Journal of General Micvobiology (197 I ) , 67,33-46
Printed in Great Britain
33
Taxonomy of the Clostridia :
Wall Composition and DNA Homologies in Clostvidium butyricum
and Other Butyric Acid-producing Clostridia
By C. S. C U M M I N S A N D J. L. J O H N S O N
Anaerobe Laboratory,
Virginia Polytechnic Institute and State University,
Blacksburg, Virginia 2406 I , U.S.A.
(Acceptedfor publication 22 April 1971)
SUMMARY
Strains designated as Clostridium butyricum were found to constitute two distinct
groups on the basis of wall sugar patterns, nucleotide-sequence similarities of
DNA preparations and nutritional requirements. Organisms in homology group I
had walls which contained only glucose and would grow in a mineral salts-glucose
medium with 0.05 pg./ml. biotin, although growth was improved by the addition
of amino acids. Some strains labelled C. multvermentans and C.fallax belonged to
this group. Organisms in homology group I1 had glucose and galactose as wall
sugars and would not grow in mineral salts-glucose medium with amino acids
and 10 vitamins unless yeast extract was also added. Strains of C. amylolyticum, C.
rubrum, C. beijerinckii, C. lacto-acetophilum and some strains labelled C. multifermentans belonged to group 11.
It is recommended that the name Clostridium butyricum be retained for the organisms in group I, while C. beijerinckii is suggested for group 11. Some of the strains
labelled C. fallax and the strains of C. acetobutylicum, C. aurantibutyricum,
C. pasteurianum and C. tyrobutyricurn did not belong to either group.
INTRODUCTION
Although many of the recognized species in the genus Clostridium produce butyric acid as
one of the major products of carbohydrate fermentation (McCoy, Fred, Peterson & Hastings,
1930; Moore, Cato & Holdeman, 1966) this property has been used as a distinguishing
characteristic for the type species of the genus, Clostridium butyricum, and for other nonpathogenic clostridia. These have at times been employed for the commercial production of
butyric acid, butanol and acetone and are known as the ‘butyric acid group’ (McCoy et al.
1926, 193o).Representatives of this group that are listed in the seventh edition of Bergey’s
Manual include C. butyricum, C. butylicum, C. beijerinckii, C. multvermentans, C. iodophilum,
C.fallax, C. tyrobutyricum, C. pasteurianum, C. amylosaccharobutylpropylicurn, C. madisonii,
C. muelleri, C. amylolyticum, C. acetobutylicum, C. saccharoacetoperbutylicum, C. roseum,
C. aurantibutyricum, C. rubrum, C. lacto-acetophilum and C. felsineum.
McCoy et al. (I 926, I 930) have subdivided the butyric acid group into those that produce
chiefly butyric and acetic acids and those that produce largely neutral end products, mainly
butanol and acetone. The first group was referred to as the ‘butyric’ organisms and the
latter as the ‘butyl’ organisms. Other phenotypic characteristics that have been used to
differentiate species within the butyric acid group of bacteria are the formation of an iodophilic substance, gelatin liquefaction, fermentation of lactose and lactate, nitrate reduction
3-2
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34
C . S. C U M M I N S AND J. L. JOHNSON
and nitrogen fixation. However, Richard (1948)found these traits to vary considerably, not
only from strain to strain but also with the same strain when tested at different times.
The purpose of the present study was to determine whether analyses of wall composition
and determination of DNA/DNA homologies could be used to characterize Clostridium
butyricum more accurately and to distinguishit more clearly from other butyric acid producing
clostridia. An outline of the wall and DNA homology results for strains of C . butyricum
has already been published (Cummins, 1970; Johnson, 1970).
METHODS
Strains of organisms
The origins and designations of the strains investigated are given in Table I. The VPI
numbers are from the culture collection of the Anaerobe Laboratory, Virginia Polytechnic
Institute and State University, and the strains will be referred to by these numbers and by
the American Type Culture Collection (ATCC) number where available. All of the strains
in our culture collection that were received labelled as Clostridium butyricum and which fitted
the description of the species were used in the study. There is some duplication in this list of
strains; for example, 2681 has the same origin as 2981 and 2968, and 2969 has the same
origin as 3266.However, since these strains came to this laboratory from different sources at
different times, they have been treated separately.
The history and particular characteristics of some strains showing high homology to the
reference strains of Clostridium butyricum, but bearing other names, are described in the
section on Results and Discussion. Phenotypic characteristics of the other strains of Clostridium (Table 2) were determined in the Anaerobe Laboratory and conform to those for
the species designated. Wherever possible, we have included the type, neotype or holotype
strain.
Growth
Anaerobic culture techniques as described by Cat0 et at. (1970)were used. The cultures
were maintained in chopped-meat medium and were grown in peptone-yeast extractglucose medium (PYG) for the inoculation of large cultures. Large cultures of the organisms
used for the preparation of walls and for the isolation of DNA were grown in a medium
containing mineral salts (Moore & Cato, 1965), 0.7 % peptone (Difco), 0.3 % trypticase
(BBL), 0.5 % yeast extract (Difco), I % glucose, 0.05 % cysteine .HCl and 0.5 % NaHCO,.
