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Applied and Environmental Microbiology, March 2006, p. 2221-2225, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.2221-2225.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

Evaluation of 23S rRNA PCR Primers for Use in Phylogenetic Studies of Bacterial Diversity{dagger}

Dana E. Hunt, Vanja Klepac-Ceraj,{ddagger} Silvia G. Acinas, Clement Gautier,§ Stefan Bertilsson, and Martin F. Polz*

Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 8 September 2005/ Accepted 19 December 2005


   ABSTRACT
Top
 Abstract
Introduction
 Evaluation of primers.
 Analysis of 23s rrna...
 Nucleotide sequence accession...
 References
 
The availability of a diverse set of 23S rRNA gene sequences enabled evaluation of the specificity of 39 previously published and 4 newly designed primers specific for bacteria. An extensive clone library constructed using an optimized primer pair resulted in similar gene richness but slightly differing coverage of some phylogenetic groups, compared to a 16S rRNA gene library from the same environmental sample.


   INTRODUCTION
Top
 Abstract
 Introduction
Evaluation of primers.
 Analysis of 23s rrna...
 Nucleotide sequence accession...
 References
 
There has been renewed interest in the use of the 23S rRNA gene with the decrease in sequencing costs and the growing popularity of techniques such as microarrays (3, 13), analysis of the 16S-23S intergenetic region (7, 9), fluorescence in situ hybridization, and quantitative PCR. The 23S rRNA gene offers the same advantages as the 16S rRNA gene (e.g., universal distribution, conserved function, and invariant and variable regions), yet it includes additional diagnostic sequence stretches due to a greater length, characteristic insertions and/or deletions (12), and possibly better phylogenetic resolution because of greater sequence variation (4, 10-12, 20). However, use of the 23S rRNA gene for bacterial community analysis is hampered by the lack of established broad-range bacterial PCR amplification and sequencing primers.

This study incorporates data from large-scale sequencing efforts to develop new and evaluate existing bacterium-specific 23S rRNA PCR amplification primers. Additionally, this study includes the first well-sampled environmental clone library of 23S rRNA sequences, greatly increasing the number of 23S rRNA gene sequences.


   Evaluation of primers.
Top
 Abstract
 Introduction
 Evaluation of primers.
Analysis of 23s rrna...
 Nucleotide sequence accession...
 References
 
To check the specificity of PCR primers, an alignment of 23S rRNA gene sequences was developed using the ARB software package (http://www.arb-home.de). Bacterial 23S rRNA sequences were obtained from published sources: the European rRNA database (22), National Center for Biotechnology Information complete bacterial genomes (as of 6 February 2005), the ARB LSU database, and environmental bacterial artificial chromosome clones (16, 18). To ensure broad environmental representation of these primers, sequences were also retrieved using BLAST from the Sargasso Sea assembled database (21) with full-length query 23S rRNA sequences from the genomes of representative organisms (Shigella flexneri 2a strain 301, Pirellula strain 1, Prochlorococcus marinus CCMP 1986, Streptomyces coelicolor A3, Bradyrhozobium japonicum USDA110, and Bacteriodes fragilis YCH46). Using this method, 1,415 nonredundant 23S rRNA sequences of >400 bp each were retrieved from the Sargasso Sea data set. Initial alignments of a total of 2,176 sequences were constructed using the ARB Fast Aligner with manual editing based on secondary structure and the existing ARB alignment. This data set was not corrected for skewing, due to overrepresentation of common laboratory organisms, pathogens, and organisms abundant in the Sargasso Sea.

The primers developed in this study (129f, 189r, 457r, and 2490r, with numbering based on Escherichia coli position) (6) show excellent correspondence to sequences in the aligned database (Table 1); additionally, some mismatches may be the result of PCR or sequencing error. Although some previously published "universal" bacterial primers display broad range, this extensive database indicates that other suggested target regions are not sufficiently conserved to serve as bacterial PCR primers (Table 2). Primers for ITS amplification show various degrees of specificity: the region corresponding to the position of primer 129f is highly conserved (8, 15), but other primers are less conserved and exclude a large fraction of bacterial diversity (7, 19).


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  		TABLE 1. Percentage mismatches to the 23S rRNA gene dataset for primers designed in this studya

 

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  		TABLE 2. Percentage mismatches to the 23S rRNA gene dataset at each position for previously described universal primersa

 
On the basis of their broad specificity, length of amplified sequence, and good amplification properties, we propose using the primers 129f (modified in this study) and 2241r for studies of bacterial 23S rRNA diversity. These primers amplify a large portion of the 23S rRNA, consistently produce only a single band of PCR product, and are highly conserved across the bacterial sequences currently available (Tables 1 and 2). Positive amplification was achieved with a diverse set of isolates under the following conditions: 3 min at 94°C; then 30 cycles, each consisting of 1 min at 94°C, 1 min at 57°C, and 2 min at 72°C; and a final 5-min extension at 72°C. All isolates used to test the primers produced PCR product of the correct size; the phyla of bacteria are listed and the number of isolates tested is given in parentheses: {alpha}-Proteobacteria (7), ß-Proteobacteria (2), {delta}-Proteobacteria (1), {varepsilon}-Proteobacteria (1), {gamma}-Proteobacteria (22), Firmicutes (7), Bacteroidetes (8), and Cyanobacteria (2).


