(Received for publication, November 23, 1994; and in revised form, May 19, 1995)
From the
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.
Hyaluronic acid is a high molecular weight linear
glycosaminoglycan composed of repeating subunits of 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. (
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 (
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
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
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
Figure 4:
Northern blot analysis of RNA isolated
from encapsulated and unencapsulated strains of group A streptococci.
Total RNA (10 µg) was probed with
[
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
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
[
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
[
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.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
1,4-linked
disaccharides of glucuronic acid
1,3-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) .
)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.
)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.
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 (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
-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 -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.
.
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-C]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
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, 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 [
-
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
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
, 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.
[
Oligo D2 (100 -
P]ATP End-labeled
Probe
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
[-
P]dATP (ICN Biomedicals, Costa Mesa, CA).
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.
, 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.
, 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.
-
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.
= 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.
-
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.
-
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).
If further experimentation confirms the
existence of the rho-independent terminator, the has operon
will be comprised of only hasA, hasB, and hasC.
We are indebted to B. Dougherty (Johns Hopkins Univ),
I. Blomfield, and D. Wozniak for their constructive comments and
advice.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.