Gutell 123.app environ micro_2013_79_1803

Published Ahead of Print 4 January 2013.
10.1128/AEM.03681-12.
2013, 79(6):1803. DOI:Appl. Environ. Microbiol.
Katrin Kellner, Robert M. Plowes and Ulrich G. Mueller
Quinn S. McFrederick, Jamie J. Cannone, Robin R. Gutell,
Rather than the Rule
Hymenopteran Hosts Is the Exception
Specificity between Lactobacilli and
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Specificity between Lactobacilli and Hymenopteran Hosts Is the
Exception Rather than the Rule
Quinn S. McFrederick,a
Jamie J. Cannone,b
Robin R. Gutell,b
Katrin Kellner,a
Robert M. Plowes,c
Ulrich G. Muellera
Section of Integrative Biology, University of Texas at Austin, Austin, Texas, USAa
; The Institute for Cellular and Molecular Biology and the Center for Computational Biology
and Bioinformatics, The University of Texas at Austin, Austin, Texas, USAb
; Brackenridge Field Laboratory, University of Texas at Austin, Austin, Texas, USAc
Lactobacilli (Lactobacillales: Lactobacillaceae) are well known for their roles in food fermentation, as probiotics, and in human
health, but they can also be dominant members of the microbiota of some species of Hymenoptera (ants, bees, and wasps).
Honey bees and bumble bees associate with host-specific lactobacilli, and some evidence suggests that these lactobacilli are im-
portant for bee health. Social transmission helps maintain associations between these bees and their respective microbiota. To
determine whether lactobacilli associated with social hymenopteran hosts are generally host specific, we gathered publicly avail-
able Lactobacillus 16S rRNA gene sequences, along with Lactobacillus sequences from 454 pyrosequencing surveys of six other
hymenopteran species (three sweat bees and three ants). We determined the comparative secondary structural models of 16S
rRNA, which allowed us to accurately align the entire 16S rRNA gene, including fast-evolving regions. BLAST searches and maxi-
mum-likelihood phylogenetic reconstructions confirmed that honey and bumble bees have host-specific Lactobacillus associates.
Regardless of colony size or within-colony oral sharing of food (trophallaxis), sweat bees and ants associate with lactobacilli that
are closely related to those found in vertebrate hosts or in diverse environments. Why honey and bumble bees associate with
host-specific lactobacilli while other social Hymenoptera do not remains an open question. Lactobacilli are known to inhibit the
growth of other microbes and can be beneficial whether they are coevolved with their host or are recruited by the host from envi-
ronmental sources through mechanisms of partner choice.
Lactobacillus is the largest genus of lactic acid bacteria (LAB),
containing species that are well known for their roles in food
production and human health (1). Lactobacilli convert sugars to
lactic acid and other acids, and some species are used in food
fermentations to preserve foods, contribute flavor, or inhibit the
growth of other bacteria (2). Other lactobacilli are used as human
probiotics; for example, L. reuteri protects children from rotavirus
gastroenteritis (3). Lactobacillus is of obvious importance to hu-
mans for both economic and health reasons.
Lactobacilli also associate with diverse nonhuman animals,
and there is evidence that some lactobacilli can protect these hosts
from pathogens (4, 5). For example, Lactobacillus reuteri is found
in the gastrointestinal tracts of pigs, rodents, chickens, and hu-
mans and aids in protection of the host from pathogens (6). Lac-
tobacillus plantarum is one of the five dominant bacterial phylo-
types found in Drosophila melanogaster intestinal tracts (7),
promoting larval growth in low-nutrient medium (8). A mixture
of LAB, including Lactobacillus species, help protect honey bee
larvae from Paenibacillus larva and Melissococcus plutonius, the
causative agents of American and European foulbrood, respec-
tively (9, 10). The Lactobacillus phylotypes Firm4 and Firm5, in
conjunction with other members of the bumble bee microbiota,
protect bumble bee workers from the trypanosome pathogen
Crithidia bombi (11, 12). LAB have also been hypothesized to play
a role in fermenting pollen stored by honey bees, thereby protect-
ing the stored bee bread from spoilage (13), although firm evi-
dence for this hypothesis is still lacking.
Several authors suggested recently that the relationships be-
tween honey and bumble bees (corbiculate apids) and their mi-
crobiotae, both of which include related Lactobacillus phylotypes,
have been shaped by coevolutionary processes (10, 14, 15). Our
recent phylogeny (16), based on a short fragment of the 16S rRNA
gene, agreed with suggestions that the Firm3, Firm4, and Firm5
honey bee- and bumble bee-associated Lactobacillus phylotypes
are host specific, which suggests that between-species transmis-
sion of these phylotypes is uncommon or absent (16). Horizontal
transmission has not been excluded, but both honey and bumble
bees can acquire microbes through within-colony social contact
(11, 17). In contrast, solitary and primitively eusocial sweat bees
associate with lactobacilli related to those that occur on flowers
(16). It is currently unknown whether sociality per se or some
unique aspect of corbiculate apid biology maintains the relation-
ship between honey and bumble bees and their host-specific lac-
tobacilli.
Here we explore the host specificity of lactobacilli that associate
with hymenopteran hosts that exhibit a range of social structures.
First, we used very accurate comparative secondary structure
models as templates to create a larger set of 16S rRNA structure
models that represent all of the major forms of structural diversity
present in the primary groups of lactobacilli. Second, we used
these structure models to construct a comprehensive and highly
accurate alignment of full-length or nearly full-length 16S rRNA
gene sequences. To determine whether host specificity is common
in associates of insects that live in large societies or is limited to
associates of corbiculate apids, we constructed two additional
alignments that included 16S-amplicon 454-pyrosequencing sur-
Received 28 November 2012 Accepted 2 January 2013
Published ahead of print 4 January 2013
Address correspondence to Quinn S. McFrederick, quinnmcfrederick@gmail.com.
Supplemental material for this article may be found at http://dx.doi.org/10.1128
/AEM.03681-12.
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
doi:10.1128/AEM.03681-12
March 2013 Volume 79 Number 6 Applied and Environmental Microbiology p. 1803–1812 aem.asm.org 1803

