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(Received for publication, August 9, 1995; and in revised form, September 27, 1995) From the
Hyaluronic acid is a high molecular weight glycosaminoglycan
composed of repeating subunits of glucuronic acid and N-acetylglucosamine. It is synthesized by the group A
streptococcal membrane-associated enzyme hyaluronate synthase. In
previous reports, the locus required for expression of hyaluronic acid,
the has operon, was identified and found to consist of two
genes, hasA and hasB encoding hyaluronate synthase
and UDP-glucose dehydrogenase, respectively. Since a transcription
terminator was not found at the end of hasB, it was the aim of
this study to identify the remaining gene(s) in the has operon. By utilizing the Tn1000 method of DNA sequencing
and inverse polymerase chain reaction, hasC, the third gene in
the has operon was shown to be 915 base pairs in length (304
amino acids) and located 192 base pairs downstream of hasB.
Sequence similarities to other genes suggested that hasC
encodes UDP-glucose pyrophosphorylase. Overexpression of hasC
using isopropyl-1-thio-
Group A streptococci (Streptococcus pyogenes) are human
pathogens that colonize the skin and mucous membranes of their host.
They cause localized infections and nonsupperative sequelae such as
post-streptococcal glomerulonephritis and rheumatic fever(1) .
Prior to 1985, the occurrence of lethal streptococcal infections was
quite rare. However, a rise in the number of streptococcal diseases
within the past decade has led to increased investigation into the
mechanisms involved in the resurgence of disease. One potential
explanation for this increased pathogenicity of group A streptococci
could be that the bacteria have evolved enhanced virulence determinants
that assist in the pathogenesis of the organism. The primary virulence
factor for group A streptococci is the M protein. M protein is a
coiled-coil dimeric molecule present on the surface of streptococci
that has been shown to interfere with phagocytic uptake of the organism
by neutrophils and protect the organism from clearance(2) . The
hyaluronic acid capsule has also been shown to be involved in the
pathogenicity of group A streptococci. It has been observed that
clinical isolates from outbreaks of rheumatic fever are highly
encapsulated as compared to nonpathogenic
strains(3, 4) . Three percent of isolates from
patients with uncomplicated pharyngitis (group I) possess a hyaluronate
capsule, whereas 21% of isolates from severe streptococcal infections
(group II), and 42% of rheumatic fever isolates (group III) exhibit a
capsule. However, in the most frequently isolated group A streptococcal
serotype, M1, the frequency of capsule production is even greater: 6%
for group I, 22% for group II, and 80% for group III. These data
support the hypothesis that the hyaluronic acid capsule is a major
virulence determinant for group A streptococci in the human host. To
demonstrate that the hyaluronic acid capsule is necessary for
pathogenesis of group A streptococci, Wessels et
al.(5, 6) have created acapsular mutants via
transposon mutagenesis that exhibited increased sensitivity to
phagocytic killing in human blood, whereas the encapsulated wild-type
strains survived killing. These same acapsular mutants were also less
virulent in mice as compared to the wild-type streptococci. In
addition, Wessels and Bronze (7) showed that the hyaluronate
capsule is necessary for early colonization of the pharynx and may be
required for invasion of group A streptococci from the pharynx to
produce a disseminated infection. These results suggested that the
hyaluronic acid capsule may provide a selective advantage over the
unencapsulated phenotype for group A streptococci. Hyaluronic acid
is a high molecular weight glycosaminoglycan that is synthesized by the
alternate addition of UDP-glucuronic acid and
UDP-N-acetylglucosamine by the streptococcal
membrane-associated enzyme hyaluronate synthase(8) . The
chromosomal locus that is necessary for the production of the
hyaluronic acid capsule (has operon) has recently been
identified(9, 10, 11, 12, 13) and is conserved in all strains of group A streptococci
and encapsulated group C streptococci(6, 14) . The has operon was found to consist of at least two genes, hasA and hasB. hasA was shown to encode
hyaluronate synthase(11, 12, 13) . DeAngelis
and Weigel (15) have created two monospecific antibodies raised
against synthetic peptides corresponding to portions of HasA. Both
antibodies recognized a 42-kDa protein from group A streptococci and a
recombinant protein from Escherichia coli containing hasA on a plasmid. hasB was shown to encode
UDP-glucose dehydrogenase(10) . Furthermore, a transposon
insertion in hasA created a polar effect on hasB
expression, providing evidence that the genes are transcribed by the
same promoter. Since sequence analysis of DNA downstream of hasA did not reveal any terminator-like
sequences(11) , the hasA promoter could regulate
transcription of the entire operon and thereby produce a polycistronic
message. Recently, it was demonstrated by Northern blot analyses that hasA, hasB, and hasC are located on the same
mRNA transcript (4.2 kb), ( In this
report, the sequence of the third gene (hasC) of the has operon is presented. hasC is located 192 nucleotides
downstream of hasB and contains a potential rho-independent terminator at the 3-prime end of the gene,
thus suggesting that hasC is the last gene in the has operon. Sequence comparisons and T7 overexpression of hasC demonstrated that hasC encodes UDP-glucose
pyrophosphorylase, an enzyme which catalyzes the production of
UDP-glucose from glucose 1-phosphate and UTP.
