Identification of the Natural Transformation Genes in Riemerella anatipestifer by Random Transposon Mutagenesis.

Shun Chen,Sai Mao,Renyong Jia,Qun Gao,Aparna Viswanathan Ammanath,Friedrich Götz,Bin Tian,Shaqiu Zhang,Mingshu Wang,Dekang Zhu,Li Huang,Mafeng Liu,Qiao Yang,Xinxin Zhao,Di Sun,Ying Wu,Xumin Ou,Juan Huang,Anchun Cheng

doi:10.3389/fmicb.2021.712198

Abstract

In our previous study, it was shown that Riemerella anatipestifer, a Gram-negative bacterium, is naturally competent, but the genes involved in the process of natural transformation remain largely unknown. In this study, a random transposon mutant library was constructed using the R. anatipestifer ATCC11845 strain to screen for the genes involved in natural transformation. Among the 3000 insertion mutants, nine mutants had completely lost the ability of natural transformation, and 14 mutants showed a significant decrease in natural transformation frequency. We found that the genes RA0C_RS04920, RA0C_RS04915, RA0C_RS02645, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, RA0C_RS09020, and RA0C_RS04870 are essential for the occurrence of natural transformation in R. anatipestifer ATCC11845. In particular, RA0C_RS04895, RA0C_RS05130, RA0C_RS05105, and RA0C_RS04870 were putatively annotated as ComEC, DprA, ComF, and RecA proteins, respectively, in the NCBI database. However, RA0C_RS02645, RA0C_RS04920, RA0C_RS04915, and RA0C_RS09020 were annotated as proteins with unknown function, with no homology to any well-characterized natural transformation machinery proteins. The homologs of these proteins are mainly distributed in the members of Flavobacteriaceae. Taken together, our results suggest that R. anatipestifer encodes a unique natural transformation machinery.

Highlights

The evolution of the bacterial genome is driven by three primary modes of horizontal gene transfer, namely, phage transduction, conjugation, and natural transformation (Soucy et al, 2015)
Riemerella anatipestifer (RA) was grown at 37°C in trypticase soy broth (TSB), on LB agar supplemented with 5% sheep blood, in GC broth (GCB), or on GCB agar plates (Liu et al, 2017)
Erm and Gen resistances were used as selectable markers for transconjugants, since RA ATCC11845 is sensitive to erythromycin but resistant to gentamycin, while E. coli S17-1 λpir is sensitive to gentamycin

Summary

Introduction

The evolution of the bacterial genome is driven by three primary modes of horizontal gene transfer, namely, phage transduction, conjugation, and natural transformation (Soucy et al, 2015). Natural transformation was first discovered in the Gram-positive bacterium Streptococcus pneumoniae in 1928 by Griffith (Antonova and Hammer, 2015). More than 82 bacterial species have been reported to be naturally transformable (Johnston et al, 2014; Liu et al, 2017). In the case of Gram-negative bacteria, the natural transformation process of Haemophilus influenzae (Dougherty and Smith, 1999), Neisseria (Sun et al, 2005), and Vibrio cholerae (Seitz and Blokesch, 2013) has been well characterized. In V. cholerae, for example, the DNA uptake machinery is composed of the type IV pilus (PilA, PilC, PilM, PilN, PilO, and PilQ, and the pilotin protein PilF), the ATPases PilT and PilB, the periplasmic DNA-binding protein ComEA, and the inner-membrane translocator proteins ComEC and ComF (Matthey and Blokesch, 2016)

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