Functional analysis of small proteins in Methanosarcina mazei focusing on those involved in nitrogen regulation or biofilm formation
Christian-Albrechts-University Kiel (Germany), Institute for General Microbiology
The methanogenic archaeon Methanosarcina mazei Gö1 is able to fix molecular nitrogen under nitrogen limitation. Using genome-wide differential RNA-seq and term-seq analysis we identified more than 70 small transcripts containing potential sORFs ranging from 12 to 61 amino acids [1,2]. Several of those so-called spRNAs are differentially transcribed under nitrogen stress conditions or in biofilms and are highly conserved in Methanosarcina strains. We succeeded in confirming the translation of three of the potential sORFs by LC-MS/MS analysis of total protein extracts , as well as in a follow up study to identify additional 13 small proteins applying a gel-free pre-fractionation approach followed by a reversed phase LC-MS analysis . Based on first characterization and detecting potential interacting proteins we hypothesize that several small proteins have a physiological function in M. mazei, particularly small proteinP36 and small proteinP26 might have a function in nitrogen regulation since interacting proteins have been detected in a nitrogen dependent way. Some experimentally verified small proteins might have a function in the establishment of M. mazei biofilms.
Consequently the aim of the proposed project is to (i) identify and characterize M. mazei proteins/complexes interacting with small proteinP26, small proteinP44 and small proteinP36 using different biochemical approaches; (ii) detect their localization in cellular fractions and complexes under different growth conditions using specific antibodies; (iii) elucidate their function by genetic approaches; (iv) identify small proteins on a global scale under different stress conditions (e.g. oxygen stress, biofilm formation) combining bioinformatic predictions, ribosome-profiling and peptidomic approaches, and (v) characterize the physiological role of selected small proteins identified in the genome-wide approach, focusing on small proteins potentially involved in biofilm formation or stress adaptation. Overall, we expect to unravel distinct physiological functions of small proteins in M. mazei and envisage uncovering novel principles in regulation.
- Jäger, D, Sharma, CM, Thomsen, J, Ehlers, C, Vogel, J, Schmitz RA (2009) ‘Deep sequencing analysis of the Methanosarcina mazei Gö1 transcriptome in response to nitrogen availability’ Proc Natl Acad Sci U S A 106: 21878-82.
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- Prasse D., Thomsen J., DeSantis R., Muntel J., Becher D., Schmitz RA (2015) First description of small proteins encoded by spRNAs in Methanosarcina mazei strain Gö1. Biochimie 117: 138-148.
- Cassidy L, Prasse D, Linke D, Schmitz RA, Tholey A (2016). Combination of bottom up 2D-LC-MS and semi-top-down GelFree-LC-MS enhances coverage of proteome and low molecular weight short open reading frame encoded peptides of the archaeon M. mazei. J Proteome Res. 15:3773-3783.