Dataset Information

Single-nucleotide resolution detection of Topo IV cleavage activity in the Escherichia coli genome with Topo-Seq.

ABSTRACT: Topoisomerase IV (Topo IV) is the main decatenation enzyme in Escherichia coli; it removes catenation links that are formed during DNA replication. Topo IV binding and cleavage sites were previously identified in the E. coli genome with ChIP-Seq and NorfIP. Here, we used a more sensitive, single-nucleotide resolution Topo-Seq procedure to identify Topo IV cleavage sites (TCSs) genome-wide. We detected thousands of TCSs scattered in the bacterial genome. The determined cleavage motif of Topo IV contained previously known cleavage determinants (-4G/+8C, -2A/+6 T, -1 T/+5A) and additional, not observed previously, positions -7C/+11G and -6C/+10G. TCSs were depleted in the Ter macrodomain except for two exceptionally strong non-canonical cleavage sites located in 33 and 38 bp from the XerC-box of the dif-site. Topo IV cleavage activity was increased in Left and Right macrodomains flanking the Ter macrodomain and was especially high in the 50-60 kb region containing the oriC origin of replication. Topo IV enrichment was also increased downstream of highly active transcription units, indicating that the enzyme is involved in relaxation of transcription-induced positive supercoiling.

SUBMITTER: Sutormin D

PROVIDER: S-EPMC10117906 | biostudies-literature | 2023

REPOSITORIES: biostudies-literature

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Single-nucleotide resolution detection of Topo IV cleavage activity in the Escherichia coli genome with Topo-Seq.

Sutormin Dmitry D Galivondzhyan Alina A Gafurov Azamat A Severinov Konstantin K

Frontiers in microbiology 20230406

Topoisomerase IV (Topo IV) is the main decatenation enzyme in Escherichia coli; it removes catenation links that are formed during DNA replication. Topo IV binding and cleavage sites were previously identified in the E. coli genome with ChIP-Seq and NorfIP. Here, we used a more sensitive, single-nucleotide resolution Topo-Seq procedure to identify Topo IV cleavage sites (TCSs) genome-wide. We detected thousands of TCSs scattered in the bacterial genome. The determined cleavage moti ...[more]

PMID: 37089538

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Project description:The objective of this study was to develop a canonical, parsimoniously-informative SNP panel for subtyping Shiga-toxin producing Escherichia coli (STEC) O157:H7 that would be consistent with epidemiological, PFGE, and MLVA clustering of human specimens. Our group had previously identified 906 putative discriminatory SNPs, which were pared down to 391 SNPs based on their prevalence in a test set. The 391 SNPs were screened using a high-throughput form of TaqMan PCR against a set of clinical isolates that represent the most diverse collection of O157:H7 isolates from outbreaks and sporadic cases examined to date. Another 30 SNPs identified by others were also screened using the same method. Two additional targets were tested using standard TaqMan PCR endpoint analysis. These 423 SNPs were reduced to a 32 SNP panel with the almost the same discriminatory value. While the panel partitioned our diverse set of isolates in a manner that was consistent with epidemiological data and PFGE and MLVA phylogenies, it resulted in fewer subtypes than either existing method and insufficient epidemiological resolution in 10 of 47 clusters. Therefore, another round of SNP discovery was undertaken using comparative genomic resequencing of pooled DNA from the 10 clusters with insufficient resolution. This process identified 4,040 potential SNPs and suggested one of the ten clusters was incorrectly grouped. After its removal, there were 2,878 SNPs, of which only 63 were previously identified and 438 occurred across multiple clusters. Among highly clonal bacteria like STEC O157:H7, linkage disequilibrium greatly limits the number of parsimoniously informative SNPs. Therefore, it is perhaps unsurprising that our panel accounted for the potential discriminatory value of numerous other SNPs reported in the literature. We concluded published O157:H7 SNPs are insufficient for effective epidemiological subtyping. However, the 438 multi-cluster SNPs we identified may provide the additional information required.

Dataset Information

Single-nucleotide resolution detection of Topo IV cleavage activity in the Escherichia coli genome with Topo-Seq.

Publications

Single-nucleotide resolution detection of Topo IV cleavage activity in the <i>Escherichia coli</i> genome with Topo-Seq.

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