Download Taxonomic Study of Anaerobic, Gram-Negative, Rod

JOURNAL OF SYSTEMATIC BACTERIOLOGY, J a n . 1990, p. 19-27
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Copyright 0 1990, International Union of Microbiological Societies
INTERNATIONAL
Vol. 40, No. 1
Taxonomic Study of Anaerobic, Gram-Negative, Rod-Shaped
Bacteria from Breweries: Emended Description of Pectinatus
cerevisiiphilus and Description of Pectinatus frisingensis sp. nov. ,
Selenomonas lacticllfex sp. nov. Zymophilus rafinosivorans gen.
nov., sp. nov., and Zymophilus paucivorans sp. nov.
KARL HEINZ SCHLEIFER,l* MONIKA LEUTERITZ,l NORBERT WEISS,2 WOLFGANG LUDWIG,l
GUDRUN KIRCHHOF,l AND HELGA SEIDEL-RUFER3
Lehrstuhl fur Mikrobiologie, Technische Universitat Miinchen, 0-8000 Munich 2, Federal Republic of Germany';
Deutsche Sammlung von Mikroorganismen und Zellkulturen, 0-3300 Braunschweig-Stockheim, Federal
Republic of Germany2; and Staatliche Brautechnische Prufi und Versuchsanstalt der Technische
Universitat Miinchen, 8050 Freising- Weihenstephan, Federal Republic of Germany,
A collection of 47 strains of obligately anaerobic, gram-negative, rod-shaped bacteria that were isolated
mainly from spoiled beer and pitching yeast was studied to learn more about their taxonomic positions. A new
species of the genus Pectinatus, PectinQtusfrisingensis, a new species of the genus Selenomonas, Selenomonas
lacticifex, and a new genus comprising two species, Zymophilus raflnosivorans and Zyrnophilus paucivorans, are
described. All of the strains contained directly cross-linked meso-diaminopimelic acid-containing peptidoglycan
and in addition the diamine cadaverine or (rarely) putrescine. The diamine was covalently linked to the
a-carboxyl group of D-glutamic acid in the peptide subunit of peptidoglycan. Lipid F was also found as a
characteristic cellular compound. The phylogenetic relationships of members of these new species were
examined by reverse transcriptase sequencing of 16s rRNA or by DNA-DNA hybridization studies or both. All
of the organisms belong to the subdivision containing species with gram-negative cell walls within the phylum
of gram-positive bacteria. This finding is in good agreement with the presence of a peptidoglycan that contains
diamine.
For a long time it was thought that gram-negative bacteria
are not important as spoilage organisms of beer. However,
more recently workers have described some anaerobic,
gram-negative bacteria that cause turbidity and off-flavors in
bottled beer. A gram-negative, anaerobic, rod-shaped bacterial contaminant of beer was isolated and described by Lee
et al. (16) as Pectinatus cerevisiiphilus. Later, bacteria fitting
the description of Lee et al. (16) were isolated from turbid
and off-flavor beer samples in the Federal Republic of
Germany (l), Scandinavia (9, lo), and Japan (22). Gramnegative, anaerobic beer spoilage cocci that were isolated by
Weiss et al. (23) were named Megasphaera cerevisiae (7).
Since the description of P. cerevisiiphilus was based on
only one strain, we decided to carry out a more thorough
taxonomic study of anaerobic, gram-negative bacteria isolated from spoiled beer or pitching yeast.
MATERIALS AND METHODS
Strains and cultivation. Strains that were isolated from
spoiled beer are listed in Table 1, and strains that were
isolated mainly from pitching yeast are shown in Table 2. P.
cerevisiiphilus ATCC 29359T (= DSM 20467T) (T = type
strain), Pectinatus sp. strain ATCC 33332T, Selenomonas
ruminantium subsp. lactilytica DSM 2872T, and Selenomonas sputigena ATCC 3 5 M T were used as reference strains.
Organisms were grown under anaerobic conditions at 30°C in
a modified MRS medium containing (per liter) 10 g of
peptone, 5 g of yeast extract, 2 g of meat extract, 10 g of
glucose, 2 g of K2HP0,, 5 g of NaCl, 0.2 g of MgSO, 7H20,
* Corresponding author
0.05 g of MnSO,, 1 ml of Tween 80, 40 ml of a salt solution
(see below), 0.5 g of cysteine hydrochloride, and 1 mg of
resazurin (pH 6.8). The salt solution contained (per liter) 2 g
of CaCl,, 0.2 g of MgSO,, 1g of K,HPO,, 1g of KH2P04,10
g of NaHCO,, and 2 g of NaC1. For MRS agar medium 10 g
of agar per liter of medium was added.
Physiological tests. Sugar fermentation tests were carried
out in microtiter plates. The wells were filled with 2 0 4
portions of filter sterilized 10% (wt/vol) solutions of sugars.
A 10-ml portion of an overnight culture grown in MRS
medium was centrifuged and suspended in 10 ml of sugarfree MRS medium. Wells containing the sugar solutions and
a control well containing sugar-free medium were inoculated
with 150-pl portions of the suspension and incubated at 30°C
for 48 h under anaerobic conditions (GasPak; BBL Microbiology Systems, Cockeysville, Md.). To evaluate acid formation, 20 p,l of a solution of bromocresol purple (10 mg
dissolved in 0.5 ml of ethanol and made up to 10 ml with
distilled water) was added. The control well containing the
sugar-free solution always showed a negative reaction. Utilization of esculin was determined in sugar-free modified
MRS medium containing 0.25% esculin, 0.05% FeCl,, and
0.25% agar. Nitrate reduction was determined in sugar-free
modified MRS medium also lacking resazurin and cysteine
but containing 0.15% KNO,, 0.2% glucose, and 0.25% agar.
Liquification of gelatin was tested in sugar- and resazurinfree modified MRS medium containing 4% gelatin and 1%
agar. For production of H2S, we used the SiM medium of E.
