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Published Ahead of Print 4 January 2013.10.1128/AEM.03681-12.2013, 79(6):1803. DOI:Appl. Environ. Microbiol.Katrin Kellner...
Specificity between Lactobacilli and Hymenopteran Hosts Is theException Rather than the RuleQuinn S. McFrederick,aJamie J. ...
veys of several bee and ant species. We then used these structure-based alignments to reconstruct the phylogenetic history...
cal base pairs, pseudoknots, and other irregular structural elements in thecomparative 16S rRNA secondary structure models...
clade (Fig. 2). Clade memberships and within-clade branchingpatterns were very similar between the full-length and the par...
flower-inhabiting and fructophilic lactobacilli (53) and lactoba-cilli used in sourdough fermentation (54). Some of the hal...
FIG 3 Secondary structure of the 16S rRNA molecule in the genus Lactobacillus. The degree of conservation in the molecule ...
terminal groups received high bootstrap support even in the par-tial 16S analysis (see Fig. S1 in the supplemental materia...
and were then vertically transmitted within these Solenopsis lin-eages or whether Lactobacillus is recruited continually f...
all of our analyses placed sequences into terminal clades with confi-dence.Other studies have found that short pyrosequenci...
27. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB,Lesniewski RA, Oakley BB, Parks DH, Robinson CJ. ...
Supplemental file S1. Supplemental text for 16S rRNA sequence alignment, secondarystructure models, and Lactobacillus phyl...
proposed for the larger dataset when two positions have similar patterns of variation (i.e.covariation) were identified. W...
minimal variance in the number of nucleotides, the information in the primary structure issufficient to align sequences wi...
alignments based on full-length sequences only, partial and complete sequencescombined, and partial sequences only, we wer...
8. Shang, L., W. Xu, S. Ozer, and R. R. Gutell. 2012. Structural constraintsidentified with covariation analysis in riboso...
22. Raftis, E. J., E. Salvetti, S. Torriani, G. E. Felis, and P. W. OToole. 2011.Genomic diversity of Lactobacillus saliva...
Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal R...
Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal R...
Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal R...
Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal R...
Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal R...
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Gutell 123.app environ micro_2013_79_1803

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McFrederick Q.S., Cannone J.J., Gutell R.R., Kellner K., Plowes R.M., and Mueller U.G. (2013).
Specificity between Lactobacilli and Hymenopteran Hosts is the Exception Rather than the Rule.
Applied and Environmental Microbiology, 79:1803-1812.

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  1. 1. 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. MuellerQuinn S. McFrederick, Jamie J. Cannone, Robin R. Gutell,Rather than the RuleHymenopteran Hosts Is the ExceptionSpecificity between Lactobacilli andhttp://aem.asm.org/content/79/6/1803Updated information and services can be found at:These include:SUPPLEMENTAL MATERIAL Supplemental materialREFERENCEShttp://aem.asm.org/content/79/6/1803#ref-list-1at:This article cites 61 articles, 23 of which can be accessed freeCONTENT ALERTSmore»articles cite this article),Receive: RSS Feeds, eTOCs, free email alerts (when newhttp://journals.asm.org/site/misc/reprints.xhtmlInformation about commercial reprint orders:http://journals.asm.org/site/subscriptions/To subscribe to to another ASM Journal go to:onFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  2. 2. Specificity between Lactobacilli and Hymenopteran Hosts Is theException Rather than the RuleQuinn S. McFrederick,aJamie J. Cannone,bRobin R. Gutell,bKatrin Kellner,aRobert M. Plowes,cUlrich G. MuelleraSection of Integrative Biology, University of Texas at Austin, Austin, Texas, USAa; The Institute for Cellular and Molecular Biology and the Center for Computational Biologyand Bioinformatics, The University of Texas at Austin, Austin, Texas, USAb; Brackenridge Field Laboratory, University of Texas at Austin, Austin, Texas, USAcLactobacilli (Lactobacillales: Lactobacillaceae) are well known for their roles in food fermentation, as probiotics, and in humanhealth, 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. Todetermine 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 otherhymenopteran species (three sweat bees and three ants). We determined the comparative secondary structural models of 16SrRNA, 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 thatare closely related to those found in vertebrate hosts or in diverse environments. Why honey and bumble bees associate withhost-specific lactobacilli while other social Hymenoptera do not remains an open question. Lactobacilli are known to inhibit thegrowth 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 foodproduction and human health (1). Lactobacilli convert sugars tolactic acid and other acids, and some species are used in foodfermentations to preserve foods, contribute flavor, or inhibit thegrowth of other bacteria (2). Other lactobacilli are used as humanprobiotics; for example, L. reuteri protects children from rotavirusgastroenteritis (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 hostsfrom pathogens (4, 5). For example, Lactobacillus reuteri is foundin 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 mixtureof LAB, including Lactobacillus species, help protect honey beelarvae from Paenibacillus larva and Melissococcus plutonius, thecausative agents of American and European foulbrood, respec-tively (9, 10). The Lactobacillus phylotypes Firm4 and Firm5, inconjunction with other members of the bumble bee microbiota,protect bumble bee workers from the trypanosome pathogenCrithidia bombi (11, 12). LAB have also been hypothesized to playa 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). Ourrecent phylogeny (16), based on a short fragment of the 16S rRNAgene, agreed with suggestions that the Firm3, Firm4, and Firm5honey bee- and bumble bee-associated Lactobacillus phylotypesare host specific, which suggests that between-species transmis-sion of these phylotypes is uncommon or absent (16). Horizontaltransmission has not been excluded, but both honey and bumblebees can acquire microbes through within-colony social contact(11, 17). In contrast, solitary and primitively eusocial sweat beesassociate with lactobacilli related to those that occur on flowers(16). It is currently unknown whether sociality per se or someunique 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 associatewith hymenopteran hosts that exhibit a range of social structures.First, we used very accurate comparative secondary structuremodels as templates to create a larger set of 16S rRNA structuremodels that represent all of the major forms of structural diversitypresent in the primary groups of lactobacilli. Second, we usedthese structure models to construct a comprehensive and highlyaccurate alignment of full-length or nearly full-length 16S rRNAgene sequences. To determine whether host specificity is commonin associates of insects that live in large societies or is limited toassociates of corbiculate apids, we constructed two additionalalignments that included 16S-amplicon 454-pyrosequencing sur-Received 28 November 2012 Accepted 2 January 2013Published ahead of print 4 January 2013Address 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.03681-12March 2013 Volume 79 Number 6 Applied and Environmental Microbiology p. 1803–1812 aem.asm.org 1803onFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  3. 3. veys of several bee and ant species. We then used these structure-based alignments to reconstruct the phylogenetic history of thegenus Lactobacillus.MATERIALS AND METHODSFull and nearly full-length Lactobacillus 16S rRNA gene sequences. Toobtain a comprehensive representation of this diverse genus, we down-loaded publicly available, full-length or nearly full-length sequences of the16S rRNA gene. First, we searched NCBI’s nucleotide database (18) forcomplete Lactobacillus 16S rRNA gene sequences. Next, we searched theRibosomal Database Project (RDP [19]) for Lactobacillus 16S sequenceslonger than 1,200 bases and of good quality, as designated by RDP. Toinclude sequences from bee-associated lactobacilli, we downloaded asso-ciated sequences from NCBI PubMed entries of bee-bacterial studies andadditionally 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 haveeliminated some sequences from type strains that were represented bylonger sequences that shared Ն97% sequence identity with the type strainsequence, we obtained the accession numbers of Lactobacillus type speci-mens from RDP. We then searched our file for the accession numbers ofthese type sequences and added missing sequences back into the align-ment. We additionally searched NCBI taxonomy for Lactobacillus speciesand added reference sequences (21), when available, for the species thatwere missing in our alignment. Finally, we searched the list of prokaryoticnames with standing in nomenclature (22) to verify that our alignmentdid indeed contain representatives from all described lactobacilli. This setof 16S rRNA sequences included identified, cultured, unidentified, anduncultured Lactobacillus species. As outgroups, we added sequences from14 bacterial species from across the Bacilli but with emphasis on the Lac-tobacillales. To investigate diversity of paralogous gene copies within agenome, 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 sequencesfrom undescribed or unidentified Lactobacillus species, and 224 sequencesfrom 160 named, but not necessarily published, Lactobacillus species for atotal 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 asecond alignment spanning the length of the 16S rRNA gene. We codedthe five prime and three prime ends of the 454 sequences as missing data.The effect of missing data on phylogenetic reconstructions is an ongoingresearch area with no simple consensus (23, 24). We therefore planned tointerpret 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-pyrosequencingstudies 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 halictiddata (16), which we obtained from one wild nest of A. pura (Virginia), onelaboratory 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 thelaboratory and sampled it again 6 months later. We collected S. invicta andS. geminata-complex samples in their native range in Argentina and intheir invasive range in Taiwan, Texas, Florida, and California (Plowes etal., unpublished). These data represent subsets of data sets from previousor forthcoming publications (see, for example, references 