Presentation Information
[2EMT-06]Reorganization of the host bacterial chromosome caused by the acquisition of the catabolic plasmid pCAR1
○Chiho Suzuki-Minakuchi1,2, Hideaki Nojiri1,2 (1. Grad. Sch. Agric. Life Sci., UTokyo (Japan), 2. CRIIM, UTokyo (Japan))
Keywords:
Plasmid,Pseudomonas,Nucleoid-associated proteins,H-NS
[Purpose]
H-NS family proteins, a type of nucleoid-associated protein, are crucial regulators of horizontally acquired genes in bacteria. These proteins have an N-terminal oligomerization domain and a C-terminal DNA-binding domain, allowing oligomer formation along DNA. H-NS family proteins encoded on both large transmissible plasmids and chromosomes play a vital role in the successful integration of new genetic sequences into host regulatory networks. Pmr is an H-NS family protein encoded on the catabolic plasmid pCAR1. Previously, we investigated the functions of Pmr and chromosomally encoded H-NS family proteins (TurA and TurB) in Pseudomonas putida KT2440. RNA-seq analysis revealed that the chromosomal genes differentially transcribed after turA or turB deletion in KT2440(pCAR1) differed from those in KT2440. We hypothesized that the binding sites of TurA and TurB change after pCAR1 carriage. To test this hypothesis, we performed ChIP-seq (chromatin immunoprecipitation sequencing) to assess the binding sites of the H-NS family proteins in KT2440 and KT2440(pCAR1). Additionally, 3C-seq (chromosome conformation capture sequencing) was performed to assess chromosomal conformational changes before and after pCAR1 carriage.
[Method]
KT2440 and KT2440(pCAR1) were grown at 30°C in a mineral medium supplemented with 0.1% (w/v) succinate as the carbon source. Cells were collected during the log and stationary phases. After formaldehyde cross-linking, the cells were lysed for ChIP. Antibodies for ChIP were prepared using synthesized peptides corresponding to residues 61–77 of TurA, residues 12–29 of TurB, and heterologously expressed Pmr as antigens. For 3C-seq, DNA was digested with Sau3AI after formaldehyde cross-linking, ligated, and sequenced.
[Results and Discussion]
ChIP-seq revealed that the binding regions of each H-NS family protein occupied approximately 5% of the total chromosomal length. TurA occupied 20% of pCAR1, while TurB and Pmr occupied 30%, indicating their preferential binding to pCAR1. The total binding length of TurA and TurB in KT2440(pCAR1) was similar to that in KT2440, suggesting that TurA and TurB relocated from the chromosome to the plasmid due to pCAR1 carriage. TurA binding sites that disappeared after pCAR1 carriage were compensated for by TurB or Pmr, whereas those of TurB were not, implying distinct functions for TurA and TurB. 3C-seq detected no dynamic conformational change in the chromosome between KT2440 and KT2440(pCAR1). Additionally, no direct contact was found between pCAR1 and the chromosome, indicating that pCAR1 is located independently of the chromosome.
[Conclusion]
ChIP-seq revealed changes in TurA and TurB chromosomal binding sites between KT2440 and KT2440(pCAR1). These changes did not affect the general conformation of the host chromosome. The reconstitution of TurA and TurB binding sites may have caused transcriptional changes in host chromosomal genes after pCAR1 carriage.
H-NS family proteins, a type of nucleoid-associated protein, are crucial regulators of horizontally acquired genes in bacteria. These proteins have an N-terminal oligomerization domain and a C-terminal DNA-binding domain, allowing oligomer formation along DNA. H-NS family proteins encoded on both large transmissible plasmids and chromosomes play a vital role in the successful integration of new genetic sequences into host regulatory networks. Pmr is an H-NS family protein encoded on the catabolic plasmid pCAR1. Previously, we investigated the functions of Pmr and chromosomally encoded H-NS family proteins (TurA and TurB) in Pseudomonas putida KT2440. RNA-seq analysis revealed that the chromosomal genes differentially transcribed after turA or turB deletion in KT2440(pCAR1) differed from those in KT2440. We hypothesized that the binding sites of TurA and TurB change after pCAR1 carriage. To test this hypothesis, we performed ChIP-seq (chromatin immunoprecipitation sequencing) to assess the binding sites of the H-NS family proteins in KT2440 and KT2440(pCAR1). Additionally, 3C-seq (chromosome conformation capture sequencing) was performed to assess chromosomal conformational changes before and after pCAR1 carriage.
[Method]
KT2440 and KT2440(pCAR1) were grown at 30°C in a mineral medium supplemented with 0.1% (w/v) succinate as the carbon source. Cells were collected during the log and stationary phases. After formaldehyde cross-linking, the cells were lysed for ChIP. Antibodies for ChIP were prepared using synthesized peptides corresponding to residues 61–77 of TurA, residues 12–29 of TurB, and heterologously expressed Pmr as antigens. For 3C-seq, DNA was digested with Sau3AI after formaldehyde cross-linking, ligated, and sequenced.
[Results and Discussion]
ChIP-seq revealed that the binding regions of each H-NS family protein occupied approximately 5% of the total chromosomal length. TurA occupied 20% of pCAR1, while TurB and Pmr occupied 30%, indicating their preferential binding to pCAR1. The total binding length of TurA and TurB in KT2440(pCAR1) was similar to that in KT2440, suggesting that TurA and TurB relocated from the chromosome to the plasmid due to pCAR1 carriage. TurA binding sites that disappeared after pCAR1 carriage were compensated for by TurB or Pmr, whereas those of TurB were not, implying distinct functions for TurA and TurB. 3C-seq detected no dynamic conformational change in the chromosome between KT2440 and KT2440(pCAR1). Additionally, no direct contact was found between pCAR1 and the chromosome, indicating that pCAR1 is located independently of the chromosome.
[Conclusion]
ChIP-seq revealed changes in TurA and TurB chromosomal binding sites between KT2440 and KT2440(pCAR1). These changes did not affect the general conformation of the host chromosome. The reconstitution of TurA and TurB binding sites may have caused transcriptional changes in host chromosomal genes after pCAR1 carriage.
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