You are on page 1of 5

International Journal of Systematic and Evolutionary Microbiology (2007), 57, 708–712 DOI 10.1099/ijs.0.

64618-0

Lactobacillus farraginis sp. nov. and Lactobacillus


parafarraginis sp. nov., heterofermentative
lactobacilli isolated from a compost of distilled
shochu residue
Akihito Endo and Sanae Okada
Correspondence NODAI Culture Collection Center, Tokyo University of Agriculture, 1-1-1 Sakuragaoka,
Akihito Endo Setagaya, Tokyo 156-8502, Japan
pegaman@hotmail.co.jp

Five strains of lactic acid bacteria were isolated from a compost of distilled shochu residue in Japan.
The isolates were separated into two groups on the basis of 16S rRNA gene sequence similarity,
and two subclusters were formed that comprised micro-organisms closely related to Lactobacillus
buchneri, L. diolivorans, L. hilgardii, L. kefiri, L. parabuchneri and L. parakefiri. DNA–DNA
relatedness results revealed that the isolates could be separated into two groups, and these groups
correlated well with the subclusters generated using the phylogenetic analysis. Moreover, the levels
of DNA–DNA relatedness showed clear separation of the two groups from their phylogenetic
relatives. Therefore, the two groups represent two novel species, for which the names Lactobacillus
farraginis sp. nov. (type strain NRIC 0676T=JCM 14108T=DSM 18382T) and Lactobacillus
parafarraginis sp. nov. (type strain NRIC 0677T=JCM 14109T=DSM 18390T) are proposed.

During a study of lactic acid bacteria originating from A sample of compost was prepared from a distilled shochu
plant materials, we isolated a novel lactic acid bacterium, residue in Miyazaki Prefecture in the southern Kyushu
Oenococcus kitaharae, from a compost of distilled shochu region of Japan. The sample was serially diluted with sterile
residue produced in Japan (Endo & Okada, 2006). Shochu is saline before being plated on MRS (Oxoid) agar containing
a traditional Japanese distilled spirit made from rice, sweet (l21) 10 mg cycloheximide, 10 mg sodium azide, 5.0 g
potato, barley or other starchy materials; Aspergillus niger calcium carbonate and 15 g agar. Plates were incubated for
and Saccharomyces cerevisiae play roles in saccharification of 5 days at 30 uC. After incubation, colonies that produced
the starch and its fermentation into alcohol. Concomitantly, clear zones were picked into MRS broth and maintained in
five strains of lactic acid bacteria were isolated from the the same medium. Of the isolates, five (NRIC 0676T, NRIC
compost and were separated into two groups on the basis of 0677T, NRIC 0678, NRIC 0679 and NRIC 0680) were used in
their 16S rRNA gene sequences: they formed two sub- the present study. Strains NRIC 0677T and NRIC 0680 grew
clusters, the members of which were related to the obligately more slowly than the other three isolates in MRS broth;
heterofermentative lactobacilli Lactobacillus buchneri, L. these isolates took 2–3 days to reach stationary phase,
diolivorans, L. hilgardii, L. kefiri, L. parabuchneri and L. whereas NRIC 0676T, NRIC 0678 and NRIC 0679 took
parakefiri. The DNA–DNA relatedness results showed clear 1–2 days. L. buchneri NRIC 1040T, L. diolivorans NRIC
separation of the isolates into two groups that correlated 0695T, L. hilgardii NRIC 1060T, L. kefiri NRIC 1693T, L.
well with the two subclusters generated from a phylogenetic parabuchneri NRIC 1780T and L. parakefiri NRIC 0217T
analysis. Moreover, the levels of DNA–DNA relatedness were obtained from the NODAI Culture Collection Center,
showed clear separation of the two groups from the Tokyo University of Agriculture (Tokyo, Japan) and were
phylogenetic relatives. This report provides a taxonomic used as reference strains in the present study. The reference
analysis of the five isolates, and proposes two novel strains were also maintained in MRS broth.
Lactobacillus species.
The 16S rRNA gene sequences of the five isolates were
determined as described previously (Endo & Okada, 2005).
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA
The closest known relatives of the isolates were determined
gene sequences of strains NRIC 0676T, NRIC 1078, NRIC 0679, NRIC by performing DataBase searches, and the sequences of
0677T and NRIC 0680 are AB262731–AB262735, respectively. closely related species were retrieved from the DDBJ database.
Maximum-likelihood and maximum-parsimony phylogenetic trees based Multiple alignments of the sequences were carried out with
on 16S rRNA gene sequences of the novel strains and related species the program CLUSTAL_X, version 1.18 (Thompson et al.,
are available as supplementary figures in IJSEM Online. 1997). Distance matrices for the aligned sequences were

