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Atypical bacterial rRNA operon structure is prevalent within the Lachnospiraceae, and use of the 16S-23S rRNA internal transcribed spacer region for the rapid identification of ruminal Butyrivibrio and Pseudobutyrivibrio strains

Atypical bacterial rRNA operon structure is prevalent within the Lachnospiraceae, and use of the... Ann Microbiol (2014) 64:1623–1631 DOI 10.1007/s13213-014-0806-2 ORIGINAL ARTICLE Atypical bacterial rRNA operon structure is prevalent within the Lachnospiraceae, and use of the 16S-23S rRNA internal transcribed spacer region for the rapid identification of ruminal Butyrivibrio and Pseudobutyrivibrio strains Dong Li & Sinead Leahy & Gemma Henderson & William Kelly & Adrian Cookson & Graeme Attwood & Christina Moon Received: 20 May 2013 /Accepted: 9 January 2014 /Published online: 29 January 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract The rumen is the fermentative forestomach of rumi- transcribed spacer (ITS) regions located between the genes. nant animals, and is host to a wide range of anaerobic bacteria However, in the rumen bacterium, Butyrivibrio proteoclasticus whose primary function is to facilitate forage degradation. B316, rRNA operons have a 16S-5S-23S rRNA gene arrange- Butyrivibrio and Pseudobutyrivibrio are closely related proteo- ment, and analysis of bacterial genome projects revealed that lytic and fibrolytic genera within the family Lachnospiraceae, this configuration was present in all publicly available complete and are commonly isolated from the rumens of animals fed genomes from members of the family Lachnospiraceae. The fibrous diets. The ribosomal RNA (rRNA) operon is an impor- 16S-23S ITS region is commonly used to identify bacterial tant phylogenetically informative locus that is present in multi- strains, thus we sought to determine the utility of this region ple copies in bacterial genomes. Ribosomal RNA genes are from rumen Butyrivibrio and Pseudobutyrivibrio isolates for typically arranged in the order 16S-23S-5S, with internal their rapid molecular identification. Polymerase chain reaction wasusedtoamplify 16S-23S ITSregions, which were assessed for length polymorphism (ITS-LP), and restriction fragment Electronic supplementary material The online version of this article (doi:10.1007/s13213-014-0806-2) contains supplementary material, length polymorphism (ITS-RFLP) using AluI, HaeIII and which is available to authorized users. HhaIonapanelof13 Butyrivibrio and Pseudobutyrivibrio : : : : : reference strains. Cluster analysis of the resulting banding D. Li S. Leahy G. Henderson W. Kelly G. Attwood C. Moon (*) patterns revealed that while the ITS-LP method did not group Animal Nutrition and Health, AgResearch Ltd, Tennent Dr, Private the strains according to major Butyrivibrio and Bag 11008, Palmerston North 4442, New Zealand Pseudobutyrivibrio clades identified via 16S rRNA gene se- e-mail: christina.moon@agresearch.co.nz quences, ITS-RFLP was more discriminative, and able to rap- D. Li idly delineate the strains into these clades. e-mail: dong.li@agresearch.co.nz S. Leahy e-mail: sinead.leahy@agresearch.co.nz . . . Keywords Butyrivibrio DNAfingerprint Lachnospiraceae . . Pseudobutyrivibrio Ribosomal RNA gene operon G. Henderson e-mail: gemma.henderson@agresearch.co.nz Ribosomal intergenic spacer W. Kelly e-mail: bill.kelly@agresearch.co.nz G. Attwood Introduction e-mail: graeme.attwood@agresearch.co.nz A. Cookson The rumen is a complex environment where plant material is Animal Nutrition and Health, degraded through the symbiotic interactions of micro- AgResearch Ltd, Grasslands Research Centre, organisms and the host. The genera Butyrivibrio and Tennent Dr, Private Bag 11008, Palmerston North 4442, Pseudobutyrivibrio, formerly collectively known as New Zealand e-mail: adrian.cookson@agresearch.co.nz Butyrivibrio fibrisolvens (Bryant and Small 1956), represent 1624 Ann Microbiol (2014) 64:1623–1631 a considerable proportion of culturable rumen anaerobic knowledge of the specific functional roles and metabolic bacteria in domestic and wild ruminants, where they con- capabilities of individual taxa will require characterisation of tribute to fibre degradation (Kopečný et al. 2003). cultivated representatives. Members of these two genera are non-spore forming, Ribosomal RNA (rRNA) gene operons are phylogenetically anaerobic, motile, curved rod-shaped bacteria that are informative loci that are typically present in multiple copies per commonly isolated from the rumens of animals fed a wide genome (rRNA genes may be present in up to 15 copies each; range of diets (Stewart et al. 1997). All ruminal Lee et al. 2009), and span ca. 5 kb in length each. They normally Butyrivibrio species studied to date can degrade the hemi- have a 16S rRNA gene–intergenic spacer I–23S rRNA gene– cellulose component of plant cell walls, and many also intergenic spacer II–5S rRNA gene arrangement in bacteria and hydrolyze other plant polysaccharides including starch and may also contain small tRNA genes (Fig. 1). However, from an pectin (Hespell et al. 1987;Kopečný et al. 2003). analysis of all available bacterial whole genome sequences (ap- Phylogenetic studies currently place Butyrivibrio and proximately 1,200), it was reported that members of the Pseudobutyrivibrio within subcluster XIVa of the Lachnospiraceae (including B. proteoclasticus B316) commonly Clostridium subphylum (Collins et al. 1994), where they are possess an alternative gene arrangement within their rRNA members of the family Lachnospiraceae. Four Butyrivibrio operons, with rRNA genes in the order 16S-5S-23S rRNA species are presently recognised: B. fibrisolvens (type strain (Kelly et al. 2010;Fig. 1). Molecular analysis of the internal D1; Bryant and Small 1956); B. crossotus (type strain Bu46 ; transcribed spacer (ITS) region, which commonly employs 16S Moore et al. 1976), B. hungatei (type strain JK615 ;Kopečný and 23S rRNA gene-specific primers (Gürtler and Stanisich et al. 2003)and B. proteoclasticus (type strain B316 ;Moon 1996), has been used for the study of microbial community et al. 2008), formerly known as Clostridium proteoclasticum structure and identification of closely related isolates for a wide (Attwood et al. 1996). To the best of our knowledge, variety of bacterial taxa. This region exhibits a high degree of Butyrivibrio crossotus strains have not been isolated from sequence and length variation (Gürtler and Stanisich 1996; the rumen, but instead from human rectal or faecal material Hassan et al. 2003; Yannarell and Triplett 2004) and is consid- (Moore et al. 1976; Willems and Collins 2009). Furthermore, ered more useful for genus- and species-level identification than molecular data indicate that, within the Lachnospiraceae phy- the 16S rRNA gene alone (Rachman et al. 2003;Kwonet al. logeny, B. crossotus occupies a very disparate position to the 2004). Recently, ITS regions from Butyrivibrio isolates from the originally described rumen-derived Butyrivibrio species reindeer rumen were sequenced following PCR-amplification (Wade 2009), and thus in principle, is not considered con- and cloning (Præsteng et al. 2011). Considerable length poly- generic. Conversely, various molecular analyses have indicat- morphism was observed between ITS copies, and similar phy- ed that there are Butyrivibrio-like isolates that do not fit into logenetic relationships were observed based on the ITS se- these species boundaries, and are likely to represent novel quences as compared to 16S rRNA gene sequences, but species within Butyrivibrio (Willems and Collins 2009). At the ITS sequence-based phylogeny appeared to provide present, the genus Pseudobutyrivibrio contains two closely more detailed resolution between the isolates (Præsteng related species: P. ruminis (type strain A12-1 ; van Gylswyk et al. 2011). ITS analyses were not performed on et al. 