The medium was prepared in 2.5 1. quantities in 3 1. Erlenmeyer flasks. Each flask was fitted
with a rubber stopper having right-angled inlet and outlet tubes. The inlet tube protruded to
approximately 2 in. from the surface of the medium, and to its outer end was attached by
rubber tubing another short section of glass tubing filled with cotton. The outlet tube
started flush with the bottom of the stopper, and its outer end was capped with a Bunsen valve.
The purpose of these arrangements was to enable gas passed in through the inlet tube to
flush air from the neck of the flask. The stopper was held in place with a clamp consisting of
a rubber-covered metal collar that extended around the neck of the flask. At opposite sides
of the collar were attached two eye-bolts which held a horizontal bar across the top of the
stopper. The medium was sterilized without the cysteine and the NaHCO,. Immediately
after sterilization the inlet tube was attached to a COz cylinder and the flask was flushed
with oxygen-free C02. The outlet tube was then clamped off and the medium allowed to
cool under I to 2 lb/in.2 of C02. A sterile solution of cysteine. HCl(1o ml. of 12.5%) and
dry sterile NaHCO, (12.5g.) were then added. After equilibration between dissolved and
gaseous COz the inlet tube was clamped off. Oxygen-free C 0 2was again flushed through the
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Taxonomy of the butyric acid clostridia
35
Table I . Sources and designations of organisms belonging to the two homology groups of
Clostridium butyricum and of other strains in the butyric acid group
VPI
no.
I536
1622
1718
2089
2389
2399
2400
2402
2403
2404
2408
2417
2419
268 I
2697
2781
2783
2793
2965
2966
2968
2969
2980
298 I
2982
2983
3005
3006
3266
4213
4419
4420
4635
4668
5481
5982
4633
2673
2675-1
2774
3 I 63-1
2766
2791-1
2670
4215
24 15-A
5729
6010
ATCC no.
859
860
6015
6014
19398
I4949
11914
17778
11914
I7777
824
&I
3
19400
Source, other numbers and designations”
CDC, KA45; C. butyricum
McClung, 2296; Kluyver, E.VI 3.2.1 ; C. butyricum (C.pectinovorum)
ATCC; Kluyver E.VI 3.6.1 ; C. butyricum (C. saccharobutyricum)
CDC, 1667; C. butyricum
Beerens, M. 6; C.multifermentans
Beerens, 1375, M. 13; C. multiferrnentans
Beerens; C. multifermentans
Beerens, 3027 ; C. multifermentans
Beerens, 71-351, ~ 1 2C.
; fallax
Beerens, F 1-1325; C. fallax
Prevot ; C. multifermentans
Prevot ; 713, C. multifermentans
Prevot; CKII, C. butyricum
McClung, I 672 ; McCoy, A 79 ; C. butyricum
Prevot, VA 6; C. amylolyticum
McClung, 2823 ; C. rubrum
McClung, 2978 ; Clostridium sp.; identified as C. butyricum
McClung, 3319 ; Clostridium sp. ;identified as C. butyricum
McClung, I I 84; E. Hellinger, Palestine; C. butyricum
McClung, I I 85 ; E. Hellinger, Palestine; C.butyricum
McClung, I 672 A ; McCoy, A 79 ; C. butyricum
McClung, 2391; NCTC, 7423; C. butyvicum
ATCC; McCoy, 75; Kral collection; C. butyvicum
ATCC; McCoy, 79; Kral collection; C. butyricum
Prevot, 7423; C. butyricum
Prevot, A 9 E I ; C.butyricum
Prevot, 962; C. butyricum
Prevot, Meb 125; C. butyricum
NCTC, 7423; McClung, 2391; C. butyricum
ATCC; H. Ng, 34; c.rubrum
ATCC ; Barker; C. lacto-acetophilum
ATCC ; McClung, I 67I ; C. beijerinckii
McClung, I 190; C. aurantibutyricum
ATCC; Barker; C. lacto-acetophilum
Hobbs, 9362; C. beijerinckii
Govoni, Baltimore City Hospital; identified as C.fallax, it was
toxic upon receipt
ATCC; McClung, 2038; C.auvantibutyricum
McClung, 633 ; C. acetobutylicum
McClung, 634; C. acetobutylicum
McClung, 2449 ; C. acetobutylicum
ATCC; Weyer (McCoy & McClung strain w); C. acetobutylicum
McClung, 557; C. felsineum
McClung, 541; C. felsineum
McClung, 308 ; McCoy, 5 ; C. pasteurianum
ATCC; McCoy, 5 ; C. pasteurianum
Prevot, ~ 2 5 C.~pseudofallax
;
ATCC; NCTC 8380; Inst. Pasteur, Paris (TRACOL);
C. fallax
PrCvot, TRACOL; C. fallax
* The first name is the person or institution from which the culture was received. €3. Beerens, Institut
Pasteur, Lille, France; A. R. Prkvot, Institut Pasteur, Paris, France; G. Hobbs, Torry Research Station,
Aberdeen, Scotland; E. McCoy, University of Wisconsin, Madison, Wisconsin, U.S.A. ; L. S . McClung,
Indiana University,Bloomington, Indiana, U.S.A. ;NCTC, National Collection of Type Cultures, Colindale,
London ; ATCC, American Type Culture Collection, Rockville, Maryland, U.S.A. ; CDC, Center for
Disease Control, Atlanta, Georgia, U.S.A.
