Mutational Analysis of the Promoter Recognized by
 Chlamydia
 and
 Escherichia coli
 σ
 28
 RNA Polymerase

Yu, Hilda Hiu Yin; Russo, Elizabeth G. Di; Rounds, Megan A.; Tan, Ming

doi:10.1128/jb.00480-06

Cited by 29 publications

(48 citation statements)

References 22 publications

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“…The frequency matrix was based on a set of 21 known bacterial 28 promoters and takes into account the frequency of occurrence of each nucleotide at each position within this promoter set. The activity matrix used functional data in the form of the relative promoter strength attributable to each nucleotide at each promoter position and was derived from a comprehensive mutational analysis of a 28 promoter (25). For each promoter position, the algorithm assigned a probability value to the four possible nucleotides, with a total probability of 1.…”

Section: Development Of a Program For Extracting Sequencesmentioning

confidence: 99%

“…C. trachomatis serovar L2 His 6 -28 and E. coli His 6 -28 were individually overexpressed in E. coli BL21(DE3) and purified, as previously described (24,25), to a concentration of 35.7 g/ml and 115.8 g/ml, respectively.…”

Section: Development Of a Program For Extracting Sequencesmentioning

confidence: 99%

“…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…”

mentioning

confidence: 99%

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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: Development Of a Program For Extracting Sequencesmentioning

confidence: 99%

Section: Development Of a Program For Extracting Sequencesmentioning

confidence: 99%

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confidence: 99%

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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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“…CT441 is predicted to be a 28 -regulated gene (26), suggesting that its expression is developmental cycle dependent. To this end, HeLa 229 cells were infected with Chlamydia LGV2 at a multiplicity of infection of 1 IFU/cell for various times, and CT441 (Fig.…”

Section: Resultsmentioning

confidence: 99%

Chlamydial CT441 Is a PDZ Domain-Containing Tail-Specific Protease That Interferes with the NF-κB Pathway of Immune Response

Lad¹,

Yang

Scott³

et al. 2007

J Bacteriol

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Chlamydia species are bacterial pathogens that affect over 140 million individuals worldwide. Ocular infection by Chlamydia trachomatis is the leading cause of preventable blindness, and urogenital tract infection by Chlamydia causes sexually transmitted disease. As obligate intracellular organisms, Chlamydia species have evolved mechanisms to evade the host immune system, including the degradation of the transcription factors regulatory factor X5 and upstream stimulation factor 1, which are required for the expression of major histocompatibility complex molecules I and II by CPAF and cleavage of p65 of the NF-B pathway by the encoded CT441 protein. Here, we report the characterization of CT441 as a tail-specific protease. CT441 contains a PDZ domain of protein-protein interactions and a Ser/Lys dyad catalytic unit. Mutation at either Ser455 or Lys481 in the active site ablated CT441 activity of p65 cleavage. In addition, we found that the production of CT441 Tsp, which was detected at the middle and late stages of an infection, correlated with p65 cleavage activity. In addition to high homology, human and mouse p65 proteins also contain an identical C-terminal tail of 22 amino acid (aa) residues. However, only human p65 was susceptible to cleavage. Using molecular biology approaches, we mapped the p65 cleavage site(s) to a region that differs from that of mouse p65 by 6 aa residues. Additionally, the substitution of T352 with a proline inhibited p65 cleavage. Together, the study demonstrates that CT441 is a tail-specific protease that is capable of interfering with the NF-B pathway of host antimicrobial and inflammatory responses.The carboxyl-terminal processing proteases (Ctp), including the bacterial tail-specific protease (Tsp), are a group of endoproteases of posttranslational protein modification, maturation, and disassembly or degradation. The Ctp proteases have been found in Archaea, plant chloroplasts, bacteria (reviewed in reference 20), and viruses (5). A well-characterized Ctp is the P1D protease that contains a PDZ domain of proteinprotein interaction and a domain of the S41B family peptidase (15,20). P1D catalyzes the C-terminal processing of the D1 protein of photosystem II, an essential event for the consequent water oxidation and the generation of oxygen molecules in oxygenic photosynthetic organisms. Tail-specific proteases have been identified from bacterial pathogens of medical importance, including Borrelia,

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“…Functional evidence has been demonstrated for several σ 66 promoters and one σ 28 promoter using mutational analysis and in vitro transcription assays (e.g. Ricci et al, 1993;Fahr et al, 1995;Douglas and Hatch, 1996;Tan et al, 1996;Tan et al, 1998;Ochiai et al, 1999;Schaumburg and Tan, 2003;Yu et al, 2003;Yu et al, 2006). Chlamydial promoters share homology with each of the eubacterial consensus sequences with certain exceptions, including the presence of a cisacting, A/T spacer region in the majority of σ 66 promoters analyzed (Schaumburg and Tan, 2000).…”

Section: Chlamydial Gene Regulationmentioning

confidence: 99%

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

AbdelRahman¹

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Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ ²⁸ RNA Polymerase

Cited by 29 publications

References 22 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

Chlamydial CT441 Is a PDZ Domain-Containing Tail-Specific Protease That Interferes with the NF-κB Pathway of Immune Response

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

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Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ 28 RNA Polymerase

Cited by 29 publications

References 22 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

Chlamydial CT441 Is a PDZ Domain-Containing Tail-Specific Protease That Interferes with the NF-κB Pathway of Immune Response

Discovery and Validation of New Regulatory RNA Elements in Chlamydia trachomatis

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Mutational Analysis of the Promoter Recognized by Chlamydia and Escherichia coli σ ²⁸ RNA Polymerase

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