Mirubactin C rescues the lethal effect of cell wall biosynthesis mutations in Bacillus subtilis

Abstract

Growth of most rod-shaped bacteria is accompanied by the insertion of new peptidoglycan into the cylindrical cell wall. This insertion, which helps maintain and determine the shape of the cell, is guided by a protein machine called the rod complex or elongasome. Although most of the proteins in this complex are essential under normal growth conditions, cell viability can be rescued, for reasons that are not understood, by the presence of a high (mM) Mg²⁺ concentration. We screened for natural product compounds that could rescue the growth of mutants affected in rod-complex function. By screening > 2,000 extracts from a diverse collection of actinobacteria, we identified a compound, mirubactin C, related to the known iron siderophore mirubactin A, which rescued growth in the low micromolar range, and this activity was confirmed using synthetic mirubactin C. The compound also displayed toxicity at higher concentrations, and this effect appears related to iron homeostasis. However, several lines of evidence suggest that the mirubactin C rescuing activity is not due simply to iron sequestration. The results support an emerging view that the functions of bacterial siderophores extend well beyond simply iron binding and uptake.

Keywords: B. subtilis; cell wall mutants; chemical biological activity; mirubactin; siderophore.

FIGURE 1

(A) Structural assignment of mirubactin C (COSY = red, selected HMBC = blue) (B) Structures of mirubactin A (C) mirubactin C.

FIGURE 2

Comparative genomic analysis between Streptomyces drozdowiczii MDA8-470, Streptomyces coelicolor A3(2) and Actinosynnema mirum DSM 43827 in regards to the genetic locus of coelichelin and mirubactin. (Top) coelichelin biosynthetic gene cluster from Streptomyces coelicolor A3(2). (Middle) Genetic locus of the mirubactin and coelichelin gene cluster in Streptomyces drozdowiczii MDA8-470. (Bottom) mirubactin gene cluster from Actinosynnema mirum DSM 43827. Homology indicated by gray blocks based on BlastP analysis.

FIGURE 3

(A) mbl-recovery assay with mirubactin C (256 μg/ml gray, 32 μg/ml magenta, 8 μg/ml black, 4 μg/ml green, 2 μg/ml yellow) 20 mM MgSO₄ (blue), no addition (red). (B) mbl-recovery assay with chemically synthesized mirubactin C (15 μg/ml purple, 3.8 μg/ml black, 0.9 μg/ml turquoise), no addition (red), 20 mM MgSO₄ (blue) (C) mbl-recovery assay with mirubactin A (256 μg/ml gray, 32 μg/ml magenta, 8 μg/ml black, 4 μg/ml green, 2 μg/ml yellow), 20 mM MgSO₄ (blue), no addition (red). (D) Activity of purified mirubactin C against Bacillus subtilis 168CA, no addition (blue) and various concentrations of mirubactin C in gray. Error bars as a result of three technical replicates are shown in gray.

FIGURE 4

Effects of divalent cations and mirubactin C on several MgSO₄-dependent cell wall mutants. (A) Growth behavior of Bacillus subtilis 168CA (wt) and Δmbl, ΔmreB, Δ4 mutant (which lacks ponA and genes for the 3 other class A PBPs) on PAB plates in the presence of 20 mM MgSO₄, 4 μg/ml mirubactin C and in the absence of any additive. (B) Phenotype of Bacillus subtilis mutants Δmbl and ΔmreB in the presence of MgSO₄ and 4 μg/ml mirubactin C.

FIGURE 5

Cellular Fe and Mg content of Bacillus subtilis 168CA and Δmbl, ΔmreB, mutant cells, as determined by ICP-MS, after culture in PAB medium with/without supplementation with 20 mM MgSO₄ or 10 μg/ml mirubactin C. P-values are given for selected conditions which were calculated using the pairwise t-test function of the ggpubr package in R.

FIGURE 6

Rescue of mirubactin C toxicity in the presence of iron. Growth behavior of Bacillus subtilis 168CA; no addition (red), 60 μg/ml mirubactin C (green), 60 μg/ml mirubactin C and 3 μM FeCl₃ (blue) and 60 μg/ml mirubactin C and 50 μM FeCl₃ (black).