An N-acyl homoserine lactone synthase in the ammonia-oxidizing bacterium Nitrosospira multiformis.

The chemolithoautotrophic bacterium Nitrosospira multiformis is involved in affecting the process of nitrogen cycling. Here we report the existence and characterization of a functional quorum sensing signal synthase in N. multiformis. One gene (nmuI) playing a role in generating a protein with high levels of similarity to N-acyl homoserine lactone (AHL) synthase protein families was identified. Two AHLs (C14-AHL and 3-oxo-C14-AHL) were detected using an AHL biosensor and liquid chromatography-mass spectrometry (LC-MS) when nmuI, producing a LuxI homologue, was introduced into Escherichia coli. However, by extracting N. multiformis culture supernatants with acidified ethyl acetate, no AHL product was obtained that was capable of activating the biosensor or being detected by LC-MS. According to reverse transcription-PCR, the nmuI gene is transcribed in N. multiformis, and a LuxR homolog (NmuR) in this ammonia-oxidizing strain showed great sensitivity to long-chain AHL signals by solubility assay. A degradation experiment demonstrated that the absence of AHL signals might be attributed to the possible AHL-inactivating activities of this strain. To summarize, an AHL synthase gene (nmuI) acting as a long-chain AHL producer has been found in a chemolithotrophic ammonia-oxidizing microorganism, and the results provide an opportunity to complete the knowledge of the regulatory networks in N. multiformis.

A novel method for RNA extraction from Andosols using casein and its application to amoA gene expression study in soil.

The lack of a universal method to extract RNA from soil hinders the progress of studies related to nitrification in soil, which is an important step in the nitrogen cycle. It is particularly difficult to extract RNA from certain types of soils such as Andosols (volcanic ash soils), which is the dominant agricultural soil in Japan, because of RNA adsorption by soil. To obtain RNA from these challenging soils to study the bacteria involved in nitrification, we developed a soil RNA extraction method for gene expression analysis. Autoclaved casein was added to an RNA extraction buffer to recover RNA from soil, and high-quality RNA was successfully extracted from eight types of agricultural soils that were significantly different in their physicochemical characteristics. To detect bacterial ammonia monooxygenase subunit A gene (amoA) transcripts, bacterial genomic DNA and messenger RNA were co-extracted from two different types of Andosols during incubation with ammonium sulfate. Polymerase chain reaction-denaturing gradient gel electrophoresis and reverse transcription polymerase chain reaction-denaturing gradient gel electrophoresis analyses of amoA in soil microcosms revealed that only few amoA, which had the highest similarities to those in Nitrosospira multiformis, were expressed in these soils after treatment with ammonium sulfate, although multiple amoA genes were present in the soil microcosms examined.

Community composition of ammonia-oxidizing bacteria and archaea in rice field soil as affected by nitrogen fertilization.

Little information is available on the ecology of ammonia-oxidizing bacteria (AOB) and archaea (AOA) in flooded rice soils. Consequently, a microcosm experiment was conducted to determine the effect of nitrogen fertilizer on the composition of AOB and AOA communities in rice soil by using molecular analyses of ammonia monooxygenase gene (amoA) fragments. Experimental treatments included three levels of N (urea) fertilizer, i.e. 50, 100 and 150 mgNkg(-1) soil. Soil samples were operationally divided into four fractions: surface soil, bulk soil deep layer, rhizosphere and washed root material. NH(4)(+)-N was the dominant form of N in soil porewater and increased with N fertilization. Cloning and sequencing of amoA gene fragments showed that the AOB community in the rice soil consisted of three major groups, i.e. Nitrosomonas communis cluster, Nitrosospira cluster 3a and cluster 3b. The sequences related to Nitrosomonas were predominant. There was a clear effect of N fertilizer and soil depth on AOB community composition based on terminal restriction fragment length polymorphism fingerprinting. Nitrosomonas appeared to be more abundant in the potentially oxic or micro-oxic fractions, including surface soil, rhizosphere and washed root material, than the deep layer of anoxic bulk soil. Furthermore, Nitrosomonas increased relatively in the partially oxic fractions and that of Nitrosospira decreased with the increasing application of N fertilizer. However, AOA community composition remained unchanged according to the denaturing gradient gel electrophoresis analyses.

