Orange I1 azoreductase [NAD(P)H : l-(4'-sulfophenylazo)-2-naphthol oxidoreductase], an enzyme catalyzing the reductive cleavage of the azo bridge of Orange I1 and related dyes, was purified to electrophoretic homogeneity from Pseudomonas species, strain KF46. This organism utilized carboxy-Orange I1 [I -(4'-carboxypheny1azo)-2-naphthol] but not Orange I1 as the sole source of carbon, energy, and nitrogen. Orange I1 azoreductase was induced 80-fold by both Orange I1 and carboxy-Orange 11. With two successive runs of affinity chromatography using two chromatographic media with different triazinyl dyes as ligands, the enzyme was purified 120-fold with 43% yield. The purified enzyme is a monomer with a molecular weight of 30000. Its K , values were 1.5 pM for both Orange I1 and carboxy-Orange 11, 5 pM for NADPH, and 180 pM for NADH. A survey of the efficiency of various Orange dyes as substrates for Orange I1 azoreductase showed that: (a) a hydroxy group in the 2-position of the naphthol ring is required; (b) charged groups in proximity to the azo group hinder the reaction; (c) a second polar substituent on the dye molecule impedes the reaction; (d) electronwithdrawing groups on the phenyl ring accelerate the reaction.

Selection for utilization of carboxy-Orange I [1-(4'-carboxyphenylazo)-4-naphthol] in the chemostat yielded Pseudomonas strain K24 which was unable to grow on carboxy-Orange II [1-(4'-carboxyphenylazo)-2-naphthol] while selection for growth on carboxy-Orange II had previously led to strain KF46 which did not utilize carboxy-Orange I. Orange I azoreductase of strain K24, the key enzyme of dye degradation, was purified 80-fold with 17% yield to electrophoretic homogeneity and compared to the previously purified Orange II azoreductase of strain KF46. Common properties of the two enzymes were their monomeric structure, their specificity for NADPH and NADH as cosubstrates, the range of their Km values for substrates and cosubstrates as well as their reactivity towards a series of substrate analogs. They differed from each other with respect to molecular weight (21,000 and 30,000) and in the absolute requirement of Orange I azoreductase for a hydroxy group in the 4'position of the naphthol ring of the substrate molecule as compared to the requirement for substrates with a 2-naphthol moiety by Orange II azoreductase. The pure enzymes did not exhibit immunological cross-reaction with each other. Crude extracts of strains K24 and KF46 and of azoreductase-negative strains isolated at different stages of the adaptation experiments, however, contained material which cross-reacted (CRM) with both anti Orange I azoreductase serum and anti Orange II azoreductase serum. The CRM may represent a common precursor protein of the azoreductases in strains K24 and KF46.

No abstract

Escherichia coli grew anaerobically on L-malate only in the presence of H2; 91% of the L-malate utilized was converted to succinate. Anaerobically isolated membrane vesicles catalyzed the reduction of fumarate with H2 and contained a b-type cytochrome. Cytochrome c552 was present in the "periplasmic space." While isolating microorganisms (from sewage) capable of growing anaerobically on malate, it was found that one of these isolates was able to utilize L-malate as carbon and energy source only when molecular hydrogen was also available. That this organism was identified as Escherichia coli (designated E. coli KA) was unexpected, as anaerobic growth on malate had never been demonstrated for this organism. It was then of interest to determine: (i) whether anaerobic growth on malate in the presence of H2 is a phenomenon common to the species of E. coli in general, or only to this one strain (KA) in particular; and (ii) how hydrogen is involved in the catabolism of malate. MATERIALS AND METHODS Bacterial strains. E. coli KA was isolated from sewage and identified by M. Busse (Technische Universitat Miinchen-Weihenstephan; Bakteriologisches Institut). The organisms listed in Table 1 were obtained from M. Busse; the DSM designation indicates the strains were originally obtained from the Deutsche Sammlung von Mikroorganismen, and the WS designation indicates strains isolated by M. Busse. WGAS parent and WGAS frd 11 (lacking fumarate reductase) were kindly provided by J. Guest; these organisms were also Gal-, TrpA-, and streptomycin resistant (27). Growth of bacteria. The medium used had the following composition (grams per liter):

The E. coli biotin (bio) operon was modified to improve biotin production by host cells: (a) the divergently transcribed wild-type bio operon was re-organized into one transcriptional unit; (b) the wild-type bio promoter was replaced with a strong artificial (tac) promoter; (c) a potential stem loop structure between bioD and bioA was removed; and (d) the wild-type bioB ribosomal binding site (RBS) was replaced with an artificial RBS that resulted in improved bioB expression. The effects of the modifications on the bio operon were studied in E. coli by measuring biotin and dethiobiotin production, and bio gene expression with mini-cells and two-dimensional polyacrylamide gel electrophoresis. The modified E. coli bio operon was introduced into a broad host-range plasmid and used to transform Agrobacterium/Rhizobium HK4, which then produced 110 mg L-1 of biotin in a 2-L fermenter, growing on a defined medium with diaminononanoic acid as the starting material. Biotin production was not growth-phase dependent in this strain, and the rate of production remained high under limiting (maintenance) and zero growth conditions.

Enterobacter aerogenes was grown in continuous culture with ammonia as the growth-limiting substrate, and changes in citrate lyase and citrate synthase activities were monitored after growth shifts from anaerobic growth on citrate to aerobic growth on citrate, aerobic growth on glucose, anaerobic growth on glucose, and anaerobic growth on glucose plus nitrate. Citrate lyase was inactivated during aerobic growth on glucose and during anaerobic growth with glucose plus nitrate. Inactivation did not occur during anaerobic growth on glucose, and, as a result of the simultaneous presence of citrate lyase and citrate synthase, growth difficulties were observed. Citrate lyase inactivation consisted of deacetylation of the enzyme. The corresponding deacetylase could not be demonstrated in cell extracts, and it is concluded that, as in a number of other inactivations, electron transport to oxygen or nitrate was required for inactivation.

No abstract

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.