Leucine-regulated self-association of leucine-responsive regulatory protein (Lrp) from Escherichia coli

Lrp is a global regulatory protein in Escherichia coli that activates expression of more than a dozen operons and represses expression of another dozen. For some operons, exogenous leucine reduces the extent of Lrp action, for others it potentiates the effect of Lrp, and for yet other operons it has no effect. In an effort to understand how leucine affects Lrp-mediated expression, we examined Lrp self-association and the effect of leucine on self-association using light scattering, chemical cross-linking, and analytical ultracentrifugation. The following results were obtained. (i) Lrp self-associates to a hexadecamer and octamer with the predominant species being hexadecamer at microM concentrations. (ii) Lrp undergoes a leucine-induced dissociation of hexadecamer to octamer. (iii) A mutant Lrp lacking 11 amino acid residues at the C terminus does not form higher-order oligomers, suggesting that the C terminus is involved in subunit association. (iv) At nM concentrations, Lrp dissociates to a dimer. It is proposed that leucine regulates the equilibrium between Lrp oligomers and thus Lrp occupancy of sites within different operons, leading to diverse regulatory patterns.     

Cooperative binding of the leucine-responsive regulatory protein (Lrp) to DNA

The leucine-responsive regulatory protein (Lrp) of Escherichia coli activates expression of a number of operons and represses expression of others. For some members of the Lrp regulon, exogenous leucine mitigates the effect of Lrp, for some it potentiates the effect of Lrp, and for others it has no effect on Lrp action. For the ilvIH operon that we study, Lrp activates expression in vivo and mediates the repression of the operon by exogenous leucine. We studied Lrp-1, a leucine-insensitive variant, to investigate mechanisms by which leucine alters Lrp action as an activator of ilvIH expression. The Asp114Glu change did not have much effect on the amount of total Lrp-1 in cells but decreased the amount of free Lrp-1 two- to threefold. Lrp monomers associate to form octamers and hexadecamers (hexadecamer form predominates at micromolar concentrations; Kd=5.27x10(-8) M), and leucine promotes the dissociation of Lrp hexadecamer to a leucine-bound octamer. By contrast, Lrp-1 exists primarily as an octamer in solution (equilibrium dissociation constant 6.5x10(-5) M) and leucine had little effect on the equilibrium. Thus, the hexadecameric form that Lrp assumes in the absence of DNA is not required for activation of the ilvIH operon. Both leucine and the lrp-1 mutation reduced the apparent affinity of Lrp binding to ilvIH DNA (contains two groups of binding sites separated by 136 bp) but they have different effects on intrinsic binding affinity and binding cooperativity. Whereas leucine reduced intrinsic binding affinities and interactions of Lrps bound at upstream and downstream regions of ilvIH DNA, it increased cooperative dimer-dimer interactions of Lrps bound to two adjacent sites. By contrast, the lrp-1 mutation did not have much effect on intrinsic binding affinities but it decreased cooperative adjacent dimer-dimer interactions and enhanced interactions of Lrps bound at upstream and downstream regions of ilvIH DNA. Our analysis is consistent with the idea that leucine enhances dimer-dimer interactions that contribute to octamer formation, concomitantly reducing dimer-dimer interactions that contribute to the longer range interactions of Lrps that are required for activation of the ilvIH promoter.     

DNA supercoiling and the Lrp protein determine the directionality of fim switch DNA inversion in Escherichia coli K-12

Site-specific recombinases of the integrase family usually require cofactors to impart directionality in the recombination reactions that they catalyze. The FimB integrase inverts the Escherichia coli fim switch (fimS) in the on-to-off and off-to-on directions with approximately equal efficiency. Inhibiting DNA gyrase with novobiocin caused inversion to become biased in the off-to-on direction. This directionality was not due to differential DNA topological distortion of fimS in the on and off phases by the activity of its resident P(fimA) promoter. Instead, the leucine-responsive regulatory (Lrp) protein was found to determine switching outcomes. Knocking out the lrp gene or abolishing Lrp binding sites 1 and 2 within fimS completely reversed the response of the switch to DNA relaxation. Inactivation of either Lrp site alone resulted in mild on-to-off bias, showing that they act together to influence the response of the switch to changes in DNA supercoiling. Thus, Lrp is not merely an architectural element organizing the fim invertasome, it collaborates with DNA supercoiling to determine the directionality of the DNA inversion event.     