1996)and P. xylanivorans (type strain Mz5 ;Kopečný Butyrivibrio type or reference strains, thus data for these et al. 2003). An examination of the phylogenetic relationships taxonomically important and highly characterised strains of a diverse range of Butyrivibrio and Pseudobutyrivibrio is lacking. Furthermore, the presence of 5S genes within strains, based on near full-length sequence analysis of 16S the ITS sequences was not reported in this study. Given rRNA genes (Wallace et al. 2006), revealed three genetically previous findings that Lachnospiraceae genomes com- distinct and well-supported (100 % bootstrap support) groups monly have 5S rRNA genes within their 16S-23S re- (Willems and Collins 2009). Group 1 included the gions, we sought to determine the extent that this rRNA B. fibrisolvens, B. proteoclasticus and B. hungatei type strains. operon arrangement is present among all bacteria, using Group 2 contained the Pseudobutyrivibrio type strains, and currently available sequence data. Furthermore, we group 3 contained B. crossotus. sought to determine the utility of these loci within Routine morphological and biochemical analyses are not rumen Butyrivibrio and Pseudobutyrivibrio strains for adequate to accurately identify Butyrivibrio and rapid (non-sequencing based) strain identification pur- Pseudobutyrivibrio isolates to the species level and molecular poses. Here, ITS length polymorphisms (ITS-LP), and analyses based on 16S rRNA gene sequences are most com- ITS region restriction fragment length polymorphism monly used for identification; however, more rapidly per- (ITS-RFLP), which provides an additional level of ITS formed molecular assays for identification would be very discrimination, was applied to a panel of reference desirable. This is particularly so as culture-independent anal- Butyrivibrio and Pseudobutyrivibrio strains, and their yses of rumen microbial communities reveal the extent of the utility in resolving phylogenetic relationships and differ- diversity of this group of organisms (Noel 2013),and entiating between strains was determined. Ann Microbiol (2014) 64:1623–1631 1625 Ala Fig. 1 a Typical 16S-23S-5S rRNA gene configuration of a bacterial as shown in c, with variant operons containing a tRNA gene (d)and a Ile rRNA operon, exemplified by the rrnJ operon of Bacillus subtilis subsp. tRNA gene (e) also present within the genome. The 5′–3′ orientation of subtilis168. Operons may also contain tRNA genes (shown in grey)and a all genes is shown from left to right, and the orientations of the conserved Ile Ala variantin168, the rrnO operon, with tRNA and tRNA genes be- 16S-2 and 23S-7 primers used for internal transcribed spacer (ITS) tween the 16S and 23S rRNA genes is shown in b. Strain 168 contains a amplification in this study are shown by half arrows above and below total of ten rRNA operons; of these, two possess tRNA genes, and the the 16S and 23S genes, respectively. Gene sizes and the positions of the remainder do not. In contrast, the six rRNA operons in Butyrivibrio primer 5′-ends are shown to scale. Scale bar1kb proteoclasticus strain B316 have a 16S-5S-23S configuration, typified Materials and methods 16S rRNA gene sequencing Strains and DNA extraction PCR and sequencing primers for the 16S rRNA gene were synthesized (Invitrogen, Auckland, New Zealand) on the basis Butyrivibrio and Pseudobutyrivibrio strains included in this of published sequence data, or primers were modified for this study are listed in Table 1. These strains were maintained study (Table 2). The 16S rRNA genes from several Butyrivibrio anaerobically in DSMZ medium 704: Butyrivibrio sp. medi- strains were amplified by PCR using the consensus primers 27f um (refer to http://www.dsmz.de) at 38 °C. Genomic DNA and 1525r* in a 25 μL volume containing 1× Platinum Taq PCR was extracted using an established protocol (de Los Reyes- buffer (Invitrogen), 1.5 mM MgCl , 0.2 mM each dNTP, 0.5 μM Gavilán et al. 1992). each primer, 1.25 U Platinum TaqDNA polymerase (Invitrogen), Table 1 List of Butyrivibrio and Pseudobutyrivibrio strains in this study a b Species Strain Other designations Source T T T Butyrivibrio fibrisolvens D1 ATCC 19171 =DSM 3071 Rod Mackie B. fibrisolvens C219a Graeme Attwood B. hungatei AR10 Ron Teather B. hungatei B835 NCDO 2398=DSM 10295 DSMZ B. hungatei JK 205 Jan Kopečný B. hungatei JK 614 Jan Kopečný T T T B. proteoclasticus B316 ATCC 51982 =DSM 14932 Graeme Attwood Butyrivibrio sp. A38 NCDO 2222=ATCC 27208 Rod Mackie T T Pseudobutyrivibrio ruminis A12-1 DSM 9787 DSMZ Pseudobutyrivibrio sp. Bu21 NCDO 2399=DSM 10317 DSMZ Pseudobutyrivibrio sp. CF1b Burk Dehority Pseudobutyrivibrio sp. CF3 Burk Dehority Pseudobutyrivibrio sp. OR79 Robert Forster ATCC American Type Culture Collection, Manassas VA; DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; NCDO National Collection of Dairy Organisms, Reading, UK Graeme Attwood, AgResearch Ltd, New Zealand; Burk Dehority, Ohio State University OH; Jan Kopečný, Institute of Animal Physiology and Genetics, Prague, Czech Republic; Rod Mackie, University of Illinois, Urbana-Champaign IL; Ron Teather and Robert Forster, Agriculture and Agri- Food, Lethbridge, Canada 1626 Ann Microbiol (2014) 64:1623–1631 Table 2 Oligonucleotides used Primer Sequence (5′–3′) Application Reference in this study 27f gagtttgatcmtggctcag PCR & sequencing of 16S rRNA Kenters et al. 2011 1525r* aaggaggtgatccarccg PCR & sequencing of 16S rRNA Modified from Lane 1991 357f ctcctacgggaggcagcag Sequencing of 16S rRNA Lane 1991 926f* aaactcaaatgaattgacgg Sequencing of 16S rRNA Modified from Lane 1991 519r* taccgcggctgctggcac Sequencing of 16S rRNA Modified from Lane 1991 907r* ccgtcaattcmtttgagttt Sequencing of 16S rRNA Modified from Lane 1991 16S-2 cttgtacacaccgcccgtc ITS amplification Modified from Gürtler and Stanisich 1996 23S-7 ggtacttagatgtttcagttc ITS amplification Gürtler and Stanisich 1996 and 50 ng genomic DNA. PCR reactions were carried out in a Phylogenetic analysis DNA Engine Peltier Thermal Cycler (BioRad, Hercules, CA) with the following thermal cycling profile: 94 °C for 3 min; Selected Clostridium subcluster XIVa 16S rRNA gene se- followed by 32 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C quences were retrieved from the SILVA database (Quast et al. for 1.5 min; with a final extension at 72 °C for 5 min. The PCR 2013) and the alignment was refined manually in ARB (version products were purified using QIAquick Spin Columns (Qiagen, 5.3; Ludwig et al. 2004). Phylogenies were inferred using Jukes Hilden, Germany), according to the manufacturer’s instructions. Cantor and Olsen distance matrix models, followed by DNA sequencing was performed using BigDye v3.1 cycle se- neighbor-joining implemented in ARB. A maximum likelihood quencing kit (Applied Biosystems, Foster City, CA) and primers phylogeny implemented in RAxML version 7.3.2 (Stamatakis listed in Table 2. Capillary separation of sequencing reactions 2006), using the GTRGAMMA nucleotide substitution model was performed on an ABI3730 (Applied Biosystems) machine at with rapid bootstrap analysis, was also constructed. Bootstrap the Massey Genome Service facility (Massey University, values were calculated from 1,000 replicates. Trees were rooted Palmerston North, New Zealand). The sequences of the 16S using the 16S rRNA gene sequence from Ruminocccus albus rRNA genes of B. hungatei strains JK205, JK614 and AR10 strain 20 (GenBank accession number AF030451) as an were deposited in GenBank under accession numbers outgroup. GU121460, GU121459 and FJ794074, respectively. Cluster analysis ITS-LP and ITS-RFLP analyses Calculation of similarity values and cluster analysis of the ITS-LP and ITS-RFLP profiles were performed using The ITS region was amplified in a final volume of 40 μLusing BioNumerics V4.0 (Applied Maths, Sint-Martens-Latem, the forward primer 16S-2, which targeted the 3′ end of the 16S Belgium). Default