| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Winogradsky Institute of Microbiology, Russian Academy of Sciences, Moscow, Russia
2 Department of Microbiology, Moscow State University, Moscow, Russia
3 Center Bioengineering, Russian Academy of Sciences, Moscow, Russia
4 Department of Biotechnology, Delft University of Technology, Delft, The Netherlands
Correspondence
Tatjana P. Tourova
ttour{at}biengi.ac.ru
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
The GenBank/EMBL/DDBJ accession numbers for the sequences reported in this paper are EF199939–EF199958 and EF202525.
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). More recently, two new genera, Thiorhodospira (Bryantseva et al., 1999) and Ectothiorhodosinus (Gorlenko et al., 2004), made up of moderately haloalkaliphilic purple sulfur bacteria isolated from soda lakes, were added to the family. All these phototrophic bacteria prefer to grow anaerobically in the light using reduced sulfur compounds as electron donors. At the same time, representatives of several genera with aerobic chemotrophic metabolism are closely related to the anaerobic phototrophs of the Ectothiorhodospiraceae, namely the heterotrophs Arhodomonas (Adkins et al., 1993) and Alkalispirillum (Rijkenberg et al., 2001), the facultative autotroph Alkalilimnicola (Yakimov et al., 2001; Sorokin et al., 2006; Hoeft et al., 2007), the obligately chemolithoautotrophic nitrifier Nitrococcus (Watson & Waterbury, 1971; Teske et al., 1994) and chemolithotrophic sulfur-oxidizing bacteria of the genera Thioalkalivibrio (Sorokin et al., 2001, 2002a, b; Banciu et al., 2004) and Thioalkalispira (Sorokin et al., 2002c). This raises several interesting questions on the evolution of the family.
The modern taxonomy of the Ectothiorhodospiraceae follows their phylogenetic relationships based on 16S rRNA gene sequences (Fig. 1). The gene encoding the 16S rRNA is widely used as a universal molecular marker for phylogenetic reconstructions and taxonomy because it has been assumed that intraspecies variation and horizontal transfer of this gene were low. Some housekeeping protein-encoding genes, e.g. gyrB, recA and rpoB (Dauga, 2002; Holmes et al., 2004), have been used as additional molecular markers for phylogenetic studies of different bacterial groups. Phylogenetic reconstructions based on sequence analyses of the 16S rRNA gene and housekeeping protein-encoding genes usually correlate quite well, but sometimes an additional analysis of functional genes is necessary in order to clarify uncertain cases. The functional genes responsible for key metabolic properties can also be used as alternative molecular markers. Sequence analysis of functional genes might help to resolve difficult taxonomic problems as well as to clarify the evolution of corresponding metabolic pathways.
|
) and chemolithotrophic species of Thioalkalivibrio (Sorokin et al., 2001, 2002a, b; Banciu et al., 2004) and Thioalkalispira (Sorokin et al., 2002c) have ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) activity. RubisCO is the key enzyme of the Calvin–Benson–Bassham reductive pentose phosphate cycle. Two different RubisCO forms are known. The large catalytic subunit of form I is encoded by cbbL, while the only subunit of form II is encoded by cbbM. The cbbL gene is in turn divided into ‘green-like’ and ‘red-like’ types. The ‘green-like’ cbbL gene has recently been found in the genomes of Ectothiorhodospira shaposhnikovii (Spiridonova et al., 2004), Thioalkalivibrio species (Tourova et al., 2005) and the Alkalispirillum–Alkalilimnicola group (Oremland et al., 2002; Sorokin et al., 2006).
The capacity for nitrogen fixation has been shown for most purple phototrophic bacteria, including some members of the Ectothiorhodospiraceae (Moshkovskii et al., 1971; Imhoff, 2005). During the process of biological nitrogen fixation, the enzyme nitrogenase catalyses the ATP-dependent reduction of dinitrogen to ammonia. Nitrogenase consists of two component metalloproteins, the iron (Fe) protein (encoded by nifH) and the molybdenum–iron (MoFe) protein (encoded by nifD and nifK). The Fe protein mediates the coupling of ATP hydrolysis to interprotein electron transfer, and its gene is highly conserved among closely related micro-organisms. This means that nifH can be used to study relationships among diazotrophic bacteria. Nevertheless, the occurrence and phylogeny of nifH in representatives of the Ectothiorhodospiraceae has not yet been investigated. The only exception is Halorhodospira halophila strain BN 9629, where the nitrogenase operon has recently been cloned and characterized (Tsuihiji et al., 2006).
The aim of the present study was to extend the evolutionary analysis of the family Ectothiorhodospiraceae based on the comparison of the 16S rRNA gene-based phylogeny with phylogenies of genes encoding the key functional enzymes RubisCO (cbbL) and nitrogenase (nifH).
|
METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. Type strains of the genera Alkalispirillum and Arhodomonas were obtained from the DSMZ. Nitrococcus mobilis ATCC 25380T was kindly provided by Dr E. Spieck (Universität Hamburg, Germany). Biomass of these bacteria was used directly for DNA extraction. Other strains were from the culture collections of the Institute of Microbiology and of the Department of Microbiology of Moscow State University (KM MGU). The cultures were maintained as described previously (Imhoff, 2005; Bryantseva et al., 1999; Sorokin et al., 2001, 2002a, b, c).
|
). The cbbL and nifH gene fragments were amplified using specially developed and previously tested primers (Spiridonova et al., 2004; Marusina et al., 2001). PCR products were purified from low-melting-point agarose using the Wizard PCR Preps kit (Promega).
Cloning and sequencing of the PCR fragments.
