Although knowledge of other families is sketchy, enough information has already accumulated to show that some characters considered hallmarks of the intracellular way of life in Chlamydiaceae are not found throughout the order.

Penicillin inhibits multiplication of Chlamydiaceae by preventing the division of RBs and their transformation into EBs. Although Chlamydiaceae do not contain structural peptidoglycan (PG), their sequenced genomes do have genes for complete or nearly complete pathways leading to its synthesis. Penicillin almost certainly acts on Chlamydiaceae by disrupting operation of the PG pathway in ways that are not yet entirely clear (Chopra et al., 1998; Ghuysen and Goffin, 1999; Bavoil et al., 2000) [PG, penicillin, and Chlamydiaceae will be discussed in more detail in Part II]. In none of the other families does penicillin inhibit multiplication (Kahane, 1993, 1999; Dilbeck et al., 1990; Horn et al., 2000; Michel et al., 1994). In fact, isolation of representatives of the other families has often been carried out in the presence of the antibiotic. Penicillin may not inhibit multiplication of Parachlamydiaceae because it does not get inside their amoebal hosts, but it nevertheless fails to stop the growth of Simkaniaceae and Waddliaceae in mammalian cell lines that penicillin is known to penetrate. Penicillin-sensitive and penicillin-insensitive Chlamydiales probably differ in important aspects of envelope structure and cell division mechanisms.

The Chlamydiaceae contains as a part of their envelopes a family-specific lipopolysaccharide with a trisaccharide of 3-deoxy-alpha-D-manno-octulosonic acid as its unique epitope (Brade et al., 1997). In Chlamydiaceae, Omp1 is the major outer membrane protein suspected of involvement in attachment and entry into host cells, and Omp2 is a cysteine-rich protein thought to be pivotal in the RB to EB conversion (Hatch, 1999). Simkania negevensis (Simkaniaceae) did not react to monoclonal antibodies to any of these 3 antigens (Kahane et al., 1993, 1999), and Waddlia chondrophila (Waddliaceae) did not react with antisera (specificity not stated) to either Chlamydia or Chlamydophila (Dilbeck et al., 1990; Henning et al., 2002). An unclassified Parachlamydia did not react with antibody to Chlamydiaceae LPS (Birtles et al., 1997). All these observations suggest that the cell envelopes of the other families are unlikely to have the same architecture as the Chlamydiaceae envelope.

There are striking similarities and differences among the developmental cycles of the 4 families. The salient features of Chlamydiaceae reproduction are that differentiation and multiplication of chlamydial bodies takes place only in membrane-bound cytoplasmic inclusions that become crowded with EBs and RBs (Hatch, 1999; Hackstadt, 1999). Simkania negevensis, the only species in Simkaniaceae, was isolated from human and simian cell lines and has been serologically implicated as a widespread human pathogen ( Kahane et al.,1993; 1995; 1999). Its natural host is unknown. By growth curve and light microscopy, the major departure of S. negevensis from the Chlamydiaceae pattern appears to be a distinct prolongation of the later stages of the developmental cycle.

Waddlia chondrophila, the sole representative of the family Waddliaceae, was isolated from an aborted calf fetus (Dilbeck et al., 1990; Henning et al., 2002). It is even more like Chlamydiaceae in the appearance of whole inclusions and of individual EBs and RBs in thin-section electron micrographs of infected mammalian cells (Kocan et al., 1990). It also progresses through the developmental cycle at the same rate. Division septae, rarely seen in Chlamydiaceae, are prominent in many dividing RBs. It would take a hard look to decide that the electron micrographs were not of C. psittaci. All this in an organism with only 85% 16S rRNA homology to Chlamydiaceae and without penicillin sensitivity (Rurangirwa et al., 1999)! This remarkable similarity between developmental cycles is strong evidence that the basic features of the developmental cycle were fixed before divergence of the families. I wonder what the W. chromophila genome looks like. If penicillin inhibition of reproduction is to be taken as evidence of participation of peptidoglycan (Chlamydiaceae), then does lack of such inhibition mean that peptidoglycan is not involved (Waddliaceae)? Thus arises the possibility of W. chondrophila multiplying in a most Chlamydiaceae-like way without synthesizing peptidoglycan. I will return to this possibility later [see: Part II. Cell division, FtsZ, and peptidoglycan]. The chances that convergent evolution in the intracellular habitat has been at least partially responsible for familial resemblances in developmental cycles cannot be totally disregarded. After all, at least 2 other obligate intracellular bacteria, Coxiella burnetii (Heinzen et al., 1999) and Ehrlichia (Cowdria) ruminantium (Jongejan et al., 1991) also grow in membrane-bound cytoplasmic vacuoles and show distinct pleomorphism.

