Characterization of <i>salA</i>, <i>syrF</i>, and <i>syrG</i> Genes and Attendant Regulatory Networks Involved in Plant Pathogenesis by <i>Pseudomonas syringae</i> pv. <i>syringae</i> B728a

<div><p><i>Pseudomonas syringae</i> pv. <i>syringae</i> B728a, causal agent of brown spot on bean, is an economically important plant pathogen that utilizes extracellular signaling to initiate a lifestyle change from an epiphyte to a pathogen. LuxR regulatory proteins play an important role in the transcriptional regulation of a variety of biological processes involving two-component signaling, quorum sensing, and secondary metabolism. Analysis of the B728a genome identified 24 LuxR-like proteins, three of which are encoded by <i>salA</i>, <i>syrF</i>, and <i>syrG</i> located adjacent to the syringomycin gene cluster. The LuxR-like proteins encoded by these three genes exhibit a domain architecture that places them in a subfamily of LuxR-like proteins associated with regulation of secondary metabolism in B728a. Deletion mutants of <i>salA</i>, <i>syrF</i>, and <i>syrG</i> failed to produce syringomycin and displayed reduction of virulence on bean. The transcriptional start sites of <i>salA</i>, <i>syrG</i>, and <i>syrF</i> were located 63, 235, and 498 bp upstream of the start codons, respectively, using primer extension analysis. The predicted -10/-35 promoter regions of <i>syrF</i> and <i>syrG</i> were confirmed using site-directed mutagenesis and GFP reporters that showed conserved promoter sequences around the -35 promoter region. Overexpression analysis and GFP reporters identified SyrG as an upstream transcriptional activator of <i>syrF</i>, where both SyrG and SyrF activate promoters of syringomycin biosynthesis genes. This study shows that <i>syrG</i> and <i>syrF</i> encode important transcriptional regulators of syringomycin biosynthesis genes.</p></div

Domain organization of LuxR proteins that are classified into four sub-families based on domain architecture and mechanism of regulatory activation.

<p><b>A.</b> GacA is a LuxR-like protein in <i>Pss</i> B728a part of a global signal transduction system characterized as having an N-terminal receiver domain activated by phosphorylated and an C-terminal HTH DNA-binding domain that is characteristic of the first sub-family of LuxR-like proteins. <b>B.</b> AhlR is part of quorum sensing system in <i>Pss</i> B728a with AhlI. It has an N-terminal auto-inducer binding domain where hexanoyl-homoserine lactone binds to activate transcription of <i>ahlI</i> and has a C-terminal HTH DNA-binding domain. This domain organization is typical of the second sub-family of LuxRs associated with quorum sensing. <b>C.</b> <i>Psyr_0993</i>, which has not been characterized in <i>Pss</i> B728a, shares homology to <i>malT</i> in <i>E</i>. <i>coli</i>. These genes encode a subfamily of LuxR-like proteins have an N-terminal AAA ATPase domain that requires ATP for transcriptional activation and has a C-terminal HTH DNA binding domain. <b>D.</b> SyrG, which has been implicated in virulence and syringomycin production in <i>Pss</i> B728a lacks any defined N-terminal regulatory domain and has a C-terminal HTH DNA binding domain. This domain organization is typically seen in the fourth subfamily of LuxR-like proteins, which have not been fully defined functionally. LuxR-like proteins characterized in this family of LuxRs have been associated with secondary metabolism in <i>Pss</i> B728a.</p

Pathogenicity assays to evaluate the contribution of <i>syrG</i> and <i>syrF</i> to virulence on bean.

