INTERNATIONAL JOURNAL OF
SYSTEMATIC
BACTERIOLOGY
O c t o b e r 197 0
Vol. 20, No. 4
Copyright 1970, Iowa S t a t e U n i v e r s i t y P r e s s
pp. 413-419
C E L L WALL COMPOSITION IN T H E CLASSIFICATION
O F GRAM POSITIVE ANAEROBES'
C. S. C u m m i n s
A n a e r o b e L a b o r a t o r y , R e s e a r c h Division
V i r g i n i a P o l y t e c h n i c Institute, B l a c k s b u r g , V i r g i n i a 2406 1
ABSTRACT. F o u r g r o u p s o f s t r a i n s o f c l o s t r i d i a a n d a n a e r o b i c c o r y n e b a c t e r i a w e r e e x a m i n e d by c h e m i c a l a n d
antigenic cell wall analysis.
I n e a c h g r o u p it w a s
p o s s i b l e t o identify m o r e t h a n one p a t t e r n of c e l l w a l l
sugar components, and these sugar patterns show a high
d e g r e e o f c o r r e l a t i o n w i t h t h e s u b g r o u p s r e v e a l e d by
cell wall agglutination t e s t s . In the two groups tested,
DNA/DNA h o m o l o g y t e s t s s h o w e d t h a t s t r a i n s w i t h t h e
s a m e c e l l w a l l s u g a r p a t t e r n h a d a h i g h d e g r e e of h o mology (80-100%).
- - - - _ - _ - - - - _
I n the l a s t ten y e a r s a g r e a t d e a l of i n f o r m a t i o n h a s b e c o m e a v a i l a b l e about t h e g e n e r a l s t r u c t u r e of the c e l l w a l l of G r a m - p o s i t i v e
b a c t e r i a , and r e p r e s e n t a t i v e s of m o s t of the commonly found g e n e r a have
been e x a m i n e d . L e s s attention h a s p e r h a p s been paid to g r a m - p o s i t i v e
a n a e r o b e s , but w h e r e v e r t h e s e have been e x a m i n e d (e.g. Salton and
Ghuysen, 1 9 5 7 ; Salton a n d P a v l i k 1960: Glendenning, 1958) t h e i r c e l l
w a l l s have shown no e s s e n t i a l d i f f e r e n c e s f r o m t h o s e of o t h e r g r a m positive o r g a n i s m s , e i t h e r i n e l e c t r o n m i c r o s c o p e a p p e a r a n c e o r i n
broad chemical structure.
In t h i s contribution, I wish to r e p o r t the r e s u l t s of s t u d i e s of c e l l
wall composition i n two g r o u p s of a n a e r o b e s . F i r s t l y , v a r i o u s m e m b e r s
of the genus C l o s t r i d i u m , and secondly, a g r o u p of " a n a e r o b i c d i p h t h e roids" c o m p o s e d of s t r a i n s of P r o p i o n i b a c t e r i u m acne^ ( C o r y n e b a c t e r i u m
a c n e s ) and s t r a i n s which a p p e a r e d t o b e m o r e o r l e s s c l o s e l y r e l a t e d t o
i t . T h e s e s t u d i e s have been m a d e p r i m a r i l y f o r taxonomic p u r p o s e s , and
two a s p e c t s only of c e l l wall c o m p o s i t i o n have b e e n c o n s i d e r e d , b r o a d
c h e m i c a l s t r u c t u r e and s e r o l o g i c a l s p e c i f i c i t y a s d e t e r m i n e d by c e l l
w a l l agglutination t e s t s . The e x p e r i m e n t a l r e s u l t s a r e given h e r e i n
outline only and will be r e p o r t e d i n d e t a i l e l s e w h e r e . In s o m e c a s e s ,
i t h a s been p o s s i b l e to c o m p a r e the r e s u l t s obtained by t h e s e m e t h o d s
with the d e g r e e of genetic homology between s t r a i n s a s d e t e r m i n e d by
DNA competition e x p e r i m e n t s . The r e s u l t s of t h e s e DNA homology
s t u d i e s a r e r e p o r t e d by D r . J . L . Johnson i n the next p a p e r .
METHODS
The b a c t e r i a w e r e grown i n a peptone - y e a s t - e x t r a c t - g l u c o s e m e d i u m ,
containing a l s o c y s t e i n e ( 0 . 0 6 % ) and b i c a r b o n a t e ( 0 . 5 % ) , u n d e r a n
a t m o s p h e r e of C 0 2 . The washed o r g a n i s m s w e r e shaken with ballotini
'Supported
by N. I. H. G r a n t No. GM 14604-04.
