Temperature and Nutrient Effects on Campylobacter jejuni Attachment on Multispecies Biofilms on Stainless Steel

2008 ◽  
Vol 71 (2) ◽  
pp. 271-278 ◽  
Author(s):  
SHERIASE Q. SANDERS ◽  
JOSEPH F. FRANK ◽  
JUDY W. ARNOLD
Keyword(s):  
Stainless Steel ◽  
Surface Area ◽  
Campylobacter Jejuni ◽  
Intestinal Tract ◽  
Fluorescent Protein ◽  
Area Coverage ◽  
Epifluorescence Microscopy ◽  
Nutrient Effects ◽  
Green Fluorescent ◽  
Multispecies Biofilms

Campylobacter jejuni is a thermophilic microaerophilic pathogen that is commonly found in the intestinal tract of chickens. In this study, attachment of C. jejuni 1221gfp in biofilms on stainless steel was assessed at various temperatures and with reduced nutrients. Bacteria collected from a saline rinse of processed broiler chicken carcasses were used to form initial biofilms. The whole carcass rinse (WCR) biofilms were formed by incubation of the bacteria for 16 h at 13, 20, 37, and 42°C on stainless steel coupons in tryptic soy broth (TSB). The resulting biofilms were stained with Hoechst 33258 stain and visualized by epifluorescence microscopy. WCR biofilms formed at 13°C yielded the highest surface area coverage (47.6%), and the lowest coverage (2.1%) was attained at 42°C. C. jejuni transformed to produce green fluorescent protein (gfp) was allowed to attach to the preexisting biofilms (from WCR incubated for 16 h) at each of the four temperatures, and attached cells were enumerated by visualization with an epifluorescence microscope. Attachment of C. jejuni 1221gfp did not significantly differ (P > 0.05) among the four temperatures. C. jejuni 1221gfp was cultured only from coupons with biofilms formed at 13 and 20°C. For nutrient limitation experiments, WCR biofilms were allowed to grow in 10- and 50-fold diluted TSB at 20 and 37°C for 48 h. The WCR biofilm surface area coverage (approximately 2%) was greater at 37°C than at 20°C for both TSB concentrations. C. jejuni 1221gfp was incubated with the WCR biofilm for 48 h at 20 and 37°C, and attached cells were enumerated. Attachment was significantly higher (P < 0.05) only for the treatments with 1:10 TSB at 20°C and 1:50 TSB at 37°C. Under reduced-nutrient conditions, C. jejuni 1221gfp was cultured only from biofilms formed at 20°C. Under the conditions tested, the attachment of C. jejuni 1221gfp on stainless steel and biofilms was affected by a combination of temperature and nutrient availability, but C. jejuni culturability was affected solely by temperature.

scholarly journals Bifunctional fusion proteins containing the sequence of the bradykinin homologue maximakinin: activities at the rat bradykinin B2 receptor

2018 ◽  
Vol 96 (5) ◽  
pp. 459-470 ◽  
Author(s):  
Xavier Charest-Morin ◽  
Robert Lodge ◽  
François Marceau
Keyword(s):  
Fusion Proteins ◽  
Enhanced Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Protein Denaturation ◽  
Epifluorescence Microscopy ◽  
Terminal Sequence ◽  
Protein Ligands ◽  
C Terminus ◽  
Bona Fide ◽  
Green Fluorescent

To support bradykinin (BK) B2 receptor (B2R) detection and therapeutic stimulation, we developed and characterized fusion proteins consisting of the BK homolog maximakinin (MK), or variants, positioned at the C-terminus of functional proteins (enhanced green fluorescent protein (EGFP), the peroxidase APEX2, or human serum albumin (HSA)). EGFP-MK loses its reactivity with anti-BK antibodies and molecular mass as it progresses in the endosomal tract of cells expressing rat B2Rs (immunoblots, epifluorescence microscopy). APEX2-(NG)15-MK is a bona fide agonist of the rat, but not of the human B2R (calcium and c-Fos signaling) and is compatible with the cytochemistry reagent TrueBlue (microscopy), a luminol-based reagent, or 3,3′,5,5′-tetramethylbenzidine (luminescence or colourimetric B2R detection, cell well plate format). APEX2-(NG)15-MK is a non-isotopic ligand suitable for drug discovery via binding competition. Affinity-purified secreted forms of HSA fused with peptides possessing the C-terminal MK or BK sequence failed to stimulate the rat B2R in the concentration range of 50–600 nmol/L. However, the non-secreted construction myc-HSA-MK is a B2R agonist, indicating that protein denaturation made the C-terminal sequence available for receptor binding. Fusion protein ligands of the B2R are stable but subjected to slow intracellular inactivation, strong species specificity, and possible steric hindrance between the receptor and large proteins.

