Journal of Microbiology

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Relaxed Cleavage Specificity of Hyperactive Variants of Escherichia coli RNase E on RNA I: Dayeong Bae , Hana Hyeon , Eunkyoung Shin , Ji&# , Kangseok Lee; J. Microbiol. 2023;61(2):211-220. Published online February 22, 2023; DOI: https://doi.org/10.1007/s12275-023-00013-z

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RNase E is an essential enzyme in Escherichia coli. The cleavage site of this single-stranded specific endoribonuclease is well-characterized in many RNA substrates. Here, we report that the upregulation of RNase E cleavage activity by a mutation that affects either RNA binding (Q36R) or enzyme multimerization (E429G) was accompanied by relaxed cleavage specificity. Both mutations led to enhanced RNase E cleavage in RNA I, an antisense RNA of ColE1-type plasmid replication, at a major site and other cryptic sites. Expression of a truncated RNA I with a major RNase E cleavage site deletion at the 5′-end (RNA I- 5) resulted in an approximately twofold increase in the steady-state levels of RNA I- 5 and the copy number of ColE1-type plasmid in E. coli cells expressing wild-type or variant RNase E compared to those expressing RNA I. These
results
indicate that RNA I- 5 does not efficiently function as an antisense RNA despite having a triphosphate group at the 5′-end, which protects the RNA from ribonuclease attack. Our study suggests that increased cleavage rates of RNase E lead to relaxed cleavage specificity on RNA I and the inability of the cleavage product of RNA I as an antisense regulator in vivo does not stem from its instability by having 5′-monophosphorylated end.

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Engineering an Escherichia coli based in vivo mRNA manufacturing platform
Edward Curry, George Muir, Jixin Qu, Zoltán Kis, Martyn Hulley, Adam Brown
Biotechnology and Bioengineering.2024; 121(6): 1912. CrossRef

Research Support, U.S. Gov't, Non-P.H.S.

Phenotypic Diversity of Escherichia coli O157:H7 Strains Associated with the Plasmid O157: Ji Youn Lim , Joon Bae Hong , Haiqing Sheng , Smriti Shringi , Rajinder Kaul , Thomas E. Besser , Carolyn J. Hovde; J. Microbiol. 2010;48(3):347-357. Published online June 23, 2010; DOI: https://doi.org/10.1007/s12275-010-9228-4

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Abstract: Escherichia coli O157:H7, a food-borne pathogen, causes hemorrhagic colitis and the hemolytic-uremic syndrome. A putative virulence factor of E. coli O157:H7 is a 60-MDa plasmid (pO157) found in 99% of all clinical isolates and many bovine-derived strains. The well characterized E. coli O157:H7 Sakai strain (Sakai) and its pO157-cured derivative (Sakai-Cu) were compared for phenotypic differences. Sakai-Cu had enhanced survival in synthetic gastric fluid, did not colonize cattle as well as wild-type Sakai, and had unchanged growth rates and tolerance to salt and heat. These results are consistent with our previous findings with another E. coli O157:H7 disease outbreak isolate ATCC 43894 and its pO157-cured (43894-Cu). However, despite the essentially sequence identical pO157 in these strains, Sakai-Cu had changes in antibiotic susceptibility and motility that did not occur in the 43894-Cu strain. This unexpected result was systematically analyzed using phenotypic microarrays testing 1,920 conditions with Sakai, 43894, and the plasmid-cured mutants. The influence of the pO157 differed between strains on a wide number of growth/survival conditions. Relative expression of genes related to acid resistance (gadA, gadX, and rpoS) and flagella production (fliC and flhD) were tested using quantitative real-time PCR and gadA and rpoS expression differed between Sakai-Cu and 43894-Cu. The strain-specific differences in phenotype that resulted from the loss of essentially DNA-sequence identical pO157 were likely due to the chromosomal genetic diversity between strains. The O157:H7 serotype diversity was further highlighted by phenotypic microarray comparisons of the two outbreak strains with a genotype 6 bovine E. coli O157:H7 isolate, rarely associated with human disease.

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