Dataset Information

The atypical OmpR/PhoB response regulator ChxR from Chlamydia trachomatis forms homodimers in vivo and binds a direct repeat of nucleotide sequences.

ABSTRACT: Two-component signal transduction systems are widespread in bacteria and are essential regulatory mechanisms for many biological processes. These systems predominantly rely on a sensor kinase to phosphorylate a response regulator for controlling activity, which is frequently transcriptional regulation. In recent years, an increasing number of atypical response regulators have been discovered in phylogenetically diverse bacteria. These atypical response regulators are not controlled by phosphorylation and exhibit transcriptional activity in their wild-type form. Relatively little is known regarding the mechanisms utilized by these atypical response regulators and the conserved characteristics of these atypical response regulators. Chlamydia spp. are medically important bacteria and encode an atypical OmpR/PhoB subfamily response regulator termed ChxR. In this study, protein expression analysis supports that ChxR is likely exerting its effect during the middle and late stages of the chlamydial developmental cycle, stages that include the formation of infectious elementary bodies. In the absence of detectable phosphorylation, ChxR formed homodimers in vitro and in vivo, similar to a phosphorylated OmpR/PhoB subfamily response regulator. ChxR was demonstrated to bind to its own promoter in vivo, supporting the role of ChxR as an autoactivator. Detailed analysis of the ChxR binding sites within its own promoter revealed a conserved cis-acting motif that includes a tandem repeat sequence. ChxR binds specifically to each of the individual sites and exhibits a relatively large spectrum of differential affinity. Taken together, these observations support the conclusion that ChxR, in the absence of phosphorylation, exhibits many of the characteristics of a phosphorylated (active) OmpR/PhoB subfamily response regulator.

SUBMITTER: Hickey JM

PROVIDER: S-EPMC3019824 | biostudies-other | 2011 Jan

REPOSITORIES: biostudies-other

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The atypical OmpR/PhoB response regulator ChxR from Chlamydia trachomatis forms homodimers in vivo and binds a direct repeat of nucleotide sequences.

Hickey John M JM Weldon Lindsey L Hefty P Scott PS

Journal of bacteriology 20101105 2

Two-component signal transduction systems are widespread in bacteria and are essential regulatory mechanisms for many biological processes. These systems predominantly rely on a sensor kinase to phosphorylate a response regulator for controlling activity, which is frequently transcriptional regulation. In recent years, an increasing number of atypical response regulators have been discovered in phylogenetically diverse bacteria. These atypical response regulators are not controlled by phosphoryl ...[more]

PMID: 21057008

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Project description:The genome sequences of Chlamydia trachomatis mouse pneumonitis (MoPn) strain Nigg (1 069 412 nt) and Chlamydia pneumoniae strain AR39 (1 229 853 nt) were determined using a random shotgun strategy. The MoPn genome exhibited a general conservation of gene order and content with the previously sequenced C.trachomatis serovar D. Differences between C.trachomatis strains were focused on an approximately 50 kb 'plasticity zone' near the termination origins. In this region MoPn contained three copies of a novel gene encoding a >3000 amino acid toxin homologous to a predicted toxin from Escherichia coli O157:H7 but had apparently lost the tryptophan biosyntheis genes found in serovar D in this region. The C. pneumoniae AR39 chromosome was >99.9% identical to the previously sequenced C.pneumoniae CWL029 genome, however, comparative analysis identified an invertible DNA segment upstream of the uridine kinase gene which was in different orientations in the two genomes. AR39 also contained a novel 4524 nt circular single-stranded (ss)DNA bacteriophage, the first time a virus has been reported infecting C. pneumoniae. Although the chlamydial genomes were highly conserved, there were intriguing differences in key nucleotide salvage pathways: C.pneumoniae has a uridine kinase gene for dUTP production, MoPn has a uracil phosphororibosyl transferase, while C.trachomatis serovar D contains neither gene. Chromosomal comparison revealed that there had been multiple large inversion events since the species divergence of C.trachomatis and C.pneumoniae, apparently oriented around the axis of the origin of replication and the termination region. The striking synteny of the Chlamydia genomes and prevalence of tandemly duplicated genes are evidence of minimal chromosome rearrangement and foreign gene uptake, presumably owing to the ecological isolation of the obligate intracellular parasites. In the absence of genetic analysis, comparative genomics will continue to provide insight into the virulence mechanisms of these important human pathogens.

