International Journal of Systematic and Evolutionary Microbiology (2015), 65, 4256–4262
DOI 10.1099/ijsem.0.000570
Lysinibacillus louembei sp. nov., a spore-forming
bacterium isolated from Ntoba Mbodi, alkaline
fermented leaves of cassava from the Republic
of the Congo
Labia Irène I. Ouoba,1,2 Alain B. Vouidibio Mbozo,3 Line Thorsen,4
Amarachukwu Anyogu,2 Dennis S. Nielsen,4 Simon C. Kobawila3 and
Jane P. Sutherland2
Correspondence
1
Labia Irène I. Ouoba
2
[email protected] or
[email protected]
Ouoba-Consulting, London, United Kingdom
Microbiology Research Unit, School of Human Sciences, Faculty of Life Sciences and Computing,
London Metropolitan University, 166–220 Holloway Road, London N7 8DB, United Kingdom
3
Faculté des Sciences, Université Marien N’Gouabi, BP: 69 Brazzaville, Republic of the Congo
4
Department of Food Science, Faculty of Science, University of Copenhagen, Rolighedsvej 26,
1958 Frederiksberg C, Denmark
Investigation of the microbial diversity of Ntoba Mbodi, an African food made from the alkaline
fermentation of cassava leaves, revealed the presence of a Gram-positive, catalase-positive,
aerobic, motile and rod-shaped endospore-forming bacterium (NM73) with unusual phenotypic
and genotypic characteristics. The analysis of the 16S rRNA gene sequence revealed that the
isolate was most closely related to Lysinibacillus meyeri WS 4626T (98.93 %), Lysinibacillus
xylanilyticus XDB9T (96.95 %) and Lysinibacillus odysseyi 34hs-1T (96.94 %). The DNA–DNA
relatedness of the isolate with L. meyeri LMG 26643T, L. xylanilyticus DSM 23493T and
L. odysseyi DSM 18869T was 41 %, 16 % and 15 %, respectively. The internal transcribed
spacer-PCR profile of the isolate was different from those of closely related bacteria. The cellwall peptidoglycan type was A4a, L -Lys-D -Asp and the major fatty acids were iso-C15 : 0,
anteiso-C15 : 0, anteiso-C17 : 0 and iso-C17 : 0 and iso-C17 : 1v10c. The polar lipids
included phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol,
phosphoaminolipid, aminolipid, two phospholipids and two unknown lipids. The predominant
menaquinones were MK-7 and MK-6.Ribose was the only whole-cell sugar detected. The DNA
G+C content was 38 mol%. Based on the results of the phenotypic and genotypic
characterization, it was concluded that the isolate represents a novel species of the genus
Lysinibacillus, for which the name of Lysinibacillus louembei sp. nov. is proposed. NM73T
(5DSM 25583T5LMG 26837T) represents the type strain.
In recent years, various novel species of the genus Lysinibacillus have been characterized, the most recently recognized
being Lysinibacillus manganicus, Lysinibacillus contaminans
and Lysinibacillus meyeri (Kämpfer et al., 2013; Liu et al.,
2013; Seiler et al., 2013). Other species include Lysinibacillus boronitolerans, Lysinibacillus sphaericus, Lysinibacillus
Abbreviations: AL, aminolipid; ITS-PCR, internal transcribed spacerPCR; PL, phospholipid; PN, phosphoaminolipid.
The GenBank/EMBL/DDBJ accession number for the 16S rRNA
gene sequence of NM73T is HG937791.
Four supplementary figures are available with the online Supplementary
Material.
4256
fusiformis, Lysinibacillus parviboronicapiens, Lysinibacillus
massiliensis, Lysinibacillus sinduriensis, Lysinibacillus odysseyi, Lysinibacillus xylanilyticus, Lysinibacillus macroides
and Lysinibacillus mangiferahumi (Ahmed et al., 2007;
Miwa et al., 2009; Lee et al., 2010; Coorevits et al., 2012;
Jung et al., 2012; Yang et al., 2012). Bacteria belonging to
the genus Lysinibacillus are aerobic, motile and endospore-forming rods that exhibit cell-wall peptidoglycan
type A4a, L -Lys-D -Asp.
