Department of Biochemistry, McMaster University, Hamilton, Ontario, CanadaL8N 3Z51
Author for correspondence: Radhey S. Gupta. Tel: +1 905 525 9140 ext. 22639. Fax: +1 905 522 9033. e-mail: gupta{at}mcmaster.ca
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: chlamydia-like organisms, lateral gene transfer, Archaea, cell-wall biosynthesis
Abbreviations: indel, insertion/deletion
a The GenBank accession numbers for the sequences reported in this paper are indicated in the text.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We have recently described a new approach based on shared conserved insertion and deletions (indels) in various proteins that has proven very useful in identifying main groups within the domain Bacteria and in understanding their relationship to each other (Gupta, 1998 , 2000a
., b
). By tracking the presence or absence of specific indels in various proteins in different phyla, this approach allows the logical deduction of the relative branching order of different groups from a common ancestor (Gupta, 1998
, 2000b
, 2001
; Griffiths & Gupta, 2001
). The use of this approach indicates that the Chlamydiaceae branch is in a similar position to the large, diverse CytophagaFlavobacteriumBacteroides group which has been placed between the Spirochaetes and the
,
-Proteobacteria (Gupta, 2000b
, 2001
). The present work describes a number of conserved indels in various proteins which provide specific molecular markers for the chlamydial group of species, and can unambiguously define and identify this group from all other groups of bacteria. It is significant that two of the largest indels identified in the present work on chlamydiae (in MurA and GlmU) are in enzymes involved in cell wall peptidoglycan biosynthesis, which could provide insights into the unusual cell wall characteristics of these organisms.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
PCR amplification and sequencing.
DNA from Chlamydiaceae strains (Chlamydophila felis FP Cello; Chlamydophila abortus EBA (EP12); Chlamydophila psittaci MN (ATCC VR 122); and Chlamydia suis R24) was generously made available to us by Dr K. Everett (University of Georgia, Athens, USA), and genomic DNA from Simkania negevensis (ATCC VR1471) and Waddlia chondrophila (ATCC 1470) was kindly provided by Dr A. Petrich (St. Josephs Hospital, Hamilton, Canada).
Oligonucleotide primers, in opposite orientations, were designed for regions that flanked three of the signatures, based on the sequences of the genes/proteins from available species of Chlamydiaceae. Degeneracy was incorporated into the primers to account for nucleotide variability at different sites in the alignment. The primers were synthesized at MOBIX, McMaster University.
PCR was performed in a Techne Techgene thermocycler. Each reaction had a final volume of 10 µl and each primer set was optimized for Mg2+ concentration (in the range of 1·54 mM) for each DNA strain tested. PCR amplification was carried out over 30 cycles (15 s at 94 °C, 15 s at 55 °C, 1 min at 72 °C) with an initial 1 min hot start at 94 °C and a final extension step (15 s at 94 °C, 15 s at 55 °C, 7 min at 72 °C). DNA fragments of the expected size were purified from 0·8% (w/v) agarose gels (using a GeneClean kit), and subcloned into the plasmid pCR2.1-TOPO using a TA cloning kit (Invitrogen). Escherichia coli JM109 cells were transformed with the ligated plasmid, and the inserts from a number of positive clones were sequenced by the dideoxy chain termination method using a T7 sequencing kit (Pharmacia). Sequences of all cloned fragments were run through a BLAST search to ensure that they were from a novel source. Attempts were made to generate mgtE, efp and murA fragments corresponding to signature regions in Chl. suis, Chlam. psittaci, Chlam. abortus, Chlam. felis, S. negevensis and W. chondrophila genes for which sequence data were not known. However, amplification was not successful in all cases. Due to the small quantity of available genomic DNA, different primer sets for PCR amplification could not be attempted, though they may prove useful in future studies. The primer sequences used for amplification of different genes were as follows.
UDP-N-acetylglucosamine 1-carboxyvinyltransferase (MurA).