veys of several bee and ant species. We then used these structure-
based alignments to reconstruct the phylogenetic history of the
genus Lactobacillus.
MATERIALS AND METHODS
Full and nearly full-length Lactobacillus 16S rRNA gene sequences. To
obtain a comprehensive representation of this diverse genus, we down-
loaded publicly available, full-length or nearly full-length sequences of the
16S rRNA gene. First, we searched NCBI’s nucleotide database (18) for
complete Lactobacillus 16S rRNA gene sequences. Next, we searched the
Ribosomal Database Project (RDP [19]) for Lactobacillus 16S sequences
longer than 1,200 bases and of good quality, as designated by RDP. To
include sequences from bee-associated lactobacilli, we downloaded asso-
ciated sequences from NCBI PubMed entries of bee-bacterial studies and
additionally searched NCBI’s nucleotide database for bee-associated lac-
tobacilli. To avoid redundant sequences and obtain a more tractable rep-
resentation of the genus, we clustered sequences of Ն97% sequence sim-
ilarity with the program CD-HIT (20). Since this clustering may have
eliminated some sequences from type strains that were represented by
longer sequences that shared Ն97% sequence identity with the type strain
sequence, we obtained the accession numbers of Lactobacillus type speci-
mens from RDP. We then searched our file for the accession numbers of
these type sequences and added missing sequences back into the align-
ment. We additionally searched NCBI taxonomy for Lactobacillus species
and added reference sequences (21), when available, for the species that
were missing in our alignment. Finally, we searched the list of prokaryotic
names with standing in nomenclature (22) to verify that our alignment
did indeed contain representatives from all described lactobacilli. This set
of 16S rRNA sequences included identified, cultured, unidentified, and
uncultured Lactobacillus species. As outgroups, we added sequences from
14 bacterial species from across the Bacilli but with emphasis on the Lac-
tobacillales. To investigate diversity of paralogous gene copies within a
genome, we included all 16S rRNA gene copies from whole-genome se-
quence of five ingroup species and one outgroup species. Our final align-
ment contained 19 outgroup sequences (from 14 species), 158 sequences
from undescribed or unidentified Lactobacillus species, and 224 sequences
from 160 named, but not necessarily published, Lactobacillus species for a
total of 401 sequences.
Partial and complete 16S rRNA gene sequences, including hyme-
nopteran associates. To integrate 454 pyrosequencing data into the align-
ment of complete or nearly complete full-length sequences, we built a
second alignment spanning the length of the 16S rRNA gene. We coded
the five prime and three prime ends of the 454 sequences as missing data.
The effect of missing data on phylogenetic reconstructions is an ongoing
research area with no simple consensus (23, 24). We therefore planned to
interpret the results of the partial and complete 16S alignment with miss-
ing data with caution.
We started with the 401 sequences described above and added se-
quences from three separate 16S rRNA-amplicon 454-pyrosequencing
studies of bacteria associated with Hymenoptera: the sweat bees (Halicti-
dae) Augochlora pura, Halictus ligatus, and Megalopta genalis (16); an at-
tine fungus-growing ant, Mycocepurus smithii (K. Kellner et al., unpub-
lished); and the fire-ants, Solenopsis invicta and Solenopsis geminata (25;
R. M. Plowes et al., unpublished). We previously published the halictid
data (16), which we obtained from one wild nest of A. pura (Virginia), one
laboratory nest of H. ligatus (Virginia), and two wild nests of M. genalis
(Barro Colorado Island, Panama). We also sampled one colony of M.
smithii in the wild in Gamboa, Panama, and then brought the nest into the
laboratory and sampled it again 6 months later. We collected S. invicta and
S. geminata-complex samples in their native range in Argentina and in
their invasive range in Taiwan, Texas, Florida, and California (Plowes et
al., unpublished). These data represent subsets of data sets from previous
or forthcoming publications (see, for example, references 16 and 25). The
three studies used the same 16S rRNA primers: Gray28F 5=-GAGTTTGA
TCNTGGCTCAG and Gray519r 5=-GTNTTACNGCGGCKGCTG,
which span V1-V3 in the secondary structure of 16S rRNA. V1-V3 are
variable regions that have been shown to accurately differentiate closely
related lactobacilli (26). Research and Testing Laboratories (Lubbock,
TX) generated the 454 sequence for the three studies at different times.
The sweat bee- and Mycocepurus-associated reads were 454 sequenced
forward from Gray28f, while the Solenopsis-associated sequences were 454
sequenced reverse from Gray519r (these later sequences were reverse
complemented for our analysis).
We used previously described pipelines to denoise and otherwise qual-
ity check the 454 16S rRNA reads (16, 25). The sweat bee-associated Lac-
tobacillus sequences were depleted of low-quality sequences and chimeras,
denoised, and assigned to phylotype as described in McFrederick et al.
(16). The Mycocepurus- and Solenopsis-associated bacterial sequences
were processed in the program mothur (27). First, we denoised the data
with the shhh.flows command. We then removed any sequences with
mismatches in the primer binding site or the barcode, or homopolymer
runs of over eight bases, and removed primers and barcodes from the
sequences. To remove chimeric sequences, we used the chimera.uchime
command, and deleted the detected chimeras. Next, we ran BLAST
searches against a 16S rRNA database curated by the Medical Biofilm
Research Institute (MBRI; Lubbock, TX). We selected sequences with top
BLAST hits against the MBRI database of at least 90% sequence identity to
Lactobacillus sequences. We chose a minimum of 90% sequence identity
in order to include novel Lactobacillus sequences, while keeping in mind
that any sequences that do not belong in the genus Lactobacillus should fall
outside of our ingroup in our phylogenetic analysis. As a validation of our
BLAST searches, we ran additional BLAST searches of the 454 sequences
against the entire nucleotide collection at NCBI. Next, to maximize the
phylogenetic signal of the short 454 reads, we removed 454 sequences that
were Ͻ350 bases long. We then clustered sequences of 97% or greater
sequence identity with the program CD-HIT (20). After quality control
and clustering, we added 84 sweat bee-associated, 79 Mycocepurus-asso-
ciated, and 56 Solenopsis-associated sequences to our alignment, for a total
of 620 sequences in the entire alignment.
The hymenopteran hosts included here vary widely in both geography
and natural history. Augochlora pura and H. ligatus both live in North
America, but A. pura is a solitary bee that nests in rotten logs (28), whereas
H. ligatus is a primitively eusocial halictid that forms small colonies in the
soil (29). Megalopta genalis is a socially polymorphic, neotropical sweat
bee that builds nests in decaying branches found in the forest understory
(30, 31). Mycocepurus smithii is a neotropical non-leaf-cutting fungus-
growing ant that forms colonies with an average of 77 workers in repro-
ductive nests in Puerto Rico (32) but can also form much larger single
colonies that occupy more than one nest in the Brazilian Amazon (33, 34).
In contrast, S. invicta is originally from South America but has become
widespread in its invasive range and can reach colony sizes of 220,000
individuals (35, 36). Solenopsis geminata variants have been introduced to
many locations, including Taiwan and possibly Florida (H. Axen, unpub-
lished data).
Partial 16S rRNA gene sequences, including hymenopteran associ-
ates. To investigate the phylogenetic utility of the short 454-pyrosequenc-
ing data, we created a third alignment spanning positions 30 to 517 in the
E. coli 16S rRNA gene sequence. We used the entire length of the 454 data
and trimmed the 401 full-length sequences to span only the regions over-
lapping the 454 pyrosequencing reads. By creating three separate align-
ments, we were able to compare results and determine differences in the
phylogenetic reconstructions based on partial versus full length versus a
combination of partial and complete 16S rRNA sequences.
Sequence alignment and RNA secondary structure. We used covari-
ation analysis to identify RNA secondary structure that is common to a set
of sequences that are known to have the same function and higher-order
structure (37). Covariation analyses identify all types of canonical and
noncanonical base pairs with a set of nucleotides that covary with one
another, regardless of their proximity to other structural elements (38,
39). Approximately 97% of the base pairs, including all of the noncanoni-
McFrederick et al.
1804 aem.asm.org Applied and Environmental Microbiology

