RNA-seq Analysis Reveals That an ECF σ Factor, AcsS, Regulates Achromobactin Biosynthesis in Pseudomonas syringae pv. syringae B728a

Abstract

Iron is an essential micronutrient for Pseudomonas syringae pv. syringae strain B728a and many other microorganisms; therefore, B728a has evolved methods of iron acquirement including the use of iron-chelating siderophores. In this study an extracytoplasmic function (ECF) sigma factor, AcsS, encoded within the achromobactin gene cluster is shown to be a major regulator of genes involved in the biosynthesis and secretion of this siderophore. However, production of achromobactin was not completely abrogated in the deletion mutant, implying that other regulators may be involved such as PvdS, the sigma factor that regulates pyoverdine biosynthesis. RNA-seq analysis identified 287 genes that are differentially expressed between the AcsS deletion mutant and the wild type strain. These genes are involved in iron response, secretion, extracellular polysaccharide production, and cell motility. Thus, the transcriptome analysis supports a role for AcsS in the regulation of achromobactin production and the potential activity of both AcsS and achromobactin in the plant-associated lifestyle of strain B728a.

Characterization of Five ECF Sigma Factors in the Genome of Pseudomonas syringae pv. syringae B728a

“…syringae strains occurred at 25°C in nutrient broth-yeast extract (NBY) liquid or agar medium [31], or on King’s B (KB) agar medium [32]. Assays for syringomycin production were conducted on Hrp minimal medium (HMM) agar [17], [33]. Mannitol glutamate-yeast extract (MGY) agar supplemented with 0.6 M sorbitol or MGY supplemented with 5% sucrose was used in assays for mucoidy [20].…”

“…Precise deletion mutants of five ECF σ factor genes, ecf5 (Psyr_1040), ecf7 (Psyr_1107), ecf6 (Psyr_4731), ecf11 (Psyr_0892), and ecf18 (Psyr_0362), were created in strain B728a using a modified [17], [38] Red recombinase deletion method [39]. This procedure utilizes homologous recombination in E. coli SW105 (http://recombineering.ncifcrf.gov), which is mediated by recombination proteins provided from a defective λ prophage inserted into the SW105 genome [40].…”

“…A limited number of ECF sigma factors, including HrpL, AlgU, SigX, PvdS, and AcsS, have been characterized in Pseudomonas [3], [10], [17]; the genome of P. syringae B728a encodes these five ECF sigma factor genes and five other ECF genes that remain uncharacterized [10]. HrpL activates the expression of the well-known hrp/hrc encoded type III secretion system and effector proteins that have a major role in plant pathogenicity of P. syringae [18], [19].…”

“…Another familiar ECF sigma factor, PvdS, is associated in fluorescent pseudomonads with the regulation of genes involved in the biosynthesis of the siderophore, pyoverdine [2], [25], [26]. Recently, Greenwald et al [17] described AcsS, an ECF sigma factor associated with controlling the biosynthesis and secretion of achromobactin, a second siderophore produced by P. syringae strain B728a.…”

“…rpoS , acsS , pvdS , and hrpL ) in P. syringae pv. syringae did not reveal a regulatory effect on expression of the phytotoxin genes [17], [30], a significant goal was to resolve whether the remaining uncharacterized ECF sigma factors included syringomycin production as a regulatory target. In addition to the three FecI-like ECF sigma factor genes, two ECF sigma factors placed in groups ECF11 and ECF18 [27] were characterized for their contributions to pathogen resistance to specific environmental stresses.…”