IJSEM Papers in Press. Published May 13, 2014 as doi:10.1099/ijs.0.063602-0
Lactobacillus bombi sp. nov., from the digestive tract of laboratory-reared bumblebee queens (Bombus terrestris)
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J. Killer 1,2*, A. Votavová 3, I. Valterová 4, E. Vlková 2, V. Rada 2, Z. Hroncová 2 1
Institute of Animal Physiology and Genetics v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4 – Krč, 142 20, Czech Republic 2 Czech University of Life Sciences, Faculty of Agrobiology, Food and Natural Resources, Department of Microbiology, Nutrition and Dietetics, Kamýcká 129, Prague 6 – Suchdol, 165 21, Czech Republic 3 Agricultural Research, Ltd., Zahradní 400/1, Troubsko, Czech Republic 4 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic. *Corresponding author. Tel.: +420 267 090 508; fax: +420 267 090 500. E-mail address: [email protected]; [email protected] Running title: Lactobacillus bombi sp. nov.; Subject category: New taxa-Firmicutes
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The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains BTLCH M1/2T, BTLCH M3/2 and M250 3MRA are KJ078643, KJ078644 and KJ078645; those for the partial rpoA and tuf gene sequences of the same strains are KJ144262, KJ144263, KJ144264, KJ144265, KJ144266 and KJ144267. The GenBank/EMBL/DDBJ accession numbers for the partial hsp60 and pheS gene sequences determined in the BTLCH M1/2T and M250 3MRA are KC294225, KJ144258, KC294226 and KJ144260. The GenBank/EMBL/DDBJ accession number for the partial hsp60 gene sequence of Lactobacillus tucceti DSM 20183T is KJ144259.
29
(Multilocus Sequence Analysis)
30 31
Three bacterial strains belonging to the genus Lactobacillus were isolated from the digestive
32
tract of laboratory-reared bumblebee queens (Bombus terrestris) using MRS agar under
33
anaerobic conditions. The isolates were identified according to 16S rRNA gene sequences as
34
yet undescribed Lactobacillus sp. with the highest 16S rRNA gene similarity (96.9 %) to
35
uncharacterized bacterial strain Lactobacillus sp. Mboho2r2 isolated from the stomach of a
36
European honeybee (Apis mellifera). Lactobacillus tucceti was found to be the closest relative
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valid species with 92.9 % 16S rRNA gene sequence similarity. However, phylogenetic
Keywords: bumblebee, Lactobacillus sp. nov., Bombus terrestris, digestive tract, MLSA
1
38
analyses based on different markers revealed that the species is phylogenetically very distant
39
from the new strains. The DNA G + C content of the type strain is 37.8 mol%. Fatty acids
40
such as C19:1
41
Presence of diphosphatidylglycerol, phosphatidylglycerol, a phospholipid, seven glycolipids
42
and two phosphoglycolipids was detected in cells. Growth at 47 oC was found. The
43
peptidoglycan type A4α L-Lys-D-Asp was determined for type strain BTLCH M1/2T.
44
Genotypic characteristics and phylogenetic analyses based on phylogenetic markers such as
45
hsp60, pheS, rpoA and tuf genes, as well as results of phenotypic characteristics and
46
chemotaxonomic analyses confirmed that new isolates belong to a new Lactobacillus species.
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The name Lactobacillus bombi sp. nov. was proposed for group of new isolates. The type
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strain is BTLCH M1/2T (=DSM 26517T = CCM 8440T).
w6c
/ cyclo C19:0
w10c/19w6,
C18:1
w9c
and C16:0 were predominant in all strains.
49 50
Representatives of the genus Lactobacillus are phylogenetically classified in the order
51
Lactobacillales within the bacterial phylum Firmicutes. Lactobacilli are found primarily in
52
environments that are rich source of carbohydrates and other organic substrates. Many species
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of lactobacilli are obligate bacterial symbionts of the digestive tract of mammals (Hammes &
54
Hertel, 2009). Digestive tract of insects is an environment that is inhabited by a wide variety
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of bacterial symbionts, many of which are among the yet undescribed species (Engel et al.,
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2012; Colman et al., 2012). Some researchers believe that the bacterial symbionts of insects,
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especially lactic acid bacteria, can to some extent positively influence the immune system and
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host health (Evans & Armstrong, 2006; Forsgren et al., 2010; Koch & Schmid-Hempel, 2012;
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Mattila et al., 2012). Many studies have been recently published on the prevalence of
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lactobacilli and other lactic acid bacteria in the digestive tract of insects, especially in the
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digestive tract of important pollinators (Mohr & Tebbe, 2006; Killer et al., 2010b; Tajabadi et
62
al., 2011; Martinson et al., 2012; Tang et al., 2012; Killer et al., 2014a). Some authors have
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shown that in the stomach of honeybees appear new, probably host-specific species of
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lactobacilli (Olofsson & Vásquez, 2008; Forsgren et al., 2010). Bacteria inhabiting the
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digestive tract of bumblebees are not sufficiently explored (Killer et al., 2010b; Koch &
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Schmid-Hempel, 2011). Studies on the isolation and characterization of bacteria present in the
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digestive tract of bumblebees have so far focused only on the representatives of the family
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Bifidobacteriaceae. Three species of bifidobacteria and a new genus within the family
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Bifidobacteriaceae were recently discovered in the digestive tract of bumblebees (Killer et al.,
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2009; Killer et al., 2010a; Killer et al., 2011).
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Isolation and detailed characterization of a new representative of the genus Lactobacillus
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occurring in the digestive tract of bumblebees is presented in this study. Based on results of
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genotypic, phylogenetic and phenotypic analyses, it was concluded that group of three
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bacterial strains represent a new Lactobacillus species.
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Bumblebees of Bombus terrestris species were bred in the laboratory of the Agricultural
76
Research, Ltd. (Troubsko, Czech Republic) in the spring of 2012. Bumblebees were kept in
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wooden hives at 26 oC and 50% RH (Relative Humidity). They were fed by fresh frozen
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honeybee pollen pellets (mix of pollen with dominance of Brassicacae, Rosaceae and Papaver
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from a local beekeeper) and sugar solution: saccharose (1000 g, white beet sugar, local
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producer) and fructose (460 g, Fructopur, Natura, Czech Republic) dissolved in water
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(1200g). Living bumblebees were transported to the Laboratory of anaerobic microbiology
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(Institute of Animal Physiology and Genetics in Prague, The Academy of Sciences of the
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Czech Republic) and immediately killed by decapitation. Fresh digestive tracts of three
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queens originated from different nests were then placed in sealed tubes containing anaerobic
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MRS Broth (Oxoid, UK) and serially diluted in the same medium. Diluted samples were
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cultivated on MRS agar under anaerobic conditions (Anaerobic jars, Oxoid) at 37 oC for 48
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hours. Genes encoding 16S rRNA were amplified in bacterial isolates originated from the 10-6
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diluted samples using primers 616V and 630R under conditions as described by Ehrmann et
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al. (2003). Three bacterial isolates originated from three bumblebee queens were then
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identified according to similarities (Killer et al., 2011) of almost complete 16S rRNA gene
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sequences (1490 bp).
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The three new isolates designed as BTLCH M1/2T, BTLCH M3/2 and M250 3MRA were
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phylogenetically most closely related to unclassified bacterial strains Lactobacillus sp.
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Mboho2r2 (GenBank a. n. HM534813) and Lactobacillus sp. AcjLac3 (GenBank a. n.
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AB810024) isolated from the digestive tract of European (Apis mellifera) and Japanese (Apis
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cerana japonica) honeybees, respectively. These new isolates were found to share 96.7-96.9
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% and 96.0–96.2 % 16S rRNA gene similarities with the mentioned unclassified isolates from
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the digestive tract of honeybees. The jPHYDIT software (Jeon et al., 2005) was used for
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calculation of sequence similarities. Lactobacillus tucceti CECT 5920T (GenBank a. n.
