The coenzyme B12 precursor 5,6-dimethylbenzimidazole is a flavin antagonist in Salmonella

Authors:

Lahiru Malalasekara1 and Jorge C. Escalante-Semerena1,*

doi: doi: 10.15698/mic2023.09.803
Volume 10, pp. 178 to 194, published 17/08/2023.

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Affiliations:

1 Department of Microbiology, University of Georgia, Athens USA.

Keywords: 

cobamide nucleobases, 5,6-dimethylbenzimidazole, CoB12 biosynthesis, benzimidazole antimicrobials, flavoenzyme inhibitors.

Corresponding Author(s):

Jorge C. Escalante-Semerena, 212C Biological Sciences Building, 120 Cedar Street, Athens, GA 30602 USA; jcescala@uga.edu

Conflict of interest statement:

The authors declare no conflict of interest with this work.

Please cite this article as:

Lahiru Malalasekara and Jorge C. Escalante-Semerena (2023). The coenzyme B12 precursor 5,6-dimethylbenzimidazole is a flavin antagonist in Salmonella. Microbial Cell 10(9): 178-194. doi: 10.15698/mic2023.09.803

© 2023 Malalasekara and Escalante-Semerena. This is an open-access article released under the terms of the Creative Commons Attribution (CC BY) license, which allows the unrestricted use, distribution, and reproduction in any medium, provided the original author and source are acknowledged.

Abstract:

Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) synthesizes adenosylcobalamin (AdoCbl, CoB12) de novo only under anoxic conditions, but it can assemble the lower ligand loop (a.k.a. the nucleotide loop) and can form the unique C-Co bond present in CoB12 in the presence or absence of molecular oxygen. During studies of nucleotide loop assembly in S. Typhimurium, we noticed that the growth of this bacterium could be arrested by the lower ligand nucleobase, namely 5,6-dimethylbenzimidazole (DMB). Here we report in vitro and in vivo evidence that shows that the structural similarity of DMB to the isoalloxazine moiety of flavin cofactors causes its deleterious effect on cell growth. We studied DMB inhibition of the housekeeping flavin dehydrogenase (Fre) and three flavoenzymes that initiate the catabolism of tricarballylate, succinate or D-alanine in S. Typhimurium. Notably, while growth with tricarballylate was inhibited by 5-methyl-benzimidazole (5-Me-Bza) and DMB, growth with succinate or glycerol was arrested by DMB but not by 5-Me-Bza. Neither unsubstituted benzimidazole nor adenine inhibited growth of S. Typhimurium at DMB inhibitory concentrations. Whole genome sequencing analysis of spontaneous mutant strains that grew in the presence of inhibitory concentrations of DMB identified mutations effecting the cycA (encodes D-Ala/D-Ser transporter) and dctA (encodes dicarboxylate transporter) genes and in the coding sequence of the tricarballylate transporter (TcuC), suggesting that increased uptake of substrates relieved DMB inhibition. We discuss two possible mechanisms of inhibition by DMB.