Mark T. Mellon

GROWTH AND NITROGEN FIXATION OF THE DIAZOTROPHIC FILAMENTOUS NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS 101 IN DEFINED MEDIA : EVIDENCE FOR A CIRCADIAN RHYTHM

Trichodesmium sp. IMS 101, originally isolated from coastal western Atlantic waters by Prufert‐Bebout and colleagues and maintained in seawater‐based media, was successfully cultivated in two artificial media. Its characteristics of growth, nitrogen fixation, and regulation of nitrogen fixation were compared to those of natural populations and Trichodesmium sp. NIBB 1067. Results indicate that the culture grown in artificial media had nitrogen fixation characteristics similar to those when the culture is grown in seawater‐based medium and to those of Trichodesmium sp. in the natural habitat. The study provides practical artificial media to facilitate the physiological studies of these important diazotrophic cyanobacteria, as well as the cultivation of other Trichodesmium species in future studies. Manipulations of the light/dark cycle were performed to determine whether or not the daily cycle of nitrogen fixation is a circadian rhythm. Cultures grown under continuous light maintained the cycle for up to 6 days. We demonstrated that the daily cycle of nitrogen fixation in Trichodesmium sp. IMS 101 was at least partially under the control of a circardian rhythm.

Expression of nifH genes in natural microbial assemblages in Lake George, New York, detected by reverse transcriptase PCR.

ABSTRACT A modified nested reverse transcriptase PCR (RT-PCR) method was used to detect the expression of nitrogenase genes in meso-oligotrophic Lake George, New York. Net (>20-μm pore size) plankton samples collected from two sites (Dome Island and Hague Marina) were extracted for total RNA and genomic DNA to determine the identity of diazotrophic organisms that were present and those that were actively expressing nitrogenase genes. Phylogenetic analysis of individual sequences cloned from PCR amplifications showed that there were phylogenetically diverse groups of bacteria that possessed a nifH gene, including representatives of unicellular and filamentous cyanobacteria, the α- and γ-subdivisions of the division Proteobacteria (α- and γ-proteobacteria), and a previously undefined group of bacteria. The phylotypes cloned from RT-PCR amplifications, which were actively expressing nifH transcripts, clustered with the unicellular and filamentous cyanobacteria, α-proteobacteria, and the novel bacterial cluster. No bacterial sequences were found which clustered with sequences from cluster II (alternative nitrogenases), III (nitrogenases in strict anaerobes), or IV (nifH-like sequences). These results indicate that there were several distinct groups of nitrogen-fixing microorganisms in the net plankton from both sampling sites and that most of the groups had representative phylotypes that were actively expressing nitrogenase genes.

Nitrogen fixation in the marine environment: relating genetic potential to nitrogenase activity

Abstract Nitrogen fixation can be an important source of nitrogen for biological productivity in the marine environment. Biological nitrogen fixation is catalyzed by the enzyme nitrogenase, which is possessed by diverse microorganisms representing virtually all phylogenetic groups. Interest in nitrogen fixation in the sea has usually been focused on rates of nitrogen fixation, but information on the types of species present with the capability for nitrogen fixation can be important for predicting nitrogen fixation rates in situ. Molecular tools for detection and characterization of the nitrogenase ( nif ) genes and immunoassays for nitrogenase protein can provide new information on the factors regulating the distribution and activity of diverse nitrogen fixing organisms in the marine environment. Amplification and characterization of nifH sequences has made it possible to identify the type(s) of organism responsible for nitrogen fixation, such as in aggregates of the cyanobacterium Trichodesmium . Differences in nitrogen fixation patterns have been linked to genetic differences between Trichodesmium strains. Further development of these approaches will provide new and powerful ways to link the genetic potential for nitrogen fixation to nitrogen fixation rates in the ocean.

ORGANIZATION OF THE nif GENES OF THE NONHETEROCYSTOUS CYANOBACTERIUM TRICHODESMIUM SP. IMS101

An approximately 16‐kb fragment of the Trichodesmium sp. IMS101 (a nonheterocystous filamentous cyanobacterium) “conventional”nif gene cluster was cloned and sequenced. The gene organization of the Trichodesmium and Anabaena variabilis vegetative (nif 2) nitrogenase gene clusters spanning the region from nif B to nif W are similar except for the absence of two open reading frames (ORF3 and ORF1) in Trichodesmium. The Trichodesmium nif EN genes encode a fused Nif EN polypeptide that does not appear to be processed into individual Nif E and Nif N polypeptides. Fused nif EN genes were previously found in the A. variabilis nif 2 genes, but we have found that fused nif EN genes are widespread in the nonheterocystous cyanobacteria. Although the gene organization of the nonheterocystous filamentous Trichodesmium nif gene cluster is very similar to that of the A. variabilis vegetative nif 2 gene cluster, phylogenetic analysis of nif sequences do not support close relatedness of Trichodesmium and A. variabilis vegetative (nif 2) nitrogenase genes.

Nitrogen Fixation in the Marine Cyanobacterium Trichodesmium

Over the past decade, the marine cyanobacteria Trichodesmiumspp. have received increasing attention [1,2], because of their ability to fix nitrogen in the presence of oxygen, and the recognition of their importance in ocean N budgets [3,4]. Trichodesmiumspp. are distributed worldwide in tropical and subtropical seas [2]. Dugdale et al. [5] first demonstrated that nitrogen fixation occurred in association with Trichodesmiumaggregates in the sea, although it was many years before it became clear that the nitrogen fixation observed was indeed due to the cyanobacterium, and not associated heterotrophic bacteria. Evidence based on DNA sequences (nifH) amplified from natural populations, and immunological studies eventually confirmed that Trichodesmiumitself was the primary nitrogen fixer [6-8], even though other diazotrophs are sometimes found associated with Trichodesmiumaggregates [9,10].