cal base pairs, pseudoknots, and other irregular structural elements in the
comparative 16S rRNA secondary structure models are in the crystal
structure of the ribosomal subunit (40). The strength of the covariation
for each predicted base pair has been quantified and used as a confidence
rating for each base pair (http://www.rna.ccbb.utexas.edu/SAE/2A/nt
_Frequency/BP/). Given this very high accuracy, as gauged with high-
resolution crystal structures, we are most confident that all or nearly all of
the base pairs in the comparative structure models for the rRNAs from
different organisms are correct, including all of the 16S rRNA secondary
structure models for the genus Lactobacillus (for a complete discussion of
covariation analysis, see File S1 in the supplemental material).
The accuracy of our predicted secondary structure models are directly
associated with the quality of the sequence alignment. Better alignments
facilitate better comparative structure models. Moreover, vice versa, very
accurate comparative structure models are the basis for the juxtaposition
of nucleotides that are components of similar and analogous structural
elements, especially in highly variable regions that can have large variances
in the number of nucleotides with little or no sequence identity. Thus, our
secondary structure-based alignments do not attempt to maximize se-
quence identity a priori. Instead, they attempt to maximize the alignment
of similar structural elements.
After the 16S rRNA sequences were semiautomatically aligned with
the template-based alignment program CRWAlign (41), we refined the
alignment manually with the alignment editor AE2 (developed by T.
Macke, Scripps Research Institute, San Diego, CA [42]). This tool was
developed for Sun Microsystems (Santa Clara, CA) workstations running
the Solaris operating system. The manual alignment process utilizes the
CRWAlign program to identify nucleotides in a column that might not
map to the same locations in the secondary and tertiary structure. A visual
inspection of the alignment determines if these flagged nucleotides should
be realigned. If necessary, this is done manually with the AE2 alignment
editor. For regions of the alignment with high sequence similarity and
minimal variance in the number of nucleotides, the information in the
primary structure is sufficient to align sequences with confidence. In con-
trast, for more variable regions in closely related sequences or between
more distantly related sequences, a high-quality alignment can only be
produced when secondary and/or tertiary structure information is in-
cluded.
Secondary structure diagrams. Secondary structure diagrams were
generated after the first secondary structure model was derived with com-
parative methods. Although the Comparative RNA Web (CRW) site
(http://www.rna.ccbb.utexas.edu/) contains more than 200 16S rRNA
secondary structure diagrams that sample the diversity within the Bacte-
ria, we generated 47 secondary structure diagrams for the present study:
38 from taxa representing all of the major phylogenetic groups of Lacto-
bacillus and nine from related organisms. We generated the secondary
structure diagrams with the interactive secondary structure program
XRNA (written in the C programming language for Sun Microsystems
workstations running the Solaris operating system by B. Weiser and H.
Noller, University of California, Santa Cruz, CA). All structure diagrams
are available as online supplemental files at http://www.rna.ccbb.utexas
.edu/.
Phylogenetic analyses. To reconstruct the phylogenetic history of the
lactobacilli, we conducted separate maximum-likelihood analyses on our
three alignments. We first determined that GTRϩIϩ⌫ was the most ap-
propriate model of sequence evolution for each of our alignments using
the Akaike Information Criterion (AIC) in Modeltest 3.7 (43) and PAUP*
4b10 (44). We then ran 20 independent search replicates on each align-
ment in the program GARLI 2.0 (45), with the genthreshfortopoterm,
stopgen, and stoptime settings set to 10,000,000. We allowed the program
to estimate all parameter values during the runs. To assess branch support
we conducted 100 bootstrap pseudoreplicates on each alignment, using a
genthreshfortopoterm setting of 5,000,000. We used Mesquite (46), to
calculate patristic distances across the entire tree. To calculate divergence,
we used the branch info tool in Mesquite (46) to measure the distance
from the base of monophyletic groups of hymenopteran associates to the
most recent common ancestor with their closest relatives.
Data availability. We deposited the 84 sweat bee-associated, 79 My-
cocepurus-associated, and 56 Solenopsis-associated sequences in the ge-
netic sequence database at the National Center for Biotechnical Informa-
tion (NCBI GenBank accession numbers KC354148 to KC354367).
RESULTS
Phylogenetic reconstructions of our three 16S rRNA gene sequence
alignmentsresultedinsimilarcladestructureforalltrees(Fig.1and2;
see also Fig. S1 and see Table S1 in the supplemental material for
simple sequence statistics for all alignments). The phylogeny based
only on full-length 16S rRNA sequences (Fig. 1) was in nearly com-
pleteagreementwiththephylogenybasedonacombinationofpartial
and complete 16S rRNA sequences (Fig. 2). The phylogeny based on
only partial 16S sequences (see Fig. S1 in the supplemental material),
however, differed in the branching patterns between and within
clades compared to the full-length phylogeny, but the terminal clade
composition remained stable. Sequence alignments and detailed
phylogenetic trees are available at TreeBase (http://purl.org/phylo
/treebase/phylows/study/TB2:S13670), and a detailed version of
Fig.2withbootstrapsupportvaluesandtaxonlabelsisavailableinthe
online supplemental files (see Fig. S2 in the supplemental material).
16S rRNA secondary structural models. We created second-
ary structural models from 16S rRNA gene sequences of 38 lacto-
bacilli and nine other Firmicutes (see File S1 in the supplemental
material). In addition, the extent of primary and secondary struc-
ture conservation for 608 16S rRNA sequences representing the
entire lactobacilli genus was mapped onto a L. acidophilus second-
ary structure diagram (Fig. 3). The first hairpin stem and loop
from the five prime end of the molecule (the V1 region) was the
most variable region in Lactobacillus 16S structure. Our structural
models suggest that next generation sequencing surveys of bacte-
rial communities aimed at elucidating the diversity of Lactobacil-
lus should use primers targeting the V1 region.
Full-length 16S rRNA gene phylogeny. Our maximum-likeli-
hood analysis of full- or nearly full-length 16S rRNA gene se-
quences recovers six major monophyletic clades, which we iden-
tify in accordance with previous studies: L. salivarius, L. delbrueckii
(acidophilus), L. casei, L. buchneri, L. plantarum, and L. reuteri
(Fig. 1) (47–50). The maximum-likelihood bootstrap support val-
ues for these clades varied greatly: the L. salivarius and L. reuteri
clades showed moderate support (82 and 85%, respectively),
while other clades showed weak support at their deepest nodes.
The deeper branches connecting these major clades uniformly
showed little support. The clade containing the greatest number of
taxa and the greatest sequence diversity was the L. delbrueckii
(acidophilus) clade, which was comprised of 160 taxa and covered
a maximum patristic distance of 1.21, out of a global maximum of
1.51 across the entire tree (Table 1).
Partial and complete 16S rRNA gene phylogeny, including
hymenopteran associates. The phylogenetic trees determined
from the analysis of the partial and complete 16S rRNA alignment
and the full-length 16S rRNA alignment were very similar (Fig. 1
and 2). The main difference between the two phylogenies was the
placement of the reuteri clade. The reuteri clade was sister to the
plantarum clade in the full-length phylogeny (Fig. 1), whereas in
the partial and complete phylogeny the members of the reuteri
clade were reconstructed paraphyletic as a series of branching lin-
eages leading to the monophyletic L. delbrueckii (acidophilus)
Rare Specificity between Hymenoptera and Lactobacilli
March 2013 Volume 79 Number 6 aem.asm.org 1805

