Localization of Proteins Within Intact Bacterial Cells Using Fluorescent Protein Fusions

2015 ◽  
pp. 51-63
Author(s):  
Veronica Wells Rowlett ◽  
William Margolin
Keyword(s):  
Fluorescent Protein ◽  
Bacterial Cells

Induced Expression of the Heat Shock Protein Genes uspA and grpE during Starvation at Low Temperatures and Their Influence on Thermal Resistance of Escherichia coli O157:H7

2003 ◽  
Vol 66 (11) ◽  
pp. 2045-2050 ◽  
Author(s):  
YI ZHANG ◽  
MANSEL W. GRIFFITHS
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Stress Responses ◽  
Thermal Tolerance ◽  
Molecular Mechanisms ◽  
Fluorescent Protein ◽  
Bacterial Cells ◽  
Induced Expression ◽  
Green Fluorescent ◽  
Shock Proteins

Heat shock proteins play an important role in protecting bacterial cells against several stresses, including starvation. In this study, the promoters for two genes encoding heat shock proteins involved in many stress responses, UspA and GrpE, were fused with the green fluorescent protein (gfp) gene. Thus, the expression of the two genes could be quantified by measuring the fluorescence emitted by the cells under different environmental conditions. The heat resistance levels of starved and nonstarved cells during storage at 5, 10, and 37°C were compared with the levels of expression of the uspA and grpE genes. D52-values (times required for decimal reductions in count at 52°C) increased by 11.5, 14.6, and 18.5 min when cells were starved for 3 h at 37°C, for 24 h at 10°C, and for 2 days at 5°C, respectively. In all cases, these increases were significant (P < 0.01), indicating that the stress imposed by starvation altered the ability of E. coli O157:H7 to survive subsequent heat treatments. Thermal tolerance was correlative with the induction of UspA and GrpE. At 5°C, the change in the thermal tolerance of the pathogen was positively linked to the induced expression of the grpE gene but negatively related to the expression of the uspA gene. The results obtained in this study indicate that UspA plays an important role in starvation-induced thermal tolerance at 37°C but that GrpE may be more involved in regulating this response at lower temperatures. An improvement in our understanding of the molecular mechanisms involved in these cross-protection responses may make it possible to devise strategies to limit their effects.

scholarly journals Stages of Infection during the Tripartite Interaction between Xenorhabdus nematophila, Its Nematode Vector, and Insect Hosts

2004 ◽  
Vol 70 (11) ◽  
pp. 6473-6480 ◽  
Author(s):  
Mathieu Sicard ◽  
Karine Brugirard-Ricaud ◽  
Sylvie Pag�s ◽  
Anne Lanois ◽  
Noel E. Boemare ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Spodoptera Littoralis ◽  
Fluorescent Protein ◽  
Bacterial Colonization ◽  
Infective Juvenile ◽  
Bacterial Cells ◽  
Xenorhabdus Nematophila ◽  
Insect Larvae ◽  
Connective Tissues ◽  
Anterior Midgut

ABSTRACT Bacteria of the genus Xenorhabdus are mutually associated with entomopathogenic nematodes of the genus Steinernema and are pathogenic to a broad spectrum of insects. The nematodes act as vectors, transmitting the bacteria to insect larvae, which die within a few days of infection. We characterized the early stages of bacterial infection in the insects by constructing a constitutive green fluorescent protein (GFP)-labeled Xenorhabdus nematophila strain. We injected the GFP-labeled bacteria into insects and monitored infection. We found that the bacteria had an extracellular life cycle in the hemolymph and rapidly colonized the anterior midgut region in Spodoptera littoralis larvae. Electron microscopy showed that the bacteria occupied the extracellular matrix of connective tissues within the muscle layers of the Spodoptera midgut. We confirmed the existence of such a specific infection site in the natural route of infection by infesting Spodoptera littoralis larvae with nematodes harboring GFP-labeled Xenorhabdus. When the infective juvenile (IJ) nematodes reached the insect gut, the bacterial cells were rapidly released from the intestinal vesicle into the nematode intestine. Xenorhabdus began to escape from the anus of the nematodes when IJs were wedged in the insect intestinal wall toward the insect hemolymph. Following their release into the insect hemocoel, GFP-labeled bacteria were found only in the anterior midgut region and hemolymph of Spodoptera larvae. Comparative infection assays conducted with another insect, Locusta migratoria, also showed early bacterial colonization of connective tissues. This work shows that the extracellular matrix acts as a particular colonization site for X. nematophila within insects.