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b e a d s i n a B r a u n d i s i n t e g r a t o r , and a f t e r f i l t r a t i o n to r e m o v e beads,
the m i x t u r e w a s d i g e s t e d with p r o n a s e f o r 1 1/2 - 2 h r s . a t 56" .
C e l l w a l l s w e r e deposited by centrifxgation a t 30.000 C, s e p a r a t e d i f
n e c e s s a r y f r o m unbroken c e l l s by d i f f e r e n t i a l centrifugation, a n d washed
X 3 i n d i s t i l l e d w a t e r . The f i n a l c e l l wall f r a c t i o n s w e r e lyophilized.
In the c a s e of s p o r i n g o r g a n i s m s , p r e c a u t i o n s w e r e t a k e n to h a r v e s t the c e l l s a t a t i m e when s p o r e f o r m a t i o n w a s m i n i m a l o r a b s e n t .
With C l . l i m o s u m a n d c l . botulinum, c u l t u r e s w e r e killed by the a d d i tion of f o r m a l i n to a f i n a l c o n c e n t r a t i o n of 2 p e r c e n t .
w hydrolysed in 6
F o r c h e m i c a l a n a l y s i s , 5 m g m of c e l l w
N HC1 f o r 1 8 h r s . a t 105°C f o r a m i n o a c i d s , and 10 nigm of c e l l w a l l s
h y d r o l y s e d i n 2 N H2SOI f o r 2 h r s . f o r s u g a r s . A f t e r s u i t a b l e n e u t r a l i zation, the h y d r o l y s a t e s w e r e examined by p a p e r c h r o m a t o g r a p h y a n d
the a m o u n t s of d i f f e r e n t components p r e s e n t r e c o r d e d roughly a s t t l i .
ttt, t+, +, 5, o r T r , depending on t h e i n t e n s i t y of t h e s p o t s . C e l l wall
agglutination t e s t s w e r e done a s d e s c r i b e d i n C u m m i n s a n d Slade, 1962.
?his brief outline of the m e t h o d s u s e d to p r e p a r e and a n a l y z e t h e
c e l l wall f r a c t i o n s i n d i c a t e s t h a t in c h e m i c a l t e r m s the a n a l y s e s a r e
c r u d e , although well s u i t e d to the e x a m i n a t i o n of l a r g e n u m b e r s of
s t r a i n s . N o examination was m a d e f o r the p r e s e n c e of teichoic a c i d s ,
lipid or h e x o s a m i n e s , and i t m u s t b e r e m e m b e r e d that the m e r e e x a m i nation of a c i d h y d r o l y s a t e s f o r s u g a r s g i v e s Little indication of the type
of p o l y m e r f r o m which the s u b s t a n c e s a r e r e l e a s e d . However, the
r e s u l t s show t h a t the d e t e r m i n a t i o n of c r u d e a m i n o a c i d and s u g a r
p a t t e r n s i n c e l l w a l l s c a n be useful i n taxonomy, provided t h a t the
Limitations of the method a r e r e a l i z e d .
RESULTS
The following g r a u p s of s t r a i n s have b e e n examined:
1 . C l o s t r i d i u m l i m o s u m , 17 s t r a i n s
2 . C l o s t r i d i u m botulinum, 22 s t r a i n s
3. C l o s t r i d i u m b u t y r i c u m , 29 s t r a i n s
4 . P r o p i o n i b a c t e r i u m E and r e l a t e d o r g a n i s m s , 4 3 s t r a i n s
1. S t r a i n s of C l o s t r i d i u m l i m o s u m .
This organism is a proteolytic anaerobic gram-positive sporing
b a c i l l u s , with oval s u b t e r m i n a l s p o r e s , which h a s b e e n i s o l a t e d f r o m a
v a r i e t y of s i t u a t i o n s including soil, bovine liver infections a n d liver
and kidney infections i n a l l i g a t o r s . It h a s proved pathogenic to m i c e in
l a b o r a t o r y t e s t s . A d e t a i l e d d e s c r i p t i o n of C l . l i m o s u m i s being p r e p a r e d f o r publication. (Cato, C u m m i n s a n d Smith, 1 9 7 0 ) .
