Table 1.
Oligodeoxynucleotide primers used in this work.
Table 2.
Plasmids used in this work.
Figure 1.
Schematics of the hetC, hetN and patS genomic regions in Anabaena sp. strain PCC 7120 and mutant derivatives.
The gene map is from [38]. The Anabaena genes are represented with grey arrows, the deleted portions (of the specified sizes) with dashed segments, and C.S3 gene-cassette insertion with a white bar. The names of the resulting mutant strains in the wild-type genetic background are indicated.
Figure 2.
Heterocyst distribution in Anabaena mutant strains.
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 Materials and Methods).
Figure 3.
Microscopy, lipids and nifHDK expression in Anabaena mutant strains.
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 nifH gene or, as a loading and transfer control, the rnpB gene. A size standard is indicated at the left and the observed transcripts at the right.
Table 3.
Spatial pattern of heterocysts in Anabaena sp. PCC 7120 mutant strains.
Figure 4.
(A) Scheme of the genomic hetC 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 hetC 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.
Table 4.
Nitrogenase activity and diazotrophic growth in Anabaena sp. PCC 7120 mutant strains.
Figure 5.
(A) Scheme of the hetP genomic region of strains CSL67 (HetP-sf-GFP fusion protein) and CSL107 (HetP-GFP-mut2 fusion protein). The pCSV3 vector portion integrated in the hetP 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.
Figure 6.
Heterocyst distribution and hetP expression levels in Anabaena mutant strains altered in hetP.
(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 hetP of the indicated strains 18 h after N step-down, measured by qRT-PCR normalized to the rnpB gene. S.E. range indicates the “standard error change” and P (the hypothesis test P) represents the probability that the difference between the sample and control groups is due only to chance [39]. Data are the mean of two independent experiments.
Figure 7.
Heterocyst distribution and asr2819 expression levels in Anabaena sp. strains PCC 7120 and CSL97.
(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 asr2819 in the indicated strains at the indicated times after N step-down, measured by qRT-PCR normalizing with the rnpB gene. Data are the mean of two to three independent experiments.