Evolution of the RNA Polymerase B' Subunit Gene (rpoB') in Halobacteriales: a Complementary Molecular Marker to the SSU rRNA Gene

David A. Walsh*, Eric Bapteste*, Masahiro Kamekura{dagger} and W. Ford Doolittle*

* Canadian Institute for Advanced Research Program in Evolutionary Biology, Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada; and {dagger} Noda Institute for Scientific Research, Noda-shi, Chiba-ken, Japan

Correspondence: E-mail: dawalsh{at}dal.ca.


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Many prokaryotes have multiple ribosomal RNA operons. Generally, sequence differences between small subunit (SSU) rRNA genes are minor (<1%) and cause little concern for phylogenetic inference or environmental diversity studies. For Halobacteriales, an order of extremely halophilic, aerobic Archaea, within-genome SSU rRNA sequence divergence can exceed 5%, rendering phylogenetic assignment problematic. The RNA polymerase B' subunit gene (rpoB') is a single-copy conserved gene that may be an appropriate alternative phylogenetic marker for Halobacteriales. We sequenced a fragment of the rpoB' gene from 21 species, encompassing 15 genera of Halobacteriales. To examine the utility of rpoB' as a phylogenetic marker in Halobacteriales, we investigated three properties of rpoB' trees: the variation in resolution between trees inferred from the rpoB' DNA and RpoB' protein alignment, the degree of mutational saturation between taxa, and congruence with the SSU rRNA tree. The rpoB' DNA and protein trees were for the most part congruent and consistently recovered two well-supported monophyletic groups, the clade I and clade II haloarchaea, within a collection of less well resolved Halobacteriales lineages. A comparison of observed versus inferred numbers of substitution revealed mutational saturation in the rpoB' DNA data set, particularly between more distant species. Thus, the RpoB' protein sequence may be more reliable than the rpoB' DNA sequence for inferring Halobacteriales phylogeny. AU tests of tree selection indicated the trees inferred from rpoB' DNA and protein alignments were significantly incongruent with the SSU rRNA tree. We discuss possible explanations for this incongruence, including tree reconstruction artifact, differential paralog sampling, and lateral gene transfer. This is the first study of Halobacteriales evolution based on a marker other than the SSU rRNA gene. In addition, we present a valuable phylogenetic framework encompassing a broad diversity of Halobacteriales, in which novel sequences can be inserted for evolutionary, ecological, or taxonomic investigations.

Key Words: Haloarchaea • RNA polymerase • molecular evolution • microbial diversity • 16S phylogeny


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
The aerobic, extremely halophilic Archaea are classified within the order Halobacteriales, family Halobacteriaceae. Members are chemo-organotrophs, utilize carbohydrates or amino acids for growth, and require at least 9% NaCl for growth; most exhibit optimal growth between 20% and 26% NaCl (Grant et al. 2001). Commonly known as haloarchaea, these organisms exist wherever hypersaline environments are found. Representatives have been isolated from natural environments such as hypersaline lakes in Antarctica (Franzmann et al. 1988) and Africa (Tindall, Ross, and Grant 1984), saline soils (Hezayen et al. 2002), the Dead Sea (Mullakhanbhai and Larsen 1975), and underground salt deposits (Vreeland et al. 2002). Solar salterns are anthropogenic environments that have provided a broad diversity of haloarchaeal isolates (Oren 2002).

Halobacteriales taxonomy is currently based upon SSU rRNA sequences and chemotaxonomic criteria, predominantly polar lipid composition (Oren, Ventosa, and Grant 1997). At present, the order Halobacteriales is the largest in the Archaea and comprises 18 genera: Haloarcula (Har.), Halobacterium (Hbt.), Halobaculum (Hbl.), Halobiforma (Hbf.), Halococcus (Hcc.), Haloferax (Hfx.), Halogeometricum (Hgm.), Halomicrobium (Hmc.), Halorhabdus (Hrd.), Halorubrum (Hrr.), Halosimplex (Hsx.), Haloterrigena (Htg.), Natrialba (Nab.), Natrinema (Nnm.), Natronobacterium (Nbt.), Natronococcus (Ncc.), Natronomonas (Nmn.), and Natronorubrum (Nrr.) (Grant et al. 2001; Hezayen et al. 2002; Oren et al. 2002; Vreeland et al. 2002). In general, each genus can be differentiated from the others based on the presence or absence of specific membrane glycolipids (Kamekura and Kates 1999). However, the recognized diversity of the Halobacteriales has increased substantially over the past decade through application of SSU rRNA gene for taxonomic placement of haloarchaeal isolates, specifically alkaliphilic haloarchaea, which lack major amounts of glycolipids in their membranes (Grant et al. 2001). On the basis of SSU rRNA phylogenetic tree reconstruction, novel genera have been created and considerable rearrangement of organisms within existing genera has occurred (Kamekura et al. 1997). Sequencing of SSU rRNA gene fragments PCR-amplified directly from hypersaline environments has identified many more novel, as yet uncultured, haloarchaeal lineages (Grant et al. 1999; Cytryn et al. 2000; Radax, Gruber, and Stan-Lotter 2001; Benlloch et al. 2002). Haloarchaea 16S phylotypes have also been identified in unexpected environments such as boreal forest soil (Jurgens, Lindstrom, and Saano 1997), deep-sea hydrothermal vents (Takai et al. 2001), and low-salt terrestrial springs (Elshahed et al. 2004). Clearly, the SSU rRNA gene has enhanced our understanding of haloarchaeal evolution, ecological distribution, and taxonomy.

SSU rRNA genes are often present in multiple copies in a single genome. In the vast majority of Bacteria and Archaea, the divergence between copies is minor (<1%) (Acinas et al. 2004). This is not the case for many haloarchaea. Significant intragenomic SSU rRNA heterogeneity is a common feature of Haloarcula species and was first identified in Haloarcula marismortui (Grant et al. 2001). Har. marismortui possesses two SSU rRNA genes that exhibit a divergence of 5% (Mylvaganam and Dennis 1992). More recently, SSU rRNA heterogeneity has been identified in the genera Halosimplex and Natrinema (Vreeland et al. 2002; Boucher et al. 2004). The two SSU rRNA genes of Halosimplex carlsbadense display 6.7% divergence, whereas one of the SSU rRNA genes in Natrinema sp. XA3-1 differs by 5% from the other three (Boucher et al. 2004). Because a divergence of approximately 2% is commonly used to delineate haloarchaeal species and 5% to 10% often distinguishes haloarchaeal genera, intragenomic heterogeneity greater than 5% complicates phylogenetic interpretations of SSU gene sequences (Montalvo-Rodriguez et al. 1998). Furthermore, the degree of intragenomic sequence heterogeneity among SSU genes affects the accuracy of SSU-based studies of microbial communities where a single SSU phylotype is assumed to represent a single organism. For haloarchaea, studies based on SSU may overestimate species diversity. This problem of overestimation is illustrated in Takai et al. (2001), where two haloarchaeal lineages within the genus Haloarcula were identified in a hydrothermal vent chimney. The apparent identification of two separate lineages is more likely a result of SSU rRNA heterogeneity in a single Haloarcula lineage.