clade (Fig. 2). Clade memberships and within-clade branching
patterns were very similar between the full-length and the partial
and complete phylogenies, as were the bootstrap support values
(Fig. 1 and 2).
Of the Hymenoptera-associated sequences, only the honey and
bumble bee-associated Firm3 and Firm4 (F3 and F4 sensu [51])
clades were host speciﬁc and highly diverged from other lactoba-
cilli (Fig. 4). Firm5 also formed a monophyletic group, but was not
as diverged from other lactobacilli as Firm3 and Firm4 (Fig. 2 and
4). In addition, two sequences from Solenopsis clustered within the
Firm5 clade. In contrast to the Firm3-Firm5 clades, most Hy-
menoptera-associated sequences were scattered throughout the
genus and either clustered with or were closely related to lactoba-
cilli known from vertebrate hosts or from diverse environmental
sources (Fig. 2; see also Fig. S2 in the supplemental material). Most
reads from the pyrosequencing surveys, however, tended to clus-
ter into several operational taxonomic units (OTUs) per host
(Table 2). The most abundant Lactobacillus OTUs associated with
Solenopsis, for example, were closely related to L. sakei and L. plan-
tarum, both of which are used in food fermentation (2). The most
abundant M. smithii-associated OTUs were closely related to sev-
eral lactobacilli that are common inhabitants of the intestinal
tracts of rodents, birds, and humans (Table 2). These M. smithii-
associated OTUs were found in both fungus-garden and worker
samples (see Table S2 in the supplemental material). As already
reported in McFrederick et al. (16), halictids associated mainly
with Lactobacillus OTUs that were related to ﬂower-inhabiting,
fructophilic lactobacilli (52) or to a clade that includes
FIG 1 16S rRNA gene maximum-likelihood phylogeny of the genus Lactobacillus, based on full or nearly full-length sequences only. Branch widths are
proportional to bootstrap support from 100 pseudoreplicates. Highlighted branches represent Lactobacillus associates of honey or bumble bees. Major clades are
indicated with curved lines and named according to previous studies (47–50).
McFrederick et al.

flower-inhabiting and fructophilic lactobacilli (53) and lactoba-
cilli used in sourdough fermentation (54). Some of the halictid-
associated OTUs formed monophyletic clades, but these clades
did not exhibit the same level of divergence as the Firm3 and
Firm4 clades (Fig. 2 and 4). Many of the terminal branches that
included Hymenoptera-associated sequences exhibited high
bootstrap support (see Fig. S2 in the supplemental material).
Partial 16S rRNA gene phylogeny, including Hymenoptera
associates. The deeper branching patterns in the partial 16S phy-
logeny did not agree with the full-length phylogeny. For example,
the partial 16S rRNA analysis recovered the L. salivarius clade as a
paraphyletic series of branching lineages leading to the monophy-
letic L. delbrueckii (acidophilus) clade (see Fig. S1 in the supple-
mental material). The L. plantarum and L. buchneri clades were
also no longer monophyletic in the partial phylogeny but instead
were both broken into two separate clades. The L. reuteri and L.
casei clades remained monophyletic in the partial 16S analysis.
Although the partial 16S analysis was unable to resolve deeper
branches in the Lactobacillus phylogeny, the membership of the
terminal clades was stable across all analyses, and many of the
FIG 2 16S rRNA gene maximum-likelihood tree of the genus Lactobacillus, based on partial and complete sequences combined. Branch widths are proportional
to bootstrap support from 100 pseudoreplicates. Highlighted branches represent Hymenoptera associated lactobacilli as indicated in the figure legend. Clades
containing lactobacilli associated with humans are indicated by a human figure, whereas clades containing lactobacilli used in food fermentation are indicated
by a piece of cheese. Major clades are indicated with curved lines and named according to previous studies (47–50).

FIG 3 Secondary structure of the 16S rRNA molecule in the genus Lactobacillus. The degree of conservation in the molecule as determined by analysis of
608 sequences is superimposed on the structure of the Lactobacillus acidophilus 16S molecule. Conservation of each nucleotide across the 608 analyzed
Lactobacillus species is indicated as follows: uppercase letters (Ͼ98% conservation), lowercase letters (90 to 98% conservation), closed circles (80 to 90%
conservation), or open circles (Ͻ80% conservation). Canonical nucleotide pairs (G-C or A-U) are represented by dashes, wobble nucleotide pairs (G-U)
are represented by small closed circles, A-G nucleotide pairs are represented by large open circles, and all other pairs are represented by large closed circles.
McFrederick et al.

terminal groups received high bootstrap support even in the par-
tial 16S analysis (see Fig. S1 in the supplemental material).
DISCUSSION
Host specificity of Hymenoptera-associated lactobacilli. Hyme-
noptera ranging from a solitary species (A. pura) to species that
live in colonies numbering in the hundreds of thousands (Sole-
nopsis) associate with lactobacilli that are either found in other
hosts or the environment. Social structure by itself does not deter-
mine whether hymenopteran hosts associate with lactobacilli that
are host-specific or that are more recent acquisitions from the
environment. Honey bees (colony sizes up to 60,000 [55]) and
bumble bees (colony sizes from 50 to Ͼ400 [55]) are the only
hymenopteran hosts examined to date that associate with lacto-
bacilli that appear to be host specific and highly diverged from
other lactobacilli.
Previous studies already suggested that the corbiculate apids
associate with host-specific lactobacilli (see, for example, refer-
ences 14, 15, and 56), but it remains unclear why honey and bum-
ble bees are special in this regard. The phylotypes associated with
Apis mellifera form a more consistent association with their hosts
compared to Bombus, and this may be related to how A. mellifera
founds new colonies (15, 56). Apis mellifera colonies are founded
via swarming, where a colony divides and approximately half of
the workers leave with the old queen to form a new colony,
whereas most ants, social wasps, and bumble bees are nonswarm-
ing (see reference 57 and references therein). The founding of
colonies by thousands of individuals may allow for the between-
generation maintenance of multiple strains that have been identi-
fied within A. mellifera-associated phylotypes (56). Why Bombus
species, in which nests are always founded by single queens (58),
maintain associations with host-specific bacteria remains to be
determined in detailed studies of dispersing Bombus queens, as
well as the changes in associated microbial communities during
colony founding. Bombus terrestris, the bumble bee whose micro-
biota has been best studied (11, 12, 59), shares nectar via honey-
pots inside the colony (60). Bombus species do not engage in oral-
oral food exchange (trophallaxis) (61), so shared food stores (e.g.,
honeypots), social contact (e.g., grooming), feces, or the nest en-
vironment may serve as important means for social transmission
of bumble bee microbiota. In contrast, trophallaxis between nest-
mates or other contact within the hive is known to be important
for the establishment of the microbiota of A. mellifera (17). Mega-
lopta and Solenopsis also share food within a colony via trophallaxis
(31,62)butdonotharborhost-specificmicrobes.Trophallaxisalone,
therefore, does not automatically lead to the maintenance of host-
specific microbes.
Our phylogenetic analyses and BLAST searches suggest likely
mechanisms regarding how environmental lactobacilli are re-
cruited into association with sweat bees and ants. As we previously
reported, sweat bees associate with lactobacilli that are related to
lactobacilli isolated from flowers, and may therefore obtain these
bacteria from flowers (16). Mycocepurus smithii belongs to the
group of attine ants that do not collect leaves to sustain growth of
their fungus gardens, but instead collect insect frass, flower parts,
seeds, and fruit-flesh (63). Two of the dominant Lactobacillus
OTUs associated with M. smithii (L. johnsonii and L. crispatus)
have been isolated from A. mellifera guts (64), indicating that these
lactobacilli may occur in the guts and frass of other insects. Myco-
cepurus smithii colonies may therefore recruit L. johnsonii, L.
crispatus, and L. salivarius from insect frass that they collect to
sustain their fungus gardens. These lactobacilli are found in both
M. smithii fungus gardens and workers, indicating that they can
occupy broad ecological niches. Alternatively, M. smithii may be
collecting fecal material of vertebrates such as mice, birds, or pigs,
all of which are hosts to the lactobacilli associated with M. smithii.
Solenopsis, on the other hand, is omnivorous (65) and may be
obtaining lactobacilli from plant, insect, or vertebrate food
sources. The most abundant Lactobacillus in Solenopsis was most
closely related to an undescribed Lactobacillus species from fer-
mented tea. This OTU was found to associate with S. invicta and S.
geminata in Argentina, North America, and Taiwan, indicating
that it might play an important role in the biology of Solenopsis.
Lactobacillus plantarum, whose comparatively large genome may
allow it to inhabit a variety of environments (66), was also found
to associate with S. invicta and S. geminata in Argentina, North
America, and Taiwan. Lactobacillus plantarum is found in dairy,
meat, plants, and the human gastrointestinal tract and may be
recruited by Solenopsis from any of these sources. It is currently
unknown whether these associations derived from an acquisition
by a Solenopsis lineage ancestral to the invasive Solenopsis lineages
TABLE 1 Maximum patristic distances of the major clades in the most
likely full-length 16S phylogenetic tree, out of a maximum patristic
distance of 1.51 across the entire tree
Clade Patristic distance
L. salivarius 0.5
L. casei 0.44
L. buchneri 0.54
L. plantarum 0.34
L. reuteri 0.55
L. delbrueckii (acidophilus) 1.21
FIG 4 Histogram of patristic distances from the base of monophyletic clades
of Hymenoptera associates to their most recent common ancestor. The honey
bee-associated Firm3 and Firm4 clades exhibited the greatest divergence, while
the honey bee-associated Firm5 clade exhibited an amount of divergence sim-
ilar to that of some other Hymenoptera associates.