Purified PCR products were cloned using the pGEM-T vector system (Promega). Plasmid DNA was extracted and purified using the Wizard MiniPrep kit (Promega). Clones containing appropriately sized inserts were sequenced from universal M13 forward and reverse primers (Sambrook et al., 1989). Sequencing was performed with an ABI 3730 sequencer using the Big Dye Terminator v. 3.1 sequencing reaction kit (Applied Biosystems).
Phylogenetic analysis.
Preliminary analysis of the new sequences was performed via the NCBI BLAST server (http://www.ncbi.nlm.nih.gov/BLAST/). Nucleotide and inferred amino acid sequences were aligned with sequences from GenBank using CLUSTAL W (Thompson et al., 1994).
Genetic distances were calculated using Kimura's two-parameter method (Kimura, 1980). To obtain synonymous and non-synonymous distances, a method of Nei & Gojobori (1986) was applied to the various sequences of protein-encoding genes using WET software (J. Dopazo; http://www.tdi.es). Phylogenetic trees were reconstructed using four different algorithms: neighbour-joining (Saitou & Nei, 1987) in the TREECONW program package (Van de Peer & De Wachter, 1994) and maximum-parsimony (Fitch, 1971), distance matrix (Fitch & Margoliash, 1967) and maximum-likelihood (Felsenstein, 1981) using PHYLIP 3.5c software (Felsenstein, 1993). Relative synonymous codon usage (RSCU) values of the cbbL and nifH genes were calculated using CodonW software (J. Peden; http://codonw.sourceforge.net). To investigate the major codon usage trends in different species, CodonW was used to carry out a correspondent analysis. Each gene produced a point in the codon space, the positions of which suggested a possible codon usage bias.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
. On the other hand, using the specific primer set for the ‘green-like’ cbbL gene, PCR products of about 750 bp were obtained from the DNAs of all strains, except the type strains of Alkalispirillum mobile and Arhodomonas aquaeolei. Clones prepared from the cbbL PCR fragments yielded a single sequence-type for all species. Results of BLAST analysis revealed a high level of similarity between the newly determined nucleotide sequences and the other cbbL sequences available in GenBank, confirming their affiliation to the same family of genes.
The nucleotide sequences obtained for RubisCO gene fragments as well as the deduced amino acid sequences of the corresponding proteins were aligned with analogous sequences of the ‘green-like’ cbbL from GenBank. Positions with gaps and ambiguous sequences were removed and the remaining 720 nucleotide and 240 amino acid positions were used for further phylogenetic analysis. The topologies of the phylogenetic trees constructed on the basis of these alignments were similar for all the methods used, the neighbour-joining (Fig. 2), maximum-parsimony, distance-matrix and maximum-likelihood (data not shown) methods.
|
), the topologies obtained did not correspond to those of the ‘ribosomal’ tree. According to 16S rRNA gene sequence analysis, all members of the family Ectothiorhodospiraceae form a monophyletic clade within the Gammaproteobacteria, consisting of several phylogenetic clusters and branches corresponding to different genera (Fig. 1). However, the cbbL-based trees suggest that the Ectothiorhodospiraceae is divided into independent clusters and branches with positions in the trees dependent on the set of chosen reference sequences (Fig. 2). These intrafamily divisions correlated only partially with the 16S rRNA gene-based tree topology. Different species of the genera Halorhodospira and Alkalispirillum–Alkalilimnicola formed monophyletic clusters with high bootstrap values (100 and 98–100 %, respectively, based on nucleotides and amino acids), and the monotypic genus Thioalkalispira formed a separate branch unrelated to other members of the family in both trees. However, the genus Ectothiorhodospira realistically formed a single cluster (with 91 % bootstrap value) in the ‘RubisCO’ tree based on nucleotides only, whereas in the tree based on amino acids this cluster was unstable (31 % bootstrap value), depending on the set of chosen reference sequences. Moreover, in the ‘RubisCO’ trees, the monotypic genus Ectothiorhodosinus formed a separate branch unrelated to Ectothiorhodospira species.
The most dramatic discrepancies between the topologies of the ‘ribosomal’ and ‘RubisCO’ trees were observed in the case of the genus Thioalkalivibrio. According to the RubisCO gene analysis, Thioalkalivibrio is not a monophyletic genus (Tourova et al., 2005). The Thioalkalivibrio species formed three independent clusters in the nucleotide-based ‘RubisCO’ tree (with 74–100 % bootstrap values) and two clusters (34–100 % bootstrap values) and one branch in the amino acid-based tree. This division correlated only partially with the inner structure of the single ‘ribosomal’ Thioalkalivibrio cluster. Only the cluster combining Thioalkalivibrio denitrificans and Thioalkalivibrio thiocyanodenitrificans (the deepest Thioalkalivibrio subcluster according to the ‘ribosomal’ tree) demonstrated a distant relationship to Ectothiorhodospira in the nucleotide-based ‘RubisCO’ tree (75 % bootstrap value). At the same time, the phylogenetic position was strongly supported in both ‘RubisCO’ trees only for the cluster Thioalkalivibrio nitratireducens/Thioalkalivibrio paradoxus. This cluster grouped with Nitrococcus mobilis with high bootstrap values (98 and 100 %, respectively, based on nucleotides and amino acids) and with one of the duplicated cbbL genes of Allochromatium vinosum (with 100 and 70 % bootstrap values, respectively, for nucleotide and amino-acid-based trees). The position of Thiorhodospira sibirica in both RubisCO trees was unexpected, because it grouped together with some representatives of the Alphaproteobacteria with strong bootstrap support (respectively 98 and 97 % based on nucleotides and amino acids).