Project description:The obligate intracellular developmental cycle of Chlamydia trachomatis presents significant challenges in defining its proteome. In this study we have applied quantitative proteomics to both the intracellular reticulate body (RB) and the extracellular elementary body (EB) from C. trachomatis. We used C. trachomatis L2 as a model chlamydial isolate for our study since it has a high infectivity:particle ratio and there is an excellent quality genome sequence. EBs and RBs (>99% pure) were quantified by chromosomal and plasmid copy number using PCR, from which the concentrations of chlamydial proteins per bacterial cell/genome were determined. RBs harvested at 15h post infection (PI) were purified by three successive rounds of gradient centrifugation. This is the earliest possible time to obtain purified RBs, free from host cell components in quantity, within the constraints of the technology. EBs were purified at 48h PI. We then used two-dimensional reverse phase UPLC to fractionate RB or EB peptides before mass spectroscopic analysis, providing absolute amount estimates of chlamydial proteins. The ability to express the data as molecules per cell gave ranking in both abundance and energy requirements for synthesis, allowing meaningful identification of rate-limiting components. The study assigned 562 proteins with high confidence and provided absolute estimates of protein concentration for 489 proteins. Interestingly, the data showed an increase in TTS capacity at 15h PI. Most of the enzymes involved in peptidoglycan biosynthesis were detected along with high levels of muramidase (in EBs) suggesting breakdown of peptidoglycan occurs in the non-dividing form of the microorganism. All the genome-encoded enzymes for glycolysis, pentose phosphate pathway and tricarboxylic acid cycle were identified and quantified; these data supported the observation that the EB is metabolically active. The availability of detailed, accurate quantitative proteomic data will be invaluable for investigations into gene regulation and function.

Project description:Chlamydia trachomatis is an obligate intracellular pathogen responsible for ocular and genital infections of significant public health importance. C. trachomatis undergoes a biphasic developmental cycle alternating between two distinct forms: the infectious elementary body (EB), and the replicative but non-infectious reticulate body (RB). The molecular basis for these developmental transitions and the metabolic properties of the EB and RB forms are poorly understood as these bacteria have traditionally been difficult to manipulate through classical genetic approaches. Using two-dimensional liquid chromatography - tandem mass spectrometry (LC/LC-MS/MS) we performed a large-scale, label-free quantitative proteomic analysis of C. trachomatis LGV-L2 EB and RB forms. Additionally, we carried out LC-MS/MS to analyse the membranes of the pathogen-containing vacuole ('inclusion'). We developed a label-free quantification approaches to measure protein abundance in a mixed-proteome background which we applied for EB and RB quantitative analysis. In this manner, we catalogued the relative distribution of > 54% of the predicted proteins in the C. trachomatis LGV-L2 proteome. Proteins required for central metabolism and glucose catabolism were predominant in the EB, whereas proteins associated with protein synthesis, ATP generation and nutrient transport were more abundant in the RB. These findings suggest that the EB is primed for a burst in metabolic activity upon entry, whereas the RB form is geared towards nutrient utilization, a rapid increase in cellular mass, and securing the resources for an impending transition back to the EB form. The most revealing difference between the two forms was the relative deficiency of cytoplasmic factors required for efficient type III secretion (T3S) in the RB stage at 18 h post infection, suggesting a reduced T3S capacity or a low frequency of active T3S apparatus assembled on a 'per organism' basis. Our results show that EB and RB proteomes are streamlined to fulfil their predicted biological functions: maximum infectivity for EBs and replicative capacity for RBs.

Dataset Information

The atypical OmpR/PhoB response regulator ChxR from Chlamydia trachomatis forms homodimers in vivo and binds a direct repeat of nucleotide sequences.

Publications

The atypical OmpR/PhoB response regulator ChxR from Chlamydia trachomatis forms homodimers in vivo and binds a direct repeat of nucleotide sequences.

Similar Datasets

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