settings were used to convert, normalise, rRNA gene, and the reverse primer 23S-7, which targeted the 5′ and subtract background. A manual inspection of the marked end of the 23S rRNA gene (Gürtler and Stanisich 1996). The bands was carried out, and corrected if necessary. Similarity reaction contained 1× HOT FIREPol Blend Master mix (Solis levels were calculated based on the Pearson product–moment BioDyne, Tartu, Estonia), 0.2 μM of each primer, and ca. 15 ng correlation coefficient, taking into account band intensities genomic DNA. Thermocycling conditions included an initial and relative band position. The unweighted pair group method cycle of 95 °C for 8 min, 30 cycles of denaturation at 95 °C for with arithmetic mean (UPGMA) clustering algorithm was 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for used to calculate differences between banding profiles on 90 s, and a final extension for 5 min at 72 °C. Amplified ITS dendrograms. regions were digested using restriction enzymes (New England Biolabs, Beverley,MA) for4hat37°Cin15 μL reaction In silico analysis of bacterial rRNA operons and ITS regions volumes containing 10 μl PCR product, 4 U enzyme, and 1× commercially supplied restriction enzyme buffer. The ITS To assess the arrangement of rRNA operons from available amplicons and resulting digestion fragments were analysed by bacterial genomes, the all.gbk.tar.gz file (ftp://ftp.ncbi.nlm. electrophoresis on 1.5 % (w/v) agarose gels in 1× TAE buffer nih.gov/genomes/Bacteria/) was downloaded from the (40 mM Tris acetate, 1 mM EDTA) and visualised by SYBR National Center for Biotechnology Information (NCBI) Safe (Life Technologies, Carlsbad, CA) staining with UV (August 2013). Ribosomal RNA operon genome co- transillumination. Images were captured with the Gel Logic ordinates were extracted from bacterial genomes (n=2,624) 200 Imaging System (Eastman Kodak, Rochester, NY). using a customised script, and the arrangements of the 16S, Ann Microbiol (2014) 64:1623–1631 1627 Table 3 Bacteria that contain 16S-5S-23S rRNA operon gene arrangements Organism Family level classification Gene copies No. operons NCBI present with 16S-5S-23S BioProject structure accession 16S 5S 23S number Brevibacillus brevis 47 Bacteria; Firmicutes; Bacilli; Bacillales; 15 14 15 14 PRJNA59175 Paenibacillaceae Thermobacillus composti KWC4 Bacteria; Firmicutes; Bacilli; Bacillales; 5 5 5 5 PRJNA74021 Paenibacillaceae Heliobacterium modesticaldum Bacteria; Firmicutes; Clostridia; Clostridiales; 8 8 8 7 PRJNA58279 Ice1 Heliobacteriaceae Desulfotomaculum acetoxidans Bacteria; Firmicutes; Clostridia; Clostridiales; 10 9 10 8 PRJNA59109 5575 Peptococcaceae Butyrivibrio proteoclasticus B316 Bacteria; Firmicutes; Clostridia; Clostridiales; 6 6 6 6 PRJNA51489 Lachnospiraceae Clostridium lentocellum RHM5 Bacteria; Firmicutes; Clostridia; Clostridiales; 11 13 12 7 PRJNA49117 Lachnospiraceae Clostridium phytofermentans Bacteria; Firmicutes; Clostridia; Clostridiales; 8 8 8 8 PRJNA58519 ISDg Lachnospiraceae Clostridium saccharolyticum Bacteria; Firmicutes; Clostridia; Clostridiales; 6 6 6 6 PRJNA51419 WM1 Lachnospiraceae Clostridium sp. SY8519 Bacteria; Firmicutes; Clostridia; Clostridiales; 4 4 4 4 PRJNA68705 Lachnospiraceae Eubacterium eligens VPI C15-48 Bacteria; Firmicutes; Clostridia; Clostridiales; 5 5 5 5 PRJNA59171 Lachnospiraceae Eubacterium rectale VPI 0990 Bacteria; Firmicutes; Clostridia; Clostridiales; 5 5 5 5 PRJNA59169 Lachnospiraceae Roseburia hominis A2-183 Bacteria; Firmicutes; Clostridia; Clostridiales; 4 4 4 4 PRJNA73419 Lachnospiraceae Organism strain names are provided here; however, the NCBI BioProject accessions list several strains by their culture collection accession numbers. These are Brevibacillus brevis 47 as NBRC 100599, Desulfotomaculum acetoxidans 5575 as DSM 771, Clostridium lentocellum RHM5 as DSM 5427, Eubacterium eligens VPI C15-48 as ATCC 27750, and Eubacterium rectale VPI 0990 as ATCC 33656 (among bacterial genome projects present in NCBI as at 22 August 2013) Classification as a member of the Lachnospiraceae according to Yutin and Galperin 2013 Classification as a member of the Lachnospiraceae according to Ludwig et al. 2009 and Yokoyama et al. 2011 Classification as a member of the Lachnospiraceae according to Ludwig et al. 2009 23S and 5S rRNA genes within each genome were assessed (Mannarelli 1988), and phylogenetic analysis of 16S rRNA manually. The rRNA gene arrangements for GenBank se- gene sequences (Willems et al. 1996; Wallace et al. 2006; quence entries containing Butyrivibrio16S-23S rRNA regions Paillard et al. 2007), differentiation of Butyrivibrio and were also examined using Infernal (Nawrocki et al. 2009)and Pseudobutyrivibrio strains using PCR amplification of the the Rfam 11.0 database (Burge et al. 2013). 16S-23S ITS region may represent an efficient and accurate In silico predictions of ITS-RFLP band sizes were made for means for typing these strains. The effect that the presence of the B. proteoclasticus strain B316. The genome sequence of strain 5S rRNA gene within this region has on differentiating strains B316 was retrieved from GenBank (accession numbers using rapid typing methods is unknown. Our previous analysis NC_014387-NC_014390), and the six sequences amplified for the prevalence of the 16S-5S-23S rRNA operon structure by primers 16S-2 and 23S-7 were extracted manually. among bacteria (Kelly et al. 2010) investigated the approxi- Sequences were restriction digested in silico with AluI, mately 1,200 bacterial genomes available at the time. Since HaeIII and HhaI using NEBcutter V2.0 (Vincze et al. 2003) then, the number of available bacterial genomes has more than to obtain expected fragment sizes. doubled, and a much greater diversity of bacteria is now represented among genome-sequenced organisms. We have thus updated the analysis to screen all complete bacterial Results and discussion genomes at the NCBI on 22 August 2013. Twelve bacterial strains, all members of the phylum Firmicutes, possessed the While there has been a great deal of progress in typing 16S-5S-23S rRNA gene arrangement (Table 3), including all eight members of the Lachnospiraceae examined. Bacterial Butyrivibrio strains using DNA–DNA hybridisation 1628 Ann Microbiol (2014) 64:1623–1631 Fig. 2 Phylogenetic tree based on nearly complete (over 1,400 nucleo- values greater than 50 % are shown. The scale bar corresponds to the tides) 16S rRNA gene sequences of selected strains belonging to clos- mean number of nucleotide substitutions per site. Strains in bold type tridial subcluster XIVa with GenBank accession numbers shown in represent those used for molecular typing in this study. Groups 1, 2 and 3 brackets. The tree was generated using the maximum likelihood method were previously identified (Willems and Collins 2009), and within group in combination with a GTRGAMMA substitution model implemented in 1, two clades with strong bootstrap support were further identified. Within RAxML. Bootstrap values were calculated from 1,000 replicates; only this study, these have been defined as sub-groups 1A and 1B strains outside of the Lachnospiraceae were also found to have Paenibacilliaceae (Brevibacillus brevis 47 and Thermobacillus this operon structure, including two members of the composti KWC4), and a member each from the Heliobacteriaceae (Heliobacterium modesticaldum Ice1) and the Peptococcaceae (Desulfotomaculum acetoxidans 5575). The evolutionary significance of this atypical rRNA operon structure is unknown. Analyses of Butyrivibrio 16S- 23S ITS sequences reported from strains isolated from rein- deer rumen (GenBank accession numbers GU452693 – GU452709; Præsteng et al. 2011) also revealed the presence of 5S sequences. Thus, it is apparent that the 16S-5S-23S rRNA operon structure is a common feature, possibly even universal, among members of the Lachnospiraceae. To determine the utility of the 16S-23S rRNA