Immunological identification and distribution of dissimilatory heme cd1 and nonheme copper nitrite reductases in denitrifying bacteria

Abstract

Polyclonal antibodies were used to identify heme or copper nitrite reductases in the following groups: 23 taxonomically diverse denitrifiers from culture collections, 100 numerically dominant denitrifiers from geographically diverse environments, and 51 denitrifiers from a culture collection not selected for denitrification. Antisera were raised against heme nitrite reductases from Pseudomonas aeruginosa and Pseudomonas stutzeri and against copper nitrite reduttase from Achromobacter cycloclastes. Nitrite reductases were identified by Western immunoblot. Diethyldithiocarbamate, which specifically inhibits copper nitrite reductases, was used to confirm the immunological characterization and determine which type was present in strains nonreactive with any antiserum. For groups in which the type of nitrite reductase has not been previously described, we found that Alcaligenes eutrophus, Bacillus azotoformans, Bradyrhizobium japonicum, Corynebacterium nephridii, and Rhizobium spp. contained copper nitrite reductase, while Aquaspirillum itersonii, Flavobacterium spp., and Pseudomonasfluorescens contained heme nitrite reductase. Heme nitrite reductases dominated, regardless of soil type or geographic origin. They occurred in 64 and 92%, respectively, of denitrifiers in the numerically dominant and nonselected collections. The two nitrite reductase types were mutually exclusive in individual bacteria, but both appeared in different strains from the Alcaligenes and Pseudomonas genera. The heme type predominated in Pseudomonas strains. The heme-type nitrite reductase appeared more conserved if judged by similarities in molecular weights and immunological reactions. The Cu type was found in more taxonomically unrelated strains and varied in molecular weight and antiserum recognition. Dissimilatory nitrite reductases (dNirs) are pivotal to the fate of combined nitrogen in the environment. They determine the point at which nitrogen is dissimilated instead of assimilated (24). Two distinct types of nitrite reductase are known: one contains a Cu center, and the other contains hemes c and dl. Both seem to carry out the same physiological reaction. NO is typically produced from NO2 during in vitro enzyme assays, but under some conditions, N20 is also produced (6, 32). Cytochrome cd, dNir has been identified in Alcaligenes faecalis (18), Micrococcus (Paracoccus) denitrificans (22), Paracoccus halodenitrificans (5), Pseudomonas aeruginosa (34), Pseudomonas stutzeri (12), and Thiobacillus denitrificans (14). Nonheme Cu dNir has been found in Achromobacter cycloclastes (8), Alcaligenes sp. (Achromobacter xylosoxidans) (17), Alcaligenesfaecalis S6 (10), Nitrosomonas europaea (25), and Rhodopseudomonas sphaeroides forma sp. denitrificans (Rhodobacter sphaeroides) (27). The distribution of the two dNir types in the environment is unknown, although cd, dNir has been identified most often in denitrifying isolates. These strains, which are typically studied in the laboratory, do not reflect the dominant denitrifying populations in nature ...

Nitrification and Denitrification Gene Abundances in Swine Wastewater Anaerobic Lagoons

“…Because the gene amoA is restricted to a total of three bacterial genera and represents a small fraction of the population in these lagoons (0.04%), it is not surprising to see it aff ected by so many environmental variables. In contrast, the gene nirK is found in a wide variety of bacterial genera (Coyne et al, 1989) and was the most well represented gene in this study (5.29%). However, it was positively correlated with lagoon conductivity, TSS, VSS, COD, and FA.…”

Physiological characterization of anaerobic ammonium oxidizing bacterium ‘Candidatus Jettenia caeni’

“…Copper is a cofactor of NirK and is required for NO2 − reduction, while it is loosely associated with the enzyme (Heylen et al, 2006;Lawton et al, 2013). Addition of DDC forms a copper chelate and suppresses the NO2 − -reducing activity of NirK-dependent heterotrophic denitrifiers (Shapleigh and Payne, 1985;Coyne et al, 1989). Therefore, inhibition by DDC suggests that 'Ca.…”

Induction of denitrifying enzymes in oxygen-limited Achromobacter cycloclastes continuous culture

“…Electrophoretically pure NOr and N20 reductase from A. cycloclastes were obtained from C. Huls¢ (C. Hulse, Ph.D. thesis, University of Virginia, 1989). Procedures [or antisera produc-tion, sample preparation and immunoblotting are described elsewhere [13]. lmmunodiffusion assays an~ Western immunobiots demonstrated the specificity of the antisera for the antigens against wMeh they were raised [13].…”