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An 18-kbp Acinetobacter calcoaceticus chromosomal segment contains the pcaIJFBDKCHG operon, which is required for catabolism of protocatechuate, and pobSRA, genes associated with conversion of p-hydroxybenzoate to protocatechuate. The genetic function of the 6.5 kbp of DNA between pcaG and pobS was unknown. Deletions in this DNA were designed by removal of fragments between restriction sites, and the deletion mutations were introduced into A. calcoaceticus by natural transformation. The mutations prevented growth with either quinate or shikimate, growth substrates that depend upon qui gene function for their catabolism to protocatechuate. The location of quL4, a gene encoding quinate-shikimate dehydrogenase, was indicated by its expression in one of the deletion mutants, and the position of the gene was confirmed by determination of its 2,427-bp nucleotide sequence. The deduced amino acid sequence of QuiA confirmed that it is a member of a family of membrane-associated, pyrrolo-quinoline quinone-dependent dehydrogenases, as had been suggested by earlier biochemical investigations. Catabolism of quinate and shikimate is initiated by NADIdependent dehydrogenases in other microorganisms, so it is evident that different gene pools were called upon to provide the ancestral enzyme for this metabolic step.Quinate and shikimate are abundant in the biosphere (17), and the ability to use these compounds as growth substrates is distributed broadly among microorganisms (7,29,36). In both eukaryotes and prokaryotes, quinate and shikimate are converted to protocatechuate which is metabolized by the P-ketoadipate pathway (Fig. 1). The first enzymatic step in the pathway, the oxidation of either quinate or shikimate, is catalyzed by a single NAD+-dependent dehydrogenase in members of the eukaryotic genera Neurospora (1) and Aspergillus (6,16). Similarly, an NAD+-dependent dehydrogenase appears to catalyze the first step in quinate catabolism by members of the gram-positive prokaryotic genus Nocardia (5).Initial investigations of quinate catabolism in the gramnegative bacterial species Acinetobacter calcoaceticus indicated that quinate dehydrogenase, the product of the quiA structural gene (Fig. 1), is a membrane-associated enzyme that acts upon either quinate or shikimate (38). The enzyme is induced by protocatechuate (8), a metabolic product of quinate metabolism (Fig. 1). The electron carrier pyrrolo-quinoline quinone (PQQ) is required for formation of a functional quinate dehydrogenase in A. calcoaceticus (23).Genetic analysis of A. calcoaceticus ADP1 is greatly facilitated by its competence for natural transformation (21). This property allowed the use of gap repair (13) to recover a 14.9-kbp chromosomal fragment. A 10.0-kbp subclone (Fig. 2) of the chromosomal fragment extends from pcaG, a structural gene for the dioxygenase that acts on protocatechuate (15), to pobA, the structural gene for the monooxygenase that converts p-hydroxybenzoate to protocatechuate (10). The pcaIJFBD-KCHG genes and pobA were known to be in separ...

We report the development and field validation of 2 ELISAs for the detection of Ancylostoma caninum or Toxocara canis coproantigens in the feces of dogs with experimental and natural infections, and evidence of cross-reactivity with respective feline counterparts. A. caninum-specific coproantigens were detected in feces of experimentally infected dogs starting at 9 d post-infection (dpi), whereas eggs were not seen until 23 dpi. T. canis-specific coproantigens were detected in 3 of 5 experimentally infected dogs by 31 dpi, and 4 of the 5 animals by 38 dpi. T. canis eggs were seen in feces of 4 of the 5 animals by 38 dpi. One dog had delayed coproantigen detection and low egg output. Additionally, 817 canine and 183 feline fecal samples from naturally infected animals tested by flotation were subjected to coproantigen ELISA testing. Of these 1,000 canine and feline samples, 13 and 23 samples, respectively, were positive for "hookworm" or "roundworm" eggs; 19 and 26 samples were ELISA positive, respectively. The T. canis ELISA detected T. cati coproantigen in cat fecal samples. Discrepant ELISA and flotation results were obtained for 16 hookworm- and 13 roundworm-positive samples. Re-examination of the egg-positive, ELISA-negative samples indicated several instances of possible misidentification or coprophagy, whereas detection of antigen in samples without egg observations is likely a reflection of true infection status with egg shedding below detection levels. There is good indication, based on accumulated field data, that these antigen tests also detect other hookworm and ascarid species.