scholarly journals Transformation and Transposon Mutagenesis of Leifsonia xyli subsp. xyli, Causal Organism of Ratoon Stunting Disease of Sugarcane

2002 ◽  
Vol 15 (3) ◽  
pp. 262-268 ◽  
Author(s):  
Stevens M. Brumbley ◽  
Lars A. Petrasovits ◽  
Robert G. Birch ◽  
Paul W. J. Taylor
Keyword(s):  
Insertional Mutagenesis ◽  
Vibrio Fischeri ◽  
Transposon Mutagenesis ◽  
Coli Strain ◽  
Causal Organism ◽  
Transposition Frequency ◽  
Ratoon Stunting Disease ◽  
Suicide Vector ◽  
Lux Operon ◽  
Cosmid Cloning

Conditions have been developed for genetic transformation and insertional mutagenesis in Leifsonia xyli subsp. xyli (Lxx), the causal organism of ratoon stunting disease (RSD), one of the most damaging and intractable diseases of sugarcane internationally. Transformation frequencies ranged from 1 to 10 colony forming units (CFU)/μg of plas-mid DNA using Clavibacter/Escherichia coli shuttle vectors pCG188, pDM302, and pDM306 and ranged from 50 to 500 CFU/μg using cosmid cloning vectors pLAFR3 and pLAFR5-km. The transformation/transposition frequency was 0 to 70 CFU/μg of DNA, using suicide vectors pUCD623 and pSUP2021 containing transposable elements Tn4431 and Tn5, respectively. It was necessary to grow Lxx in media containing 0.1% glycine for electroporation and to amplify large plasmids in a dam¯/dcm¯ E. coli strain and purify the DNA by anion exchange. To keep selection pressure at an optimum, the transformants were grown on nitrocellulose filters (0.2-μm pore size) on media containing the appropriate antibiotics. Transposon Tn4431 containing a promoterless lux operon from Vibrio fischeri and a tetra-cycline-resistance gene was introduced on the suicide vector pUCD623. All but 1% of the putative transposon mutants produce light, indicating transposition into functional Lxx genes. Southern blot analysis of these transformants indicates predominantly single transposon insertions at unique sites. The cosmid cloning vector pLAFR5-km was stably maintained in Lxx. The development of a transformation and transposon mutagenesis system opens the way for molecular analysis of pathogenicity determinants in Lxx.

scholarly journals Colonization of Host Plants by the Fire Blight Pathogen Erwinia amylovora Marked with Genes for Bioluminescence and Fluorescence

1998 ◽  
Vol 88 (5) ◽  
pp. 416-421 ◽  
Author(s):  
Jochen Bogs ◽  
Iris Bruchmüller ◽  
Claudia Erbar ◽  
Klaus Geider
Keyword(s):  
Host Plants ◽  
Fire Blight ◽  
Erwinia Amylovora ◽  
Vibrio Fischeri ◽  
Bacterial Invasion ◽  
Leaf Hairs ◽  
Lux Operon ◽  
And Migration ◽  
Green Fluorescent ◽  
Lac Promoter

To follow the movement of Erwinia amylovora in plant tissue without dissection, this bacterium was marked with either the lux operon from Vibrio fischeri or the gfp gene from the jellyfish Aequorea victoria, both carried on multicopy plasmids and expressed under the control of the lac promoter from Escherichia coli. Movement of the pathogen was visualized in leaves, stems, and roots of apple seedlings, and migration of E. amylovora was traced from inoculation sites in the stem to as far as the roots. Green fluorescent E. amylovora cells were observed in the xylem and later appeared to break out of the vessels into the intercellular spaces of the adjacent parenchyma. Inoculation in the intercostal region of leaves caused a zone of slow necrosis that finally resulted in bacterial invasion of the xylem vessels. Labeled bacteria could also be seen in association with the anchor sites of leaf hairs. Distortion of the epidermis adjacent to leaf hairs created openings that were observed by scanning electron microscopy. As the intercostal region, the bases of leaf hairs provided E. amylovora access to intact xylem vessels, which allowed further distribution of the pathogen in the host plant.