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NR_042194) was found to be the closest relative valid species with 92.9 % 16S rRNA gene
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sequence similarity. Three new bacterial isolates had high 99.7 % gene similarity among each
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other. These results suggest that the new bacterial isolates belong to the same bacterial
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phylotype probably representing a new bacterial species of the genus Lactobacillus
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(Stackebrandt & Ebers, 2006). However, MLST (Multilocus Sequence Typing) method,
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phylogenetic, phenotypic and chemotaxonomic analysis were then used to confirm this
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assumption.
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Genes encoding the heat shock protein 60 kDa (hsp60), phenyl-alanyl t-RNA synthase alpha
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subunit (pheS), RNA polymerase alpha subunit (rpoA) and translation elongation factor Tu
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(tuf) were sequenced in three strains as the additional phylogenetic markers. Detailed
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information about the primers and PCR parameters for amplification of these genes were
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previously published by Goh et al. (2000), Dobson et al. (2002), Naser et al. (2005, 2007) and
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Ventura et al. (2003). Amplified fragments were subsequently checked by electrophoresis on
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1.5 % PCR agarose gel (Top-Bio, Czech Republic), purified using PCR purification kit
114
(Qiagen) and sequenced by automatic genetic analyser ABI PRISM 3130xl (Applied
115
Biosystems). Defined sequences of phylogenetic markers were compared with sequences of
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type bacterial strains of closest related species based on the scan results in gene database
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through the BLAST (Basic Local Alignment Search Tool) program. Results of hsp60, pheS,
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rpoA and tuf gene sequence similarity testing revealed closest relatives sequences of L. tucceti
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DSM 20183T (81.6–82.3 %; GenBank accession number KJ144259), L. nagelii LMG 21593T
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(78.7–79.2 %; AM087708), L. curvatus LMG 9198T (73.3–73.6 %; AM087783) and L.
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crustorum LMG 23699T (83.2–83.6 %; FN395011), respectively. Authors, who designed the
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above phylogenetic markers indicate much higher values of inter-species sequence similarity
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(Goh et al., 2000; Naser et al., 2007). Identical hsp60 and pheS gene sequences were found in
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BTLCH M1/2T and BTLCH M3/2 strains. For this reason, only the BTLCH M1/2T and M250
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3MRA bacterial strains were further implied in phylogenetic analyses based on these
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phylogenetic markers. Similarities of hsp60, pheS, rpoA and tuf gene sequences among three
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strains were 99.0, 98.6, 99.4–99.7 and 99.5–100 %, respectively. These results suggest that
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new strains represent a new species within the genus Lactobacillus. Phylogenetic analyses
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subsequently confirmed this assumption.
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Similar procedure that has been described recently (Killer et al., 2013) was used for
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construction of phylogenetic trees based on partial 16S rRNA, hsp60, pheS, rpoA and tuf gene
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sequences. Phylogenetic trees were constructed based on sequences of type strains of
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Lactobacillus species by maximum-likelihood algorithm using the MEGA 5.05 program
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(Tamura et al., 2011) and the Jukes-Cantor model. Topology of trees was checked also by
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neighbour-joining and maximum-parsimony algorithms. Alignments provided by CLUSTAL
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W algorithm were improved by removing hypervariable positions using the program Gblocks
137
(Castresana, 2000). Phylogenetic tree constructed based on 16S rRNA gene sequences of
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138
Lactobacillus species present in the digestive tract of animals revealed that three strains
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BTLCH M1/2T, BTLCH M3/2 and M250 3MRA are situated together with uncharacterized
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Lactobacillus strains from the digestive tract of honeybees in a separate phylogenetic cluster
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(Fig. 1). These lactobacilli from the digestive tract of pollinators represent a new phylogenetic
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lineage within the genus Lactobacillus. Thus, new lactobacilli are phylogenetically distant to
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the L. alimentarius cluster (Chenoll et al., 2006) including the species L. tucceti, the most
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closely valid taxon based on 16S rRNA gene sequence similarity. Significant differences
145
between this species and isolates from the digestive tract of bumblebees was further
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demonstrated by biochemical characteristics. For these reasons, type strain of L. tucceti was
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not used for comparative analyses of other phenotypic characteristics.
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Phylogenetic trees reconstructed using hsp60, pheS, rpoA and tuf gene sequences confirmed
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that three studied strains can be classified as a new Lactobacillus taxon. They are positioned
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on separate phylogenetic branches among Lactobacillus species (Fig. S1-4). However,
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topology of the trees did not match to that obtained on the basis of 16S rRNA gene sequences.
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It is due to shorter gene fragments used and absence of relevant gene sequences for
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unclassified probably new taxa of lactobacilli from the digestive tract of pollinators.
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The PCR–DGGE (Denaturing Gradient Gel Electrophoresis) has been chosen as a tool to
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demonstrate the presence of the described new Lactobacillus species in the digestive tract of
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laboratory-reared bumblebee queens (Bombus terrestris) originated from four different
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localities in Moravia (Czech Republic). Total bacterial DNA from the digestive tract of
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bumblebee queens was isolated and analysed exactly as described previously (Killer et al.,
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2014). Amplified fragments (200 bp) of 16S rRNA gene belonging to the L. bombi BTLCH
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M1/2T (99-100% sequence similarities) strain were observed in all samples (Fig. S5).
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The modified enzymatic degradation method (Killer et al., 2011) was used for determination
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of the DNA G + C contents in strain BTLCH M1/2T, BTLCH M3/2 and M250 3MRA strains.
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The obtained values 37.8 (mean of three experiments, SD = 0.4), 37.2 (SD = 0.1) and 38.0
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(SD = 0.6) mol% are in the interval of values (32-55 mol%) defined for different species of
165
lactobacilli (Hammes & Hertel, 2009).
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API 50 CHL, Rapid ID 32A and API ZYM commercial kits (all bioMérieux, France) were
167
applied for determination of biochemical characteristics in three tested strains and L. tucceti
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DSM 20183T. Tests were performed according to the manufacturer's instructions, except that
169
the API 50 CHL test strips were incubated under anaerobic conditions (Anaerobic jars,
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Oxoid) at 37 oC for 48 hrs. Bacterial strains were also tested for oxidase activity (Lui &
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Jurtshuk, 1986) and hydrolysis of gelatine by API 20E system (bioMérieux, France).
172
Physiological properties such as the ability to grow in the range of different temperatures, pH
173
values and environments with varying oxygen tension were determined by the methods
174
described previously (Killer et al., 2013). Production of D- and L-lactic acid by the D-/L-
175
lactic acid kit (Megazyme, Ireland) was also tested. Durham tube in MRS broth was used for
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testing of gas production from glucose. Tested strains differed in utilization of eighteen
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substrates and production of ten enzymes (Table 1). L. tucceti DSM 20183T had a very
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different substrate utilization and enzyme activity pattern in comparison to three new strains
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what is in agreement with phylogenetic distance (Fig. 1). Analysed Lactobacillus strains from
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the digestive tract of bumblebees differed among themselves in utilization of L-arabinose, D-
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galactose, L-rhamnose, cellobiose and production of urease, arginine dihydrolase, alkaline
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phosphatase, acid phosphatase, tyrosine arylamidase and alanine arylamidase. Substrate
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utilization and enzyme activity patterns tested by the API 50 CHL and Rapid ID 32A kits did
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not reveal any similarity to profiles of Lactobacillus species deposited at Apiweb database
185
(https://apiweb.biomerieux.com/servlet/Authenticate). D,L-lactic acids were produced by
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cells of all analysed bacterial strains. No gas production from glucose was found. Growth at
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the temperature from 20 to 47 oC and at the pH from 4 to 8.5 ranges was observed in new
7
188
isolates from the digestive tract of bumblebees. Growth at higher temperatures than 45 oC was
189
detected only in some species of lactobacilli (Pedersen et al., 2004; Hammes & Hertel, 2009).