Of the four families, developmental cycles in Parachlamydiaceae have diverged the most from the others. Some members of this family multiply outside of the context of the familiar communal inclusion of Chlamydiaceae. Parachlamydiaceae are endobionts of free-living amoebae. There are 2 monospecific genera. Parachlamydia acanthamoebae is an intracellular parasite of amoeba of the genus Acanthamoeba (Michel et al., 1994; Amman et al., 1997; Fritsche et al., 2000). The host for Neochlamydia hartmanellae is Hartmanella vermiformis (Horn et al., 2000). Numerous other amoeba-dwelling Parachlamydiaceae have been described but not assigned to definite taxons (Fritsche et al., 2000). In P. acanthamoebae, membrane-bound inclusions with many EBs and RBs occur throughout the host cytoplasm (Amman et al., 1997). However, in N. hartmanellae, electron micrographs show EBs and RBs in the cytoplasm of H. vermiformis that are not enclosed in vacuoles and are without inclusion membranes (Horn et al., 2000). Did these neochlamydiae multiply in inclusions and then escape into the cytoplasm or did they actually multiply there? An as yet unclassified Parachlamydiaceae exhibits still another variation. There are vacuoles, but not vacuoles with many chlamydial bodies. Instead, individual EBs and RBs, each within its own membrane-bound vacuole, are seen in the amoebal cytoplasm (Fritsche et al., 2000). What happens when one of these isolated RBs divides? Does its vacuole divide in synchrony? It is surprizing to find so much variation in a single Chlamydiales family. I have already suggested that Parachlamydiaceae are descended from a familial LCA that multiplied in communal inclusions like the other Chlamydiales. Thus, P. acanthamoebae would resemble the LCA, whereas N. hartmanellae and the unclassified isolate would represent major divergences. In any event, they provide variations on the inclusion membrane-developmental cycle relationship that may provide insights on the evolutionary origins of the complex interplay between inclusion membrane and chlamydial cells in Chlamydiaceae [See: Part II. The pathway from the first intracellular ancestor to modern chlamydiae].

Developmental cycles in Parachlamydiaceae have been studied in free-living amoebae while cycles in other chlamydiae have been studied in mammalian cell lines. Do the differences reflect evolutionary adaptation to very different eukaryotic hosts? It would be interesting to examine vacuole-Parachlamydiaceae relations in a mammalian cell; and what of the reverse situation? Limited investigations suggest that passage from protist to mammalian cell and back again does indeed happen. 16S rRNA sequences resembling those of Parachlamydia have been isolated from respiratory tracts, monocytes, and blood vessels of humans (Ossewaarde and Meijer, 1999; Corsaro et al., 2001). Hall's coccus, which appears to be a Parachlamydia, has been found in humans (Birtles et al., 1997). P. acanthamoebae reproduces in Vero cells (Michel et al., 1994), C. pneumoniae grows in A. castellani (Essig et al., 1997) and S. negevensis multiplies and survives in Acanthamoeba polyphaga (Kahane et al., 2001). It may be that free-living amoebae not only serve as reservoirs for vertebrate chlamydiae but also that chlamydiae whose natural hosts are amoebae also cause disease in people. Whether amoebae will prove as important in the natural history of human chlamydial infections as they are to those caused by legionellae (Harb et al., 2000) remains to be seen.
[JWM]