Diversity of ammonia oxidising bacteria in a vertical flow constructed wetland.

Vertical flow constructed wetlands (VFCWs) with intermittent loading are very suitable for nitrification. Ammonia oxidising bacteria (AOB) are the limiting step of nitration. Therefore the AOB community of a full-scale VFCW, receiving municipal wastewater, was investigated within this study. The diversity of the functional gene encoding the alpha-subunit of the ammonia monooxygenase (amoA), present only in AOB, was assessed by denaturing gradient gel electrophoresis (DGGE). Only very few amoA sequence types dominated the wetland filter substrate; nevertheless a stable nitrification performance could be observed. During the cold season the nitrification was slightly reduced, but it has been shown that the same AOB could be identified. No spatial AOB pattern could be observed within the filter body of the VFCW. The most prominent bands were excised from DGGE gels and sequenced. Sequence analyses revealed two dominant AOB lineages: Nitrosomonas europaea/"Nitrosococcus mobilis" and Nitrosospira. Species of the Nitrosomonas lineage are commonly found in conventional wastewater treatment plants (WWTPs). In contrast, members of the Nitrosospira lineage are rarely present in WWTPs. Our observations indicate that the AOB community in this VFCW is similar to that found in horizontal flow constructed wetlands, but differs from common WWTPs regarding the presence of Nitrosospira.

Ammonia-oxidizing bacterial community composition in estuarine and oceanic environments assessed using a functional gene microarray.

The relationship between environmental factors and functional gene diversity of ammonia-oxidizing bacteria (AOB) was investigated across a transect from the freshwater portions of the Chesapeake Bay and Choptank River out into the Sargasso Sea. Oligonucleotide probes (70-bp) designed to represent the diversity of ammonia monooxygenase (amoA) genes from Chesapeake Bay clone libraries and cultivated AOB were used to construct a glass slide microarray. Hybridization patterns among the probes in 14 samples along the transect showed clear variations in amoA community composition. Probes representing uncultivated members of the Nitrosospira-like AOB dominated the probe signal, especially in the more marine samples. Of the cultivated species, only Nitrosospira briensis was detected at appreciable levels. Discrimination analysis of hybridization signals detected two guilds. Guild 1 was dominated by the marine Nitrosospira-like probe signal, and Guild 2's largest contribution was from upper bay (freshwater) sediment probes. Principal components analysis showed that Guild 1 was positively correlated with salinity, temperature and chlorophyll a concentration, while Guild 2 was positively correlated with concentrations of oxygen, dissolved organic carbon, and particulate nitrogen and carbon, suggesting that different amoA sequences represent organisms that occupy different ecological niches within the estuarine/marine environment. The trend from most diversity of AOB in the upper estuary towards dominance of a single type in the polyhaline region of the Bay is consistent with the declining importance of AOB with increasing salinity, and with the idea that AO-Archaea are the more important ammonia oxidizers in the ocean.

Phylogeny of nitrite reductase (nirK) and nitric oxide reductase (norB) genes from Nitrosospira species isolated from soil.

Ammonia-oxidizing bacteria are believed to be an important source of the climatically important trace gas nitrous oxide (N(2)O). The genes for nitrite reductase (nirK) and nitric oxide reductase (norB), putatively responsible for nitrous oxide production, have been identified in several ammonia-oxidizing bacteria, but not in Nitrosospira strains that may dominate ammonia-oxidizing communities in soil. In this study, sequences from nirK and norB genes were detected in several cultured Nitrosospira species and the diversity and phylogeny of these genes were compared with those in other ammoniaoxidizing bacteria and in classical denitrifiers. The nirK and norB gene sequences obtained from Nitrosospira spp. were diverse and appeared to be less conserved than 16S rRNA genes and functional ammonia monooxygenase (amoA) genes. The nirK and norB genes from some Nitrosospira spp. were not phylogenetically distinct from those of denitrifiers, and phylogenetic analysis suggests that the nirK and norB genes in ammonia-oxidizing bacteria have been subject to lateral transfer.

