L-serine degradation in Escherichia coli K-12: cloning and sequencing of the sdaA gene

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).

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

“…Wild-type E. coZi does not use L-serine as a carbon source, but can do so when the L-SD level is increased, either by mutation or by cloning sduA on a multi-copy plasmid (Su et al, 1989). L-SD2 is sufficiently similar so that one would expect it to serve the same function as sduA.…”

“…DNA sequences were determined by the dideoxy chain termination method of Sanger et al (1977), according to methods described in Su et al (1989). Both DNA strands were sequenced with dGTP and one with dITP.…”

“…To examine the nature of regulation of the wild-type gene, we made an in-vitro insertion of the lucZ gene in-frame into the clone derived from the Kohara phage. Using methods previously used for s d d (Su et al, 1989), we ligated lac2 from pMC1871 to an EcoRV fragment from pWBT6, which should result in an in-frame fusion of lac2 at codon 63 of the Kohara-phage derived clone of sduB.…”

“…Expression of sduA is subject to at least two global regulatory systems (Lin et al, 1990;Newman et al, 1981) and is affected by a large number of environmental factors. The s d d gene has been cloned and sequenced (Su et al, 1989) and its product purified (Su et al, 1993).The existence of the second L-SD was first suggested by the fact that there is a considerable amount of L-SD activity in LB-grown cells carrying a null mutation in s d d (Su and Newman, 1991). L-SD2 is very similar in biochemical properties to L-SD1, but it is coded by the sduB gene, mapped at 60.1 min (Su and Newman, 1991).…”

“…Expression of sduA is subject to at least two global regulatory systems (Lin et al, 1990;Newman et al, 1981) and is affected by a large number of environmental factors. The s d d gene has been cloned and sequenced (Su et al, 1989) and its product purified (Su et al, 1993).…”