Acquisition of new metabolic capabilities: multicopy suppression by cloned transaminase genes in Escherichia coli K-12.

The four general transaminases of Escherichia coli K-12 have overlapping, but discrete, substrate specificities and participate in the final step in the synthesis of at least seven different amino acids. Through the use of strains that have mutations in one or more transaminase genes and carry a different wild-type (wt) gene on a multicopy plasmid, it was possible to detect instances in which an amplified wt gene suppressed nonallelic mutations. In these cases, overproduction of the enzyme permitted a broader range of substrates to be used at physiologically significant levels, either because a low catalytic efficiency (in the case analyzed here) or a low affinity of the enzyme towards the substrate prevented its effective utilization under normal conditions. Consequently, by compensating for a low catalytic reaction rate, enzyme overproduction circumvents the original lesion and restores biosynthetic activity to the mutant strain. The suppression of a mutation in one gene by amplified copies of a different wt gene is termed 'multicopy suppression'. This phenomenon is useful for detecting poorly expressed genes, for detecting duplicate genes, for identifying secondary functions of the products of known genes, and for elucidating the metabolic role of the product of the suppressed gene.

The m gamma delta-1 element, a small gamma delta (Tn1000) derivative useful for plasmid mutagenesis, allele replacement and DNA sequencing.

Transposon gamma delta (Tn1000), a 6-kb member of the Tn3 family, is widely used for plasmid mutagenesis. A 1.8-kb derivative of gamma delta was constructed that contains the kan gene from Tn5 and the resolution (res) site from gamma delta cloned between 40-bp inverted repeats of gamma delta's delta (delta) end. This element, named m gamma delta-1, lacks the genes encoding transposase and resolvase, and therefore depends on its host to supply transposition and resolution functions. Thus, in strains lacking gamma delta, m gamma delta-1 will not transpose. The m gamma delta-1 element is shown to be useful for mutagenesis of plasmids, DNA sequencing, and allele replacement (in Streptomyces avermitilis).

GelStar nucleic acid gel stain: high sensitivity detection in gels.

GelStar nucleic acid gel stain can be used for sensitive fluorescent detection of both double-stranded (ds) and single-stranded (ss) DNAs, oligonucleotides and RNA in gels. The stain can be added to agarose gels at casting for immediate imaging after electrophoresis or can be used after electrophoresis with both agarose and acrylamide gels. GelStar stain is highly fluorescent only when bound to nucleic acids thus giving superior signal-to-noise ratios and obviating the need to destain the gel. The detection limits of GelStar strain are 20 pg for dsDNA, 25 pg for ssDNA and 10 ng for native or glyoxal-treated RNA.

Sequence and evolution of the FruR protein of Salmonella typhimurium: a pleiotropic transcriptional regulatory protein possessing both activator and repressor functions which is homologous to the periplasmic ribose-binding protein.

The repressor of the fructose (fru) operon of Salmonella typhimurium (FruR) has been implicated in the transcriptional regulation of dozens of genes concerned with central metabolic pathways of carbon utilization. We here report the nucleotide sequence of the gene encoding FruR and analyse both its operator-promoter region and its deduced amino acyl sequence. The FruR protein was overexpressed and was shown to have a molecular weight of about 36 kDa in agreement with the molecular weight deduced from the gene sequence. Sequence analyses revealed that FruR is homologous to 9 distinct bacterial DNA-binding proteins, most of which recognize sugar inducers and all of which possess helix-turn-helix motifs within their N-terminal regions and exhibit sequence identity throughout most of their lengths. FruR is also homologous to the periplasmic ribose-binding protein which serves as a constituent of the ribose transport/chemoreception system. The ribose-binding protein is in turn homologous to binding proteins specific for arabinose and galactose. The periplasmic binding proteins, the structures of some of which have been elucidated in three dimensions, lack the N-terminal helix-turn-helix region, but instead possess N-terminal hydrophobic signal sequences which target them to the periplasm. A phylogenetic tree for the more closely related proteins of this superfamily was constructed, and a signature motif was identified which should facilitate future detection of additional transcriptional regulatory proteins belonging to this family.

A new pleiotropic mutation causing defective carbohydrate uptake in Escherichia coli K-12: isolation, mapping, and preliminary characterization.

A new pleiotropic mutation, designated cup-1 (for carbohydrate uptake), which impairs the ability of Escherichia coli cells to grow on a large number of phosphotransferase system (PTS) and non-PTS carbohydrates by blocking their entry into the cells, has been isolated, partially characterized, and mapped. The mutants grew poorly even on rich and glucose minimal media. Fast-growing revertants rapidly accumulated in cultures grown on either of the above two media and made stable maintenance of the mutation difficult. Several extragenic suppressor mutations that permitted cup cells to grow on specific single sugars or groups of sugars have been isolated. One such suppressor, which enabled cup cells to grow as well on glycerol minimal medium as their wild-type parent, has been helpful in stably maintaining these cells in this medium. cup-1 has been mapped to 97 min on the standard E. coli map. It cotransduced with a transposon Tn10 inserted clockwise to it and (very weakly) with uxuA. Surprisingly, it failed to cotransduce with pyrB, argI, or valS, three markers located nearby but counterclockwise to it. In F' merodiploids, cup-1 was dominant over its cup+ allele. Cyclic AMP permitted growth of cup-1 cells on some sugars but not all. Apparently, reduced cyclic AMP level and therefore noninduction of several sugar operons is one but not the only effect of cup.

