Functional Characterization of the mazEF Toxin-Antitoxin System in the Pathogenic Bacterium Agrobacterium tumefaciens

Abstract

Agrobacterium tumefaciens is a pathogen of various plants which transfers its own DNA (T-DNA) to the host plants. It is used for producing genetically modified plants with this ability. To control T-DNA transfer to the right place, toxin-antitoxin (TA) systems of A. tumefaciens were used to control the target site of transfer without any unintentional targeting. Here, we describe a toxin-antitoxin system, Atu0939 (mazE-at) and Atu0940 (mazF-at), in the chromosome of Agrobacterium tumefaciens. The toxin in the TA system has 33.3% identity and 45.5% similarity with MazF in Escherichia coli. The expression of MazF-at caused cell growth inhibition, while cells with MazF-at co-expressed with MazE-at grew normally. In vivo and in vitro assays revealed that MazF-at inhibited protein synthesis by decreasing the cellular mRNA stability. Moreover, the catalytic residue of MazF-at was determined to be the 24th glutamic acid using site-directed mutagenesis. From the results, we concluded that MazF-at is a type II toxin-antitoxin system and a ribosome-independent endoribonuclease. Here, we characterized a TA system in A. tumefaciens whose understanding might help to find its physiological function and to develop further applications.

Helicobacter macacaeMazF interplays with Escherichia coli homologs and enhances antibiotic tolerance

“…21,22 MazFhm shared 42.5% amino acid identity with MazFec, and MazEhm shared 30.6% amino acid identity with MazEec, which is comparable to similarities of MazF homologs from other species with MazFec. 21,22,30…”

“…22 The isoelectric point of MazEhm and MazFhm are 9.06 and 9.38, respectively, both higher than pH 7.0, which is different from classical MazEF systems, whose toxin MazF is usually acidic, and the cognate antitoxin are basic at neutral pH. 21,22 MazFhm shared 42.5% amino acid identity with MazFec, and MazEhm shared 30.6% amino acid identity with MazEec, which is comparable to similarities of MazF homologs from other species with MazFec. 21,22,30…”

“…In many studies, MazF homologs were cloned to araBAD promoter-controlled expression system (for example pBAD vector system) to evaluate the toxicity, and no interference of endogenous E. coli MazEF was reported. [20][21][22]32,33 The MazF homolog of Legionella pneumophila and Agrobacterium tumefaciens cloned to pBAD24 showed significant growth arrest of E. coli strain BW25113. 22,32 For generation of large amount of proteins, in some studies, MazF homologs were cloned into high expression vectors such as pColdTF, which contains a large chaperone tag to help solubility.…”

“…[20][21][22]32,33 The MazF homolog of Legionella pneumophila and Agrobacterium tumefaciens cloned to pBAD24 showed significant growth arrest of E. coli strain BW25113. 22,32 For generation of large amount of proteins, in some studies, MazF homologs were cloned into high expression vectors such as pColdTF, which contains a large chaperone tag to help solubility. The tag may also hinder the toxic activity of MazF.…”

“…No interplay between them and MazF from E. coli has been reported. [20][21][22] Here we reported a MazF homolog from Helicobacter macacae (MazFhm), a bacterium colonizing rhesus monkey and causes digestive symptoms. H. macacae infection of rhesus was used as a model for human H. pylori infection, 23 and a systemic search of MazF homolog in the latter species resulted no subject.…”