Merck AG (Darmstadt, Federal Republic of Germany) enriched with 5 g of yeast extract per liter, salt solution (see
above), 0.05 g of cysteine hydrochloride per liter, and 1g of
glucose per liter.
19
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INT.J. SYST.BACTERIOL.
SCHLEIFER ET AL.
TABLE 1. Characteristics of strains of Pectinatus species isolated from spoiled beef
~~
Strain
Group I ( P . cerevisiiphilus)
ATCC 29359T
WK1
WK2
WK3
WK4
BH1
BH2
Walc2
Walb
Wa3l1
Wa312
Group I1 (P.frisingenesis)
ATCC 33332T
BB1
BB2
DS 1
DS2
DU1
DU2
MKKl
MKK2
MKPl
MU1
MU2
WL1
WL2
G+C
content
(mol%)
40
40
38
39
ND
40
40
40
41
40
40
39
41
40
40
ND
39
ND
40
38
40
39
39
ND
40
Production of acid from:
Xylose
Mannitol
Dulcitol
Adonitol
Xylitol
Inositol
Cellobiose
Melibiose
g~~-$$e
Gluconate
Esculin
-
-
-
+2
-
-
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+W
+W
+
+W
+
+W
+W
+
+
+
+W
+W
+
+
All strains (except the strains indicated in parentheses) produce acid from ribose (strains DS1 and DS2), arabinose, rhamnose, glycerol, erythritol, sorbitol
(strain ATCC 29359), fructose, mannose, glucose, and galactose (strain ATCC 33332); all strains (except the strains indicated in parentheses) fail to produce acid
from sucrose, trehalose, melezitose, inulin, glycogen, lactose (strain Wa3/1), maltose (strains ATCC 33332T, DS1, DS2, and MU2), &nose (strains MKK 1 and
MKP l), salicin (strains ATCC 29359T, MKK1, WL1, and WL2), and starch (strains MKKl and MKP1).
+, Positive reaction; -, negative reaction; +w, weak positive reaction.
Fermentation products. Fermentation products were determined as described by Holdeman et al. (11).A model 7620
gas chromatograph (Hewlett-Packard Co., Palo Alto, Calif.)
equipped with a Chromosorb 101 column and a flame ionization detector was used for analysis of volatile and nonvolatile acids.
Cell walls. Preparation of cell walls and determination of
the peptidoglycan type were carried out as described by
Schleifer and Kandler (20). Cadaverine and putrescine could
be separated by gas chromatography, using a column filled
with Chromosorb 103 (Sigma Chemical Co., Deisenhofen,
Federal Republic of Germany).
Quinones and quinonelike compounds. Analyses of quinones and quinonelike compounds were performed as described by Collins et al. (2).
Determination of G+C contents of DNAs and DNA homology studies. DNA was isolated as described by Meyer and
Schleifer (18). The melting point of the purified DNA was
determined by using the method of Marmur and Doty (17),
and the guanine-plus-cytosine (G + C) content was calculated
by using the method of DeLey et al. (5). DNA from Escherichiu coli strain B (Sigma Chemical Co., St. Louis, Mo.),
with a G + C content of 51.7 mol%, was used as the reference. DNA-DNA hybridization studies were carried out by
spectrzphotometrically determining renaturation rates, as
previously described (5, 13).
16s rRNA analysis. RNA was isolated as described previously (6). Sequencing of 16s rRNA by using reverse tran-
scriptase was done as described by Lane et al. (15). Oligonucleotide primers were synthesized by standard methods,
using a Biosearch Cyclone DNA synthesizer. The sequences
of these primers are complementary to highly conserved
regions of 16s rRNA. Sequence ambiguities were resolved
by using terminal transferase (4). Alignment of the sequences was done with respect to conserved primary structures, as well as secondary structures. From this alignment
binary dissimilarity values were calculated and subsequently
transformed into phylogenetic distance (Knuc) values (12).
Only those positions which were present in all sequences
and were invariant in 30 to 90% of the aligned sequences
were included. Thus, universal positions, as well as highly
variable positions, were excluded. An unrooted phylogenetic tree was constructed from the distance values by using
the method of Fitch and Margoliash (8).
RESULTS
Morphology. Group I and I1 strains were slightly curved,
gram-negative, rod-shaped bacteria with rounded ends.
They occurred predominantly as single cells or sometimes in
pairs. They were 0.7 to 0.9 by 3 to 20 pm. The occurrence of
longer, slightly helical filaments was characteristic for stationary-phase cultures. Younger cells were usually motile.
Group I11 strains were straight to slightly curved, motile,
gram-negative rod-shaped organisms with rounded ends.
They were 0.7 to 0.9 by 3 to 15 pm. They occurred
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ROD-SHAPED BACTERIA FROM BREWERIES
21
TABLE 2. Physiological characteristics and G+C contents of gram-negative, anaerobic, slightly curved to helical or crescent-shaped
bacteria isolated from pitching yeast and brewery wastea
Strain
Group 111 (Z. rafinosivorans)
OW1311
OW1312
ow1111
ow1112
0w 10/1
ow1012
OW9
OW8
IB1
IB2
RG1
RG2
VB2
SH2T (= DSM 207GT)
Group IIIa (Z. paucivorans)
VB 1
VB3a
AAIT (= DSM 20756*)
AA2
Group IV (Selenomonas lacticifex)
VB4bT (= DSM 20757T)
VB6
VB7
VB8
G+C
Production of acid from:
content
(mol%) Ribose Xylose Rhamnose Mannitol Adonitol Erythritol Xylitol Inositol Glycerol Salicin Amygdalin
41
41
41
39
38
41
40
38
39
40
40
40
39
41
39
41
40
40
51
51
52
52
+b
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+W
-
+W
+W
+W
+W
+W
+W
-
+
+W
+
+
-
-
-
-
-
-
+
+
+
+
+
-
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+W
+W
+W
+W
+W
+W
+W
+W
+W
+W
+W
+W
+
+
-
+-
-
+W
-
-
+W
+W
-
-
+
+
+
+
+
+
+
+
+
+
+
+W
+
+
+W
-
+W
+W
+
-
+
a All strains (except the strains indicated in parentheses) produce acid from arabinose (strain VBl), sorbitol (group IV strains and strain VB3a), dulcitol (group
IIIa and group IV strains), fructose, rnannose, glucose, galactose (strains VB1 and VB3a), lactose (strains VB1, VB3a, and AAIT), maltose (strain VB4b), sucrose,
cellobiose (strains VBS, VB7, and VB6), raffinose (strains OW8 and OW9 and group IIIa strains), and melibiose (strains OW8 and OW9 and group IIIa strains);
all strains (except the strain indicated in parentheses) fail to produce acid from trehalose, rnelezitose, inulin, glycogen, or starch (strain OW10/2).
