Structural and Functional Insights into Bacillus subtilis Sigma Factor Inhibitor CsfB

摘要

class="head no_bottom_margin" id="sec1title">IntroductionEukaryotic and prokaryotic cells alike possess the ability to alter their phenotypes through global changes in gene expression. In bacteria, these transitions enable survival during stress conditions, drive developmental programs, and promote infection of host organisms. One common mechanism bacteria utilize to effect large-scale changes in gene expression is through alternative sigma (σ) factor subunits of RNA polymerase (RNAP). The dissociable RNAP sigma factor subunit is responsible for recognition of promoter DNA and the subsequent initiation of transcription. Most sigma factors are members of the σ⁷⁰ superfamily, which is subdivided into four classes based upon conservation and the presence/absence of the conserved sigma domains (σ1.1, σ2, σ3, and σ4) that mediate interactions with RNAP and/or promoter DNA (reviewed in , ). All bacteria employ an essential primary sigma factor (class I) that directs transcription of housekeeping genes; many bacteria also possess alternative sigma factors (classes II, III, and IV) that compete for binding to RNAP and redirect it to transcribe sets of genes required for adaptive responses. Hence, the suite of genes expressed in a bacterial cell can be reprogrammed by manipulating the levels, activity, or availability of alternative sigma factors (reviewed in ).One prevalent form of post-translational regulation of alternative sigma factors occurs via anti-sigma factors: proteins that bind to and prevent their cognate sigma factor from interacting with RNAP. Unlike sigma factors, which share sequence, structural, and functional conservation, anti-sigma factors are more diverse in their sequences, structures, and/or mode of sigma factor inhibition (reviewed in ). A number of structural and bioinformatics analyses have revealed that anti-sigma factors for the class IV extracytoplasmic function (ECF) sigma factors often share one of two conserved anti-sigma domain structures, despite little sequence conservation (reviewed in ). Less is known, however, of the structural features of anti-sigma factors that antagonize non-class IV alternative sigma factors, given the limited number of structures determined to date (, , ).Here, we have structurally analyzed CsfB (also called Gin), a small, Zn²⁺-binding anti-sigma factor that inhibits two class III alternative sigma factors during spore formation by the model bacterium Bacillus subtilis (A) (, , , , , ). In the forespore cell (the nascent spore), CsfB binds and inhibits the late-acting sigma factor σ^G, helping to ensure that it does not become active before the early-acting sigma factor σ^F has completed its program of gene expression (href="#bib22" rid="bib22" class=" bibr popnode">Karmazyn-Campelli et al., 2008, href="#bib36" rid="bib36" class=" bibr popnode">Rhayat et al., 2009). In the mother cell, which helps support the development of the forespore, CsfB binds the early-acting sigma factor σ^E, helping to inactivate it after the switch to σ^K (href="#bib39" rid="bib39" class=" bibr popnode">Serrano et al., 2015). Here, we report the structure of CsfB and characterize its interaction with σ^G and σ^E.href="/pmc/articles/PMC5890618/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">class="inline_block ts_canvas" href="/core/lw/2.0/html/tileshop_pmc/tileshop_pmc_inline.html?title=Click%20on%20image%20to%20zoom&p=PMC3&id=5890618_gr1.jpg" target="tileshopwindow">target="object" href="/pmc/articles/PMC5890618/figure/fig1/?report=objectonly">Open in a separate windowclass="figpopup" href="/pmc/articles/PMC5890618/figure/fig1/" target="figure" rid-figpopup="fig1" rid-ob="ob-fig1">Figure 1The Anti-sigma Factor CsfB Helps to Orchestrate the Switch from Early to Late Gene Expression during B. subtilis Sporulation(A) Cartoon depiction of the role of the dual-specificity anti-sigma factor CsfB in regulating the transition from early to late gene expression during B. subtilis sporulation. Early in sporulation (reviewed in href="#bib45" rid="bib45" class=" bibr popnode">Tan and Ramamurthi, 2014), an asymmetric cell division event produces two cells: a smaller forespore (the nascent spore) and a larger mother cell. Initially, these two cells lie side-by-side; the mother cell then engulfs the forespore in a phagocytic-like process. At early times, σ^F and σ^E drive gene expression in the forespore and mother cell, respectively. Among the genes activated by σ^F and σ^E are those encoding the late-acting sigma factors, σ^G and σ^K, respectively (dashed arrows). The anti-sigma factor CsfB is expressed in both compartments under the control of σ^F and σ^K (dashed arrows). In the forespore, CsfB antagonizes σ^G at early times (barred line). In the mother cell, CsfB antagonizes σ^E at later times (barred line).(B) ¹H-¹⁵N HSQC spectrum of CsfB (orange). Full assignment of the cleaved CsfB version appears in black (CsfB¹⁻⁴⁸), partial assignment of the residual full-length CsfB in blue and the tag residues in gray; sc denotes side chain resonances. The C-terminal residue from the cleaved version (A48) is highlighted by a green square.