Immunocytochemical localization of membrane-bound ammonia monooxygenase in cells of ammonia oxidizing bacteria.

The intracellular location of the membrane-bound ammonia monooxygenase (AMO) in all genera of ammonia oxidizing bacteria (Nitrosomonas, Nitrosococcus and Nitrosospira) was determined by electron microscopic immunocytochemistry. Polyclonal antibodies recognizing the two subunits, AmoA- and AmoB-proteins, were used for post-embedding labeling. Ultrathin sections revealed that the AmoB-protein was located in all genera on the cytoplasmic membrane. In cells of Nitrosomonas and Nitrosococus additional but less AmoB-labeling was found on the intracytoplasmic membrane (ICM). In contrast to the detection of AmoB-protein, the AmoA-antibodies failed to detect the AmoA-protein. Based on quantitative immunoblots the extent of ICM in Nitrosomonas eutropha was correlated with the amount of AmoA in the cells. The highest extent of ICM and amount of AmoA was found at low ammonium substrate concentrations.

Loss of diversity of ammonia-oxidizing bacteria correlates with increasing salinity in an estuary system.

Ammonia-oxidizing bacteria (AOB) play an important role in nitrogen cycling in estuaries, but little is known about AOB diversity, distribution and activity in relation to the chemical and physical changes encountered in estuary systems. Although estuarine salinity gradients are well recognized to influence microbial community structure, few studies have examined the influence of varying salinity on the diversity and stability of AOB populations. To investigate these relationships, we collected sediment samples from low-, mid- and high-salinity sites in Plum Island Sound estuary, MA, during spring and late summer over 3 years. Ammonia-oxidizing bacteria distribution and diversity were assessed by terminal restriction fragment length polymorphism (TRFLP) analysis of the ammonia monooxygenase (amoA) gene, and fragments were identified by screening amoA clone libraries constructed from each site. Most striking was the stability and low diversity of the AOB community at the high-salinity site, showing little variability over 3 years. Ammonia-oxidizing bacteria at the high-salinity site were not closely related to any cultured AOB, but were most similar to Nitrosospira spp. Ammonia-oxidizing bacteria at the mid- and low-salinity sites were distributed among Nitrosospira-like sequences and sequences related to Nitrosomonas ureae/oligotropha and Nitrosomonas sp. Nm143. Our study suggests that salinity is a strong environmental control on AOB diversity and distribution in this estuary.

Emergence of competitive dominant ammonia-oxidizing bacterial populations in a full-scale industrial wastewater treatment plant.

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.

Urease-encoding genes in ammonia-oxidizing bacteria.

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.

Use of amoB as a new molecular marker for ammonia-oxidizing bacteria.

Specific molecular determination and classification of ammonia-oxidizing bacteria have relied on the use of conventional markers such as 16S rDNA. However, this gene does not satisfactorily provide a wide vision of all phylogenetic lineages. Despite the initial expectations, the use of functional genes as for example amoA has only been useful to corroborate the established taxonomy. Ammonia-oxidizing bacteria constitute a physiological group that crosses over principal phylogenetic radiations. Therefore, it is necessary to look for novel functional markers, which are needed for both diversity and taxonomic studies. In this work, the available amoB sequences have been used to design a new degenerate set of primers flanking a ca. 500-bp region. Partial amoB gene sequences of up to 16 AOB strains (5 Nitrosomonas, 10 Nitrosospira, and 1 Nitrosococcus) belonging to both the beta- and the gamma-Proteobacteria have been obtained. Comparison of both DNA and deduced amino acid sequences results in three subgroups, two of them of the beta-Proteobacteria and a third one of the gamma-Proteobacteria displaying 75% and 35% homology in their deduced amino acid sequences, respectively. This gene has proven to be a suitable molecular marker to study AOB, as well as providing a new insight into the classification of this group.