ELSEVIER
MICROBIOLOGY
REVIEWS
FEMS MicrobiologyReviews 17 (1995) 233-240
Taxonomic relationships among strains of Clostridium
acetobutylicum and other phenotypically similar organisms
J.L. Johnson, J.-S. Chen *
Department of Biochemistry and Anaerobic Microbiology, Virginia Polytechnic Institute and State University, Blacksburg, VA 24061-0305,
USA
Received 26 October 1994; accepted 9 March 1995
Abstract
Most of the presently studied acetone-butanol (solvent)-producing bacteria are labelled as Clostridium acetobutylicum.
This situation contrasts what was experienced by investigators of the 1940s who faced a plurality of names for
solvent-producing bacteria. Significant phenotypic differences, however, exist among the presently studied strains of C.
acetobutylicum, which raised the question of whether or not these organisms can truly be considered as members of one
species. Furthermore, two cultures (ATCC 824 and NCIMB 8052) that are thought to be equivalent in serving as the type
strain of C. acetobutylicum have significantly different properties. To assess the relatedness of these bacteria as members of
a species, a comparison of similarity of their genomic DNA is most effective. DNAs from cultures of clostridia labelled as
C. acetobutylicum, 'C. butylicum', and C. saccharoperbutylacetonicum from several collections have been compared with
DNAs from reference strains, including the type strain of C. acetobutylicum and C. beijerinckii. Based on DNA
reassociation, which measures sequence similarities, four distinct groups or species (with inter-group similarities below 30%)
were identified: (i) those having > 80% DNA sequence similarity with the type strain of C. acetobutylicum; (ii) those,
including NCIMB 8052, having > 70% DNA sequence similarity with the type strain of C. beijerinckii; (iii) two cultures
(NRRL B643 and NCP 262) having 94% similarity between them; and (iv) C. saccharoperbutylacetonicum. Identification of
four species from these solvent-producing clostridia explains the discrepancies reported by different laboratories, and
classification of these bacteria on the basis of their genomic relatedness should facilitate future genetic experiments. It is
noteworthy that after the carbon source was switched from starch (corn mash) to sugars (molasses), the industrial solvent
fermentation indeed utilized organisms (represented by groups 2, 3, and 4) genetically distinct from C. acetobutylicum.
Keywords: Taxonomyof solvent-producingclostridia; Clostridium acetobutylicum; Clostridium beijerinckii; Clostridium saccharoperbutylacetonicum; DNA sequence similarities
I. Introduction
During the past 15 years, much new research has
been conducted with the acetone-butanol (solvent)-
* Corresponding author. Tel.: + 1 (540) 231 7129; Fax: + 1
(540) 231 7126; E-mail: [email protected].
producing clostridia. Organisms used in these studies
include Clostridium acetobutylicum (see recent reviews in [1]), C. aurantibutyricum [2], C. beijerinckii
(syn. C. butylicum, [2]), C. puniceum [3], C.
tetanomorphum [4], C. saccharoperbutylacetonicum
[5], and C. thermosaccharolyticum [6]. The most
widely used organisms, however, have been several
strains of C. acetobutylicum that are available from
0168-6445/95//$29.00 © 1995 Federationof European MicrobiologicalSocieties. All rights reserved
SSD1 0168-6445(95)00016-X
J.L. Johnson, J.-S. Chen / FEMS Microbiology ReL,iews 17 (1995) 233-240
234
culture collections such as ATCC (American Type
Culture Collection), DSM (German Collection of
Microorganisms or Deutsche Sammlung von
Mikroorganismen), NCIMB (National Collections of
Industrial & Marine Bacteria Ltd), and NRRL (Midwest Area National Center for Agriculture Utilization Research, US Department of Agriculture).
Apparently equivalent cultures are maintained in
different collections. For example, the type strain for
C. acetobutylicum (ATCC 824, DSM 792, NCIMB
8052 (see below for further information on this
strain), and NRRL B527) is available from each of
these collections, and it has been used more than any
other strains in recent investigations. As might be
expected, studies involving different strains of C.
