Diversity and distribution of DNA sequences with affinity to ammonia-oxidizing bacteria of the beta subdivision of the class Proteobacteria in the Arctic Ocean

Abstract

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.

FIG. 1

Location of Arctic Ocean stations where DNA samples were collected during SCICEX 95 (circle), 96 (triangle), and 97 (square).

FIG. 2

Distribution of ammonia-oxidizing bacteria in the Arctic Ocean at depths of 5 m (A), 55 m (B), 133 m (C), and 235 m (D). Symbols are defined in the legend to Fig. 1; open and filled symbols indicate the presence and lack of detection of ammonia oxidizers, respectively.

FIG. 3

Image of representative DGGE gels containing samples from the Arctic Ocean taken on the SCICEX 95, SCICEX 96, and SCICEX 97 cruises. Lane 1: 55 m, 71°49′N, 152°23′W; lane 2: 55 m, 72°16′N, 154°24′W; lane 3: 55 m, 72°34′N, 155°47′W; lane 4: 55 m, 73°36′N, 161°80′W; lane 6: 55 m, 74°27′N, 163°30′W; lane 7: 55 m, 73°32′N, 160°56′W; lane 8: 55 m, 74°30′N, 164°32′W; lane 9: 55 m, 76°60′N, 162°36′W; lane 10: 132 m, 74°20′N, 163°60′W; lane 11: 55 m, 74°20′N, 163°60′W; lane 12: 55 m, 77°21′N, 150°41′W; lane 14: 132 m, 80°28′N, 156°53′W; lane 15: 55 m, 80°29′N, 156°54′W; lane 16: 55 m, 76°57′N, 161°44′W; lane 17: 55 m, 83°38′N, 131°16′E; lane 18: 131 m, 70°53′N, 141°50′W; lane 19: 235 m, 70°53′N, 141°51′W; lane 20: 55 m, 72°08′N, 154°16′W; lane 22: 55 m, 75°17′N, 170°11′W; lane 23: 55 m, 75°31′N, 179°35′W; lane 24: 55 m, 79°00′N, 189°37′W; lane 25: 55 m, 78°19′N, 164°55′W; lanes 5, 13, and 21 are standards containing a mixture of Clostridium perfringens and Bacillus thuringiensis genomic DNA (Sigma). The nitAB PCR products shown in lanes 2, 3, 14, and 17 were used to generate the clone libraries. The nitAB PCR product shown in lane 17 was also sequenced directly. Band A is the common band present in all samples.

FIG. 4

Distribution of samples from SCICEX 95, 96, and 97 cruises according to the number of bands in the DGGE gel. Filled circles represent samples that have one major band, and open circles represent samples that have more than one major band. Arrows indicate samples which were used to generate clone libraries 95A (A), 95B (B), 96A (C), and 96B (D).

FIG. 5

Images of DGGE gels comparing bands from clones (which were sequenced) with the band pattern of the PCR product used to generate the clone library. Bands A to E represent the clones with their respective bands in the original samples. Bands of clones 95A-4, 95B-22, 95B-7, 95B-3, 96A-11, and 96A-19 were not detected in the original samples. Clone 95A-21 represents three other clones (95A-13, 95A-14, and 95A-40) that were sequenced, and 96A-4 also represents 96A-17.

FIG. 6

Neighbor-joining tree showing phylogenetic relationship of Arctic Ocean sequences to closely related β-Proteobacteria ammonia oxidizer sequences. The tree was generated by using a 1,040-bp region of the 16S rDNA. Clones from this study are indicated in boldface type, and the direct sequence is underlined. Clusters are numbered according to the method of Stephens et al. (36). Bootstrap values higher than 50% are shown. The tree is unrooted, and E. coli is used as an out group. The bar indicate a Jukes-Cantor distance of 0.03. The accession numbers of the sequences used to make the phylogenetic tree are given in Table 2.