Bacterial Functions Involved in Cell Growth Control

Division Of Basic Sciences - Nci

Investigators

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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. We have also examined the mechanism by which Hfq operates to allow sRNAs to act, using in vivo studies of mutations in the RNA binding faces of Hfq. In a series of studies, in collaboration with G. Storz (NICHD) and with S. Woodson (JHU), we have carried out in vivo and in vitro dissection of Hfq that has changed our understanding of how this protein acts with sRNAs. This work demonstrated that Hfq-dependent sRNAs fall into two classes, defined by their behavior in different Hfq mutants. Class I sRNAs are rapidly degraded when used, most likely dependent upon pairing; their targets bind to the distal face. Class II sRNAs are generally more stable than Class I sRNAs, and their targets bind to rim sites in Hfq. These results help to explain previously observed competition between sRNAs, differential effects of different hfq alleles on different sRNA:mRNA pairs and provide insights into regulatory networks. 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 collaboration with the lab of G. Storz, we have examined the global effect of deleting the CTD of Hfq, and find 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. Our results define two independent roles for the CTD, one involved in reinforcing the distal face RNA binding activity of Hfq and the second defined by mutations at the C-terminal tip of Hfq. These different roles help to explain why previous studies came to conflicting conclusions about the role of the CTD. In a genetic screen using a recently developed bifunctional fluorescent reporter, we have identified novel regulators of sRNA stability and function, including a new RNA sponge and two previously uncharacterized proteins. One of the new proteins, the founding member of a family of proteins highly conserved in bacteria, specifically targets a subset of sRNAs for degradation. It requires polynucleotide phosphorylase (PNPase) to do this, suggesting that it may work in a complex with the PNPase. Work by others demonstrated a novel endonuclease activity for this family of proteins. The other new protein has global rather than sRNA-specific effects on sRNA-based regulation when overproduced.. Unexpectedly, this protein, a member of the broad transacetylase family. Our results show that this protein, 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. 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 regulatory cascade affecting capsule synthesis, in a collaboration with S. Buchanan and NCATs. 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. 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. Recent work has demonstrated that signaling to this cascade can also take place independently of the best-characterized pathway. 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.