Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels

The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure. Biofilm development initiates when bacteria attach to and sense a surface. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, the transition to the biofilm phenotype is controlled by the intracellular signal and second messenger cyclic-di-GMP (c-di-GMP). It is not known how biomedical materials might be adjusted to impede c-di-GMP signalling, and there are few extant methods for conducting such studies. Here, we develop such a method. We allowed P. aeruginosa to attach to the surfaces of poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These bacteria contained a plasmid for a green fluorescent protein (GFP) reporter for c-di-GMP. We used laser-scanning confocal microscopy to measure the dynamics of the GFP reporter for 3 h, beginning 1 h after introducing bacteria to the hydrogel. We controlled for the effects of changes in bacterial metabolism using a promoterless plasmid for GFP, and for the effects of light passing through different hydrogels being differently attenuated by using fluorescent plastic beads as ‘standard candles’ for calibration. We demonstrate that this method can measure statistically significant differences in c-di-GMP signalling associated with different PEGDA gel types and with the surface-exposed protein PilY1.

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Peanut Clump Virus RNA-1-Encoded P15 Regulates Viral RNA Accumulation but Is Not Abundant at Viral RNA Replication Sites

Journal of Virology ◽

10.1128/jvi.75.4.1941-1948.2001 ◽

2001 ◽

Vol 75 (4) ◽

pp. 1941-1948 ◽

Cited By ~ 23

Author(s):

Patrice Dunoyer ◽

Etienne Herzog ◽

Odile Hemmer ◽

Christophe Ritzenthaler ◽

Christiane Fritsch

Keyword(s):

Laser Scanning ◽

Fluorescent Protein ◽

Laser Scanning Confocal Microscopy ◽

Viral Rna ◽

Epifluorescence Microscopy ◽

Scanning Confocal Microscopy ◽

Virus Rna ◽

Green Fluorescent ◽

Rna Accumulation

ABSTRACT RNA-1 of peanut clump pecluvirus (PCV) encodes N-terminally overlapping proteins which contain helicase-like (P131) and polymerase-like (P191) domains and is able to replicate in the absence of RNA-2 in protoplasts of tobacco BY-2 cells. RNA-1 also encodes P15, which is expressed via a subgenomic RNA. To investigate the role of P15, we analyzed RNA accumulation in tobacco BY-2 protoplasts inoculated with RNA-1 containing mutations in P15. For all the mutants, the amount of progeny RNA-1 produced was significantly lower than that obtained for wild-type RNA-1. If RNA-2 was included in the inoculum, the accumulation of both progeny RNAs was diminished, but near-normal yields of both could be recovered if the inoculum was supplemented with a small, chimeric viral replicon expressing P15, demonstrating that P15 has an effect on viral RNA accumulation. To further analyze the role of P15, transcripts were produced expressing P15 fused to enhanced green fluorescent protein (EGFP). Following inoculation to protoplasts, epifluorescence microscopy revealed that P15 accumulated as spots around the nucleus and in the cytoplasm. Intracellular sites of viral RNA synthesis were visualized by laser scanning confocal microscopy of infected protoplasts labeled with 5-bromouridine 5′-triphosphate (BrUTP). BrUTP labeling also occured in spots distributed within the cytoplasm and around the nucleus. However, the BrUTP-labeled RNA and EGFP/P15 very rarely colocalized, suggesting that P15 does not act primarily at sites of viral replication but intervenes indirectly to control viral accumulation levels.

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Monitoring Tritrophic Biocontrol Interactions Between Bacillus spp., Fusarium oxysporum f. sp. cubense, Tropical Race 4, and Banana Plants in vivo Based on Fluorescent Transformation System

Frontiers in Microbiology ◽

10.3389/fmicb.2021.754918 ◽

2021 ◽

Vol 12 ◽

Author(s):

Ping He ◽

Shu Li ◽

Shengtao Xu ◽

Huacai Fan ◽

Yongfen Wang ◽

...

Keyword(s):

