Characterization of the ModABC Molybdate Transport System of Pseudomonas putida in Nicotine Degradation.

Zhenyuan Xia,Hai-Lei Wei,Hong-Yue Zhang,Liping Lei

doi:10.3389/fmicb.2018.03030

Abstract

Pseudomonas putida J5 is an efficient nicotine-degrading bacterial strain that catabolizes nicotine through the pyrrolidine pathway. In our previous study, we used Tn5 transposon mutagenesis to investigate nicotine metabolism-associated genes, and 18 nicotine degradation-deficient mutants were isolated from 16,324 Tn5-transformants. Three of the mutants were Tn5 inserts into the modABC gene cluster that encoded an ABC-type, high-affinity, molybdate transporter. In-frame deletion of the modABC genes abolished the nicotine-degrading ability of strain J5, and complementation with modABC either from P. putida or Arthrobacter oxidans restored the degrading activity of the mutant to wild-type level. Nicotine degradation of J5 was inhibited markedly by addition of tungstate, a specific antagonist of molybdate. Molybdate at a non-physiologically high concentration (100 μM) fully restored nicotine-degrading activity and recovered growth of the modABC mutant in a nicotine minimal medium. Transcriptional analysis revealed that the expression of modABC was up-regulated at low molybdate concentrations and down-regulated at high moybdate concentrations, which indicated that at least one other system was able to transport molybdate, but with lower affinity. These results suggested that the molybdate transport system was essential to nicotine metabolism in P. putida J5.

Highlights

Molybdenum (Mo) plays essential roles in bacteria, because it serves as a cofactor for a number of enzymes that catalyze a variety of oxidation/reduction reactions, and it is involved in microbial metabolism of carbon, nitrogen, and sulfur (Hille, 1996; Kisker et al, 1997)
Pseudomonas putida was grown in Luria-Bertani (LB) medium or NI medium at 28◦C (Wei et al, 2009; Xia et al, 2015), and Escherichia coli was grown at 37◦C in LB medium
Similar results were obtained from complementation of mod with modABC genes of A. oxidans J4 (Figures 3A,B), which is an efficient nicotine-degrading, grampositive bacterium that was isolated from tobacco rhizospheres. These results indicated that the molybdate transport system was involved in nicotine metabolism in P. putida J5

Summary

Introduction

Molybdenum (Mo) plays essential roles in bacteria, because it serves as a cofactor for a number of enzymes that catalyze a variety of oxidation/reduction reactions, and it is involved in microbial metabolism of carbon, nitrogen, and sulfur (Hille, 1996; Kisker et al, 1997). In Escherichia coli, the first identified Mo transporter was the high affinity ModABC transport system, in which ModA was responsible for molybdate binding, ModB was the transmembrane component of the permease, and ModC provided the energizer function on the cytoplasmic side of the membrane (Maupin-Furlow et al, 1995). Mod homologs can be identified in ≥50 bacteria (Leimkuhler and Iobbi-Nivol, 2015). Among these bacteria, mod genes have been functionally characterized in only a few species, which include Rhodobacter capsulatus (Wang et al, 1993), Azotobacter vinelandii (Luque et al, 1993), Staphylococcus carnosus (Neubauer et al, 1999), and Bradyrhizobium japonicum (Delgado et al, 2006). Purified quinoline dehydrogenase from P. putida Chin IK indicated the presence of favin and molybdenum-binding pterin, and the enzyme activity was wholly dependent on the availability of molybdate in the growth medium (Blaschke et al, 1991)

Methods

Results

Conclusion