16 and 25). Thethree studies used the same 16S rRNA primers: Gray28F 5=-GAGTTTGATCNTGGCTCAG and Gray519r 5=-GTNTTACNGCGGCKGCTG,which span V1-V3 in the secondary structure of 16S rRNA. V1-V3 arevariable regions that have been shown to accurately differentiate closelyrelated 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 sequencedforward from Gray28f, while the Solenopsis-associated sequences were 454sequenced reverse from Gray519r (these later sequences were reversecomplemented 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 sequenceswere processed in the program mothur (27). First, we denoised the datawith the shhh.flows command. We then removed any sequences withmismatches in the primer binding site or the barcode, or homopolymerruns of over eight bases, and removed primers and barcodes from thesequences. To remove chimeric sequences, we used the chimera.uchimecommand, and deleted the detected chimeras. Next, we ran BLASTsearches against a 16S rRNA database curated by the Medical BiofilmResearch Institute (MBRI; Lubbock, TX). We selected sequences with topBLAST hits against the MBRI database of at least 90% sequence identity toLactobacillus sequences. We chose a minimum of 90% sequence identityin order to include novel Lactobacillus sequences, while keeping in mindthat any sequences that do not belong in the genus Lactobacillus should falloutside of our ingroup in our phylogenetic analysis. As a validation of ourBLAST searches, we ran additional BLAST searches of the 454 sequencesagainst the entire nucleotide collection at NCBI. Next, to maximize thephylogenetic signal of the short 454 reads, we removed 454 sequences thatwere Ͻ350 bases long. We then clustered sequences of 97% or greatersequence identity with the program CD-HIT (20). After quality controland clustering, we added 84 sweat bee-associated, 79 Mycocepurus-asso-ciated, and 56 Solenopsis-associated sequences to our alignment, for a totalof 620 sequences in the entire alignment.The hymenopteran hosts included here vary widely in both geographyand natural history. Augochlora pura and H. ligatus both live in NorthAmerica, but A. pura is a solitary bee that nests in rotten logs (28), whereasH. ligatus is a primitively eusocial halictid that forms small colonies in thesoil (29). Megalopta genalis is a socially polymorphic, neotropical sweatbee 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 singlecolonies that occupy more than one nest in the Brazilian Amazon (33, 34).In contrast, S. invicta is originally from South America but has becomewidespread in its invasive range and can reach colony sizes of 220,000individuals (35, 36). Solenopsis geminata variants have been introduced tomany 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 theE. coli 16S rRNA gene sequence. We used the entire length of the 454 dataand 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 thephylogenetic reconstructions based on partial versus full length versus acombination 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 setof sequences that are known to have the same function and higher-orderstructure (37). Covariation analyses identify all types of canonical andnoncanonical base pairs with a set of nucleotides that covary with oneanother, 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 MicrobiologyonFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  4. 4. cal base pairs, pseudoknots, and other irregular structural elements in thecomparative 16S rRNA secondary structure models are in the crystalstructure of the ribosomal subunit (40). The strength of the covariationfor each predicted base pair has been quantified and used as a confidencerating 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 ofthe base pairs in the comparative structure models for the rRNAs fromdifferent organisms are correct, including all of the 16S rRNA secondarystructure models for the genus Lactobacillus (for a complete discussion ofcovariation analysis, see File S1 in the supplemental material).The accuracy of our predicted secondary structure models are directlyassociated with the quality of the sequence alignment. Better alignmentsfacilitate better comparative structure models. Moreover, vice versa, veryaccurate comparative structure models are the basis for the juxtapositionof nucleotides that are components of similar and analogous structuralelements, especially in highly variable regions that can have large variancesin the number of nucleotides with little or no sequence identity. Thus, oursecondary structure-based alignments do not attempt to maximize se-quence identity a priori. Instead, they attempt to maximize the alignmentof similar structural elements.After the 16S rRNA sequences were semiautomatically aligned withthe template-based alignment program CRWAlign (41), we refined thealignment manually with the alignment editor AE2 (developed by T.Macke, Scripps Research Institute, San Diego, CA [42]). This tool wasdeveloped for Sun Microsystems (Santa Clara, CA) workstations runningthe Solaris operating system. The manual alignment process utilizes theCRWAlign program to identify nucleotides in a column that might notmap to the same locations in the secondary and tertiary structure. A visualinspection of the alignment determines if these flagged nucleotides shouldbe realigned. If necessary, this is done manually with the AE2 alignmenteditor. For regions of the alignment with high sequence similarity andminimal variance in the number of nucleotides, the information in theprimary structure is sufficient to align sequences with confidence. In con-trast, for more variable regions in closely related sequences or betweenmore distantly related sequences, a high-quality alignment can only beproduced when secondary and/or tertiary structure information is in-cluded.Secondary structure diagrams. Secondary structure diagrams weregenerated 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 rRNAsecondary 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 secondarystructure diagrams with the interactive secondary structure programXRNA (written in the C programming language for Sun Microsystemsworkstations running the Solaris operating system by B. Weiser and H.Noller, University of California, Santa Cruz, CA). All structure diagramsare available as online supplemental files at http://www.rna.ccbb.utexas.edu/.Phylogenetic analyses. To reconstruct the phylogenetic history of thelactobacilli, we conducted separate maximum-likelihood analyses on ourthree alignments. We first determined that GTRϩIϩ⌫ was the most ap-propriate model of sequence evolution for each of our alignments usingthe 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 programto estimate all parameter values during the runs. To assess branch supportwe conducted 100 bootstrap pseudoreplicates on each alignment, using agenthreshfortopoterm setting of 5,000,000. We used Mesquite (46), tocalculate patristic distances across the entire tree. To calculate divergence,we used the branch info tool in Mesquite (46) to measure the distancefrom the base of monophyletic groups of hymenopteran associates to themost 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).RESULTSPhylogenetic reconstructions of our three 16S rRNA gene sequencealignmentsresultedinsimilarcladestructureforalltrees(Fig.1and2;see also Fig. S1 and see Table S1 in the supplemental material forsimple sequence statistics for all alignments). The phylogeny basedonly on full-length 16S rRNA sequences (Fig. 1) was in nearly com-pleteagreementwiththephylogenybasedonacombinationofpartialand complete 16S rRNA sequences (Fig. 2). The phylogeny based ononly partial 16S sequences (see Fig. S1 in the supplemental material),however, differed in the branching patterns between and withinclades compared to the full-length phylogeny, but the terminal cladecomposition remained stable. Sequence alignments and detailedphylogenetic trees are available at TreeBase (http://purl.org/phylo/treebase/phylows/study/TB2:S13670), and a detailed version ofFig.2withbootstrapsupportvaluesandtaxonlabelsisavailableintheonline 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 supplementalmaterial). In addition, the extent of primary and secondary struc-ture conservation for 608 16S rRNA sequences representing theentire lactobacilli genus was mapped onto a L. acidophilus second-ary structure diagram (Fig. 3). The first hairpin stem and loopfrom the five prime end of the molecule (the V1 region) was themost variable region in Lactobacillus 16S structure. Our structuralmodels 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. reutericlades showed moderate support (82 and 85%, respectively),while other clades showed weak support at their deepest nodes.The deeper branches connecting these major clades uniformlyshowed little support. The clade containing the greatest number oftaxa and the greatest sequence diversity was the L. delbrueckii(acidophilus) clade, which was comprised of 160 taxa and covereda maximum patristic distance of 1.21, out of a global maximum of1.51 across the entire tree (Table 1).Partial and complete 16S rRNA gene phylogeny, includinghymenopteran associates. The phylogenetic trees determinedfrom the analysis of the partial and complete 16S rRNA alignmentand the full-length 16S rRNA alignment were very similar (Fig. 1and 2). The main difference between the two phylogenies was theplacement of the reuteri clade. The reuteri clade was sister to theplantarum clade in the full-length phylogeny (Fig. 1), whereas inthe partial and complete phylogeny the members of the reutericlade were reconstructed paraphyletic as a series of branching lin-eages leading to the monophyletic L. delbrueckii (acidophilus)Rare Specificity between Hymenoptera and LactobacilliMarch 2013 Volume 79 Number 6 aem.asm.org 1805onFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  5. 