An immunological approach to detect phosphate stress in populations and single cells of photosynthetic picoplankton

Abstract

In the marine cyanobacterium Synechococcus sp. strain WH7803, PstS is a 32-kDa cell wall-associated phosphate-binding protein specifically synthesized under conditions of restricted inorganic phosphate (P i) availability (D. J. Scanlan, N. H. Mann, and N. G. Carr, Mol. Microbiol. 10:181-191, 1993). We have assessed its use as a potential diagnostic marker for the P status of photosynthetic picoplankton. Expression of PstS in Synechococcus sp. strain WH7803 was observed when the P i concentration fell below 50 nM, demonstrating that the protein is induced at concentrations of P i typical of oligotrophic conditions. PstS expression could be specifically detected by use of standard Western blotting (immunoblotting) techniques in natural mesocosm samples under conditions in which the N/P ratio was artificially manipulated to force P depletion. In addition, we have developed an immunofluorescence assay that can detect PstS expression in single Synechococcus cells both in laboratory cultures and natural samples. We show that antibodies raised against PstS cross-react with P-depleted Prochlorococcus cells, extending the use of these antibodies to both major groups of prokaryotic photosynthetic picoplankton. Furthermore, DNA sequencing of a Prochlorococcus pstS homolog demonstrated high amino acid sequence identity (77%) with the marine Synechococcus sp. strain WH7803 protein, including those residues in Escherichia coli PstS known to be directly involved in phosphate binding.

Molecular markers define progressing stages of phosphorus limitation in the nitrogen‐fixing cyanobacterium, Crocosphaera

“…The variable background levels of pstS in C. watsonii make it difficult to identify whether this organism is responding to phosphorus stress from pstS expression alone; pstS transcripts may be detected in cells that are not under phosphorus stress, depending on the time of day. Since the detection of pstS is a common tool for predicting phosphorus stress in natural populations of picocyanobacteria (Scanlan et al 1997, Fuller et al 2005, Hung et al 2013, this finding has implications for the use of pstS as an environmental biomarker for Crocosphaera. When working with natural populations, late dark phase (D7-D11) is the ideal time to sample for pstS, since diel expression is lowest at that time (Fig.…”

Molecular ecology of the marine cyanobacterial genera Prochlorococcus and Synechococcus

“…The ability to routinely isolate and maintain in culture marine bacteria is relatively rare and so the relative ease with which both Synechococcus and Prochlorococcus strains can be isolated has not only allowed development of phylogenetic probes but also those molecular markers where precise physiological information was needed (e.g. see [9]). We describe in detail now the recent progress that has been made elucidating marine cyanobacterial diversity taking culture studies and natural populations in turn.…”

“…Thus, the axenic HLI Prochlorococcus PCC9511 is capable of growth on Na 2 L-glycerophosphate, Na 4 -pyrophosphate, glucose-6phosphate and ATP in addition to inorganic phosphate (Pi) [36], whilst the marine Synechococcus strain WH7803 has been shown to utilise Pi, dCTP, p-nitrophenyl phosphate, glucose-6-phosphate and glycerol phosphate as a sole P source [9]. Interestingly, the ability to utilise cAMP as sole P source may be a clade-speci¢c trait amongst marine Synechococcus strains ( [9] ; L. Moore, personal communication), and whilst the one axenic Prochlorococcus isolate that has been investigated can utilise this substrate (L. Moore, personal communication) it will require further work to assess whether this re£ects the genera as a whole. The ability to utilise nucleotides as a P source suggests hydrolysis by alkaline phosphatase or the presence of a speci¢c 5P-nucleotidase, but certainly utilisation of cAMP would suggest the presence of a speci¢c high a⁄nity transport system for this substrate.…”

“…They allow an indication of the factors that can constrain productivity, factors which in turn may a¡ect community structure and species succession. With respect to Synechococcus and Prochlorococcus and the macronutrient P, markers which are useful have been based on responses that either induce high a⁄nity uptake systems for the preferred source of the nutrient [9,44] or utilise alternative sources of the nutrient, but which may be energetically more costly to acquire [45]. Alternatively, markers may target a component of the regulatory system itself.…”

“…In combination with P status markers this will allow dual nutrient status information to be obtained from the same organisms. In addition, the fact that for one of these P status markers, PstS, encoding a high-a⁄nity P binding protein, a single cell im-muno£uorescence assay has been developed [9], whilst ntcA gene expression uses ntcA primers of di¡erent taxonomic speci¢city, allows assessment of N and P status at various taxonomic levels. Given the likely importance of clade-speci¢c di¡erences in nutrient physiology, which we have already seen with respect to utilisation of speci¢c N and P substrates, such speci¢city is clearly important, though interpretation of this molecular data will require a clear understanding of the regulation of nutrient physiology across the genera.…”