No abstract

The circadian clock is a timekeeping mechanism that enables anticipation of daily environmental changes. In the plant Arabidopsis thaliana, the circadian system is a multiloop series of interlocked transcription-translation feedbacks. Several genes have been arranged in these oscillation loops, but the position of the core-clock gene ELF4 in this network was previously undetermined. ELF4 lacks sequence similarity to known domains, and functional homologs have not yet been identified. Here we show that ELF4 is functionally conserved within a subclade of related sequences, and forms an alpha-helical homodimer with a likely electrostatic interface that could be structurally modeled. We support this hypothesis by expression analysis of new elf4 hypomorphic alleles. These weak mutants were found to have expression level phenotypes of both morning and evening clock genes, implicating multiple entry points of ELF4 within the multiloop network. This could be mathematically modeled. Furthermore, morning-expression defects were particular to some elf4 alleles, suggesting predominant ELF4 action just preceding dawn. We provide a new hypothesis about ELF4 in the oscillator-it acts as a homodimer to integrate two arms of the circadian clock.

The first 54 promoters in Chlamydia trachomatis L2 were mapped upstream of hypothetical proteins CT652.1 and CT683. Comparative genomics indicated that these 54 promoters and potential upstream activation binding sites are conserved in orthologous C. trachomatis D, C. trachomatis mouse pneumonitis strain, and Chlamydia pneumoniae (CWL029 and AR39) genes.Chlamydia is an organism of major medical and veterinary significance; however, its obligate intracellular existence makes genetic investigations a challenge. The unique developmental cycle of Chlamydia involves the interconversion between the infectious elementary body and the metabolically active reticulate body (23). Although the key morphological stages of chlamydial development are understood (19, 23) and the developmental expression of over 20 genes has been determined (12, 18), the elements which regulate this developmental gene expression are yet to be elucidated. Our recent investigations (18) 28 and 54 . The present study utilized the complete Chlamydia trachomatis genome sequence (27) and a modified fluorescence-based primer extension (PE) assay to identify and map the first 54 promoters for Chlamydia.Transcription initiation from 54 promoters is a multistep process involving the recognition of the promoter by 54 , binding of the core RNA polymerase to the 54 to form a closed complex, and subsequent activation to an open complex following binding by an enhancer binding protein (EBP) (20,21). In most cases the EBP binds an upstream activator sequence (UAS) located within 200 bp of the promoter (15, 21) and is brought into contact with the 54 -RNA polymerase complex by DNA looping, an event mediated by the integration host factor (IHF) or intrinsic DNA bends (9). In addition to the 54 gene (rpoN), recently identified in the C. trachomatis genome, genes for the NtrC family EBP (ntrC) and IHF (ihfA) were also found to be present (27). More detailed analysis of the translated amino acid sequences identified that RpoN ( 54 ) contains a perfect RpoN box (ARRTVAKYR), which is responsible for recognition of the cognate promoter (30), and the chlamydial NtrC homolog has an exact match to the 54 -binding domain, GAFTGA (7). Furthermore, the chlamydial NtrC has six of seven conserved amino acids of the UAS binding domain, GESGCGK (7) (the underlined amino acid is nonconserved). We previously reported the late-stage-specific expression of rpoN (18) and subsequently confirmed that ntrC was transcribed by reverse transcription-PCR analysis (data not shown). These observations led us to hypothesize that some chlamydial genes would be regulated by NtrC-activated 54 -mediated transcription initiation. Of the cognate promoters for all eubacterial sigma factors, 54 promoters are the most highly conserved (2) and hence lend themselves to a computational search of the full chlamydial genome. We therefore used the Findpatterns database searching program of the Australian National Genome Information Service to search the C. trachomatis D genome for sequences corresponding to ...

Zoonotic infections are a growing threat to global health. Chlamydia pneumoniae is a major human pathogen that is widespread in human populations, causing acute respiratory disease, and has been associated with chronic disease. C. pneumoniae was first identified solely in human populations; however, its host range now includes other mammals, marsupials, amphibians, and reptiles. Australian koalas (Phascolarctos cinereus) are widely infected with two species of Chlamydia, C. pecorum and C. pneumoniae. Transmission of C. pneumoniae between animals and humans has not been reported; however, two other chlamydial species, C. psittaci and C. abortus, are known zoonotic pathogens. We have sequenced the 1,241,024-bp chromosome and a 7.5-kb cryptic chlamydial plasmid of the koala strain of C. pneumoniae (LPCoLN) using the whole-genome shotgun method. Comparative genomic analysis, including pseudogene and single-nucleotide polymorphism (SNP) distribution, and phylogenetic analysis of conserved genes and SNPs against the human isolates of C. pneumoniae show that the LPCoLN isolate is basal to human isolates. Thus, we propose based on compelling genomic and phylogenetic evidence that humans were originally infected zoonotically by an animal isolate(s) of C. pneumoniae which adapted to humans primarily through the processes of gene decay and plasmid loss, to the point where the animal reservoir is no longer required for transmission.

Chlamydia trachomatis is an important human pathogen which possesses a unique bi-phasic developmental cycle. We used lightcycler methodology to quantitatively measure gene transcript levels in C. trachomatis strain L2. By measuring 16S rRNA transcript levels, we determined C. trachomatis L2 to have a generation time of approximately 3 h and an inclusion burst size of 200^300 particles. The three chlamydial c c factor genes rpoD (c c 66 ), rpsD (c c 28 ) and rpoN (c c 54 ) exhibited different patterns of temporal expression. rpoD was central to early chlamydial development, whereas rpsD and rpoN were temporally expressed, coinciding with elementary body (EB) to reticulate body (RB) conversion and RB to EB conversion, respectively.z 1999 Federation of European Biochemical Societies.

Background: Chlamydia trachomatis, an obligate intracellular human pathogen, is the most prevalent bacterial sexually transmitted infection worldwide and a leading cause of preventable blindness. HtrA is a virulence and stress response periplasmic serine protease and molecular chaperone found in many bacteria. Recombinant purified C. trachomatis HtrA has been previously shown to have both activities. This investigation examined the physiological role of Chlamydia trachomatis HtrA.

No abstract

Characterization of the protease, HtrA, from pathogen Chlamydia trachomatis is presented. The purified recombinant protein was a serine endoprotease, specific for unfolded proteins, and temperature activated above 34°C. Chaperone activity was observed, although this appeared target-dependent. Inactive protease (S247A) was able to chaperone insulin B-chain, irrespective of temperature, but at 30°C only HtrA and not S247A displayed significant chaperone activity for a-lactalbumin. These data demonstrate that chaperone activity may involve functional protease domain and that C. trachomatis HtrA functions as both a chaperone and protease at 37°C. These properties are consistent with the developmental cycle of this obligate intracellular bacterium.