The anaerobic degradation of l-serine and l-threonine in enterobacteria: networks of pathways and regulatory signals

The mechanisms controlling the biosynthesis and degradation of l-serine and l-threonine are remarkably complex. Their metabolism forms a network of pathways linking several amino acids, central primary metabolites such as pyruvate, oxaloacetate and 3-phosphoglycerate, and C₁ metabolism. Studies on the degradation of these amino acids in Escherichia coli have revealed the involvement of fascinating enzymes that utilise quite diverse catalytic mechanisms. Moreover, it is emerging that both environmental and metabolic signals have a major impact in controlling enzyme synthesis. This is exemplified by the anaerobically regulated tdc operon, which encodes a metabolic pathway for the degradation of serine and threonine. Studies on this pathway are beginning to provide insights into how an organism adapts its genetic makeup to meet the physiological demands of the cell. Received: 30 August 1998 / Accepted: 9 October 1998     

Identification of a Salmonella virulence gene required for formation of filamentous structures containing lysosomal membrane glycoproteins within epithelial cells

Salmonella species are facultative intracellular pathogens that invade epithelial cells and reside within lysosomal membrane glycoprotein (Igp)-containing vacuoles. Coincident with the onset of bacterial replication inside these vacuoles, Salmonella induce the formation of stable Igp-containing filamentous structures that connect with the Salmonella-containing vacuoles. Salmonella typhimurium SL1344::Tn 10dCm mutant strains unable to induce these structures were isolated. All contained insertions within a novel Salmonella induced filament gene A (sifA). sifA is present only in Salmonella species and encodes a protein with a predicted molecular mass of 38kDa and an apparent molecular mass of 35kDa. sifA is flanked by ～300 base pairs, and sifA and its flanking DNA show no homology to sequences in DNA databases. sifA is located within the potABCD operon, a housekeeping locus involved in periplasmic transport of polyamines. Fourteen-base-pair direct repeats mark the probable site of integration of sifA and its flanking DNA at the 3 end of potB. sifA and its flanking DNA have a significantly reduced G+C content (41%) when compared with the potABCD operon (51%) and the Salmonella genome (52–54%). Deletion mutant strains     

Construction of an arginine-producing strain of Serratia marcescens

Summary In Serratia marcescens Sr41, l-canavanine was demonstrated to be a weak cell growth inhibitor in minimal medium containing glucose as the sole carbon source. The inhibition of cell growth was enhanced by changing the carbon source from glucose to l-glutamic acid. An arginine regulatory mutant (i.e., argR mutant) in which formation of l-arginine biosynthetic enzymes was genetically derepressed was isolated by selecting for l-canavanine resistance on the glutamate medium. Furthermore, an l-arginine-producing strain was constructed by introducing the mutation leading to feedback-resistant N-acetylglutamate synthase into the argR mutant. The resulting transductant produced about 40 g/l of l-arginine, whereas the wild strain produced no l-arginine and the argR mutant only 3 g/l.     

Proline production by regulatory mutants of Serratia marcescens

Summary Proline-producing strains of Serratia marcescens Sr41 were constructed by three rounds of mutagenesis. A strain SP103 which did not degrade l-proline carried the putA mutation leading to lack of proline oxidase. A 3,4-dehydroproline-resistant mutant SP105, derived from strain SP103, carried the dpr-1 mutation which resulted in desensitization of the feedback inhibition of glutamate kinase. Strain SP103 produced 5.5 mg of l-proline per ml of fermentation medium containing sucrose and urea. Growth inhibition by proline analogs was enhanced when succinate was used as a carbon source in the medium. A thiazolidine-4-carboxylate-resistant mutant SP126 derived from strain SP105 produced 20.5 mg of l-proline per ml of medium. The mutation carried by strain SP126 might be distant from dpr-1 and putA mutations on the chromosome. Pyrroline-5-carboxylate reductase was not repressed by proline in S. marcescens Sr41.     

Isolation and mapping of a uracil-sensitive mutant of Salmonella typhimurium

Summary A uracil-sensitive mutant of Salmonella typhimurium was isolated by diethyl sulfate mutagenesis and penicillin counterselection. This mutation identifies a new Salmonella gene that is well separated from the structural genes for arginine and pyrimidine biosynthesis. The use-1 mutation was located between the ilv gene cluster (isoleucine-valine operon) and hisR (structural gene for histidine tRNA) at 83 map units.     

