Undecaprenyl Pyrophosphate Involvement in Susceptibility of Bacillus subtilis to Rare Earth Elements

Abstract

bThe rare earth element scandium has weak antibacterial potency. We identified a mutation responsible for a scandium-resistant phenotype in Bacillus subtilis. This mutation was found within the uppS gene, which encodes undecaprenyl pyrophosphate synthase, and designated uppS86 (for the Thr-to-Ile amino acid substitution at residue 86 of undecaprenyl pyrophosphate synthase). The uppS86 mutation also gave rise to increased resistance to bacitracin, which prevents cell wall synthesis by inhibiting the dephosphorylation of undecaprenyl pyrophosphate, in addition to enhanced amylase production. Conversely, overexpression of the wild-type uppS gene resulted in increased susceptibilities to both scandium and bacitracin. Moreover, the mutant lacking undecaprenyl pyrophosphate phosphatase (BcrC) showed increased susceptibility to all rare earth elements tested. These results suggest that the accumulation of undecaprenyl pyrophosphate renders cells more susceptible to rare earth elements. The availability of undecaprenyl pyrophosphate may be an important determinant for susceptibility to rare earth elements, such as scandium. Rare earth elements consist of 17 elements, including scandium, yttrium, and the lanthanides (15 elements from lanthanum to lutetium in the periodic table). As rare earth elements have useful physical and chemical properties, these elements are of considerable importance in various industries. Although there have been many studies concerning their useful features, little is known about their biological effects in living cells. We recently reported that rare earth elements, especially scandium, activate secondary metabolism and extracellular enzyme production in certain microorganisms (12,15,31). In the Gram-positive model bacterium Bacillus subtilis, the addition of scandium to the growth medium stimulates production of an extracellular enzyme, amylase, and the dipeptide antibiotic, bacilysin (12). This effect was found to be exerted at the transcriptional level during the late stationary growth phase. These previous results suggest that the rare earth elements (especially scandium) have remarkable biological effects on microorganisms.Rare earth elements have long been known to have weak antimicrobial potency. For example, lanthanum has been reported to inhibit the growth of Streptococcus faecalis due to depletion of phosphate from the medium (34). It has also been reported that scandium and indium complexes of enterochelin have bacteriostatic effects on both Klebsiella pneumoniae (25) and Escherichia coli (26). However, there have been no reports addressing mutations conferring rare earth element resistance. In the present study, we isolated a scandium-resistant B. subtilis mutant and successfully identified the mutation responsible for its phenotype. The results presented here suggest that undecaprenyl pyrophosphate (C 55 -PP) is involved in susceptibility to rare earth elements, including scandium. This is the first report describing a key molecule involved in rare earth element susceptibility. MA...

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

“…The CRISPRi system has recently been adapted as a tool to repress bacterial gene expression and facilitate the analysis of essential gene functions (16). Our inability to construct a uppP bcrC double mutant suggested that these two genes might be a synthetic lethal pair (in contrast with a previous report [22,39]), although they could both be deleted when a third candidate phosphatase, YodM, was overexpressed. The ability of YodM to rescue cell growth in a uppP bcrC double mutant and its homology to other UPP-Pases support a possible role for YodM as a UPP-Pase that is active under growth conditions not yet identified.…”

“…While we could construct yodM bcrC and yodM uppP double mutants, we were unable to obtain a bcrC uppP double mutant in multiple attempts, despite previous suggestions that a double mutant had been obtained (22,39). For example, when chromosomal DNA from a bcrC::mls (macrolide-lincosamide-streptogramin B) strain was used to transform a uppP::spc (spectinomycin) strain, we recovered MLS r transformants, but only if the cells simultaneously acquired a functional copy of uppP (and therefore lost Spc r ).…”

“…UppP (YubB) was also speculated to function as a UPP-Pase based on homology but played a less important role than BcrC in bacitracin resistance (22). Mutations that affect UPP levels were later found to affect the sensitivity of B. subtilis to rare earth metals and, based on this phenotype, it was also suggested that BcrC was the major enzyme, with UppP having a minor role (39). However, this assay may report specifically on the level of extracellular UPP that is accessible for binding by rare earth metals.…”

“…Because of the nature of Und-P synthesis and recycling, it is reasonable to speculate that one UPP-Pase has a cytosol-facing active site and functions to convert UPP produced by UppS into Und-P for use in cell wall synthesis, while the other one recycles UPP produced as a product of the transglycosylase reaction catalyzed by class A PBPs. We speculate that the former activity is UppP, which might account for the minor role of this enzyme with respect to sensitivity to rare earth metals (39). After each addition to the growing peptidoglycan (or WTA) polymer, UPP is released and must be dephosphorylated and flipped back to the cytoplasmic face of the membrane for reuse.…”

Reducing the Level of Undecaprenyl Pyrophosphate Synthase Has Complex Effects on Susceptibility to Cell Wall Antibiotics

“…Comparison of the whole-genome sequences revealed a single-base substitution (A to C) in the RBS of the uppS gene that encodes an undecaprenyl pyrophosphate synthase, a key enzyme in cell wall peptidoglycan biosynthesis. The uppS gene is essential for the survival of B. subtilis (46), and the five-gene cluster, including uppS comprises a single operon (Fig. 1C) (47).…”