The type II secretion (T2S) system of Vibrio cholerae is a multiprotein complex that spans the cell envelope and secretes proteins important for pathogenesis as well as survival in different environments. Here we report that, in addition to the loss of extracellular secretion, removal or inhibition of expression of the T2S genes, epsC-N, results in growth defects and a broad range of alterations in the outer membrane that interfere with its barrier function. Specifically, the sensitivity to membrane-perturbing agents such as bile salts and the antimicrobial peptide polymyxin B is increased, and periplasmic constituents leak out into the culture medium. As a consequence, the E stress response is induced. Furthermore, due to the defects caused by inactivation of the T2S system, the ⌬eps deletion mutant of V. cholerae strain N16961 is incapable of surviving the passage through the infant mouse gastrointestinal tract. The growth defect and leaky outer membrane phenotypes are suppressed when the culture medium is supplemented with 5% glucose or sucrose, although the eps mutants remain sensitive to membrane-damaging agents. This suggests that the sugars do not restore the integrity of the outer membrane in the eps mutant strains per se but may provide osmoprotective functions.Gram-negative bacteria possess highly sophisticated and organized cell envelopes that consist of inner and outer membranes separated by the periplasmic compartment and the peptidoglycan layer. The outer membrane is made up of a lipopolysaccharide (LPS)-phospholipid asymmetric bilayer and functions as a barrier preventing entry of toxic substances, including antibiotics, dyes, and detergents (60, 72). At the same time, the outer membrane allows nutrient acquisition and transport of molecules in and out of the cell. Several dedicated transport systems have evolved for this purpose, and at least six pathways are required for extracellular protein secretion alone (43,67). One such system is the type II secretion (T2S) system, which has been identified in a wide variety of Proteobacteria, including many pathogens (for reviews, see references 12 and 74). Many of the proteins secreted by the T2S pathway, such as proteases, lipases, cellulases, pectinases, phospholipases, lipases, and toxins, contribute to virulence. These secreted proteins are synthesized with signal peptides and are first transported into the periplasmic compartment by Sec-or Tat-dependent processes and then cross the outer membrane through the T2S machinery (66, 89).In recent years, much attention has been paid to the interactions between individual components and the mechanism by which the multiprotein T2S complex is assembled. The present model for T2S machines includes a secretion pore in the outer membrane, a multiprotein subcomplex localized in the cytoplasmic membrane, a pseudopilus that spans the periplasmic compartment, and an ATPase in the cytoplasm (38). Besides the interactions within the T2S complex, it appears that T2S components also interact with other cell envelope con...

Recent advances in microscopy and protein localization techniques have provided new insights into the remarkable complexity of the bacterial cell. Although bacteria lack discrete cellular compartments such as organelles, they possess an impressive scheme of subcellular organization at the level of protein localization. There are a growing number of examples of bacterial proteins for which specific intracellular localizations are essential for proper function and regulation. Dynamic polar localization of proteins critical for cell division, chromosome partitioning, and cell cycle control in Escherichia coli, Bacillus subtilis, and Caulobacter crescentus have recently been described (see Table 1). These exciting observations establish that bacterial polarity plays a critical cellular role and that prokaryotic organization is much more complex than previously believed.Clearly, many proteins and protein complexes are able to navigate the bacterial cell and ultimately recognize their appropriate destinations. The current challenge is to uncover the mechanisms, both active and passive, by which proteins are localized and then maintained at the proper intracellular location. The goal of this minireview is to explore a variety of examples of bacterial polarity, to expand upon the current models of polar localization, and to shed light on the spectrum of ways that bacteria may distinguish the polar cellular membrane from the lateral membrane. Several excellent reviews covering recent observations of dynamic polar protein localization have recently been published (7,37,38,41,76,82,83,92,93,102). Here we focus on other aspects of bacterial polarity, including the ultrastructural differences at the cell pole, the modes of polarity in actin-based motility and chemotaxis, and the implications of polarity in bacterial cellular function. MORPHOLOGICAL DIFFERENCES AT CELL ENDSThe diversity of bacterial shapes extends well beyond the basic sphere, rod, and spirochete forms. Many bacteria are decorated with pili, flagella, and/or stalks, which are often found exclusively at one or both cell poles. The presence of such polar structures reveals that at least some bacteria display a complicated organization scheme, since biogenesis of polar structures clearly demands an asymmetry of their components.In addition to these external polar structures, early ultrastructural studies revealed internal differences at the cell poles of some bacteria. One striking example is the polar organelle found at the flagellated pole of diverse gram-negative bacteria such as Aquaspirillum (65), Sphaerotilus (96), Rhodopseudomonas (95), Campylobacter (9, 75), and Helicobacter (63). In each case, the polar organelle is subpolarly located near the cytoplasmic membrane adjacent to the flagella (Fig. 1), suggesting a relationship between the polar flagella and the polar organelle. Further supporting this model, Sphaerotilus natans swarm cells have a polar organelle, whereas nonmotile cells do not (34). The polar organelle has not been identified in all polarl...