The forward and reverse primers used to amplify this gene were: 5'-CCAGATAARATYGARGCDGCYGGWATGGCYGCRGTWGT-3' and 5'-GCAATVAGWGCKGCCATRACATARGCAAAYCCYGCDCGYAAATC-3', R represents A or G; Y represents C or T; D represents G, A or T; W represents A or T; V represents G, C or A; K represents G or T; M represents A or C; S represents G or C. This primer set was successfully used to amplify approximately 0·5 kb fragments of the murA gene from Chlam. abortus, Chlam. psittaci and Chlam. felis genomic DNA. A 690 bp fragment from W. chondrophila was generated using a forward primer for the conserved amino acid sequence VGATEN (5'-GTNGGNGCNACNGARAA-3') and a reverse primer for the amino acid sequence AGFAYVMA (5'-GCCATNACRTANGCRAANCCNGC-3'), where N is A, C, G or T. murA sequence data were submitted to GenBank under the accession numbers AY038586, AY038585, AY038587 and AF468694 for Chlam. abortus, Chlam. psittaci, Chlam. felis and W. chondrophila respectively.
Translation elongation factor P (EF-P) protein.
The forward and reverse primers for this gene were: 5'-TTRMGMRTWRARATYATGG-3' and 5'-WACDCGRGASTCRTARCTYC-3'. These primers successfully amplified an approximately 0·5 kb fragment of the efp gene from Chl. suis and Chlam. abortus. The primers used to amplify an approximately 0·4 kb fragment from S. negevensis genomic DNA were based on the conserved amino acid sequences VKPGKG (forward primer: 5'-GTNAARCCNGGNAARGG-3') and TGAKIMVP (reverse primer: 5'-GGNACCATDATYTTNGCNCCNGT-3'). Sequence information was submitted to GenBank under the accession numbers AY038589, AY038588 and AF468693 for Chl. suis, Chlam. abortus and S. negevensis respectively.
Mg2+ transport (MgtE) protein.
A 0·5 kb fragment from Chl. suis genomic DNA was amplified using the following forward and reverse primers: 5'-GTKKCBRCYTGYATWCGMARTAATCCTGGSRTTGA-3' and 5'-GTWGCCATACTMCGVACWARAATHGTGCT-3', where B is G, C or T; and H is A, C or T. Sequence data for this gene fragment were submitted to GenBank under the accession number AY038584.
Phylogenetic analysis.
Phylogenetic analyses based on MurA or GlmU protein sequences were carried out as described in our earlier work (Gupta & Singh, 1994 ; Gupta et al., 1997
). The aligned sequences were analysed using the programs SEQBOOT, PROTDIST, NEIGHBOR and CONSENSE from the PHYLIP 3.5 program software (Felsenstein, 1994
). The sequences for these proteins were analysed both with and without the large inserts found in these proteins to exclude the effects of these regions on the observed topology.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
We have also performed phylogenetic analyses based on an alignment of MurA protein sequences. A consensus neighbour-joining phylogenetic tree based on 100 bootstrap replicates of the MurA protein sequences is presented in Fig. 2. All Chlamydiaceae species grouped together in this tree with a bootstrap confidence score of 95 out of 100. Interestingly, and as expected based on 16S and 23S rRNA trees (Everett et al., 1999
; Bush & Everett, 2001
), W. chondrophila formed an outgroup of the Chlamydiaceae family and the clade consisting of these species was recovered 99 times out of 100 in bootstrap replicates. The association of W. chondrophila with the Chlamydiaceae was not dependent upon the large common insert shared by these species, as omission of this region from the alignment did not affect the bootstrap score of the node leading to these species. These results provide strong evidence that W. chondrophila is specifically related to the Chlamydiaceae. The phylogenetic trees based on MurA sequences also supported a strong and specific relationship of the chlamydiae to the Streptomyces species. These two groups of species formed a clade, exclusive of any other bacteria, which was recovered 100% of the time in phylogenetic trees constructed either with or without the large insert (Fig. 2
). The unusual branching of Streptomyces with chlamydiae, instead of with other Gram-positive bacteria, strongly suggests that murA gene has been laterally transferred between these two groups of species.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The chlamydial group of species until very recently consisted of a single family, the Chlamydiaceae, which contained one genus and four species then named Chlamydia trachomatis, Chlamydia pecorum, Chlamydia pneumoniae and Chlamydia psittaci (Fields & Barnes, 1992 ; Bush & Everett, 2001
