Cell division, one-carbon metabolism and methionine synthesis in a metK-deficient Escherichia coli mutant, and a role for MmuM.

An E. coli K-12 mutant deficient in S-adenosylmethionine (SAM) synthesis, i.e ΔmetK, but expressing a rickettsial SAM transporter, can grow in glucose minimal medium if provided with both SAM and methionine. It uses the externally provided (R)-enantiomer of SAM as methyl donor to produce most but not all of its methionine, by methylation of homocysteine catalysed by homocysteine methyltransferase (MmuM). The ΔmetK cells are also altered in growth and are twice as long as those of the parent strain. When starved of SAM, the mutant makes a small proportion of very long cells suggesting a role of SAM and of methylation in the onset of crosswall formation.

Studies on the role of the metK gene product of Escherichia coli K-12.

We show here that the metK gene is essential to the growth of Escherichia coli K-12 and can be deleted only in the presence of a rescue plasmid carrying a functional metK gene. When metK expression was limited, genomic DNA methylation decreased and cell division was hampered. Through primer extension, the transcription start site of metK was located at 140 bp upstream of the translation start site. The frequently used metK84 mutant has been shown to carry an A(r)G transition in the -10 region of the metK promoter. This accounts for its low level of S-adenosylmethionine (SAM) synthetase and SAM deficiency.

Lack of S-adenosylmethionine results in a cell division defect in Escherichia coli.

The enzyme S-adenosylmethionine (SAM) synthetase, the Escherichia coli metK gene product, produces SAM, the cell's major methyl donor. We show here that SAM synthetase activity is induced by leucine and repressed by Lrp, the leucine-responsive regulatory protein. When SAM synthetase activity falls below a certain critical threshold, the cells produce long filaments with regularly distributed nucleoids. Expression of a plasmid-carried metK gene prevents filamentation and restores normal growth to the metK mutant. This indicates that lack of SAM results in a division defect.

Comparison of the sensitivities of two Escherichia coli genes to in vivo variation of Lrp concentration.

Transcription of the Escherichia coli genes serA and gltBDF depends on the leucine-responsive regulatory protein, Lrp, and is very much decreased in an lrp mutant. By the use of an Lrp-deficient host and the lrp gene cloned under a plasmid-borne arabinose pBAD promoter, we varied the amount of Lrp present in the cell and showed that both genes were transcribed in proportion to the amount of Lrp synthesized. The affinity of serA for Lrp was four to five times greater than the affinity of gltD. Overproduction of Lrp was lethal to the cell.

Metabolic regulation of lrp gene expression in Escherichia coli K-12.

Expression of the lrp gene is regulated in part by the nutrients available to the cell, and is decreased in rich medium, in glucose minimal media enriched with amino acids, and in minimal medium with alternative carbon sources, such as acetate and succinate. When Lrp production is increased in a given medium, expression of its target genes is also increased. However, when the medium is changed from glucose to acetate, the response of the target genes is governed by many factors.

Identification of Lrp-regulated genes by inverse PCR and sequencing: regulation of two mal operons of Escherichia coli by leucine-responsive regulatory protein.

We have used the technique of inverse PCR to identify Escherichia coli chromosomal genes carrying Lrp-regulated inserts. This technique revealed that malT, malEFG, and malB-lamB-malK are all activated two- to fivefold by Lrp and confirmed that Lrp regulates expression of the leuDBCA and livHJKG operons. lacZ transcription is also increased in the presence of Lrp. However, the growth rate of the Lrp mutant on maltose and lactose is not decreased by Lrp deficiency.

Leucine-responsive regulatory protein: a global regulator of gene expression in E. coli.

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.

Altered amino acid metabolism in lrp mutants of Escherichia coli K12 and their derivatives.

