Journal Article

Role of PumB antitoxin as a transcriptional regulator of the PumAB type-II toxin–antitoxin system and its endoribonuclease activity on the PumA (toxin) transcript

Hernández-Ramírez, K. C., Valle-Maldonado, M. I., Patiño-Medina, J. A., Calo, S., Garre, V., Ramírez-Díaz, M. I., Jácome-Galarza, I. E., Meza-Carmen, V.

2023

Overview

The PumAB type-II toxin–antitoxin (TA) system is encoded by pum AB genes that are organized into an operon. This system is encoded by the pUM505 plasmid, isolated from a Pseudomonas aeruginosa clinical strain. The pum A gene encodes a putative RelE toxin protein (toxic component), whereas the pum B gene encodes a putative HTH antitoxin protein. The expression of the PumAB system in Escherichia coli confers plasmid stability. In addition, PumA toxin overexpression in P. aeruginosa possesses the capability to increase bacterial virulence, an effect that is neutralized by the PumB antitoxin. The aim of this study was to establish the mechanism of regulation of the PumAB toxin–antitoxin system from pUM505. By an in silico analysis of the putative regulatory elements, we identified two putative internal promoters, P pumB and P pumB-AlgU (in addition to the already reported P pumAB ), located upstream of pum B. By RT-qPCR assays, we determined that the pum AB genes are transcribed differentially, in that the mRNA of pum B is more abundant than the pum A transcript. We also observed that pum B could be expressed individually and that its mRNA levels decreased under oxidative stress, during individual expression as well as co-expression of pum AB. However, under stressful conditions, the pum A mRNA levels were not affected. This suggests the negative regulation of pum B by stressful conditions. The PumB purified protein was found to bind to a DNA region located between the P pumAB and the pum A coding region, and PumA participates in PumB binding, suggesting that a PumA–PumB complex co-regulates the transcription of the pum AB operon. Interestingly, the pum A mRNA levels decreased after incubation in vitro with PumB protein. This effect was repressed by ribonuclease inhibitors, suggesting that PumB could function as an RNAse toward the mRNA of the toxin. Taken together, we conclude that the PumAB TA system possesses multiple mechanisms to regulate its expression, as well as that the PumB antitoxin generates a decrease in the mRNA toxin levels, suggesting an RNase function. Our analysis provides new insights into the understanding of the control of TA systems from mobile plasmid-encoded genes from a human pathogen.

• Publisher: Springer Berlin Heidelberg,Springer Nature B.V
• Subject: Animal Genetics and Genomics / animal pathogens / antitoxins / Antitoxins - genetics / Antitoxins - metabolism / Apoptosis Regulatory Proteins - metabolism / Bacterial Proteins - genetics / Bacterial Proteins - metabolism / Bacterial Toxins - genetics / Biochemistry / Biomedical and Life Sciences / computer simulation / Endoribonucleases - genetics / Endoribonucleases - metabolism / Escherichia coli / Escherichia coli - genetics / Escherichia coli - metabolism / Gene Expression Regulation, Bacterial / Genes / genomics / Human Genetics / Humans / Life Sciences / Microbial Genetics and Genomics / operon / Original Article / Oxidative stress / Plant Genetics and Genomics / plasmids / Proteins / Pseudomonas aeruginosa / Puma / Regulatory sequences / Ribonuclease / Ribonuclease inhibitors / ribonucleases / Ribonucleases - genetics / Ribonucleases - metabolism / RNA, Messenger / toxicity / Toxin-Antitoxin Systems - genetics / toxins / transcription factors / Virulence