Characterization of an Archaeal Two-Component System That Regulates Methanogenesis in <i>Methanosaeta harundinacea</i>

<div><p>Two-component signal transduction systems (TCSs) are a major mechanism used by bacteria in response to environmental changes. Although many sequenced archaeal genomes encode TCSs, they remain poorly understood. Previously, we reported that a methanogenic archaeon, <i>Methanosaeta harundinacea</i>, encodes FilI, which synthesizes carboxyl-acyl homoserine lactones, to regulate transitions of cellular morphology and carbon metabolic fluxes. Here, we report that <i>filI</i>, the cotranscribed <i>filR2</i>, and the adjacent <i>filR1</i> constitute an archaeal TCS. FilI possesses a cytoplasmic kinase domain (histidine kinase A and histidine kinase-like ATPase) and its cognate response regulator. FilR1 carries a receiver (REC) domain coupled with an ArsR-related domain with potential DNA-binding ability, while FilR2 carries only a REC domain. In a phosphorelay assay, FilI was autophosphorylated and specifically transferred the phosphoryl group to FilR1 and FilR2, confirming that the three formed a cognate TCS. Through chromatin immunoprecipitation–quantitative polymerase chain reaction (ChIP-qPCR) using an anti-FilR1 antibody, FilR1 was shown to form <i>in vivo</i> associations with its own promoter and the promoter of the <i>filI-filR2</i> operon, demonstrating a regulatory pattern common among TCSs. ChIP-qPCR also detected FilR1 associations with key genes involved in acetoclastic methanogenesis, <i>acs4</i> and <i>acs1</i>. Electrophoretic mobility shift assays confirmed the <i>in vitro</i> tight binding of FilR1 to its own promoter and those of <i>filI-filR2</i>, <i>acs4</i>, and <i>mtrABC</i>. This also proves the DNA-binding ability of the ArsR-related domain, which is found primarily in Archaea. The archaeal promoters of <i>acs4</i>, <i>filI</i>, <i>acs1</i>, and <i>mtrABC</i> also initiated FilR1-modulated expression in an <i>Escherichia coli lux</i> reporter system, suggesting that FilR1 can up-regulate both archaeal and bacterial transcription. In conclusion, this work identifies an archaeal FilI/FilRs TCS that regulates the methanogenesis of <i>M. harundinacea</i>.</p></div

Expression of the archaeal promoters in the <i>E. coli ex vivo</i> reporter system.

a.<p>Values are shown as relative light units and the average of at least three independent readings.</p>b.<p>Genes and operons shown in each reporter plasmid are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095502#pone.0095502.s004" target="_blank">Table S1</a>.</p>c.<p>Fold difference of the luciferase activity are calculated from those determined for <i>E. coli</i> strain carrying plasmid pairs of FilR1-vacant p184 plus pO^x-lux over that of the strain carrying pOx-lux alone (+p184/−), strain with FilR1 plus pO^x-lux over that with pO^x-lux alone (+pFilR1/−), and strain with FilR1 plus pO^x-lux over that with FilR1-vacant p184 plus pO^x-lux (+pFilR1/+p184).</p

Schematic representation of the domain structures of FilI and FilR proteins.

<p>(A) Domain structure of FilI analyzed using programs of Pfam and NCBI blast. (B) Location analysis of domains in FilI through the programs of TMHMM, TMpred and SOSUI. (C) Domain structures of FilR1 and FilR2 were analyzed by programs of Pfam and NCBI blast. In addition, the amino acid identity (%) for the aligned fragments of FilR1 and FilR2 is shown on the right. (D) Protein sequence alignment of the REC domains of FilR1 (C-terminal 276–446aa) and FilR2 (the whole length) by software GeneDoc. Identical amino acids are shown with a black background, while similar amino acids are shown with a gray background.</p

ChIP assays showed FilR1 binding to the promoters of its own (P<i>_filR1</i>) and <i>filI-filR2</i> operon (P<i>_filR1-filR2</i>) inside the cells of <i>M. hurandiacea</i> 6AC.

<p>(A) PCR products of the promoters of its own (P<i>_filR1</i>) and <i>filI-filR2</i> operon (P<i>_filR1-filR2</i>) were amplified from the anti-FilR1 antibody immunoprecipitated DNA (Ab_FilR1), and input DNA sample before immunoprecipitation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0095502#s4" target="_blank">Material and Methods</a>) as a positive control (Input). Almost no PCR products were amplified from the mock-IP DNA (CK) samples. (B) qPCR detected the enrichment folds of the DNA fragments in anti-FilR1 antibody immunoprecipitated DNA (Ab_FilR1, gray bar) over mock-IP control (CK, black bar). PCR amplifications were performed using the specific primers for the promoter regions of <i>filR1</i> (P<i>_filR1</i>) and <i>filI-filR2</i> operon (P<i>_filI-R2</i>). An intragenic DNA fragment of the16S rRNA gene (16 s) was included as the negative control.</p

EMSAs showed FilR1 binding to the promoters of its own and the operon <i>filI-filR2</i>.