Mechanism of l-methionine overproduction by Escherichia coli: the replacement of Ser-54 by Asn in the MetJ protein causes the derepression of l-methionine biosynthetic enzymes

We derived l-methionine-analogue-resistant mutants from Escherichia coli JM109 strain by mutagenesis with N-methyl-N′-nitro-N-nitrosoguanidine and selected the potent l-methionine-overproducing strains by microbioassay using lactic acid bacteria. One of the mutants, strain TN1, produced approximately 910 mg l-methionine/l following the addition of 0.1% yeast extract to fundamental medium containing glucose and ammonium sulfate. The l-methionine biosynthetic enzymes, cystathionine γ-synthase and cystathionine β-lyase, of the l-methionine-overproducing mutants were little repressed by l-methionine. To analyse the mechanism of l-methionine overproduction in the mutant strains, the metJ gene coding for the E. colimet repressor, MetJ protein, was cloned and sequenced by the polymerase chain reaction. The same single-amino-acid subsitution (wild-type Ser → Asn) at position 54 was observed in four independent l-methionine-producing mutants. When the wild-type metJ gene was then introduced into strain TN1 having the mutant metJ gene, the level of enzyme synthesis and the l-methionine productivity in the transformants were found to revert to those of the wild-type. It was therefore considered that only one point mutation in the metJ gene occurred in the l-methionine-producing mutants. These results demonstrate the important role of residue 54 of the MetJ protein in l-methionine overproduction, probably because of the derepression of l-methionine biosynthetic enzymes. Received: 6 January 1999 / Received last revision: 19 February 1999 / Accepted: 26 February 1999     

Amplification of the tryptophan operon gene in Escherichia coli chromosome to increase l-tryptophan biosynthesis

Classical mutagenesis could desensitize the feedback inhibition of l-tryptophan (l-Trp) biosynthesis. Among the mutants, a5-fluorotryptophan-resistant strain, Escherichia coli EMS4-C25 produced 3 g/l of l-Trp within 18 h. The feedback-resistant l-Trp operon gene (trp) prepared from E. coli EMS4-C25 was inserted into pUC19 and pHSG576 to generate pTC701 and pTC576, respectively. When pHSG576 and pTC701 were introduced into E. coli EMS4-C25, chromosomal integration occured through homologous recombination. By using Souther hybridization, we demostrated that the integrated plasmids existed as multicopies. The strains with integrated foreign trp operon gene had higher activities of anthranilate synthase and Trp synthase than those found for the host strain and produced 9.2 g/l of l-Trp with 13% conversion yield from d-glucose. The integration and implification of the trp-operon-beraing plasmid avoided the plasmid instability and increased l-TRp production. Correspondence to: E.-C. Chan     

Mutation-Screening in <Emphasis Type="SmallCaps">l</Emphasis>-(+)-Lactic Acid Producing Strains by Ion Implantation

In this paper, in order to obtain some industrial strains with high yield of l-(+)-lactic acid, the wild type strain Lactobacillus casei CICC6028 was mutated by nitrogen ions implantation. By study, it was found that the high positive mutation rate was obtained when the output power was 10 keV and the dose of N⁺ implantation was 50 × 2.6 × 10¹³ ions/cm². In addition, the initial screening methods were also studied, and it was found that the transparent halos method was unavailable, for some high yield strains of l-(+)-lactic acid were missed. Then a mutant strain which was named as N-2 was isolated, its optimum fermentation temperature was 40°C and the l-(+)-lactic acid yield was 136 g/l compared to the original strain whose optimum fermentation temperature was 34°C and l-(+)-lactic acid production was 98 g/l. Finally, High Performance Liquid Chromatography method was used to analyze the purity of l-(+)-lactic acid that was produced by the mutant N-2, and the result showed the main production of N-2 was l-(+)-lactic acid.     

Biochemical and genetic characterization of a mutant of Salmonella typhimurium defective in a locus for glutamate dehydrogenase activity

Summary By two consecutive treatments with N-methyl-N-nitro-N-nitrosoguanidine we obtained mutant SM151 of Salmonella typhimurium which differs from the parental LT2 strain in: a) is able to use l-glutamate as carbon source (first mutation), and b) requires that amino acid for growth (second mutation). It was found that the requirement of mutant SM151 for glutamate was due to a very low activity of glutamate dehydrogenase. Both glutamate-oxaloacetate transaminase and aspartase activities were present at normal levels. Glutamate dehydrogenase activity was strongly repressed by glutamate; aspartase activity was under severe catabolite (glucose) repression, while glutamate-oxaloacetate transaminase was partially repressed by glutamate. By conjugation and transduction the locus gdh, responsible for the low activity of the glutamate dehydrogenase of mutant SM151, was located at about minute 128 of the bacterial chromosome and found to be linked to the argC, argF, and metB loci.     

Altered control of chorismate mutase leads to tryptophan auxotrophy of Pichia guilliermondii

We have isolated the tryptophan auxotrophic mutant strain, PK101, of Pichia guilliermondii. This strain is not defective in any of the tryptophan biosynthetic enzymes, but its chrismate mutase, an enzyme of the phenylalanine-tyrosine biosynthesis, is changed. In comparison with the wild type chorismate mutase, the enzyme of PK101 is characterized by a complete loss of sensitivity to l-phenylalanine inhibition and to a considerable loss of sensitivity to l-tryptophan activation. Furthermore, the chorismate mutase activity of the mutant is more than 7-fold higher in the absence of l-tryptophan than in the wild type. The PK101 enzyme is also changed in the pH optimum and in some kinetic constants. We found an increased intracellular pool of both phenylalanine and tyrosine and a reduced contents of tryptophan in the mutant cells. Our genetic data indicate that the mutant phenotype is dominant over the wild type.     

