Editorial: The Parachlamydia genome sequence: milestone in chlamydial research

The recent publication of the complete genome sequence for "Parachlamydia-related symbiont UWE25" [Horn et al., 2004] represents a milestone in chlamydial and, particularly Chlamydiales research, for three main reasons.

Firstly, this is the first genome sequence for an endosymbiotic environmental chlamydial species, organisms which are thought to have diverged from the Chlamydiaceae about 700 million years ago. Adapted to relatively primitive unicellular hosts (acanthamoebae), it might be expected that UWE25 is closer in evolutionary terms to the last common ancestor of the chlamydiae than the more familiar Chlamydiaceae. The latter have undergone the significant genomic degradation characteristic of intracellular pathogens adapting to multicellular eukaryotic hosts. In support of this, the genome of UWE25 is twice as large as that of any pathogenic chlamydiae. Moreover with one notable exception (the type 4 secretion genes) there is little evidence for recent lateral gene transfer events in UWE25. Thus it is reasonable to suppose that the genome sequence of UWE25 offers insights into the genetic makeup of the last common ancestor of the chlamydiae [Horn et al., 2004].

Secondly, comparison of the genomes of UWE25 and Chlamydiaceae provides insights into how the chlamydiae might have evolved from endosymbionts into highly successful pathogens. Primitive amoeba-like eukaryotic cells may have played an important role in this. A number of bacteria are known to be able to survive phagocytosis and even to multiply within the amoebal cell, even though acanthamoebae trophozoites feed on many bacteria. Studies of the replication of Legionella pneumophila in free living amoebae gave rise to the concept that amoebae might be a kind of "biological gymnasium" in which free-living bacteria acquire the necessary "fitness" to become adapted intracellular pathogens [Harb et al., 2000; Marciano-Cabral & Cabral, 2003; Marrie et al., 2001]. This adaptation was probably accompanied by substantial genome reorganisation as shown by the sparse conservation of gene order between the EWE25 and Chlamydiaceae genomes. However, the 1,093 coding sequences present in UWE25 but not the pathogenic Chlamydiaceae are fairly evenly spread throughout the genome, indicating that the observed genomic reorganisation was not primarily determined by lateral gene transfer events [Horn et al., 2004].

Thirdly, the UWE25 genome sequence may add insight into the evolution of the mitochondrion and chloroplast. It is widely believed that mitochondria and chloroplasts evolved from free living bacteria related to cyanobacteria, that colonised free living protists prior to the divergence of protist and eukaryotic cells some 1500 million years ago. The Chlamydiaceae genome sequences had already revealed an unexpectedly high number of cyanobacterial and plant gene homologues [see Chlamydia - plant gene relationships]. This is even more apparent in the UWE25 genome sequence: [Table 1].

Though originally cautiously called Parachlamydia-related symbiont UWE25, this organism is now reclassified within the family Parachlamydiaceae as Candidatus Protochlamydia amoebophila, reflecting sufficient differences to its closest known relative, Parachlamydia acanthamoebae Bn(₉)(^T) to warrant its classification in a new genus [Collingro et al., 2005]. The 'Candidatus' status reflects the fact the organism has not been grown in cell-free media and is deposited in one (ATCC PRA-7) rather than two cell culture collections.

Table 1: The relationship of predicted UWE25 coding sequences to other taxonomic groups as predicted by first BLAST hits. Adapted from Fig S5, Horn et al., 2004.

Many of the plant genes homologous with UWE25 are located in the chloroplast, suggesting cyanobacterial ancestry. However some of these homologous plant genes are phylogenetically more closely related to chlamydiae than to cyanobacteria, indicating complex ancestral transfers between plants and both these bacterial groups [Horn et al., 2004]. It is now considered that a chlamydial ancestor played a key role in enabling an ingested cyanobacterium to eventually become the first primitive plant plastid; for further details of this intriguing story see plant-chlamydia relationships.

Details of the UWE25 genome may be found in the on-line paper of Horn et al., 2004 and in the excellent, extensively annotated UWE 25 genome web site. Included in individual gene annotations are relevant links to this web site. The table below summarises some of the distinguishing features of the UWE25 and available Chlamydiaceae genome sequences: [Table 2]:

Table 2: Differences and similarities between the UWE25 and Chlamydiaceae genome sequences.

