A central role for the transcriptional regulator VtlR in small RNA-mediated gene regulation in\xa0Agrobacterium tumefaciens

James A Budnick,Li Luo,Miranda J Ginder,Lauren M Sheehan,Kevin C Failor,Julia M Perkowski,Jason E Heindl,Lin Kang,Clayton C Caswell,Kirsten A Kohl,John F Pinto,Pawel Michalak

doi:10.1038/s41598-020-72117-0

Abstract

LysR-type transcriptional regulators (LTTRs) are the most common type of transcriptional regulators in prokaryotes and function by altering gene expression in response to environmental stimuli. In the class Alphaproteobacteria, a conserved LTTR named VtlR is critical to the establishment of host-microbe interactions. In the mammalian pathogen Brucella abortus, VtlR is required for full virulence in a mouse model of infection, and VtlR activates the expression of abcR2, which encodes a small regulatory RNA (sRNA). In the plant symbiont Sinorhizobium meliloti, the ortholog of VtlR, named LsrB, is involved in the symbiosis of the bacterium with alfalfa. Agrobacterium tumefaciens is a close relative of both B. abortus and S. meliloti, and this bacterium is the causative agent of crown gall disease in plants. In the present study, we demonstrate that VtlR is involved in the ability of A. tumefaciens to grow appropriately in artificial medium, and an A. tumefaciens vtlR deletion strain is defective in motility, biofilm formation, and tumorigenesis of potato discs. RNA-sequencing analyses revealed that more than 250 genes are dysregulated in the ∆vtlR strain, and importantly, VtlR directly controls the expression of three sRNAs in A. tumefaciens. Taken together, these data support a model in which VtlR indirectly regulates hundreds of genes via manipulation of sRNA pathways in A. tumefaciens, and moreover, while the VtlR/LsrB protein is present and structurally conserved in many members of the Alphaproteobacteria, the VtlR/LsrB regulatory circuitry has diverged in order to accommodate the unique environmental niche of each organism.

Highlights

LysR-type transcriptional regulators (LTTRs) are well represented in the three domains of life, and encompass the most common type of transcriptional regulator in prokaryotes[1]
Northern blot analysis revealed no change in the expression of the AbcRs in the absence of lsrB; indicating that lsrB does not regulate the expression of AbcR1 or AbcR2 in S. meliloti (Fig. S1)
This work further characterizes the regulatory capabilities of the AbcR small regulatory RNA (sRNA) in Agrobacterium pathogenesis (Fig. 3; Supplementary Tables S1, S2), as well as a novel sRNA called VrsA (Supplementary Fig. S3; Supplementary Table S6)

Summary

Introduction

LysR-type transcriptional regulators (LTTRs) are well represented in the three domains of life, and encompass the most common type of transcriptional regulator in prokaryotes[1]. LTTRs can undergo conformational changes without binding to a substrate This is the case with OxyR, a redox-sensing LTTR responsible for activating genes important for responding to reactive oxygen s pecies[16,17]. LTTRs play important regulatory roles in bacteria, allowing organisms to sense environmental cues and, in turn, swiftly alter gene expression through transcriptional activation and/or repression. The AbcR sRNAs regulate target mRNAs encoding ABC-type transport systems, many of which are responsible for transporting nutrients in specific environmental c onditions[23,24,25,26,27,28,29] Several of these mRNA targets encode transport systems found in A. tumefaciens, S. meliloti and B. abortus, and have been shown to be regulated by one or both of the AbcR sRNAs. Several of these mRNA targets encode transport systems found in A. tumefaciens, S. meliloti and B. abortus, and have been shown to be regulated by one or both of the AbcR sRNAs It was unknown if the conservation of the AbcR system includes the transcriptional regulation of the sRNAs by VtlR/LsrB

Methods

Results

Conclusion