<p>Bean leaves were inoculated by vacuum infiltration with bacterial suspensions containing 10⁷ CFU/cm² of either B728a, B728aΔ<i>syrG</i>, B728aΔ<i>syrF</i>, B728aΔ<i>syrGΔsyrF</i>, B728aΔ<i>gacS</i>, B728aΔ<i>syrG</i> (pKT::<i>syrG</i>), B728aΔ<i>syrF</i> (pKT::<i>syrF</i>), or B728aΔ<i>syrG</i>Δ<i>syrF</i> (pKT::<i>syrG</i>). Plants were maintained at room temperature in a growth chamber for 72 h. Necrotic lesion surface areas were calculated using ImageJ software. This experiment was performed in triplicate, and representative results are shown.</p

Analysis of the promoter regions of <i>syrG</i> (A) and <i>syrF</i> (B) by testing the effect deletion mutants have on the expression of <i>syrG</i>::<i>gfp</i> and <i>syrF</i>::<i>gfp</i> transcriptional fusions.

<p>All <i>gfp</i> constructs were electroporated into cells of <i>Pss</i> B728a to measure GFP fluorescence (AU). All measurements were averages over three technical replicates of three biological samples. Horizontal bars represent the standard error of the average values.</p

Effect of overexpression of N-terminal region (NTR) of SyrG and SyrF on syringomycin production in <i>Pss</i> B728a.

<p>Bacterial strains were grown on HMM for 4 days. Plates were oversprayed with <i>Geotrichum candidum</i> and incubated 24 h at 26°C to observe zones of inhibition indicative of syringomycin production. The experiment was repeated in triplicate.</p

Expression analysis in the apoplast of bean of genes encoding LuxR-like proteins in <i>Pss</i> B728a.

<p>The genes that encode proteins that are classified in the first subfamily of LuxR (Class I) are shown in the green and are typically associated with two component signal transduction systems. Shown in blue are genes that encode LuxR-like proteins implicated in quorum sensing based on domain architecture. Psyr_0993, which is shown as gray, is the only gene that encodes a protein characterized as a LAL or LuxR-like proteins that require ATP for activation. The final subfamily of LuxR-like proteins are encoded on genes shown in orange bars, which lack an N-terminal regulatory domain and are associated with secondary metabolism. Out of all 24 LuxR-like proteins found in the genome of <i>Pss</i> B728a, the genes encoding SyrG and SyrF are the most highly expressed in the apoplast when compared to HMM liquid medium. The values are represented as the average fold change of three technical replicates of three biological samples. Gene expression was normalized to the <i>16s-rRNA</i> and <i>recA</i> internal control genes. Vertical bars indicate standard errors of the average values over triplicate runs.</p

Bioassy to evaluate syringomycin production in parental strain B728a and derivative mutants.

<p>Bacterial strains were grown on HMM for 4 days. Plates were oversprayed with <i>Geotrichum candidum</i> and incubated 24 h at 26°C to observe zones of inhibition indicative of syringomycin production. The experiment was repeated in triplicate.</p

Alignment of <i>syrG</i> and <i>syrF</i> promoter sequences in <i>Pss</i> B728a.

<p>The predicted promoter sequences of <i>syrG</i> and <i>syrF</i> were aligned using T-COFFEE and conserved sites are shown as asterisks. The color code is based on CORE index, using consistency among pairwise alignments for estimating reliability. Sequences shown in red indicate high reliability, where green is indicative of low reliability.</p

Quantitative real-time PCR analysis of LuxR-like genes in Δ<i>salA</i>, Δ<i>syrG</i>, and Δ<i>syrF</i> mutants of <i>Pss</i> B728a.

<p>The values represent the average fold change in gene expression from parental strain <i>Pss</i> B728a; the results are the averages of three technical replicates from three biological samples grown in HMM liquid medium. Gene expression levels were normalized to <i>16s-rRNA</i> and <i>recA</i> internal control genes, and vertical bars indicate standard errors of the average values over triplicate runs. Negative values indicate a decrease in transcript abundance by taking the negative inverse of a fold change value less than 1.</p

Putative pilus assembly/fimbrial genes downstream of <i>ecf7</i> in B728a.^a

a<p>Percent amino acid identities of orthologs are shown in parentheses.</p