During the previous funding cycle a 5-gene operon named sat, for secretion and acid tolerance, was identified in Streptococcus mutans. This operon, located immediately downstream from the opuA osmotolerance operon, contains ffh that encodes Ffh, a key component of the bacterial signal recognition particle (SRP). S. mutans ffh mutants displayed acid intolerance and an acid inducible promoter was identified that allowed coordinate expression of the entire sat locus as a single mRNA transcript. Mutants displayed markedly reduced levels of H+/ATPase in isolated membranes leading to the belief that an acid tolerance response involves cotranslational translocation of membrane proteins by the SRP. In the present proposal, we will study further regulation of the sat locus, and its regulatory relationship to opuA, by means of Northern blot and real time RT-PCR analyses and investigate the role of YlxM, encoded by y/xM immediately upstream offfh, as a putative regulatory protein. We will confirm the presence of the SRP in S. mutans and determine its similarity to SRPs described from other prokaryotes by searching for the known complexed components, Ffh and the small cytoplasmic (4.5S) RNA, along with the membrane receptor, FtsY, in complexes immunoprecipitated from strain NG8 cellular extracts. We will employ the ffh mutant, MK4, and the wild-type strain NG8 to identify membrane proteins that require the SRP for translocation. Cells grown under steady-state (chemostat) conditions at pH 7 or 5 will serve as the source of cell fractions. Two-D gel electrophoresis, yeast two-hybrid, Ffh-GST affinity chromatography and affinity chromatography will be employed. Major physiologic parameters, including glycolysis and pH homeostasis, and genetic competence will be studied in this mutant which has been shown to have major alterations in these characteristics. Also during the previous funding cycle, the necessity of the central proline-rich repeat domain for surface expression of adhesin P1 (I/II) in S. mutans, but not secretion in E coli, was described. The role of gene products encoded within the clp, dnaK and groE operons in the intra-cellular turnover and chaperone-mediated surface expression of P1 will be assessed and intra-molecular interactions of P1 domains necessary for stability and translocation will be evaluated. By complementation of E. coli Sec-pathway mutants with an NG8 gene library, S. mutans homologues/analogues of Sec components will be identified. Immunoprecipitation of P1 and associated cellular proteins with anti-P1 antibodies will be used to identify chaperones or modifying factors involved in the export of this key adhesin. The project described in this proposal continues studies of membrane biogenesis and translocation of a major surface-localized protein of S. mutans.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
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Special Emphasis Panel (ZRG1-OBM-2 (05))
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Lunsford, Dwayne
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University of Florida
Schools of Dentistry
United States
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Binepal, Gursonika; Gill, Kamal; Crowley, Paula et al. (2016) Trk2 Potassium Transport System in Streptococcus mutans and Its Role in Potassium Homeostasis, Biofilm Formation, and Stress Tolerance. J Bacteriol 198:1087-100
Crowley, P J; Brady, L J (2016) Evaluation of the effects of Streptococcus mutans chaperones and protein secretion machinery components on cell surface protein biogenesis, competence, and mutacin production. Mol Oral Microbiol 31:59-77
Tang, Wenxing; Bhatt, Avni; Smith, Adam N et al. (2016) Specific binding of a naturally occurring amyloidogenic fragment of Streptococcus mutans adhesin P1 to intact P1 on the cell surface characterized by solid state NMR spectroscopy. J Biomol NMR 64:153-64
Sullan, Ruby May A; Li, James K; Crowley, Paula J et al. (2015) Binding forces of Streptococcus mutans P1 adhesin. ACS Nano 9:1448-60
Heim, Kyle P; Sullan, Ruby May A; Crowley, Paula J et al. (2015) Identification of a supramolecular functional architecture of Streptococcus mutans adhesin P1 on the bacterial cell surface. J Biol Chem 290:9002-19
Lewis, N E; Brady, L J (2015) Breaking the bacterial protein targeting and translocation model: oral organisms as a case in point. Mol Oral Microbiol 30:186-97
Williams, Matthew L; Crowley, Paula J; Hasona, Adnan et al. (2014) YlxM is a newly identified accessory protein that influences the function of signal recognition particle pathway components in Streptococcus mutans. J Bacteriol 196:2043-52
Liao, Sumei; Klein, Marlise I; Heim, Kyle P et al. (2014) Streptococcus mutans extracellular DNA is upregulated during growth in biofilms, actively released via membrane vesicles, and influenced by components of the protein secretion machinery. J Bacteriol 196:2355-66
Heim, Kyle P; Crowley, Paula J; Long, Joanna R et al. (2014) An intramolecular lock facilitates folding and stabilizes the tertiary structure of Streptococcus mutans adhesin P1. Proc Natl Acad Sci U S A 111:15746-51
Heim, Kyle P; Crowley, Paula J; Brady, L Jeannine (2013) An intramolecular interaction involving the N terminus of a streptococcal adhesin affects its conformation and adhesive function. J Biol Chem 288:13762-74

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