Bacterial Functions Involved in Cell Growth Control

Division Of Basic Sciences - Nci

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Abstract

Complex and rapidly adaptable regulatory networks allow bacteria such as E. coli to change metabolism to optimize growth and survival, in mammalian hosts and 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 two global searches for non-coding RNAs in E. coli, contributing significantly to the 100-200 regulatory RNAs that are now known. The majority of these small RNAs (sRNAs) bind tightly to the RNA chaperone Hfq. We and others have shown that sRNAs that bind tightly to Hfq act by pairing with multiple target mRNAs, regulating stability and translation of the mRNA, either positively or negatively, although some of these sRNAs also have additional roles. 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. In a genetic screen using a fluorescent reporter, we identified novel regulators of sRNA stability and function, including a new RNA sponge and two previously uncharacterized proteins. One of the new proteins disrupts all Hfq-based regulation. Our results show that this protein, a member of the broad transacetylase family and now named HqbA, directly interacts with the distal face of Hfq, helping in the quality control of RNA binding. Because this effect is independent of acetylation, the results suggest this is a bifunctional protein and its discovery suggests that yet other such previously unknown regulators may exist. In collaboration with Sarah Woodson (Johns Hopkins) and Aravind Iyer (NCBI) we are further examining the in vitro and in vivo activities and evolution of this protein. In other studies, in collaboration with Gisela Storz (NICHD), we are using a powerful global approach (RIL-Seq) to define which sRNAs interact with which mRNAs, extending this approach for the first time to cells carrying mutant alleles of Hfq that block RNA binding to a given face of this chaperone. These results are helping to define new sRNAs, how RNAs compete on Hfq, and provide a better understanding of Hfq function in vivo. Overall, we have developed highly efficient in vivo tools for studying sRNAs and the networks they reside in. Our focus is increasingly on the role of the sRNAs in complex bacterial behavior, investigations into the mechanism of sRNA function, and dissecting of novel mechanisms for regulating translation initiation. We have also returned to our interest in the phosphorelay regulatory cascade affecting capsule synthesis. This regulatory cascade falls into the family of "two-component systems", but is significantly more complex. Antimicrobial peptides and antibiotics induce this system, dependent upon an outer membrane lipoprotein, RcsF, and its interaction with a novel negative regulator. In one set of studies, we have investigated RcsF-independent signaling in this system and dissected three different inducing conditions, each of which works at a different stage in the phosphorelay. One acts on the periplasmic domain of the histidine kinase, not previously implicated in signaling. The proteins in this cascade also regulate aspects of the bacterial response to membrane stress, are needed for in vivo establishment of commensal growth, and are important virulence factors in Klebsiella. We are collaborating with S. Buchanan (NIDDK) to determine structures for this signalling system. 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 both better understand how the cell senses and responds to cell membrane stress and to investigate the development of novel antibiotics that act by perturbing this important regulon.