MICROBIOLOGICAL IMAGE

https://doi.org/10.1016/j.ram.2018.09.006

Endophytic microorganisms Agrobacterium tumefaciens 6N2 and Meyerozyma guilliermondii 6N serve as models for the study of microbial interactions in colony biofilms

Los microorganismos endofíticos Agrobacterium tumefaciens 6N2 y Meyerozyma guilliermondii 6N sirven como modelos para el estudio de las interacciones microbianas en biofilms en colonia

 

Elisa Violeta Bertini^a, Ana Carolina del V. Leguina^a, Lucía I. Castellanos de Figueroa^a,b, Carlos G. Nieto-Peñalver^a,b,1

^a PROIMI, CONICET (Planta Piloto de Procesos Industriales Microbiológicos), Tucumán, Argentina
^b Instituto de Microbiología, Facultad de Bioquímica, Química y Farmacia, Universidad Nacional de Tucumán, Tucumán, Argentina

Received 18 June 2018; accepted 7 September 2018
Available online 14 January 2019

*Corresponding author.

E-mail address: cgnieto@proimi.org.ar (C.G. Nieto-Peñalver).

0325-7541/© 2018 Asociación Argentina de Microbiología. Published by Elsevier España, S.L.U. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

 

We analyzed the morphology of colonies of endophytic bacteria and yeasts by fluorescence. Agrobacterium tume-faciens 6N2 (formerly 197MX) is a non-pathogenic isolate from sugarcane with features of relevance for microbial interactions¹. Meyerozyma guilliermondii 6N was obtained from the same host³.

A. tumefaciens 6N2 was tagged with gfp. AAV-a, allowing the detection of cells actively expressing the fluorescence². GFP-tagged 6N2 and M. guilliermondii 6N were cultured at 30 °C in nutrient broth until late exponential phase¹. Cell densities were adjusted to ~0.1 OD_600nm, and spot inoculated on nutrient agar (NA) and YPD agar plates⁵, as pure or mixed cultures (proportion 1:1). Plates were incubated at 30°C for 72 h; colonies were imbibed with 10 µl of 25

Calcofluor White M2R (CW) that binds chitin and cellulose, and observed with a 4x objective lens under a Zeiss SteREO Lumar.V12 Stereomicroscope with GFP and DAPI filters.

While 6N2 colonies exhibited a wrinkled surface on YPD agar (Fig. 1A and C), they were smooth and convex on NA (Fig. 2A and C). Under interaction conditions 6N2 showed singular patterns: 6N2 was located at the edges and the center on YPD agar (Fig. 1D), and was evenly distributed on NA (Fig. 2D). In both media 6N seemed to be located on top of the bacterial growth (Fig. 1B and B). 6N development was granular with lobate borders on YPD agar (Fig. 1B and F), and was smooth and dotted on NA (Fig. 2B and F). Noteworthy, 6N2 was not stained with CW (Fig. 1E and E). Beyond the effect of the interactions on the host plant, the images show the potential of the strains and the techniques for the study of interactions in a colony biofilm, even if the true location of each strain requires other techniques (e.g., confocal microscopy)⁴.

Figure 1 Colony biofilms of A. tumefaciens 6N2 and M. guilliermondii 6N growing on YPD agar. A, C and E: pure colony of 6N2 observed with visible illumination, GFP filter and DAPI filter, respectively. B, D and F: colony of mixed cultures of 6N2 and 6N observed with visible illumination, GFP filter and DAPI filter, respectively.

Figure 2 Colony biofilms of A. tumefaciens 6N2 and M. guilliermondii 6N growing on NA. A, C and E: pure colony of 6N2 observed with visible illumination, GFP filter and DAPI filter, respectively. B, D and F: colony of mixed cultures of 6N2 and 6N observed with visible illumination, GFP filter and DAPI filter, respectively.

Funding

This work was supported by the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET, PIP 946, PU-E 22920160100012CO), Agencia Nacional de Promoción Científica y Tecnológica (PICT 2016 No. 0532; PICT 2016 No. 2013), and Secretaría de Ciencia, Arte e Innovación Tecnológica from the Universidad Nacional de Tucumán (PIUNT D609).

Conflict of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

We thank Jean-Michel Camadro (Institut Jacques Monod, UMR7592, CNRS Université Paris Diderot, Paris, France) for kindly providing access to the stereomicroscope facility.

References

1. Bertini EV, Leguina AC, del V, Castellanos de Figueroa LI, Nieto Penalver CG. Caracterización de bacterias endofíticas de cana de azúcar productoras de N-acil homoserina lactonas. Archivos de Bioquímica, Química y Farmacia. 2016;25:5-19.         [ Links ]

2. Lambertsen L, Sternberg C, Molin S. Mini-Tn7 transposons for site-specific tagging of bacteria with fluorescent proteins. Environ Microbiol. 2004;6:726-32.         [ Links ]

3. Leguina AC, del V, Nieto C, Pajot HM, Bertini EV, Mac Cormack W, Castellanos de Figueroa LI, Nieto-Peñalver CG. Inactivation of bacterial quorum sensing signals N-acyl homoserine lactones is widespread in yeasts. Fungal Biol. 2018;122:52-62.         [ Links ]

4. Merritt JH, Kadouri DE, O'Toole GA. Growing and analyzing static biofilms. In: Curr Protoc Microbiol; 2005. pp. 1B.1.1 -1B.1.18.         [ Links ]

5. Yeast Extract-Peptone-Dextrose (YPD) Medium (Liquid or Solid). In: Cold Spring Harb Protoc; 2017, http://dx.doi.org/10.1101/pdb.rec090563.         [ Links ]