; Meijer et al., 1999
; Pettersson et al., 1997
; Fukushi & Hirai, 1992
; Grayston et al., 1989
; Moulder et al., 1984
; Page, 1968
). However, the taxonomy of this group has undergone a major revision within the last three years. Based on DNADNA hybridization, and 16S rRNA and 23S rRNA sequence analyses, Everett et al. (1999)
have proposed a new taxonomic classification for this group. The proposal recognizes nine species within the Chlamydiaceae, which are placed into two different genera, Chlamydia and Chlamydophila. Chlamydia consists of three species, Chl. trachomatis, Chl. suis and Chl. muridarum, whereas the genus Chlamydophila is made up of six species, Chlam. abortus, Chlam. psittaci. Chlam. felis, Chlam. caviae, Chlam. pneumoniae and Chlamydophila pecorum (Bush & Everett, 2001
; Herrmann et al., 2000
). In addition to Chlamydiaceae, the proposal recognizes two new families of chlamydia-like organisms, Parachlamydiaceae and Simkaniaceae (Bush & Everett, 2001
; Everett et al., 1999
; Kahane et al., 1999
; Ossewaarde & Meijer, 1999
). A fourth family of chlamydia-like organisms, Waddliaceae, was recognized in a separate proposal (Rurangirwa et al., 1999
). The assignment of distinct family status to these chlamydia-like organisms is presently based on very limited sequence information, consisting primarily of differences in their 16S and 23S rRNA sequences (Everett et al., 1999
). The proposal to identify new families and genera within chlamydiae based mainly on arbitrary degree of differences in the 16S and 23S rRNA sequences has met with strong opposition from many scientists (Schachter et al., 2001
). It has been argued that since all known chlamydiae species share a unique and highly conserved biological replication cycle, they should be retained within a single genus unless there is a compelling reason to do otherwise.
The signatures described here should prove helpful in clarifying the classification of the chlamydial species. For all eight signatures described here, sequence information is known for at least two species from each of the two proposed genera within Chlamydiaceae. For some signatures, sequence data are known for six to seven out of a possible nine species. Since the two genera, Chlamydia and Chlamydophila, are indicated to be monophyletic in their 16S and 23S rRNA trees (with 100% bootstrap scores) (Everett et al., 1999 ; Bush & Everett, 2001
), the presence of these signatures in at least two species from each genus strongly suggests that they will also be found in other species of Chlamydiaceae for which sequence information is lacking at present. Therefore, all of the signatures described here are at least distinctive of the Chlamydiaceae group of species. For the MurA and EF-P proteins, we were also successful in amplifying a gene fragment from the two chlamydia-like organisms, W. chondrophila and S. negevensis, respectively. The sequences from both of these species contained the indicated indels. In phylogenetic trees based on MurA sequences, Waddlia formed an outgroup of the Chlamydiaceae species and the clade consisting of these species was supported at a very high degree of bootstrap confidence level (99 out of 100). These results provide strong molecular and phylogenetic evidence that these species are specifically related to the traditional chlamydiae (i.e., Chlamydiaceae) species. The presence of these signatures in these organisms, which have been assigned to different families within the order Chlamydiales, is strongly suggestive that these signatures may prove distinctive for the entire Chlamydiales order. It is unclear at present whether the other identified signatures are also present in these chlamydia-like organisms. It is quite possible that of these signatures, some may prove specific for the Chlamydiaceae, while others will be commonly shared by the entire Chlamydiales order. In future studies, it should then be possible to unambiguously identify species belonging to the Chlamydiaceae family or the Chlamydiales order from all other bacteria, simply by determining the presence or absence of these molecular signatures. Further studies should also reveal whether some of these signatures are commonly shared by the Chlamydiaceae and one or more of the Parachlamydiaceae, Simkaniaceae and Waddliaceae families. This should provide insight as to how the various proposed families within Chlamydiales have evolved and are related to each other.
