Quantitative Selection Analysis of Bacteriophage φCbK Susceptibility in Caulobacter crescentus

Abstract

Classical molecular genetics uses stringent selective conditions to identify mutants with distinct phenotypic responses. Mutations giving rise to less pronounced phenotypes are often missed. However, to gain systems-level insights into complex genetic interaction networks requires genome-wide assignment of quantitative phenotypic traits. In this paper, we present a quantitative selection approach coupled with transposon sequencing (QS-TnSeq) to globally identify the cellular components that orchestrate susceptibility of the cell cycle model bacterium Caulobacter crescentus toward bacteriophage φCbK infection. We found that 135 genes representing 3.30% of the Caulobacter genome exhibit significant accumulation of transposon insertions upon φCbK selection. More than 85% thereof consist of new factors not previously associated with phage φCbK susceptibility. Using hierarchical clustering of dose-dependent TnSeq datasets, we grouped these genes into functional modules that correlate with different stages of the φCbK infection process. We assign φCbK susceptibility to eight new genes that represent novel components of the pilus secretion machinery. Further, we demonstrate that, from 86 motility genes, only seven genes encoding structural and regulatory components of the flagellar hook increase phage resistance when disrupted by transposons, suggesting a link between flagellar hook assembly and pili biogenesis. In addition, we observe high recovery of Tn5 insertions within regulatory sequences of the genes encoding the essential NADH:ubiquinone oxidoreductase complex indicating that intact proton motive force is crucial for effective phage propagation. In sum, QS-TnSeq is broadly applicable to perform quantitative and genome-wide systems-genetics analysis of complex phenotypic traits.

Gene Transfer Agent Promotes Evolvability within the Fittest Subpopulation of a Bacterial Pathogen

“…TnSeq couples hypersaturated transposon (Tn) mutagenesis with next-generation sequencing to profile changes in genome-wide insertion patterns occurring upon selection of large mutant pools (Christen et al., 2011, Christen et al., 2016). We constructed a himar-based Tn probe and generated large input mutant libraries encompassing several hundreds of thousands of insertion events (STAR Methods).…”

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

“…The resulting strain expresses a bipolarly localized and truncated ZitP-GFP fusion protein and is viable (Hughes et al, 2010). Consistent with this result, genome-wide transposon insertion sequencing (Tn-Seq) revealed that ZitP is not essential for viability, but required for pilus biogenesis and motility (Christen et al, 2011, 2016; Hughes et al, 2010)(Mignolet et al, 2016). The predicted ZitP protein harbors an N-terminal zinc-finger domain (zinc_ribbon_5 or PF13719 superfamily, residues 1–37, henceforth ZnR, Figure 1B–C) and a trans-membrane segment (TM, residues 106–127) preceding the C-terminal DUF3426 (residues 128–245).…”

“…Here, we unearth a reciprocal, physical and conserved interaction between PopZ and the cytoplasmic N-terminal zinc-finger domain (ZnR) from ZitP (Hughes et al, 2010), a bifunctional and bipolar membrane protein whose C-terminal DUF3426 domain is required for polar pilus biogenesis (Mignolet et al, 2016)(Christen et al, 2016), but dispensable for motility. We locate the structural determinants governing PopZ•ZitP complex formation and we show that this interaction is required to control cytokinesis and centromere positioning from the membrane.…”

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

“…As pili are necessary for infection by the lytic caulophage CbK (φCbK) (Skerker and Shapiro, 2000), we specifically sought mutants in pilus assembly factors encoded outside of the major pilus assembly cpa gene locus ( pilA-cpaA-K) (Christen et al, 2016; Skerker and Shapiro, 2000). To this end, we conducted himar1 -transposon ( Tn ) mutagenesis of wild-type ( WT ) Caulobacter in the presence of φCbK (see Methods) and recovered mutants with Tninsertions in CCNA_02298, renamed here zitP (z inc-finger targeting the poles) because of the pleiotropic roles detailed below, or in cpaM (CCNA_03552 ) (Figure 1C) (Marks et al, 2010).…”

“…To this end, we conducted himar1 -transposon ( Tn ) mutagenesis of wild-type ( WT ) Caulobacter in the presence of φCbK (see Methods) and recovered mutants with Tninsertions in CCNA_02298, renamed here zitP (z inc-finger targeting the poles) because of the pleiotropic roles detailed below, or in cpaM (CCNA_03552 ) (Figure 1C) (Marks et al, 2010). 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).…”