708 64618 G 2007 IUMS Printed in Great Britain


Lactobacillus farraginis and L. parafarraginis spp. nov.

calculated by using the two-parameter method of Kimura were obtained by using the maximum-likelihood and
(1980). The neighbour-joining method was used to maximum-parsimony methods (see Supplementary Figs S1
construct a phylogenetic tree (Saitou & Nei, 1987). The and S2 available in IJSEM Online). L. buchneri, L. hilgardii
robustness of individual branches was estimated by using and L. kefiri were classified within the L. buchneri phylo-
bootstrapping with 1000 replicates (Felsenstein, 1985). genetic group, together with Lactobacillus fructivorans, L.
Phylogenetic trees were also constructed by using the lindneri and L. sanfransiscensis, by Schleifer & Ludwig (1995).
maximum-likelihood (Cavalli-Sforza & Edwards, 1967) Our phylogenetic trees showed that the L. buchneri phylo-
and maximum-parsimony (Kluge & Farris, 1969) methods genetic group was divided into three subclusters: the L.
with PHYLIP version 3.65 (Felsenstein, 2005). The 16S rRNA buchneri subcluster (L. buchneri, L. diolivorans, L. hilgardii, L.
gene sequences of the isolates were compared with one kefiri, L. parabuchneri, L. parakefiri and the five novel
another, and the sequences of strains NRIC 0676T and NRIC isolates), the Lactobacillus brevis subcluster (Lactobacillus
0677T were used to search for sequence similarity using acidifarinae, L. brevis, L. parabrevis, L. spicheri and L. zymae)
DataBase. Approximately 1390 bp of the 16S rRNA gene and the L. fructivorans subcluster (L. fructivorans, L.
sequences of the isolates and related species were used to homohiochii, L. lindneri and L. sanfransiscensis) (Fig. 1).
construct the phylogenetic tree. After the sequence compar- The sequence similarities of the members of the L. buchneri
isons, the isolates were separated into two groups. The 16S subcluster ranged from 96.7 to 99.1 %, and the sequence
rRNA gene sequence similarities between strains NRIC similarities between the members of the L. buchneri
0676T, NRIC 0678 and NRIC 0679 ranged from 99.9 to subcluster and the members of the L. brevis and L.
100 %, and the sequence similarity between NRIC 0677T and fructivorans subclusters were 92.6–95.2 % and 90.7–94.0 %,
NRIC 0680 was 100 %. The sequence similarity between respectively. The sequence similarities among the members
strains NRIC 0676T and NRIC 0677T was 97.6 %. The highest of the L. brevis subcluster were 96.1–99.7 %, and those
levels of sequence similarity between NRIC 0676T and strains between the members of the L. brevis and L. fructivorans
of known species of lactic acid bacteria were obtained with subclusters were 90.7–94.0 %. The sequence similarities
the type strains of L. hilgardii, L. buchneri, L. diolivorans, L. among the members of the L. fructivorans subcluster ranged
parabuchneri, L. kefiri and L. parakefiri (98.2, 97.5, 97.3, 96.8, from 93.6 to 99.2 %.
96.7 and 96.7 %, respectively); for NRIC 0677T the highest
values were with respect to the type strains of L. hilgardii, L, Because of the high levels of 16S rRNA gene sequence
buchneri, L. diolivorans, L. parabuchneri, L. kefiri and L. similarity between the isolates and the species of the L.
parakefiri (98.0, 97.7, 97.6, 97.8, 97.5 and 97.1 %, respec- buchneri subcluster, the levels of DNA–DNA relatedness
tively). The strains in the two groups formed two subclusters, between the isolates and L. buchneri NRIC 1040T, L.
the members of which were closely related to obligately diolivorans NRIC 0695T, L. hilgardii NRIC 1060T, L. kefiri
heterofermentative lactobacilli (L. buchneri, L. diolivorans, L. NRIC 1693T, L. parabuchneri NRIC 1780T and L. parakefiri
hilgardii, L. kefiri, L. parabuchneri and L. parakefiri) in the NRIC 0217T were determined. Members of the L. brevis and
neighbour-joining analysis (Fig. 1). Identical tree topologies L. fructivorans subclusters were not used in determinations