Detection and phylogenetic analysis of nitrogenase reductase genes
PCR products of the expected size (about 450 bp) were obtained with the nifH-specific primers using the DNAs of all investigated species of the phototrophic genera Ectothiorhodospira, Halorhodospira and Thiorhodospira, but not of the genus Ectothiorhodosinus. Unexpectedly, nifH fragments were also obtained for the chemotrophic species Thioalkalispira microaerophila and Alkalilimnicola halodurans, for which diazotrophic potential was not suspected previously. Preliminary screening in the GenBank database demonstrated that all newly determined nucleotide sequences belong to the nifH gene family. Moreover, the nifH gene fragment obtained from the type strain of Hlr. halophila was almost identical to the analogous fragment of the previously sequenced nifH gene from Hlr. halophila BN 9629 (GenBank accession no. AB189641).
The nucleotide sequences of nifH gene fragments as well as the deduced amino acid sequences of the corresponding proteins were aligned, positions with gaps and ambiguous sequences were removed and the remaining 444 nucleotide and 148 amino acid positions were used for further phylogenetic analysis. The topologies of the phylogenetic trees constructed on the basis of these alignments were similar for all methods used: the neighbour-joining (Fig. 3), maximum-parsimony, distance-matrix and maximum-likelihood (data not shown) methods.
|
The nifH phylogeny of Thioalkalispira microaerophila and Alkalilimnicola halodurans was of particular interest. In both ‘nitrogenase’ trees, they formed two separate branches with an uncertain branching point position, and were slightly related only to Azoarcus and Azovibrio, representing the Betaproteobacteria (bootstrap values did not exceed 56 %). Thus, their nifH genes do not have a clear relationship to the nifH of the other members of the Ectothiorhodospiraceae.
Comparison of genetic distances among the cbbL, nifH and 16S rRNA genes
Nucleotide substitutions within protein-encoding regions are divided into two classes: synonymous (silent), which are largely invisible to natural selection, and non-synonymous (resulting in amino acid replacement), which may be under strong selective pressure. Synonymous distances in the cbbL and nifH genes were examined for all possible combinations of the investigated species of the Ectothiorhodospiraceae. A significant correlation between the synonymous distances in the cbbL genes and those in the nifH genes was observed, with a correlation coefficient (r) of 0.91. This result is in agreement with the assumption that the synonymous substitution rate is constant for many chromosomal genes in many organisms and that it can serve as a suitable molecular indicator of their evolution (Lawrence et al., 1991).
The total numbers of substitutions used for the calculation of genetic distances for cbbL and nifH allowed estimation of the ranges of genomic variation (Table 2). The intrafamily genetic distances between the cbbL, nifH and 16S rRNA gene sequences were up to 0.414, 0.295 and 0.134, respectively. This indicated a variability order cbbL>nifH>>16S rRNA gene.
|
).
On the basis of DNA–DNA hybridization analysis, Ventura et al. (2000) proposed to consider Ect. vacuolata and Ectothiorhodospira marismortui as junior synonyms of Ect. shaposhnikovii and Ect. mobilis, respectively. However, the similarities of cbbL gene sequences of the pairs Ect. vacuolata–Ect. shaposhnikovii and Ect. marismortui–Ect. mobilis were at the same level as for the pair Ect. shaposhnikovii–Ect. mobilis, and much higher than for different strains of Alkalispirillum mobile (Table 2). On the other hand, although the nifH gene sequences of Ect. vacuolata and Ect. shaposhnikovii were found to be very similar (0.010), the same (high) level of similarity was shown for the sequences of Ect. shaposhnikovii and Ect. mobilis (0.007). These data are in good agreement with the current taxonomy of the genus Ectothiorhodospira, where Ect. vacuolata, Ect. shaposhnikovii, Ect. marismortui and Ect. mobilis are considered as four different species, and they disagree with the reclassification proposed by Ventura et al. (2000).
Nucleotide composition and codon usage of the cbbL and nifH genes
Genes in closely related species tend to be rather similar in their G+C content as well as in synonymous codon usage, in contrast to genes acquired by horizontal transfer, which often have atypical G+C content and codon usage bias (Medigue et al., 1991). Therefore, it was interesting to compare the G+C content and codon usage of the RubisCO and nitrogenase reductase genes within the family Ectothiorhodospiraceae to detect the possible role of gene transfer in their evolution.
The total G+C content of all analysed cbbL and nifH gene fragments was close to the genomic G+C content for each species of the family (56.2–70.7 and 56.2–66.3 against 52.9–68.4 mol%, respectively; Table 1). The G+C3 content (in the third position of codons) of these genes (57.7–96.9 and 75.8–93.7 mol%) was higher than their total G+C content and the overall genomic G+C content; it is typical of G+C-biased micro-organisms that they preferentially use G or C in the third position of the codons (Ohtaka & Ishikawa, 1993).
The broadest intragenus range of G+C content variation in cbbL and nifH genes (62.7–68.6 and 60.9–68.0 mol%, respectively) was found for the Halorhodospira species, consistent with the variation of their genomic G+C content (52.9–68.4 mol%). Among the other representatives of the Ectothiorhodospiraceae, the lowest G+C contents of the nifH and cbbL genes (less than 60.3 mol%) were found for the monotypic genera Ectothiorhodosinus, Thioalkalispira and Thiorhodospira, and were also comparable to their overall genomic G+C content. Interestingly, among the species of the Alkalispirillum–Alkalilimnicola group, the G+C content of cbbL differed significantly from the genomic G+C content and was the highest in the Ectothiorhodospiraceae (up to 71.0 mol%). At the same time, the G+C content of the nifH gene of Alkalilimnicola halodurans (which is the only representative of the Alkalispirillum–Alkalilimnicola group that has a nifH gene) was similar to the overall genomic G+C content.
