Three genes controlling streptomycin susceptibility in Agrobacterium fabrum.

Streptomycin (Sm) is a commonly used antibiotic for its efficacy against diverse bacteria. The plant pathogen Agrobacterium fabrum is a model for studying pathogenesis and interkingdom gene transfer. Streptomycin-resistant variants of A. fabrum are commonly employed in genetic analyses, yet mechanisms of resistance and susceptibility to streptomycin in this organism have not previously been investigated. We observe that resistance to a high concentration of streptomycin arises at high frequency in A. fabrum, and we attribute this trait to the presence of a chromosomal gene (strB) encoding a putative aminoglycoside phosphotransferase. We show how strB, along with rpsL (encoding ribosomal protein S12) and rsmG (encoding a 16S rRNA methyltransferase), modulates streptomycin sensitivity in A. fabrum. IMPORTANCE The plant pathogen Agrobacterium fabrum is a widely used model bacterium for studying biofilms, bacterial motility, pathogenesis, and gene transfer from bacteria to plants. Streptomycin (Sm) is an aminoglycoside antibiotic known for its broad efficacy against gram-negative bacteria. A. fabrum exhibits endogenous resistance to somewhat high levels of streptomycin, but the mechanism underlying this resistance has not been elucidated. Here, we demonstrate that this resistance is caused by a chromosomally encoded streptomycin-inactivating enzyme, StrB, that has not been previously characterized in A. fabrum. Furthermore, we show how the genes rsmG, rpsL, and strB jointly modulate streptomycin susceptibility in A. fabrum.

Esters of Glucose-2-Phosphate: Occurrence and Chemistry.

Phosphodiesters of glucose-2-phosphate (G2P) are found only in few natural compounds such as agrocinopine D and agrocin 84. Agrocinopine D is a G2P phosphodiester produced by plants infected by Agrobacterium fabrum C58 and recognized by the bacterial periplasmic binding protein AccA for being transported into the bacteria before cleavage by the phosphodiesterase AccF, releasing G2P, which promotes virulence by binding the repressor protein AccR. The G2P amide agrocin 84 is a natural antibiotic produced by the non-pathogenic Agrobacterium radiobacter K84 strain used as a biocontrol agent by competing with Agrobacterium fabrum C58. G2P esters are also found in irregular glycogen structures. The rare glucopyranosyl-2-phophoryl moiety found in agrocin 84 is the key structural signature enabling its action as a natural antibiotic. Likewise, G2P and G2P esters can also dupe the Agrobacterium agrocinopine catabolism cascade. Such observations illustrate the importance of G2P esters on which we have recently focused our interest. After a brief review of the reported phosphorylation coupling methods and the choice of carbohydrate building blocks used in G2P chemistry, a flexible access to glucose-2-phosphate esters using the phosphoramidite route is proposed.

The plant defense signal galactinol is specifically used as a nutrient by the bacterial pathogen Agrobacterium fabrum.

The bacterial plant pathogen Agrobacterium fabrum uses periplasmic-binding proteins (PBPs) along with ABC transporters to import a wide variety of plant molecules as nutrients. Nonetheless, how A. fabrum acquires plant metabolites is incompletely understood. Using genetic approaches and affinity measurements, we identified here the PBP MelB and its transporter as being responsible for the uptake of the raffinose family of oligosaccharides (RFO), which are the most widespread d-galactose-containing oligosaccharides in higher plants. We also found that the RFO precursor galactinol, recently described as a plant defense molecule, is imported into Agrobacterium via MelB with nanomolar range affinity. Structural analyses and binding mode comparisons of the X-ray structures of MelB in complex with raffinose, stachyose, galactinol, galactose, and melibiose (a raffinose degradation product) revealed how MelB recognizes the nonreducing end galactose common to all these ligands and that MelB has a strong preference for a two-unit sugar ligand. Of note, MelB conferred a competitive advantage to A. fabrum in colonizing the rhizosphere of tomato plants. Our integrative work highlights the structural and functional characteristics of melibiose and galactinol assimilation by A. fabrum, leading to a competitive advantage for these bacteria in the rhizosphere. We propose that the PBP MelB, which is highly conserved among both symbionts and pathogens from Rhizobiace family, is a major trait in these bacteria required for early steps of plant colonization.

Agrobacterium fabrum gene atu1420 regulates the pathogenicity by affecting the degradation of growth- and virulence-associated phenols.

