Colloidal Gold Labels for Immunocytochemical Analysis of Microbes

Smit, J; Todd, William J.

doi:10.1007/978-1-4684-5119-1_17

Cited by 22 publications

(17 citation statements)

References 87 publications

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“…However, preparations of membrane vesicles, devoid of most cytoplasmic proteins, appear to contain some enzyme I and HPr (4,5), suggesting that these proteins may be membrane associated. The studies reported here used immunoelectron microscopy (6)(7)(8) to visualize the precise in vivo distribution of enzyme I. The results support the idea that enzyme I is localized at the inner membrane by interaction with a limited number of binding sites.…”

supporting

confidence: 73%

“…We show here that at least one-third of the cellular enzyme I of wild-type E. coli (Fig. 2) The use of immunochemistry coupled with electron microscopy of cryosections has evolved as a powerful tool for determining the physiological location of proteins in microbes (6). Lazdunski has effectively used this approach for the localization in E. coli of elongation factor Tu (27) and colicins (28,29).…”

Section: Discussionmentioning

confidence: 93%

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Localization to the inner surface of the cytoplasmic membrane by immunoelectron microscopy of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Ghosh

Owens

Pietri

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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The phosphoenolpyruvate:sugar phospho- MATERIALS AND METHODS Growth of E. coil Strains. The strains of E. coli used in this study are listed in Table 1. The plasmid pDIA100 (10) encodes the gene for adenylate cyclase; the plasmids pEL06 (9) and pDS20 (11) encode genes for the PTS proteins HPr, enzyme I, and a fragment of enzyme iiiGIc; and plasmid pDS48 (11) encodes the gene for enzyme IIIGIc. The plasmid pEL06 resident in strain 600 results in an approximately 20-to 70-fold overexpression of enzyme I (9, 11). Strains were grown in LB medium (12) at 370C with aeration. For studies involving the localization of 83-galactosidase, cells were grown to midlogarithmic phase, at which point 5 mM isopropyl B-D-thiogalactopyranoside was added and the cultures were allowed to grow for another hour. Plasmids were maintained in the appropriate strains by including in the cultures ampicillin (30 ,Ag/ml), kanamycin (50 jug/ml), or tetracycline (10 kg/ml). Overnight cultures of the strains were diluted 1:10 with fresh medium and grown for 4 hr. The cells were collected by centrifugation (10,000 x g) and then washed with phosphate-buffered saline (PBS) at pH 7.2. The washed cells were resuspended in 0.5% glutaraldehyde/0.6% tannic acid in PBS for 30 min at room temperature (7,8). The fixed cells were washed once with PBS followed by several washes in 30% (vol/vol) glycerol in PBS or 2.3 M sucrose in PBS for cryoprotection (13). After the final wash, the wet pellet was thoroughly mixed with a Vortex mixer. Drops of the thick suspension were placed on cryomicrotome specimen pins and quickly frozen by plunging them into liquid nitrogen-cooled propane (14, 15). These specimens were stored in a liquid nitrogen reservoir for future sectioning.Enzyme I and Antibody Reagents. Enzyme I was purified from E. coli strain 599 (9) as described (16). Samples of the purified protein were used for production of rabbit polyclonal antibody by standard procedures.fi-Galactosidase. The f3-galactosidase activity of strains KL16 and 600 were measured by the method of Miller (17). The activities were strain KL16 = 185 Miller units and strain 600 = 69 Miller units. Rabbit antibody against E. coli ,3-galactosidase was from Cappell Laboratories.Immunoelectron Microscopy. Specimen pins were mounted on the holder of a Cryonova cryomicrotome (LKB) and 60-to 80-nm-thick sections were cut with a freshly broken glass knife at -98°C. Ribbons of dry sections were collected from the edge of the knife on freezing drops of sucrose (13); they were then transferred to Formvar-coated electron microscope grids, which were placed on liquefied gelatin. Immunolabeling of the sections for revealing antigenic sites and staining of the sections on the grids for generating contrast of the cellular structures were accomplished by floating the grids on drops of solutions placed in Petri dishes. The treatments were done in the following sequence (all solutions were made in PBS): 0.02 M glycine, preimmune serum (1: 1000 dilution), immune serum (1:1000 dilution), protein A...

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supporting

confidence: 73%

Section: Discussionmentioning

confidence: 93%

Localization to the inner surface of the cytoplasmic membrane by immunoelectron microscopy of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Ghosh

Owens

Pietri

et al. 1989

Proc. Natl. Acad. Sci. U.S.A.

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“…There is also evidence in C. crescentus for a cytoplasmic pool of pilin (32). The observation that adsorption of bacteriophage 4CbS to the C. crescentus pili prevented the loss of these structures at the time of stalk formation ( Fig.…”

Section: Resultsmentioning

confidence: 88%

Sequential regulation of developmental events during polar morphogenesis in Caulobacter crescentus: assembly of pili on swarmer cells requires cell separation

