Plasmid gene for putative integral membrane protein affects formation of lipopolysaccharide and motility in Azospirillum brasilense Sp245

Abstract

The bacterium Azospirillum brasilense can swim and swarm owing to the work of polar and lateral flagella. Its major surface glycopolymers consist of lipopolysaccharides (LPS) and Calcofluor-binding polysaccharides (Cal+ phenotype). Motility and surface glycopolymers are important for the interactions of plant-associated bacteria with plants. The facultative plant endophyte A. brasilense Sp245 produces two antigenically different LPS, LpsI, and LpsII, containing identical O-polysaccharides. Previously, using vector pJFF350 for random Omegon-Km mutagenesis, we constructed a mutant of Sp245 named KM018 that still possessed flagella, although paralyzed. The mutant was no longer able to produce Calcofluor-binding polysaccharides and LpsII. Because of the limited experimental data on the genetic aspects of surface glycopolymer production and flagellar motility in azospirilla, the aim of this study was to identify and examine in more detail the coding sequence of strain Sp245, inactivated in the mutant. We found that pJFF350 was integrated into a coding sequence for a putative integral membrane protein of unknown function (AZOBR_p60025) located in the sixth plasmid of Sp245. To clarify the role of the putative protein, we cloned AZOBR_p60025 in the expression vector pRK415 and used it for the genetic complementation of mutant KM018. The SDS–PAGE, immunodiffusion, and linear immunoelectrophoresis analyses showed that in strain KM018 (pRK415–p60025), the wild-type LpsI+ LpsII+ profile was restored. The complemented mutant had a Cal+ phenotype and it was capable of swimming and swarming motility. Thus, the AZOBR_p60025-encoded protein significantly affects the composition of the major cell-surface glycopolymers and the single-cell and social motility of azospirilla.

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Abbreviations

CBPS:

Calcofluor-binding polysaccharides

CDS:

Coding sequence

CPS:

Capsular polysaccharides

EPS:

Exopolysaccharides

Km:

Kanamycin

LB:

Luria–Bertani

LPS:

Lipopolysaccharides

MSM:

Malate–salt medium

OPS:

O-polysaccharide

PDB:

Protein Data Bank

Tc:

Tetracycline

TSA:

Trypton soya agar

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Acknowledgments

The authors thank Dmitry I. Mokeev and Andrei M. Burov for the technical support, Dmitry N. Tychinin for correcting our English, and the IBPPM RAS Symbiosis Centre for the Collective Use of Research Equipment (Saratov, Russia) for access to the Libra 120 microscope.

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L.P. Petrova: conduct of research, data analysis.

S.S. Yevstigneyeva: conduct of research, data analysis.

Yu.A. Filip’echeva: conduct of research, data analysis.

A.V. Shelud’ko: study design, conduct of research, data analysis.

G.L. Burygin: conduct of research, data analysis.

E.I. Katsy: study design and coordination, conduct of research, data analysis, writing of the manuscript.

Corresponding author

Correspondence to Elena I. Katsy.

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Petrova, L.P., Yevstigneyeva, S.S., Filip’echeva, Y.A. et al. Plasmid gene for putative integral membrane protein affects formation of lipopolysaccharide and motility in Azospirillum brasilense Sp245. Folia Microbiol (2020). https://doi.org/10.1007/s12223-020-00805-5

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