To sequence the remaining 3-prime
fragment of hasC, chromosomal DNA from streptococcal strain
WF51 was digested with XbaI and BglII and
electrophoresed, and a 3-5.5-kb region was extracted from the gel
and purified using The complete sequence of hasC was determined by utilizing
the fragment assembly program of the GCG software package(21) .
BESTFIT and PILEUP, additional GCG programs, were used to align other
sequences in the data base with hasC. FOLDRNA was used to
determine the secondary structure of hasC.
pGAC315 was transformed into E. coli strains BL-21(DE3),
BL-21(DE3)(pLysE), and BL-21(DE3)(pLysS) in order for T7 expression
experiments. As a plasmid control, pET11a was also transformed into the
above strains. T7 overexpression of hasC was achieved as per
Studier et al.(19) . Briefly, TYPG (5 ml) was
inoculated with a single microcolony from the above transformations,
and grown at 37 °C until the optical density of the culture at 600
nm reached 0.6. At this point, 1 ml of the culture was removed as a
preinduction sample, and IPTG (1 mM final concentration) was
added to the remaining culture followed by a further incubation period.
Additional 1-ml samples were taken at 2 and 3 h post-IPTG induction,
and the samples were sedimented at 13,000 For overexpression of larger quantities of
HasC for enzymatic assays, TYPG media (50 ml) was inoculated with 500
µl of an overnight culture of BL-21(DE3)(pLysS)(pGAC315) or
BL-21(DE3)(pLysS)(pET11a) and grown to OD
Figure 1:
Nucleotide sequence of hasC
gene. The nucleotide sequence of hasC and the surrounding
regions (including the 3-prime end of hasB) is presented and
the predicted amino acid sequence is shown below the open reading
frame. A potential ribosome binding site (AGTGAGGAG) is underlined and
restriction sites are noted above the nucleotides. A potential rho-independent transcription terminator is shown in bold. The
dashed arrows represent the PCR primers (D-34a and D-37a) that were
used to synthesize hasC for T7
expression.
hasC was demonstrated
to consist of 915 base pairs (304 amino acids, approximately 33.7 kDa)
and possesses a potential Shine-Delgarno sequence (Fig. 1; RBS,
base pairs 176-184) capable of base pairing with the 3-prime
terminus of the streptococcal 16 s rRNA(27) . Weak -35
and -10 sites were observed upstream of the hasC start
codon; however, primer extension analysis did not identify a
transcription start site directly upstream of hasC (data not
shown), and Northern analyses demonstrated that hasC was part
of the 4.2-kb has operon transcript(14) . A potential
transcriptional terminator was located within the 3-prime end of hasC spanning an additional 24 base pairs past the hasC termination codon. These data suggested that hasC is the third and last gene in the has operon.
Figure 2:
Alignment of the HasC and Cps3U amino acid
sequences. An optimal alignment of HasC and Cps3U proteins was
generated using BESTFIT program from GCG software package. Bars represent identical amino acids; single dots,
similarities of <0.5 but >1.0; and double dots,
similarities that are >0.5 between the residues. The shaded
region indicates the region of identity around the active site
lysine with potato tuber enzyme. The bold lysine residue
identifies the catalytic lysyl residue defined by Katsube et
al.(33) .
Figure 3:
Analysis of bacterial lysates from T7
expression by SDS-PAGE. BL-21(DE3)(pLysS) cells containing either
pGAC315 or pET11a vector alone were grown to an optical density at 600
nm of 0.6 and induced with 1 mM IPTG. Lane 0,
bacteria removed prior to induction with IPTG. Lanes 2 and 3, samples removed 2 and 3 h postinduction, respectively. The
size of the overexpressed HasC, 36 kDa, is indicated at the left.
