©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Hyaluronic Acid Synthesis Operon (has) Expression in Group A Streptococci (*)

(Received for publication, November 23, 1994; and in revised form, May 19, 1995)

Dinene L. Crater (§) I. van de Rijn (¶)

From the Wake Forest University Medical Center, Winston-Salem, North Carolina 27157

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The has operon is composed of three genes, hasA, hasB, and hasC that encode hyaluronate synthase, UDP-glucose dehydrogenase, and presumptively UDP-glucose pyrophosphorylase, respectively. Expression of the has operon was shown to be required for the synthesis of the hyaluronic acid capsule in group A streptococci. Previous studies indicated that some group A and group C streptococcal strains produce the hyaluronic acid capsule, while others do not. In addition, it was observed that encapsulated strains cultured in stationary phase of growth lose the hyaluronic acid capsule. Therefore, the molecular mechanisms controlling the expression of the hyaluronic acid capsule in group A streptococci was investigated. In this study, it was determined that all encapsulated and unencapsulated strains of group A streptococci as well as encapsulated group C streptococci analyzed possess the has operon locus. The acapsular phenotype was accounted for by the absence of hyaluronate synthase activity in the membrane and not the production of extracellular hyaluronidase. A has operon mRNA transcript was not expressed by unencapsulated strains of group A streptococci, whereas encapsulated strains of group A streptococci grown to mid to late exponential phase produced the hyaluronate capsule, as well as has operon mRNA. However, as the streptococci entered the stationary phase of growth, they became acapsular and this was concomitant with the loss of has operon mRNA transcript. These results were confirmed by primer extension analyses of RNA isolated from encapsulated and unencapsulated strains of group A streptococci as well as RNA prepared from encapsulated strains cultured in exponential and stationary phases of growth. Thus, the loss of has operon mRNA in unencapsulated group A streptococci, as well as growth phase regulation occurs at the previously mapped has operon promoter. These data suggested that the synthesis of the hyaluronic acid capsule for group A streptococci may be controlled by transcriptional mechanisms.


INTRODUCTION

Hyaluronic acid is a high molecular weight linear glycosaminoglycan composed of repeating subunits of beta1,4-linked disaccharides of glucuronic acid beta1,3-linked to N-acetylglucosamine. Data suggests that this polymer is synthesized by a membrane-associated hyaluronate synthase from the precursors UDP-glucuronic acid and UDP-N-acetylglucosamine (1) . Group A streptococci (Streptococcus pyogenes) have been shown to possess a hyaluronic acid capsule that is identical to the hyaluronic acid found in human connective tissue(2) . This may be the cause of its poor immunogenicity in the human host(3) .

The ability to infect a host requires resistance to the host's immune system and capsules have been shown to facilitate survival of the organism by interfering with antibodies, complement, and phagocytosis-mediated host defense mechanisms(4) . The presence of a polysaccharide capsule was shown to play a role in the pathogenesis of organisms such as Staphylococcus aureus(5) , Escherichia coli(6) , Pseudomonas aeruginosa(7) , Haemophilus influenzae(8) and Streptococcus pneumoniae(9) . Recently, Wessels et al.(10) demonstrated that acapsular mutants of group A streptococci resulted in a loss of virulence(10) . van de Rijn (11) previously demonstrated that strains of group A streptococci only expressed hyaluronate synthase activity and hyaluronic acid capsule during the exponential phase of growth. Loss of capsule formation during the stationary phase of growth correlated with the loss of hyaluronate synthase activity.

Attempts by our laboratory and others to purify hyaluronate synthase to homogeneity resulted in loss of enzyme activity. Therefore, an alternative approach involving identification of the genes necessary for expression of the hyaluronate capsule was devised. Dougherty and van de Rijn (12) created acapsular mutants via transposon mutagenesis that proved to be negative for hyaluronate synthase activity as compared to wild type strains of group A streptococci. Additional investigations led to the definition of a genetic locus for hyaluronic acid synthesis (has). The has operon is composed of at least 3 genes: hasA, hasB, and hasC (see Fig. 1). The first gene in the operon, hasA, codes for hyaluronate synthase(3, 13) . Amino acid sequence comparison indicated that hasA possesses homology to a family of proteins involved in polysaccharide production(12) . DeAngelis and Weigel (14) recently created polyclonal antibodies against synthetic peptides of hasA that recognize a 42-kDa protein and these antibodies depleted hyaluronate synthase activity from functional detergent extracts of streptococcal membranes. The second gene, hasB, encodes UDP-glucose dehydrogenase, the enzyme that catalyzes the conversion of UDP-glucose to UDP-glucuronic acid(15) . The third gene, hasC, possesses sequence homology to UDP-glucose pyrophosphorylase. (^1)Furthermore, DeAngelis et al.(16) performed complementation analyses which indicated that hasA and hasB are sufficient for hyaluronic acid production in their acapsular mutants, as well as in Enterococcus faecalis and E. coli(16) . Deletions in either of the genes resulted in loss of capsule synthesis. Together, these data suggested that the has operon contains the genes necessary for the synthesis of the hyaluronic acid capsule.


