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1 Cornell University, Ithaca, NY
2 Woods Hole Oceanographic Institution, Woods Hole, MA
* Corresponding author: sdyhrman{at}whoi.edu
Cyanobacteria of the genus Trichodesmium are abundant in tropical and subtropical regions and significantly contribute to carbon and nitrogen fixation in these environments (1). Recent studies suggest that phosphorus (P) supply may influence carbon and nitrogen fixation by Trichodesmium in the Atlantic (2, 3). However, evidence of phosphorus deficiency in field populations differs among Trichodesmium species (2, 3), suggesting that the individual species may have unique scavenging mechanisms. Here we examine several genes involved in phosphorus physiology within four species, T. erythraeum, T. tenue, T. thiebautii, and the related species Katagnymene spiralis.
Three genes thought to be related to phosphorus physiology were examined in this study: phoA and two copies of pstS, designated pstS1 and pstS2. These genes are common components of P-regulated scavenging mechanisms in other cyanobacteria and heterotrophic bacteria (4, 5). The phoA gene codes for a predicted alkaline phosphatase, an enzyme that hydrolyzes inorganic phosphorus from organic phosphorus, which can then be used by the organism for growth. The activity of this enzyme has been used as an indicator of phosphorus stress in field populations (2). PstS is a high-affinity binding protein for inorganic phosphorus and is part of a phosphate-induced high-affinity scavenging system.
The sequenced genome of T. erythraeum (http://genome.jgi-psf.org/draft_microbes/trier/trier.home.html) was used to identify phoA and two copies of pstS (pstS1 and pstS2) on the basis of their similarity to characterized genes in GenBank as part of the ongoing Trichodesmium annotation effort (unpubl. data). Multiple primer pairs were designed to amplify internal fragments (referred to as internal) or the complete phoA, pstS1, or pstS2 gene (referred to as external) (Table 1). DNA was extracted following the method described by Orcutt et al. (6). PCR amplification conditions were performed with PfuTurbo DNA polymerase (Stratagene, La Jolla, CA) using conditions optimized for each gene and species (Table 1). To optimize amplification, the annealing temperature was varied for each gene and species over a range of at least 25 °C. Other variables included magnesium and enzyme concentration.
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, in order to identify the activity associated with the putative gene. However, activity and expression work is still ongoing.
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Preliminary sequencing data for the genes and gene fragments indicate that pstS1, pstS2, and phoA in T. tenue as well as pstS2 in Kategnymene are approximately 98% identical to the corresponding genes in T. erythraeum. Given the morphological distinction of the Trichodesmium species, this high degree of similarity was surprising. However, coverage of the entire gene in each species may reveal regions within the genes that are less highly conserved. This hypothesis is consistent with the results for the only other functional gene to be sequenced from multiple Trichodesmium species, hetR, which shows a lower degree of similarity, 91%–95% (7).
Despite extensive efforts to amplify all the genes by varying annealing temperature, primers, and concentrations of magnesium, enzyme, and template, we were unable to amplify pstS2 or phoA from T. thiebautii. This is particularly interesting because T. thiebautii has been shown to have alkaline phosphatase activity (2), and because of the degree of similarity of phoA among the other species. Additionally, T. thiebautii’s evolutionary relationship, inferred from Trichodesmium ITS sequences (6), indicates T. tenue and K. spiralis are more closely related to each other than to T. thiebautii. It may be that phoA in T. thiebautii is very different compared to the other species or that a different gene and gene product are used in the hydrolysis of dissolved organic phosphorus for this species.
Future work is needed to determine whether pstS2 and phoA are present in T. thiebautii. Although other species and isolates of Trichodesmium should be analyzed, our initial identification of putative P-regulated genes in these Trichodesmium species has improved our knowledge of phosphorus scavenging mechanisms in this important genus.
This work was funded by NSF grant OCE-0220945, and by NSF Research Experience for Undergraduates grant OCE-0097498.
Literature Cited
This article has been cited by other articles:
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  S. T. Dyhrman and S. T. Haley Phosphorus Scavenging in the Unicellular Marine Diazotroph Crocosphaera watsonii Appl. Envir. Microbiol., February 1, 2006; 72(2): 1452 - 1458. [Abstract] [Full Text] [PDF]
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