Secretome analysis of Anabaena sp. PCC 7120 and the involvement of the TolC‐homologue HgdD in protein secretion

Abstract

Secretion of proteins is a central strategy of bacteria to influence and respond to their environment. Until now, there has been very few discoveries regarding the cyanobacterial secrotome or the secretion machineries involved. For a mutant of the outer membrane channel TolC-homologue HgdD of Anabaena sp. PCC 7120, a filamentous and heterocyst-forming cyanobacterium, an altered secretome profile was reported. To define the role of HgdD in protein secretion, we have developed a method to isolate extracellular proteins of Anabaena sp. PCC 7120 wild type and an hgdD loss-of-function mutant. We identified 51 proteins of which the majority is predicted to have an extracellular secretion signal, while few seem to be localized in the periplasmic space. Eight proteins were exclusively identified in the secretome of wild-type cells, which coincides with the distribution of type I secretion signal. We selected three candidates and generated hemagglutinin-tagged fusion proteins which could be exclusively detected in the extracellular protein fraction. However, these proteins are not secreted in the hgdD-mutant background, where they are rapidly degraded. This confirms a direct function of HgdD in protein secretion and points to the existence of a quality control mechanism at least for proteins secreted in an HgdD-dependent pathway.

Multiple modes of iron uptake by the filamentous, siderophore‐producing cyanobacterium, Anabaena sp. PCC 7120

“…Standard growth of cultures was performed as described in (Moslavac et al, 2007b;Tripp et al, 2012;Hahn et al, 2015). For short-term uptake experiments Anabaena sp.…”

The versatile TolC‐like Slr1270 in the cyanobacterium Synechocystis sp. PCC 6803

“…There is evidence, though, that Synechocystis sp. PCC 6803 (Sergeyenko and Los, ) and other cyanobacteria (Vilhauer et al ., ; Christie‐Oleza et al ., 2015a,b; Hahn et al ., ; Oliveira et al ., ) are capable of secreting proteins, some of which retain enzymatic activity in the extracellular milieu (Shirkey et al ., ; Vilhauer et al ., ; Oliveira et al ., ). Nevertheless, the molecular mechanisms determining translocation of those proteins from the cytoplasm across the plasma and outer membranes remain elusive.…”

HesF, an exoprotein required for filament adhesion and aggregation in Anabaena sp. PCC 7120

“…HgdD has been implicated in the secretion across the outer membrane of a number of essential factors, including components of the cell wall (Moslavac et al ., ), elements required for the uptake of iron (Nicolaisen et al ., ; Stevanovic et al ., ) and export of toxins in a proton gradient‐dependent manner (Hahn et al ., 2012; 2013). In addition to this wide variety of substrates, HgdD has also been reported as an important factor in modulating the protein secretion pattern (Moslavac et al ., ; Hahn et al ., ). In the present work, we show that BSMP3 is unable to translocate HesF to the extracellular milieu (Fig.…”

“…Nevertheless, the TolClike protein HgdD in Anabaena sp. PCC 7120 (Moslavac et al, 2007) has been suggested to mediate the translocation, among other things, of proteinaceous substrates as a component of the type I export system (Moslavac et al, 2007;Hahn et al, 2014). Thus, an hgdD mutant (BSMP3) was generated in Anabaena sp.…”

“…The subset of proteins present in the extracellular milieu, the exoproteome (Desvaux et al, 2009), has not been thoroughly investigated in cyanobacteria, even though there is evidence that cyanobacteria translocate proteins to the surrounding environment (e.g. Sergeyenko and Los, 2000;Hahn et al, 2014;Vilhauer et al, 2014). In our group, we have started to identify and to characterize the exoproteome of the filamentous cyanobacterium Anabaena (also known as Nostoc) sp.…”