Laser Scanning ◽

Fluorescent Protein ◽

Laser Scanning Confocal Microscopy ◽

Transformation System ◽

Scanning Confocal Microscopy ◽

Bacillus Spp ◽

Green Fluorescent ◽

Race 4

Bacillus spp. is effective biocontrol agents for Fusarium wilt of banana (FWB), tropical race 4 (TR4). This study explores the colonization by Bacillus subtilis, Bacillus velezensis, and Bacillus amyloliquefaciens of host banana plants and elucidates the mechanism of antagonistic TR4 biocontrol. The authors selected one B. subtilis strain, three B. velezensis strains, and three B. amyloliquefaciens strains that are proven to significantly inhibit TR4 in vitro, optimized the genetic transformation conditions and explored their colonization process in banana plants. The results showed that we successfully constructed an optimized fluorescent electro-transformation system (OD600 of bacteria concentration=0.7, plasmid concentration=50ng/μl, plasmid volume=2μl, transformation voltage=1.8kV, and transformation capacitance=400Ω) of TR4-inhibitory Bacillus spp. strains. The red fluorescent protein (RFP)-labeled strains were shown to have high stability with a plasmid-retention frequency above 98%, where bacterial growth rates and TR4 inhibition are unaffected by fluorescent plasmid insertion. In vivo colonizing observation by Laser Scanning Confocal Microscopy (LSCM) and Scanning Electron Microscopy (SEM) showed that Bacillus spp. can colonize the internal cells of banana plantlets roots. Further, fluorescent observation by LSCM showed these RFP-labeled bacteria exhibit chemotaxis (chemotaxis ratio was 1.85±0.04) toward green fluorescent protein (GFP)-labeled TR4 hyphae in banana plants. We conclude that B. subtilis, B. velezensis, and B. amyloliquefaciens can successfully colonize banana plants and interact with TR4. Monitoring its dynamic interaction with TR4 and its biocontrol mechanism is under further study.

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Penetration of Surface-Inoculated Bacteria as a Result of Hydrodynamic Shock Wave Treatment of Beef Steaks

Journal of Food Protection ◽

10.4315/0362-028x-65.4.616 ◽

2002 ◽

Vol 65 (4) ◽

pp. 616-620 ◽

Cited By ~ 11

Author(s):

T. A. LORCA ◽

M. D. PIERSON ◽

J. R. CLAUS ◽

J. D. EIFERT ◽

J. E. MARCY ◽

...

Keyword(s):

Shock Wave ◽

Laser Scanning ◽

Fluorescent Protein ◽

Laser Scanning Confocal Microscopy ◽

Exterior Surface ◽

Safety Hazard ◽

E Coli ◽

Scanning Confocal Microscopy ◽

Beef Steaks ◽

Green Fluorescent

The top surface of the raw eye of round steaks was inoculated with either green fluorescent protein (GFP)-labeled Escherichia coli (E. coli-GFP) or rifampin-resistant E. coli (E. coli-rif). Cryostat sampling in concert with laser scanning confocal microscopy (LSCM) or plating onto antibiotic selective agar was used to determine if hydrodynamic shock wave (HSW) treatment resulted in the movement of the inoculated bacteria from the outer inoculated surface to the interior of intact beef steaks. HSW treatment induced the movement of both marker bacteria into the steaks to a maximum depth of 300 μm (0.3 mm). Because popular steak-cooking techniques involve the application of heat from the exterior surface of the steak to achieve internal temperatures ranging from 55 to 82°C, the extent of bacterial penetration observed in HSW-treated steaks does not appear to pose a safety hazard to consumers.

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The 25-kDa Movement Protein of PVX Elicits Nb-Mediated Hypersensitive Cell Death in Potato

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1999.12.6.536 ◽

1999 ◽

Vol 12 (6) ◽

pp. 536-543 ◽

Cited By ~ 60

Author(s):

Isabelle Malcuit ◽

María Rosa Marano ◽

Tony A. Kavanagh ◽

Walter De Jong ◽

Alec Forsyth ◽

...

Keyword(s):

Laser Scanning ◽

Fluorescent Protein ◽

Movement Protein ◽

Potato Virus X ◽

Laser Scanning Confocal Microscopy ◽

Hypersensitive Resistance ◽

Hypersensitive Cell Death ◽

Scanning Confocal Microscopy ◽

Gus Reporter ◽

Green Fluorescent

The potato gene Nb confers hypersensitive resistance to potato virus X (PVX). To characterize the viral elicitor of this resistance we introduced modifications into the genome of avirulent strains (ROTH1 and CP2) and virulent strains (UK3 and CP4) of PVX. From the analysis of the modified viral genomes, the Nb avirulence determinant was mapped in the PVX 25K gene coding for the 25-kDa movement protein. Furthermore, we showed that the isoleucine residue at position 6 of this protein was required for activation of the Nb response. Transient co-expression of the avirulent 25K gene with the β-glucuronidase (GUS) reporter gene introduced by particle bombardment in resistant and susceptible potato cells confirmed that the elicitor activity provided by the 25-kDa protein did not require other PVX-encoded proteins. To study cellular events associated with the Nb response, the 25-kDa proteins of PVX strains ROTH1 and UK3 were tagged with the green fluorescent protein (GFP) so that the dynamics of the subcellular distribution of the 25KGFP fusion proteins could be followed in living potato epidermal cells by laser scanning confocal microscopy. Using this method, we showed that the Nb-mediated response is associated with degradation of subcellular structures.

