Appl Environ Microbiol, 1998 Oct, 64(10), 3584 - 90 Effects of pH and oxygen and ammonium concentrations on the community structure of nitrifying bacteria from wastewater Princic A, Mahne I I, Megusar F, Paul EA, Tiedje JM. Shifts in nitrifying community structure and function in response to different ammonium concentrations (50, 500, 1,000, and 3,000 mg of N liter-1), pH values (pH 6.0, 7.0, and 8.2), and oxygen concentrations (1, 7, and 21%) were studied in experimental reactors inoculated with nitrifying bacteria from a wastewater treatment plant . The abilities of the communities selected for these conditions to regain their original structures after conditions were returned to the original conditions were also determined . Changes in nitrifying community structure were determined by performing an amplified ribosomal DNA (rDNA) restriction analysis of PCR products obtained with ammonia oxidizer-specific rDNA primers, by phylogenetic probing, by small-subunit (SSU) rDNA sequencing, and by performing a cellular fatty acid analysis . Digestion of ammonia-oxidizer SSU rDNA with five restriction enzymes showed that a high ammonium level resulted in a great community structure change that was reversible once the ammonium concentration was returned to its original level . The smaller changes in community structure brought about by the two pH extremes, however, were irreversible . Sequence analysis revealed that the highest ammonium environment stimulated growth of a nitrifier strain that exhibited 92.6% similarity in a partial SSU rRNA sequence to its nearest relative, Nitrosomonas eutropha C-91, although the PCR product did not hybridize with a general phylogenetic probe for ammonia oxidizers belonging to the beta subgroup of the class Proteobacteria . A principal-component analysis of fatty acid methyl ester data detected changes from the starter culture in all communities under the new selective conditions, but after the standard conditions were restored, all communities produced the original fatty acid profiles. Appl Environ Microbiol, 1998 Oct, 64(10), 3656 - 62 A bioluminescence assay using Nitrosomonas europaea for rapid and sensitive detection of nitrification inhibitors; Iizumi T et al.; An expression vector for the luxAB genes, derived from Vibrio harveyi, was introduced into Nitrosomonas europaea . Although the recombinant strain produced bioluminescence due to the expression of the luxAB genes under normal growing conditions, the intensity of the light emission decreased immediately, in a time-and dose-dependent manner, with the addition of ammonia monooxygenase inhibitors, such as allylthiourea, phenol, and nitrapyrin . When whole cells were challenged with several nitrification inhibitors and toxic compounds, a close relationship was found between the change in the intensity of the light emission and the level of ammonia-oxidizing activity . The response of bioluminescence to the addition of allylthiourea was considerably faster than the change in the ammonia-oxidizing rate, measured as both the O2 uptake and NO2- production rates . The bioluminescence of cells inactivated by ammonia monooxygenase inhibitor was recovered rapidly by the addition of certain substrates for hydroxylamine oxidoreductase . These results suggested that the inhibition of bioluminescence was caused by the immediate decrease of reducing power in the cell due to the inactivation of ammonia monooxygenase, as well as by the destruction of other cellular metabolic pathways . We conclude that the assay system using luminous Nitrosomonas can be applied as a rapid and sensitive detection test for nitrification inhibitors, and it will be used to monitor the nitrification process in wastewater treatment plants. J Biochem (Tokyo), 1998 Oct, 124(4), 811 - 5 Effect of long-term ammonia starvation on the oxidation of ammonia and hydroxylamine by Nitrosomonas europaea; Wilhelm R et al.; Axenic cultures of the ammonia-oxidizing bacterium Nitrosomonas europaea were starved of ammonia (energy source) for up to 342 d . During this time the bacteria retained the ability to respond instantly to ammonia (1 mM) or hydroxylamine (0.1 mM) amendment by oxidizing it to nitrite without initial protein synthesis . In vivo, the ability to oxidize amended ammonia stayed almost constant during the starvation period, but a drop in the hydroxylamine oxidation rate (to 33%) was observed after 4 wk of starvation when exogenous hydroxylamine was supplied as sole energy source . In contrast, it has been shown that the level and in vitro activity of hydroxylamine oxidoreductase were not significantly affected during the starvation period . Only minor changes were detected between the protein patterns on one-dimensional SDS-PAGE of growing and starved cells . Thus, it is concluded that the activities of the energy-generating enzymes in N . europaea were not affected during long-term ammonia starvation. Biophys J, 1998 Oct, 75(4), 1964 - 72 Primary sequence and solution conformation of ferrocytochrome c-552 from Nitrosomonas europaea; Timkovich R et al.; Cytochrome c-552 from Nitrosomonas europaea is a 9.1-kDa monoheme protein that is a member of the bacterial cytochrome c-551 family . The gene encoding for c-552 has been cloned and sequenced and the primary sequence of the product deduced . Proton resonance assignments were made for all main-chain and most side-chain protons in the diamagnetic, reduced form by two-dimensional NMR techniques . Distance constraints (1056) were determined from nuclear Overhauser enhancements, and torsion angle constraints (88) were determined from scalar coupling estimates . Solution conformations for the protein were computed by the hybrid distance geometry-simulated annealing approach . For 20 computed structures, the root mean squared deviation from the average position of equivalent atoms was 0.84 A (sigma = 0.12) for backbone atoms over all residues . Analysis by residue revealed there were three regions clearly less well defined than the rest of the protein: the first two residues at the N-terminus, the last two at the C-terminus, and a loop region from residues 34 to 40 . Omitting these regions from the comparison, the root mean squared deviation was 0.61 A (sigma = 0.13) for backbone atoms, 0.86 A (sigma = 0.12) for all associated heavy atoms, and 0 . 43 A (sigma = 0.17) for the heme group . The global folding of the protein is consistent with others in the c-551 family . A deletion at the N-terminus relative to other family members had no impact on the global folding, whereas an insertion at residue 65 did affect the way the polypeptide packs against the methionine-ligated side of the heme . The effects of specific substitutions will be discussed . The structure of c-552 serves to delineate essential features of the c-551 family. Curr Microbiol, 1998 Oct, 37(4), 281 - 8 Physiological and molecular biological characterization of ammonia oxidation of the heterotrophic nitrifier Pseudomonas putida; Daum M et al.; The heterotrophic nitrifier Pseudomonas putida aerobically oxidized ammonia to hydroxylamine, nitrite, and nitrate . Product formation was accompanied by a small but significant release of NO, whereas N2O evolution could not be detected under the assay conditions employed . The isolate reduced nitrate to nitrite and partially further to NO under anaerobic conditions . Aerobically grown cells utilized gamma-aminobutyrate as a carbon source and as a N-source by ammonification . The physiological experiments, in particular the inhibition pattern by C2H2, indicated that P . putida expressed an ammonia monooxigenase . DNA-hybridization with an amoA gene probe coding for the smaller subunit of the ammonia monooxigenase of Nitrosomonas europaea allowed us to identify, to clone, and to sequence a region with an open reading frame showing distinct sequence similarities to the amoA gene of autotrophic ammonia oxidizers. Appl Environ Microbiol, 1998 Sep, 64(9), 3264 - 9 Biosensor determination of the microscale distribution of nitrate, nitrate assimilation, nitrification, and denitrification in a diatom-inhabited freshwater sediment Lorenzen J, Larsen LH, Kjaer T, Revsbech NP. High-resolution NO3- profiles in freshwater sediment covered with benthic diatoms were obtained with a new microscale NO3- biosensor characterized by absence of interference from chemical species other than NO2- and N2O . Analysis of the microprofiles obtained indicated no nitrification during darkness, high rates of nitrification and a tight coupling between nitrification and denitrification during illumination, and substantial rates of NO3- assimilation during illumination . Nitrification during darkness could be induced by purging the bulk water with O2 gas, indicating that the stimulatory effect on nitrification by illumination was caused by algal production of O2 . NH4+ addition did not stimulate nitrification during darkness when O2 was restricted to the upper 1-mm layer, and there was thus a low nitrification potential in the permanently oxic top 1 mm of the sediment. Appl Environ Microbiol, 1998 Sep, 64(9), 3480 - 5 Identification and activities in situ of Nitrosospira and Nitrospira spp . as dominant populations in a nitrifying fluidized bed reactor; Schramm A et al.; Bacterial aggregates from a chemolithoautotrophic, nitrifying fluidized bed reactor were investigated with microsensors and rRNA-based molecular techniques . The microprofiles of O2, NH4+, NO2-, and NO3- demonstrated the occurrence of complete nitrification in the outer 125 microgram of the aggregates . The ammonia oxidizers were identified as members of the Nitrosospira group by fluorescence in situ hybridization (FISH) . No ammonia- or nitrite-oxidizing bacteria of the genus Nitrosomonas or Nitrobacter, respectively, could be detected by FISH . To identify the nitrite oxidizers, a 16S ribosomal DNA clone library was constructed and screened by denaturing gradient gel electrophoresis and selected clones were sequenced . The organisms represented by these sequences formed two phylogenetically distinct clusters affiliated with the nitrite oxidizer Nitrospira moscoviensis . 16S rRNA-targeted oligonucleotide probes were designed for in situ detection of these organisms . FISH analysis showed that the dominant populations of Nitrospira spp . and Nitrosospira spp . formed separate, dense clusters which were in contact with each other and occurred throughout the aggregate . A second, smaller, morphologically and genetically different population of Nitrospira spp . was restricted to the outer nitrifying zones. FEMS Microbiol Lett, 1998 Aug 1, 165(1), 153 - 7 Linkage of genes encoding enolase (eno) and CTP synthase (pyrG) in the beta-subdivision proteobacterium Nitrosomonas europaea; Mahony TJ et al.; The gene encoding enolase (eno) from the ammonia oxidising bacterium Nitrosomonas europaea has been cloned and sequenced . The deduced amino acid sequence for enolase from N . europaea was 65% identical (76% similar) to its Bacillus subtilis orthologue . An incomplete open reading frame located 432 bp 5' of eno was identified as pyrG, which encodes CTP synthase . These two genes are therefore organised in N . europaea, a beta-subdivision proteobacterium, in the same way as in the gamma-subdivision proteobacterium Escherichia coli. Syst Appl Microbiol, 1998 Jun, 21(2), 321 - 30 Recovery of a Nitrosomonas-like 16S rDNA sequence group from freshwater habitats; Speksnijder AG et al.; In order to study the diversity of ammonia-oxidising bacteria in freshwater habitats, including sediments, a molecular approach focused on the sequencing of 16S rDNA was adopted . 