The r e s u l t s of c e l l wall a n a l y s i s of 1 7 s t r a i n s of 0.
Limosum a r e
shown i n Table 1, and i t can be s e e n that 1 3 of the 17 s t r a i n s f o r m a
v e r y homogeneous g r o u p both by s u g a r p a t t e r n a n d s e r o l o g i c a l t e s t s .
The a g r e e m e n t between t h e s e two s e t s of r e s u l t s i s p e r h a p s not s u r p r i s ing s i n c e i t is v e r y likely t h a t t h e a n t i g e n i c d e t e r m i n a n t s r e s p o n s i b l e
f o r c e l l wall agglutination a r e t h o s e of the c e l l wall p o l y s a c c h a r i d e which
i s the s o u r c e of the s u g a r s obtained on c e l l wall h y d r o l y s i s . The r e s u l t s
of c r o s s a b s o r p t i o n t e s t s (not r e c o r d e d in the t a b l e ) have shown that the
two s t r a i n s u s e d to p r e p a r e a n t i s e r a to t h i s g r o u p (1936-C a n d 2485)
a r e s e r o l o g i c a l l y i d e n t i c a l . Of the o t h e r 4 s t r a i n s , two have a d i s t i n c tive c e l l w a l l s u g a r p a t t e r n ( G a l a c t o s e t++, G l u c o s e t, R h a m n o s e t ) and
s e e m to be s e r o l o g i c a l l y i d e n t i c a l when t e s t e d a g a i n s t a s e r u m to one of
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S Y ST E MAT1C
TABLE I.
415
BAC T E R I O LOGY
COMPOSITION & SEROLOGICAL REACTIONS OF
CELL WALLS OF CL. LIMOSUM
Number of
GrouI
Strains
1.
(includes strains
Gal.
CW Sugars
Glu.
+
+
+
+
Mann.
CW agglutinating litres
%am.
2
with sera to strains:
1928
*-
2755
1937-C
2485
-
160-640
160-640
1936-C & 2485)
1
2.
-
-
2
20
20
2
3.
(includes strain
1928)
-
1
4.
(2755)
-
Major CW aminoacids in all strains; ala, glu, e - D A P
*-
=
no reaction at dilution 1 / 2 0 .
t h e m (1928). One of the 2 r e m a i n i n g s t r a i n s (Group 2) a p p e a r to be
somewhat i n t e r m e d i a t e between the f i r s t two groups, while the Last
s t r a i n (2755) s e e m s quite u n r e l a t e d t o the o t h e r s . It i s w o r t h noting
that i f s u s p e n s i o n s of f o r m a l i n - k i l l e d i n t a c t c e l l s a r e u s e d i n p l a c e of
s u s p e n s i o n s of purified c e l l wall f r a g m e n t s a totally different p i c t u r e
i s obtained. The i n t a c t c e l l s u s p e n s i o n s a g g l u t i n a t e with the homologous
s e r a but t h e r e i s tittle o r no c r o s s - r e a c t i o n with o t h e r s t r a i n s and no
s e r o l o g i c a l g r o u p s c a n be identified. R e s u l t s f o r DNA homologies a r e
not yet availabie f o r t h e s e s t r a i n s , and i n t h e i r a b s e n c e , i t is difficult
t o a s s e s s the p o s s i b l e r e l a t i o n s h i p s between t h e s e s e r o l o g i c a l g r o u p s .
S t r a i n s of C_. botulinum
T h r e e well-defined c e l l wall s u g a r p a t t e r n s w e r e found i n the 2 2
s t r a i n s examined, a s shown i n Table II. The r e s u l t s of c e l l wall
agglutination t e s t s a r e a l s o given i n the Table and i t c a n be s e e n that
with s u g a r p a t t e r n s LI and 111, the two s e t s of r e s u l t s a g r e e v e r y s a t i s f a c t o r i l y . Unfortunately, a s a t i s f a c t o r y s e r u m h a s not yet been p r e p a r e d
f o r s t r a i n s showing s u g a r p a t t e r n I (proteolytic s t r a i n s of toxigenic t y p e s
A, B, a n d F). However, i t i s c l e a r that t h e s e s t r a i n s differ s e r o l o g i catty f r o m the o t h e r two g r o u p s .