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C. S . C U M M I N S A N D J. L. JOHNSON
36
flask when 20 ml. of inoculum was added. After the initiation of growth, the clamp was
removed from the Bunsen valve to allow the release of fermentation gas. The cultures were
mixed with a Teflon-coated magnetic stirring bar during incubation at 37".
Labelled DNA was obtained from the reference organisms by growing them in the BMAA
medium (see section below on methods for determination of nutritional requirements)
containing biotin and 3H adenine (250 pCi/200 ml., New England Nuclear, Boston, Mass.,
U.S.A.). About 5 to 10ml. of PYG medium were also included for reference strain 2983 which
had complex growth requirements.
Preparation and analysis of walls
Growth was stopped in late log phase by adding 2 % (vjv) formalin. The bacteria were
centrifuged, washed once with distilled water and again resuspended in distilled water.
Twenty ml. amounts of 0.1mm./diam. glass beads and 20 ml. volumes of bacterial suspension
were placed in 50 ml. bottles and shaken for 5 min. at 4000 cyc./min. in a Braun mechanical cell
homogenizer. The disrupted bacteria were separated from the glass beads by fiItration
through a no. 2 porosity sintered glass filter. Pronase (Calbiochem) was added to the
bacterial material (about 50 pg./mL) and the mixture incubated at 56" for I to 2 h. The walls
were pelleted by centrifuging at 48,000 g for 20 min., washed twice by resuspending in
distilled water and centrifuging, checked microscopically, and preparations showing intact
organisms were shaken and digested again. When a preparation of thoroughly disrupted
bacteria was obtained, debris was removed by slow-speed centrifugation (480 g , 10 min.),
and the walls lyophilized.
Wall amino acids were detected by hydrolysis and paper chromatography. Five mg.
amounts of walls were suspended in 6 ml. of 6 N-HCl and hydrolysed in sealed tubes for 18 h.
at TOO to 105". The hydrolysates were filtered, evaporated to dryness and the residue taken
up in 0-25 to 0.3 ml. of distilled water. Two-dimensional ascending chromatography was
done on 8 in. squares of Whatman no. I paper in a Shandon DDH 8 in. Chromatank.
Normally 10pl. of hydrolysate (equivalent to approximately 200 pg. of original wall) was
applied at the origin. The first solvent was n-butanol + acetic acid +water (120 :30 :50) and
the second solvent phenolfwater (go: 10)with I ml. of NH,OH (sp.gr. 0-880)/200ml. of
solvent. The spots were made visible by dipping the papers in a solution of 0.1% ninhydrin
in 95 % acetone containing a few drops of pyridine and heating them at 80 to 100' for I to
2 min. Permanent preparations were made by dipping the ninhydrin-treated papers in 5 yo
NiSO,. 6H20, blotting and drying.
The isomeric forms of a-s-diaminopimelic acid (DAP) were determined by one-dimensional chromatography using Schleicher and Schuell paper no. 2043A and the methanol +
pyridine HCl+ water solvent (80 :10:2.5 :17'5) of Rhuland, Work, Denman & Hoare
(1955).
The sugars were determined by hydrolysing 10mg. amounts of walls in 5 ml. of 2 s-H,SO,
for 2 h. at 100'. After cooling, the acid was neutralized with solid BaCO, to a pH of approximately 7.0. The mixture was centrifuged, the barium sulphate deposit washed with 2 ml.
distilled water and the combined supernatants evaporated to dryness in vacuo over phosphorus pentoxide. The soluble material was taken up in 0.25 to 0.3 ml. of distilled water, and
if necessary centrifuged to remove residual barium sulphate or other insoluble material.
The hydrolysates (25 PI.) were spotted on Whatman no. I paper and developed wing an
ethylacetate pyridine + water solvent (80 :20 :10).The sugars were detected by the aniline
hydrogen phthalate method or with alkaline silver (Trevelyan & Harrison, I 952).
+
+
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Taxonomy of the butyric acid clostridia
37
Determination of nutritional requirements
The basal medium (BM) used for testing nutritional requirements contained the mixture
of salts described by Pittman & Bryant (1964) to which was added 0.05 % cysteine, 1-0%
glucose and 0.5 % NaHCO,. The medium was adjusted to pH 7.0, dispensed in 5 ml.
amounts into tubes flushed with CO, and stoppered before autoclaving at 15 lb/in.2 for
15 min. Basal medium + amino acids (BMAA) included vitamin-free, salt-free acid hydrolysed casein (Difco) and 0.0033 % tryptophane.
