In pathogenic bacteria, complex systems of control have evolved that activate defenses against the various toxic agents used by immune cells to combat infection. These systems regulate important virulence determinants, which are targets of drugs designed to alleviate infectious disease. The low GC Gram-positive bacteria have a unique system of transcriptional control, mediated by the protein Spx, that functions in the response to toxic oxidants, including those produced by phagocytic immune cells. Spx is a transcriptional regulator that we discovered in the spore-forming bacterium, Bacillus subtilis, but is also found in Staphylococcus aureus, Streptococcus pneumoniae, Listeria monocytogenes, and Bacillus anthracis. Spx has been shown to control the expression of virulence-associated activities in S. aureus, and is produced to high levels in Listeria monocytogenes and B. anthracis during host cell invasion. Spx defines a family of regulators that structurally resemble the enzyme arsenate reductase. It bears an N-terminal CXXC redox disulfide center that regulates its activity. Its mechanism of control is unique in that free Spx does not exhibit DNA-binding activity, but when bound to RNA polymerase (RNAP) in its oxidized disulfide form, it directs the enzyme to transcribe specific genes that function in oxidative stress defense. Spx concentration is reduced to low levels after recovery from stress by the protease ClpXP and the Spx-binding protein YjbH, which accelerates Spx proteolysis. The goal of the proposed research is to learn how B. subtilis Spx activates transcription initiation and how YjbH functions to mediate Spx proteolysis. Specific nucleotide sequence elements have been identified in the promoter regions of genes controlled by Spx. These sequences bind a complex of Spx and the C-terminal domain of RNAP subunit (CTD). The promoter DNA sequence and the structural requirements of CTD/Spx for promoter DNA binding will be determined. Furthermore, the dynamics of subunit positioning in RNAP holoenzyme within the Spx-activated transcription complex will be explored. Efficient proteolysis requires YjbH, a zinc-binding protein that interacts with Spx and accelerates ClpXP-catalyzed Spx proteolysis. Studies are proposed to investigate the oxidant-induced stabilization of Spx and redox control of YjbH activity. This study will focus on the Zn-binding site of the adaptor to test the model that oxidation of Zn-coordinating cysteine residues causes inactivation of YjbH and release of Spx. Differential thiol labeling and mass spectrometry will be performed to investigate the extent and nature of thiol oxidation of Spx and YjbH in vivo. A small protein factor, YirB that interacts with YjbH, will be studied to determine if it is a member of a growing list of small proteins that prevent protease-catalyzed degradation by inhibiting protease adaptor proteins.

Public Health Relevance

Complex systems of control have evolved in disease-causing bacteria to mobilize defenses that counter the toxic oxidizing agents produced by phagocytic immune cells. The regulatory protein Spx controls important components of the bacterial response to oxidative attack, and the study of its mechanism of action will uncover targets for neutralizing infectious microorganisms.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM045898-21
Application #
8528615
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Reddy, Michael K
Project Start
1992-02-01
Project End
2014-07-31
Budget Start
2013-08-01
Budget End
2014-07-31
Support Year
21
Fiscal Year
2013
Total Cost
$396,453
Indirect Cost
$130,243
Name
Oregon Health and Science University
Department
Engineering (All Types)
Type
Schools of Medicine
DUNS #
096997515
City
Portland
State
OR
Country
United States
Zip Code
97239
Chan, Chio Mui; Hahn, Erik; Zuber, Peter (2014) Adaptor bypass mutations of Bacillus subtilis?spx suggest a mechanism for YjbH-enhanced proteolysis of the regulator Spx by ClpXP. Mol Microbiol 93:426-38
Nakano, Michiko M; Kominos-Marvell, Wren; Sane, Bhagyashree et al. (2014) spxA2, encoding a regulator of stress resistance in Bacillus anthracis, is controlled by SaiR, a new member of the Rrf2 protein family. Mol Microbiol 94:815-27
Lin, Ann A; Walthers, Don; Zuber, Peter (2013) Residue substitutions near the redox center of Bacillus subtilis Spx affect RNA polymerase interaction, redox control, and Spx-DNA contact at a conserved cis-acting element. J Bacteriol 195:3967-78
Chan, Chio Mui; Garg, Saurabh; Lin, Ann A et al. (2012) Geobacillus thermodenitrificans YjbH recognizes the C-terminal end of Bacillus subtilis Spx to accelerate Spx proteolysis by ClpXP. Microbiology 158:1268-78
Lin, Ann A; Zuber, Peter (2012) Evidence that a single monomer of Spx can productively interact with RNA polymerase in Bacillus subtilis. J Bacteriol 194:1697-707
Zuber, Peter; Chauhan, Shefali; Pilaka, Praseeda et al. (2011) Phenotype enhancement screen of a regulatory spx mutant unveils a role for the ytpQ gene in the control of iron homeostasis. PLoS One 6:e25066
Kommineni, Sushma; Garg, Saurabh K; Chan, Chio Mui et al. (2011) YjbH-enhanced proteolysis of Spx by ClpXP in Bacillus subtilis is inhibited by the small protein YirB (YuzO). J Bacteriol 193:2133-40
Nakano, Michiko M; Lin, Ann; Zuber, Cole S et al. (2010) Promoter recognition by a complex of Spx and the C-terminal domain of the RNA polymerase alpha subunit. PLoS One 5:e8664
Garg, Saurabh K; Kommineni, Sushma; Henslee, Luke et al. (2009) The YjbH protein of Bacillus subtilis enhances ClpXP-catalyzed proteolysis of Spx. J Bacteriol 191:1268-77
Zuber, Peter (2009) Management of oxidative stress in Bacillus. Annu Rev Microbiol 63:575-97

Showing the most recent 10 out of 27 publications