Figure 1: Restriction map of group A streptococcal DNA for the locus involved in hyaluronic acid capusle synthesis (the has operon). A, restriction sites are abbreviated as follows: X = XbaI; H = HindIII; C = ClaI; EI = EcoRI; EV = EcoRV; B = BstXI. The solid arrow designates the transcription start site and the dotted arrow represents the length of the has operon transcript. The solid lines indicate the probes used for Southern and Northern blot analyses; 1, 1.4-kb XbaI/ClaI polymerase chain reaction fragment; 2, 0.9-kb EcoR I/HindIII fragment of pGAC144(15) ; 3, 1.4-kb XhoI/EcoRI fragment of pGAC146(15) ; 4, 0.6-kb HindIII internal fragment of pGAC126(12) ; 5, 1.9-kb BstXI/HindIII fragment of pGAC130(12) ; 6, 2.6-kb XbaI/HindIII fragment of pGAC112(12) . B, schematic representation of restriction fragments observed by Southern blot analyses. Dashed bars represent heterogeneity of restriction fragments of designated strains.



The above data indicated that the has operon is responsible for the synthesis of the hyaluronate capsule in group A streptococci. However, the regulation of expression of the has operon remained to be determined. Since the genes that encode other streptococcal virulence factors (i.e. M protein and C5a peptidase) were shown to be transcriptionally regulated(17) , one can reason that the has operon is controlled in a similar manner. Primer extension analysis identified the transcription start site of hasA, as well as upstream promoter consensus sequences. Sequence analysis of DNA immediately downstream of hasA did not reveal any terminator-like structures(3) , indicating that the has operon promoter could regulate transcription of the entire operon, thus producing a polycistronic mRNA. In addition, the transposon insertion into hasA exhibited a polar effect on hasB expression, providing further evidence that hasA, hasB, and hasC are transcribed by the same promoter.

In this paper, we show that all encapsulated strains of group A streptococci possess the has operon as demonstrated by a single 4.1-kb (^2)mRNA product. Northern blot and primer extension analyses indicated that the level of this transcript decreased as the bacteria entered the stationary phase of growth where upon the strains lost their hyaluronic acid capsules. In addition, unencapsulated strains of group A streptococci possess the has operon genes but do not exhibit measurable amounts of has operon mRNA. Taken together, these data imply that the synthesis of the hyaluronic acid capsule may be regulated by transcriptional mechanisms.


EXPERIMENTAL PROCEDURES

Bacterial Strains and Media

The bacterial strains used in these studies are listed as follows. Encapsulated group A streptococci: A486 (T26), A995 (T57), B438 (T18), B915 (T49), B920 (T4), S43/192/1 (T6), T9 (T9), T12/126 (T12), T27A(T27), 4-64 (T3), 5-19 (T3) WF50 (T18), WF51 (T18); unencapsulated group A streptococci: B361 (T2), B429 (T4), B931 (T2), D420 (T41), D471 (T6), D480 (T1), D678 (T11), F301 (T49), F302 (T12), GT8670 (T49), IRP41 (T28), NZ131 (T49), T22 (T22), T25(3) (T25), T4/95/3 (T4), WF62 (T18), WF200 (Not typed), WF210 (T56); encapsulated group C streptococci: D181; unencapsulated group C streptococci: C-74, 26RP66; other unencapsulated streptococci: A580 (group B), 090R (group B), D166B (group G), K208 (group H), D167B (group L), D167A (group M), SBE2 (S. faecium), SBE3 (E. faecalis), SBE4 (S. salivarius), SBE8 (S. milleri), SBE9 (S. bovis). Unless otherwise noted, streptococci were grown at 37 °C in chemically defined media (CDM(18) ). Growth of bacteria was measured by optical density using a Spectronic 20 (Bausch & Lomb, Rochester, NY) at a wavelength of 650 nm.