A functional leuABCD operon is required for leucine synthesis by the tyrosine-repressible transaminase in Escherichia coli K-12.

In Escherichia coli K-12, two enzymes, encoded by ilvE and tyrB, catalyze the amination of 2-ketoisocaproate (2-KIC) to form leucine. Although leucine-requiring derivatives of an ilvE strain that are unable to grow on 2-KIC were expected to have mutations only in tyrB, mapping studies showed that one such mutation was tightly linked to the leu operon (at 1.5 min), not to tyrB (at 92 min). Chromosomal fragments cloned because they complemented this mutation were found to complement leu mutations, and vice versa, but none of these fragments complemented a tyrB mutation. The Tn5 insertion and flanking host DNA from this anomalous mutant was cloned in vivo, using Mu dII4042, and an in vivo procedure was developed to isolate deletion derivatives of Tn5-containing plasmids. These deletion plasmids were used to determine the DNA sequences flanking the transposon. The data showed that Tn5 was inserted between bp 122 and 132 in the leu leader. In addition, other ilvE leu double mutants were found to be unable to grow on 2-KIC in place of leucine. The accumulation of 2-ketoisovalerate in ilvE leu double mutants was shown to interfere with 2-KIC amination by the tyrB-encoded transaminase and also by the aspC- and avtA-encoded transaminases (which are able to catalyze this reaction in vivo when the corresponding genes are present on multicopy plasmids).

Glucose metabolism in 'Sphingomonas elodea': pathway engineering via construction of a glucose-6-phosphate dehydrogenase insertion mutant.

'Sphingomonas (formerly Pseudomonas) elodea' produces the industrially important polysaccharide gellan when grown in media containing glucose. Glucose catabolic enzymes and enzymes of central carbon metabolism were assayed in crude extracts of glucose-grown cultures of this bacterium. Based on these analyses it was concluded that glucose is converted to either gluconate or glucose 6-phosphate and that both of these products are converted to 6-phosphogluconate, a precursor for the Entner-Doudoroff (ED) and pentose phosphate pathways. Phosphoglucoisomerase (Pgi) activity was detected, but the lack of phosphofructokinase activity indicated that the Embden-Meyerhof glycolytic pathway is non-functional for glucose degradation. Thus, this bacterium utilizes glucose mainly via the ED and pentose phosphate pathways. Enzyme analyses suggested the involvement of glucose-6-phosphate dehydrogenase (Zwf) in glucose utilization and CO2 production. The zwf gene was cloned from 'S. elodea' and partially sequenced, and a null zwf mutant was constructed. This mutant exhibited no Zwf activity in in vitro assays, grew normally on glucose minimal medium and accumulated biomass (cells plus gellan) and produced CO2 at the same rates as the parental strain. Potential explanations for this finding are provided. Clones carrying the pgi gene were isolated fortuitously.

Mevalonate kinase is predominantly localized in peroxisomes and is defective in patients with peroxisome deficiency disorders.

We reported recently that mevalonate kinase (EC 2.7.1.36; ATP:mevalonate 5-phosphotransferase) that was isolated from rat liver and believed to be a cytosolic protein was localized in rat liver peroxisomes. In addition, we found that the mevalonate kinase monoclonal antibody used in the study also reacted with several other proteins present in the mitochondrial and cytosolic fractions. These findings raised the prospect of the presence of several isoenzymes of mevalonate kinase localized in different compartments of the cell. In the current study we produced four new polyclonal antibodies against different epitopes of mevalonate kinase to investigate the subcellular localization of the protein by several different approaches: (i) by analytical subcellular fractionation and immunoblotting of mevalonate kinase in the isolated subcellular fractions with the monospecific antibodies; (ii) by immunocryoelectron microscopy techniques; and (iii) by expressing the cDNA encoding mevalonate kinase in mammalian cells. The data obtained demonstrate that there is only one mevalonate kinase protein that is predominantly localized in peroxisomes. We also illustrate that the protein is targeted to and imported into peroxisomes. In addition, we show that in cells and tissues obtained from patients with peroxisomal deficiency diseases mevalonate kinase protein and its activity are severely reduced.

pBR322-derived multicopy plasmids harboring large inserts are often dimers in Escherichia coli K-12.

pBR322-related plasmids that are 2.3 to 5.1 kb were found predominantly as monomers, while plasmids that are 7.7 to 15.2 kb were found predominantly as dimers in rec+ cells of Escherichia coli K-12.

In vitro asymmetric binding of the pleiotropic regulatory protein, FruR, to the ace operator controlling glyoxylate shunt enzyme synthesis.

The fruR gene of Escherichia coli, which encodes the regulatory protein FruR, was cloned in the pT7-5 expression vector so as to overproduce a protein tagged with 6 histidine residues. By using a one-step chromatographic procedure, FruR was purified to near-homogeneity. Analysis of the protein under both denaturing and nondenaturing conditions indicated that it is a tetramer with a molecular mass of about 150 kilodaltons. The positions of interference between FruR and the operator of the acetate operon were examined. The number and nature of the nucleotides essential for FruR binding were determined by several different techniques: base methylation with dimethyl sulfate, base removal by formic acid and hydrazine, uracil interference, and hydroxyl radical footprinting. It was observed that FruR asymmetrically binds to a 16-base pair DNA sequence located 170 base pairs upstream from the transcriptional start point of the ace operon.