Structural and Functional Characterizations of SsgB, a Conserved Activator of Developmental Cell Division in Morphologically Complex Actinomycetes

SsgA-like proteins (SALPs) are a family of homologous cell division-related proteins that occur exclusively in morphologically complex actinomycetes. We show that SsgB, a subfamily of SALPs, is the archetypal SALP that is functionally conserved in all sporulating actinomycetes. Sporulation-specific cell division of Streptomyces coelicolor ssgB mutants is restored by introduction of distant ssgB orthologues from other actinomycetes. Interestingly, the number of septa (and spores) of the complemented null mutants is dictated by the specific ssgB orthologue that is expressed. The crystal structure of the SsgB from Thermobifida fusca was determined at 2.6 ? resolution and represents the first structure for this family. The structure revealed similarities to a class of eukaryotic “whirly” single-stranded DNA/RNA-binding proteins. However, the electro-negative surface of the SALPs suggests that neither SsgB nor any of the other SALPs are likely to interact with nucleotide substrates. Instead, we show that a conserved hydrophobic surface is likely to be important for SALP function and suggest that proteins are the likely binding partners.The mechanisms governing the correct timing and localization of cell division is one of the most studied topics in cell biology. In unicellular bacteria like Escherichia coli, cell division occurs at the mid-cell position, away from the chromosomes (1–3). The key step in this process is the appropriate timing and localization of cell division protein FtsZ to the future septum site, followed by polymerization to the Z-ring and sequential recruitment of the divisome components (1, 4). One of the major advances in our understanding of the cell division process and, in particular, of the function of FtsZ came from elucidation of the three-dimensional structure of FtsZ, which showed striking similarity to the eukaryotic protein tubulin, despite very low sequence similarity (5). Such prokaryotic ancestry was later also revealed for the cytoskeletal proteins MreB and Mbl, which belong to the actin family (6, 7), and underscored the notion of generally conserved principles in cytokinesis.Spore-forming Gram-positive Streptomyces bacteria are an important source of clinically useful antibiotics and anticancer agents (8). In these morphologically complex microorganisms, cell division is distinctly different from that in unicellular bacteria in several ways. For one, they are the only known organisms where FtsZ and MreB are both dispensable for growth (9, 10), which makes streptomycetes ideal for the study of cytokinesis. Streptomycetes have a complex life cycle that is mechanistically very similar to filamentous fungi, in producing a mycelium and propagating by sporulation (11, 12). During sporulation, the cell division machinery produces up to 100 septa simultaneously, spaced at around 1 μm, resulting in long chains of uniform and unigenomic spores (10, 13, 14). Besides the simultaneous production of multiple septa, cell division in mycelial actinomycetes also differs from that in other bacteria at the molecular level; actinomycetes lack orthologues of MinC and MinE for septum site localization (15, 16) as well as the nucleoid occlusion system Noc and Z-ring anchoring proteins, such as FtsA and ZipA (1, 6). Instead, several unique protein families have been identified that play a role in the control of cell division, including CrgA and the SsgA-like proteins (17–19). However, molecular details of their mode of action are so far lacking.The SsgA (sporulation of Streptomyces griseus)-like proteins (SALPs)³ are small (around 130–140 residues), actinomycete-specific proteins, which control sporulation-related processes in streptomycetes (17, 20). Streptomyces coelicolor contains seven SALP paralogues (SsgA to SsgG). SsgA and SsgB are essential for sporulation of S. coelicolor (21, 22). SALPs are involved in the control of cell wall-related events, such as septum localization and synthesis, thickening of the spore wall, and autolytic spore separation (17, 20), and SsgA itself directly activates sporulation-specific cell division (22, 23). The morphological complexity of actinomycetes apparently correlates to the number of SALP homologues in each organism, with one paralogue in single spore-forming actinomycetes (e.g. Salinispora or Thermobifida) and up to seven in multispore formers (two in erythromycin producer Saccharopolyspora erythraea, 3–5 in Frankia, and 6–7 in Streptomyces) (17). Most SALPs can be assigned to three subfamilies (SsgA, SsgBG, and SsgDE) based on phylogenetic analysis (17). At present, there are no functional homologues for the SALPs, and structural information is lacking. To advance our understanding of how SALPs function at the molecular level and to provide a structural template for a unique protein family without obvious relatives in any other organism, we selected the single SALP homologue from the thermophilic soil bacterium Thermobifida fusca (a major degrader of plant cell walls used in waste remediation (24)) for detailed structural analysis by x-ray crystallography, as part of our structural genomics program.In this work, we show that SsgB is most likely the archetypical SALP that occurs in morphologically complex actinomycetes, with an evolutionarily conserved function in the control of development. The three-dimensional structure of the SsgB orthologue from T. fusca was determined and revealed significant structural similarity to a eukaryotic family of ssDNA/gRNA-binding proteins. However, the structure and experimental data both suggest that SALPs probably interact with protein ligands through a hydrophobic region on their surface.