16S rDNA sequences showing affinity with the beta-subgroup of ammonia-oxidising bacteria were recovered by specific PCR of directly isolated DNA from freshwater samples, and samples from brackish water and Glyceria maxima rhizosphere were included in the analysis for comparison . The ammonia oxidiser-like sequences recovered from several locations, which exhibit differences in the composition of their total microbial communities as indicated by denaturing gradient gel electrophoresis, formed a strong monophyletic cluster including Nitrosomonas ureae . This is the first report presenting sequences from an apparently dominant group of Nitrosomonas-like organisms among the beta-subdivision of ammonia-oxidising bacteria in freshwater environments . This group of sequences extends the known diversity within the beta-subgroup of ammonia-oxidisers . The new sequences related to Nitrosomonas ureae do not match with some published primers and probes designed for the detection of Nitrosomonas species, which may explain why these sequences have not previously been detected in freshwater habitats . The sequence diversity detected within this group of sequences was minimal across the environments examined, and no patterns of distribution were indicated with respect to environmental factors such as sediment depth or location. Syst Appl Microbiol, 1998 Jun, 21(2), 315 - 20 Monitoring the denitrifying Hyphomicrobium DNA/DNA hybridization group HG 27 in activated sludge and lake water using MPN cultivation and subsequent screening with the gene probe Hvu-1; Gliesche CG et al.; Gene probe Hvu-1 is specific for the Hyphomicrobium DNA/DNA-hybridization group HG 27 . This group is one of the dominant populations of denitrifying hyphomicrobia in the activated sludge of the sewage treatment plant Plon . The wastewater treatment process of this treatment plant can be characterized as a combination of simultaneous and intermittent nitrification and denitrification . Using this specific probe Hvu-1 (combined with the most-probable-number method and colony hybridization) the abundance of this denitrifying Hyphomicrobium population in activated sludge and in the adjacent receiving Lake Kleiner Ploner See was investigated as a subfraction of total facultatively anaerobic hyphomicrobia . A 15-month monitoring of the activated sludge and of the lake water was conducted to determine temporal variations of the occurrence of this specific population . During the sampling period total facultatively anaerobic hyphomicrobia remained very constant over a year (9 x 10(4) to 6 x 10(5) ml-1) . The population of the denitrifying Hyphomicrobium DNA/DNA-hybridization group HG 27 amounted to approximately 30% of the total facultatively anaerobic hyphomicrobia found in the activated sludge . Significant correlations to environmental background parameters or seasonal variations of this population were not found . Furthermore, this specific denitrifying Hyphomicrobium population has no importance for the lake ecosystem. Appl Environ Microbiol, 1998 Aug, 64(8), 2958 - 65 Analysis of beta-subgroup proteobacterial ammonia oxidizer populations in soil by denaturing gradient gel electrophoresis analysis and hierarchical phylogenetic probing Stephen JR, Kowalchuk GA, Bruns MAV, McCaig AE, Phillips CJ, Embley TM, Prosser JI. A combination of denaturing gradient gel electrophoresis (DGGE) and oligonucleotide probing was used to investigate the influence of soil pH on the compositions of natural populations of autotrophic beta-subgroup proteobacterial ammonia oxidizers . PCR primers specific to this group were used to amplify 16S ribosomal DNA (rDNA) from soils maintained for 36 years at a range of pH values, and PCR products were analyzed by DGGE . Genus- and cluster-specific probes were designed to bind to sequences within the region amplified by these primers . A sequence specific to all beta-subgroup ammonia oxidizers could not be identified, but probes specific for Nitrosospira clusters 1 to 4 and Nitrosomonas clusters 6 and 7 (J . R . Stephen, A . E . McCaig, Z . Smith, J . I . Prosser, and T . M . Embley, Appl . Environ . Microbiol . 62:4147-4154, 1996) were designed . Elution profiles of probes against target sequences and closely related nontarget sequences indicated a requirement for high-stringency hybridization conditions to distinguish between different clusters . DGGE banding patterns suggested the presence of Nitrosomonas cluster 6a and Nitrosospira clusters 2, 3, and 4 in all soil plots, but results were ambiguous because of overlapping banding patterns . Unambiguous band identification of the same clusters was achieved by combined DGGE and probing of blots with the cluster-specific radiolabelled probes . The relative intensities of hybridization signals provided information on the apparent selection of different Nitrosospira genotypes in samples of soil of different pHs . The signal from the Nitrosospira cluster 3 probe decreased significantly, relative to an internal control probe, with decreasing soil pH in the range of 6.6 to 3.9, while Nitrosospira cluster 2 hybridization signals increased with increasing soil acidity . Signals from Nitrosospira cluster 4 were greatest at pH 5.5, decreasing at lower and higher values, while Nitrosomonas cluster 6a signals did not vary significantly with pH . These findings are in agreement with a previous molecular study (J . R . Stephen, A . E . McCaig, Z . Smith, J . I . Prosser, and T . M . Embley, Appl . Environ . Microbiol 62:4147-4154, 1996) of the same sites, which demonstrated the presence of the same four clusters of ammonia oxidizers and indicated that selection might be occurring for clusters 2 and 3 at acid and neutral pHs, respectively . The two studies used different sets of PCR primers for amplification of 16S rDNA sequences from soil, and the similar findings suggest that PCR bias was unlikely to be a significant factor . The present study demonstrates the value of DGGE and probing for rapid analysis of natural soil communities of beta-subgroup proteobacterial ammonia oxidizers, indicates significant pH-associated differences in Nitrosospira populations, and suggests that Nitrosospira cluster 2 may be of significance for ammonia-oxidizing activity in acid soils. Appl Environ Microbiol, 1998 Aug, 64(8), 3042 - 51 Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations; Juretschko S et al.; The ammonia-oxidizing and nitrite-oxidizing bacterial populations occurring in the nitrifying activated sludge of an industrial wastewater treatment plant receiving sewage with high ammonia concentrations were studied by use of a polyphasic approach . In situ hybridization with a set of hierarchical 16S rRNA-targeted probes for ammonia-oxidizing bacteria revealed the dominance of Nitrosococcus mobilis-like bacteria . The phylogenetic affiliation suggested by fluorescent in situ hybridization (FISH) was confirmed by isolation of N . mobilis as the numerically dominant ammonia oxidizer and subsequent comparative 16S rRNA gene (rDNA) sequence and DNA-DNA hybridization analyses . For molecular fine-scale analysis of the ammonia-oxidizing population, a partial stretch of the gene encoding the active-site polypeptide of ammonia monooxygenase (amoA) was amplified from total DNA extracted from ammonia oxidizer isolates and from activated sludge . However, comparative sequence analysis of 13 amoA clone sequences from activated sludge demonstrated that these sequences were highly similar to each other and to the corresponding amoA gene fragments of Nitrosomonas europaea Nm50 and the N . mobilis isolate . The unexpected high sequence similarity between the amoA gene fragments of the N . mobilis isolate and N . europaea indicates a possible lateral gene transfer event . Although a Nitrobacter strain was isolated, members of the nitrite-oxidizing genus Nitrobacter were not detectable in the activated sludge by in situ hybridization . Therefore, we used the rRNA approach to investigate the abundance of other well-known nitrite-oxidizing bacterial genera . Three different methods were used for DNA extraction from the activated sludge . For each DNA preparation, almost full-length genes encoding small-subunit rRNA were separately amplified and used to generate three 16S rDNA libraries . By comparative sequence analysis, 2 of 60 randomly selected clones could be assigned to the nitrite-oxidizing bacteria of the genus Nitrospira . Based on these clone sequences, a specific 16S rRNA-targeted probe was developed . FISH of the activated sludge with this probe demonstrated that Nitrospira-like bacteria were present in significant numbers (9% of the total bacterial counts) and frequently occurred in coaggregated microcolonies with N . mobilis. Mikrobiologiia, 1998 Mar-Apr, 67(2), 274 - 80 {Ecological consequences of radioactive pollution for soil bacteria within the 10-km region around the Chernobyl Atomic Energy Station}; Romanovskaia VA et al.; The diversity of aerobic chemoorganotrophic (capable of growing on nutrient agar) bacteria in radioactive soil (0.3-17.0 microCi/kg soil) sampled in the 10-km zone around the Chernobyl Nuclear Power Plant (CNPP) was found to be lower than that observed in control, uncontaminated soil with a radioactivity of 0.002-0.006 microCi/kg soil . All the radioactive soil samples contained the bacteria Bacillus cereus and Methylobacterium extorquens or M . mesophillicum, which exhibited a high tolerance to 0.3-1.0 M hydrogen peroxide, whose action can to a certain extent simulate the effect of ionizing radiation . Some of the contaminated soil samples contained other species of chemoorganotrophic bacteria with a low tolerance to H2O2 . The survival of bacteria in the Chernobyl accident zone is probably due to the functioning of mechanisms efficiently neutralizing peroxide compounds and repairing radiation-damaged DNA . The population of cellulolytic, nitrifying, and sulfate-reducing bacteria in contaminated soil was found to be 1-2 orders of magnitude less than in control soil, indicating the unfavorable effect of anthropogenic radiation on the abundance and diversity of soil bacteria. Appl Environ Microbiol, 1998 Jul 1, 64(7), 2528 - 32 Molecular Analysis of Bacterial Communities in a Three-Compartment Granular Activated Sludge System Indicates Community-Level Control by Incompatible Nitrification Processes; Holben WE et al.; Bacterial community structure and the predominant nitrifying activities and populations in each compartment of a three-compartment activated sludge system were determined . Each compartment was originally inoculated with the same activated sludge community entrapped in polyethylene glycol gel granules, and ammonium nitrogen was supplied to the system in an inorganic salts solution at a rate of 5.0 g of N liter of granular activated sludge-1 day-1 . After 150 days of operation, the system was found to comprise a series of sequential nitrifying reactions (K . Noto, T . Ogasawara, Y . Suwa, and T . Sumino, Water Res . 32:769-773, 1998), presumably mediated by different bacterial populations . Activity data showed that all NH4-N was completely oxidized in compartments one and two (approximately half in each), but no significant nitrite oxidation was observed in these compartments . In contrast, all available nitrite was oxidized to nitrate in compartment three . To study the microbial populations and communities in this system, total bacterial DNA isolated from each compartment was analyzed for community structure based on the G+C contents of the component populations . Compartment one showed dominant populations having 50 and 67% G+C contents . Compartment two was similar in structure to compartment one . The bacterial community in compartment three had dominant populations with 62 and 67% G+C contents and retained the 50% G+C content population only at a greatly diminished level . The 50% G+C content population from compartment one hybridized strongly with amo (ammonia monooxygenase) and hao (hydroxylamine oxidoreductase) gene probes from Nitrosomonas europaea . However, the 50% G+C content population from compartment two hybridized strongly with the hao probe but only weakly with the amo probe, suggesting that the predominant ammonia-oxidizing populations in compartments one and two might be different . Since different activities and populations come to dominate in each compartment from an identical inoculum, it appears that the nitrification processes may be somewhat incompatible, resulting in a series of sequential reactions and different communities in this three-compartment system. J Bacteriol, 1998 Jul, 180(13), 3353 - 9 Mutagenesis and expression of amo, which codes for ammonia monooxygenase in Nitrosomonas europaea; Hommes NG et al.; Nitrosomonas europaea has two copies of the operon encoding ammonia monooxygenase (AMO) . The nucleotide sequences of the two copies of amoA were obtained, and they were found to differ by one nucleotide . To determine if both copies of amoA were functional, insertional mutagenesis was performed to inactivate either copy of amoA alone . A DNA cassette containing the lacZ and kan genes inserted into amoA was constructed . Mutagenesis was done by using transformation and homologous recombination to mobilize the cassette into the chromosomal copies of amoA . Mutations were obtained in both copies of amoA . Either copy of amoA was sufficient to support growth when the other copy was disrupted . However, inactivation of one copy of amoA, but not the other, resulted in slower growth . Measurements of ammonia-dependent O2 consumption, which depends on AMO, confirmed that the slower-growing mutant had lower activity while the faster-growing mutant had near wild-type levels of activity . Similarly, as measured by {14C}acetylene label incorporation, there was less active AMO present in the slower-growing mutant than in the faster-growing mutant or in the wild type . Northern blot analysis of transcription likewise showed that the slower-growing mutant had less full-sized AMO mRNA. Arch Microbiol, 1998 Apr, 169(4), 282 - 6 High rate of aerobic nitrification and denitrification by Nitrosomonas eutropha grown in a fermentor with complete biomass retention in the presence of gaseous NO2 or NO; Zart D et al.; A pure culture of the obligately lithoautotrophic ammonia-oxidizer Nitrosomonas eutropha was grown in a laboratory-scale bioreactor with complete biomass retention . The air supply was supplemented with nitrogen dioxide (NO2; 25 or 50 ppm) or nitric oxide (NO; 25 or 50 ppm) . Compared to cultures grown without these nitrogenous oxides, the addition of NO2 or NO to the culture resulted in a significant increase of the nitrification rate, specific activity of ammonia oxidation, growth rate, and maximum cell densities . In contrast, the growth yield slightly decreased in the presence of NO or NO2 . Maximum cell densities of about 2 x 10(10) cells ml-1 and a maximum nitrification rate of about 221 mmol NH4+ l-1 day-1 were obtained after 3 weeks in the presence of 50 ppm NO2 . Furthermore, in the stationary phase about 50% of the nitrite produced was aerobically denitrified to dinitrogen (N2) and traces of nitrous oxide (N2O) . When cells were supplemented with NO, a high rate of aerobic denitrification occurred only during the first days of the exponential growth phase. Appl Environ Microbiol, 1998 Jun, 64(6), 2266 - 8 Estimation of nitrifying bacterial activities by measuring oxygen uptake in the presence of the metabolic inhibitors allylthiourea and azide Ginestet P, Audic JM, Urbain V V, Block JC. The effects of two metabolic inhibitors on an enriched nitrifying biomass during incubation for short periods of time were investigated by determining respirometric measurements . Allylthiourea (86 &mgr;M) and azide (24 &mgr;M) were shown to be strong, selective inhibitors of ammonia and nitrite oxidation, respectively . Consequently, a differential respirometry method for estimating nitrifying and heterotrophic bacterial activities within a mixed biomass is proposed. Appl Environ Microbiol, 1998 May, 64(5), 1878 - 83 Microbiology of a nitrite-oxidizing bioreactor; Burrell PC et al.; The microbiology of the biomass from a nitrite-oxidizing sequencing batch reactor (NOSBR) fed with an inorganic salts solution and nitrite as the sole energy source that had been operating for 6 months was investigated by microscopy, by culture-dependent methods, and by molecular biological methods, and the seed sludge that was used to inoculate the NOSBR was investigated by molecular biological methods . The NOSBR sludge comprised a complex and diverse microbial community containing gram-negative and gram-positive rods, cocci, and filaments . By culture-dependent methods (i.e., micromanipulation and sample dilution and spread plate inoculation), 16 heterotrophs (6 gram positive and 10 gram negative) were identified in the NOSBR sludge (RC), but no autotrophs were isolated . 