scholarly journals Visualizing Microtubule-Dependent Vasopressin Type 2 Receptor Trafficking Using a New High-Affinity Fluorescent Vasopressin Ligand

Endocrinology ◽  
2011 ◽  
Vol 152 (10) ◽  
pp. 3893-3904 ◽  
Author(s):  
Sylvia Chen ◽  
Matthew J. Webber ◽  
Jean-Pierre Vilardaga ◽  
Ashok Khatri ◽  
Dennis Brown ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Receptor Trafficking ◽  
Epifluorescence Microscopy ◽  
Intracellular Camp ◽  
Green Fluorescent ◽  
Perinuclear Area ◽  
Major Target ◽  
High Binding Affinity ◽  
Dependent Mechanism

The vasopressin receptor type 2 (V2R) is the major target of vasopressin (VP) in renal epithelial cells. Although it is known that VP induces V2R internalization, accumulation in the perinuclear area, and degradation, the V2R intracellular trafficking pathways remain elusive. We visualized this process by developing a new fluorescent VP analog tagged by tetramethylrhodamine (TMR)-[Lys-(PEG)2-Suc-TMR8]VP or (VPTMR). This ligand is fully functional as revealed by its high binding affinity toward V2R [(Kd) =157 ± 52 nm] and ability to increase intracellular cAMP 32-fold. VPTMR induced V2R internalization in LLC-PK1 cells expressing either a FLAG-tagged receptor (FLAG-V2R) or V2R C-terminally tagged with green fluorescent protein (GFP) (V2R-GFP). After internalization, VPTMR and V2R-GFP colocalized in the perinuclear area, suggesting that the hormone and receptor traffic along the same pathway. VPTMR and V2R colocalized initially with the early endosome markers EEA1 and Rab5, and later with the recycling and late endosome markers Rab11 and Rab25. Epifluorescence microscopy of LLC-PK1 cells expressing GFP-tagged microtubules (MT) showed that VPTMR-containing vesicles travel along the MT network, and even remain attached to MT during the metaphase and anaphase of mitosis. Colchicine, a MT-depolymerizing agent, abolished perinuclear accumulation of VPTMR, and Western blot analysis showed that VP-induced V2R-GFP degradation is markedly retarded, but not abolished, by colchicine (10 μM). We conclude that the new VPTMR ligand is suitable for dissecting V2R and VP internalization and trafficking in cells, and that V2R trafficking and down-regulation is an MT-dependent mechanism.

An enhanced GFP reporter system to monitor gene expression in Borrelia burgdorferi

Microbiology ◽  
2003 ◽  
Vol 149 (7) ◽  
pp. 1819-1828 ◽  
Author(s):  
James A. Carroll ◽  
Philip E. Stewart ◽  
Patricia Rosa ◽  
Abdallah F. Elias ◽  
Claude F. Garon
Keyword(s):  
Gene Expression ◽  
Borrelia Burgdorferi ◽  
Fluorescent Protein ◽  
Regulation Of Gene Expression ◽  
Epifluorescence Microscopy ◽  
Reporter System ◽  
Environmental Signals ◽  
Gfp Reporter ◽  
Green Fluorescent

Borrelia burgdorferi regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the ospC, ospA and flaB promoters from B. burgdorferi B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature in vitro. GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5′ of ospC was sufficient to regulate the reporter gfp in vitro in response to pH and temperature in B. burgdorferi B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B. burgdorferi B31 to regulate ospC in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two B. burgdorferi strains.

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

2001 ◽  
Vol 75 (4) ◽  
pp. 1941-1948 ◽  
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.