The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport

Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine. A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize l-leucine as sole nitrogen source and very low levels of [¹⁴C]l-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [¹⁴C]l-leucine uptake. Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine. The complementing gene, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation. The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin mutant containing the allele act1-1. Sac3p has been previously shown to be localized in the nucleus. When a lep1 mutant was crossed with a sac3 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic. Received: 6 January 1999 / Accepted: 4 June 1999     

Nucleotide sequence of the Escherichia coli hisD gene and of the Escherichia coli and Salmonella typhimurium hisIE region

Summary In this paper we report the nucleotide sequence of the hisD gene of Escherichia coli and of the hisIE region of both E. coli and Salmonella typhimurium. The hisD gene codes for a bifunctional enzyme, l-histidinol: NAD⁺ oxidoreductase, of 434 amino acids with a molecular mass of 46,199 daltons. We established that the hisIE region of both S. typhimurium and E. coli is composed of a single gene and not, as previously believed, of two separate genes. The derived amino acid sequence indicates that the hisIE gene codes for a bifunctional protein of 203 amino acids with an approximate molecular mass of 22,700 daltons. We also determined the nucleotide sequence of a deletion mutant in S. typhimurium which abolishes the hisF and hisI functions but retains the hisE function. We deduced that the mutant produces a chimeric protein fusing the aminoterminal region of the upstream hisF gene to the carboxylterminal domain of the hisIE gene which encodes for the hisE function. In view of these results the structural and functional organization of the histidine operon in enteric bacteria needs to be revised. The operon is composed of only 8 genes and the pathway leading to the biosynthesis of the amino acid requires 11 enzymatic steps.     

An evolvant ofEscherichia coli that employs thel-fucose pathway also for growth onl-galactose andd-arabinose

Summary l-Galactose,d-arabinose, andl-fucose form six-membered rings with identical stereoconfigurations. However, onlyl-fucose can serve as the sole carbon and energy source of wild-typeEscherichia coli K-12. A mutant that can grow onl-galactose andd-arabinose was isolated by alternate selection on the two sugars. Thel-fucose pathway became inducible by all three sugars. Transduction into the mutant of the wild-type fuc⁺ region containing both the regulatory and structural genes abolished the novel growth abilities onl-galactose andd-arabinose, whereas transduction into the mutant of a fuc deletion abolished the growth abilities on all three sugars. Introduction of the wild-type fucR⁺ (which encodes the activator protein for the fuc regulon) on a multicopy plasmid depressed the growth abilities of the mutant onl-galactose andd-arabinose, but not onl-fucose. The results suggest that the effector specificity of the activator protein in the mutant was broadened. It is proposed that an adaptive response of an activator-controlled system is more likely than that of a repressor-controlled system to achieve fixation in a population, because the first variant to emerge in response to a novel metabolic demand has a good chance of having an altered specificity of regulation. Such a change entails little or no metabolic liability during the absence of the novel substrate. In contrast, the first variant of a negatively controlled system to emerge has an overwhelming chance of being the result of a random mutation that destroys repressor function. Although negatively controlled systems can be more opportunistic in exploiting new conditions than positively controlled systems, an adaptive change is less likely to become fixed because of the cost associated with gratuitous constitutive gene expression in the absence of the substrate.     

Double mutation of the <Emphasis Type="Italic">PDC1</Emphasis> and <Emphasis Type="Italic">ADH1</Emphasis> genes improves lactate production in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> expressing the bovine lactate dehydrogenase gene

Expression of a heterologous l-lactate dehydrogenase (l-ldh) gene enables production of optically pure l-lactate by yeast Saccharomyces cerevisiae. However, the lactate yields with engineered yeasts are lower than those in the case of lactic acid bacteria because there is a strong tendency for ethanol to be competitively produced from pyruvate. To decrease the ethanol production and increase the lactate yield, inactivation of the genes that are involved in ethanol production from pyruvate is necessary. We conducted double disruption of the pyruvate decarboxylase 1 (PDC1) and alcohol dehydrogenase 1 (ADH1) genes in a S. cerevisiae strain by replacing them with the bovine l-ldh gene. The lactate yield was increased in the pdc1/adh1 double mutant compared with that in the single pdc1 mutant. The specific growth rate of the double mutant was decreased on glucose but not affected on ethanol or acetate compared with in the control strain. The aeration rate had a strong influence on the production rate and yield of lactate in this strain. The highest lactate yield of 0.75 g lactate produced per gram of glucose consumed was achieved at a lower aeration rate.