Plasmids and DNA Manipulations

Plasmids used in this study include pGAC112(12) , pGAC126(12) , pGAC130(12) , pGAC142(3) , pGAC144 (15) , and pGAC146(15) . For streptococcal chromosomal DNA isolation, CDM (50 ml) supplemented with an additional 0.02 M glycine was inoculated with 0.5 ml of streptococci (OD of 0.4) and grown overnight at 37 °C. The culture was then sedimented at 10,000 g for 5 min and treated as per Dougherty and van de Rijn(12) . The final DNA preparation was resuspended in 0.2 ml of TE and stored at 4 °C. In order to create the 1.4-kb probe for the region downstream of hasC, chromosomal DNA from WF51 was digested with XbaI and BglII, electrophoresed, and a 3-5.5-kb region was extracted from the gel and purified using beta-agarase (New England Biolabs). The ends of the fragment were blunted and ligated which created a circular piece of DNA that spans from the BglII site in the 5`-region of hasA to the XbaI site downstream of hasC. The DNA was then subjected to inverse polymerase chain reaction using D-10 (5`-CTTAGAACACCCACAGGTC-3`) and D-11 (5`-CATTTGGATAGATATAAGTATC-3`) as primers. DNA restriction enzymes were obtained from Promega Corp. (Madison, WI) and used according to the manufacture's suggestions.

Southern Blot Analysis

Restriction enzyme-digested DNA from agarose gels was transferred to Magnagraph nylon membranes (MSI, Westboro, MA) by capillary action(19) . The membranes were then subjected to DNA/DNA hybridizations according to the Genius system (Boehringer Mannheim) and modified as follows: after incubation with alpha-digoxigenin alkaline phosphatase antibody, the excess antibody was removed by washing the filter in 1 POST-SAAP (0.05 M Tris, pH 10.0, 0.1 M NaCl), 4 times for 20 min at room temperature with a 5-min wash in distilled water between each POST-SAAP wash. The hybridization was detected using Lumi-Phos 530 and bands were visualized by exposure of the treated membranes to XAR-5 film (Eastman Kodak Co., Rochester, NY) at room temperature for 5 min.

Hyaluronidase Assays

Bacteria were grown to either exponential or stationary phase of growth, sedimented at 10,000 g, and the supernatant was saved for further experimentation. Two different assays were used to test for the presence of hyaluronidase. The agar plate assay utilized a Petri dish which contained Noble agar (1%), hyaluronic acid (400 µg/ml, Miles Laboratories), bovine serum albumin (1%, fraction V, Sigma), and sodium azide (0.1%)(20) . Holes were punched into the agar and supernatant (10 µl) was placed in each well. A positive control consisted of purified bovine testes hyaluronidase (1 unit, Worthington Biochemical Corp.), whereas uninoculated medium served as a negative control. The plate was then incubated at 37 °C overnight and analyzed for a zone of clearing around the well indicating the presence of hyaluronidase.

The dye binding assay of Hotez et al.(21) was used as a second method to determine whether hyaluronidase was present in bacterial supernatants. Briefly, supernatant (20 µl) was incubated with purified hyaluronic acid (4 µg, Miles) in a sodium acetate buffer (0.05 M NaOAC, ph 6.0; final volume = 100 µl). The reaction was then incubated at 37 °C for 0-24 h, stopped by the addition of 0.9 ml of Stains-All solution (17 mg Stains-All (Eastman Kodak), 50% formamide, 0.06% glacial acetic acid in a final volume of 100 ml) and the absorbance was then read at A.

Hyaluronic Acid Synthesis

The presence of a hyaluronic acid capsule was initially identified by India ink staining(11) . In order to detect hyaluronate synthase activity, membranes were prepared by phage lysin treatment of group A streptococci and solubilized according to the procedure by van de Rijn and Drake(22) . Membranes were detergent extracted and the analysis of the transfer of UDP-[U-^14C]glucuronic acid to hyaluronic acid was monitored by a spin column assay as described by Dougherty and van de Rijn(12) .

Protein Determination

Protein quantitations were accomplished using the bicinchoninic acid assay (BCA; Pierce Chemical Co.). Since buffer A from the membrane extraction procedure interferes with the BCA protein assay, all samples were treated according to the protocol used by Dougherty and van de Rijn(12) .

RNA Isolation

CDM (100 ml, supplemented with an additional 0.02 M glycine) was inoculated with 1 ml of a streptococcal culture grown to an optical density of 0.4 at A. The bacteria were incubated at 37 °C until the desired OD was reached. At this time, hyaluronidase was added to a final concentration of 35 µg/ml and the culture was chilled on ice for 5 min. The bacteria were then sedimented at 10,000 g and the pellet resuspended in 10 ml of protoplasting buffer (30% raffinose with 0.01% MgCl(2) and 0.05 M sodium phosphate buffer, pH 6.1) per gram of bacteria. Phage lysin was added (50,000 units/g of bacteria) and the cultures were incubated at 37 °C for 5 min and then immediately placed on ice. The mixture was then sedimented at 8,000 g and the pellet was resuspended in RNAsol B (3 ml/0.5-g pellet; Tel-Test, Inc., Friendswood, TX). RNA was extracted according to the manufacturer's suggestion. The final RNA pellet was resuspended in EDTA (0.3 ml, 1 mM, pH 8.0) and stored at -70 °C. RNA concentrations were measured by reading the absorbance at an optical density of 260 nm.