+, Positive reaction; -, negative reaction; +w,weak positive reaction.
predominantly as single cells or sometimes in pairs or short
chains. Group IIIa strains were curved or helical, motile,
gram-negative rod-shaped bacteria with rounded ends. They
were 0.8 to 1.0 by 5 to 30 pm. They occurred singly or in
pairs or short chains. Group IV strains were curved, crescent-shaped, motile, gram-negative rods with rounded ends.
They were 0.6 to 0.9 by 5 to 15 km. They occurred
predominantly as single cells or (rarely) in pairs. In some
strains motility was lost after several subcultivations.
Physiological and chemical properties. All of the strains
which we studied were obligately anaerobic. The physiological properties of the strains belonging to the morphologically similar groups I and I1 are listed in Table 1, and the
properties of group 111, IIIa, and IV strains are shown in
Table 2. The G+C contents of the DNAs of group I and I1
strains were 38 to 41 mol% (Table I). Similar G+C contents
were found for DNAs of representatives of groups I11 and
IIIa (Table 2), whereas strains belonging to group IV had
distinctly higher G+C contents (51 to 52 mol%) (Table 2). All
of the strains which we studied contained the directly
cross-linked meso-diaminopimelic acid peptidoglycan type.
In addition, the a-carboxyl group of D-glutamic acid was
substituted predominantly by cadaverine, and in some
strains a minor amount of cadaverine was replaced by
putrescine. Such a peptidoglycan type containing a diaminesubstituted glutamic acid was previously found in strains of
Megasphaera, Selenomonas, and Sporornusa species (7,
14). Neither menaquinones nor ubiquinones were detected.
However, as in Megasphaera, Selenomonas, and Sporomusa species, lipid F (21) was found in all of the strains
studied. The major fermentation products in modified MRS
medium were acetic and propionic acids. All group IV
strains also produced major amounts of lactic acid, predominantly D-lactic acid. Distinct amounts of lactic acid were
also formed by Selenomonas ruminantium subsp. lactilytica
DSM 2872=, whereas all of the other strains formed little or
no lactic acid.
DNA-DNA hybridization studies. DNA-DNA hybridization
studies showed clearly that group I, 11, 111, and IV strains
form distinct genospecies. Within each group the DNA
homology values were in the range of 70 to loo%, whereas
no significant level of homology (<25%) was found between
representatives of the different groups. Strains belonging to
group IIIa not only exhibited high homology values among
each other (>75%), but also exhibited a distinct relationship
(homology values between 44 and 48%) to representatives of
group 111.
16s rRNA sequences and phylogenetic relationships. The
partial 16s rRNA sequences of Selenomonas ruminantium
subsp. lactilytica, Selenomonas sputigena, Selenomonas
lacticifex, Zymophilus paucivorans, P . cerevisiiphilus, and
Pectinatus frisingensis are aligned in Fig. 1. Our sequences
were compared with the 16s rRNA sequences of Megasphaera elsdenii, Sporornusa paucivorans (sequences kindly
provided by C. R. Woese), Bacillus subtilis, Streptomyces
coelicolor, and E . coli (3). An evolutionary distance analysis
was performed as described above. An unrooted phylogenetic tree is shown in Fig. 2.
DISCUSSION
The comparison of the 16s rRNA sequences of the strains
which we isolated with the sequences of reference strains
revealed that our strains belong to the subdivision of species
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INT. J. SYST.BACTERIOL.
SCHLEIFER ET AL.
1
S.r.
72
s.l.
TTaATGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn
..AACGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGGAAC
..ATTGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn
Z.p.
P.f.
P.C.
TTAATGGAGAGTTTGATCCTGGCTCAGGACAAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn
TTTATGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn
S.S.
..aTTGGAGAGTTTGATCCTGGCTCAGGACGAACGCTGGCGGCGTGCTnnnnnnnnnnnnnnnnnnnnnnnn
73
144
S.r. nnnnnnnnnnnnnnnnnnnnnnnnnnnnnACGGGTGAGTAACACGTAGGCAACCTGCCGCAAAGATGGGGAC
S.S.
CTTTATTTCGGTAAAGGTGAGAGTGGCAAACGGGTGAGTAACACGTAGGCAACCTGCCGACAGGATGGGGAC
s . l . nnnnnnnnnnnnnnnnnnnnnaGTnGCaaaCGGGTGAGTAACnC~TAGGCAACCTGCCTTTGAGATGGGGAC
z.p. nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnGGTGaGTAACaCGTAGGCAACCTGCCTTTTAGATGGGGAC
P . f . nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnTGAGTAAcnnnTAggCAACCTGCCTCcAAGATGGGGAC
P.C. nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnncaacctGCCTncaAGATGGGGAC
145
21 6
S.r. AACAGTCCGAAAGGACTGCTAATACCGAATGTTGTCaGaCTCCCGCATGGGAGCCTGATTAAAGATGGCCTC
S . S . AACATTCCGAAAGGAATGCTAATACCGAATGGAGTCCGGGGATGGCATCATCCCCGGATAAAAGATGGCCTC
s.l. AACAGTTCGAAAGGGCTGCTAATACCGaATGTTGTATATTTTCCACATGGAAGATATATTAAAGATGGCCTC
z.p. AACATCGCGAAAGCGGTGCTAATACCGAATGTTGTGTCTTTCCCACATGGGAAAGCTAC~AAAGATGGCCTC
P . f . AACATCtCGAAAGGGGTGCTAATACCGAATGTTGTAATGTTGTAAAGCgATTGCATAATGGCTTTACCAAAGGCGGC
P.C. AACACTCCGAAAGGGGTgCTAATACCGAATGTTGTAATGCTGCTGCAnnnCAGTATTACCAAAGGtggC
...
