HISTORIA DE LA COPIA PRIVADA EN ESPAÑA

Although N. spumigena is ecologically important in the Baltic Sea, where it contributes substantially to both primary production and the annual nitrogen input. Few studies have addressed the physiology and genetics of N2- fixation and heterocyst differentiation in this

cyanobacterium. To date, most studies have focused on environmental conditions promoting bloom formation in general, and toxin production in particular.

In general, ammonia and nitrate inhibit N2-fixation in cyanobacteria, with nitrate being

less effective and strain dependent [43,44,45,46]. This is because, as explained above, ammonia is readily incorporated into the GS/GOGAT pathway while nitrate needs to be first reduced to ammonia before entering the pathway. In the Baltic N. spumigena strain AV1, ammonia was found to inhibit N2- fixation activity [14,46,47], but not to sustain growth for a

long period of time [46,47]. Indeed, unlike “model” cyanobacteria, there are no reports showing clearly that Baltic strains of N. spumigena can grow for several generations by utilizing ammonia in the absence of N2-fixation activity [46]. In addition, it was also found

that nitrate does not exert any negative effect on N2-fixation activity [14,46,47]. Accordingly,

N. spumigena seems unable to utilize neither ammonia nor nitrate to support growth. Thus,

the Baltic cyanobacterium N. spumigena seems to be an “obligatory” N2-fixer, if we can use

such a term in this context. Expression analysis of nifH transcription has confirmed the results obtained through measuring growth and N2-fixation activity and showed that expression of

nifH responds negatively to ammonia supplementation and does not respond to nitrate

supplementation in N. spumigena stain AV1 [46,47]. In a mesocosm experiment conducted by Vuorio et al. [48], it was demonstrated that N. spumgiena enriched with dissolved inorganic nitrogen (DIN) did not show any response while Anabaena spp. grew better. In synthesis, it seems that the Baltic Sea N. spumigena is not as efficient in DIN uptake/assimilation as other “model” cyanobacteria. This partially explains the observation that blooms of N. spumigena in the Baltic Sea develop when nitrogen is limited and are more affected by abiotic factors, such as temperature, salinity and phosphorus level.

Investigations about heterocyst formation and frequency in Baltic Sea N. spumigena strain AV1 in the presence of ammonia has revealed that the filaments form heterocysts and maintain heterocyst frequency in the presence of ammonia and in the absence of any detectable N2-fixation activity [46,47]. Moreover, the expression patterns of ntcA, the key

regulator of nitrogen metabolism, and hetR, the master regulator of heterocyst differentiation, were not affected [46]. Thus, Baltic Sea N. spumigena strain AV1 exhibits what Vintila and El-Shehawy [46] called “uncoupling” between N2-fixation and heterocyst differentiation. As

far as we know, such a behavior was never reported in any of the previously studied heterocystous cyanobacteria.

A detailed proteomic analysis of a Baltic Sea N. spumigena strain AV1 during ammonia supplementation has revealed that the cells exhibit low energy metabolism and carbon fixation [49]. The authors suggested that the inefficiency of the Baltic Sea N. spumigena in utilizing ammonia as an external nitrogen source could be due to ammonia toxicity that causes damage to PSII [49]. In absence of a repair mechanism, energy production goes down, leading to lower carbon fixation and lower growth rate [49]. It was previously suggested that the toxicity of ammonia to cyanobacteria may be due to rapid photo-damage to PSII [50]. Proteomic analysis thus confirmed the results obtained from the transcriptional analysis, morphological observations, and activity measurements, and showed that N. spumigena is not

efficient in utilizing externally supplied ammonia or nitrate. Accordingly, Baltic Sea N.

spumigena does not replace the energy-intensive N2-fixation by assimilation of DIN and

continues to form heterocysts in the presence of DIN and in the absence of any detectable N2-

activity.

It is then important to investigate if such “uncoupling” behavior is characteristic to the species N. spumigena or only to the strains living in the Baltic Sea. Vintila and El-Shehawy [47] have analyzed the response of three non-Baltic Sea N. spumigena isolates (two strains were isolated in Australia and one strain was isolated in Canada) as well as five Baltic Sea isolates. Their results demonstrated that the non-Baltic Sea isolates exhibited a classical response to ammonia supplementation; cessation of N2-fixation activity and loss of

heterocysts, while the Baltic Sea isolates exhibited the “uncoupling” response [47].

There is a clear need to analyze more isolates before drawing a general conclusion. However, for now we can ask the question: what makes Baltic Sea N. spumigena peculiar? Is it the variations in its genetic background? Is it a genome reduction? Or is it impairment in the regulatory network at the level of nitrogen/carbon metabolism?

Analysis of hetR sequence of the Baltic Sea N. spumigena strain CCY9141 versus the sequence of the same gene in Anabaena variabilis, Anabaena sp. PCC 7120 and Nostoc

punctiforme revealed that there are three amino acids substituted in hetR of Nodularia versus

the other hetR sequences. Nevertheless, these amino acids were not shown to be among the ones that are required for hetR function in Anabaena sp. PCC 7120 [51,46]. Moreover, analysis of N. spumigena CCY9141 genome revealed the presence of amt, nir, nar and nrtP, which code for ammonium permease, nitrite reductase, nitrate reductase and nitrate/nitrite permease, respectively. Hence the peculiarity of N. spumigena is not likely to be due to variations in the genetic background of Baltic Sea N. spumigena in regards to hetR sequence or as a result of a genome reduction in N uptake/assimilation machinery.

During heterocyst development, DNA-rearrangements take place within the nif-operon. In vegetative cells of Anabaena sp. PCC 7120, Anabaena variabilis and Nostoc punctiforme, the nifD gene is interrupted by an excision element that varies in size depending on species [52, 53,54]. During late stages in heterocyst differentiation, site-specific recombination between repeated sequences at the ends of the excision element causes deletion of the element leading to the transcription of nifH. Vintila et al. [55] have found that, unlike “model” cyanobacteria, the Baltic Sea N. spumigena contains an insertion element in the nifH of the

nifHDK operon and an operating DNA rearrangement mechanism. They showed that this

insertion element is present only in the tested Baltic Sea strains that exhibit “uncoupling” between N2-fixation and heterocyst differentiation, while it is absent in the tested non-Baltic

Sea strains. Furthermore, they have analyzed NifH protein using Western Blot and 2-D electrophoresis coupled to Maldi TOF/TOF and demonstrated the unusual presence of three NifH protein bands.

Two of these bands seem to be a result of an unknown modification in response to the light/dark growth regime, while the third seems to be a membrane bound and does not respond to the light regime. The presence of two NifH protein forms that are modified have been previously reported in nitrogen fixing cyanobacteria [56,57,58], while membrane bound nitrogenase was not demonstrated. In fact some studies have demonstrated that nitrogenase is randomly distributed in heterocyst [59, 60], nevertheless, localization of nitrogenase close to the membranes that are filling up heterocyst cannot be excluded.

Altogether, the data gathered demonstrate that the regulatory circuit regulating N2-

fixation in the Baltic Sea N. spumigena seems, at least in some key features, different than what is known to operate in “model” cyanobacteria [5].