Components of intestinal epithelial hypoxia activate the virulence circuitry of Pseudomonas

2005 ◽  
Vol 288 (5) ◽  
pp. G1048-G1054 ◽  
Author(s):  
Jonathan E. Kohler ◽  
Olga Zaborina ◽  
Licheng Wu ◽  
Yingmin Wang ◽  
Cindy Bethel ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Surgical Stress ◽  
Recovery Period ◽  
Intestinal Lumen ◽  
Bacterial Cells ◽  
Intestinal Epithelial ◽  
Promoter Activation ◽  
Fusion Construct ◽  
Green Fluorescent ◽  
Ambient Hypoxia

We have previously shown that a lethal virulence trait in Pseudomonas aeruginosa, the PA-I lectin, is expressed by bacteria within the intestinal lumen of surgically stressed mice. The aim of this study was to determine whether intestinal epithelial hypoxia, a common response to surgical stress, could activate PA-I expression. A fusion construct was generated to express green fluorescent protein downstream of the PA-I gene, serving as a stable reporter strain for PA-I expression in P. aeruginosa. Polarized Caco-2 monolayers were exposed to ambient hypoxia (0.1–0.3% O2) for 1 h, with or without a recovery period of normoxia (21% O2) for 2 h, and then inoculated with P. aeruginosa containing the PA-I reporter construct. Hypoxic Caco-2 monolayers caused a significant increase in PA-I promoter activity relative to normoxic monolayers (165% at 1 h; P < 0.001). Similar activation of PA-I was also induced by cell-free apical, but not basal, media from hypoxic Caco-2 monolayers. PA-I promoter activation was preferentially enhanced in bacterial cells that physically interacted with hypoxic epithelia. We conclude that the virulence circuitry of P. aeruginosa is activated by both soluble and contact-mediated elements of the intestinal epithelium during hypoxia and normoxic recovery.

Green fluorescent protein (GFP)-labeling of enterobacteria associated with fruit flies (Diptera: Tephritidae) and persistence in their natural hostRhagoletis completaCresson.

2011 ◽  
Vol 57 (11) ◽  
pp. 969-973 ◽  
Author(s):  
Isabel Martinez-Sañudo ◽  
Claudia Savio ◽  
Luca Mazzon ◽  
Vincenzo Girolami ◽  
Silvia Ciolfi ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Insect Pest ◽  
Klebsiella Oxytoca ◽  
Management Strategies ◽  
Pupal Stage ◽  
Fruit Flies ◽  
Economic Damage ◽  
Bacterial Cells ◽  
Oesophageal Bulb ◽  
Green Fluorescent

Fruit flies (Diptera: Tephritidae) are a highly successful, widespread group of insects that cause economic damage in agriculture. Data available so far on the composition of the bacterial community associated with their digestive tract indicate that members of Enterobacteriaceae are the species most often isolated. Bacteria naturally occurring in insect guts may be engineered and used to study the spatial and functional interactions of microbes within the insect system and offer one route to meet the demand for novel insect pest management strategies. With this aim we introduced by conjugation the gfp gene carried by the suicide plasmid pTn5gfpmut1 into Klebsiella oxytoca and Raoultella (formerly Klebsiella ) spp. strains isolated from the oesophageal bulb of the fruit flies Ceratitis capitata (Wiedemann) and Rhagoletis completa Cresson, respectively. The GFP-encoding gene was stably maintained in two tested transgenic strains, both originally isolated from R. completa. In one case, GFP-labeled bacterial cells were used to feed larvae and adults of the original host. Genetically modified bacteria were able to colonize the gut of larvae and persisted through all larval instars to pupal stage.

scholarly journals Modifying TIMER, a slow-folding DsRed derivative, for optimal use in quickly-dividing bacteria

2021 ◽  
Author(s):  
Pavan Patel ◽  
Brendan J. O’Hara ◽  
Emily Aunins ◽  
Kimberly M. Davis
Keyword(s):  
Nitric Oxide ◽  
Cell Division ◽  
Stationary Phase ◽  
Yersinia Pseudotuberculosis ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Bacterial Cells ◽  
E Coli ◽  
Slow Growing ◽  
Host Tissues