Sequence and homology analyses demonstrated that hasC encoded UDP-glucose pyrophosphorylase, the enzyme that in
the presence of UTP catalyzes the reaction of glucose 1-phosphate to
UDP-glucose. UDP-glucose is the substrate for the gene product of hasB, UDP-glucose dehydrogenase, responsible for the
production of UDP-glucuronic acid in the presence of NAD. Finally,
UDP-glucuronic acid is a component of the hyaluronic acid capsule for
group A streptococci which is synthesized by the membrane associated
enzyme hyaluronate synthase encoded by hasA. All three genes
have been shown to be components of the has operon(10, 11, 14) . It has previously
been shown by Southern analysis that the three genes that comprise the
operon are located on the same 8.4-kb XbaI restriction
fragment. In addition, Northern blot analyses demonstrated that hasA, hasB, and hasC are contained within a
4.2-kb mRNA transcript. Sequence data revealed that hasC is
located 192 nucleotides downstream of hasB. This is a
substantial distance for sequential genes in an operon in streptococci
where the distance between hasA and hasB is only 37
nucleotides. Potential transcription initiation sites were observed
between hasB and hasC; however, primer extension
analyses of the entire 192 nucleotide region using multiple primers did
not reveal an initiation site (data not shown) providing further
evidence that hasC is a constituent of the has operon. Since hasC is not transcribed from its own
promoter, the distance between hasB and hasC may be
important for structural requirements or interaction with proteins
involved in translation machinery. Our previous Northern analysis
data (14) and the presence of a potential rho-independent terminator at the 3-prime end of hasC
gene (Fig. 1, bold) provides strong evidence that hasC is the last gene comprising the has operon.
Characteristics of a strong terminator include dyad symmetry, an
inverted repeat containing 6-8 uracil residues, and a stem-loop
rich in G and C nucleotides. The stem of the terminator for hasC is 25 nucleotides in length and contains 6 A-T pairs, the
loop consists of 5 nucleotides (CAAAGT), and there are 16 G or C
nucleotides (Fig. 4). Although the terminator of hasC
complies with all of the requirements for a rho-independent
terminator, further experimentation is required to confirm that this
potential structure is the transcription termination site of the has operon.
Figure 4:
Hypothetical structure of the rho-independent transcription terminator of the has operon. The RNA secondary structure of nucleotides 1072-1129
of hasC was created by using GCG software FOLDRNA
program.
To overexpress hasC in E.
coli, the pET-T7 expression system was utilized. This system
employed the use of various plasmids to enhance the expression of the
gene of interest, especially genes that may be toxic to the cell. For
overexpression of hasC, transformants harboring pLysS were
found to produce maximal amounts of HasC (Fig. 3), as compared
to transformants containing pLysE or BL-21(DE3) alone (data not shown).
The higher level of lysozyme produced from pLysE has been shown to
interfere with the growth rate of the bacteria and may decrease the
amount of transcription after IPTG induction. As with the previous
expression of hasB (10) it was important to use low
copy number plasmids under tight transcription control for expression
of the gene product in E. coli. The predicted size for HasC
was approximately 33.7 kDa which is in good agreement with the product
of the T7 overexperssion of hasC in E. coli (36 kDa).
Variation in the predicted size from the size demonstrated by SDS-PAGE
also was seen with other bacterial UDP-glucose pyrophosphorylases. GalU
was shown to be 40 kDa (predicted size, 32.9 kDa) (22, 30) and CelA was 30 kDa (predicted size, 30.9
kDa) (31) . The size difference between the predicted and the
actual size determined by SDS-PAGE could be due to secondary structure
of the protein causing the protein to migrate through the gel slower
than expected. Also as with the expression analysis of group A
streptococcal UDP-glucose dehydrogenase (HasB)(10) , induction
of group A streptococcal UDP-glucose pyrophosphorylase in E. coli appears to have led to a greater expression of protein (Fig. 3) than enzyme activity. Analysis of crude extracts
indicated that enzyme activity versus protein concentration
appeared approximately linear (data not shown) and that no NADPH
oxidation was demonstrated. One important consideration is that the
enzyme is labile and/or inhibited by components of the assay system.