...
21 7
288
S.r. TACTTGTAAgcTATCGCTTTGCGATGGGTnTGCGTCTGATTAGCTaGTTGGTG.GGGTAACGGCCTACCAAG
S.S. TGAATAT..gCTATCGCCTGTCGATGGGCCTnCGTCTGATTAGCCAGTTGGCG.GGGTAACGGCCTACCAAA
s . l . TATTTATAAgCTATCGCTCAAAGATGGGTnTGCGTCTGATTAGCTaGTTGGTG.GGGTAAcGGCCTACCAAG
z.p. TATTTATAAGCTGTCACTAAAAGATGGGTCTGCGTCTGATTAGCTaGTTGGCG.GGGTAAATGCCCACCAAG
P . f . .TTTTA...gCTGTCGCTTGGAGATGGGCCTaCGTCTGATTAGCTaGTTGGTGACGGTAACGGCGCACCAAG
P.C. .TTTTA...gCTATCGCTTGGAGATGGGCCTaCGTCTGATTAGCTaGTTGGTGACGGTAACGGCGCACCAAG
289
S.r.
S.S.
360
GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGAACTGAGACACGGTCCaGACTCCTACGG
GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAGACTCCTACGG
s.l.
GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACACATTGGGACTGAGACACGGCCCAGACTCCTACGG
z.p.
P.f.
P.C.
GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGG
GCAACGATCAGTAGCCGGTCTGAGAGGATGGACGGCCACATTGGGACTGAGACACGGCCCAaACTCCTACGG
GCGACGATCAGTAGCCGGTCTGAGAGGATGAACGGCCACATTGGGACTGAGACACGGCCCAgACTCCTACGG
S.r.
GAGGCAGCAGTGGGGAATCTTcCGCAATGGGCGAAAGcCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGGT
361
S.S.
s.l.
z.p.
P.f.
P.C.
432
GAGGCAGCAGTGGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGTC
GAGGCAGCAGTGGGGAATCTTCCACAATGGACGACGAAAGTCTGATGGAGCAACG~CGCGTGAGTGAAGAAGGGT
GAGGCAGCAGTGGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGAAGAAGGTT
GAGGCAGCaGTGGGGAATCTTcCGCAATGGGCGAAAGcCTGACGGAGCAACGCCGCGTGAACGAGGAAGGTC
GAGGCAGCAGTggGGAATCTTCCGCAATGGGCGAAAGCCTGACGGAGCAACGCCGCGTGAACGAGGAAGGTC
43 3
S.r.
S.S.
s.l.
z.p.
P.f.
P.C.
504
TTCGGCTCGTaAAGCTCTGTTGACGGGGACGAACGTGCGTGCGAGATGCGAATAGTTTCTTGCAATGACGGTACCC
TTCGGATCGTAAAGCTCTGTTGCACGGGACGAAAACCGAGCTTGAGAATATTGAGTTTGGGTGACGGTACCG
TTCGGCTCGTAAAGCTCTGTTGTCGGGGACGAAtGTGCAGTATTTGAATAAAGTACTGCAATGACGGTACCT
TTCGGATCGTtAAGCTCTGTTGTTTGAGACGAACGTGCAGTATACGAATAATGTGCTGTAATGACGGTATCA
TTCGGATCGTAAAGTTCTGTTGCAGGGGACGAATGGCAGTAGTGTT~TACCACTATTGAATGACGGTACCC
TTCGGATCGTtAAGTTCTGTTGCAGGGGACGAACGGCACTATAGCCAATAAGTATAGTGAATGACGGTACCC
505
S.r.
S.S.
s.l.
z.p.
P.f.
P.C.
576
GTcGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCCAGCGTTGTCCGGAATT
AGCGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCCAGCGTTGTCCGGAATT
GACGAGGAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCaAGCGTTGTCCGGAATT
AACTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATT
TGTTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGCGGCAAGCGTTGTCCGGAATT
TGTTAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCgGTAATACGTAGGCGGCAAGCGTTGTCCGGAATt
FIG. 1. Alignment of 16s rRNA partial sequences of Selenomonas ruminantium (S.r.), Sefenomonas sputigena ( S . S . ) , Sefenomonas
lacticifex (S.l.), Zymophilus paucivorans (Z.P.), Pectinatus cerevisiiphifus (P.c.), and P . frisingensis (P.f.). The lower-case letters indicate
nucleotides whose assignment is uncertain; n indicates a nucleotide of unknown composition. Dots were inserted for purposes of alignment.
The 3’-terminalparts (about 55 bases) of the sequences were not determined.
with gram-negative walls within the phylum of gram-positive
eubacteria (24). This agrees very well with the occurrence of
the diamine-substituted, directly cross-linked meso-diaminopimelic acid peptidoglycan type among this group of bacteria. The same peptidoglycan type has been found in
Veilonella species and Centipedu periodontii (Weiss, unpublished data), indicating the relationship of these organisms to
this subdivision within the gram-positive eubacteria. The
16s rRNA analysis showed that a representative of group I1
is closely related to P . cerevisiiphilus DSM 20467T (Fig. 2).
