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, both aerobically and anaerobically, 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. A large number 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. 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 our previously developed approaches for screening targets of interest and the sRNAs regulating them, 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, somewhat surprisingly, directly by Hfq in the absence of sRNAs, dependent upon sites in the mutS 5'UTR. Hfq repression of MutS translation in stationary phase cells allows mutagenesis, considered to be a form of bet-hedging, as cells run out of nutrients. We are investigating whether Hfq regulates other genes in this sRNA-independent fashion, using global RNA seq results in a set of Hfq mutants, to find changes independent of the sRNA-binding face of Hfq. In another project, a small RNA processed from the 3' UTR of an operon encoding TCA proteins was found to regulate levels of the signaling molecule acetyl phosphate and change flux through the "acetate switch". This work demonstrates the importance of 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 not 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. 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 and differential effects of different hfq alleles on different sRNA:mRNA pairs. 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 recent work in our lab, 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. We identify genetic changes (mutations or overexpression) that perturb expression of the RpoS general stress factor, and use those to investigate regulatory pathways. In a screen of a plasmid library for negative regulation of RpoS, we identified two novel regulators, both of which appear to act by blocking the ability of sRNAs to activate RpoS translation. In the first case, the mRNA for a gene of the ribose catabolism operon was found to act as a small RNA decoy; this mRNA is expressed at high levels whenever the cell encounters ribose. Thus RpoS is down-regulated when ribose is present. A poorly characterized transcription factor was also found to counteract translational activation of RpoS, likely indirectly. 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 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 effects on sRNA-based regulation when overproduced. This is likely due to its direct interaction with the distal face of Hfq. Unexpectedly, this protein, a member of the broad transacetylase family, is needed under anaerobic conditions for resistance to high levels of Nickel. Our results suggest multiple important activities for this protein. Each of these 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. 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 perturb *TRUNCATED*