Lambda placMu insertions in genes of the leucine regulon: extension of the regulon to genes not regulated by leucine

Abstract

The leucine regulon coordinates the expression of several Escherichia coli genes according to the presence of exogenous leucine, which interacts with the bp gene product, Lrp. We isolated and characterized 22 strains with k placMu insertions in Lrp-regulated genes. Lrp and leucine influenced gene expression in a surprising variety of ways. We identified two genes that are regulated by Lrp and not affected by L-leucine. We therefore rename this the leucine-fp regulon. Genes coding for glycine cleavage and leucine biosynthesis enzymes have been identified as members of the leucine4rp regulon. We suggest that the brp gene product activates genes needed for growth in minimal medium, and we show that the gene is repressed by its own product and is highly repressed during growth in rich medium.We recently showed that in Escherichia coli the branchedchain amino acid L-leucine is the effector of a global response that governs a group of genes known as the leucine regulon (11). Regulation of the synthesis of a number of enzymes from different metabolic pathways is affected by the presence of L-leucine in the growth medium (11,16). This response to exogenous L-leucine was altered in an E. coli mutant carrying an insertion in a gene at 20 min (11). This gene, referred to originally as rbl, was renamed lip by consensus among interested investigators (11,20).The lip gene codes for a 38-kDa dimer, the leucineresponsive regulatory protein (Lrp), which has been purified extensively (31). Under the name of the ilvIH binding protein, it was shown to bind to two sites upstream of ilvIH (22), for which it is a positive regulator. A mutation in lrp made the strain constitutive for ilvIH expression (20) and changed only one nucleotide, resulting in a substitution of Glu for Asp. The lip gene was shown to be identical to oppI (2) and is also thought to be identical to livR (12,20).Until now, the genes of the leucine regulon have been identified by guesswork or by the accident of a previously known regulatory gene proving to be identical to lip. As a result of a somewhat different accident, lysU has now been added to the regulon. We present here the results of a screening among random X placMu9 insertions for leucineand Lrp-regulated expression of lacZ. We used a system based on that used for the isolation of din genes (8). We also show that some genes are regulated by Lrp without leucine, and therefore we rename the regulon as the leucine-Lrp regulon. MATERIALS AND METHODSCultures. The strains used in this study-all derivatives of E. coli K-12-are described in Table 1. The plasmids used are also listed in Table 1. Cultures were grown as described previously (11) with L-isoleucine and L-valine (each at 50 ,g/ml) added to the minimal medium used to grow strain CU1008 and all its derivatives to compensate for the ilvA mutation carried by these strains. Other genetic techniques. Plasmid isolations, DNA manipulations, transductions, and transformations were performed as described previously (13, 14).Enzyme assays. L-Serine deaminase was as...

Glycine binds the transcriptional accessory protein GcvR to disrupt a GcvA/GcvR interaction and allow GcvA-mediated activation of the Escherichia coli gcvTHP operon

“…Transcription of the gcv operon is under the control of three global regulatory proteins [Lrp, PurR and cAMPreceptor protein (CRP)] and two gcv-specific transcriptional regulatory proteins (GcvA and GcvR) (Ghrist & Stauffer, 1995 ;Lin et al, 1992 ;Wilson et al, 1993a ;Wilson & Stauffer, 1994 ;Wonderling & Stauffer, 1999). Together these Fig.…”

The leucine-responsive regulatory protein of Escherichia coli negatively regulates transcription of ompC and micF and positively regulates translation of ompF

“…Previous studies have shown that micF expression is increased when cells are shifted from a glucose minimal medium to LB and that increased micF transcription does not require a functional OmpR protein (10). Since the levels of Lrp in LB are known to be decreased compared with those in glucose minimal media (6,12,27), it will be interesting to determine whether the effect of LB on micF expression requires a functional Lrp protein.…”

“…The finding that expression of the OmpC and OmpF porins is regulated by Lrp provides useful insight into the physiological role of the Lrp regulon. We, and others, have previously postulated that Lrp modulates metabolic pathways in E. coli in response to the availability of nutrients in the medium (6,12,27) and that it may mediate changes necessary for survival outside an animal host. Genes and proteins that are involved in catabolism of amino acids and oligopeptide transport are negatively regulated by Lrp, while genes and proteins involved in amino acid biosynthesis and the assimilation of ammonia are positively regulated by Lrp.…”

“…An increase in temperature or osmolarity leads to increased expression of ompC and decreased expression of ompF (24), as does an lrp mutation. Furthermore, studies performed in our laboratory (12) and in the laboratories of Elaine Newman (27) and Joseph Calvo (6) have demonstrated that Lrp levels are lower during growth in rich media like LB than during growth in minimal media. These observations led us to investigate the relationship between regulation of porin expression by osmolarity, by micF, and by Lrp.…”

GcvA binding site 1 in the gcvTHP promoter of Escherichia coli is required for GcvA-mediated repression but not for GcvA-mediated activation

“…amino acid metabolic pathways (Calvo & Matthews, 1994), is required for expression of a gcvT ::lacZ fusion (Lin et al, 1992 ;. Lrp binds to and protects from DNase I digestion a large region of DNA upstream of the gcv promoter from about base k92 to k229 .…”