scholarly journals Isolation and Characterization of FecA- and FeoB-Mediated Iron Acquisition Systems of the Spirochete Leptospira biflexa by Random Insertional Mutagenesis

2005 ◽  
Vol 187 (9) ◽  
pp. 3249-3254 ◽  
Author(s):  
Hélène Louvel ◽  
Isabelle Saint Girons ◽  
Mathieu Picardeau
Keyword(s):  
Insertional Mutagenesis ◽  
Iron Acquisition ◽  
Transposon Mutagenesis ◽  
Ecological Niches ◽  
Acid Biosynthesis ◽  
Gene Encoding ◽  
Isolation And Characterization ◽  
Transposon Mutants

ABSTRACT The specific mechanisms by which Leptospira spp. acquire iron from their ecological niches are unknown. A major factor contributing to our ignorance of spirochetal biology is the lack of methods for genetic analysis of these organisms. In this study, we have developed a system for random transposon mutagenesis of Leptospira biflexa using a mariner transposon, Himar1. To demonstrate the validity of Himar1 in vivo transposon mutagenesis in L. biflexa, a screen of mutants for clones impaired in amino acid biosynthesis was first performed, enabling the identification of tryptophan and glutamate auxotrophs. To investigate iron transporters, 2,000 L. biflexa transposon mutants were screened onto media with and without hemin, thus allowing the identification of five hemin-requiring mutants, and the putative genes responsible for this phenotype were identified. Three mutants had distinct insertions in a gene encoding a protein which shares homology with the TonB-dependent receptor FecA, involved in ferric citrate transport. We also identified two mutants with a Himar1 insertion into a feoB-like gene, the product of which is required for ferrous iron uptake in many bacterial organisms. Interestingly, the growth inhibition exhibited by the fecA and feoB mutants was relieved by deferoxamine, suggesting the presence of a ferric hydroxamate transporter. These results confirm the importance of iron for the growth of Leptospira and its ability to use multiple iron sources.

scholarly journals A new Vibrio fischeri lux gene precedes a bidirectional termination site for the lux operon.

1990 ◽  
Vol 172 (12) ◽  
pp. 6797-6802 ◽  
Author(s):  
A Swartzman ◽  
S Kapoor ◽  
A F Graham ◽  
E A Meighen
Keyword(s):  
Vibrio Fischeri ◽  
Lux Operon

Involvement of host factors in the regulation of the Vibrio fischeri lux operon in Escherichia coli cells

Microbiology ◽  
2006 ◽  
Vol 75 (4) ◽  
pp. 452-458 ◽  
Author(s):  
I. V. Manukhov ◽  
V. Yu. Kotova ◽  
G. B. Zavil’gel’sky
Keyword(s):  
Escherichia Coli ◽  
Vibrio Fischeri ◽  
Host Factors ◽  
Escherichia Coli Cells ◽  
Lux Operon

scholarly journals Recent advances in the molecular biology ofLeifsonia xylisubsp.xyli, causal organism of ratoon stunting disease

2006 ◽  
Vol 35 (6) ◽  
pp. 681 ◽  
Author(s):  
S. M. Brumbley ◽  
L. A. Petrasovits ◽  
S. R. Hermann ◽  
A. J. Young ◽  
B. J. Croft
Keyword(s):  
Molecular Biology ◽  
Causal Organism ◽  
Ratoon Stunting Disease ◽  
Recent Advances

scholarly journals A Highly Efficient Transposon Mutagenesis System for the Tomato Pathogen Clavibacter michiganensis subsp. michiganensis

2001 ◽  
Vol 14 (11) ◽  
pp. 1312-1318 ◽  
Author(s):  
Oliver Kirchner ◽  
Karl-Heinz Gartemann ◽  
Eva-Maria Zellermann ◽  
Rudolf Eichenlaub ◽  
Annette Burger
Keyword(s):  
Transposon Mutagenesis ◽  
Saturation Mutagenesis ◽  
Clavibacter Michiganensis ◽  
Insertion Element ◽  
Suicide Vector ◽  
Clavibacter Michiganensis Subsp ◽  
Transposon Mutants ◽  
Insertion Sites ◽  
Tomato Pathogen ◽  
Electroporation Efficiency