Culture and Detection of Campylobacter jejuni within Mixed Microbial Populations of Biofilms on Stainless Steel

2007 ◽  
Vol 70 (6) ◽  
pp. 1379-1385 ◽  
Author(s):  
SHERIASE Q. SANDERS ◽  
DOROTHY H. BOOTHE ◽  
JOSEPH F. FRANK ◽  
JUDY W. ARNOLD
Keyword(s):  
Stainless Steel ◽  
Campylobacter Jejuni ◽  
Laser Scanning ◽  
The United States ◽  
Laser Scanning Microscopy ◽  
Epifluorescence Microscopy ◽  
Confocal Laser ◽  
Atmospheric Conditions ◽  
Culture Methods ◽  
Bacterial Populations

Campylobacter jejuni is the most frequently reported cause of foodborne illness in the United States, but its survival outside the host is poor. The objective of this research was to examine the formation and composition of biofilms by C. jejuni alone and within mixed bacterial populations from the poultry-processing environment. C. jejuni growth was assessed with four media, two temperatures, and two atmospheric conditions to develop culture methods for liquid media that would allow growth within the biofilms. Growth kinetics was followed at four cell densities to determine temporal compatibility within biofilm mixtures. Analysis of the biofilms by confocal laser scanning microscopy showed that C. jejuni formed a biofilm when incubated without other bacteria. The average surface area of stainless steel covered by C. jejuni increased by 50% from 24 to 48 h, remained level to 96 h, and then decreased by 88% by 168 h. C. jejuni and mixed bacterial populations formed biofilms during incubation periods of up to 7 days. The area of the mixture was significantly greater than for C. jejuni alone at 24 h, was approximately the same at 48 h, and was significantly less by 168 h. When incubated with either of two initial inoculum densities of other bacteria, the number of C. jejuni was enhanced after 24 h. The intensity of fluorescence and cell viability were monitored by epifluorescence microscopy. This study provides the basis for studying interactions of Campylobacter spp. with other bacteria in the environment, which will aid in the design of effective intervention strategies.

scholarly journals Development and Application of an Insertional System for Gene Delivery and Expression in Campylobacter jejuni

2005 ◽  
Vol 71 (7) ◽  
pp. 4004-4013 ◽  
Author(s):  
A. V. Karlyshev ◽  
B. W. Wren
Keyword(s):  
Gene Delivery ◽  
Campylobacter Jejuni ◽  
Fluorescent Protein ◽  
Gene Clusters ◽  
Target Cells ◽  
Rrna Gene ◽  
Efficient System ◽  
Universal Procedure ◽  
Species Specific ◽  
Green Fluorescent

ABSTRACT The genetic investigation of Campylobacter jejuni, an important gastrointestinal pathogen, has been hampered by the lack of an efficient system for introduction of exogenous genetic information, as commonly used vectors designed for Escherichia coli and other bacteria cannot be maintained in Campylobacter cells. Additionally, gene expression in Campylobacter requires the presence of species-specific promoters. In this study we exploited the availability of several conserved copies of rRNA gene clusters for insertion of various genes into the chromosome by homologous recombination. The high conservation of the rRNA sequences means that the procedure can be applied to other Campylobacter strains. The presence of a Campylobacter-derived promoter in this vector ensures expression of exogenous genes in target cells. The efficiency of the procedure was demonstrated by complementation of mutations in two strains of Campylobacter. In addition, we applied the system for introduction and expression of a green fluorescent protein (GFP). GFP-expressing Campylobacter allowed visualization of sessile bacteria attached to a glass surface in stationary liquid culture. The study demonstrated that the attached bacteria contained an assemblage of coccoid and spiral forms with liquid channels preserving viable highly motile cells. We demonstrate a novel universal procedure for gene delivery and expression that can be used as an efficient tool to study this poorly understood pathogen. The principles developed in this study could be more widely applied for the manipulation of other bacteria that are refractory to genetic analysis.