There is an additional problem with relying solely on a single molecular marker, such as the SSU rRNA, to determine evolutionary relationships between haloarchaea. In light of lateral gene transfer (LGT), the evolutionary history of a single gene may not necessarily reflect the evolutionary history of the organism as a whole (Gogarten, Doolittle, and Lawrence 2002). Gene exchange between lineages can result in mosaic genomes, whose constituent genes have different evolutionary histories. Comparative analysis of complete genome sequences has shown that LGT is a central force in prokaryotic evolution (Nesbo, Boucher, and Doolittle 2001; Boucher et al. 2003). Indeed, the genome sequence of Halobacterium sp. NRC-1 is a chimerical structure and appears to have acquired a substantial number of genes from bacteria, including many respiratory genes (Ng et al. 2000; Boucher et al. 2003). A mosaic gene structure indicating gene exchange by homologous recombination has also been reported for rRNA operons in several organisms (Yap, Zhang, and Wang 1999; Iredell et al. 2003; Schouls, Schot, and Jacobs 2003; van Berkum et al. 2003), including the haloarchaeon Natrinema sp. XA3-1 (Boucher et al. 2004). To understand the history of haloarchaea and the extent to which their genomes are mosaic, it is important to compare and contrast the history of different genes from these organisms.

The approximately 1,100–amino acid B-subunit of RNA polymerase, RpoB, and the gene encoding it, rpoB, have become popular phylogenetic markers. The rpoB gene has many of the characteristics that have made the SSU rRNA gene itself so useful since its introduction as a universal phylogenetic marker by Woese and Fox (1977). These characteristics include a universal distribution, conserved function, and possession of regions of conserved and variable regions. The RpoB protein has been used to infer relationships between archaeal orders (Matte-Tailliez et al. 2002) and Gram-positive and Gram-negative bacteria (Morse, O'Hanlon, and Collins 2002), whereas the rpoB gene has been used to infer relationships between {alpha}-proteobacterial genera (Taillardat-Bisch, Raoult, and Drancourt 2003) and among members of the Enterobacteriaceae (Mollet, Drancourt, and Raoult 1997) and to identify Staphylococcus species (Drancourt and Raoult 2002). In haloarchaea and some other Archaea, the rpoB gene is fragmented into two genes, rpoB' and rpoB'. The encoded polypeptides, RpoB'' and RpoB' correspond to the N-terminus (~500 amino acids) and C-terminus (~600 amino acids) of RpoB, respectively (Leffers et al. 1989).

Most importantly and in contrast to the SSU rRNA, rpoB has not been detected in multiple copies in any completely sequenced prokaryotic genome. The benefit of the rpoB gene over the SSU rRNA in bacterial community analysis has been previously realized (Dahllof, Baillie, and Kjelleberg 2000), and studies have successfully implemented the rpoB gene to investigate bacterial diversity in marine and soil environments (Peixoto et al. 2002; Taylor et al. 2004). Therefore, the rpoB gene may be a valuable alternative to the SSU rRNA gene in ecological studies of haloarchaea communities.

In this study, we expanded the number of available rpoB' sequences by obtaining an approximately 1.3-kb fragment of the rpoB' gene from 21 haloarchaeal species, encompassing 15 genera. From the rpoB' nucleotide sequences and corresponding amino acid sequences, we inferred phylogenetic trees by a maximum-likelihood method. Approximately Unbiased (AU) tests of tree selection indicated the haloarchaea rpoB' phylogeny is not congruent with the SSU rRNA phylogeny. In other words, the SSU rRNA and rpoB' genes seem to exhibit different evolutionary histories in the haloarchaea. Besides investigating the evolution of haloarchaea, the rpoB' data set made available through this study provides a valuable framework in which novel rpoB' sequences can be inserted, whether for taxonomic, evolutionary, or ecological investigations.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Haloarchaeal Strains and Genomic DNA
All haloarchaeal strains used in this study are listed in table 1. Genomic DNA was extracted from these strains using the protocol from Wilson (1994).


View this table:
[in this window]
[in a new window]
 
Table 1 Halobacteriales Strains and Accession Numbers of rpoB' and SSU rRNA Sequences Used in This Study

 
PCR Amplification of rpoB' Gene Fragments
An approximately 1,300-bp segment of the rpoB' gene was amplified by PCR from purified genomic DNA using forward primer rpoB-444F (5'-TCC CGT ACC CNG ARC AYA AY-3') and reverse primer rpoB-1752R (5'AGT AGA AGY TTR AAN GCR TA-3'). Two separate PCR protocols were required to obtain specific amplification from all desired organisms. All PCR thermal cycling was carried out in 0.2 ml reaction tubes in a Mastercycle gradient thermocycler (Eppendorf). Amplification from Har. aidinensis, Hrd. utahensis, Htg. sp. GSL-11, Nab. asiatica, Nnm. pallidum, Nrr. sp. Tenzan-10, Nmn. pharaonis, Har. japonica, and Hfx. mediterranei was performed in a 50 µl reaction volume containing 5 µl 10x Taq PCR buffer, 0.8 mM deoxynucleoside triphosphates, 1.5 mM MgCl2, 0.5 µM each primer, 2.5 U Taq DNA polymerase, and 1 ml purified genomic DNA. The thermal profile for amplification began with an initial denaturation step (3 min, 94°C) followed by 35 cycles of denaturation (1 min, 94°C), annealing (30 s, 48°C), and extension (2 min, 68°C). Thermal cycling was followed by a final terminal extension step (10 min, 72°C). Amplification from Hgm. borinquense, Hbf. haloterrestris, Hbl. gomorrense, Hrr. distributum, Hrr. coriense, Hcc. morrhuae, Hcc. saccharolyticus, Hfx. denitrificans, Htg. turkmenica, Nbt. gregoryi, Nbt. sp. SSL6, Hsx. carlsbadense was performed in a 50 µl reaction volume containing 10 ml Pfx Amplification Buffer, 10 µl PCRx Enhancer Solution, 1.6 mM deoxynucleoside triphosphates, 1 mM MgSO4, 1 mM each primer, 2 U Pfx DNA polymerase, and 1 ml purified genomic DNA. The thermal profile for amplification began with an initial denaturation step (5 min, 94°C) followed by 35 cycles of denaturation (45 s, 94°C), annealing (45 s, 48°C), and extension (1 min 20 s, 72°C). Thermal cycling was followed by a final terminal extension step (5 min, 72°C).

Gel Purification and Cloning
Aliquots (5 µl) of the PCR reaction were analyzed by agarose (0.7%) gel electrophoresis to confirm primer specificity. The remainder of the PCR reaction (45 µl) was then subjected to electrophoresis and the desired DNA fragment (~1300 bp) was eluted from the gel using a MiniElute Gel Extraction Kit (Qiagen). Purified DNA fragments were cloned using a TOPO TA Cloning Kit (Invitrogen) using pCR 2.1-TOPO as a vector. Transformants were analyzed for insert using M13 forward and M13 reverse primers as outlined in the TOPO TA Cloning Manual. Two positive clones were selected from each PCR reaction for DNA sequencing.

DNA Sequencing and Assembly
Clones were sequenced using MegaBase technology and BigDye chemistry. Full coverage of the DNA insert was ensured through the use of multiple sequencing primers. In addition to the vector-specific primers M13F and M13R, a forward primer (rpoB-863F, 5'-GAC GGC CTC GTC AAC CCC GA-3') and reverse primer (rpoB-1092R, 5'-CGG CTC GCR AAY TTR TCN CC-3') targeted to conserved regions of the rpoB' gene were used for sequencing. Sequencher version 4.1.2 (Gene Codes Corporation) was used to visualize sequence chromatograms and assemble/edit DNA sequence fragments.