Fig. 1. Neighbour-joining phylogenetic tree,


based on the 16S rRNA gene sequences,
showing the relationships of the five novel iso-
lates and related species. Leuconostoc mesen-
teroides subsp. dextranicum NRIC 1539T was
used as an outgroup. Bootstrap percentages
above 70 % are given at the branching points.

http://ijs.sgmjournals.org 709
A. Endo and S. Okada

of levels of DNA–DNA relatedness because the 16S rRNA Primer D Primer E Primer F
gene sequence similarities between the isolates and the M 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 M
members of these subclusters were considerably lower
than the values recommended for species differentiation
(Stackebrandt & Goebel, 1994). The DNA G+C contents of
the novel isolates were also determined. Extraction and
isolation of bacterial DNA were performed by using the
method of Marmur (1961), as modified by Ezaki et al.
(1983). DNA–DNA hybridization was carried out by using
the microdilution-well technique, with photobiotin for
labelling of the DNA (Ezaki et al., 1989). The G+C contents
of the strains tested were determined by HPLC as described
by Tamaoka & Komagata (1984). The levels of DNA–DNA
relatedness showed clear separation of the isolates into two
groups. Strains NRIC 0676T, NRIC 0678 and NRIC 0679 Fig. 2. RAPD PCR fingerprinting of the five novel isolates.
shared high levels of DNA–DNA relatedness (87–100 %), Primers D, E and F were used. Lanes: M, size markers (500 bp
and strains NRIC 0677T and NRIC 0680 shared 95 % DNA– DNA ladder; Takara-bio); 1, NRIC 0676T; 2, NRIC 0678; 3,
DNA relatedness. In contrast, strain NRIC 0676T showed NRIC 0679; 4, NRIC 0677T; 5, NRIC 0680.
low levels of DNA–DNA relatedness (42–46 %) with respect
to NRIC 0677T and NRIC 0680, and NRIC 0677T also
showed low levels of DNA–DNA relatedness (43–48 %) with were not used for comparisons with the isolates because of
respect to NRIC 0676T, NRIC 0678 and NRIC 0679. the low levels of 16S rRNA gene sequence similarity to the
Therefore, we concluded that the two groups were novel strains. The isolates were separated into two groups on
genetically distinguishable from each other; these groups the basis of phenotypic characteristics, and these groups
correlated well with the two subclusters obtained using correlated well with the two groups separated on the basis of
phylogenetic analysis. The levels of DNA–DNA relatedness phylogenetic analysis, levels of DNA–DNA relatedness and
for NRIC 0676T with respect to L. hilgardii NRIC 1060T, RAPD fingerprinting. Strains NRIC 0676T, NRIC 0677T and
L. diolivorans NRIC 0695T, L. buchneri NRIC 1040T, L. NRIC 0678 grew at 45 uC and grew slowly at 15 uC, but
parabuchneri NRIC 1780T, L. kefiri NRIC 1693T and strains NRIC 0677T and NRIC 0680 did not grow at 15 or
L. parakefiri NRIC 0217T were 47, 35, 30, 29, 23 and 45 uC. Growth at 45 uC was not found in other species of
20 %, respectively; those for NRIC 0677T with respect to
these reference strains were 37, 32, 25, 25, 30 and 21 %,
respectively. The G+C contents of strains NRIC 0676T,
NRIC 0678, NRIC 0679 ranged from 40 to 41 mol% and Table 1. Differential characteristics among the novel iso-
that of strains NRIC 0677T and NRIC 0680 was 40 mol%. lates and closely related species

