Characterization of the Streptomyces lavendulae IMRU 3455 linear plasmid pSLV45
MICROBIOLOGY-SGM
Authors: Hosted, TJ; Wang, T; Horan, AC
Abstract
Streptomyces lavendulae IMRU 3455 contains two large linear plasmids designated pSLV45 (45 kb) and pSLV195 (195 kb). A cosmid, pSPRX604, containing 42 kb from pSLV45 was cloned and sequenced. pSLV45 was tagged with a hygromycin-resistance marker by homologous recombination to generate the derivatives pSLV45.680 and pSLV45.681. An apramycin-resistance marker was introduced into S. lavendulae IMRU 467 using the pSPR910 integration vector to yield the recipient strain SPW910. The self-transmissible nature of pSLV45 was determined by transfer of pSLV45.680 and pSLV45.681 from the donor strains SPW680 and SPW681 into the recipient strain SPW910. Southern analysis indicated the presence of hygromycin- and pSLV45-hybridizing sequences within SPW910 exconjugants. PFGE analysis confirmed pSLV45.680 and pSLV45.681 were transferred intact and formed freely replicating linear plasmids. Sequence analysis of pSPRX604 revealed genes predicted to be involved in plasmid transfer, partitioning and regulation. The transfer of the linear plasmid pSLV45 from S. lavendulae IMRU3455 into S. lavendulae IMRU 467 may allow the development of pSLV45 as an actinomycete-to-actinomycete conjugative shuttle vector.
Next Generation of Tn7-Based Single-Copy Insertion Elements for Use in Multi- and Pan-Drug-Resistant Strains of Acinetobacter baumannii
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
Authors: Ducas-Mowchun, Kaleigh; De Silva, P. Malaka; Crisostomo, Leandro; Fernando, Dinesh M.; Chao, Tzu-Chiao; Pelka, Peter; Schweizer, Herbert P.; Kumar, Ayush
Abstract
The purpose of this study was to create single-copy gene expression systems for use in genomic manipulations of multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of Acinetobacter baumannii. In this study, mini-Tn7 vectors with zeocin and apramycin selection markers were created by cloning the We and aac(3)-IV genes, respectively, enabling either inducible gene expression (pUC18T-mini-Tn7T-Zeo-LAC and pUC18T-mini-Tn7T-Apr-LAC) or expression from native or constitutive promoters (pUC18T-mini-Tn7T-Zeo and pUC18T-mini-Tn7T-Apr). The selection markers of these plasmids are contained within a Flp recombinase target (FRT) cassette, which can be used to obtain unmarked mini-Tn7 insertions upon introduction of a source of Flp recombinase. To this end, site-specific excision vectors pFLP2A and pFLP2Z (containing apramycin and zeocin selection markers, respectively) were created in this study as an accessory to the mini-Tn7 vectors described above. Combinations of these novel mini-Tn7 plasmids and their compatible pFLP2Z or pFLP2A accessory plasmid were used to generate unmarked insertions in MDR clinical isolates of A. baumannii. In addition, several fluorescent markers were cloned and inserted into MDR and XDR clinical isolates of A. baumannii via these apramycin and zeocin mini-Tn7 constructs to demonstrate their application. IMPORTANCE Acinetobacter baumannii is a high-priority pathogen for which research on mechanisms of resistance and virulence is a critical need. Commonly used antibiotic selection markers are not suitable for use in MDR and XDR isolates of A. baumannii due to the high antibiotic resistance of these isolates, which poses a barrier to the study of this pathogen. This study demonstrates the practical potential of using apramycin and zeocin mini-Tn7- and Flp recombinase-encoded constructs to carry out genomic manipulations in clinical isolates of A. baumannii displaying MDR and XDR phenotypes.