NITRATE TRANSPORTER GENES FROM THE DIATOM CYLINDROTHECA FUSIFORMIS (BACILLARIOPHYCEAE): mRNA LEVELS CONTROLLED BY NITROGEN SOURCE AND BY THE CELL CYCLE

Abstract

The molecular characterization of components involved in nitrate uptake and assimilation in phytoplankton is likely to provide new insights into these processes, their regulation, and their effect on primary production. We report the cloning and initial characterization of the first nitrate transporter genes in a marine organism, from the diatom Cylindrotheca fusiformis Reimann et Lewin. A clone isolated from a silicon-responsive cDNA library was shown by sequence comparison to encode a homolog of high-affinity nitrate transporters. The C. fusiformis nitrate transporter cDNA was named NAT (NitrAte Transporter). The NAT cDNA was used to isolate a genomic clone that contained two additional nitrate transporter genes, NAT1 and NAT2, arranged in tandem. The cDNA and two genomic sequences were highly conserved, and only 18 of 1446 nucleotides in the coding region differed. At least four copies of NAT genes were present in C. fusiformis and as shown by hybridization, multiple copies were present in other diatom species. The transcript abundance of NAT genes in cultures with different nitrogen sources was monitored by RNase protection assays. NAT mRNA levels were high in the presence of nitrate, at nearly the same level during nitrogen starvation, and also high in urea-containing cultures. Lower mRNA levels occurred in nitrite-grown cultures. NAT transcript levels were highly repressed with NH Cl or NH NO as the nitrogen source, although very low amounts were detected. These results suggested that monitoring NAT mRNA levels could serve as a marker for (1) nitrate uptake in nitrate medium, (2) nitrogen starvation, and (3) ammonium use by virtue of absence of expression. NAT mRNA levels were not directly regulated by light or dark, but were apparently related to cellular growth and protein synthesis. Using light/dark synchronized cultures to monitor cell cycle responses, NAT mRNA levels were high in early G phase, decreased through the remainder of G , then increased during DNA synthesis in S phase and into G , and finally decreased after M phase. In silicon-starvation synchronized cultures, levels were high at the G /S phase boundary, high throughout S and G , and finally decreased after M phase. It was clear that NAT expression, and by inference nitrate uptake, did not occur at continuous levels throughout the cell cycle. The results of the RNase protection experiments suggested that transcriptional regulation is a major contributing factor in the control of diatom nitrate uptake. The cloning of the C. fusiformis nitrate transporter genes provides a new tool for investigating diatom nitrogen uptake and metabolism. In addition, the regulation of NAT expression by nitrogen source is likely to be useful in developing techniques to specifically control the expression of genes fused to NAT regulatory sequences in transgenic diatoms.

Dynamic model of flexible phytoplankton nutrient uptake

“…A crucial factor in this regulation is the cell's own internal nutrient content (9,28,29). For instance, the internal levels of nutrients such as nitrate are the main inducers of genes that encode membrane transporters and enzymes involved in assimilation pathways (4,6,30). Concretely, repression of regulation must affect transport capabilities, minimizing synthesis when the cell contains a sufficient amount of the incoming nutrient element (29); on the other hand, when nutrient is low, the cell activates the synthesis of sites (5,9,31) to maximize the probability of occurrence of an encounter with the scarce amount of nutrient ions arriving at the cell surface.…”

Role of urea in microbial metabolism in aquatic systems: a biochemical and molecular review

“…Previous investigators have noted that some diatoms cannot use urea as a source of N for growth (Neilson & Larsson 1980). More recently, Hildebrand & Dahlin (2000) reported complete growth arrest of the diatom Cylindrotheca fusiformis when its sole N source was switched from NO 3 -to urea and its NO 3 -transporter was concomitantly up-regulated to the same level as in N-starved cultures. Some phytoplankton may not grow on urea, because they do not possess enzymes essential for urea uptake and/or catabolism.…”

“…To better understand how urea uptake and urease activity are regulated by N availability and other environmental factors, work on regulation similar to that done on NO 3 -transporters and NO 3 -reductase (Lomas & Glibert 1999, Hildebrand & Dahlin 2000, Parker & Armbrust 2005) is needed for urea transport, urease, UALase, and urea cycle enzymes. The abundance of transcripts encoding urea transport and urease and/or UALase enzymes may be regulated by global N regulators (such as NtcA, AmtR) in bacteria in response to N availability (NO 3 -, NH 4 + , glutamine, and amino acids) in many bacteria and eukaryotic phytoplankton.…”

The place of diatoms in the biofuels industry

“…The individual parts are relatively easily disrupted, and organic solvents readily extract lipid and pigments [137,138]. The diatom cell wall is actually easier to break open than the wall of some green algae; for example, RNA isolation procedures for diatoms involve simply vortexing cells in the presence of Tri Reagent [139], whereas for some green algae frozen cells have to be ground in a mortar and pestle prior to extraction. Furthermore, pigments are easily extracted from diatoms and blue-green algae using 90% acetone, whereas harsher solvents are required for the extraction of pigments from coccoid green algae [140].…”