5. clade (Fig. 2). Clade memberships and within-clade branchingpatterns were very similar between the full-length and the partialand complete phylogenies, as were the bootstrap support values(Fig. 1 and 2).Of the Hymenoptera-associated sequences, only the honey andbumble 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 notas diverged from other lactobacilli as Firm3 and Firm4 (Fig. 2 and4). In addition, two sequences from Solenopsis clustered within theFirm5 clade. In contrast to the Firm3-Firm5 clades, most Hy-menoptera-associated sequences were scattered throughout thegenus and either clustered with or were closely related to lactoba-cilli known from vertebrate hosts or from diverse environmentalsources (Fig. 2; see also Fig. S2 in the supplemental material). Mostreads 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 withSolenopsis, for example, were closely related to L. sakei and L. plan-tarum, both of which are used in food fermentation (2). The mostabundant M. smithii-associated OTUs were closely related to sev-eral lactobacilli that are common inhabitants of the intestinaltracts of rodents, birds, and humans (Table 2). These M. smithii-associated OTUs were found in both fungus-garden and workersamples (see Table S2 in the supplemental material). As alreadyreported in McFrederick et al. (16), halictids associated mainlywith Lactobacillus OTUs that were related to flower-inhabiting,fructophilic lactobacilli (52) or to a clade that includesFIG 1 16S rRNA gene maximum-likelihood phylogeny of the genus Lactobacillus, based on full or nearly full-length sequences only. Branch widths areproportional to bootstrap support from 100 pseudoreplicates. Highlighted branches represent Lactobacillus associates of honey or bumble bees. Major clades areindicated with curved lines and named according to previous studies (47–50).McFrederick et al.1806 aem.asm.org Applied and Environmental MicrobiologyonFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  6. 6. 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 cladesdid not exhibit the same level of divergence as the Firm3 andFirm4 clades (Fig. 2 and 4). Many of the terminal branches thatincluded Hymenoptera-associated sequences exhibited highbootstrap support (see Fig. S2 in the supplemental material).Partial 16S rRNA gene phylogeny, including Hymenopteraassociates. 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 aparaphyletic 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 werealso no longer monophyletic in the partial phylogeny but insteadwere 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 deeperbranches in the Lactobacillus phylogeny, the membership of theterminal clades was stable across all analyses, and many of theFIG 2 16S rRNA gene maximum-likelihood tree of the genus Lactobacillus, based on partial and complete sequences combined. Branch widths are proportionalto bootstrap support from 100 pseudoreplicates. Highlighted branches represent Hymenoptera associated lactobacilli as indicated in the figure legend. Cladescontaining lactobacilli associated with humans are indicated by a human figure, whereas clades containing lactobacilli used in food fermentation are indicatedby a piece of cheese. Major clades are indicated with curved lines and named according to previous studies (47–50).Rare Specificity between Hymenoptera and LactobacilliMarch 2013 Volume 79 Number 6 aem.asm.org 1807onFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  7. 7. FIG 3 Secondary structure of the 16S rRNA molecule in the genus Lactobacillus. The degree of conservation in the molecule as determined by analysis of608 sequences is superimposed on the structure of the Lactobacillus acidophilus 16S molecule. Conservation of each nucleotide across the 608 analyzedLactobacillus 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.1808 aem.asm.org Applied and Environmental MicrobiologyonFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  8. 8. terminal groups received high bootstrap support even in the par-tial 16S analysis (see Fig. S1 in the supplemental material).DISCUSSIONHost specificity of Hymenoptera-associated lactobacilli. Hyme-noptera ranging from a solitary species (A. pura) to species thatlive in colonies numbering in the hundreds of thousands (Sole-nopsis) associate with lactobacilli that are either found in otherhosts or the environment. Social structure by itself does not deter-mine whether hymenopteran hosts associate with lactobacilli thatare host-specific or that are more recent acquisitions from theenvironment. Honey bees (colony sizes up to 60,000 [55]) andbumble bees (colony sizes from 50 to Ͼ400 [55]) are the onlyhymenopteran hosts examined to date that associate with lacto-bacilli that appear to be host specific and highly diverged fromother lactobacilli.Previous studies already suggested that the corbiculate apidsassociate 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 withApis mellifera form a more consistent association with their hostscompared to Bombus, and this may be related to how A. melliferafounds new colonies (15, 56). Apis mellifera colonies are foundedvia swarming, where a colony divides and approximately half ofthe 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 ofcolonies 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 Bombusspecies, in which nests are always founded by single queens (58),maintain associations with host-specific bacteria remains to bedetermined in detailed studies of dispersing Bombus queens, aswell as the changes in associated microbial communities duringcolony 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 transmissionof bumble bee microbiota. In contrast, trophallaxis between nest-mates or other contact within the hive is known to be importantfor 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 likelymechanisms regarding how environmental lactobacilli are re-cruited into association with sweat bees and ants. As we previouslyreported, sweat bees associate with lactobacilli that are related tolactobacilli isolated from flowers, and may therefore obtain thesebacteria from flowers (16). Mycocepurus smithii belongs to thegroup of attine ants that do not collect leaves to sustain growth oftheir fungus gardens, but instead collect insect frass, flower parts,seeds, and fruit-flesh (63). Two of the dominant LactobacillusOTUs associated with M. smithii (L. johnsonii and L. crispatus)have been isolated from A. mellifera guts (64), indicating that theselactobacilli 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 tosustain their fungus gardens. These lactobacilli are found in bothM. smithii fungus gardens and workers, indicating that they canoccupy broad ecological niches. Alternatively, M. smithii may becollecting 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 beobtaining lactobacilli from plant, insect, or vertebrate foodsources. The most abundant Lactobacillus in Solenopsis was mostclosely 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, indicatingthat it might play an important role in the biology of Solenopsis.Lactobacillus plantarum, whose comparatively large genome mayallow it to inhabit a variety of environments (66), was also foundto associate with S. invicta and S. geminata in Argentina, NorthAmerica, and Taiwan. Lactobacillus plantarum is found in dairy,meat, plants, and the human gastrointestinal tract and may berecruited by Solenopsis from any of these sources. It is currentlyunknown whether these associations derived from an acquisitionby a Solenopsis lineage ancestral to the invasive Solenopsis lineagesTABLE 1 Maximum patristic distances of the major clades in the mostlikely full-length 16S phylogenetic tree, out of a maximum patristicdistance of 1.51 across the entire treeClade Patristic distanceL. salivarius 0.5L. casei 0.44L. buchneri 0.54L. plantarum 0.34L. reuteri 0.55L. delbrueckii (acidophilus) 1.21FIG 4 Histogram of patristic distances from the base of monophyletic cladesof Hymenoptera associates to their most recent common ancestor. The honeybee-associated Firm3 and Firm4 clades exhibited the greatest divergence, whilethe honey bee-associated Firm5 clade exhibited an amount of divergence sim-ilar to that of some other Hymenoptera associates.Rare Specificity between Hymenoptera and LactobacilliMarch 2013 Volume 79 Number 6 aem.asm.org 1809onFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  9. 9. and were then vertically transmitted within these Solenopsis lin-eages or whether Lactobacillus is recruited continually from theenvironment by Solenopsis. Notably, we found several Firm5 se-quences, which are thought to be specific to honey and bumblebees (15), associated with Solenopsis. These sequences were notabundant, suggesting that they may be transient and perhapspicked up by Solenopsis when scavenging dead A. mellifera or Bom-bus workers.The function of lactobacilli in hymenopteran hosts outside ofthe honey and bumble bees is still relatively unexplored, but theavailable evidence suggests that lactobacilli facilitate digestion ofsugars 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 asexaptations for the symbiotic habit. For example, lactic acid bac-teria (LAB) digest a variety of sugars, with the main end productbeing 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 alsoknown for the secretion of bacteriocins, which are compoundsthat inhibit the growth of other bacteria (68). For example, L. sakeiproduces several bacteriocins that inhibit pathogenic and spoilageorganisms (69). These general properties of lactobacilli mean thatthey are likely to be beneficial to hymenopteran hosts, and furtherresearch should determine the relative importance of acidificationversus bacteriocin-secretion in the biology of ant- and bee-asso-ciated Lactobacillus.Utility of the 16S rRNA gene for Lactobacillus identificationand phylogeny. Although our analysis of 16S rRNA did not re-solve the deeper branches in the Lactobacillus phylogeny, 16SrRNA 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 inprevious 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 ofmissing data or 454 pyrosequencing length data on phylogeneticreconstructions. Although the analysis using only partial sequencesperformed 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 speciesare binned as halictids)aHost Source of read No. of reads Closest phylogenetic relative Best BLAST hitSequenceidentity (%)Solenopsis Brood geminata TX 5,993 Undescribed L. sakei 92Brood geminata TW 2,118 L. plantarum L. plantarum 99Brood invicta TX 935 L. curvatus L. curvatus 99Brood invicta TX 756 L. brevis L. brevis 99Pooled invicta TW 661 L. plantarum L. plantarum 99Pooled invicta TW 324 L. plantarum L. plantarum 99Pooled geminata TW 261 L. paracasei L. casei 99Brood geminata TW 167 L. kimchicus L. odoratitofui 99Brood geminata TW 156 Undescribed L. sakei 93Brood geminata TW 116 L. vaccinostercus L. vaccinostercus 99Mycocepurus smithii Garden 14,152 L. johnsonii L. johnsonii 99Worker 3,748 L. crispatus L. crispatus 99Garden 2,794 L. salivarius L. salivarius 97Garden 1,059 L. aviarius L. aviarius 99Garden 806 L. crispatus L. crispatus 98Worker 598 L. salivarius L. salivarius 99Garden 586 L. reuteri L. reuteri 99Worker 431 L. salivarius L. salivarius 98Worker 420 L. aviarius/L. johnsonii L. aviarius 90Garden 408 L. johnsonii L. johnsonii 95Halictids Larva 20,669 L. kunkeei/L. ozensis L. kunkeei 93Pollen 15,761 L. fructivorans L. fructivorans 91Larva 3,923 L. kunkeei/L. ozensis L. kunkeei 93Pollen 3,631 L. kunkeei/L. ozensis L. kunkeei 94Pollen 1,991 L. kunkeei/L. ozensis L. kunkeei 93Pollen 1,557 L. fructivorans L. fructivorans 90Larva 1,455 L. kunkeei/L. ozensis L. kunkeei 93Pollen 1,285 L. kunkeei/L. ozensis L. kunkeei 94Frass 1,102 L. paracasei L. casei 99Pollen 397 L. kunkeei/L. ozensis L. kunkeei 94aThe 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 ofreads 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 aparticular 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 sequenceidentity 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% sequenceidentity to each other.McFrederick et al.1810 aem.asm.org Applied and Environmental MicrobiologyonFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  10. 