Chlamydiologists are by now accustomed to the high quality papers on chlamydial cell biology which emanate from Ted Hackstadt's NIH laboratory in the outback of Hamilton, Montana (where the skies are wide, men are men, the knapweed roams wild and the beer comes in satisfyingly large pitchers). Afficionados of this site will know that, in my review of the papers presented at the Memphis meeting of the chlamydial basic research society, I was particularly impressed by a paper presented by Nicole Grieshaber from Ted's laboratory, which was selected for the chlamydiae.com gold star! I have been looking ever since for this paper to appear in print; finally it has appeared [Grieshaber et al., 2004]. It was well worth the wait.

It has long been known that chlamydial elementary bodies are characterised by a condensed chromatin, with the chlamydial DNA bound to two homologues of eukaryotic histone H1, Hc1 and Hc2 which are encoded by the genes hctA and hctB respectively. Hc1 is conserved among all chlamydiae, whereas Hc2 is a more heterogenous protein which is absent from some chlamydiae. HctA and hctB are transcribed late in the chlamydial developmental cycle concomitant with nucleoid condensation as reticulate bodies are transformed into the precursors of elementary bodies. Hc1 expression in E. coli results in condensation of E. coli DNA into a nucleoid and the shut down of transcriptional and translational activity. Thus expression of Hc1 in E. coli is effectively lethal, as E. coli lacks what chlamydiae have, the means to release the DNA histone complex. Nicole and colleagues therefore devised a clever heterologous genetic screen to identify any members of a C. trachomatis serovar L2 genebank that were capable of rescuing E. coli from HCt1-induced lethal DNA condensation. Essentially, their screen was a two plasmid system to coexpress in E. coli both Hc1 (under a tet promoter) and the chlamydial gene library (under a lac promoter). A predicted kinase encoded by CT 804, a gene homologous to ispE (ychB) in E. coli, and involved in the non-mevalonate methyl-erythritol-4-phosphate (MEP) pathway of isoprenoid biosynthesis was protective, despite continued synthesis of Hc1. IspE catalyses the ATP-dependent phosphorylation of 4-diphosphocytidyl-2-C-methyl-D-erythritol to the corresponding 2-phosphate. There were two possibilities; either the effect might be mediated by a small metabolite produced from the activity of CT804, or it might result from direct action of the enzyme on the histone.

To explore these options, E. coli expressing Hc1 alone, or with CT804, were grown in the presence of fosfidomycin, a novel herbicide and anti-malaria drug that inhibits IspC, an enzyme involved in an early step of the MEP pathway. It was reasoned that if a small molecule produced downstream of IspC was involved, rescue by CT804 over-expression would be abolished by fosfidomycin, whereas a direct protein - protein interaction would not be affected. Treatment with fosfidomycin did block the protective effect of CT 804, indicating involvement of a MEP pathway metabolite.

To verify this, deproteinated detergent-extracts of E. coli expressing CT804 in the presence or absence of fosfidomycin were applied to authentic chlamydial nucleoid ghosts prepared by zwittergent extraction. The ability of the extracts to protect the chromatin from DNAse1 digestion was then assessed. Extracts of E. coli expressing CT804 in the absence of fosfidomycin released chlamydial DNA from the nucleoid, rendering it sensitive to DNAse1 digestion. This did not occur of the E. coli expressed CT804 in the presence of fosfidomycin, elegant proof that chlamydial nucleoid decondensation is mediated by a small metabolite in the MEP pathway.

Orthologs of the entire MEP pathway are present in the chlamydial and E. coli genomes. In chlamydiae it was shown that each of these genes is within 2 hours of infection. To identify the MEP metabolite involved in decondensation, the MEP pathway was partially reconstructed by sequentially adding the expressed and partially purified orthologous chlamydial enzyme proteins, IspD (CT462), IspE (CT804) and IspF (CT434) to commercially available MEP. Only when all three enzymes were present together with MEP was the chlamydial chromatin dissociated from Hc1. Thus the actual molecule dissociating the DNA-Hc1 interaction appears to be 2-C-methyl-D-erythritol 2,4 cyclodiphosphate (MEC) [Grieshaber et al., 2004] although MEC has not yet been directly identified in germinating elementary bodies. MEC might (i) be directly competitive with DNA binding; (ii) covalently modify the histone (there was no evidence for this from MALDI-TOF mass spec studies); or (iii) cause the condensed chromosome to unravel. It was considered most likely that the small molecule acts as a competitive inhibitor or as an allosteric effector on Hc1.

It is a paradox that decondensation of the chlamydial nucleoid, a critical step in chlamydial development, is itself dependent on de novo transcription and translation of CT804. The question therefore is how CT804 is available for transcription given the condensed state of the chlamydial DNA in the nucleoid? Furthermore what happens to the potentially inhibitory MEC during nucleoid formation as reticulate bodies begin to form elementary bodies? [Grieshaber et al., 2004].

[Comment: This paper describes a stunning piece of detective work among rare molecules, embodying both technical and intellectual virtuosity. It tantalises with new questions while providing fresh insight. The interaction of DNA binding proteins with DNA usually involves phosphorylation, methylation, acetylation, proteolysis or changes in DNA topology. Disruption of chlamydial Hc1-DNA interaction by a small molecule is an unusual mechanism for releasing histone from chromatin. The authors point out that the MEP pathway may be a new target for antichlamydial chemotherapy, as this pathway is critical for chlamydial differentiation but it is not present in mammalian cells. In its published form this paper is a tour de force and richly deserves the sparingly awarded three chlamydiae.com gold stars! I look forward to an update of this story at the Budapest meeting]

[MEW] June 2004

SEE ALSO: Chlamydial development in pictures ; Developmental regulation.

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