A TolC-like protein is required for heterocyst development in Anabaena sp. strain PCC 7120

Abstract

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N2 as the sole nitrogen source. The transition from vegetative cells to heterocysts requires marked metabolic and morphological changes. We show that a trimeric pore-forming outer membrane beta-barrel protein belonging to the TolC family, Alr2887, is up-regulated in developing heterocysts and is essential for diazotrophic growth. Mutants defective in Alr2887 did not form the specific glycolipid layer of the heterocyst cell wall, which is necessary to protect nitrogenase from external oxygen. Comparison of the glycolipid contents of wild-type and mutant cells indicated that the protein is not involved in the synthesis of glycolipids but might instead serve as an exporter for the glycolipid moieties or enzymes involved in glycolipid attachment. We propose that Alr2887, together with an ABC transporter like DevBCA, is part of a protein export system essential for assembly of the heterocyst glycolipid layer. We designate the alr2887 gene hgdD (heterocyst glycolipid deposition protein).

FIG. 1.

HgdD is up-regulated in proheterocysts upon nitrogen step-down. (A) RNA was isolated from NH₄⁺-grown cells (lane 1) and from cells that had been starved for combined nitrogen for 6, 9, or 12 h (lanes 2 to 4). Samples contained 30 μg RNA. The blots were successively hybridized with ³²P-labeled probes for hgdD (upper panel) and rnpB (lower panel), which were used as loading and transfer controls, respectively. The sizes of RNA standards are indicated on the left. (B) N-terminal HgdD-GFP translational fusion in strain NME-alr2887-GFP as visualized by confocal microscopy. The cyanobacterial autofluorescence (AUF.), the GFP fluorescence (GFP), the overlay of the two signals (Overl.), and a bright-field image (BFI) of the NME-alr2887-GFP strain 16 h after nitrogen step-down are shown.

FIG. 2.

Sequence of HgdD is related to those of TolC proteins. (A) Model of HgdD generated as described in the text, showing structural homology to the outer membrane protein TolC. Side, top, and bottom views are shown. Further details are available in the supplemental material. The coordinates are available upon request. (B) Tree of 178 sequences of members of the TolC family (for details, see Fig. S2 in the supplemental material), representing a maximum likelihood phylogeny, assuming the WAG model and constant rates across sites. The tree is based on a multiple alignment. Bar = 0.1 amino acid substitution. The functional categories were assigned as described by Andersen et al. (3). (C) Isolated cell walls (CW) from lysed heterocysts of strain NMP-alr2887-GFP (lane 1) were treated with 8 M urea (lane 2), 0.1 M sodium carbonate (lane 3), or 1 M NaCl (lane 4). The pelleted membrane fraction (P) or the supernatant (S) was probed with antibodies against the outer membrane protein Alr2269 (αalr2269), which was used as a control, or GFP (αGFP). The migration positions of some molecular mass markers (in kDa) are indicated on the right for the pellet fraction probed with anti-GFP.

FIG. 3.

Ultrastructure of heterocysts of hgdD mutants: transmission electron micrographs of ultrathin sections of a connection of a heterocyst and a vegetative cell of the wild-type Anabaena sp. strain (A and B) and mutant DR181 (C and D). Magnifications of the cell wall and heterocyst envelope, indicated by squares in panels A and C, are shown for the wild-type strain and DR181 in panels B and D, respectively. In panel A, the white space surrounding the cell wall resulted from dehydration of the protoplast during sample preparation. Bar = 1 μm for panels A and C. GL, laminated layer; P, homogeneous layer; CM, cytoplasmic membrane; OM, outer membrane.

FIG. 4.

Progress of HGL synthesis and HGL layer formation. (A) Thin-layer chromatography of lipids of the wild-type Anabaena sp. strain before (lane 1) or at indicated times after (lanes 2 to 7) transfer to BG11₀ medium. (B and C) Representative transmission electron micrographs of ultrathin sections of filaments of the wild-type Anabaena sp. strain 14 h (B) or 20 h (C) after transfer to BG11₀ medium. The arrowhead in panel C indicates the HGL layer after 20 h. Bar = 1 μm. (D) The fluorescence of the NME-alr2887-GFP strain was determined, and the difference from the wild-type strain background for three independent measurements and three independent NME-alr2887-GFP clones is shown for the indicated times after nitrogen step-down. A.U., arbitrary units; ex, excitation; em, emission.

FIG. 5.

Heterocyst-specific glycolipids and secreted proteins in hgdD mutants. (A) Thin-layer chromatography of lipids of the wild-type Anabaena sp. strain (wt) (lanes 1, 3, 5, and 8), mutant NMΔ-alr2887 (Δ) (lanes 2, 4, 6, and 9), and mutant DR181 (DR) (lanes 7 and 10). Lipids were extracted from filaments grown in BG11 medium (lanes 1 and 2), from filaments 3 days after transfer to BG11₀ medium (lanes 3 and 4), from isolated heterocysts (Het) (lanes 5, 6, and 7), or from isolated heterocyst cell walls (CW) (lanes 8, 9, and 10). The lipids of the cell wall fractions show a slightly altered migration behavior due to edge effects of the thin-layer chromatography plate. The HGL lipids are indicated by arrows (open arrows, HGL1; gray arrows, HGL2). In lanes 9 and 10 the area where HGL2 would be expected is also indicated. (B) Proteins secreted from the wild type or NMΔ-alr2887 mutant 9 h after nitrogen step-down were concentrated and subjected to SDS-PAGE, followed by silver staining. The positions of molecular weight standards (MW) are indicated on the right, and proteins not secreted from the mutant are indicated by arrows.

FIG. 6.

Proposed functional arrangement of HgdD (Alr2887). The structural composition of the HgdD/DevABC complex is shown, as discussed in the text. HgdD was modeled as shown in Fig. 2A, DevA was modeled using MJ0796, a bacterial ATP binding cassette (PDB:1L2T), as the template, and sections of DevB were modeled using MexA (PDB:1VF7) as the template. The pink boxes represent an extension of the two-helix coiled coil of DevB bridging the periplasm and attaching to HgdD (Alr2887). The ellipse with a question mark indicates that further elements stabilizing the complex might exist. OM, outer membrane; PM, cytoplasmic membrane.