Cholera toxin (CT) and the Escherichia coli heat-labile toxin (LT) are functionally, structurally and immunologically similar enterotoxins. Both toxins cause the elevation of cyclic AMP levels in gut epithelial cells by catalysing the NAD-dependent ADP ribosylation of membrane proteins. Each toxin is composed of two dissimilar subunits. The A subunit has an enzymatic activity and is the adenylate cyclase-activating component of the enterotoxin. The B subunit recognizes membrane components and binds the holotoxin to the target call juxtaposing the A subunit with its substrates. Binding studies and competition experiments indicate that the membrane receptors for cholera toxin B subunit (CT-B) and LT-B are similar but not identical (these studies were performed before by LT was purified to homogeneity). The monosialosylganglioside GMI has been shown to be the receptor for the cholera toxin, and it probably composes part of the receptor for LT. Gyles and Barnum, first reported that LT and cholera toxin were immunologically related, and it has subsequently been shown that they share common antigenic determinants in both A and B subunits. The primary structure of CT-B has been determined. We report here a comparison between the amino acid sequences of LT-B and CT-B. The nucleotide sequence of the LT-B cistron (eltB) was determined using a recombinant plasmid encoding LT. Translation of this sequence revealed that LT-B and CT-B show significant amino acid sequence homology. In addition, several features of the eltB cistron were revealed by the sequence analysis.

The structure and products of the two cistrons encoding the Escherichia coli heat-labile toxin (LT) were studied. The LT deoxyribonucleic acid (DNA) region had been isolated as part of a DNA fragment from the plasmid P307, and this fragment was joined to the cloning vector pBR313. Deletion mutations of various lengths were introduced into the LT DNA region and into the adjacent DNA sequences. Analysis of the deletions indicated that the maximum size of the LT DNA region was 1.2 x 10(6) daltons. Two proteins of 11,500 daltons and 25,500 daltons had been shown to be encoded by the LT DNA region. The functions of these LT gene products were investigated. The 11,500-dalton protein had an adsorption activity for Y-1 adrenal cells, and this protein was shown to form aggregates of four or five monomers. The 25,500-dalton protein was shown to have an adenylate cyclase-activating activity. The two cistrons encoding for each of the LT proteins have been located on a genetic map of the LT DNA region. Both cistrons are probably transcribed from the same promoter.

The Escherichia coli gene coding for dihydropteroate synthase (DHPS) has been cloned and sequenced. The protein has 282 amino acids and a compositional molecular mass of 30,314 daltons. Increased expression of the enzyme was realized by using a T7 expression system. The enzyme was purified and crystallized. A temperature-sensitive mutant was isolated and found to express a DHPS with a lower specific activity and lower affinities for para-aminobenzoic acid and sulfathiazole. The allele had a point mutation that changed a phenylalanine codon to a leucine codon, and the mutation was in a codon that is conserved among published DHPS sequences.Dihydropteroate synthase (DHPS) (EC 2.5.1.15) catalyzes the condensation of para-aminobenzoic acid (pAB) with 7,8-dihydro-6-hydroxymethylpterin-pyrophosphate, forming 7,8-dihydropteroate (39, 44). This intermediary metabolite is subsequently converted to tetrahydrofolic acid, essential for the syntheses of purines, thymidylate, glycine, methionine, pantothenic acid, and N-formylmethionyl-tRNA. Sulfonamides are pAB analogs that are recognized by DHPS as alternate substrates (4,7,45,57). In the presence of sulfonamides, DHPS forms a sulfa-pterin adduct that is metabolically inert and diffuses from the cell (40). Folate cofactor depletion results in growth inhibition and in the appropriate environment, cell death (55).DHPS activity was first identified in crude cell extracts of several organisms by Shiota and coworkers (44, 45) and was identified in Escherichia coli by Brown et al. (8). The kinetic characteristics of DHPS have been studied by using partially purified extracts. Recently, a purification procedure for DHPS was published which showed that this enzyme constituted less than 0.01% of all the proteins in a cell (54). DHPS was purified to homogeneity, the sequence of the first 28 amino acids was determined, and the protein was shown to be a homodimer of two 30-kDa subunits. However, the purification procedure yielded less than 2 mg of purified protein from 1 kg of starting material, emphasizing the need to clone and overexpress this important chemotherapeutic target.Recently, the chromosomal gene that codes for DHPS in Streptococcus pneumoniae was cloned, sequenced, and shown to code for a protein of 34 kDa (28). A similar sequence was also identified in a Bacillus subtilis folic acid biosynthetic operon (48). Two other genes (sulI and sulII) that code for plasmid-borne sulfonamide-resistant DHPSs have also been sequenced (38, 52). However, there is no information about the E. coli DHPS gene. In this communication, we report the cloning, sequencing, and enhanced expression of the E. coli DHPS gene, designated folP. We also report conditions that allow crystallization of this enzyme.

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