Isolation and characterization of insertion sequence elements from gram-negative bacteria by using new broad-host-range, positive selection vectors.

Sec (selenocysteine) is a rare amino acid in proteins. It is co-translationally inserted into proteins at UGA codons with the help of SECIS (Sec insertion sequence) elements. A full set of selenoproteins within a genome, known as the selenoproteome, is highly variable in different organisms. However, most of the known eukaryotic selenoproteins are represented in the mammalian selenoproteome. In addition, many of these selenoproteins have cysteine orthologues. Here, we describe a new selenoprotein, designated Fep15, which is distantly related to members of the 15 kDa selenoprotein (Sep15) family. Fep15 is absent in mammals, can be detected only in fish and is present in these organisms only in the selenoprotein form. In contrast with other members of the Sep15 family, which contain a putative active site composed of Sec and cysteine, Fep15 has only Sec. When transiently expressed in mammalian cells, Fep15 incorporated Sec in an SECIS- and SBP2 (SECIS-binding protein 2)-dependent manner and was targeted to the endoplasmic reticulum by its N-terminal signal peptide. Phylogenetic analyses of Sep15 family members suggest that Fep15 evolved by gene duplication.

Download Full-text

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.

Download Full-text

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

Download Full-text

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.

Download Full-text

Isolation and Characterization of Multidrug-Resistant Gram-Negative Bacteria Found in Free-Ranging Long-Eared Hedgehogs (Erinaceus concolor) From Tabriz, Iran

Journal of Exotic Pet Medicine ◽

10.1053/j.jepm.2015.04.014 ◽

2015 ◽

Vol 24 (2) ◽

pp. 235-239

Author(s):

Payman Zare ◽

Hassan Ghorbani-Choboghlo

Keyword(s):

Multidrug Resistant ◽

Gram Negative Bacteria ◽

Gram Negative ◽

Isolation And Characterization ◽

Free Ranging

Download Full-text

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.

Download Full-text