An Escherichia coli lrp mutant, lacking the leucine-responsive regulatory protein and the global response it controls, is deregulated in the expression of many genes, but is nevertheless able to grow in glucose-minimal medium at 37 degrees C. In the presence of isoleucine and valine, the growth rate of the lrp mutant at 37 degrees C is significantly increased by exogenous L-serine or L-leucine (or both), suggesting that synthesis of these amino acids is limiting. In the absence of isoleucine and valine, however, growth is severely inhibited by both L-serine and L-leucine. A shift to 42 degrees C or to anaerobiosis makes the lrp mutant auxotrophic for L-serine. Three double mutants carrying lrp and another known mutation, acquire new auxotrophies: lrp relA, lacking the stringent response to amino acid limitation, requires leucine; lrp ssd with numerous metabolic perturbations and antibiotic resistances, requires serine and leucine; and lrp pnt, lacking pyridine nucleotide transhydrogenase, requires glutamate or aspartate (or the corresponding amides). The lrp mutant, although able to achieve balanced growth in some conditions, is clearly on the edge of a metabolic precipice, unable to tolerate many physiological and genetic perturbations which are inocuous to wild-type E. coli.

Sequencing and characterization of the sdaC gene and identification of the sdaCB operon in Escherichia coli K12.

We describe here the regulatory and coding region, and DNA sequence, for a newly recognized gene, sdaC, which codes for a hydrophobic protein with several predicted membrane-spanning domains. sdaC and sdaB form a single operon, with 57 bp between the end of sdaC and the start of sdaB. Expression of the sdaCB operon is regulated mainly by catabolite repression, but is also slightly sensitive to regulation by leucine-responsive regulatory protein. Cells carrying sdaC on a multicopy plasmid have increased L-serine transport capacity, insensitive to threonine, suggesting that sdaC might code for a recently described highly specific serine transporter [Kayahara, T., Thelen, P., Ogawa, W., Inaba, K., Tsuda, M., Goldberg, E. B. & Tsuchiya, T. (1992) J. Bacteriol. 174, 7482-7485].

The leucine-responsive regulatory protein: more than a regulator?

The leucine-responsive regulatory protein (Lrp) is the regulator of the recently discovered leucine/Lrp regulon in Escherichia coli. Like other global regulators, it regulates the expression of 35 or more specific target operons. Studies of this global response have led to the suggestion that Lrp--and perhaps some other gene regulators--may also participate in the maintenance of chromosome structure and organization.

Sequencing and characterization of the sdaB gene from Escherichia coli K-12.

The sdaB gene which codes for the second L-serine deaminase (L-SD) of Escherichia coli K-12 has been sequenced and shown to be very similar to the sdaA gene which codes for the first L-serine deaminase. sdaB is transcribed in rich medium, particularly in the absence of glucose, and is under the control of catabolite activator protein. A mutation which established expression of the sdaB gene and synthesis of L-serine deaminase 2 in minimal medium has been demonstrated to result in a change in the ribosome-binding site of the sdaB gene.

Use of gene fusions of the structural gene sdaA to purify L-serine deaminase 1 from Escherichia coli K-12.

The purification by affinity chromatography of beta-galactosidase from strains carrying sdaA/lacZ gene fusions results in the copurification of L-serine deaminase 1. We conclude that sdaA is the structural gene for the latter enzyme. The purified L-serine deaminase 1 obtained after collagenase treatment of an sdaA-collagen-lacZ fusion differs from the native enzyme by the addition of several amino acids at the C-terminal. Like the enzyme in crude extracts, this purified enzyme is catalytically inactive, and is activated by incubation with iron and dithiothreitol.

The lrp gene product regulates expression of lysU in Escherichia coli K-12.

In Escherichia coli K-12, expression of the lysU gene is regulated by the lrp gene product, as indicated by an increase in the level of lysyl-tRNA synthetase activity and LysU protein in an lrp mutant. Comparison of the patterns of protein expression visualized by two-dimensional gel electrophoresis indicated that LysU is present at higher levels in an lrp strain than in its isogenic lrp+ parent. The purified lrp gene product was shown to bind to sites upstream of the lysU gene and to protect several sites against DNase I digestion. A region extending over 100 nucleotides, between 60 and 160 nucleotides upstream from the start of the lysU coding sequence, showed altered sensitivity to DNase I digestion in the presence of the Lrp protein. The extent of protected DNA suggests a complex interaction of Lrp protein and upstream lysU DNA.