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The best growth at strictly anaerobic conditions on TPY (Scardovi, 1986) and MRS agar was
191
found. Nevertheless, poor growth was observed also in microaerophilic conditions.
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Determination of the end products of hexose catabolism in the strain BTLCH M1/2T was
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performed using capillary isotachophoresis (Killer et al., 2011). Lactic, acetic and propionic
194
acids were determined in concentration of 85.4 mmol l-1 (65% of all short-chain fatty acids
195
produced), 32.7 mmol l-1 (25%) and 13.2 mmol l-1 (10%), respectively. These results, along
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with the ability to utilize some pentoses, suggest that the novel strain belongs to the
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facultatively heterofermentative lactobacilli (group B according to Hammes & Hertel, 2009).
198
Cellular fatty acids profiles were determined in three strains representing new Lactobacillus
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taxon using methods described by Kämpfer & Kroppenstedt (1996) and Miller (1982).
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Summed C19:1 w6c / cyclo C19:0 w10c/19w6; C18:1 w9c and C16:0 acids were detected as the major
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fatty acids in cells (Table 2). These fatty acids have been previously identified as the main in
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lactobacilli (Gomez Zavaglia et al., 2000).
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Profile of cellular polar lipids and structure of peptidoglycan were determined in the BTLCH
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M1/2T strain by the Identification Service of the DSMZ by methods described previously
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(Killer et al., 2010a). Polar lipids detected in the strain were diphosphatidylglycerol,
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phosphatidylglycerol, a phospholipid, seven glycolipids and two phosphoglycolipids (Fig. 2).
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Phosphatidylglycerol, phospholipids and glycolipids seem to be widely distributed among
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different Lactobacillus species (Arbogast & Henderson, 1975; Kim et al., 2011; Killer et al.,
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2014a; Killer et al., 2014b). On the other hand, occurence of phosphoglycolipids, lipids and
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phosphatidylethanolamine can differ in cells of different taxa of lactobacilli (Kim et al., 2011;
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Liang et al., 2011; Killer et al., 2014a; Killer et al., 2014b). Chemical analysis revealed
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peptidoglycan type A4α L-Lys-D-Asp (type A11.31 according to DSMZ www.peptidoglycan-
8
213
types.info). The molar ratio of the amino acids in the peptidoglycan hydrolysate was as
214
follows: 2.9 Ala : 0.9 Asp : 1.0 Glu : 0.7 Lys. This peptidoglycan structure was determined
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for most species of lactobacilli and representatives of the order Lactobacillales (Schumann,
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2011).
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Provided results allow to classify group of analyzed bacterial strains as a new species within
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the genus Lactobacillus for which the name Lactobacillus bombi sp. nov. is proposed.
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Description of Lactobacillus bombi sp. nov.
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Lactobacillus bombi (L. n. bombus a boom, a deep hollow noise, buzzing, also the zoological
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genus name of the bumblebee; N.L. gen. n. bombi of Bombus, of a bumblebee).
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Cells growing on soft 0.5% MRS agar under anaerobic conditions are Gram-stain-positive,
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catalase- and oxidase-negative, regular, more or less curved long rods with rounded ends
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organized mostly singly and in pairs. They reach 0.7-1.0 µm in width and 2.2.-7.2 µm in
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length. Best growth was observed in anaerobic TPY and MRS broth or agar, lower growth in
227
microaerophilic conditions on the same agars. Colonies on MRS agar under anaerobic
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conditions after 72 h are cream in color with sharp edges. Colony morphology is a disc-
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shaped in profile but also triangular in approximately one third of colonies. Colony size is
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1.21 to 2.82 mm in diameter. The DNA G + C content of the type strain is 37.8 mol%.
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Optimum temperature for growth is 37 oC, with a minimum of 20 oC and a maximum of 47 oC.
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Growth occurs at pH values of 4-8.5. Utilize D-glucose, D-fructose, D-mannose, D-xylose, N-
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acetylglucosamin, amygdalin, arbutin, esculin, salicin, melibiose, sucrose, trehalose, raffinose
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and gentiobiose. Variable in utilization of L-arabinose, D-galactose, L-rhamnose and
235
cellobiose. Negative for utilization of glycerol, erythritol, D-arabinose, D-ribose, L-xylose, D-
236
mannitol, D-Adonitol, methyl-βD-xylopyranoside, L-sorbose, dulcitol, inositol, D-sorbitol,
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237
methyl-αD-mannopyranoside,
methyl-αD-glucopyranoside,
238
melezitose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose,
239
D-arabitol, L-arabitol, potassium gluconate, potassium 2-keto-gluconate and potassium 5-
240
keto-gluconate. Produces α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-
241
glucosaminidase, glutamic acid decarboxylase, arginine arylamidase, proline arylamidase,
242
phenylalanine arylamidase, leucine arylamidase, hystidine arylamidase, valine arylamidase,
243
serine arylamidase and naphthol-AS-BI-phosphohydrolase. Negative for β-galactosidase-6-
244
phosphate, α-glucosidase, α-arabinosidase, β-glucuronidase, α-fucosidase, reduction of
245
nitrates, production indole from L-tryptophan, leucyl-glycine arylamidase, pyroglutamic acid
246
arylamidase, glutamyl glutamic acid arylamidase, esterase lipase (C8), lipase (C14), cystine
247
arylamidase, trypsin, α-chymotrypsin, α-mannosidase, gelatin hydrolysis, catalase and oxidase.
248
Variable in production of urease, arginine dihyrolase, alkaline phosphatase, acid phosphatase,
249
tyrosine arylamidase and alanine arylamidase. The determined peptidoglycan structure type is
250
A4α L-Lys-D-Asp. Major fatty acids in cells are summed C19:1 w6c / cyclo C19:0 w10c/19w6; C18:1
251
w9c
252
diphosphatidylglycerol, phosphatidylglycerol, a phospholipid, seven glycolipids and two
253
phosphoglycolipids.
254
The type strain, BTLCH M1/2T (=DSM 26517T = CCM 8440T) was isolated from the
255
digestive tract of bumblebee queen (Bombus terrestris) laboratory-reared at the Agricultural
256
Research, Ltd. (Troubsko, Czech Republic) in 2012. Additional strains of the species are
257
BTLCH M3/2 and M250 3MRA.
lactose,
maltose,
inulin,
and C16:0, respectively. Profile of cell polar lipids revealed the presence of
258 259
Acknowledgments
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This study was primarily supported by the Technological Agency of the Czech Republic
261
(project No. TA01020969) and by institutional funding on long-term conceptual development
10
262
of research organization, then by the projects No. 20132023 and No. 20132013 of the Internal
263
Grant Agency of the Czech University of Life Sciences Prague and by the Czech National
264
Agency for Agricultural Research (NAZV QJ 1210047).
265 266 267
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Killer, J., Kopečný, J., Mrázek, J., Koppová, I., Havlík, J., Benada, O. & Kott, T. (2011).
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Bifidobacterium actinocoloniiforme sp. nov. and Bifidobacterium bohemicum sp. nov., from
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Killer, J., Kopečný, J., Mrázek, J., Rada, V., Benada, O., Koppová, I., Havlík, J. &
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Straka, J. (2009). Bifidobacterium bombi sp. nov., from the bumblebee digestive tract. Int J
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Killer, J., Kopečný, J., Mrázek, J., Rada, V., Dubná, S. & Marounek, M. (2010b).