The vitamin solutions (Pittman & Bryant, 1964) were made separately in distilled water
at x I ooo concentration and included thiamine-HCI, Ca-D-pantothenate, nicotinamide,
riboflavin and pyridoxal at 2 mg./ml.; p-aminobenzoic acid at I mg./ml.; biotin, folic acid
and DL-thioctic acid at 0.5 mg./ml.; and vitamin B,, at 0.2 mg./ml. For use, the desired
vitamin solutions were mixed, diluted tenfold, and sterilized by filtration through a cellulose
acetate filter (0.22 pm. pore size, Millipore). One drop of the x IOO sterile solution was
added aseptically by Pasteur pipette to 5 ml. of BM or BMAA medium. This gave an approximately I/IOOO dilution of the original vitamin stock solutions.
The amount and rate of growth at 37" was estimated with a Spectronic 20 spectrophotometer at a wavelength of 560 nm.
D N A isolation
The bacteria from late log phase cultures were suspended in 0.15 M-NaCl and 0.01
M-EDTA (pH 8-0) and incubated with lysozynie (Marmur, 1961). One yo sodium lauryl
sulphate (SLS) was then added to complete the lysis. Some of the clostridial species were not
susceptible to lysozyme and were disrupted mechanically. After a preliminary extraction
with chromatography-grade liquid phenol, the DNA was isolated according to the method
of Marmur (1961).
Base ratios
The base compositions of the DNA preparations were determined by their thermal
melting point (T,) using an automatic recording spectrophotometer (Gilford Instrument
Laboratories, Oberlin, Ohio, U.S.A.). Escherichia coli B DNA was used as the standard
and the percentage guanine + cytosine (% GC) was calculated from the equation of Marmur
& Doty (1962).
DNA 12 om0 logy experiments
The methods employed in the DNA homology experiments have been described (Johnson
& Ordal, 1968; Johnson, Anderson & Ordal, 1970). The small filters were 3 x 9 mm. and
held 7 to 8 pg. of immobilized DNA. One pg. amounts of 3H DNA were used and competitor levels of 75 and 150 pg. were employed. The incubation temperature was 56".
RESULTS AND DISCUSSION
Strains labelled Clostridium butyricum were tested by the routine methods used in the
Anaerobe Laboratory and conformed to the description of the species in that they were
Gram-positive, anaerobic, sporing bacilli which were actively saccharolytic and produced
large amounts of gas in media containing fermentable carbohydrate. The main products of
fermentation with all strains were butyric and acetic acids. No strain produced indol or
showed proteolytic activity in chopped meat, and none produced detectable amounts of
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C. S . CUMMINS A N D J. L. J O H N S O N
Table 2 . Wall sugars, DNA base composition and DNA homology values
Name as designated
Homology group I
C. butyricum
C. butyricum
C. butyricum
C. butyricum
C. butyricum
C. butyricum
C. butyricum
C. butyricum
C. butyricum
C. butyricum
C. multifermentans
C. multifermentans
C. rnultifermentans
C. multijermentans
C. multifermentans
C. fallax
C. fallax
C.fallax
ATCC
VPI
no.
no.
DAP
isomer
Wall sugars
r
p
Glc
A
-
Gal
>
Rha
Man
"/o
?& Homoiogy
GC 1718
3266
2983
I00
I00
17
34
24
Clostvidium butyricum and butyric acid clostridia
1718
3266
I536
I 622
2089
2419
2969
3005
3006
4752
2398
2399
2400
2402
2417
2403
2404
5982
860
19398
19398
Homology group I1
C. butyricum
2983
2681
C. butyricum
C. butyricum
2783
C. butyricum
2793
C. butyricum
2798
C. butyricum
2965
C. butyricum
2966
C. butyricum
2968
C. butyricum
2980
C. butyricum
298 I
6014
C. multifermentans
2408
C. rnultifermentans
5708
C. amylolyticum
2697
C. beijerinckii
548 1
25751
C. beijerinckii
4420
17778
C. rubrum
4123
I4949
C. rubrum
2781
C. lacto-acetophilum 4419
11914
C. lacto-acetophilum 4668 E-I 1914
C. aurantibutyricum 4635
Other strains in 'the butyric acid group '
C. aurantibutyricum 4633
17777
C. pasteurianum
42 I 5
6013
C. pasteurianum
2670
C. acetobutylicum
2673
C. acetobutylicum
2675-1
.
C. acetobutylicurn
2774
C. acetobutylicum
3I 63-1
824
C. tyrobutyricum
5392
25755
C. pseudojallax
2410
C. pseudofallax
2415-A
.
C.felsineum
2791-1
.