16S ribosomal DNA clone libraries of the two microbial communities revealed that the seed sludge (GC) comprised a complex microbial community dominated by Proteobacteria (29% beta subclass; 18% gamma subclass) and high G + C gram-positive bacteria (10%) . Three clones (4%) were closely related to the autotrophic nitrite-oxidizer Nitrospira moscoviensis . The NOSBR sludge was overwhelmingly dominated by bacteria closely related to N . moscoviensis (89%) . Two clone sequences were similar to those of the genus Nitrobacter . Near-complete insert sequences of eight RC and one GC N . moscoviensis clone were determined and phylogenetically analyzed . This is the first report of the presence of bacteria from the Nitrospira phylum in wastewater treatment systems, and it is hypothesized that these bacteria are the unknown nitrite oxidizers in these processes. Z Naturforsch {C}, 1998 Jan-Feb, 53(1-2), 69 - 81 DNA-probing for genes coding for denitrification, N2-fixation and nitrification in bacteria isolated from different soils; Kloos K et al.; Bacteria isolated from different layers of four soils of the Cologne area were analyzed for denitrifying, nitrifying and N2-fixing isolates by colony hybridization using gene probes . In the soils tested, the percentage of denitrifying bacteria among the total population isolated was 3-8% (in one case exceptionally 15%) and thus small . Denitrifying bacteria were particularly enriched in the upper layer (depth approximately 5 cm) and were present only in low amounts at 25 cm depth in two gleysol soils . Nitrate apparently did not determine the distribution of denitrifying bacteria in these soils . The potential denitrification activity of different soil layers coincided with the distribution pattern of isolates assessed by DNA-probing . The total number of bacteria and of denitrifying isolates was considerably higher in or at the roots of plants than in the bulk, root-free soil adjacent to the plants . The percentage of the isolated aerobic N2-fixing bacteria varied between 0-3%, and these bacteria could be isolated mainly from the upper 5 cm layer . A small portion of the isolates hybridized with the probe coding for part of one subunit of ammonia monooxygenase from Nitrosomonas europaea . The investigation showed that DNA-probing can provide useful information about the relative distribution of denitrifying and N2-fixing bacteria in different soils and their layers. Arch Microbiol, 1998 Mar, 169(3), 225 - 30 Isolation and immunocytochemical location of the nitrite-oxidizing system in nitrospira moscoviensis Spieck E, Ehrich S, Aamand J, Bock E. A membrane-associated nitrite-oxidizing system of Nitrospira moscoviensis was isolated from heat-treated membranes . The four major proteins of the enzyme fraction had apparent molecular masses of 130, 62, 46, and 29 kDa, respectively . The nitrite-oxidizing activity was dependent on the presence of molybdenum . In contrast to the nitrite oxidoreductase of Nitrobacter hamburgensis X14, the activity of the nitrite-oxidizing system of Ns . moscoviensis increased when solubilized by heat treatment . Electron microscopy of the purified enzyme revealed uniform particles with a size of approximately 7 x 9 nm . SDS-immunoblotting analysis of crude extracts showed that the monoclonal antibodies Hyb 153-3, which recognize the beta-subunit of the nitrite oxidoreductase from Nitrobacter, reacted with a protein of 50 kDa in Ns . moscoviensis . This protein corresponded to the protein of 46 kDa of the purified enzyme and contained a b-type cytochrome . Using electron microscopic immunocytochemistry and the monoclonal antibodies Hyb 153-3, the nitrite-oxidizing system of Ns . moscoviensis was shown to be located in the periplasmic space . Here a periodic arrangement of membrane-associated particles was found on the outside of the cytoplasmic membrane in the form of a hexagonal pattern . It is supposed that these particles represent the nitrite-oxidizing system in Nitrospira. Biochemistry, 1998 Jan 13, 37(2), 523 - 9 Correlation of optical and EPR signals with the P460 heme of hydroxylamine oxidoreductase from Nitrosomonas europaea; Arciero DM et al.; Hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea catalyzes the four-electron oxidation of NH2OH to NO2- . Each subunit of the trimeric enzyme contains seven c-hemes and one heme P460 . In previous work {Hendrich, M . P., et al . (1994) J . Am . Chem . Soc . 116, 11961-11968}, an integer-spin EPR signal at g = 7.7 was discovered from the active site of the resting enzyme . This new signal was assigned to an exchange-coupled cluster containing ferric heme P460 and a ferric c-heme . An electrochemical titration of HAO is presented here in which EPR signals and optical bands, believed to be associated with the P460 heme, are monitored . In the EPR titration, as a redox center with Em8 = -140 mV becomes reduced, the integer-spin signal disappears . Then, upon reduction of a redox center with Em8 = -190 mV, a g = 6 type signal, which has been previously assigned to a high-spin form of the ferric P460 heme of HAO, appears . However, in the -140 to -190 mV range, we have been unable to identify an additional EPR signal attributable to the P460 center . Thus, the electronic environment of oxidized P460 heme of HAO appears to pass through three states before reduction in a titration experiment, with an intermediate state that is not readily detectable by X-band EPR . The best candidate for the c-heme partner of the P460 heme is the heme at -190 mV, which would correspond to heme 6 of the crystal structure . A possible function of the exchange-coupled heme cluster is to facilitate two-electron oxidation steps of the substrate . An earlier spectropotentiometric titration of HAO {Collins, M . J., et al . (1993) J . Biol . Chem . 268, 14655-14662} identified a broad, weak optical band, centered near 740 nm, that was tentatively assigned to the oxidized P460 heme . This assignment has been strengthened by additional spectropotentiometric titrations at several values of pH and also by rapid kinetic experiments following the reduction of HAO by dithionite . The 740 nm band is not observed in fully oxidized HAO . In the spectropotentiometric titrations, its appearance cannot be correlated with reduction of a specific c-heme nor modeled to a Nernstian one-electron redox center . Instead, the range of potential in which the 740 nm band is present depends on whether the titration is carried out in an oxidative or reductive direction . One possible interpretation is that the 740 nm band is a property of the oxidized high-spin P460 heme but not of the low-spin state, and that the transition between the two spin states occurs at different potentials depending on the direction of the electrochemical titration. Microbiology, 1997 Dec, 143 ( Pt 12), 3775 - 83 Heterologous expression of heterotrophic nitrification genes; Crossman LC et al.; Paracoccus denitrificans is a heterotrophic organism capable of oxidizing ammonia to nitrite during growth on an organic carbon and energy source . This pathway, termed heterotrophic nitrification, requires the concerted action of an ammonia monooxygenase (AMO) and hydroxylamine oxidase (HAO) . The genes required for heterotrophic nitrification have been isolated by introducing a Pa . denitrificans genomic library into Pseudomonas putida and screening for the accumulation of nitrite . In contrast to the situation in chemolithoautotrophic ammonia oxidizers, the genes encoding AMO and HAO are present in single linked copies in the genome of Pa . denitrificans . AMO from Pa . denitrificans expressed in Ps . putida is capable of oxidizing ethene (ethylene) to epoxyethane (ethylene oxide), which is indicative of a relaxed substrate specificity . Further, when expressed in the methylotroph Methylobacterium extorquens AM1, the AMO endows on this organism the ability to grow on ethene and methane . Thus, the Pa . denitrificans AMO is capable of oxidizing methane to methanol, as is the case for the AMO from Nitrosomonas europaea . The heterotrophic nitrification genes are moderately toxic in M . extorquens, more toxic in Ps . putida, and non-toxic in Escherichia coli . Toxicity is due to the activity of the gene products in M . extorquens, and both expression and activity in Ps . putida . This is the first time that the genes encoding an active AMO have been expressed in a heterologous host. Biotechnol Prog, 1997 Nov-Dec, 13(6), 794 - 8 Biotreatment of ammonia from air by an immobilized Arthrobacter oxydans CH8 biofilter; Chung YC et al.; A heterotrophic Arthrobacter oxydans CH8 that was capable of removing NH3 from NH3 containing gas was isolated from livestock farming wastewater . The A . oxydans CH8 was immobilized with calcium alginate packed into filter column . Metered NH3-containing gas was partially humidified and passed through the glass column . Extensive tests including the removal characteristics, the removal efficiencies, and the metabolic products of NH3 by A . oxydans CH8 were conducted . Additionally, the operation criteria for the biofilter was also established . NH3 removel capacities were elevated by the immobilized-cell (biological conversion) method and the BDST (bed depth service time) method (physical adsorption), respectively . The optium temperature for removing NH3 was 30 degrees C, while the nitrification ability remained 80% at 40 degrees C . The high efficiency (> 97%) in the removal of NH3 was attained at 36 L/h with pH control and was not decreased because of high NH3 inlet concentration . In addition, the high maximum removal rate (1.22 g of N/day (kg of bead)) enhanced the use of the biofilter in industrial-scale NH3(g) pollution control . The ability to remove NH3 at high inlet concentration and temperature suggested that the immobilized A . oxydans CH8 biofilter has potential in processing NH3 gas. Appl Environ Microbiol, 1997 Dec, 63(12), 4704 - 12 The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations; Rotthauwe JH et al.; The naturally occurring genetic heterogeneity of autotrophic ammonia-oxidizing populations belonging to the beta subclass of the Proteobacteria was studied by using a newly developed PCR-based assay targeting a partial stretch of the gene which encodes the active-site polypeptide of ammonia monooxygenase (amoA) . The PCR yielded a specific 491-bp fragment with all of the nitrifiers tested, but not with the homologous stretch of the particulate methane monooxygenase, a key enzyme of methane-oxidizing bacteria . The assay also specifically detected amoA in DNA extracted from various aquatic and terrestrial environments . The resulting PCR products retrieved from rice roots, activated sludge, a freshwater sample, and an enrichment culture were used for the generation of amoA gene libraries . No false positives were detected in a set of 47 randomly selected clone sequences that were analyzed further . The majority of the environmental sequences retrieved from rice roots and activated sludge grouped within the phylogenetic radiation defined by cultured strains of the genera Nitrosomonas and Nitrosospira . The comparative analysis identified members of both of these genera in activated sludge; however, only Nitrosospira-like sequences with very similar amino acid patterns were found on rice roots . Further differentiation of these molecular isolates was clearly possible on the nucleic acid level due to the accumulation of synonymous mutations, suggesting that several closely related but distinct Nitrosospira-like populations are the main colonizers of the rhizosphere of rice . Each of the amoA gene libraries obtained from the freshwater sample and the enrichment culture was dominated by a novel lineage that shared a branch with the Nitrosospira cluster but could not be assigned to any of the known pure cultures . Our data suggest that amoA represents a very powerful molecular tool for analyzing indigenous ammonia-oxidizing communities due to (i) its specificity, (ii) its fine-scale resolution of closely related populations, and (iii) the fact that a functional trait rather