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Killer, J., Mrázek, J., Bunešová, V., Havlík, J., Koppová, I., Benada, O., Rada, V.,
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Kopečný, J. & Vlková, E. (2013). Pseudoscardovia suis gen. nov., sp. nov., a new member
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Kim, H. J., Eom, S. J., Park, S. J., Cha, C. J. & Kim, G. B. (2011). Lactobacillus alvi sp.
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Koch, H. & Schmid-Hempel, P. (2011). Bacterial communities in central European
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Koch, H. & Schmid-Hempel P. (2012). Gut microbiota instead of host genotype drive the
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Liang, Z. Q., Srinivasan, S., Kim, Y. J., Kim, H. B., Wang, H. T. & Yang, D. C. (2011).
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Lactobacillus kimchicus sp. nov., a β-glucosidase-producing bacterium isolated from kimchi.
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Lui, J. - K., & Jurtshuk, P. (1986). N,N,N’-N’-tetramethyl-p-phenylenediamine-dependent
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cytochrome oxidase analyses of Bacillus species. Int J Syst Bacteriol 36, 38–46.
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Martinson, V. G., Moy, J. & Moran, N. A. (2012). Establishment of characteristic gut
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Mattila, H. R., Rios, D., Walker-Sperling, V. E., Roeselers, G. & Newton, I. L. (2012).
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Mohr, K. I. & Tebbe, C. C. (2006). Diversity and phylotype consistency of bacteria in the
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guts of three bee species (Apoidea) at an oilseed rape field. Environ Microbiol 8, 258-272.
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Miller, L. T. (1982). A single derivatization method for bacterial fatty acid methyl esters
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including hydroxy acids. J Clin Microbiol 16, 584-586.
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Swings, J. (2005). Application of multilocus sequence analysis (MLSA) for rapid
349
identification of Enterococcus species based on rpoA and pheS genes. Microbiology 151,
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2141-2150.
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Naser, S. M., Dawyndt, P., Hoste, B., Gevers, D., Vandemeulebroecke, K., Cleenwerck,
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I., Vancanneyt, M. & Swings, J. (2007). Identification of lactobacilli by pheS and rpoA gene
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sequence analyses. Int J Syst Evol Microbiol 57, 2777-2789.
354
Olofsson, T. & Vásquez, A. (2008). Detection and identification of a novel lactic acid
355
bacterial flora within the honey stomach of the honeybee Apis mellifera. Curr Microbiol 57,
356
356-363.
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Pedersen, C., Jonsson, H., Lindberg, J. E. & Roos, S. (2004). Microbiological
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characterization of wet wheat distillers' grain, with focus on isolation of lactobacilli with
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potential as probiotics. Appl Environ Microbiol 70, 1522-1527.
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Scardovi, V. (1986). Genus Bifidobacterium. In: Sneath, P.H.A., Mair, N.S., Sharp, M.E.,
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Holt, J.G. (Eds.), Bergey´s manual of systematic bacteriology, vol. 2, Williams and Wilkins,
362
Baltimore, pp. 1418-1434.
363
Schumann, P. (2011). In: Rainey, F. & Oren, A. (Eds.), Methods in Microbiology, vol. 38,
364
Taxonomy of Prokaryotes, Peptidoglycan Structure, Academic Press, London, pp. 101-129.
365
Stackebrandt, E. & Ebers, J. (2006). Taxonomic parameters revisited: tarnished gold
366
standarts. Microbiology Today 33, 152-155.
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Tajabadi, N., Mardan, M., Manap, M. Y. A., Shuhaimi, M., Meimandipour, A. &
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Nateghi, L. (2011). Detection and identification of Lactobacillus bacteria found in the honey
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stomach of the giant honeybee Apis dorsata. Apidologie 42, 642-649.
370
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M., Kumar, S. (2011). MEGA5:
371
Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary
372
Distance, and Maximum Parsimony Methods. Mol Biol Evol 28, 2731-2739.
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Tang, X., Freitak, D., Vogel, H., Ping, L., Shao, Y., Cordero, E. A., Andersen, G.,
374
Westermann, M., Heckel, D. G. & Boland, W. (2012). Complexity and variability of gut
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commensal microbiota in polyphagous lepidopteran larvae. PLoS One 7:e36978.
376
Vásquez, A., Forsgren, E., Fries, I., Paxton, R. J., Flaberg, E., Szekely, L. & Olofsson, T.
377
C. (2012). Symbionts as major modulators of insect health: lactic acid bacteria and
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honeybees. PLoS One 7(3):e33188.
379
Ventura, M., Canchaya, C., Meylan, V., Klaenhammer, T. R. & Zink, R. (2003).
380
Analysis, characterization, and loci of the tuf genes in lactobacillus and bifidobacterium
381
species and their direct application for species identification. Appl Environ Microbiol 69,
382
6908-6922.
383
15
384
Table 1. Differences in biochemical characteristics among strains representing a new species
385
within the genus Lactobacillus and their related valid species based on 16S rRNA gene
386
similarity. Taxa: 1, L. bombi BTLCH M1/2T; 2, L. bombi BTLCH M3/2; 3, L. bombi M250
387
3MRA; 4, L. tucceti DSM 20183T.
388 389 Characteristic Utilization of : L-Arabinose D-Xylose D-Galactose L-Rhamnose D-Mannitol N-Acetylglucosamine Cellobiose Amygdalin Arbutin Esculin Salicin Maltose Melibiose Sucrose Trehalose Raffinose Gentiobiose L-Fucose Production of : Urease Arginine dihydrolase α-Galactosidase β-Galactosidase N-Acetyl-β-glucosaminidase Alkaline phosphatase Acid phosphatase Tyrosine arylamidase Alanine arylamidase Glycine arylamidase
390 391 392 393 394 395 396 397 398 399 400 401
1
2
3
4
+ + w w + + + + + + + w + + -
+ + + + + + + + + + + + + -
+ + w w + + + + + + + + + -
+ + + + +
+ + + + w w -
+ + + w + + -
+ + + + + w w -
+ +
Notes: All strains utilize D-glucose, D-fructose, D-mannose. None produce acids from glycerol, erythritol, D-arabinose, Dribose,
L-xylose,
D-Adonitol,
methyl-βD-xylopyranoside,
L-sorbose, dulcitol,
inositol,
D-sorbitol,
methyl-αD-
mannopyranoside, methyl-αD-glucopyranoside, lactose, inulin, melezitose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-keto-gluconate and potassium 5-ketogluconate. All tested strains are able to produce glutamic acid decarboxylase, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, leucine arylamidase, hystidine arylamidase, valine arylamidase, serine arylamidase and naphtholAS-BI-phosphohydrolase. All are negative for production of β-galactosidase-6-phosphate, α-glucosidase, α-arabinosidase, βglucuronidase, α-fucosidase, reduction of nitrates, indole from L-tryptophan, leucyl-glycine arylamidase, pyroglutamic acid arylamidase, glutamyl glutamic acid arylamidase, esterase lipase (C8), lipase (C14), cystine arylamidase, trypsin, αchymotrypsin, α-mannosidase, gelatin hydrolysis, catalase and oxidase. +, positive reaction; w, weakly positive reaction; -, negative reaction. Data are from this study.
402 16
403
Table 2. Cellular fatty acid profiles of the L. bombi strains. Relative concentrations (%; w/v)
404
of fatty acids were calculated. Data are from this study.
