Genome-Wide Transcriptional Regulation and Chromosome Structural Arrangement by GalR in E. coli.

The regulatory protein, GalR, is known for controlling transcription of genes related to D-galactose metabolism in Escherichia coli. Here, using a combination of experimental and bioinformatic approaches, we identify novel GalR binding sites upstream of several genes whose function is not directly related to D-galactose metabolism. Moreover, we do not observe regulation of these genes by GalR under standard growth conditions. Thus, our data indicate a broader regulatory role for GalR, and suggest that regulation by GalR is modulated by other factors. Surprisingly, we detect regulation of 158 transcripts by GalR, with few regulated genes being associated with a nearby GalR binding site. Based on our earlier observation of long-range interactions between distally bound GalR dimers, we propose that GalR indirectly regulates the transcription of many genes by inducing large-scale restructuring of the chromosome.

Genome scale patterns of supercoiling in a bacterial chromosome.

DNA in bacterial cells primarily exists in a negatively supercoiled state. The extent of supercoiling differs between regions of the chromosome, changes in response to external conditions and regulates gene expression. Here we report the use of trimethylpsoralen intercalation to map the extent of supercoiling across the Escherichia coli chromosome during exponential and stationary growth phases. We find that stationary phase E. coli cells display a gradient of negative supercoiling, with the terminus being more negatively supercoiled than the origin of replication, and that such a gradient is absent in exponentially growing cells. This stationary phase pattern is correlated with the binding of the nucleoid-associated protein HU, and we show that it is lost in an HU deletion strain. We suggest that HU establishes higher supercoiling near the terminus of the chromosome during stationary phase, whereas during exponential growth DNA gyrase and/or transcription equalizes supercoiling across the chromosome.

Metabolite changes signal genetic regulatory mechanisms for robust cell behavior.

UNLABELLED: Exploiting mechanisms of utilizing the sugar d-galactose in Escherichia coli as a model system, we explored the consequences of accumulation of critical intermediates of the d-galactose metabolic pathways by monitoring cell growth, metabolites, and transcript profiles. These studies revealed both metabolic network changes far from the d-galactose pathway and changes in the global gene regulatory network. The concentration change of a critical intermediate disturbs the equilibrium state, generating a ripple effect through several metabolic pathways that ends up signaling up- or downregulation of specific sets of genes in a programmed manner to cope with the imbalance. Such long-range effects on metabolites and genetic regulatory mechanisms not only may be a common feature in bacteria but very likely operate during cellular development and differentiation in higher organisms as well as in disease cells, like cancer cells. IMPORTANCE: Metabolite accumulation can create adverse intracellular conditions that are relieved by compensatory immediate changes of metabolite pools and later changes of transcript levels. It has been known that gene expression is normally regulated by added catabolic substrates (induction) or anabolic end products (repression). It is becoming apparent now that change in the concentration of metabolic intermediates also plays a critical role in genetic regulatory networks for metabolic homeostasis. Our study provides new insight into how metabolite pool changes transduce signals to global gene regulatory networks.

Noncoding RNAs binding to the nucleoid protein HU in Escherichia coli.

Some unidentified RNA molecules, together with the nucleoid protein HU, were suggested to be involved in the nucleoid structure of Escherichia coli. HU is a conserved protein known for its role in binding to DNA and maintaining negative supercoils in the latter. HU also binds to a few RNAs, but the full spectrum of its binding targets in the cell is not known. To understand any interaction of HU with RNA in the nucleoid structure, we immunoprecipitated potential HU-RNA complexes from cells and examined bound RNAs by hybridization to whole-genome tiling arrays. We identified associations between HU and 10 new intragenic and intergenic noncoding RNAs (ncRNAs), 2 of which are homologous to the annotated bacterial interspersed mosaic elements (BIMEs) and boxC DNA repeat elements. We confirmed direct binding of HU to BIME RNA in vitro. We also studied the nucleoid shape of HU and two of the ncRNA mutants (nc1 and nc5) by transmission electron microscopy and showed that both HU and the two ncRNAs play a role in nucleoid morphology. We propose that at least two of the ncRNA species complex with HU and help the formation or maintenance of the architecture of the E. coli chromosome. We also observed binding of HU with rRNA and tRNA segments, a few small RNAs, and a distinct small set of mRNAs, although the significance, if any, of these associations is not known.

Cellular stress created by intermediary metabolite imbalances.

Small molecules generally activate or inhibit gene transcription as externally added substrates or as internally accumulated end-products, respectively. Rarely has a connection been made that links an intracellular intermediary metabolite as a signal of gene expression. We report that a perturbation in the critical step of a metabolic pathway--the D-galactose amphibolic pathway--changes the dynamics of the pathways leading to accumulation of the intermediary metabolite UDP-galactose. This accumulation causes cell stress and transduces signals that alter gene expression so as to cope with the stress by restoring balance in the metabolite pool. This underscores the importance of studying the global effects of alterations in the level of intermediary metabolites in causing stress and coping with it by transducing signals to genes to reach a stable state of equilibrium (homeostasis). Such studies are an essential component in the integration of metabolomics, proteomics, and transcriptomics.

A phage display system designed to detect and study protein-protein interactions.

Analysing protein-protein interactions is critical in proteomics and drug discovery. The usage of 2-Hybrid (2lambda) systems is limited to an in vivo environment. We describe a bacteriophage 2-Hybrid system for studying protein interactions in vitro. Bait and prey are displayed as fusions to the surface of phage lambda that are marked with different selectable drug-resistant markers. An interaction of phages in vitro through displayed proteins allows bacterial infection by two phages resulting in double drug-resistant bacterial colonies at very low multiplicity of infections. We demonstrate interaction of the protein sorting signal Ubiquitin with the Vps9-CUE, a Ubiquitin binding domain, and by the interaction of (Gly-Glu)(4) and (Gly-Arg)(4) peptides. Interruptions of the phage interactions by non-fused (free) bait or prey molecules show how robust and unique our approach is. We also demonstrate the use of Ubiquitin and CUE display phages to find binding partners in a lambda-display library. The unique usefulness to 2lambda is also described.

Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin-resistant Enterococcus faecium.

Colonization of the gastrointestinal tract with vancomycin-resistant Enterococcus faecium (VRE) has become endemic in many hospitals and nursing homes in the United States. Such colonization predisposes the individual to VRE bacteremia and/or endocarditis, and immunocompromised patients are at particular risk for these conditions. The emergence of antibiotic-resistant bacterial strains requires the exploration of alternative antibacterial therapies, which led our group to study the ability of bacterial viruses (bacteriophages, or phages) to rescue mice with VRE bacteremia. The phage strain used in this study has lytic activity against a wide range of clinical isolates of VRE. One of these VRE strains was used to induce bacteremia in mice by intraperitoneal (i.p.) injection of 10(9) CFU. The resulting bacteremia was fatal within 48 h. A single i.p. injection of 3 x 10(8) PFU of the phage strain, administered 45 min after the bacterial challenge, was sufficient to rescue 100% of the animals. Even when treatment was delayed to the point where all animals were moribund, approximately 50% of them were rescued by a single injection of this phage preparation. The ability of this phage to rescue bacteremic mice was demonstrated to be due to the functional capabilities of the phage and not to a nonspecific immune effect. The rescue of bacteremic mice could be effected only by phage strains able to grow in vitro on the bacterial host used to infect the animals, and when such strains are heat inactivated they lose their ability to rescue the infected mice.