than a phylogenetic trait is detected. Biodegradation, 1997, 8(1), 21 - 30 Enhancement of ethene removal from waste gas by stimulating nitrification; de heyder B et al.; The treatment of poorly water soluble waste gas compounds, such as ethene, is associated with low substrate concentration levels in the liquid phase . This low concentration level might hamper the optimal development of a microbial population . In this respect, the possible benefit of introducing nitrifying activity in the heterotrophic removal of ethene at moderate concentrations (< 1000 ppm) from a waste gas was investigated . Nitrifying activity is known to be associated with (i) the production of soluble microbial products, which can act as (co-)substrates for heterotrophic micro-organisms and (ii) the co-oxidation of ethene . The used reactor configuration was a packed granular activated carbon biobed inoculated with the heterotrophic strain Mycobacterium E3 . The nitrifying activity was introduced by regular submersion in a nitrifying medium prepared from (i) compost or (ii) activated sludge . In both cases a clear enhancement of the volumetric removal rate of ethene could be observed . When combined with a NH3 dosage on a daily basis, a gradual increase of the volumetric removal rate of ethene could be observed . For a volumetric loading rate of 3 kg ethene-COD.m-3.d-1, the volumetric removal rate could thus be increased with a factor 1.8, i.e . from 0.72 to a level of 1.26 kg ethene-COD.m-3.d-1. FEBS Lett, 1997 Jun 30, 410(2-3), 457 - 60 Evidence for a crosslink between c-heme and a lysine residue in cytochrome P460 of Nitrosomonas europaea; Arciero DM et al.; Cytochrome P460 and hydroxylamine oxidoreductase (HAO) of Nitrosomonas europaea catalyze the oxidation of hydroxylamine . Cytochrome P460 contains an unidentified heme-like chromophore whose distinctive spectroscopic properties are similar to those for the P460 heme found in HAO . The heme P460 of HAO has previously been shown by protein chemistry and NMR structural analysis to be a c-heme with an additional covalent crosslink between the C2 ring carbon of a tyrosine residue of the polypeptide chain and a meso carbon of the porphyrin {Arciero, D.M . et al . (1993) Biochemistry 32, 9370-9378} . The recent determination of the gene sequence for cytochrome P460 {Bergmann, D.J . and Hooper, A.B . (1994) FEBS Lett . 353, 324-326} indicates that the heme in this protein also possesses a c-heme binding site and provides the basis for determining whether an HAO-like crosslink exists to the porphyrin . Sequence analysis of a purified heme-containing tryptic chromopeptide from cytochrome P460 revealed two predominant amino acid residues per cycle . Two peptides present in the chromopeptide with the sequences NLPTAEXAAXHK and DGTVTVXELVSV . Comparison of the data to the gene sequence for the protein revealed that the gaps in the first peptide (indicated by X's) code for C residues, confirming the prediction of a c-heme binding motif . The gap in the sequence in the second peptide at cycle 7 is predicted by the gene sequence to be a K . The results suggest that the lysine residue is crosslinked in some manner to the porphyrin macrocycle, possibly mimicking the tyrosine crosslink found for the heme P460 of HAO . While a common role for the crosslinked residues in HAO and cytochrome P460 is difficult to ascertain due to the dissimilarities in side chain structure, it may be related to the similar pKa values for lysine and tyrosine. Can J Microbiol, 1997 Jun, 43(6), 595 - 8 Pyridine degradation and heterocyclic nitrification by Bacillus coagulans; Uma B et al.; A gram-positive, pyridine-degrading microorganism identified as Bacillus coagulans has been isolated from contaminated soil by enrichment culture technique . Pyridine was used as sole source of carbon, nitrogen, and energy . Bacillus coagulans has a unique potential to reduce nitrogen from aromatic ring to ammonia and subsequently heterotrophically to nitrite and nitrate . The maximum degradation of pyridine was 94.1% within 72 h at 30 degrees C with a 7.57-h doubling time . The study suggests possible existence of aromatic degradation and heterotrophic nitrification in Bacillus coagulans. Appl Environ Microbiol, 1997 Jun, 63(6), 2397 - 402 Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology; Guschin DY et al.; The utility of parallel hybridization of environmental nucleic acids to many oligonucleotides immobilized in a matrix of polyacrylamide gel pads on a glass slide (oligonucleotide microchip) was evaluated . Oligonucleotides complementary to small-subunit rRNA sequences of selected microbial groups, encompassing key genera of nitrifying bacteria, were shown to selectively retain labeled target nucleic acid derived from either DNA or RNA forms of the target sequences . The utility of varying the probe concentration to normalize hybridization signals and the use of multicolor detection for simultaneous quantitation of multiple probe-target populations were demonstrated. Appl Environ Microbiol, 1997 Jun, 63(6), 2281 - 6 Cell density-regulated recovery of starved biofilm populations of ammonia-oxidizing bacteria; Batchelor SE et al.; The speed of recovery of cell suspensions and biofilm populations of the ammonia oxidizer Nitrosomonas europaea, following starvation was determined . Stationary-phase cells, washed and resuspended in ammoniumfree inorganic medium, were starved for periods of up to 42 days, after which the medium was supplemented with ammonium and subsequent growth was monitored by measuring nitrite concentration changes . Cultures exhibited a lag phase prior to exponential nitrite production, which increased from 8.72 h (no starvation) to 153 h after starvation for 42 days . Biofilm populations of N . europaea colonizing sand or soil particles in continuous-flow, fixed column reactors were starved by continuous supply of ammonium-free medium . Following resupply of ammonium, starved biofilms exhibited no lag phase prior to nitrite production, even after starvation for 43.2 days, although there was evidence of cell loss during starvation . Biofilm formation will therefore provide a significant ecological advantage for ammonia oxidizers in natural environments in which the substrate supply is intermittent . Cell density-dependent phenomena in a number of gram-negative bacteria are mediated by N-acyl homoserine lactones (AHL), including N-(3-oxohexanoyl)-L-homoserine lactone (OHHL) . Addition of both ammonium and OHHL to cell suspensions starved for 28 days decreased the lag phase in a concentration-dependent manner from 53.4 h to a minimum of 10.8 h . AHL production by N . europaea was detected by using a luxR-luxAB AHL reporter system . The results suggest that rapid recovery of high-density biofilm populations may be due to production and accumulation of OHHL to levels not possible in relatively low-density cell suspensions. J Biochem (Tokyo), 1997 May, 121(5), 957 - 60 Effect of ammonia starvation on hydroxylamine oxidoreductase activity of Nitrosomonas europaea; Nejidat A et al.; A technique for detection of the activity of hydroxylamine oxidoreductase (HAO) involving denaturing SDS-polyacrylamide gels was developed . The activity of HAO of Nitrosomonas europaea was assayed using this technique, which revealed a single active band of 140 kDa . The HAO activity of other ammonia-oxidizers was also resistant to SDS, the molecular weights being identical to that of N . europaea . N . europaea cells starved of ammonia for up to 72 h retained a considerable amount of HAO, as detected on Western blot analysis, and a significant level of its activity, as found on assaying at the end of the starvation period . Only after 4 h incubation of starved N . europaea cells with 2.0 mM ammonia was some increase in the HAO level observed . The results indicate that HAO remains highly stable during ammonia starvation of N . europaea. FEMS Microbiol Lett, 1997 May 1, 150(1), 65 - 73 A gene encoding a membrane protein exists upstream of the amoA/amoB genes in ammonia oxidizing bacteria: a third member of the amo operon? Klotz MG, Alzerreca J, Norton JM. The gene cluster encoding ammonia monooxygenase (AMO) in the chemolithotrophic soil bacterium Nitrosospira sp . NpAV was found to contain a third open reading frame, termed amoC, upstream of the genes amoA and amoB that encode the subunits of AMO . The amoC gene and its flanking regions were isolated and sequenced from a 4.4 kb EcoRI fragment that contains one of three copies of the ammonia monooxygenase gene cluster . The presence of this gene upstream of the other two amoA gene copies in Nitrosospira NpAV as well as upstream of amoA genes in the genomes of other ammonia oxidizing nitrifiers (strains in the genera Nitrosomonas, Nitrosopira, Nitrosolobus and Nitrosovibrio) was confirmed using genomic DNA, oligodeoxyribonucleotide primers and the PCR . The amoC gene in Nitrosospira sp . NpAV encodes a 270 amino acid polypeptide of approximately 36 kDa . Topological analysis of the predicted primary structure revealed 6 membrane spanning domains . The amoC gene was expressed in recombinant Escherichia coli from its indigenous promoter. Appl Environ Microbiol, 1997 May, 63(5), 1777 - 84 Cloning, nucleotide sequence, and regulatory analysis of the Nitrosomonas europaea dnaK gene; Iizumi T et al.; The dnaK gene of an ammonia-oxidizing bacterium, Nitrosomonas europaea, was cloned and sequenced . It was found that the dnaK gene product was highly homologous to previously analyzed dnaK gene products from other organisms at the amino acid level . Two partial open reading frames located upstream and downstream of the dnaK gene were also found and identified as grpE and dnaJ genes, respectively, by the predicted amino acid homology of their gene products to other bacterial GrpE and DnaJ proteins . Transcription of the dnaK gene was strongly induced by a heat shock from 30 to 37 degrees C . An analysis of the expression of the dnaK gene fused to the lacZ translational reporter gene also showed eightfold increase in beta-galactosidase activity after the heat shock induction . Heat-inducible transcription start sites of the dnaK gene, revealed by primer extension analysis, were located 16 and 17 nucleotides upstream from the translational start codon of the dnaK gene, and the predicted promoter sequence showed a homology to the consensus sequence of sigma 32-dependent heat shock promoters of gram-negative bacteria . The upstream region of the dnaK gene did not contain the inverted repeat structure that was involved in the regulation of the heat shock gene of several gram-negative and gram-positive bacteria . Therefore, we conclude that the heat shock regulatory mechanism of the N . europaea dnaK gene may be similar to the sigma 32-dependent mechanism observed in other gram-negative bacteria. Microsc Res Tech, 1997 Apr 15, 37(2), 162 - 70 Evidence for the microbial basis of a chemoautotrophic invertebrate community at a whale fall on the deep seafloor: bone-colonizing bacteria and invertebrate endosymbionts; Deming JW et al.; To explore the microbial basis for a remarkable macrofaunal community at the site of a whale skeleton on the seafloor of the Santa Catalina Basin, we obtained samples of whale bone, bone-colonizing invertebrates, microbial mats, and the dominant fauna in the adjacent sulfide-rich sediments during Alvin expeditions in 1988 and 1991 . Invertebrate tissues were examined by transmission electron microscopy (TEM) and mats and bone-penetrating bacteria by epifluorescence microscopy (EM) . Tissues from the dominant bivalve Vesicomya c.f . gigas, the mytilid mussel Idasola washingtonia, and selected gastropods and limpets were also assayed chemically for enzymes diagnostic of sulfur- and methane-based chemoautotrophy and for stable carbon isotopic composition . Results of all analyses were consistent with dominant sulfur-based endosymbioses in the clam and mussel (the first record of endosymbiosis in the genus Idasola) and the general absence of methane symbioses at the site, strengthening the analogy of the whale-skeleton faunal community to those known from distant Pacific hydrothermal vent sites . Examples of minor endosymbionts, either nitrifying or methanotrophic cells according to internal membrane structures by TEM, raised the possibility of a supplemental mode of nutrition to the clam, or means to remove ammonia in the gill tissue, in the event of significant changes in the chemical environment. Appl Environ Microbiol, 1997 Apr, 63(4), 1489 - 97 Analysis of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in coastal sand dunes by denaturing gradient gel electrophoresis and sequencing of PCR-amplified 16S ribosomal DNA fragments; Kowalchuk GA et al.; Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations . DGGE was evaluated for the identification of ammonia oxidizers of the beta subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes . Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 5' GC clamp, were designed to amplify a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains . The primers were tested against a representative selection of clones and cultures encompassing the currently recognized beta-subdivision ammonia oxidizer 16S rDNA sequence diversity . Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used . The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations of differing in pH and distance from the beach . The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs . Ammonia oxidizer-like sequences from different phylogenetic groupings isolated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples . Bands from the DGGE gels of environmental samples were excised, reamplified, and directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones . Six of the seven sequenced clusters of beta-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure between some sample sites were demonstrated . The most seaward dune site contained sequences showing affinity with sequence clusters previously isolated only from marine environments and was the only site where sequences relate to Nitrosomonas genes could be detected . Nitrosospira-like sequences were present in all sites, and there was some evidence of differences between Nitrosospira populations in acid and alkaline dune soils . Such differences in community structure may affect physiological differences within beta-subdivision ammonia oxidizers, with consequent effects on nitrification rates in response to key environmental factors. Nat Struct Biol, 1997 Apr, 4(4), 276 - 84 The 2.8 A structure of hydroxylamine oxidoreductase from a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea; Igarashi N et al.; The 2.8 A crystal structure of hydroxylamine oxidoreductase of a nitrifying chemoautotrophic bacterium, Nitrosomonas europaea, is described . Twenty-four haems lie in the centre bottom of the trimeric molecule, localized in four clusters within each monomer . The haem clusters within the trimer are aligned to form a ring that has inlet and outlet sites . The inlet is occupied by a novel haem, P460, and there are two possible outlet sites per monomer formed by paired haems lying within a cavity or cleft on the protein surface . The structure suggests pathways by which electron transfer may occur through the precisely arranged haems and provides a framework for the interpretation of previous and future biochemical and genetic observations. Arch Microbiol, 1997 Mar 7, 167(2/3), 106 - 11 Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha Schmidt I I, Bock E. Nitrosomonas eutropha, an obligately lithoautotrophic bacterium, was able to nitrify and denitrify simultaneously under anoxic conditions when gaseous nitrogen dioxide (NO2) was supplemented to the atmosphere . In the presence of gaseous NO2, ammonia was oxidized, nitrite and nitric oxide (NO) were formed, and hydroxylamine occurred as an intermediate . Between 40 and 60% of the produced nitrite was denitrified to dinitrogen (N2) . Nitrous oxide (N2O) was shown to be an intermediate of denitrification . Under an N2 atmosphere supplemented with 25 ppm NO2 and 300 ppm CO2, the amount of cell protein increased by 0.87 mg protein per mmol ammonia oxidized, and the cell number of N . eutropha increased by 5.8 x 10(9) cells per mmol ammonia oxidized . In addition, the ATP and NADH content increased by 4.3 mumol ATP (g protein)-1 and 6.3 mumol NADH (g protein)-1 and was about the same in both anaerobically and aerobically grown cells . Without NO2, the ATP content decreased by 0.7 mumol (g protein)-1, and the NADH content decreased by 1.2 mumol (g protein)-1 . NO was shown to inhibit anaerobic ammonia oxidation. Arch Microbiol, 1997 Feb-Mar, 167(2-3), 106 - 11 Anaerobic ammonia oxidation with nitrogen dioxide by Nitrosomonas eutropha; Schmidt I et al.; Nitrosomonas eutropha, an obligately lithoautotrophic bacterium, was able to nitrify and denitrify simultaneously under anoxic conditions when gaseous nitrogen dioxide (NO2) was supplemented to the atmosphere . In the presence of gaseous NO2, ammonia was oxidized, nitrite and nitric oxide (NO) were formed, and hydroxylamine occurred as an intermediate . Between 40 and 60% of the produced nitrite was denitrified to dinitrogen (N2) . Nitrous oxide (N2O) was shown to be an intermediate of denitrification . Under an N2 atmosphere supplemented with 25 ppm NO2 and 300 ppm CO2, the amount of cell protein increased by 0.87 mg protein per mmol ammonia oxidized, and the cell number of N . eutropha increased by 5.8 x 10(9) cells per mmol ammonia oxidized . In addition, the ATP and NADH content increased by 4.3 micromol ATP (g protein)-1 and 6.3 micromol NADH (g protein)-1 and was about the same in both anaerobically and aerobically grown cells . Without NO2, the ATP content decreased by 0.7 micromol (g protein)-1, and the NADH content decreased by 1.2 micromol (g protein)-1 . NO was shown to inhibit anaerobic ammonia oxidation. Antonie Van Leeuwenhoek, 1997 Feb, 71(1-2), 75 - 93 Novel principles in the microbial conversion of nitrogen compounds; Jetten MS et al.; Some aspects of inorganic nitrogen conversion by microorganisms like N2O emission and hydroxylamine metabolism studied by Beijerinck and Kluyver, founders of the Delft School of Microbiology, are still actual today . In the Kluyver Laboratory for Biotechnology, microbial conversion of nitrogen compounds is still a central research theme . In recent years a range of new microbial processes and process technological applications have been studied . This paper gives a review of these developments including, aerobic denitrification, anaerobic ammonium oxidation, heterotrophic nitrification, and formation of intermediates (NO2-, NO, N2O), as well as the way these processes are controlled at the genetic and enzyme level. Antonie Van Leeuwenhoek, 1997 Feb, 71(1-2), 69 - 74 Hydroxylamine metabolism in Pseudomonas PB16: involvement of a novel hydroxylamine oxidoreductase; Jetten MS et al.; Pseudomonas strain PB16, a Gram-negative heterotrophic nitrifying bacterium closely related to Pseudomonas azalaica on the basis of 16 S rDNA analysis, was able to use hydroxylamine as an additional energy source during growth in acetate limited chemostat cultures giving an increased biomass yield . In aerobically growing cells of Pseudomonas PB16 only 50% of supplemented hydroxylamine could be recovered as nitrite . In addition to nitrite, N2O could be detected in the chemostat off-gas, indicating combined heterotrophic nitrification and aerobic denitrification . The maximum specific hydroxylamine oxidizing activity observed was 450 nmol per min per mg dry weight, with a Ks of approximately 40 microM . Upon addition of hydroxylamine to the medium, Pseudomonas PB16 induced a soluble 132 KDa dimeric hydroxylamine oxidoreductase . The enzyme had a pH optimum of 9, and did not contain spectroscopic features typical for cytochromes, which is in contrast to hydroxylamine oxidoreductases found in autotrophic bacteria. Antonie Van Leeuwenhoek, 1997 Feb, 71(1-2), 33 - 41 Emerging principles of inorganic nitrogen metabolism in Paracoccus denitrificans and related bacteria; Stouthamer AH et al.; The taxonomy of Paracoccus denitrificans and related bacteria is discussed . Evidence is given which shows that the physiological differences between P . denitrificans and Thiosphaera pantotropha are less fundamental than previously thought . A proposal to consider a species P . pantotropha is mentioned . The properties of the denitrifying enzymes and the genes involved in their formation in P . denitrificans is discussed . The synthesis of the membrane-bound-nitrate reductase is regulated by FNR, that of the nitrite- and nitric oxide reductase by NNR . Evidence is given that FNR acts as a redox sensor rather than an oxygen sensor . The occurrence of aerobic denitrification and coupled heterotrophic nitrification-denitrification in the original strain of Thiosphaera pantotropha are explained by a limiting respiratory activity which activates FNR . Aerobic denitrification leads to a lower growth yield and an increase in mumax in batch culture when a limiting respiratory activity is assumed and when excess substrate is present . Coupled heterotrophic nitrification-denitrification gives a smaller increase in mumax and a more drastic reduction in yield . Both processes are thus advantageous to the organism . In a chemostat with limiting substrate these processes are disadvantageous . T . pantotropha has lost the ability for aerobic denitrification during extended cultivation . Possibly the substrate concentration was limiting during extended cultivation giving a selective advantage to variants which have lost these properties . The calculations predict that P . denitrificans should be able to grow chemolithotrophically with hydroxylamine. J Bacteriol, 1997 Jan, 179(1), 31 - 40 Metal selectivity of in situ microcolonies in biofilms of the Elbe river; Lunsdorf H et al.; The ultrastructure of natural complex biofilm communities of the Elbe river grown in situ on microscopic glass coverslips was studied by using transmission electron microscopy and energy-dispersive x-ray (EDX) analysis . Characteristic microcolonies which measured between 3.3 and 9.3 microm in diameter were frequently observed . They had an outer envelope and harbored 6 to 30 cells . The cells formed short rods measuring 1.09 +/- 0.28 microm (n = 10) in length and 0.55 + 0.07 microm (n = 21) in width . They were surrounded by a thick layer of electron-transparent, nonosmicated matter, 120 to 300 nm thick . Individual cells exhibited a unique ultrastructural trait, namely, a concentric membrane stack which completely surrounded the cytoplasm . It consisted of three membrane doublets, which showed an overall thickness of 57 to 66 nm . The center-to-center spacing between two membrane doublets was 22.2 +/- 1.0 nm (n = 12) . The bacterial cell wall seemed to be of the gram-negative type . The fact that upon shrinkage hexagonal clefts appeared proved the cells to be tightly packed, and septum formation by binary fissions was observed . All of these morphological details indicate that the cells within these microcolonies were actively growing and did not represent spore-like states . EDX analysis showed that only the electron-dense surface deposit of the microcolonies contained Mn and Fe in significant amounts, while these two elements were absent from the intercellular space and the cytoplasm of the microorganisms . In contrast, aluminum ions were able to penetrate the outer envelope of the microcolonies and were detected in the intercellular space . They were, however, completely absent from the microbial cytoplasm, indicating a filter cascade with respect to aluminum . From the ultrastructural data together with the deposition of iron and manganese on the microcolony surface, it appears that these organisms may belong to the genus Siderocapsa or Nitrosomonas . They do not precisely match any of the described species and may therefore represent a new species. Microbiol Rev, 1996 Dec, 60(4), 609 - 40 Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO); Conrad R; Production and consumption processes in soils contribute to the global cycles of many trace gases (CH4, CO, OCS, H2, N2O, and NO) that are relevant for atmospheric chemistry and climate . Soil microbial processes contribute substantially to the budgets of atmospheric trace gases . The flux of trace gases between soil and atmosphere is usually the result of simultaneously operating production and consumption processes in soil: The relevant processes are not yet proven with absolute certainty, but the following are likely for trace gas consumption: H2 oxidation by abiontic soil enzymes; CO cooxidation by the ammonium monooxygenase of nitrifying bacteria; CH4 oxidation by unknown methanotrophic bacteria that utilize CH4 for growth; OCS hydrolysis by bacteria containing carbonic anhydrase; N2O reduction to N2 by denitrifying bacteria; NO consumption by either reduction to N2O in denitrifiers or oxidation to nitrate in heterotrophic bacteria . Wetland soils, in contrast to upland soils are generally anoxic and thus support the production of trace gases (H2, CO, CH4, N2O, and NO) by anaerobic bacteria such as fermenters, methanogens, acetogens, sulfate reducers, and denitrifiers . Methane is the dominant gaseous product of anaerobic degradation of organic matter and is released into the atmosphere, whereas the other trace gases are only intermediates, which are mostly cycled within the anoxic habitat . A significant percentage of the produced methane is oxidized by methanotrophic bacteria at anoxic-oxic interfaces such as the soil surface and the root surface of aquatic plants that serve as conduits for O2 transport into and CH4 transport out of the wetland soils . The dominant production processes in upland soils are different from those in wetland soils and include H2 production by biological N2 fixation, CO production by chemical decomposition of soil organic matter, and NO and N2O production by nitrification and denitrification . The processes responsible for CH4 production in upland soils are completely unclear, as are the OCS production processes in general . A problem for future research is the attribution of trace gas metabolic processes not only to functional groups of microorganisms but also to particular taxa . Thus, it is completely unclear how important microbial diversity is for the control of trace gas flux at the ecosystem level . However, different microbial communities may be part of the reason for differences in trace gas metabolism, e.g., effects of nitrogen fertilizers on CH4 uptake by soil; decrease of CH4 production with decreasing temperature; or different rates and modes of NO and N2O production in different soils and under different conditions. Appl Environ Microbiol, 1996 Dec, 62(12), 4641 - 7 Structure and function of a nitrifying biofilm as determined by in situ hybridization and the use of microelectrodes; Schramm A et al.; Microprofiles of O2 and NO3- were measured in nitrifying biofilms from the trickling filter of an aquaculture water recirculation system . By use of a newly developed biosensor for NO3-, it was possible to avoid conventional interference from other ions . Nitrification was restricted to a narrow zone of 50 microns on the very top of the film . In the same biofilms, the vertical distributions of members of the lithoautotrophic ammonia-oxidizing genus Nitrosomonas and of the nitrite-oxidizing genus Nitrobacter were investigated by applying fluorescence in situ hybridization of whole fixed cells with 16S rRNA-targeted oligonucleotide probes in combination with confocal laser-scanning microscopy . Ammonia oxidizers formed a dense layer of cell clusters in the upper part of the biofilm, whereas the nitrite oxidizers showed less-dense aggregates in close vicinity to the Nitrosomonas clusters . Both species were not restricted to the oxic zone of the biofilm but were also detected in substantially lower numbers in the anoxic layers and even occasionally at the bottom of the biofilm. FEBS Lett, 1996 Nov 11, 397(1), 35 - 8 Evidence for an iron center in the ammonia monooxygenase from Nitrosomonas europaea; Zahn JA et al.; Binding of the ligand, nitric oxide, in the presence of reductant was used to identify a ferrous S = 3/2 signal, characteristic of a ferrous nitrosyl complex, and a g= 2.03 copper or iron signal in membranes of the ammonia-oxidizing bacterium, Nitrosomonas europaea . The same ferrous S = 3/2 signal is thought to be a component of the membrane-associated methane monooxygenase (pMMO) of Methylococcus capsulatus Bath, since it is seen in the membrane fraction of cells expressing pMMO and in the purified enzyme, but not in the membrane fraction of cells expressing the soluble MMO {Zahn, J.A . and DiSpirito, A.A . (1996) J . Bacteriol . 