Insight into posttranscriptional regulatory mechanisms and global regulatory circuitry will be sought through the study of the carbon storage regulatory system (Csr) of Escherichia coli. Csr includes: CsrA, an RNA binding protein that regulates translation and/or the stability of a large number of target mRNAs;CsrB and CsrC, noncoding sRNAs that use molecular mimicry to sequester and antagonize CsrA;BarA-UvrY, a two- component signal transduction system that activates transcription of csrB and csrC;and CsrD, a protein that specifically targets CsrB and CsrC RNAs for degradation by RNase E. In E. coli, CsrA regulates metabolism, motility, and multicellular behavior on a broad scale. Our recently published RNA-seq studies identified 712 different RNAs that bind to CsrA, including mRNAs for >40 regulatory factors. Our published and preliminary findings further reveal that Csr is reciprocally linked to a number of other global regulatory circuits, implying tht Csr affects bacterial physiology on a global scale. We hypothesize that this complex circuitry allows Csr to reinforce the transcriptional effects of stress response systems at a posttranscriptional level.
The specific aims of this proposal are: 1) Identify reciprocal regulatoy interactions of Csr with other global regulatory systems and elucidate novel CsrA-mediated regulatory mechanisms, which have come to light from the results of genetic screens and RNA-seq studies. We will focus on CsrA-mediated regulation of two stress-response sigma factors (?S and ?E), and two ribonucleases that participate in bulk mRNA turnover (RNase E and PNPase). 2) Define the CsrB/C RNA turnover pathway, focusing on the mechanism by which CsrD promotes RNase E-dependent cleavage. Of particular interest, our preliminary results indicate that CsrD specifically couples CsrB synthesis with targeted turnover by RNase E, suggesting that the influence of RNA synthesis on RNA decay may be an underappreciated feature of RNA biology. 3) Determine the molecular mechanisms by which DeaD, a DEAD-box RNA helicase, positively regulates CsrB/C RNA levels. Our preliminary results indicate that DeaD directly affects expression of the response regulator UvrY, which is required for csrB/C transcription. Our results further suggest that DeaD unwinds a secondary structure that inhibits translation of uvrY. Thus, these studies will determine the mechanism of an important regulatory influence on the Csr system and provide fundamental insight into the role of DeaD helicase in translation. The long-range objectives of these studies are to fully understand the regulatory components, genetic circuitry, molecular mechanisms, and biological functions of the Csr system, thereby defining basic principles that underpin a regulatory super-network. Highly conserved regulatory systems homologous to Csr control the expression of virulence factors and/or transmission traits in numerous human, animal, and plant pathogens, responsible for diverse infections. Thus, these studies will provide fundamental understanding of the regulation of bacterial metabolism and pathogenesis and may suggest novel therapeutic and/or vaccine strategies.

Public Health Relevance

Insight into global regulatory circuitry will be sought through the study of a novel paradigm in post- transcriptional genetic regulation, the carbon storage regulatory (Csr) system of Escherichia coli. The Csr system controls bacterial metabolism, physiology and cellular behavior on a broad scale, and regulates the expression of virulence factors in plant, animal and human pathogens. Thus, these studies will provide fundamental understanding of the regulation of bacterial physiology and pathogenesis, and may suggest novel preventative or therapeutic approaches for bacterial infections.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM059969-15A1
Application #
8439814
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Reddy, Michael K
Project Start
1999-08-01
Project End
2017-05-31
Budget Start
2013-08-01
Budget End
2014-05-31
Support Year
15
Fiscal Year
2013
Total Cost
$413,901
Indirect Cost
$79,026
Name
University of Florida
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
969663814
City
Gainesville
State
FL
Country
United States
Zip Code
32611
Leng, Yuanyuan; Vakulskas, Christopher A; Zere, Tesfalem R et al. (2016) Regulation of CsrB/C sRNA decay by EIIA(Glc) of the phosphoenolpyruvate: carbohydrate phosphotransferase system. Mol Microbiol 99:627-39
Mukherjee, Sampriti; Oshiro, Reid T; Yakhnin, Helen et al. (2016) FliW antagonizes CsrA RNA binding by a noncompetitive allosteric mechanism. Proc Natl Acad Sci U S A 113:9870-5
Chowdhury, Nityananda; Kwan, Brian W; McGibbon, Louise C et al. (2016) Toxin MqsR cleaves single-stranded mRNA with various 5' ends. Microbiologyopen 5:370-7
Pannuri, Archana; Vakulskas, Christopher A; Zere, Tesfalem et al. (2016) Circuitry Linking the Catabolite Repression and Csr Global Regulatory Systems of Escherichia coli. J Bacteriol 198:3000-3015
Vakulskas, Christopher A; Leng, Yuanyuan; Abe, Hazuki et al. (2016) Antagonistic control of the turnover pathway for the global regulatory sRNA CsrB by the CsrA and CsrD proteins. Nucleic Acids Res 44:7896-910
Wang, Yan; Andole Pannuri, Archana; Ni, Dongchun et al. (2016) Structural Basis for Translocation of a Biofilm-supporting Exopolysaccharide across the Bacterial Outer Membrane. J Biol Chem 291:10046-57
Vakulskas, Christopher A; Potts, Anastasia H; Babitzke, Paul et al. (2015) Regulation of bacterial virulence by Csr (Rsm) systems. Microbiol Mol Biol Rev 79:193-224
Abbott, Zachary D; Yakhnin, Helen; Babitzke, Paul et al. (2015) csrR, a Paralog and Direct Target of CsrA, Promotes Legionella pneumophila Resilience in Water. MBio 6:e00595
Zere, Tesfalem R; Vakulskas, Christopher A; Leng, Yuanyuan et al. (2015) Genomic Targets and Features of BarA-UvrY (-SirA) Signal Transduction Systems. PLoS One 10:e0145035
Camacho, Martha I; Alvarez, Adrian F; Chavez, Ricardo Gonzalez et al. (2015) Effects of the global regulator CsrA on the BarA/UvrY two-component signaling system. J Bacteriol 197:983-91

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