Therefore, group I and I1 organisms should be placed in the
genus Pectinatus. From 16s rRNA analyses it is also quite
clear that the genus Pectinatus does not belong in the family
Bacteriodaceae, as suggested by Lee et al. (16), since the
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ROD-SHAPED BACTERIA FROM BREWERIES
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23
648
ATTGGGCGTAAAGGGAGCGCAGGCGGGAAGGCAAGTCAGTCTTAAAAGTGCGGGgCTcAACCCCGTgATGGG
S.S. ATTGGGCGTAAAGGGAGCGCAGGCGGACATATAAGTCCATCTTAAAAGTGCGGGGCTcAACCCCGTgAGGGG
S.1. ATTGGGCGTAAAGGGAGCGCAGGCGGGAAGGTAAGTCGGTCTTAAAAGTGCGGGGCTcAACCCCGTgATGGG
Z.p. ATTGGGCGTAAAGGGAGCGCAGGTGGGAATGTAAGTCAGTCAGTCTTAAAAGTGCGAGGCTcAACCTCGTGATGGG
P . f . ATTGGGCGTAAAGGGAGCGCAGGCGGATGACTAAGCGGATCTTAAAAGTGCGGGGCTcaACCCCGTgATGGG
P.c. ATTGGGCGTAAAGGGAGCGCAGGCGGAACATTAAGCGGATCTTAAAAGTGCGGGGCTcAACCCCGTGATGGG
S.r.
649
720
4 . r . ATTGAAACTGTCTTTCTTGAGTGCAGGAGAGGAAAGCGGAATTCCTaGTGTAGCGGTGAAATGCGTAGATAT
S.S. ATGGAAACTGTATGCCTTGAGTgCAGGAGAGGAAAGCGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATAT
S.l.
ATCGAAACTATCTTTCTTGAGTGCAGGAGAGGAAAGTGGAATTCCTaGTGTAGCGGTGAAATGCGTAGATAT
Z.p. ACAGAAACTACATTTCTTGAGTGCAGGAGAGGAAAGTGGAATTCCTaGTGTAGCGGTGAAATGCGTAGATAT
P.f.
P.C.
ATTCGAACTGGTCATCTTGAGTGCAGGAGAGGAAAGCGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATAT
S.r.
TgGGAGGAACACCaGTGGCGAAGGCGGCTTTCTGGACTGTaACTGACGCTGAGGCTCGAAAGCGTGGGGAGC
TGGGAGGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCCAGGGGAGC
TAGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCAAGGGGAGC
TAGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGACTGTAACTGACACTGAGGCTCGAAAGCTAGGGGAGC
GTCCGAACTGAGGTTCTTGAGTGCAGGAGAGGAAAGCGGAATTCCCAGTGTAGCGGTGAAATGCGTAGATAT
721
S.S.
S.1.
2.p.
P.f.
P.c.
792
TGGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCCAGGGTAGC
TGGGAAGAACACCAGTGGCGAAGGCGGCTTTCTGGACTGTAACTGACGCTGAGGCTCGAAAGCCAGGGTAGC
793
864
S.r.
GAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCTAGGTGTAGGAGGTATTGACCCCTT
S . S . GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGAATGCTAGGTGTAGGAGGTATTGACCCCTC
S.l.
GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGAATACTAGGTGTGGGAGGTATTGACCcTTT
Z.p. GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGAATACTAGGTGTAGGGGGTATTGACCCTTT
P . f . GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGGATACTAGGTGTAGGGGGTATTGACCCtnC
P.c. GAACGGGATTAGATACCCCGGTAGTCCTnGCCGTAAACGATGGATACTAGGTGTAGGGGGTATTGACCcTTt
865
S.r.
936
CTGTGCCGGAGTTAACGCAATAAGCATTCCGCATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTG
S.S. CTGTGCCGGAGTTAACGCAATAAGCATTCCGCCTGGGGAGTACGGTCGCAAGCATTCCGACTGAAACTCAAAGGAATTG
S.1. CCGTGCCGGAGTTAACGCAATAAGTATTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTG
Z.p. CTGTGCCGGAGCTAACGCAATAAGTATTCCGCCTGGGGAGTACGGCCGCAAGGTTG~ACTCAAAGGAATTG
P.f.
P.c.
CTGTGCCGGAGTTAACGCAATAAGTATCCCGCCTGGGGAGTACGGCCGCAAGTCAGGCTGAAACTCAAAGGAATTG
CTGTGCCGGAGCAAACGCAATAAGTATCCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTG
S.r.
ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGnnGCAACGCGAAGAACCTTACCAGGGCTTGA
S.S.
S.l.
ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGaaGCAAGCATTCCCGCGAAGAACCTTACCAGGGCTTGA
ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGnnGCAACGCGAAGAACCTTACCAGGGCTTGA
937
1008
Z.P.
ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGanGCAACGCGAAGAACCTTACCAGGGCTTGA
P.f.
P.C.
ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGnnGCAACGCGAAGAACCTTACCAGGGCTTGA
S.r.
S.S.
CATTGAGTGAAAGctCTAGAGATAGAGCCC.TCtCTTCGGAGA.CAcGAAAACAGGTGGTGCATGGCTGTCG
S.1.
CATTGAGTGAAAGGTGCAGAGATGCATCCC.TCTCTTCGGAGA.CACGAAAACAGGTGGTGCATGGCTGTCG
ACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGAaGCAACGCGAAGAACCTTACCAGGGCTTGA
1009
1080
CATTGAGTGAAAGAGCTAGAGATAGCTTCc.nCTCTTCGGGGA.CACGAA~CAGGTGGTGCATGGCTGTCG
Z.p. CATTGAGTGAAAGGCTAAGCATTCCGAGATTAGTCCCTCTTCTTCGGAAGACACAAAAACAGGTGGTGCATGGCTGTCG
CATTGATTGaCGTATGCAGAGATGCATATTTTCTCTTCGGAGGACAAGAAAACAGGTGGTGCATGGCTGTCG
CATTGATTGACGTATCCAGAGATGGATATTTTCTCTTCGGAGGACAAGAAAACAGGTGGTGCATGGCTGTCG
P.f.
P.c.
1 1 52
1081
S.r.
S.S.
TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCATTTGTTGCCAGCACGT
TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTGTCCTTTGTTGCCAGCGCGT
S.l.
TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTTTGTTGCCAGCACGT
Z a p . TCAGCTCGTGTC~TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTATGTTGCCAGCACGT
P . f . TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTAT&ATTTGTTGCCAGCACGT
P.C. TCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCATTTGTTGCCAGCACGT
FIG. l-Continued
genus Bacteroides and related genera represent a distinct
phylum within the phylogenetic tree of eubacteria (24). A
representative of group IV is distinctly related to Selenomonas ruminantium and Selenomonas sputigena and should
therefore be assigned to the genus Selenomonas. A representative of group IIIa is only distantly related to any of the
other anaerobic, gram-negative eubacteria. Group IIIa
strains, together with group I11 strains (see below), form the
basis for a new genus.
DNA-DNA hybridization studies showed that all of the
group I strains are closely related to P . cerevisiiphilus ATCC
29359T,whereas group I1 strains shared high DNA homology
values with Pectinatus sp. strain ATCC 33332T. DNA-DNA
hybridization studies between strains ATCC 29359T and
ATCC 33332T showed a DNA homology value of 16%. The
two Pectinatus species can be distinguished phenotypically.
Differences were found in the degradation of xylose, cellobiose, melibiose, and N-acetylglucosamine. However, the
phenotypic properties of P. cerevisiiphilus ATCC 29359Tare
not in agreement with the original description (16). To be
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24
INT. J. SYST.BACTERIOL.
SCHLEIFER ET AL.
1 1 53
1224
S.r.
CAAGGTGGGAACTCAAATGAGACTGCCGCGGACAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC
S.S.
AATGGCGGGAACTCAAAGGAGACTGCCGCGGAGAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC
s.l. AATGGTGGGAACTCAAAAGAGACTGCCGCGGACAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC
z . p . AATGGTGGGAACTCATGGGAGACTGCCGCGGATAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC
P.f. CAAGGTGGGAACTCAAATGAGACTGCCGCGGATAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC
P.C. AAAGGTGGGAACTCAAGTGAGACTGCCGCGGACAACGCGGAGGAAGGCGGGGATGACGTCAAGTCATCATGC
1225
1296
S.r.
S.S.
CCCTTATGTCCTGGGCTACACACGTACTACAATGGGATGGACAGAGgGaAGCGAaCCCgcgAGGGCAAGCGA
CCCTTATGTCCTGGGCTACACACGTACTACAATGGGATGGACAGAGGGAAGCGAAGGCGTGAGCCGGAGCGG
s . l . CCCTTATGTCCTGGGCTACACACGTACTACAATGGGATGGACACgggGCAGCGAAGCCGCGAGGCCAAGCGA
z . p . CCCTTACGTCCTGGGCTACACACGTACTACAATGGGACACACAGAGGGAAGCGAAGGAgTGATCTGGAGCGG
P.f. CCCTTACGTCCTGGGCTACACACGTACTACAATGGGATACACAGAGGGAAGCGAAGGAGTGATCTgGAGCGG
P.C. CCCTTATGTCCTGGGCTACACACGTACTACAATGGGATAAACAGAGGGAAGCGAGACCGCGAGGTGGAGCGA
1297
1368
S.r. ACCCCATtAACCATCTCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGTCGGAATCGCTAGTAAT
S.S. ACCCCACAAACCATCCCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGTCGGAATCGCTAGTAAT
s.l. ACCCCATAAACCATCCCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTTGGAATCGCTAGTAAT
z . p . ACCCCAAAAAATGTCTCCCAGTTCGGATTGCAGGCTGCAACCCGCCTGCATGAAGTCGGAATCGCTAGTAAT
P.f. AACCCAAAAAATATCCCCCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATG~GTCGGAATCGCTAGTAAT
P.C. AACCCATAAATTATCTCTCAGTTCGGATTGCAGGCTGCAACTCGCCTGCATGAAGTCGGAATCGCTACTAAT
1369
1440
CGCTGGTCAGCATACAGCGGTGAATACGTTCCCGGGCCTTGTACACAccgccCGTCACACCACGGAAGTCAT
S . S . CGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACACcgCcCGTCACACCACGGAAGTCAT
s . l . CGCTGGTCAGCATACAGCGGTGAATACGTTCCCGgGCCTTGTACACACcgccCGTCACACCACGGAAGTCAT
z . p . CGCAGGTCAGCATACTGCGGTGAATACGTTCCCggGTCTTGTACACACcgccCGTCACACCACGAAAGTCAT
P.f. CGCAGGTCAGCATACTGCGGTGAATACGTTCCCGGGCCTTGTACACAccgccCGTCACACCACGAAAGTCAT
P.C. CGCAGGTCAGCATACTGCGGTGAATACGTTCCCgggTCTTGTACACAccgccCGTCACACCACGAAAGTCAT
S.r.
1441
1508
S.r. TCACACCCGAAGCCGG.TGGGTAAACCGCAAGGAtATAGGAtATAGCCGTCTAAGGTnGGGGCGATGACCGGGGn
TCACGCCCAAAGCCGG.TGGGCtAACCGCAAGGAGGCAGCCGTCTAA~GCGGGGGCGATGACTGGGGn
S.S.
s. 1. TCACACCCGAAGCCGG.TGGGtAAACCGCAAGGACACAGCAAGGACACAGCCGTCtAAGGTnGGGGCGATGACCGGGGn
z . p . TCACACCCGAAGCCGG.TGGGGTAACCGCAAGGAGCCAGCCGTCTAAGGTnGGGGCGATGACtGGGGT
P.f. CCACACCCGAAGCCGGCTAAGGG..CCGCAAGGAACCGACCGTCTAAGGTnGGGGCGATGAccGGGGn
P.C. CCACACCCGAAGCCGGCTAAGGG..cCGCAAGGCACCGACCGTCTAAGGTnGGGGCGATGAccGGGGn
FIG. l-Continued
Escherichia coli
\
Selenomonas
mmimntium
Selenomonas sputigena
Zymophilus paucivorans
Pectinatus cerevisiiphilus
Megasphaera elsdenii
Sporomusa paucivorans
Bacillus subtilis
Streptomyces
coelicolor
FIG. 2. Unrooted phylogenetic tree showing the relationships among the organisms investigated in this study and reference organisms. Bar
unit.
