Construction of Mobilizable Mini-Tn7Vectors for Bioluminescent Detection of Gram-Negative Bacteria and Single-Copy PromoterluxReporter Analysis

ABSTRACTWe describe the construction of mini-Tn7-based broad-host-range vectors encodingluxgenes as bioluminescent reporters. These constructs can be mobilized into the desired host(s) by conjugation for chromosomal mini-Tn7-luxintegration and are useful for localization of bacteria during infections or for characterizing regulation of promoters of interest in Gram-negative bacteria.

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Construction of a Broad-Host-Range Tn7-Based Vector for Single-Copy PBAD-Controlled Gene Expression in Gram-Negative Bacteria

Applied and Environmental Microbiology ◽

10.1128/aem.02926-12 ◽

2012 ◽

Vol 79 (2) ◽

pp. 718-721 ◽

Cited By ~ 20

Author(s):

F. Heath Damron ◽

Elizabeth S. McKenney ◽

Herbert P. Schweizer ◽

Joanna B. Goldberg

Keyword(s):

Gene Expression ◽

Pseudomonas Aeruginosa ◽

Host Range ◽

Single Copy ◽

Broad Host Range ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Content Type ◽

Delivery Vector ◽

Inducible Gene

ABSTRACTWe describe a mini-Tn7-based broad-host-range expression cassette for arabinose-inducible gene expression from the PBADpromoter. This delivery vector, pTJ1, can integrate a single copy of a gene into the chromosome of Gram-negative bacteria for diverse genetic applications, of which several are discussed, usingPseudomonas aeruginosaas the model host.

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Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

Genetics ◽

10.1093/genetics/130.1.27 ◽

1992 ◽

Vol 130 (1) ◽

pp. 27-36 ◽

Cited By ~ 2

Author(s):

A Greener ◽

S M Lehman ◽

D R Helinski

Keyword(s):

Host Range ◽

Transcription Initiation ◽

Promoter Activity ◽

Firefly Luciferase ◽

Broad Host Range ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Broad Host Range Plasmid ◽

Transcriptional Start Sites ◽

Plasmid Rk2

Abstract A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

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A Standardized Inverter Package Borne by Broad Host Range Plasmids for Genetic Circuit Design in Gram-Negative Bacteria

ACS Synthetic Biology ◽

10.1021/acssynbio.0c00529 ◽

2020 ◽

Author(s):

Huseyin Tas ◽

Ángel Goñi-Moreno ◽

Víctor de Lorenzo

Keyword(s):

Host Range ◽

Circuit Design ◽

Genetic Circuit ◽

Broad Host Range ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Broad Host Range Plasmids

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Gene Targeting in Gram-Negative Bacteria by Use of a Mobile Group II Intron (“Targetron”) Expressed from a Broad-Host-Range Vector

Applied and Environmental Microbiology ◽

10.1128/aem.02829-06 ◽

2007 ◽

Vol 73 (8) ◽

pp. 2735-2743 ◽

Cited By ~ 37

Author(s):

Jun Yao ◽

Alan M. Lambowitz

Keyword(s):

Host Range ◽

Gene Targeting ◽

Inducible Promoter ◽

Broad Host Range ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Efficient Gene ◽

Range Vector ◽

Group Ii ◽

Broad Host Range Vector

ABSTRACT Mobile group II introns (“targetrons”) can be programmed for insertion into virtually any desired DNA target with high frequency and specificity. Here, we show that targetrons expressed via an m-toluic acid-inducible promoter from a broad-host-range vector containing an RK2 minireplicon can be used for efficient gene targeting in a variety of gram-negative bacteria, including Escherichia coli, Pseudomonas aeruginosa, and Agrobacterium tumefaciens. Targetrons expressed from donor plasmids introduced by electroporation or conjugation yielded targeted disruptions at frequencies of 1 to 58% of screened colonies in the E. coli lacZ, P. aeruginosa pqsA and pqsH, and A. tumefaciens aopB and chvI genes. The development of this broad-host-range system for targetron expression should facilitate gene targeting in many bacteria.

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