Agrobacterium fabrum is a phytopathogen that causes the crown gall disease. Some plant-derived molecules, e.g. phenols, directly affect A. fabrum-plant interactions. Here, we characterize a phenolic catabolism-related gene, atu1420, that affects the pathogenicity of A. fabrum. Atu1420 is predicted to be an O-demethylase with high structural homology to Sphingomonas paucimobilis LigM. The HPLC-UV analysis showed that atu1420 affected the degradation of acetosyringone (AS). The deletion of atu1420 gene significantly enhanced the AS-induced virulence (vir) gene expression. atu1420 was shown to relieve the inhibitory effect of vanillic acid on the AS-induced vir gene expression and the growth of A. fabrum. The expression of atu1420 and the degradation of AS in A. fabrum C58 was up-regulated by the addition of indole acetic acid (IAA). The inhibitory effect of IAA on the AS-induced vir gene expression was partially relieved by the deletion of atu1420 gene, indicating that accelerating the degradation of AS is one of the ways that IAA inhibits vir genes induction. Furthermore, atu1420 mutant produced more pronounced tumors on kalanchoe leaves than the wild-type strain. These findings reveal the role of atu1420 in A. fabrum-host interactions and will broaden our understanding of the regulatory network of the interactions.

Pan-Chromosome and Comparative Analysis of Agrobacterium fabrum Reveal Important Traits Concerning the Genetic Diversity, Evolutionary Dynamics, and Niche Adaptation of the Species.

Agrobacterium fabrum has been critical for the development of plant genetic engineering and agricultural biotechnology due to its ability to transform eukaryotic cells. However, the gene composition, evolutionary dynamics, and niche adaptation of this species is still unknown. Therefore, we established a comparative genomic analysis based on a pan-chromosome data set to evaluate the genetic diversity of A. fabrum. Here, 25 A. fabrum genomes were selected for analysis by core genome phylogeny combined with the average nucleotide identity (ANI), amino acid identity (AAI), and in silico DNA-DNA hybridization (DDH) values. An open pan-genome of A. fabrum exhibits genetic diversity with variable accessorial genes as evidenced by a consensus pan-genome of 12 representative genomes. The genomic plasticity of A. fabrum is apparent in its putative sequences for mobile genetic elements (MGEs), limited horizontal gene transfer barriers, and potentially horizontally transferred genes. The evolutionary constraints and functional enrichment in the pan-chromosome were measured by the Clusters of Orthologous Groups (COG) categories using eggNOG-mapper software, and the nonsynonymous/synonymous rate ratio (dN/dS) was determined using HYPHY software. Comparative analysis revealed significant differences in the functional enrichment and the degree of purifying selection between the core genome and non-core genome. We demonstrate that the core gene families undergo stronger purifying selection but have a significant bias to contain one or more positively selected sites. Furthermore, although they shared similar genetic diversity, we observed significant differences between chromosome 1 (Chr I) and the chromid in their functional features and evolutionary constraints. We demonstrate that putative genetic elements responsible for plant infection, ecological adaptation, and speciation represent the core genome, highlighting their importance in the adaptation of A. fabrum to plant-related niches. Our pan-chromosome analysis of A. fabrum provides comprehensive insights into the genetic properties, evolutionary patterns, and niche adaptation of the species. IMPORTANCE Agrobacterium spp. live in diverse plant-associated niches such as soil, the rhizosphere, and vegetation, which are challenged by multiple stressors such as diverse energy sources, plant defenses, and microbial competition. They have evolved the ability to utilize diverse resources, escape plant defenses, and defeat competitors. However, the underlying genetic diversity and evolutionary dynamics of Agrobacterium spp. remain unexplored. We examined the phylogeny and pan-genome of A. fabrum to define intraspecies evolutionary relationships. Our results indicate an open pan-genome and numerous MGEs and horizontally transferred genes among A. fabrum genomes, reflecting the flexibility of the chromosomes and the potential for genetic exchange. Furthermore, we observed significant differences in the functional features and evolutionary constraints between the core and accessory genomes and between Chr I and the chromid, respectively.

The Only Chemoreceptor Encoded by che Operon Affects the Chemotactic Response of Agrobacterium to Various Chemoeffectors.

Chemoreceptor (also called methyl-accepting chemotaxis protein, MCP) is the leading signal protein in the chemotaxis signaling pathway. MCP senses and binds chemoeffectors, specifically, and transmits the sensed signal to downstream proteins of the chemotaxis signaling system. The genome of Agrobacterium fabrum (previously, tumefaciens) C58 predicts that a total of 20 genes can encode MCP, but only the MCP-encoding gene atu0514 is located inside the che operon. Hence, the identification of the exact function of atu0514-encoding chemoreceptor (here, named as MCP514) will be very important for us to understand more deeply the chemotaxis signal transduction mechanism of A. fabrum. The deletion of atu0514 significantly decreased the chemotactic migration of A. fabrum in a swim plate. The test of atu0514-deletion mutant (Δ514) chemotaxis toward single chemicals showed that the deficiency of MCP514 significantly weakened the chemotactic response of A. fabrum to four various chemicals, sucrose, valine, citric acid and acetosyringone (AS), but did not completely abolish the chemotactic response. MCP514 was localized at cell poles although it lacks a transmembrane (TM) region and is predicted to be a cytoplasmic chemoreceptor. The replacement of residue Phe328 showed that the helical structure in the hairpin subdomain of MCP514 is a direct determinant for the cellular localization of MCP514. Single respective replacements of key residues indicated that residues Asn336 and Val353 play a key role in maintaining the chemotactic function of MCP514.