An important question that needs to be understood in future work concerns the functional significance of the identified indels on the biochemical and physiological characteristics of the chlamydial species. Since the identified signatures are present in all available chlamydial sequences but not in any other bacteria, it strongly suggests that they were introduced in a common ancestor of this group of bacteria at the time of its evolution. Because these indels have not been lost in any of the chlamydial species examined to date, they likely play an important role in the biology of these organisms and could have been important in their evolution. Thus, it is of much interest to understand the functional significance of these site-specific alterations in these conserved and widely distributed genes. Although all of these signatures are potentially interesting, the two containing the most prominent indels are both in proteins involved in cell-wall-peptidoglycan biosynthesis. The MurA protein, which contains the 16 aa insert, is responsible for carrying out the first essential and committed step in peptidoglycan biosynthesis in various bacteria (Brown et al., 1995 ; Du et al., 2000
). The large insert seen in MurA is also present in the two streptomyces species Streptomyces coelicolor and Streptomyces lividans, but not any other Gram-positive or Gram-negative bacteria. The shared presence of this common insert as well as the phylogenetic studies based on MurA protein sequences strongly suggests that a lateral transfer of this gene has occurred between these two groups of species. The biochemical significance of this lateral gene transfer event and whether the exchange occurred from chlamydiae to Streptomyces or vice versa is unclear at present.
Similar to the MurA protein, the GlmU protein that contains the 17 aa indel is also involved in peptidoglycan biosynthesis. Interestingly, the large insert in this protein is also present in all archaeal homologues and phylogenetic studies of GlmU protein sequences indicate that the gene containing this indel has been laterally acquired by chlamydiae from an archaeon. The above observations are of much interest because although the genomes of chlamydial species contain a full complement of the genes involved in peptidoglycan synthesis (Stephens et al., 1998 ; Hatch, 1998
; Kalman et al., 1999
; Read et al., 2000
), these species are generally believed to lack, or be deficient in, peptidoglycan (Hua et al., 1985
; Fox et al., 1990
; Hatch, 1996
, 1998
; Ghuysen & Goffin, 1999
). It has been suggested that chlamydiae synthesize a defective peptidoglycan or an atypical cell wall differing from other bacteria (Hatch, 1996
; Ghuysen & Goffin, 1999
). However, the biochemical basis of this phenomenon remains to be understood. In this context, the presence of large inserts in essential enzymes involved in cell wall synthesis has the potential to inactivate or modify the cellular functions of such proteins. It should be pointed out in this regard, that GlmU is a bifunctional protein which catalyses two of the essential steps leading to the synthesis of UDP-N-acetylglucosamine, a fundamental precursor for bacterial cell wall synthesis (Gehring et al., 1996
; Pompeo et al., 2001
). It has been shown that the N-terminal domain of this protein (residues 3227 in E. coli) catalyses the uridyltransferase activity whereas the C-terminal domain is responsible for the acetyltransferase activity (Brown et al., 1999
; Pompeo et al., 2001
). The insert in GlmU is in the C-terminal region and hence its main effect should be on the acetyltransferase activity. The acetyltransferase domain of GlmU is made up of 10 regular coils, with an inter-coil distance of generally 17 aa (Brown et al., 1999
; Sulzenbacher et al., 2001
). The observed 17 aa insert in GlmU is expected to add an extra coil in the protein structure. How this may affect the function of the protein remains to be determined. It is also of much interest that the GlmU homologues from chlamydiae are much shorter than those from other species (about 205 aa compared to 450 aa from other bacteria) and they seem to lack the N-terminal region. Thus, the enzyme from chlamydial species should be lacking the uridyltransferase activity. Similar to chlamydiae, the cell wall composition/structure in the Archaea also differs from the Bacteria (Woese, 1987
; Kandler & Konig, 1993
). If there have been lateral gene transfers between Archaea and chlamydiae, or chlamydiae and Streptomyces, as strongly suggested by the results presented here, then it becomes of much interest to determine the functional significance of these lateral gene-transfer events in these divergent prokaryotes.
