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...

SummaryThe loss of the ability to deaminate L-serine severely impairs growth and cell division in Escherichia coli K-12. A strain from which the three genes (sdaA, sdaB, tdcG) coding for this organism's three L-serine deaminases had been deleted grows well in glucose minimal medium but, on subculture into minimal medium with glucose and casamino acids, it makes very large, abnormally shaped cells, many of which lyse. When inoculated into Luria-Bertani (LB) broth with or without glucose, it makes very long filaments. Provision of S-adenosylmethionine restores cell division in LB broth with glucose, and repairs much of the difficulty in growth in medium with casamino acids. We suggest that replication of E. coli is regulated by methylation, that an unusually high intracellular L-serine concentration, in the presence of other amino acids, starves the cell for Sadenosylmethionine and that it is the absence of S-adenosylmethionine and/or of C1-tetrahydrofolate derivatives that prevents normal cell division.

No abstract

A new mutant of Escherichia coli K-12 unable to grow with L-serine, glycine, and L-leucine has been isolated by A plac Mu insertion and shown to be deficient in L-serine deaminase activity. The corresponding gene, sdaA, has been cloned from a prototrophic strain, and the clone has been characterized and sequenced. The evidence is consistent with the hypothesis that sdaA is the structural gene for L-serine deaminase. However, other possibilities are also considered. No significant homology with previously reported DNA or protein sequences was detected.The enzyme activity L-serine deaminase (L-SD) converts L-serine to pyruvate (19). It is present in Escherichia coli cells grown in glucose-minimal medium and is induced to higher levels by growth with glycine and/or L-leucine but not its substrate, L-serine (10, 19).There Although the L-SD-deficient mutants are clearly lacking in a physiological ability, the ability to grow with L-serine, glycine, and L-leucine as the carbon source, they are not totally unable to synthesize L-SD (18). When grown in Luria broth (LB), the mutants showed a great deal of L-SD activity (18). It is possible, as we suggested earlier, that a second L-serine deaminase is made in E. coli K-12 (18).Very little is known about the physical nature of either L-SD or its posttranslational activating system. As part of our studies on this subject, we report the characterization of a new mutant deficient in the L-SD synthesized in minimal medium. We have cloned and sequenced the relevant gene, sdaA. The evidence suggests that sdaA codes for either the structure of L-SD or a regulator of L-SD activity. * Corresponding author. MATERIALS AND METHODSStrains, bacteriophages, and plasmids. The strains, bacteriophages, and plasmids used in this study are listed in Table 1.Cultures, media, and growth conditions. The minimal medium used, neutralized to pH 7, has been previously described (18). All derivatives of strain MEW1 required Lisoleucine and L-valine for growth, and so these were added to all media at 50 p.g/ml each.Medium with L-serine, glycine, and L-leucine as sole carbon sources other than L-isoleucine and L-valine is called SGL medium, and strains unable to grow in this medium are termed SGL-. L-Serine, glycine, and L-leucine were usually provided at 2,000, 300, and 300 [Lg/ml, respectively.Enzyme assays. L-SD was assayed as previously described in toluene-treated whole cells (10) and in crude extracts (16).

The leucine-responsive regulatory protein (Lrp) regulates transcription of the many genes of the Lrp regulon, repressing some and activating others, some in response to L-leucine and some independent of it. The physiology and molecular biology of the regulon in Escherichia coli are summarized here. However, the high degree of conservation of the protein suggests that it has an important role in all enterobacteria. We suggest that this role is not only as a transcriptional regulator but also as a determinant of chromosome structure.