Characterization of an archaeal two-component system that regulates methanogenesis in Methanosaeta harundinacea

Abstract

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, Methanosaeta harundinacea, encodes FilI, which synthesizes carboxyl-acyl homoserine lactones, to regulate transitions of cellular morphology and carbon metabolic fluxes. Here, we report that filI, the cotranscribed filR2, and the adjacent filR1 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 in vivo associations with its own promoter and the promoter of the filI-filR2 operon, demonstrating a regulatory pattern common among TCSs. ChIP-qPCR also detected FilR1 associations with key genes involved in acetoclastic methanogenesis, acs4 and acs1. Electrophoretic mobility shift assays confirmed the in vitro tight binding of FilR1 to its own promoter and those of filI-filR2, acs4, and mtrABC. This also proves the DNA-binding ability of the ArsR-related domain, which is found primarily in Archaea. The archaeal promoters of acs4, filI, acs1, and mtrABC also initiated FilR1-modulated expression in an Escherichia coli lux 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 M. harundinacea.

Figure 1. Schematic representation of the domain structures of FilI and FilR proteins.

(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.

Figure 2. Cotranscription of filI and filR2 in M. harundinacea.

(A) Schematic arrangement of filI and fliR2 in the genome. (B) Agarose gel electrophoresis of PCR products amplified from the intergenic region between filI and filR2. (C) The intergenic spacer between filR2 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 M. harundinacea cells; cDNA, reverse transcripts from the total RNA; gDNA, genomic DNA of M. harundinacea. 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.

Figure 3. Assays of the autophosphorylation of FilI and phosphotransfer from FilI to FilR1 and FilR2.

(A) SDS-PAGE of the recombinant his-tagged FilI, FilR1 and FilR2 purified from E. coli. (B) An autoradiogram visualized the autophosphorylation of the recombinant entire FilI protein (2 µg) after incubation for 45 min at 37°C in the presence of [γ-³²P]ATP (lane 2) and without FilI as the negative control (lane 1). Phosphotransfer was assayed by addition of 4 µg of His-tagged RRs into the autophosphorylation reaction of FilI for certain times before being visualized on SDS-PAGE. Lane 3 and 6, negative controls of the phosphotransfer reactions without FilI included. Lane 4 and 5, FilR1 phosphorylated for 2 and 5 min, respectively; lane 7 and 8, FilR2 phosphorylated for 2 and 5 min, respectively. Dotted arrows indicate nonspecific bands generated from [γ-³²P]ATP. (C) An autoradiogram visualized the autophosphorylation of the N-terminal truncated FilI (FilI-C) and phosphotransfer assay under the same treatments as the entire protein. Lane 1, no truncated FilI included; lane 2, FilI-C incubated with [γ-³²P]ATP for 45 min; lane 3, FilR2 incubated with [γ-³²P]ATP for 10 min; lane 4, 5 and 6, FilR2 phosphorylated for 2, 5 and 10 min, respectively. (D) An autoradiogram visualized the autophosphorylation of FilI-C and phosphotransfer assay to other regulators under the same treatments as the entire protein. Lane 1, no truncated FilI included, lane 2, FilI-C incubated with [γ-³²P]ATP for 45 min; lane 3 and 6, proteins Mhar_0169 and Mhar_1520 incubated with [γ-³²P]ATP for 10 min, respectively; lane 4 and 5, Mhar_0169 phosphorylated for 10 and 30 min, respectively; lane 7 and 8, Mhar_1520 phosphorylated for 10 and 30 min, respectively. Solid arrows indicate the phosphorylated proteins: Pi-FilI, Pi-FilI-C, Pi-FilR1 and Pi-FilR2.

Figure 4. ChIP assays showed FilR1 binding to the promoters of its own (P_filR1) and filI-filR2 operon (P_filR1-filR2) inside the cells of M. hurandiacea 6AC.

(A) PCR products of the promoters of its own (P_filR1) and filI-filR2 operon (P_filR1-filR2) were amplified from the anti-FilR1 antibody immunoprecipitated DNA (Ab_FilR1), and input DNA sample before immunoprecipitation (Material and Methods) 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 filR1 (P_filR1) and filI-filR2 operon (P_filI-R2). An intragenic DNA fragment of the16S rRNA gene (16 s) was included as the negative control.

Figure 5. ChIP-PCR and ChIP-qPCR detected FilR1 associations with the promoters of genes for methanogenesis inside M. harundinacea cells.

(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_acs1, promoter region of operon acs1; P_acs4, promoter region of acs4; P_mtr, promoter region of operon mtr; P_fwd, promoter region of the operon fwdCABD; P_omp, promoter region of omp and 16s, intragenic DNA fragment of 16S rDNA used as the control.

Figure 6. EMSAs showed FilR1 binding to the promoters of its own and the operon filI-filR2.

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 filI-filR2 promoter (NP_filI-filR2) 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_filI-R2, promoter of the filI-filR2 operon; (B) P_filR1, promoter of filR1, and (C) U_filI, an internal DNA fragment of gene filI.

Figure 7. EMSAs showed FilR1 binding to the promoters of genes key to methanogenesis in M. harundinacea.

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_acs1, promoter of the acs1 operon; (B) P_acs4, promoter of acs4; (C) P_mtr, promoter of the mtr operon; (D) P_fwdCABD, promoter of the fwdCABD operon; (E), P_omp, promoter of omp; and (F) P_{Mhar_0449}, promoter of Mhar_0449.