Multiple Sequence Alignment and Phylogenetic Analysis
Accession numbers for all rpoB' nucleotide sequences and SSU rRNA used in this study are presented in table 1. Nucleotide sequence of the rpoB' gene for Halobacterium sp. NRC-1 and all SSU rRNA genes were retrieved from the GenBank database. Nucleotide sequence of the rpoB' gene for Har. marismortui and Hfx. volcanii were retrieved from the Halophiles Genomes Web page (zdna2.umbi.umd.edu). Multiple sequence alignments were constructed using ClustalW (Thompson, Higgins, and Gibson 1994) and, if required, edited manually to remove gaps and ambiguously aligned characters. Phylogenetic analyses of DNA sequences were performed with PAUP* version 4.0b10 (Swofford 1998) using the heuristic search option and the TBR branch-swapping algorithm. Maximum likelihood (ML) was employed as the tree reconstruction method. The nucleotide substitution model, {alpha} parameter of the {Gamma} distribution, fraction of invariable sites, and nucleotide frequencies were estimated for each data set using Modeltest (Posada and Crandall 1998). The confidence of each node was determined by assembling a consensus tree of 100 bootstrap replicates. Protein alignments were analyzed by ML using PROML with a JTT model of substitution, a mixed model of rate heterogeneity ({Gamma}8), global rearrangments, and randomized input order of sequences. Model parameters were estimated from the data set using PUZZLE version 5.1 (Schmidt et al. 2002). ML bootstrap support values represent a consensus (calculated using CONSENSE) of 100 ML trees obtained using SEQBOOT and PROML. CONSENSE, PROML, and SEQBOOT are included in the PHYLIP version 3.6a software package (Felsenstein 2000).

Identification of Mutational Saturation of the rpoB' Gene in Haloarchaea
Partial rpoB' sequences of Htg. turkmenica and Hcc. morrhuae were eliminated from the alignment before analyses. Saturation analyses of the rpoB' gene were done for each codon position of the rpoB' gene using comp_mat from MUST version 3.0 (Philippe 1993). The proportion of pairwise observed differences between rpoB' sequences was calculated with an uncorrected NJ method using MUST 3.0 and was plotted against the proportion of inferred substitutions estimated by maximum parsimony using PAUP* 4.0b10 (Swofford 1998).

AU Test of Tree Selection
A set of topologies including the best trees for the haloarchaeal alignments of the rpoB' gene, RpoB' protein, rpoB' gene without the third codon position, and all possible SSU data sets (differing in combinations of sequences for species with multiple divergent SSU genes: Har. marismortui and Hsx. carlsbadense) were used as user-tree in PUZZLE 5.1, option–wsl, with a JTT+{Gamma}8+I model of evolution to estimate the likelihood of each site of a given data set and global tree likelihoods for each tree. These two sets of likelihood values were used as input for CONSEL (Shimodaira and Hasegawa 2001) to perform the AU test (Shimodaira 2002). Trees were rejected at P < 0.05.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
Amplification of rpoB' Gene Fragments and Alignment of Nucleotide Sequences and Putative Amino acid Sequences
Several degenerate primer sets were designed based on a nucleotide alignment of rpoB' genes from Halobacterium sp. NRC-1, Hfx. volcanii, and Har. marismortui. Primer pair rpoB-444F/rpoB-1752R, chosen because it produced a single PCR product of the expected size (~1,300 bp), was used to clone and sequence rpoB' gene fragments from 21 species encompassing 15 genera of haloarchaea. Because of DNA sequencing difficulty, only partial sequence data was retrieved from the approximately 1,300-bp cloned rpoB' fragment of Halococcus morrhuae and Haloterrigena turkmenica. All rpoB' gene fragments reported here, except for these, are 1,305 bp in length and were aligned with those of Halobacterium sp. NRC-1, Hfx. volcanii, and Har. marismortui. As evident from the alignment, the primary structure of this portion of the rpoB' gene is highly conserved in haloarchaea. The DNA sequences can be unambiguously aligned without gaps. Natronobacterium gregoryi and Natronobacterium sp. SSL6 were found to have identical rpoB' sequences, and the latter was eliminated from further phylogenetic analyses. This alignment, comprising 21 haloarchaeal rpoB' gene fragments, contained 1,305 sites, of which 752 (58%) were constant across taxa. Addition of the partial rpoB' sequence data of Hcc. morrhuae and Htg. turkmenica identified the regions of rpoB' sequence data missing from these species. The first 616 bp of sequence data of the rpoB' alignment were missing for Htg. turkmenica, and an internal region of rpoB' sequence data, corresponding to bp 514 to 606 of the haloarchaeal rpoB' DNA alignment, was missing for Hcc. morrhuae.

Upon translation of the rpoB' DNA alignment, several groups of species were found to have identical RpoB' amino acid sequences: Natrinema pallidum, and Haloterrigena sp. GSL-11; Hfx. volcanii and Haloferax denitrificans; Halorubrum distributum and Halorubrum coriense; and Har. marismortui, Haloarcula japonica, and Haloarcula aidinensis. RpoB' amino acid sequences for Haloterrigena sp. GSL-11, Haloferax denitrificans, Halorubrum coriense, Haloarcula japonica, and Haloarcula aidinensis were eliminated from the RpoB' alignment to expedite subsequent phylogenetic analyses. This putative amino acid alignment composed of 16 partial RpoB' amino acid sequences contained 435 sites, of which 310 (71%) were constant across taxa. The translated rpoB' fragments from Hcc. morrhuae and Htg. turkmenica were added to this alignment, resulting in an RpoB' alignment of 18 unique RpoB' amino acid sequences.

Nucleotide and Amino Acid Composition of Haloarchaeal rpoB' Genes
Unidentified sequence heterogeneity can result in systematic error if the model of evolution employed in the analysis incorrectly assumes that the same sequence composition exists in all lineages (Swofford et al. 1996). The G+C content for rpoB' gene fragments is presented in table 1 and was found to vary between 63.2% in Natronorubrum sp. Tenzan-10 and 69.2% in Hrr. distributum. The rpoB' DNA alignment containing all 23 rpoB' DNA sequences passed a chi-square test of base frequency homogeneity (P = 0.74, df = 66). Furthermore, all nucleotide and translated rpoB' sequences passed the chi-square test employed in Tree-Puzzle that compares either the nucleotide or amino acid composition of each sequence to the frequency distribution assumed under the maximum-likelihood model of evolution. Therefore, we are reasonably confident that compositional bias is not affecting our phylogenetic analyses.

Location of Variable and Conserved Regions of Haloarchaeal rpoB' Genes
We plotted the relative evolutionary rate for each amino acid position, as calculated by Tree-Puzzle, against its position in the rpoB' gene. A graphical representation of the results obtained from this analysis is presented in figure 1. Two hypervariable regions, HvR-1 and HvR-2, were located towards the 3' end of the rpoB' gene. These regions correspond approximately to nucleotide positions 1200 to 1400 and 1550 to 1750 of the Halobacterium sp. NRC-1 rpoB' gene, respectively. Within HvR-1, 24 of 63 amino acid positions are variable, whereas 36 of 67 amino acid positions are variable within HvR-2. The most highly conserved region of rpoB' is located between nucleotide positions 850 and 1200. Within this region, only 21 of 116 amino acid positions are variable. The 5' region of rpoB' exhibits relatively high amino acid sequence conservation interspersed with very short variable regions.