The Divergent Key Residues of Two Agrobacterium fabrum (tumefaciens) CheY Paralogs Play a Key Role in Distinguishing Their Functions.

The chemotactic response regulator CheY, when phosphorylated by the phosphoryl group from phosphorylated CheA, can bind to the motor switch complex to control the flagellar motor rotation. Agrobacterium fabrum (previous name: Agrobacterium tumefaciens), a phytopathogen, carries two paralogous cheY genes, cheY1 and cheY2. The functional difference of two paralogous CheYs remains unclear. Three cheY-deletion mutants were constructed to test the effects of two CheYs on the chemotaxis of A.fabrum. Phenotypes of three cheY-deletion mutants show that deletion of each cheY significantly affects the chemotactic response, but cheY2-deletion possesses more prominent effects on the chemotactic migration and swimming pattern of A. fabrum than does cheY1-deletion. CheA-dependent cellular localization of two CheY paralogs and in vitro pull-down of two CheY paralogs by FliM demonstrate that the distinct roles of two CheY paralogs arise mainly from the differentiation of their binding affinities for the motor switch component FliM, agreeing with the divergence of the key residues on the motor-binding surface involved in the interaction with FliM. The single respective replacements of key residues R93 and A109 on the motor-binding surface of CheY2 by alanine (A) and valine (V), the corresponding residues of CheY1, significantly enhanced the function of CheY2 in regulating the chemotactic response of A. fabrum CheY-deficient mutant Δy to nutrient substances and host attractants. These results conclude that the divergence of the key residues in the functional subdomain is the decisive factor of functional differentiation of these two CheY homologs and protein function may be improved by the substitution of the divergent key residues in the functional domain for the corresponding residues of its paralogs. This finding will help us to better understand how paralogous proteins sub-functionalize. In addition, the acquirement of two CheY2 variants, whose chemotactic response functions are significantly improved, will be very useful for us to further explore the mechanism of CheY to bind and regulate the flagellar motor and the role of chemotaxis in the pathogenicity of A. fabrum.

Agrobacterium fabrum atu0526-Encoding Protein Is the Only Chemoreceptor That Regulates Chemoattraction toward the Broad Antibacterial Agent Formic Acid.

Soil-born plant pathogens, especially Agrobacterium, generally navigate their way to hosts through recognition of the root exudates by chemoreceptors. However, there is still a lack of appropriate identification of chemoreceptors and their ligands in Agrobacterium. Here, Atu0526, a sCache-type chemoreceptor from Agrobacterium fabrum C58, was confirmed as the receptor of a broad antibacterial agent, formic acid. The binding of formic acid to Atu0526 was screened using a thermo shift assay and verified using isothermal titration calorimetry. Inconsistent with the previously reported antimicrobial properties, formic acid was confirmed to be a chemoattractant to A. fabrum and could promote its growth. The chemotaxis of A. fabrum C58 toward formic acid was completely lost with the knock-out of atu0526, and regained with the complementation of the gene, indicating that Atu0526 is the only chemoreceptor for formic acid in A. fabrum C58. The affinity of formic acid to Atu0526LBD significantly increased after the arginine at position 115 was replaced by alanine. However, in vivo experiments showed that the R115A mutation fully abolished the chemotaxis of A. fabrum toward formic acid. Molecular docking based on a predicted 3D structure of Atu0526 suggested that the arginine may provide "an anchorage" for formic acid to pull the minor loop, thereby forming a conformational change that generates the ligand-binding signal. Collectively, our findings will promote an understanding of sCache-type chemoreceptors and their signal transduction mechanism.

Evidence for weak interaction between phytochromes Agp1 and Agp2 from Agrobacterium fabrum.

During bacterial conjugation, plasmid DNA is transferred from cell to cell. In Agrobacterium fabrum, conjugation is regulated by the phytochrome photoreceptors Agp1 and Agp2. Both contribute equally to this regulation. Agp1 and Agp2 are histidine kinases, but, for Agp2, we found no autophosphorylation activity. A clear autophosphorylation signal, however, was obtained with mutants in which the phosphoaccepting Asp of the C-terminal response regulator domain is replaced. Thus, the Agp2 histidine kinase differs from the classical transphosphorylation pattern. We performed size exclusion, photoconversion, dark reversion, autophosphorylation, chromophore assembly kinetics and fluorescence resonance energy transfer measurements on mixed Agp1/Agp2 samples. These assays pointed to an interaction between both proteins. This could partially explain the coaction of both phytochromes in the cell.