178, 1018-1029} . Treatment of resting membranes or cells of N . europaea with nitrapyrin, 2-chloro,6-trichloromethylpyridine, resulted in the increase in magnitude of a g = 6, high-spin ferric iron signal . In the presence of NO and reductant, nitrapyrin prevented the formation of the S = 3/2 nitrosyl-iron complex while increasing the intensity of the g = 6 signal . Nitrapyrin is a specific inhibitor of, and is reduced by, the ammonia monoxygenase (AMO) {Bedard, C . and Knowles, R . (1989) Microbiol . Rev . 53, 68-83} . Taken together the data suggest that iron capable of forming the S = 3/2 complex is a catalytic component of AMO of N . europaea, possibly a part of the oxygen-activating center . Inactivation of the membrane-associated AMO with acetylene did not diminish the S = 3/2 nitrosyl-iron signal, the g = 6 signal, or the g = 6 signal. Biochem J, 1996 Nov 1, 319 ( Pt 3), 823 - 7 The biochemical characterization of a novel non-haem-iron hydroxylamine oxidase from Paracoccus denitrificans GB17; Moir JW et al.; The characterization of the hydroxylamine oxidase from the heterotrophic nitrifier Paracoccus denitrificans GB17 indicates the enzyme to be entirely distinct from the hydroxylamine oxidase from the autotrophic nitrifier Nitrosomonas europaea . Hydroxylamine oxidase from P . denitrificans contains three to five non-haem, non-iron-sulphur iron atoms as prosthetic groups, predominantly co-ordinated by carboxylate ligands . The interaction of the enzyme with the electron-accepting proteins cytochrome C556 and pseudoazurin is mainly hydrophobic . The catalytic mechanism of hydroxylamine oxidase from P . denitrificans is different from the enzyme from N . europaea because the production of nitrite by the former requires molecular oxygen . Under anaerobic conditions the enzyme makes nitrous oxide as a sole product. Appl Environ Microbiol, 1996 Nov, 62(11), 4224 - 8 Nitrogen removal by tubular gel containing Nitrosomonas europaea and Paracoccus denitrificans; Uemoto H et al.; A new bioreactor for the removal of nitrogen from wastewater is described which consists of a tubular polymeric gel containing Nitrosomonas europaea and Paracoccus denitrificans . The outer surface of the tube is in aerobic contact with wastewater containing ammonia, while the inside of the tube is in anaerobic contact with ethanol flowing through the tube . N . europaea oxidizes ammonia to nitrite in the gel, and then P . denitrificans reduces the nitrite to nitrogen gas in the same gel . This concept would be effective for simplifying nitrogen removal systems requiring aerobic and anaerobic operations. Appl Environ Microbiol, 1996 Nov, 62(11), 4147 - 54 Molecular diversity of soil and marine 16S rRNA gene sequences related to beta-subgroup ammonia-oxidizing bacteria; Stephen JR et al.; We have conducted a preliminary phylogenetic survey of ammonia-oxidizing beta-proteobacteria, using 16S rRNA gene libraries prepared by selective PCR and DNA from acid and neutral soils and polluted and nonpolluted marine sediments . Enrichment cultures were established from samples and analyzed by PCR . Analysis of 111 partial sequences of c . 300 bases revealed that the environmental sequences formed seven clusters, four of which are novel, within the phylogenetic radiation defined by cultured autotrophic ammonia oxidizers . Longer sequences from 13 cluster representatives support their phylogenetic positions relative to cultured taxa . These data suggest that known taxa may not be representative of the ammonia-oxidizing beta-proteobacteria in our samples . Our data provide further evidence that molecular and culture-based enrichment methods can select for different community members . Most enrichments contained novel Nitrosomonas-like sequences whereas novel Nitrosospira-like sequences were more common from gene libraries of soils and marine sediments . This is the first evidence for the occurrence of Nitrosospira-like strains in marine samples . Clear differences between the sequences of soil and marine sediment libraries were detected . Comparison of 16S rRNA sequences from polluted and nonpolluted sediments provided no strong evidence that the community composition was determined by the degree of pollution . Soil clone sequences fell into four clusters, each containing sequences from acid and neutral soils in varying proportions . Our data suggest that some related strains may be present in both samples, but further work is needed to resolve whether there is selection due to pH for particular sequence types. Microb Ecol, 1996 Nov, 32(3), 247 - 61 Nitrification and Denitrification: Probing the Nitrogen Cycle in Aquatic Environments Ward BB. Methods designed to detect microorganisms involved in the biogeochemistry of nitrogen in the marine environment are rapidly being developed and deployed in ecological investigations . Probes based on phylogenetic sequences (usually rRNA) and those based on the sequences of functional genes or proteins have both been demonstrated in the nitrogen cycle . The most progress has been made for ammonia oxidizers; several sets of PCR primers have been described and their specificity may be optimized to allow detection of genetically and ecologically meaningful groups . For denitrifying bacteria, functional probes based on nitrite reductase show most promise . These approaches should complement the more familiar, but no less sophisticated, methods that focus on quantification of in situ transformation rates . Both approaches in combination will be useful in understanding regulation and environmental control of biogeochemical processes. Mutat Res, 1996 Oct 28, 358(1), 7 - 14 A study on carcinogenesis of endogenous nitrite and nitrosamine, and prevention of cancer; Huang YG et al.; Cancer patients show a salivary NO2- density that is twice that of healthy individuals . This indicates that endogenous NO2- is correlated with the cancer . By applying a culture medium modified by vitamin B1 or KCIO3, we have found that many bacteria in the human body (especially the bowels) can nitrify NH4+ to NO2- . NO2- can be combined with a second amine to form nitrosamine which is a carcinogen . We fed mice using G+ coccus, which can lead to a precancerous lesion and mammary carcinoma, and proved that nitrification in vivo is carcinogenic . We have tested a method to prevent cancer in 7392 workers . Patients with a salivary density of NO2- over 10 ppm for 3 consecutive days and with light symptoms were our targets for preventive treatment using antibiotics and nitrosamine destroyers . The result shows that the average cancer incidence rate in the control group is 58.4% higher than in the experimental group over 9 years . The difference between the two groups is statistically significant (x2 = 8.5, p < 0.01) . This is reliable evidence that preventive treatment is effective when based on the idea that endogenous NO2- and nitrosamine are the carcinogens. Mikrobiologiia, 1996 Sep-Oct, 65(5), 621 - 6 {Biotransformation of nitrogen-containing compounds in the process of the continuous final purification of effluents from swine-breeding farms by immobilized cells}; Arkhipenko IA et al.; After their two-step purification in aeration tanks, effluents from a swine-breeding farm were subjected to final purification in a flow bioreactor with immobilized cells derived from activated sludge . The dynamics of nitrogen compounds were studied at the reactor inlet and outlet at three flow rates . The use of a simple mathematical model of the nitrogen balance allowed the rates of nitrogen assimilation-mineralization two nitrification stages, and nitrogen fixation-denitrification to be estimated . The first stage of nitrification was found to proceed intensely and produce high nitrite concentrations (up to 100 mg N/l, accounting for about half of the total N at the inlet) . The second nitrification stage was inhibited, and nitrate was only produced after 3-4 weeks of continuous operation . The strategy of the final purification of effluents from nitrogen compounds is discussed on the basis of the known regulatory mechanisms of microbial activities in activated sludge. Environ Manage, 1996 Sep, 20(5), 649 - 66 Issues, Impacts, and Implications of Shrimp Aquaculture in Thailand Dierberg FE, Kiattisimkul W. Water quality impacts to and from intensive shrimp aquaculture in Thailand are substantial . Besides the surface and subsurface salinization of freshwaters, loadings of solids, oxygen-consuming organic matter, and nutrients to receiving waters are considerable when the cumulative impacts from water exchange during the growout cycle, pond drainage during harvesting, and illegal pond sediment disposal are taken into account . Although just beginning to be considered in Thailand, partial recirculating and integrated intensive farming systems are producing promising, if somewhat limited, results . By providing on-site treatment of the effluent from the shrimp growout ponds, there is less reliance on using outside water supplies, believed to be the source of the contamination.The explosion in the number of intensively operated shrimp farms has not only impacted the coastal zone of Thailand, but has also resulted in an unsustainable aquaculture industry . Abandonment of shrimp ponds due to either drastic, disease-caused collapses or more grandual, year-to-year reductions in the productivity of the pond is common . To move Thailand towards a more sustainable aquaculture industry and coastal zone environment, integrated aquaculture management is needed . Components of integrated aquaculture management are technical and institutional . The technical components involve deployment of wastewater treatment and minimal water-use systems aimed at making aquaculture operations more hydraulically closed . Before this is possible, technical and economic feasibility studies on enhanced nitrification systems and organic solids removal by oxidation between production cycles and/or the utilization of plastic pond liners need to be conducted . The integration of semi-intensive aquaculture within mangrove areas also should be investigated since mangrove losses attributable to shrimp aquaculture are estimated to be between 16 and 32 % of the total mangrove area destroyed betweeen 1979 and 1993.Government policy needs to devote as much attention to sustainability issues as it has on promoting intensive pond culture . Such a balanced policy would include training and education monitoring and enforcement, rehabilitating abandoned ponds, managing land use within the coastal zone, more community involvement, and government reorganization to eliminate overlapping jurisdictions among agencies.As integrated aquaculture management becomes more the practice than the exception, less risk of crop failure to the industry and reduced discharge loadings from intensively managed shrimp ponds to receiving waters can be expected . Projected limitations on growing and marketing shrimp in the future, such as scarcity of land and broodstock, continued disease outbreaks, negative publicity, regulatory enforcement, water treatment and solids disposal costs, and increased competition from growers in other Asian countries will also drive the government and the industry towards adopting integrated aquaculture management.KEY WORDS: Shrimp aquaculture; Thailand; Historical practices; Environmental impacts; Sustainability; Integrated management; Water treatment; Institutional aspects Appl Environ Microbiol, 1996 Aug, 62(8), 2888 - 96 Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria; Hovanec TA et al.; Three nucleic acid probes, two for autotrophic ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria and one for alpha subdivision nitrite-oxidizing bacteria, were developed and used to study nitrifying bacterial phylotypes associated with various freshwater and seawater aquarium biofilters . Nitrosomonas europaea and related species were detected in all nitrifying seawater systems and accounted for as much as 20% of the total eubacterial rRNA . In contrast, nitrifying bacteria belonging to the beta-proteobacterial subdivision were detected in only two samples from freshwater aquaria showing vigorous nitrification rates . rRNA originating from nitrite-oxidizing alpha subdivision proteobacteria was not detected in samples from either aquarium environment . The data obtained indicate that chemolithotrophic ammonia oxidation in the freshwater aquaria was not due to beta-proteobacterial phylotypes related to members of the genus Nitrosomonas and their close relatives, the organisms usually implicated in freshwater nitrification . It is likely that nitrification in natural environments is even more complex than nitrification in these simple systems and is less well characterized with regard to the microorganisms responsible. Appl Environ Microbiol, 1996 Jul, 62(7), 2352 - 5 Monoclonal antibodies recognizing nitrite oxidoreductase of Nitrobacter hamburgensis, N . winogradskyi, and N . vulgaris; Aamand J et al.; Three monoclonal antibodies (MAbs) against nitrite oxidoreductase (NOR) of Nitrobacter hamburgensis were produced . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting analysis of the purified enzyme showed that the MAbs named Hyb 153.1 and Hyb 153.3 both recognized a protein with a molecular mass of 64,000 Da, while Hyb 153.2 recognized a protein with a molecular mass of 115,000 Da . The molecular masses of these proteins are in the same range as those of the proteins of the alpha (115,000-Da) or beta (65,000-Da) subunit of the NOR . By using the antibodies, the amount of NOR was shown to be dependent on the growth conditions . The highest level of NOR was observed in N . hamburgensis when cells were growing mixotrophically . Analysis of whole-cell extracts of N . hamburgensis, N . winogradskyi, and N . vulgaris indicated serological homology of the NORs from these species of the genus Nitrobacter . The immunological analysis enables detection of the key enzyme of the genus Nitrobacter. Microb Ecol, 1996 Jul, 32(2), 171 - 84 Effects of Toluene on Microbially-Mediated Processes Involved in the Soil Nitrogen Cycle Fuller ME, Scow KM. The effects of toluene on indigenous microbial populations involved in the soil nitrogen cycle were examined . Ammonia oxidation potential (AOP) and nitrite oxidation potential (NOP) were both reduced after incubation with high toluene concentrations for 45 days, with the former activity showing greater sensitivity . KCl-extractable ammonium (NH4+ext) levels increased dramatically in soil exposed to high toluene levels, and arginine ammonification was not significantly affected . Alfalfa-amended soil incubated in the presence of 200 &mgr;g toluene ml-1 showed progressive accumulation of NH4+ext over 37 days, indicating that mineralization of plant-associated nitrogen was not hindered by toluene . AOP in treated soil was much less than in control soil on days 7 and 37, but the MPN of ammonia oxidizers in control and exposed soil were not significantly different . Soil incubated with 100 &mgr;g toluene ml-1for 28 days, vented and allowed to incubate for an additional 7 to 30 days, exhibited only slight increases in AOP and NOP, while NH4+ext returned to control levels within a week . Soil exposed to 200 &mgr;g toluene ml-1 and treated in the same manner showed no increases in either AOP or NOP, and NH4+extremained elevated for the duration of the experiment, indicating more long-term effects on soil nitrogen cycling had occurred . Ammonia oxidizer levels in control soil and soil incubated with 100 &mgr;g toluene ml-1 were not appreciably different, whereas levels of ammonia oxidizers were very low in soil exposed to 200 &mgr;g toluene ml-1 and increased only slightly by 30 days post vent . Experiments to determine how toluene affects the AOP of soil indicated a competitive inhibition mechanism, with an effective concentration causing 50% reduction in activity (EC50) of 11 &mgr;M toluene, and a competitive inhibition constant (Ki) of 0.1&plusmn; 0.05 &mgr;M toluene . These results indicate the potential for toluene to adversely impact nitrogen cycling in the terrestrial ecosystem by affecting indigenous soil nitrifiers, which are sensitive to lower levels of toluene than has been previously reported. J Bacteriol, 1996 Jul, 178(13), 3710 - 4 Mutagenesis of hydroxylamine oxidoreductase in Nitrosomonas europaea by transformation and recombination; Hommes NG et al.; Mutagenesis of Nitrosomonas europaea was achieved by electroporation and recombination . To demonstrate this, an aminoglycoside 3'-phosphotransferase (kan) gene was specifically inserted into each of the three gene copies of hao individually . Southern hybridizations and PCR analysis showed the incorporation of the kan gene at the chosen genetic loci . The isolation of mutant strains was achieved in 7 to 14 days when the strains were grown on solid medium . The induced mutations were stable even in the absence of kanamycin-selective pressure for periods of up to 45 days in culture . The mutant strains did not show an observable phenotype different from that of the wild type when grown under the same conditions. J Air Waste Manag Assoc, 1996 Jun, 46(6), 502 - 9 Aerobic treatment of a nitrogen-limited chemical process wastewater; Smith DP; Nitrogen transformations and their effect on aerobic suspended growth treatment of an industrial wastewater were studied in three parallel bench-scale reactors operated at 5 degrees C at mean cell residence times (MCRT) of 15, 30, and 60 days . In normal process wastewater, the bulk of influent nitrogen was in organic form, and the fraction transformed was almost totally incorporated into synthesized biomass . Assimilative control by heterotrophs maintained ammonia-nitrogen levels below permitted effluent levels, and nitrification was not significant . Although volatile suspended solids had a nitrogen content of only 5% to 8%, effective organics removal was maintained, and total organic carbon and filtered daily average five-day biochemical oxygen demand (BOD5) were below permitted effluent levels . A marked improvement in settleability and lower effluent total suspended solids was achieved by adding ammonia-nitrogen to the wastewater in excess of stoichiometric growth requirements . During a batch production cycle of a cationic chemical, the ratio of nitrogen to chemical oxygen demand and the fraction of the total influent nitrogen in soluble form increased in the wastewater . Reactor effluent ammonia levels increased to above permit levels at all three MCRTs during treatment of wastewater containing cationic production effluents . The magnitude of ammonia increase was greater for longer MCRTs, suggesting that synthesis of cell mass was not capable of assimilating the increased ammonia supply under these non-steady conditions . The experimental results suggest several potential strategies for operating the aerobic process at the treatment facility, including adding nitrogen to improve settleability and discontinuing these additions when wastewater contains a high ratio of nitrogen to chemical oxygen demand and an elevated soluble nitrogen fraction. Appl Environ Microbiol, 1996 Jun, 62(6), 2156 - 62 Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria; Mobarry BK et al.; A hierarchical set of five 16S rRNA-targeted oligonucleotide DNA probes for phylogenetically defined groups of autotrophic ammonia- and nitrite-oxidizing bacteria was developed for environmental and determinative studies . Hybridization conditions were established for each probe by using temperature dissociation profiles of target and closely related nontarget organisms to document specificity . Environmental application was demonstrated by quantitative slot blot hybridization and whole-cell hybridization of nitrifying activated sludge and biofilm samples . Results obtained with both techniques suggested the occurrence of novel populations of ammonia oxidizers . In situ hybridization experiments revealed that Nitrobacter and Nitrosomonas species occurred in clusters and frequently were in contact with each other within sludge flocs. FEMS Microbiol Lett, 1996 Jun 1, 139(2-3), 181 - 8 The gene encoding ammonia monooxygenase subunit A exists in three nearly identical copies in Nitrosospira sp . NpAV; Norton JM et al.; The gene encoding ammonia monooxygenase subunit A (AmoA) was found in three copies of the genome of the chemolithotrophic soil bacterium, Nitrosospira sp . NpAV . The open reading frame and flanking regions of the three copies were isolated from digested size fractionated genomic DNA using oligodeoxyribonucleotide primers and polymerase chain reaction . The three gene copies of amoA were sequenced and the sequences compared to each other . The open reading frames and the upstream and downstream flanking regions were nearly identical in the three copies . All three copies were expressed in recombinant Escherichia coli strains from the indigenous promoter producing a product of approximately 30 kDa . All amoA copies encode 274 amino acid polypeptides which have similarity to the ammonia monooxygenase acetylene-binding protein from Nitrosomonas europaea. FEBS Lett, 1996 May 27, 387(1), 71 - 4 The purification of ammonia monooxygenase from Paracoccus denitrificans; Moir JW et al.; The heterotrophic nitrifier Paracoccus denitrificans expresses a membrane-associated ammonia monooxygenase . The active enzyme has been solubilized in the detergent dodecyl-beta-D-maltoside and purified by standard chromatographic techniques . This is the first purification of an ammonia monooxygenase . The enzyme consists of two subunits with molecular masses of 38 and 46 kDa . The purified enzyme is a quinol oxidase, is inhibited by light and a variety of chelating agents and is activated by cupric ions . These properties indicate that this enzyme has similarities to a family of enzymes including the ammonia monooxygenase from Nitrosomonas europaea and the particulate methane monooxygenase from Methylococcus capsulatus (Bath). Antonie Van Leeuwenhoek, 1996 May, 69(4), 305 - 15 Nitrobacter winogradskyi cytochrome c oxidase genes are organized in a repeated gene cluster; Berben G; Cytochrome c oxidase (EC 1.9.3.1) is one of the components of the electron transport chain by which Nitrobacter, a facultative lithoautotrophic bacterium, recovers energy from nitrite oxidation . The genes encoding the two catalytic core subunits of the enzyme were isolated from a Nitrobacter winogradskyi gene library . Sequencing of one of the 14 cloned DNA segments revealed that the subunit genes are side by side in an operon-like cluster . Remarkably the cluster appears to be present in at least two copies per genome . It extends over a 5-6 kb length including, besides the catalytic core subunit genes, other cytochrome oxidase related genes, especially a heme O synthase gene . Noteworthy is the new kind of gene order identified within the cluster . Deduced sequences for the cytochrome oxidase subunits and for the heme O synthase look closest to their counterparts in other alpha-subdivision Proteobacteria, particularly the Rhizobiaceae . This confirms the phylogenetic relationships established only upon 16S rRNA data . Furthermore, interesting similarities exist between N . winogradskyi and mitochondrial cytochrome oxidase subunits while the heme O synthase sequence gives some new insights about the other similar published alpha-subdivision proteobacterial sequences. J Struct Biol, 1996 May-Jun, 116(3), 429 - 31 Crystallization and preliminary crystallographic analysis of cytochrome c553 peroxidase from Nitrosomonas europaea; Pappa H et al.; The di-heme peroxidase (cytochrome c553 peroxidase) from Nitrosomonas europaea has been crystallized in a form suitable for high-resolution X-ray structure determination . A complete data set was obtained to 2.5A and the data were indexed in space group P2(1) with a = 88.79 A, b = 55.93 A, c = 144.37 A, beta = 103.87 degrees . The self-rotation function indicates one homodimer per asymmetric unit. Mol Microbiol, 1996 May, 20(3), 541 - 8 Induction of ammonia monooxygenase and hydroxylamine oxidoreductase mRNAs by ammonium in Nitrosomonas europaea; Sayavedra-Soto LA et al.; In Nitrosomonas europaea, ammonia monooxygenase (AMO) and hydroxylamine oxidoreductase (HAO) catalyse the oxidation of ammonia (NH3) to nitrite (NO2-) . A transcript of 3500 bases hybridizes to probes for amoA and amoB (genes that code for AMO proteins) . A transcript of 2100 bases hybridizes to probes for hao (the gene that codes for HAO) . Induction of the mRNAs detected by amo and hao probes required the presence of ammonium (NH4+) . To correlate new levels of mRNA with de novo activity, existent mRNA pools and AMO activity were depleted prior to induction by NH4+ . The mRNAs of AMO and HAO were depleted by depriving the cells of energy for at least 8 h; AMO activity was inactivated with acetylene (C2H2) after mRNA depletion . In cells treated this way, levels of new AMO mRNA and de novo AMO enzyme activity were correlated with increased NH4+ concentrations up to 1 mM after 3 h of incubation . HAO mRNA also increased in the NH4(+)-treated cells . Other proteins and RNAs induced by NH4+ were detected in 14CO2-labelling experiments . The AMO and HAO mRNAs were preferentially synthesized during