Salmonella infection is a common bacterial disease that affects the intestinal tract. It is estimated to cause about 1.2 million illnesses in the Unitd States each year, with about 23,000 hospitalizations and 450 deaths. A deep knowledge of its physiology and pathogenesis is important for identifying novel targets to develop new antimicrobial agents due to the emergence of multidrug-resistant Salmonella strains. Currently, the genetic code expansion strategy has been widely used to incorporate various unnatural amino acids (UAAs) into target proteins to solve the problems which are difficult or impossible to address by most classical methods due to the limited chemical diversity of the 20 natural amino acids. However, very few studies have been reported to incorporate UAAs in human pathogens such as Salmonella. Here, we will expand the genetic code in Salmonella by introducing an orthogonal translation system (OTS) which can efficiently incorporate various UAAs into target proteins to form labels and probes for confocal microscopy, paramagnetic resonance spectroscopy (EPR), Fourier transform infrared spectroscopy (FTIR), or fluorescence resonance energy transfer (FRET) (Aim 1). For proof-of-concept, we will use this OTS to investigate the spatial organization of bacterial microcompartments (BMCs) in Salmonella which has not been studied before (Aim 2). BMCs are large protein complexes containing viral capsid-like shells and encapsulated enzymes involved in various metabolic pathways. Studies have implicated 1,2-propanediol and ethanolamine degradations, which occur within BMCs, in Salmonella pathogenesis. We will incorporate p-azido-phenylalanine into the shell proteins of BMCs in Salmonella, followed by a Cu-free click reaction to form small-sized fluorescent probes for dynamic imaging to overcome the potential interference with the assemblies and functions of BMCs from the commonly used fluorescent protein tags. The spatial distribution of BMCs, the segregation of BMCs during cell divisions, key proteins of BMCs responsible for the spatial organization, and the interactions between BMC proteins and cytoskeletal components will be determined. These studies will provide new information and may reveal new paradigms in our knowledge of BMCs. In summary, we will show a practical application of the genetic code expansion strategy in studies of bacteria. Since Salmonella is an important model pathogen, the OTS we developed in this proposal could be broadly used by many laboratories to facilitate studies of microbial pathogenesis.

Public Health Relevance

Salmonella infection is a common bacterial disease that affects the intestinal tract and causes about 1.2 million illnesses in the United States each year, with about 23,000 hospitalizations and 450 deaths. We will expand the genetic code in Salmonella to investigate the spatial organization of bacterial microcompartments which have been implicated in pathogenesis. The proposed studies here can be broadly used by many laboratories to facilitate the studies in microbial pathogenesis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Exploratory/Developmental Grants (R21)
Project #
1R21AI119813-01
Application #
8950306
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Alexander, William A
Project Start
2015-06-15
Project End
2017-05-31
Budget Start
2015-06-15
Budget End
2016-05-31
Support Year
1
Fiscal Year
2015
Total Cost
$249,750
Indirect Cost
$99,750
Name
Yale University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06510
Venkat, Sumana; Chen, Hao; Stahman, Alleigh et al. (2018) Characterizing Lysine Acetylation of Isocitrate Dehydrogenase in Escherichia coli. J Mol Biol 430:1901-1911
Venkat, Sumana; Sturges, Jourdan; Stahman, Alleigh et al. (2018) Genetically Incorporating Two Distinct Post-translational Modifications into One Protein Simultaneously. ACS Synth Biol 7:689-695
Venkat, Sumana; Gregory, Caroline; Sturges, Jourdan et al. (2017) Studying the Lysine Acetylation of Malate Dehydrogenase. J Mol Biol 429:1396-1405
Gan, Qinglei; Fan, Chenguang (2017) Increasing the fidelity of noncanonical amino acid incorporation in cell-free protein synthesis. Biochim Biophys Acta Gen Subj 1861:3047-3052
Venkat, Sumana; Gregory, Caroline; Gan, Qinglei et al. (2017) Biochemical Characterization of the Lysine Acetylation of Tyrosyl-tRNA Synthetase in Escherichia coli. Chembiochem 18:1928-1934
Bryson, David I; Fan, Chenguang; Guo, Li-Tao et al. (2017) Continuous directed evolution of aminoacyl-tRNA synthetases. Nat Chem Biol 13:1253-1260
Venkat, Sumana; Nannapaneni, Dharma Theja; Gregory, Caroline et al. (2017) Genetically encoding thioacetyl-lysine as a non-deacetylatable analog of lysine acetylation in Escherichia coli. FEBS Open Bio 7:1805-1814
Venkat, Sumana; Gregory, Caroline; Meng, Kexin et al. (2017) A Facile Protocol to Generate Site-Specifically Acetylated Proteins in Escherichia Coli. J Vis Exp :
Fan, Chenguang; Ip, Kevan; Söll, Dieter (2016) Expanding the genetic code of Escherichia coli with phosphotyrosine. FEBS Lett 590:3040-7
Gan, Qinglei; Lehman, Brent P; Bobik, Thomas A et al. (2016) Expanding the genetic code of Salmonella with non-canonical amino acids. Sci Rep 6:39920

Showing the most recent 10 out of 13 publications