Northern Blot Analysis

Total RNA (10 µg) isolated from strains of group A streptococci was separated on a 1.1% agarose-formaldehyde gel, transferred onto Magnagraph nylon membranes (23) , and then bound to the nylon membrane by UV cross-linking (UV Stratalinker 2400, Stratagene). The filters were prehybridized in Church-Gilbert solution (0.5 M NaPO(4), pH 7.0, 0.01 M EDTA, 7% SDS, 1% bovine serum albumin) (24) for 2 h at 65 °C. Filters were hybridized in Church-Gilbert solution containing [alpha-P]dCTP-labeled random primed DNA probes (Boehringer Mannheim). The probes used for these experiments are shown in Fig. 1A. Following hybridization the filters were washed 2 times at room temperature and 2 times at 65 °C in 2 SSC, 0.1% SDS. The resulting RNA filters were finally analyzed by autoradiography after a 30-min exposure at -70 °C.

Primer Extension

RNA (30 µg) was ethanol precipitated and resuspended in diethyl pyrocarbonate-treated water (7 µl). The RNA or control (RNA absent) reactions were then annealed to end-labeled oligonucleotide D2 (complementary to the hasA gene: 5`-CCTACAGTTGATGTTCC-3`(3) ; 5 10^4 counts/min) in annealing buffer (1.0 M KCl, 100 mM Tris-HCl, pH 8.3; final volume of 10 µl). The reactions were heated at 80 °C for 5 min and slow cooled to 42 °C. The reactions were then incubated at 42 °C for 2 h. After the annealing reaction, 9.9 µl of extension mixture (2 µl elongation buffer (0.9 M Tris-HCl, pH 8.3, 100 mM MgCl(2), 100 mM dithiothreitol), 1 µl of dNTP (4 mM each dATP, dCTP, dGTP, dTTP), 0.5 µl of RNAsin (Promega, 20 units), 5 µl of diethyl pyrocarbonate-treated water, 1.0 µl of avian myeloblastosis virus reverse transcriptase (Promega)) was mixed with the annealing reactions and incubated at 42 °C for an additional 30 min. The reactions were stopped by the addition of phenol/chloroform/isoamyl alcohol (25:24:1), extracted 1 time, and finally precipitated with ethanol. The final pellets were resuspended in formamide stop solution (1:1 with diethyl pyrocarbonate-treated water, 10 µl), heated to 65 °C for 10 min, and run adjacent to a sequence ladder of DNA primed with the same oligonucleotide in order to size the product.

[-P]ATP End-labeled Probe

Oligo D2 (100 g) was incubated with [-P]ATP (100 µCi, ICN Biomedicals, Costa Mesa, CA), 10 polynucleotide kinase buffer (2.5 µl), and T4 polynucleotide kinase (4 units, Promega, Madison, WI) at 37 °C for 30 min, then 65 °C for 5 min to inactivate the kinase. Unincorporated radiolabel was removed by passing the mixture through Sephadex G-25 spin columns (5 Prime-3 Prime, Boulder, CO). One microliter of the reaction was used to measure specific activity of the probe.

DNA Sequencing

pGAC142 (5 µg) served as the template for sequencing. The DNA was denatured in 0.2 M NaOH at 37 °C for 30 min, and then precipitated with ethanol. This DNA was subsequently sequenced by the chain termination method (25) using Sequenase 2.0 kit (U. S. Biochemical Corp.) and [alpha-P]dATP (ICN Biomedicals, Costa Mesa, CA).


RESULTS

Southern Blot Analysis of Streptococcal Strains

Previously, our laboratory and others have identified a chromosomal locus (has) of encapsulated group A streptococci that is necessary for the production of a hyaluronic acid capsule(3, 10, 12, 13, 15, 16) (Fig. 1A). It was yet to be determined if the has locus is ubiquitously present in all strains of streptococci or solely in encapsulated strains of group A streptococci. To examine this, chromosomal DNA was isolated from 13 strains of encapsulated and 18 strains of unencapsulated group A streptococci and the DNAs were digested with XbaI. Southern blot analysis of the DNA from both encapsulated and unencapsulated group A streptococcal strains when probed separately with the hasA, hasB (data not shown), and hasC genes demonstrated that all three genes hybridized to the same restriction fragment, which confirmed the hypothesis that hasA, hasB, and hasC were linked (Fig. 2). However, when the filters were hybridized with hasC, the appearance of minor hybridizing bands were evident in both encapsulated and unencapsulated strains (Fig. 2). This indicated the possibility that either multiple copies of hasC exist on the chromosome of these strains or that they possess a gene that is similar in sequence to hasC.


