Gel electrophoresis was performed as described in the text. Lane 1, Dye markers; lane 2, cdLTA from S. agalactiae (1 ,ug of organic phosphate); lane 3, cdLTA from S. agalactiae plus CAM (1 pLg of organic phosphate); lane 4, cdLTA from E. hirae (1 jig of organic phosphate); lane 5, cdLTA from E. hirae plus CAM (1 jig of organic phosphate); lane 6, cdLTA from E. hirae plus CAM (2 jig of organic phosphate).

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J. BACTERIOL.

TABLE 2. Turnover of LTA in S. agalactiae Sampling time

(min)

[3H]glycerol [kdpm (% of total incorporated)] in: LTA and

LTA from

andolTA whole froms cells

dLTA (min) dLTA

0 15 30 60

5.72 5.11 6.11 6.61

3.10 (54.2) 2.44 (47.8) 2.37 (38.8) 2.02 (30.6)

dLTA from whole cells and

supernatant

2.62 (45.8) 2.67 (52.2) 3.74 (61.2) 4.59 (69.4)

weight range in S. agalactiae (Fig. 7, lanes 2 and 3). Although there was an apparent shift in the electrophoretic migration of cdLTA from CAM-treated cells of S. agalactiae, no significant change was observed for cells of E. hirae (Fig. 7, lanes 4, 5, and 6). Any increased glucosylation of LTA in E. hirae did not seem to alter the electrophoretic migration of the cdLTA in this system. This result suggested that the apparent change in the distribution of cdLTA from CAMtreated cells did not appear to be a consequence of increased glucosyl substitution, but may reflect an inability to synthesize the larger polymers or inhibition of further production of LTA and degradation of existing polymers. Turnover of LTA in S. agalactiae. It has been demonstrated that not only does inhibition of protein synthesis prevent the synthesis of LTA, but turnover continues in the absence of further protein synthesis. The fate of this polymer was examined as cells made the transition into the stationary phase of growth. S. agalactiae 110 was labeled with [3H]glycerol and allowed to grow to mid-exponential phase, at which point cells were washed free of the label and suspended in radiolabel-free, conditioned, mid-exponentialphase supernatant fluid containing 15 ,ug of unlabeled glycerol per ml. Aliquots (100 ml) were taken at 15- and 30-min intervals until the culture reached stationary phase. After a 60-min chase with unlabeled glycerol, 30% of the total [3H]glycerol was present in the LTA, compared with 50% present at the initial time point (Table 2). The decrease in label present in LTA with a concomitant increase of label into the dLTA fraction (cell surface and extracellular) suggests that this amphiphilic molecule may be turned over by a deacylation event. DISCUSSION

By adapting a procedure developed for analysis of the Kl a method was developed for examining the polymer length of a negatively charged polyol molecule, LTA. This technique involved separation of purified native or cdLTA by gel electrophoresis with a 30 to 40% linear polyacrylamide gradient. The polymers could then be visualized with an alcian bluesilver stain protocol that stains negatively charged polysaccharides. Native LTA migrated as a smear in the upper two-thirds of the gel. Better resolution was obtained if the fatty acids were first removed from the polymer to disrupt the ability of the amphiphile to form micelles. cdLTA migrated as a ladder of discrete bands in the bottom third of the gel and resembled the well-described banding pattern reported for lipopolysaccharide (13) and, recently, teichuronic acid from Micrococcus luteus (39). However, the polymer differed from other polysaccharides (13, 34) and macromolecules (22) in that it migrated ahead of the bromophenol blue dye front, which is normally used in other