Ntoba Mbodi is a traditional food obtained by alkaline fermentation of cassava leaves in the Republic of the Congo
(Congo-Brazzaville, Central Africa). During an investigation of the diversity of species of Bacillus involved in
the fermentation, a spore-forming isolate with unusual
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Lysinibacillus louembei sp. nov.
phenotypic and genotypic properties was isolated and
characterized. Preliminary phenotypic and genotypic investigations revealed that the isolate, NM73T, belonged to the
genus Lysinibacillus but was different from species of the
genus Lysinibacillus with validly published names and,
thus, potentially represented a novel species. This manuscript describes the phenotypic and genotypic characteristics of this bacterium for the first time.
For investigations of the Bacillus population of Ntoba
Mbodi, about 500 bacteria were isolated from samples of
the product, originating from different production sites
in various localities of the Republic of the Congo. Appropriate dilutions of the samples were spread on nutrient
agar (NA; Oxoid CM0003) plates, which were incubated
at 37 8C for 48 h. After enumeration, individual colonies
were subcultured and purified. Spore-forming, catalasepositive and Gram-positive bacteria were maintained in
nutrient broth (NB; Oxoid CM0001) supplemented with
30 % glycerol (v/v) for further phenotypic and genotypic
characterizations. From the identification tests, it was possible to assign most of the bacteria to known Bacillus
species, but one isolate (NM73T) had unusual phenotypic
and genotypic properties and, although identified as
belonging to the genus Lysinibacillus, it was not possible
to allocate a defined species name to this isolate. Additional
samples of Ntoba Mbodi were analysed for detection of
further isolates with the same phenotypic and genotypic
characteristics as NM73T, but the screening was unsuccessful. Analysis of the 16S rRNA sequence revealed that
NM73T was similar (100 % similarity for the 16S rRNA
gene sequence) to an unidentified isolate of the genus Bacillus (Bacillus sp. 2B273-6XC, GenBank accession number
AB243843), which was also isolated as a sole contaminant
during tea processing in Japan. Consequently, it was
intended to include that isolate in this study, but unfortunately the holder of the isolate was not able to revive the bacterium to allow collaborative research. Various species of the
genus Lysinibacillus such as L. odysseyi 34hs-1T (DSM
18869T), L. meyeri WS 4626T (LMG 26643T),
L. manganicus Mn1-7T (DSM 26584T) and L. contaminans
FSt3AT (DSM 25560T) were also characterized and recognized as single isolates (La Duc et al., 2004; Kämpfer et al.,
2013; Liu et al., 2013; Seiler et al., 2013).
Phenotypic characterization of NM73T included a wide
range of procedures and was carried out mainly according
to protocols described by Schleifer (2009) and standards set
by Logan et al. (2009) for describing novel endosporeforming species. Type strains of bacteria closely related to
NM73T (L. meyeri LMG 26643T, L. xylanilyticus DSM
23493T and L. odysseyi DSM 18869T) were simultaneously
screened for comparison. The morphology of vegetative
cells and endospores was screened after 24–72 h growth
at 37 8C in NA, NA/MnSO4, NB and brain heart infusion
agar (BHI-A; Oxoid CM1136) using a phase-contrast
microscope (Olympus BH2). For determination of the
temperature range for growth, isolates were inoculated on
NA, BHI-A and tryptone soya agar (TSA; Oxoid
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CM0131) and incubated at different temperatures (5, 10,
15, 20, 30, 37, 40, 45, 48, 50 and 55 8C) for times ranging
from 24 h to 4 weeks (at the lower temperatures). Growth
at different pH values (2.5, 4, 5, 5.5, 6, 7, 8, 9, 9.5 and 10)
and NaCl concentrations (w/v: 2, 5, 7 and 10 %) was determined in NB, tryptone soya broth (TSB; Oxoid CM0129)
and brain heart infusion (BHI; Oxoid CM1135) medium.
The inoculated media were incubated in a shaking water
bath at 37 8C for times ranging from 24 h to 2 weeks.
Growth was determined by observation of the turbidity
of the media and plate counting, which also allowed a
purity check. Growth in anaerobic conditions at 37 8C in
NA, TSA and BHI-A was performed in both anaerobic
jars and an anaerobic cabinet (Don Whitley) using Bacillus
licheniformis and Bacillus subtilis as positive and negative
controls, respectively. Plates were incubated for times ranging from 72 h to 4 weeks.