operon region for fingerprinting rumen Butyrivibrio and Pseudobutyrivibrio strains, a selection of type and reference strains were used (Table 1). Initially, to determine the evolu- tionary relationships between these strains, distance and max- imum likelihood-based phylogenetic trees were constructed based on near full length 16S rRNA gene sequences of Fig. 3 unweighted pair group method with arithmetic mean (UPGMA) Butyrivibrio and Pseudobutyrivibrio type and reference strain dendrogram generated from ITS length polymorphism (ITS-LP) patterns of Butyrivibrio and Pseudobutyrivibrio strains. The dendrogram was sequences (obtained from GenBank), as well as the three generated using Bionumerics software with the optimization tolerance B. hungatei strains: JK205, JK614 and AR10 (obtained in this set at 0.5 % and the position tolerance for band analysis set at 1 %, and the study). The phylogenies showed general agreement in topol- Pearson coefficient of correlation is indicated above. The gel showing the ogy, with the maximum likelihood tree shown in Fig. 2. banding patterns of each strain is to the right of the dendrogram, with the band size markers indicated below Within both the distance and maximum likelihood trees, the Ann Microbiol (2014) 64:1623–1631 1629 Fig. 4 UPGMA dendrogram analysis of concatenated AluI, HaeIII and HhaI ITS-RFLP profiles of Butyrivibrio and Pseudobutyrivibrio strains. Cluster analyses were performed as described in Fig. 3 and three main groups were identified that corresponded to the groups and subgroups identified in the 16S rRNA gene phylogeny (Fig. 2). The gels show restriction digestion of ITS amplicons with AluI, HaeIII and HhaI, with band size markers shown below three major groups previously recognised (Willems and In contrast to ITS-LP, ITS-RFLP utilises both length and Collins 2009) were identified, each with 100 % bootstrap sequence variation in the ITS region and this is predicted to be support. In addition, two well-defined subgroups within group more discriminatory. ITS amplicons were initially subjected to 1, which we have denoted subgroup 1A and subgroup 1B digestion by enzymes AluI, HaeIII, HhaI, MspI, RsaI, Sau3AI (Fig. 2), were further identified (with 90 % and 100 % boot- and XbaI to determine which were most informative for the strap support, respectively), which contained B. fibrisolvens Butyrivibrio and Pseudobutyrivibrio strains. Digestion with sequences (subgroup 1A), and B. hungatei, B. proteoclasticus AluI, HaeIII and HhaI were deemed sufficiently discriminato- and A38 sequences (subgroup 1B). Group 2 contained se- ry for subsequent restriction profiling and cluster analysis (see quences from all of the Pseudobutyrivibrio strains included in Online Resource 1 for the original gel images). Furthermore, the analysis. While two species of Pseudobutyrivibrio are in silico analyses of predicted RFLP band sizes for formally recognised, the strains do not clearly form monophy- B. proteoclasticus B316—the only strain for which a complete letic and well-supported subgroups representing each species genome sequence is available—corresponded well to the ob- (Fig. 2; Kopečný et al. 2003;Grilli et al. 2013). Furthermore, a tained profiles (Online Resource 2). BioNumerics analysis of 16S rRNA gene pairwise analysis of all strains within group 2, the concatenated ITS-RFLP profiles from AluI, HaeIII and showed that all strains exhibited >97 % unambiguous se- HhaI(Fig. 4) resulted in three well-defined clusters, with the quence identity with one another. Thus, within this study we distribution of strains within each group consistent with those have referred to these strains as Pseudobutyrivibrio sp., apart obtained via the 16S rRNA gene phylogeny (Fig. 2). However from the type strains, which are referred to by their specific the branching order of the clusters differed, this time with epithets. This phylogeny served as a reference for cluster subgroup 1A forming a sister clade to group 2, and subgroup analyses from the ITS-LP and ITS-RFLP fingerprinting data. 1B being basal to both of these (Fig. 4). ITS amplifications of the panel of strains examined in this Of the typing methods, ITS-LP was the fastest to per- study generated diverse banding patterns consisting of ap- form, requiring only amplification and gel electrophoresis, proximately two to five bands per isolate, ranging in size from in contrast to ITS-RFLP, which required additional restric- ca. 700–1,200 bp (Fig. 3). The UPGMA cluster analysis of tion digestion. The reproducibility of the ITS-based profiles the ITS-LP profiles (Fig. 3) separated the strains into was very good. Although agarose gel electrophoresis is a groups that did not reflect the groups and subgroups convenient method with which to rapidly visualise finger- identified in the 16S gene phylogeny (Fig. 2). While prints, the resolution of ITS-LP and ITS-RFLP banding most of the Pseudobutyrivibrio strains clustered together, patterns may be improved using more sophisticated separa- P. ruminis strain A12-1 clustered with B. proteoclasticus and tion methods such as capillary electrophoresis, which en- two of the B. hungatei strains. The B. hungatei strains were ables discrimination of fragments differing only one base distributed between two different clades with Pearson corre- pair in length (Baele et al. 2002), or RIS-denaturing gradi- lation coefficients (PCCs) of 50 % among the strains exam- ent gel electrophoresis (RIS-DGGE; Kan et al. 2011), ined. Overall, the ITS-LP method appeared to be of limited which differentiates sequence variants in the RIS region, utility in rapidly classifying the Butyrivibrio and and therefore confers high resolving power. Indeed, RIS- Pseudobutyrivibrio strains to the group level. DGGE was able to distinguish closely related strains of 1630 Ann Microbiol (2014) 64:1623–1631 proteoclasticus B316 highlights adaptation to a polysaccharide- Shewanella that were not differentiated by 16S rRNA gene rich environment. PLoS One 5(8):e11942 sequences alone (Kan et al. 2011). Kenters N, Henderson G, Jeyanathan J, Kittelmann S, Janssen PH (2011) In conclusion, we have found that the 16S-5S-23S rRNA Isolation of previously uncultured rumen bacteria by dilution to operon structure that is atypical among bacteria, appears to be extinction using a new liquid culture medium. J Microbiol Methods 84(1):52–60 highly prevalent (and potentially universal) among Kopečný J, Zorec M, Mrázek J, Kobayashi Y, Marinšek-Logar R (2003) Butyrivibrio strains and members of the Lachnospiraceae. Butyrivibrio hungateisp.nov.and Pseudobutyrivibrio xylanivorans Length polymorphisms revealed by amplification of the sp. nov., butyrate-producing bacteria from the rumen. Int J Syst Evol 16S-23S ITS region were found to be of limited value for Microbiol 53(1):201–209 Kwon HS, Yang EH, Yeon SW, Kang BH, Kim TY (2004) Rapid distinguishing ruminal Butyrivibrio and Pseudobutyrivibrio identification of probiotic Lactobacillus species by multiplex PCR strains. In contrast, ITS-RFLP provided a useful method with using species-specific primers based on the region extending from which to rapidly identify strains to the group level as defined 16S rRNA through 23S rRNA. 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Int J Syst Bacteriol 44:812–826 Bacteroidaceae and Butyrivibrio and description of Desulfomonas de Los Reyes-Gavilán CG, Limsowtin GK, Tailliez P, Séchaud L, gen. nov. and ten new species in the genera Desulfomonas, Accolas JP (1992) A Lactobacillus helveticus-specific DNA probe Butyrivibrio, Eubacterium, Clostridium,and Ruminococcus.Int J detects restriction fragment length polymorphisms in this species. Syst Bacteriol 26(2):238–252 Appl Environ Microbiol 58(10):3429–3432 Nawrocki EP, Kolbe DL, Eddy SR (2009) Infernal 1.0: inference of RNA Grilli DJ, Ceron ME, Paez S, Egea V, Schnittger L, Cravero S, Escudero alignments. Bioinformatics 25(10):1335–1337 MS, Allegretti L, Arenas GN (2013) Isolation of Pseudobutyrivibrio Noel SJ (2013) Cultivation and community analysis of plant‐adherent ruminis and Pseudobutyrivibrio xylanivorans from rumen of Creole rumen bacteria. PhD thesis, Massey University, Palmerston North, goats fed native forage diet. 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Atypical bacterial rRNA operon structure is prevalent within the Lachnospiraceae, and use of the 16S-23S rRNA internal transcribed spacer region for the rapid identification of ruminal Butyrivibrio and Pseudobutyrivibrio strains