C. felsineum
2766
C. fallax
5729
C. fallax
6010
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
meso
L
meso
meso
meso
L
L
+
+
3+
3+
2+
2+
3+
+
+
2+
2+
28
28
28
28
28
28
27
28
I00
92
88
96
91
85
I00
89
2+
28
I00
+
2+
2+
k
k
&
+
+
+_
2+
I
+
+
+
I
+
+
3+
+
-t
+
+
+
i:
2+
3+
2 f
2 f
2+
+
+
+
2
+
t
+
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84
78
98
77
79
31
23
29
I00
+
3+
3+
85
80
28
28
28
28
28
28
28
28
27
28
28
26
28
27
27
26
28
27
26
28
29
28
28
28
28
26
26
26
22
38
20
45
37
34
35
40
40
35
41
87
86
85
LOO
88
72
80
85
47
37
13
22
I00
15
20
21
26
30
26
25
25
I8
22
25
29
21
28
22
23
21
I8
I7
2
I
6
6
I4
0
78
77
78
76
80
82
87
98
81
79
84
80
91
79
93
90
79
79
92
20
I0
0
I
4
3
4
15
I
22
9
7
24
5
21
I2
5
27
2
5
Taxonomy of the butyric acid clostridia
Table
VPI
Name as designated
no.
C. putri’cum
C. putrificum
C. perfringens
C. perfringens
C. novyi (type A)
C. novyi (type A)
C . botulinum (type A)
C. botulinum (type B)
C. pectinovorum
C. sporogenes
C. sordellii
C. haemolyticum
C. haemolyticum
C. tetani
C. septicum
C. septicum
C. paraputrijkum
C. paraputri8cum
C. tertium
C, tertium
C. innocuum
C. innocuum
2720
4440-1
I537
5694
1619
1626
2 I 30
2 I 3I
4269
3046
2027
2 I 67
0566
2010
1526
2017
1584
I 586
1565
2033
1614
2019
ATCC
no.
DAP
isomer
2
(cunt.)
Wall sugars
meso
meso
meso
meso
L
I 2464
.
.
.
.
meso
meso
meso
meso
meso
NoDAP
NoDAP
NoDAP
NoDAP
. NoDAP
14501 NoDAP
. NoDAP
+
“/o
yo Homology
<
p
h
,
Glc
Gal
Rha
Other strains of Clostridium
meso
25784
meso
L
L
39
-
+
Tr
-
+
+
+
+
2 f
+
+
++
+
+
+
+
-
-
+
+
Tr
-
Tr
+
+
Tr
-
+
+
+-
-
-
-
+
-
-
+
+
+
-
GC 1718
3266
2983
22
16
24
23
26
28
22
2
8
I2
I4
24
I1
I1
25
9
17
18
15
21
I1
21
I3
29
I4
25
24
0
I1
13
27
26
26
24
43
43
29
I9
23
16
I
23
I4
21
26
0
2
0
0
Tr = trace. 3 , 2 4,
etc. indicate the relative amounts present.
lecithinase or lipase on McClung-Toabe egg-yolk agar. In addition, the walls of all these
strains contained the meso-isomer of DAP, and the DNA from all of the strains was 27 to
28% GC (Table 2).
The wall sugar compositions and the DNA homology results agree in showing that
strains labelled Clostridium butyricum fall into two groups, one containing glucose as the
only wall sugar, and the other glucose and galactose (Table 2). The group I organisms
(wall sugar: glucose) showed from 7 2 to 100% homology with reference DNA prepared
from two strains in that group (1718 and 3266). Similarly, DNA preparations from group I1
strains (wall sugars : glucose and galactose) showed 77 to 98 % homology with the reference
DNA from a group I1 strain (2983). However, when competitor DNA preparations from
each group were tested against reference DNA from the other, the degree of homology was
much lower, usually of the order of 25 to 35 yo with extremes of 15 and 47 yo.
As well as strains originally designated as Clostridium butyricum, strains labelled C. multifermentans, C .fatlax, C . amylolyticum, C. beijerinckii, C. lacto-acetophilum, C. rubrum and
C. aurantibutyricum fell clearly into one or the other of the two C. butyricum groups. The
important question regarding these cultures, and a difficult one to answer, is whether they
are characteristic of the species whose name they bear, or are mislabelled cultures. A great
deal of phenotypic similarity has long been recognized among these organisms, and there
has been a corresponding degree of uncertainty about their nomenclature. As far as possible,
we have tried to determine the origins and authenticity of those organisms, belonging to the
two homology groups, that bear specific epithets other than C. butyricum. This information
is given below:
Clostridium multifermentans. It is usually stated (Breed, Murray & Smith, I 957; Beerens,
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40
Caste1 & Put, 1962) that C. multifermentans ferments glycerol but not mannitol, while
C. butyricum ferments mannitol but not glycerol. Furthermore, C. multifermentans is said
to be haemolytic, while C. butyricum is not. All except one (5708) of our seven strains of
C. multifermentans did ferment glycerol (pH of less than 5-90 after I week) and none
fermented mannitol. However, only eight out of 20 strains of C. butyricum fermented mannitol, while four were found to ferment glycerol, so that the distinction between the two
groups on this basis was difficult to apply. Moreover, eight out of 20 strains of C. butyricum
on horse blood agar produced either slight clearing of the blood under the colony, or some
degree of greenish discoloration. In fact, it was not possible to detect any consistent difference
between strains of ‘butyricum’ and those of ‘multifermentans’.