= 0.1 K,,,
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VOL.40, 1990
ROD-SHAPED BACTERIA FROM BREWERIES
sure that the present type strain of P. cerevisiiphilus is
distinctly different from the original description of this
species, we studied not only strain DSM 20467T but also
strain ATCC 29359T. Both of these strains had the same
phenotypic characteristics, had high levels of DNA homology (95%), and differed significantly from the original description of P. cerevisiiphilus. L. H . Moore has studied three
different lyophilized cultures of strain ATCC 29359T (two in
1981 and one in 1989) and found the same characteristics as
we did (L. H. Moore, personal communication). Therefore,
the description of P. cerevisiiphilus is emended below, and
strains belonging to group I1 are described as new species, P.
frzsingensis. Groups 111 and IIIa were related to one another
but were genetically distinct from other anaerobic gramnegative, rod-shaped bacteria (Fig. 2). They should be
assigned to different species of a new genus, Zymophilus
rafinosivorans and 2.paucivorans. Genetic, morphological,
and physiological data indicate a distant relationship between group IV strains and Selenomonas ruminantium.
Therefore, these strains should form a new species within
the genus Selenomonas, namely Selenomonas lacticijkx. On
the basis of the G+C content of its DNA (51 to 52 mol%),
this organism can be clearly separated from other species of
Selenomonas, such as Selenomonas sputigena, Selenomonas noxia, Selenomonas flueggei, Selenomonas infelix, Selenomonas dianae, and Selenomonas artemidis, that have
significantly higher G+C contents (56 to 58 mol%) (19).
Differential characteristics of the newly described species
are given in Table 3.
Emended description of Pectinadus cerevisiiphilus. The
emended description below of Pectinatus cerevisiiphilus is
based on the descriptions given by Lee et al. (16) and by
Moore (personal communication) and our results. Cells are
gram-negative, slightly curved rods that are 0.7 to 0.9 Frn in
diameter and 2 to 30 p,m or more long. They occur singly, in
pairs, and only rarely in short chains. The occurrence of
longer, helical filaments is characteristic for older cells. The
cells are usually motile. These organisms are obligately
anaerobic, nonsporeforming mesophiles. Flagella emanate
from only one side of the cell (comblike). They do not
produce catalase and cytochromes. Colonies are circular,
entire, beige to white, glistening, and opaque. The optimum
temperature for growth is 30°C. Glucose is fermented to
acetic and propionic acids. In contrasts to the original
description given by Lee et al. (16), both Moore (for the type
strain) and we (also for other strains) found that xylose and
melibiose are fermented, whereas maltose and cellobiose are
not. Cadaverine and putrescine are found as characteristic
components of the cell wall peptidoglycan type, directly
cross-linked meso-diaminopimelicacid. Lipid F is also found
as a characteristic cellular compound. The G+C content of
the DNA is 38 to 41 mol%. The type strain is strain ATCC
29359 (= DSM 20467). The phenotypic properties of the type
strain are shown in Table 1.
Description of Pectinutus fisingemis sp. nov. Pectinatus
jbingensis (frisin-gen’sis. L. adj. fnsingensis, of Frisinga,
the Latin name of Freising, a town in the Federal Republic of
Germany where the organism was isolated). The description
below is based on 11 strains which were isolated from
spoiled beer. These organisms are obligately anaerobic,
usually motile, nonsporeforming gram-negative rods. Cells
are slightly curved and occur predominantly as single cells or
sometimes in pairs. They are 0.7 to 0.9 by 3 to 20 pm. The
occurrence of longer, slightly helical filaments is characteristic for older cultures.
Colonies on modified MRS agar medium are shiny,
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26
SCHLEIFER ET AL.
INT. J. SYST.BACTERIOL.
TABLE 4. Characteristics for the differentiation of Selenomonas species
Acid produced from:
Species
Cellobiose
Lactose
-a
-
-
Selenomonas artemidis
Selenomonas dianae
Selenomonas flueggei
Selenomonas infelix
Selenomonas lacticifPx
Selenomonas noxia
Selenomonas ruminantium
Selenomonas sputigena
a
+ , Positive reaction;
+
+
+
-
-
-
+-
Mannitol
Sucrose
+
+
+
+
+
+
+
+
+
+
+
-
+-
V
+
Lactic acid
as a major
fermentation
product
G+C content
of DNA
(mol%)
-
58
53
56
58
51-52
57
48-53
57
-
+V
-
-, negative reaction; v, variable reaction.
opaque, circular, slightly yellow, and 1to 2 mm in diameter
after 3 days at 30°C. The optimum temperature for growth is
30°C. Glucose is fermented to acetic and propionic acids,
acetoin, and sometimes minor amounts of succinic acid.
Phenotypic characteristics of the different group I1 strains
are shown in Table 1.
Cadaverine and rarely putrescine are found as characteristic components of the cell wall peptidoglycan. The peptidoglycan type is directly cross-linked meso-diaminopimelic
acid. Lipid F is also found as a characteristic cellular
compound. The G+C content of the DNA is 38 to 41 mol%.
The type strain is strain ATCC 33332. The phenotypic
properties of the type strain are shown in Table 1.
P.frisingensis can be distinguished from P.cerevisiiphilus
by its usual ability to ferment cellobiose, inositol, and
N-acetyl-glucosamine and its inability to utilize xylose and
melibiose.
Descriptionof Selenomonas lacticifex sp. nov. Selenomonas
lacticifex (1ac.ti’ci.fex. M. L. n. acidum lacticum, lactic
acid; L. suffix fex, a maker; L.n. lacticifex, a maker of lactic
acid). The description below is based on four strains that
were isolated from pitching yeast. These organisms are
obligately anaerobic, usually motile, nonsporeforming,
gram-negative rods. The cells are curved and crescent
shaped and occur predominantly as single cells or (rarely) in
pairs. They are 0.6 to 0.9 by 5 to 15 pm. Colonies on
modified MRS agar medium are smooth, opaque, circular,
yellowish, and 2 to 3 mm in diameter after 3 days at 30°C.
