Whole Genome Sequencing and Tn 5-Insertion Mutagenesis of Pseudomonas taiwanensis CMS to Probe Its Antagonistic Activity Against Rice Bacterial Blight Disease

Abstract

The Gram-negative bacterium Pseudomonas taiwanensis is a novel bacterium that uses shrimp shell waste as its sole sources of carbon and nitrogen. It is a versatile bacterium with potential for use in biological control, with activities including toxicity toward insects, fungi, and the rice pathogen Xanthomonas oryzae pv.oryzae (Xoo). In this study, the complete 5.08-Mb genome sequence of P. taiwanensis CMS was determined by a combination of NGS/Sanger sequencing and optical mapping. Comparison of optical maps of seven Pseudomonas species showed that P. taiwanensis is most closely related to P. putida KT 2400. We screened a total of 11,646 individual Tn5-transponson tagged strains to identify genes that are involved in the production and regulation of the iron-chelator pyoverdine in P. taiwanensis, which is a key anti-Xoo factor. Our results indicated that the two-component system (TCS) EnvZ/OmpR plays a positive regulatory role in the production of pyoverdine, whereas the sigma factor RpoS functions as a repressor. The knowledge of the molecular basis of the regulation of pyoverdine by P. taiwanensis provided herein will be useful for its development for use in biological control, including as an anti-Xoo agent.

Keywords: Pseudomonas taiwanensis; Rice bacterial blight; Xanthomonas oryzae pv.oryzae (Xoo); biocontrol agent.

Figure 1

Circular representation of the P. taiwanensis genome (NCBI accession no. CP011858). The outermost circle shows the scale, with a resolution of 10 Kb. The genome map of P. taiwanensis starts with gene dnaA. Circles 1 and 2 show predicted coding regions color coded on the forward and reverse strands by COG assignment: red, RNA processing and modification; green, chromatin structure and dynamics; blue, energy production and conversion; purple, cell cycle control and mitosis; yellow, amino acid metabolism and transport; orange, nucleotide metabolism and transport; grey, carbohydrate metabolism and transport; dark red, coenzyme metabolism; dark green, lipid metabolism; dark blue, translation; dark purple, transcription; dark yellow, replication and repair; dark orange, cell wall/membrane/envelop biogenesis; dark grey, cell motility; light red, post-translational modification, protein turnover, chaperone functions; light green, inorganic ion transport and metabolism; light blue, secondary structure; light purple, general functional prediction only; light yellow, function unknown; light orange, signal transduction; light grey, intracellular trafficking and secretion; black, not in COGs. Circle 3 shows rRNA, tRNA, and ncRNA. Circle 4 shows two-component system envZ genes. Circle 5 shows GC skew in a 1000-bp window. Circles 6 and 7 show GC content (purple) and AT content (yellow) in a 1000-bp window, respectively.

Figure 2

Phylogenetic analysis of P. taiwanensis compared with representative Pseudomonas species and other anti-insect and anti-microbial bacteria. (A) Phylogenetic analysis of the BamH1 whole-genome optical map of P. taiwanensis compared to in silico BamH1-digestion maps of other Pseudomonas species based on the unweighted-pair group method with arithmetic averages (UPGMA). (B) Neighbor-joining (NJ) tree analysis of several representative Pseudomonas species and anti-insect or anti-microbial bacteria by multilocus sequence typing (MLST) based on seven housekeeping genes (rpoD, gyrB, acnB, cts, gap, pgi, and pfk). The branch support of the NJ tree is calculated using a set of 1000 bootstrap replicates and the p-distance method. The unit for branch length is substitutions/site. Gram-positive bacteria Bacilli are used as an outgroup.

Figure 3

Pairwise genome comparison based on the Artemis comparison tool (ACT). Visualization of comparisons between genomes uses Circos. Blue and purple lines are reverse orientation regions between genomes. Web-based implementation of the Artemis Comparison Tool (WebACT) with default values was used.

Figure 4

Venn diagram illustrating the distribution of gene families among three genomes, P. taiwanensis, P. putida KT2400, and P. entomophila L48. Homologous genes in P. taiwanensis, P. putida KT2400, and P. entomophila L48 were clustered into gene families. Each division of the Venn diagram shows orthoMCL groups and total number of clustered genes.

Figure 5

Clusters of orthologous groups (COG) analysis of P. taiwanensis involved in anti-Xoo activity. (A) A total of 913 genes were determined to have decreased anti-Xoo activity among 11,646 Tn5-inserted mutants (B) 19 genes were determined to have increased toxicity against Xoo. All genes were annotated by COG database. The number on the X axes represent the number of genes.

Figure 6

Pyoverdine secreted by type VI secretion system and regulated by EnvZ/OmpR. (A) We compared the capabilities of the wild type and 12 TCS mutants (bvgS, bvgA, envZ, atoS, gacS, zraR, glnG, dctD, gseC, liaS, creC, zraS) of P. taiwanensis against rice pathogen Xanthomonas oryzae pv. oryzae (Xoo). (B) Quantification of extracellular mature pyoverdine was achieved by measuring fluorescence at excitation 405 nm and emission 460 nm. Pyoverdine values were normalized against the cell optical densities (Ex405, Em 460/OD₆₀₀). Among the mutants being examined, only envZ::Tn5 mutant showed significantly lower pyoverdine production. * p < 0.05. (C) MALDI-IMS analysis reveals secreted pyoverdine concentration around wild-type and envZ mutant colonies. (D) Growth curves of wild type and envZ mutant were measured by optical density at 600 nm. Bacteria were grown for 24 h on iron-limited agar plates. Intensity gradients for pyoverdine by color histograms (maximum, white; minimum, black). Scale bar, 2 mm.

Figure 7

Negative regulation of pyoverdine by RpoS. (A) Wild type and rpoS mutant were incubated for 72 h at 28 °C with 200 rpm in iron-limited medium, and (B) numbers of CFU of wild type and rpoS mutant were measured during 72 h incubation. (C) In antagonistic assay, total broth of wild type and mutant after 72 h incubation were placed into the hole of Xoo-containg 1/2 agar plate, and then the inhibition zone was measured (cm). (D) MALDI-IMS of pyoverdine from wild type and rpoS mutant on the surface of an iron-limited agar plate after 72 h incubation. (E) Quantification of pyoverdine from culture supernatant of wild type and rpoS mutant after 72 h incubation using LTQ-Orbitrap mass spectrometer. Intensity gradients for pyoverdine as color histograms (maximum, white; minimum, black). Scale bar, 2 mm.

Figure 8

Proposed scheme for synthesis, secretion and regulation of pyoverdine.Pyoverdine precursors are synthesized in the cytoplasm and secreted into the periplasm by the inner membrane transporter PvdE, and processed into mature pyoverdine in the periplasmic space. T6SS is involved in the secretion of pyoverdine via an unknown mechanism. EnvZ/OmpR positively regulates pyoverdine production, whereas the sigma factor RpoS negatively affects pyoverdine production.