16S rRNA and amoA-based phylogeny of 12 novel betaproteobacterial ammonia-oxidizing isolates: extension of the dataset and proposal of a new lineage within the nitrosomonads.
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The phylogenetic relationship of 12 ammonia-oxidizing isolates (eight nitrosospiras and four nitrosomonads), for which no gene sequence information was available previously, was investigated based on their genes encoding 16S rRNA and the active site subunit of ammonia monooxygenase (AmoA). Almost full-length 16S rRNA gene sequences were determined for the 12 isolates. In addition, 16S rRNA gene sequences of 15 ammonia-oxidizing bacteria (AOB) published previously were completed to allow for a more reliable phylogeny inference of members of this guild. Moreover, sequences of 453 bp fragments of the amoA gene were determined from 15 AOB, including the 12 isolates, and completed for 10 additional AOB. 16S rRNA gene and amoA-based analyses, including all available sequences of AOB pure cultures, were performed to determine the position of the newly retrieved sequences within the established phylogenetic framework. The resulting 16S rRNA gene and amoA tree topologies were similar but not identical and demonstrated a superior resolution of 16S rRNA versus amoA analysis. While 11 of the 12 isolates could be assigned to different phylogenetic groups recognized within the betaproteobacterial AOB, the estuarine isolate Nitrosomonas sp. Nm143 formed a separate lineage together with three other marine isolates whose 16S rRNA sequences have not been published but have been deposited in public databases. In addition, 17 environmentally retrieved 16S rRNA gene sequences not assigned previously and all originating exclusively from marine or estuarine sites clearly belong to this lineage. (+info)
Urease-encoding genes in ammonia-oxidizing bacteria.
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Many but not all ammonia-oxidizing bacteria (AOB) produce urease (urea amidohydrolase, EC 3.5.1.5) and are capable of using urea for chemolithotrophic growth. We sequenced the urease operons from two AOB, the beta-proteobacterium Nitrosospira sp. strain NpAV and the gamma-proteobacterium Nitrosococcus oceani. In both organisms, all seven urease genes were contiguous: the three structural urease genes ureABC were preceded and succeeded by the accessory genes ureD and ureEFG, respectively. Green fluorescent protein reporter gene fusions revealed that the ure genes were under control of a single operon promoter upstream of the ureD gene in Nitrosococcus oceani. Southern analyses revealed two copies of ureC in the Nitrosospira sp. strain NpAV genome, while a single copy of the ure operon was detected in the genome of Nitrosococcus oceani. The ureC gene encodes the alpha subunit protein containing the active site and conserved nickel binding ligands; these conserved regions were suitable primer targets for obtaining further ureC sequences from additional AOB. In order to develop molecular tools for detecting the ureolytic ecotype of AOB, ureC genes were sequenced from several beta-proteobacterial AOB. Pairwise identity values ranged from 80 to 90% for the UreC peptides of AOB within a subdivision. UreC sequences deduced from AOB urease genes and available UreC sequences in the public databases were used to construct alignments and make phylogenetic inferences. The UreC proteins from beta-proteobacterial AOB formed a distinct monophyletic group. Unexpectedly, the peptides from AOB did not group most closely with the UreC proteins from other beta-proteobacteria. Instead, it appears that urease in beta-proteobacterial autotrophic ammonia oxidizers is the product of divergent evolution in the common ancestor of gamma- and beta-proteobacteria that was initiated before their divergence during speciation. Sequence motifs conserved for the proteobacteria and variable regions possibly discriminatory for ureC from beta-proteobacterial AOB were identified for future use in environmental analysis of ureolytic AOB. These gene sequences are the first publicly available for ure genes from autotrophic AOB. (+info)
Emergence of competitive dominant ammonia-oxidizing bacterial populations in a full-scale industrial wastewater treatment plant.
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Ammonia-oxidizing bacterial populations in an industrial wastewater treatment plant were investigated with amoA and 16S rRNA gene real-time PCR assays. Nitrosomonas nitrosa initially dominated, but over time RI-27-type ammonia oxidizers, also within the Nitrosomonas communis lineage, increased from below detection to codominance. This shift occurred even though nitrification remained constant. (+info)
Influence of starvation on potential ammonia-oxidizing activity and amoA mRNA levels of Nitrosospira briensis.