Chlamydial infections are responsible or have been implicated in a wide variety of diseases affecting many different systems and organs (Moulder et al., 1984 ). Hence, identification of the known chlamydial species and other novel species related to this group in different pathological conditions is of importance. The dependence of the Chlamydiaceae on host cells for their growth, and the presence of other contaminating bacteria in clinical specimens, have presented problems in their definitive identification in clinical situations. Some of the large chlamydial-specific signatures described here could prove useful in this regard. For example, the MurA protein contain a 16 aa or 48 nt insert that is distinctive for the chlamydial species and not found in any other bacteria. The homologues of MurA are also not found in eukaryotes. Based on alignment of the murA gene sequences from various chlamydial species, several conserved regions both within this large insert, and in regions flanking it, can be identified. PCR amplification utilizing such sequences should exhibit a high degree of specificity in detecting chlamydial species. Likewise, the large 17 aa or 51 nt insert in the GlmU protein could also prove useful for similar purposes.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Brown, E. D., Vivas, E. I., Walsh, C. T. & Kolter, R. (1995). MurA (MurZ), the enzyme that catalyzes the first committed step in peptidoglycan biosynthesis, is essential in Escherichia coli. J Bacteriol 177, 4194-4197.[Abstract]
Brown, K., Pompeo, F., Dixon, S., Mengin-Lecreulx, D., Cambillau, C. & Bourne, Y. (1999). Crystal structure of the bifunctional N-acetylglucosamine 1-phosphate uridyltransferase from Escherichia coli: a paradigm for the related pyrophosphorylase superfamily. EMBO J 18, 4096-4107.
Bush, R. M. & Everett, K. D. E. (2001). Molecular evolution of the Chlamydiaceae. Int J Syst Evol Microbiol 51, 203-220.[Abstract]
Daian, C. M., Wolff, A. H. & Bielory, L. (2000). The role of atypical organisms in asthma. Allergy Asthma Proc 21, 107-111.[Medline]
Du, W., Brown, J. R., Sylvester, D. R., Huang, J., Chalker, A. F., So, C. Y., Holmes, D. J. & Wallis, N. G. (2000). Two active forms of UDP-N-acetylglucosamine enolpyruvyltransferase in gram-positive bacteria. J Bacteriol 182, 4146-4152.
Everett, K. D., Bush, R. M. & Andersen, A. A. (1999). Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simikaniacae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol 49, 415-440.[Abstract]
Felsenstein, J. (1994). PHYLIP version 3.5. Seattle, WA: University of Washington.
Fields, P. I. & Barnes, R. C. (1992). The genus Chlamydia. In The Prokaryotes, pp. 36913709. Edited by A. Balows and others. New York: Springer.
Fox, A., Rogers, J. C., Gilbart, J., Morgan, S., Davis, C. H., Knight, S. & Wyrick, P. B. (1990). Muramic acid is not detectable in Chlamydia psittaci or Chlamydia trachomatis by gas chromatography-mass spectrometry. Infect Immun 58, 835-837.[Medline]
Fukushi, H. & Hirai, K. (1992). Proposal of Chlamydia pecorum sp. nov. for Chlamydia strains derived from ruminants. Int J Syst Bacteriol 42, 306-308.[Abstract]
Gehring, A. M., Lees, W. J., Mindiola, D. J., Walsh, C. T. & Brown, E. D. (1996). Acetyltransferase precedes uridyl transfer in the formation of UDP-N-acetylglucosamine in separable active sites of the bifunctional GlmU proteins of Escherichia coli. Biochemistry 35, 579-585.[Medline]
Ghuysen, J.-M. & Goffin, C. (1999). Lack of cell wall peptidoglycan versus penicillin sensitivity: new insights into the chlamydial anomaly. Antimicrob Agents Chemother 43, 2339-2344.
