Purification of recombinant <i>Chlamydia trachomatis</i> histone H1‐like protein Hc2, and comparative functional analysis of Hc2 and Hc1

Pedersen, Lotte B.; Birkelund, Svend; Christiansen, Gunna

doi:10.1111/j.1365-2958.1996.tb02618.x

Cited by 24 publications

(24 citation statements)

References 32 publications

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“…hctB encodes Hc2, a histone-like protein that causes DNA condensation (1,2,4,14,15). Tsp is a predicted protease with similarity to CPAF, a secreted chlamydial protease that cleaves host transcription factors involved in major histocompatibility complex class I and class II antigen expression (18,26).…”

Section: Discussionmentioning

confidence: 99%

In Silico Prediction and Functional Validation of σ ²⁸ -Regulated Genes in Chlamydia and Escherichia coli

Kibler

Tan

2006

J Bacteriol

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28 RNA polymerase is an alternative RNA polymerase that has been proposed to have a role in late developmental gene regulation in Chlamydia, but only a single target gene has been identified. To discover additional 28 -dependent genes in the Chlamydia trachomatis genome, we applied bioinformatic methods using a probability weight matrix based on known 28 Genome sequencing has indicated that all Chlamydia species encode two alternative sigma factors, suggesting a role for alternative forms of RNA polymerase in chlamydial gene regulation. We have demonstrated that one of these alternative RNA polymerases, 28 RNA polymerase, transcribes hctB (24), a gene whose transcript is detectable only at late time points in the chlamydial developmental cycle (6, 16). hctB encodes Hc2, one of two histone-like proteins in Chlamydia that have been shown to be responsible for the condensation of DNA during conversion of the metabolically active form of chlamydiae, known as a reticulate body, to the infectious extracellular form, the elementary body (8). To date, hctB is the only 28 -regulated gene that has been identified in Chlamydia, and it is not known whether the role of 28 RNA polymerase is confined to the regulation of late gene expression in the developmental cycle.To identify additional 28 -regulated genes in Chlamydia, we have combined the use of bioinformatics, to predict 28 -regulated promoters in the chlamydial genome, with testing of promoter activity in a chlamydial 28 in vitro transcription assay. We used two in silico approaches, identifying candidate promoters on the basis of sequences that either resemble the consensus bacterial 28 promoter (9, 10) or are predicted to be highly transcribed by 28 RNA polymerase based on functional studies (25). Using information from both approaches, we have a developed a computer algorithm to identify candidate 28 promoters in the chlamydial genome and have shown that five promoters are transcribed by chlamydial 28 RNA polymerase. This method can be applied to other bacterial genomes, and we have also identified five new 28 -regulated genes in Escherichia coli. MATERIALS AND METHODS Development of a program for extracting sequences.We developed a program called SequenceExtractor to extract user-defined DNA sequences from a genome. The program requires two input files, consisting of a genome sequence file and a file containing the start and stop coordinates for each gene within the DNA sequence being examined. We applied this program to extract two files in fasta format from each of the genomes of C. trachomatis serovar D, E. coli K-12, and Salmonella enterica serovar Typhimurium using sequences obtained from TIGR (http://www.tigr.org). For each organism, the first output file contained 200 bp of sequence upstream for each gene ("200 bp upstream"). The second output file was more restrictive and contained up to 200 bp of upstream sequence for each gene, provided that these sequences were in the intergenic region and not within the coding region of the nearest upstream gene ("200...

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Section: Discussionmentioning

confidence: 99%

In Silico Prediction and Functional Validation of σ ²⁸ -Regulated Genes in Chlamydia and Escherichia coli

Kibler

Tan

2006

J Bacteriol

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“…Chlamydia species alternate between two morphologically distinct forms: a spore-like form called the elementary body (EB) and a vegetative form termed the reticulate body (RB). EBs are small (ϳ0.2 m) and are characterized by the presence of a dense nucleoid structure that consists of DNA tightly packed by bacterial histone-like proteins (34)(35)(36)(37)(38)(39)(40)(41). EBs are infectious, and their rigid cell walls aid in their dissemination throughout the extracellular environment.…”

Section: The Chlamydia Life Cyclementioning

confidence: 99%

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

Bastidas

Valdivia

2016

Microbiol Mol Biol Rev

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SUMMARYChlamydiaspecies infect millions of individuals worldwide and are important etiological agents of sexually transmitted disease, infertility, and blinding trachoma. Historically, the genetic intractability of this intracellular pathogen has hindered the molecular dissection of virulence factors contributing to its pathogenesis. The obligate intracellular life cycle ofChlamydiaand restrictions on the use of antibiotics as selectable markers have impeded the development of molecular tools to genetically manipulate these pathogens. However, recent developments in the field have resulted in significant gains in our ability to alter the genome ofChlamydia, which will expedite the elucidation of virulence mechanisms. In this review, we discuss the challenges affecting the development of molecular genetic tools forChlamydiaand the work that laid the foundation for recent advancements in the genetic analysis of this recalcitrant pathogen.