The grouping of s t r a i n s a s shown by t h e s e t e s t s a g r e e s welt with
that found by o t h e r w o r k e r s . F o r example, Walker and B a t t y (1964),
using a fluorescent-antibody technique found that t y p e s A , B , a n d F
(proteolytic s t r a i n s ) c r o s s - r e a c t e d and that t y p e s C a n d D c r o s s r e a c t e d , but that t h e r e was no c r o s s - r e a c t i o n between the A , B , a n d F
s t r a i n s and the C a n d D s t r a i n s : type E s t r a i n s r e a c t e d only with
Type E a n t i s e r u m .
2.
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INTERNATIONAL J O U R N A L
416
TABLE 11. CELL WALL COMPOSITION IN
OF CLOSTRIDIUM BOWLINOM
(22 strains)
Mucopeptide Aminoacids.
Ala, Glu, m e s o - P A P , all strains
Cell Wall Agglutination
with sera to:
Cell Wall Sugars.
I. Glucose.
Type C Type D Type E
(2)
Type A
(1)
Type B, Prot.
(2)
Tr.
Type F, Prot.
-
-
11.
No sugar detected,
(or trace Glucose only)
( 41
Type c
Type D
(3)
111. Galactose and Glucose
Type
(81
Type F, Non-Prot. ( 2 )
+
+
+
+
-
-
-
+
+
-
TABLE 111. CELL WALL COMPOSITION IN CL. BUTYRICUM~
Results in 29 strains
Mucopeptide amino acids. Ala, Glu,meso-DAP; all strains
Cell wall sugars.
Group I. CW sugar; Glucose.
16 strains, received as:
C1. butyricum
9
C1. multifermentans 5
c1. fallax
2
-
-
Group 11. CW sugars; Galactose, Glucose
13 strains, received as:
C1. butyricum
9
C1. multifermentans 1
C1. amylolyticum
1
c1. D.
2
-
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SYSTEMATIC
417
BACTERIOLOGY
S t r a i n s of 5. butyricum.
Cell w a l l a n a l y s e s w e r e c a r r i e d out on 29 s t r a i n s which had been
definitely o r tentatively identified a s 5 . butyricum by the r e s u l t s of
routine t e s t s c a r r i e d out i n Anaerobe Laboratory, Virginia Polytechnic
Institute. The r e s u l t s a r e shown i n Table III. A s c a n be s e e n f r o m the
Table, not a l l of these s t r a i n s w e r e listed a s CL. butyricum when
r e c e i v e d ; however, a l l except 5 w e r e listed e i t h e r a s C 1 . butyricum o r
GI. multifermentans. Two groups w e r e identified on t h e b a s i s of c e l l
wall s u g a r p a t t e r n s , (see Table 111) and on re-examining the r e s u l t s of
fermentation and other t e s t s on these s t r a i n s , s o m e d i f f e r e n c e s could
be found between t h e 2 cell wall groups. T h e s e differences a r e not absolute, but i n g e n e r a l s t r a i n s of Group I ( c e l l wall s u g a r , glucose) a p p e a r
to grow m o r e vigorously and produce a lower final p k i n dextrin, glycogen
and s t a r c h , t h a n do s t r a i n s in Group 11 ( c e l l wall s u g a r s , galactose and
glucose).
3.
W L E IV.
Group I
CELL WALL COMPOSITION IN ANAEROBIC DIPHTHEROIDS
Mucopeptide
Sugars
Ala, Glu, Gly
LL-Dap
Galactose
Glucose
Mannose
Group I1 Ala, Glu, Gly
LL-DAP
Glucose
Mannose
Serological Reactions*
Serum R15
Serum
0162
+
+
320-1280
Number of Strains
<20
35
40-80
6
.r
+
2
<20-80
*Serum R15 was prepared against a Group I s t r a i n , and
serum 0162 against a Group I1 s t r a i n .
The r e s u l t s of DNA homology studies on these s t r a i n s , r e p o r t e d
i n detail by D r . Johnson i n the next p a p e r , s e e m to confirm the
existence of the 2 groups. The d e g r e e of homology between s t r a i n s
within each group ranged between 7570 and 95% , while if s t r a i n s of one
group a r e tested f o r homology against a r e f e r e n c e DNA p r e p a r e d f r o m
a m e m b e r of the other group, the d e g r e e of homology was considerably
lower, varying between 20% and 4oY0
It h a s not been possible to produce satisfactory a n t i s e r a for t h e s e
s t r a i n s and we do not yet know whether each group is c h a r a c t e r i z e d by
a distinctive cell wall antigen. However, t h e r e i s s o m e p r e l i m i n a r y
evidence f r o m immunodiffusion t e s t s that s t r a i n s i n Group II do have
such a n antigen i n common.
