Selection and characterization of Spiroplasma citri mutants by random transposome mutagenesis

Area of Science:

• Microbiology
• Genetics
• Plant Pathology

Background:

• Phytopathogenic spiroplasmas exhibit a dual-host life cycle, infecting both plants and insects.
• Understanding spiroplasma genes is crucial for deciphering their complex life cycle and pathogenicity.
• Random mutagenesis is a key strategy for identifying essential genes in microorganisms.

Purpose of the Study:

• To evaluate the efficacy of the EZ::TN™ Tnp transposome™ system for generating random insertional mutations in Spiroplasma citri.
• To establish a reliable method for genetic manipulation of the fastidious spiroplasma species.

Main Methods:

• Electroporation-mediated transformation was used to introduce the EZ::TN™ transposome™ into Spiroplasma citri BR3-3X.
• Selection of transformants was performed on agar plates, followed by broth culture for growth assessment.
• Polymerase Chain Reaction (PCR), Southern blot hybridization, and nucleotide sequencing were employed to confirm transposon insertion and analyze its genomic location.

Main Results:

• The EZ::TN™ transposome™ system demonstrated efficient transformation of Spiroplasma citri.
• While initial growth of transformants was impaired, optimizing broth volume facilitated robust growth.
• Transposon insertions were confirmed to be random, stable, and primarily single-site integrations within the chromosome.
• All mutants retained the ability to adhere to the Circulifer tenellus (CT-1) cell line.

Conclusions:

• The EZ::TN™ Tnp transposome™ system is a viable tool for random insertional mutagenesis in Spiroplasma citri.
• This system expands the genetic manipulation toolkit for studying fastidious spiroplasmas.
• Further research can utilize this system to identify genes critical for spiroplasma survival and pathogenicity in their dual hosts.