Novel mechanism for scavenging of hypochlorite involving a periplasmic methionine-rich Peptide and methionine sulfoxide reductase

Abstract

Reactive chlorine species (RCS) defense mechanisms are important for bacterial fitness in diverse environments. In addition to the anthropogenic use of RCS in the form of bleach, these compounds are also produced naturally through photochemical reactions of natural organic matter and in vivo by the mammalian immune system in response to invading microorganisms. To gain insight into bacterial RCS defense mechanisms, we investigated Azospira suillum strain PS, which produces periplasmic RCS as an intermediate of perchlorate respiration. Our studies identified an RCS response involving an RCS stress-sensing sigma/anti-sigma factor system (SigF/NrsF), a soluble hypochlorite-scavenging methionine-rich periplasmic protein (MrpX), and a putative periplasmic methionine sulfoxide reductase (YedY1). We investigated the underlying mechanism by phenotypic characterization of appropriate gene deletions, chemogenomic profiling of barcoded transposon pools, transcriptome sequencing, and biochemical assessment of methionine oxidation. Our results demonstrated that SigF was specifically activated by RCS and initiated the transcription of a small regulon centering around yedY1 and mrpX. A yedY1 paralog (yedY2) was found to have a similar fitness to yedY1 despite not being regulated by SigF. Markerless deletions of yedY2 confirmed its synergy with the SigF regulon. MrpX was strongly induced and rapidly oxidized by RCS, especially hypochlorite. Our results suggest a mechanism involving hypochlorite scavenging by sacrificial oxidation of the MrpX in the periplasm. Reduced MrpX is regenerated by the YedY methionine sulfoxide reductase activity. The phylogenomic distribution of this system revealed conservation in several Proteobacteria of clinical importance, including uropathogenic Escherichia coli and Brucella spp., implying a putative role in immune response evasion in vivo.

Importance: Bacteria are often stressed in the environment by reactive chlorine species (RCS) of either anthropogenic or natural origin, but little is known of the defense mechanisms they have evolved. Using a microorganism that generates RCS internally as part of its respiratory process allowed us to uncover a novel defense mechanism based on RCS scavenging by reductive reaction with a sacrificial methionine-rich peptide and redox recycling through a methionine sulfoxide reductase. This system is conserved in a broad diversity of organisms, including some of clinical importance, invoking a possible important role in innate immune system evasion.

FIG 1

(A) Impact of chlorite treatment on wild-type and ΔsigF strains during log-phase growth in ALP medium. Chlorite was added in two successive spikes as indicated by the black arrows. (B) Difference in growth in ALP medium between wild-type and ΔnrsF cells when 160 µM chlorite is added immediately after inoculation during lag phase. OD600, optical density at 600 nm.

FIG 2

Anaerobic growth of wild-type, ΔsigF, and ΔnrsF strains in minimal medium containing 30 mM lactate and 20 mM chlorate.

FIG 3

Average per-base coverage (y axis) across all RNA-seq replicates in the region of the sigF-nrsF and yedY1Z1-mrpX operons. The inset shows the transcriptional start sites that were identified and the SigF promoter found upstream from the start sites. The nucleotides also conserved in Caulobacter crescentus and Bradyrhizobium japonicum are indicated in bold.

FIG 4

Heatmap generated using MeV to show the fitness values for all 17 genes that make up the core of the PRI (A), the cluster of genes that contains yedY1Z1 and yedY2Z2 (B), and the cluster of genes with the strongest defects specific to the chlorite stress condition (C). The gradient at the top indicates the magnitude of the growth defect; exact numbers and estimates of error can be found in Data Set S3 in the supplemental material.

FIG 5

Anaerobic growth curve showing the slight defect of the ΔyedY1 ΔyedY2 strain with 20 mM chlorate and severe defects of the ΔsigF ΔyedY2 strain with 2.5 mM or 20 mM chlorate. This experiment was carried out using minimal medium containing 30 mM lactate with a wild-type control.

FIG 6

(A) Western blot of total protein extracted from the wild type using an anti-myc tag primary antibody and HRP-conjugated secondary antibody. The samples run were from strains with several genotypes: ΔmrpX (negative control), ΔnrsF mrpX::myc (positive control), and mrpX::myc (experiment). Aliquots of the mrpX::myc strain were withdrawn at various times following a chlorite treatment (from 0 to 60 min, indicated). The arrow shows the migration of MrpX in the untreated positive control. (B) Reactions of purified MrpX with various ratios of RCS. All ratios were calculated based on the methionine molarity of the purified protein. [Met_MrpX], the concentration of methionine residues in MrpX, calculated by multiplying the number of predicted methionines found in MrpX by the total concentration of the MrpX peptide.

FIG 7

The genomic organization of genes of interest from several organisms, including perchlorate reducers and host-associated organisms.