Nitrogenase activity and diazotrophic growth in <i>Anabaena</i> sp. PCC 7120 mutant strains.

<p>Nitrogenase activity was determined in ammonium-grown filaments incubated in the absence of combined nitrogen for 24 or 48 h and expressed in µmol ethylene produced (mg Chl)⁻¹ h⁻¹. Data are the mean and standard deviation of the mean (number of independent experiments indicated in parenthesis). Diazotrophic growth was tested in solid BG11₀ medium.</p

Heterocyst distribution and <i>asr2819</i> expression levels in <i>Anabaena</i> sp. strains PCC 7120 and CSL97.

<p>(A) Heterocyst distribution in the indicated strains grown in bubbled cultures with ammonium and incubated for the indicated times in the absence of combined nitrogen under culture conditions (see legend to Fig. 2 for details). (B) Ratios of the expression levels of <i>asr2819</i> in the indicated strains at the indicated times after N step-down, measured by qRT-PCR normalizing with the <i>rnpB</i> gene. Data are the mean of two to three independent experiments.</p

Plasmids used in this work.

<p>Plasmids used in this work.</p

Oligodeoxynucleotide primers used in this work.

<p>Oligodeoxynucleotide primers used in this work.</p

Microscopy, lipids and <i>nifHDK</i> expression in <i>Anabaena</i> mutant strains.

<p>Filaments from bubbled, ammonium-supplemented cultures of the indicated strains were washed three times with BG11₀ medium, resuspended in BG11₀ medium and incubated under the same culture conditions for the times indicated in h. (A) Samples taken at 24 h were stained with Alcian Blue prior to being photographed under a light microscope. (B) Lipids (GI and GIII are heterocyst envelope glycolipids) were isolated and separated by TLC. (C) Total RNA was isolated and used in northern blot analysis with a probe of the <i>nifH</i> gene or, as a loading and transfer control, the <i>rnpB</i> gene. A size standard is indicated at the left and the observed transcripts at the right.</p

Localization of HetC-GFP.

<p>(A) Scheme of the genomic <i>hetC</i> region of strains CSM1 (expressing a HetC-GFP-mut2 fusion protein) and CSL33 (expressing a HetC-p-GFP-mut2 fusion protein). The pCSV3 vector portion integrated in the <i>hetC</i> locus is represented as a thin line. (B) Scheme of the putative domains of the HetC protein. (C) Confocal microscopy of filaments of strains CSM1 and CSL33 grown in bubbled, ammonium-supplemented medium and incubated for 24 h in medium containing no combined nitrogen. Cyanobacterial autofluorescence (red) is shown in the right-hand images, and merged autofluorescence and GFP fluorescence (green) in the left-hand images. Heterocysts (indicated with white arrows) are identified by their greatly diminished autofluorescence.</p

Heterocyst distribution in <i>Anabaena</i> mutant strains.

<p>Filaments from bubbled, ammonium-supplemented cultures of the indicated strains were washed three times with BG11₀ medium, resuspended in BG11₀ and incubated under the same culture conditions for 24, 48 or 72 h (as indicated). Cells were counted after staining with Alcian Blue. Data are the mean and standard deviation of the mean of two to six independent experiments (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104571#s2" target="_blank">Materials and Methods</a>).</p

Spatial pattern of heterocysts in <i>Anabaena</i> sp. PCC 7120 mutant strains.

<p>Filaments were grown with ammonium and incubated for the indicated times in the absence of combined nitrogen. Heterocyst frequency (as percentage of total cells), the mean size of vegetative cell intervals between heterocysts, and the percentage of contiguous heterocysts (interval size  = 0, percentage of total intervals) were calculated (for the strains included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104571#pone-0104571-g002" target="_blank">Figs. 2</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104571#pone-0104571-g006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104571#pone-0104571-g007" target="_blank">7</a> values are from the data in the figures).</p

Localization of HetP-GFP.

<p>(A) Scheme of the <i>hetP</i> genomic region of strains CSL67 (HetP-sf-GFP fusion protein) and CSL107 (HetP-GFP-mut2 fusion protein). The pCSV3 vector portion integrated in the <i>hetP</i> locus is represented as a thin line. (B) Scheme of the HetP protein showing a predicted transmembrane segment (TMS). (C) Confocal microscopy of filaments of strain CSL67 grown in BG11₀ solid medium (upper part) or deconvoluted fluorescence microscopy image of filaments of strain CSL67 grown in bubbled ammonium-supplemented medium and incubated for 40 h in medium containing no combined nitrogen (lower part). (D) Fluorescence microscopy of filaments of strain CSL107 grown in BG11₀ solid medium. Merged images of autofluorescence and GFP fluorescence are shown at the left side, and of bright field and GFP fluorescence at the right side. Heterocysts and some proheterocysts are indicated with white arrows.</p

Heterocyst distribution and <i>hetP</i> expression levels in <i>Anabaena</i> mutant strains altered in <i>hetP</i>.

<p>(A) Heterocyst distribution in the indicated strains grown in bubbled cultures with ammonium and incubated for the indicated times in the absence of combined nitrogen under culture conditions (see legend to Fig. 2 for details). (B) Ratios of the expression levels of <i>hetP</i> of the indicated strains 18 h after N step-down, measured by qRT-PCR normalized to the <i>rnpB</i> gene. S.E. range indicates the “standard error change” and <i>P</i> (the hypothesis test P) represents the probability that the difference between the sample and control groups is due only to chance <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0104571#pone.0104571-Pfaffl1" target="_blank">[39]</a>. Data are the mean of two independent experiments.</p