Ecological Aspects of ntcA Gene Expression and Its Use as an Indicator of the Nitrogen Status of Marine Synechococcus spp

Abstract

Nitrogen nutrition in cyanobacteria is regulated by NtcA, a transcriptional activator that is subject to negative control by ammonium. Using Synechococcus sp. strain WH7803 as a model organism, we show that ntcA expression was induced when cells were exposed to nitrogen stress but not when they were subjected to phosphorus or iron deprivation. Transcript levels accumulated in cells grown on a variety of inorganic and organic nitrogen sources, with the sole exception of ammonium. ntcA transcription was induced when ammonium levels dropped below 1 M and reached maximal levels within 2 h. Furthermore, the addition of more than 1 M ammonium led to a rapid decline in ntcA mRNA. The negative effect of ammonium was prevented by the addition of Lmethionine-D,L-sulfoximine (MSX) and azaserine, inhibitors of ammonium assimilation. Thus, basal ntcA transcript levels are indicative of ammonium utilization. Conversely, the highest ntcA transcript levels were found in cells lacking a nitrogen source capable of supporting growth. Therefore, maximal ntcA expression would indicate nitrogen deprivation. This state of nitrogen deprivation was induced by a 1-h incubation with MSX. The rapid response of ntcA gene expression to the addition of ammonium and MSX was used to design a protocol for assessing relative ntcA transcript levels in field populations of cyanobacteria, from which their nitrogen status can be inferred. ntcA was basally expressed in Synechococcus at a nutrient-enriched site at the northern tip of the Gulf of Aqaba, Red Sea. Therefore, these cyanobacteria were not nitrogen stressed, and their nitrogen requirements were met by regenerated nitrogen in the form of ammonium.

Decoupling of ammonium regulation and ntcA transcription in the diazotrophic marine cyanobacterium Trichodesmium sp. IMS101

“…The use of the glutamine synthetase inhibitor MSX indicated that metabolized (as opposed to ambient) ammonium suppressed the transcription of N genes in the Trichodesmium filaments, similar to our findings in marine Synechococcus (Lindell and Post, 2001;Lindell et al, 2005). This is consistent with the concept that a-oxoglutarate accumulates and acts as the NtcA activator molecule when ammonium supplies are limiting Tanigawa et al, 2002).…”

“…Uptake rates of ammonium often exceed rates of N 2 fixation (Mulholland et al, 2001). Although both organisms thrive in N-depleted environments, N assimilation is thus not as stringently controlled in Trichodesmium as was found in marine Synechococcus (Lindell and Post 2001;Lindell et al, 1998Lindell et al, , 2005. This led us to hypothesize that NtcA control over N assimilation may be principally different in the non-heterocystous filaments of Trichodesmium spp.…”

“…Surprisingly, ammonium did not repress the transcription of the N-regulatory gene ntcA, and its mRNA accumulated to high levels, even in the presence of 2-2000 mM ammonium. In Synechococcus WH7803, ntcA transcript levels dropped below detection when ammonium concentrations exceed 1 mM (Lindell and Post, 2001;Lindell et al, 2005).…”

“…Ammonium addition immediately downregulated nitrate assimilation and N 2 fixation. In the non-N 2 -fixing Synechococcus WH7803, ntcA transcription is below detection when ammonium concentrations exceed 1 mM, and transcript levels are upregulated in nitrate-grown cells and become maximal under N-deplete conditions (Lindell and Post 2001;Lindell et al, 1998Lindell et al, , 2005. It has been reported that ambient ammonium accumulates to B2 mM in N 2 -fixing ( ¼ N-deplete) cultures of Trichodesmium IMS101 (Mulholland et al, 2001), thus raising questions about the mechanisms of ammonium control over N assimilation and N 2 fixation in this organism.…”

“…Its gene product, NtcA, is a 24 kD DNA-binding protein that belongs to the cAMP receptor protein (CRP) family of transcriptional activators (VegaPalas et al, 1992;Flores and Herrero, 1994). Freshwater and marine unicellular, non-N 2 -fixing cyanobacteria show a clear, direct relationship between ntcA expression and low ammonium levels (Frias et al, 1994;Lee et al, 1999;Sauer et al, 1999;Lindell and Post, 2001;Lindell et al, 2005) Marine Synechococcus WH7803 expresses ntcA at basic levels when grown on ammonium. These levels increase to intermediate when exposed to alternative N sources and maximum transcript levels are attained under N starvation (Lindell and Post, 2001;Lindell et al, 2005).…”