Randomly amplified polymorphic DNA (RAPD) finger- Taxa: 1, strains NRIC 0676T, NRIC 0678 and NRIC 0679 (L. farragi-
nis sp. nov.); 2, strains NRIC 0677T and NRIC 0680 (L. parafarraginis
printing was performed to differentiate the five isolates by
sp. nov.); 3, L. buchneri (data from Kandler & Weiss, 1986); 4, L. dio-
a method described previously (Endo & Okada, 2006).
livorans (Krooneman et al., 2002); 5, L. hilgardii (Kandler & Weiss,
Primers D (59-GAGGACAAAG), E (59-GGCGTCGGTT)
1986); 6, L. kefiri (Kandler & Kunath, 1983); 7, L. parabuchneri
and F (59-GGCCACGGAA) were used in this study. The
(Farrow et al., 1988); 8, L. parakefiri (Takizawa et al., 1994). +,
fingerprinting indicated that the isolates were separated into
Positive; 2, negative; V, variable; ND, no data available.
two groups. The fingerprints of strains NRIC 0676T, NRIC
0678 and NRIC 0679 differed slightly, and those of strains
Characteristic 1 2 3 4 5 6 7 8
NRIC 0677T and NRIC 0680 were similar to each other
(Fig. 2). This demonstrated that the isolates were separated Acid from:
at the strain level within each group. The two groups L-Arabinose + + + + 2 + + +
produced fingerprints that were quite different. The groups D-Xylose 2 + V + + 2 2 2
correlated well with the separate groups generated by Galactose + + V + V 2 + +
phylogenetic analysis or levels of DNA–DNA relatedness. Lactose 2 2 V 2 V + 2 +
Melibiose + + + + 2 + + V
Morphological, physiological and biochemical character- Sucrose + + V 2 V 2 + 2
istics of the isolates were determined by using a method Raffinose + + V V 2 2 2 2
described previously (Endo & Okada, 2005); MRS broth was Melezitose + + + 2 V 2 + V
used as a basal medium. The characteristics of the isolates Growth at:
(described in detail in the species descriptions below) were 10 uC 2 2 ND 2 ND + + ND
compared with those of closely related species (Table 1). 45 uC + 2 2 2 2 2 2 2
Members of the L. brevis and L. fructivorans subclusters

710 International Journal of Systematic and Evolutionary Microbiology 57


Lactobacillus farraginis and L. parafarraginis spp. nov.

