p-Aminobenzoate biosynthesis in Escherichia coli. Purification of aminodeoxychorismate lyase and cloning of pabC

p-Aminobenzoate, a component of the vitamin folate, is one of seven compounds derived from the aromatic precursor chorismate in Escherichia coli. Historically the gene products of pabA and pabB were assumed to be sufficient for de novo p-aminobenzoate biosynthesis. Recent studies, however, have shown that these proteins, as nonidentical subunits of a single enzyme, act on chorismate to form a diffusible intermediate, most likely 4-amino-4-deoxychorismate. This intermediate is then converted to p-aminodeoxychorismate lyase (Nichols, B. P., Seibold, A. S., and Doktor, S. Z. (1989) J. Biol. Chem. 264, 8597-8601). Here we describe partial characterization of the intermediate and the purification of aminodeoxychorismate lyase 4100-fold to near homogeneity. Further purification of this enzyme by high pressure liquid chromatography permitted isolation of a pure sample that yielded N-terminal sequence. A 64-fold redundant oligonucleotide probe was used to identify a lambda clone containing the gene encoding aminodeoxychorismate lyase. The aminodeoxychorismate lyase gene, designated pabC, was mapped to 25 min on the E. coli chromosome and lies on a 7.5-kilobase pair EcoRI fragment. A strain harboring a pACYC184 recombinant containing pabC overproduced aminodeoxychorismate lyase activity 77-fold.     

Production of Hexagenia limbata nymphs in contaminated sediments in the Upper Great Lakes Connecting Channels

In April through October 1986, we sampled sediments and populations of nymphs of the burrowing mayfly, Hexagenia limbata (Serville), at 11 locations throughout the connecting channels of the upper Great Lakes, to determine if sediment contaminants adversely affected nymph production. Production over this period was high (980 to 9231 mg dry wt m^-2) at the five locations where measured sediment levels of oil, cyanide, and six metals were below the threshold criteria of the U.S. Environmental Protection Agency and the Ontario Ministry of Environment for contaminated or polluted sediments, and also where the criterion for visible oil given in the Water Quality Agreement between the U.S.A. and Canada for connecting waters of the Great Lakes was not exceeded. At the other six locations where sediments were polluted, production was markedly lower (359 to 872 mg dry wt m^-2). This finding is significant because it indicates that existing sediment quality criteria can be applied to protect H. limbata from oil, cyanide, and metals in the Great Lakes and connecting channels where the species fulfills a major role in secondary production and trophic transfer of energy.Contribution 733, of the National Fisheries Research Center-Great Lakes, U.S. Fish and Wildlife Service, 1451 Green Road, Ann Arbor, MI 48105.     

Cloning and sequencing of Escherichia coli ubiC and purification of chorismate lyase.

In Escherichia coli, chorismate lyase catalyzes the first step in ubiquinone biosynthesis, the conversion of chorismate to 4-hydroxybenzoate. 4-Hydroxybenzoate is converted to 3-octaprenyl-4-hydroxybenzoate by 4-hydroxybenzoate octaprenyltransferase. These two enzymes are encoded by ubiC and ubiA, respectively, and have been reported to map near one another at 92 min on the E. coli chromosome. We have cloned the ubiCA gene cluster and determined the nucleotide sequence of ubiC and a portion of ubiA. The nucleotide sequence abuts with a previously determined sequence that encodes a large portion of ubiA. ubiC was localized by subcloning, and overproducing plasmids were constructed. Overexpression of ubiC allowed the purification of chorismate lyase to homogeneity, and N-terminal sequence analysis of chorismate lyase unambiguously defined the beginning of the ubiC coding region. Although chorismate lyase showed no significant amino acid sequence similarity to 4-amino-4-deoxychorismate lyase (4-amino-4-deoxychroismate----4-aminobenzoate), the product of E. coli pabC, chorismate lyase overproduction could complement the growth requirement for 4-aminobenzoate of a pabC mutant strain. Of the several enzymes that convert chorismate to intermediates of E. coli biosynthetic pathways, chorismate lyase is the last to be isolated and characterized.     