All media used for the phenotypic characterization were
suitable for sustaining growth of the novel species. However, the best results were obtained with TSA/TSB and
BHI-A/BHI. After 24 h incubation at 37 8C, cells of
NM73T were highly motile and were Gram-positive, catalase- and oxidase-positive rods (0.7–0.864–14 mm).
Additionally, spherical endospores in terminal swollen
sporangia were observed after 72 h (Fig. S1, available in
the online Supplementary Material). NM73T only grew
aerobically at 10–48 8C (optimum 37 8C), pH 6–9.5 (optimum pH 7–9) and in the presence of up to 7 % (w/v)
NaCl. Growth at 10 8C and in 7 % (w/v) NaCl occurred
after 6 days and 6–9 days respectively. The minimum
growth temperature for the other organisms used for comparison was 15 8C, but variable maximum growth temperatures (45 8C for L. meyeri LMG 26643T, 40 8C for
L. xylanilyticus DSM 23493T and 50 8C for L. odysseyi
DSM 18869T) were observed. For growth in different
NaCl concentrations, the maximum concentration allowing growth was 5 % (w/v) for L. meyeri LMG 26643T and
L. odysseyi DSM 18869T, and 2 % (w/v) for
L. xylanilyticus DSM 23493T. The pH range for growth
was 6–9.5 for L. meyeri LMG 26643T, and 5.5–9.5 for
both L. odysseyi DSM 18869T and L. xylanilyticus DSM
23493T. In some cases, different temperatures, NaCl concentrations and pH ranges for growth have been reported
in earlier studies; e.g. temperature ranges for growth of
10–42 8C and 25–42 8C were reported for L. meyeri and
L. odysseyi, respectively (La Duc et al., 2004; Seiler et al.,
2013), and 5.5 % (w/v) was reported as the maximum
NaCl concentration allowing growth of L. xylanilyticus.
The differences observed might be related to different
growth environments, such as the media used.
The Voges–Proskauer reaction, nitrate reduction, H2S production, urea hydrolysis and other enzymatic activities and
carbohydrate assimilation were tested using the API 20 E
and API 50 CHB systems (bioMérieux). Citrate utilization
was tested using the API 20 E and the method described by
Reddy et al. (2007). Hydrolysis of pectin was carried out
according to Bertheau et al. (1984). Phenotypic
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L. I. I. Ouoba and others
Table 1. Different phenotypic characteristics of NM73T and closely related strains of species of the genus Lysinibacillus
Strains: 1, NM73T; 2, L. meyeri LMG 26643T; 3, L. xylanilyticus DSM 23493T; 4, L. odysseyi DSM 18869T. All isolates exhibited negative reactions in
all other substrates in the API 50 CH and API 20E systems. All data were obtained from the current study, except for the chemotaxonomic data for
the reference isolates, which were retrieved from Seiler et al. (2013) for L. meyeri LMG 26643T (WS 4626T), Lee et al. (2010) for L. xylanilyticus DSM
23493T (XDB9T) and Jung et al. (2012) for L. odysseyi DSM 18869T (34hs-1T). AL, Aminolipid; DPG, diphosphglycerol; PE, phosphoethanolamine;
PG, phosphoglycerol; PL, phospholipid; PN, phosphoaminolipid.+, Positive; 2, negative; W , weakly positive.
Characteristic
1
2
3
4
pH range for growth
6–9.5
6–9.5
5.5–9
5.5–9.5
Temperature range for growth (8C)
10–48
15–45
15–40
15–50
NaCl range for growth (%, w/v)
0–7
0–5
0–2
0–5
W
Voges–Proskauer reaction
+
+
+
Hydrolysis of starch
+
2
2
2
Hydrolysis of gelatin
+
+
+
2
W
2
Citrate utilization
+
2
W
2
2
2
Tryptophan deaminase
Acid production from:
W
+
+
2
N-Acetyl-b-glucosamine
D -Fructose
+
+
2
2
Glycerol
W
2
+
2
D -Mannitol
W
2
2
2
Chemotaxonomic characteristics
Polar lipids
DPG, PG, PE, PN, AL, PL DPG, PG, PE, PN,
DPG, PG, PE, PN, DPG, PG, PE, PL
Quinones
MK-7, MK-6
MK-7, MK-6 MK-5, MK-4, MK-3 MK-7
MK-7
characteristics of NM73T and related bacteria are shown in
Table 1, with similarities observed in some cases. Ability to
utilize N-acetyl-b-glucosamine, D -fructose, D -mannitol
and glycerol was variable according to the isolate and the
substrate screened. Production of acid from all other substrates in the API 50 CH system was negative for all isolates.