10. all of our analyses placed sequences into terminal clades with confi-dence.Other studies have found that short pyrosequencing data canbe 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 sizeddata can accurately place sequences into terminal clades. Our sec-ondary structural models of 16S rRNA indicate that the hairpinstem and loop in V1 is the most variable region in Lactobacillus.Next-generation sequencing surveys targeting V1 should providethe best taxonomic resolution for closely related lactobacilli.Conclusions. We created highly accurate comparative modelsof 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 usingour structurally informed sequence alignments revealed that honeyand bumble bees associate with host-specific Lactobacillus. In con-trast, regardless of colony size or within-colony oral sharing offood (trophallaxis), sweat bees and ants associate with lactobacillithat are closely related to those found in vertebrate hosts or indiverse environments. Host specificity therefore appears to be theexception rather than the rule for Hymenoptera-associated lacto-bacilli. Nest founding by colony fission has been proposed as alikely means for long-term transmission of microbiota withinhoney bee lineages (15). M. smithii may facultatively found nestsby colony fissioning (34), and yet we found that M. smithii doesnot 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 queensmay disrupt long-term associations because new microbiota arefrequently acquired by a social-insect lineage during that stage;and (iii) obligate colony fissioning is more likely than facultativecolony fissioning to maintain long-term host-microbe specifici-ties in a social insect. Studies of lactobacilli associated with otherobligate colony-fissioning Hymenoptera such as meliponine beesand related solitary and communal nesters such as euglossine bees(55) will help determine whether colony-fissioning or some othercharacteristic of apid biology promotes host specificity. Regardlessof how they are acquired, lactobacilli are known to inhibit thegrowth of other microbes and may be beneficial whether they arecoevolved with their host or are recruited by the host from envi-ronmental sources through mechanisms of partner choice.ACKNOWLEDGMENTSThis material is based on work supported by National Science Foundationgrant PRFB-1003133 awarded to Q.S.M. and grant DEB-0919519awarded to U.G.M., Texas Higher Education Coordinating Board grant01923 awarded to L. E. Gilbert, and National Institutes of Health grantGM067317 awarded to R.R.G.We appreciate technical assistance from D. A. Estrada in the initialprocessing of the Solenopsis sequences. We thank three anonymous re-viewers for their helpful comments.REFERENCES1. Dellaglio F, Felis GE. 2005. Taxonomy of lactobacilli and bifidobacteria,p 25. In Tannock G (ed), Probiotics and prebiotics: scientific aspects.Caister Aademic Press, Norfolk, United Kingdom.2. Leroy F, De Vuyst L. 2004. Lactic acid bacteria as functional startercultures for the food fermentation industry. Trends Food Sci. Technol.15:67–78.3. Shornikova AV, Casas IA, Mykkanen H, Salo E, Vesikari T. 1997.Bacteriotherapy with Lactobacillus reuteri in rotavirus gastroenteritis.Pediatr. Infect. Dis. J. 16:1103–1107.4. Walter J, Britton RA, Roos S. 2011. Host-microbial symbiosis in thevertebrate gastrointestinal tract and the Lactobacillus reuteri paradigm.Proc. Natl. Acad. Sci. U. S. A. 108:4645–4653.5. Cross ML. 2002. Microbes versus microbes: immune signals generated byprobiotic lactobacilli and their role in protection against microbial patho-gens. FEMS Immunol. Med. Microbiol. 34:245–253.6. Casas IA, Dobrogosz WJ. 2000. Validation of the probiotic concept:Lactobacillus reuteri confers broad-spectrum protection against disease inhumans and animals. Microb. Ecol. Health Dis. 12:247–285.7. Wong CNA, Ng P, Douglas AE. 2011. Low diversity bacterial communityin the gut of the fruitfly Drosophila melanogaster. Environ. Microbiol.13:1889–1900.8. Storelli G, Defaye A, Erkosar B, Hols P, Royet J, Leulier F. 2011.Lactobacillus plantarum promotes Drosophila systemic growth by modu-lating hormonal signals through TOR-dependent nutrient sensing. CellMetab. 14:403–414.9. Forsgren E, Olofsson TC, Vasquez A, Fries I. 2010. Novel lactic acidbacteria inhibiting Paenibacillus larvae in honey bee larvae. Apidologie41:99–108.10. Vasquez A, Forsgren E, Fries I, Paxton RJ, Flaberg E, Szekely L,Olofsson TC. 2012. Symbionts as major modulators of insect health: lacticacid bacteria and honeybees. PLoS One 7:e33188. doi:10.1371/journal.pone.0033188.11. Koch H, Schmid-Hempel P. 2011. Socially transmitted gut microbiotaprotect bumble bees against an intestinal parasite. Proc. Natl. Acad. Sci.U. S. A. 108:19288–19292.12. Koch H, Schmid-Hempel P. 2012. Gut microbiota instead of host geno-type drive the specificity in the interaction of a natural host-parasite sys-tem. Ecol. Lett. 15:1095–1103.13. Vasquez A, Olofsson TC. 2009. The lactic acid bacteria involved in theproduction of bee pollen and bee bread. J. Apicult. Res. 48:189–195.14. Koch H, Schmid-Hempel P. 2011. Bacterial communities in CentralEuropean bumblebees: low diversity and high specificity. Microb. Ecol.62:121–133.15. Martinson VG, Danforth BN, Minckley RL, Rueppell O, Tingek S,Moran NA. 2011. A simple and distinctive microbiota associated withhoney bees and bumble bees. Mol. Ecol. 20:619–628.16. McFrederick QS, Wcislo WT, Taylor DR, Ishak HD, Dowd SE, MuellerUG. 2012. Environment or kin: whence do bees obtain acidophilic bacte-ria? Mol. Ecol. 21:1754–1768.17. Martinson VG, Moy J, Moran NA. 2012. Establishment of characteristicgut bacteria during development of the honey bee worker. Appl. Environ.Microbiol. 78:2830–2840.18. Benson DA, Boguski MS, Lipman DJ, Ostell J. 1997. GenBank. NucleicAcids Res. 25:1–6.19. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM. 2009.The Ribosomal Database Project: improved alignments and new tools forrRNA analysis. Nucleic Acids Res. 37:D141–D145.20. Huang Y, Niu B, Gao Y, Fu L, Li W. 2010. CD-HIT Suite: a web serverfor clustering and comparing biological sequences. Bioinformatics 26:680–682.21. Pruitt KD, Tatusova T, Brown GR, Maglott DR. 2012. NCBI ReferenceSequences (RefSeq): current status, new features, and genome annotationpolicy. Nucleic Acids Res. 40:D130–D135. doi:10.1093/nar/gkr1079.22. Euzeby JP. 2012. List of prokaryotic names with standing in nomencla-ture. http://www.bacterio.cict.fr.23. Lemmon AR, Brown JM, Stanger-Hall K, Lemmon EM. 2009. The effectof ambiguous data on phylogenetic estimates obtained by maximum like-lihood and Bayesian inference. Syst. Biol. 58:130–145.24. Wiens JJ, Morrill MC. 2011. Missing data in phylogenetic analysis: rec-onciling results from simulations and empirical data. Syst. Biol. 60:719–731.25. Ishak HD, Plowes R, Sen R, Kellner K, Meyer E, Estrada DA, Dowd SE,Mueller UG. 2011. Bacterial diversity in Solenopsis invicta and Solenopsisgeminata ant colonies characterized by 16S amplicon 454 pyrosequencing.Microb. Ecol. 61:821–831.26. Kullen MJ, Sanozky-Dawes RB, Crowell DC, Klaenhammer TR. 2000.Use of the DNA sequence of variable regions of the 16S rRNA gene forrapid and accurate identification of bacteria in the Lactobacillus acidophi-lus complex. J. Appl. Microbiol. 89:511–516.Rare Specificity between Hymenoptera and LactobacilliMarch 2013 Volume 79 Number 6 aem.asm.org 1811onFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  11. 11. 27. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB,Lesniewski RA, Oakley BB, Parks DH, Robinson CJ. 2009. Introducingmothur: open-source, platform-independent, community-supportedsoftware for describing and comparing microbial communities. Appl. En-viron. Microbiol. 75:7537–7541.28. Stockhammer KA. 1966. Nesting habits and life cycle of a sweat bee,Augochlora pura (Hymenoptera: Halictidae). J. Kans. Entomol. Soc. 39:157–192.29. Michener CD, Bennett FD. 1977. Geographical variation in nesting bi-ology and social organization of Halictus ligatus. Univ. Kans. Sci. Bull.51:233–260.30. Wcislo WT, Arneson L, Roesch K, Gonzalez V, Smith A, Fernandez H.2004. The evolution of nocturnal behaviour in sweat bees, Megalopta ge-nalis and M. ecuadoria (Hymenoptera: Halictidae): an escape from com-petitors and enemies? Biol. J. Linn. Soc. Lond. 83:377–387.31. Wcislo WT, Gonzalez VH. 2006. Social and ecological contexts of tro-phallaxis in facultatively social sweat bees, Megalopta genalis and M. ecua-doria (Hymenoptera, Halictidae). Insectes Sociaux 53:220–225.32. Fernandez-Marin H, Zimmerman JK, Wcislo WT, Rehner SA. 2005.Colony foundation, nest architecture, and demography of a basal fungus-growing ant, Mycocepurus smithii (Hymenoptera, Formicidae). J. Nat.Hist. 39:1735–1743.33. Rabeling C, Gonzales O, Schultz TR, Bacci M, Garcia MVB, VerhaaghM, Ishak HD, Mueller UG. 2011. Cryptic sexual populations account forgenetic diversity and ecological success in a widely distributed, asexualfungus-growing ant. Proc. Natl. Acad. Sci. U. S. A. 108:12366–12371.34. Rabeling C, Lino-Neto J, Cappellari SC, Dos-Santos IA, Mueller UG,Bacci M. 2009. Thelytokous parthenogenesis in the fungus-gardening antMycocepurus smithii (Hymenoptera: Formicidae). PLoS One 4:e6781. doi:10.1371/journal.pone.0006781.35. Tschinkel WR. 1988. Colony growth and the ontogeny of worker poly-morphism in the fire ant, Solenopsis invicta. Behav. Ecol. Sociobiol. 