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

Abstract

MazF is an endoribonucleolytic toxin that cleaves intracellular RNAs in sequence-specific manners. It is liberated in bacterial cells in response to environmental changes and is suggested to contribute to bacterial survival by inducing translational regulation. Thus, determining the cleavage specificity provides insights into the physiological functions of MazF orthologues. Nitrospira, detected in a wide range of environments, is thought to have evolved the ability to cope with their surroundings. To investigate the molecular mechanism of its environmental adaption, a MazF module from Nitrospira strain ND1, which was isolated from the activated sludge of a wastewater treatment plant, is examined in this study. By combining a massive parallel sequencing method and fluorometric assay, we detected that this functional RNA-cleaving toxin specifically recognizes the AACU, AACG, and AAUU motifs. Additionally, statistical analysis suggested that this enzyme regulates various specific functions in order to resist environmental stresses.

Conserved Amino Acid Moieties of Candidatus Desulforudis audaxviator MazF Determine Ribonuclease Activity and Specificity

“…The amino acid sequences of MazFs from several species, the cleavage sequences (ACA, UAC, AACU, UACA, and UACAU) of which are similar to the UACAAA recognition sequence of MazF-Da ( Zhang et al, 2003b ; Zhu et al, 2009 ; Park et al, 2011 ; Rothenbacher et al, 2012 ; Yamaguchi et al, 2012 ; Schuster et al, 2013 ; Miyamoto et al, 2016a , b , 2017 , 2018 ; Shaku et al, 2018 ; Ishida et al, 2019 ; Aoi et al, 2020 ), were selected for multiple alignment. We identified Gly6 (G6), Pro15 (P15), Gly18 (G18), Glu20 (E20), Arg25 (R25), Pro26 (P26), Asn36 (N36), and Arg85 (R85) as highly conserved sites ( Figure 5A ).…”

“…Haloquadratum walsbyi, isolated from a hypersaline pool in the Sinai Peninsula, possesses a unique MazF that recognizes the longest known recognition sequence, consisting of seven bases (UUACUCA) ( Yamaguchi et al, 2012 ). More recently, the MazF homologs of Methanohalobium evestigatum and the Nitrospira strain ND1 have been shown to recognize not just one but multiple sequences ( Ishida et al, 2019 ; Aoi et al, 2020 ). These studies show that MazF toxins, which are conserved in various species, are unique ribonucleases with different recognition sequences and lengths.…”

MazEF Homologs in Symbiobacterium thermophilum Exhibit Cross-Neutralization with Non-Cognate MazEFs

“…Considering the above homology, the cross-neutralization between MazF and non-cognate MazE was examined, revealing the obvious suppression of MazE-bs on MazF-sth. The structure mechanism was analyzed by multiple sequence alignment (MSA) [ 11 , 13 , 14 , 16 , 17 , 18 , 19 , 20 , 25 , 26 , 41 , 42 , 43 , 44 , 45 , 46 , 47 ] ( Figure S5 ) combined with the simulation of the MazEF-sth complex structure via AlphaFold2 [ 48 ] ( Figure S6 ). Focus was placed on the interface; next, regions of the MazF-sth dimer interacting with MazE-sth were estimated as follows and highlighted with red/blue squares on MSA ( Figures S5 and S6 ).…”

A Bias in the Reading of the Genetic Code of Escherichia coli is a Characteristic for Genes that Specify Stress-induced MazF-mediated Proteins

“…Recently, Aoi et al reported that, in Nitrospira strain ND1, the MazF toxin specifically recognizes the motifs AACU, AACG, and AAUU [ 15 ]. However, since, in this case, MazF target is not a coding triplet, a bias in the reading of the genetic code should not be characteristic for genes that specify stress-induced MazF mediated proteins of Nitrospira strain ND.…”