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The effect of short-term ammonia starvation on Nitrosospira briensis was investigated. The ammonia-oxidizing activity was determined in a concentrated cell suspension with a NOx biosensor. The apparent half-saturation constant [Km(app)] value of the NH3 oxidation of N. briensis was 3 microM NH3 for cultures grown both in continuous and batch cultures as determined by a NOx biosensor. Cells grown on the wall of the vessel had a lower Km(app) value of 1.8 microM NH3. Nonstarving cultures of N. briensis showed potential ammonia-oxidizing activities of between 200 to 250 microM N h(-1), and this activity decreased only slowly during starvation up to 10 days. Within 10 min after the addition of fresh NH4+, 100% activity was regained. Parallel with activity measurements, amoA mRNA and 16S rRNA were investigated. No changes were observed in the 16S rRNA, but a relative decrease of amoA mRNA was observed during the starvation period. During resuscitation, an increase in amoA mRNA expression was detected simultaneously. The patterns of the soluble protein fraction of a 2-week-starved culture of N. briensis showed only small differences in comparison to a nonstarved control. From these results we conclude that N. briensis cells remain in a state allowing fast recovery of ammonia-oxidizing activity after addition of NH4+ to a starved culture. Maintaining cells in this kind of active state could be the survival strategy of ammonia-oxidizing bacteria in nature under fluctuating NH4+ availability. (+info)
Response of Nitrosospira sp. strain AF-like ammonia oxidizers to changes in temperature, soil moisture content, and fertilizer concentration.
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Very little is known regarding the ecology of Nitrosospira sp. strain AF-like bacteria, a unique group of ammonia oxidizers within the Betaproteobacteria. We studied the response of Nitrosospira sp. strain AF-like ammonia oxidizers to changing environmental conditions by applying molecular methods and physiological measurements to Californian grassland soil manipulated in the laboratory. This soil is naturally high in Nitrosospira sp. strain AF-like bacteria relative to the much-better-studied Nitrosospira multiformis-like ammonia-oxidizing bacteria. Increases in temperature, soil moisture, and fertilizer interacted to reduce the relative abundance of Nitrosospira sp. strain AF-like bacteria, although they remained numerically dominant. The overall abundance of ammonia-oxidizing bacteria increased with increasing soil moisture and decreased with increasing temperature. Potential nitrification activity was altered by interactions among temperature, soil moisture, and fertilizer, with activity tending to be higher when soil moisture and temperature were increased. The increase in potential nitrification activity with increased temperature was surprising, given that the overall abundance of ammonia-oxidizing bacteria decreased significantly under these conditions. This observation suggests that (i) Nitrosospira sp. strain AF-like bacteria may respond to increased temperature with an increase in activity, despite a decrease in abundance, or (ii) that potential nitrification activity in these soils may be due to organisms other than bacteria (e.g., archaeal ammonia oxidizers), at least under conditions of increased temperature. (+info)
Ammonia-oxidizing beta-proteobacteria from the oxygen minimum zone off northern Chile.
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The composition of ammonia-oxidizing bacteria from the beta-Proteobacteria subclass (betaAOB) was studied in the surface and upper-oxycline oxic waters (2- to 50-m depth, approximately 200 to 44 microM O(2)) and within the oxygen minimum zone (OMZ) suboxic waters (50- to 400-m depth, < or =10 microM O(2)) of the eastern South Pacific off northern Chile. This study was carried out through cloning and sequencing of genes coding for 16S rRNA and the ammonia monooxygenase enzyme active subunit (amoA). Sequences affiliated with Nitrosospira-like cluster 1 dominated the 16S rRNA gene clone libraries constructed from both oxic and suboxic waters. Cluster 1 consists exclusively of yet-uncultivated betaAOB from marine environments. However, a single clone, out of 224 obtained from the OMZ, was found to belong to Nitrosospira lineage cluster 0. To our knowledge, cluster 0 sequences have been derived from betaAOB isolated only from sand, soil, and freshwater environments. Sequences in clone libraries of the amoA gene from the surface and upper oxycline could be grouped in a marine subcluster, also containing no cultured representatives. In contrast, all 74 amoA sequences originating from the OMZ were either closely affiliated with cultured Nitrosospira spp. from clusters 0 and 2 or with other yet-uncultured betaAOB from soil and an aerated-anoxic Orbal process waste treatment plant. Our results reveal the presence of Nitrosospira-like betaAOB in both oxic and suboxic waters associated with the OMZ but with a clear community shift at the functional level (amoA) along the strong oxygen gradient. (+info)
Complete genome sequence of Nitrosospira multiformis, an ammonia-oxidizing bacterium from the soil environment.
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Nitrospira community composition in nitrifying reactors operated with two different dissolved oxygen levels.
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Nitrospira is a dominant member of nitrite-oxidizing bacteria (NOB) in nitrifying bioreactors as well as in natural habitats. In this study, Nitrospira NOB were investigated in the two nitrifying reactors operated with high and low dissolved oxygen (DO) concentrations for a period of 300 days. Phylogenetic and terminal restriction fragment length polymorphism analyses based on 16S rRNA gene sequences revealed that the Nitrospira community compositions of the two reactors during the early period related to group 1 and half of the Nitrospira community composition shifted to group 2 in the high-DO reactor after day 179, although there was no significant change in the low-DO reactor. These results suggested that DO was an important factor affecting Nitrospira community compositions in the nitrifying reactors. (+info)