FEMS Microbiology Letters 30 (1985) 37-41
Published by Elsevier
37
FEM 02232
Cloning of sucrase genes from Streptococcus mutans in
bacteriophage lambda
(Genetic engineering; sugar metabolism; streptococci)
R.R.B. Russell *, P. M o r r i s s e y and G. D o u g a n
* Royal College of Surgeons of England, Downe, Kent BR67JJ, and Department of Bacteriology, Wellcome Research Laboratories,
Beckenham, Kent, U.K.
Received 24 June 1985
Accepted 3 July 1985
1. SUMMARY
Chromosomal DNA from Streptococcus mutans
strain Ingbritt (serotype c) was cloned into the
bacteriophage X replacement vector L47.1. The
bank of recombinant phage was screened for the
presence of plaques in which sucrose-hydrolysing
(sucrase) activity was expressed. Two distinct
sucrase-expressing recombinants were identified.
In one type, designated scr, the product is an
invertase-like enzyme. This enzyme was purified
from lysates of recombinants and shown to have
an apparent M r of 59000. The second class of
recombinants was found to express a glucosyltransferase, identical in size, as well as in enzymatic and antigenic properties, to the previously
described product of gtfA.
2. I N T R O D U C T I O N
The wide variety of pathways for metabolising
sucrose which Streptococcus mutans possesses is
believed to be an important factor in the ability of
the organism to cause dental caries [1]. Not only
can S. mutans use sucrose as a source of carbon
and energy but also as a substrate for a range of
enzymes synthesising extracellular polymers. The
plethora of enzymes involved in metabolism of
sucrose, both within the cell and extracellularly,
has greatly complicated the task of characterising
and purifying them [2]. Modern techniques of
recombinant DNA manipulation are well-suited to
analysis of such complex systems because the genes
responsible for individual functions can be isolated and cloned into a novel genetic background.
Thus, transferring genes for sucrose-metabolising
enzymes from S. mutans to Escherichia coli K-12,
a strain which cannot utilise sucrose, can allow
detailed study of their particular products. Curtiss
and his colleagues first described such an approach to investigations of S. mutans and, using a
plasmid vector system, identified a gene for a
glucosyltransferase which they named gtfA [3,4].
In this paper we described the application of a
bacteriophage vector system for the cloning of S.
mutans genes, and the identification of recombinants specifying specific sucrose-degrading enzymes
(sucrases). A preliminary account of this work has
appeared [5].
0378-1097/85/$03.30 © 1985 Federation of European Microbiological Societies
38
3. MATERIALS
AND
METHODS
3.1. Construction of gene bank
The DNA manipulation
techniques
used here
have been described
in detail elsewhere [6,7].
Briefly, chromosomal
DNA from S. mutans strain
Ingbritt (serotype c) was partially digested with
restriction endonuclease
Sau 3A to give fragments
which were cloned into the replacement
bacteriophage vector h L47.1 [B] and packaged into
phase particles in vitro.
3.2. Screening of gene bank
The recombinant
phage gene bank was plated
with E. coli C600 in soft agar made with Luria
Broth on top of M9 minimal salts agar medium [9]
containing
sucrose as sole carbon source and the
supplements
(thiamin,
threonine
and leucine) required by C600. Plates were incubated 2 or 3 days
at 37’C. Recombinant
plaques suspected of containing sucrase genes were picked and checked by
restreaking.
3.3. Chromatography
Reaction products
from the action of cloned
sucrases on sucrose were separated by thin-layer
chromatography
(TLC) [lo] or descending
paper
chromatography
on Whatman
No. 1 paper in
pyridine : ethyl acetate : water (12 : 9 : 4). Spots on
TLC were detected with diphenylamine
reagent
[ll] spots on paper with ammoniacal
silver nitrate
[12]. When 14C-labelled sucrose was used, spots
were detected by autoradiography,
using Kodak
X-Omat AR film. For quantitation,
spots were cut
from paper chromatograms
and radioactivity
determined by liquid scintillation
counting.
3.4. Purification of sucrases
Soft agar from 20 plates on which confluent
lysis of E. co/i C600 had been induced by recombinant phage was harvested by adding 50 mM
Tris-HCl
(pH 7.5) and scraping off the soft agar
layer. The agar suspension was disrupted by sonication then clarified by centrifugation.
Solid ammonium sulphate was added to a final concentration of 1 M and the preparation
kept overnight at
4°C. The precipitate (which included intact phage
particles) was removed by centrifugation
and the
supernatant
fraction applied directly to a column
containing
8 ml phenyl-Sepharose
4B, previously
equilibrated
with 50 mM Tris-HCl
(ph 7.5) containing 1 M ammonium
sulphate. The column was
eluted with a gradient,
total volume 40 ml, decreasing from 50 mM Tris-HCl
1 M ammonium
sulphate to 10 mM Tris-HCl.