View larger version (8K):
[in this window]
[in a new window]
 
FIG. 1.— Identification of variable and conserved regions of the rpoB' gene in haloarchaea. The relative evolutionary rate of each amino acid position was calculated from the translated rpoB' gene in haloarchaea using four rate categories (category 1 = slowest, category 4 = fastest). A sliding window method was used to calculate the average relative evolutionary rate. The window size was 20 amino acids and was slid in 1–amino acid increment along the length of the RpoB alignment. HvR-1, hypervariable region 1; HvR-2, hypervariable region 2

 
Detection of Mutational Saturation of the Haloarchaeal rpoB' DNA Alignment
Mutational saturation can be identified when, for each pairwise comparison of taxa, the number of inferred substitutions estimated by maximum parsimony is plotted against the number of observed substitutions estimated by uncorrected neighbor joining (Philippe et al. 1994). A highly saturated data set displays a characteristic plateau where the number of observed substitutions appears globally constant whatever the actual phylogenetic distance between species. Using this method, we investigated the prevalence of saturation at each codon position in the haloarchaeal rpoB' alignment to construct a tree with the less saturated, but still informative, data set (fig. 2).



View larger version (13K):
[in this window]
[in a new window]
 
FIG. 2.— Degree of mutational saturation at the (A) first, (B) second, and (C) third codon positions of the haloarchaea rpoB' alignment. Observed differences between pairs of haloarchaea were calculated by an uncorrected NJ method. Inferred differences were calculated by maximum parsimony. Highly saturated data sets display a characteristic plateau.

 
The first codon position exhibits the beginning of a saturation process (fig. 2A). The number of observed differences between rpoB' sequences begins to plateau when the distance between taxa inferred by parsimony reaches 0.19 substitutions per site (fig. 2A). Interestingly, all pairwise comparisons above 0.19 inferred substitutions per site are either between members of two distantly related haloarchaeal clades, clade I and clade II, (see below) or between members of clade I and Halobacterium sp. NRC-1. At the second codon position, the observed difference between species increases proportionally with the number of substitutions inferred by parsimony (fig. 2B), indicating this position is nearly free of mutational saturation. In contrast, analysis of the third codon position reveals a high degree of saturation (fig. 2C). Only between the most closely related taxa, such as Haloferax species or Natrinema species, is the effect of saturation not observed at the third codon position.

Comparison of rpoB' Phylogenetic Trees Obtained from DNA and Putative Protein Alignments
ML phylogenetic analysis was performed on both the haloarchaeal rpoB' DNA alignment and the haloarchaeal RpoB' protein alignment. The haloarchaeal RpoB' protein alignment was supplemented with six nonhaloarchaeal, euryarchaeal RpoB sequences to provide an outgroup for the haloarchaea. Euryarchaeal sequences included in this outgroup were carefully chosen so as to not violate the amino acid frequency assumed under the ML model of evolution. The best ML trees inferred from the haloarchaeal rpoB' DNA data set and the haloarchaeal RpoB' protein data set plus outgroup are presented in figure 3A and B, respectively. Only ML bootstrap values greater than 60% are presented in the trees.



View larger version (38K):
[in this window]
[in a new window]
 
FIG. 3.— (A) Best ML tree inferred from the rpoB' DNA alignment for the Halobacteriales. The evolutionary model and parameters employed in the analyses were GTR+ {Gamma} 4+I, {alpha} = 0.96, I = 0.48. (B) Best ML tree inferred from the RpoB' protein for the Halobacteriales. The evolutionary model and parameters employed in the analyses were JTT+ {Gamma} 8, {alpha} = 0.46. Support for nodes in both trees correspond to bootstrap values for 100 pseudoreplicates. Only values greater than 60% are displayed. The euryarchaeal outgroup was composed of RpoB sequences from Methanosarcina acetivorans, Methanosarcina barkeri, Methanosarcina mazei, Archaeoglobus fulgidus, Methanothermobacter thermautotrophicus, and Methanococcus vannielii.

 
Regardless of data set utilized, phylogenetic analysis of rpoB' consistently recovered two monophyletic groups with high bootstrap support, imbedded within a collection of less well resolved haloarchaeal lineages. These groups have been highlighted as the clade I and clade II haloarchaea in figure 3. Exclusion of certain taxa from clade I or clade II was done because their position in respect to clade I or clade II is either poorly resolved or questionable (fig. 3). In figure 3A, a weakly supported cluster (<60%) corresponding to Halorhabdus utahensis, Halosimplex carlsbadense, and Natronomonas pharaonis forms a monophyletic group with Haloarcula species. However, this clade is not reconstructed from the RpoB' protein alignment (fig. 3B). Instead, there is sequential branching of Halosimplex, Haloarcula, Halorhabdus, and Natronomonas from the root of the tree towards the clade I haloarchaea. The position of Halobacterium sp. NRC-1 adjacent to the clade II haloarchaea is consistent and well supported in trees constructed from the complete rpoB' DNA alignment (94% [fig. 3A]) and the RpoB' protein alignment (84% [fig. 3B]). However, Halobacterium sp. NRC-1 was not included as a member of the clade II haloarchaea, because this relationship was not supported in SSU rRNA phylogenies. On the other hand, the monophyly of the clade I and clade II, as defined here, were supported in SSU rRNA phylogenies (fig. 4) (see below).



View larger version (31K):
[in this window]
[in a new window]
 
FIG. 4.— Comparison of best ML trees inferred from the RpoB' protein and SSU gene for Halobacteriales. The evolutionary model and parameters employed in the analyses for the RpoB' gene were JTT+{Gamma}8, {alpha} = 0.20. For the SSU gene they were GTR+{Gamma}4+I, {alpha} = 0.72, I = 0.58. Support for nodes in all trees correspond to bootstrap values for 100 pseudoreplicates. Only values greater than 60% are displayed. For the species Hsx. carlsbadense and Har. marismortui, both their SSU genes were included in the trees. Trees have been arbitrarily rooted on the clade I haloarchaea for display purposes.

 
The clade I haloarchaea is dominated by alkaliphilic haloarchaea and contains species assigned to the genera Halobiforma, Haloterrigena, Natrialba, Natrinema, Natronobacterium, and Natronorubrum. The bootstrap support for clade I inferred from the rpoB' DNA alignment is 100% (fig. 3A). The bootstrap support for clade I inferred from the RpoB' protein alignment is 99% (fig. 3B). Within clade I, two highly supported relationships are recovered in the tree inferred from the rpoB' DNA alignment. They are the sister relationship between Nnm. pallidum and Htg. sp. GSL-11 (100%) and the basal position of Natrialba asiatica (83%) (fig. 3A). In the RpoB' protein tree there is strong support for the branching of Nnm. pallidum and Htg. turkmenica (84%). However, the position of Nab. asiatica at the base of clade I is no longer apparent (fig. 3B). Instead, Nab. asiatica branches with Nrr. sp. Tenzan-10 with moderate bootstrap support (71%) (fig. 3B). This discrepancy in the position of Nab. asiatica between the DNA and protein phylogenies is addressed later, taking into consideration the results of the phylogenetic analysis performed on the rpoB' DNA alignment without the third codon position.