AbstractIt is now well appreciated that members of pathogenic bacterial populations exhibit heterogeneity in growth rates and metabolic activity, and it is known this can impact the ability to eliminate all members of the bacterial population during antibiotic treatment. It remains unclear which pathways promote slowed bacterial growth within host tissues, primarily because it has been difficult to identify and isolate slow growing bacteria from host tissues for downstream analyses. To overcome this limitation, we have developed a novel variant of TIMER, a slow-folding fluorescent protein, to identify subsets of slowly dividing bacteria within host tissues. The original TIMER folds too slowly for fluorescence accumulation in quickly replicating bacterial species (Escherichia coli, Yersinia pseudotuberculosis), however this TIMER42 variant accumulates signal in late stationary phase cultures of E. coli and Y. pseudotuberculosis. We show TIMER42 signal also accumulates during exposure to sources of nitric oxide (NO), suggesting TIMER42 signal detects growth-arrested bacterial cells. In a mouse model of Y. pseudotuberculosis deep tissue infection, TIMER42 signal is clearly detected, and primarily accumulates in bacteria expressing markers of stationary phase growth. There was not significant overlap between TIMER42 signal and NO-exposed subpopulations of bacteria within host tissues, suggesting NO stress was transient, allowing bacteria to recover from this stress and resume replication. This novel TIMER42 variant represents a new faster folding TIMER that will enable additional studies of slow-growing subpopulations of bacteria, specifically within bacterial species that quickly divide.Author SummaryWe have generated a variant of TIMER that can be used to mark slow-growing subsets of Yersinia pseudotuberculosis, which has a relatively short division time, similar to E. coli. We used a combination of site-directed and random mutagenesis to generate the TIMER42 variant, which has red fluorescent signal accumulation in post-exponential or stationary phase cells. We found that nitric oxide (NO) stress is sufficient to promote TIMER42 signal accumulation in culture, however within host tissues, TIMER42 signal correlates with a stationary phase reporter (dps). These results suggest NO may cause an immediate arrest in bacterial cell division, but during growth in host tissues exposure to NO is transient, allowing bacteria to recover from this stress and resume cell division. Thus instead of indicating a response to host stressors, TIMER42 signal accumulation within host tissues appears to identify slow-growing cells that are experiencing nutrient limitation.

scholarly journals EmrE, a Small Escherichia coli Multidrug Transporter, Protects Saccharomyces cerevisiae from Toxins by Sequestration in the Vacuole

1999 ◽  
Vol 181 (3) ◽  
pp. 949-956 ◽  
Author(s):  
Rodrigo Yelin ◽  
Dvir Rotem ◽  
Shimon Schuldiner
Keyword(s):  
Escherichia Coli ◽  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Methyl Viologen ◽  
Fluorescent Protein ◽  
Copper Ions ◽  
Yeast Cells ◽  
Multidrug Transporter ◽  
Uptake System ◽  
Bacterial Cells

ABSTRACT In this report we describe the functional expression of EmrE, a 110-amino-acid multidrug transporter from Escherichia coli, in the yeast Saccharomyces cerevisiae. To allow for phenotypic complementation, a mutant strain sensitive to a series of cationic lipophilic drugs was first identified. A hemagglutinin epitope-tagged version of EmrE (HA-EmrE) conferring resistance to a wide variety of drugs, including acriflavine, ethidium, methyl viologen, and the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), was functionally expressed in this strain. HA-EmrE is expressed in yeast at relatively high levels (0.5 mg/liter), is soluble in a mixture of organic solvents, and can be functionally reconstituted in proteoliposomes. In bacterial cells, EmrE removes toxic compounds by active transport through the plasma membrane, lowering their cytosolic concentration. However, yeast cells expressing HA-EmrE take up 14C-methyl viologen as well as control cells do. Thus, we investigated the basis of the enhanced resistance to the above compounds. Using Cu2+ ions or methylamine, we could selectively permeabilize the plasma membrane or deplete the proton electrochemical gradients across the vacuolar membrane, respectively. Incubation of yeast cells with copper ions caused an increase in 14C-methyl viologen uptake. In contrast, treatment with methylamine markedly diminished the extent of uptake. Conversely, the effect of Cu2+ and methylamine on a plasma membrane uptake system, proline, was essentially the opposite: while inhibited by the addition of Cu2+, it remained unaffected when cells were treated with methylamine. To examine the intracellular distribution of HA-EmrE, a functional chimera between HA-EmrE and the green fluorescent protein (HA-EmrE-GFP) was prepared. The pattern of HA-EmrE-GFP fluorescence distribution was virtually identical to that of the vacuolar marker FM 4-64, indicating that the transporter is found mainly in this organelle. Therefore, HA-EmrE protects yeast cells by lowering the cytoplasmic concentrations through removal of the toxin to the vacuole. This novel way of detoxification has been previously suggested to function in organisms in which a large vacuolar compartment exists. This report represents the first molecular description of such a mechanism.

