1 Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton L8N 3Z5, Canada
2 Department of Pathology and Molecular Medicine, McMaster University, Hamilton L8N 3Z5, Canada
Correspondence
Radhey S. Gupta
gupta{at}mcmaster.ca
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ABSTRACT |
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INTRODUCTION |
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The task of identifying novel molecular characteristics that can provide reliable markers for chlamydiae is facilitated by the availability of genomic sequences. The complete genome sequences of five chlamydiae species, Chlamydophila (Chlam.) pneumoniae (four different strains: AR39, CWL029, J139, TW-183) (Read et al., 2000), Chlamydophila caviae GPIC (Read et al., 2003
), Chlamydia (Chl.) trachomatis (serovar D) (Stephens et al., 1998
), Chlamydia muridarum (Read et al., 2000
) and an environmental chlamydia, Parachlamydia sp. UWE25 (Horn et al., 2004
), are now available. The comparative analyses of these genomes provide a powerful means for identifying novel molecular characteristics that are distinctive to different groups of bacteria (Hatch, 1998
; Kalman et al., 1999
). In an earlier study, we reported a number of conserved inserts and deletions (indels) in widely distributed proteins that were uniquely shared by Chlamydiaceae species (Griffiths & Gupta, 2002
). However, most of these indels were small (1 aa), and for most of them sequence information from other chlamydiae families was not available. In the present work, which extends this earlier study, we have identified a number of prominent chlamydiae-specific conserved indels in essential proteins which are found in all bacteria, and in some cases in species from all three domains (Olsen & Woese, 1997
). The proteins which contain chlamydiae-specific signatures include RNA polymerase
subunit (RpoA), elongation factor Tu (EF-Tu), DNA gyrase
subunit (GyrB), elongation factor P (EF-P) and lysyl-tRNA synthetase (LyrRS). Sequence information for most of these proteins (RpoA, EF-Tu, EF-P and GyrB) was obtained from species (Simkania, Waddlia and Neochlamydia) covering all of the known chlamydiae families. The presence of these signatures in all of these species provides evidence that they are distinctive characteristics of the phylum Chlamydiales. For the Simkania, Waddlia and Neochlamydia species, apart from the 16S and 23S rRNA, very little sequence information is available in the databases. Hence, we have carried out a phylogenetic analysis based on a concatenated dataset of sequences from these genes (RpoA, GyrB, EF-P and EF-Tu), to determine the interrelationships among different chlamydiae species.
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METHODS |
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Identification of chlamydiae-specific signatures.
Multiple sequence alignments for a large number of proteins have been created in our earlier work (Gupta, 1998, 2000
, 2004
; Griffiths & Gupta, 2001
, 2002
, 2004
; Gupta et al., 2003
) (see also www.bacterialphylogeny.com). To search for chlamydiae-specific signatures, these alignments were visually inspected to identify any indel that was uniquely present in all available chlamydiae homologues and flanked by conserved sequences. The indels which were not flanked by conserved regions, and/or which were not present in all chlamydiae, were omitted from further consideration in this study. The chlamydiae specificity of potentially useful indels was further evaluated by carrying out additional BLAST searches (Altschul et al., 1997
) on short sequence segments (usually 60100 aa) containing the indel and the flanking conserved regions. The purpose of these BLAST searches was to obtain sequence information from all available species to ensure that the identified signatures were only present in the chlamydiae homologues. The sequence information for various useful signatures, which were chosen for further investigation, was compiled into signature files, such as those shown in Figs 15
.
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RESULTS |
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The core subunits of RNA polymerase, ,
and
', are highly conserved in species from all three domains (Olsen & Woese, 1997
). In the
subunit (RpoA), which is encoded by the rpoA gene, a 17 aa insert is present in the N-terminal region in various Chlamydiaceae homologues (Fig. 1
). A slightly smaller insert of 15 aa is also present in the same position in Parachlamydia sp. UWE25. The insert sequences in all of these species are very similar, indicating that they are of common origin. This insert is not found in any species other than chlamydiae, indicating that it is highly specific for this group. To determine whether this indel is present in Sim. negevensis, Wad. chondrophila and Neo. hartmanellae, PCR amplification of DNA from these species was carried out using the primers indicated in Table 1
. For Simkania and Waddlia, this led to specific amplification of 0·5 kb fragments, which were cloned and sequenced. Both species were found to contain the 17 aa insert (sequence information included in Fig. 1
), providing evidence that this large insert is a distinctive characteristic of various Chlamydiales. Although these primer sets were not successful in amplifying the target fragments from Neochlamydia under our experimental conditions, it is likely that Neo. hartmanellae will contain the insert, as it has been shown to be part of the genus Parachlamydia.