Figure 2: Southern hybridization of encapsulated and unencapsulated strains of group A streptococci. Whole cell DNA was digested with XbaI and probed with digoxigenin-UTP-labeled hasA, hasB (data not shown) or hasC. Cap, encapsulated strains (lanes 1-3); Cap, unencapsulated strains (lanes 4-7). Lane 1, WF51; lane 2, S43/192; lane 3, T12/126; lane 4, WF200; lane 5, WF210; lane 6, NZ131; lane 7, GT8760. The sizes of the respective bands are indicated at the left in kilobases.



Some heterogeneity of the size of the XbaI fragment was evident between strains (8.4-11 kb), but this did not correlate with the capsular phenotype (Fig. 2). In order to confirm that there were no deletions or insertions within the operon coding region and surrounding sequences, DNA was isolated from representative strains and digested with restriction enzymes (HindIII/XbaI, EcoRI/HindIII, and EcoRV/EcoRI; see Fig. 1A). The digested DNAs were then electrophoresed, blotted onto nylon filters, and probed independently with six probes that span the entire XbaI restriction fragment (see Fig. 1A). Analysis of the various restriction digests demonstrated that DNA from the encapsulated and unencapsulated strains possessed identical fragments within and downstream of the has operon coding region (Fig. 1B). Heterogeneity of two fragments was observed upstream of the promoter region. Strain T12/126 possessed a 3.1-kb HindIII/XbaI fragment as compared to a 2.6-kb fragment for all other strains tested and strain S43/192 exhibited a 1.2-kb insertion upstream of the promoter. Since both of these strains are encapsulated, the heterogeneity does not appear to effect the capsular phenotype.

DNA was also isolated from 14 strains representing other groups of streptococci and probed with hasA, hasB, and hasC to determine if they possess the has operon. Only DNA isolated from the encapsulated group C streptococcal strain hybridized to the has operon probes. The encapsulated strain (D181) contained a 6.2-kb XbaI fragment as compared to the 8.4-kb fragment in group A streptococci (Fig. 3). However, the has operon probes did not hybridize to DNA isolated from unencapsulated strains of group C and G streptococci. These data indicated that the has locus may be conserved between encapsulated group A and group C streptococci.


Figure 3: Comparison by Southern hybridization of an encapsulated group A streptococcal strain to encapsulated and unencapsulated strains of group C and G streptococci. Whole cell DNA was digested with XbaI and probed with digoxigenin-UTP-labeled hasA. Cap, encapsulated; Cap, unencapsulated. Lane 1, WF51; lane 2, D181; lane 3, 26RP66; lane 4, D166B. The sizes of the respective bands are indicated at the left in kilobases.



Determination of Streptococcal Hyaluronidase Production

The above data indicated that the acapsular phenotype of unencapsulated strains of group A streptococci was not due to a major deletion within the has operon. The question remained as to why strains that encode the has locus did not produce a hyaluronate capsule. Since unencapsulated strains may secrete a higher concentration of hyaluronidase than encapsulated strains, representatives of these strains were tested for their ability to secrete this enzyme. Preliminary studies using a hyaluronic acid digestion in agar assay demonstrated that supernatants from both encapsulated and unencapsulated strains grown to exponential or stationary phase did not cause a zone of clearance to form as compared to the positive control, indicating the absence of secretion of hyaluronidase into the supernatant by these strains (data not shown). Similar results were obtained using the supernatants in the dye binding assay of Hotez et al.(21) . In addition, concentrates of the supernatants by ammonium sulfate precipitation did not demonstrate the presence of hyaluronidase. Taken together, these data provide evidence to suggest that the unencapsulated phenotype of encapsulated strains during stationary phase or unencapsulated strains containing the has operon was not due to the production of hyaluronidase.

Determination of an Active Hyaluronate Synthase in the Streptococcal Membrane

Another possibility as to why strains that encode the has locus did not produce a hyaluronate capsule was the absence of hyaluronate synthase activity in the membranes. Therefore, membranes and detergent extracts of encapsulated and unencapsulated strains of group A streptococci were prepared and assayed for hyaluronate synthase activity. As shown in Table 1, the extracts isolated from encapsulated strains WF13 and S43/192/1 at exponential phase of growth exhibited hyaluronate synthase activity (13.9 and 36.3 nmol/h/mg protein, respectively). However, the membrane extracts isolated from unencapsulated strains of group A streptococci did not possess hyaluronate synthase activity (<0.3 nmol/h/mg protein). In addition, the extracts obtained from encapsulated strains isolated during stationary phase showed negligible activity (0.3 and <0.3 nmol/h/mg protein, respectively). These results indicated that both unencapsulated strains as well as encapsulated strains of group A streptococci grown to stationary phase do not possess an active hyaluronate synthase complex in the membrane allowing for the acapsular phenotype.