Lrp, a leucine-responsive protein, regulates branched-chain amino acid transport genes in Escherichia coli.

We investigated the relationship between two regulatory genes, livR and lrp, that map near min 20 on the Escherichia coli chromosome. livR was identified earlier as a regulatory gene affecting high-affinity transport of branched-chain amino acids through the LIV-I and LS transport systems, encoded by the livJ and livKHMGF operons. lrp was characterized more recently as a regulatory gene of a regulon that includes operons involved in isoleucine-valine biosynthesis, oligopeptide transport, and serine and threonine catabolism. The expression of each of these livR- and lrp-regulated operons is altered in cells when leucine is added to their growth medium. The following results demonstrate that livR and lrp are the same gene. The lrp gene from a livR1-containing strain was cloned and shown to contain two single-base-pair substitutions in comparison with the wild-type strain. Mutations in livR affected the regulation of ilvIH, an operon known to be controlled by lrp, and mutations in lrp affected the regulation of the LIV-I and LS transport systems. Lrp from a wild-type strain bound specifically to several sites upstream of the ilvIH operon, whereas binding by Lrp from a livR1-containing strain was barely detectable. In a strain containing a Tn10 insertion in lrp, high-affinity leucine transport occurred at a high, constitutive level, as did expression from the livJ and livK promoters as measured by lacZ reporter gene expression. Taken together, these results suggest that Lrp acts directly or indirectly to repress livJ and livK expression and that leucine is required for this repression. This pattern of regulation is unusual for operons that are controlled by Lrp.     

Global gene expression profiling in Escherichia coli K12. The effects of leucine-responsive regulatory protein

Leucine-responsive regulatory protein (Lrp) is a global regulatory protein that affects the expression of multiple genes and operons in bacteria. Although the physiological purpose of Lrp-mediated gene regulation remains unclear, it has been suggested that it functions to coordinate cellular metabolism with the nutritional state of the environment. The results of gene expression profiles between otherwise isogenic lrp(+) and lrp(-) strains of Escherichia coli support this suggestion. The newly discovered Lrp-regulated genes reported here are involved either in small molecule or macromolecule synthesis or degradation, or in small molecule transport and environmental stress responses. Although many of these regulatory effects are direct, others are indirect consequences of Lrp-mediated changes in the expression levels of other global regulatory proteins. Because computational methods to analyze and interpret high dimensional DNA microarray data are still an early stage, much of the emphasis of this work is directed toward the development of methods to identify differentially expressed genes with a high level of confidence. In particular, we describe a Bayesian statistical framework for a posterior estimate of the standard deviation of gene measurements based on a limited number of replications. We also describe an algorithm to compute a posterior estimate of differential expression for each gene based on the experiment-wide global false positive and false negative level for a DNA microarray data set. This allows the experimenter to compute posterior probabilities of differential expression for each individual differential gene expression measurement.     

Escherichia coli cells lacking methylation-blocking factor (leucine-responsive regulatory protein) have precise timing of initiation of DNA replication in the cell cycle.

A protein that is required for specific methylation inhibition of two GATC sites in the papBA pilin promoter region, known as methylation-blocking factor (Mbf) and recently shown to be identical to the leucine-responsive regulatory protein (Lrp), is not responsible for the delayed methylation at oriC implicated in an eclipse period following initiation of DNA replication. Cells containing a transposon mutation within the mbf (lrp) gene initiate DNA replication at the correct time during the cell cycle, whereas cells with increased amounts of the Dam methyltransferase initiate DNA replication randomly throughout the cell cycle.     

An Escherichia coli mutant unable to support site-specific recombination of bacteriophage lambda

We report the isolation of mutations in, and the characterization of, an Escherichia coli gene, hip, that is required for site-specific recombination of phage lambda. hip mutants are recessive and are located near minute 20 on the linkage map. The gene product is not vital to bacterial growth, since deletion mutants are viable. The absence of hip product reduces lambda integration to barely detectable levels and also reduces prophage excision, but less drastically. Certain mutations in the lambda int gene partially restore integration and excision in hip- hosts. Homologous recombination promoted by recA does not require hip function. In addition to their defect in site-specific recombination, hip mutants are unable to support lytic growth of phage Mu or of certain lambda mutants. Their pleiotropic phenotype closely resembles that of himA mutants, but complementation, mapping and DNA sequencing show that hip and himA are different genes.     