Use of Green Fluorescent Protein and Image Analysis to Quantify Proliferation of Trichoderma harzianum in Nonsterile Soil

2004 ◽  
Vol 94 (12) ◽  
pp. 1383-1389 ◽  
Author(s):  
K. A. Orr ◽  
G. R. Knudsen
Keyword(s):  
Biological Control ◽  
Image Analysis ◽  
Green Fluorescent Protein ◽  
Trichoderma Harzianum ◽  
Fluorescent Protein ◽  
Biological Control Agent ◽  
Hyphal Growth ◽  
Epifluorescence Microscopy ◽  
Nonsterile Soil ◽  
Green Fluorescent

One drawback of traditional methods for fungal biomass measurement is the inability to distinguish biomass of an introduced fungus from that of the indigenous microbial community in nonsterile soil. We quantified biomass of a specific fungal biological control agent in nonsterile soil using epifluorescence microscopy and image analysis of green fluorescent protein (GFP)-expressing Trichoderma harzianum (ThzID1-M3). Numbers of colony forming units on a semiselective medium were compared with biomass estimates from image analysis, after ThzID1-M3 was incubated in soil that either remained moist (-0.05 MPa) for 14 to 21 days or remained moist for approximately 5 days and then was allowed to dry to <-3.0 MPa. Recovery of significant numbers of ThzID1-M3 propagules lagged approximately 3 days behind initiation of hyphal growth. Reductions in both colony counts and biomass were observed over time when soil was allowed to dry. However, in soil that remained moist, colony counts increased over a 14- to 21-day period even though biomass declined after approximately 3 to 5 days. Our results confirm that use of GFP, along with epifluorescence microscopy, is a useful tool to distinguish active hyphal biomass, the form of the fungus that is functional for biological control, from inactive propagules such as conidia or chlamydospores that are enumerated by plate counts.

376 VECTOR-MEDIATED GENE TRANSFER INTO RHESUS MACAQUE EMBRYOS

2006 ◽  
Vol 18 (2) ◽  
pp. 295
Author(s):  
H. M. Kubisch ◽  
C. Gagliardi ◽  
D. G. Romero ◽  
B. A. Bunnell ◽  
M. S. Ratterree
Keyword(s):  
Rhesus Macaque ◽  
Viral Vectors ◽  
Fluorescent Protein ◽  
Epifluorescence Microscopy ◽  
Vitro Fertilization ◽  
Mosaic Expression ◽  
Morula Stage ◽  
Series Of Experiments ◽  
Green Fluorescent

A series of experiments was performed to assess the suitability of various viral vectors for transformation of rhesus macaque (Macaca mulatta) embryos. Viral vectors included the adenovirus-associated virus (AAV) containing a CMV-EGFP transgene and a lentivirus (FUGW) carrying the green fluorescent protein (GFP) gene from either the jellyfish or a humanized version from renilla linked to an ubiquitin promoter. Embryos were generated by in vitro fertilization of oocytes retrieved by laparoscopy from superovulated females. Resulting zygotes were injected under the zona pellucida (ZP) with varying viral concentrations. Embryos were subsequently cultured for 5 days and thereafter analyzed by epifluorescence microscopy. A total of 62 zygotes were injected with one of three vectors AAV2 (25), AAV2.1 (15), or AAV2.7 (22). Of these 22, 9 and 16, respectively, reached the blastocyst and morula stage. There was no difference in the percentage of embryos expressing GFP between vectors (24, 53.2, and 36.4%, respectively). However, all of the positive embryos proved to be mosaics. In a second set of experiments, FUGW was injected under the ZP of 155 embryos. Of these, 76 received virus carrying renilla GFP, while the remaining 79 were injected with virus carrying the jellyfish GFP. Following injection with renilla, 52 reached the blastocyst/morula stage on Day 7, while 43 containing jellyfish GFP proceeded to this stage. Expression of jellyfish GFP could be seen in 65% of the embryos of which 35.6% were mosaics, whereas renilla GFP was found in only 15.8% of the embryos, although none of these were mosaic. To determine whether the mosaic expression was caused by transgene silencing, three mosaic embryos were dissociated on Day 3 and 10; 10 and 8 blastomeres, respectively, were obtained. Analysis by PCR showed all but one blastomere carrying the vector. Similarly, the presence of the vector was identified by PCR in 17 of 19 non-expressing embryos injected with renilla. These results show that AAV and lentivirus can transform rhesus embryos, which can subsequently continue in development. However, identification of positive embryos by epifluorescence alone may not be sufficient. Funding was provided by NIH/NCRR grant RR000164-13.

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