Calcium regulation of the human PMN cytosolic 15-lipoxygenase

Addition of tracer (pg) amounts of [3H]arachidonic acid to the 120,000 x g cytosolic fraction of human polymorphonuclear leukocytes (PMNs) produced [3H]-15-HETE, the product of the 15-lipoxygenase, as the major metabolite. In the presence of nanomolar and low micromolar amounts of calcium, [3H]-15-HETE formation was increased as much as 15-fold which corresponded to 17% conversion of added substrate. This enhancement of the cytosolic 15-lipoxygenase activity, which was reversible by EGTA, was inhibited by phosphatidyl serine and phosphatidyl choline. Millimolar levels of calcium inhibited the cytosolic 15-lipoxygenase and the 5-lipoxygenase product 5-HETE could reverse this inhibition. These results indicate that calcium is an important modulator of the PMN 15-lipoxygenase when the enzyme is in a cytosolic milieu.     

Effects of pipette geometry on the time course of solution change in patch clamp experiments.

The time course of change in current through KATP channels in inside-out membrane patches, after step change of permeant ion (K+) concentration, was measured. A simple model of the patch as a membrane disc at the base of a cone with the apex removed, was able to describe the time course of channel activity after step change of [K+]. By measuring pipette geometry and using jumps of [permeant ion], it was then possible to estimate the time course of concentration at the membrane for jumps of any other ion or gating ligand. A simple channel block mechanism was used to simulate experiments with concentration jumps of a blocking ligand. The rate constants for ligand-channel interaction were extracted by least-squares fitting of computed mass action responses to those observed in simulated experiments. The simulations showed that even with diffusion delays of hundreds of milliseconds (as may occur in inside-out patch experiments), ligand association and dissociation rates of up to 1,000 s-1 could be accurately extracted by this approach. The approach should be generally applicable to the analysis of ligand concentration jump experiments on any ion channel whose activity is modulated by intracellular ligand.     

Nucleotide sequence of the trpB gene in Escherichia coli and Salmonella typhimurium

We have obtained the entire nucleotide sequence of the penultimate gene of the tryptophan operon, trpB, in Escherichia coli and Salmonella typhimurium. The amino acid sequence deduced for the E. coli gene product is in agreement with earlier, fragmentary protein sequence results. The trpB nucleotide sequences for the two bacterial species are perfectly colinear and show 85% identity. Most of the nucleotide differences found are without consequence for the amino acid sequence, which shows greater than 96% identity. The degree of conservation of both the nucleotide and amino acid sequences is significantly greater than for trpA, the adjacent gene encoding the other subunit of the same enzyme. When synonymous third codon position nucleotide differences are examined, they seem to be distributed at random throughout trpB and trpA, except for one completely conserved 66 basepair long region within trpB.     

Nucleotide sequences of the trpG regions of Escherichia coli,Shigella dysenteriae,Salmonella typhimurium and Serratia marcescens

The nucleotide sequences of Serratia marcescens trpG and the corresponding regions of Escherichia coli, Shigella dysenteriae and Salmonella typhimurium trpD have been determined. Analysis of the nucleotide sequence divergence suggests the following evolutionary relationships: Serratia-[Salmonella, (Escherichia, Shigella)]. Partial reconstruction of ancestral nucleotide sequences and subsequent analysis of nucleotide substitutions show that the majority of nucleotide substitutions in the evolution of trp(G)D are transitions that result in a reduction of G + C content. Since most of the nucleotide substitutions are in the third position of codons, bias in synonymous codon usage also reflects G + C content. The trpE-trp(G)D junction in the four organisms is characterized by overlapping translation termination and initiation codons. The relative positions of trpE and trp(G)D thus became fixed in evolution before the fusion of trpG and trpD. Nucleotide sequences representing the fusion of trpG and trpD in Escherichia, Shigella and Salmonella are not more nor less divergent than other portions of the trp(G)D coding sequences.     

Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.