Protochlamydia amoebophila UWE 25 genome	Chlamydiaceae genomes
Approx 2.4 MBp (ca 3KBp of sequence not available)	Approx 1.1 MBp
35.8% GC content	Approx 40% GC content
2,031 open reading frames	Approx 1,000 open reading frames
Complete TCA (tricarboxylic acid) cycle	Incomplete TCA (tricarboxylic acid) cycle
Synthesizes glycine, serine, glutamine, proline but not tryptophan	Ability of some biovars to synthesize tryptophan affects interferon sensitivity Cannot synthesize glycine, serine, glutamine and proline
Possesses glucokinase Complete pentose phosphate pathway Generates 4 ATP molecules during glycolysis	No glucokinase: dependent on host for phosphorylated sugars Complete pentose phosphate pathway Generates 4 ATP molecules during glycolysis
Encodes complete Type III secretion system	Encodes complete Type III secretion system
Recent gain of Type IV secretion system	No Type IV secretion system
Additional F-type ATPase, cytochrome c + its oxidase, H+ translocating NADH oxidoreductase Can use menaquinone in respiratory pathway	Less energy from oxidative phosphorylation
5 nucleotide transporters including ATP import Conserved ATP/ADP translocase [Schmitz-Esser et al., 2004]	2 nucleotide transporters Conserved ATP/ADP translocase [Schmitz-Esser et al., 2004]
Outer envelope: cysteine rich OmcA & OmcB present NO outerenvelope OmpA, PorB or pmps Inc proteins present	Outer envelope: cysteine rich OmcA & OmcB also present OmpA, PorB and pmps present and important Inc proteins present
CPAF present but histocompatability antigens absent from amoebal cells	CPAF present and thought to play a role in degrading mammalian histocompatability antigens
Encodes an F pilus conjugative system	No genes for DNA transfer by conjugation

Interestingly UWE25, which already possessed a type III, virulence associated secretion system, has additionally acquired a type IV secretion system by probable recent gene transfer from an unknown donor with a G + C content of >42% [Horn et al., 2004]. The basic Chlamydiaceae architecture of an elementary body whose wall is stabilised by S-S bridged proteins rather than peptidoglycan is also seen in UWE25 and is probably an ancient Chlamydiales feature. However the acquisition of OmpA (the major outer membrane protein) and of the polymorphic membrane proteins by the Chlamydiaceae has likely played a key role in their adaptation as pathogens of avians and mammals.

Genomic islands and conjugation

Greub, Collyn et al., 2004 noted that the genome of Protochlamydia amoebophila UWE25 contained a G+C-rich 19-kb segment forming part of a 100-kb chromosome region, containing 100 highly co-oriented open reading frames, flanked by two 17-bp direct repeats. Two identical gly-tRNA genes in tandem were present at the proximal end of this genetic element while several mobility genes encoding transposases and bacteriophage-related proteins were located within this chromosome region. Thus, this region largely fulfillled the criteria for a genomic island, which they named Pam100G. The G+C content analysis shows that several modules compose this island. Surprisingly, one of them encodes all the genes (traF, traG, traH, traN, traU, traW, and trbC) essential for F-like conjugative DNA transfer and involved in sex pilus retraction and mating pair stabilization. This strongly suggests that, like other F-like operons, the parachlamydial tra unit is devoted to DNA transfer. The close relatedness of this tra unit to F-like tra operons involved in conjugative transfer is confirmed by phylogenetic analyses performed on concatenated genes and gene order conservation. These analyses and that of the gly-tRNA distribution in 140 genomic islands suggest a proteobacterial origin for the parachlamydial tra unit. This is the first hint of a putative conjugative system in chlamydiae and it was postulated that conjugation most probably occurs within free-living amoebae which might contain hundreds of parachlamydiae tightly packed in vacuoles. This conjugative system is not present in published Chlamydiaceae genomes. The authors suggest that it was acquired after the divergence between Parachlamydiaceae and Chlamydiaceae, when the Parachlamydia-related symbiont was an intracellular bacteria. Perhaps this heterologous DNA was acquired from a phylogenetically-distant bacteria sharing an amoebal vacuole. However it seems more likely to this reviewer that Chlamydiaceae lost their conjugative ability through the characteristic genomic degradation consequent upon increasing adaptation to intracellular life (the genome of the chlamydiaceae is much smaller than that of P. amoebophila). It has been suggested that in human parachlamydial infections genomic islands might be involved in pathogenicity and their conjugative systems might be developed as genetic tools for the Chlamydiales.

