Bacterial Functions Involved in Cell Growth Control

参与细胞生长控制的细菌功能

• 批准号: 10262026
• 负责人: SUSAN GOTTESMAN
• 金额: $ 117.89万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262026
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Acetates; Affect; Alleles; Amino Acids; Antibiotics; Area; Bacteria; Bacteria sigma factor KatF protein; Bacterial Genes; Behavior; Binding; Binding Sites; Biological Assay; C-terminal; Carbon; Cell Communication; Cells; Collaborations; Complex; Core Protein; Development; Dissection; Distal; Equilibrium; Escherichia coli; Eukaryotic Cell; Face; Family; Genetic Transcription; Genetic Translation; Goals; Growth; In Vitro; Klebsiella; Laboratories; Literature; Mediating; Membrane; Messenger RNA; Metabolism; Methods; Mismatch Repair; Molecular Chaperones; Mutagenesis; Mutation; National Institute of Child Health and Human Development; Nutrient; Operon; Organism; Pathway interactions; Phase; Physiological; Play; Porifera; Post-Transcriptional Regulation; Process; Proteins; Publishing; RNA; RNA Binding; RNA Splicing; RNA Stability; Regulation; Regulatory Pathway; Regulon; Reporter; Resistance; Ribonucleases; Role; Running; Series; Sigma Factor; Signal Transduction; Signaling Molecule; Site; Small RNA; Stress; Surface; System; Untranslated RNA; Virulence Factors; Work; acetyl phosphate; base; biological adaptation to stress; capsule; cell behavior; cell growth; falls; genetic regulatory protein; in vivo; insight; interest; mutant; novel; pathogenic bacteria; response; screening; small molecule; symbiont; tool; transcription factor

Complex and rapidly adaptable regulatory networks allow bacteria such as E. coli to cComplex and rapidly adaptable regulatory networks allow bacteria such as E. coli to change metabolism to optimize growth and survival in mammalian hosts, outside of the host and in response to a variety of stresses. In the last twenty years, the important roles of small non-coding RNAs in regulation in all organisms have been recognized. Our laboratory, in collaboration with others, undertook multiple global searches for non-coding RNAs in E. coli, contributing significantly to the 100-200 regulatory RNAs that are now identified. A large number of these small RNAs (sRNAs) bind tightly to the RNA chaperone Hfq and require Hfq for their function. We and others have shown that sRNAs that bind to Hfq act by pairing with multiple target mRNAs, regulating stability and translation of the mRNAs, either positively or negatively. Our lab has studied many of these sRNAs in detail. Each sRNA is regulated by different stress conditions, suggesting that the sRNA plays an important role in adapting to stress. We have also examined the mechanism by which Hfq operates to allow sRNAs to act. The lab continues to investigate the in vivo roles of small RNAs, identifying the regulatory networks they participate in and their roles in those networks. Using approaches for screening targets of interest and the sRNAs regulating them that were previously developed in the laboratory, we continue to investigate regulatory pathways for sRNAs. mutS, encoding a component of the mismatch repair system, was found to be regulated by a small RNA, ArcZ, and, unexpectedly, directly by Hfq in the absence of sRNAs, dependent upon sites in the mutS 5'UTR. Mutation of these sites leads to increased levels of MutS protein in stationary phase cells and decreased mutagenesis, demonstrating the role of post-transcriptional regulation in allowing mutagenesis as cells run out of nutrients. We are now investigating approaches to identifying other mRNAs regulated by Hfq in the absence of sRNAs. In another project, a small RNA processed from the 3' UTR of an operon encoding TCA proteins has been found to regulate levels of the signaling molecule acetyl phosphate and change flux through the "acetate switch". Lessons learned from this project suggest the importance of many other previously unappreciated sRNAs made from 3' UTRs. The action of these small RNAs depends on the RNA chaperone Hfq, a protein with homology to the Lsm and Sm families of eukaryotic proteins involved in RNA splicing and other functions. Hfq binds both to sRNAs and to mRNAs, and stimulates pairing, but exactly how it does this has been clear. In a series of studies in collaboration with G. Storz (NICHD) and with S. Woodson (JHU), we have carried out an in vivo dissection of Hfq that has changed our understanding of how this protein acts with sRNAs. We have found that the Hfq-dependent sRNAs fall into two classes, defined by their behavior in different Hfq mutants. All of these sRNAs depend on the known sRNA binding site on the proximal face of Hfq for in vivo stability. Class I sRNAs are rapidly degraded when used, most likely dependent upon pairing; their targets bind to the distal face of Hfq. Class II sRNAs are generally more stable than Class I sRNAs, and their targets bind to rim sites on Hfq. These results help to explain previously observed competition between sRNAs and differential effects of different hfq alleles on different sRNA:mRNA pairs. The major activities of Hfq are dependent upon the highly conserved core of the protein. The C-terminus of E. coli Hfq (CTD) is unstructured, and its role has been unclear. In collaboration with S. Woodson, we defined in vivo and in vitro roles for the CTD in stabilization and release of Class II sRNAs. In subsequent work in our laboratory in collaboration with the laboratory of G. Storz, we examined the global effect of deleting the CTD of Hfq, and found only subtle effects on RNA accumulation. However, in combination with mutations on the RNA binding faces of Hfq, loss of the CTD can have synergistic effects, leading us to define two independent functions for this region. Amino acids right beyond the core appear to contribute to the activity of the distal face of Hfq, while sequences near the C-terminal tip and upstream linker of the Hfq CTD synergize with the rim of Hfq, affecting sRNA accumulation and function. These separate functions help to explain some of the contradictions in the published literature. In another collaboration with the Storz lab, we combined the newly developed RIL-Seq method for global identification of sRNA-mRNA pairs with selected Hfq mutant alleles to dissect the roles of the various Hfq RNA binding surfaces on pairing. This further allows us to understand more about how Class I and Class II sRNAs differ. Using a newly developed bi-functional fluorescent reporter we have identified novel regulators of sRNA stability and function, including a new RNA sponge and two previously uncharacterized proteins, one of which has global effects on sRNA-based regulation and another of which has effects on the stability of specific sRNAs. The specific regulator appears to function as a unique adaptor for a major ribonuclease. Each of these novel functions opens up previously unknown levels of regulation of sRNA function. Overall, we have developed highly efficient in vivo tools for studying sRNAs and the networks they reside in. We have also returned to our interest in the regulatory cascade affecting capsule synthesis, in a collaboration with S. Buchanan and NCATs. The proteins in this cascade also regulate many bacterial genes involved in the response to membrane stress, are needed for in vivo establishment of commensal growth, and are important virulence factors in Klebsiella. Studies on the Interactions of the components of the regulatory cascade have changed our understanding of signal transduction through this system, demonstrating that a critical negative regulator of signaling acts by interaction with a phosphorelay protein, leading to a major revision in our understanding of signaling in this system and providing new insight into the general principles affecting related and widespread signaling systems. We have developed an efficient assay for screening for small molecules that activate or inactivate the cascade and have found evidence for effects of a variety of antibiotics in inducing the system. The long-term goal of this is to investigate the development of novel antibiotics that act by perturbing this important regulon.