Identification of Nitrite-Oxidizing Bacteria with Monoclonal Antibodies Recognizing the Nitrite Oxidoreductase

Abstract

Immunoblot analyses performed with three monoclonal antibodies (MAbs) that recognized the nitrite oxidoreductase (NOR) of the genus Nitrobacter were used for taxonomic investigations of nitrite oxidizers. We found that these MAbs were able to detect the nitrite-oxidizing systems (NOS) of the generaNitrospira, Nitrococcus, andNitrospina. The MAb designated Hyb 153-2, which recognized the α subunit of the NOR (α-NOR), was specific for species belonging to the genus Nitrobacter. In contrast, Hyb 153-3, which recognized the β-NOR, reacted with nitrite oxidizers of the four genera. Hyb 153-1, which also recognized the β-NOR, bound to members of the generaNitrobacter and Nitrococcus. The molecular masses of the β-NOR of the genusNitrobacter and the β subunit of the NOS (β-NOS) of the genus Nitrococcus were identical (65 kDa). In contrast, the molecular masses of the β-NOS of the generaNitrospina and Nitrospira were different (48 and 46 kDa). When the genus-specific reactions of the MAbs were correlated with 16S rRNA sequences, they reflected the phylogenetic relationships among the nitrite oxidizers. The specific reactions of the MAbs allowed us to classify novel isolates and nitrite oxidizers in enrichment cultures at the genus level. In ecological studies the immunoblot analyses demonstrated thatNitrobacter or Nitrospira cells could be enriched from activated sludge by using various substrate concentrations. Fluorescence in situ hybridization and electron microscopic analyses confirmed these results. Permeated cells of pure cultures of members of the four genera were suitable for immunofluorescence labeling; these cells exhibited fluorescence signals that were consistent with the location of the NOS.

Nitrite-oxidizing bacteria guild ecology associated with nitrification failure in a continuous-flow reactor

“…In fact, the two guilds almost always coexist in WWTPs, although Nitrospira spp. are most prevalent during periods of optimal performance (Bartosch et al, 1999;Wagner et al, 2002), particularly Type I and Type II Nitrospira sublineages (Daims et al, 2001). It is reported here that unstable behaviour in nitrification can result from occasional, but sometimes dramatic shifts in the NOB guild, which can cause sudden and complete loss of function.…”

Isolation of sublineage I Nitrospira by a novel cultivation strategy

“…Therefore, the physiological and biochemical properties of NOB have been characterized by observations of Nitrobacter strains (Starkenburg et al, 2011). More recently, cultivation-independent molecular methods (Wagner et al, 1996;Burrell et al, 1998;Hovanec et al, 1998;Juretschko et al, 1998) and immunological techniques (Bartosch et al, 1999) have indicated that the genus Nitrospira is more widespread than Nitrobacter and most likely plays a primary role in the global nitrogen cycle, and natural nitrite conversion is an important part of the global nitrogen cycle. The genus Nitrospira represents a monophyletic lineage within the deep-branching bacterial phylum Nitrospirae, which is distinct from that of proteobacterial NOB (Ehrich et al, 1995).…”

Graph‐based deconvolution analysis of multiplex sandwich microarray immunoassays: applications for environmental monitoring

“…The identification and monitoring of microorganisms and bioanalytes from complex natural samples is of general interest in biomedical, bioterrorism, environmental, industrial or astrobiological applications (Schloter et al, 1995;Luppa et al, 2001;Lim et al, 2005;Parro et al, 2008;Van Dorst et al, 2010). For environmental applications, immunoassays have been developed for identification of organisms, detection of key metabolic enzymes and serotyping bacterial pathogens (Bartosch et al, 1999;Fiencke and Bock, 2004;Marimon et al, 2010). O'Connor and Coates (2002) reported a good example of the usefulness of immunoassays for the description of physiological functionality of complex microbial populations regardless of their phylogenetic affiliations: they developed a universal immunoprobe for (per)chlorate-reducing bacteria by producing antibodies against the key enzyme for (per)chlorate reduction (chlorite dismutase).…”