Phenotype Enhancement Screen of a Regulatory spx Mutant Unveils a Role for the ytpQ Gene in the Control of Iron Homeostasis

Abstract

Spx is a global regulator of genes that are induced by disulfide stress in Bacillus subtilis. The regulon that it governs is comprised of over 120 genes based on microarray analysis, although it is not known how many of these are under direct Spx control. Most of the Spx-regulated genes (SRGs) are of unknown function, but many encode products that are conserved in low %GC Gram-positive bacteria. Using a gene-disruption library of B. subtilis genomic mutations, the SRGs were screened for phenotypes related to Spx-controlled activities, such as poor growth in minimal medium and sensitivity to methyglyoxal, but nearly all of the SRG mutations showed little if any phenotype. To uncover SRG function, the mutations were rescreened in an spx mutant background to determine which mutant SRG allele would enhance the spx mutant phenotype. One of the SRGs, ytpQ was the site of a mutation that, when combined with an spx null mutation, elevated the severity of the Spx mutant phenotype, as shown by reduced growth in a minimal medium and by hypersensitivity to methyglyoxal. The ytpQ mutant showed elevated oxidative protein damage when exposed to methylglyoxal, and reduced growth rate in liquid culture. Proteomic and transcriptomic data indicated that the ytpQ mutation caused the derepression of the Fur and PerR regulons of B. subtilis. Our study suggests that the ytpQ gene, encoding a conserved DUF1444 protein, functions directly or indirectly in iron homeostasis. The ytpQ mutant phenotype mimics that of a fur mutation, suggesting a condition of low cellular iron. In vitro transcription analysis indicated that Spx stimulates transcription from the ytpPQR operon within which the ytpQ gene resides. The work uncovers a link between Spx and control of iron homeostasis.

Transcriptional and Phenotypic Characterization of Novel Spx-Regulated Genes in Streptococcus mutans

“…Recently, the Zuber lab utilized such an approach to uncover the role of Spx-regulated genes of unknown function in B . subtilis [ 40 ]. Specifically, double mutant strains were created by introducing mutations on selected Spx-regulated genes into the spx mutant background followed by a screen for enhanced oxidative stress sensitivity when compared to the spx single mutant.…”

Evidence that a Single Monomer of Spx Can Productively Interact with RNA Polymerase in Bacillus subtilis

“…His-tagged, A -depleted RNAP (SAd-RNAP) was purified from A mutant B. subtilis strain ORB5853 [rpoC-His 10 sigA(L366A)], in which the Leu366 substitution in A weakens the interaction with the RNAP core enzyme (52). B. subtilis cells were grown in 2ϫYT liquid containing chloramphenicol and neomycin at 37°C until the OD 600 of the culture reached 0.8 to 0.9 and were then harvested by centrifugation at 2,000 ϫ g. The pellets were frozen at Ϫ80°C prior to purification.…”

“…Spx governs a large regulon of genes that includes those encoding thioredoxin (trxA), thioredoxin reductase (trxB) (34), and methionine sulfoxide reductase (49) and genes whose products function in bacillithiol biosynthesis (9), cysteine biosynthesis (35), and iron uptake and metabolism (52). Spx has also been linked to control of biofilm-and virulence-related functions (19,39).…”

Methylglyoxal resistance in Bacillus subtilis: contributions of bacillithiol‐dependent and independent pathways

“…As a result, the Spx (thiol homeostasis), CtsR (misfolded protein response) and CymR (cysteine formation) regulons are induced. The Spx regulon includes ytpQ, which has been implicated in both iron homeostasis and prevention of oxidative protein damage after exposure to MG (Zuber et al, 2011).…”