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Springer Journals
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Copyright © 2014 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
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1590-4261
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1869-2044
DOI
10.1007/s13213-014-0806-2
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Abstract

Ann Microbiol (2014) 64:1623–1631 DOI 10.1007/s13213-014-0806-2 ORIGINAL ARTICLE Atypical bacterial rRNA operon structure is prevalent within the Lachnospiraceae, and use of the 16S-23S rRNA internal transcribed spacer region for the rapid identification of ruminal Butyrivibrio and Pseudobutyrivibrio strains Dong Li & Sinead Leahy & Gemma Henderson & William Kelly & Adrian Cookson & Graeme Attwood & Christina Moon Received: 20 May 2013 /Accepted: 9 January 2014 /Published online: 29 January 2014 Springer-Verlag Berlin Heidelberg and the University of Milan 2014 Abstract The rumen is the fermentative forestomach of rumi- transcribed spacer (ITS) regions located between the genes. nant animals, and is host to a wide range of anaerobic bacteria However, in the rumen bacterium, Butyrivibrio proteoclasticus whose primary function is to facilitate forage degradation. B316, rRNA operons have a 16S-5S-23S rRNA gene arrange- Butyrivibrio and Pseudobutyrivibrio are closely related proteo- ment, and analysis of bacterial genome projects revealed that lytic and fibrolytic genera within the family Lachnospiraceae, this configuration was present in all publicly available complete and are commonly isolated from the rumens of animals fed genomes from members of the family Lachnospiraceae. The fibrous diets. The ribosomal RNA (rRNA) operon is an impor- 16S-23S ITS region is commonly used to identify bacterial tant phylogenetically informative locus that is present in multi- strains, thus we sought to determine the utility of this region ple copies in bacterial genomes. Ribosomal RNA genes are from rumen Butyrivibrio and Pseudobutyrivibrio isolates for typically arranged in the order 16S-23S-5S, with internal their rapid molecular identification. Polymerase chain reaction wasusedtoamplify 16S-23S ITSregions, which were assessed for length polymorphism (ITS-LP), and restriction fragment Electronic supplementary material The online version of this article (doi:10.1007/s13213-014-0806-2) contains supplementary material, length polymorphism (ITS-RFLP) using AluI, HaeIII and which is available to authorized users. HhaIonapanelof13 Butyrivibrio and Pseudobutyrivibrio : : : : : reference strains. Cluster analysis of the resulting banding D. Li S. Leahy G. Henderson W. Kelly G. Attwood C. Moon (*) patterns revealed that while the ITS-LP method did not group Animal Nutrition and Health, AgResearch Ltd, Tennent Dr, Private the strains according to major Butyrivibrio and Bag 11008, Palmerston North 4442, New Zealand Pseudobutyrivibrio clades identified via 16S rRNA gene se- e-mail: christina.moon@agresearch.co.nz quences, ITS-RFLP was more discriminative, and able to rap- D. Li idly delineate the strains into these clades. e-mail: dong.li@agresearch.co.nz S. Leahy e-mail: sinead.leahy@agresearch.co.nz . . . Keywords Butyrivibrio DNAfingerprint Lachnospiraceae . . Pseudobutyrivibrio Ribosomal RNA gene operon G. Henderson e-mail: gemma.henderson@agresearch.co.nz Ribosomal intergenic spacer W. Kelly e-mail: bill.kelly@agresearch.co.nz G. Attwood Introduction e-mail: graeme.attwood@agresearch.co.nz A. Cookson The rumen is a complex environment where plant material is Animal Nutrition and Health, degraded through the symbiotic interactions of micro- AgResearch Ltd, Grasslands Research Centre, organisms and the host. The genera Butyrivibrio and Tennent Dr, Private Bag 11008, Palmerston North 4442, Pseudobutyrivibrio, formerly collectively known as New Zealand e-mail: adrian.cookson@agresearch.co.nz Butyrivibrio fibrisolvens (Bryant and Small 1956), represent 1624 Ann Microbiol (2014) 64:1623–1631 a considerable proportion of culturable rumen anaerobic knowledge of the specific functional roles and metabolic bacteria in domestic and wild ruminants, where they con- capabilities of individual taxa will require characterisation of tribute to fibre degradation (Kopečný et al. 2003). cultivated representatives. Members of these two genera are non-spore forming, Ribosomal RNA (rRNA) gene operons are phylogenetically anaerobic, motile, curved rod-shaped bacteria that are informative loci that are typically present in multiple copies per commonly isolated from the rumens of animals fed a wide genome (rRNA genes may be present in up to 15 copies each; range of diets (Stewart et al. 1997). All ruminal Lee et al. 2009), and span ca. 5 kb in length each. They normally Butyrivibrio species studied to date can degrade the hemi- have a 16S rRNA gene–intergenic spacer I–23S rRNA gene– cellulose component of plant cell walls, and many also intergenic spacer II–5S rRNA gene arrangement in bacteria and hydrolyze other plant polysaccharides including starch and may also contain small tRNA genes (Fig. 1). However, from an pectin (Hespell et al. 1987;Kopečný et al. 2003). analysis of all available bacterial whole genome sequences (ap- Phylogenetic studies currently place Butyrivibrio and proximately 1,200), it was reported that members of the Pseudobutyrivibrio within subcluster XIVa of the Lachnospiraceae (including B. proteoclasticus B316) commonly Clostridium subphylum (Collins et al. 1994), where they are possess an alternative gene arrangement within their rRNA members of the family Lachnospiraceae. Four Butyrivibrio operons, with rRNA genes in the order 16S-5S-23S rRNA species are presently recognised: B. fibrisolvens (type strain (Kelly et al. 2010;Fig. 1). Molecular analysis of the internal D1; Bryant and Small 1956); B. crossotus (type strain Bu46 ; transcribed spacer (ITS) region, which commonly employs 16S Moore et al. 1976), B. hungatei (type strain JK615 ;Kopečný and 23S rRNA gene-specific primers (Gürtler and Stanisich et al. 2003)and B. proteoclasticus (type strain B316 ;Moon 1996), has been used for the study of microbial community et al. 2008), formerly known as Clostridium proteoclasticum structure and identification of closely related isolates for a wide (Attwood et al. 1996). To the best of our knowledge, variety of bacterial taxa. This region exhibits a high degree of Butyrivibrio crossotus strains have not been isolated from sequence and length variation (Gürtler and Stanisich 1996; the rumen, but instead from human rectal or faecal material Hassan et al. 2003; Yannarell and Triplett 2004) and is consid- (Moore et al. 1976; Willems and Collins 2009). Furthermore, ered more useful for genus- and species-level identification than molecular data indicate that, within the Lachnospiraceae phy- the 16S rRNA gene alone (Rachman et al. 2003;Kwonet al. logeny, B. crossotus occupies a very disparate position to the 2004). Recently, ITS regions from Butyrivibrio isolates from the originally described rumen-derived Butyrivibrio species reindeer rumen were sequenced following PCR-amplification (Wade 2009), and thus in principle, is not considered con- and cloning (Præsteng et al. 2011). Considerable length poly- generic. Conversely, various molecular analyses have indicat- morphism