This project will test two overarching hypotheses about bioenergetic work in respiratory bacteria, i.e., membrane-associated processes that are energized by an electrochemical gradient of protons across the membrane, the protonmotive force (pmf). The experimental system for much of the work is an alkaliphilic Bacillus that exhibits constitutive readiness to meet the challenge of a high bioenergetic work-load. Alkaliphilic Bacillus pseudofirmus OF4 also has special adaptations that facilitate its bioenergetic work under conditions of low pmf. The first overarching hypothesis is that specific structural features enhance the ability of the alkaliphile ATP synthase and its uniquely complex Mrp-type Na? antiporter to respectively support respiration- dependent ATP synthesis and cytoplasmic pH homeostasis at low pmf. Adaptive features are hypothesized to enable these pmf-user complexes to take advantage of sequestered paths of proton transfer from respiratory pumps and to gather protons in proton-poor environments. Other features of bioenergetic machinery, including ATP synthase, prevent proton leaks during oxidative phosphorylation (OXPHOS). The first two Specific Aims focus on structural-functional features of alkaliphile machinery.
Specific Aim #1 tests the hypothesis that the unique P51XXEXXP motif of the F1F0-ATP synthase c-rotor prevents proton leakiness. This will be assessed through comparative structural studies of the stable c-rotor rings from wild-type and a cP51A mutant. 2-D projection maps will be analyzed and high resolution structural information from 3D crystals will be sought.
Specific Aim #2 tests the hypothesis that the 7-protein Mrp hetero-oligomer is a consortium of antiporters and other transporters that are interdependent and synergistic, e.g. jointly presenting a large external surface engineered for proton-gathering. Proposed experiments include a screen for a putative anion transport function using a new Mrp mutant that confers high Na+resistance but lacks antiport activity. The second overarching hypothesis is that major pmf-consuming complexes such as ATP synthase and Mrp antiporter draw upon a network of cell components and regulators to meet bioenergetic challenges. In non-alkaliphiles that are not "hard-wired" for heavy bioenergetic work-loads, activation of a major pmf-consumer by sodium-alkali challenge elicits a systems response that facilitates management of challenge. The third and fourth Specific Aims focus on physiological contexts of bioenergetic work.
Specific Aim #3 probes the interplay between alkaliphile respiratory chain supercomplexes, cardiolipin and ATP synthase in OXPHOS at low pmf.
Specific Aim #4 will model and test a hypothesized systems response to Mnh (a Mrp homologue) activity in Staphylococcus aureus that results in a net increase in the transmembrane potential and impacts membrane lipid fluidity as well as sensitivity to antimicrobial peptides. The model will be tested and built via a panel of genetic, biochemical and molecular assays. The overall goal is to fill in gaps in our understanding of the mechanisms and plasticity of bioenergetic work and of the cell-wide responses that manage changes in bioenergetic work-load.

Public Health Relevance

Oxidative phosphorylation, whose mechanism will be probed in an established model system, is a central physiological process in which malfunctions are associated with inborn genetic disorders and age-associated diseases. The very unusual properties and physiological impacts of the Mrp-type antiporters, the other focus of the proposed work, are incompletely understood but are of importance because these membrane transporters are widespread in Gram-negative and Gram-positive pathogens where they have impacts on virulence and resistance to antimicrobial agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM028454-28
Application #
8265669
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Anderson, Vernon
Project Start
1981-09-01
Project End
2014-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
28
Fiscal Year
2012
Total Cost
$419,513
Indirect Cost
$172,013
Name
Icahn School of Medicine at Mount Sinai
Department
Pharmacology
Type
Schools of Medicine
DUNS #
078861598
City
New York
State
NY
Country
United States
Zip Code
10029
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DeCaen, Paul G; Takahashi, Yuka; Krulwich, Terry A et al. (2014) Ionic selectivity and thermal adaptations within the voltage-gated sodium channel family of alkaliphilic Bacillus. Elife 3:
Preiss, Laura; Langer, Julian D; Hicks, David B et al. (2014) The c-ring ion binding site of the ATP synthase from Bacillus pseudofirmus?OF4 is adapted to alkaliphilic lifestyle. Mol Microbiol 92:973-84
Morino, Masato; Suzuki, Toshiharu; Ito, Masahiro et al. (2014) Purification and functional reconstitution of a seven-subunit mrp-type na+/h+ antiporter. J Bacteriol 196:28-35
Preiss, Laura; Klyszejko, Adriana L; Hicks, David B et al. (2013) The c-ring stoichiometry of ATP synthase is adapted to cell physiological requirements of alkaliphilic Bacillus pseudofirmus OF4. Proc Natl Acad Sci U S A 110:7874-9
Price-Whelan, Alexa; Poon, Chun Kit; Benson, Meredith A et al. (2013) Transcriptional profiling of Staphylococcus aureus during growth in 2 M NaCl leads to clarification of physiological roles for Kdp and Ktr K+ uptake systems. MBio 4:
Liu, Jun; Hicks, David B; Krulwich, Terry A (2013) Roles of AtpI and two YidC-type proteins from alkaliphilic Bacillus pseudofirmus OF4 in ATP synthase assembly and nonfermentative growth. J Bacteriol 195:220-30
Krulwich, Terry A; Sachs, George; Padan, Etana (2011) Molecular aspects of bacterial pH sensing and homeostasis. Nat Rev Microbiol 9:330-43
Liu, Jun; Fackelmayer, Oliver J; Hicks, David B et al. (2011) Mutations in a helix-1 motif of the ATP synthase c-subunit of Bacillus pseudofirmus OF4 cause functional deficits and changes in the c-ring stability and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochemistry 50:5497-506
Janto, Benjamin; Ahmed, Azad; Ito, Masahiro et al. (2011) Genome of alkaliphilic Bacillus pseudofirmus OF4 reveals adaptations that support the ability to grow in an external pH range from 7.5 to 11.4. Environ Microbiol 13:3289-309

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