Grayston, J. T., Kuo, C. C., Campbell, A. & Wang, S. P. (1989). Chlamydia pneumoniae sp. nov. for Chlamydia sp. strain TWAR. Int J Syst Bacteriol 39, 88-90.
Griffiths, E. & Gupta, R. S. (2001). The use of signature sequences in different proteins to determine the relative branching order of bacterial divisions: evidence that Fibrobacter diverged at a similar time to Chlamydia and the CytophagaFlavobacteriumBacteroides division. Microbiology 147, 2611-2622.
Gupta, R. S. (1998). Protein phylogenies and signature sequences: a reappraisal of evolutionary relationships among archaebacteria, eubacteria, and eukaryotes. Microbiol Mol Biol Rev 62, 1435-1491.
Gupta, R. S. (2000a). The natural evolutionary relationships among prokaryotes. CRC Crit Rev Microbiol 26, 111-131.
Gupta, R. S. (2000b). The phylogeny of proteobacteria: relationships to other eubacterial phyla and eukaryotes. FEMS Microbiol Rev 24, 367-402.[Medline]
Gupta, R. S. (2001). The branching order and phylogenetic placements of species from completed bacterial genomes based on conserved indels found in various proteins. Int Microbiol 4, 187-202.[Medline]
Gupta, R. S. & Johari, V. (1998). Signature sequences in diverse proteins provide evidence of a close evolutionary relationship betweeen the Deinococcus/Thermus group and Cyanobacteria. J Mol Evol 46, 716-720.[Medline]
Gupta, R. S. & Singh, B. (1994). Phylogenetic analysis of 70 kDa protein sequences suggests a chimeric origin of the eukaryotic cell nucleus. Curr Biol 4, 1104-1114.[Medline]
Gupta, R. S., Bustard, K., Falah, M. & Singh, D. (1997). Sequencing of heat shock protein 70 (DnaK) homologs from Deinococcus proteolyticus and Thermomicrobium roseum and their integration in a protein based phylogeny of prokaryotes. J Bacteriol 179, 345-357.[Abstract]
Hatch, T. P. (1996). Disulfide cross-linked envelope proteins: the functional equivalent of peptidoglycan in chlamydiae? J Bacteriol 178, 1-5.
Hatch, T. P. (1998). Chlamydia: old ideas crushed, new mysteries bared. Science 282, 638-639.
Herrmann, B., Pettersson, B., Everett, K. D. E., Mikkelsen, N. E. & Kirsebom, L. A. (2000). Characterization of the rnpB gene and RNase P RNA in the order Chlamydiales. Int J Syst Evol Microbiol 50, 149-158.[Abstract]
Hua, S., Youxum, Z. & Rungte, L. (1985). Presence of muramic acid in Chlamydia trachomatis proved by liquid chromatography-mass spectrometry. Kexue Tongbao (Chinese Science Bulletin), 30, 695-699.
Kahane, S., Everett, K. D. E., Kimmel, N. & Friedman, M. G. (1999). Simkania negevensis strain ZT: growth, antigenic and genome characteristics. Int J Syst Bacteriol 49, 815-820.[Abstract]
Kalman, S., Mitchell, W., Marathe, R. & 7 other authors (1999). Comparative genomes of Chlamydia pneumonia and C. trachomatis. Nat Genet 21, 385389.
Kandler, O. & Konig, H. (1993). Cell envelopes of archaea: structure and chemistry. In The Biochemistry of Archaea (Archaebacteria) , pp. 223-259. Edited by M. Kates, D. J. Kushner & A. T. Matheson. New York:Elsevier.