The clade II haloarchaea comprises species assigned to the genera Halobaculum, Haloferax, Halogeometricum, and Halorubrum. In the rpoB' DNA tree, bootstrap support for clade II is 95% (fig. 3A). In the RpoB' protein tree, bootstrap support for clade II is 100% (fig. 3B). Within clade II, both trees exhibit high support for the branching of Haloferax species and Halogeometricum borinquense together to the exclusion of Halobaculum gomorrense and Halorubrum species (fig. 3). Interestingly, there is only very weak support (46%) for the monophyly of Haloferax inferred from the rpoB' DNA alignment (fig. 3A). In fact, bootstrap analysis reveals equivalent support (54%) for an alternate topology where Hfx. mediterranei and Hgm. borinquense branch together as sister taxa. The apparent monophyly of Haloferax is however, recovered in the tree inferred from the RpoB' protein alignment with high statistical support (99%) (fig. 3B). A further, but not robust, discrepancy between trees inferred from the rpoB' DNA alignment and the RpoB' protein alignment is the branching order of Halorubrum species and Hbl. gomorrense. In the rpoB' DNA tree, Halorubrum species are positioned at the base of clade II with moderate bootstrap support (65%) (fig. 3A). Contrarily, in the RpoB' protein tree, Hbl. gomorrense is at the base of clade II, although with little support (54%) (fig. 3B).

The Effect of Halobacterium sp. NRC-1 rpoB' on the Branching Pattern Within Clade II Haloarchaea
The branch leading to Halobacterium sp. NRC-1 appears to be the longest in all trees, particularly the rpoB' DNA tree (fig. 3A). This suggests the rpoB' gene of Halobacterium sp. NRC-1 gene has an elevated rate of evolution relative to other haloarchaeal rpoB' genes. A known artifact of phylogenetic analysis is long-branch attraction (LBA) that tends to group long branches together, irrespective of their true phylogenetic positions (Felsenstein 2004). In respect to the clade II haloarchaea, we reasoned the difference in branching pattern between trees inferred from the rpoB' DNA and protein data sets may be caused by a local long-branch effect in the rpoB' DNA tree between Halobacterium sp. NRC-1 and Halorubrum species. To investigate this hypothesis, we removed the Halobacterium sp. NRC-1 rpoB' sequence from all data sets and repeated the phylogenetic analyses.

Removal of Halobacterium sp. NRC-1 from the haloarchaeal RpoB' protein data set (no outgroup) resulted in an increase in bootstrap support from 72% to 86% for the position of Hrr. coriense next to Hgm. borinquense, Hfx. volcanii, and Hfx. mediterranei. More notably, removal of Halobacterium sp. NRC-1 from the rpoB' DNA data set resulted in a switch in branching order of Halorubrum species and Hbl. gomorrense to that observed in the RpoB' protein tree (fig. 3B). This topology was relatively well supported by bootstrap analysis (81%) and is probably a better reflection of the true evolutionary history of the rpoB' gene in haloarchaea.

Haloarchaeal rpoB' Phylogeny of First and Second Codon Positions
To decrease the impact of third codon position saturation on the rpoB' phylogeny, we also constructed a rpoB' phylogeny using only the first and second codon positions. In trees inferred from the first and second codon alone, the monophyly of the clade I and clade II haloarchaea is robustly upheld by bootstrap values greater than 90%. However, there are two strongly supported relationships in the complete rpoB' phylogeny that are not recovered with high support upon removal of the third codon position from analysis. The bootstrap value for the position of Halobacterium sp. NRC-1 sister to the clade II haloarchaea dropped from 94% to 59%. Similarly, bootstrap support for the position of Nab. asiatica at the base of the clade I haloarchaea decreased from 83% to 44%, demonstrating the basal position of Nab. asiatica in clade I is heavily dependent on the third codon position. Because we have established that the third position of the haloarchaea data set is saturated and the position of Nab. asiatica is not the same in the RpoB' protein tree, a basal position of Nab. asiatica in clade I is unlikely to represent a realistic evolutionary relationship.

Comparison Between Haloarchaea Phylogenies Obtained from the rpoB' Gene and SSU rRNA Gene
Two data sets were used to infer phylogenetic trees from a 1,427-nt alignment of haloarchaeal SSU rRNA genes by the ML method. The first data set contained the same taxa as the tree inferred from the rpoB' DNA alignment, and the second data set contained the same taxa as the tree inferred from the RpoB' protein alignment. For those organisms with known intragenomic SSU rRNA heterogeneity, namely Hsx. carlsbadense and Har. marismortui (Mylvaganam and Dennis 1992; Boucher et al. 2004), both SSU rRNA paralogs from each species were included in phylogenetic trees.

The haloarchaea phylogeny inferred from the RpoB' protein alignment (fig. 4) is arrayed against that obtained from the SSU rRNA gene in figure 4. The two monophyletic groups, herein labeled as the clade I and clade II haloarchaea, inferred from rpoB' phylogenies are preserved in the SSU rRNA tree. The bootstrap support for the clade I haloarchaea is 100% (fig. 4). The bootstrap support for the clade II haloarchaea is 87% (fig. 4).

Many relationships between haloarchaea are consistent in the RpoB' protein and SSU rRNA phylogenies. However, the trees are not identical. There are several well-supported relationships in the SSU rRNA tree that are not apparent in the RpoB' tree (fig. 4): (1) Halococcus branches adjacent to the clade I haloarchaea with 89% bootstrap support, (2) Nnm. pallidum, Htg. turkmenica, Nrr. sp. Tenzan 10, and Nab. asiatica form a robustly supported clade (88%), and (3) Hrr. coriense and Hbl. gomorrense branch together as sister taxa (100%). In addition, the "doubtful" position of Halobacterium sp. NRC-1 adjacent to the clade II haloarchaea in the rpoB' trees is not supported in the SSU rRNA tree (fig. 4).

To determine whether the extent of incongruence between rpoB' trees and SSU rRNA trees was statistically significant, we used the AU test of tree selection (Shimodaira 2002). SSU rRNA trees were inferred from all four possible combinations of Har. marismortui and Hsx. carlsbadense SSU rRNA genes to minimize the probability of tree rejection as a sole result of differential SSU rRNA paralog sampling. Because SSU rRNA heterogeneity is unknown, yet likely, in Har. japonica and Har. aidinensis, sequences from these organisms were removed to avoid tree rejection caused by unidentified SSU rRNA paralog sampling in these organisms.

Independent of the Har. marismortui and Hsx. carlsbadense SSU rRNA genes included in the tree, all four SSU rRNA trees were strongly rejected (P < 0.001) by the rpoB' DNA data set, the RpoB' protein data set, and the rpoB' DNA alignment with the third codon position removed (table 2). Likewise, the trees inferred from the rpoB' DNA data set, the RpoB' protein data set, and the rpoB' DNA alignment with the third codon position removed were strongly rejected (P < 0.001) by all four SSU rRNA data sets (table 2). These results demonstrate the haloarchaea rpoB' phylogenies and the SSU rRNA phylogenies are significantly incompatible with one another. Furthermore, tree incongruence was not caused solely by the fast-evolving Halobacterium sp. NRC-1 rpoB' sequence, nor was it caused by Nab. asiatica or Halococcus sp. sequences, because removal of these sequences singly and as a group did not have a significant effect on the AU test results.