22:103–115.36. Morrison LW, Porter SD, Daniels E, Korzukhin MD. 2004. Potentialglobal range expansion of the invasive fire ant, Solenopsis invicta. Biol.Invasions 6:183–191.37. Woese CR, Magrum LJ, Gupta R, Siegel RB, Stahl DA, Kop J, CrawfordN, Brosius R, Gutell R, Hogan JJ, Noller HF. 1980. Secondary structuremodel for bacterial 16S rRNA: phylogenetic, enzymatic and chemical ev-idence. Nucleic Acids Res. 8:2275–2294.38. Gutell R, Power A, Hertz G, Putz E, Stormo G. 1992. Identifyingconstraints on the higher-order structure of RNA: continued develop-ment and application of comparative sequence analysis methods. NucleicAcids Res. 20:5785–5795.39. Shang L, Xu W, Ozer S, Gutell RR. 2012. Structural constraints identi-fied with covariation analysis in rRNA. PLoS One 7:e39383. doi:10.1371/journal.pone.0039383.40. Gutell RR, Lee JC, Cannone JJ. 2002. The accuracy of rRNA comparativestructure models. Curr. Opin. Struct. Biol. 12:301–310.41. Gardner DP, Xu W, Miranker DP, Ozer S, Cannone JJ, Gutell RR. 2012.An accurate scalable template-based alignment algorithm, p 237–243. InProceedings of the 2012 IEEE International Conference on Bioinformaticsand Biomedicine. IEEE Computer Society, Washington, DC. doi:10.1109/BIBM.2012.6392676.42. Larsen N, Olsen GJ, Maidak BL, McCaughey MJ, Overbeek R, MackeTJ, Marsh TL, Woese CR. 1993. The ribosomal database project. NucleicAcids Res. 21:3021–3023.43. Posada D, Crandall KA. 1998. Modeltest: testing the model of DNAsubstitution. Bioinformatics 14:817–818.44. Swofford DL. 2002. PAUP*: phylogenetic analysis using parsimony (*andother methods), v.4b10. Sinauer Associates, Sunderland, MA.45. Zwickl DJ. 2006. Genetic algorithm approaches for the phylogenetic anal-ysis of large biological sequence datasets under the maximum likelihoodcriterion. University of Texas at Austin, Austin, TX.46. Maddison WP, Maddison DR. 2010. Mesquite: a modular system forevolutionary analysis, version 2.74. http://mesuiteproject.org.47. Canchaya C, Claesson MJ, Fitzgerald GF, Van Sinderen D, O’ToolePW. 2006. Diversity of the genus Lactobacillus revealed by comparativegenomics of five species. Microbiology 152:3185–3196.48. Felis GE, Dellaglio F. 2007. Taxonomy of lactobacilli and bifidobacteria.Curr. Issues Intest. Microbiol. 8:44–61.49. Pot B, Tsakalidou E. 2009. Taxonomy and metabolism of Lactobacillus, p3–58. In Ljungh A, Wadstrom T (ed), Lactobacillus molecular biology:from genomics to probiotics. Caister Academic Press, Norfolk, UnitedKingdom.50. Salvetti E, Torriani S, Felis GE. 2012. The genus Lactobacillus: a taxo-nomic update. Probiotics Antimicrob. Proteins 4:217–226.51. Babendreier D, Joller D, Romeis J, Bigler F, Widmer F. 2007. Bacterialcommunity structures in honeybee intestines and their response to twoinsecticidal proteins. FEMS Microbiol. Ecol. 59:600–610.52. Endo A, Irisawa T, Futagawa-Endo Y, Takano K, du Toit M, Okada S,Dicks LMT. 2012. Characterization and emended description of Lactoba-cillus kunkeei as a fructophilic lactic acid bacterium. Int. J. Syst. Evol.Microbiol. 62:500–504.53. Endo A, Futagawa-Endo Y, Sakamoto M, Kitahara M, Dicks LMT.2010. Lactobacillus florum sp. nov., a fructophilic species isolated fromflowers. Int. J. Syst. Evol. Microbiol. 60:2478–2482.54. Corsetti A, Settanni L. 2007. Lactobacilli in sourdough fermentation.Food Res. Int. 40:539–558.55. Michener CD. 1974. The social behavior of the bees. Belknap Press, Cam-bridge, MA.56. Engel P, Martinson VG, Moran NA. 2012. Functional diversity withinthe simple gut microbiota of the honey bee. Proc. Natl. Acad. Sci. U. S. A.109:11002–11007.57. Ratnieks FLW, Reeve HK. 1991. The evolution of queen-rearing nepo-tism in social Hymenoptera: effects of discrimination costs in swarmingspecies. J. Evol. Biol. 4:93–115.58. Goulson D. 2003. Bumblebees: their behaviour and ecology. Oxford Uni-versity Press, Oxford, United Kingdom.59. Koch H, Cisarovsky G, Schmid-Hempel P. 2012. Ecological effects ongut bacterial communities in wild bumblebee colonies. J. Anim. Ecol.81:1202–1210.60. Dornhaus A, Chittka L. 2005. Bumble bees (Bombus terrestris) store bothfood and information in honeypots. Behav. Ecol. 16:661–666.61. Schmid-Hempel P. 2001. On the evolutionary ecology of host-parasiteinteractions: addressing the question with regard to bumblebees and theirparasites. Naturwissenschaften 88:147–158.62. Sorensen A, Busch TM, Vinson SB. 1985. Trophallaxis by temporalsubcastes in the fire ant, Solenopsis invicta, in response to honey. Physiol.Entomol. 10:105–111.63. Mehdiabadi NJ, Schultz TR. 2010. Natural history and phylogeny of thefungus-farming ants (Hymenoptera: Formicidae: Myrmicinae: Attini).Myrmecol. News 13:37–55.64. Carina Audisio M, Torres MJ, Sabate DC, Ibarguren C, Apella MC.2011. Properties of different lactic acid bacteria isolated from Apis mel-lifera L. bee-gut. Microbiol. Res. 166:1–13.65. Lofgren CS, Banks WA, Glancey BM. 1975. Biology and control ofimported fire ants. Annu. Rev. Entomol. 20:1–30.66. Kleerebezem M, Boekhorst J, Van Kranenburg R, Molenaar D, KuipersOP, Leer R, Tarchini R, Peters SA, Sandbrink HM, Fiers MWEJ. 2003.Complete genome sequence of Lactobacillus plantarum WCFS1. Proc.Natl. Acad. Sci. U. S. A. 100:1990–1995.67. Schloss PD, Westcott SL. 2011. Assessing and improving methods used inoperational taxonomic unit-based approaches for 16S rRNA gene se-quence analysis. Appl. Environ. Microbiol. 77:3219–3226.68. Naidu A, Bidlack W, Clemens R. 1999. Probiotic spectra of lactic acidbacteria (LAB). Crit. Rev. Food Sci. Nutr. 39:13–126.69. Champomier-Verges MC, Chaillou S, Cornet M, Zagorec M. 2001.Lactobacillus sakei: recent developments and future prospects. Res. Micro-biol. 152:839–848.70. Liu Z, Lozupone C, Hamady M, Bushman FD, Knight R. 2007. Shortpyrosequencing reads suffice for accurate microbial community analysis.Nucleic Acids Res. 35:e120. doi:10.1093/nar/gkm541.McFrederick et al.1812 aem.asm.org Applied and Environmental MicrobiologyonFebruary26,2013byUnivofTexasAustinhttp://aem.asm.org/Downloadedfrom
  12. 12. Supplemental file S1. Supplemental text for 16S rRNA sequence alignment, secondarystructure models, and Lactobacillus phylogenetics. 16S rRNA secondary structuremodels 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 smallclosed circles, A-G nucleotide pairs are represented by large open circles, and all otherpairs are represented by large closed circles.The role of 16S rRNA secondary structure in Lactobacillus phylogeneticsIntroductionThe 16S rRNA gene has so far failed to yield robust phylogenies for the genusLactobacillus (1), despite an abundance of 16S rDNA sequence information (68,044sequences, National Center for Biotechnology Information nucleotide database, accessed14 June 2012). Previous 16S Lactobacillus phylogenies, however, did not utilize thehigher order structure of the 16S RNA molecule to optimize the sequence alignment, anessential step to reconstruct the best phylogenetic tree (2, 3). Comparative structuralmodels of the Lactobacillus 16S rRNAs will improve the quality of sequence alignmentsand improve phylogenetic reconstructions and the analysis of 16S-based next-generationsequencing surveys of bacterial communities. Of the nine hypervariable regions of the16S gene, some windows (e.g.V3-V5, positions ~400- 860 Escherichia coli numbering)are not sufficient for classification of most human-associated Lactobacillaceae sequencesto the genus level (4). Other 16S regions, such as V1-V2 (positions ~60- 220 E. colinumbering), may allow for better resolution (5). In addition, better alignments thatexplicitly juxtapose analogous secondary structure may also improve taxonomicassignment of next-generation 16S amplicon sequences. Due to the ubiquitous use of16S rRNA information for bacterial identification and phylogenetic reconstruction,knowledge of 16S secondary structure and its utility in DNA sequence alignments isclearly needed.MethodsSequence Alignment and RNA Secondary StructureComparative analysis is used to identify an RNA secondary structure that iscommon to a set of sequences that are known to have the same function and higher-orderstructure. Starting in the early 1980s, covariation analysis, a specific type ofcomparative analysis, was applied to a sequence alignment to predict a Bacterial 16SrRNA secondary structure (6). Initially the simplier covariation methods only identifiedcanonical base pairs (ie. G:C, A:U, and G:U) that occurred within a regular helixcomposed of at least two base pairs that are consecutive and anti-parallel with oneanother. However, more sophisticated covariation methods now identify all types ofcanonical and non-canonical base pairs with a set of nucleotides that covary with oneanother, regardless of their proximity to other structural elements (7, 8). In parallel withthe contribution of more accurate and sensitive covariation methods, the originalsecondary structure models were refined as the number and phylogenetic and structuraldiversity of 16S rRNA sequences increased. A few of the originally proposed base pairswere 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
  13. 13. 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 canonicalG: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 arrangedadjacent and anti-parallel with one another to form nested helices, while a few helicesformed pseudoknots and other base pairs were arranged in parallel (3).In addition to the refinements in the comparative structure models, our confidencein the proposed comparative structure model were quantified and increased with thelarger datasets that were analyzed with more sophisticated computational methods (9).With a sufficiently broad sampling of Bacterial 16S rRNAs, strong and robust patterns ofsequence and secondary structure conservation and variation were discovered. Patterns ofstructural variation at specific regions of the 16S rRNA were identified and shown to bediagnostic 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 therRNA comparative structure models were substantiated with site directed mutagenesis,chemical modifications, and other experiments, the most authoritative assessment of theaccuracy was determined from the high resolution crystal structures of the 30S and 50Sribosomal subunits (11, 12). Approximately 97% of the base pairs, including all of thenon-canonical base pairs, psuedoknots, and other irregular structural elements in thecomparative 16S rRNA secondary structure models are in the crystal structure (13). Thestrength of the covariation for each predicted base pair has been quantified and used as aconfidence rating for each base pair(http://www.rna.ccbb.utexas.edu/SAE/2A/nt_Frequency/BP/). Given this very highaccuracy, as gauged with the high-resolution crystal structures, we are most confidentthat all or nearly all of the base pairs in the comparative structure models for the rRNAsfrom different organisms are also correct, including all of the 16S rRNA secondarystructure models for the Lactobacillus.The accuracy of these predicted secondary structure models are directlyassociated with the quality of the sequence alignment. Better alignments facilitate bettercomparative structure models. And vice a versa, very accurate comparative structuremodels are the basis for the juxtaposition of nucleotides that are components of similarand analogous structural elements, especially in those highly variable regions that canhave large variances in the number of nucleotides with little or no sequence identity.Thus these secondary structure-based alignments do not attempt to maximize sequenceidentity a priori. Instead they attempt to maximize the alignment of similar structuralelements.After the 16S rRNA sequences were semi-automatically aligned with thetemplate-based alignment program - CRWAlign (14). We refined the alignmentmanually with the alignment editor ‘‘AE2’’ (developed by T. Macke, Scripps ResearchInstitute, San Diego, CA; 15). This tool was developed for Sun Microsystems’ (SantaClara, CA, USA) workstations running the Solaris operating system.The manual alignment process utilizes the CRWAlign program to identifynucleotides in a column that might not map to the same locations in the secondary andtertiary structure. A visual inspection of the alignment determines if these flaggednucleotides should be realigned. If necessary, this is done manually with the AE2alignment editor. For regions of the alignment with high sequence similarity and
