Identification of Pathogenicity-Related Genes in Biofilm-Defective Acidovorax citrulli by Transposon Tn5 Mutagenesis

Abstract

Biofilm formation is important for virulence of a large number of plant pathogenic bacteria. Indeed, some virulence genes have been found to be involved in the formation of biofilm in bacterial fruit blotch pathogen Acidovorax citrulli. However, some virulent strains of A. citrulli were unable to format biofilm, indicating the complexity between biofilm formation and virulence. In this study, virulence-related genes were identified in the biofilm-defective strain A1 of A. citrulli by using Tn5 insertion, pathogenicity test, and high-efficiency thermal asymmetric interlaced PCR (hiTAIL-PCR). Results from this study indicated that 22 out of the obtained 301 mutants significantly decreased the virulence of strain A1 compared to the wild-type. Furthermore, sequence analysis indicated that the obtained 22 mutants were due to the insertion of Tn5 into eight genes, including Aave 4244 (cation diffusion facilitator family transporter), Aave 4286 (hypothetical protein), Aave 4189 (alpha/beta hydrolase fold), Aave 1911 (IMP dehydrogenase/GMP reductase domain), Aave 4383 (bacterial export proteins, family 1), Aave 4256 (Hsp70 protein), Aave 0003 (histidine kinase, DNA gyrase B, and HSP90-like ATPase), and Aave 2428 (pyridoxal-phosphate dependent enzyme). Furthermore, the growth of mutant Aave 2428 was unaffected and even increased by the change in incubation temperature, NaCl concentration and the pH of the LB broth, indicating that this gene may be directly involved in the bacterial virulence. Overall, the determination of the eight pathogenicity-related genes in strain A1 will be helpful to elucidate the pathogenesis of biofilm-defective A. citrulli.

Light Modulates Virulence Factors in Strain Aac5 of Acidovorax citrulli

“…For example, Bahar et al (2011) reported that the fliC mutant of A. citrulli, which is involved in swimming motility, showed a significant reduction in virulence in seed transmission assays and stem and foliage inoculations, compared to the wild-type. Consistently, Luo reported that a biofilm-defective A. citrulli mutant showed reduced virulence [34]. Additionally, Wang et al [28] also reported that swimming motility and biofilm formation were important for the virulence of A. citrulli.…”

Identification of Genes Involved in Antifungal Activity of Burkholderia seminalis Against Rhizoctonia solani Using Tn5 Transposon Mutation Method

“…It has been well known that biofilm formation is a dynamic and complex process involving signal transduction systems, transcriptional regulation, and stress response, which make it important for both beneficial bacteria and plant pathogenic bacteria [ 15 , 35 ]. In agreement with the results from some other studies, most of the mutants significantly reduced biofilm formation, which may, at least partially, be attributed to the reduced antifungal activity against R. solani .…”

“…A library of Tn5 transposon mutants of B. seminalis R456 was constructed using the MA138E-EZ-Tn5-KAN-2 TNP Transposome Kit, which was carried out according to the manufacturer’s protocol (Epicentre Biotechnologies, Madison, WI, USA). The competent cells and electroporation transformation were prepared using a previously described method [ 15 ] with slight modifications. In brief, following precooling for 40 min, bacterial cells were suspended in 40 μL sterile water to obtain a final concentration of 1 × 10 10 CFU/mL, and then added to 1 μL of the EZ-Tn5 transposon (Epicentre Biotechnologies, Madison, WI, USA).…”

“…Bacterial growth was determined according to the method of [ 15 ] by measuring the OD600 values of the wild type and Tn5 insertion mutants of strain R456 using a microplate spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). In brief, after inoculating 50 μL of overnight-cultured bacterial suspension (OD600 = 1.0) into 5 mL of LB broth, the mixtures were then incubated at 30 °C, 200 rpm/min for 6, 12, 18 and 24 h, respectively.…”

“…Furthermore, this modified EZ-TN5 system is superior to other transposon systems in consistently delivering only a single copy per chromosome [ 14 ]. Indeed, using this technique, we have not only constructed the mutant library, but also have identified the virulence-related genes from the watermelon fruit blotch pathogen Acidovorax citrilli by evaluating the pathogenicity of each mutant to seedlings [ 15 ].…”