Septal protein SepJ from the heterocyst‐forming cyanobacterium Anabaena forms multimers and interacts with peptidoglycan

Félix Ramos‐León,Enrique Flores,Natalia Battchikova,Eva‐Mari Aro,Vicente Mariscal

doi:10.1002/2211-5463.12280

Abstract

Heterocyst‐forming cyanobacteria grow as filaments that can be hundreds of cells long. Proteinaceous septal junctions provide cell–cell binding and communication functions in the filament. In Anabaena sp. strain PCC 7120, the SepJ protein is important for the formation of septal junctions. SepJ consists of integral membrane and extramembrane sections – the latter including linker and coiled‐coil domains. SepJ (predicted MW, 81.3 kDa) solubilized from Anabaena membranes was found in complexes of about 296–334 kDa, suggesting that SepJ forms multimeric complexes. We constructed an Anabaena strain producing a double‐tagged SepJ protein (SepJ‐GFP‐His10) and isolated the tagged protein by a two‐step affinity chromatography procedure. Analysis of the purified protein preparation provided no indication of the presence of specific SepJ partners, but suggested that SepJ is processed to remove an N‐terminal fragment. Additionally, pull‐down experiments showed that His6‐tagged versions of SepJ and of the SepJ coiled‐coil domain interact with Anabaena peptidoglycan (PG). Our results indicate that SepJ forms multimers, that it interacts with PG, and that the coiled‐coil domain is involved in this interaction. These observations support the idea that SepJ is a component of the septal junctions that join the cells in the Anabaena filament.

Highlights

Heterocyst-forming cyanobacteria grow as filaments that can be hundreds of cells long
The membranes were solubilized with 0.5–5% dodecyl b-D-maltoside (DDM), and the solubilized material was analyzed by blue native (BN)-PAGE and western blotting performed with antibodies raised against the coiled-coil domain of SepJ
This analysis showed that a complex that reacted with anti-SepJ protein and its coiled-coil domain (SepJ-CC) was solubilized from the membranes by using 2% DDM, whereas lower DDM concentrations were insufficient to solubilize the complex and higher DDM concentrations apparently destabilized the complex releasing material of lower MW

Summary

Introduction

Heterocyst-forming cyanobacteria grow as filaments that can be hundreds of cells long. Our results indicate that SepJ forms multimers, that it interacts with PG, and that the coiled-coil domain is involved in this interaction These observations support the idea that SepJ is a component of the septal junctions that join the cells in the Anabaena filament. Inactivation of the corresponding genes results in a filament fragmentation phenotype, which is strongest in the case of sepJ [13,14,15] Both SepJ and the FraCD proteins are required in Anabaena to produce a normal number of nanopores [16], there is evidence for the existence of at least two types of septal junctions, those related to SepJ and those related to FraCD [17]

Methods

Results

Discussion

Conclusion