The wzm gene located on the pRhico plasmid of Azospirillum brasilense Sp7 is involved in lipopolysaccharide synthesis

Abstract

Several genes involved in the interaction between Azospirillum brasilense Sp7 and plants are located on the pRhico plasmid. Here we report the characterization of an Sp7 mutant strain with impairment of the pRhico-located gene wzm. This gene encodes an inner-membrane component of an ATP-binding cassette (ABC) transporter with similarity to transporters involved in surface polysaccharide export. Indeed, SDS-PAGE revealed that LPS synthesis is affected in the wzm mutant. No significant differences were observed between wild-type and mutant strains in exopolysaccharide (EPS) amount; however, several differences were observed between them in EPS monosaccharide composition, and only wild-type colonies stained positively with Congo red. Microscopy revealed that wzm mutant cells are longer and thinner, and exhibit several differences in their cell surface relative to the wild-type. The wzm mutant was more resistant to oxidative stress, starvation, desiccation, heat and osmotic shock than the wild-type. In contrast, the mutant was more susceptible than the wild-type to UV radiation and saline stress. The strains also differed in their susceptibility to different antibiotics. Differences between the strains were also observed in their outer-membrane protein composition. No differences were observed between strains in their ability to attach to sweet corn roots and seeds, and to promote growth under the tested conditions. As LPS plays an important role in cell envelope structural integrity, we propose that the pleiotropic phenotypic changes observed in the wzm mutant are due to its altered LPS relative to the wild-type. INTRODUCTIONThe Azospirillum genus belongs to the alpha-proteobacteria and comprises free-living, nitrogen-fixing, vibrio-or spirillum-shaped rods that exert beneficial effects on plant growth and yield of many crops of agronomic importance (Dobbelaere et al., 2001). Plant growth promotion by Azospirillum is attributed to morphological and physiological changes in inoculated plant roots, which enhance water and mineral uptake. Plant growth substances, such as auxins, cytokinins and gibberellins (Steenhoudt & Vanderleyden, 2000;Dobbelaere et al., 2001), as well as nitric oxide (Creus et al., 2005) produced by the bacteria are at least partially responsible for the plant growth promotion.Within the Azospirillum genus, one of the most studied species is Azospirillum brasilense (Tarrand et al., 1978). A. brasilense cells are surrounded by a dense, tightly cellbound layer of capsular polysaccharides (CPSs), and by outer exopolysaccharides (EPSs), which are loosely bound to the cell and easily detached by centrifugation (Burdman et al., 2000c). EPSs and CPSs, as well as extracellular proteins, have been shown to play important roles in bacterial aggregation and in the establishment of the bacterium-plant association (Burdman et al., 1999(Burdman et al., , 2000cSteenhoudt & Vanderleyden, 2000). It has been suggested that EPSs and CPSs are involved in anchoring the bacteria to the surface of wheat roots , and in t...

The Azospirillum brasilense Sp7 noeJ and noeL genes are involved in extracellular polysaccharide biosynthesis

“…Fucose is found to be one of the main sugars in this fraction, while mannose was detected in small traces. Both sugars are also detected in the EPS of this strain; however, in EPS, while mannose is often one of the main sugars, only small traces of fucose have been detected (Burdman et al, 2000a;Lerner et al, 2009).…”

“…Cloning, transformation procedures and Southern blot analyses were performed as described previously (Lerner et al, 2009). Restriction enzymes and DNA ligase were purchased from New England BioLabs.…”

“…EPS composition and concentration were determined as described by Lerner et al (2009). LPS extraction was performed using two methods.…”

“…LPS extraction was performed using two methods. In one procedure, LPS were extracted and analysed by SDS-PAGE as previously described (Lerner et al, 2009). In a second procedure, LPS were extracted with the LPS extraction kit (Intron Biotechnology) according to the manufacturer's instructions and samples were run on a sodium deoxycholate (DOC)-PAGE gel This study Fig.…”

“…As changes in EPS and/or LPS can affect the bacterial response to environmental changes (Mao et al, 2001;Touze et al, 2004;Lerner et al, 2009), the mutants were also characterized for their survival abilities in response to several stresses, and their biofilm formation ability was evaluated.…”