The ability of Methylobacterium extorquens to grow on methanol as the sole carbon and energy source has been the object of intense research activity. Unquestionably, the bacterial cell envelope serves as a defensive barrier against such an environmental stressor, with a decisive role played by the membrane lipidome, which is crucial for stress resistance. However, the chemistry and the function of the main constituent of the M. extorquens outer membrane, the lipopolysaccharide (LPS), is still undefined. Here, we show that M. extorquens produces a rough-type LPS with an uncommon, non-phosphory-lated, and extensively O-methylated core oligosaccharide, densely substituted with negatively charged residues in the inner region, including novel monosaccharide derivatives such as O-methylated Kdo/Ko units. Lipid A is composed of a non-phosphorylated trisaccharide backbone with a distinctive, low acylation pattern; indeed, the sugar skeleton was decorated with three acyl moieties and a secondary very long chain fatty acid, in turn substituted by a 3 -O-acetyl-butyrate residue. Spectroscopic, conformational, and biophysical analyses on M. extorquens LPS highlighted how structural and tridimensional features impact the molecular organization of the outer membrane. Furthermore, these chemical features also impacted and improved membrane resistance in the presence of methanol, thus regulating membrane ordering and dynamics.

Methylobacterium extorquens is a facultative methylotrophic Gram-negative bacterium, often associated with plants, that exhibits a unique ability to grow in the presence of high methanol concentrations, which serves as a single carbon energy source. We found that M. extorquens strain PA1 secretes a mixture of different exopolysaccharides (EPSs) when grown in reference medium or in presence of methanol, that induces the secretion of a peculiar and heterogenous mixture of EPSs, with different structure, composition, repeating units, bulk and a variable degree of methylation. These factors influenced 3D structure and supramolecular assets, diffusion properties and hydrodynamic radius, and likely contribute to increase methanol tolerance and cell stability. No direct methanol involvement in the EPSs solvation shell was detected, indicating that the polymer exposure to methanol is water mediated. The presence of methanol induces no changes in size and shape of the polymer chains, highlighting how water-methanol mixtures are a good solvent for refEPS and metEPS.

Lipopolysaccharides, the major outer membrane components of Gram-negative bacteria, are crucial actors of the host-microbial dialogue. They can contribute to the establishment of either symbiosis or bacterial virulence, depending on the bacterial lifestyle. Plant microbiota shows great complexity, promotes plant health and growth and assures protection from pathogens. How plants perceive LPS from plant-associated bacteria and discriminate between beneficial and pathogenic microbes is an open and urgent question. Here, we report on the structure, conformation, membrane properties and immune recognition of LPS isolated from the Arabidopsis thaliana root microbiota member Herbaspirillum sp. Root189. The LPS consists of an O-methylated and variously acetylated D-rhamnose containing polysaccharide with a rather hydrophobic surface. Plant immunology studies in A. thaliana demonstrate that the native acetylated O-antigen shields the LPS from immune recognition whereas the O-deacylated one does not. These findings highlight the role of Herbaspirillum LPS within plant-microbial crosstalk, and how O-antigen modifications influence membrane properties and modulate LPS host recognition.

Cupriavidus metallidurans is a Gram-negative bacterium that has attracted the attention of the scientific community since its discovery back in 1976. It was initially studied as a model organism for bioremediation processes due to its ability to survive in heavy metal-rich environments. However, in recent years, there is evidence that this bacterium can be a potential pathogen for humans. How C. metallidurans can survive in such different environments is unknown and prompted the following work. Its great adaptability could be explained by the structural and conformational studies of the O-antigen portion of the lipopolysaccharide, the main constituent of the outer membrane of Gram-negative bacteria, which is the one in direct contact with the external environment. Therefore, a combination of chemical and spectroscopic analyses was used to define the O-antigen structure, disclosing that it is a polysaccharide constituted of a linear tetrasaccharide repeating unit that does not resemble other structures already reported for bacteria: [4)-α-d-GalNAc-(1→3)-α-d-Qui2NAc4NHBA-(1→3)-α-l-Rha-(1→3)-α-l-Rha-(1→]. Interestingly, the molecular dynamics studies revealed that the three-dimensional structure of the O-antigen is highly flexible: it might adopt three different right-handed helix conformations described by a two, three, or four-fold symmetry. This conformational behavior could represent the reason behind the survival of C. metallidurans in different environments.

The ability to produce an extracellular matrix and form multicellular communities is an adaptive behavior shared by many bacteria. In Bacillus subtilis, the model system for spore-forming bacteria, matrix production is one of the possible differentiation pathways that a cell can follow when vegetative growth is no longer feasible. While in B. subtilis the genetic system controlling matrix production has been studied in detail, it is still unclear whether other spore formers utilize similar mechanisms. We report that SF214, a pigmented strain of Bacillus pumilus isolated from the marine environment, can produce an extracellular matrix relying on orthologs of many of the genes known to be important for matrix synthesis in B. subtilis. We also report a characterization of the carbohydrates forming the extracellular matrix of strain SF214. The isolation and characterization of mutants altered in matrix synthesis, pigmentation, and spore formation suggest that in strain SF214 the three processes are strictly interconnected and regulated by a common molecular mechanism.

Rhodopseudomonas palustris is a purple photosynthetic bacterium characterized by a versatile nature and a remarkable ability to adapt to various environments. In this work, we focused our attention to its membrane characteristics and defined the structural and conformational features of the O-chain polysaccharide of LPS isolated from R. palustris strain BisA53. This strain produces a polymer with a trisaccharide repeating unit characterized by d-rhamnose, 3-deoxy-d-lyxo-2-heptulosaric acid (Dha), and a novel C-branched monosaccharide, a 4-amino-4,6-dideoxy-3-C-methyl-2-O-methyl-α-l-glucopyranose whose absolute configuration has been determined by a combination of 2D NMR spectroscopy and molecular mechanic and dynamic simulation.