Downloaded from www.microbiologyresearch.org by IP: 23.22.50.124 On: Sat, 14 May 2016 14:18:46 A calcium signal is involved in heterocyst differentiation in the cyanobacterium Anabaena sp. PCC7120 I. Torrecilla, F. Legane ´ s, I. Bonilla and F. Ferna ´ ndez-Pin ˜ as Correspondence F. Ferna ´ ndez-Pin ˜ as francisca.pina@uam.es Departamento de Biologı ´a, Facultad de Ciencias, Universidad Auto ´ noma de Madrid, Madrid 28049, Spain Received 15 June 2004 Revised 22 July 2004 Accepted 3 August 2004 The impact of calcium signals in virtually all cells has led to the study of their role in prokaryotic organisms as stress response modulators. Cell differentiation in adverse conditions is a common Ca 2+ -requiring response. Nitrogen starvation induces the differentiation of N 2 -fixing heterocysts in the filamentous cyanobacterium Anabaena sp. PCC7120. This paper reports the use of a recombinant strain of this organism expressing the photoprotein aequorin to monitor the intracellular free-calcium concentration during the course of heterocyst differentiation. A specific calcium signature that is triggered exclusively when cells are deprived of combined nitrogen and generated by intracellular calcium stores was identified. The intracellular calcium signal was manipulated by treatment with specific calcium drugs, and the effect of such manipulation on the process of heterocyst differentiation was subsequently assessed. Suppression, magnification or poor regulation of this signal prevented the process of heterocyst differentiation, thereby suggesting that a calcium signal with a defined set of kinetic parameters may be required for differentiation. A hetR mutant of Anabaena sp. PCC7120 that cannot differentiate into heterocysts retains, however, the capacity to generate the calcium transient in response to nitrogen deprivation, strongly suggesting that Ca 2+ may be involved in a very early step of the differentiation process. INTRODUCTION The prospect of Ca 2+ having an important role as an intracellular second messenger in prokaryotic, as well as in eukaryotic, organisms is currently supported by a sub- stantial amount of literature. Since Ca 2+ was first studied in bacteria fifty years ago (Norris & Jensen, 1957) it has been implicated in a broad range of physiological pro- cesses in prokaryotes that include chemotaxis and motility (Tisa & Adler, 1992, 1995; Watkins et al., 1995; Pitta et al., 1997), pathogenesis (Rose et al., 1993; Straley et al., 1993), the cell cycle and the control of the initiation of replica- tion (Jime ´nez-Sanchez et al., 1993; Yu & Margolin, 1997), quorum sensing (Wherten & Lundgren, 2001) and spore and fruiting body formation (Inouye et al., 1983; O’Hara & Hageman, 1990). In many cases, the involvement of Ca 2+ in the regulation of cellular processes has been roughly described in terms of an influx or efflux of Ca 2+ from the cytosol. The application in bacteria of the calcium detec- tion method based on the calcium-binding luminescent photoprotein aequorin (Knight et al., 1991) has allowed the monitoring of intracellular free-calcium concentration ([Ca 2+ ] i ) in such a way that fluxes of free calcium in prokaryotic cells can be precisely portrayed. In this regard, our group has been the first to report direct evidence of Ca 2+ signalling in cyanobacteria (Torrecilla et al., 2000, 2001, 2004). Cyanobacteria are a large group of photosynthetic, oxygen- evolving prokaryotes. Many of them are also capable of fixing atmospheric N 2 , a process that requires nitrogenase to be protected from oxygen. The filamentous cyano- bacterium Anabaena sp. PCC7120 differentiates specialized cells called heterocysts that create a microoxic environment for nitrogen fixation; heterocysts normally form at semi- regular intervals along the filaments, following a develop- mental pattern, as a response to nitrogen deprivation. Heterocyst differentiation follows a specific scheme and requires global changes in gene expression (reviewed by Wolk et al., 1994; Wolk, 1996, 2000, Golden & Yoon, 1998, 2003) but an overall model of the regulatory networks controlling development remains elusive. In the regula- tion of early events in the process of differentiation, the expression patterns of several genes have been studied and a few have been placed into an ordered sequence; among Abbreviations: BAPTA-AM, 1,2-bis(2-aminophenoxy)ethane-N,N,N9,N9- tetraacetic acid tetrakis(acetoxymethyl ester); [Ca 2+ ] i , intracellular free- Ca 2+ concentration; EGTA, ethylene glycol-bis(2-aminoethylether)- N,N,N9,N9-tetraacetic acid; TFP, trifluoperazine. 0002-7403 G 2004 SGM Printed in Great Britain 3731 Microbiology (2004), 150, 3731–3739 DOI 10.1099/mic.0.27403-0