  14. 14. minimal variance in the number of nucleotides, the information in the primary structure issufficient to align sequences with confidence. In contrast, for more variable regions inclosely related sequences or between more distantly related sequences, a high-qualityalignment can only be produced when secondary and/or tertiary structure information isincluded.Secondary Structure DiagramsSecondary structure diagrams were generated after the first secondary structure modelwas derived with comparative methods. These diagrams were refined as the model wasmodified based on increased data and improved covariation methods (see above). Whilethe Comparative RNA Web (CRW) Site (http://www.rna.ccbb.utexas.edu/) contains morethan 200 16S rRNA secondary structure diagrams that sample the diversity within theBacteria, 47 secondary structure diagrams for each of the major phylogenetic groups ofLactobacillus and a few related organisms were generated for this study. The secondarystructure diagrams were generated with the interactive secondary structure program -XRNA (written in the C programming language for Sun Microsystems’ workstationsrunning the Solaris operating system by B. Weiser and H. Noller, University ofCalifornia, Santa Cruz). All structure diagrams are available at:http://www.rna.ccbb.utexas.edu/.Discussion:Utility of 16S rDNA for Lactobacillus identification and phylogenyOur secondary structural models of 16S rRNA indicate that the hairpin stem andloop in V1 is the most variable region in Lactobacillus. Accordingly, 16S primerstargeting this region for next-generation sequencing surveys of Lactobacillus shouldprovide the best taxonomic resolution for closely related taxa. Other regions that exhibitvariation 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 structuralmodels that we present here will also serve as valuable resources for aligningLactobacillus 16S rRNA.Our 16S rRNA phylogenetic tree had high support values for terminal branchingpatterns but not deeper branching patterns in the Lactobacillus phylogeny. Even with ourhighly accurate and structurally informed sequence alignment, the 16S rRNA gene didnot confidently resolve the deeper branches in the Lactobacillus phylogeny. TheLactobacillus phylogeny has so far been reconstructed using both 16S rDNA sequenceinformation (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 developedfor several lactobacilli and their relatives (19-24), and sequences from these studies maybe useful in designing primers for single copy protein coding genes conserved across thegenus Lactobacillus, thereby providing greater phylogenetic resolution and coverage ofthe genus.While our analysis of 16S rRNA did not resolve the deeper branches in theLactobacillus phylogeny, 16S rRNA does place sequences into terminal clades withconfidence. Clade membership in all of our analyses was generally well supported, andlargely agrees with the clade structures suggested in previous studies (16-18). By using
  15. 15. alignments based on full-length sequences only, partial and complete sequencescombined, and partial sequences only, we were able to assess the influence of missingdata or 454 pyrosequencing length data on phylogenetic reconstructions. Although thefull-length and partial and complete analyses differed in the placement of some deeperbranches, the inclusion of partial sequences had little effect on the overall results. Theplacement of the partial and complete sequences into their respective clades was almostcompletely congruent with the phylogeny based on full-length sequences. The analysisusing only partial sequences performed poorly with regard to the placement of deeperbranches, but placed sequences robustly into terminal clades remarkably well. Otherstudies have found that short pyrosequencing data can be placed accurately into anexisting full-length phylogeny (25), and our results suggest that if a highly variableregion (i.e., the V1 region for Lactobacillus) can be accurately aligned, alignments builton short pyrosequencing sized data can accurately place sequences into terminal clades.Implications for Lactobacillus systematicsBy gathering all publicly available 16S sequences and clustering sequences thatshared 97% or greater sequence identity, we created an updated, complete representationof the diversity of the genus Lactobacillus. In agreement with previous studies (17), wedetermined that the L. delbrueckii (acidophilus) clade represents a significant portion ofLactobacillus diversity. Also in agreement with previous studies (1, 26), we determinedthat Paralactobacillus and Pediococcus are within Lactobacillus, and that the overalltaxonomy of the genus Lactobacillus needs significant revisionary work.References:1. Claesson, M. J., D. van Sinderen, and P. W. OToole. 2008. Lactobacillusphylogenomics-towards a reclassification of the genus. International Journal ofSystematic and Evolutionary Microbiology 58:2945-2954.2. Woese, C. R. 1987. Bacterial evolution. Microbiological Reviews 51:221.3. Gutell, R. R., N. Larsen, and C. R. Woese. 1994. Lessons from an evolvingrRNA: 16S and 23S rRNA structures from a comparative perspective.Microbiological Reviews 58:10-26.4. Jumpstart Consortium Human Microbiome Project Data GenerationWorking Group. 2012. Evaluation of 16S rDNA-Based Community Profiling forHuman Microbiome Research. PLoS ONE 7:e39315.5. Kullen, M. J., R. B. Sanozky-Dawes, D. C. Crowell, and T. R. Klaenhammer.2000. Use of the DNA sequence of variable regions of the 16S rRNA gene forrapid and accurate identification of bacteria in the Lactobacillus acidophiluscomplex. Journal of Applied Microbiology 89:511-516.6. Woese, C. R., L. J. Magrum, R. Gupta, R. B. Siegel, D. A. Stahl, J. Kop, N.Crawford, R. Brosius, R. Gutell, J. J. Hogan, and H. F. Noller. 1980.Secondary structure model for bacterial 16S ribosomal RNA: phylogenetic,enzymatic and chemical evidence. Nucleic Acids Research 8:2275-2294.7. Gutell, R., A. Power, G. Hertz, E. Putz, and G. Stormo. 1992. Identifyingconstraints on the higher-order structure of RNA: continued development andapplication of comparative sequence analysis methods. Nucleic Acids Research20:5785-5795.
  16. 16. 8. Shang, L., W. Xu, S. Ozer, and R. R. Gutell. 2012. Structural constraintsidentified with covariation analysis in ribosomal RNA. PLoS ONE 7:e39383.9. Cannone, J., S. Subramanian, M. Schnare, J. Collett, L. DSouza, Y. Du, B.Feng, N. Lin, L. Madabusi, K. Mueller, N. Pande, Z. Shang, N. Yu, and R. R.Gutell. 2002. The comparative RNA web (CRW) site: an online database ofcomparative sequence and structure information for ribosomal, intron, and otherRNAs. BMC Bioinformatics 3:2.10. Gutell, R. R. 1992. Presented at the The Symposium--The Origin and Evolutionof Prokaryotic and Eukaryotic Cells, Shimoda, Japan.11. Wimberly, B. T., D. E. Brodersen, W. M. Clemons, R. J. Morgan-Warren, A.P. Carter, C. Vonrhein, T. Hartschk, and V. Ramakrishnan. 2000. Structureof the 30S ribosomal subunit. Nature 407:327-339.12. Ban, N., P. Nissen, J. Hansen, P. B. Moore, and T. A. Steitz. 2000. Thecomplete atomic structure of the large ribosomal subunit at 2.4 a resolution.Science 289:905-920.13. Gutell, R. R., J. C. Lee, and J. J. Cannone. 2002. The accuracy of ribosomalRNA comparative structure models. Current Opinion in Structural Biology12:301-310.14. Gardner, D. P., W. Xu, D. P. Miranker, S. Ozer, J. J. Cannone, and R. R.Gutell. In Press. Presented at the IEEE International Conference onBioinformatics and Biomedicine (BIBM 2012), Philadelphia, USA.15. Larsen, N., G. J. Olsen, B. L. Maidak, M. J. McCaughey, R. Overbeek, T. J.Macke, T. L. Marsh, and C. R. Woese. 1993. The ribosomal database project.Nucleic Acids Research 21:3021-3023.16. Canchaya, C., M. J. Claesson, G. F. Fitzgerald, D. Van Sinderen, and P. W.OToole. 2006. Diversity of the genus Lactobacillus revealed by comparativegenomics of five species. Microbiology 152:3185-3196.17. Felis, G. E., and F. Dellaglio. 2007. Taxonomy of Lactobacilli andBifidobacteria. Current Issues in Intestinal Microbiology 8:44-61.18. Pot, B., and E. Tsakalidou. 2009. Taxonomy and metabolism of Lactobacillus,p. 3-58. In A. Ljungh and T. Wadstrom (ed.), Lactobacillus Molecular Biology:from Genomics to Probiotics. Caister Academic Press, Norfolk, UK.19. Ruiz-Garbajosa, P., M. J. M. Bonten, D. A. Ronbinson, J. Top, S. R.Nallapareddy, C. Torres, T. M. Coque, R. Canton, F. Baquero, B. E. Murray,R. del Campo, and R. J. L. Willems. 2006. Multilocus sequence typing schemefor Enterococcus faecalis reveals hospital-adapted genetic complexes in abackground of high rates of recombination. Journal of Clinical Microbiology44:2220-2228.20. Berger, B., D. P. Pridmore, C. Barretto, F. Delmas-Julien, K. Schreiber, F.Arigoni, and H. Brussow. 2007. Similarity and differences in the Lactobacillusacidophilus group identified by polyphasic analysis and comparative genomics.Journal of Bacteriology 189:1311-1321.21. Cai, H., B. T. Rodriguez, W. Zhang, J. R. Broadbent, and J. L. Steele. 2007.Genotypic and phenotypic characterization of Lactobacillus casei strains isolatedfrom different ecological niches suggests frequent recombination and nichespecificity. Microbiology 153:2655-2665.
  17. 17. 22. Raftis, E. J., E. Salvetti, S. Torriani, G. E. Felis, and P. W. OToole. 2011.Genomic diversity of Lactobacillus salivarius. Applied and EnvironmentalMicrobiology 77:954-965.23. Picozzi, C., G. Bonacina, I. Vigentini, and R. Foschino. 2010. Genetic diversityin Italian Lactobacillus sanfranciscensis strains assessed by multilocus sequencetyping and pulsed-field gel electrophoresis analyses. Microbiology 156:2035-2045.24. Buhnik-Rosenblau, K., V. Matsko-Efimov, M. Jung, H. Shin, Y. Danin-Poleg,and Y. Kashi. 2012. Indication for co-evolution of Lactobacillus johnsonii withits host. BMC Microbiology 12:149.25. Liu, Z., C. Lozupone, M. Hamady, F. D. Bushman, and R. Knight. 2007.Short pyrosequencing reads suffice for accurate microbial community analysis.Nucleic Acids Research 35:e120.26. Haakensen, M., V. Pittet, and B. Ziola. 2011. Reclassification ofParalactobacillus selangorensis Leisner et al. 2000 as Lactobacillusselangorensis comb. nov. International Journal of Systematic and EvolutionaryMicrobiology 61:2979-2983.  