The ArcA/ArcB two-component regulatory system of Escherichia coli is essential for Xer site-specific recombination at psi

Two recombinases, XerC and XerD, act at the recombination sites psi and cer in plasmids pSC101 and ColE1 respectively. Recombination at these sites maintains the plasmids in a monomeric state and helps to promote stable plasmid inheritance. The accessory protein PepA acts at both psi and cer to ensure that only intramolecular recombination takes place. An additional accessory protein, ArgR, is required for recombination at cer but not at psi . Here, we demonstrate that the ArcA/ArcB two-component regulatory system of Escherichia coli , which mediates adaptation to anaerobic growth conditions, is required for efficient recombination in vivo at psi . Phosphorylated ArcA binds to psi in vitro and increases the efficiency of recombination at this site. Binding of ArcA to psi may contribute to the formation of a higher order synaptic complex between a pair of psi sites, thus helping to ensure that recombination is intramolecular.     

Deletion of an essential gene in Escherichia coli by site-specific recombination with linear DNA fragments

Deletion of an essential gene in Escherichia coli was accomplished by transformation of linear DNA fragments that have a Kanr gene segment flanked by sequences homologous to closely spaced regions on the E. coli chromosome. Selection for a double crossover within homologous sequences can effectively delete an entire gene. Cell viability is maintained by provision of the essential gene on a plasmid with a temperature-sensitive replicon, resulting in cells which have a temperature-sensitive phenotype.     

Multicopy plasmid stability in Escherichia coli requires host-encoded functions that lead to plasmid site-specific recombination

Summary The heritable stability of the multicopy plasmid ColE1 and its natural relatives, requires the presence in the plasmid of a site (cer in ColE1) that acts as a substrate for site-specific recombination, thereby maintaining plasmids in the monomeric state. Multimerization, promoted by homologous recombination, leads to plasmid loss. Here we show that the Escherichia coli chromosome encodes at least two unlinked functions that act on cer and its analogous sites, to promote stabilizing site-specific recombination. One of these functions is encoded by a gene residing on a cosmid that also contains the argI and pyrB genes, mapping it to the 96–97 min region of the E. coli map.     

Probing the regulatory site of Escherichia coli aspartate transcarbamoylase by site-specific mutagenesis.

The effector binding site of Escherichia coli aspartate transcarbamoylase, composed of the triphosphate and ribose-base subsites, is located on the regulatory (r) chains of the enzyme. In order to probe the function of amino acid side chains at this nucleotide triphosphate site, site-specific mutagenesis was used to create three mutant versions of the enzyme. On the basis of the three-dimensional structure of the enzyme with CTP bound, three residues were selected. Specifically, Arg-96r was replaced with Gln, and His-20r and Tyr-89r were both replaced with Ala. Analyses of these mutant enzymes indicate that none of these substitutions significantly alter the catalytic properties of the enzyme. However, the mutations at His-20r and Tyr-89r produced altered response to the regulatory nucleotides. For the His-20r----Ala enzyme, the affinities of the enzyme for ATP and CTP are reduced 40-fold and 10-fold, respectively, when compared with the wild-type enzyme. Furthermore, CTP is able to inhibit the His-20r----Ala enzyme 40% more than the wild-type enzyme. In the case of the Tyr-89r----Ala enzyme. ATP can increase the mutant enzyme's activity 181% compared to 157% for the wild-type enzyme, while simultaneously the affinity of this enzyme for ATP decreases about 70%. These results suggest that Tyr-89r does have an indirect role in the discrimination between ATP and CTP. The His-20r----Ala enzyme shows no UTP synergistic inhibition in the presence of CTP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of molecular crowding on the interaction between DNA and the Escherichia coli regulatory protein TyrR

Fluorescence quenching has been used to measure quantitatively the effects of sucrose and triethylene glycol on the interaction between the Escherichia coli regulatory protein TyrR and a 30-basepair oligonucleotide containing the strong TyrR box of the TyrR operon. It was observed that the apparent binding constant increased in the presence of co-solutes, the dependence of the logarithm of the apparent binding constant on molar concentration being indistinguishable and essentially linear for both co-solutes. This activation of the TyrR-oligonucleotide interaction is attributed to thermodynamic nonideality arising from molecular crowding, an interpretation which is supported by the reasonable agreement observed between the experimental extent of reaction enhancement and that predicted on the statistical-mechanical basis of excluded volume.