sRNA154 a newly identified regulator of nitrogen fixation in Methanosarcina mazei strain Gö1

Abstract

Trans-encoded sRNA₁₅₄ is exclusively expressed under nitrogen (N)-deficiency in Methanosarcina mazei strain Gö1. The sRNA₁₅₄ deletion strain showed a significant decrease in growth under N-limitation, pointing toward a regulatory role of sRNA₁₅₄ in N-metabolism. Aiming to elucidate its regulatory function we characterized sRNA₁₅₄ by means of biochemical and genetic approaches. 24 homologs of sRNA₁₅₄ were identified in recently reported draft genomes of Methanosarcina strains, demonstrating high conservation in sequence and predicted secondary structure with two highly conserved single stranded loops. Transcriptome studies of sRNA₁₅₄ deletion mutants by an RNA-seq approach uncovered nifH- and nrpA-mRNA, encoding the α-subunit of nitrogenase and the transcriptional activator of the nitrogen fixation (nif)-operon, as potential targets besides other components of the N-metabolism. Furthermore, results obtained from stability, complementation and western blot analysis, as well as in silico target predictions combined with electrophoretic mobility shift-assays_, argue for a stabilizing effect of sRNA₁₅₄ on the polycistronic nif-mRNA and nrpA-mRNA by binding with both loops. Further identified N-related targets were studied, which demonstrates that translation initiation of glnA₂-mRNA, encoding glutamine synthetase2, appears to be affected by sRNA₁₅₄ masking the ribosome binding site, whereas glnA₁-mRNA appears to be stabilized by sRNA₁₅₄. Overall, we propose that sRNA₁₅₄ has a crucial regulatory role in N-metabolism in M. mazei by stabilizing the polycistronic mRNA encoding nitrogenase and glnA₁-mRNA, as well as allowing a feed forward regulation of nif-gene expression by stabilizing nrpA-mRNA. Consequently, sRNA₁₅₄ represents the first archaeal sRNA, for which a positive posttranscriptional regulation is demonstrated as well as inhibition of translation initiation.

Keywords: Methanosarcina mazei; RNA stability; nitrogen fixation; nitrogenase; regulatory RNAs.

Figure 1.

Characterization of sRNA₁₅₄ (A) Genomic context of sRNA₁₅₄, promotor and terminator region of sRNA₁₅₄. Potential TATA- and BRE box, the transcriptional start site (TSS) (+1), as well as the termination site (TT) are indicated. The 5´and 3´end of sRNA₁₅₄ was determined by RACE analysis (Ambion, Thermo Scientific, Darmstadt, Germany). (B) RNA-seq analysis of total RNA of M. mazei wt - grown under nitrogen sufficient (+NH₄⁺) and fixing (N₂) conditions - using the Illumina technique revealed an absence of sRNA₁₅₄ transcript under +NH₄⁺ and high induction under N₂-conditions. (C) Conservation of promotor regions of sRNA₁₅₄ homologues from those various Methanosarcina isolates described in Fig. 2. The regions upstream of the TSS were aligned using the ClustalW multiple alignment tool.

Figure 2.

Conservation of sRNA₁₅₄ in Methanosarcina species Multiple secondary structure alignment of sRNA₁₅₄ homologues in related Methanosarcina species performed with LocARNA, MM, Methanosarcina mazei strains S-6, Go1, WWM610, TMA, SarPi, LYC, C16, Tuc01; MMHB, Methanosarcina horonobensis HB1; MA, Methanosarcina acetivorans strain C2A; Msp, Methanosarcina sp strains Naples 100, WWM596, WH1, MTP4, Kolksee; Msiciliae, Methanosarcina siciliae strains T4M, HI350, C2J; Mvacuolata, Methanosarcina vacuolata Z761; MB, Methanosarcina barkeri strains fusaro, Wiesmoor, MS, 227, 3; Mlacustris, Methanosarcina lacustris strains ZS, Z7289. (B) Consensus secondary structure prediction by RNAlifold. Conserved single stranded loop RNA regions are indicated in blue (loop 1) and purple (loop 2).

Figure 3.