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“…Expression of Hc1 in E. coli leads to a down regulation of transcription, translation, and bacterial replication in conjunction with condensation of the chromatin (1). Hc1 and Hc2 have both been found to strongly repress transcription and translation in vitro by modulating DNA topology and/or binding to DNA and RNA (1,10,11).…”

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“…Hc2 binds to and condenses DNA in vitro and condenses the chromatin upon expression in E. coli (4,11). hctB, the gene encoding Hc2, was cloned into the pTet vector described previously (5) and expressed in E. coli DH5␣PRO to verify condensation of the E. coli chromatin and determine the effects on viability.…”

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confidence: 99%

Regulation of the Chlamydia trachomatis Histone H1-Like Protein Hc2 Is IspE Dependent and IhtA Independent

Grieshaber¹,

Sager²,

Dooley³

et al. 2006

J Bacteriol

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The chlamydial histone-like proteins, Hc1 and Hc2, function as global regulators of chromatin structure and gene expression. Hc1 and Hc2 expression and activity are developmentally regulated. A small metabolite that disrupts Hc1 interaction with DNA also disrupts Hc2 interactions; however, the small regulatory RNA that inhibits Hc1 translation is specific to Hc1.The obligate intracellular bacterium Chlamydia trachomatis is the leading cause of infectious blindness worldwide (19) and the most prevalent bacterial agent of sexually transmitted disease in industrialized and developing countries (20). Chlamydiae undergo a unique biphasic developmental cycle, alternating between two developmental forms. Infection of the host cell is initiated by the metabolically inert elementary body (EB), which possesses a characteristic core of condensed chromatin. Following internalization, the chromatin becomes dispersed, transcription and translation are initiated, and EBs differentiate to the metabolically active reticulate bodies (RBs). RBs replicate for 18 to 48 h within a membrane-bound vacuole called an inclusion until they asynchronously differentiate to EBs, a process characterized by compaction of the chromatin. The EBs are released from the host cell, and a new round of infection begins (9, 15).Two DNA binding proteins, Hc1 and Hc2, are believed to be responsible for the compaction of the chlamydial genome (4,8,12,17). Hc1 and Hc2 (encoded by hctA and hctB, respectively) bear primary amino acid similarity to eukaryotic histone H1 and are expressed late in the developmental cycle concomitantly with RB-to-EB conversion and chromatin condensation (3, 15). Hc1 is conserved among the chlamydiae, while Hc2 displays variable molecular weight depending on the species (7,13,14,16,18). Overexpression of either of these two proteins in Escherichia coli results in compaction of the bacterial chromatin, although the ultrastructure of the DNA-protein complex is distinct (2, 4). In particular, overexpression of Hc1 in E. coli causes chromatin condensation ultrastructurally similar in appearance to the spherical condensed nucleoids of EBs (2), while overexpression of Hc2 induces a condensed nucleoid with a more toroidal appearance. Expression of Hc1 in E. coli leads to a down regulation of transcription, translation, and bacterial replication in conjunction with condensation of the chromatin (1). Hc1 and Hc2 have both been found to strongly repress transcription and translation in vitro by modulating DNA topology and/or binding to DNA and RNA (1, 10, 11).The observed effects on chromatin structure and activity in E. coli are believed to reflect the native function of the histones in chlamydiae as they are considered to act as global regulators of chlamydial gene expression during the terminal differentiation of RBs to EBs at the end of the developmental cycle. Expression of the chlamydial histones in E. coli is effectively lethal, as E. coli apparently lacks the ability to release its chromatin from the constraints imposed by Hc1 or Hc...

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Purification of recombinant Chlamydia trachomatis histone H1‐like protein Hc2, and comparative functional analysis of Hc2 and Hc1

Cited by 24 publications

References 32 publications

In Silico Prediction and Functional Validation of σ ²⁸ -Regulated Genes in Chlamydia and Escherichia coli

In Silico Prediction and Functional Validation of σ ²⁸ -Regulated Genes in Chlamydia and Escherichia coli

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

Regulation of the Chlamydia trachomatis Histone H1-Like Protein Hc2 Is IspE Dependent and IhtA Independent

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Purification of recombinant Chlamydia trachomatis histone H1‐like protein Hc2, and comparative functional analysis of Hc2 and Hc1

Cited by 24 publications

References 32 publications

In Silico Prediction and Functional Validation of σ 28 -Regulated Genes in Chlamydia and Escherichia coli

In Silico Prediction and Functional Validation of σ 28 -Regulated Genes in Chlamydia and Escherichia coli

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

Regulation of the Chlamydia trachomatis Histone H1-Like Protein Hc2 Is IspE Dependent and IhtA Independent

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In Silico Prediction and Functional Validation of σ ²⁸ -Regulated Genes in Chlamydia and Escherichia coli

In Silico Prediction and Functional Validation of σ ²⁸ -Regulated Genes in Chlamydia and Escherichia coli