acetobutylicum yielded different results, and properties of some strains differ significantly (see below)
that one could question the taxonomic relationship
among 'these strains. More surprisingly, studies using
the type strain from two different collections also
gave contradictory results. It has thus prompted examinations of the history of the strains in the culture
collections and also investigations to determine the
genetic relatedness of these strains. This paper re-
Table 1
Apparent relationship among strains of C. acetobutylicum, C. beijerinckii, and related clostridia in culture collections and laboratories ~
ATCC
NRRL
DSM
NCIMB
McCoy
B466
824
(862) c
3625
4259
8529
10132
B527
B528
B529
B530
B591
B592
B593
B594
Reid 39-90
526
792
8052 ~
1737
1731
1738
619
1739
9380
6445
8049
B596
B643
A-77
W, B-16
D, B-15
H, A-49
T, A-50
48
A-8
A-39
A-21
A- 14
Weyer
Donker
Hall
Thaysen
A-38
1732
1733
(13564)
17778; 25752
27021
27022
35702
39057
39058
39236
43084
Other source
791
2951
6441
6442
6443
6444
9362
B-14
B-3
B-4
B-10
A-13
A-67
CSC
Weizmann
P262; NCP262
N1-4
VP15481
NI-4
N 1-504 (from N 1-4)
Blaschek (from 824)
IFP 903
IFP 904
CPC B11-3
Benassi
The apparent relationship is based on information contained in the catalogs published by American Type Culture Collection (ATCC),
National Collection of Industrial and Marine Bacteria (NCIMB), and Deutsche Sammlung yon Mikroorganismen (DSM). History of NRRL
strains was from L.K. Nakamura (Midwest Area National Center for Agricultural Utilization Research, US Department of Agriculture).
History of McCoy strains was from L.S. McClung (Indiana University) via E. Cato.
b NCIMB 8052 has been shown to be different from ATCC 824, DSM 792, and NRRL B527.
c Parentheses denote deaccessioned strains.
a
J.L. Johnson, J.-S. Chen / FEMS Microbiology' Re~,iews l 7 (1995) 233-240
views the current status of the taxonomy of strains of
C. acetobutylicum and related organisms in major
culture collections.
2. Historic background
strains
of
C. acetobutylicum
The Weizmann process for solvent production
used starchy material as the carbon source, and the
organism for the process was C. acetobutylicum [7].
Following the expiration of the Weizmann patent in
1936, molasses was found to be a more desirable raw
material than potato or corn mashes. The isolation of
new organisms capable of fermenting molasses to
solvents led to the proliferation of names for
solvent-producing clostridia, as patents for these molasses-based processes contained a multitude of
names for organisms that were associated with these
processes (see Table 1 in [8]). Several of these
processes produced isopropanol instead of acetone as
a major end product (see Table 2 in [8]). During that
period, new species were proposed on the basis of
raw materials used, the ratio of end products formed,
and other distinct growth characteristics. Using the
current criteria of molecular taxonomy, the species
status of many of these organisms would be questionable because the genetic properties of these organisms were largely unexplored, and the ratio of
end products formed is not a useful criterion for the
classification of solvent-producing clostridia [2].
Some of these molasses-fermenting and solventproducing clostridia were later grouped with C. acetobutvlieum. For example, 'C. acetobutylicum' NRRL
B591 was strain A8 of the former McCoy collection
at the University of Wisconsin, and it had the name
'Clostridium saccharoacetobutylicum' in the US
Patent 2,110,109 [9].
In an effort to obtain molasses-fermenting and
high-butanol-producing clostridia, Hongo and Nagata
in 1959 isolated from soil a useful bacterium and
named it C. saccharoperbutylacetonicum [10,11].
This organism is distinct from other clostridia in its
susceptibility to phages [12]. Reysset and co-workers
[13] temporarily included C. saccharoperbutylacetonicum in C. acetobutylicum because the species
name was not validly published according to the
rules of the Code of Nomenclature. Consequently,
235
this organism and its derivatives became known as
C. acetobutylicum in more recent literature [14-16].
The majority of C. acetobutylicum strains in culture collections today can be traced, directly or
indirectly, to the McCoy collection, who obtained
some of them from other investigators. The type
strain for C. acetobutylicum (ATCC 824, DSM 792,
and NRRL B527) can be traced to McCoy strain W
(for Weyer) or strain BI6. Several other strains from
different collections also seem equivalent (see Table
1). Excluding mutant strains and equivalent strains,
these culture collections appear to have a total of 16
potentially distinct strains of C. acetobutylicum.
3. Significant differences among strains resembling C. acetobutylicum
During the past 15 years, a substantial amount of
research was performed on several strains resembling C. acetobutylicum (see recent reviews in [1]),
which include ATCC 824, DSM 792, DSM 1731,
DSM 1732, NCIMB 6444, NCIMB 8052, NCP 262,
and NRRL B643. Several other strains from these
collections were also used but to a lesser degree.
