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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.

ABSTRACT: 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.

SUBMITTER: Kowalchuk GA

PROVIDER: S-EPMC168443 | biostudies-other | 1997 Apr

REPOSITORIES: biostudies-other

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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 G A GA Stephen J R JR De Boer W W Prosser J I JI Embley T M TM Woldendorp J W JW

Applied and environmental microbiology 19970401 4

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 ...[more]

PMID: 9097446

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Project description:The aim of this study was to determine if there were differences between the types of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria associated with particulate material and planktonic samples obtained from the northwestern Mediterranean Sea. A nested PCR procedure performed with ammonia oxidizer-selective primers was used to amplify 16S rRNA genes from extracted DNA. The results of partial and full-length sequence analyses of 16S rRNA genes suggested that different groups of ammonia-oxidizing bacteria were associated with the two sample types. The particle-associated sequences were predominantly related to Nitrosomonas eutropha, while the sequences obtained from the planktonic samples were related to a novel marine Nitrosospira group (cluster 1) for which there is no cultured representative yet. A number of oligonucleotide probes specific for different groups of ammonia oxidizers were used to estimate the relative abundance of sequence types in samples of clone libraries. The planktonic libraries contained lower proportions of ammonia oxidizer clones (0 to 26%) than the particulate material libraries (9 to 83%). Samples of the planktonic and particle-associated libraries showed that there were depth-related differences in the ammonia oxidizer populations, with the highest number of positive clones in the particle-associated sample occurring at a depth of 700 m. The greatest difference between planktonic and particle-associated populations occurred at a depth of 400 m, where only 4% of the clones in the planktonic library were identified as Nitrosomonas clones, while 96% of these clones were identified as clones that were related to the marine Nitrosospira species. Conversely, all ammonia oxidizer-positive clones obtained from the particle-associated library were members of the Nitrosomonas group. This is the first indication that Nitrosomonas species and Nitrosospira species may occupy at least two distinct environmental niches in marine environments. The occurrence of these groups in different niches may result from differences in physiological properties and, coupled with the different environmental conditions associated with these niches, may lead to significant differences in the nature and rates of nitrogen cycling in these environments.

Project description:The spatial distribution and diversity of ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria (hereinafter referred to as ammonia oxidizers) in the Arctic Ocean were determined. The presence of ammonia oxidizers was detected by PCR amplification of 16S rRNA genes using a primer set specific for this group of organisms (nitA and nitB, which amplifies a 1.1-kb fragment between positions 137 and 1234, corresponding to Escherichia coli 16S rDNA numbering). We analyzed 246 samples collected from the upper water column (5 to 235 m) during March and April 1995, September and October 1996, and September 1997. Ammonia oxidizers were detected in 25% of the samples from 5 m, 80% of the samples from 55 m, 88% of the samples from 133 m, and 50% of the samples from 235 m. Analysis of nitA-nitB PCR product by nested PCR-denaturing gradient gel electrophoresis (DGGE) showed that all positive samples contained the same major band (band A), indicating the presence of a dominant, ubiquitous ammonia oxidizer in the Arctic Ocean basin. Twenty-two percent of the samples contained additional major bands. These samples were restricted to the Chukchi Sea shelf break, the Chukchi cap, and the Canada basin; areas likely influenced by Pacific inflow. The nucleotide sequence of the 1.1-kb nitA-nitB PCR product from a sample that contained only band A grouped with sequences designated group 1 marine Nitrosospira-like sequences. PCR-DGGE analysis of 122 clones from four libraries revealed that 67 to 71% of the inserts contained sequences with the same mobility as band A. Nucleotide sequences (1.1 kb) of another distinct group of clones, found only in 1995 samples (25%), fell into the group 5 marine Nitrosomonas-like sequences. Our results suggest that the Arctic Ocean beta-proteobacterial ammonia oxidizers have low diversity and are dominated by marine Nitrosospira-like organisms. Diversity appears to be higher in Western Arctic Ocean regions influenced by inflow from the Pacific Ocean through the Bering and Chukchi seas.

