Use of digitized video microscopy with a fluorogenic enzyme substrate to demonstrate cell‐ and compartment‐specific gene expression in <i>Salmonella enteritidis</i> and <i>Bacillus subtilis</i>

Lewis, Peter J.; Nwoguh, C. E.; Barer, Michael R.; Harwood, Colin R.; Errington, Jeff

doi:10.1111/j.1365-2958.1994.tb00459.x

Cited by 35 publications

(38 citation statements)

References 22 publications

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“…Samples were prepared from cultures harvested 2.5 and 4 h after the onset of sporulation and probed with anti-SafA antisera and a secondary anti-rabbit immunoglobulin G (IgG) fluorescent conjugate. We used a nucleoid stain (DAPI) in our immunofluorescence experiments as a marker for the approximate stage of sporulation of each sporangium and to distinguish between the mother cell and forespore compartments (13,22,23,31,34). Differential interference contrast images were captured to mark the position of the whole cell.…”

Section: Resultsmentioning

confidence: 99%

SpoVID Guides SafA to the Spore Coat in Bacillus subtilis

et al. 2001

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Bacteria assemble complex structures by targeting proteins to specific subcellular locations. The protein coat that encases Bacillus subtilis spores is an example of a structure that requires coordinated targeting and assembly of more than 24 polypeptides. The earliest stages of coat assembly require the action of three morphogenetic proteins: SpoIVA, CotE, and SpoVID. In the first steps, a basement layer of SpoIVA forms around the surface of the forespore, guiding the subsequent positioning of a ring of CotE protein about 75 nm from the forespore surface. SpoVID localizes near the forespore membrane where it functions to maintain the integrity of the CotE ring and to anchor the nascent coat to the underlying spore structures. However, it is not known which spore coat proteins interact directly with SpoVID. In this study we examined the interaction between SpoVID and another spore coat protein, SafA, in vivo using the yeast two-hybrid system and in vitro. We found evidence that SpoVID and SafA directly interact and that SafA interacts with itself. Immunofluorescence microscopy showed that SafA localized around the forespore early during coat assembly and that this localization of SafA was dependent on SpoVID. Moreover, targeting of SafA to the forespore was also dependent on SpoIVA, as was targeting of SpoVID to the forespore. We suggest that the localization of SafA to the spore coat requires direct interaction with SpoVID.Proteins are targeted to specific subcellular locations during the assembly of a variety of bacterial structures. The structures assembled by prokaryotes include, for example, the cell division septum (3), surface protein layers (S-layers) (36), and a number of surface appendages such as flagella (24) and pili (17). Here we are concerned with the assembly of the Bacillus subtilis spore coat, a proteinaceous structure that encases spores (reviewed in references 7 and 16). The B. subtilis spore is a metabolically dormant cell type that is formed as an adaptive response to nutrient depletion (9, 30, 37). The spore coat provides protection against physical and chemical insults and can sense and respond to nutrients that trigger germination (9, 26).Spore morphogenesis commences by the placement of an asymmetric septum that divides the rod-shaped cell into a larger mother cell and a smaller prespore, each containing a copy of the chromosome. The septal membranes migrate around the prespore, in a process known as engulfment, which eventually results in the formation of a free-floating protoplast (the forespore) separated from the mother cell cytoplasm by a double-membrane system. Cell wall material (the cortex) is deposited between the membranes of the forespore (9, 30, 37). A thick coat composed of more than two dozen proteins is assembled around the outer forespore membrane. The majority of the proteins that make up the coat are synthesized in the mother cell under the direction of mother cell-specific RNA polymerase sigma factors (7,16,30).

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Section: Resultsmentioning

confidence: 99%

SpoVID Guides SafA to the Spore Coat in Bacillus subtilis

et al. 2001

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“…This region corresponded to the forespore, as determined by DNA staining with propidium iodide in separate experiments. The forespore can be recognized as an area of intense DNA staining, because of the more (14,20,33). It should be noted that, because of spectral crossover, propidium iodide is not optimal for use with GFP.…”

