scholarly journals Broad-Host-Range Expression Reveals Native and Host Regulatory Elements That Influence Heterologous Antibiotic Production in Gram-Negative Bacteria

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Jia Jia Zhang ◽  
Xiaoyu Tang ◽  
Michelle Zhang ◽  
Darlene Nguyen ◽  
Bradley S. Moore
Keyword(s):  
Heterologous Expression ◽  
Host Range ◽  
Regulatory Elements ◽  
Cloning And Expression ◽  
Broad Host Range ◽  
Implicit Assumption ◽  
Gram Negative ◽  
E Coli ◽  
Expression Platform ◽  
Violacein Production

ABSTRACTHeterologous expression has become a powerful tool for studying microbial biosynthetic gene clusters (BGCs). Here, we extend the transformation-associated recombination cloning and heterologous expression platform for microbial BGCs to include Gram-negative proteobacterial expression hosts. Using a broad-host-range expression platform, we test the implicit assumption that biosynthetic pathways are more successfully expressed in more closely related heterologous hosts. Cloning and expression of the violacein BGC fromPseudoalteromonas luteoviolacea2ta16 revealed robust production in two proteobacterial hosts,Pseudomonas putidaKT2440 andAgrobacterium tumefaciensLBA4404, but very little production of the antibiotic in various laboratory strains ofEscherichia coli, despite their closer phylogenetic relationship. We identified a nonclustered LuxR-type quorum-sensing receptor fromP. luteoviolacea2ta16, PviR, that increases pathway transcription and violacein production inE. coliby ∼60-fold independently of acyl-homoserine lactone autoinducers. AlthoughE. coliharbors the most similar homolog of PviR identified from all of the hosts tested, overexpression of variousE. colitranscription factors did not result in a statistically significant increase in violacein production, while overexpression of twoA. tumefaciensPviR homologs significantly increased production. Thus, this work not only introduces a new genetic platform for the heterologous expression of microbial BGCs, it also challenges the assumption that host phylogeny is an accurate predictor of host compatibility.IMPORTANCEAlthough Gram-positive heterologous hosts such asStreptomyceshave been developed and optimized to support diverse secondary metabolic reactions, there has been comparatively less work on Gram-negative hosts, some of which grow faster and are easier to work with. This work presents a new genetic platform for direct cloning and broad-host-range heterologous expression of BGCs in Gram-negative proteobacterial expression hosts, and we leverage this platform to uncover regulatory elements that influence violacein expression fromPseudoalteromonas. Although it is often assumed that BGCs will be more successfully expressed in more closely related hosts, our work suggests that this may not be a general rule of thumb, as heterologous production of natural products can be influenced by specific host regulatory and/or biosynthetic elements, and the identity and effectiveness of those elements are difficult to predict. We argue for the use of a diverse set of heterologous hosts, which may also provide insights into the BGC biosynthetic mechanism and the biological function of BGCs.

scholarly journals A standardized broad host range inverter package for genetic circuitry design in Gram-negative bacteria

2020 ◽  
Author(s):  
Huseyin Tas ◽  
Ángel Goñi-Moreno ◽  
Víctor de Lorenzo
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Logic Gates ◽  
Building Blocks ◽  
Broad Host Range ◽  
Gram Negative ◽  
E Coli ◽  
Environmental Bacterium ◽  
High Copy Number Plasmid ◽  
Specific Strain

ABSTRACTGenetically encoded logic gates, especially inverters—NOT gates—are the building blocks for designing circuits, engineering biosensors or decision-making devices in synthetic biology. However, the repertoire of inverters readily available for different species is rather limited. In this work, a large whole of NOT gates that was shown to function previously in a specific strain of Escherichia coli, was recreated as broad host range (BHR) collection of constructs assembled in low, medium and high copy number plasmid backbones of the SEVA (Standard European Vector Architecture) collection. The input/output function of each of the gates was characterized and parameterized in the environmental bacterium and metabolic engineering chassis Pseudomonas putida. Comparisons of the resulting fluorescence cytometry data with those published for the same gates in Escherichia coli provided useful hints on the portability of the corresponding gates. The hereby described BHR inverter package (20 different versions of 12 distinct gates) thus becomes a toolbox of choice for designing genetic circuitries in a variety of Gram-negative species other than E. coli.

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

Genetics ◽  
1992 ◽  
Vol 130 (1) ◽  
pp. 27-36 ◽  
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.

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

2012 ◽  
Vol 79 (2) ◽  
pp. 718-721 ◽  
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.

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

2007 ◽  
Vol 73 (8) ◽  
pp. 2735-2743 ◽  
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.

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

2013 ◽  
Vol 79 (13) ◽  
pp. 4149-4153 ◽  
Author(s):  
F. Heath Damron ◽  
Elizabeth S. McKenney ◽  
Mariette Barbier ◽  
George W. Liechti ◽  
Herbert P. Schweizer ◽  
...  
Keyword(s):  
Host Range ◽  
Single Copy ◽  
Broad Host Range ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Bioluminescent Reporters

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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