405 Bacterial strains / Fatty acid
BTLCH M1/2T
BTLCH M3/2
M250 3MRA
Summed C19:1 w6c / cyclo C19:0 w10c/19w6
37.1
29.9
27.8
C18:1 w9c
35.6
33.3
29.4
C16:0
13.2
18.3
20.5
C18:1 w7c
4.3
3.8
5.5
C14: 0
4.1
5.8
6.2
Summed C16:1 w7c / iso C15 2OH
2.5
2.3
1.3
C18:0
1.8
< 0.1
1.7
C17:1 w7c
0.6
1.5
0.9
C15:0
0.5
< 0.1
< 0.1
406 407
17
408
Fig. 1. Unrooted phylogenetic tree of species of the genus Lactobacillus that occur in the
409
digestive tract of humans and animals, showing the postition of strains representing
410
Lactobacillus bombi sp. nov. They occur in a separate cluster together with uncharacterized
411
lactobacilli originating from digestive tract of honeybees. The tree was reconstructed by the
412
maximum-likelihood method based on 16S rRNA gene sequences (lenght of 1336
413
nucleotides) using MEGA version 5.05 software and the Jukes-Cantor model. Bootstap
414
values, expressed as percentages of 1000 datasets, are given et nodes. Numbers in parentheses
415
correspond to the GenBank accession numbers. Bar, 0.01 substitutions per nucleotide
416
position.
417 418 419
Fig. 2. Profile of cellular polar lipids detected in the strain Lactobacillus bombi BTLCH
420
M1/2 . DPG = Diphosphatidylglycerol, PG = Phosphatidylglycerol, PL = Phospholipid, GL =
421
Glycolipid, PGL = Phosphoglycolipids.
T
422
18
Fig. 1. Unrooted phylogenetic tree of species of the genus Lactobacillus that occur in the digestive tract of humans and animals, showing the postition of strains representing Lactobacillus bombi sp. nov. They occur in a separate cluster together with uncharacterized lactobacilli originating from digestive tract of honeybees. The tree was reconstructed by the maximum-likelihood method based on 16S rRNA gene sequences (lenght of 1336 nucleotides) using MEGA version 5.05 software and the Jukes-Cantor model. Bootstap values, expressed as percentages of 1000 datasets, are given et nodes. Numbers in parentheses correspond to the GenBank accession numbers. Bar, 0.01 substitutions per nucleotide position.
Fig. 2. Profile of cellular polar lipids detected in the strain Lactobacillus bombi BTLCH M1/2T. DPG = Diphosphatidylglycerol, PG = Phosphatidylglycerol, PL = Phospholipid, GL = Glycolipid, PGL = Phosphoglycolipids.
Lactobacillus bombi sp. nov., from the digestive tract of laboratory-reared bumblebee queens (Bombus terrestris)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
J. Killer 1,2*, A. Votavová 3, I. Valterová 4, E. Vlková 2, V. Rada 2, Z. Hroncová 2 1
Institute of Animal Physiology and Genetics v.v.i., Academy of Sciences of the Czech Republic, Vídeňská 1083, Prague 4 – Krč, 142 20, Czech Republic 2 Czech University of Life Sciences, Faculty of Agrobiology, Food and Natural Resources, Department of Microbiology, Nutrition and Dietetics, Kamýcká 129, Prague 6 – Suchdol, 165 21, Czech Republic 3 Agricultural Research, Ltd., Zahradní 400/1, Troubsko, Czech Republic 4 Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 10 Prague 6, Czech Republic. *Corresponding author. Tel.: +420 267 090 508; fax: +420 267 090 500. E-mail address: [email protected]; [email protected] Running title: Lactobacillus bombi sp. nov.; Subject category: New taxa-Firmicutes
19 20 21 22 23 24 25 26 27 28
The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequences of strains BTLCH M1/2T, BTLCH M3/2 and M250 3MRA are KJ078643, KJ078644 and KJ078645; those for the partial rpoA and tuf gene sequences of the same strains are KJ144262, KJ144263, KJ144264, KJ144265, KJ144266 and KJ144267. The GenBank/EMBL/DDBJ accession numbers for the partial hsp60 and pheS gene sequences determined in the BTLCH M1/2T and M250 3MRA are KC294225, KJ144258, KC294226 and KJ144260. The GenBank/EMBL/DDBJ accession number for the partial hsp60 gene sequence of Lactobacillus tucceti DSM 20183T is KJ144259.
29
(Multilocus Sequence Analysis)
30 31
Three bacterial strains belonging to the genus Lactobacillus were isolated from the digestive
32
tract of laboratory-reared bumblebee queens (Bombus terrestris) using MRS agar under
33
anaerobic conditions. The isolates were identified according to 16S rRNA gene sequences as
34
yet undescribed Lactobacillus sp. with the highest 16S rRNA gene similarity (96.9 %) to
35
uncharacterized bacterial strain Lactobacillus sp. Mboho2r2 isolated from the stomach of a
36
European honeybee (Apis mellifera). Lactobacillus tucceti was found to be the closest relative
37
valid species with 92.9 % 16S rRNA gene sequence similarity. However, phylogenetic
Keywords: bumblebee, Lactobacillus sp. nov., Bombus terrestris, digestive tract, MLSA
1
38
analyses based on different markers revealed that the species is phylogenetically very distant
39
from the new strains. The DNA G + C content of the type strain is 37.8 mol%. Fatty acids
40
such as C19:1
41
Presence of diphosphatidylglycerol, phosphatidylglycerol, a phospholipid, seven glycolipids
42
and two phosphoglycolipids was detected in cells. Growth at 47 oC was found. The
43
peptidoglycan type A4α L-Lys-D-Asp was determined for type strain BTLCH M1/2T.
44
Genotypic characteristics and phylogenetic analyses based on phylogenetic markers such as
45
hsp60, pheS, rpoA and tuf genes, as well as results of phenotypic characteristics and
46
chemotaxonomic analyses confirmed that new isolates belong to a new Lactobacillus species.
47
The name Lactobacillus bombi sp. nov. was proposed for group of new isolates. The type
48
strain is BTLCH M1/2T (=DSM 26517T = CCM 8440T).
w6c
/ cyclo C19:0
w10c/19w6,
C18:1
w9c
and C16:0 were predominant in all strains.
49 50
Representatives of the genus Lactobacillus are phylogenetically classified in the order
51
Lactobacillales within the bacterial phylum Firmicutes. Lactobacilli are found primarily in
52
environments that are rich source of carbohydrates and other organic substrates. Many species
53
of lactobacilli are obligate bacterial symbionts of the digestive tract of mammals (Hammes &
54
Hertel, 2009). Digestive tract of insects is an environment that is inhabited by a wide variety
55
of bacterial symbionts, many of which are among the yet undescribed species (Engel et al.,
56
2012; Colman et al., 2012). Some researchers believe that the bacterial symbionts of insects,
57
especially lactic acid bacteria, can to some extent positively influence the immune system and
58
host health (Evans & Armstrong, 2006; Forsgren et al., 2010; Koch & Schmid-Hempel, 2012;
59
Mattila et al., 2012). Many studies have been recently published on the prevalence of
60
lactobacilli and other lactic acid bacteria in the digestive tract of insects, especially in the
61
digestive tract of important pollinators (Mohr & Tebbe, 2006; Killer et al., 2010b; Tajabadi et
62
al., 2011; Martinson et al., 2012; Tang et al., 2012; Killer et al., 2014a). Some authors have
2
63
shown that in the stomach of honeybees appear new, probably host-specific species of
64
lactobacilli (Olofsson & Vásquez, 2008; Forsgren et al., 2010). Bacteria inhabiting the
65
digestive tract of bumblebees are not sufficiently explored (Killer et al., 2010b; Koch &
66
Schmid-Hempel, 2011). Studies on the isolation and characterization of bacteria present in the
67
digestive tract of bumblebees have so far focused only on the representatives of the family
68
Bifidobacteriaceae. Three species of bifidobacteria and a new genus within the family
69
Bifidobacteriaceae were recently discovered in the digestive tract of bumblebees (Killer et al.,
70
2009; Killer et al., 2010a; Killer et al., 2011).