and were then vertically transmitted within these Solenopsis lin-
eages or whether Lactobacillus is recruited continually from the
environment by Solenopsis. Notably, we found several Firm5 se-
quences, which are thought to be specific to honey and bumble
bees (15), associated with Solenopsis. These sequences were not
abundant, suggesting that they may be transient and perhaps
picked up by Solenopsis when scavenging dead A. mellifera or Bom-
bus workers.
The function of lactobacilli in hymenopteran hosts outside of
the honey and bumble bees is still relatively unexplored, but the
available evidence suggests that lactobacilli facilitate digestion of
sugars and inhibit the growth of other microbes through acidifi-
cation, thereby benefiting their hosts. Although 16S rRNA phylo-
type does not translate well into functional phenotypes (67), lac-
tobacilli exhibit some general properties which may serve as
exaptations for the symbiotic habit. For example, lactic acid bac-
teria (LAB) digest a variety of sugars, with the main end product
being lactic acid, although other acids and ethanol are also com-
mon end products (49). By lowering the pH of their environment,
LAB inhibit the growth of many other bacteria (68). LAB are also
known for the secretion of bacteriocins, which are compounds
that inhibit the growth of other bacteria (68). For example, L. sakei
produces several bacteriocins that inhibit pathogenic and spoilage
organisms (69). These general properties of lactobacilli mean that
they are likely to be beneficial to hymenopteran hosts, and further
research should determine the relative importance of acidification
versus bacteriocin-secretion in the biology of ant- and bee-asso-
ciated Lactobacillus.
Utility of the 16S rRNA gene for Lactobacillus identification
and phylogeny. Although our analysis of 16S rRNA did not re-
solve the deeper branches in the Lactobacillus phylogeny, 16S
rRNA did place sequences into terminal clades with confidence.
Clade membership in all of our analyses was generally well sup-
ported and largely agrees with the clade structures suggested in
previous studies (47–50). By using alignments based on full-
length sequences only, partial and complete sequences combined,
and partial sequences only, we were able to assess the influence of
missing data or 454 pyrosequencing length data on phylogenetic
reconstructions. Although the analysis using only partial sequences
performed poorly with regard to the placement of deeper branches,
TABLE 2 Ten most abundant Lactobacillus OTUs (clustered at Ն97% sequence identity) associated with three ant and three bee species (bee species
are binned as halictids)a
Host Source of read No. of reads Closest phylogenetic relative Best BLAST hit
Sequence
identity (%)
Solenopsis Brood geminata TX 5,993 Undescribed L. sakei 92
Brood geminata TW 2,118 L. plantarum L. plantarum 99
Brood invicta TX 935 L. curvatus L. curvatus 99
Brood invicta TX 756 L. brevis L. brevis 99
Pooled invicta TW 661 L. plantarum L. plantarum 99
Pooled invicta TW 324 L. plantarum L. plantarum 99
Pooled geminata TW 261 L. paracasei L. casei 99
Brood geminata TW 167 L. kimchicus L. odoratitofui 99
Brood geminata TW 156 Undescribed L. sakei 93
Brood geminata TW 116 L. vaccinostercus L. vaccinostercus 99
Mycocepurus smithii Garden 14,152 L. johnsonii L. johnsonii 99
Worker 3,748 L. crispatus L. crispatus 99
Garden 2,794 L. salivarius L. salivarius 97
Garden 1,059 L. aviarius L. aviarius 99
Garden 806 L. crispatus L. crispatus 98
Worker 598 L. salivarius L. salivarius 99
Garden 586 L. reuteri L. reuteri 99
Worker 431 L. salivarius L. salivarius 98
Worker 420 L. aviarius/L. johnsonii L. aviarius 90
Garden 408 L. johnsonii L. johnsonii 95
Halictids Larva 20,669 L. kunkeei/L. ozensis L. kunkeei 93
Pollen 15,761 L. fructivorans L. fructivorans 91
Larva 3,923 L. kunkeei/L. ozensis L. kunkeei 93
Pollen 3,631 L. kunkeei/L. ozensis L. kunkeei 94
Pollen 1,557 L. fructivorans L. fructivorans 90
Larva 1,455 L. kunkeei/L. ozensis L. kunkeei 93
Frass 1,102 L. paracasei L. casei 99
Pollen 397 L. kunkeei/L. ozensis L. kunkeei 94
a
The source of read indicates the types of samples from which the OTUs were isolated (TX, Texas; TW, Taiwan; pooled, workers and brood from five colonies). The number of
reads represents the number of reads that cluster into each OTU. The closest phylogenetic relative is the closest Lactobacillus species in our phylogenetic reconstruction with which a
particular read clustered (Fig. 2). The best BLAST hit is the top hit to a named Lactobacillus species from a BLAST search of NCBI’s entire nucleotide collection. The sequence
identity is the percent sequence identity of the query to the best BLAST hit. Several OTUs shared top BLAST hits to the same Lactobacillus species but shared Ͻ97% sequence
identity to each other.
McFrederick et al.

