Domain architecture and structure of the bacterial cell division protein DivIB

Abstract

Bacterial cytokinesis requires the coordinated assembly of a complex of proteins, collectively known as the divisome, at the incipient division site. DivIB͞FtsQ is a conserved component of the divisome in bacteria with cell walls, suggesting that it plays a role in synthesis and͞or remodeling of septal peptidoglycan. We demonstrate that the extracytoplasmic region of DivIB comprises three discrete domains that we designate ␣, ␤, and ␥ from the N to C terminus. The ␣-domain is proximal to the cytoplasmic membrane and coincident with the polypeptide transport-associated domain that was proposed previously to function as a molecular chaperone. The ␤-domain has a unique 3D fold, with no eukaryotic counterpart, and we show that it interconverts between two discrete conformations via cis-trans isomerization of a Tyr-Pro peptide bond. We propose that this isomerization might modulate protein-protein interactions of the flanking ␣-and ␥-domains. The C-terminal ␥-domain is unstructured in the absence of other divisomal proteins, but we show that it is critical for DivIB function.bacterial cytokinesis ͉ divisome ͉ NMR ͉ protein structure B acterial cytokinesis requires spatiotemporal coordination of the assembly of a complex of cell division proteins, collectively known as the divisome, at the incipient division site (1, 2). Divisome assembly is initiated by the formation of a circumferential ring of FtsZ (the Z ring) around the inner surface of the cytoplasmic membrane (3, 4). FtsZ, the precursor of eukaryotic tubulin, is a GTPase that most likely provides at least part of the contractile force for septal invagination through dynamic remodeling of the Z ring (5-7). In addition, the Z ring promotes the switch from lateral to preseptal murein synthesis (8), and it provides a scaffold for recruitment of downstream divisomal proteins.In Escherichia coli the known divisomal proteins are thought to be recruited to the division site in the following hierarchical order: FtsZ4ZipA,FtsA4FtsEX4FtsK4FtsQ4(FtsL, FtsB)4FtsW4FtsI 4FtsN4AmiC, where the arrow indicates that recruitment of a particular protein requires prior assembly of all previously listed components, and parentheses enclose proteins whose recruitment to the divisome is interdependent (2, 9, 10). However, recent studies suggest that some subcomplexes, such as that formed among FtsQ, FtsL, and FtsB, might be preassembled before Z ring formation and trafficked to the developing division site as a preformed complex (2, 11). This latter situation is likely more akin to that in Bacillus subtilis, where the observed interdependent localization of most divisomal proteins argues for a more concerted recruitment to the division site (1,12).Whereas the earliest recruits to the division site are either wholly or mostly cytoplasmic (e.g., FtsZ, FtsA, and FtsK), the later recruits are typically bitopic membrane proteins with a short N-terminal cytoplasmic region, a single transmembrane segment, and a larger extracytoplasmic (ec) domain (i.e., FtsQ, FtsL, FtsB, FtsI, and FtsN). Rema...

Evidence from Artificial Septal Targeting and Site-Directed Mutagenesis that Residues in the Extracytoplasmic β Domain of DivIB Mediate Its Interaction with the Divisomal Transpeptidase PBP 2B

“…Construction of the divIB null strain RSA8 (⌬divIB::cat::ermC) and RSA8-derived strains carrying an inducible copy of divIB at the amyE locus has been described previously (31). Strains produced for this study are numbered RSA11 through RSA19, RSA21, RSA22, RSA24 through RSA28, RSA30, and RSA31.…”

“…Cells were plated on NZY (N-Z amine, yeast extract) plates supplemented with agar, 100 g/ml spectinomycin (Spc), and 25 g/ml ampicillin (Amp) for selection. Point mutations were introduced into divIB for incorporation at the amyE chromosomal locus of B. subtilis as described previously, using pSAR50 as a template and pDG364 as the cloning vector (31).…”

“…We have chosen to refer to this region as the ␥ tail to be consistent with recent work on Streptococcus pneumoniae DivIB (25) and to highlight the fact that this region is unstructured, at least in the absence of other divisomal proteins (25,31). In B. subtilis DivIB, the three extracytoplasmic regions were originally defined as residues 51 to 122 (␣), 123 to 228 (␤), and 229 to 263 (␥) (31,38). As will be discussed in more detail below, there is some debate about the exact boundary between the ␤ and ␥ domains in DivIB (25,36), but we used the originally defined domain boundaries for initial studies.…”

“…2F) were also incapable of recruiting PBP 2B to division septa, as evidenced by the halo of fluorescence around the cell periphery and the absence of midcell localization. Previous work suggests that the absence of PBP 2B recruitment by these ZapA-DivIB fusions was not due to misfolding, low levels of expression, and/or instability of the fusion proteins (31,38). The separated ␣ and ␤ domains of DivIB used in this study (i.e., residues 51 to 122 of B. subtilis DivIB [DivIB ] and DivIB 123-228 , respectively) were previously shown to be functional (38), and, as demonstrated by Western blotting (Fig.…”

“…Although the unstructured ␥ "domain" in B. subtilis DivIB might be as short as ϳ20 residues (see discussion below), this region comprises Ͼ60 residues in some species. We have chosen to refer to this region as the ␥ tail to be consistent with recent work on Streptococcus pneumoniae DivIB (25) and to highlight the fact that this region is unstructured, at least in the absence of other divisomal proteins (25,31). In B. subtilis DivIB, the three extracytoplasmic regions were originally defined as residues 51 to 122 (␣), 123 to 228 (␤), and 229 to 263 (␥) (31,38).…”