Deep sequencing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability

Abstract

Methanosarcina mazei and related mesophilic archaea are the only organisms fermenting acetate, methylamines, and methanol to methane and carbon dioxide, contributing significantly to greenhouse gas production. The biochemistry of these metabolic processes is well studied, and genome sequences are available, yet little is known about the overall transcriptional organization and the noncoding regions representing 25% of the 4.01-Mb genome of M. mazei. We present a genome-wide analysis of transcription start sites (TSS) in M. mazei grown under different nitrogen availabilities. Pyrosequencing-based differential analysis of primary vs. processed 5' ends of transcripts discovered 876 TSS across the M. mazei genome. Unlike in other archaea, in which leaderless mRNAs are prevalent, the majority of the detected mRNAs in M. mazei carry long untranslated 5' regions. Our experimental data predict a total of 208 small RNA (sRNA) candidates, mostly from intergenic regions but also antisense to 5' and 3' regions of mRNAs. In addition, 40 new small mRNAs with ORFs of < or = 30 aa were identified, some of which might have dual functions as mRNA and regulatory sRNA. We confirmed differential expression of several sRNA genes in response to nitrogen availability. Inspection of their promoter regions revealed a unique conserved sequence motif associated with nitrogen-responsive regulation, which might serve as a regulator binding site upstream of the common IIB recognition element. Strikingly, several sRNAs antisense to mRNAs encoding transposases indicate nitrogen-dependent transposition events. This global TSS map in archaea will facilitate a better understanding of transcriptional and posttranscriptional control in the third domain of life.

Fig. 1.

Visualization of the clone distribution of cDNAs mapped to the chromosomal glnA₁ region. An extraction of the screenshot of the Integrated Genome Browser (Affymetrix) of the mapped cDNAs is shown for nitrogen fixation (NF+) and ammonium conditions (NS+) of the M. mazei glnA₁ region. The y axis indicates the relative score of clone numbers per nucleotide. The nitrogen-regulated TSS and a potential second TSS of glnA₁ are indicated. The respective Northern blot analysis is shown (Right).

Fig. 2.

Transcription of spRNA₀₉ and sRNA₆₀. (A) Chromosomal localization; (B) cDNA clone distribution for nitrogen fixation (NF+) and ammonium conditions (NS+); (C) confirmatory Northern blot analysis; the calculated fold induction (NF vs. NS) is given below the blot.

Fig. 3.

Expression of selected sRNA candidates in response to nitrogen. Total RNA was isolated from M. mazei grown under nitrogen fixation (NF) and ammonium conditions (NS) and subjected to Northern blot analysis. Blots showing stable sRNAs transcripts, the lower line the 5S rRNA transcripts of the respective RNA preparations. The calculated fold induction (NF vs. NS) of at least 2 independent experiments is given below the blots.

Fig. 4.

Promoter region of sRNA₁₅₄ (A) and sRNA₁₅₉ (B). The NrpR binding boxes are indicated by gray boxes.

Fig. 5.

Sequence-logo of promotor regions of sRNAs candidates differentially transcribed in response to nitrogen. The regions upstream of the TSS (up to 500 nt) were aligned using the ClustalW multiple alignment tool (50) and the consensus visualized with WebLogo (51).

Fig. 6.

Transcription of MM2686 and the corresponding cis-encoded asRNA₃₆. cDNA clone distribution for nitrogen fixation (NF+) and ammonium (NS+) conditions of the M. mazei transposase gene (MM2686) region is shown for the forward and reverse strand.

Fig. 7.

Genomic map showing the localization of the verified IGR transcripts. The 400-kb fragment (nt 11,700–407,000) thoroughly analyzed is enlarged. Red, new ORFs; green, spRNAs; dark blue, asRNAs; and light blue, sRNAs.