was observed between ITS copies, and similar phy- ed that there are Butyrivibrio-like isolates that do not fit into logenetic relationships were observed based on the ITS se- these species boundaries, and are likely to represent novel quences as compared to 16S rRNA gene sequences, but species within Butyrivibrio (Willems and Collins 2009). At the ITS sequence-based phylogeny appeared to provide present, the genus Pseudobutyrivibrio contains two closely more detailed resolution between the isolates (Præsteng related species: P. ruminis (type strain A12-1 ; van Gylswyk et al. 2011). ITS analyses were not performed on et al. 1996)and P. xylanivorans (type strain Mz5 ;Kopečný Butyrivibrio type or reference strains, thus data for these et al. 2003). An examination of the phylogenetic relationships taxonomically important and highly characterised strains of a diverse range of Butyrivibrio and Pseudobutyrivibrio is lacking. Furthermore, the presence of 5S genes within strains, based on near full-length sequence analysis of 16S the ITS sequences was not reported in this study. Given rRNA genes (Wallace et al. 2006), revealed three genetically previous findings that Lachnospiraceae genomes com- distinct and well-supported (100 % bootstrap support) groups monly have 5S rRNA genes within their 16S-23S re- (Willems and Collins 2009). Group 1 included the gions, we sought to determine the extent that this rRNA B. fibrisolvens, B. proteoclasticus and B. hungatei type strains. operon arrangement is present among all bacteria, using Group 2 contained the Pseudobutyrivibrio type strains, and currently available sequence data. Furthermore, we group 3 contained B. crossotus. sought to determine the utility of these loci within Routine morphological and biochemical analyses are not rumen Butyrivibrio and Pseudobutyrivibrio strains for adequate to accurately identify Butyrivibrio and rapid (non-sequencing based) strain identification pur- Pseudobutyrivibrio isolates to the species level and molecular poses. Here, ITS length polymorphisms (ITS-LP), and analyses based on 16S rRNA gene sequences are most com- ITS region restriction fragment length polymorphism monly used for identification; however, more rapidly per- (ITS-RFLP), which provides an additional level of ITS formed molecular assays for identification would be very discrimination, was applied to a panel of reference desirable. This is particularly so as culture-independent anal- Butyrivibrio and Pseudobutyrivibrio strains, and their yses of rumen microbial communities reveal the extent of the utility in resolving phylogenetic relationships and differ- diversity of this group of organisms (Noel 2013),and entiating between strains was determined. Ann Microbiol (2014) 64:1623–1631 1625 Ala Fig. 1 a Typical 16S-23S-5S rRNA gene configuration of a bacterial as shown in c, with variant operons containing a tRNA gene (d)and a Ile rRNA operon, exemplified by the rrnJ operon of Bacillus subtilis subsp. tRNA gene (e) also present within the genome. The 5′–3′ orientation of subtilis168. Operons may also contain tRNA genes (shown in grey)and a all genes is shown from left to right, and the orientations of the conserved Ile Ala variantin168, the rrnO operon, with tRNA and tRNA genes be- 16S-2 and 23S-7 primers used for internal transcribed spacer (ITS) tween the 16S and 23S rRNA genes is shown in b. Strain 168 contains a amplification in this study are shown by half arrows above and below total of ten rRNA operons; of these, two possess tRNA genes, and the the 16S and 23S genes, respectively. Gene sizes and the positions of the remainder do not. In contrast, the six rRNA operons in Butyrivibrio primer 5′-ends are shown to scale. Scale bar1kb proteoclasticus strain B316 have a 16S-5S-23S configuration, typified Materials and methods 16S rRNA gene sequencing Strains and DNA extraction PCR and sequencing primers for the 16S rRNA gene were synthesized (Invitrogen, Auckland, New Zealand) on the basis Butyrivibrio and Pseudobutyrivibrio strains included in this of published sequence data, or primers were modified for this study are listed in Table 1. These strains were maintained study (Table 2). The 16S rRNA genes from several Butyrivibrio anaerobically in DSMZ medium 704: Butyrivibrio sp. medi- strains were amplified by PCR using the consensus primers 27f um (refer to http://www.dsmz.de) at 38 °C. Genomic DNA and 1525r* in a 25 μL volume containing 1× Platinum Taq PCR was extracted using an established protocol (de Los Reyes- buffer (Invitrogen), 1.5 mM MgCl , 0.2 mM each dNTP, 0.5 μM Gavilán et al. 1992). each primer, 1.25 U Platinum TaqDNA polymerase (Invitrogen), Table 1 List of Butyrivibrio and Pseudobutyrivibrio strains in this study a b Species Strain Other designations Source T T T Butyrivibrio fibrisolvens D1 ATCC 19171 =DSM 3071 Rod Mackie B. fibrisolvens C219a Graeme Attwood B. hungatei AR10 Ron Teather B. hungatei B835 NCDO 2398=DSM 10295 DSMZ B. hungatei JK 205 Jan Kopečný B. hungatei JK 614 Jan Kopečný T T T B. proteoclasticus B316 ATCC 51982 =DSM 14932 Graeme Attwood Butyrivibrio sp. A38 NCDO 2222=ATCC 27208 Rod Mackie T T Pseudobutyrivibrio ruminis A12-1 DSM 9787 DSMZ Pseudobutyrivibrio sp. Bu21 NCDO 2399=DSM 10317 DSMZ Pseudobutyrivibrio sp. CF1b Burk Dehority Pseudobutyrivibrio sp. CF3 Burk Dehority Pseudobutyrivibrio sp. OR79 Robert Forster ATCC American Type Culture Collection, Manassas VA; DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen, Braunschweig, Germany; NCDO National Collection of Dairy Organisms, Reading, UK Graeme Attwood, AgResearch Ltd, New Zealand; Burk Dehority, Ohio State University OH; Jan Kopečný, Institute of Animal Physiology and Genetics, Prague, Czech Republic; Rod Mackie, University of Illinois, Urbana-Champaign IL; Ron Teather and Robert Forster, Agriculture and Agri- Food, Lethbridge, Canada 1626 Ann Microbiol (2014) 64:1623–1631 Table 2 Oligonucleotides used Primer Sequence (5′–3′) Application Reference in this study 27f gagtttgatcmtggctcag PCR & sequencing of 16S rRNA Kenters et al. 2011 1525r* aaggaggtgatccarccg PCR & sequencing of 16S rRNA Modified from Lane 1991 357f ctcctacgggaggcagcag Sequencing of 16S rRNA Lane 1991 926f* aaactcaaatgaattgacgg Sequencing of 16S rRNA Modified from Lane 1991 519r* taccgcggctgctggcac Sequencing of 16S rRNA Modified from Lane 1991 907r* ccgtcaattcmtttgagttt Sequencing of 16S rRNA Modified from Lane 1991 16S-2 cttgtacacaccgcccgtc ITS amplification Modified from Gürtler and Stanisich 1996 23S-7 ggtacttagatgtttcagttc ITS amplification Gürtler and Stanisich 1996 and 50 ng genomic DNA. PCR reactions were carried out in a Phylogenetic analysis DNA Engine Peltier Thermal Cycler (BioRad, Hercules, CA) with the following thermal cycling profile: 94 °C for 3 min; Selected Clostridium subcluster XIVa 16S rRNA gene se- followed by 32 cycles of 94 °C for 30 s, 57 °C for 30 s, and 72 °C quences were retrieved from the SILVA database (Quast et al. for 1.5 min; with a final extension at 72 °C for 5 min. The PCR 2013) and the alignment was refined manually in ARB (version products were purified using QIAquick Spin Columns (Qiagen, 5.3; Ludwig et al. 2004). Phylogenies were inferred using Jukes Hilden, Germany), according to the manufacturer’s instructions. Cantor and Olsen distance matrix models, followed by DNA sequencing was performed using BigDye v3.1 cycle se- neighbor-joining implemented in ARB. A maximum likelihood quencing kit (Applied Biosystems, Foster City, CA) and primers phylogeny implemented in RAxML version 7.3.2 (Stamatakis listed in Table 2. Capillary separation of sequencing reactions 2006), using the GTRGAMMA nucleotide substitution model was performed on an ABI3730 (Applied Biosystems) machine at with rapid bootstrap analysis, was also constructed. Bootstrap the Massey Genome Service facility (Massey University, values were calculated from 1,000 replicates. Trees were rooted Palmerston North, New