Measurement of the Level and Size of has Operon Transcript

In the preceding experiments, it was shown that strains of group A streptococci, either encapsulated or unencapsulated, possess the has operon. Recently, many bacterial virulence genes have been shown to be regulated via transcriptional mechanisms(17, 26, 27, 28) . Therefore experiments were devised to determine the amount of has operon transcript in both encapsulated and unencapsulated strains of group A streptococci that possess the has operon. As shown in Fig. 4, the hasA probe hybridized to a 4.1-kb mRNA from RNA isolated from encapsulated strains of group A streptococci during exponential phase of growth (lanes 1a, b and 2a, b), whereas the probe did not hybridize to unencapsulated strains (lanes 3-6) indicating the absence of has operon mRNA. An additional 7.2-kb mRNA was observed with only one encapsulated strain (S43/192/1) at early exponential phase (Fig. 4, lane 2a) which was absent during mid-exponential and stationary phase (lanes 2b and 2c). Additionally, probing with hasB and hasC resulted in the same hybridization pattern for all strains. This suggested that hasA, hasB, and hasC were contained within the same mRNA transcript.


Figure 4: Northern blot analysis of RNA isolated from encapsulated and unencapsulated strains of group A streptococci. Total RNA (10 µg) was probed with [alpha-P]dCTP-labeled hasA, hasB, or hasC as indicated on the right. Lane 1a, WF51 OD = 0.3; lane 1b, WF51 OD = 0.6; lane 1c, WF51 OD = 1.2; lane 2a, S43/192/1 OD = 0.3; lane 2b, S43/192/1 OD = 0.86; lane 2c, S43/192/1 OD = 1.0; lane 3, WF210; lane 4, WF200; lane 5, NZ131; lane 6, GT8760. RNA run in lanes 3-6 was isolated from strains grown to mid-exponential phase of growth. The size of the major band is represented on the left in kilobases.



In an attempt to observe whether the has operon transcript was regulated between exponential and stationary phase of growth, it was necessary to identify the level of has operon transcript in encapsulated strains of group A streptococci. The capsular phenotype was established and RNA was isolated from strain WF51 at different time points in the growth curve (Fig. 5A). India ink preparations demonstrated that the capsule remained present throughout the exponential phase of growth and was totally absent approximately 2 h into stationary phase. The RNAs were then electrophoresed on an agarose-formaldehyde gel, blotted onto nylon membranes, and the hasA probe was hybridized to the membranes to detect the size and amount of has operon transcript that was present at each stage of growth (Fig. 5B). During the exponential phase of growth (A = 0.1-0.8), the hyaluronic acid capsule was present and a 4.1-kb has operon transcript was expressed. However, as the bacteria entered stationary phase, the capsular phenotype and expression of the has operon mRNA were lost (Fig. 5B, A = 1.0-1.3; Fig. 4, lanes 1c and 2c). Identical results were achieved when the filters were probed with the hasB and hasC genes (data not shown). In addition, RNA isolated from other encapsulated strains gave similar findings. These data indicated that the growth phase regulation of group A streptococci hyaluronic acid capsule synthesis may occur at the level of transcription.


Figure 5: Correlation between the presence of hyaluronate capsule and the has operon transcript in encapsulated streptococcal strain WF51. A, growth curve of WF51. Time represents hours after 1% inoculum in fresh medium. Capsule: + = encapsulated; - = unencapsulated (determined by India ink stain). B, Northern blot of RNA isolated at different ODs in the growth curve. Total RNA (10 µg) was probed with [alpha-P]dCTP-labeled hasA. The specific ODs are indicated at the top. The size of the major band (4.1) is represented at the left in kilobases.



Recently, the promoter region for hasA was identified(3) . Since no termination-like sequence was observed for hasA or hasB, it was possible that this promoter directed transcription for the entire operon. To further support the hypothesis that the has operon is regulated by transcriptional mechanisms, primer extension analysis was performed. A decrease or absence of capsule may be due to a shift to a weak promoter, resulting in a reduced rate of transcription of the has operon. The results from this approach would provide evidence as to which promoter is used during the various phases of growth in encapsulated and unencapsulated strains of group A streptococci.

Therefore, labeled oligonucleotide D2 was annealed to RNA isolated during exponential or stationary phase of growth from the different strains of group A streptococci. As shown in Fig. 6(lanes 1-3), primer extension products were present only in encapsulated strains grown to exponential phase and the product was the same size as demonstrated by Dougherty and van de Rijn(3) . RNA from strain WF51 was isolated at mid-exponential phase (OD = 0.4) and late exponential phase (OD = 0.6) (Fig. 6, lanes 1 and 2, respectively) and correlated with the presence of capsule and mRNA (Fig. 5). However, when RNA was isolated from a group A streptococcal strain (S43/192/1) grown to mid-exponential phase and stationary phase, a primer extension product was only observed for the exponential phase as compared to the stationary phase culture (Fig. 6, lanes 3 and 4). Additionally, RNA isolated from unencapsulated strains of group A streptococci during exponential phase did not produce a primer extension product (Fig. 6, lanes 5-9) which correlated with the absence of mRNA on Northern blots (Fig. 4). These data therefore provided evidence that no transcription occurred from the has operon promoter in the unencapsulated strains of group A streptococci. Taken together, these results support the hypothesis that the synthesis of the hyaluronic acid capsule is regulated via transcriptional mechanisms.