Toward this hypothesis it was determined that both imidazole-HCl and
Tricine-NaOH buffers greatly inhibited the streptococcal enzyme (data
not shown). Both of these buffers have previously been used for other
UDP-glucose pyrophosphorylase assays(24, 25) .
Finally, the specific activity of various preparations of the
streptococcal UDP-glucose pyrophosphorylase varied 2-4-fold when
expressed in E. coli (data not shown). This did not appear to
be due to granule formation since all of the enzyme activity could be
retrieved from the cytoplasmic fraction. UDP-glucose
pyrophosphorylase is found in a wide variety of organisms (plant,
animals, and bacteria) and provides UDP-glucose for the biosynthetic
pathways of many carbohydrates (sucrose, cellulose, and glycogen) as
well as hyaluronic acid. The most detailed work on UDP-glucose
pyrophosphorylase has been done using the cDNA from potato
tuber(34) . The cDNA has been cloned and the recombinant
protein expressed in E. coli(33) . The active site of
the enzyme was shown to include 5 key lysine residues, as shown by
site-directed mutagenesis and affinity labeling(33) . Hossain et al.(22) have demonstrated that prokaryotic
UDP-glucose pyrophosphorylases are structurally diverse from the
eukaryotic enzyme. Although HasC and other prokaryotic UDP-glucose
pyrophosphorylases do not possess sequence homology with the potato
tuber enzyme or other eukaryotic pyrophosphorylases, many contain the
lysine residue found to be indespensible for enzyme function (Fig. 2, Lys-367 from potato tuber). However, further structural
analysis of the catalytic sites of these enzymes are required in order
to confirm whether this lysine is necessary for enzymatic activity in
the prokaryote UDP-glucose pyrophosphorylases. In summary, our
previous Northern analysis data demonstrating a single 4.2-kb
transcript (14) and the observed putative rho-independent terminator at the 3-prime end of hasC
would indicate that the has operon is composed of hasA, hasB, and hasC. On going experiments
include analysis of regulatory factors that may assist in the control
of has operon transcription during exponential phase of
growth. Finally, purification of the expressed HasC protein will be
required to determine its enzyme kinetics and an analysis of its active
site is required for comparison to other prokaryote and plant and
animal UDP-glucose pyrophosphorylases to better understand the
evolutionary differences between these enzymes.
The nucleotide
sequence(s) reported in this paper has been submitted to the
GenBank(TM)/EMBL Data Bank with accession number(s)
U33452[GenBank].
Volume 270,
Number 48,
Issue of December 1, 1995 pp. 28676-28680
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
DEMONSTRATION OF UDP-GLUCOSE PYROPHOSPHORYLASE ACTIVITY (*)
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-D-galactopyranoside induction of
the T7 promoter in the pET translation system allowed for the
production of bacterial extracts from Escherichia coli that
possessed increased UDP-glucose pyrophosphorylase activity as compared
to nondetectable levels in extracts with vector alone. In addition,
expression of HasC resulted in a protein of approximately 36 kDa as
shown by SDS-polyacrylamide gel electrophoresis. These data as well as
complementation analysis of hasC in an E. coli galU
mutant confirmed that hasC encodes UDP-glucose
pyrophosphorylase. Finally, since sequence analysis identified a
potential rho-independent transcription terminator at the
3-prime terminus of the gene, hasC is the third and probably
the final gene in the has operon.
)thus confirming the observation
that the genes are components of an operon(14) .