Further details of the phenotypic characteristics of the
novel isolate, NM73T, and closely related bacteria are presented in Table 1 and in the species description.
Analyses of cellular fatty acids, diamino acids of cell-wall
peptidoglycans, polar lipids, respiratory quinones and
whole-cell sugar composition were carried out by the identification service of the Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ, Braunschweig,
Germany) as described by Staneck & Roberts (1974), Kuykendall et al. (1988), Tindall (1990a, b), Tindall et al.
(2007) and Schumann (2011). Cellular fatty acids detected
in NM73T were: iso-C14 : 0 (0.2 %), iso-C15 : 0 (24.0 %),
anteiso-C15 : 0 (17.8 %), C15 : 0 (0.2 %), C16 : 1v7c alcohol (3.4 %), iso-C16 : 0 (6.0 %), C16 : 1 v11c (2.5 %),
C16 : 0 (1.2 %), iso-C17 : 1v10c (10.3 %), iso-C17 : 0
(12.7 %), anteiso-C17 : 0 (19.1 %) and fatty acids that
could not be separated by the microbial identification
(summed feature 4, 2.6 %). Investigation of the cellular
fatty acid profile of NM73T was performed simultaneously
with those of L. meyeri LMG 26643T, L. xylanilyticus DSM
23493T and L. odysseyi DSM 18869T, showing that the
NM73T profile was closer to the profile of L. meyeri
(Table 2). It was also noted that C10 : 0, iso-C13 : 0 and
C14 : 0 were not detected in NM73T, whereas C10 : 0 was
detected in L. xylanilyticus, iso-C13 : 0 in L. xylanilyticus
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and L. odysseyi and C14 : 0 in L. meyeri. The amounts of
some other fatty acids varied significantly according to the
isolate. Analysis of the cell-wall peptidoglycan of NM73T
revealed the presence of the amino acids alanine, aspartic
acid, glutamic acid and lysine (1.1 Ala: 0.8 Asp: 1 Glu: 0.9
Lys) and the peptides L -Lys-D -Asp, L -Ala-D -Glu, L -Lys-D Ala, D -Ala-L -Lys-D -Asp. These results indicate a cell-wall
peptidoglycan type A4a, L -Lys-D -Asp, which is characteristic
of species of the genus Lysinibacillus (Lee et al., 2010). Two
menaquinones were detected in NM73T: MK-7 (78 %) and
MK-6 (22 %). This profile is different from that of the
most closely related bacterium L. meyeri LMG 26643T,
which includes five menaquinones: MK-7 (52 %), MK-6
(38 %), MK-5 (5 %), MK-4 (3 %) and MK-3 (2 %) (Seiler
et al., 2013). The polar lipids of NM73T included phosphatidylethanolamine, diphosphatidylglycerol, phosphatidylglycerol, phosphoaminolipid (PN), aminolipid (AL), two
phospholipids (PL) and two unknown lipids (Fig. S2). This
profile differs from those of L. meyeri LMG 26643T and
L. xylanilyticus DSM 23493T, in which PN, AL and PL were
not reported (Lee et al., 2010; Seiler et al., 2013). The polar
lipids PN and AL were not detected in L. odysseyi (Jung
et al., 2012). Ribose was detected in NM73T as the sole
whole-cell sugar. This constitutes an important difference
between NM73T and related species such as L. meyeri and
L. odysseyi, which have xylose as the predominant cell
sugar (Seiler et al., 2013).
The ability of the novel bacterium to produce toxins was
investigated by detection of genes encoding production of
Bacillus haemolysin BL (hblA, hblC, hblD), non-haemolytic
enterotoxin (nheA, nheB, nheC), cytotoxin K (cytK) and
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Lysinibacillus louembei sp. nov.
