The anaerobic metabolism of phthalate and other aromatic compounds by the denitrifying bacterium Pseudomonas sp. strain P136 was studied. Benzoate, cyclohex-1-ene-carboxylate, -2-hydroxycyclohexanecarboxylate, and pimelate were detected as predominant metabolic intermediates during the metabolism of three isomers of phthalate, m-hydroxybenzoate, p-hydroxybenzoate, and cyclohex-3-ene-carboxylate. Inducible acyl-coenzyme A synthetase activities for phthalates, benzoate, cyclohex-1-ene-carboxylate, and cyclohex-3-ene-carboxylate were detected in the cells grown on aromatic compounds. Simultaneous adaptation to these aromatic compounds also occurred. A similar phenomenon was observed in the aerobic metabolism of aromatic compounds by this strain. A new pathway for the anaerobic metabolism of phthalate and a series of other aromatic compounds by this strain was proposed. Some properties of the regulation of this pathway were also discussed.Anaerobic metabolism of aromatic compounds by bacteria is regarded as a quite different process from general aerobic metabolism. The occurrence of anaerobic utilization of aromatic compounds has been reported for several bacterial cultures (7); however, only a few pathways were proposed for the anaerobic metabolism of aromatic compounds on the basis of biochemical evidence.Especially for denitrifying bacteria, there had been only one instance in which a pathway was proposed: that of the anaerobic metabolism of benzoate by Moraxella sp. (24). Although several cultures of denitrifying bacteria have been shown to utilize benzoate and its derivatives anaerobically (5,19,22), the metabolic pathways occurring in such bacteria have not been studied with emphasis on biochemical aspects.The bacterial strains mentioned above failed to utilize phthalate under anaerobic conditions, and little is known about the anaerobic metabolism .of phthalate. It is well known that phthalate is metabolized aerobically via a pathway which is quite different from that for benzoate utilization (9). However, the anaerobic metabolic pathway of phthalate is still unclear. Although two strains of denitrifying bacteria have been recently reported to utilize phthalate anaerobically denitrification with phthalate (1) and positive growth on phthalate (5) have been only briefly described. These investigations lacked detailed biochemical studies on the metabolic intermediates, and there had been little information about the pathway based on the substantial experimental evidence on the anaerobic metabolism of phthalate.We selective enrichment for the anaerobic utilization of ophthalate by nitrate respiration. Strain P136 was cultivated under aerobic and anaerobic conditions in basal medium containing appropriate substrates, and cell suspensions for the biochemical studies were prepared as described previously (17).Isolation of metabolic intermediates. For the anaerobic accumulation of metabolites, reactions were carried out in a series of 25-ml Thunberg tubes. The main compartment of each tube contained 5 ml of cell susp...

Protein disulfide-isomerase (PDI), which reactivates inactive scrambled RNase, was purified from Saccharomyces cerevisiae. The enzyme was purified 1,850-fold to apparent homogeneity by five purification steps: 30-70% ammonium sulfate fractionation, DEAE Toyopearl-650S and Butyl Toyopearl-650S chromatographies, and differential Phenyl-5PW HPLC with or without cysteine. The native enzyme had an apparent Mr of 140,000 on gel filtration chromatography, and its NH2-terminal was blocked. The Mr of its subunits were estimated to be 70,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis, indicating that the enzyme is probably composed of two identical subunits. The Mr of the subunits changed to 60,000 on endoglucosaminidase H treatment, indicating that the enzyme is transported into the endoplasmic reticulum. The enzyme has a pH optimum of 8.5, and pI of 4.02. Its enzymic properties were compared with those of purified bovine liver PDI. The Km values of yeast and bovine PDIs for scrambled RNase were 1 x 10(-5) and 2 x 10(-5) M, and their Vmax values were 6 and 7 units/mg protein, respectively. The two enzymes showed no significant differences in Km or Vmax values with respect to thiol compounds. Bacitracin inhibited both PDIs in the same fashion. These results indicate that this yeast PDI corresponds to mammalian PDI.

Marine nitrogen-fixing bacteria distributed in the eelgrass bed and seawater of Aburatsubo Inlet, Kanagawa, Japan were investigated using anaerobic and microaerobic enrichment culture methods. The present enrichment culture methods are simple and efficient for enumeration and isolation of nitrogen-fixing bacteria from marine environments. Mostprobable-number (MPN) values obtained for nitrogen-fixing bacteria ranged from 1.1×10(2) to 4.6×10(2)/ml for seawater, 4.0×10(4) to 4.3×10(5)/g wet wt for eelgrass-bed sediment, and 2.1 × 10(5) to 1.2 × 10(7)/g wet wt for eelgrass-root samples. More than 100 strains of halophilic, nitrogen-fixing bacteria belonging to the family Vibrionaceae were isolated from the MPN tubes. These isolates were roughly classified into seven groups on the basis of their physiological and biochemical characteristics. The majority of the isolates were assigned to the genusVibrio and one group to the genusPhotobacterium. However, there was also a group that could not be identified to the generic level. All isolates expressed nitrogen fixation activities under anaerobic conditions, and no organic growth factors were required for their activities.