Bacterial Strains and Plasmids
The streptococcal
strain used in this study (WF51, T18) was grown in CDM(16) . E. coli strains used were JM109, BL21(DE3), DPWC,
BW26(17) , and DEV6 (gift from the E. coli Genetic
Stock Center, Yale University; lacZ105, , galU65, relA1, spoT1, thi-1) (18) grown in Luria broth or
TYPG (16 g of tryptone, 16 g of yeast extract, 5 g of NaCl, 2.5 g of
K
PO
, and 5 g of glucose/liter of
HO; see (10) ) medium as indicated. Plasmids used
in this study include pGAC144(10) , pGAC312 (this study),
pGAC315 (this study), pMOB(17) , pBluescript (Stratagene),
pET11a(19) , pLysE(19) , and pLysS(19) .DNA Purification and Manipulations
Streptococcal
chromosomal DNA was purified as per Dougherty and van de
Rijn(9) . Plasmid DNA was purified from E. coli cultures (3 ml) by the alkaline lysis procedure (20) or
Wizard Mini-Preps (Promega, Madison, WI) and resuspended in a final
volume of 100 µl of TE (100 µl, 10 mM Tris-HCl, pH
7.5, 1 mM EDTA). DNA restriction enzymes were purchased from
Promega and used according to the manufacturer's suggestions.Cloning and DNA Sequence of the hasC Gene
The
0.9-kb EcoRI/HindIII of pGAC144 was subcloned into
the miniplasmid pMOB for sequencing utilizing the Tn1000 protocol as per Strathmann et al.(17) . Briefly,
pMOB containing the 0.9-kb EcoRI/HindIII fragment was
transformed into the host strain DPWC (which contains Tn1000 on the F factor) by the one-step method(20) . The F factor
was then transferred to the recipient strain BW26 by conjugation and
positive colonies were selected for on LB agar plates containing
carbenicillin and kanamycin (100 µg/ml and 50 µg/ml,
respectively). Twenty individual colonies were subjected to PCR to
determine the position of insertion of the transposon within the target
gene. Akaline lysis miniprep DNA (40 µl) was used as the template
for double-stranded sequencing with Sequenase version 2.0 (U. S.
Biochemical Corp., Cleveland, OH).-agarase (New England Biolabs, Beverly, MA).
The ends of the isolated fragments were blunted and ligated, creating a
cirular piece of DNA that spans from the BglII site in the
5-prime region of hasA (11) to the XbaI site
downstream of hasC. The DNA was then subjected to inverse PCR
using oligonucleotides D-10 (5`-CTTAGAACACCCACAGGTC-3`) and D-11
(5`-CATTTGGATAGATATAAGTATC-3`) as primers. The 1.4-kb PCR product was
gel-purified by -agarase and subjected to double-stranded DNA
sequencing using Sequenase version 2.0. This process starting with PCR
was repeated three times with identical results obtained each time. Complementation of E. coli galU Mutation
pGAC315
or pET11a was transformed into the E. coli galU mutant strain
DEV6 (18) , and single colonies were grown in chromogenic
medium (3 ml; 1% polypeptone, 0.5% yeast extract, 2% galactose, 0.003%
bromthymol blue, 0.5 mM IPTG, 50 µg/ml ampicillin) (22) at 37 °C. Complementation was judged successful by a
color change of the culture supernatant from green to yellow that
demonstrated the ability of the hasC-complemented mutant to
produce acid from growth in galactose.T7 Overexpression of hasC in E. coli
In order to
express hasC, chromosomal DNA isolated from WF51 was subjected
to PCR using oligonucleotide D-34a (5`-GCACATATGACCAAAGTCAGAAAAG-3`) to
incorporate an NdeI site at the 5-prime end of hasC
and oligonucleotide D-37a (5`-GCAGGATCCAAGTACCAACACATTACTTTG-3`) to
incorporate a BamHI site at the 3-prime end for further
subcloning into the translation vector pET11a. The 925-base pair PCR
product was subjected to T4 polynucleotide kinase (Promega) in order to
phosphorylate the ends and then subcloned into SmaI-digested
pBluescript II (KS) to create pGAC312. Next, hasC was removed from pGAC312 by restriction digestion with NdeI and BamHI and finally subcloned into NdeI/BamHI-digested pET11a to create pGAC315.
g for 10 s
and finally resuspended in TE. Sample buffer (60 µl) was added to
each sample and boiled for 5 min to solubilize the cells. Thirty
microliters of preinduction lysates and 10 µl of 2- and 3-h lysates
were loaded per lane on a 10% SDS-PAGE gel and electrophoresed as per
Laemmli (23) . = 0.9. A
preinduction sample (8.5 ml) was removed from the culture, IPTG (1
mM final concentration) was added to the remaining culture,
and the incubation was resumed. Aliquots (8.5 ml) were removed from
each of the cultures at 3 h postinduction. The samples were sedimented
at 10,000
g for 10 min, and the pellet was resuspended
in enzyme buffer (1 ml, 50 mM potassium phosphate, pH 8.7) for
subsequent enzyme assays.Detection of hasC Enzyme Activity
Bacterial cells
were disrupted by glass bead breakage as per Dougherty and van de
Rijn(10) . Modifications of the procedure included the buffer
used for prewashing the glass beads and resuspension of the bacterial
cells (50 mM potassium phosphate, pH 8.7), and the cells were
broken only two times in the minibead breaker. UDP-glucose
pyrophosphorylase activity was monitored using the coupled reaction
with the enzymes phosphoglucomutase and glucose-6-phosphate
dehydrogenase as described by Joshi (24) and modified by
Fjaervik et al.(25) . Potassium phosphate (50
mM, pH 8.7) was substituted for the reaction buffer since the
imidazole-HCl buffer inhibited the streptococcal enzyme. The reaction
was initiated upon addition of crude enzyme (10 µl) to a 1-ml
cuvette containing assay buffer (750 µl, prewarmed to 30 °C).