Table 2. Fatty acid composition (as a percentage of total present) of NM73T and strains of closely related species of the
genus Lysinibacillus
Strains: 1, NM73T; 2, L. meyeri LMG 26643T; 3, L. xylanilyticus DSM
23493T; 4, L. odysseyi DSM 18869T. All data were obtained in this
study under comparable conditions. 2, Not detected.
Fatty acids
C10 : 0
Iso-C13 : 0
Iso-C14 : 0
C14 : 0
Iso-C15 : 0
Anteiso-C15 : 0
C15 : 0
C16 : 1v7c alcohol
Iso-C16 : 0
C16 : 1v11c
C16 : 0
Iso-C17 : 1v10c
Iso-C17 : 0
Anteiso-C17 : 0
Summed feature 4*
1
2
3
4
2
2
0.2
2
24.0
17.8
0.2
3.4
6.0
2.5
1.2
10.3
12.7
19.1
2.6
2
2
0.2
0.3
35.4
12.8
0.7
2.6
3.1
5.0
1.8
13.7
10.5
11.6
2.4
0.2
0.4
3.5
1.5
44.1
11.3
3.2
10.4
7.6
3.1
0.6
2.3
3.9
5.0
3.0
2
0.4
8.7
0.4
42.0
10.2
0.6
12.5
16.7
2.1
0.6
1.1
2.6
1.8
0.5
*Summed features represent fatty acids that cannot be separated by
the microbial identification system. Summed feature 4 consisted of
iso-C17 : 1, I/anteiso B and/or anteiso-C17 : 1 B/iso I.
emetic toxin (cesB) using PCR (Ouoba et al., 2008). Moreover,
a Bacillus cereus Enterotoxin Reverse Passive Latex Agglutination test kit (BCET-RPLA; Oxoid D0950A) was used to
screen the ability of the isolate to produce the L2 component
of the tripartite haemolysin BL enterotoxin in broth. None
of the genes investigated was detected in NM73T and the
isolate was not able to produce the L2 component.
For molecular characterization, the novel isolate was first
investigated by 16S rRNA gene sequencing. Amplification
was performed using the primers 0011f [AGAGTTTGAT
(C/T) (A/C)TGGCTCAG] and 1510r [ACGG(C/T)TACC
TTGTTACGACTT]. After purification using the QIAquick
PCR Purification kit (Qiagen 28104), the PCR products
were sequenced (Source Bioscience) in both directions
with the primers mentioned above. The sequences were
aligned using Vector NTI Suite 10 software (Informax)
and compared to 16S rRNA gene sequences in the GenBank
database using the BLAST algorithm (Altschul et al., 1997)
and Eztaxon (Kim et al., 2012). Sequences of NM73T and
those of its closely related bacteria, retrieved from the
GenBank database, were aligned and phylogenetic trees
were reconstructed by the maximum-likelihood, maximum-parsimony and neighbour-joining methods, as
described by Liu et al. (2013) using MEGA 6 software
(Tamura et al., 2013). Analysis of the almost complete
sequence of the 16S rRNA gene sequence (1405 bp)
revealed that the novel isolate is closely related to species
of the genus Lysinibacillus, with L. meyeri WS 4626T
http://ijs.microbiologyresearch.org
(98.93 %, 14 bp difference) being the most closely related
species. According to the Eztaxon analysis, the other closely
related bacteria were L. xylanilyticus XDB9T (96.95 %,
41 bp difference) and L. odysseyi 34hs-1T (96.94 %, 43 bp
difference). The phylogenetic analysis demonstrated that
NM73T is distinct from other species of the genus Lysinibacillus and placed the isolate next to L. meyeri WS 4626T
(Fig. 1). L. odysseyi was also placed next to NM73T and
L. meyeri, but L. xylanilyticus was more distantly related
as reported also by Seiler et al. (2013). The results of the
phylogenetic analyses using the maximum-likelihood
(Fig. 1), neighbour-joining (Fig. S3) and maximumparsimony (Fig. S4) procedures were similar.
The DNA G+C content (mol%) of the novel species
was determined by the identification service of the
Belgian Coordinated Collections of Microorganisms/LMG
(BCCM/LMG, Gent, Belgium) using the DNA isolation
method described by Cleenwerck et al. (2002) and HPLC
(Mesbah et al., 1989). Isolate NM73T exhibited a G+C
content of 38 mol%. DNA–DNA hybridizations were conducted to determine the degree of relatedness between
NM73T and affiliated species. This was also performed by
BCCM/LMG using modified methods of Gevers et al.