Clostridium fallax. The taxonomic position of C. fallax is complicated by many phenotypic similarities with C. butyricum. The major characteristic that differentiates it from C.
butyricum is toxicity, but this is rapidly lost after isolation. Two strains labelled C. fallax
(2403 and 2404) were obtained from H. Beerens; however, both wall results and the
homology data demonstrate that they belong to C. butyricum homology group I (Table 2).
Prkvot & Loth (1941) determined the fermentation products produced by three strains of
C. fallax including the Tracol strain which was used by Weinberg & Skguin (1915) to
describe the species. Prkvot & Loth (1941) found the phenotypic characteristics of the
Tracol strain to be identical with the original description and identified the major fermentation products as acetic, valeric and lactic acids. We have received transfers of the Tracol
strain from the ATCC ( A T C C I ~ ~ VPI5729)
OO,
and from Prevot (VPI~OIO).
Both contain
L-DAP, have the same wall sugars and show low levels of DNA homology with the reference
organisms (Table 2). Analysis of the fermentation products in the Anaerobe Laboratory
showed that both strains produced acetic and lactic acids but butyric instead of valeric
acid. One strain labelled C. pseudofallax (2410) appears to be similar to the Tracol cultures
(Table 2).
Clostridium rubrum. The two strains of C. rubrum represent two subcultures of the original
strain 34 of Ng & Vaughn (1963)~one of which (4213) came to us from the ATCC (ATCC
14949) while the other (2781) came from McClung’s collection (MCCLUNG
2823). The
phenotypic characters of both as determined in the Anaerobe Laboratory were essentially
the same, except that 2781 did not produce pigment, while 4213 did produce a distinct
pinkish sediment in fermentation tubes where growth was good (i.e. where carbohydrate had
been utilized).
Clostridium amylolyticum. This strain (2697) came from the collection of Professor
Prkvot, and appears from our copy of his records to be the original strain isolated by him
(Prkvot & Saissac, 1950). Apart from the fact that our culture of it appears to be nonmotile, the characteristics of the strain agree with those given by Prevot.
Clostridium aurantibutyricum. The two strains of this species (4633 and 4635) appear
from our records to have been derived from the original w45 strain of Hellinger (1947).
However, they now differ rather sharply in their reactions. Strain 4633 ( ~ ~ ~ ~ 1 7 7 7 7 ;
MCCLUNG2038) is almost certainly authentic, since it is motile, produces gelatinase, and was
found by McClung in 1955 to give a deeply pigmented growth in maize mash and colonies
with pigmented centres on yeast extract dextrose agar (L. S. McClung, personal communication), although it does not now produce pigment. The other strain (4635) is non-motile,
and does not produce gelatinase, and so has presumably become mislabelled at some point.
It is of interest that the two strains also differ markedly in the degree of homology to the
reference DNA from 2983 and in wall composition (Table 2).
One of the Clostridium lacto-acetophilum strains (4668) studied by Bhat & Barker (1947)
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Taxonomy of the butyric acid clostridia
was obtained through the ATCC. The phenotypic characteristics of this strain, obtained in
the Anaerobe Laboratory, were similar to those in the original description.
In summary, it seems that the properties of Clostridium rubrum 4213 and 2781, C.
amylolyticum 2967, C. aurantibutyricum 4633 and C. lacto-acetophilum 4668 correspond
sufficiently closely to the original descriptions to make it reasonable to accept them as
representative strains. In the case of the seven strains of C. rnultifermentans it appears that
in terms of fermentation pattern at least six of the seven strains agree with previously
published descriptions of C. multijermentans, although it must be mentioned that the
characters used would not, from our results, have made it possible to distinguish clearly
between ‘mult$ermentans ’ and ‘butyricum ’.
The results in Table 2 show that there are a number of ‘butyric acid group ’ organisms
which are distinct from either group of Clostridium butyricum. For example, most of the
strains listed as C. pasteurianum, C. acetobutylicum, C. aurantibutyricum, C. fallax and
C. pseudofallax show a relatively low degree of homology to the reference C. butyricum
strains. In some cases, as with the two strains of C. pusteurianum, the degree of homology is
very low indeed. Most of the strains also show differences in wall sugar patterns, which in
some cases appear to be well defined and distinctive (e.g. C . pasteurianum) and in two strains
the L-isomer of DAP was found instead of the meso-isomer. In short, the wall results suggest
that further investigation would show distinctive groups, but the number of strains examined
is so far too small to identify these clearly.
As well as other ‘butyric acid’ clostridia, we have examined representatives of other
species, many of which are predominantly proteolytic rather than saccharolytic and might
therefore be regarded as less closely related to Clostridium butyricum. Many of these strains
do in fact produce butyric acids as a major end product of fermentation, but may do this
from nitrogenous sources rather than from sugars. The results obtained on these strains are
given in the second part of Table 2.