Figure 6: Primer extension analysis of RNA isolated from encapsulated and unencapsulated strains of group A streptococci. Streptococcal RNA (30 µg) was annealed to [-P]ATP-labeled oligonucleotide D2 and extended in the presence of avian myeloblastosis virus reverse transcriptase and dNTPs. Oligonucleotide D2 was also used to sequence the hasA promoter and the sequence reactions (lanes G, A, T, and C) were electrophoresed alongside the primer extension reactions. Lane 1, WF51, OD = 0.4; lane 2, WF51, OD = 0.6; lane 3, S43/192/1 OD = 0.4; lane 4, S43/192/1 OD = 1.0; lane 5, WF210; lane 6, NZ131; lane 7, WF200; lane 8, GT8760; lane 9, T22; lane 10, control (no RNA). RNA run in lanes 5-9 was isolated from strains grown to mid-exponential phase of growth. The arrow at the right represents the location of the primer extension product which correlates with the initiation of transcription (GGTCCTGTCTTT).




DISCUSSION

Group A streptococci (S. pyogenes) express a polysaccharide capsule composed of hyaluronic acid which previously was demonstrated to be encoded by a specific locus, the has operon. It was unknown whether this locus was located only on the chromosome of encapsulated group A streptococci or present in all streptococci. Wessels et al.(10) previously demonstrated that 11 group A streptococcal strains contained a 16-kb BamHI fragment which hybridized with a probe that included the has operon. The data presented in this report demonstrate that encapsulated strains as well as unencapsulated strains of group A streptococci and an encapsulated strain of group C streptococcus contain the genes necessary for hyaluronic acid capsule synthesis on a XbaI fragment. In addition, the genes required for hyaluronic acid synthesis (hasA, hasB, hasC) are located in an operon. The size of the XbaI fragment was shown to possess polymorphism between the encapsulated strains (Fig. 2). Restriction analyses indicated that encapsulated and unencapsulated strains of group A streptococci possess identical fragments immediately upstream, within, and downstream of the has operon coding region. The encapsulated strian T12/126 contains additional sequences in the 5` HindIII/XbaI fragment (0.5 kb) and strain S43/192 possesses a 1.2-kb insertion directly 3` to the HindIII site upstream of hasA (Fig. 1B). However, the unencapsulated strains contain the has operon on the same 8.4-kb XbaI fragment as one of the encapsulated strains (Fig. 2, lanes 1 and 4-7). The possibilities still exist that a minor deletion not visible by gel electrophoresis analysis was not present in the has operon of unencapsulated strains or that an essential unlinked gene required for capsule synthesis contained a deletion or mutation. These possibilities and the role of the additional sequences found within the two encapsulated strains in capsule production are currently under investigation.

Considering the fact that unencapsulated strains of group A streptococci contain the has operon, the question remained as to why they do not express a hyaluronic acid capsule. Since encapsulated strains grown to stationary phase and unencapsulated strains of group A streptococci do not produce hyaluronidase or possess hyaluronate synthase activity in membrane extracts (Table 1), it was hypothesized that the production of the hyaluronic acid capsule was controlled by gene expression. Transcriptional regulation has been established for the genes that encode for the polysaccharide capsules of E. coli and P. aeruginosa. E. coli K12 expresses a capsule that is composed of colanic acid and the mechanism of the regulatory system which includes RcsA, RcsB, RcsC, and the Lon protease has been established(6) . In P. aeruginosa, transcriptional control of alginate biosynthesis is regulated by a two-component system (algR, algB) and histone-like proteins (algP, IHF)(29) .

It has been observed that several virulence factors for group A streptococci are controlled by transcriptional regulation(17, 26, 30, 31, 32) . In preliminary experiments to determine if the production of a hyaluronic acid capsule is controlled via transcription, the capsular phenotype was correlated with the presence or absence of the has operon RNA transcript. As shown in Fig. 4, only encapsulated strains of group A streptococci grown to exponential phase exhibited the has operon mRNA. However, in all unencapsulated strains tested, the acapsular phenotype correlated with the absence of has operon mRNA. Primer extension analyses confirmed the hypothesis that transcription did not occur from the has operon promoter in unencapsulated strains. Together, the data suggested that the acapsular phenotype was due to a lack of has operon transcription from the hasA promoter. To account for the difference in capsular phenotype between encapsulated and unencapsulated strains of group A streptococci, an additional regulatory factor may be present in either of the strains. For example, the unencapsulated strains may possess a negative regulator that inhibits transcription of the has operon. Also, these strains may synthesize a factor that decreases the stability of the has operon transcript. However, in contrast, the encapsulated strains could possess additional cis- or trans-acting factors that are not present in the unencapsulated strains, but are required for the expression of the hyaluronic acid capsule.

