A major facilitator superfamily protein, HepP, is involved in formation of the heterocyst envelope polysaccharide in the cyanobacterium Anabaena sp. strain PCC 7120

Abstract

Some filamentous cyanobacteria such as Anabaena sp. strain PCC 7120 produce cells, termed heterocysts, specialized in nitrogen fixation. Heterocysts bear a thick envelope containing an inner layer of glycolipids and an outer layer of polysaccharide that restrict the diffusion of air (including O(2)) into the heterocyst. Anabaena sp. mutants impaired in production of either of those layers show a Fox(-) phenotype (requiring fixed nitrogen for growth under oxic conditions). We have characterized a set of transposon-induced Fox(-) mutants in which transposon Tn5-1063 was inserted into the Anabaena sp. chromosome open reading frame all1711 which encodes a predicted membrane protein that belongs to the major facilitator superfamily (MFS). These mutants showed higher nitrogenase activities under anoxic than under oxic conditions and altered sucrose uptake. Electron microscopy and alcian blue staining showed a lack of the heterocyst envelope polysaccharide (Hep) layer. Northern blot and primer extension analyses showed that, in a manner dependent on the nitrogen-control transcription factor NtcA, all1711 was strongly induced after nitrogen step-down. Confocal microscopy of an Anabaena sp. strain producing an All1711-green fluorescent protein (All1711-GFP) fusion protein showed induction in all cells of the filament but at higher levels in differentiating heterocysts. All1711-GFP was located in the periphery of the cells, consistent with All1711 being a cytoplasmic membrane protein. Expression of all1711 from the P(glnA) promoter in a multicopy plasmid led to production of a presumptive exopolysaccharide by vegetative cells. These results suggest that All1711, which we denote HepP, is involved in transport of glycoside(s), with a specific physiological role in production of Hep.

Fig 1

Mutants of all1711. (A) Schematic of the all1711 locus in strain PCC 7120, with indication of the insertion sites and orientation of transposon Tn5-1063 in mutant strains FQ163, FQ296, and FQ880. The locations of the primers used to generate the probe for Northern blot analysis (Fig. 3 and 8) are indicated. (B and C) Electron micrograph of a defective heterocyst (Het) and its adjacent vegetative cell (VC) in mutant strain FQ880 (B) and further magnification of the area shown by a rectangle in panel B (C).

Fig 2

Wild-type Anabaena sp. strain PCC 7120 (two samples of which are shown), all1711 mutants, and complemented mutant strains. Filaments from bubbled BG11C cultures of the indicated strains (see genotypes in Table 1) were incubated in BG11₀C medium for 24 h. Samples from these cultures were then supplemented with a solution of 1% alcian blue 8GX (Sigma) and visualized by light microscopy. The heterocyst envelope polysaccharide (Hep) layer of the wild type and of the complemented mutants is stained blue. Some examples of heterocysts or, in the case of the mutants, presumptive heterocysts are indicated by arrowheads. Bar, 25 μm.

Fig 3

Expression of all1711. (A) Northern blot analysis performed with the all1711 probe indicated in Fig. 1A and RNA from strains PCC 7120 (wild type), CSE2 (ntcA), and 216 (hetR). RNA was isolated from bubbled ammonium-grown cultures (with Sm and Sp for the CSE2 mutant) incubated for the indicated number of hours without combined nitrogen (and without antibiotics). The different images correspond to different parts of the same filter and therefore can be compared. The size of the identified all1711 transcript is indicated. Hybridization with a probe of the Anabaena rnpB gene was used as a loading and transfer control (lower panels). (B) Primer extension analysis performed with oligodeoxynucleotide primer all1711-9 and the RNA samples from the induction times (indicated in hours) of the same preparations used for the Northern blot analysis (see panel A). The sequencing ladder shown at right corresponds to the all0862 gene promoter (37). (C) Sequence of the all1711 promoter region, with indication of primer all1711-9 used for the analysis shown in panel B, the detected RNA 5′ ends (nucleotides enclosed in ovals), two putative −10 promoter hexamers (shaded boxes), and two putative NtcA binding sites identified by the underlined triplets (GAA/TAC and GCA/TGC, respectively).

