Temporal patterns of nitrogenase gene (nifH) expression in the oligotrophic North Pacific Ocean

Abstract

Dinitrogen (N(2))-fixing microorganisms (diazotrophs) play important roles in ocean biogeochemistry and plankton productivity. In this study, we examined the presence and expression of specific planktonic nitrogenase genes (nifH) in the upper ocean (0 to 175 m) at Station ALOHA in the oligotrophic North Pacific Ocean. Clone libraries constructed from reverse-transcribed PCR-amplified mRNA revealed six unique phylotypes. Five of the nifH phylotypes grouped with sequences from unicellular and filamentous cyanobacteria, and one of the phylotypes clustered with gamma-proteobacteria. The cyanobacterial nifH phylotypes retrieved included two sequence types that phylogenetically grouped with unicellular cyanobacteria (termed groups A and B), several sequences closely related (97 to 99%) to Trichodesmium spp. and Katagnymene spiralis, and two previously unreported phylotypes clustering with heterocyst-forming nifH cyanobacteria. Temporal patterns of nifH expression were evaluated using reverse-transcribed quantitative PCR amplification of nifH gene transcripts. The filamentous and presumed unicellular group A cyanobacterial phylotypes exhibited elevated nifH transcription during the day, while members of the group B (closely related to Crocosphaera watsonii) unicellular phylotype displayed greater nifH transcription at night. In situ nifH expression by all of the cyanobacterial phylotypes exhibited pronounced diel periodicity. The gamma-proteobacterial phylotype had low transcript abundance and did not exhibit a clear diurnal periodicity in nifH expression. The temporal separation of nifH expression by the various phylotypes suggests that open ocean diazotrophic cyanobacteria have unique in situ physiological responses to daily fluctuations of light in the upper ocean.

FIG. 1.

Neighbor-joining phylogenetic relationships of nifH nucleic acid sequences obtained from upper ocean plankton samples at Station ALOHA. The polygons represent nifH sequences exhibiting >95% similarity; the numbers of sequences retrieved from RT-PCR clone libraries from this study are indicated inside the polygons. Sequences in boldface type were used to develop phylotype-specific QPCR primers and probes. The accession numbers of representative sequences are indicated next to the phylotypes. The trees in boxes include the sequence types targeted by QPCR. Distances were determined using a Jukes-Cantor correction (22); trees were bootstrapped 1,000 times, and bootstrap values of >50% are indicated at the nodes.

FIG. 2.

Depth profiles for nifH transcript abundance (nifH cDNA copies liter⁻¹) for five different nifH phylotypes. Samples were obtained from midnight (solid symbols) and noon (open symbols) depth profiles in December 2002 at Station ALOHA. Transcript abundance was determined by QPCR amplification of reverse-transcribed nifH cDNA; the symbols indicate mean nifH cDNA concentrations from triplicate QPCR assays. (A) Group A transcripts; (B) group B phylotype; (C) Trichodesmium spp.; (D) heterocyst-1; (E) γ-proteobacterial phylotypes.

FIG. 3.

Temporal patterns of nifH transcription (nifH cDNA copies liter⁻¹) by five phylotypes in the upper ocean (25 m) at Station ALOHA in December 2002. (A) Unicellular cyanobacterial transcription for group A and group B phylotypes; (B) γ-proteobacterial transcripts; (C) transcript concentrations for filamentous cyanobacterial phylotypes, including Trichodesmium spp. and heterocyst-1. The symbols indicate mean cDNA concentrations from triplicate QPCRs, and the error bars indicate ±1 standard deviation.

FIG. 4.

Temporal patterns of cyanobacterial nifH transcript abundance normalized to nifH gene copy abundance (nifH cDNA copies/gene copy). nifH expression was normalized to the gene copy abundance for each phylotype; nifH gene abundance was determined for midnight on 12 December 2002. Gene abundance was normalized to nifH expression for unicellular group A (A), unicellular group B (B), and Trichodesmium spp. (C). The symbols indicate the mean number of nifH cDNA copies (reverse-transcribed RNA) divided by the average number of nifH gene copies (from DNA); the error bars indicate ±1 standard deviation (including propagated error) of the sample mean.