A TolC-Like Protein Is Required for Heterocyst Development inAnabaenasp. Strain PCC 7120

Abstract

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 forms heterocysts in a semiregular pattern when it is grown on N 2 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 ␤-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).Gram-negative bacteria use a type I export system to transfer proteins or other molecules, like siderophores or fatty acids, to the cell surface (37, 50, 55). The proteinaceous substrates contain a C-terminal secretion signal essential and sufficient for export. The information for targeting and translocation, however, seems to be presented in the form of a secondary structure element rather than in the primary sequence as no clear amino acid motif has been identified. The machinery of type I export systems bridges the periplasm, allowing transfer of their substrates beyond the outer membrane. The export system is composed of a translocase spanning the plasma membrane with an ␣-helical, pore-forming domain and a cytosolic domain energizing the translocation process. Additionally, an adaptor protein associates with the translocase and, after substrate association, induces an interaction with an outer membrane TolC-like exit tunnel to conduct the secretion. Whereas TolC-like proteins from gram-negative proteobacteria have been amply investigated (37,50,54,55), nothing is known about these systems in cyanobacteria. Based on sequence similarity (40) and proteomic analysis of the cell wall fraction (42), we have recently proposed the existence of a protein of this type, Alr2887, in Anabaena sp. strain PCC 7120, for which a function in lipid transfer has previously been proposed (40).Anabaena sp. strain PCC 7120 is a filamentous cyanobacterium which starts a program of cell differentiation upon nitrogen starvation that results in the appearance of nitrogen-fixing cells. These cells are called heterocysts and are arranged in a semiregular pattern along the filament (63). Three mechanisms protect the oxygen-labile nitrogenase from irreversible inactivation by O 2 produced by oxygenic photosynthesis: (i) formation of extra envelope layers outside the gram-negative cell wall, (ii) degradation of photosystem II antennae and a d...

The TolC-like Protein HgdD of the Cyanobacterium Anabaena sp. PCC 7120 Is Involved in Secondary Metabolite Export and Antibiotic Resistance

“…PCC 7120 (referred to hereafter as Anabaena sp.) has been identified by proteomic analysis (18,19), and its relation to the TolC family was confirmed by homology modeling and analysis of the corresponding mutant (5,7,20). Anabaena sp.…”

“…Furthermore, the mutants of the other two Omp85-like genes (alr0075 and alr4893) (54,57,58) do not show an overlapping phenotype with the hgdD mutant (59) with respect to heterocyst development (5) or the sensitivity of AFS-I-hgdD to 30 mM lysozyme (59). A function of Alr0075 in HgdD insertion and assembly is further unlikely because this Omp85-like protein only exists in vegetative cells (54), whereas the expression of hgdD is enhanced in heterocysts (5). This could suggest that in Anabaena sp., the function of the Omp85 is not required for the assembly of HgdD, which, however, has to be further investigated.…”

“…We observed uptake of ethidium (394 Da) and erythromycin (734 Da; Figs. [3][4][5][6][7][8][9][10][11], and a permeability coefficient for Anabaena sp. (Fig.…”

“…In line with this, the mutant resulting in a loss of function displays an impairment in heterocyst development due to the missing heterocyst-specific glycolipid layer and was therefore termed HgdD (heterocyst glycolipid depositioning D) (5). Preliminary secretome analysis demonstrated the correlation of this phenotype with an altered secretome, leading to the proposal that HgdD is involved in protein secretion (5). Subsequent studies demonstrated an impairment of the same mutant in siderophore and glycolipid secretion (7,21).…”

“…It was shown that the transcript of the TolC-like gene is enhanced upon nitrogen starvation. In line with this, the mutant resulting in a loss of function displays an impairment in heterocyst development due to the missing heterocyst-specific glycolipid layer and was therefore termed HgdD (heterocyst glycolipid depositioning D) (5). Preliminary secretome analysis demonstrated the correlation of this phenotype with an altered secretome, leading to the proposal that HgdD is involved in protein secretion (5).…”