The subsequent reduction of NADP at 340 nm was monitored using a
Spectronic 2000 (Bausch & Lomb), and the reaction rate was
calculated from the initial linear portion of the curve (reduction of
NADP versus time). Only trace amounts, if any, of reducing
activity were found in the extracts in the absence of UDP-glucose, and
the values were subtracted from the data. Protein concentration was
determined by the Markwell et al.(26) assay using
duplicate samples and BSA as standard. A unit of UDP-glucose
pyrophosphorylase activity was defined as the amount of enzyme required
to produce 1 µmol of NADPH/min at 30 °C.
Cloning and DNA Sequence of the hasC
Gene
Further analysis of pGAC144, a clone that contained the
3-prime region of hasB and approximately 1 kb of additional
DNA(10) , identified an open reading frame 192 nucleotides
downstream of hasB. In order to sequence this open reading
frame, the 0.9-kb EcoRI/HindIII fragment of pGAC144
(beginning 130 nucleotides downstream of hasB) was subcloned
into the minivector pMOB and subjected to Tn1000 sequencing as
per Strathmann et al.(17) . The open reading frame,
designated hasC, was shown to initiate 62 nucleotides
downstream of the EcoRI site (Fig. 1); however, the
3-prime end of the gene (up to the HindIII site) did not
contain a stop codon. Additional experiments were initiated to
determine the sequence of the remaining portion of hasC.
Chromosomal DNA was isolated from WF51 (the prototype encapsulated
strain of group A streptococci), cloned to form a circular piece of
DNA, and subjected to inverse PCR using primers D-10 and D-11 (see
``Experimental Procedures''). The 1.4-kb PCR product was then
sequenced using hasC primers to complete the sequence of hasC. A termination codon (TAA) was found 39 nucleotides (12
amino acids) downstream of the HindIII site of pGAC144, thus
identifying the end of hasC.
Comparison of hasC to Other Bacterial UDP-glucose
Pyrophosphorylases
Sequence comparisons using the FASTA program
of the Genetics Computer Group software package (21) revealed
that hasC shows homology to other genes that encode
UDP-glucose pyrophosphorylase. HasC displayed 71.2% identity
(initn, 1682) to Cps3U from type 3 Streptococcus
pneumoniae(28) (Fig. 2), 63.5% identity
(initn, 667) to GtaB from Bacillus
subtilis(29) , 57.5% identity (initn, 432) to E. coli GalU(30) , 53.6% identity (initn,
218) to CelA of Acetobacter xylinum(31) , and 52.8%
identity (initn, 178) to Rhizobium meliloti ExoN(32) . The lysine residue essential for catalytic
activity of UDP-glucose pyrophosphorylase from potato tuber has
previously been determined(33) . Alignment of the surrounding 9
amino acid residues with HasC and Cps3U demonstrated homology with 6
residues including the catalytic lysine (Fig. 2). Further
experimentation is required to determine whether this lysine is
involved in catalysis in the streptococcal enzyme.
Complementation of E. coli GalU Mutation
To
confirm that hasC encodes UDP-glucose pyrophosphorylase,
pGAC315 or pET11a were transformed into DEV6, an E. coli K-12
strain that has a mutation in galU and is therefore deficient
in UDP-glucose pyrophosphorylase activity(18) . After growth in
chromogenic medium containing bromthymol blue, the clone containing
pGAC315 produced acid from galactose as exhibited by the color change
from green to yellow. JM109 cells (galU)
served as a positive control. However, the clone that contained the
vector without hasC or untransformed DEV6 were unable to
ferment galactose. These observations further indicated that hasC encodes UDP-glucose pyrophosphorylase.