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Characterization and imaging of A6 epithelial cell clones expressing fluorescently labeled ENaC subunits

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.2.c624 ◽

2001 ◽

Vol 281 (2) ◽

pp. C624-C632 ◽

Cited By ~ 20

Author(s):

Bonnie L. Blazer-Yost ◽

Michael Butterworth ◽

Amy D. Hartman ◽

Gretchen E. Parker ◽

Carla J. Faletti ◽

...

Keyword(s):

Laser Scanning ◽

Fluorescent Protein ◽

Laser Scanning Confocal Microscopy ◽

Scanning Confocal Microscopy ◽

Parent Line ◽

Rate Limiting Step ◽

Cell Clones ◽

Clonal Lines ◽

Green Fluorescent ◽

Rate Limiting

A6 model renal epithelial cells were stably transfected with enhanced green fluorescent protein (EGFP)-tagged α- or β-subunits of the epithelial Na+channel (ENaC). Transfected RNA and proteins were both expressed in low abundance, similar to the endogenous levels of ENaC in native cells. In living cells, laser scanning confocal microscopy revealed a predominately subapical distribution of EGFP-labeled subunits, suggesting a readily accessible pool of subunits available to participate in Na+ transport. The basal level of Na+ transport in the clonal lines was enhanced two- to fourfold relative to the parent line. Natriferic responses to insulin or aldosterone were similar in magnitude to the parent line, while forskolin-stimulated Na+ transport was 64% greater than control in both the α- and β-transfected lines. In response to forskolin, EGFP-labeled channel subunits traffic to the apical membrane. These data suggest that channel regulators, not the channel per se, form the rate-limiting step in response to insulin or aldosterone stimulation, while the number of channel subunits is important for basal as well as cAMP-stimulated Na+transport.

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Dynamics of Actin-Based Movement byRickettsia rickettsii in Vero Cells

Infection and Immunity ◽

10.1128/iai.67.8.4201-4207.1999 ◽

1999 ◽

Vol 67 (8) ◽

pp. 4201-4207 ◽

Cited By ~ 80

Author(s):

Robert A. Heinzen ◽

Scott S. Grieshaber ◽

Levi S. Van Kirk ◽

Clinton J. Devin

Keyword(s):

Laser Scanning ◽

Fluorescent Protein ◽

Average Rate ◽

Time Lapse ◽

Vero Cells ◽

Laser Scanning Confocal Microscopy ◽

Phenotypic Differences ◽

Rickettsia Rickettsii ◽

Scanning Confocal Microscopy ◽

Green Fluorescent

ABSTRACT Actin-based motility (ABM) is a virulence mechanism exploited by invasive bacterial pathogens in the genera Listeria,Shigella, and Rickettsia. Due to experimental constraints imposed by the lack of genetic tools and their obligate intracellular nature, little is known about rickettsial ABM relative toListeria and Shigella ABM systems. In this study, we directly compared the dynamics and behavior of ABM ofRickettsia rickettsii and Listeria monocytogenes. A time-lapse video of moving intracellular bacteria was obtained by laser-scanning confocal microscopy of infected Vero cells synthesizing β-actin coupled to green fluorescent protein (GFP). Analysis of time-lapse images demonstrated that R. rickettsii organisms move through the cell cytoplasm at an average rate of 4.8 ± 0.6 μm/min (mean ± standard deviation). This speed was 2.5 times slower than that of L. monocytogenes, which moved at an average rate of 12.0 ± 3.1 μm/min. Although rickettsiae moved more slowly, the actin filaments comprising the actin comet tail were significantly more stable, with an average half-life approximately three times that of L. monocytogenes (100.6 ± 19.2 s versus 33.0 ± 7.6 s, respectively). The actin tail associated with intracytoplasmic rickettsiae remained stationary in the cytoplasm as the organism moved forward. In contrast, actin tails of rickettsiae trapped within the nucleus displayed dramatic movements. The observed phenotypic differences between the ABM of Listeria andRickettsia may indicate fundamental differences in the mechanisms of actin recruitment and polymerization.

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Streamlined Embedding of Cell Monolayers on Gridded Glass-Bottom Imaging Dishes for Correlative Light and Electron Microscopy

Microscopy and Microanalysis ◽

10.1017/s1431927610094092 ◽

2010 ◽

Vol 16 (6) ◽

pp. 747-754 ◽

Cited By ~ 22

Author(s):

Hugo H. Hanson ◽

James E. Reilly ◽

Rebecca Lee ◽

William G. Janssen ◽

Greg R. Phillips

Keyword(s):

Electron Microscopy ◽

Laser Scanning ◽

Fluorescent Protein ◽

Light And Electron Microscopy ◽

Laser Scanning Confocal Microscopy ◽

Step Method ◽

Cell Monolayers ◽

Scanning Confocal Microscopy ◽

Transmission Electron ◽

Green Fluorescent

AbstractCorrelative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.