the L. buchneri subcluster (Table 1). However, as the major Cells are Gram-positive, non-motile rods, measuring
characteristics of the strains in the two groups were similar 0.862–4 mm. Cells occur singly or in pairs and chains.
to those of the phylogenetic relatives, precise identification Facultatively anaerobic and catalase-negative. Colonies on
among the two groups and their phylogenetic relatives based MRS agar are beige, smooth and approximately 1–2 mm in
on phenotypic features is difficult. Therefore, determina- diameter after incubation for 4 days. Heterofermentative
tions of DNA–DNA relatedness are needed for precise and produces lactic acid, carbon dioxide and ethanol or
identification of the species in the L. buchneri subcluster. acetic acid from D-glucose. D- and L-lactate are produced in
The same has been observed for the species in the the ratio 3 : 2. Nitrate is not reduced. Acid is produced from
Lactobacillus acidophilus group (Fujisawa et al., 1992; D-glucose, D-fructose, D-galactose, L-arabinose, D-ribose,
Naser et al., 2006). D-xylose, maltose, melibiose, sucrose, raffinose and D-
melezitose, weakly from D-gluconate, but not from
The data showed that the five isolates could be divided into D-mannose, L-rhamnose, cellobiose, lactose, D-salicin, D-
two genetically distinct groups; these groups were also trehalose, D-sorbitol or starch. Acid production from D-
genetically distinguishable from known species of lactic acid mannitol is variable depending on the strain. Dextran is not
bacteria. Thus, these two groups represent two novel species, formed from sucrose. Cells grow at temperatures between 20
for which the names Lactobacillus farraginis sp. nov. (strains and 37 uC but not at 15 or 45 uC. All strains grow at
NRIC 0676T, NRIC 0678 and NRIC 0679) and Lactobacillus pH 4.0–8.5. Growth is observed in MRS broth containing
parafarraginis sp. nov. (strains NRIC 0677T and NRIC 0680) 5 % (w/v) NaCl. Cells do not contain meso-diaminopimelic
are proposed. acid in their peptidoglycan. The DNA G+C content is
40 mol%.
Description of Lactobacillus farraginis sp. nov.
The type strain is NRIC 0677T (=JCM 14109T=DSM
Lactobacillus farraginis (far.ra9gi.nis. L. gen. n. farraginis of
18390T), and has a G+C content of 40 mol%. Both known
mash, pertaining to shochu mash, an ingredient of a
strains were isolated from a compost of distilled shochu
compost material from which the type strain was isolated).
residue collected at a shochu distillery in Miyazaki
Cells are Gram-positive, non-motile rods, measuring Prefecture, southern Kyushu area, Japan, in 2003.
0.863–6 mm. Cells occur singly or in pairs and chains.
Facultatively anaerobic and catalase-negative. Colonies on
MRS agar are beige, smooth and approximately 2 mm in Acknowledgements
diameter after incubation for 4 days. Heterofermentative We thank the owner and staff of Akashi Distillery Ltd, Miyazaki, Japan,
and produces lactic acid, carbon dioxide and ethanol or for providing the fermentation samples. We also thank K. Komagata
acetic acid from D-glucose. D- and L-lactate are produced in for valuable discussions and R. Tsuji (NODAI Culture Collection
a ratio of 1 : 1. Nitrate is not reduced. Acid is produced from Center, Faculty of Applied Bioscience, Tokyo University of Agriculture,
Tokyo, Japan) for technical assistance.
D-glucose, D-fructose, D-galactose, L-arabinose, D-ribose,
maltose, melibiose, sucrose, raffinose and D-melezitose,
weakly from D-gluconate, but not from D-mannose, D-
xylose, L-rhamnose, cellobiose, lactose, D-salicin, D-treha-
References
lose, D-mannitol, D-sorbitol or starch. Dextran is not Cavalli-Sforza, L. L. & Edwards, A. W. F. (1967). Phylogenetic
formed from sucrose. Cells grow at temperatures between 15 analysis models and estimation procedures. Am J Hum Genet 19,
and 45 uC but not at 10 or 50 uC. All strains grow at 233–257.
pH 4.0–8.5; some strains grow at pH 9.0. No growth is Endo, A. & Okada, S. (2005). Lactobacillus satsumensis sp. nov.,
isolated from mashes of shochu, a traditional Japanese distilled spirit
observed in MRS broth containing 5 % (w/v) NaCl. Cells do
made from fermented rice and other starchy materials. Int J Syst Evol
not contain meso-diaminopimelic acid in their peptidogly- Microbiol 55, 83–85.
can. The DNA G+C content ranges from 40 to 41 mol%.
Endo, A. & Okada, S. (2006). Oenococcus kitaharae sp. nov., a non-
acidophilic and non-malolactic-fermenting oenococcus isolated from
The type strain is NRIC 0676 (=JCM 14108 =DSM
T T
a composting distilled shochu residue. Int J Syst Evol Microbiol 56,
18382T), and has a G+C content of 41 mol%. All three 2345–2348.
known strains were isolated from a compost of distilled
Ezaki, T., Yamamoto, N., Ninomiya, K., Suzuki, S. & Yabuuchi, E.
shochu residue collected at a shochu distillery in Miyazaki (1983). Transfer of Peptococcus indolicus, Peptococcus asaccharolyticus,
Prefecture, southern Kyushu region, Japan, in 2003. and Peptococcus magnus to the genus Peptostreptococcus and proposal
of Peptostreptococcus tetradius sp. nov. Int J Syst Bacteriol 33,
Description of Lactobacillus parafarraginis 683–698.
sp. nov. Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric
deoxyribonucleic acid-deoxyribonucleic acid hybridization in micro-
Lactobacillus parafarraginis (pa.ra.far.ra9gi.nis. Gr. prep. dilution wells as an alternative to membrane filter hybridization in
para beside; L. gen. n. farraginis of mash, the specific epithet which radioisotopes are used to determine genetic relatedness among
of Lactobacillus farraginis; N.L. gen. n. parafarraginis bacterial strains. Int J Syst Bacteriol 39, 224–229.
beside farraginis, pertaining to the close relationship to L. Farrow, J. A. E., Phillips, B. A. & Collins, M. D. (1988). Nucleic acid
farraginis). studies on some heterofermentative lactobacilli: description of