energy-limiting conditions. Chemosphere, 1996 Apr, 32(8), 1563 - 74 Biodegradability of fluorinated surfactants under aerobic and anaerobic conditions; Remde A et al.; The "ready biodegradability" of three fluorinated surfactants was determined under aerobic and anaerobic conditions . Surfactant 1, a solution of a fluorinated surfactant in water, was easily degradable under both aerobic and anaerobic conditions during the incubation periods of 28 and 60 days, respectively . Surfactant 2, a nonionic fluorinated surfactant, was degraded under aerobic conditions in a range of 35-77% during 28 days depending on the source of activated sludge . Aerobic degradation was inhibited by the nitrification-inhibitor dicyandiamide indicating that ammonium oxidizing bacteria may play a role in degradation of surfactant 2 . Under anaerobic conditions surfactant 2 was not degraded . Surfactant 3, an anionic fluorinated surfactant, was degraded neither under aerobic nor under anaerobic conditions . Under anaerobic conditions, surfactant 3 inhibited the methane production rate of sludge from a digester . The EC50, i.e . the concentration of surfactant 3 that inhibited 50% of methanogenesis, was determined at 160 mg/l. FEMS Microbiol Lett, 1996 Mar 15, 137(1), 9 - 12 Isolation and characterization of a functional promoter from Nitrosomonas europaea; Nejidat A et al.; A functional promoter from the obligate autotrophic ammonia oxidizing bacterium Nitrosomonas europaea was identified by expression in Escherichia coli, using a promoterless reporter gene . Transcription initiation site of this promoter was determined by primer extension analysis . The sequences at -35 and -10 have low similarity to the -10/-35 consensus sequence of known prokaryotic promoters. Microbiol Res, 1996 Mar, 151(1), 105 - 11 Influence of the herbicide bentazon on soil microbial community; Allievi L et al.; Changes in microbial numbers and activities in a soil in response to bentazon applied at 10 and 100 ppm were studied after 4 and 30 weeks of incubation in laboratory conditions . As regards the eight general and functional microbial groups studied (aerobic and anaerobic bacteria, fungi, aerobic and anaerobic N2-fixing bacteria, nitrifiers, aerobic and anaerobic cellulolytic microorganisms), only the number of anaerobic N2-fixing bacteria significantly decreased, in the presence of the highest herbicide concentration for 30 weeks . At both the incubation times, only the higher dose of bentazon markedly inhibited soil nitrification and CO2 emission . Methanogenesis was inhibited by 1000 ppm bentazon added to anaerobic liquid cultures containing 5% soil for at least 2 weeks . There was an incomplete recovery of the herbicide at the two incubation times: < 5% of 10 ppm after 4 weeks and about 30% of 100 ppm after 30 weeks . No biodegradation of the compound was observed in liquid cultures under aerobic or anaerobic conditions . It is concluded that a bentazon concentration no higher than the field rate distributed within a 2-cm layer of soil does not considerably affect the microflora even in the absence of microbial degradation. Antonie Van Leeuwenhoek, 1996 Jan, 69(1), 41 - 6 The influence of anaerobic pretreatment on the nitrogen removal from biosynthetic pharmaceutical wastewaters; Jenicek P et al.; The C:N ratio of the pharmaceutical wastewaters is usually suitable for a combination of the anaerobic pretreatment with the high COD removal and aerobic posttreatment with the efficient biological N removal . This kind of anaerobic-aerobic process was tested in semipilot scale by using a UASB reactor and an activated sludge system with a predenitrification (total volume 100 l) . It was found that at a total HRT of 2.3 days an average of 97.5% of COD and 73.5% of total N was removed . The UASB reactor was operated at 30 degrees C with a volumetric loading rate of 8.7 kg.m-3.d-1, the efficiency of COD removal was 92.2% . The processes, which take part in the biological removal of nitrogen, especially the nitrification, were running with lower rates than usually observed in aerobic treatment systems. Appl Biochem Biotechnol, 1996 Spring, 57-58, 869 - 76 The effect of antibiotics on nitrification processes . Batch assays; Gomez J et al.; The effect of different antibiotics at several concentrations of ampicillin (0-250 mg/L), benzylpenicillin (0-250 mg/L), novobiocine (0-150 mg/L), oxytetracycline (0-250 mg/L), and chloramphenicol (0-50 mg/L) on a stabilized nitrifying sludge was evaluated under aerated and lithoautotrophic conditions . No effect resulting from the presence of antibiotics on the biomass and nitrate production was noticed . The specific growth rate and volumetric nitrification rate average values for the controls were 8.28 x 10-3/h-1 and 2.74 x 10-3 g/L.h, respectively . Similar rate values were found when different kinds of antibiotic and concentrations were tested . These results may be explained by the nature of the floc or the instability of the antibiotics. Arch Biochem Biophys, 1995 Dec 1, 324(1), 53 - 8 Interaction with membranes of cytochrome c554 from Nitrosomonas europaea; McTavish H et al.; Two c-cytochromes extrinsically bound to the membranes of Nitrosomonas europaea have been identified . One is the tetraheme cytochrome c554, a protein previously described as soluble and periplasmic . Depending on the concentration of Fe and Cu in the growth medium, from 50 to 100% of the total cellular cytochrome c554 is membrane-associated . The cytochromes c554 found in the soluble or membrane fractions are identical in the spectroscopic, chromatographic, or primary structural properties examined . The interaction of cytochrome c554 with membranes is ionic in nature; it is disrupted by high concentrations of salt . Both membrane-derived and periplasmic forms of cytochrome c554 rebind tightly to membranes which have been washed free of the cytochrome . Cytochrome c554 binds to phospholipid vesicles, suggesting that phospholipids may play a role in the interaction of this cytochrome with the membrane . During the oxidation of NH2OH, the ability of the soluble hydroxylamine oxidoreductase (HAO) to transfer electrons to its natural electron acceptor, cytochrome c554, is substantially impaired when the latter is bound to phospholipid vesicles . The second c-cytochrome associated with membranes in N . europaea is identified as HAO based on its catalytic activity and the presence of a 464-nm ferrous absorption band . A small fraction of HAO is found to be membrane-bound and only in cells grown under low Fe/low Cu . This subpopulation of HAO can be released from the membranes without detergents. FEMS Microbiol Lett, 1995 Nov 1, 133(1-2), 131 - 5 Comparative analysis of gene sequences encoding ammonia monooxygenase of Nitrosospira sp . AHB1 and Nitrosolobus multiformis C-71; Rotthauwe JH et al.; DNA encoding ammonia monooxygenase from two phylogenetically related autotrophic nitrifying bacteria, Nitrosospira sp . AHB1 and Nitrosolobus multiformis C-71, was amplified by PCR . The resulting products were cloned into the vector pCR-Script . A continuous region of DNA of about 1.5 kb for strain AHB1 and 1.24 kb for N . multiformis C-71 was analysed . These comprised the major part of the gene amoA encoding the active site polypeptide and, directly downstream, the 5' portion of the amoB gene . The identity values for these sequences at the amino acid level were 93.0% for amoA and 96.1% for amoB . The corresponding values for the nucleic acid sequences were 86.7% and 88.8%, respectively . The identity of the 16S rRNA gene of strain AHB1 to that of N . multiformis C-71 was at least 98.5% . The different degree of sequence conservation between the 16S rDNA and the genes encoding for ammonia monooxygenase facilitates the application of the latter as a molecular tool for a fine-scale differentiation of autotrophic nitrifying bacteria, at the species or strain level, in both environmental and cultivation studies. Microbiology, 1995 Nov, 141 ( Pt 11), 2793 - 800 Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of nitrosospiras in the environment; Hiorns WD et al.; Oligonucleotide sequences selected from the 16S rRNA genes of various species of ammonia-oxidizing bacteria were evaluated as specific PCR amplification primers and probes . The specificities of primer pairs for eubacterial, Nitrosospira and Nitrosomonas rRNA genes were established with sequence databases, and the primer pairs were used to amplify DNA from laboratory cultures and environmental samples . Eubacterial rRNA genes amplified from samples of soil and activated sludge hybridized with an oligonucleotide probe specific for Nitrosospira spp., but not with a Nitrosomonas-specific probe . Lakewater and sediment samples were analysed using a nested PCR technique in which eubacterial rRNA genes were subjected to a secondary amplification with Nitrosomonas or Nitrosospira specific primers . Again, the presence of Nitrosospira DNA, but not Nitrosomonas DNA, was detected and this was confirmed by hybridization of the amplified DNA with an internal oligonucleotide probe . Enrichments of lakewater and sediment samples, incubated for two weeks in the presence of ammonium, produced nitrite and were found to contain DNA from both Nitrosospira and Nitrosomonas as determined by nested PCR amplification and probing of 16S rRNA genes . This demonstrates that Nitrosospira spp . are widespread in the environment . The implications of the detection of Nitrosomonas DNA only after enrichment culture are discussed. FEMS Microbiol Lett, 1995 Oct 15, 132(3), 203 - 8 Evidence that particulate methane monooxygenase and ammonia monooxygenase may be evolutionarily related; Holmes AJ et al.; Genes encoding particulate methane monooxygenase and ammonia monooxygenase share high sequence identity . Degenerate oligonucleotide primers were designed, based on regions of shared amino acid sequence between the 27-kDa polypeptides, which are believed to contain the active sites, of particulate methane monooxygenase and ammonia monooxygenase . A 525-bp internal DNA fragment of the genes encoding these polypeptides (pmoA and amoA) from a variety of methanotrophic and nitrifying bacteria was amplified by PCR, cloned and sequenced . Representatives of each of the phylogenetic groups of both methanotrophs (alpha- and gamma-Proteobacteria) and ammonia-oxidizing nitrifying bacteria (beta- and gamma-Proteobacteria) were included . Analysis of the predicted amino acid sequences of these genes revealed strong conservation of both primary and secondary structure . Nitrosococcus oceanus AmoA showed higher identity to PmoA sequences from other members of the gamma-Proteobacteria than to AmoA sequences . These results suggest that the particulate methane monooxygenase and ammonia monooxygenase are evolutionarily related enzymes despite their different physiological roles in these bacteria. Gene, 1995 Sep 22, 163(1), 159 - 60 Sequence of an ammonia monooxygenase subunit A-encoding gene from Nitrosospira sp . NpAV; Klotz MG et al.; One of three gene copies encoding ammonia monooxygenase subunit A (AmoA) and flanking sequences was isolated from genomic DNA of the chemolithotrophic soil bacterium Nitrosospira sp . NpAV using oligodeoxyribonucleotide primers and the polymerase chain reaction (PCR) . The gene (amoA) encodes a 274-amino-acid polypeptide which has similarity to the ammonia monooxygenase acetylene-binding protein from Nitrosomonas europaea. Biochemistry, 1995 Sep 19, 34(37), 11743 - 9 NIH shift in the hydroxylation of aromatic compounds by the ammonia-oxidizing bacterium Nitrosomonas europaea . Evidence against an arene oxide intermediate; Vannelli T et al.; The migration of deuterium and hydrogen was observed in the aromatic hydroxylation of specifically deuterated, monosubstituted benzenes catalyzed by ammonia monooxygenase of Nitrosomonas europaea . The phenolic products of the hydroxylation of aromatics containing ortho-/para-directing substituents (F, Cl, Br, I, OH, NH2, CH3, CH2CH3, and OCH3) were primarily para-phenols . In contrast, with aromatics containing meta-directing substituents (NO2 and CN), the phenolic products were a more even mixture of meta-and para-phenols . ortho-Fluorophenol was the only ortho-phenolic product observed . The nature of the products suggested that the reaction involved an enzyme-specific, electrophilic addition to the aromatic ring so as to favor hydroxylation at either the meta- or para-positions . With the fluoro-, chloro-, and bromobenzene substrates, the values for the migration and retention of deuterium during hydroxylation (NIH shift) were nearly identical when the deuterium was either at the site of hydroxylation or at an adjacent site, indicating a possible common intermediate . The values of the NIH shift with the nitrobenzene substrate were significantly lower when the deuterium was at the site of hydroxylation than at an adjacent site, indicating the operation of a direct loss mechanism . The present results suggest that the aromatic hydroxylation involved a radical or carbocation intermediate which decayed, without the formation of an arene oxide, to form phenolic products with the accompanying direct loss of deuterium at the site of hydroxylation or the shift of the deuterium to an adjacent site. J Bacteriol, 1995 Sep, 177(17), 4974 - 9 Effects of ammonia on the de novo synthesis of polypeptides in cells of Nitrosomonas europaea denied ammonia as an energy source; Hyman MR et al.; The effects of ammonium on the de novo synthesis of polypeptides in the soil-