Codon usage analysis for the cbbL and nifH genes was carried out on the RSCU data. Correspondence analysis of the results (Fig. 4) identified the major trends in codon usage: the horizontal axis is associated with G+C3, whereas the vertical axis is correlated with the frequencies of codons ending in C or U versus A or G (Fennoy & Bailey-Serres, 1993). Codon usage analysis of cbbL genes is compatible with the formation of a common group on the plot for most representatives of the Ectothiorhodospiraceae: species of Ectothiorhodospira, Halorhodospira, Thioalkalivibrio and the Alkalispirillum–Alkalilimnicola group and Nitrococcus mobilis (Fig. 4). However, the codon usage of Halorhodospira abdelmalekii was similar to that of alphaproteobacteria. The cbbL codon usage bias of Trs. sibirica, Thioalkalispira microaerophila and Ectothiorhodosinus mongolicus was consistent with their G+C content bias.
|
). It appears that the codon usage bias in both cbbL and nifH suggests their origin from an intrafamily genome divergence rather than from lateral gene transfer. |
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
). At the same time, the topology of the ‘nitrogenase’ tree exhibits significant correlation with reconstructions based on ‘ribosomal’ gene analysis (Achouak et al., 1999; Tourova et al., 2006). This divergence can be useful to resolve particular problems of the evolution of autotrophic and diazotrophic bacteria. The cbbL-, nifH- and 16S rRNA gene-based trees reconstructed in this study for the species of the Ectothiorhodospiraceae were not highly congruent in their branching patterns. While all species formed a monophyletic clade in the ‘ribosomal’ tree, in the ‘RubisCO’ and ‘nitrogenase’ trees, this clade disintegrated into a number of broadly distributed clusters and branches. However, most of the differences between the trees were in areas of low bootstrap values. The low resolution of the deep branches in the cbbL- and nifH-based trees may be due to accelerated rates of sequence divergence or poor representation of the taxa in databases in comparison with 16S rRNA gene sequences. Therefore, considering the similar codon usage in the cbbL and nifH genes, these phylogenetic data may be regarded as evidence of the monophyletic origin of most cbbL and nifH genes within the family Ectothiorhodospiraceae.
Nevertheless, in some cases, the ‘RubisCO’-based trees showed that relationships inside the Ectothiorhodospiraceae were inconsistent both with ‘ribosomal’ phylogeny and phenotypic properties. This may be due to either inaccuracy in the cbbL trees (phylogenetic construction bias) or occurrence of lateral gene transfer. The example is the phylogenetic position of Trs. sibirica: in both ‘RubisCO’ trees, it rooted with some alphaproteobacteria while, in the ‘ribosomal’ tree and in the ‘nitrogenase’ amino-acid-based tree, it clustered together with the species of the Ectothiorhodospiraceae. However, the G+C content of the cbbL gene of Trs. sibirica (58.9 mol%) was comparable to that of the nifH gene (56.2 mol%) and the total genome (56.7 mol%), but was significantly lower than in the available alphaproteobacterial gene sequences (64.3–65.8 mol%). The codon usage patterns of nifH and cbbL of Trs. sibirica were also similar and differed from the alphaproteobacterial pattern (Fig. 4). Therefore, the unusual rooting of Trs. sibirica on the ‘RubisCO’ tree might originate from a higher rate of non-synonymous nucleotide substitutions rather than from lateral gene transfer.
The cbbL phylogeny of the cluster including the chemolithoautotrophic sulfur-oxidizers Thioalkalivibrio nitratireducens and Thioalkalivibrio paradoxus and the nitrifier Nitrococcus mobilis is of particular interest. Phylogenetic analysis of both nucleotide and amino acid sequences showed that the RubisCO genes in this group have a common origin different from the origin of the analogous genes in other species of the family. These three species clustered with strong bootstrap support with one of the duplicated cbbL genes of the purple sulfur bacterium Alc. vinosum, a member of the family Chromatiaceae. One of the possible evolutionary mechanisms that could have taken place in this case is lateral gene transfer, which has presumably played a significant role in the evolution of the genes belonging to the RubisCO family (Delwiche & Palmer, 1996; Watson & Tabita, 1997). Similarities in codon usage (Fig. 4) and G+C content between the duplicated cbbL genes of Alc. vinosum (65.1–66.3 mol%) and genes of Thioalkalivibrio and Nitrococcus (63.9–66.5 mol%) do not contradict this suggestion. Interestingly, both of these Thioalkalivibrio species are morphologically similar to Allochromatium (large coccoid rods with sulfur inclusions) but strikingly different from the other Thioalkalivibrio species and members of the Ectothiorhodospiraceae in general (vibrio/spirilla that deposit elemental sulfur outside the cell). It is noteworthy that the formation of intracellular sulfur globules is a characteristic feature of members of the Chromatiaceae and Allochromatium in particular. Moreover, biochemical and recent genetic studies have demonstrated that a reverse dissimilatory sulfite reductase complex encoded by a large gene cluster is responsible for further oxidation of intracellular sulfur to sulfate (Dahl et al., 2005). Preliminary tests with primers specific for the Alc. vinosum reverse dsr gene complex indicated the presence of some of the genes in Thioalkalivibrio nitratireducens and Thioalkalivibrio paradoxus (C. Dahl, personal communication). Cells of Nitrococcus mobilis also have a spherical shape. The tubular membranes of Nitrococcus mobilis are quite similar to the internal photosynthetic membrane system of Thiococcus pfennigii, a member of the Chromatiaceae (Imhoff, 2005). These observations may indicate that there was an exchange of important genetic information between these different types of autotrophic bacteria. This case is just one of many examples of inconsistency between the taxonomic position determined on the basis of 16S rRNA gene sequences and other essential characteristics of the organism in question. Taking into consideration the striking structural similarity between species of the Chromatiaceae and these three representatives of the Ectothiorhodospiraceae (Thioalkalivibrio nitratireducens, Thioalkalivibrio paradoxus and Nitrococcus mobilis), it might be speculated that gene transfer between these phylogenetically distant (based on 16S rRNA gene analysis) organisms might have involved not only individual genes (i.e. cbbL) but also genetic blocks. The alternative and simpler assumption may be lateral transfer of the 16S rRNA gene. Although it is generally accepted that this is a rare event, such a possibility cannot be excluded (Tourova, 2003).