How rod-shaped bacteria form and maintain their shape is an important question in bacterial cell biology. Results from fluorescent light microscopy have led many to believe that the actin homolog MreB and a number of other proteins form long helical filaments along the inner membrane of the cell. Here we show using electron cryotomography of six different rod-shaped bacterial species, at macromolecular resolution, that no long (>80 nm) helical filaments exist near or along either surface of the inner membrane. We also use correlated cryo-fluorescent light microscopy (cryo-fLM) and electron cryo-tomography (ECT) to identify cytoplasmic bundles of MreB, showing that MreB filaments are detectable by ECT. In light of these results, the structure and function of MreB must be reconsidered: instead of acting as a large, rigid scaffold that localizes cell-wall synthetic machinery, moving MreB complexes may apply tension to growing peptidoglycan strands to ensure their orderly, linear insertion.

Secretion of cholera toxin and other virulence factors from Vibrio cholerae is mediated by the type II secretion (T2S) apparatus, a multiprotein complex composed of both inner and outer membrane proteins. To better understand the mechanism by which the T2S complex coordinates translocation of its substrates, we are examining the protein-protein interactions of its components, encoded by the extracellular protein secretion (eps) genes. In this study, we took a cell biological approach, observing the dynamics of fluorescently tagged EpsC and EpsM proteins in vivo. We report that the level and context of fluorescent protein fusion expression can have a bold effect on subcellular location and that chromosomal, intraoperon expression conditions are optimal for determining the intracellular locations of fusion proteins. Fluorescently tagged, chromosomally expressed EpsC and EpsM form discrete foci along the lengths of the cells, different from the polar localization for green fluorescent protein (GFP)-EpsM previously described, as the fusions are balanced with all their interacting partner proteins within the T2S complex. Additionally, we observed that fluorescent foci in both chromosomal GFP-EpsC-and GFP-EpsM-expressing strains disperse upon deletion of epsD, suggesting that EpsD is critical to the localization of EpsC and EpsM and perhaps their assembly into the T2S complex.The type II secretion (T2S) pathway is widely used by pathogenic gram-negative bacteria for delivery of virulence factors into the extracellular milieu (11,17,46). Proteins destined for release through this pathway are first translocated across the cytoplasmic membrane via the Sec (24, 42) or Tat (59) machinery. Following folding and assembly in the periplasm, the proteins are transported across the outer membrane via the T2S machinery, a complex composed of 12 to 16 different gene products, depending on the species. In Vibrio cholerae, the elements of the T2S apparatus are encoded by the extracellular protein secretion (eps) genes, epsC through epsN and pilD (vcpD) (18,31,39,49,50). Together these proteins coordinate the outer membrane translocation of the major virulence factor, cholera toxin, as well as chitinase, lipase, hemagglutinin/ protease, and other proteases (12,27,49). Our studies are focused on better understanding how the T2S complex assembles in the cell envelope of V. cholerae to begin to elucidate the mechanism by which extracellular secretion is accomplished.

The chemosensory complexes in Escherichia coli are localized predominantly in large aggregates at one or both of the cell poles, however, neither the role of the polar localization nor the role of the clustering is understood. In E. coli, the two classes of chemoreceptors or transducers, high-and low-abundance, differ in their ability to support chemotaxis when expressed as the sole chemoreceptor type in the cell. In this study, we examined both the contribution of individual chemoreceptors to polar clustering and the ability of each chemoreceptor type to cluster in the absence of all others. We found that polar clustering of methyl-accepting chemotaxis proteins (MCPs) is not dependent on any one chemoreceptor type. Remarkably, when expressed individually at similar levels, the chemoreceptors display differential clustering abilities. The high-abundance transducers cluster at the cell pole almost as well as do the MCPs in cells expressing all four species, whereas the low-abundance transducers, although polar, are not particularly clustered. CheA and CheW distributions in strains expressing only one chemoreceptor type coincide with MCP localization, indicating that the low-abundance chemoreceptors are competent for ternary complex formation but are defective in aggregation. These studies reveal that, in contrast to our previous model, polarity of the chemoreceptors is independent of clustering, suggesting that the polar localization of the chemoreceptors is not simply caused by diffusion limitations on large protein aggregates.