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 #
5R01GM059969-16
Application #
8706890
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
2014-06-01
Budget End
2015-05-31
Support Year
16
Fiscal Year
2014
Total Cost
$402,900
Indirect Cost
$68,025
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
Ali, Mohamed M; Newsom, David L; González, Juan F et al. (2014) Fructose-asparagine is a primary nutrient during growth of Salmonella in the inflamed intestine. PLoS Pathog 10:e1004209
Vakulskas, Christopher A; Pannuri, Archana; Cortés-Selva, Diana et al. (2014) Global effects of the DEAD-box RNA helicase DeaD (CsdA) on gene expression over a broad range of temperatures. Mol Microbiol 92:945-58
Sterzenbach, Torsten; Nguyen, Kim T; Nuccio, Sean-Paul et al. (2013) A novel CsrA titration mechanism regulates fimbrial gene expression in Salmonella typhimurium. EMBO J 32:2872-83
Patterson-Fortin, Laura M; Vakulskas, Christopher A; Yakhnin, Helen et al. (2013) Dual posttranscriptional regulation via a cofactor-responsive mRNA leader. J Mol Biol 425:3662-77
Yakhnin, Alexander V; Baker, Carol S; Vakulskas, Christopher A et al. (2013) CsrA activates flhDC expression by protecting flhDC mRNA from RNase E-mediated cleavage. Mol Microbiol 87:851-66
Romeo, Tony; Vakulskas, Christopher A; Babitzke, Paul (2013) Post-transcriptional regulation on a global scale: form and function of Csr/Rsm systems. Environ Microbiol 15:313-24
Pannuri, Archana; Yakhnin, Helen; Vakulskas, Christopher A et al. (2012) Translational repression of NhaR, a novel pathway for multi-tier regulation of biofilm circuitry by CsrA. J Bacteriol 194:79-89
Yakhnin, Helen; Yakhnin, Alexander V; Baker, Carol S et al. (2011) Complex regulation of the global regulatory gene csrA: CsrA-mediated translational repression, transcription from five promoters by Eýýýýýýýý and Eýý(S), and indirect transcriptional activation by CsrA. Mol Microbiol 81:689-704
Martinez, Luary C; Yakhnin, Helen; Camacho, Martha I et al. (2011) Integration of a complex regulatory cascade involving the SirA/BarA and Csr global regulatory systems that controls expression of the Salmonella SPI-1 and SPI-2 virulence regulons through HilD. Mol Microbiol 80:1637-56
Yakhnin, Helen; Baker, Carol S; Berezin, Igor et al. (2011) CsrA represses translation of sdiA, which encodes the N-acylhomoserine-L-lactone receptor of Escherichia coli, by binding exclusively within the coding region of sdiA mRNA. J Bacteriol 193:6162-70

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