The possibility of chemolithoheterotrophic aerobic growth has been shown for many representatives of the purple sulfur bacteria, from both the Chromatiaceae and the Ectothiorhodospiraceae, and some members of the Chromatiaceae can grow chemolithoautotrophically in the presence of oxygen, as colourless sulfur bacteria do (Kondratieva et al., 1976; Kämpf & Pfennig, 1980). In the case of Thioalkalivibrio species, which are currently classified as members of the Ectothiorhodospiraceae, it might be speculated that these alkaliphilic, aerobic, sulfur-oxidizing, chemolithoautotrophic bacteria represent direct aerobic descendants of the purple sulfur bacteria that have lost the genes responsible for photosynthesis. The results of the cbbL gene analysis did not contradict this suggestion as a whole, but they demonstrated that the putative phototrophic ancestors might be different for some groups of Thioalkalivibrio species.
The genera Alkalispirillum and Alkalilimnicola were originally described as non-phototrophic, aerobic, heterotrophic relatives of the Ectothiorhodospira–Halorhodospira group. However, it has been shown recently that some novel strains and the type strain of Alkalilimnicola halodurans are capable of lithoautotrophy and have cbbL genes (Oremland et al., 2002; Sorokin et al., 2006). The cbbL-based phylogenetic trees and codon usage analysis confirmed the relatedness of the Alkalispirillum–Alkalilimnicola group to the Ectothiorhodospiraceae. However, in spite of the high DNA–DNA relatedness and the 16S rRNA gene and cbbL sequence similarity, they are significantly different in details of their autotrophic metabolism (Oremland et al., 2002; Sorokin et al., 2006). For example, hydrogen-based autotrophy was found only in two strains and could be lost easily during cultivation (Sorokin et al., 2006). This might indicate the location of the RubisCO genes on a plasmid (which may be lost during heterotrophic growth). Such a loss may be a reason for the absence (temporary or constant) of the cbbL genes in the genome of the type strain of Alkalispirillum mobile.
The absence of RubisCO genes in Arhodomonas aquaeolei was in accordance with the original description of this bacterium as an obligate heterotroph (Adkins et al., 1993).
Nitrogen fixation is considered to be a characteristic property of purple sulfur bacteria, including the Ectothiorhodospiraceae (Moshkovskii et al., 1971; Imhoff, 2005). Moreover, nitrogenase-mediated hydrogen production was shown for representatives of this group (Chadwick & Irgens, 1991; Tsuihiji et al., 2006). Therefore, it is not surprising that nifH genes were detected in all phototrophic members of the Ectothiorhodospiraceae with the sole exception of Ers. mongolicus. This bacterium was isolated from a habitat enriched in organic compounds and had only a weak capacity for photoautotrophic growth (Gorlenko et al., 2004); growth under such conditions might also be accompanied by a loss of the nitrogenase genes. Since many members of the Ectothiorhodospiraceae do not have this gene and the topologies of the nifH-based trees correlate only partially with the topology of the 16S rRNA gene tree, nifH does not seem to be suitable for basic phylogenetic assessments. However, its analysis may help to understand the relationship of these bacteria, especially over small phylogenetic distances.
The occurrence of nifH genes in the aerobic chemotrophic Alkalilimnicola halodurans and Thioalkalispira microaerophila is more difficult to interpret. Although analysis of these nifH genes confirmed their relatedness to nifH of other members of the Ectothiorhodospiraceae, measurement of nitrogenase activity in Alkalilimnicola halodurans and Thioalkalispira microaerophila is necessary in order to confirm whether these genes are functional. Several examples of ancient altered genes (‘pseudogenes’), which can become non-functional but may still retain sufficient similarity to functional genes, are well documented (Ochman & Davalos, 2006).
The conclusion that most of the cbbL and nifH genes within the family Ectothiorhodospiraceae have a monophyletic origin allows them to be used them for analyses of microbial communities in situ. The substantial limitation of in situ analyses based on functional genes is the poor representation of many taxa (including the Ectothiorhodospiraceae) in databases. This limits identification of the obtained sequences or leads to their misidentification. The database obtained in this study could provide good support for in situ studies (for example, in haloalkaline lakes, where members of the Ectothiorhodospiraceae thrive).
In general, molecular phylogenies based on a single gene may be misleading, because of the complexity of the evolutionary process. Thus, data from several genes encoding different cellular functions are more suitable for realistic phylogenetic reconstructions.
|
ACKNOWLEDGEMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
|---|
Adkins, J. P., Madigan, M. T., Mandelco, L., Woese, C. R. & Tanner, R. S. (1993). Arhodomonas aquaeolei gen. nov., sp. nov., an aerobic, halophilic bacterium isolated from a subterranean brine. Int J Syst Bacteriol 43, 514–520.