Among the more extensively studied strains, ATCC
824, DSM 792, and NCIMB 8052 were considered
equivalent as the latter two cultures were propagated
from ATCC 824, which is the type strain for the
species (see catalogs of strains published by these
culture collections). Significant differences in genetic
and physiological properties, however, have been
detected between strains NCIMB 8052 and ATCC
824 (e.g. [16,17]). In addition, strains NCP 262 and
NRRL B643 also differ from the type strain. These
differences are briefly reviewed below.
3.1. Degeneration of the ability to produce solcents
'C. acetobutylicum' NCIMB 8052 (formerly NCIB
8052) readily loses its ability to produce solvents or
to form spores in continuous cultures [18,19]. On the
other hand, strains ATCC 824 and DSM 1731 retain
their ability to produce solvents in continuous cultures, although asporogenous mutants become dominant in such cultures [20,21]. ATCC 824 and DSM
792 retain their ability to produce solvents after
repeated subculturing at 24-h intervals, but for some
unknown reasons a larger volume of inoculum be-
236
J.L. Johnson,J.-S. Chen/ FEMSMicrobiologyReviews17 (1995)233-240
came required for the batch culture to produce solvents once a certain number of subculturing was
surpassed [22].
3.2. pH and the solventogenic switch
The pH of the culture medium has long been
considered an important factor in the control of
solvent production [23]. A low pH was believed
necessary for the onset of solventogenesis or for
sustaining solvent production by C. acetobutylicum.
Experiments performed with C. acetobutylicum
ATCC 824, DSM 792, and DSM 1731 showed that
sustained solvent production occurred only at or
below pH 5.5, with or without the supplementation
of butyrate [24-27]. However, butanol production
was initiated at pH 6, although it was not sustained,
with the strain ATCC 824 [24,28]. An optimal pH of
4.3 was found for DSM 792 and DSM 1731 [25,27],
and when the pH was not controlled, solvent production by DSM 792 occurred even when the pH fell to
3.8 during active growth [25]. In contrast, the strain
NCIMB 8052 will produce solvents in cultures maintained at pH 7 [29], which resembles the behavior of
C. beijerinckii VPI 13436 ( = NRRL B592) [30]. It
is important to note that industrial solvent production
using cultures other than the 'Weizmann organism'
requires the maintenance of the pH above 5.5, especially when molasses served as the raw material [31].
With respect to the pH requirement, 'C. acetobutylicum' P262 represents another class because
good levels of solvent production can be obtained
within the pH range of 5.0-6.5, and no solvent is
produced if the pH of the culture is allowed to drop
below 4.5 during the early part of the fermentation
[23].
3.3. Structure and organization of acid-and solventproduction genes
Most of the structural genes encoding enzymes
for acid-and solvent formation in C. acetobutylicum
ATCC 824 have been cloned and sequenced [32],
and corresponding genes have also been cloned from
strains DSM 792 and NCIMB 8052 and analysed.
Between ATCC 824 [33] and NCIMB 8052 [34],
significant sequence differences are present in the
region of chromosome encompassing the two genes
(ptb and buk) that code for the enzymes -phos-
photransbutyrylase (PTB) and butyrate kinase (BK)
-for the synthesis of butyric acid. The deduced
amino acid sequence for PTB had an identity of 68%
between the two strains, whereas the sequence for
BK had an identity of 64.2%. For PTB, the initiation
codon is GUG in ATCC 824, whereas it is AUG in
NCIMB 8052. The relative position of ptb and buk
is the same in the two organisms, but the length of
the intergenic region differs by 100 bp and there is
no sequence similarity in the flanking regions. Unless the analysed ptb and buk genes are coding for
different sets of isozymes and are from different
regions of the chromosome, the sequence data indicate that ATCC 824 and NCIMB 8052 are not
equivalent.
In contrast, ATCC 824 and DSM 792 are equivalent in terms of the structure and organization of the
genes encoding the solvent-forming enzymes acetoacetate decarboxylase, acetoacetate:acetate/
butyrate coenzyme A-transferase, and the putative
aldehyde/alcohol dehydrogenase [35-38].
In 'C. acetobutylicum' P262, the genes (adh-1
and hbd) that encode an alcohol dehydrogenase
(ADH) and the 3-hydroxybutyryl-CoA dehydrogenase (HBDH) occur next to each other [39]. DNA
sequences related to adh-1 were not detected in
ATCC 824 [40], and the gene for HBDH is not close
to genes for ADH in ATCC 824 (G. Bennett, personal communication).