A transposon mutagenesis system for Clavibacter michiganensis subsp. michiganensis was developed based on antibiotic resistance transposons that were derived from the insertion element IS1409 from Arthrobacter sp. strain TM1 NCIB12013. As a prerequisite, the electroporation efficiency was optimized by using unmethylated DNA and treatment of the cells with glycine such that about 5 × 106 transformants per μg of DNA were generally obtained. Electroporation of C. michiganensis subsp. michiganensis with a suicide vector carrying transposon Tn1409C resulted in approximately 1 × 103 transposon mutants per μg of DNA and thus is suitable for saturation mutagenesis. Analysis of Tn1409C insertion sites suggests a random mode of transposition. Transposition of Tn1409C was also demonstrated for other subspecies of C. michiganensis.

Bacteriophage-Based Bioluminescent Bioreporter for the Detection of Escherichia coli O157:H7

2007 ◽  
Vol 70 (6) ◽  
pp. 1386-1392 ◽  
Author(s):  
JENNIFER R. BRIGATI ◽  
STEVEN A. RIPP ◽  
COURTNEY M. JOHNSON ◽  
POLINA A. IAKOVA ◽  
PATRICIA JEGIER ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pathogenic Bacteria ◽  
Vibrio Fischeri ◽  
Homoserine Lactone ◽  
Escherichia Coli O157 ◽  
Recombinant Phage ◽  
E Coli ◽  
Lux Operon ◽  
Multiple Sample ◽  
New Bioassay

The rapid detection of pathogenic bacteria in food and water is vital for the prevention of foodborne illness. In this study, the lux reporter genes were used in a new bioassay that allows pathogen monitoring without multiple sample manipulations or the addition of exogenous substrate. A recombinant phage specific for Escherichia coli O157:H7 was constructed that, upon infection, catalyzes the synthesis of N-(3-oxohexanoyl)-L-homoserine lactone (OHHL). This phage PP01 derivative carries the luxI gene from Vibrio fischeri under the control of the phage promoter PL. OHHL produced by infected E. coli O157:H7 induces bioluminescence in bioreporter cells carrying the V. fischeri lux operon. The ability of phage PP01-luxI to detect several strains of E. coli O157:H7 was confirmed in a 96-well plate assay. In this assay, luxCDABE bioreporter cells capable of detecting OHHL were mixed with phage PP01-luxI and E. coli O157:H7, and luminescence was monitored. Reporter phages induced light in bioreporter cells within 1 h when exposed to 104 CFU/ml of E. coli O157:H7 and were able to detect 10 CFU/ml in pure culture with a preincubation step (total detection time, 4 h). The detection method was also applied to contaminated apple juice and was able to detect 104 CFU/ml of E. coli O157:H7 in 2 h after a 6-h preincubation.

scholarly journals Effective Mutagenesis of Vibrio fischeri by Using Hyperactive Mini-Tn5 Derivatives

2008 ◽  
Vol 74 (22) ◽  
pp. 7059-7063 ◽  
Author(s):  
Noreen L. Lyell ◽  
Anne K. Dunn ◽  
Jeffrey L. Bose ◽  
Susan L. Vescovi ◽  
Eric V. Stabb
Keyword(s):  
Vibrio Fischeri ◽  
Transposon Mutagenesis ◽  
Inexpensive Technique ◽  
Single Insertion

ABSTRACT We have developed a transposon mutagenesis system for Vibrio fischeri ES114 that utilizes a hyperactive mutant Tn5 transposase (E54K and M56A) and optimized transposon ends. Using a conjugation-based procedure, we obtained independent single-insertion mini-Tn5 mutants at a rate of ∼10−6. This simple and inexpensive technique represents a significant improvement over previous methods for transposon mutagenesis of V. fischeri and should be applicable to many other bacteria.

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