The presence of a hyaluronic acid capsule was also correlated with the amount of has operon mRNA at different periods in the growth curve of encapsulated strains of group A streptococci (Fig. 5). Throughout exponential phase of growth, the bacteria retained the hyaluronic acid capsule and has operon mRNA was produced. However, as the bacteria entered stationary phase of growth, the level of RNA began to decrease and was absent after 2 h into stationary phase. This corresponded to a disappearance of hyaluronic acid capsule and a loss in hyaluronate synthase activity that was evident during stationary phase of growth. In addition, primer extension analyses indicated that no transcription was observed from the has operon promoter during stationary phase. These results suggested that the absence of capsule production in encapsulated strains of streptococci as they entered stationary phase of growth was due to a decrease in has operon mRNA.

The decrease in the level of the has operon mRNA that was evident in encapsulated strains of group A streptococci grown to stationary phase may be due in part to a decrease in mRNA stability in addition to a decrease in the level of transcription. There may exist a factor(s) produced during stationary phase of growth that inhibits transcription of the has operon. Additionally, the has operon mRNA may become extremely unstable at this point in the growth curve. It was previously observed that differential expression of certain operons in bacteria can be attributed to differences in segment stabilities of their polycistronic transcripts (33) . mRNA stability has been shown to control the expression of Bacillus subtilis sdh operon transcript(34) . The sdh operon consists of 3 genes: sdhC, sdhA, and sdhB, which encode the 3 subunits of the membrane-bound succinate dehydrogenase. During exponential phase of growth, the 3 cistrons are all stably transcribed in the polycistronic mRNA; however, during stationary phase, a rapid decay of the 5` end of the transcript occurs, causing a decrease in the level of sdhC transcript observed in primer extension analyses. This decrease in the amount of SdhC is the cause of lower levels of succinate dehydrogenase observed during stationary phase of growth in B. subtilis. Additionally, the 5` non-coding region of ompA in E. coli is critical in controlling the stability of ompA mRNA(33) . Although there presently is no evidence to disprove the role of mRNA stability in the regulation of the has operon, our findings still support the hypothesis that the expression of the hyaluronic acid capsule is controlled by transcriptional mechanisms.

Measurement of the has operon mRNA gave a value of approximately 4.1 kb ( Fig. 4and Fig. 5). The size of the DNA from the transcription start sight upstream of hasA to the end of hasC is 3.6 kb. The 500-base pair difference between the two results probably was due to a running anomaly of the gel or folding characteristics of the mRNA since further primer extension analyses did not detect an additional transcription start site (up to 550 base pairs upstream) of the hasA promoter (data not shown). This would indicate that the hasA promoter previously identified by Dougherty and van de Rijn (3) is the sole transcription start site for the has operon. In addition, it was observed that a 7.2-kb RNA species was present in a single encapsulated strain. Currently it is under investigation whether or not the has operon is present in this transcript or the band is due to nonspecific binding. When probing the blots with hasC, no additional transcripts were observed indicating that the minor hybridizing bands seen in the Southern analyses (Fig. 2) might have been due to nonspecific binding of the probe or that an additional copy of hasC or another gene that possesses sequence homology to hasC is not transcribed. Finally, preliminary results indicate the presence of a potential rho-independent transcription terminator at the 3-prime end of hasC.^2 If further experimentation confirms the existence of the rho-independent terminator, the has operon will be comprised of only hasA, hasB, and hasC.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grant AI37320 from the National Institutes of Health (to I. v. d. R.), North Carolina Heart Association Grant-in-Aid NC-94-SA-09 (to D. L. C.), and Oligonucleotide Core Laboratory of the Comprehensive Cancer Center of Wake Forest University Grant CA12107. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Predoctoral trainee (T32-AI-07401) of the National Institutes of Health.

To whom correspondence should be addressed. Tel.: 910-716-2263; Fax: 910-716-4204.

^1
D. L. Crater and I. van de Rijn, unpublished results.

^2
The abbreviation used is: kb, kilobase pair(s).


ACKNOWLEDGEMENTS

We are indebted to B. Dougherty (Johns Hopkins Univ), I. Blomfield, and D. Wozniak for their constructive comments and advice.


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