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Investigate the roles of an ancestral SMC protein in bacterial chromosome segregation

Lead Research Organisation: John Innes Centre
Department Name: UNLISTED

Abstract

Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.

Technical Summary

Bacterial chromosomes must be organized to be compatible with a myriad of DNA-based processes including transcription, replication, segregation and repair. Structural Maintenance of Chromosome proteins (SMC) participate in organizing chromosomes in virtually all living organisms. We were the first to show by chromosome conformation capture (Hi-C) that Caulobacter crescentus lacking SMC has reduced interactions between loci at approximately equivalent positions on opposite chromosomal arms, suggesting a role of SMC in chromosome organization in this bacterium. However, how SMC binds the chromatin and shapes the chromosome structure remain poorly understood. To answer this question, we will (i) define the genome-wide DNA binding sites of SMC, (ii) systematically determine the identities of DNA loci that are brought spatially close together by SMC, and finally (iii) determine DNA loading sites and the protein loader of SMC in Caulobacter. Since SMC is highly conserved from bacteria to humans, knowledge generated from this project is applicable to understand the chromosome organizations in other bacterial species as well. We are especially interested in applying these knowledge to industrial-important species such as Streptomyces.

Planned Impact

unavailable
 
Description 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.

More recently, we reconstituted a parS-dependent ParB spreading event using purified proteins from Caulobacter crescentus and showed that CTP is required for spreading. We further showed that ParB spreading requires a closed DNA substrate, and a DNA-binding transcriptional regulator can act as a roadblock to attenuate spreading unidirectionally in vitro. Our biochemical reconstitutions recapitulate many observed in vivo properties of ParB and opens up avenues to investigate the interactions between ParB-parS with ParA and SMC. We are now extending the work to investigate the interaction between Noc and CTP. Noc is a paralog of ParB in Firmicutes. ParB-parS systems have been widely exploited to label and image DNA loci in vivo, in both bacteria and eukaryotes. Recently, the ParB-parS system has been utilized in synthetic biology, for example, as part of a genetic circuit to enable asymmetric cell division in E. coli (Molinari et al Nature Chemical Biology 2019 PMID:31406375).
Exploitation Route The outputs of our proposed research have the potential to deliver impact at four levels:

1. the advancement of our fundamental knowledge of bacterial chromosome organisation, the molecular mechanism of bacterial SMC and their essential homologs in humans such as cohesin and condensin.

2. providing insights into the functions of human cohesin and condensin and how their malfunctions result in disease. Defective cohesin, condensin or associated genes contributes to tumour formation such as in colorectal cancer. Defective cohesin was also thought to cause the genetic disease Cornelia deLange syndrome. The affected children have defects such as missing fingers, mental retardation, growth failure, heart defects, and other impairments. In the near future, this research may not only elucidate the pathology underlying some of these diseases, but may also provide insights leading to new treatment.

3. informing synthetic biologists and relevant industries of rules to design total synthetic chromosome (for example, synthetic yeast Sc 2.0 project) and minimal genome (for example, SMC-encoding gene is one of 473 essential genes in the synthetic bacterium M. mycoides JCVI-syn1.0, Craig Venter 2016).

4. training of next-generation scientists and postdoctoral researcher directly employed in this project.

5. Research on ParB might result in applications in synthetic biology. ParB-parS systems have been widely exploited to label and image DNA loci in vivo, in both bacteria and eukaryotes. Recently, the ParB-parS system has been utilized in synthetic biology, for example, as part of a genetic circuit to enable asymmetric cell division in E. coli (Molinari et al Nature Chemical Biology 2019 PMID:31406375).
Sectors Chemicals

Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology
 
Description Research Fellows Enhanced Research Expenses
Amount £167,949 (GBP)
Funding ID RF\ERE\210039 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2022 
End 12/2023
 
Description Royal Society University Research Fellowship Renewal
Amount £483,734 (GBP)
Funding ID URF\R\201020 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2021 
End 12/2023
 
Description Wellcome Trust Investigator Awards
Amount £1,271,158 (GBP)
Funding ID 221776/Z/20/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 05/2021 
End 06/2026