Sequential fractions
of 1 ml volume were collected from the column
and analysed for sucrase activity by assaying the
release of reducing sugar with the neocuproin
assay of Dygert et al. [13] or by a microscale assay
for glucose release with the glucose oxidase/peroxidase linked reaction previously
used to assay
fructosyltransferase
[14]. Active fractions
were
pooled, dialysed against 10 mM Tris-HCl,
and
applied to a column containing
8 ml DEAE-Trisacryl M equilibrated
with the same buffer. This
column was eluted with an increasing salt gradient
of O-50 mM NaCl in 10 mM Tris-HCl.
Active
fractions were again collected and pooled.
3.5. Immunological techniques
Procedures
for preparation
of antiserum
to
purified proteins
and Western blotting
were as
described
previously
[15]. Staining of blots was
with 4-chloro-1-naphthol
[16].
4. RESULTS
4.1. Detection of sucrase recombinants
The detection method relies upon the fact that
E. coli K12 is unable to utilise sucrose though it
will ferment monosaccharides
(glucose or fructose)
released by cleavage of sucrose. The nutrients
in
the soft agar layer allowed a faint lawn of E. coli
to grow, sufficient to support phage multiplication
and formation of plaques. However, the release of
monosaccharides
in plaques where sucrase genes
were expressed supported
much more vigorous
growth and so such plaques were surrounded
by
‘haloes’ of E. cofi (Fig. 1). Approximately
1 in
every 400 plaques from the gene bank showed
such cross-feeding.
4.2. Characterisation of cloned sucrases
The enzymic activity of lysates prepared from
cross-feeding recombinants
was examined by TLC
39
~iiilii~!iiii~i
Fig. 1. Appearance of plaques of recombinant h scr plated on
E. coli C600 on minimal medium with sucrose as carbon
source.
of the products formed following incubation with
sucrose. Two classes of recombinants were found.
The lysates of one split sucrose into glucose and
fructose. When uniformly labelled [14C]sucrose was
used as substrate, it was found that equimolar
amounts of each monosaccharide were produced.
The enzyme specified thus has fl-fructosanosidase
activity, i.e. is an invertase-type enzyme. Until its
enzymatic properties have been more thoroughly
investigated however, we regard it as premature to
refer to it as invertase but refer to it as sucrase and
propose scr as the symbol for the relevant structural gene in S. m u t a n s .
Lysates of the second class of recombinants
acted on sucrose to release free fructose and a
polymer which remained at the point of application. The fact that there was little radioactivity in
the free glucose spot suggested that the polymer
was a glucan and so the enzyme was concluded to
be a glucosyltransferase.
4. 3. P u r i f i c a t i o n o f s u c r a s e s
The sucrase activity from both classes of recombinant was readily purified by t h e sequence of
steps given in MATERIALSAND METHODS. The scr
gene product had an M r of 59000 (Fig. 2) while
that of the glucosyltransferase was 55 000.
Fig. 2. SDS-PAGE of proteins present at different stages of
purification of scr enzyme. (a) Mr standards: /3-galactosidase
(116000), phosphorylase (94000), bovine albumin (69000),
pyruvate kinase (60000), ovalbumin (43000), carbonic
anhydrase (30000). (b) Crude lysate of E. coil infected with h
scr. (c) Peak from phenyl-Sepharose column. (d) Peak from
DEAE-Trisacryl column.
4. 4. I m m u n o l o g i c a l s t u d i e s
When antiserum raised against the purified scr
enzyme was used in Western blot experiments, it
reacted with an antigen present in cell extracts of
S. m u t a n s strain Ingbritt which was of identical
electrophoretic mobility to the antigen produced
in E. coli (Fig. 3). No antigen could be detected in
concentrated culture supernatant. In contrast, the
M r 55 000 glucosyltransferase was detected in both
cell and supernatant fractions of S. m u t a n s . The
size, enzymatic properties and cell location of the
55 000 M r enzyme suggested that it was identical
to G T F - A described by Curtiss et al. [3,4] and this
identity was confirmed by Western blot experiments which showed that antiserum kindly provided by R. Curtiss reacted with the enzyme purified by us. Conversely, our antiserum reacted with
an M r 55 000 antigen made in E. coli carrying the
40
Fig. 3. Western blot of: (a) scr enzyme prepared from E. coli
lysate; (b) sonic extract o f S. mutans lngbritt cells; (c) concentrated culture supernatant of S. mutans. Bands were detected
with antiserum to scr enzyme purified from E. coli lysate.
recombinant plasmid pYA 601 (gtfA) also made
available by R. Curtiss.
syltransferase, by the cross-feeding method. GtfA
has recently been described by Curtiss et al. [3,4]
and our data confirm their observations in its
properties, In addition, we used the sensitive Western blotting technique to seek an immunological
relationship between the gtfA product and the
high M r glucosyltransferases [19], but found none.
