Versatile use of Azospirillum brasilense strains tagged with egfp and mCherry genes for the visualization of biofilms associated with wheat roots

Microbiological Research
Alberto Ramirez-MataBeatriz E Baca

Abstract

This study reports the introduction of egfp or mCherry markers to the Sp245, Sp7, and M40 wild-type strains of Azospirillum brasilense and the hhkB (encoding for a putative hybrid histidine kinase) minus mutant an isogenic strain of A. brasilense Sp245 to monitor colonization of wheat (Triticum aestivum). Two plasmids were constructed: (1) the pJMS-2 suicide plasmid derived from pSUP202 and harboring the mCherry gene expressed under the constitutive kanamycin resistance promoter to create a cis tag and (2) the broad-range plasmid pMP2449-5 that carries the mCherry gene under the lac promoter, which is derived from the plasmid pMP2444; to create the in trans tag. The stability of the plasmids encoding egfp and mCherry were confirmed in vitro for seven days of bacterial growth, and then, the A. brasilense strains harboring the plasmids were studied under nonselective conditions for adherence to seeds and, at seven or 14 days post-inoculation, for wheat root colonization. The utility of the labeled strains was proven by observation, using fluorescence microscopy and confocal laser scanning microscopy (CLSM) in wheat plants inoculated with the labeled strains and compared with the CFU g-1 for seed and wheat root. The method was sui...Continue Reading

Citations

Feb 28, 2020·FEMS Microbiology Letters·Víctor I Viruega-GóngoraAlberto Ramírez-Mata
Jan 14, 2021·Scientific Reports·José Francisco Cruz-PérezAlberto Ramírez-Mata
Feb 5, 2021·Environmental Microbiology Reports·Hayley E KnightsPhilip S Poole
Dec 24, 2020·Letters in Applied Microbiology·M L PuenteF Cassán
Apr 17, 2021·Frontiers in Plant Science·Maria Chiara GuerrieriEdoardo Puglisi

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