Kuo, C. C., Jackson, L. A., Campbell, L. A. & Grayston, J. T. (1995). Chlamydia pneumoniae (TWAR). Clin Microbiol Rev 8, 451-461.[Abstract]
Laga, M., Nzila, N. & Goeman, J. (1991). The interrelationship of sexually transmitted diseases and HIV infection: implications for the control of both epidemics in Africa. AIDS 5, S55-S63.[Medline]
Ludwig, W. & Klenk, H.-P. (2001). Overview: a phylogenetic backbone and taxonomic framework for procaryotic systematics. In Bergeys Manual of Systematic Bacteriology, vol. 1, The Archaea and the deeply branching and phototrophic Bacteria, pp 4965. Edited by D. R. Boone and R. W. Castenholz. Berlin: Springer.
Meijer, A., Morre, S. A., van den Brule, A. J. C., Savelkoul, P. H. M. & Ossewaarde, J. M. (1999). Genomic relatedness of chlamydia isolates determined by amplified fragment length polymorphism analysis. J Bacteriol 181, 4469-4475.
Moulder, J. W., Hatch, T., Kuo, C. C., Schachter, J. & Storz, J. (1984). Genus Chlamydia. In Bergeys Manual of Systematic Bacteriology , pp. 729-739. Edited by N. R. Krieg & J. G. Holt. Baltimore, MD:Williams & Wilkins.
Ossewaarde, J. M. & Meijer, A. (1999). Molecular evidence for the existence of additional members of the order Chlamydiales. Microbiology 145, 411-417.[Abstract]
Page, L. A. (1968). Proposal for the recognition of two species in the genus Chlamydia Jones, Rake and Stearns, 1945. Int J Syst Bacteriol 18, 51-66.
Pettersson, B., Andersson, A., Leitner, T., Olsvik, O., Uhlen, M., Storey, C. & Black, C. M. (1997). Evolutionary relationships among members of the genus Chlamydia based on 16S ribosomal DNA analysis. J Bacteriol 179, 4195-4205.[Abstract]
Pompeo, F., Bourne, Y., Heijenoor, J. V., Fassy, F. & Mengin-Lecreulx, D. (2001). Dissection of the bifunctional Escherichia coli N-aectylglucosamine-1-phosphate uridyltransferase enzyme into autonomously functional domains and evidence that trimerization is absolutely required for glucosamine-1-phosphate acetyltransferase activity and cell growth. J Biol Chem 276, 3883-3889.
Read, T. D., Brunham, R. C., Shen, C. & 22 other authors (2000). Genome sequences of Chlamydia trachomatis MoPn and Chlamydia pneumoniae AR39. Nucleic Acids Res 28, 13971406.
Rurangirwa, F. R., Dilbeck, P. M., Crawford, T. B., McGuire, T. C. & McElwain, T. F. (1999). Analysis of the 16S rRNA gene of micro-organism WSU 86-1044 from an aborted bovine foetus reveals that it is a member of the order Chlamydiales: proposal of Waddliaceae fam. nov., Waddlia chondrophila gen. nov., sp. nov. Int J Syst Bacteriol 49, 577-581.[Abstract]
Saikku, P. (2000). Chlamydia pneumoniae in atherosclerosis. J Intern Med 247, 396.
Schachter, J. (1999). Infection and disease epidemiology. In Chlamydia: Intracellular Biology, Pathogenesis, and Immunity, pp. 139169. Washington, DC: American Society for Microbiology.
Schachter, J., Stephens, R. S., Timms, P. & 29 other authors (2001). Radical changes to chlamydial taxonomy are not necessary just yet. Int J Syst Evol Microbiol 51, 249.
Stephens, R. S., Kalman, S., Lammel, C. & 9 other authors (1998). Genome sequence of an obligate intracellular pathogen of humans: Chlamydia trachomatis. Science 282, 754759.
Sulzenbacher, G., Gal, L., Peneff, C., Fassy, F. & Bourne, Y. (2001). Crystal structure of Streptococcus pneumoniae N-acetylglucosamine-1-phosphate uridyltransferase bound to acetyl-coenzyme A reveals a novel active site architecture. J Biol Chem, 276, 11844-11851.
Woese, C. R. (1987). Bacterial evolution. Microbiol Rev 51, 221-271.
Received 28 September 2001;
revised 26 March 2002;
accepted 3 April 2002.