The wall sugar patterns of these organisms are variable, and in most cases quite distinct
from those of either of the Clostridium butyricum groups. More interesting is the variation
in diamino acid of the mucopeptide. It has been well established that strains of C. perfringens have L-DAP in place of the meso-isomer (Hoare & Work, 1957; Salton & Ghuysen,
1957; Haythornthwaite, 1968) and the presence of the L-isomer has been previously reported
in C. pectinovorum (Glendenning, 1958). As mentioned above, it appears from the present
results that other species may also contain the L-isomer. The most unexpected finding was
that a group of strains, representing several different species, have no DAP in their vegetative
walls. A qualitative examination of formamide-extracted wall fractions from the two strains
of C. innocuum and one of C. tertium showed only alanine, glutamic acid and lysine, so that
in these strains lysine has replaced DAP as the diamino acid of mucopeptide. We have not yet
determined whether DAP is present in the spores from these strains, as in the case of the aerobic
aerobic sporing bacillus Bacillus sphaericus (Powell & Strange, 1957 ; Hungerer & Tipper,
1969).
The results for GC content and DNA homology for these strains are also given in the
second part of Table 2 . In terms of yoGC in the DNA most of them show the characteristically low figures found in clostridia. The DNA from the two strains of Clostridium innocuum,
however, have a GC content of 43 %, which is much higher than the rest of the clostridial
DNA preparations, but similar to that found in Bacillus. The DNA homology results for
the strains listed in the second part of Table 2 show that most of them have a definite, but
low, degree of homology to the reference DNAs from the two C. butyricum groups. The
figures vary from about 10% to about 30 % homology, which is essentially the same range
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42
C. S. C U M M I N S AND J. L. J O H N S O N
of values as was found when testing the other butyric acid clostridia against the same
reference DNA preparations, and is very similar to the range found when the two groups of
C. butyricum are tested against each other. In a few cases-for example, the strain of
C. tetani (2010) and the two strains of C. innocuum - there appears to be no homology to the
reference preparations. This is not surprising in the case of the C. innocuum strains in view of
their very different GC content.
From all of these results we may conclude that the nucleotide sequences in the DNA of
the two groups of Clostridium butyricum differ from each other almost to the same extent as
the sequences in either group differ from those in other species of Clostridium. The results
do not suggest that the ‘butyric acid group’ of organisms as a whole constitute a distinct
homology subgroup within the genus Clostridiurn.
Lactate fermentation. Bhat & Barker (I947) chose the acetate-dependent fermentation of
lactate as the distinguishing characteristic for differentiating Clostridium Zucto-acetophilum
from C. butyricum. Bryant & Burkey (1956) observed the same type of fermentation in
several strains of C . butyricum and therefore considered C. lacto-acetophilum invalid. We
tested the ability of several strains from each homology group to grow in the lactateacetate medium of Bryant & Burkey (1956) and found that in each group some strains were
able to grow well in this medium, while some either failed to grow or showed only marginal
growth. We would therefore agree with Bryant & Burkey (1956) that C. lacto-acetophilum
should not be regarded as a separate species on the basis of the acetate-dependent fermentation of lactate.
Nutritional requirements. Since the phenotypic characteristics that have been used to
differentiate the species of the butyric acid bacteria have failed to distinguish the two homology groups, it was considered useful to determine their nutritional requirements. Previous
investigators (Snell & Williams, 1939; Lampen & Peterson, 1943; Wiken & Richard, 1952)
have found that on media containing amino acids or hydrolysed casein, a majority of the
strains of butyric acid bacteria required biotin for luxuriant growth. Some strains required
both biotin and p-aminobenzoic acid, and others p-aminobenzoic acid alone. Wiken &
Richard (1952) found that occasional strains required no vitamins. In the present investigation strains from each homology group have been tested for their ability to grow in a basal
glucose-mineral salts medium (BM) and in the same medium supplemented with amino acids
(BMAA), or with amino acids and vitamins. In general, the organisms in homology group
I would grow in BM+ biotin (0.5 pg./ml.), but grew much faster when Casamino acids+
tryptophane were included also. Growth curves, through three transfers, for strains 3266 and
1622 are shown in Fig. I . Strain 3266 started to grow rapidly on its first transfer from
chopped-meat medium to BM, but when the transmission value had dropped to about 85 %
(initially IOO %) growth stopped and subsequent transfers failed to grow. In BM +biotin the
initial growth was again rapid to about 80% transmission, but, following a lag period,
growth was resumed at a slower rate (Fig. I 6 ) and the two subsequent transfers in BM
biotin showed the slower rate of growth. It may be seen from Fig. r b that although the
growth rate in BM +biotin was slow, the final density of the cultures approached that found
in the complete medium. In BMAA+biotin, however, growth in the first and subsequent
transfers was complete in about 8 h., and the results were virtually identical with those found
in the peptone-glucose-yeast extract medium. Strain 1622 differed from 3266 in that some
growth regularly occurred in BM alone, even at the third transfer, and that in BM +biotin
the slowing of growth was less, so that it was complete in 24 h.
