9506911 Matthews The leucine-responsive regulatory protein (Lrp) is a recently discovered transcriptional regulator in Escherichia coli that mediates changes in the expression of more than 30 known genes and operons, and at least as many more unidentified genes and operons. In attempting to understand the response of E. coli and other unicellular organisms to changes in nutrient availability, it is important to understand how shifts in the regulation of whole blocks of genes are mediated, and how a small number of regulatory proteins can work together to integrate physiological responses. Lrp induces a particularly diverse set of regulatory responses among operons in the regulon, inhibiting the expression of some operons and activating the expression of others. In addition, target operons differ greatly with respect to their sensitivity to the co-regulator, leucine; leucine antagonizes the effect of Lrp on some operons, is required for the effect of Lrp on others; and has little or no effect on regulation of still other operons by Lrp. Lrp thus has unusual properties as a DNA-binding regulatory protein. A long term goal of the research is to understand at a molecular level how Lrp mediates these diverse effects. These studies should also clarify the role of Lrp in regulating metabolic processes in E. coli and other Gram-negative bacteria. A major focus of the research will be the comparison of in vivo and in vitro studies on regulation of genes and operons by Lrp. These comparisons initially will focus on the regulation by Lrp of the gltBDF operon, which codes for the subunits of glutamate synthase. This enzyme plays an essential role in the assimilation of low levels of ammonia during growth of E. coli in glucose minimal medium. Under these conditions, the net rate of transcription of gltBDF is 44-fold higher in an lrpt strain than in an isogenic Irp::Tn10 strain. In vivo studies of gltBDF regulation will employ a strain, AAEC546, in which the Irp gene has been placed under the control of the lacUVS promoter so that the synthesis of Lrp is controlled by addition of an inducer (IPTG) to the medium, and which contains a transcriptional fusion of the gltBDF promoter to the lacZ+ gene coding for (-galactosidase. In this strain, the effect of varying concentrations of Lrp on the expression of gltBDF can be studied, and compared with effects of Lrp on transcriptional activation of gltBDF in vitro. Preliminary studies have established that Lrp binds to the promoter region of gltBDF both in the presence and in the absence of leucine, but that the ability of bound Lrp to activate transcription is markedly reduced (~4-fold) in the presence of leucine. Further studies will seek to define the differences between the interaction of Lrp with gltBDF in the presence and in the absence of leucine. Using derivatives of strain AAEC546 containing transcriptional lacZ+ fusions to other genes in the Lrp regulon, the effect of leucine on the transcriptional regulation of these genes will be compared with results obtained with gltBDF. Strain AAEC546 has also been used to identify genes and operons in the Lrp regulon by mutational analysis following random insertions of (placmu into the chromosome. In contrast to the methods used to identify such genes in other laboratories, the strategy employed does not depend on effects of leucine on the Lrp-dependent regulation of gene expression. Ten fusions, displaying highly varied patterns of regulation by Lrp and by leucine, have been isolated. These fusions will now be cloned, permitting the identification of the operons into which the fusion has been inserted. Future research will involve characterization of the effect of Lrp and leucine on the expression of these operons in vitro and in vivo. In the classical models for prokaryotic transcriptional regulation, for instance in regulation by LacI or Crp, the coregulator primarily modulates the affinity of the regulatory protein for its specific binding sites on the target DNA. The initial studies of trans criptional regulation by Lrp fit a very different paradigm, in which the coregulator, leucine, affects both the affinity of Lrp for specific sites upstream of target genes and the efficacy of bound Lrp as a transcriptional regulator. In this way, Lrp resembles AraC and MerR more than it does Crp and LacI. The studies seek to provide an understanding of this phenomenon at the molecular level. By these means, our understanding of the way in which a single regulatory protein modulates the activity of so many other genes and operons in E. coli, with such large variations in the type and degree of modulation of expression, should be improved. %%% The growth of bacteria is affected by the environment in which they are growing. These studies will lead to an understanding of how large groups of genes are regulated by environmental signals. This knowledge is potentially useful in modulating the growth of bacteria for applications to biotechnology, as well as understanding how bacteria function in a variety of environments. ***
