We have investigated DNA segregation in E. coli by inserting multiple lac operator sequences into the chromosome near the origin of replication (oriC), in the hisC gene, a terminus marker, and into plasmids P1 and F. Expression of a GFP-LacI fusion protein allowed visualization of lac operator localization. oriC was shown to be specifically localized at or near the cell poles, and when duplicated, one copy moved to the site of new pole formation near the site of cell division. In contrast, P1 and F localized to the cell center and on duplication appeared to move rapidly to the quarter positions in the cell. Our analysis suggests that different active processes are involved in movement and localization of the chromosome and of the two plasmids during segregation.

To investigate chromosome segregation in B. subtilis, we introduced tandem copies of the lactose operon operator into the chromosome near the replication origin or terminus. We then visualized the position of the operator cassettes with green fluorescent protein fused to the Lac1 repressor. In sporulating bacteria, which undergo asymmetric cell division, origins localized near each pole of the cell whereas termini were restricted to the middle. In growing cells, which undergo binary fission, origins were observed at various positions but preferentially toward the poles early in the cell cycle. In contrast, termini showed little preference for the poles. These results indicate the existence of a mitotic-like apparatus that is responsible for moving the origin regions of newly formed chromosomes toward opposite ends of the cell.

SummaryWe describe the use of time-lapse fluorescence microscopy to visualize the movement of the DNA replication origin and terminus regions on the Bacillus subtilis chromosome during the course of the cell cycle. The origin and terminus regions were tagged with a cassette of tandem lac operator repeats and visualized through the use of a fusion of the green fluorescent protein to the LacI repressor. We have discovered that origin regions abruptly move apart towards the cell poles during a brief interval of the cell cycle. This movement was also seen in the absence of cell wall growth and in the absence of the product of the parB homologue spo0J. The origin regions moved apart an average distance of 1.4 m in an 11 min period of abrupt movement, representing an average velocity of 0.17 m min ¹1. and reaching a maximum velocity of greater than 0.27 m min ¹1 . The terminus region also exhibited a striking pattern of movement but not as far or a rapid as the origin region. These results provide evidence for a mitotic-like motor that is responsible for segregation of the origin regions of the chromosomes.

The antifungal susceptibilities of 232 pathogenic bloodstream Candida isolates collected during a recently completed trial comparing fluconazole (400 mg/day) with amphotericin B (0.5 mg/kg of body weight per day) as treatment for candidemia in the nonneutropenic patient were determined both by the National Committee for Clinical Laboratory Standards M27-P macrobroth methodology and by a less cumbersome broth microdilution methodology. For amphotericin B, M27-P yielded a very narrow range of MICs (0.125 to 1 g/ml) and there were no susceptibility differences among species. For fluconazole, a broad range of MICs were seen (0.125 to >64 g/ml), with characteristic MICs seen for each species in the rank order Candida albicans < C. parapsilosis Х Х C. lusitaniae < C. glabrata Х Х C. krusei Х Х C. lipolytica. The MIC distribution for C. tropicalis was bimodal and could not be ranked. Broth microdilution MICs were within one tube dilution of the M27-P MIC for Ն Ն90% of isolates with amphotericin B and for Ն Ն77% of isolates with fluconazole. For both methods, elevated MICs did not predict treatment failure. In the case of amphotericin B, the MIC range was too narrow to permit identification of resistant isolates. In the case of fluconazole, MICs for isolates associated with failure to clear the bloodstream consistently were equivalent to the median MIC for the given species. Successful courses of therapy were seen with four isolates from four patients despite MICs of Ն Ն32 g/ml. As MICs obtained by M27-P and similar methods correlate with responsiveness to fluconazole therapy in animal models and in AIDS patients with oropharyngeal candidiasis, the lack of correlation in this setting suggests that the MICs for these isolates are at or below the relevant fluconazole breakpoint for this dose of fluconazole and patient setting and that host factors such as failure to exchange intravenous catheters were more important than MIC in predicting outcome.Antifungal susceptibility testing remains in evolution. Recent work by the National Committee for Clinical Laboratory Standards has focused on the development of standard, reproducible methods for testing of yeast. The proposed method M27-P is a broth macrodilution method that has good interand intralaboratory reproducibility (5, 10). As MICs obtained by methods similar to M27-P have generally correlated well with outcome in various animal models of infection, it is anticipated that M27-P (or a method utilizing the principles of M27-P) will prove useful in prediction of the likelihood of response to a given antifungal agent (16). However, no breakpoints have yet been established for M27-P. During our recently completed trial of fluconazole versus amphotericin B for therapy of candidemia in nonneutropenic patients, we collected Candida bloodstream isolates from our patients (15). We now report the distribution of M27-P MICs seen in this collection of pathogenic Candida isolates, the correlation of these MICs with an easily performed broth microdilution variation of M27-P, and the ...

Ion current rectification with quartz nanopipette electrodes was investigated through the control of the surface charge. The presence and absence of a positively charged poly-L-lysine (PLL) coating resulted in the rectified current with opposite polarity. The results agreed with the theories developed for current-rectifying conical nanopores, suggesting the similar underlying mechanism among asymmetric nanostructure in general. This surface condition dependence can be used as the fundamental principle of multi-purpose real-time in vivo biosensors. Nanomaterials are being widely exploited by recent technologies because of their extraordinary properties. Although the development of fabrication processes for particular nanostructures poses great challenges by itself, the practical use of these nanomaterials is also of great interest for biology and medicine. 1 Because of their structural diversity, these materials are often categorized and referred to as nanoparticles, 2 nanowires, 3 nanotubes, 4 nanopores, 5 or nanopatterned surfaces. 6 Nanopipettes are among these; a nanopipette is defined as a pipette with a very fine tip that has a nanoscale opening. Nanolithography is one of the typical applications of nanopipettes as a delivery tool of a tiny amount of chemicals. 7,8 Nanopipettes are versatile enough to be used as a tool for sensitive detection in biomedical applications. Optical detection of fluorescently labeled macromolecules such as DNA or proteins with nanopipettes has been reported. 9,10 Fully electrical detection has also been shown with similarly sized nanoparticles, whose flow through the nanopipette opening creates temporal current blockades. 11 The ultimate goal of these efforts, the label-free real-time electrical detection of single molecules, could be achieved eventually by a deeper understanding of the fundamental characteristics of nanopipette electrodes under an external electric field. It not only helps to unveil the dynamics of biological systems but can also have a remarkable impact on drug screening and pathogen detection. Interestingly, although a general understanding of nanopipette electrodes can be based on the understanding of microelectrodes, the unique nanoscale geometry often causes characteristic behavior that requires further focused studies. For example, related studies have examined the physicochemical properties of nanopipettes under varying conditions such as electrolyte concentrations and pH, 12 or polyethylene glycol polymer coatings. 13 Similarly shaped goldplated conical nanopores have been studied in a more detailed manner, involving observations of the role of surface charge in the ionic current. 14 Our target here is the effect of cationic polymer coating on a glass nanopipette surface, providing the basis for functionalized nanopipettes that will be used as sensitive biosensors. By understanding how ions flow through the nanometer-sized opening, how these ions interact with the surface inside and outside the tip, and what happens if the surface is modified by...

Single DNA molecules labeled with nanoparticles can be detected by blockades of ionic current as they are translocated through a nanopipette tip formed by a pulled glass capillary. The nanopipette detection technique can provide not only tools for detection and identification of single DNA and protein molecules but also deeper insight and understanding of stochastic interactions of various biomolecules with their environment.

No abstract

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...