  18. 18. Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal RNA5ʼ3ʼLactobacillus acidophilus(GenBank accession number M58802)1.cellular organisms 2.Bacteria 3.Firmicutes4.Bacilli 5.Lactobacillales 6.Lactobacillaceae7.LactobacillusJune 2012NNAAAACGAG A G U UU GAUCCUGGCUCAGGACGAACGCUGGCGGCGUGCCUAAUACAUGCAAG U CG AG C G A G C U G AACCAACAGAUUCACUUCGGUGAUGACGUUGGGNAACGCUAGCGGCGGAUGGGUGAGUAACACGUGGGGAACCUGCCCCAUAGUC U G GGA UAC C A C U U G GAAACAGGUGCUAAUACCGGAUAAGAAAGCAGAUCGCAUGAUCAGCUUAUAAAAGGCGGCGUAA G C U G U C GCUAUGGNNUGGCCCCGCGGUGCAUUAGCUAGUUGGUAGGGUAACGG CCUACCAAGGCAAUG AUGCAUAGCCGAGUUGAG AGA C UG AUC GGC CACAUUGGGACUGAGAC A CGG C C C A AACUCCUAC GGGAGGC A GCNGUAGGGAAUCUUCCACAAUGGACGAAA G U C U GAU G G A GCAA CGCCGCGUGAGUGAAGAAGGUUUUCG G A U CGU A AAG C U CUGUUGUUGGUGAAGAAGGAUAGAGGUAGUAA CUGGCCUUUA U UUGAC G GUAAUCAACCAGAAAGUCACGGCU A ACUACGUGCCAGCAG C CGC GGUAAUACGUAGGUGGCNAGCGUUGUCCGGAUUUAUUG GGCGUAAAGCGAGCGCAGGCGGAAGAAUAAGUCUGAUGUGAAAGCCCUCGGCUUAA C C G A G G A A C U G C A U C G G AAAC U G U U U U U CUUGAGUGCAGAAGAGGAGAGUGGAACUCCAUGUGUAGCGGUGGA A U G CGUA GAUA U A U G G A A G A A CAC CAGU GG C GAAGGCGGCUCUCUGGUCUGCAACUGACGCUGAGGCUC NNAAGCAUGGGUA GCGAACAGGAUUA G AUACCCUGGUAGUCCAUGC C G UAAACGAUG A G U G C U A AGUGUUGGGAGGUU UCCGCCUCUCAGUGCU GCAGCUAACGCAUUAAGCACUCCGCCUG G GGA G U ACG A C C GCAAGGUUGAAACUCAAAG G A A U U G A C GGG G N C C C GCA C A A GCGGUGGAGCAUGUGGUUUAAUUCGAAGCAACG CGAAGAAC C U UACCAGGUCUUGACAUCUAGUGCAAUCCGUAGAGA U A C G G N G U UCCCUU CGGGGACACUA AGAC AGGUGGUGCA UGGCUGUCGUCAGCUCGUGUCGUGAGAUGUUGGGUUA AGUCCCG CAA C G A G UGC A ACC C U U G U C A U U A GU U GC CA GC AU UAAGUUGGGCACUCUAAUGAGACUGCCGGUGACAAACCGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGCCCC UUAUGACCUGGGCUACACACGUGCUAC A AUGGACAGUACA A C GAGGA GCAA GCCUG CGAAGGCAAGCGAAUCUCUUAAAGCUGUUCUCAGUUCGGACUGCAGUCUGCAACUCGACUGCACGAAGCUGGAAUCGCUAGUAAUCGCGGAUCAGCACGCCGCGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUCUGCAAUGCCCAAAGCCGGUGGCCUAA CCUU CGGGAAGGAGCCGUCUAAGGCAGGGCAGAUGACNNNNNNNNNNNNGUAACA AGNN N N N N N N N N N NGAACCUGNNNNNNGAUCACCUCCUUUCUACanonical nucleotide pairs (G-C or A-U) arerepresented by dashes, wobble nucleotide pairs (G-U)are represented by small closed circles, A-G nucleotidepairs are represented by large open circles, and allother pairs are represented by large closed circles.Taxonomy:
  19. 19. Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal RNA5’3’Lactobacillus delbrueckii subsp.bulgaricus 2038(CP000156)1.cellular organisms 2.Bacteria 3.Firmicutes4.Bacilli 5.Lactobacillales 6.Lactobacillaceae7.Lactobacillus 8.Lactobacillus delbrueckii9.Lactobacillus delbrueckii subsp. bulgaricusJuly 2012AAAUUGAG A G U UU GAUCCUGGCUCAGGACGAACGCUGGCGGCGUGCCUAAUACAUGCAAG U CG AGCGAGCUGAAUUCAAA GA UUCC UUCGGGAUGAUUUGUUGGACGCUAGCGGCGGAUGGGUGAGUAACACGUGGGC AAUCUGCCCUAAAGAC U G GGA UAC C A C U U G GAAACAGGUGCUAAUACCGGAUAACAACAUGAAUCGCAUGAUUCAAGUUUGAAAGGCGGCGCAA G C U G U C ACUUUAGGAUGAGCCCGCGGCGCAUUAGCUAGUUGGUGGGGUAAAGG CCUACCAAGGCAAUG AUGCGUAGCCGAGUUGAG AGA C UG AUC GGC CACAUUGGGACUGAGAC A CGG C C C A AACUCCUAC GGGAGGC A GCAGUAGGGAAUCUUCCACAAUGGACGCAA G U C U G A U G G A GCAA CGCCGCGUGAGUGAAGAAGGUUUUCG G A U CGU A AAG C U CUGUUGUUGGUGAAGAAGGAUAGAGGCAGUAA CUGGUCUUUA U UUGAC G GUAAUCAACCAGAAAGUCACGGCU A ACUACGUGCCAGCAG C CGC GGUAAUACGUAGGUGGCAAGCGUUGUCCGGAUUUAUUG GGCGUAAAGCGAGCGCAGGCGGAAUGAUAAGUCUGAUGUGAAAGCCCACGGCUCAA C C G U G G A A C U G C A U C G G AAAC U G U C A U U CUUGAGUGCAGAAGAGGAGAGUGGAAUUCCAUGUGUAGCGGUGGA A U G CGUA GAUA U A U G G A AG AA CAC CAGU GG C GAAGGCGGCUCUCUGGUCUGCAACUGACGCUGAGGCUC GAAAGCAUGGGUA GCGAACAGGAUUA G AUACCCUGGUAGUCCAUGC C G UAAACGAUG A G C G CU A GGUGUUGGGGACUU UCCGGUCCUCAGUGCC GCAGCAAACGCAUUAAGCGCUCCGCCUG G GGA G U ACG A C C GCAAGGUUGAAACUCAAAG G A A U U G A C GGG G G C C C GCA C A A GCGGUGGAGCAUGUGGUUUAAUUCGAAGCAACG CGAAGAAC C U UACCAGGUCUUGACAUCCUGUGCUACACCUAGAGA U A G G U G G U UCCCUU CGGGGACGCAG AGAC AGGUGGUGCA UGGCUGUCGUCAGCUCGUGUCGUGAGAUGUUGGGUUA AGUCCCG CAA C G A G CGC A ACC C U U G U C U U U A GU U GC CA UC AU UAAGUUGGGCACUCUAAAGAGACUGCCGGUGACAAACCGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGCCCC UUAUGACCUGGGCUACACACGUGCUAC A AUGGGCAGUACA A C GAGAA GCGA A CCCG CGAGGGUAAGCGGAUCUCUUAAAGCUGUUCUCAGUUCGGACUGCAGGCUGCAACUCGCCUGCACGAAGCUGGAAUCGCUAGUAAUCGCGGAUCAGCACGCCGCGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGAAGUCUGCAAUGCCCAAAGUCGGUGGGAUAA CCUUU AUAGGAGUCAGCCGCCUAAGGCAGGGCAGAUGACUGGGGUGAAGUCGUAACA AGGU A G C C G U A G G AGAACCUGCGGCUGGAUCACCUCCUUUCUAAGGAA
  20. 20. Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal RNA5’3’Lactobacillus amylovorus GRL 1112(CP002338)1.cellular organisms 2.Bacteria 3.Firmicutes4.Bacilli 5.Lactobacillales 6.Lactobacillaceae7.Lactobacillus 8.Lactobacillus amylovorusJuly 2012UUUAAAAUGAG A G U UU GAUCCUGGCUCAGGACGAACGCUGGCGGCGUGCCUAAUACAUGCAAG U CG AGCGAGCGGAACCA ACA GA UUUAC UUCGGUAAUGACGUUGGGAAAGCGAGCGGCGGAUGGGUGAGUAACACGUGGGGAACCUGCCCCUAAGUC U G GGA UAC C A U U U G GAAACAGGUGCUAAUACCGGAUAAUAAAGCAGAUCGCAUGAUCAGCUUUUGAAAGGCGGCGUAA G C U G U C GCUAAGGGAUGGCCCCGCGGUGCAUUAGCUAGUUGGUAAGGUAACGG CUUACCAAGGCGACG AUGCAUAGCCGAGUUGAG AGA C UG AUC GGC CACAUUGGGACUGAGAC A CGG C C C A AACUCCUAC GGGAGGC A GCAGUAGGGAAUCUUCCACAAUGGACGCAA G U C U G A U G G A GCAA CGCCGCGUGAGUGAAGAAGGUUUUCG G A U CGU A AAG C U CUGUUGUUGGUGAAGAAGGAUAGAGGUAGUAA CUGGCCUUUA U UUGAC G GUAAUCAACCAGAAAGUCACGGCU A ACUACGUGCCAGCAG C CGC GGUAAUACGUAGGUGGCAAGCGUUGUCCGGAUUUAUUG