   Analysis of 23S rRNA clone library.
Top
 Abstract
 Introduction
 Evaluation of primers.
 Analysis of 23s rrna...
Nucleotide sequence accession...
 References
 
The 129f-2241r primer set was subsequently used to construct a clone library to evaluate coverage and relative distribution of phyla in comparison with a 16S rRNA clone library constructed from a parallel sample (1). A surface seawater sample from the marine end of Plum Island Sound estuary (northeastern Massachusetts) was collected as previously described (1). Cells were lysed using bead beading (5), and DNA was purified using phenol:chloroform:isoamyl alcohol extraction, sodium acetate and ethanol precipitation, and RNase I treatment (17). DNA was amplified in 10 replicate 20-µl PCRs, each reaction mixture containing 50 ng of purified DNA template. PCR conditions were as follows: 3 min at 94°C; then 15 cycles, each consisting of 1 min at 94°C, 1 min at 57°C, and 2 min at 72°C; and a final 5-min extension at 72°C. PCR products were pooled, precipitated with ethanol, and gel extracted (QIAGEN gel extraction kit). Amplicons were cloned using the TOPO-TA kit (Invitrogen).

A total of 535 operational taxonomic units (OTUs) were identified, based on sequential digests with restriction enzymes HhaI and MspI of cloned inserts amplified using internal plasmid primers (M13). Inserts with restriction patterns adding up to >2,500 nucleotides were excluded, as they were assumed to originate from more than one cloned 23S rRNA gene insert. To determine the phylogenetic coverage, at least one member of each OTU was sequenced and grouped into higher taxonomic groups (subphylum or phylum). Both 129f and 457r were used as sequencing primers on plasmids extracted using a QIAprep Spin Miniprep kit (QIAGEN) and M13-amplified PCR products, respectively. A total of 614 clone library sequences were edited using Sequencher, and phylum-level identification of the OTUs was made using discontinuous megaBLAST with a scoring metric (match = 4; mismatch = –5) to allow identification of sequences highly divergent from those present in the database. The cutoff for categorization of a sequence was a sequence length of 300 bp of at least 85% similarity to an organism of known phylogeny.

A comparison of 23S and 16S rRNA (1) gene clone libraries constructed from replicate water samples yielded gross similarities but also some important differences (Fig. 1). The observed levels of richness in the two libraries were comparable when the digestion-defined OTUs in the 23S library were approximated by 99% sequence identity clusters (1, 14) in the 16S rRNA library (535 versus 520 for the 23S and 16S rRNA gene libraries, respectively). In both libraries, Bacteroidetes and {alpha}-Proteobacteria were the most abundant groups (2). However, the 23S library displayed a higher percentage of Bacteriodetes (42.8% versus 32.5%) and lower percentages of {gamma}-Proteobacteria (3.9% versus 22.8%), Actinobacteria, and minor groups. This comparison is of interest because it may reflect the primer bias of either 23S or 16S rRNA primers, a shallower depth of sequence coverage in the 23S library masking rare variants, or a limited 23S rRNA database preventing identification of certain groups. Planctomycetales were probably excluded by these 23S rRNA primers because the forward primer targets a region not present in their 23S rRNA gene. Additionally, >5% mismatches were observed at position 10 of primer 129f to the set of aligned sequences (Table 1); these mismatches occurred primarily in environmental sequences rather than in cultured isolates, confirming the value of incorporating environmental shotgun sequences in the alignment. This mismatch may explain the low level of abundance of {gamma}-Proteobacteria in the clone library, as this alternate sequence is present in the SAR-86 group (16, 18) and other {gamma}-Proteobacteria in the database, as well as members of other phyla. This problem can be remedied by adding an additional degeneracy to primer 129f with the final sequence as CYGAATGGGRVAACC; this modified primer paired with 2241r positively amplified a subset of 14 isolates from diverse phyla. The large number of unknowns is due to the difficulty of 23S rRNA sequence identification because of the poor depth of sequence coverage, especially for less-well-studied phyla (i.e., ß-, {delta}-, and {varepsilon}-Proteobacteria).


Figure 1
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  		FIG. 1. Relative frequency distribution of major phylogenetic groups detected among the environmental sequences from a 16S rRNA library (black) and a 23S rRNA library (gray).

 
Applications using 23S primers, especially techniques such as automated rRNA intergenic spacer analysis that are highly sensitive to the primers chosen (7), should be reevaluated and perhaps modified in light of this data. Nonetheless, this comparison of 16S and 23S rRNA gene sequences shows that reasonable coverage and agreement between broad-range primer pairs can be achieved.

The alignment used to check the primers is available online (see the supplemental material and the ARB database, available for download at http://web.mit.edu/polz/seq=align.html).


   Nucleotide sequence accession numbers.
Top
 Abstract
 Introduction
 Evaluation of primers.
 Analysis of 23s rrna...
 Nucleotide sequence accession...
References
 
Sequences were submitted to GenBank with accession numbers DQ312516 to DQ313129.


   ACKNOWLEDGMENTS
 
This research was supported by grants from NSF, NIEHS, and DOE (Genomes to Life).

Thanks to Ivan Ceraj for programming assistance and Patricia McAndrew for help with initial clone library screening.


   FOOTNOTES
 
* Corresponding author. Mailing address: Massachusetts Institute of Technology, 48-421, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-7128. Fax: (617) 258-8850. E-mail: mpolz{at}mit.edu. Back

{dagger} Supplemental material for this article may be found at http://aem.asm.org/. Back

{ddagger} Present address: Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, MA 02115. Back

§ Present address: Laboratory for Optics and Biosciences, Institut National de la Santé et de la Recherche Médicale, Ecole Polytechnique, 91128 Palaiseau Cedex, France. Back

Present address: Limnology/Department of Ecology and Evolution, Norbyvägen, Uppsala University, SE-75236 Uppsala, Sweden. Back


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 Introduction
 Evaluation of primers.
 Analysis of 23s rrna...
 Nucleotide sequence accession...
 References
 

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Applied and Environmental Microbiology, March 2006, p. 2221-2225, Vol. 72, No. 3
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.3.2221-2225.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.




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