NDLI: Detection of recombinant Pseudomonas putida in the wheat rhizosphere by fluorescence in situ hybridization targeting mRNA and rRNA

Content Provider	Springer Nature Link
Author	Wu, Cindy H. Hwang, Yu Chen Lee, Wonkyu Mulchandani, Ashok Wood, Thomas K. Yates, Marylynn V. Chen, Wilfred
Copyright Year	2008
Abstract	A method was developed to detect a specific strain of bacteria in wheat root rhizoplane using fluorescence in situ hybridization and confocal microscopy. Probes targeting both 23S rRNA and messenger RNA were used simultaneously to achieve detection of recombinant Pseudomonas putida (TOM20) expressing toluene o-monooxygenase (tom) genes and synthetic phytochelatin (EC20). The probe specific to P. putida 23S rRNA sequences was labeled with Cy3 fluor, and the probe specific to the tom genes was labeled with Alexa647 fluor. Probe specificity was first determined, and hybridization temperature was optimized using three rhizosphere bacteria pure cultures as controls, along with the P. putida TOM20 strain. The probes were highly specific to the respective targets, with minimal non-specific binding. The recombinant strain was inoculated into wheat seedling rhizosphere. Colonization of P. putida TOM20 was confirmed by extraction of root biofilm and growth of colonies on selective agar medium. Confocal microscopy of hybridized root biofilm detected P. putida TOM20 cells emitting both Cy3 and Alexa647 fluorescence signals.
Starting Page	511
Ending Page	518
Page Count	8
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	79
Issue Number	3
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2008-04-04
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	FISH mRNA Rhizosphere bacteria In situ gene expression Microbial Genetics and Genomics Microbiology Biotechnology
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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