Establishing a Markerless Genetic Exchange System forMethanosarcina mazeiStrain Gö1 for Constructing Chromosomal Mutants of Small RNA Genes

Abstract

A markerless genetic exchange system was successfully established in Methanosarcina mazei strain Gö1 using the hpt gene coding for hypoxanthine phosphoribosyltransferase. First, a chromosomal deletion mutant of the hpt gene was generated conferring resistance to the purine analog 8-aza-2,6-diaminopurine (8-ADP). The nonreplicating allelic exchange vector (pRS345) carrying the pac-resistance cassette for direct selection of chromosomal integration, and the hpt gene for counterselection was introduced into this strain. By a pop-in and ultimately pop-out event of the plasmid from the chromosome, allelic exchange is enabled. Using this system, we successfully generated a M. mazei deletion mutant of the gene encoding the regulatory non-coding RNA sRNA154. Characterizing M. mazeiΔsRNA 154 under nitrogen limiting conditions demonstrated differential expression of at least three cytoplasmic proteins and reduced growth strongly arguing for a prominent role of sRNA154 in regulation of nitrogen fixation by posttranscriptional regulation.

The transcriptional activator NrpA is crucial for inducing nitrogen fixation in Methanosarcina mazei Gö1 under nitrogen‐limited conditions

“…Moreover, additional activation by direct interaction of MM1708 with RNA polymerase also appears to be possible; however, we currently have no experimental evidence for interactions between MM1708 and RNA polymerase. We have further indication that sRNA 154 , which is another regulator, positively effecting nif gene transcription . It is tempting to speculate that the stability of the MM1708 transcript or the respective translation initiation of the transcript is positively affected by sRNA 154 (Fig.…”

Contribution of Transcriptomics to Systems-Level Understanding of MethanogenicArchaea

“…The presence of 135 of the sRNAs depended upon nitrogen availability indicating that they play a regulatory role in nitrogen metabolism of M. mazei . One of the sRNAs, designated sRNA 154 , was subsequently shown to be important for optimal growth rate in nitrogen fixation conditions [58]. This indicates that sRNAs play an important role in posttranscriptional regulation of nitrogen metabolism in M. mazei .…”

sRNA41 affects ribosome binding sites within polycistronic mRNAs in Methanosarcina mazei Gö1

“…A genome‐wide differential RNA sequencing (dRNA‐seq) approach identified 248 sRNAs to be differentially expressed in response to N‐availability, suggesting that sRNA‐mediated regulation in nitrogen and general stress response plays an important role in M. mazei (Jäger et al ., ). Analyzing sRNA 154 , highly upregulated under N 2 ‐fixing conditions, demonstrated its central regulatory role in N‐metabolism affecting several components, including nitrogenase and glutamine synthetase by masking the RBSs or positively affecting transcript stabilities as reported recently (Ehlers et al ., ; Prasse et al ., ). Concerning its metabolic versatility, M. mazei can utilize a range of carbon and energy sources (e.g., methanol, methylamine or acetate), however, its utilization underlies a likewise strictly regulated system (Hovey et al ., ).…”