Ancient co-option of an amino acid ABC transporter locus in Pseudomonas syringae for host signal-dependent virulence gene regulation

Abstract

Pathogenic bacteria frequently acquire virulence traits via horizontal gene transfer, yet additional evolutionary innovations may be necessary to integrate newly acquired genes into existing regulatory pathways. The plant bacterial pathogen Pseudomonas syringae relies on a horizontally acquired type III secretion system (T3SS) to cause disease. T3SS-encoding genes are induced by plant-derived metabolites, yet how this regulation occurs, and how it evolved, is poorly understood. Here we report that the two-component system AauS-AauR and substrate-binding protein AatJ, proteins encoded by an acidic amino acid-transport (aat) and-utilization (aau) locus in P. syringae, directly regulate T3SS-encoding genes in response to host aspartate and glutamate signals. Mutants of P. syringae strain DC3000 lacking aauS, aauR or aatJ expressed lower levels of T3SS genes in response to aspartate and glutamate, and had decreased T3SS deployment and virulence during infection of Arabidopsis. We identified an AauR-binding motif (Rbm) upstream of genes encoding T3SS regulators HrpR and HrpS, and demonstrated that this Rbm is required for maximal T3SS deployment and virulence of DC3000. The Rbm upstream of hrpRS is conserved in all P. syringae strains with a canonical T3SS, suggesting AauR regulation of hrpRS is ancient. Consistent with a model of conserved function, an aauR deletion mutant of P. syringae strain B728a, a bean pathogen, had decreased T3SS expression and growth in host plants. Together, our data suggest that, upon acquisition of T3SS-encoding genes, a strain ancestral to P. syringae co-opted an existing AatJ-AauS-AauR pathway to regulate T3SS deployment in response to specific host metabolite signals.

Signal transduction schemes in Pseudomonas syringae

“…In the presence of sugars, a cluster of amino acids from plant extracts are capable of inducing the expression of T3SS genes in the Pto DC3000 strain [62] . A recent Tn 5 transposon mutagenesis screening study identifies that the TCS AauRS (encoded by the amino acid uptake locus) and the substrate-binding protein AatJ together activate the transcription of T3SS genes by sensing acidic amino acid signals in host cells [63] . AauRS activates the transcription of the adjacent aatJQMP operon in the presence of external acidic amino acid signals [63] .…”

“…A recent Tn 5 transposon mutagenesis screening study identifies that the TCS AauRS (encoded by the amino acid uptake locus) and the substrate-binding protein AatJ together activate the transcription of T3SS genes by sensing acidic amino acid signals in host cells [63] . AauRS activates the transcription of the adjacent aatJQMP operon in the presence of external acidic amino acid signals [63] . Among 15 amino acids tested in ligand profile, l -Asp and l -Glu show the highest levels of induction of the aatJ promoter [63] .…”

“…AauRS activates the transcription of the adjacent aatJQMP operon in the presence of external acidic amino acid signals [63] . Among 15 amino acids tested in ligand profile, l -Asp and l -Glu show the highest levels of induction of the aatJ promoter [63] . Interestingly, l -Asp and l -Glu are the ligands of MCP PSPTO_2480 in Pto DC3000, which assist Pto DC3000 to invade apoplasts of tomato leaves (see details in Section 5 ) [64] .…”

“…The response regulator AauR binds to an AauR-binding motif (TTCGG-N 4 -CCGAA) in the promoter of the hrpRS operon, thus directly activating its transcription and promoting bacterial virulence in Arabidopsis ( Fig. 1 A, process 6) [63] . The AauR-binding motif is highly conserved in the hrpRS promoter sequences among 17 bacterial strains with a canonical T3SS, indicating that this activation function is ancient [63] .…”

“…1 A, process 6) [63] . The AauR-binding motif is highly conserved in the hrpRS promoter sequences among 17 bacterial strains with a canonical T3SS, indicating that this activation function is ancient [63] . Similar AauR regulation of hrpRS transcription and virulence has also been shown in the Pss B728a strain [63] .…”