Project description:Plant-parasitic nematode species have been reported on several occasions from coastal sand dunes, including Longidorus and Rotylenchus species (Vovlas et al., 2008; De Luca et al., 2009; Mateille et al., 2014). In April 2016, 10 soil samples of 3 to 4 kg from the rhizosphere of Tamarix smyrnensis with different vegetation around (viz. Elymus farctus, Lycium schweinfurthii, Crithmum maritimum, and Arthrocnemum sp.) were collected for diagnosis of plant-parasitic nematodes. The area of sampling was a coastal sand dune near the archeological site of Komos, southwest of Crete, Greece. Low soil populations of a needle and a spiral nematode were detected (3 and 8 individuals/1,000 cm3 of soil, respectively), which prompted us to undertake a detailed morphological and molecular comparative study with previous reported data. Nematodes were extracted from soil with the wet sieving and decanting method (Cobb, 1918). Morphological and molecular analyses of females identified these species as Longidorus kuiperi Brinkman, Loof and Barbez, 1987, and Rotylenchus eximius Siddiqi, 1964. The morphology of L. kuiperi females (six specimens studied) was characterized by having a slender body; very broad lip region (27 ± 1.5 [25 to 30] ?m in width); short, hemispherical tail; body length of (7.1 ± 0.8 [6.5 to 8.5] mm); vulva position at 47% to 55% of body length; odontostyle length of (105 ± 6.5 [90 to 115] ?m); males very common (but less frequent than females [45% vs 55%]); tail region with 15 to 20 supplements and bulged terminal cuticle. The morphology of R. eximius females (four specimens studied) was characterized by having a hemispherical lip region clearly set off; with four annuli; body without longitudinal striations; lateral fields areolated in the pharyngeal region only; stylet 36 to 38 ?m; and broadly rounded tail. The morphology of the isolated nematodes agreed with previous descriptions of L. kuiperi (Brinkman et al., 1987; De Luca et al., 2009), and R. eximius (Siddiqi, 1964; Castillo and Vovlas, 2005). A single individual was used for DNA extraction. Primers and polymerase chain reaction conditions used in this research were specified in Cantalapiedra et al. (2013) and Archidona-Yuste et al. (2016), and a single amplicon of 800 and 1,100 bp was obtained and sequenced, respectively. D2-D3 (KX398055-KX398056) and ITS sequence alignments (751 and 648 bp, respectively) from L. kuiperi (KX398057) showed 98% to 99% similarity, differing in 4, and from 6 to 12 nucleotides, respectively, to other sequences of L. kuiperi deposited in GenBank from Italy and Spain (AM911623, AM905267-AM905270, respectively), with a query coverage of 99%. Similarly, D2-D3 sequence alignment from R. eximius (KX398058) showed 100% to 99% to another sequence of R. eximius deposited in GenBank from Italy and Spain (EU280794, DQ328741), differing in zero to three nucleotides, respectively, and a query coverage of 81%. Phylogenetic analyses using Bayesian Inference placed L. kuiperi in a highly supported (100%) clade that included all L. kuiperi sequences deposited in GenBank (Archidona-Yuste et al., 2016), and R. eximius in a highly supported (100%) clade that included all R. eximius sequences deposited in GenBank (Cantalapiedra-Navarrete et al., 2013). All identification methods were consistent with L. kuiperi and R. eximius. To our knowledge, this is the first report of L. kuiperi and R. eximius in Greece. Consequently, all these data suggest that coastal sand dunes in Europe constitute environmental conditions optimal for colonization and development of L. kuiperi, as previously reported (De Luca et al., 2009). Similarly, R. eximius has been reported in several Mediterranean countries, including Italy, Morocco, Spain, and Tunisia (Castillo and Vovlas, 2005), and this report extend the geographical distribution of this species.

Dataset Information

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.

Publications

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.

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