Modularity and determinants of a (bi-)polarization control system from free-living and obligate intracellular bacteria

Abstract

Although free-living and obligate intracellular bacteria are both polarized it is unclear whether the underlying polarization mechanisms and effector proteins are conserved. Here we dissect at the cytological, functional and structural level a conserved polarization module from the free living α-proteobacterium Caulobacter crescentus and an orthologous system from an obligate intracellular (rickettsial) pathogen. The NMR solution structure of the zinc-finger (ZnR) domain from the bifunctional and bipolar ZitP pilus assembly/motility regulator revealed conserved interaction determinants for PopZ, a bipolar matrix protein that anchors the ParB centromere-binding protein and other regulatory factors at the poles. We show that ZitP regulates cytokinesis and the localization of ParB and PopZ, targeting PopZ independently of the previously known binding sites for its client proteins. Through heterologous localization assays with rickettsial ZitP and PopZ orthologs, we document the shared ancestries, activities and structural determinants of a (bi-)polarization system encoded in free-living and obligate intracellular α-proteobacteria.DOI: http://dx.doi.org/10.7554/eLife.20640.001

Functional dichotomy and distinct nanoscale assemblies of a cell cycle-controlled bipolar zinc-finger regulator

“…This is intriguing and hints at a potentially important and conserved role of such zinc-finger domain proteins in developmental processes that rely on protein polarization in bacteria and polar matrix proteins such as PopZ to interact with them. In a complementary study, we additionally show in vitro and in vivo that zinc-bound ZitP binds PopZ directly and regulates PopZ localization without the periplasmic DUF3426 domain (Bergé et al, 2016), suggesting that this activity in ZitP may underlie the aforementioned CtrA-independent role in motility.…”

“…The zinc_ribbon_5 domain promotes PopZ-dependent localization of ZitP to the stalked pole and efficient swarming motility by an unknown mechanism. Interestingly, in a related study, we recently found that ZitP controls PopZ localization independently of the DUF3426 (Bergé et al, 2016). …”

“…While both genes have previously been implicated in polar functions and their transcription is cell cycle-regulated (Christen et al, 2016; Fioravanti et al, 2013; Fumeaux et al, 2014; Hughes et al, 2010; McGrath et al, 2007), they are poorly characterized. The zitP gene is predicted to encode a 311-residue bitopic trans-membrane (TM) protein harbouring a CXXC-(X) 19 -CXXC motif that binds a zinc ion (zinc_ribbon_5 or PF13719 superfamily, residues 1-37) at the cytoplasmic N-terminus (Bergé et al, 2016) and a conserved domain-of-unknown function (DUF3426, residues 128-245) in the C-terminal region that is predicted to reside in the periplasm (Figure 1C). The cpaM gene codes for a 394-residue protein harbouring a single N-terminal TM domain and a C-terminal CE4_DAC2-like polysaccharide deacetylase domain predicted to be periplasmic (Figure 1C).…”

“…To identify the determinants within ZitP governing the differential polar localization and to test if they support specific functions, we first constructed a mutant variant of ZitP in which all four zinc-coordinating cysteine residues in the zinc-finger domain (Bergé et al, 2016) are replaced by serine residues (henceforth ZitP CS , Figure 1C). The motility of Δ zitP cells expressing ZitP CS or Dendra2-ZitP CS is reduced compared to those expressing the WT version of ZitP (ZitP or Dendra2-ZitP; Figure 2G and K).…”

“…ZitP CS also confers (CpaM-dependent) firing of CtrA-activated promoters with similar efficiency as WT ZitP (Figure 3—figure supplement 8A). Since Dendra2-CpaM is also still monopolar in ∆ popZ cells, zinc-binding within the zinc_ribbon_5 domain is necessary for the interaction between PopZ and ZitP (Bergé et al, 2016), but not for CpaM localization/interaction. Thus, inactivation of the zinc-coordinating residues in ZitP effectively mimics the monopolar localization of Dendra2-ZitP in ∆ popZ cells and functions as unmodified ZitP with respect to the functions that depend on CpaM.…”