all of our analyses placed sequences into terminal clades with confi-
dence.
Other studies have found that short pyrosequencing data can
be placed accurately into an existing full-length phylogeny (70),
and our results suggest that if a highly variable region can be ac-
curately aligned, alignments built on short pyrosequencing sized
data can accurately place sequences into terminal clades. Our sec-
ondary structural models of 16S rRNA indicate that the hairpin
stem and loop in V1 is the most variable region in Lactobacillus.
Next-generation sequencing surveys targeting V1 should provide
the best taxonomic resolution for closely related lactobacilli.
Conclusions. We created highly accurate comparative models
of 16S rRNA secondary structure and used these models to pre-
cisely align the entire 16S rRNA gene, including fast-evolving re-
gions. Maximum-likelihood phylogenetic reconstructions using
our structurally informed sequence alignments revealed that honey
and bumble bees associate with host-specific Lactobacillus. In con-
trast, regardless of colony size or within-colony oral sharing of
food (trophallaxis), sweat bees and ants associate with lactobacilli
that are closely related to those found in vertebrate hosts or in
diverse environments. Host specificity therefore appears to be the
exception rather than the rule for Hymenoptera-associated lacto-
bacilli. Nest founding by colony fission has been proposed as a
likely means for long-term transmission of microbiota within
honey bee lineages (15). M. smithii may facultatively found nests
by colony fissioning (34), and yet we found that M. smithii does
not associate with host-specific lactobacilli. This suggests that (i)
facultative colony fissioning is not sufficient to maintain long-
term associations between a social-insect lineage and all host-spe-
cific microbiota; (ii) regular colony founding by single queens
may disrupt long-term associations because new microbiota are
frequently acquired by a social-insect lineage during that stage;
and (iii) obligate colony fissioning is more likely than facultative
colony fissioning to maintain long-term host-microbe specifici-
ties in a social insect. Studies of lactobacilli associated with other
obligate colony-fissioning Hymenoptera such as meliponine bees
and related solitary and communal nesters such as euglossine bees
(55) will help determine whether colony-fissioning or some other
characteristic of apid biology promotes host specificity. Regardless
of how they are acquired, lactobacilli are known to inhibit the
growth of other microbes and may be beneficial whether they are
coevolved with their host or are recruited by the host from envi-
ronmental sources through mechanisms of partner choice.
ACKNOWLEDGMENTS
This material is based on work supported by National Science Foundation
grant PRFB-1003133 awarded to Q.S.M. and grant DEB-0919519
awarded to U.G.M., Texas Higher Education Coordinating Board grant
01923 awarded to L. E. Gilbert, and National Institutes of Health grant
GM067317 awarded to R.R.G.
We appreciate technical assistance from D. A. Estrada in the initial
processing of the Solenopsis sequences. We thank three anonymous re-
viewers for their helpful comments.
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McFrederick et al.

Supplemental file S1. Supplemental text for 16S rRNA sequence alignment, secondary
structure models, and Lactobacillus phylogenetics. 16S rRNA secondary structure
models for 38 lactobacilli and 9 other Firmicutes. Canonical nucleotide pairs (G-C or A-
U) are represented by dashes, wobble nucleotide pairs (G-U) are represented by small
closed circles, A-G nucleotide pairs are represented by large open circles, and all other
pairs are represented by large closed circles.
The role of 16S rRNA secondary structure in Lactobacillus phylogenetics
Introduction
The 16S rRNA gene has so far failed to yield robust phylogenies for the genus
Lactobacillus (1), despite an abundance of 16S rDNA sequence information (68,044
sequences, National Center for Biotechnology Information nucleotide database, accessed
14 June 2012). Previous 16S Lactobacillus phylogenies, however, did not utilize the
higher order structure of the 16S RNA molecule to optimize the sequence alignment, an
essential step to reconstruct the best phylogenetic tree (2, 3). Comparative structural
models of the Lactobacillus 16S rRNAs will improve the quality of sequence alignments
and improve phylogenetic reconstructions and the analysis of 16S-based next-generation
sequencing surveys of bacterial communities. Of the nine hypervariable regions of the
16S gene, some windows (e.g.V3-V5, positions ~400- 860 Escherichia coli numbering)
are not sufficient for classification of most human-associated Lactobacillaceae sequences
to the genus level (4). Other 16S regions, such as V1-V2 (positions ~60- 220 E. coli
numbering), may allow for better resolution (5). In addition, better alignments that
explicitly juxtapose analogous secondary structure may also improve taxonomic
assignment of next-generation 16S amplicon sequences. Due to the ubiquitous use of
16S rRNA information for bacterial identification and phylogenetic reconstruction,
knowledge of 16S secondary structure and its utility in DNA sequence alignments is
clearly needed.
Methods
Sequence Alignment and RNA Secondary Structure
Comparative analysis is used to identify an RNA secondary structure that is
common to a set of sequences that are known to have the same function and higher-order
structure. Starting in the early 1980's, covariation analysis, a specific type of
comparative analysis, was applied to a sequence alignment to predict a Bacterial 16S
rRNA secondary structure (6). Initially the simplier covariation methods only identified
canonical base pairs (ie. G:C, A:U, and G:U) that occurred within a regular helix
composed of at least two base pairs that are consecutive and anti-parallel with one
another. However, more sophisticated covariation methods now identify all types of
canonical and non-canonical base pairs with a set of nucleotides that covary with one
another, regardless of their proximity to other structural elements (7, 8). In parallel with
the contribution of more accurate and sensitive covariation methods, the original
secondary structure models were refined as the number and phylogenetic and structural
diversity of 16S rRNA sequences increased. A few of the originally proposed base pairs
were removed when the variation at the two base paired positions were not coordinated
(ie. variation but no covariation) in the larger dataset. In addition, new base pairs were

proposed for the larger dataset when two positions have similar patterns of variation (i.e.
covariation) were identified. While the vast majority of these base pairs were canonical
G:C, A:U, and G:U, a few were non-canonical, including the base pair exchanges - A:A
<> G:G, A:G <> G:A, and U:U <> C:C. The vast majority of the base pairs are arranged
adjacent and anti-parallel with one another to form nested helices, while a few helices
formed pseudoknots and other base pairs were arranged in parallel (3).
In addition to the refinements in the comparative structure models, our confidence
in the proposed comparative structure model were quantified and increased with the
larger datasets that were analyzed with more sophisticated computational methods (9).
With a sufficiently broad sampling of Bacterial 16S rRNAs, strong and robust patterns of
sequence and secondary structure conservation and variation were discovered. Patterns of
structural variation at specific regions of the 16S rRNA were identified and shown to be
diagnostic of the organisms on specific branches of the phylogenetic tree (2, 9, 10)
(www.rna.ccbb.utexas.edu/SAE/2B/ConsStruc/). While the accuracy of regions of the
rRNA comparative structure models were substantiated with site directed mutagenesis,
chemical modifications, and other experiments, the most authoritative assessment of the
accuracy was determined from the high resolution crystal structures of the 30S and 50S
ribosomal subunits (11, 12). Approximately 97% of the base pairs, including all of the
non-canonical base pairs, psuedoknots, and other irregular structural elements in the
comparative 16S rRNA secondary structure models are in the crystal structure (13). The
strength of the covariation for each predicted base pair has been quantified and used as a
confidence rating for each base pair
(http://www.rna.ccbb.utexas.edu/SAE/2A/nt_Frequency/BP/). Given this very high
accuracy, as gauged with the high-resolution crystal structures, we are most confident
that all or nearly all of the base pairs in the comparative structure models for the rRNAs
from different organisms are also correct, including all of the 16S rRNA secondary
structure models for the Lactobacillus.
The accuracy of these predicted secondary structure models are directly
associated with the quality of the sequence alignment. Better alignments facilitate better
comparative structure models. And vice a versa, very accurate comparative structure
models are the basis for the juxtaposition of nucleotides that are components of similar
and analogous structural elements, especially in those highly variable regions that can
have large variances in the number of nucleotides with little or no sequence identity.
Thus these secondary structure-based alignments do not attempt to maximize sequence
identity a priori. Instead they attempt to maximize the alignment of similar structural
elements.
After the 16S rRNA sequences were semi-automatically aligned with the
template-based alignment program - CRWAlign (14). We refined the alignment
manually with the alignment editor ‘‘AE2’’ (developed by T. Macke, Scripps Research
Institute, San Diego, CA; 15). This tool was developed for Sun Microsystems’ (Santa
Clara, CA, USA) workstations running the Solaris operating system.
The manual alignment process utilizes the CRWAlign program to identify
nucleotides in a column that might not map to the same locations in the secondary and
tertiary structure. A visual inspection of the alignment determines if these flagged
nucleotides should be realigned. If necessary, this is done manually with the AE2
alignment editor. For regions of the alignment with high sequence similarity and