All of the organisms from homology group I1 which were tested (12 strains) failed to
grow, even in the first transfer in BMAA supplemented with 10vitamins (thiamine, panto-
+
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Taxonomy of the butyric acid clostridia
43
thenate, nicotinamide, riboflavin, pyridoxal, p-aminobenzoic acid, biotin, folic acid,
thioctic acid and vitamin Biz). The organisms only grew when a complex mixture of growth
factors, such as yeast extract, was included.
These results demonstrate that previous studies on the vitamin requirements of butyric
acid bacteria (e.g. Wiken & Richard, 1952) have dealt with organisms similar to those in
our homology group I, i.e. those which will grow luxuriantly in basal medium with amino
acids and biotin. It is of interest to note, however, that Bhat & Barker (1947) reported that
strains of Clostridium Zacto-acetophilum would not grow unless yeast extract was added to
the medium. Results of wall analysis and DNA homology tests on two different subcultures
(4419 and 4668) of one of their strains are included in Table 2 and both fall clearly into
homology group 11.
100
90
80
70
60
50
40
g
.
3
10
._
v)
E
$
2
100
90
80
70
60
50
40
30
20
10
0
Incubation time (h.)
Fig. I . Growth response of two Clostridium butyricum strains. 0, First transfer; A, second sequential transfer; 0,third sequential transfer. (a) Growth of strain 3266 on mineral salts-glucose
medium (BM); (b]same as (a)but medium supplemented with biotin (BM+biotin); (c) growth of
strain I 622 on BM; (6) growth of strain 1622 on BM biotin. In (b)and (4, = growth in mineral
salts-glucose medium supplemented with biotin, casein hydrolysate and tryptophane (BMAA+
biotin).
+
To further characterize the butyric acid organisms that have a low level of nucleotide
similarity with homology groups I and 11, additional competition experiments were run using
reference DNA from Clostridium tyrobutyricum (5392), C . pasteurianum (4215) and C.
acetobutylicum (3163-1). It is clear from the results (Table 3) that these organisms, in
addition to C. aurantibutyricum, have only a low level of nucleotide similarity with one
another.
Taxonomic considerations. Because of many phenotypic similarities and the limited
original descriptions, two distinct groups of organisms have been included in Clostridium
butyricum. We consider that the nutritional requirements, the wall sugar compositions and
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C . S. C U M M I N S A N D J. L. JOHNSON
44
the DNA homology data justify the consideration of them as separate species. Since
C. butyricum is the oldest name (Prazmowski, I 880) and designates the type species, it should
be retained. For the following reasons it appears reasonable that C. butyricum should be
represented by the homology group I organisms.
Table 3. Nucleotide similarities among other butyric organisms
% Homology
VPI
no.
5392
4215
2670
3 I 63-1
2673
2774
2675-1
4633
3266
2983
548 1
ATCC no.
Name
25755
6013
C. tyrobutyricum
C. pasteurianum
C. pasteurianurn
C. acetobutylicum
C. acetobutylicum
C. acetobutylicurn
C. acetobutylicum
C. aurantibutyvicum
C. butyricum (group I)
C. butyricum (group 11)
C. beijerinckii
824
I 7777
19398
2575 1
h
I
5392
\
4215
3163-1
I0
I00
I00
I0
I0
I0
I00
I0
I2
96
92
90
I00
4
I2
7
9
8
12
8
7
I4
I5
18
16
2
18
I0
I
I5
4
(I) Because of the ease with which the group I organisms can be cultured, it is more likely
that a representative of this type was isolated first.
(2) The production of butyric acid during fermentation and the requirement for biotin
are common to many of the species within the genus Clostridium. On these grounds, m embers
of homology group I would represent a good type species. On the other hand, organisms
with more complex requirements may reflect varying degrees of loss in biosynthetic capabilities and would be less typical of the genus as a whole.
(3) Previous studies on the vitamin requirements of Clostridium butyricum (Snell &
Williams, 1939; Lampen & Peterson, 1943; Wiken & Richard, 1952)indicate that the homology group I organisms were used. Certain strains of C. multifermentans and C .fallax should
be considered in this group.
Among the organisms which we have found to belong to homology group I1 are strains
bearing several specific names other than Clostridium butyricum. These are C. beijerinckii,
C. amylolyticum, C. rubrum, C. multifermentans and C. lacto-acetophilum. The on1y specific
name which seems suitable for the group is C. beijerinckii. Strains of C. multferment ans have
been found in both homology groups, and the names amylolyticum, rubrum and lactoacetophilum describe particular properties not characteristic of homology group I1 as a
whole. We would propose therefore that the homo1 ogy group I1 organisms be regarded as a
separate species, and that they should bz called C. beijerinckii (Donker, 1926) and that
rubrum, amylolyticum and lacto -acetophilum be regarded as synonyms of beijerinckii.
This investigation was supported by the National Institutes of Health, Institute of General
Medical Sciences, grant number GM 14604. We thank Professors A. R. Prkvot, L. S.
McClung, L. V. Holdeman and W. E. C. Mo ore for supplying us with unpublished phenotypic data on many of the strains. The technical assistance of Wendy Barrick, Patricia
Lahoda and Habiba Najafi was appreciated.
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Taxonomy of the butyric acid clostridia
45
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