Sommer

Newton

1988

J Bacteriol

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Pili, along with the flagellum and DNA bacteriophage receptors, are structural markers for polar morphogenesis in Caulobacter crescentus. Pili act as primary receptors for a number of small, C. crescentusspecific DNA and RNA bacteriophages, and the timing of pilus-dependent adsorption of bacteriophage 4Cb5 in synchronized cell populations has led to the general conclusion that pili are formed coordinately with the flagellum and other polar surface structures in the predivisional cell. The use of rotary platinum shadow casting and electron microscopy as a direct assay for formation of flagella and pili in synchronous cell cultures now shows, however, that when expressed as fractions of the swarmer cell cycle, flagella are assembled on the predivisional cells at approximately 0.8 and that pili are assembled on the new swarmer cells at approximately 0.1 of the next cell cycle. Adsorption of pilus-specific bacteriophage 4Cb5 prevented the loss of pili from swarmer cells during development, which suggests that these structures are retracted at the time of stalk formation. Examination of temperature-sensitive cell division mutants showed that the assembly of pili depends on completion of cell separation. These results indicate that the stage-specific events required for polar morphogenesis in C. crescentus occur sequentially, rather than coordinately in the cell cycle, and that the timing of these events reflects the order of underlying cell cycle steps.Morphogenesis in the aquatic bacterium Caulobacter crescentus is characterized by stage-specific assembly and loss of polar surface structures. During each cell cycle, the nonmotile stalked cell assembles a flagellum and receptor sites for DNA bacteriophages (CbK and 4XLC72 at the cell pole distal to the stalk. These structures become part of the motile swarmer cell at division and are lost again when the swarmer cell differentiates into a stalked cell (for reviews, see references 15 and 25). The pili, which are also assembled at the flagellated cell pole (22), are another stage-specific marker for polar morphogenesis. The pili are flexible filaments with a diameter of 4 nm and a length from 1 to 4 ,um and are similar in appearance to the common polar pili of Pseudomonas aeruginosa (1). The C. crescentus pilin has a molecular weight of 8,000 and an amino-terminal sequence similar in hydrophobicity to the P. aeruginosa pilin (31), which is in turn homologous to the chromosomally encoded pilins of a number of unrelated organisms, including Neisseria gonorrhoeae and Moraxella nonliquefaciens (11,19).The pili act as primary receptors for a number of small DNA and RNA bacteriophages that are specific for the caulobacters (22,23). The time of the pilus-dependent RNA phage XCb5 adsorption in synchronized cell populations is restricted to the period that includes cell division and the early swarmer cell stage (26)(27)(28). This observation has led to the general conclusion that pili are formed coordinately with the flagellum and other polar surface structures in the predivisio...

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“…Colloidal gold was prepared as 5-nm-diameter particles by the phosphorus reduction method (49). Antibody labeling of cells for fluorescence microscopy was done by standard indirect labeling methods, using rabbit primary antisera and fluorescein-conjugated anti-rabbit serum, raised in a goat.…”

Section: Methodsmentioning

confidence: 99%

Characterization of mutants of Caulobacter crescentus defective in surface attachment of the paracrystalline surface layer

et al. 1994

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Strains of Caulobacter crescentus express a paracrystalline surface layer (S-layer) consisting of the protein RsaA. Mutants of C. crescentus NA1000 and CB2, isolated for their ability to grow in the absence of calcium ions, uniformly no longer had the S-layer attached to the cell surface. However, RsaA was still produced, and when colonies grown on calcium-sufficient medium were examined, large two-dimensional arrays of S-layer were found intermixed with the cells. Such arrays were not found in calcium-deficient medium even when high levels of magnesium ions were provided. The arrays could be disrupted with divalent ion chelators and more readily with the calcium-selective ethylene glycol-bis(P-aminoethyl ether)NNN',N'-tetraacetic acid (EGTA).Thus, the outer membrane surface was not needed as a template for self-assembly, but calcium likely was. The cell surface and S-layer gene of assembly-defective mutants of NA1000 were examined to determine the basis of the S-layer surface attachment defect. Mutants had no detectable alteration in the rough lipopolysaccharide (LPS) or a characterized capsular polysaccharide, but another polysaccharide molecule was greatly reduced or absent in all calcium-independent mutants. The molecule was shown to be a smooth LPS with a core sugar and fatty acid complement identical to those of the rough LPS and an 0 polysaccharide of homogeneous length, tentatively considered to be composed of 4,6-dideoxy-4-amino hexose, 3,6-dideoxy-3-amino hexose, and glycerol in equal proportions. This molecule (termed SLPS) was detectable by surface labeling with a specific antiserum only when the S-layer was not present. The rsaA genes from three calcium-independent mutants were cloned and expressed in an S-layer-negative, SLPS-positive strain. A normal S-layer was produced, ruling out defects in rsaA4 in these cases. It is proposed that SLPS is required for S-layer surface attachment, possibly via calcium bridging. The data support the possibility that calcium binding is required to prevent an otherwise lethal effect of SLPS. If true, mutations that eliminate the 0 polysaccharide of SLPS eliminate the lethal effects of calcium-deprived SLPS, at the expense of S-layer attachment.Surface layers (S-layers) are a common feature on the surface of eubacteria and archaebacteria (34). The structures are generally composed of repeated units of a single protein or glycoprotein (44). It is likely that in many bacteria the layer is the outermost layer, directly exposed to the environment (44). In such a position, S-layers may well have roles of protection for the cell or be involved with pathogenesis in the case of pathogenic bacteria (34). Many aspects of this general bacterial structure were summarized in a recent international meeting devoted to such structures (5).Most often, S-layer proteins are assembled on (rather than within) the underlying layer, whether it is a bilayer membrane or a complex of peptidoglycan or pseudomurein (34). Thus, S-layer structural subunits are secreted proteins and must retain b...

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Colloidal Gold Labels for Immunocytochemical Analysis of Microbes

Cited by 22 publications

References 87 publications

Localization to the inner surface of the cytoplasmic membrane by immunoelectron microscopy of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Localization to the inner surface of the cytoplasmic membrane by immunoelectron microscopy of enzyme I of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli.

Sequential regulation of developmental events during polar morphogenesis in Caulobacter crescentus: assembly of pili on swarmer cells requires cell separation

Characterization of mutants of Caulobacter crescentus defective in surface attachment of the paracrystalline surface layer

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