Abstract
Antibiotic-producing microorganisms have developed several self-resistance mechanisms to protect them from autotoxicity. Transporters belonging to the resistance- nodulation-division (RND) superfamily commonly confer multidrug resistance in Gram-negative bacteria. Phenazines are heterocyclic, nitrogen-containing and redox-active compounds that exhibit diverse activities. We previously identified six phenazines from Lysobacter antibioticus OH13, a soil bacterium emerging as a potential biocontrol agent. Among these phenazines, myxin, a di-N-oxide phenazine, exhibited potent activity against a variety of microorganisms. In this study, we identified a novel RND efflux pump gene cluster, designated lexABC, which is located far away in the genome from the myxin biosynthesis gene cluster. We found a putative LysR-type transcriptional regulator encoding gene lexR, which was adjacent to lexABC. Deletion of lexABC or lexR gene resulted in significant increasing susceptibility of strains to myxin and loss of myxin production. The results demonstrated that LexABC pump conferred resistance against myxin. The myxin produced at lower concentrations in these mutants was derivatized by deoxidation and O-methylation. Furthermore, we found that the abolishment of myxin with deletion of LaPhzB, which is an essential gene in myxin biosynthesis, resulted in significant downregulation of the lexABC. However, exogenous supplementation with myxin to LaPhzB mutant could efficiently induce the expression of lexABC genes. Moreover, lexR mutation also led to decreased expression of lexABC, which indicates that LexR potentially positively modulated the expression of lexABC. Our findings reveal a resistance mechanism against myxin of L. antibioticus, which coordinates regulatory pathways to protect itself from autotoxicity.
Highlights
Phenazines are a large class of heterocyclic, nitrogen-containing and redox-active natural products exhibiting a wide range of biological activities including antibiotic, antitumor, biofilm modulation and so on
Bioinformatics analysis of L. antibioticus OH13 genomic sequence revealed three genes encoding RND efflux pump proteins, which were similar to MexAB-OprM and MexGHIOpmD from P. aeruginosa, located distant from the phenazine biosynthetic gene cluster
The inhibition circles of myxin against the above mentioned mutants increased with the growing concentrations of myxin (0.125, 0.25, 0.5 μg), while 0.5 μg of myxin had little effect on the growth of the wild type (Supplementary Figure S3). These results demonstrated the important roles of the RND system LexABC and the regulator LexR in self-resistance of L. antibioticus OH13 against myxin
Summary
Phenazines are a large class of heterocyclic, nitrogen-containing and redox-active natural products exhibiting a wide range of biological activities including antibiotic, antitumor, biofilm modulation and so on. Natural phenazines have been identified as secondary metabolites primarily from Pseudomonas and Streptomyces (Laursen and Nielsen, 2004; Cimmino et al, 2012; Guttenberger et al, 2017). We previously isolated six phenazines from Lysobacter antibioticus OH13, of which myxin (Figure 1), an N-oxide phenazine, drew much attention due to its potent activity against microbes (Weigele et al, 1970; Zhao et al, 2016) and human cancer cells (Viktorsson et al, 2017). The unique feature of myxin is that it belongs to the class of phenazine compounds and to a class known as heterocyclic N-oxides with bioreductively activated, hypoxia-selective DNA-damaging properties (Mfuh and Larionov, 2015). The unique mode of action and strong biological activities of myxin make it an attractive target of study and development
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