.
"Anaerobic Diphtheroiddl
In this collection of 43 s t r a i n s , two c l e a r - c u t groups could be d i s tinguished on the b a s i s of c e l l wall s u g a r p a t t e r n s and c e l l wall agglutination t e s t s (TableIV). It a p p e a r s f r o m the serological r e s u l t s that
both g r o u p s s h a r e a common antigenic determinant i n t h e i r c e l l walls,
since s t r a i n s i n Group I1 c r o s s - r e a c t a t r a t h e r lower t i t r e s with a
s e r u m p r e p a r e d against a s t r a i n belonging to group I . However,
absorption of this s e r u m with cell walls of a' group II s t r a i n abolished
the c r o s s - r e a c t i o n while leaving the t i t r e against group. I s t r a i n s
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undiminished. The r e s u l t s suggest that the m a j o r antigenic d e t e r m i nant of the group I s t r a i n s i s connected with the p r e s e n c e of galactose
i n the c e l l wall polysaccharide, since this sugar i s not p r e s e n t in
s t r a i n s of Group 11.
The DNA homology r e s u l t s appear to c o r r e l a t e with the cell wall
sugar groupings. DNA homology values of group I s t r a i n s a r e f r o m
95-100 p e r c e n t with a r e f e r e n c e o r g a n i s m f r o m group I, w h e r e a s DNA's
f r o m group I1 s t r a i n s showed i n general a slightly lower d e g r e e of
homology (89-92 percent) to the s a m e r e f e r e n c e DNA.
A s indicated i n Table IV, the c h a r a c t e r i s t i c mucopeptide amino
acid pattern i n these s t r a i n s i s alanine, glutamic acid, glycine and
LL-DAP. However, i n a t l e a s t one s i m i l a r s t r a i n only m e s o - D A P w a s
present, a n d the amount of glycine was minimal. B y c e l l wall sugar
pattern, this s t r a i n belonged to Group 11, and i t behaved serologically
i n the s a m e way a s other group I1 s t r a i n s . I t i s of considerable i n t e r e s t
that this s t r a i n showed the s a m e relatively high d e g r e e of homology to
the group I r e f e r e n c e DNA as do other group I1 s t r a i n s , despite the f a c t
that i t s mucopeptide contains a different i s o m e r of DAP.
DISCUSSION
I t i s v e r y likely, a s pointed out above, that both c e l l well sugar
patterns and cell wall serological reactions a r e functions of the s a m e
polymer, namely the c e l l wall polysaccharide. Sugar p a t t e r n s m a d e
up of one o r two of the common hexoses (e.g. glucose alone, galactose
alone, o r glucose and galactose) a r e s o common i n the Gram-positive
bacteria that they can be of little value i n assigning o r g a n i s m s to the
b r o a d e r taxonomic groups (genus, family o r above). Even i n the c a s e
of species i n the s a m e genus, i t may be noted that i n the p r e s e n t
r e s u l t s both Clostridium butyricum and Clostridium botulinum have
subgroups within each which a r e distinguished by having glucose alone
o r glucose a n d galactose a s t h e i r c e l l wall sugars, but this does not
n e c e s s a r i l y indicate that s t r a i n s of G. botulinum which have glucose
a s cell wall s u g a r a r e m o r e closely r e l a t e d than t h e i r fellows to those
s t r a i n s of C L butyricum whose walls contain glucose. Immunological
techniques a r e valuable i n this situation, because the specificity of
antigen-antibody reactions i s such that they will distinguish c l e a r l y
between c e l l wall polysaccharides which a p p e a r to b e s i m i l a r i n t e r m s
of crude sugar patterns.
At the moment the main value of the type of analysis proposed h e r e
i s i n the f u r t h e r investigation of a group of s t r a i n s which have a l r e a d y
been grouped on morphological and/or physiological grounds. In
such a collection of s t r a i n s , the a n a l y s i s of c e l l wall sugar p a t t e r n s
may be a useful indication of the homogeneity o r otherwise of the group.
This i s well illustrated by the r e s u l t s quoted h e r e f o r G.butyricum,
where the finding of 2 distinct c e l l wall s u g a r p a t t e r n s among s t r a i n s
tentatively assigned to this s p e c i e s prompted a f u r t h e r study of the
strains.
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