GGCGUAAAGCGAGCGCAGGCGGAAAAAUAAGUCUAAUGUGAAAGCCCUCGGCUUAA C C G A G G A A C U G C A U C G G AAAC U G U U U U U CUUGAGUGCAGAAGAGGAGAGUGGAACUCCAUGUGUAGCGGUGGA A U G CGUA GAUA U A U G G A AG AA CAC CAGU GG C GAAGGCGGCUCUCUGGUCUGCAACUGACGCUGAGGCUC GAAAGCAUGGGUA GCGAACAGGAUUA G AUACCCUGGUAGUCCAUGC C G UAAACGAUG A G U G CU A AGUGUUGGGAGGUU UCCGCCUCUCAGUGCU GCAGCUAACGCAUUAAGCACUCCGCCUG G GGA G U ACG A C C GCAAGGUUGAAACUCAAAG G A A U U G A C GGG G G C C C GCA C A A GCGGUGGAGCAUGUGGUUUAAUUCGAAGCAACG CGAAGAAC C U UACCAGGUCUUGACAUCUAGUGCAAUCUGUAGAGA U A C G G A G U UCCCUU CGGGGACGCUA AGAC AGGUGGUGCA UGGCUGUCGUCAGCUCGUGUCGUGAGAUGUUGGGUUA AGUCCCG CAA C G A G CGC A ACC C U U G U U A U U A GU U GC CA GC AU UAAGUUGGGCACUCUAAUGAGACUGCCGGUGACAAACCGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGCCCC UUAUGACCUGGGCUACACACGUGCUAC A AUGGGCAGUACA A C GAGAA GCAA GCCUG CGAAGGCAAGCGAAUCUCUGAAAGCUGUUCUCAGUUCGGACUGCAGUCUGCAACUCGACUGCACGAAGCUGGAAUCGCUAGUAAUCGCGGAUCAGCACGCCGCGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGGAGUCUGCAAUGCCCAAAGCCGGUGGCCUAA CCUU CGGGAAGGAGCCGUCUAAGGCAGGGCAGAUGACUGGGGUGAAGUCGUAACA AGGU A G C C G U A G G AGAACCUGCGGCUGGAUCACCUCCUUUCUAAGGAAG
  21. 21. Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal RNA5’3’Lactobacillus gasseri ATCC 33323(AF519171)1.cellular organisms 2.Bacteria 3.Firmicutes4.Bacilli 5.Lactobacillales 6.Lactobacillaceae7.Lactobacillus 8.Lactobacillus gasseriJuly 2012AAAAUGAG A G U UU GAUCCUGGCUCAGGACGAACGCUGGCGGCGUGCCUAAUACAUGCAAG U CG AGCGAGCUUG CCUAGAUGAAUUUGGUGCUUGCACCAGAUGAAACUAGAUACAAGCGAGCGGCGGACGGGUGAGUAACACGUGGGU AACCUGCCCAAGAGAC U G GGA UAA C A C C U G GAAACAGAUGCUAAUACCGGAUAACAACACUAGACGCAUGUCUAGAGUUUAAAAGAUGGUUCU G C U A U C ACUCUUGGAUGGACCUGCGGUGCAUUAGCUAGUUGGUAAGGUAACGG CUUACCAAGGCAAUG AUGCAUAGCCGAGUUGAG AGA C UG AUC GGC CACAUUGGGACUGAGAC A CGG C C C A AACUCCUAC GGGAGGC A GCAGUAGGGAAUCUUCCACAAUGGACGCAA G U C U G A U G G A GCAA CGCCGCGUGAGUGAAGAAGGGUUUCG G C U CGU A AAG C U CUGUUGGUAGUGAAGAAAGAUAGAGGUAGUAA CUGGCCUUUA U UUGAC G GUAAUUACUUAGAAAGUCACGGCU A ACUACGUGCCAGCAG C CGC GGUAAUACGUAGGUGGCAAGCGUUGUCCGGAUUUAUUG GGCGUAAAGCGAGUGCAGGCGGUUCAAUAAGUCUGAUGUGAAAGCCUUCGGCUCAA C C G G A G A A U U G C A U C A G AAAC U G U U G A A CUUGAGUGCAGAAGAGGAGAGUGGAACUCCAUGUGUAGCGGUGGA A U G CGUA GAUA U A U G G A AG AA CAC CAGU GG C GAAGGCGGCUCUCUGGUCUGCAACUGACGCUGAGGCUC GAAAGCAUGGGUA GCGAACAGGAUUA G AUACCCUGGUAGUCCAUGC C G UAAACGAUG A G U G CU A AGUGUUGGGAGGUU UCCGCCUCUCAGUGCU GCAGCUAACGCAUUAAGCACUCCGCCUG G GGA G U ACG A C C GCAAGGUUGAAACUCAAAG G A A U U G A C GGG G G C C C GCA C A A GCGGUGGAGCAUGUGGUUUAAUUCGAAGCAACG CGAAGAAC C U UACCAGGUCUUGACAUCCAGUGCAAACCUAAGAGA U U A G G U G U UCCCUU CGGGGACGCUG AGAC AGGUGGUGCA UGGCUGUCGUCAGCUCGUGUCGUGAGAUGUUGGGUUA AGUCCCG CAA C G A G CGC A ACC C U U G U C A U U A GU U GC CA UC AU UAAGUUGGGCACUCUAAUGAGACUGCCGGUGACAAACCGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGCCCC UUAUGACCUGGGCUACACACGUGCUAC A AUGGACGGUACA A C GAGAA GCGA A CCUG CGAAGGCAAGCGGAUCUCUGAAAGCCGUUCUCAGUUCGGACUGUAGGCUGCAACUCGCCUACACGAAGCUGGAAUCGCUAGUAAUCGCGGAUCAGCACGCCGCGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGAGAGUCUGUAACACCCAAAGCCGGUGGGAUAA CCUUU AUAGGAGUCAGCCGUCUAAGGUAGGACAGAUGAUUAGGGUGAAGUCGUAACA AGGU A G C C G U A G G AGAACCUGCGGCUGGAUCACCUCCUUU
  22. 22. Citation and related information available at http://www.rna.ccbb.utexas.eduSecondary Structure: small subunit ribosomal RNA5’3’Lactobacillus delbrueckii subsp. lactis(M58823)1.cellular organisms 2.Bacteria 3.Firmicutes4.Bacilli 5.Lactobacillales 6.Lactobacillaceae7.Lactobacillus 8.Lactobacillus delbrueckiiJune 2012NNCAAAUUGAG A G U UU GAUCCUGGCUCAGGACGAACGCUGGCGGCGUGCCUAAUACAUGCAAG U CG AGCGA GCUGA AUUCA AA GAUUCCUUCGGGAGGAUUUGUUGGANNNNAGCGGCGGAUGGGUGAGUAACACGUGGGC AAUCUGCCCUAAAGAC U G GGA UAC C A C U U G GAAACAGGUGCUAAUACCGGAUAACAACAUGAAUCGCAUGAUUCAAGUUUGAAAGGCGGCGCAA G C U G U C ACUUUAGGAUGAGCCCGCGGCGCAUUAGCUAGUUGGUGGGGUAAAGG CCUACCAAGGCAAUG AUGCGUAGCCGAGUUGAG AGA C UG AUC GGC CACAUUGGGACUGAGAC A CGG C C C A AACUCCUAC GGGAGGC A GCAGUAGGGAAUCUUCCACAAUGGACGCAA G U C U G A U G G A GCAA CGCCGCGUGAGUGAAGAAGGUCUUCG G A U CGU A AAG C U CUGUUGUUGGUGAAGAAGGAUAGAGGCAGUAA CUGGUCUUUA U UUGAC R GUAAUCAACCAGAAAGUCACGGCU A ACUACGUGCCAGCAG C CGC GGUAAUACGUAGGUGGCAAGCGUUGUCCGGAUUUAUUG GGCGUAAAGCGAGCGCAGGCGGAAUGAUAAGUCUGAUGUGAAAGCCCACGGCUCAA C C G U G G A A C U G C A U C G G AAAC U G U C A U U CUUGAGUGCAGAAGAGGAGAGUGGAACUCCAUGUGUAGCGGUGGA A U G CGUA GAUA U A U G G A AG AA CAC CAGU GG C GAAGGCGGCUCUCUGGUCUGCAACUGACGCUGAGGCUC GAAAGCAUGGGUA GCGAACAGGAUUA G AUACCCUGGUAGUCCAUGC C G UAAACGAUG A G C G CU A GGUGUUGGGGACUU UCCGGUCCUCAGUGCC GCAGCAAACGCAUUAAGCGCUCCGCCUG G GGA G U ACG A C C GCAAGGUUGAAACUCAAAG G A A U U G A C GGG G G C C N GCA C A A GCGGUGGAGCAUGUGGUUUAAUUCGAAGCAACG CGAAGAAC C U UACCAGGUCUUGACAUCCUGCGCUACACCUAGAGA U A G G C C G U UCCCUU CGGGGACGCAG AGAC AGGUGGUGCA UGGCUGUCGUCAGCUCGUGUCGUGAGAUGUUGGGUUA AGUCCCG CAA C G A G CGC A ACC C U U G U C U U U A GU U GC CA UC AU UAAGUUGGGCACUCUAGAGAGACUGCCGGUGACAAACCGGAGGAAGGUGGGGAUGACGUCAAGUCAUCAUGCCCC UUAUGACCUGGGCUACACACGUGCUAC A AUGGGCAGUACA A C GUGAA GCGA A CCCG CGAGGGUAAGCGGAUCUCUUAAAGCUGCUCUCAGUUCGGACUGCAGGCUGCAACUCGCCUGCACGAAGCUGGAAUCGCUAGUAAUCGCGGAUCAGCACGCCGCGGUGAAUACGUUCCCGGGCCUUGUACACACCGCCCGUCACACCAUGGAAGUCUGCAAUGCCCAAAGUCGGUGGGAUAA CCUUU AUAGGAGUCAGCCGCCUAAGGCAGGGCAGAUGACUGGGGNNNNNNNGUAACA AGNN N N N N N N N N N NGAACCUGNNNNNNGAUCACCUCCUUUCUA

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