View this table:
[in this window]
[in a new window]
 
Table 2 Summary of AU Test Results for rpoB' and SSU rRNA Trees

 
In addition to incongruence between rpoB' and SSU rRNA phylogenies in haloarchaea, several of the SSU rRNA trees, which differed in the SSU rRNA gene chosen to represent Har. marismortui and Hsx. carlsbadense, were recognized as incongruent with one another. For example, the SSU rRNA tree constructed from the data set that contains the Har. marismortui rrnB gene and the Hsx. carlsbadense rrnA gene is strongly rejected (P < 0.005) by data sets that contain, alternatively, the Hsx. carlsbadense rrnB gene (table 2). This is a strong indication that the two divergent SSU rRNA paralogs of Hsx. carlsbadense do not contain congruent phylogenetic signal, and paralog selection has a significant effect on the position of Hsx. carlsbadense in the SSU rRNA tree.


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
This study was motivated by the recent identification of significant intragenomic SSU rRNA heterogeneity in three genera of Halobacteriales (Mylvaganam and Dennis 1992; Boucher et al. 2004). Unidentified intragenomic SSU rRNA heterogeneity can have a profound effect on the observed topology of the SSU rRNA tree. For a species such as Hsx. carlsbadense, which harbors two divergent SSU genes, we have demonstrated that its position in the SSU phylogeny is highly dependent on choice of SSU gene included in the analysis. The position of Hsx. carlsbadense inferred from rrnA is not congruent with the position inferred from rrnB. Given that the phenomenon of heterogeneity is scattered among distant Halobacteriales genera, it may be present in additional genera. For the majority of haloarchaea, only a single SSU rRNA gene is available in the public database. If some of these organisms harbor multiple divergent SSU genes, their position in the SSU tree is contingent upon which SSU gene has been amplified from the genome.

Our objective was to complement the haloarchaea SSU rRNA phylogeny with an alternative molecular marker unlikely to be paralogous, namely the RNA polymerase B'-subunit gene (rpoB'), to investigate the evolutionary history of haloarchaea. We have demonstrated the rpoB' gene provides a similar degree of phylogenetic resolution as the SSU rRNA gene, yet does not bear the problem of paralogy. For example, the relationship between Haloarcula species is difficult to distinguish by SSU rRNA phylogeny because of known paralogy in this genus (Grant et al. 2001). Conversely, the relationship between Haloarcula species is apparent in the rpoB' phylogeny. Har. japonica and Har. marismortui group together to the exclusion of Har. aidinensis (fig. 3A). This demonstrates an advantage of using the rpoB' gene in addition to the SSU rRNA gene to infer phylogenetic relationships among the haloarchaea.

The rpoB gene appears to be a suitable candidate for a molecular marker in microbial community analysis (Dahllof, Baillie, and Kjelleberg 2000). The nature of the evolutionary rate along the rpoB' gene in haloarchaea makes this gene ideal for such studies in haloarchaea. PCR primers with a minimal amount of degeneracy can be targeted to the conserved regions bracketing the region HvR-1 (fig. 1). HvR-1 may be the optimal region for analysis because its relatively high sequence variability should provide phylogenetic resolution for fine scale or "microdiversity" studies. Clusters of microdiversity can then be placed in a larger phylogenetic context using the translated HvR-1 region. In this study, we present a rpoB' phylogenetic framework encompassing a broad diversity of haloarchaea, in which these novel sequences can be placed.

Mutational saturation is apparent in the haloarchaea rpoB' DNA alignment, particularly at the third codon position (fig. 2C). This level of saturation may lead to artifactually resolved relationships, such as the basal position of Nab. asiatica in the clade I haloarchaea (fig. 3A). However, the identification of mutational saturation is not necessarily detrimental to the use of rpoB' as a phylogenetic marker in haloarchaea as the data set can be tailored to the phylogenetic question under address. The RpoB' protein alignment is well suited for revealing deeper relationships such as between distant haloarchaeal genera, whereas the faster evolving rpoB' DNA alignment can uncover finer scale evolutionary relationships such as between members of a single genus or several closely related genera. We also note that although trees inferred from the rpoB' DNA and protein data sets exhibited slightly different topologies, these differences are not statistically significant, as trees were deemed to be congruent in AU tests (P < 0.05).

Trees inferred from all rpoB' data sets (i.e., DNA, protein, and DNA without third codon position) were incongruent with SSU trees. Incongruent phylogenies indicate either the SSU or rpoB' phylogenies or both are incorrect because of artifacts of phylogenetic tree reconstruction methods, and/or the trees are accurate and the differences are biologically relevant. We did not detect any sequence composition bias in the rpoB' DNA or protein alignment. Nor did we detect sequence compositional bias in the haloarchaea SSU rRNA alignment (data not shown).

SSU rRNA and rpoB' tree incongruence could result from hidden SSU rRNA heterogeneity and paralog sampling effects in some haloarchaeal lineages, specifically within the clade I haloarchaea. Intragenomic SSU heterogeneity is documented in the genus Natrinema (Boucher et al. 2004). Taxonomic problems between this genus and Haloterrigena are known to exist and may be a direct consequence of SSU rRNA heterogeneity (Tindall 2003). The taxonomic confusion between haloarchaea currently classified as Haloterrigena and Natrinema could potentially be reduced through phylogenetic analysis of the rpoB' gene in these lineages.

Perhaps the incongruities between the SSU and rpoB' gene phylogenies are also a result of LGT between haloarchaeal lineages. Although LGT of either rpoB' or SSU rRNA is a distinct possibility, there are two factors making it difficult to clearly identify the candidate gene effected by LGT: (1) only two gene phylogenies are available for the haloarchaea, and (2) there are no conflicting nodes robustly supported in both phylogenies.

Does the distribution of any chemotaxonomic characters among haloarchaea suggest one gene was transferred over the other? One such characteristic is the presence/absence of C20C25 core membrane lipids. C20C25 lipids are a defining feature of all clade I haloarchaeal genera plus Halococcus and Natronomonas. Assuming the ability to synthesize C20C25 arose only once during haloarchaeal evolution and has not been lost, the presence of C20C25 should be a shared characteristic of all haloarchaea lineages that diverged after the appearance of C20C25 lipids. A gene that has not been transferred since this event should accurately reconstruct the monophyly of C20C25-containing haloarchaea. In neither the RpoB' or SSU rRNA phylogenies are C20C25-containing haloarchaea monophyletic. However, Natronomonas and clade I form a monophyletic group in the rpoB' phylogeny (fig. 4, left side), and Halococcus and clade I form a monophyletic group in the SSU rRNA phylogeny (fig. 4, right side). An hypothesis that may explain this observation could include LGT of rpoB' between an ancestor of Halococcus and a C20C25-lacking haloarchaeon or LGT of a SSU rRNA gene between an ancestor of Natronomonas and a C20C25-lacking haloarchaeon. Investigation into the validity of this interpretation would benefit from a multigenic approach in which several additional genes were sequenced from haloarchaea and their evolutionary histories compared with the rpoB' and SSU rRNA phylogenies.

We also note the presence of C20C25 core lipids in a single Halorubrum species, Halorubrum vacuolatum. Originally classified in the genus Natronobacterium, this organism was transferred to the genus Halorubrum based on SSU sequence similarity and the existence of genus-specific signature bases (Kamekura et al. 1997). Generally, members of the genus Halorubrum are neutrophilic, lack C20C25 lipids, and contain membrane glycolipids (Grant et al. 2001). Besides the presence of C20C25 core lipids, Hrr. vacuolatum is alkaliphilic and lacks substantial glycolipids (Mwatha and Grant 1993), rendering this organism more alike the clade I haloarchaea than other Halorubrum species. Phylogenetic analysis of the rpoB' gene from Hrr. vacuolatum may propose an alternative phylogenetic position than the SSU gene, suggesting a mosaic genome structure in this organism.