71
Isolation and detailed characterization of a new representative of the genus Lactobacillus
72
occurring in the digestive tract of bumblebees is presented in this study. Based on results of
73
genotypic, phylogenetic and phenotypic analyses, it was concluded that group of three
74
bacterial strains represent a new Lactobacillus species.
75
Bumblebees of Bombus terrestris species were bred in the laboratory of the Agricultural
76
Research, Ltd. (Troubsko, Czech Republic) in the spring of 2012. Bumblebees were kept in
77
wooden hives at 26 oC and 50% RH (Relative Humidity). They were fed by fresh frozen
78
honeybee pollen pellets (mix of pollen with dominance of Brassicacae, Rosaceae and Papaver
79
from a local beekeeper) and sugar solution: saccharose (1000 g, white beet sugar, local
80
producer) and fructose (460 g, Fructopur, Natura, Czech Republic) dissolved in water
81
(1200g). Living bumblebees were transported to the Laboratory of anaerobic microbiology
82
(Institute of Animal Physiology and Genetics in Prague, The Academy of Sciences of the
83
Czech Republic) and immediately killed by decapitation. Fresh digestive tracts of three
84
queens originated from different nests were then placed in sealed tubes containing anaerobic
85
MRS Broth (Oxoid, UK) and serially diluted in the same medium. Diluted samples were
86
cultivated on MRS agar under anaerobic conditions (Anaerobic jars, Oxoid) at 37 oC for 48
87
hours. Genes encoding 16S rRNA were amplified in bacterial isolates originated from the 10-6
3
88
diluted samples using primers 616V and 630R under conditions as described by Ehrmann et
89
al. (2003). Three bacterial isolates originated from three bumblebee queens were then
90
identified according to similarities (Killer et al., 2011) of almost complete 16S rRNA gene
91
sequences (1490 bp).
92
The three new isolates designed as BTLCH M1/2T, BTLCH M3/2 and M250 3MRA were
93
phylogenetically most closely related to unclassified bacterial strains Lactobacillus sp.
94
Mboho2r2 (GenBank a. n. HM534813) and Lactobacillus sp. AcjLac3 (GenBank a. n.
95
AB810024) isolated from the digestive tract of European (Apis mellifera) and Japanese (Apis
96
cerana japonica) honeybees, respectively. These new isolates were found to share 96.7-96.9
97
% and 96.0–96.2 % 16S rRNA gene similarities with the mentioned unclassified isolates from
98
the digestive tract of honeybees. The jPHYDIT software (Jeon et al., 2005) was used for
99
calculation of sequence similarities. Lactobacillus tucceti CECT 5920T (GenBank a. n.
100
NR_042194) was found to be the closest relative valid species with 92.9 % 16S rRNA gene
101
sequence similarity. Three new bacterial isolates had high 99.7 % gene similarity among each
102
other. These results suggest that the new bacterial isolates belong to the same bacterial
103
phylotype probably representing a new bacterial species of the genus Lactobacillus
104
(Stackebrandt & Ebers, 2006). However, MLST (Multilocus Sequence Typing) method,
105
phylogenetic, phenotypic and chemotaxonomic analysis were then used to confirm this
106
assumption.
107
Genes encoding the heat shock protein 60 kDa (hsp60), phenyl-alanyl t-RNA synthase alpha
108
subunit (pheS), RNA polymerase alpha subunit (rpoA) and translation elongation factor Tu
109
(tuf) were sequenced in three strains as the additional phylogenetic markers. Detailed
110
information about the primers and PCR parameters for amplification of these genes were
111
previously published by Goh et al. (2000), Dobson et al. (2002), Naser et al. (2005, 2007) and
112
Ventura et al. (2003). Amplified fragments were subsequently checked by electrophoresis on
4
113
1.5 % PCR agarose gel (Top-Bio, Czech Republic), purified using PCR purification kit
114
(Qiagen) and sequenced by automatic genetic analyser ABI PRISM 3130xl (Applied
115
Biosystems). Defined sequences of phylogenetic markers were compared with sequences of
116
type bacterial strains of closest related species based on the scan results in gene database
117
through the BLAST (Basic Local Alignment Search Tool) program. Results of hsp60, pheS,
118
rpoA and tuf gene sequence similarity testing revealed closest relatives sequences of L. tucceti
119
DSM 20183T (81.6–82.3 %; GenBank accession number KJ144259), L. nagelii LMG 21593T
120
(78.7–79.2 %; AM087708), L. curvatus LMG 9198T (73.3–73.6 %; AM087783) and L.
121
crustorum LMG 23699T (83.2–83.6 %; FN395011), respectively. Authors, who designed the
122
above phylogenetic markers indicate much higher values of inter-species sequence similarity
123
(Goh et al., 2000; Naser et al., 2007). Identical hsp60 and pheS gene sequences were found in
124
BTLCH M1/2T and BTLCH M3/2 strains. For this reason, only the BTLCH M1/2T and M250
125
3MRA bacterial strains were further implied in phylogenetic analyses based on these
126
phylogenetic markers. Similarities of hsp60, pheS, rpoA and tuf gene sequences among three
127
strains were 99.0, 98.6, 99.4–99.7 and 99.5–100 %, respectively. These results suggest that
128
new strains represent a new species within the genus Lactobacillus. Phylogenetic analyses
129
subsequently confirmed this assumption.
130
Similar procedure that has been described recently (Killer et al., 2013) was used for
131
construction of phylogenetic trees based on partial 16S rRNA, hsp60, pheS, rpoA and tuf gene
132
sequences. Phylogenetic trees were constructed based on sequences of type strains of
133
Lactobacillus species by maximum-likelihood algorithm using the MEGA 5.05 program
134
(Tamura et al., 2011) and the Jukes-Cantor model. Topology of trees was checked also by
135
neighbour-joining and maximum-parsimony algorithms. Alignments provided by CLUSTAL
136
W algorithm were improved by removing hypervariable positions using the program Gblocks
137
(Castresana, 2000). Phylogenetic tree constructed based on 16S rRNA gene sequences of
5
138
Lactobacillus species present in the digestive tract of animals revealed that three strains
139
BTLCH M1/2T, BTLCH M3/2 and M250 3MRA are situated together with uncharacterized
140
Lactobacillus strains from the digestive tract of honeybees in a separate phylogenetic cluster
141
(Fig. 1). These lactobacilli from the digestive tract of pollinators represent a new phylogenetic
142
lineage within the genus Lactobacillus. Thus, new lactobacilli are phylogenetically distant to
143
the L. alimentarius cluster (Chenoll et al., 2006) including the species L. tucceti, the most
144
closely valid taxon based on 16S rRNA gene sequence similarity. Significant differences
145
between this species and isolates from the digestive tract of bumblebees was further
146
demonstrated by biochemical characteristics. For these reasons, type strain of L. tucceti was
147
not used for comparative analyses of other phenotypic characteristics.
148
Phylogenetic trees reconstructed using hsp60, pheS, rpoA and tuf gene sequences confirmed
149
that three studied strains can be classified as a new Lactobacillus taxon. They are positioned
150
on separate phylogenetic branches among Lactobacillus species (Fig. S1-4). However,
151
topology of the trees did not match to that obtained on the basis of 16S rRNA gene sequences.
152
It is due to shorter gene fragments used and absence of relevant gene sequences for
153
unclassified probably new taxa of lactobacilli from the digestive tract of pollinators.