We have not yet performed the necessary extensive enzymological studies of the scr enzyme which
might allow us to determine its spectrum of action
and hence deduce its funtion in sucrose utilization
by S. mutans but its M r (59 000) is quite distinct
from that of the invertase studied by Maynard and
Kuramitsu [20]. Tanzer et al. [21] and Chassy and
Porter [22], all of whom reported sizes of
40000-50000. Chassy and Porter [22] provided
evidence that the intracellular invertase, although
it can cleave sucrose, is actually a sucrose-6-phosphate, hydrolase and H.K. Kuramitsu (personal
communication) has evidence for sucrose-6-phosphate hydrolase activity in E. coli recombinants
carrying a gene apparently the same as scr.
The characterisation of these various enzymes
and analysis of the complex pathways of sugar
metabolism will be greatly helped by the development of techniques for cloning and expression of
streptococcal genes in E. coli.
5. DISCUSSION
E. coli K12 is normally unable to transport
sucrose into the cell so that cloning systems based
upon plasmid vectors require the devising of ingenious strategies for uptake of the sugar [3,4].
One of the advantages of the X system, however, is
that sucrose-metabolising enzymes from S. mutans
carried by recombinant phage are released during
the lytic cycle and so one can detect their activity
within the resultant plaques. A screening procedure similar to that described here, based on
cross-feeding of auxotrophs, was earlier used by
Franklin [17] to detect 2~ trp recombinants and by
Drew and Clarke [18] in studies of Pseudornonas
amidases but does not appear to have been applied
to carbohydrate metabolism before.
Two separate sucrase enzymes were detected in
our first attempts at screening the gene bank
though we have subsequently discovered others,
including high M r glucosyltransferase and fructo-
REFERENCES
[1] Hamada, S. and Slade, H.D. (1980) Microbiol. Rev. 44,
331-384.
[2] Chassy, B.M. (1983) in Glucosyltransferases, glucans,
sucrose and dental caries (Doyle, R.J. and Ciardi, J.E.,
Eds.), pp. 3-10, IRL Press, Washington.
[3] Curtiss, R., Robeson, J.P., Barletta, R., Abiko, Y. and
Smorawinska, M. (1982) in Microbiology 1982 (Schlessinger, D., Ed.), pp. 253-257, American Society for Microbiology, Washingon.
[4] Robeson, J.P., Barletta, R.G. and Curtiss, R. (1983) J.
Bacteriol. 153, 211-221.
[5] Morrissey, P., Dougan, G., Gilpin, M.L. and Russell,
R.R.B. (1985) in Vaccines 85 (Lerner, R.A., Chanock,
R.M. and Brown, F., Eds.), pp. 117-120, Cold Spring
Harbor Laboratories, New York.
[6] Kehoe, M., Duncan, J., Foster, T., Fairweather, N. and
Dougan, G. (1983) Infect,-Immun. 41, 1105-1111.
17] Russell, R.R.B., Coleman, D. and Dougan, G. (1985) J.
Gen. Microbiol. 131, 195-299.
[8] Loenen, W.A.M. and Brammar, W.J. (1980) Gene 20,
249-259.
41
[9] Maniatis, T., Fritsch, E.F. and Sambrook, J. (1982) Molecular Cloning. A Laboratory
Manual., Cold Spring Harbor
Laboratory,
Cold Spring Harbor, NY.
[lo] Zilic, Z., Blau, N. and Knob, M. (1979) J. Chromatogr.
164, 91-94.
[II] Walkley, J.W. and Tillamn, J. (1977) J. Chromatogr.
132,
172-174.
[12] Dawson, R.M.C., Elliott, D.C., Elliott, W.H. and Jones,
K.M. (1969) Data for Biochemical Research, Oxford University Press, London.
[13] Dygert. S.. Li, L.H., Florida, D. and Thoma, J.A. (1965)
Anal. Biochem. 13, 367-374.
[14] Russell, R.R.B., Donald, A.C. and Douglas, C.W.I. (1983)
J. Gen. Microbial. 139, 3243-3250.
[15] Russell, R.R.B.. Peach, S.L., Colman, G. and Cohen, B.
(1983) J. Gen. Microbial. 129, 865-875.
[16] Hawkes,
B&hem.
117) Franklin,
(Hershey.
Laboratory,
R.E., Niday, E. and Gordon,
J. (1982) Anal.
119, 142-147.
N.C. (1971) in The Bacteriophage
Lambda
A.D.. Ed.), pp. 621-638,
Cold Spring Harbor
Cold Spring Harbor, NY.
[18] Drew, R.E. and Clarke, P.H. (1980) Mol. Gen. Genet. 177,
311-320.
[19] Russell, R.R.B. (1979) Microbios 23. 135-146.
[20] Maynard,
M.T. and Kuramitsu,
H.K. (1979) Infect Immun. 23, 873-883.
[21] Tamer, J.M., Brown, A.T., McInerney. M.F. and Woodiel,
F.N. (1976) Infect. Immun. 16, 318-327.
1221 Chassy, B.M. and Porter, E.V. (1979) Biochem. Biophys.
Res. Commun. 89, 307-314.