3.4. Presence of a specific restriction endonuclease
Strains ATCC 824 and NCIMB 8052 can be
distinguished by the presence in the former of a
restriction endonuclease recognizing the sequence
5'-GCNGC-3' [41]. Thus, unless properly methylated, plasmids prepared from Escherichia coli
cannot transform ATCC 824 [42]. On the other hand,
NCIMB 8052 [43] (and C. saccharoperbutylacetonicum N1-4082 [15]) apparently lacks the restriction
system because they can be transformed by shuttle
vectors prepared in E. coli without the ~b3T I methyltransferase.
3.5. Profile of restriction fragments
Wilkinson and Young [16] analysed the profile of
restriction fragments and the minimum size of the
J.L. Johnson, J.-S. Chen/ FEMSMicrobiologyReviews17 (1995) 233-240
genome of C. saccharoperbutylacetonicum and four
strains of 'C. acetobutylicum'. After cleavage of
their DNA with SmaI or ApaI, the restriction profile
of ATCC 824 and NCIMB 8052 is clearly different.
Based on their restriction profiles and minimum
genome sizes, three discrete groups were identified:
Group 1, strains ATCC 824 and DSM 1731; group 2,
NCIMB 8052 and C. saccharoperbutylacetonicum
N1-4081; group 3, NCP 262.
237
and hence for the butanol and butyraldehyde dehydrogenase activities of this organism. A 96 000-Da
polypeptide has been identified as the product of the
aad gene [38], which suggests that the butyraldehyde
dehydrogenase of C. acetobutylicum strains ATCC
824 and DSM 792 is different from the ALDH
purified from 'C. acetobutylicum' NRRL B643.
3.6. Properties of solvent-forming enzymes
4. Distinct groups of solvent-producing clostridia
as defined by DNA sequence similarities
Among the differences in the solvent-forming enzymes between strains, the property of the alcohol
and aldehyde dehydrogenases is perhaps the most
remarkable. The complex picture of alcohol dehydrogenases (ADHs) found in solvent-producing bacteria
will be reviewed elsewhere [44]. Aldehyde dehydrogenase (ALDH) is a key enzyme for the formation of
butyraldehyde and acetaldehyde, which are reduced
by ADHs to form butanol and ethanol, respectively.
From 'C. acetobutylicum' NRRL B643, an inducible coenzyme A-linked ALDH was purified [45],
which has a native molecular mass of 115 000 and a
subunit molecular mass of 56000. A very similar
ALDH was purified from C. beijerinckii NRRL
B592 [46], and the purified ALDH from this organism can account for all acetaldehyde-and butyraldehyde-linked activities in the cell extract, indicating
that the ALDH is the major, if not the only, ALDH
in this organism. These results would suggest that
ALDH is a conserved enzyme in solvent-producing
clostridia. More recent findings with C. acetobutylicum ATCC 824 and DSM 792, however, reveal the presence of different ALDHs in solvent-producing clostridia.
In an analysis of Tn916-induced mutants of C.
acetobutylicum DSM 792, acetaldehyde dehydrogenase and butyraldehyde dehydrogenase activities
were found to be affected differently in some mutant
strains, indicating the presence of at least two ALDHs
in this organism [47]. It was later found that an
apparently fused gene, named adhE for the strain
DSM 792 [36] or aad for the strain ATCC 824 [38],
encodes a polypeptide containing two domains, one
corresponding to an ADH and the other to an ALDH.
Transcriptional properties of the adhE gene suggest
that its product is responsible for butanol formation
Similarities in phenotypic characteristics have
been the traditional and the most accessible criteria
for bacterial classification. However, genetically distinct but phenotypically similar (in terms of practically measurable traits) organisms cannot be distinguished by this approach. This problem is solved by
using genomic relatedness as the basis for classification [48]. Two solvent-producing species, C. acetobutylicum and C. beijerinckii, are unambiguously
distinguished by a comparison of their DNA sequence similarity [49]. On the other hand, solvent
production is not a useful trait for classification [2].
A group of bacteria is readily recognized as members of a species when their DNA sequence similarities are above a proposed cut-off value of 60-70%,
which is based on comparisons of natural bacterial
groups (conventional species) sharing many phenotypic characteristics [48]. Classifications based on
genomic relatedness are conceptually unified and are
less susceptible to frequent or radical changes.