154
The PCR–DGGE (Denaturing Gradient Gel Electrophoresis) has been chosen as a tool to
155
demonstrate the presence of the described new Lactobacillus species in the digestive tract of
156
laboratory-reared bumblebee queens (Bombus terrestris) originated from four different
157
localities in Moravia (Czech Republic). Total bacterial DNA from the digestive tract of
158
bumblebee queens was isolated and analysed exactly as described previously (Killer et al.,
159
2014). Amplified fragments (200 bp) of 16S rRNA gene belonging to the L. bombi BTLCH
160
M1/2T (99-100% sequence similarities) strain were observed in all samples (Fig. S5).
161
The modified enzymatic degradation method (Killer et al., 2011) was used for determination
162
of the DNA G + C contents in strain BTLCH M1/2T, BTLCH M3/2 and M250 3MRA strains.
6
163
The obtained values 37.8 (mean of three experiments, SD = 0.4), 37.2 (SD = 0.1) and 38.0
164
(SD = 0.6) mol% are in the interval of values (32-55 mol%) defined for different species of
165
lactobacilli (Hammes & Hertel, 2009).
166
API 50 CHL, Rapid ID 32A and API ZYM commercial kits (all bioMérieux, France) were
167
applied for determination of biochemical characteristics in three tested strains and L. tucceti
168
DSM 20183T. Tests were performed according to the manufacturer's instructions, except that
169
the API 50 CHL test strips were incubated under anaerobic conditions (Anaerobic jars,
170
Oxoid) at 37 oC for 48 hrs. Bacterial strains were also tested for oxidase activity (Lui &
171
Jurtshuk, 1986) and hydrolysis of gelatine by API 20E system (bioMérieux, France).
172
Physiological properties such as the ability to grow in the range of different temperatures, pH
173
values and environments with varying oxygen tension were determined by the methods
174
described previously (Killer et al., 2013). Production of D- and L-lactic acid by the D-/L-
175
lactic acid kit (Megazyme, Ireland) was also tested. Durham tube in MRS broth was used for
176
testing of gas production from glucose. Tested strains differed in utilization of eighteen
177
substrates and production of ten enzymes (Table 1). L. tucceti DSM 20183T had a very
178
different substrate utilization and enzyme activity pattern in comparison to three new strains
179
what is in agreement with phylogenetic distance (Fig. 1). Analysed Lactobacillus strains from
180
the digestive tract of bumblebees differed among themselves in utilization of L-arabinose, D-
181
galactose, L-rhamnose, cellobiose and production of urease, arginine dihydrolase, alkaline
182
phosphatase, acid phosphatase, tyrosine arylamidase and alanine arylamidase. Substrate
183
utilization and enzyme activity patterns tested by the API 50 CHL and Rapid ID 32A kits did
184
not reveal any similarity to profiles of Lactobacillus species deposited at Apiweb database
185
(https://apiweb.biomerieux.com/servlet/Authenticate). D,L-lactic acids were produced by
186
cells of all analysed bacterial strains. No gas production from glucose was found. Growth at
187
the temperature from 20 to 47 oC and at the pH from 4 to 8.5 ranges was observed in new
7
188
isolates from the digestive tract of bumblebees. Growth at higher temperatures than 45 oC was
189
detected only in some species of lactobacilli (Pedersen et al., 2004; Hammes & Hertel, 2009).
190
The best growth at strictly anaerobic conditions on TPY (Scardovi, 1986) and MRS agar was
191
found. Nevertheless, poor growth was observed also in microaerophilic conditions.
192
Determination of the end products of hexose catabolism in the strain BTLCH M1/2T was
193
performed using capillary isotachophoresis (Killer et al., 2011). Lactic, acetic and propionic
194
acids were determined in concentration of 85.4 mmol l-1 (65% of all short-chain fatty acids
195
produced), 32.7 mmol l-1 (25%) and 13.2 mmol l-1 (10%), respectively. These results, along
196
with the ability to utilize some pentoses, suggest that the novel strain belongs to the
197
facultatively heterofermentative lactobacilli (group B according to Hammes & Hertel, 2009).
198
Cellular fatty acids profiles were determined in three strains representing new Lactobacillus
199
taxon using methods described by Kämpfer & Kroppenstedt (1996) and Miller (1982).
200
Summed C19:1 w6c / cyclo C19:0 w10c/19w6; C18:1 w9c and C16:0 acids were detected as the major
201
fatty acids in cells (Table 2). These fatty acids have been previously identified as the main in
202
lactobacilli (Gomez Zavaglia et al., 2000).
203
Profile of cellular polar lipids and structure of peptidoglycan were determined in the BTLCH
204
M1/2T strain by the Identification Service of the DSMZ by methods described previously
205
(Killer et al., 2010a). Polar lipids detected in the strain were diphosphatidylglycerol,
206
phosphatidylglycerol, a phospholipid, seven glycolipids and two phosphoglycolipids (Fig. 2).
207
Phosphatidylglycerol, phospholipids and glycolipids seem to be widely distributed among
208
different Lactobacillus species (Arbogast & Henderson, 1975; Kim et al., 2011; Killer et al.,
209
2014a; Killer et al., 2014b). On the other hand, occurence of phosphoglycolipids, lipids and
210
phosphatidylethanolamine can differ in cells of different taxa of lactobacilli (Kim et al., 2011;
211
Liang et al., 2011; Killer et al., 2014a; Killer et al., 2014b). Chemical analysis revealed
212
peptidoglycan type A4α L-Lys-D-Asp (type A11.31 according to DSMZ www.peptidoglycan-
8
213
types.info). The molar ratio of the amino acids in the peptidoglycan hydrolysate was as
214
follows: 2.9 Ala : 0.9 Asp : 1.0 Glu : 0.7 Lys. This peptidoglycan structure was determined
215
for most species of lactobacilli and representatives of the order Lactobacillales (Schumann,
216
2011).
217
Provided results allow to classify group of analyzed bacterial strains as a new species within
218
the genus Lactobacillus for which the name Lactobacillus bombi sp. nov. is proposed.
219 220
Description of Lactobacillus bombi sp. nov.
221
Lactobacillus bombi (L. n. bombus a boom, a deep hollow noise, buzzing, also the zoological
222
genus name of the bumblebee; N.L. gen. n. bombi of Bombus, of a bumblebee).
223
Cells growing on soft 0.5% MRS agar under anaerobic conditions are Gram-stain-positive,
224
catalase- and oxidase-negative, regular, more or less curved long rods with rounded ends
225
organized mostly singly and in pairs. They reach 0.7-1.0 µm in width and 2.2.-7.2 µm in
226
length. Best growth was observed in anaerobic TPY and MRS broth or agar, lower growth in
227
microaerophilic conditions on the same agars. Colonies on MRS agar under anaerobic
228
conditions after 72 h are cream in color with sharp edges. Colony morphology is a disc-
229
shaped in profile but also triangular in approximately one third of colonies. Colony size is
230
1.21 to 2.82 mm in diameter. The DNA G + C content of the type strain is 37.8 mol%.
231
Optimum temperature for growth is 37 oC, with a minimum of 20 oC and a maximum of 47 oC.