The conjugation-encoding genes of the tra unit occur together with lgrE, a 5.6-kb gene similar to five others of P. amoebophila: lgrA to lgrD, & lgrF. These are all "Large G+C-Rich" genes encoding so-called LGR proteins. There were no homologs to the whole protein sequence of the LGRs present in other organisms. Phylogenetic analyses suggested that serial duplications producing the six LGRs occurred relatively recently and nucleotide usage analyses showed that lgrB, lgrE and lgrF were relocated on the chromosome. The C-terminal part of LGRs is homologous to Leucine-Rich Repeats domains (LRRs). Defined by a cumulative alignment score, the 5 to 18 concatenated 28-meric LRRs of LGRs have a predicted alpha-helix conformation. Their closest homologs are the 28-residue RI-like LRRs of mammalian NODs and the 24-meres of some Ralstonia and Legionella proteins. Interestingly, lgrE, exhibited Pfam domains related to DNA metabolism. A parsimonious evolutionary scenario of these domains was proposed, driven by adjacent concatenations of LRRs [Eugster et al., 2007].

The genomes of Rickettsia felis and Rickettsia bellii also have a set of putative conjugal DNA transfer genes most similar to homologues found in Protochlamydia amoebophila UWE25. Their genomes exhibits many other genes highly similar to homologues in intracellular bacteria of amoebae. Sex pili-like cell surface appendages were observed on R. bellii, an organism which multiplies efficiently in the nucleus of eukaryotic cells and survives in the phagocytic amoeba, Acanthamoeba polyphaga. These results suggest that amoeba-like ancestral protozoa could have served as a genetic "melting pot" where the ancestors of rickettsiae and other bacteria promiscuously exchanged genes, eventually leading to their adaptation to an intracellular lifestyle within eukaryotic cells [Ogata et al., 2006].

Nucleotide transporters

The genome of P. amoebophila encodes five paralogous carrier proteins belonging to the nucleotide transporter family. Three P. amoebophila nucleotide transporter isoforms, PamNTT2, PamNTT3 and PamNTT5, possess several conserved amino acid residues known to be critical for nucleotide transport. Hafferkamp et al., 2006 showed that these carrier proteins are able to transport nucleotides, although substrate specificities and mode of transport differ were unique among known nucleotide transporters. PamNTT2 was a counter exchange transporter exhibiting submillimolar apparent affinities for all four RNA nucleotides. PamNTT3 catalysed an unidirectional proton-coupled transport confined to UTP, whereas PamNTT5 mediated a proton-energized GTP and ATP import. All nucleotide transporter genes of P. amoebophila were transcribed during intracellular multiplication in acanthamoebae. These protochlamydial nucleotide transporters appeared to be intimately connected with their host cell's metabolism in a surprisingly complex manner. [Hafferkamp et al., 2006].

The ATP/ADP transporter PamNTT1 was reconstituted into artificial lipid vesicles. These revealed high import velocities for ATP and an unexpected and previously unobserved stimulating effect of the luminal ADP on nucleotide import affinities. Preference of the nucleotide hetero-exchange is independent of the membrane potential, and therefore, PamNTT1 not only structurally but also functionally differs from the well-characterized mitochondrial ADP/ATP carriers. Reconstituted PamNTT1 exhibited a bidirectional orientation in lipid vesicles, but interestingly, only carriers inserted with the N-terminus directed to the proteoliposomal interior are functional. Data in the paper indicate the functional basis of how the intracellular P. amoebophila manages to exploit the energy pool of its host cell effectively by using PamNTT1. This membrane protein mediates a preferred import of ATP, which is additionally stimulated by a high internal (bacterial) ADP/ATP ratio, while the orientation-dependent functionality of the transporter ensures that it is not working in a mode that is detrimental to P. amoebophila. Heterologous expression and purification of high amounts of PamNTT1 provides the basis for its crystallization and detailed structure/function analyses. Furthermore, functional reconstitution of this essential chlamydial protein paves the way for high-throughput uptake studies in order to screen for specific inhibitors potentially suitable as anti-chlamydial drugs. [Trentman et al., 2007].

