Nitrosomonas europaea MazF Specifically Recognises the UGG Motif and Promotes Selective RNA Degradation

Abstract

Toxin-antitoxin (TA) systems are implicated in prokaryotic stress adaptation. Previously, bioinformatics analysis predicted that such systems are abundant in some slowly growing chemolithotrophs; e.g., Nitrosomonas europaea. Nevertheless, the molecular functions of these stress-response modules remain largely unclear, limiting insight regarding their physiological roles. Herein, we show that one of the putative MazF family members, encoded at the ALW85_RS04820 locus, constitutes a functional toxin that engenders a TA pair with its cognate MazE antitoxin. The coordinate application of a specialised RNA-Seq and a fluorescence quenching technique clarified that a unique triplet, UGG, serves as the determinant for MazF cleavage. Notably, statistical analysis predicted that two transcripts, which are unique in the autotroph, comprise the prime targets of the MazF endoribonuclease: hydroxylamine dehydrogenase (hao), which is essential for ammonia oxidation, and a large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (rbcL), which plays an important role in carbon assimilation. Given that N. europaea obtains energy and reductants via ammonia oxidation and the carbon for its growth from carbon dioxide, the chemolithotroph might use the MazF endoribonuclease to modulate its translation profile and subsequent biochemical reactions.

The Variety in the Common Theme of Translation Inhibition by Type II Toxin–Antitoxin Systems

“…Subsets of toxins sharing similar target sequences are boxed with different colors. Protein identifiers and cleavage specificity were taken from: E. coli MazF (ChpAK) (NP_417262.1) (Culviner and Laub, 2018;Mets et al, 2019), ChpBK (NP_418646.1) (Zhang et al, 2005), Kid (PemK) (YP_003937673.1) (Munoz-Gomez et al, 2004;Zhang et al, 2004); Bacillus subtilis MazF bs (AOR96854.1) (Park et al, 2011); Bacillus anthracis MoxT (NP_842807.1) (Verma and Bhatnagar, 2014); Staphylococcus aureus MazF sa (BBJ19047.1) (Zhu et al, 2009); Staphylococcus equorum MazF seq (AFV93478.1) (Schuster et al, 2013); Clostridium difficile MazF cd (YP_001089981.1) (Rothenbacher et al, 2012); Legionella pneumophila MazF lp (CCD10720.1) (Shaku et al, 2018); Deinococcus radiodurans MazF DR0417 (AAF09995.1) (Miyamoto et al, 2017); Haloquadratum walsbyi MazF hw (WP_048066888.1) (Yamaguchi et al, 2012); Pseudomonas putida MazF pp (NP_742932.1) (Miyamoto et al, 2016a); Myxococcus xanthus MazF mx (SDX28280.1) (Nariya and Inouye, 2008); Methanohalobium evestigatum MazF me (WP_013195679.1); Nitrosomonas europaea MazF ne1 (WP_011111532.1) (Miyamoto et al, 2018), MazF NE1181 (CAD85092.1) (Miyamoto et al, 2016b); Mycobacterium tuberculosis MazF-mt1 (NP_217317.1) (Zhu et al, 2006), MazF-mt3 (NP_216507.1) (Zhu et al, 2008;Schifano et al, 2014), MazF-mt6 (NP_215618.1) (Schifano et al, 2013), MazF-mt7 (NP_216011.1) (Zhu et al, 2008), MazF-mt9 (YP_004837055.2) (Barth et al, 2019).…”

MazF Endoribonucleolytic Toxin Conserved in Nitrospira Specifically Cleaves the AACU, AACG, and AAUU Motifs

“…It is well known that MazF homologues are widespread across the bacterial and archaeal domains [36]. They cleave single-stranded RNAs at specific three to seven base motifs, thereby modulating translation through degradation of intracellular RNA pools or silencing of specific transcripts (Table S3) [11,15,[17][18][19]26,28,29,31,[37][38][39][40][41][42][43][44][45]. Here, we demonstrated that MazF-nd1 cuts AACU, AACG, and AAUU motifs.…”

“…Nitrifying bacteria are generally sensitive to environmental fluctuations and easily enter a dormant state. Previously, we showed the MazF endoribonucleases (referred to as MazF NE1181 and MazFne1 in the previous papers) in N. europaea are functional growth regulators, and their sequence-specificities may allow N. europaea to alter its translation profile and survive under certain stressful conditions [18,19]. MazF-nd1 shares only 34.2% and 23.5% identities with MazF NE1181 and MazFne1, respectively ( Figure S3).…”

“…Previously, we detected the recognition sequences of two MazF enzymes conserved in Nitrosomonas europaea, a representative ammonia-oxidizing bacterium, and performed statistical analysis to predict their main regulation targets [18,19]. The results suggested that one of the MazF enzymes improves heavy metal resistance by leaving the related transcripts uncleaved [18], while another downregulates cellular activities by digesting mRNAs essential to ammonia oxidation and carbon fixation [19]. As the other crucial contributor to complete nitrification, the environmental adaption of nitrite-oxidizing bacteria (NOB) is also of interest.…”

YjbH Solubility Controls Spx in Staphylococcus aureus: Implication for MazEF Toxin-Antitoxin System Regulation

“…It is found also in other clinically important bacteria (Mittenhuber, 1999;Nguyen et al, 2011;Schifano et al, 2013;Cho et al, 2017). Several studies were conducted to characterize the MazEF locus by studying its transcriptional activation and function Fu et al, 2009;Zhu et al, 2009;Zorzini et al, 2011Zorzini et al, , 2014Miyamoto et al, 2018). MazEF is composed of MazF toxin and its activity is modulated by the MazE antitoxin (Figure 1).…”