A set of shuttle plasmids for gene expression in Acinetobacter baumannii.

Jing Jie,Zhaoqing Luo,Xiao Chu,Dan Li,Anirudh K Singh

doi:10.1371/journal.pone.0246918

Jing Jie, Zhaoqing Luo + Show 3 more

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https://doi.org/10.1371/journal.pone.0246918

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Journal: PloS one	Publication Date: Feb 10, 2021
Citations: 13	License type: CC BY 4.0

Affiliation: First Hospital of Jilin University, Jilin University

Abstract

Infections caused by the emerging opportunistic bacterial pathogen Acinetobacter baumannii are occurring at increasingly alarming rates, and such increase in incidence is further compounded by the development of wide spread multidrug-resistant strains. Yet, our understanding of its pathogenesis and biology remains limited which can be attributed in part to the scarce of tools for molecular genetic analysis of this bacterium. Plasmids based on pWH1277 originally isolated from Acinetobacter calcoaceticus are the only vehicles currently available for ectopic gene expression in Acinetobacter species, which restricts experiments that require simultaneous analysis of multiple genes. Here, we found that plasmids of the IncQ group are able to replicate in A. baumannii and can stably co-reside with derivatives of pWH1277. Furthermore, we have constructed a series of four plasmids that allow inducible expression of Flag-tagged proteins in A. baumannii by arabinose or isopropyl β-d-1-thiogalactopyranoside. Together with constructs previously developed, these plasmids will accommodate the need in genetic analysis of this increasingly important pathogen.

Highlights

Acinetobacter baumannii is a Gram-negative bacterium known for its pleomorphism and the lack of motility; it is an important opportunistic pathogen that can colonize the skin and often is isolated from the respiratory and oropharynx secretions of infected individuals [1]
To identify additional cloning vehicles capable of replicating in A. baumannii autonomously, we examined a number of broad-host-range plasmids for their ability to transform this bacterium, including pRK415K of the IncP group [38], pDSK519 of the IncQ group [27] derived from RSF1010 [39] and pBBR1MCS2, which is of an unknown Inc group but can coexist with plasmids from many different Inc groups [40]
When DNA of each of these plasmids was introduced into A. baumannii by electroporation, transformants resistant to kanamycin only appeared in samples receiving pDSK519, in which an average of approximately 2.3x103 colonies were obtained in transformation with 0.2 μg plasmid DNA, suggesting that this vehicle can autonomously replicate in this bacterium

Summary

Introduction

Acinetobacter baumannii is a Gram-negative bacterium known for its pleomorphism and the lack of motility; it is an important opportunistic pathogen that can colonize the skin and often is isolated from the respiratory and oropharynx secretions of infected individuals [1]. Infection by A. baumannii normally occurs in immunocompromised populations and is of high incidence in hospital environments, in intensive care unit (ICU) wards with chronically ill patients [2]. The high incidence of infection by A. baumannii is compounded by the dramatic increase in the development of multidrug-resistant (MDR) strains, which has generated alarms in the medical community [2, 3]. Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Enterobacter species [4]. These bacteria cause the majority of nosocomial infections, and represent distinct paradigms of pathogenesis, transmission, and resistance posing grave threat to hospitals [5]

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