http://ijs.sgmjournals.org 711
A. Endo and S. Okada

Lactobacillus malefermentans sp. nov. and Lactobacillus parabuchneri 1,2-propanediol-degrading bacterium isolated from aerobically
sp. nov. FEMS Microbiol Lett 55, 163–168. stable maize silage. Int J Syst Evol Microbiol 52, 639–646.
Felsenstein, J. (1985). Confidence limits on phylogenies: an Marmur, J. (1961). A procedure for the isolation of deoxyribonucleic
approach using the bootstrap. Evolution 39, 783–791. acid from microorganisms. J Mol Evol 3, 208–218.
Felsenstein, J. (2005). PHYLIP (phylogeny inference package), version Naser, S. M., Hagen, K. E., Vancanneyt, M., Cleenwerck, I., Swings, J. &
3.65. Distributed by the author. Department of Genome Sciences, Tompkins, T. A. (2006). Lactobacillus suntoryeus Cachat and Priest
University of Washington, Seattle, USA. 2005 is a later synonym of Lactobacillus helveticus (Orla-Jensen 1919)
Fujisawa, T., Benno, Y., Yaeshima, T. & Mitsuoka, T. (1992). Bergey et al. 1925 (Approved Lists 1980). Int J Syst Evol Microbiol 56,
Taxonomic study of the Lactobacillus acidophilus group, with 355–360.
recognition of Lactobacillus gallinarum sp. nov. and Lactobacillus Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new
johnsonii sp. nov. and synonymy of Lactobacillus acidophilus group method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.
A3 (Johnson et al. 1980) with the type strain of Lactobacillus Schleifer, K. H. & Ludwig, W. (1995). Phylogeny of the genus
amylovorus (Nakamura 1981). Int J Syst Bacteriol 42, 487–491. Lactobacillus and related genera. Syst Appl Microbiol 18, 461–467.
Kandler, O. & Kunath, P. (1983). Lactobacillus kefir sp. nov., a Stackebrandt, E. & Goebel, B. M. (1994). Taxonomic note: a place
component of the microflora of kefir. Syst Appl Microbiol 4, 286–294. for DNA-DNA reassociation and 16S rRNA sequence analysis in the
Kandler, O. & Weiss, N. (1986). Genus Lactobacillus Beijerinck 1901, present species definition in bacteriology. Int J Syst Bacteriol 44, 846–849.
212AL. In Bergey’s Manual of Systematic Bacteriology, vol. 2, Takizawa, S., Kojima, S., Tamura, S., Fujinaga, S., Benno, Y. &
pp. 1209–1234. Edited by P. H. A. Sneath, N. S. Mair, M. E. Nakase, T. (1994). Lactobacillus kefirgranum sp. nov. and
Sharpe & J. G. Holt. Baltimore: Williams & Wilkins. Lactobacillus parakefir sp. nov., two new species from kefir grains.
Kimura, M. (1980). A simple method for estimating evolutionary Int J Syst Bacteriol 44, 435–439.
rates of base substitutions through comparative studies of nucleotide Tamaoka, J. & Komagata, K. (1984). Determination of DNA base
sequences. J Mol Evol 16, 111–120. composition by reversed-phase high-performance liquid chromato-
Kluge, A. G. & Farris, J. S. (1969). Quantitative phyletics and the graphy. FEMS Microbiol Lett 25, 125–128.
evolution of the anurans. Syst Zool 18, 1–32. Thompson, J. D., Gibson, T. J., Plewniak, F., Jeanmougin, F. &
Krooneman, J., Faber, F., Alderkamp, A. C., Oude Elferink, S. J. H. W., Higgins, D. G. (1997). The CLUSTAL_X windows interface: flexible
Deiehuis, F., Cleenwerck, I., Swings, J., Gottschal, J. C. & strategies for multiple sequence alignment aided by quality analysis
Vancanneyt, M. (2002). Lactobacillus diolivorans sp. nov., a tools. Nucleic Acids Res 25, 4876–4882.

712 International Journal of Systematic and Evolutionary Microbiology 57