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 
We thank Yan Boucher, Thane Papke, and members of the W. F. Doolittle laboratory for stimulating discussion and two anonymous reviewers for helpful comments. This work was supported by grant MT4467 to W.F.D. from the Canadian Institute for Health Research.


    Footnotes
 
Jonathan Eisen, Associate Editor


    References
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 References
 

    Acinas, S. G., L. A. Marcelino, V. Klepac-Ceraj, and M. F. Polz. 2004. Divergence and redundancy of 16s rrna sequences in genomes with multiple rrn operons. J. Bacteriol. 186:2629–2635.[Abstract/Free Full Text]

    Benlloch, S., A. Lopez-Lopez, E. O. Casamayor et al. (12 co-authors). 2002. Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern. Environ. Microbiol. 4:349–360.[CrossRef][ISI][Medline]

    Boucher, Y., C. J. Douady, R. T. Papke, D. A. Walsh, M. E. Boudreau, C. L. Nesbo, R. J. Case, and W. F. Doolittle. 2003. Lateral gene transfer and the origins of prokaryotic groups. Annu. Rev. Genet. 37:283–328.[CrossRef][ISI][Medline]

    Boucher, Y., C. J. Douady, A. K. Sharma, M. Kamekura, and W. F. Doolittle. 2004. Intragenomic heterogeneity and intergenomic recombination among haloarchaeal ribosomal RNA genes. J. Bact. (in press).

    Cytryn, E., D. Minz, R. S. Oremland, and Y. Cohen. 2000. Distribution and diversity of archaea corresponding to the limnological cycle of a hypersaline stratified lake (Solar lake, Sinai, Egypt). Appl. Environ. Microbiol. 66:3269–3276.[Abstract/Free Full Text]

    Dahllof, I., H. Baillie, and S. Kjelleberg. 2000. rpoB-based microbial community analysis avoids limitations inherent in 16S rRNA gene intraspecies heterogeneity. Appl. Environ. Microbiol. 66:3376–3380.[Abstract/Free Full Text]

    Drancourt, M., and D. Raoult. 2002. rpoB gene sequence-based identification of Staphylococcus species. J. Clin. Microbiol. 40:1333–1338.[Abstract/Free Full Text]

    Elshahed, M. S., F. Z. Najar, B. A. Roe, A. Oren, T. A. Dewers, and L. R. Krumholz. 2004. Survey of Archaeal diversity reveals an abundance of halophilic Archaea in a low-salt, sulfide- and sulfur-rich spring. Appl. Environ. Microbiol. 70:2230–2239.[Abstract/Free Full Text]

    Felsenstein, J. 2000. PHYLIP (Phylogeny inference package). Version 3.6a. Distributed by the author, University of Washington, Seattle.

    ———. 2004. Inferring phylogenies. Sinauer Associates, Sunderland, Mass.

    Franzmann, P. D., E. Stackebrandt, K. Sanderson, J. K. Volkman, D. E. Cameron, P. L. Stevenson, T. A. McMeekin, and H. R. Burton. 1988. Halobacterium lacusprofundi sp. nov., a halophilic bacterium isolated from Deep Lake, Antarctica. Syst. Appl. Microbiol. 11:20–27.[ISI]

    Gogarten, J. P., W. F. Doolittle, and J. G. Lawrence. 2002. Prokaryotic evolution in light of gene transfer. Mol. Biol. Evol. 19:2226–2238.[Abstract/Free Full Text]

    Grant, S., W. D. Grant, B. E. Jones, C. Kato, and L. Li. 1999. Novel archaeal phylotypes from an East African alkaline saltern. Extremophiles 3:139–145.[CrossRef][ISI][Medline]

    Grant, W. D., M. Kamekura, T. J. McGenity, and A. Ventosa. 2001. Class III. Halobacteria class. nov. Pp. 294–334 in D. R. Boone and R. W. Castenholz, eds. Bergey's manual of systematic bacteriology. Springer, New York.

    Hezayen, F. F., B. J. Tindall, A. Steinbuchel, and B. H. Rehm. 2002. Characterization of a novel halophilic archaeon, Halobiforma haloterrestris gen. nov., sp. nov., and transfer of Natronobacterium nitratireducens to Halobiforma nitratireducens comb. nov. Int. J. Syst, Evol. Microbiol. 52:2271–2280.[Abstract/Free Full Text]

    Iredell, J., D. Blanckenberg, M. Arvand, S. Grauling, E. J. Feil, and R. J. Birtles. 2003. Characterization of the natural population of Bartonella henselae by multilocus sequence typing. J. Clin. Microbiol. 41:5071–5079.[Abstract/Free Full Text]

    Jurgens, G., K. Lindstrom, and A. Saano. 1997. Novel group within the kingdom Crenarchaeota from boreal forest soil. Appl. Environ. Microbiol. 63:803–805.[Abstract]

    Kamekura, M., M. L. Dyall-Smith, V. Upasani, A. Ventosa, and M. Kates. 1997. Diversity of alkaliphilic halobacteria: proposals for transfer of Natronobacterium vacuolatum, Natronobacterium magadii, and Natronobacterium pharaonis to Halorubrum, Natrialba, and Natronomonas gen. nov., respectively, as Halorubrum vacuolatum comb. nov., Natrialba magadii comb. nov., and Natronomonas pharaonis comb. nov., respectively. Int. J. Syst. Bacteriol. 47:853–857.[Abstract/Free Full Text]

    Kamekura, M., and M. Kates. 1999. Structural diversity of membrane lipids in members of Halobacteriaceae. Biosci. Biotechnol. Biochem. 63:969–972.[ISI][Medline]

    Leffers, H., F. Gropp, F. Lottspeich, W. Zillig, and R. A. Garrett. 1989. Sequence, organization, transcription and evolution of RNA polymerase subunit genes from the archaebacterial extreme halophiles Halobacterium halobium and Halococcus morrhuae. J. Mol. Biol. 206:1–17.[ISI][Medline]

    Matte-Tailliez, O., C. Brochier, P. Forterre, and H. Philippe. 2002. Archaeal phylogeny based on ribosomal proteins. Mol. Biol. Evol. 19:631–639.[Abstract/Free Full Text]

    Mollet, C., M. Drancourt, and D. Raoult. 1997. rpoB sequence analysis as a novel basis for bacterial identification. Mol. Microbiol. 26:1005–1011.[ISI][Medline]

    Montalvo-Rodriguez, R., R. H. Vreeland, A. Oren, M. Kessel, C. Betancourt, and J. Lopez-Garriga. 1998. Halogeometricum borinquense gen. nov., sp. nov., a novel halophilic archaeon from Puerto Rico. Int. J. Syst. Bacteriol. 48(pt 4):1305–1312.[Abstract/Free Full Text]

    Morse, R., K. O'Hanlon, and M. D. Collins. 2002. Phylogenetic, amino acid content and indel analyses of the beta subunit of DNA-dependent RNA polymerase of gram-positive and gram-negative bacteria. Int. J. Syst. Evol. Microbiol 52:1477–1484.[Abstract/Free Full Text]

    Mullakhanbhai, M. F., and H. Larsen. 1975. Halobacterium volcanii spec. nov., a Dead Sea halobacterium with a moderate salt requirement. Arch. Microbiol. 104:207–214.[ISI][Medline]

    Mwatha, W. E., and W. D. Grant. 1993. Natronobacterium vacuolatum sp. nov., a haloalkaliphilic archaeon isolated from Lake Magadi, Kenya. Int. J. Syst. Bacteriol. 43:401–404.

