The soil amoeba Dictyostelium discoideum is a haploid eukaryote that, upon starvation, aggregates and enters a developmental cycle to produce fruiting bodies. In this study, we infected single‐cell stages of D. discoideum with different Legionella species. Intracellular growth of Legionella in this new host system was compared with their growth in the natural host Acanthamoeba castellanii. Transmission electron microscopy of infected D. discoideum cells revealed that legionellae reside within the phagosome. Using confocal microscopy, it was observed that replicating, intracellular, green fluorescent protein (GFP)‐tagged legionellae rarely co‐localized with fluorescent antibodies directed against the lysosomal protein DdLIMP of D. discoideum. This indicates that the bacteria inhibit the fusion of phagosomes and lysosomes in this particular host system. In addition, Legionella infection of D. discoideum inhibited the differentiation of the host into the multicellular fruiting stage. Co‐culture studies with profilin‐minus D. discoideum mutants and Legionella resulted in higher rates of infection when compared with infections of wild‐type amoebae. Because the amoebae are amenable to genetic manipulation as a result of their haploid genome and because a number of cellular markers are available, we show for the first time that D. discoideum is a valuable model system for studying intracellular pathogenesis of microbial pathogens.

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Abstract, Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin Ltti did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.

Bacteriophage K139 was recently characterized as a temperate phage of O1 Vibrio cholerae. In this study we have determined the phage adsorption site on the bacterial cell surface. Phage-binding studies with purified lipopolysaccharide (LPS) of different O1 serotypes and biotypes revealed that the O1 antigen serves as the phage receptor. In addition, phage-resistant O1 El Tor strains were screened by using a virulent isolate of phage K139. Analysis of the LPS of such spontaneous phage-resistant mutants revealed that most of them synthesize incomplete LPS molecules, composed of either defective O1 antigen or core oligosaccharide. By applying phage-binding studies, it was possible to distinguish between receptor mutants and mutations which probably caused abortion of later steps of phage infection. Furthermore, we investigated the genetic nature of O1-negative strains by Southern hybridization with probes specific for the O antigen biosynthesis cluster (rfb region). Two of the investigated O1 antigen-negative mutants revealed insertions of element IS1004 into the rfb gene cluster. Treating one wbeW::IS1004 serum-sensitive mutant with normal human serum, we found that several survivors showed precise excision of IS1004, restoring O antigen biosynthesis and serum resistance. Investigation of clinical isolates by screening for phage resistance and performing LPS analysis of nonlysogenic strains led to the identification of a strain with decreased O1 antigen presentation. This strain had a significant reduction in its ability to colonize the mouse small intestine.Vibrio cholerae strains from serogroups O1 and O139 are the etiologic agents of cholera, a life-threatening acute diarrhea. The O1 serogroup is divided into the main serotypes Inaba and Ogawa, and O1 is subdivided into two distinct biotypes, designated classical and El Tor (22). Lipopolysaccharide (LPS) is the major integral component of the outer membrane and chemically consists of an O antigen, a core oligosaccharide, and lipid A. The O antigen of O1 V. cholerae consists of a homopolymer of approximately 18 (132) linked linear 4-(3-deoxy-L-glycero-tetronamido)-4,6-dideoxy-D-mannose) (23, 36). The LPS also contains the carbohydrate quinovosamine, which at the present time cannot be precisely defined as a component of either the O antigen or the core oligosaccharide (45). The Ogawa and Inaba serotypes differ by the presence of a 2-Omethyl group in the nonreducing terminal carbohydrate in the Ogawa O antigen (19,21). It was shown that Ogawa and Inaba O1 LPS can interconvert and that this serotype variation is due to spontaneous mutations in the wbeT gene (47). Strains of the serogroup O139 contain only a short O antigen but, in contrast to O1 strains, are encapsulated (51). Molecular and epidemiological analyses as well as phage typing revealed that O139 strains are very similar to O1 El Tor strains (2,17,18). One characteristic difference is the replacement of the 22-kb O1 rfb region with a 35-kb DNA fragment encoding the O139 O antigen and capsule (4,5,10,48)...

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