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Insulin targeting to the regulated secretory pathway after fusion with green fluorescent protein and firefly luciferase

Biochemical Journal ◽

10.1042/bj3310669 ◽

1998 ◽

Vol 331 (2) ◽

pp. 669-675 ◽

Cited By ~ 55

Author(s):

Aristea E. POULI ◽

Helen J. KENNEDY ◽

J. George SCHOFIELD ◽

Guy A. RUTTER

Keyword(s):

Endoplasmic Reticulum ◽

Green Fluorescent Protein ◽

Laser Scanning ◽

Secretory Pathway ◽

Fluorescent Protein ◽

Firefly Luciferase ◽

Laser Scanning Confocal Microscopy ◽

Detectable Effect ◽

Scanning Confocal Microscopy ◽

Green Fluorescent

We have prepared recombinant cDNAs encoding chimaeras between human preproinsulin (sp.B.C.A., for B-, Connecting- and A-peptides) and a thermostable mutant of green fluorescent protein (GFPS65T, V163A, GFP*). The subcellular localization of the expressed chimaeras was monitored in living insulin-secreting INS-1 β-cells by laser scanning confocal microscopy. When GFP* was fused at the immediate N-terminus of the B-chain (sp.[GFP*].B.C.A.myc) two distinct patterns of fluorescence were apparent. In 1530/1740 cells examined, fluorescence was confined to a reticular, exclusively extranuclear structure, and closely co-localized with the endoplasmic reticulum marker, calreticulin. However, 210/1740 (12.1%) of cells displayed punctate fluorescence, which partially co-localized with the trans-Golgi network marker, TGN 38, and with the dense core secretory granule marker, phogrin. Since secretion of GFP* fluorescence into the medium could not readily be measured, we prepared a chimaera in which firefly luciferase was fused at the C-terminus of proinsulin (sp.B.C.A.myc.[Luc]). This chimaera displayed a distribution closely similar to that of sp.[GFP*].B.C.A.myc, but with a lower proportion (15/310, 4.8%) of the cells showing clear punctate distribution. At substimulatory glucose concentrations (3 mM) secretion of sp.B.C.A.myc.[Luc] could not be detected (rate of release into the medium identical with that of the cytosolic Renilla reniformis luciferase), indicating that the chimaera did not enter the constitutive secretory pathway. However, elevated (30 mM) glucose stimulated the release of the sp.B.C.A.myc.[Luc] luciferase chimaera, without a detectable effect on R. reniformis luciferase release. These data suggest that fusion of insulin, and the much larger photoproteins GFP* and luciferase, leads predominantly to misfolding and retention in the endoplasmic reticulum. However, the properly folded chimaeras are apparently still correctly targeted to the regulated, rather than the constitutive, secretory pathway. These chimaeras should therefore be valuable tools to monitor the exocytosis of insulin in real time.

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Laser Scanning Confocal Microscopy and Three-Dimesnsional Reconstruction of the Mitotic Spindles of Unequally Dividing Embryonic Cells

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100158376 ◽

1990 ◽

Vol 48 (3) ◽

pp. 166-167

Author(s):

J. Holy ◽

G. Schatten

Keyword(s):

Confocal Microscopy ◽

Mitotic Spindle ◽

Laser Scanning ◽

Three Dimensional ◽

Laser Scanning Confocal Microscopy ◽

Two Dimensions ◽

Embryonic Cells ◽

Mitotic Spindles ◽

Cleavage Division ◽

Scanning Confocal Microscopy

One of the classic limitations of light microscopy has been the fact that three dimensional biological events could only be visualized in two dimensions. Recently, this shortcoming has been overcome by combining the technologies of laser scanning confocal microscopy (LSCM) and computer processing of microscopical data by volume rendering methods. We have employed these techniques to examine morphogenetic events characterizing early development of sea urchin embryos. Specifically, the fourth cleavage division was examined because it is at this point that the first morphological signs of cell differentiation appear, manifested in the production of macromeres and micromeres by unequally dividing vegetal blastomeres.The mitotic spindle within vegetal blastomeres undergoing unequal cleavage are highly polarized and develop specialized, flattened asters toward the micromere pole. In order to reconstruct the three-dimensional features of these spindles, both isolated spindles and intact, extracted embryos were fluorescently labeled with antibodies directed against either centrosomes or tubulin.

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