Transcript patterns of a sRNA₁₅₄ chromosomal deletion mutant using an RNA-seq approach RNA sequence analysis (using the Illumina technique) was performed using RNA isolated from M. mazei wt and sRNA₁₅₄ chromosomal deletion mutants (ΔsRNA₁₅₄:: pac) growing under N-fixing conditions. For each strain two biological replicates were analyzed, representing two independent wt clones and two independent generated mutant clones (ΔsRNA₁₅₄:: pac). Visualization of the distribution of cDNA reads of selected genes involved in the N-metabolism (glnA₁, glnA₂, glnK₁, amtB₁, nifH and nrpA) are exemplarily shown for one biological replicate. The fold change (ΔsRNA₁₅₄:: pac vs. wt) indicated below represent the average change of both independent biological replicates.

Figure 4.

mRNA stability assay comparing M. mazei Δ sRNA₁₅₄ with the parental strain. To validate the stabilizing effects of sRNA₁₅₄ on its target mRNAs we performed an mRNA half-life assay, using 100 µg/ml actinomycin D to inhibit transcription. Cells were harvested before (at time point zero) and after 30 and 60 min supplementing actinomycin D, followed by RNA isolation and qRT-PCR analysis to verify mRNA decay in the chromosomal deletion strain compared to the wt (for primer sets see Table S3). Fold changes in the sRNA₁₅₄ deletion mutant vs. wt are given by mean values of two biologically independent experiments.

Figure 5.

NifH protein expression patterns in the absence of sRNA₁₅₄ under N- limitation Cell extracts were prepared from exponentially growing cultures of M. mazei wt, M. mazei sRNA₁₅₄::pac-mutant, M. mazei nrpA::pac mutant and M. mazei sRNA₁₅₄::markerless mutant strains under N-limitation. Defined amounts of cell extracts were separated by SDS PAGE followed by western blot analysis using polyclonal antibodies generated against NifH. Relative amounts of NifH in the M. mazei sRNA₁₅₄ deletion and nrpA deletion-mutant strain compared to M. mazei wt strain were calculated using the Aida image analyzer for three independent biological replicates. The average fold-expression changes are depicted, the lower panel represents one exemplarily chosen original western blot. A): lane 1–3, His-NifH standards (20 ng, 10 ng, 5 ng); lane 4, M. mazei ΔnrpA::pac-mutant (100 µg); lane 5, M. mazei wt cell extract (100 µg); lane 6, M. mazei ΔsRNA₁₅₄::markerless-mutant strain (100 µg); B lane 1–3, His-NifH standards (20 ng, 10 ng, 5 ng); lane 4, M. mazei ΔnrpA::pac-mutant strain (50 µg); lane 5, M. mazei wt cell extract (50 µg); lane 6, M. mazei ΔsRNA₁₅₄::pac-mutant (50 µg). X, protein bands which are also present under NH₄⁺ sufficient growth conditions under which NifH protein is not translated.

Figure 6.

Target predictions for sRNA₁₅₄ (IntaRNA) In silico prediction of potential interactions between sRNA₁₅₄ and mRNA targets performed with IntaRNA. Selected predicted interactions of genes involved in N-metabolism are classified into two classes. class I: mRNA targets interacting with both loops of sRNA₁₅₄, targeting several sites of the mRNA including the ribosome binding site (RBS). class II: mRNA targets interacting with one loop of sRNA₁₅₄ at several positions of the target mRNA. Loop1 of sRNA₁₅₄ = indicated in turquoise, Loop2 of sRNA₁₅₄ = indicated in purple.

Figure 7.

Electrophoretic mobility shift assays Electrophoretic mobility shift assays (EMSAs) were performed using approximately 5 nM of radioactively 5′end labeled sRNA₁₅₄ or additionally added 2 µM cold sRNA₁₅₄ (retardation experiment). The assays were performed with increasing concentrations of unlabeled target mRNAs. After 15 min incubation, samples were run on a native 6% PAA gel. The respective autoradiographs of the gels are shown for: glnA₁ short fragment of the first 400 nt; for glnA₂ short fragments from 1–100 nt, 100–200 nt and from 1–200 nt; nrpA full length transcript. The respective retardation of sRNA₁₅₄ is indicated on the left site of the corresponding EMSAs.