Zealand). The sequences of the 16S using the 16S rRNA gene sequence from Ruminocccus albus rRNA genes of B. hungatei strains JK205, JK614 and AR10 strain 20 (GenBank accession number AF030451) as an were deposited in GenBank under accession numbers outgroup. GU121460, GU121459 and FJ794074, respectively. Cluster analysis ITS-LP and ITS-RFLP analyses Calculation of similarity values and cluster analysis of the ITS-LP and ITS-RFLP profiles were performed using The ITS region was amplified in a final volume of 40 μLusing BioNumerics V4.0 (Applied Maths, Sint-Martens-Latem, the forward primer 16S-2, which targeted the 3′ end of the 16S Belgium). Default settings were used to convert, normalise, rRNA gene, and the reverse primer 23S-7, which targeted the 5′ and subtract background. A manual inspection of the marked end of the 23S rRNA gene (Gürtler and Stanisich 1996). The bands was carried out, and corrected if necessary. Similarity reaction contained 1× HOT FIREPol Blend Master mix (Solis levels were calculated based on the Pearson product–moment BioDyne, Tartu, Estonia), 0.2 μM of each primer, and ca. 15 ng correlation coefficient, taking into account band intensities genomic DNA. Thermocycling conditions included an initial and relative band position. The unweighted pair group method cycle of 95 °C for 8 min, 30 cycles of denaturation at 95 °C for with arithmetic mean (UPGMA) clustering algorithm was 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for used to calculate differences between banding profiles on 90 s, and a final extension for 5 min at 72 °C. Amplified ITS dendrograms. regions were digested using restriction enzymes (New England Biolabs, Beverley,MA) for4hat37°Cin15 μL reaction In silico analysis of bacterial rRNA operons and ITS regions volumes containing 10 μl PCR product, 4 U enzyme, and 1× commercially supplied restriction enzyme buffer. The ITS To assess the arrangement of rRNA operons from available amplicons and resulting digestion fragments were analysed by bacterial genomes, the all.gbk.tar.gz file (ftp://ftp.ncbi.nlm. electrophoresis on 1.5 % (w/v) agarose gels in 1× TAE buffer nih.gov/genomes/Bacteria/) was downloaded from the (40 mM Tris acetate, 1 mM EDTA) and visualised by SYBR National Center for Biotechnology Information (NCBI) Safe (Life Technologies, Carlsbad, CA) staining with UV (August 2013). Ribosomal RNA operon genome co- transillumination. Images were captured with the Gel Logic ordinates were extracted from bacterial genomes (n=2,624) 200 Imaging System (Eastman Kodak, Rochester, NY). using a customised script, and the arrangements of the 16S, Ann Microbiol (2014) 64:1623–1631 1627 Table 3 Bacteria that contain 16S-5S-23S rRNA operon gene arrangements Organism Family level classification Gene copies No. operons NCBI present with 16S-5S-23S BioProject structure accession 16S 5S 23S number Brevibacillus brevis 47 Bacteria; Firmicutes; Bacilli; Bacillales; 15 14 15 14 PRJNA59175 Paenibacillaceae Thermobacillus composti KWC4 Bacteria; Firmicutes; Bacilli; Bacillales; 5 5 5 5 PRJNA74021 Paenibacillaceae Heliobacterium modesticaldum Bacteria; Firmicutes; Clostridia; Clostridiales; 8 8 8 7 PRJNA58279 Ice1 Heliobacteriaceae Desulfotomaculum acetoxidans Bacteria; Firmicutes; Clostridia; Clostridiales; 10 9 10 8 PRJNA59109 5575 Peptococcaceae Butyrivibrio proteoclasticus B316 Bacteria; Firmicutes; Clostridia; Clostridiales; 6 6 6 6 PRJNA51489 Lachnospiraceae Clostridium lentocellum RHM5 Bacteria; Firmicutes; Clostridia; Clostridiales; 11 13 12 7 PRJNA49117 Lachnospiraceae Clostridium phytofermentans Bacteria; Firmicutes; Clostridia; Clostridiales; 8 8 8 8 PRJNA58519 ISDg Lachnospiraceae Clostridium saccharolyticum Bacteria; Firmicutes; Clostridia; Clostridiales; 6 6 6 6 PRJNA51419 WM1 Lachnospiraceae Clostridium sp. SY8519 Bacteria; Firmicutes; Clostridia; Clostridiales; 4 4 4 4 PRJNA68705 Lachnospiraceae Eubacterium eligens VPI C15-48 Bacteria; Firmicutes; Clostridia; Clostridiales; 5 5 5 5 PRJNA59171 Lachnospiraceae Eubacterium rectale VPI 0990 Bacteria; Firmicutes; Clostridia; Clostridiales; 5 5 5 5 PRJNA59169 Lachnospiraceae Roseburia hominis A2-183 Bacteria; Firmicutes; Clostridia; Clostridiales; 4 4 4 4 PRJNA73419 Lachnospiraceae Organism strain names are provided here; however, the NCBI BioProject accessions list several strains by their culture collection accession numbers. These are Brevibacillus brevis 47 as NBRC 100599, Desulfotomaculum acetoxidans 5575 as DSM 771, Clostridium lentocellum RHM5 as DSM 5427, Eubacterium eligens VPI C15-48 as ATCC 27750, and Eubacterium rectale VPI 0990 as ATCC 33656 (among bacterial genome projects present in NCBI as at 22 August 2013) Classification as a member of the Lachnospiraceae according to Yutin and Galperin 2013 Classification as a member of the Lachnospiraceae according to Ludwig et al. 2009 and Yokoyama et al. 2011 Classification as a member of the Lachnospiraceae according to Ludwig et al. 2009 23S and 5S rRNA genes within each genome were assessed (Mannarelli 1988), and phylogenetic analysis of 16S rRNA manually. The rRNA gene arrangements for GenBank se- gene sequences (Willems et al. 1996; Wallace et al. 2006; quence entries containing Butyrivibrio16S-23S rRNA regions Paillard et al. 2007), differentiation of Butyrivibrio and were also examined using Infernal (Nawrocki et al. 2009)and Pseudobutyrivibrio strains using PCR amplification of the the Rfam 11.0 database (Burge et al. 2013). 16S-23S ITS region may represent an efficient and accurate In silico predictions of ITS-RFLP band sizes were made for means for typing these strains. The effect that the presence of the B. proteoclasticus strain B316. The genome sequence of strain 5S rRNA gene within this region has on differentiating strains B316 was retrieved from GenBank (accession numbers using rapid typing methods is unknown. Our previous analysis NC_014387-NC_014390), and the six sequences amplified for the prevalence of the 16S-5S-23S rRNA operon structure by primers 16S-2 and 23S-7 were extracted manually. among bacteria (Kelly et al. 2010) investigated the approxi- Sequences were restriction digested in silico with AluI, mately 1,200 bacterial genomes available at the time. Since HaeIII and HhaI using NEBcutter V2.0 (Vincze et al. 2003) then, the number of available bacterial genomes has more than to obtain expected fragment sizes. doubled, and a much greater diversity of bacteria is now represented among genome-sequenced organisms. We have thus updated the analysis to screen all complete bacterial Results and discussion genomes at the NCBI on 22 August 2013. Twelve bacterial strains, all members of the phylum Firmicutes, possessed the While there has been a great deal of progress in typing 16S-5S-23S rRNA gene arrangement (Table 3), including all eight members of the Lachnospiraceae examined. Bacterial Butyrivibrio strains using DNA–DNA hybridisation 1628 Ann Microbiol (2014) 64:1623–1631 Fig. 2 Phylogenetic tree based on nearly complete (over 1,400 nucleo- values greater than 50 % are shown. The scale bar corresponds to the tides) 16S rRNA gene sequences of selected strains belonging to clos- mean number of nucleotide substitutions per site. Strains in bold type tridial subcluster XIVa with GenBank accession numbers shown in represent those used for molecular typing in this study. Groups 1, 2 and 3 brackets. The tree was generated using the maximum likelihood method were previously identified (Willems and Collins 2009), and within group in combination with a GTRGAMMA substitution model implemented in 1, two clades with strong bootstrap support were further identified. Within RAxML. Bootstrap values were calculated from 1,000 replicates; only this study, these have been defined as sub-groups 1A and 1B strains outside of the Lachnospiraceae were also found to have Paenibacilliaceae (Brevibacillus brevis 47 and Thermobacillus this operon structure, including two