Besides comparing genomic relatedness, useful
taxonomic information is also obtained by comparing sequence similarities between ribosomal RNAs
[48]. Cistrons for rRNA represent a subset of the
genome, and nucleotide sequences of rRNAs are
more conserved than the complete genome among
related organisms [50]. Therefore, comparisons of
rRNA sequence similarities are more useful in distinguishing taxa above the species level as well as in
identifying groups of more related species. Thus,
several groups of clostridia can be recognized based
on the similarity of their 23S rRNA, and the following solvent-producing species belong to distinct
groups: group IA, C. beijerinckii; group IB, C. aurantibutyricum; and group IJ, C. acetobutylicum [50].
Because of the differences observed among cultures
238
J.L. Johnson,
J.-S. Chen/ FEMS MicrobiologyReviews 17 (1995)233-240
labelled as C. acetobutylicum, it is desirable to measure their DNA sequence similarities. Based on results obtained to date, the currently available 'C.
acetobutylicum' cultures from major culture collections and laboratories can be separated into four
distinct groups with inter-group similarities below
30% (Johnson, Toth, and Chen, unpublished results).
The intra-group similarity is at least 73%, which
supports a species status for each of these four
groups. A listing of cultures within each group is as
follows:
Group 1. This group contains cultures having a
DNA sequence similarity between 84 and 99% with
the type strain (ATCC 824) of C. acetobutylicum:
ATCC 4259, DSM 792, DSM 1731, NCIMB 619,
NCIMB 2951, NCIMB 6441, NCIMB 6442, NCIMB
6443, NRRL B527, NRRL B528, and NRRL B529.
These cultures are therefore strains of C. aceto-
butylicum.
Group 2. The group contains cultures of 'C.
acetobutylicum' having a DNA sequence similarity
between 73 and 84% with the type strain (VPI
5481 = A T C C 25752) of C. beijerinckii: ATCC
10132, NCIMB 6444, NCIMB 6445, NCIMB 8049,
NCIMB 8052, NRRL B591, and NRRL B594. These
cultures are therefore strains of C. beijerinckii. In
addition, the following cultures, whose current labels
are in parentheses, have also been identified as strains
of C. beijerinckii: DSM 526 ('Clostridium pasteurianum'), NRRL B466 ('C. butylicum'), NRRL B592
('C. butylicum'), NRRL B593 ('C. butylicum'),
NRRL B596 ('C. butylicum'), and McCoy strain
A77 ( = NRRL B598, 'C. pasteurianum').
Group 3. Two cultures of 'C. acetobutylicum',
NRRL B643 and NCP 262 ( = P262), had 94% of
similarity. This group is yet to be named as a
species.
Group 4. This group is represented by C. saccharoperbutylacetonicum strain N1-4 and its derivative
N1-408i; the latter strain has been known as 'C.
acetobutylicum' NI-4081 in recent literature [13-15].
5. Concluding remarks
Using DNA reassociation as the basis of classification, the currently investigated cultures of 'C. acetobutylicum' can be identified as strains of four
species. This finding can account for the significant
differences previously contrasting different strains of
'C. acetobutylicum'. The identification of NCIMB
8052 as a strain of C. beijerinckii solves the problem
associated with its previous designation as the type
strain of C. acetobutylicum. The reported close phylogenetic relationship between C. acetobutylicum and
C. beijerinckii as well as C. butyricum [51,52] contradicts the findings of two earlier studies [49,50].
This discrepancy is now resolved because the recently reported sequence of 16S rRNA of 'C. acetobutylicum' [51] was obtained with strain NCIMB
8052, which is C. beijerinckii. Classification of solvent-producing clostridia on the basis of genome
relatedness should facilitate future genetic experiments. It is noteworthy that after the carbon source
was switched from starch (potato or corn mashes) to
sugars (molasses) in the late 1930s, the industrial
solvent fermentation indeed utilized organisms genetically distinct from C. acetobutylicum. Organisms
for the molasses-based processes include C. beijerinckii, C. saccharoperbutylacetonicum, and a species
represented by NCP 262 and NRRL B643.
Acknowledgements
We thank Peter Diirre, Robert Gherna, Lawrence
K. Nakamura, Ronald S. Pirrie, Gilles Reysset, David
Woods, Sadazo Yoshino, and Michael Young for
providing cultures used in the measurement of DNA
reassociation in authors' laboratories. The work of
Johnson, Toth, and Chen reported here was supported by US Department of Energy grant DE-FG0585-ER13368 (L-S.C.) and by the Cooperative State
Research Service, US Department of Agriculture,
under project number 6129960 (J.-S.C. and J.L.J.).
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