(2001) for DNA extraction and Ezaki et al. (1989) for the
hybridizations. DNA–DNA reassociation values between
NM73T and L. meyeri LMG 26643T (WS 4626T),
L. xylanilyticus DSM 23493T (XDB9T) and L. odysseyi
DSM 18869T (34hs-1T) were 41 %, 16 % and 15 %,
respectively. These values are well below the 70 %
threshold value commonly recommended for the delineation of a novel species (Wayne et al., 1987; Tindall et al.,
2010) and support the distinctiveness of isolate NM73T.
Furthermore, NM73T and the type strains of closely related
species were genotypically characterized by internal transcribed spacer-PCR (ITS-PCR) using primers S-D-Bact1494-a-S-20 (59GTCGTAACAAGGTAGCCGTA-39) and
L-D-Bact-0035-a-A-15
(59-CAAGGCATCCACCGT-39).
The profiles were compared by cluster analysis in BioNumerics 4.50: Dice’s Coefficient/UPGMA (Applied Maths,
Sint-Martens-Latem, Belgium). The ITS-PCR fingerprint
of NM73T was distinct from those of L. meyeri LMG
26643T (WS 4626T), L. xylanilyticus DSM 23493T
(XDB9T), L. odysseyi DSM 18869T (34hs-1T) and other
species of the genus Lysinibacillus (Fig. 2). L. meyeri LMG
26643T exhibited the closest ITS-PCR fingerprint to the
NM73T profile. However, differences represented by the presence of two bands positioned at 490 and 510 bp in NM73T
and the presence of a band of 350 bp in L. meyeri LMG
26643T were observed. ITS-PCR has been reported to differentiate some species within the same genus, including
Bacillus (Gürtler & Stanisich, 1996; Johnson et al., 2000;
Ouoba et al., 2004; Ahaotu et al., 2013).
The results obtained in the present study clearly show that
isolate NM73T is genetically and phenotypically different
from all species with validly published names, with L. meyeri
being the most closely related species. Phenotypically,
NM73T can be differentiated from L. meyeri LMG 26643T
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L. I. I. Ouoba and others
95 Lysinibacillus sphaericus C3-41 (CP000817)
72
Lysinibacillus fusiformis NBRC 15717T (AB271743)
0.05
Lysinibacillus mangiferihumi M-GX18T (JF731238)
72
Lysinibacillus contaminans FSt3AT (KC254732)
94
Lysinibacillus parviboronicapiens BAM-582T (AB300598)
Lysinibacillus xylanilyticus XDB9T (FJ477040)
Lysinibacillus boronitolerans 10aT (AB199591)
92
99 Lysinibacillus macroides LMG 18474T (AJ628749)
52
Lysinibacillus odysseyi 34hs-1T (AF526913)
Lysinibacillus meyeri WS 4626T (HE577173)
100
Lysinibacillus louembei NM73T (HG937791)
Lysinibacillus sinduriensis BLB-1T (FJ169465)
Lysinibacillus massiliensis 4400831T (AY677116)
96
99
Lysinibacillus manganicus Mn1-7T (JX993821)
Bacillus cecembensis PN5T (AM773821)
90
Bacillus isronensis B3W22T (AMCK01000046)
100 Solibacillus silvestris HR3-23T (AJ006086)
Bacillus subtilis subsp. subtilis NCIB 3610T (ABQL01000001)
Fig. 1. Maximum-likelihood tree based on almost complete 16S rRNA gene sequences (1405 bp) showing the phylogenetic
position of isolate NM73T within the closely related group of species of the genus Lysinibacillus (GenBank/EMBL/DDBJ
accession numbers in parentheses). Bacillus subtilis subsp. subtilis was included as an out-group. Bootstrap values based
on 1000 replications are given on each node. Only bootstrap values .50 % are shown at branch points.