minimal variance in the number of nucleotides, the information in the primary structure is
sufficient to align sequences with confidence. In contrast, for more variable regions in
closely related sequences or between more distantly related sequences, a high-quality
alignment can only be produced when secondary and/or tertiary structure information is
included.
Secondary Structure Diagrams
Secondary structure diagrams were generated after the first secondary structure model
was derived with comparative methods. These diagrams were refined as the model was
modified based on increased data and improved covariation methods (see above). While
the Comparative RNA Web (CRW) Site (http://www.rna.ccbb.utexas.edu/) contains more
than 200 16S rRNA secondary structure diagrams that sample the diversity within the
Bacteria, 47 secondary structure diagrams for each of the major phylogenetic groups of
Lactobacillus and a few related organisms were generated for this study. The secondary
structure diagrams were generated with the interactive secondary structure program -
XRNA (written in the C programming language for Sun Microsystems’ workstations
running the Solaris operating system by B. Weiser and H. Noller, University of
California, Santa Cruz). All structure diagrams are available at:
http://www.rna.ccbb.utexas.edu/.
Discussion:
Utility of 16S rDNA for Lactobacillus identification and phylogeny
Our secondary structural models of 16S rRNA indicate that the hairpin stem and
loop in V1 is the most variable region in Lactobacillus. Accordingly, 16S primers
targeting this region for next-generation sequencing surveys of Lactobacillus should
provide the best taxonomic resolution for closely related taxa. Other regions that exhibit
variation are bases 180-200, 440-480, 997-1043, and 1242-1294 (E. coli numbering),
although these regions will not provide the same resolution as V1-V2. The structural
models that we present here will also serve as valuable resources for aligning
Lactobacillus 16S rRNA.
Our 16S rRNA phylogenetic tree had high support values for terminal branching
patterns but not deeper branching patterns in the Lactobacillus phylogeny. Even with our
highly accurate and structurally informed sequence alignment, the 16S rRNA gene did
not confidently resolve the deeper branches in the Lactobacillus phylogeny. The
Lactobacillus phylogeny has so far been reconstructed using both 16S rDNA sequence
information (1, 16-18) or with phylogenomic data, using information from many genes
(1, 16). The phylogenomic approach, while resolving deeper branches with confidence,
results in trees with few taxa. Multilocus sequence typing schemes have been developed
for several lactobacilli and their relatives (19-24), and sequences from these studies may
be useful in designing primers for single copy protein coding genes conserved across the
genus Lactobacillus, thereby providing greater phylogenetic resolution and coverage of
the genus.
While our analysis of 16S rRNA did not resolve the deeper branches in the
Lactobacillus phylogeny, 16S rRNA does place sequences into terminal clades with
confidence. Clade membership in all of our analyses was generally well supported, and
largely agrees with the clade structures suggested in previous studies (16-18). By using

alignments based on full-length sequences only, partial and complete sequences
combined, and partial sequences only, we were able to assess the influence of missing
data or 454 pyrosequencing length data on phylogenetic reconstructions. Although the
full-length and partial and complete analyses differed in the placement of some deeper
branches, the inclusion of partial sequences had little effect on the overall results. The
placement of the partial and complete sequences into their respective clades was almost
completely congruent with the phylogeny based on full-length sequences. The analysis
using only partial sequences performed poorly with regard to the placement of deeper
branches, but placed sequences robustly into terminal clades remarkably well. Other
studies have found that short pyrosequencing data can be placed accurately into an
existing full-length phylogeny (25), and our results suggest that if a highly variable
region (i.e., the V1 region for Lactobacillus) can be accurately aligned, alignments built
on short pyrosequencing sized data can accurately place sequences into terminal clades.
Implications for Lactobacillus systematics
By gathering all publicly available 16S sequences and clustering sequences that
shared 97% or greater sequence identity, we created an updated, complete representation
of the diversity of the genus Lactobacillus. In agreement with previous studies (17), we
determined that the L. delbrueckii (acidophilus) clade represents a significant portion of
Lactobacillus diversity. Also in agreement with previous studies (1, 26), we determined
that Paralactobacillus and Pediococcus are within Lactobacillus, and that the overall
taxonomy of the genus Lactobacillus needs significant revisionary work.
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Citation and related information available at http://www.rna.ccbb.utexas.edu
Secondary Structure: small subunit ribosomal RNA
5ʼ
3ʼ
Lactobacillus acidophilus
(GenBank accession number M58802)
1.cellular organisms 2.Bacteria 3.Firmicutes
4.Bacilli 5.Lactobacillales 6.Lactobacillaceae
7.Lactobacillus
June 2012
N
N
A
A
A
A
C
G
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A
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A
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G
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A
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G
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A
G
G
G
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A
G
A
U
G
A
C
N
N
N
N
N
N
N
N
N
N
N
N
G
U
AA
C
A A
G
N
N N N N N N N N N N N
G
A
ACCUGNNNNNN
G
A
U
C
A
C
C
U
C
C
U
U
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C
U
A
Canonical nucleotide pairs (G-C or A-U) are
represented by dashes, wobble nucleotide pairs (G-U)
are represented by small closed circles, A-G nucleotide
pairs are represented by large open circles, and all
other pairs are represented by large closed circles.
Taxonomy:

