Bacteria lacking trans-translation activity have defects in development, differentiation, virulence, stress responses, and viability, but the reasons trans-translation is required for these processes are not known. Our long-term goal is to understand the mechanism of action and physiological role of trans-translation in bacteria. The overall objective of this application is to understand how regulation of trans-translation activity and substrate selectivity is used to control genetic pathways responsible for differentiation and cell cycle progression in Caulobacter crescentus. Our central hypothesis is that bacteria regulate the generation of key substrates for trans-translation, as well as the availability of tmRNA and SmpB, to control genetic circuits responsible for initiation of DNA replication and other physiological processes. The rationale for the proposed research is to establish a paradigm for post-transcriptional regulation through controlled trans-translation. The central hypothesis will be tested by pursuing the following specific aims: 1) identify the mechanism for generation of trans-translation substrates in C. crescentus;2) identify mechanisms for regulation of trans- translation activity;and 3) determine the role of trans-translation in co-translational secretion. Under the first aim, genetic and biochemical approaches will be used to identify cis- and trans-acting factors responsible for substrate selectivity through a nucleic acid motif found in 66% of trans-translation substrates. In the second aim, genetic and biochemical assays will be used to identify the molecular interactions responsible for cell- cycle regulated SmpB proteolysis and tmRNA degradation by RNase R. In the third aim, the molecular basis for the genetic interaction between trans-translation and the SecYEG translocator will be tested. The proposed research is significant because it will provide a paradigm for post-transcriptional gene regulation by trans- translation that is required for bacterial growth and development. This paradigm will open the door to a more detailed understanding of how bacteria rapidly change their gene expression profiles in response to environmental and developmental cues. Elucidation of this process in a model system for bacterial development is expected to provide a framework for interpreting a wide array of data from other species, and a basis for understanding how trans-translation is used by bacteria in the environment and during pathogenesis.

Public Health Relevance

The proposed studies are of a ubiquitous pathway for post-transcriptional gene regulation that is crucial for bacterial physiology, including development and pathogenesis. These studies are expected to reveal the molecular mechanisms for regulating trans-translation activity and substrate generation that are required for differentiation and cell-cycle control in Caulobacter crescentus. These mechanisms are likely to function in other species that require trans-translation for differentiation, virulence, or viability, and will be tested in the pathogenic bacterium Shigella flexneri. Our results will enable the investigation of these pathways in other medically important bacteria, and the targeting of these systems for antibacterial agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM068720-08
Application #
8478129
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Bender, Michael T
Project Start
2004-05-01
Project End
2015-05-31
Budget Start
2013-06-01
Budget End
2014-05-31
Support Year
8
Fiscal Year
2013
Total Cost
$239,355
Indirect Cost
$77,235
Name
Pennsylvania State University
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
003403953
City
University Park
State
PA
Country
United States
Zip Code
16802
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Goralski, Tyler D P; Dewan, Kalyan K; Alumasa, John N et al. (2016) Inhibitors of Ribosome Rescue Arrest Growth of Francisella tularensis at All Stages of Intracellular Replication. Antimicrob Agents Chemother 60:3276-82
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El-Mowafi, S A; Sineva, E; Alumasa, J N et al. (2015) Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay. Antimicrob Agents Chemother 59:193-205
Keiler, Kenneth C; Feaga, Heather A (2014) Resolving nonstop translation complexes is a matter of life or death. J Bacteriol 196:2123-30
Feaga, Heather A; Viollier, Patrick H; Keiler, Kenneth C (2014) Release of nonstop ribosomes is essential. MBio 5:e01916
El-Mowafi, Shaima A; Alumasa, John N; Ades, Sarah E et al. (2014) Cell-based assay to identify inhibitors of the Hfq-sRNA regulatory pathway. Antimicrob Agents Chemother 58:5500-9
Keiler, Kenneth C; Alumasa, John N (2013) The potential of trans-translation inhibitors as antibiotics. Future Microbiol 8:1235-7
Ramadoss, Nitya S; Zhou, Xin; Keiler, Kenneth C (2013) tmRNA is essential in Shigella flexneri. PLoS One 8:e57537
Ramadoss, Nitya S; Alumasa, John N; Cheng, Lin et al. (2013) Small molecule inhibitors of trans-translation have broad-spectrum antibiotic activity. Proc Natl Acad Sci U S A 110:10282-7

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