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.
- MeSH
- Azospirillum brasilense genetika metabolismus MeSH
- bakteriální proteiny genetika MeSH
- flagella MeSH
- lipopolysacharidy metabolismus MeSH
- membránové proteiny genetika metabolismus MeSH
- mutace MeSH
- mutageneze MeSH
- plazmidy genetika MeSH
- polysacharidy metabolismus MeSH
- výpočetní biologie MeSH
- Publikační typ
- časopisecké články MeSH
A derivative of Azospirillum brasilense Sp245, Sp245.5, which spontaneously lost 85 and 120 MDa replicons upon the formation of a new megaplasmid, has been shown to produce a novel lipopolysaccharide and to lose Calcofluor-binding polysaccharides. As compared to Sp245, the derivative displays notably increased heavy metal tolerance. The phenotypes of Sp245 and Sp245.5 are characterized by the following minimal inhibitory concentrations (MICs) of heavy metals: 0.5 and 0.9 μmol l(-1) of Ag(+), 0.4 and 0.7 mmol l(-1) of Co(2+), 0.9 and 4.7 mmol l(-1) of Cu(2+), and 3.1 and 11.5 mmol l(-1) of Zn(2+), respectively. In Sp245, in the presence of a nonlethal concentration (0.625 μmol l(-1)) of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP), the MIC of cobalt, copper, and zinc drop 1.3- to 1.6-fold, but the low tolerance to silver is unaffected. In Sp245.5, CCCP does not affect cobalt tolerance, suppresses tolerance to copper and silver to the wild-type levels, and causes a 1.4-fold decrease in resistance to zinc. Therefore, significant elevation of heavy metal tolerance in Sp245.5 seems caused by the induction/overexpression of the proton-dependent efflux of certain metal ions. The novel cell surface and other unknown factors could also be responsible for the increased tolerance of A. brasilense Sp245.5 to heavy metals.
