Enabling Efficient Genetic Manipulations in a Rare Actinomycete Pseudonocardia alni Shahu.

Jie Li,Han Si,Baiyang Wang,Wenfang Peng,Yanli Zheng,Yuting Zhao,Qing Yang

doi:10.3389/fmicb.2022.848964

Abstract

Pseudonocardia species are emerging as important microorganisms of global concern with unique and increasingly significant ecological roles and represent a prominent source of bioactive natural products, but genetic engineering of these organisms for biotechnological applications is greatly hindered due to the limitation of efficient genetic manipulation tools. In this regard, we report here the establishment of an efficient genetic manipulation system for a newly isolated strain, Pseudonocardia alni Shahu, based on plasmid conjugal transfer from Escherichia coli to Pseudonocardia. Conjugants were yielded upon determining the optimal ratio between the donor and recipient cells, and designed genome modifications were efficiently accomplished, including exogenous gene integration based on an integrative plasmid and chromosomal stretch removal by homologous recombination using a suicidal non-replicating vector. Collectively, this work has made the P. alni Shahu accessible for genetic engineering, and provided an important reference for developing genetic manipulation methods in other rare actinomycetes.

Highlights

Bacteria in the rare genus of Pseudonocardia belong to Actinomycetes that are represented by Gram-positive bacteria with high G + C DNA content (Tiwari and Gupta, 2012)
Pseudonocardia alni Shahu cells were grown at 30°C in an ISP4 medium (10 g/L soluble starch, 1 g/L K2HPO4, 1 g/L MgSO4·7H2O, 1 g/L NaCl, 2 g/L (NH4)2SO4, 2 g/L CaCO3, 0.001 g/L FeSO4·7H2O, 0.001 g/L MnCl2·4H2O, and 0.001 g/L ZnSO4·7H2O, and pH 7.0–7.4) or on ISP4 agar plates (15 g/L agar in ISP4), while E. coli strains at 37°C in Luria broth or on Luria agar plates
To further detail the species of this isolate, its genome sequence was subjected to a taxonomic analysis with genomes of Pseudonocardia available in the NCBI Genome database to estimate pairwise Average Nucleotide Identity (ANI) score using the FastANI method

Summary

Introduction

Bacteria in the rare genus of Pseudonocardia belong to Actinomycetes that are represented by Gram-positive bacteria with high G + C DNA content (Tiwari and Gupta, 2012). The generally poor efficiency or perhaps lack of genetic manipulation tools for Pseudonocardia has largely impeded deeper research and further application of this genus. Easy and efficient DNA introduction from Echerichia coli to actinomycetes can be achieved via conjugal transfer. This method represents probably the most straightforward strategy for genetic manipulation in actinomycetes. The other two types of vectors, integrative plasmids and suicidal non-replicating vectors, can introduce stable genomic alterations in many actinomycetes. The suicidal non-replicating vectors have been extensively used to generate stable DNA inserts into any neutral genomic region, or deletion of genes of interest, via homologous recombination

Methods

Results

Conclusion