Spatial distribution and transcriptional activity of an uncultured clade of planktonic diazotrophic gamma-proteobacteria in the Arabian sea

Abstract

The spatial distribution of an uncultured clade of marine diazotrophic gamma-proteobacteria in the Arabian Sea was investigated by the development of a specific primer pair to amplify an internal fragment of nifH by PCR. These organisms were most readily detected in highly oligotrophic surface waters but could also be found in deeper waters below the nutricline. nifH transcripts originating from this clade were detected in oligotrophic surface waters and, in addition, in the deeper and the more productive near-coastal waters. The nifH sequences most closely related to the unidentified marine bacterial group are from environmental clones amplified from the Atlantic and Pacific Oceans. These findings suggest that these gamma-proteobacteria are widespread and likely to be an important component of the heterotrophic diazotrophic microbial community of the tropical and subtropical oceans.

FIG. 1.

Location of the stations (1 to 11) occupied in the Arabian Sea during the AMBITION cruise in September 2001.

FIG. 2.

Phylogram showing the relationship between the nifH gene of the UMB cluster and other diazotrophic members of the γ-proteobacteria. The percentages (>50%) of 1,000 bootstrap replicates supporting the topography of the tree are shown at the nodes. The nifH gene from the δ-proteobacterium Desulfovibrio gigas was used as an outgroup. The inset phylogram shows the relationship between the nifH gene of the UMB clade including four previously reported environmental clones. The tree is based on all 242 bp of the nifH DNA sequences recovered and the corresponding coding regions of the previously reported clones.

FIG. 3.

Alignment of the UMB primers with the corresponding regions of nifH from the γ-proteobacterium V. natriegens (top). The nucleotide positions where the V. natriegens sequence differs are indicated by the corresponding bases below the UMB sequences. The analytical agarose gel shows the PCR products obtained from either V. natriegens or the UMB clone 01/11/2O with either the universal nested primer set (U) or UMB primers (C) (bottom). The same template concentrations were used for each reaction.

FIG. 4.

Depth distribution of nifH-containing organisms at station 1 detected with either the universal (U) or UMB-specific (C) nested primers (upper panel) and Southern blot of the same gel probed with the UMB probe (lower panel).

FIG. 5.

Depth distribution of transcriptionally active nifH-containing organisms at station 1 amplified by RT-PCR with the universal nested primers (upper panel) and Southern blot of nifH cDNAs probed with the UMB probe (lower panel).

FIG. 6.

Horizontal distribution of nifH-containing organisms in the Arabian Sea amplified by using either the universal (U) or UMB-specific (C) nested primers (upper panel). The station and cast numbers are shown (as station number/cast number) for each sample and are followed by the depth from which the sample was obtained. Lower panel, Southern blot of the same gel probed with the UMB probe.

FIG. 7.

Horizontal distribution of transcriptionally active nifH-containing organisms in the Arabian Sea amplified by RT-PCR with the universal nested primers (upper panel) and Southern blot of nifH cDNAs probed with the UMB probe (lower panel).