Thesis Abstract Marco Dignum 2003

Phosphate uptake proteins as markers for the nutrient status of freshwater cyanobacteria

The persistence of algal blooms in lakes depends on the nutrient status and light climate in the water. Phosphorus (P), mainly incorporated in the form of dissolved inorganic orthophosphate (Pi), is a key element for the growth of freshwater algae (phytoplankton). Measures to reduce the external P-load in heavily eutrophied lakes have brought Pi-concentrations below the detection limits of current analytical methods. The often-encountered dominance of cyanobacteria in such lakes is due to their excellent adaptation capabilities, allowing them to thrive at extremely low Pi concentrations and light intensities. Efficiency of P-incorporation at low concentrations depends on three processes: conversion of organic P-esters (Po) outside the cells (scavenging strategy), Pi-transport through the cell envelope (affinity strategy), and saving of surplus P (polyphosphate) inside the cells (storage strategy). Because the nutrient status cannot be inferred from Pi-measurements, water quality research will benefit from the recently renewed interest in the use of biochemical characteristics of the algae as indicator for their physiological status. In addition to determinations of the species composition, a desired 'toolbox' of nutrient status indicators contributes to improved understanding of the state of a lake. A large part of the work in this thesis has been devoted to the design of methods for the marking of cells.

The model cyanobacterium Synechocystis sp. PCC 6803 shows successive responses to diminishing availability of Pi, consisting of specific adaptations (variation of protein synthesis) as well as general stress responses (attenuation of photosynthetic activity and growth rate). The genome of Synechocystis encodes tandem high-affinity Pi-uptake systems (Pst), one of which is specifically expressed in Pi-deficient conditions. This Pst-system contains a periplasmic Pi-binding protein, and is co-regulated with alkaline phosphatase (AP); two pore-forming proteins in the outer membrane (porins) are also under control of Pi-availability. Each of these proteins is a candidate to serve as marker protein for diagnosing Pi-deficiency in algae; Pi-binding protein PstS is widespread among cyanobacteria; the porins SomA and SomB have surface exposed loops that may serve as antigenic markers; the activity of AP enzyme PhoA can be easily made visible. In spite of its shortcomings, AP activity was proven to be a useful indicator for Pi-deficiency in algae. The ELF-97 phosphatase substrate yields highly fluorescent precipitates at the site of enzymatic activity (AP induced fluorescence). This allows recognition of the nutrient status of individual cells in a flow cytometer.

In a case-study performed in Lake Loosdrecht, The Netherlands, we have applied the ELF-method in conjunction with separation of the present phytoplankton groups on the basis of their endogenous fluorescent pigments. The nutrient status of the cells could be related to their actual growth rate, determined from the relative incorporation of natural carbon-isotopes in the membrane fatty acids.