Section: Resultsmentioning

confidence: 99%

Use of green fluorescent protein for visualization of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis

et al. 1995

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We report the use of the green fluorescent protein (GFP) of Aequorea victoria to visualize cell-specific gene expression and protein subcellular localization during sporulation in Bacillus subtilis. Sporangia bearing the gene (gfp) for the green fluorescent protein fused to genes under the control of the sporulation transcription factor F exhibited a forespore-specific pattern of fluorescence. Forespore-specific fluorescence could be detected with fusions to promoters that are utilized with low (csfB) and high (sspE-2G) efficiency by Fcontaining RNA polymerase. Conversely, a mother cell-specific pattern of fluorescence was observed in sporangia bearing a transcriptional fusion of gfp to a spore coat protein gene (cotE) under the control of E and an in-frame fusion to a regulatory gene (gerE) under the control of K . An in-frame fusion of gfp to cotE demonstrated that GFP can also be used to visualize protein subcellular localization. In sporangia producing the CotE-GFP fusion protein, fluorescence was found to localize around the developing spore, and this localization was dependent upon SpoIVA, a morphogenetic protein known to determine proper localization of CotE.The green fluorescent protein (GFP) of the bioluminescent jellyfish Aequorea victoria has elicited much interest as a reporter protein for the visualization of gene expression and protein subcellular localization and has been used in a wide range of organisms, including Escherichia coli, Caenorhabditis elegans, and Drosophila melanogaster (3,37). GFP is a 238-amino-acid (27-kDa) protein that emits green light when excited with blue light, the source of which in A. victoria is a calcium-activated photoprotein, aequorin (24, 25). The fluorophore is formed by an autocatalytic cyclization of three amino acid residues within GFP (4). Here we report the use of GFP to visualize cell-specific gene expression and protein localization during sporulation in Bacillus subtilis.Sporulation in B. subtilis involves the formation of an asymmetrically positioned septum, which partitions the developing cell into forespore and mother cell compartments. Initially, the forespore and mother cell lie side by side, but later in development the forespore is engulfed by the mother cell, resulting in a cell within a cell. Differential gene expression between the mother cell and forespore is initially directed by RNA polymerase containing E and F , respectively (2,10,14,21,23,35), and later K and G , respectively (16-19, 22, 36). Two examples of promoters that are under the control of F are the promoters for csfB, a weakly expressed gene of unknown function (9), and sspE-2G, an artificial promoter that supports a high level of transcription (35). An example of a promoter under the control of E is that of cotE, a gene that encodes a protein involved in the formation of the spore coat (11). An example of a promoter under the control of K is that of gerE, a regulatory gene encoding a DNA-binding protein that activates late expression of spore coat genes (7, 39). These four promo...

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“…The only reliable way to determine the fraction of the population capable of resuscitation, besides advanced microscopic detection methods (Lewis et al, 1994) and flow cytometry, is the application of the MPN method, which is also called dilution extinction or fraction-negative method. In this type of assay the population is dissected in multiple samples of dilutions containing ultimately one or less than one cell per sample.…”

Section: Recovery Of Vbnc Bacteriamentioning

confidence: 99%

Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibrio vulnificus

Weichart

Kjelleberg

1996

Microbiology

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The estuarine, human-pathogenic bacterium Vibrio vulnificus responds to low temperature by the formation of viable but nonculturable (VBNC) cells, while Starvation a t moderate temperatures allows for maintenance O f culturability of this organism. Recovery of cold-incubated populations of V. vulnificus was restricted to the culturable fraction in slide cultures and most probable number assays. These populations, however, gave between 1.1-and 8-fold higher c.f.u. counts on soft agar plates than on ordinary agar plates, indicating that a small and variable fraction of the cell population was injured rather than nonculturable. Thus, the population of cold-incubated cells is composed of culturable, injured and nonculturable cells, with the numbers of the culturable and injured cells rapidly decreasing during cold incubation. Recovery of nonculturable cells of the organism, however, could not be obtained by any combination of temperature and nutrient shifts in any of the assays. VBNC cells of the organism were assessed with regard to their persistence and stress resistance in comparison to growing and starved cells. The sonication resistance of VBNC cells was initially similar to that of growing cells, but increased during prolonged cold incubation. The final resistance of cold-incubated VBNC cells was equal to the markedly increased resistance of starving cells, which also displayed increased resistance against exposure to ethanol and mechanical stress. Our results indicate that in spite of the apparent absence of recovery under a wide range of laboratory conditions, VBNC cells of V. vulnificus undergo changes at low temperature which potentially allow them to persist for extended periods.

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Use of digitized video microscopy with a fluorogenic enzyme substrate to demonstrate cell‐ and compartment‐specific gene expression in Salmonella enteritidis and Bacillus subtilis

Cited by 35 publications

References 22 publications

SpoVID Guides SafA to the Spore Coat in Bacillus subtilis

SpoVID Guides SafA to the Spore Coat in Bacillus subtilis

Use of green fluorescent protein for visualization of cell-specific gene expression and subcellular protein localization during sporulation in Bacillus subtilis

Stress resistance and recovery potential of culturable and viable but nonculturable cells of Vibrio vulnificus

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