<p>Purified recombinant FilR1 protein was incubated with 0.5-labeled DNA in the standard binding reaction mixture at 25°C for 20 min, and then run on a native PAGE. Concentration of purified FilR1 protein was shown at the top of each lane. Unlabeled <i>filI-filR2</i> promoter (NP<i>_filI-filR2</i>) was used as a competitor substrate of FilR1, which was added at the final concentrations of 5, 25, 125 and 250 nM in lane 6 and 15, 7 and 16, 8 and 17, and 9 and 18, respectively. (A) P<i>_filI-R2</i>, promoter of the <i>filI-filR2</i> operon; (B) P<i>_filR1</i>, promoter of <i>filR1</i>, and (C) U<i>_filI</i>, an internal DNA fragment of gene <i>filI</i>.</p

Cotranscription of <i>filI</i> and <i>filR2</i> in <i>M. harundinacea</i>.

<p>(A) Schematic arrangement of <i>filI</i> and <i>fliR2</i> in the genome. (B) Agarose gel electrophoresis of PCR products amplified from the intergenic region between <i>filI</i> and <i>filR2</i>. (C) The intergenic spacer between <i>filR2</i> and its upstream gene encoding a ferredoxin using the respective template labeled at the top of each gel: -, no DNA; RNA, total RNA extracted form <i>M. harundinacea</i> cells; cDNA, reverse transcripts from the total RNA; gDNA, genomic DNA of <i>M. harundinacea</i>. M, DL2000 marker with the sizes shown at the right. Primers spacer-IR2-R/F and spacer-gen-F/R were used for PCR reactions in (B) and (C), respectively.</p

ChIP-PCR and ChIP-qPCR detected FilR1 associations with the promoters of genes for methanogenesis inside <i>M. harundinacea</i> cells.

<p>(A) PCR products amplified with the indicated primers using anti-FilR1 antibody immunoprecipitated DNA (AbFilR1) as a template and mock-IP DNA as a negative control (CK). (B) qPCR assays detected the enrichment of DNA fragments in anti-FilR1 antibody immunoprecipitated samples (AbFilR1, gray bar) over mock-IP negative control samples (CK, black bar). P<i>_acs1</i>, promoter region of operon <i>acs1</i>; P<i>_acs4</i>, promoter region of <i>acs4</i>; P<i>_mtr</i>, promoter region of operon <i>mtr</i>; P<i>_fwd</i>, promoter region of the operon <i>fwdCABD</i>; P<i>_omp</i>, promoter region of <i>omp</i> and 16s, intragenic DNA fragment of 16S rDNA used as the control.</p

EMSAs showed FilR1 binding to the promoters of genes key to methanogenesis in <i>M. harundinacea</i>.

<p>Purified recombinant FilR1 protein was incubated with 0.5-labeled DNA in the standard binding reaction mixture for 20 min at 25°C and then run on a native PAGE. Concentration of purified FilR1 protein used was shown at the top of each lane. (A) P<i>_acs1</i>, promoter of the <i>acs1</i> operon; (B) P<i>_acs4</i>, promoter of <i>acs4</i>; (C) P<i>_mtr</i>, promoter of the <i>mtr</i> operon; (D) P<i>_fwdCABD</i>, promoter of the <i>fwdCABD</i> operon; (E), P<i>_omp</i>, promoter of <i>omp</i>; and (F) P<i>_{Mhar_0449}</i>, promoter of Mhar_0449.</p

Real-time PCR and Western blot analysis of THOP1 expression in NSCLC.

<p>A. Expression of THOP1 mRNA in the NSCLC tissues was lower than that in the normal lung tissues (<i>P</i><0.01). B. Western blot analysis suggested that NSCLC specimens had decreased THOP1 protein expression compared to corresponding normal tissues. N: Normal lung tissue, T: NSCLC specimens.</p

Kaplan–Meier curves of disease-free and overall survival stratified according to THOP1 protein, lymph node metastasis and TNM stage.

<p>Kaplan–Meier curves of disease-free and overall survival stratified according to THOP1 protein, lymph node metastasis and TNM stage.</p