In the protein synthesis elongation factor-Tu (EF-Tu), which is again a highly conserved protein found in species from all three domains (Hashimoto & Hasegawa, 1996), two different chlamydiae-specific signatures are present in the same region of the protein. One of these signatures consists of a 2 aa insert, whereas the other is a 2 aa deletion, separated from the first by a conserved region of 24 aa (Fig. 2
). These inserts are again specific for chlamydiae and not found in any other bacteria. Upon sequencing of a 0·4 kb fragment from Wad. chondrophila, and 0·8 kb fragments from Sim. negevensis and Neo. hartmanellae, both these signatures were found to be present in these species (see Fig. 2
). These signatures are thus again indicated to be distinctive characteristics of various chlamydiae, which were likely introduced in a common ancestor of this lineage.
DNA gyrase is a type II topoisomerase, which makes transient double strand breaks in DNA to allow another DNA strand to pass through (Levine et al., 1998). DNA gyrase function is essential in bacteria for maintaining the appropriate levels of supercoiling in the chromosome and for transcription and replication processes. The enzyme consists of two subunits, A and B, that combine to form a functional A2B2 complex. The B subunit of DNA gyrase (GyrB) contains a 6 aa insert that is uniquely present in various available chlamydia homologues (Fig. 3
). The specificity and distinctiveness of this insert were again tested by PCR amplification of 0·9 kb fragments of gyrB from Wad. chondrophila, Sim. negevensis and Neo. hartmanellae. The cloning and sequencing of these fragments revealed that the indicated signature insert was present in all three species (see Fig. 3
), confirming that it is a reliable molecular marker for all chlamydiae.
We have previously described a 1 aa insert in EF-P (Griffiths & Gupta, 2002) that is found in all Chlamydiaceae species, including Chlamydophila abortus and Chlamydia suis, which were sequenced in our previous work, as well as Sim. negevensis. This insert is also present in Parachlamydiae sp. UWE25, and its sequence is completely conserved in all species. Further work that we have carried out indicates that this insert is also present in Wad. chondrophila (Fig. 4
), indicating that in a similar manner to the inserts in RpoA, EF-Tu and GyrB, this insert is also a distinctive characteristic of various chlamydiae species.
Another signature that is specific for various chlamydiae was identified in the enzyme lysyl-tRNA synthetase, which catalyses the correct attachment of lysine to its cognate tRNA in the protein synthesis process (Woese et al., 2000). The chlamydial LysRS proteins (from the known Chlamydiaceae and Parachlamydia) were found to contain a 6 aa insert which is unique to this group of species (Fig. 5
). Using the PCR primers described in Table 1
, we were also successful in amplifying a 0·6 kb LysRS fragment from Wad. chondrophila that was found to contain the indel. However, our attempts to PCR-amplify the corresponding fragment from Sim. negevensis and Neo. hartmanellae using this set of primers were unsuccessful, indicating that the primer region may have undergone mutational changes in these species. Since species representing other chlamydiae families contain this insert, it is likely that this indel will also be found in Simkania and Neochlamydia.
Phylogenetic analysis of chlamydiae species based on concatenated protein sequences
Our understanding of the evolutionary relationships among chlamydiae is based solely on 16S and 23S rRNA sequences (Everett et al., 1999a; Bush & Everett, 2001
; Corsaro et al., 2003
; Horn et al., 2004
). The sequence information for different Chlamydiales species is currently not available for any protein-coding genes. Since in our work we have sequenced fragments for a number of protein-coding genes from different chlamydiae families, it was of interest to carry out a phylogenetic analysis based on these protein sequences and compare it with the 16S rRNA tree. As the amplified fragments generated in this work were small, to obtain a reliable phylogenetic tree we have combined (i.e. concatenated) sequence information for GyrB, RpoA, EF-Tu and EF-P proteins from Wad. chondrophila, Sim. negevensis, Parachlamydia sp. UWE25, Chl. trachomatis, Chl. muridarum, Chlam. pneumoniae, Chlam caviae and Thermus aquaticus. This concatenated dataset containing 529 aa positions was used to construct a neighbour-joined tree which was bootstrapped (100 times) for statistical analysis (Felsenstein, 1988
). Thermus aquaticus was used as an outgroup to root the tree (Fig. 6A
). The resulting topology showed that the Chlamydiales appear to have evolved in two distinct groups, the Chlamydiaceae and the chlamydiae-like organisms. Within the Chlamydiaceae, the genus Chlamydia appears to have diverged later than Chlamydophila. Among the chlamydiae-like species, Simkania branches the earliest, followed by a bifurcating clade including Waddlia and Parachlamydia. All branches were resolved with significant bootstrap scores, and the observed branching pattern was not affected by the presence or absence of the signature inserts (not shown). These findings were also corroborated by individual protein phylogenies based on these protein sequences (data not shown). It should be mentioned that the amino acid sequences of the insert in the EF-Tu protein (Fig. 2
) also distinguish the clades consisting of Chlamydia (insert sequence SE), Chlamydophila (insert sequence SQ) and Waddlia/Parachlamydia (insert sequence GE). We also constructed a neighbour-joined phylogenetic tree based on full-length 16S rRNA sequences (Maidak et al., 2001
) (Fig. 6B
). This tree showed a similar topology to that seen in the concatenated protein tree, and branches separating the chlamydiae-like species from the Chlamydiaceae were resolved with high statistical support (bootstraps >90 %). 16S rRNA trees constructed by other investigators have resulted in topologies very similar to that obtained with our concatenated protein dataset (Everett et al., 1999a
; Bush & Everett, 2001
; Corsaro et al., 2003
; Horn et al., 2004
). Thus, it is likely that the chlamydiae-like species (Waddlia, Parachlamydia and Simkania) form a distinct clade within the phylum Chlamydiae.
