Bacillus subtilis biofilm matrix components target seed oil bodies to promote growth and anti-fungal resistance in melon.

Beneficial microorganisms are used to stimulate the germination of seeds; however, their growth-promoting mechanisms remain largely unexplored. Bacillus subtilis is commonly found in association with different plant organs, providing protection against pathogens or stimulating plant growth. We report that application of B. subtilis to melon seeds results in genetic and physiological responses in seeds that alter the metabolic and developmental status in 5-d and 1-month-old plants upon germination. We analysed mutants in different components of the extracellular matrix of B. subtilis biofilms in interaction with seeds and found cooperation in bacterial colonization of seed storage tissues and growth promotion. Combining confocal microscopy with fluorogenic probes, we found that two specific components of the extracellular matrix, amyloid protein TasA and fengycin, differentially increased the concentrations of reactive oxygen species inside seeds. Further, using electron and fluorescence microscopy and metabolomics, we showed that both TasA and fengycin targeted the oil bodies in the seed endosperm, resulting in specific changes in lipid metabolism and accumulation of glutathione-related molecules. In turn, this results in two different plant growth developmental programmes: TasA and fengycin stimulate the development of radicles, and fengycin alone stimulate the growth of adult plants and resistance in the phylloplane to the fungus Botrytis cinerea. Understanding mechanisms of bacterial growth promotion will enable the design of bespoke growth promotion strains.

An Alternative and Conserved Cell Wall Enzyme That Can Substitute for the Lipid II Synthase MurG.

The cell wall is a stress-bearing structure and a unifying trait in bacteria. Without exception, synthesis of the cell wall involves formation of the precursor molecule lipid II by the activity of the essential biosynthetic enzyme MurG, which is encoded in the division and cell wall synthesis (dcw) gene cluster. Here, we present the discovery of a cell wall enzyme that can substitute for MurG. A mutant of Kitasatospora viridifaciens lacking a significant part of the dcw cluster, including murG, surprisingly produced lipid II and wild-type peptidoglycan. Genomic analysis identified a distant murG homologue, which encodes a putative enzyme that shares only around 31% amino acid sequence identity with MurG. We show that this enzyme can replace the canonical MurG, and we therefore designated it MglA. Orthologues of mglA are present in 38% of all genomes of Kitasatospora and members of the sister genus Streptomyces CRISPR interference experiments showed that K. viridifaciens mglA can also functionally replace murG in Streptomyces coelicolor, thus validating its bioactivity and demonstrating that it is active in multiple genera. All together, these results identify MglA as a bona fide lipid II synthase, thus demonstrating plasticity in cell wall synthesis.IMPORTANCE Almost all bacteria are surrounded by a cell wall, which protects cells from environmental harm. Formation of the cell wall requires the precursor molecule lipid II, which in bacteria is universally synthesized by the conserved and essential lipid II synthase MurG. We here exploit the unique ability of an actinobacterial strain capable of growing with or without its cell wall to discover an alternative lipid II synthase, MglA. Although this enzyme bears only weak sequence similarity to MurG, it can functionally replace MurG and can even do so in organisms that naturally have only a canonical MurG. The observation that MglA proteins are found in many actinobacteria highlights the plasticity in cell wall synthesis in these bacteria and demonstrates that important new cell wall biosynthetic enzymes remain to be discovered.