Banciu, H., Sorokin, D. Y., Galinski, E. A., Muyzer, G., Kleerebezem, R. & Kuenen, J. G. (2004). Thialkalivibrio halophilus sp. nov., a novel obligately chemolithoautotrophic, facultatively alkaliphilic, and extremely salt-tolerant, sulfur-oxidizing bacterium from a hypersaline alkaline lake. Extremophiles 8, 325–334.[Medline]
Boulygina, E. S., Kuznetsov, B. B., Marusina, A. I., Tourova, T. P., Kravchenko, I. K., Bykova, S. A., Kolganova, T. V. & Galchenko, V. F. (2002). A study of nucleotide sequences of nifH genes of some methanotrophic bacteria. Microbiology English translation of Mikrobiologiia 71, 425–432.[CrossRef]
Bryantseva, I., Gorlenko, V. M., Kompantseva, E. I., Imhoff, J. F., Suling, J. & Mityushina, L. (1999). Thiorhodospira sibirica gen. nov., sp. nov., a new alkaliphilic purple sulfur bacterium from a Siberian soda lake. Int J Syst Bacteriol 49, 697–703.
Chadwick, L. J. & Irgens, R. L. (1991). Hydrogen gas production by an Ectothiorhodospira vacuolata strain. Appl Environ Microbiol 57, 594–596.
Dahl, C., Engels, S., Pott-Sperling, A. S., Schulte, A., Sander, J., Lübbe, Y., Deuster, O. & Brune, D. C. (2005). Novel genes of the dsr gene cluster and evidence for close interaction of Dsr proteins during sulfur oxidation in the phototrophic sulfur bacterium Allochromatium vinosum. J Bacteriol 187, 1392–1404.
Dauga, C. (2002). Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies. Int J Syst Evol Microbiol 52, 531–547.[Abstract]
Delwiche, C. F. & Palmer, J. D. (1996). Rampant horizontal transfer and duplication of RubisCO genes in eubacteria and plastids. Mol Biol Evol 13, 873–882.[Abstract]
Felsenstein, J. (1981). Evolutionary trees from DNA sequences: a maximum likelihood approach. J Mol Evol 17, 368–376.[CrossRef][Medline]
Felsenstein, J. (1993). PHYLIP (phylogeny inference package), version 3.53c. Distributed by the author. Department of Genome Sciences, University of Washington, Seattle, USA.
Fennoy, S. L. & Bailey-Serres, J. (1993). Synonymous codon usage in Zea mays L. nuclear genes is varied by levels of C and G-ending codons. Nucleic Acids Res 21, 5294–5300.
Fitch, W. M. (1971). Toward defining the course of evolution: minimum change for a specific tree topology. Syst Zool 20, 406–416.[Abstract]
Fitch, W. M. & Margoliash, E. (1967). Construction of phylogenetic trees. Science 155, 279–284.
Gorlenko, V. M., Bryantseva, I. A., Panteleeva, E. E., Tourova, T. P., Kolganova, T. V., Makhneva, Z. K. & Moskalenko, A. A. (2004). Ectothiorhodosinus mongolicum gen. nov., sp. nov., a new purple bacterium from a soda lake in Mongolia. Microbiology English translation of Mikrobiologiia 73, 66–73.[CrossRef]
Hoeft, S. E., Switzer Blum, J., Stolz, J. F., Tabita, F. R., Witte, B., King, G. M., Santini, J. M. & Oremland, R. S. (2007). Alkalilimnicola ehrlichii sp. nov., a novel arsenite-oxidizing, haloalkaliphilic gammaproteobacterium capable of chemoautotrophic or heterotrophic growth with nitrate or oxygen as the electron acceptor. Int J Syst Evol Microbiol 57, 504–512.
Holmes, D. E., Nevin, K. P. & Lovley, D. R. (2004). Comparison of 16S rRNA, nifD, recA, gyrB, rpoB and fusA genes within the family Geobacteraceae fam. nov. Int J Syst Evol Microbiol 54, 1591–1599.
Imhoff, J. F. (2005). Family II. Ectothiorhodospiraceae Imhoff. In Bergey's Manual of Systematic Bacteriology, 2nd edn, vol. 2, part B, The Gammaproteobacteria, pp. 41–52. Edited by D. J. Brenner, N. R. Krieg, J. T. Staley & G. M. Garrity. New York: Springer.
Imhoff, J. F. (2006). The family Ectothiorhodospiraceae. In The Prokaryotes: a Handbook on the Biology of Bacteria, 3rd edn, vol. 6, pp. 874–886. Edited by M. Dworkin, S. Falkow, E. Rosenberg, K. H. Schleifer & E. Stackebrandt. New York: Springer.
