Person:

Sherman, David

The cell surface of most Gram-negative bacteria is covered with lipopolysaccharide (LPS). The network of charges and sugars provided by the dense packing of LPS molecules in the outer leaflet of the outer membrane interferes with the entry of hydrophobic compounds into the cell, including many antibiotics. In addition, LPS can be recognized by the immune system and plays a crucial role in many interactions between bacteria and their animal hosts. LPS is synthesized in the inner membrane of Gram-negative bacteria, so it must be transported across their cell envelope to assemble at the cell surface. Over the past two decades, much of the research on LPS biogenesis has focused on the discovery and understanding of Lpt, a multi-protein complex that spans the cell envelope and functions to transport LPS from the inner membrane to the outer membrane. This paper focuses on the early steps of the transport of LPS by the Lpt machinery: the extraction of LPS from the inner membrane. The accompanying paper (May JM, Sherman DJ, Simpson BW, Ruiz N, Kahne D. 2015 Phil. Trans. R. Soc. B 370, 20150027. (doi:10.1098/rstb.2015.0027)) describes the subsequent steps as LPS travels through the periplasm and the outer membrane to its final destination at the cell surface.

Gram-negative bacteria possess an outer membrane (OM) containing lipopolysaccharide (LPS). Proper assembly of the OM not only prevents certain antibiotics from entering the cell, but also allows others to be pumped out. To assemble this barrier, the seven-protein lipopolysaccharide transport (Lpt) system extracts LPS from the outer leaflet of the inner membrane (IM), transports it across the periplasm and inserts it selectively into the outer leaflet of the OM. As LPS is important, if not essential, in most Gram-negative bacteria, the LPS biosynthesis and biogenesis pathways are attractive targets in the development of new classes of antibiotics. The accompanying paper (Simpson BW, May JM, Sherman DJ, Kahne D, Ruiz N. 2015 Phil. Trans. R. Soc. B 370, 20150029. (doi:10.1098/rstb.2015.0029)) reviewed the biosynthesis of LPS and its extraction from the IM. This paper will trace its journey across the periplasm and insertion into the OM.

Gram-negative bacteria contain an unusual outer membrane that prevents the entry of most currently available antibiotics. This membrane contains a complex glycolipid, LPS, on the exterior. It is not understood how such a large molecule, which can contain hundreds of sugars and six fatty acyl chains, is transported across the cell envelope from its site of synthesis in the cytoplasmic membrane to the cell surface. Using a combination of genetics, biochemistry, and structural biology, we characterized residues in the protein that powers LPS transport to gain mechanistic insight into how ATP hydrolysis is coupled to the biological function of the transporter. These tools help us understand how to design antibiotics targeting this essential pathway.

A wide variety of enzymatic pathways that produce specialized metabolites in bacteria, fungi and plants are known to be encoded in biosynthetic gene clusters. Information about these clusters, pathways and metabolites is currently dispersed throughout the literature, making it difficult to exploit. To facilitate consistent and systematic deposition and retrieval of data on biosynthetic gene clusters, we propose the Minimum Information about a Biosynthetic Gene cluster (MIBiG) data standard.

Background: Molecular interaction Information is a key resource in modern biomedical research. Publicly available data have previously been provided in a broad array of diverse formats, making access to this very difficult. The publication and wide implementation of the Human Proteome Organisation Proteomics Standards Initiative Molecular Interactions (HUPO PSI-MI) format in 2004 was a major step towards the establishment of a single, unified format by which molecular interactions should be presented, but focused purely on protein-protein interactions. Results: The HUPO-PSI has further developed the PSI-MI XML schema to enable the description of interactions between a wider range of molecular types, for example nucleic acids, chemical entities, and molecular complexes. Extensive details about each supported molecular interaction can now be captured, including the biological role of each molecule within that interaction, detailed description of interacting domains, and the kinetic parameters of the interaction. The format is supported by data management and analysis tools and has been adopted by major interaction data providers. Additionally, a simpler, tab-delimited format MITAB2.5 has been developed for the benefit of users who require only minimal information in an easy to access configuration. Conclusion: The PSI-MI XML2.5 and MITAB2.5 formats have been jointly developed by interaction data producers and providers from both the academic and commercial sector, and are already widely implemented and well supported by an active development community. PSI-MI XML2.5 enables the description of highly detailed molecular interaction data and facilitates data exchange between databases and users without loss of information. MITAB2.5 is a simpler format appropriate for fast Perl parsing or loading into Microsoft Excel.

The surface of Gram-negative bacteria is largely composed of lipopolysaccharide (LPS), a complex glycolipid that contains multiple fatty acyl chains and up to hundreds of sugars. LPS is transported from its site of synthesis at the cytoplasmic inner membrane (IM), across an aqueous compartment (the periplasm), and through the outer membrane (OM) to the cell surface. LPS is essential for the survival of most Gram-negative bacteria. Seven essential and conserved lipopolysaccharide transport (Lpt) proteins in Escherichia coli are responsible for assembling LPS. These proteins form a continuous bridge from the IM to the OM, but it is unknown how they function in LPS transport. This dissertation describes the development of a reconstitution of LPS transport using purified Lpt proteins. The first two steps of this process are extraction of LPS from the IM by LptBFGC and transport along the periplasmic bridge comprising LptA. We obtained high-resolution crystal structures of the ATPase LptB, which taught us how to overexpress and purify LptBFGC as a stable complex that is highly active in proteoliposomes. Crystallographic snapshots of LptB bound to ATP and ADP provided insight into how energy is used to extract the fatty acyl chains of LPS from the IM. Using site-specific photocrosslinking, we monitored ATP-dependent extraction of LPS from proteoliposomes by LptC and release to LptA. This reconstitution of LptBFGC led to a hypothesis about a role for LptC in regulating the activity of the Lpt IM complex. We also developed methods to reconstitute the late steps of transport by monitoring LPS transit from LptA to the OM components, LptDE, contained in a different proteoliposome. One important observation from this reconstitution is that it appears as though the OM translocon can change activity depending on the amount of LPS in the OM. These studies provide us with the first clues as to how the cell might regulate LPS flux to the cell surface. This reconstitution will allow for mechanistic studies of LPS transport and will aid in the discovery and development of antibiotics targeting this essential process.