Determining the chemical composition of biological materials is paramount to the study of natural phenomena. Here, we describe the composition of model gram-negative outer membranes, focusing on the predominant assembly, an asymmetrical bilayer of lipid molecules. We also give an overview of lipid biosynthetic pathways and molecular mechanisms that organize this material into the outer membrane bilayer. An emphasis is placed on the potential of these pathways as targets for antibiotic development. We discuss deviations in composition, through bacterial cell surface remodeling, and alternative modalities to the asymmetric lipid bilayer. Outer membrane lipid alterations of current microbiological interest, such as lipid structures found in commensal bacteria, are emphasized. Additionally, outer membrane components could potentially be engineered to develop vaccine platforms. Observations related to composition and assembly of gram-negative outer membranes will continue to generate novel discoveries, broaden biotechnologies, and reveal profound mysteries to compel future research.

The bacterial cell surface is the first structure the host immune system targets to prevent infection. Cationic antimicrobial peptides of the innate immune system bind to the membrane of Gram-negative pathogens via conserved, surface-exposed lipopolysaccharide (LPS) molecules. We recently reported that modern strains of the global intestinal pathogen Vibrio cholerae modify the anionic lipid A domain of LPS with a novel moiety, amino acids. Remarkably, glycine or diglycine addition to lipid A alters the surface charge of the bacteria to help evade the cationic antimicrobial peptide polymyxin. However, the regulatory mechanisms of lipid A modification in V. cholerae are unknown. Here, we identify a novel two-component system that regulates lipid A glycine modification by responding to important biological cues associated with pathogenesis, including bile, mildly acidic pH, and cationic antimicrobial peptides. The histidine kinase Vc1319 (VprB) and the response regulator Vc1320 (VprA) respond to these signals and are required for the expression of the almEFG operon that encodes the genes essential for glycine modification of lipid A. Importantly, both the newly identified two-component system and the lipid A modification machinery are required for colonization of the mammalian host. This study demonstrates how V. cholerae uses a previously unknown regulatory network, independent of well-studied V. cholerae virulence factors and regulators, to respond to the host environment and cause infection.

Here we implement ultraviolet photodissociation (UVPD) in an online liquid chromatographic tandem mass spectrometry (MS/MS) strategy to support analysis of complex mixtures of lipid A combinatorially modified during development of vaccine adjuvants. UVPD mass spectrometry at 193 nm was utilized to characterize the structures and fragment ion types of lipid A from Escherichia coli, Vibrio cholerae, and Pseudomonas aeruginosa using an Orbitrap mass spectrometer. The fragment ions generated by UVPD were compared to those from collision induced dissociation (CID) and higher energy collision dissociation (HCD) with respect to the precursor charge state. UVPD afforded the widest array of fragment ion types including acyl chain C–O, C–N, and C–C bond cleavages and glycosidic C–O and cross ring cleavages, thus providing the most comprehensive structural analysis of the lipid A. UVPD exhibited virtually no dependence on precursor ion charge state and was best at determining lipid A structure including acyl chain length and composition, giving it an advantage over collision based methods. UVPD was incorporated into an LC–MS/MS methodology for the analysis of a number of structural variants in a complex mixture of combinatorially engineered Escherichia coli lipid A.

N-1 Methylation of the nearly invariant purine residue found at position 9 of tRNA is a nucleotide modification found in multiple tRNA species throughout Eukarya and Archaea. First discovered in Saccharomyces cerevisiae, the tRNA methyltransferase Trm10 is a highly conserved protein both necessary and sufficient to catalyze all known instances of m 1 G 9 modification in yeast. Although there are 19 unique tRNA species that contain a G at position 9 in yeast, and whose fully modified sequence is known, only 9 of these tRNA species are modified with m 1 G 9 in wild-type cells. The elements that allow Trm10 to distinguish between structurally similar tRNA species are not known, and sequences that are shared between all substrate or all nonsubstrate tRNAs have not been identified. Here, we demonstrate that the in vitro methylation activity of yeast Trm10 is not sufficient to explain the observed pattern of modification in vivo, as additional tRNA species are substrates for Trm10 m 1 G 9 methyltransferase activity. Similarly, overexpression of Trm10 in yeast yields m 1 G 9 containing tRNA species that are ordinarily unmodified in vivo. Thus, yeast Trm10 has a significantly broader tRNA substrate specificity than is suggested by the observed pattern of modification in wildtype yeast. These results may shed light onto the suggested involvement of Trm10 in other pathways in other organisms, particularly in higher eukaryotes that contain up to three different genes with sequence similarity to the single TRM10 gene in yeast, and where these other enzymes have been implicated in pathways beyond tRNA processing.

SHORT ABSTRACT Isolation and characterization of the lipid A domain of lipopolysaccharide (LPS) from Gram-negative bacteria provides insight into cell surface based mechanisms of antibiotic resistance, bacterial survival and fitness, and how chemically diverse lipid A molecular species differentially modulate host innate immune responses. LONG ABSTRACT Lipopolysaccharide (LPS) is the major cell surface molecule of Gram-negative bacteria, deposited on the outer leaflet of the outer membrane bilayer. LPS can be subdivided into three components: the distal O-polysaccharide, a core oligosaccharide, and the lipid A hydrophobic anchor, where lipid A is the only component essential for bacterial cell survival. Following its synthesis, lipid A is chemically modified in response to environmental stresses such as pH or temperature, to promote resistance to antibiotic compounds, and to evade recognition by mediators of the host innate immune response. The following protocol details the small- and large-scale isolation of lipid A from Gram-negative bacteria. Isolated material is then chemically characterized by thin layer chromatography (TLC) or mass-spectrometry (MS). In additional to matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS we also describe tandem MS protocols for analyzing lipid A molecular species using electrospray ionization (ESI) coupled to collision induced dissociation (CID) and newly employed ultraviolet photodissociation (UVPD) methods. Our MS protocols allow for unequivocal determination of chemical structure, paramount to characterization of lipid A molecules that contain unique or novel chemical modifications. We also describe the radioisotopic labeling, and subsequent isolation, of lipid A from bacterial cells for analysis by TLC. Relative to MS-based protocols, TLC provides a more economical and rapid characterization method, but cannot be used to unambiguously assign lipid A chemical structures without the use of standards of known chemical structure. Over the last two decades isolation and characterization of lipid A has led to numerous exciting discoveries that have improved our understanding of the physiology of Gram-negative bacteria, mechanisms of antibiotic resistance, the human innate immune response and have provided many new targets in the development of antibacterial compounds.