100
80
60
40
20
(WS 4626T) by characteristics such as the ability to utilize
citrate, hydrolyse starch, grow at 10 8C, 48 8C and in 7 % (w/
v) NaCl, the presence of AL, PN and PL in its polar lipid profile,
the absence of the menaquinones MK-5, MK-4 and MK-3 in its
respiratory quinone profile and the absence of C14 : 0 in its
fatty acid profile. Additionally, ribose was recorded as the
sole whole-cell sugar in NM73T, whereas xylose plus traces of
mannose and glucose were detected in L. meyeri. Genotypically,
NM73T can be differentiated from L. meyeri LMG 26643T (WS
4626T) by a DNA–DNA hybridization combination of 41 %,
substitutions of 14 bp in the 16S RNA gene sequence and a
different ITS-PCR fingerprint.
Isolate NM73T represents a novel species belonging to the
genus Lysinibacillus. The name Lysinibacillus louembei
sp. nov. is proposed with NM73T (5DSM 25583T5LMG
26837T) being the type strain.
Description of Lysinibacillus louembei
Lysinibacillus louembei (lou.em’be.i. N.L. gen. masc. n.
louembei Louembe) named in honour of Professor
Delphin Louembe from the Republic of the Congo for
his substantial contribution to a better understanding of
510 bp
490 bp
350 bp
Lysinibacillus louembei NM73T
Lysinibacillus meyeri LMG 26643T
Lysinibacillus odysseyi DSM 18869T
Lysinibacillus massiliensis CIP 108446T
Lysinibacillus xylanilyticus DSM 23493T
Lysinibacillus fusiformis DSM 2898T
Lysinibacillus sphaericus NCIMB 9370T
Fig. 2. Cluster analysis of ITS-PCR fingerprints of isolate NM73T and closely related bacteria. The dendrogram is based on
the Dice’s Coefficient/UPGMA.
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Lysinibacillus louembei sp. nov.
the microbial diversity of Congolese traditional fermented
foods.
After 24 h incubation at 37 uC in NA, NB and BHI-A, cells
are highly motile, Gram-positive, catalase-positive and oxidase-positive rods (agar: 0.6–0.7|2–6 mm; broth: 0.7–
0.8|4–14 mm). After 72 h incubation on NA/MnSO4 or
BHI-A, terminal and spherical endospores in swollen sporangia are observed. Colonies are cream to light brown,
shiny, smooth, convex and round, with regular margins
after 48 h incubation at 37 uC on NA (1–2 mm) and TSA
(4–5 mm). On blood agar, colonies (3–4 mm) are greyish
and exhibit a white coloration in the middle. Cells grow
only aerobically at 10–48 uC (optimum 37 uC), pH 6–9.5
(optimum 7–9) and in the presence of up to 7 % (w/v)
NaCl. The micro-organism is positive for the Voges–Proskauer test, citrate utilization, tryptophan deaminase (weak)
and hydrolysis of gelatin, starch, pectin and aesculin
(weak). It is negative for the reduction of nitrate to nitrite,
production of indole, hydrogen sulphide, urea hydrolysis,
beta-galactosidase, arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase and b-galactosidase.
It produces acid from D -fructose, D -mannitol (weakly),
glycerol (weakly) and N-acetyl-b-glucosamine (weakly).
The other substrates in the API 50 CH or 20E systems
are not assimilated. The cell-wall peptidoglycan type is
A4a, L -Lys-D -Asp. It contains the amino acids alaninine,
aspartic acid, glutamic acid and lysine (1.1 Ala: 0.8 Asp:
1 Glu: 0.9 Lys), and the peptides L -Lys-D -Asp, L -Ala-D Glu, L -Lys-D -Ala and D -Ala-L -Lys-D -Asp. Major fatty
acids are iso-C15 : 0, anteiso-C15 : 0, anteiso-C17 : 0, isoC17 : 0,and iso-C17 : 1v10c. Respiratory quinones are
MK-6 (22 %) and MK-7 (78 %) and the polar lipids
include phosphatidylethanolamine, diphosphatidylglycerol,
phosphatidylglycerol, phosphoaminolipid, aminolipid, two
types of phospholipid and two unknown lipids. Ribose is
the only whole-cell sugar. The DNA G+C content of the
type strain is 38 mol%.
The type strain, NM73T (5DSM 25583T5LMG 26837T)
was isolated from fermented cassava leaves from Brazzaville, Republic of the Congo.
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