The optimum temperature for growth is 30°C. Glucose is
fermented to acetic, lactic, and propionic acids. Phenotypic
characteristics of the different group IV strains are shown in
Table 2.
Cadaverine and rarely putrescine are found as characteristic components of cell wall peptidoglycan. The peptidoglycan type is directly cross-linked meso-diaminopimelic acid.
Lipid F is also found as a characteristic cellular compound.
The G+C content,of the DNA is 51 to 52 mol%. The type
strain is strain DSM 20757 (= VB4b). The phenotypic
properties of the type strain are shown in Table 2.
The differentiation of Selenomonas lacticifex from the
previously described Selenomonas species is shown in Table
4. Selenomonas lacticifex is distinguished from most other
Selenomonas species by its ability to ferment glucose not
only to acetic and propionic acids but also to lactic acid as
major products. Moreover, the G+C content of its DNA is
distinctly lower. Only Selenomonas ruminantium subsp.
lactilytica also produces major amounts of lactic acid and
has a DNA G+C content similar to that of Selenomonas
lacticifex. Selenomonas lacticifex can be distinguished from
this subspecies by its inability to utilize mannitol and dulcitol.
Description of Zymophilus gen. nov. Zymophilus
(Zy.mo’phi.lus. Gr.n. zyme, leaven, yeast; Gr. adj. philus,
lover; M.L. masc. n. Zyrnophilus, yeast lover). Slightly
curved to helical rods, 0.7 to 1.0 by 3 to 30 pm, occurring
singly, in pairs, or in short chains. No resting stages are
known. Gram negative. Motile. Mobility can be lost after
several subcultivations. Strictly anaerobic. Chemoorganotrophic, having a fermentative type of metabolism. Glucose
is fermented to acetic and propionic acids. These organisms
occur in pitching yeast. Cadaverine and rarely putrescine are
found as characteristic components of cell wall peptidoglycan. The peptidoglycan type is directly cross-linked mesodiaminopimelic acid. Lipid F is found as a characteristic
cellular compound. The G+C content of the DNA is 38 to 41
mol%. All of the organisms belonging to this new genus were
isolated from pitching yeast or brewery waste. The type
species is Zymophilus rafinosivorans. Major differences
between Zymophilus and other similar genera are shown in
Table 5.
Description of Zymophilus racffinosivorans sp. nov. Zymophilus rafinosivorans (raf.fi.no.si’vo.rans . M.L. n.
rafinosum, raffinose; L.v. vorare, to eat. M.L. part. adj.
rafinosivorans, raffinose devouring). The description below
is based on 10 strains that were isolated from pitching yeast
and four strains (strains OWS, OW9, OW10/1, and OW10/2)
that were isolated from brewery waste. These organisms are
obligately anaerobic, motile, nonsporeforming, gram-nega-
TABLE 5. Characteristics of the genus Zymophilus and other gram-negative, nonsporeforming, anaerobic genera
Genus
dr::‘
Zymophilus
Pectinatus
Selenomonas
Megasphaera
a
+, Positive reaction;
Products of glucose or fructose fermentationa
G+C
cocci
+
+
+-
-
+
Acetate
Acetoin
Butyrate
+
+
+
-
+
-
ND
-
+
(+>
-
Lactate
-
- or
-
+
Succinate
+
+
+
-
(+>
____
-, negative reaction;
Propionate
________~
(+), minor products; ND, not determined.
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-
Valerate
caprateand
-
+
‘Ontent
(mol%)
38-41
38-41
48-58
4453
VOL. 40, 1990
ROD-SHAPED BACTERIA FROM BREWERIES
tive rods. Cells are straight to slightly curved and occur
predominantly as single cells or sometimes in pairs or short
chains. They are 0.7 to 0.9 by 3 to 15 pm.
Colonies on modified MRS agar medium are circular,
smooth, opaque, slightly yellow, and 1 to 2 mm in diameter
after 3 days at 30°C. The optimum temperature for growth is
30°C. Glucose is fermented to acetic and propionic acids.
Phenotypic characteristics of the different group I11 strains
are shown in Table 2.
The G+C content of the DNA is 38 to 41 mol%. The type
strain is strain DSM 20765 (= SH2). The phenotypic properties of the type strain are shown in Table 2.
Descriptionof Zymophilus paucivorans sp. nov. Zymophilus
paucivorans (pau.ci’vo.rans. L. adj. paucus, little; L. v.
vorare, to eat. ; M . L . adj. paucivorans, utilizing relatively
few carbohydrates). The description below is based on four
strains that were isolated from pitching yeast. These organisms are obligately anaerobic, motile, nonsporeforming,
gram-negative rods. The cells are curved or helically shaped
and occur singly, in pairs, or in short chains. They are 0.8 to
1.0 by 5 to 30 pm. In two of the strains motility disappeared
after several subcultivations.
Colonies on modified MRS agar medium are circular,
smooth, slightly yellow, and 1 to 2 mm in diameter after 3
days at 30°C. Glucose is fermented to acetic and propionic
acids and trace amounts of lactic acid. Phenotypic characteristics of the different group IIIa strains are shown in Table
2.
Cadaverine and rarely putrescine are found as characteristic components of the cell wall peptidoglycan.
The peptidoglycan type is directly cross-linked mesodiaminopimelic acid, Lipid F is also found as a characteristic
cellular compound. The G+C content of the DNA is 39 to 41
mol%. The type strain is strain DSM 20756 (= AA1). The
phenotypic properties of the type strain are shown in Table
2.
2. ra$nosivorans and 2. paucivorans are closely related
species with homology values of 44 to 48%. However, these
organisms are phenotypically so different that separation
into two species is warranted. These organisms are not only
morphologically quite different, but they also differ in the
utilization of xylose, rhamnose, xylitol, raffinose, melibiose,
and inositol (Tables 2 and 3).
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