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

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.

A LysR‐type transcriptional regulator controls the expression of numerous small RNAs in Agrobacterium tumefaciens

“…A similar pervasive role of the corresponding LTTR was reported in Brucella where the vtlR mutant was attenuated in macrophages and mice (Sheehan et al, 2015). The Agrobacterium homolog VtlR/LsrB is equally important for efficient attachment to and transformation of host plants finally resulting in tumor formation, as was shown by various assays and in different strains in the present and previous studies (Budnick et al, 2020;Tang et al, 2018). Other phenotypic defects of the vtlR/lsrB mutant include succinoglycan production, biofilm formation, and resistance to oxidative stress, iron limitation, sucrose, and various antibiotics.…”

“…Despite a stringent cutoff (p value of 0.05; at least threefold differences compared to WT), this number is much higher than in previous reports (Budnick et al, 2020;Tang et al, 2018) (Figure 6a), which might in part be due to different strains. Tang et al used the disarmed F I G U R E 3 Phenotypic characterization of the vtlR/lsrB mutant.…”

“…Promoter sequences upstream of the transcription start site (+1) are shown. Putative-binding sites for VtlR/LsrB in positively regulated genes are marked in green, whereas putative-binding sites in negatively regulated genes are labeled in yellow B. abortus or A. tumefaciens indeed resulted in drastically reduced expression of AbcR2 or AbcR1, respectively (Budnick et al, 2020;Sheehan et al, 2015). We recently reported that A. tumefaciens VtlR/LsrB controls the expression of seven genes coding for small arginine-rich proteins (Kraus et al, 2020).…”

“…Given the enormous number of genes in the VtlR/LsrB regulons of Sinorhizobium, Brucella, and Agrobacterium somewhere in the range of several hundreds to more than one thousand, it is currently unclear which specific LTTR targets are responsible for the observed growth and symbiosis/virulence defects. The successful heterologous complementation of some growth defects of vtlR/lsrB mutants with orthologs from other organisms (Budnick et al, 2020;Tang et al, 2018) suggests some common and some divergent functionalities of these LTTRs. 1 and 5), and of two other substantially regulated sRNAs with unknown function are indicated and some other proteins (Li et al, 2019).…”

Chemical ecology of an apex predator life cycle

“…For instance, LysR-type transcription factors, which are conserved across proteobacteria, are characterized by a conserved N -terminal DNA-binding domain and a C -terminal domain that varies among LysR-type regulator homologs. The latter domain is responsible for ligand metabolite binding and dictates the response specificity of the transcription factor {Budnick, 2020; Henikoff, 1988; Maddocks, 2008}. In a range of bacteria, LysR-type regulators modulate various phenotypes, including virulence, nutrient uptake and metabolic homeostasis, motility, quorum sensing, and antibiotic resistance {Abdelhamed, 2020}.…”