Imhoff, J. F. & Süling, J. (1996). The phylogenetic relationship among Ectothiorhodospiraceae. A re-evaluation of their taxonomy on the basis of 16S rDNA analyses. Arch Microbiol 165, 106–113.[CrossRef][Medline]
Kämpf, C. & Pfennig, N. (1980). Capacity of Chromatiaceae for chemotrophic growth. Specific respiration rates of Thiocystis violacea and Chromatium vinosum. Arch Microbiol 127, 125–135.[CrossRef]
Kimura, M. (1980). A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. J Mol Evol 16, 111–120.[CrossRef][Medline]
Kondratieva, E. N., Zhukov, V. G., Ivanovsky, R. N., Petushkova, U. P. & Monosov, E. Z. (1976). The capacity of phototrophic sulfur bacterium Thiocapsa roseopersicina for chemosynthesis. Arch Microbiol 108, 287–292.[CrossRef][Medline]
Lawrence, J. G., Hartl, D. L. & Ochman, H. (1991). Molecular considerations in the evolution of bacterial genes. J Mol Evol 33, 241–250.[CrossRef][Medline]
Marusina, A. I., Boulygina, E. S., Kuznetsov, B. B., Tourova, T. P., Kravchenko, I. K. & Gal'chenko, V. F. (2001). A system of oligonucleotide primers for the amplification of nifH genes of different taxonomic groups of prokaryotes. Microbiology English translation of Mikrobiologiia 70, 73–78.[CrossRef]
Medigue, C., Rouxel, T., Vigier, P., Henaut, A. & Danchin, A. (1991). Evidence for horizontal gene transfer in Escherichia coli speciation. J Mol Biol 222, 851–856.[CrossRef][Medline]
Moshkovskii, Iu. Sh., Uspenskaia, N. Ia. & Mardanian, S. S. (1971). Effect of nitrogen source in the culture medium on the capability for nitrogen fixation and the Mossbauer spectra of Ectothiorhodospira shaposhnikovii cells. Biofizika 16, 933–936 (in Russian).[Medline]
Nei, M. & Gojobori, T. (1986). Simple methods for estimating the numbers of synonymous and nonsynonymous nucleotide substitutions. Mol Biol Evol 3, 418–426.[Abstract]
Ochman, H. & Davalos, L. M. (2006). The nature and dynamics of bacterial genomes. Science 311, 1730–1733.
Ohtaka, C. & Ishikawa, H. (1993). Accumulation of adenine and thymine in groE-homologous operon of an intracellular symbiont. J Mol Evol 36, 121–126.[CrossRef][Medline]
Oremland, R. S., Hoeft, S. E., Santini, J. M., Bano, N., Hollibaugh, R. A. & Hollibaugh, J. T. (2002). Anaerobic oxidation of arsenite in Mono Lake water and by a facultative, arsenite-oxidizing chemoautotroph, strain MLHE-1. Appl Environ Microbiol 68, 4795–4802.
Rijkenberg, M. J., Kort, R. & Hellingwerf, K. J. (2001). Alkalispirillum mobile gen. nov., spec. nov., an alkaliphilic non-phototrophic member of the Ectothiorhodospiraceae. Arch Microbiol 175, 369–375.[CrossRef][Medline]
Saitou, N. & Nei, M. (1987). The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4, 406–425.[Abstract]
Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
Sorokin, D. Y., Lysenko, A. M., Mityushina, L. L., Tourova, T. P., Jones, B. E., Rainey, F. A., Robertson, L. A. & Kuenen, G. J. (2001). Thioalkalimicrobium aerophilum gen. nov., sp. nov. and Thioalkalimicrobium sibiricum sp. nov., and Thioalkalivibrio versutus gen. nov., sp. nov., Thioalkalivibrio nitratis sp. nov. and Thioalkalivibrio denitrificans sp. nov., novel obligately alkaliphilic and obligately chemolithoautotrophic sulfur-oxidizing bacteria from soda lakes. Int J Syst Evol Microbiol 51, 565–580.[Abstract]
Sorokin, D. Y., Tourova, T. P., Lysenko, A. M., Mityushina, L. L. & Kuenen, J. G. (2002a). Thioalkalivibrio thiocyanoxidans sp. nov. and Thioalkalivibrio paradoxus sp. nov., novel alkaliphilic, obligately autotrophic, sulfur-oxidizing bacteria capable of growth on thiocyanate, from soda lakes. Int J Syst Evol Microbiol 52, 657–664.[Abstract]
Sorokin, D. Y., Gorlenko, V. M., Tourova, T. P., Tsapin, A. I., Nealson, K. H. & Kuenen, G. J. (2002b). Thioalkalimicrobium cyclicum sp. nov. and Thioalkalivibrio jannaschii sp. nov., novel species of haloalkaliphilic, obligately chemolithoautotrophic sulfur-oxidizing bacteria from hypersaline alkaline Mono Lake (California). Int J Syst Evol Microbiol 52, 913–920.[Abstract]
Sorokin, D. Y., Tourova, T. P., Kolganova, T. V., Sjollema, K. A. & Kuenen, J. G. (2002c). Thioalkalispira microaerophila gen. nov., sp. nov., a novel lithoautotrophic, sulfur-oxidizing bacterium from a soda lake. Int J Syst Evol Microbiol 52, 2175–2182.[Abstract]
Sorokin, D. Y., Zhilina, T. N., Lysenko, A. M., Tourova, T. P. & Spiridonova, E. M. (2006). Metabolic versatility of haloalkaliphilic bacteria from soda lakes belonging to the Alkalispirillum-Alkalilimnicola group. Extremophiles 10, 213–220.[CrossRef][Medline]
Spiridonova, E. M., Berg, I. A., Kolganova, T. V., Ivanovskii, R. N., Kuznetsov, B. B. & Tourova, T. P. (2004). An oligonucleotide primer system for amplification of the ribulose-1,5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups. Microbiology English translation of Mikrobiologiia 73, 377–387.[Medline]
Teske, A., Alm, E., Regan, J. M., Toze, S., Rittmann, B. E. & Stahl, D. A. (1994). Evolutionary relationship among ammonia- and nitrite-oxidizing bacteria. J Bacteriol 176, 6623–6630.
Thompson, J. D., Higgins, D. G. & Gibson, T. J. (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.