    Mylvaganam, S., and P. P. Dennis. 1992. Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic archaebacterium Haloarcula marismortui. Genetics 130:399–410.[Abstract/Free Full Text]

    Nesbo, C. L., Y. Boucher, and W. F. Doolittle. 2001. Defining the core of nontransferable prokaryotic genes: the euryarchaeal core. J. Mol. Evol. 53:340–350.[CrossRef][ISI][Medline]

    Ng, W. V., S. P. Kennedy, G. G. Mahairas et al. (43 co-authors). 2000. Genome sequence of Halobacterium species NRC-1. Proc. Natl. Acad. Sci. USA 97:12176–12181.[Abstract/Free Full Text]

    Oren, A. 2002. Halophilic microorganisms and their environments. Kluwer Academics, Dordrecht, The Netherlands.

    Oren, A., R. Elevi, S. Watanabe, K. Ihara, and A. Corcelli. 2002. Halomicrobium mukohataei gen. nov., comb. nov., and amended description of Halomicrobium mukohataei. Int. J. Syst. Evol. Microbiol. 52:1831–1835.[Abstract/Free Full Text]

    Oren, A., A. Ventosa, and W. D. Grant. 1997. Proposed minimal standards for description of new taxa in the order Halobacteriales. Int. J. Syst. Bacteriol. 47:233–238.

    Peixoto, R. S., H. L. da Costa Coutinho, N. G. Rumjanek, A. Macrae, and A. S. Rosado. 2002. Use of rpoB and 16S rRNA genes to analyse bacterial diversity of a tropical soil using PCR and DGGE. Lett. Appl. Microbiol. 35:316–320.[CrossRef][ISI][Medline]

    Philippe, H. 1993. MUST, a computer package of management utilities for sequences and trees. Nucleic Acids Res. 21:5264–5272.[Abstract]

    Philippe, H., U. Sorhannus, A. Barion, R. Perasso, F. Gasse, and A. Adoutte. 1994. Comparison of molecular and paleontological data in diatoms suggegsts a major gap in the fossil record. J. Evol. Biol. 7:247–265.[ISI]

    Posada, D., and K. A. Crandall. 1998. MODELTEST: testing the model of DNA substitution. Bioinformatics 14:817–818.[Abstract]

    Radax, C., C. Gruber, and H. Stan-Lotter. 2001. Novel haloarchaeal 16S rRNA gene sequences from Alpine Permo-Triassic rock salt. Extremophiles 5:221–228.[CrossRef][ISI][Medline]

    Schmidt, H. A., K. Strimmer, M. Vingron, and A. von Haeseler. 2002. TREE-PUZZLE: maximum likelihood phylogenetic analysis using quartets and parallel computing. Bioinformatics 18:502–504.[Abstract/Free Full Text]

    Schouls, L. M., C. S. Schot, and J. A. Jacobs. 2003. Horizontal transfer of segments of the 16S rRNA genes between species of the Streptococcus anginosus group. J. Bacteriol. 185:7241–7246.[Abstract/Free Full Text]

    Shimodaira, H. 2002. An approximately unbiased test of phylogenetic tree selection. Syst. Biol. 51:492–508.[CrossRef][ISI][Medline]

    Shimodaira, H., and M. Hasegawa. 2001. CONSEL: for assessing the confidence of phylogenetic tree selection. Bioinformatics 17:1246–1247.[Abstract/Free Full Text]

    Swofford, D. L. 1998. PAUP*: phylogenetic analysis using parsimony (*and other methods). Sinauer Associates, Sunderland, Mass.

    Swofford, D., G. Olsen, P. Waddell, and D. Hills. 1996. Phylogenetic inference. Pp. 495–503 in D. Hillis, C. Moritz, and B. Mable, eds. Molecular systematics. Sinauer Associates, Sunderland, Mass.

    Taillardat-Bisch, A. V., D. Raoult, and M. Drancourt. 2003. RNA polymerase beta-subunit-based phylogeny of Ehrlichia spp., Anaplasma spp., Neorickettsia spp. and Wolbachia pipientis. Int. J. Syst. Evol. Microbiol. 53:455–458.[Abstract/Free Full Text]

    Takai, K., T. Komatsu, F. Inagaki, and K. Horikoshi. 2001. Distribution of archaea in a black smoker chimney structure. Appl. Environ. Microbiol. 67:3618–3629.[Abstract/Free Full Text]

    Taylor, M. W., P. J. Schupp, I. Dahllof, S. Kjelleberg, and P. D. Steinberg. 2004. Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity. Environ. Microbiol. 6:121–130.[CrossRef][ISI][Medline]

    Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22:4673–4680.[Abstract]

    Tindall, B. J. 2003. Taxonomic problems arising in the genera Haloterrigena and Natrinema. Int. J. Syst. Evol. Microbiol. 53:1697–1698.[Abstract/Free Full Text]

    Tindall, B. J., H. N. M. Ross, and W. D. Grant. 1984. Natronobacterium, gen. nov. and Natronococcus, gen. nov., two new genera of haloalkaliphilic archaeabacteria. Syst. Appl. Microbiol. 5:41–57.[ISI]

    van Berkum, P., Z. Terefework, L. Paulin, S. Suomalainen, K. Lindstrom, and B. D. Eardly. 2003. Discordant phylogenies within the rrn loci of Rhizobia. J. Bacteriol. 185:2988–2998.[Abstract/Free Full Text]

    Vreeland, R. H., S. Straight, J. Krammes, K. Dougherty, W. D. Rosenzweig, and M. Kamekura. 2002. Halosimplex carlsbadense gen. nov., sp. nov., a unique halophilic archaeon, with three 16S rRNA genes, that grows only in defined medium with glycerol and acetate or pyruvate. Extremophiles 6:445–452.[CrossRef][ISI][Medline]

    Wilson, K. 1994. Preparation of genomic DNA from bacteria. Pp. 2.4.1–2.4.5 in F. M. Ausubel, R. Brent, R. E. Kingston, D. D. Moore, J. G. Seidman, J. A. Smith, and K. Struhl, eds. Current protocols in molecular biology. John Wiley and Sons, New York.

    Woese, C. R., and G. E. Fox. 1977. Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc. Natl. Acad. Sci. USA 74:5088–5090.[Abstract]

    Yap, W. H., Z. Zhang, and Y. Wang. 1999. Distinct types of rRNA operons exist in the genome of the actinomycete Thermomonospora chromogena and evidence for horizontal transfer of an entire rRNA operon. J. Bacteriol. 181:5201–5209.[Abstract/Free Full Text]

Accepted for publication August 30, 2004.





This Article
Abstract
FREE Full Text (PDF)
All Versions of this Article:
21/12/2340    most recent
msh248v1
Alert me when this article is cited
Alert me if a correction is posted
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Add to My Personal Archive
Download to citation manager
Search for citing articles in:
ISI Web of Science (1)
Request Permissions
Google Scholar
Articles by Walsh, D. A.
Articles by Doolittle, W. F.
PubMed
PubMed Citation
Articles by Walsh, D. A.
Articles by Doolittle, W. F.