polysaccharide capsule of Escherichia coli (34),

systems to mark the extent of migration. In addition, this technique required a higher percentage (>15%) of polyacrylamide to resolve the cdLTA into individual bands. The mass/charge ratio appeared to be equal and similar to the mass/charge ratio of DNA, and therefore LTA was separated in this system on the basis of size, since limited acid hydrolysis resulted in the disappearance of slower-migrating bands concomitant with the appearance of faster-migrating bands. This is consistent with the hydrolysis of highermolecular-weight polymers to lower-molecular-weight and faster-migrating molecules. This was further supported by analysis of LTA fractionated according to size by gel filtration column chromatography. The LTA peak that eluted from the Sephadex G-100 column in the 4,000- to 8,000-Da range migrated diagonally when fractions were electrophoresed on a 30 to 40% polyacrylamide gradient gel. This result is similar to those observed for lipopolysaccharide from E. coli (35), Salmonella typhimurium (35), Salmonella minnesota (35), and Pseudomonas aeruginosa (36). Using this method, we conducted a survey of LTAs from other gram-positive bacteria for which this molecule has been fairly well characterized. In all organisms examined, the cdLTA appeared as a heterogeneous population of molecules. The electrophoretic migration profiles of cdLTAs among the streptococci appeared to be similar in the molecular weight range exhibited by this polymer, although differences were seen in the staining intensity within the population of molecules. However, E. hirae produced highermolecular-weight cdLTA, and the lower-molecular-weight bands detected in the other bacteria were not evident in this organism. It is quite evident from these results that LTA is heterogeneous in size and that past reports of polymer length are actually an average of chain length (12). It is apparent from this study that PEN and CAM have markedly different effects on LTA synthesis in S. agalactiae. Inhibition of peptidoglycan synthesis with PEN resulted in the enhanced synthesis and release of this amphipathic molecule. While PEN has been shown to promote release of LTA in a number of gram-positive bacteria (1, 4, 16, 18, 25), it has different effects on synthesis of the polymer. In the oral streptococci, PEN inhibited synthesis of LTA in S. sanguis (16) while augmenting synthesis in S. mutans (3). Recently, it was shown that secretion of LTA in Lactobacillus casei was the result of continued phospholipid synthesis following cessation of growth and blebbing of the cytoplasmic membrane from the septum of these cells (35a). Enhanced or continued phospholipid synthesis would supply substrate for polymerization, since diphosphatidylglycerol has been shown to serve as the precursor in the polymerization of LTA (5, 11). In marked contrast to these results, CAM was found to inhibit LTA synthesis and result in its eventual loss from the cell surface as a consequence of deacylation. Cessation of lipopolysaccharide synthesis as a result of inhibition of protein synthesis has also been reported in E. coli (17). Synthesis of lipopolysaccharide was shown to be regulated by the stringent response, since CAM could "relax" the inhibition in amino acid-starved cells (17). It was established in the present study that CAM caused an apparent shift in the electrophoretic migration profile of cdLTA from S. agalactiae towards the lower-molecular-weight range. CAM appeared to change the electrophoretic migration of LTA in E. hirae, causing an apparent increase in the molecular weight of this polymer (21). However, the apparent shift was attributed to the increased carbohydrate substitution that occurred as a consequence of protein synthesis inhibition

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(21). In this study, no apparent change in the electrophoretic migration of the polymer could be seen for E. hirae LTA (Fig. 7). These results suggest that cessation of protein synthesis during the transition from exponential to stationary phase may be the consequence of inhibition of enzyme synthesis necessary for production of the higher-molecularweight form of the polymer or enhanced degradation and/or release of the high-molecular-weight LTA. Release of LTA from S. agalactiae during growth appeared to occur by deacylation, as suggested from pulse-chase data (Table 2). The amount of total (supernatant and cell surface) dLTA increased with a concomitant decrease in native LTA. This result is similar to those of pulse-chase studies examining the precursor-product relationship between intracellular and extracellular LTA in E. hirae (20). E. hirae also appeared to release LTA in this form because of a deacylase present in the cytoplasmic membrane (19). These results differ from the data obtained for S. pyogenes and S. mutans, which demonstrate that both forms of the polymer (native and dLTA) are present in the supernatant fluid (1, 18). It is anticipated that this procedure will prove useful in future studies of LTA, particularly in the physiological and genetic analysis of the regulation of LTA synthesis and the influence of environmental factors on its expression and biological activities. ACKNOWLEDGMENTS We thank Elizabeth K. Eskew and Ellen Snow for their valuable technical assistance. This research was supported by grant AI-22380 from the National Institutes of Health. J.J.M. was supported by Public Health Service training grant T32 AI-07271 from the National Institutes of Health. REFERENCES 1. Alkan, M. L., and E. H. Beachey. 1978. Excretion of lipoteichoic acid by group A streptococci: influence of penicillin on excretion and loss of ability to adhere to human oral epithelial cells. J. Clin. Invest. 61:671-677. 2. Beachey, E. H., and H. S. Courtney. 1987. Bacterial adherence: the attachment of group A streptococci to mucosal surfaces. Rev. Infect. Dis. 9(Suppl.):S475-S481. 3. Brissette, J. L., and R. A. Pieringer. 1985. The effect of penicillin on fatty acid synthesis and excretion in Streptococcus mutans BHT. Lipids 20:173-179. 4. Brissette, J. L., G. D. Shockman, and R. A. Pieringer. 1982. Effects of penicillin on synthesis and excretion of lipid and lipoteichoic acid from Streptococcus mutans BHT. J. Bacteriol. 151:838-844. 5. Cabacungan, E., and R. A. Pieringer. 1981. Mode of elongation of the glycerolphosphate polymer of membrane lipoteichoic acid of Streptococcus faecalis ATCC 9790. J. Bacteriol. 147:75-79. 6. Chan, R. C. Y., G. Reid, R. T. Irvin, A. W. Bruce, and J. W. Costerton. 1985. Competitive exclusion of uropathogens from human uroepithelial cells by Lactobacillus whole cells and cell

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