Overexpression of hasC in E. coli
To determine the
molecular size of its gene product, hasC was subcloned under
the control of the T7 promoter in the pET11a translation vector (see
``Experimental Procedures''). For T7 expression,
transformants harboring pGAC315 or pET11a were grown in TYPG and
induced with 1 mM IPTG. Transformants containing pLysS and
pGAC315 produced the maximum amount of overexpression as compared to
transformants harboring pLysE or BL-21(DE3) alone (data not shown).
Control cells harboring pET11a did not exhibit overexpression of new
proteins (Fig. 3; pET11a, lanes 0, 2, and 3) as compared to cells containing pGAC315 (lanes 2 and 3) which demonstrated overexpression of a 36-kDa
protein at 2 and 3 h postinduction with IPTG. This band was not
enhanced in extracts of cells harboring pGAC315 prior to IPTG induction (Fig. 3, lane 0).
Analysis of UDP-Glucose Pyrophosphorylase
Activity
Following the expression of hasC in E.
coli, cell extracts were assayed for UDP-glucose pyrophosphorylase
activity as described under ``Experimental Procedures'' in
order to confirm that hasC coded for UDP-glucose
pyrophosphorylase. In these experiments, BL-21(DE3)(pLysS) harboring
the pET11a expression plasmid did not demonstrate UDP-glucose
pyrophosphorylase activity before or after induction. Similarly treated
BL-21(DE3)(pLysS) harboring pGAC315 demonstrated UDP-glucose
pyrophosphorylase activity only after IPTG induction (specific activity
0.13 unit/mg of protein). Substitution of UDP-galactose for UDP-glucose
in the assays demonstrated no activity in uninduced or IPTG-induced
organisms (data not shown).
)-D-galactopyranoside; PCR, polymerase
chain reaction; PAGE, polyacrylamide gel electrophoresis.
We are indebted to I. Blomfield who assisted with the
pET-T7 expression system and R. Paul Ross and D. Ward who provided aid
with Tn1000 sequencing.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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P. N. Ward, T. R. Field, W. G. F. Ditcham, E. Maguin, and J. A. Leigh Identification and Disruption of Two Discrete Loci Encoding Hyaluronic Acid Capsule Biosynthesis Genes hasA, hasB, and hasC in Streptococcus uberis Infect. Immun., January 1, 2001; 69(1): 392 - 399. [Abstract] [Full Text] [PDF]
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 G. G. Hardy, M. J. Caimano, and J. Yother Capsule Biosynthesis and Basic Metabolism in Streptococcus pneumoniae Are Linked through the Cellular Phosphoglucomutase J. Bacteriol., April 1, 2000; 182(7): 1854 - 1863. [Abstract] [Full Text]
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R. T. Okinaka, K. Cloud, O. Hampton, A. R. Hoffmaster, K. K. Hill, P. Keim, T. M. Koehler, G. Lamke, S. Kumano, J. Mahillon, et al. Sequence and Organization of pXO1, the Large Bacillus anthracis Plasmid Harboring the Anthrax Toxin Genes J. Bacteriol., October 15, 1999; 181(20): 6509 - 6515. [Abstract] [Full Text]
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 B. Bernish and I. van de Rijn Characterization of a Two-component System in Streptococcus pyogenes Which Is Involved in Regulation of Hyaluronic Acid Production J. Biol. Chem., February 19, 1999; 274(8): 4786 - 4793. [Abstract] [Full Text] [PDF]
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 M. Mollerach, R. Lopez, and E. Garcia Characterization of the galU Gene of Streptococcus pneumoniae Encoding a Uridine Diphosphoglucose Pyrophosphorylase: A Gene Essential for Capsular Polysaccharide Biosynthesis J. Exp. Med., December 7, 1998; 188(11): 2047 - 2056. [Abstract] [Full Text] [PDF]
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C. D. Ashbaugh, S. Albertí, and M. R. Wessels Molecular Analysis of the Capsule Gene Region of Group A Streptococcus: the hasAB Genes Are Sufficient for Capsule Expression J. Bacteriol., September 15, 1998; 180(18): 4955 - 4959. [Abstract] [Full Text]
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P. H. Weigel, V. C. Hascall, and M. Tammi Hyaluronan Synthases J. Biol. Chem., May 30, 1997; 272(22): 13997 - 14000. [Full Text] [PDF]
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