5’
3’
Lactobacillus delbrueckii subsp.
bulgaricus 2038
(CP000156)
7.Lactobacillus 8.Lactobacillus delbrueckii
9.Lactobacillus delbrueckii subsp. bulgaricus
July 2012
A
A
A
U
U
G
A
G A G U U
U G
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G
A
C
GA
A
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5’
3’
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(CP002338)
7.Lactobacillus 8.Lactobacillus amylovorus
July 2012
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5’
3’
Lactobacillus gasseri ATCC 33323
(AF519171)
7.Lactobacillus 8.Lactobacillus gasseri
July 2012
A
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G
G
A
U
U
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A
U
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G G
G
C
GU
A
A
A
G
C
G
A
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UG
C
A
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CGGUUCAAU
A
AGUCUGAUGUG
A
AA
G
CCUUCGGCU
C
A
A C C G G A G A A U U G C A U C A G A
AA
C U G U U G A A C
U
U
G
A
G
U
G
C
A
G
A
A
G
A
G
G
A
G
A
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GG
AA
CUCCAUGU
GUA
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G
G
A A U G C
G
U
A G
A
U
A U A U G G A A
G A
A C
A
C C
A
G
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G C G
A
A
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G
C
U
C
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C
U
G
G
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C
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C
A
A
C
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A
C
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A
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A
A
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C
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C
C
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G
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C
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C C G U
A
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G
AU
G A G U G CU A A
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C
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C
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A
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AC
G
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C
G
C
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AGGUU
GAAA
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A
A
A
G G A A U U G A C G
G
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C
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A
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A
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CU
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C
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GCAAACCUAAG
A
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A U U A G G U G U U
C
C
C
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G
G
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A
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C
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A
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C
C
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A
A C G A G C
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GUUGGG
C
AC
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G
A
C
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G
C
C
G
G
U
G
ACA
A
A
C
C
G
G
A
G
G
A
A
G
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UGGGGA
U
G
A
C
G
U
C
A
A
G
UC
A
UC
A
U
G
C
C
C
C U
U
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A
C
C
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G
G
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A
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AC
A
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G
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A
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C
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C
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A
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G
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AA
G
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C
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G
C
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A
G
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UC
G
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A
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G
C
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U
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C
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A
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A
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GC
U
G
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A
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C
G
C
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A
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A
A
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C
G
C
G
G
A
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A
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C
G
C
C
G
C
G
G
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G
A
A
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A
C
GU
U
C
C
CGGGCCUUGU
A
CA
C
A
C
C
G
C
C
C
G
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C
A
C
A
C
C
A
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G
A
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A
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A
A
C
A
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C
C
A
A
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C
C
G
G
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G
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A
UA
A C
C
U
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U A
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A
G
G
A
G
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C
A
G
C
C
G
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C
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A
A
G
G
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A
G
G
A
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A
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A
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G
A
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A
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G
G
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G
A
A
G
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C
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AA
C
A A
G
G
G
A
ACCUGCGGCUG
G
A
U
C
A
C
C
U
C
C
U
U
U

5’
3’
Lactobacillus delbrueckii subsp. lactis
(M58823)
7.Lactobacillus 8.Lactobacillus delbrueckii
June 2012
N
N
C
A
A
A
U
U
G
A
G A G U U
U G
A
UCCUGGCUCAG
G
A
C
GA
A
C
G
C
U
GG
C
G
G
C
G
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G
C
C
UA
A
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A
CA
U
G
C
A
A
G U C
G A
GC
G
A G
CU
G
A A
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C
A A
A G
A
UU
CC
U
U
CG
GG
AG
G
AU
UU
G
UU
GG
A
NN
NN
A
GCGGC
G
G
A
UGG
G
U
G
A
G
U
A
ACA
C
G
U
G
G
G
C A
AU
C
U
G
C
C
C
U
A
A
A
G
AC U G G
G
A U
A
C C A C U U G G
A
A
ACAGGUGC
U
AAU
A
CCGG
A
U
A
A
C
A
A
C
A
U
G
A
A
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C
G
CA
U
G
A
U
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C
A
A
G
U
U
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G
A
A
A
GGCGGCG
C
A
A G C U G U C A
C
U
U
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A
G
G
A
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A
G
C
C
C
G
C
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GC
G
C
A
UU
A
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GU
U
G
G
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G
G
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A
A
G
G C
C
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A
C
C
A
A
G
G
C
A
A
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G
C
G
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AGUU
G
A
G A
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A
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CUG
A
G
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C A C
G
G C C C A A
A
C
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C
C
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C G
G
G
A
G
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C
A
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A
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G
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A
A
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CU
U
CCA
CAA
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C
A
A
A C
G
C
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CGUGAGU
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A
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U
C
G G A U C
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UG
A
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AG
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G
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A
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GU
A
A
A
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G
A
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CG
C
A
G
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CGGAAUGAU
A
AGUCUGAUGUG
A
AA
G
CCCACGGCU
C
A
A C C G U G G A A C U G C A U C G G A
AA
C U G U C A U U C
U
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G
A
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A
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AA
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G
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A
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G
C
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A
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A
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A
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A
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C
C
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G
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A
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A
A
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G
AU
G A G C G CU A G
G
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G
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G
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A
C
U
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C
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U
C
C
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C
A
G
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G
C
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C
A
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C
A
A
AC
G
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A
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C
G
C
C
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AGGUU
GAAA
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A
A
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A
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A
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C
AC
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G
A
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A
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A
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U
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C
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C
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G
C
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A
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G
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A
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C
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U
C
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C
C
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A
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A
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GC
U
G
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A
A
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G
C
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A
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A
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C
G
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A
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C
G
C
C
G
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A
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GU
U
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CGGGCCUUGU
A
CA
C
A
C
C
G
C
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A
C
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A
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G
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UA
A C
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A
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N
N
N
N
N
N
N
GU
AA
C
A A
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N
N N N N N N N N N N N
G
A
ACCUGNNNNNN
G
A
U
C
A
C
C
U
C
C
U
U
U
C
U
A

5’
3’
Lactobacillus sp. 61D
(FR681902)
7.Lactobacillus
July 2012
A
C
C
U
A
C
G
U
A
G A G U U
U G
A
UCAUGGCUCAG
G
A
C
GA
A
C
G
C
U
GG
C
G
G
C
G
U
G
C
C
UA
A
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CA
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G
C
A
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G U C G A
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C
G
A
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C
U
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C
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A
G
A
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A
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C
G
G
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C
U
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C
A
C
C
G
A
A
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A
A
A
C
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A
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A
C
A
A
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C
G
A
G
CGGC
G
G
A
CGG
G
U
G
A
G
U
A
ACA
C
G
U
G
G
G
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AC
C
U
G
C
C
C
A
A
G
A
G
AC U G G
G
A U
A
A C A C C U G G
A
A
ACAGAUGC
U
AAU
A
CCGG
A
U
A
A
C
A
A
C
A
U
C
U
A
G
C
G
CA
U
G
C
U
A
G
A
A
G
U
U
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G
A
A
A
GAUGGG
U
U
U C U A U C A
C
U
C
U
U
G
G
A
U
G
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A
C
C
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G
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A
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A
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A
C
G
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A
C
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A
A
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A
A
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C
A
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A
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AGUU
G
A
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A
C
A
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CUG
A
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A
C A C
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A
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U
C
C
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A
C G
G
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A
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C A G
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A
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A
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CU
U
CCA
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C
A
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CGUGAGU
G
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A
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U
C
G G C U C
G
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A
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GAA
A
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G
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U A A
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A
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A
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A
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G
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A
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A
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A
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G
C
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G
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A
A
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A
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A
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A
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A
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U
A G
C
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A
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A
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C
C
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G
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A
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G
AU
G A G U G CU A A
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A
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G
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C
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AGGUU
GAAA
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A
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G G A A U U G A C G
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C
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A
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A U U A G G U G U U
C
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C
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G
G
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G
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AA
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G
G
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A
ACCUGCGGUUG
G
A
U
C
A
C
C
U
C
C
U
U
A

5’
3’
uncultured bacterium
(DQ922980)
1.cellular organisms 2.Bacteria 3.environmental samples
July 2012
U
U
U
A
A
U
C
G
A
G A G U U
U G
A
UCCUGGCUCAG
G
A
C
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G
A
U
C
A
C
C
U
C
C
U
U
U
C
U
A

5’
3’
Lactobacillus salivarius
(AB186340)
7.Lactobacillus
June 2012
N
N
N N N N N
N N
N
NNNNNNNNNNN
N
N
N
NN
N
N
A
C
U
GG
C
G
G
C
G
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C
UA
A
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A C G A A A C U U U
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C
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G G A U C
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C
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G
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A A
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A G C C G
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Gutell 123.app environ micro_2013_79_1803

Gutell 123.app environ micro_2013_79_1803

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Gutell 123.app environ micro_2013_79_1803