members of the composti KWC4), and a member each from the Heliobacteriaceae (Heliobacterium modesticaldum Ice1) and the Peptococcaceae (Desulfotomaculum acetoxidans 5575). The evolutionary significance of this atypical rRNA operon structure is unknown. Analyses of Butyrivibrio 16S- 23S ITS sequences reported from strains isolated from rein- deer rumen (GenBank accession numbers GU452693 – GU452709; Præsteng et al. 2011) also revealed the presence of 5S sequences. Thus, it is apparent that the 16S-5S-23S rRNA operon structure is a common feature, possibly even universal, among members of the Lachnospiraceae. To determine the utility of the 16S-23S rRNA operon region for fingerprinting rumen Butyrivibrio and Pseudobutyrivibrio strains, a selection of type and reference strains were used (Table 1). Initially, to determine the evolu- tionary relationships between these strains, distance and max- imum likelihood-based phylogenetic trees were constructed based on near full length 16S rRNA gene sequences of Fig. 3 unweighted pair group method with arithmetic mean (UPGMA) Butyrivibrio and Pseudobutyrivibrio type and reference strain dendrogram generated from ITS length polymorphism (ITS-LP) patterns of Butyrivibrio and Pseudobutyrivibrio strains. The dendrogram was sequences (obtained from GenBank), as well as the three generated using Bionumerics software with the optimization tolerance B. hungatei strains: JK205, JK614 and AR10 (obtained in this set at 0.5 % and the position tolerance for band analysis set at 1 %, and the study). The phylogenies showed general agreement in topol- Pearson coefficient of correlation is indicated above. The gel showing the ogy, with the maximum likelihood tree shown in Fig. 2. banding patterns of each strain is to the right of the dendrogram, with the band size markers indicated below Within both the distance and maximum likelihood trees, the Ann Microbiol (2014) 64:1623–1631 1629 Fig. 4 UPGMA dendrogram analysis of concatenated AluI, HaeIII and HhaI ITS-RFLP profiles of Butyrivibrio and Pseudobutyrivibrio strains. Cluster analyses were performed as described in Fig. 3 and three main groups were identified that corresponded to the groups and subgroups identified in the 16S rRNA gene phylogeny (Fig. 2). The gels show restriction digestion of ITS amplicons with AluI, HaeIII and HhaI, with band size markers shown below three major groups previously recognised (Willems and In contrast to ITS-LP, ITS-RFLP utilises both length and Collins 2009) were identified, each with 100 % bootstrap sequence variation in the ITS region and this is predicted to be support. In addition, two well-defined subgroups within group more discriminatory. ITS amplicons were initially subjected to 1, which we have denoted subgroup 1A and subgroup 1B digestion by enzymes AluI, HaeIII, HhaI, MspI, RsaI, Sau3AI (Fig. 2), were further identified (with 90 % and 100 % boot- and XbaI to determine which were most informative for the strap support, respectively), which contained B. fibrisolvens Butyrivibrio and Pseudobutyrivibrio strains. Digestion with sequences (subgroup 1A), and B. hungatei, B. proteoclasticus AluI, HaeIII and HhaI were deemed sufficiently discriminato- and A38 sequences (subgroup 1B). Group 2 contained se- ry for subsequent restriction profiling and cluster analysis (see quences from all of the Pseudobutyrivibrio strains included in Online Resource 1 for the original gel images). Furthermore, the analysis. While two species of Pseudobutyrivibrio are in silico analyses of predicted RFLP band sizes for formally recognised, the strains do not clearly form monophy- B. proteoclasticus B316—the only strain for which a complete letic and well-supported subgroups representing each species genome sequence is available—corresponded well to the ob- (Fig. 2; Kopečný et al. 2003;Grilli et al. 2013). Furthermore, a tained profiles (Online Resource 2). BioNumerics analysis of 16S rRNA gene pairwise analysis of all strains within group 2, the concatenated ITS-RFLP profiles from AluI, HaeIII and showed that all strains exhibited >97 % unambiguous se- HhaI(Fig. 4) resulted in three well-defined clusters, with the quence identity with one another. Thus, within this study we distribution of strains within each group consistent with those have referred to these strains as Pseudobutyrivibrio sp., apart obtained via the 16S rRNA gene phylogeny (Fig. 2). However from the type strains, which are referred to by their specific the branching order of the clusters differed, this time with epithets. This phylogeny served as a reference for cluster subgroup 1A forming a sister clade to group 2, and subgroup analyses from the ITS-LP and ITS-RFLP fingerprinting data. 1B being basal to both of these (Fig. 4). ITS amplifications of the panel of strains examined in this Of the typing methods, ITS-LP was the fastest to per- study generated diverse banding patterns consisting of ap- form, requiring only amplification and gel electrophoresis, proximately two to five bands per isolate, ranging in size from in contrast to ITS-RFLP, which required additional restric- ca. 700–1,200 bp (Fig. 3). The UPGMA cluster analysis of tion digestion. The reproducibility of the ITS-based profiles the ITS-LP profiles (Fig. 3) separated the strains into was very good. Although agarose gel electrophoresis is a groups that did not reflect the groups and subgroups convenient method with which to rapidly visualise finger- identified in the 16S gene phylogeny (Fig. 2). While prints, the resolution of ITS-LP and ITS-RFLP banding most of the Pseudobutyrivibrio strains clustered together, patterns may be improved using more sophisticated separa- P. ruminis strain A12-1 clustered with B. proteoclasticus and tion methods such as capillary electrophoresis, which en- two of the B. hungatei strains. The B. hungatei strains were ables discrimination of fragments differing only one base distributed between two different clades with Pearson corre- pair in length (Baele et al. 2002), or RIS-denaturing gradi- lation coefficients (PCCs) of 50 % among the strains exam- ent gel electrophoresis (RIS-DGGE; Kan et al. 2011), ined. Overall, the ITS-LP method appeared to be of limited which differentiates sequence variants in the RIS region, utility in rapidly classifying the Butyrivibrio and and therefore confers high resolving power. Indeed, RIS- Pseudobutyrivibrio strains to the group level. DGGE was able to distinguish closely related strains of 1630 Ann Microbiol (2014) 64:1623–1631 proteoclasticus B316 highlights adaptation to a polysaccharide- Shewanella that were not differentiated by 16S rRNA gene rich environment. PLoS One 5(8):e11942 sequences alone (Kan et al. 2011). Kenters N, Henderson G, Jeyanathan J, Kittelmann S, Janssen PH (2011) In conclusion, we have found that the 16S-5S-23S rRNA Isolation of previously uncultured rumen bacteria by dilution to operon structure that is atypical among bacteria, appears to be extinction using a new liquid culture medium. J Microbiol Methods 84(1):52–60 highly prevalent (and potentially universal) among Kopečný J, Zorec M, Mrázek J, Kobayashi Y, Marinšek-Logar R (2003) Butyrivibrio strains and members of the Lachnospiraceae. Butyrivibrio hungateisp.nov.and Pseudobutyrivibrio xylanivorans Length polymorphisms revealed by amplification of the sp. nov., butyrate-producing bacteria from the rumen. Int J Syst Evol 16S-23S ITS region were found to be of limited value for Microbiol 53(1):201–209 Kwon HS, Yang EH, Yeon SW, Kang BH, Kim TY (2004) Rapid distinguishing ruminal Butyrivibrio and Pseudobutyrivibrio identification of probiotic Lactobacillus species by multiplex PCR strains. In contrast, ITS-RFLP provided a useful method with using species-specific primers based on the region extending from which to rapidly identify strains to the group level as defined 16S rRNA through 23S rRNA. 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