Fig 4

Expression of an All1711-GFP fusion protein. (A) Schematic of the all1711 locus in strain CSRL22 carrying the all1711-gfp-mut2 fusion. Note that the fusion gene was incorporated into the chromosome through a single-recombination event between the Anabaena sp. DNA cloned in plasmid pCSRL88 and the corresponding chromosomal region; therefore, no wild-type copy of all1711 remained. The locations of the primers used to study the genetic structure of CSRL22 (see Materials and Methods) are shown. The wavy line denotes the junction of linearized pCSRL88 with the 3′ end of all1711 that was interrupted by the insertion of pCSRL88. (B) GFP fluorescence (left panels) and bright-field (right panels) micrographs of filaments of strain CSRL22 grown with ammonium and incubated without combined nitrogen (in the presence of Sm and Sp for the mutant) for the times indicated (in hours). Arrowheads point to proheterocysts (at 9 and 12 h) or heterocysts (at 24 h). Bar, 10 μm. Identical microscope settings were used for the different time points.

Fig 5

GFP fluorescence along examples of filaments of strain CSRL22 incubated without combined nitrogen for 12 h (A) or 24 h (B). The lower panels present the quantification of the fluorescence from each whole cell along the filaments shown in the upper panels. Relative levels of GFP fluorescence are presented in arbitrary units that are comparable for panels A and B. Arrowheads point to a proheterocyst (A) or a heterocyst (B).

Fig 6

Growth of Anabaena sp. PCC 7120 wild type, all1711 mutants, and complemented mutant strains with nitrate as the nitrogen source (BG11 medium) or without combined nitrogen (BG11₀ medium). Spots of each indicated strain (see genotypes in Table 1) were inoculated with an amount of nitrate-grown cells resuspended in BG11₀ medium containing (from left to right) 100, 10, 1, or 0.1 ng Chl. The plates were then incubated under culture conditions for 15 days and photographed. Where present, Nm was at 30 μg ml⁻¹ and Em at 5 μg ml⁻¹. Complemented strains were pregrown with Nm with or without Em as appropriate.

Fig 7

Extracellular polysaccharides in strain FQ163-C3. (Top panels) Sample colonies of strains FQ163 and FQ163-C3 (from BG11-agar plates containing 30 μg Nm ml⁻¹ without and with 5 μg Em ml⁻¹, respectively) were supplemented with a solution of 1% alcian blue 8GX (Sigma) and visualized by light microscopy. (Middle panels) Filaments from spots atop BG11-agar plates (with antibiotics as described above) were suspended in 150 μl of BG11 medium, supplemented with 10 μl of 1% alcian blue 8GX, and visualized by light microscopy. (Bottom panels) Biomass from the surface of BG11-agar plates (with antibiotics as described above) was harvested and suspended in 9 ml of BG11₀ medium. These filament suspensions were supplemented with 1 ml of 0.02% alcian blue–0.06% acetic acid and, after 5 min at room temperature, harvested by centrifugation (4,000 × g, 5 min). Samples from the sediment were visualized by light microscopy.

Fig 8

Expression of all1711 in the wild type, two all1711 mutants, and corresponding complemented all1711 mutant strains. Northern blot analysis was performed with the all1711 probe indicated in Fig. 1A and RNA from cells grown in bubbled BG11C medium containing Nm for FQ163 and FQ296 and Nm plus Em for the complemented mutants (see genotypes in Table 1). The approximate sizes of different all1711 transcripts are indicated. Because the gene is expressed from a set of promoters in the plasmid complementing the mutants that differ from the promoter in the wild-type chromosome, the observed sizes differ from that in Fig. 3. Hybridization with a probe of the Anabaena rnpB gene was used as a loading and transfer control (lower panel).