232
Growth occurs at pH values of 4-8.5. Utilize D-glucose, D-fructose, D-mannose, D-xylose, N-
233
acetylglucosamin, amygdalin, arbutin, esculin, salicin, melibiose, sucrose, trehalose, raffinose
234
and gentiobiose. Variable in utilization of L-arabinose, D-galactose, L-rhamnose and
235
cellobiose. Negative for utilization of glycerol, erythritol, D-arabinose, D-ribose, L-xylose, D-
236
mannitol, D-Adonitol, methyl-βD-xylopyranoside, L-sorbose, dulcitol, inositol, D-sorbitol,
9
237
methyl-αD-mannopyranoside,
methyl-αD-glucopyranoside,
238
melezitose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, L-fucose,
239
D-arabitol, L-arabitol, potassium gluconate, potassium 2-keto-gluconate and potassium 5-
240
keto-gluconate. Produces α-galactosidase, β-galactosidase, β-glucosidase, N-acetyl-β-
241
glucosaminidase, glutamic acid decarboxylase, arginine arylamidase, proline arylamidase,
242
phenylalanine arylamidase, leucine arylamidase, hystidine arylamidase, valine arylamidase,
243
serine arylamidase and naphthol-AS-BI-phosphohydrolase. Negative for β-galactosidase-6-
244
phosphate, α-glucosidase, α-arabinosidase, β-glucuronidase, α-fucosidase, reduction of
245
nitrates, production indole from L-tryptophan, leucyl-glycine arylamidase, pyroglutamic acid
246
arylamidase, glutamyl glutamic acid arylamidase, esterase lipase (C8), lipase (C14), cystine
247
arylamidase, trypsin, α-chymotrypsin, α-mannosidase, gelatin hydrolysis, catalase and oxidase.
248
Variable in production of urease, arginine dihyrolase, alkaline phosphatase, acid phosphatase,
249
tyrosine arylamidase and alanine arylamidase. The determined peptidoglycan structure type is
250
A4α L-Lys-D-Asp. Major fatty acids in cells are summed C19:1 w6c / cyclo C19:0 w10c/19w6; C18:1
251
w9c
252
diphosphatidylglycerol, phosphatidylglycerol, a phospholipid, seven glycolipids and two
253
phosphoglycolipids.
254
The type strain, BTLCH M1/2T (=DSM 26517T = CCM 8440T) was isolated from the
255
digestive tract of bumblebee queen (Bombus terrestris) laboratory-reared at the Agricultural
256
Research, Ltd. (Troubsko, Czech Republic) in 2012. Additional strains of the species are
257
BTLCH M3/2 and M250 3MRA.
lactose,
maltose,
inulin,
and C16:0, respectively. Profile of cell polar lipids revealed the presence of
258 259
Acknowledgments
260
This study was primarily supported by the Technological Agency of the Czech Republic
261
(project No. TA01020969) and by institutional funding on long-term conceptual development
10
262
of research organization, then by the projects No. 20132023 and No. 20132013 of the Internal
263
Grant Agency of the Czech University of Life Sciences Prague and by the Czech National
264
Agency for Agricultural Research (NAZV QJ 1210047).
265 266 267
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382
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383
15
384
Table 1. Differences in biochemical characteristics among strains representing a new species
385
within the genus Lactobacillus and their related valid species based on 16S rRNA gene
386
similarity. Taxa: 1, L. bombi BTLCH M1/2T; 2, L. bombi BTLCH M3/2; 3, L. bombi M250
387
3MRA; 4, L. tucceti DSM 20183T.
388 389 Characteristic Utilization of : L-Arabinose D-Xylose D-Galactose L-Rhamnose D-Mannitol N-Acetylglucosamine Cellobiose Amygdalin Arbutin Esculin Salicin Maltose Melibiose Sucrose Trehalose Raffinose Gentiobiose L-Fucose Production of : Urease Arginine dihydrolase α-Galactosidase β-Galactosidase N-Acetyl-β-glucosaminidase Alkaline phosphatase Acid phosphatase Tyrosine arylamidase Alanine arylamidase Glycine arylamidase
390 391 392 393 394 395 396 397 398 399 400 401
1
2
3
4
+ + w w + + + + + + + w + + -
+ + + + + + + + + + + + + -
+ + w w + + + + + + + + + -
+ + + + +
+ + + + w w -
+ + + w + + -
+ + + + + w w -
+ +
Notes: All strains utilize D-glucose, D-fructose, D-mannose. None produce acids from glycerol, erythritol, D-arabinose, Dribose,
L-xylose,
D-Adonitol,
methyl-βD-xylopyranoside,
L-sorbose, dulcitol,
inositol,
D-sorbitol,
methyl-αD-
mannopyranoside, methyl-αD-glucopyranoside, lactose, inulin, melezitose, starch, glycogen, xylitol, D-turanose, D-lyxose, D-tagatose, D-fucose, D-arabitol, L-arabitol, potassium gluconate, potassium 2-keto-gluconate and potassium 5-ketogluconate. All tested strains are able to produce glutamic acid decarboxylase, arginine arylamidase, proline arylamidase, phenylalanine arylamidase, leucine arylamidase, hystidine arylamidase, valine arylamidase, serine arylamidase and naphtholAS-BI-phosphohydrolase. All are negative for production of β-galactosidase-6-phosphate, α-glucosidase, α-arabinosidase, βglucuronidase, α-fucosidase, reduction of nitrates, indole from L-tryptophan, leucyl-glycine arylamidase, pyroglutamic acid arylamidase, glutamyl glutamic acid arylamidase, esterase lipase (C8), lipase (C14), cystine arylamidase, trypsin, αchymotrypsin, α-mannosidase, gelatin hydrolysis, catalase and oxidase. +, positive reaction; w, weakly positive reaction; -, negative reaction. Data are from this study.
402 16
403
Table 2. Cellular fatty acid profiles of the L. bombi strains. Relative concentrations (%; w/v)
404
of fatty acids were calculated. Data are from this study.
405 Bacterial strains / Fatty acid
BTLCH M1/2T
BTLCH M3/2
M250 3MRA
Summed C19:1 w6c / cyclo C19:0 w10c/19w6
37.1
29.9
27.8
C18:1 w9c
35.6
33.3
29.4
C16:0
13.2
18.3
20.5
C18:1 w7c
4.3
3.8
5.5
C14: 0
4.1
5.8
6.2
Summed C16:1 w7c / iso C15 2OH
2.5
2.3
1.3
C18:0
1.8
< 0.1
1.7
C17:1 w7c
0.6
1.5
0.9
C15:0
0.5
< 0.1
< 0.1
406 407
17
408
Fig. 1. Unrooted phylogenetic tree of species of the genus Lactobacillus that occur in the
409
digestive tract of humans and animals, showing the postition of strains representing
410
Lactobacillus bombi sp. nov. They occur in a separate cluster together with uncharacterized
411
lactobacilli originating from digestive tract of honeybees. The tree was reconstructed by the
412
maximum-likelihood method based on 16S rRNA gene sequences (lenght of 1336
413
nucleotides) using MEGA version 5.05 software and the Jukes-Cantor model. Bootstap
414
values, expressed as percentages of 1000 datasets, are given et nodes. Numbers in parentheses
415
correspond to the GenBank accession numbers. Bar, 0.01 substitutions per nucleotide
416
position.
417 418 419
Fig. 2. Profile of cellular polar lipids detected in the strain Lactobacillus bombi BTLCH
420
M1/2 . DPG = Diphosphatidylglycerol, PG = Phosphatidylglycerol, PL = Phospholipid, GL =
421
Glycolipid, PGL = Phosphoglycolipids.
T
422
18
Fig. 1. Unrooted phylogenetic tree of species of the genus Lactobacillus that occur in the digestive tract of humans and animals, showing the postition of strains representing Lactobacillus bombi sp. nov. They occur in a separate cluster together with uncharacterized lactobacilli originating from digestive tract of honeybees. The tree was reconstructed by the maximum-likelihood method based on 16S rRNA gene sequences (lenght of 1336 nucleotides) using MEGA version 5.05 software and the Jukes-Cantor model. Bootstap values, expressed as percentages of 1000 datasets, are given et nodes. Numbers in parentheses correspond to the GenBank accession numbers. Bar, 0.01 substitutions per nucleotide position.
Fig. 2. Profile of cellular polar lipids detected in the strain Lactobacillus bombi BTLCH M1/2T. DPG = Diphosphatidylglycerol, PG = Phosphatidylglycerol, PL = Phospholipid, GL = Glycolipid, PGL = Phosphoglycolipids.
