One of the most exciting developments in the field of bacterial pathogenesis in recent years is the discovery that many pathogens utilized complex nanomachines to deliver bacterially encoded effector proteins into target eukaryotic cells. These effector proteins modulate a variety of cellular functions for the pathogen’s benefit. One of these protein-delivery machines is the type III secretion system (T3SS). T3SSs are widespread in nature and are encoded not only by bacteria pathogenic to vertebrates or plants, but also by bacteria that are symbiotic to plants or insects. A central component of T3SSs is the needle complex, a supramolecular structure that mediates the passage of the secreted proteins across the bacterial envelope. Working in conjunction with several cytoplasmic components, the needle complex engages specific substrates in sequential order, moves them across the bacterial envelope, and ultimately delivers them into eukaryotic cells. The central role of T3SSs in pathogenesis makes them great targets for novel antimicrobial strategies.

A simple generic method for optimizing membrane protein overexpression in Escherichia coli is still lacking. We have studied the physiological response of the widely used ''Walker strains'' C41(DE3) and C43(DE3), which are derived from BL21(DE3), to membrane protein overexpression. For unknown reasons, overexpression of many membrane proteins in these strains is hardly toxic, often resulting in high overexpression yields. By using a combination of physiological, proteomic, and genetic techniques we have shown that mutations in the lacUV5 promoter governing expression of T7 RNA polymerase are key to the improved membrane protein overexpression characteristics of the Walker strains. Based on this observation, we have engineered a derivative strain of E. coli BL21(DE3), termed Lemo21(DE3), in which the activity of the T7 RNA polymerase can be precisely controlled by its natural inhibitor T7 lysozyme (T7Lys). Lemo21(DE3) is tunable for membrane protein overexpression and conveniently allows optimizing overexpression of any given membrane protein by using only a single strain rather than a multitude of different strains. The generality and simplicity of our approach make it ideal for highthroughput applications.engineering ͉ systems biotechnology ͉ proteomics T he natural abundance of membrane proteins is typically too low to isolate sufficient amounts of material for functional and structural studies. Therefore, membrane proteins must be obtained by overexpression, and the bacterium E. coli is the most widely used vehicle for this purpose (1). Although many membrane proteins can be overexpressed in inclusion bodies, their refolding into functional proteins is often not successful (2). To avoid the refolding problem, overexpression of membrane proteins by accumulation in the cytoplasmic membrane is needed. However, overexpression is often toxic to the cell, thereby preventing biomass formation and severely reducing yields (1). Thus, membrane protein overexpression has to be optimized, but no systematic, generic, and high-throughput-compatible method is available for the optimization process.Bacteriophage T7 RNA polymerase (T7RNAP) is often used to drive recombinant protein production in E. coli (3). In BL21(DE3) and its derivatives, the gene encoding T7RNAP is under control of the lacUV5 promoter, a strong variant of the wild-type lac promoter. It is insensitive to catabolite repression and, therefore, controlled only by the lac repressor, LacI, which binds to the lac operator (4). T7RNAP exclusively recognizes the T7 promoter and it transcribes eight times faster than E. coli RNAP allowing high yield protein production (5). Most T7 expression vectors employ a T7lac hybrid promoter that combines the strong T7 10 promoter with a lac operator to diminish leaky expression. On addition of the inducer isopropyl ␤-Dthiogalactoside (IPTG), lacI repression is relieved, resulting in recombinant protein production. If toxicity due to leaky expression is a problem, T7RNAP activity can be further dampened with the T7RNAP inhibit...

Bacterial type III protein secretion systems deliver effector proteins into eukaryotic cells in order to modulate cellular processes. Central to the function of these protein delivery machines is their ability to recognize and secrete substrates in a defined order. Here, we describe a mechanism by which a type III secretion system from the bacterial enteropathogen Salmonella enterica serovar Typhimurium can sort its substrates prior to secretion. This mechanism involves a cytoplasmic sorting platform that is sequentially loaded with the appropriate secreted proteins. The sequential loading of this platform, facilitated by customized chaperones, ensures the hierarchy in type III protein secretion. Given the presence of these machines in many important pathogens, these findings can serve as the bases for the development of novel antimicrobial strategies.

Export of proteins through type III secretion systems is critical for motility and virulence of many major bacterial pathogens. Three putative integral membrane proteins (FliP, FliQ, FliR) are suggested to form the core of an export gate in the inner membrane, but their structure, assembly and location within the final nanomachine remain unclear. Here, we present the cryoelectron microscopy structure of the Salmonella Typhimurium FliP-FliQ-FliR complex at 4.2 Å. None of the subunits adopt canonical integral membrane protein topologies, and common helix-turn-helix structural elements allow them to form a helical assembly with 5:4:1 stoichiometry. Fitting of the structure into reconstructions of intact secretion systems, combined with cross-linking, localize the export gate as a core component of the periplasmic portion of the machinery. This study thereby identifies the export gate as a key element of the secretion channel and implies that it primes the helical architecture of the components assembling downstream.