Permissive zones for the centromere-binding protein ParB on the Caulobacter crescentus chromosome

Ngat T Tran,Clare E Stevenson,Tung B K Le,Nicolle F Som,Anyarat Thanapipatsiri,Adam S B Jalal

doi:10.1093/nar/gkx1192

Abstract

Proper chromosome segregation is essential in all living organisms. In Caulobacter crescentus, the ParA–ParB–parS system is required for proper chromosome segregation and cell viability. The bacterial centromere-like parS DNA locus is the first to be segregated following chromosome replication. parS is bound by ParB protein, which in turn interacts with ParA to partition the ParB-parS nucleoprotein complex to each daughter cell. Here, we investigated the genome-wide distribution of ParB on the Caulobacter chromosome using a combination of in vivo chromatin immunoprecipitation (ChIP-seq) and in vitro DNA affinity purification with deep sequencing (IDAP-seq). We confirmed two previously identified parS sites and discovered at least three more sites that cluster ∼8 kb from the origin of replication. We showed that Caulobacter ParB nucleates at parS sites and associates non-specifically with ∼10 kb flanking DNA to form a high-order nucleoprotein complex on the left chromosomal arm. Lastly, using transposon mutagenesis coupled with deep sequencing (Tn-seq), we identified a ∼500 kb region surrounding the native parS cluster that is tolerable to the insertion of a second parS cluster without severely affecting cell viability. Our results demonstrate that the genomic distribution of parS sites is highly restricted and is crucial for chromosome segregation in Caulobacter.

Highlights

All plasmids and primers used in strain and plasmid construction are listed in Supplementary Table S2
2.5 μL of each DNA fragment at equimolar concentration was added to 5 μL Gibson master mix (NEB), and the mixture was incubated at 50°C for 60 minutes. 5 μL was used to transform chemically-competent E. coli DH5α cells
The resulting PCR product was purified by gel extraction

Summary

Introduction

Binding of ParB to parS site was recorded and expressed as response unit (RU). The final PCR product was gel-purified and assembled to an NdeI-NheI-cut pMT675 (1) using a 2x Gibson master mix (NEB). The final PCR product was gel-purified and assembled to an NdeI-HindIII-cut pET21b using a 2x Gibson master mix (NEB).

Results

Conclusion