Tourova, T. P. (2003). Copy number of ribosomal operons in prokaryotes and its effect on phylogenetic analyses. Microbiology English translation of Mikrobiologiia 72, 389–402.[CrossRef]
Tourova, T. P., Spiridonova, E. M., Berg, I. A., Kuznetsov, B. B. & Sorokin, D. Yu. (2005). Phylogeny of ribulose-1,5-bisphosphate carboxylase/oxygenase genes in haloalkaliphilic obligately autotrophic sulfur-oxidizing bacteria of the genus Thioalkalivibrio. Microbiology English translation of Mikrobiologiia 74, 321–328.[CrossRef]
Tourova, T. P., Spiridonova, E. M., Slobodova, N. V., Boulygina, E. S., Keppen, O. I., Kuznetsov, B. B. & Ivanovsky, R. N. (2006). Phylogeny of anoxygenic filamentous phototrophic bacteria of the family Oscillochloridaceae as inferred from comparative analyses of the rrs, cbbL, and nifH genes. Microbiology English translation of Mikrobiologiia 75, 192–200.[CrossRef]
Tsuihiji, H., Yamazaki, Y., Kamikubo, H., Imamoto, Y. & Kataoka, M. (2006). Cloning and characterization of nif structural and regulatory genes in the purple sulfur bacterium, Halorhodospira halophila. J Biosci Bioeng 101, 263–270.[CrossRef][Medline]
Van de Peer, Y. & De Wachter, R. (1994). TREECON for Windows: a software package for the construction and drawing of evolutionary trees for the Microsoft Windows environment. Comput Appl Biosci 10, 569–570.
Ventura, S., Viti, C., Pastorelli, R. & Giovannetti, L. (2000). Revision of species delineation in the genus Ectothiorhodospira. Int J Syst Evol Microbiol 50, 583–591.[Abstract]
Watson, G. M. & Tabita, F. R. (1997). Microbial ribulose-1,5-bisphosphate carboxylase/oxygenase: a molecule for phylogenetic and enzymological investigation. FEMS Microbiol Lett 146, 13–22.[CrossRef][Medline]
Watson, S. W. & Waterbury, J. B. (1971). Characteristics of two marine nitrite oxidizing bacteria, Nitrospira gracilis nov. gen. nov. sp. and Nitrococcus mobilis nov. gen. nov. sp. Arch Mikrobiol 77, 203–230.[CrossRef]
Yakimov, M. M., Giuliano, L., Chernikova, T. N., Gentile, G., Abraham, W.-R., Lünsdorf, H., Timmis, K. N. & Golyshin, P. N. (2001). Alcalilimnicola halodurans gen. nov., sp. nov., an alkaliphilic, moderately halophilic and extremely halotolerant bacterium, isolated from sediments of soda-depositing Lake Natron, East Africa Rift Valley. Int J Syst Evol Microbiol 51, 2133–2143.[Abstract]
This article has been cited by other articles:
|
|
 |
|
  V. M. Gorlenko, I. A. Bryantseva, S. Rabold, T. P. Tourova, D. Rubtsova, E. Smirnova, V. Thiel, and J. F. Imhoff Ectothiorhodospira variabilis sp. nov., an alkaliphilic and halophilic purple sulfur bacterium from soda lakes Int J Syst Evol Microbiol, April 1, 2009; 59(4): 658 - 664. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I. D. Sorokin, I. K. Kravchenko, T. P. Tourova, T. V. Kolganova, E. S. Boulygina, and D. Yu. Sorokin Bacillus alkalidiazotrophicus sp. nov., a diazotrophic, low salt-tolerant alkaliphile isolated from Mongolian soda soil Int J Syst Evol Microbiol, October 1, 2008; 58(10): 2459 - 2464. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| INT J SYST EVOL MICROBIOL | MICROBIOLOGY | J GEN VIROL |
| J MED MICROBIOL | ALL SGM JOURNALS | |
), cbbL genes of other micro-organisms (
), nifH genes of the studied group (
) and nifH genes of other micro-organisms (
). Abbreviations: Emo, Ect. mobilis; Esh, Ect. shaposhnikovii; Eva, Ect. vacuolata; Eha, Ect. haloalkaliphila; Ema, Ect. marismortui; Hab, Hlr. abdelmalekii; Hhc, Hlr. halochloris; Hha, Hlr. halophila; Trsi, Trs. sibirica; Ersm, Ectothiorhodosinus mongolicus; Tsmi, Thioalkalispira microaerophila; Tve, Thioalkalivibrio versutus; Tde, Thioalkalivibrio denitrificans; Tth, Thioalkalivibrio thiocyanoxidans; Tha, Thialkalivibrio halophilus; Tja, Thioalkalivibrio jannaschii; Tnr, Thialkalivibrio nitratireducens; Tni, Thioalkalivibrio nitratis; Tpa, Thioalkalivibrio paradoxus; Ttc, Thialkalivibrio thiocyanodenitrificans; Amo1, Alkalispirillum mobile AGDZ; Amo2, Alkalispirillum mobile Z-0008; Amo3, Alkalispirillum mobile ALPs2; Aha, Alkalilimnicola halodurans; Als, Alkalilimnicola sp. AHN 1; Nmo, Nitrococcus mobilis; AviA, Alc. vinosum rbcA; AviL, Alc. vinosum rbcL; Rca, Rhodobacter capsulatus; Rvs, Rhodovulum sulfidophilum; Tcl, Thioclava pacifica. Genes are plotted at their co-ordinates on the two axes produced by the analysis; the axes are described in Results.