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

The leucine regulon coordinates the expression of several Escherichia coli genes according to the presence of exogenous leucine, which interacts with the lrp gene product, Lrp. We isolated and characterized 22 strains with lambda 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-lrp regulon. Genes coding for glycine cleavage and leucine biosynthesis enzymes have been identified as members of the leucine-lrp regulon. We suggest that the lrp 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.

A novel L-serine deaminase activity in Escherichia coli K-12.

We demonstrate here that Escherichia coli K-12 synthesizes two different L-serine deaminases (L-SD) catalyzing the nonoxidative deamination of L-serine to pyruvate, one coded for by the previously described sdaA gene and a second, hitherto undescribed enzyme which we call L-SD2. A strain carrying a null mutation in sdaA made no detectable L-SD in minimal medium, but had activity in Luria broth. We describe a mutation, sdaX, which affects the regulation of L-SD2 and permits its expression in minimal medium, and an insertion mutation, sdaB, which abolishes L-SD2 activity completely. Both mutations lie near 60.5 min on the E. coli genetic map. The two L-SD enzymes have similar enzyme parameters, and both require posttranslational activation.

The leucine regulon of Escherichia coli K-12: a mutation in rblA alters expression of L-leucine-dependent metabolic operons.

We have isolated and characterized a highly pleiotropic Escherichia coli mutant affected in the activity of a number of enzymes involved in different metabolic pathways, all of which are regulated by leucine. Selected for its ability to grow with L-serine as sole carbon source, the rbl-1::Tn10 mutant had high levels of L-serine deaminase activity (due to increased transcription of the structural gene) and of another amino acid-degrading enzyme, L-threonine dehydrogenase, and decreased transcription of the operons serA and ilvIH, coding for biosynthetic enzymes. The rbl mutation suppressed the slow growth of a metK mutant, deficient in S-adenosylmethionine synthetase. Furthermore, metK mutants spontaneously accumulated faster-growing rbl-like derivatives, and a commonly used metK strain, RG62, carries such a mutation. The rbl gene is located near 20 min on the E. coli genetic map. All phenotypes of the rbl mutant could be observed in rbl+ strains cultivated in the presence of L-leucine, and exogenous L-leucine had little further effect on the rbl strains. We propose that the rbl gene product is the regulator of a global response to leucine.

A relationship between L-serine degradation and methionine biosynthesis in Escherichia coli K12.

While wild-type Escherichia coli K12 cannot grow with L-serine as carbon source, two types of mutants with altered methionine metabolism can. The first type, metJ mutants, in which the methionine biosynthetic enzymes are expressed constitutively, are able to grow with L-serine as carbon source. Furthermore, a plasmid carrying the metC gene confers ability to grow on L-serine. These observations suggest that in these mutants, L-serine deamination may be a result of a side-reaction of the metC gene product, cystathionine beta-lyase. The second type is exemplified by two newly isolated strains carrying mutations mapping between 89.6 and 90 min. These mutants use L-serine as carbon source, and also require methionine for growth with glucose at 37 degrees C and above. The phenotypes of the new mutants resemble those of both met and his constitutive mutants in some respects, but have been differentiated from both of them.

A possible mechanism for the in vitro activation of L-serine deaminase activity in Escherichia coli K12.

L-Serine deaminase is inactive in crude extracts of Escherichia coli K12, but can be activated by incubation with iron and dithiothreitol. This activation requires oxygen, and is inhibited by free radical scavengers and by diethylene triamine pentaacetic acid, which prevents Fe cycling. We suggest that in vitro activation of L-serine deaminase is catalyzed by an oxidant (perhaps hydroxyl radicals). Also, activation may be accompanied by a decrease in molecular weight and involve both a cleavage of the polypeptide chain and a reversible reduction of the molecule.