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DISCUSSION |
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All well-studied chlamydiae species are characterized by a unique two-stage life cycle (Fields & Barnes, 1992; Kalayoglu & Byrne, 2001
). However, for most new reports of chlamydiae-like species (sequences), which are from non-culturable sources, it is difficult to obtain information in this regard. The primary means used for identifying novel chlamydiae species in different hosts and environmental samples involves PCR amplification using primers based on 16S rRNA sequences, and the clustering of any amplified sequence with the known chlamydiae species in the rRNA trees (Rurangirwa et al., 1999
; Everett et al., 1999b
; Horn et al., 2000
, 2004
; Corsaro et al., 2002
; Thao et al., 2003
). Although this method has proved very useful in advancing our understanding of chlamydial diversity, the branching patterns in phylogenetic trees are known to be affected by a large number of factors (Felsenstein, 1988
; Gupta, 1998
; Moreira & Philippe, 2000
; Baldauf, 2003
). Other than their branching pattern in the tree, no distinctive molecular signature for chlamydiae is present in the 16S rRNA sequences. Hence, the availability of other specific and reliable molecular markers for confirming the presence of chlamydiae species in environmental samples should be very useful.
In the present work, we have described several distinctive molecular markers for chlamydiae species in highly conserved proteins. These markers consists of prominent inserts and deletions in highly conserved regions of a number of important proteins (RpoA, EF-Tu, GyrB, EF-P and LysRS) that are unique to various chlamydiae homologues and not found in any other bacteria. The sequence information for most of these proteins (except LysRS) has been obtained from Waddlia and Simkania, and for EF-Tu and GyrB from Neochlamydia as well. The shared presence of these signatures in all of the chlamydiae families strongly indicates that these signatures were introduced in a common ancestor of the Chlamydiales and that they are distinctive characteristics of the entire phylum. The proteins in which these chlamydiae-specific signatures are present are all involved in essential functions related to information-transfer processes. Most of these proteins are ubiquitous in bacteria, and some (RpoA, EF-Tu and LysRS) are present in species from all three domains. Because of their essential functions, the primary structures of these proteins are highly conserved. The PCR primers for EF-Tu and gyrase B used in our work were successful in amplifying the corresponding gene fragments from Sim. negevensis, Wad. chondrophila and Neo. hartmanellae. Because these primers are based on sequence information for Chlamydiaceae and Parachlamydia sp. UWE25, they are also expected to work for these species. The PCR primers for RpoA and EF-P also successfully amplified the corresponding fragments from Sim. negevensis and Wad. chondrophila, but they were not successful with Neo. hartmanellae under the experimental conditions used in the present study. However, based on the fact that these sequences are highly conserved, it should be possible to design other primers that would work in cases where PCR amplification was not successful in the present study. These signatures and the PCR primers provide novel means, in addition to the 16S rRNA-based primers, for identifying and confirming the presence of chlamydiae-related sequences in environmental samples. Because of the chlamydiae specificities of these signatures, if any of the amplified sequences are found to contain these signatures, this will provide reliable evidence for the presence of a chlamydiae-like organism in a given sample.
Phylogenetic analysis based on a combined dataset of protein fragments from RpoA, EF-Tu, Ef-P and GyrB proteins indicates that the environmental chlamydiae (Simkania, Waddlia and Parachlamydia) and the traditional Chlamydiaceae (Chlamydophila and Chlamydia) species form distinct clades in the resulting tree. Similar relationships were also noted in individual protein phylogenies as well as in a tree for these species based on the 16S rRNA sequences. Thus, it is highly likely that the chlamydiae-like species (Waddlia, Parachlamydia and Simkania) have diverged from the traditional Chlamydiaceae species at a very early stage in the evolution of this group of bacteria. If this is the case, then the chlamydiae-like species may contain a very different range of metabolic, infective and virulence capabilities, as evidenced by the large difference in genome size between Parachlamydia sp. UWE25 and all other sequenced Chlamydiaceae (Hatch, 1998; Kalman et al., 1999
; Read et al., 2000
, 2003
; Horn et al., 2004
).
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ACKNOWLEDGEMENTS |
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Received 21 March 2005;
revised 17 May 2005;
accepted 27 May 2005.
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