What next?

Given the widespread host spectrum of the Chlamydiales and the probable association of their ancestors and cyanobacteria with protists which evolved into chloroplast bearing cells, this reviewer predicts that Chlamydiales will eventually be found in lower plant cells. Finding them in an unambiguous way will be a considerable challenge to researchers, as any search will inevitably be confounded by the widespread occurrence of environmental chlamydiae in aquatic environments.

There are significant differences among members of the Chlamydiales outside the Chlamydiaceae. In the Parachlamydiaceae, N. hartmanellae is frequently found free in the amoeba cytoplasm [Horn et al., 2000], whereas UWE25 is not [Essig et al., 1997; Greub & Raoult, 2002b]. Rhabdochlamydia have a rod-like form. Both Simkania and Waddlia are pathogens of higher eukaryote cells [Dilbeck et al., 1990; Greub & Raoult 2002a; ibid 2003; Kahane et al., 1998; Lieberman et al., 2002]. Simkania has a distinct prolonged late stage in its growth cycle [Kahane et al., 2002] whereas Waddlia chondrophila has unusually prominent division septae [Henning et al., 2002; Kocan et al., 1990]. The adaptations made by these organisms to pathogenicity may differ in some respects from those made by the Chlamydiaceae justifying the need for genomic sequences of representatives of these families too; chlamydial biology needs to be viewed in its broader Chlamydiales context. In this respect the current taxonomy [Everett et al., 1999], while undoubtedly incomplete [Meijer & Ossewaarde, 2002; Ossewaarde & Meijer, 2001] is helpful in facilitating appreciation of the overall picture. Genomic sequencing the chlamydiae is a relatively easy given the small genome size of these organisms and new genomic sequences are appearing quite frequently; see the links section for updated links to published Chlamydiales genome sequences.

References

Horn, M. (2001). Molecular ecology of free-living amoebae and their bacterial endosymbionts: diversity and interactions. PhD thesis Technical University Munich

Horn, M., Collingro, A., Schmitz-Esser, S., Beier, C. L., Purkhold, U., Fartmann, B., Brandt, P., Nyakatura, G. J., Droege, M., Frishman, D., Rattei, T., Mewes, H. W. & Wagner M (2004). Illuminating the evolutionary history of Chlamydiae. Science 304(5671):728 - 730. Epub 2004 Apr 08.

Meijer, A. & Ossewaarde, J. M. (2002). Description of a wider diversity within the order Chlamydiales than currently classified. pp. 13 - 16, in: Chlamydial Infections – Proceedings of the Tenth International Symposium on Human Chlamydial Infections. Eds. Schachter, J. et al. San Francisco: International Chlamydia Symposium. [See: presentation this site: chlamydiales diversity]

Schmid, E. N., Müller, K. D. & Michel, R. (2001). Evidence for bacteriophages within Neochlamydia hartmannellae, an obligate endoparasitic bacterium of the free-living amoeba Hartmannella vermiformis. Endocytobiosis Cell Research 14, 115 - 119.

Visvesvara, G. S. (1995). Pathogenic and opportunistic free-living amoebae. In: Manual of clinical microbiology, pp. 1383-1390. Edited by P. R. Murray, E. J. Baron, M. A. Pfaller, F. C. Tenover & R. H. Yolken. Washington, D.C., ASM Press.

The Protochlamydia amoebophila UWE25 genome sequence: a milestone in Chlamydiales research

Genomic islands and conjugation

Nucleotide transporters

What next?

References