Multidrug resistant bacteria are a global threat for human and animal health. However, they are only part of the problem of antibiotic failure. Another bacterial strategy that contributes to their capacity to withstand antimicrobials is the formation of biofilms. Biofilms are associations of microorganisms embedded a self-produced extracellular matrix. They create particular environments that confer bacterial tolerance and resistance to antibiotics by different mechanisms that depend upon factors such as biofilm composition, architecture, the stage of biofilm development, and growth conditions. The biofilm structure hinders the penetration of antibiotics and may prevent the accumulation of bactericidal concentrations throughout the entire biofilm. In addition, gradients of dispersion of nutrients and oxygen within the biofilm generate different metabolic states of individual cells and favor the development of antibiotic tolerance and bacterial persistence. Furthermore, antimicrobial resistance may develop within biofilms through a variety of mechanisms. The expression of efflux pumps may be induced in various parts of the biofilm and the mutation frequency is induced, while the presence of extracellular DNA and the close contact between cells favor horizontal gene transfer. A deep understanding of the mechanisms by which biofilms cause tolerance/resistance to antibiotics helps to develop novel strategies to fight these infections.

Plant growth-promoting rhizobacteria (PGPR) can protect plants against pathogenic microbes through a diversity of mechanisms including competition for nutrients, production of antibiotics, and stimulation of the host immune system, a phenomenon called induced systemic resistance (ISR). In the past 30 years, the Pseudomonas spp. PGPR strains WCS358, WCS374 and WCS417 of the Willie Commelin Scholten (WCS) collection have been studied in detail in pioneering papers on the molecular basis of PGPR-mediated ISR and mechanisms of biological control of soil-borne pathogens via siderophore-mediated competition for iron. The genomes of the model WCS PGPR strains were sequenced and analyzed to unearth genetic cues related to biological questions that surfaced during the past 30 years of functional studies on these plant-beneficial microbes. Whole genome comparisons revealed important novel insights into iron acquisition strategies with consequences for both bacterial ecology and plant protection, specifics of bacterial determinants involved in plant-PGPR recognition, and diversity of protein secretion systems involved in microbe-microbe and microbe-plant communication. Furthermore, multi-locus sequence alignment and whole genome comparison revealed the taxonomic position of the WCS model strains within the Pseudomonas genus. Despite the enormous diversity of Pseudomonas spp. in soils, several plant-associated Pseudomonas spp. strains that have been isolated from different hosts at different geographic regions appear to be nearly isogenic to WCS358, WCS374, or WCS417. Interestingly, all these WCS look-a-likes have been selected because of their plant protective or plant growth-promoting properties. The genome sequences of the model WCS strains revealed that they can be considered representatives of universally-present plant-beneficial Pseudomonas spp. With their well-characterized functions in the promotion of plant growth and health, the fully sequenced genomes of the WCS strains provide a genetic framework that allows for detailed analysis of the biological mechanisms of the plant-beneficial traits of these PGPR. Considering the increasing focus on the role of the root microbiome in plant health, functional genomics of the WCS strains will enhance our understanding of the diversity of functions of the root microbiome.

Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a well-established imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution.

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract. The outer membrane protects Gram-negative bacteria against harmful compounds from the environment. Nutrients usually pass this barrier by passive diffusion via pore-forming proteins. However, nutrients that are scarce in the environment are taken up via an active, receptor-mediated process. The vast majority of Gram-negative bacterial receptors described to date are involved in iron acquisition. Since free iron is scarce in the human host, these receptors constitute important virulence factors. In a search for putative vaccine components, we have characterized here a new receptor of Neisseria meningitidis, a resident of the nasopharynx that occasionally causes sepsis and meningitis. We show that expression of this receptor is induced under zinc limitation and that the protein is involved in the uptake of zinc. Homologues of this protein are present in many other Gram-negative pathogens, particularly in those residing in the respiratory tract, suggesting that receptor-mediated zinc acquisition is important for bacteria residing in this niche. We also found that the protein is highly conserved among N. meningitidis isolates and that it induces bactericidal antibodies upon immunization of mice. Therefore, the protein appears an excellent candidate for the development of a vaccine against N. meningitidis, for which no universal vaccine is available yet.

The cell envelope of Gram-negative bacteria consists of two membranes separated by the periplasm. In contrast with most integral membrane proteins, which span the membrane in the form of hydrophobicα-helices, integral outer-membrane proteins (OMPs) formβ-barrels. Similarβ-barrel proteins are found in the outer membranes of mitochondria and chloroplasts, probably reflecting the endosymbiont origin of these eukaryotic cell organelles. How theseβ-barrel proteins are assembled into the outer membrane has remained enigmatic for a long time. In recent years, much progress has been reached in this field by the identification of the components of the OMP assembly machinery. The central component of this machinery, called Omp85 or BamA, is an essential and highly conserved bacterial protein that recognizes a signature sequence at the C terminus of its substrate OMPs. A homologue of this protein is also found in mitochondria, where it is required for the assembly ofβ-barrel proteins into the outer membrane as well. Although accessory components of the machineries are different between bacteria and mitochondria, a mitochondrialβ-barrel OMP can be assembled into the bacterial outer membrane and, vice versa, bacterial OMPs expressed in yeast are assembled into the mitochondrial outer membrane. These observations indicate that the basic mechanism of OMP assembly is evolutionarily highly conserved.

β‐Barrel proteins are present in the outer membranes of Gram‐negative bacteria, mitochondria and chloroplasts. The central component of their assembly machinery is called Omp85 in bacteria. Omp85 is predicted to consist of an integral membrane domain and an amino‐terminal periplasmic extension containing five polypeptide‐transport‐associated (POTRA) domains. We have addressed the function of these domains by creating POTRA domain deletions in Omp85 of Neisseria meningitidis. Four POTRA domains could be deleted with only slight defects in Omp85 function. Only the most carboxy‐terminal POTRA domain was essential, as was the membrane domain. Thus, similar to the mitochondrial Omp85 homologue, the functional core of bacterial Omp85 consists of its membrane domain and a single POTRA domain, that is, POTRA5.