scholarly journals Enzymatic Responses to Low-Intensity Radiation of Tritium

2020 ◽  
Vol 21 (22) ◽  
pp. 8464
Author(s):  
Tatiana V. Rozhko ◽  
Elena V. Nemtseva ◽  
Maria V. Gardt ◽  
Alexander V. Raikov ◽  
Albert E. Lisitsa ◽  
...  
Keyword(s):  
Proton Transfer ◽  
Fluorescent Protein ◽  
Tritiated Water ◽  
Coupled System ◽  
Enzymatic Reactions ◽  
Bacterial Cells ◽  
Initial Stage ◽  
Hormetic Response ◽  
Intensity Radiation

The present study considers a possible role of enzymatic reactions in the adaptive response of cells to the beta-emitting radionuclide tritium under conditions of low-dose exposures. Effects of tritiated water (HTO) on the reactions of bacterial luciferase and NAD(P)H:FMN-oxidoreductase, as well as a coupled system of these two reactions, were studied at radioactivity concentrations ≤ 200 MBq/L. Additionally, one of the simplest enzymatic reactions, photobiochemical proton transfer in Coelenteramide-containing Fluorescent Protein (CLM-FP), was also investigated. We found that HTO increased the activity of NAD(P)H:FMN-oxidoreductase at the initial stage of its reaction (by up to 230%); however, a rise of luciferase activity was moderate (<20%). The CLM-FP samples did not show any increase in the rate of the photobiochemical proton transfer under the exposure to HTO. The responses of the enzyme systems were compared to the ‘hormetic’ response of luminous marine bacterial cells studied earlier. We conclude that (1) the oxidoreductase reaction contributes significantly to the activation of the coupled enzyme system and bacterial cells by tritium, and (2) an increase in the organization level of biological systems promotes the hormesis phenomenon.

scholarly journals Dominance of Vibrio fischeri in Secreted Mucus outside the Light Organ of Euprymna scolopes: the First Site of Symbiont Specificity

2003 ◽  
Vol 69 (7) ◽  
pp. 3932-3937 ◽  
Author(s):  
Spencer V. Nyholm ◽  
Margaret J. McFall-Ngai
Keyword(s):  
Fluorescent Protein ◽  
Vibrio Fischeri ◽  
Signal Molecules ◽  
Natural Seawater ◽  
Gram Negative Bacteria ◽  
Light Organ ◽  
Bacterial Cells ◽  
Euprymna Scolopes ◽  
Gram Negative ◽  
Green Fluorescent

ABSTRACT Previous studies of the Euprymna scolopes-Vibrio fischeri symbiosis have demonstrated that, during colonization, the hatchling host secretes mucus in which gram-negative environmental bacteria amass in dense aggregations outside the sites of infection. In this study, experiments with green fluorescent protein-labeled symbiotic and nonsymbiotic species of gram-negative bacteria were used to characterize the behavior of cells in the aggregates. When hatchling animals were exposed to 103 to 106 V. fischeri cells/ml added to natural seawater, which contains a mix of approximately 106 nonspecific bacterial cells/ml, V. fischeri cells were the principal bacterial cells present in the aggregations. Furthermore, when animals were exposed to equal cell numbers of V. fischeri (either a motile or a nonmotile strain) and either Vibrio parahaemolyticus or Photobacterium leiognathi, phylogenetically related gram-negative bacteria that also occur in the host's habitat, the symbiont cells were dominant in the aggregations. The presence of V. fischeri did not compromise the viability of these other species in the aggregations, and no significant growth of V. fischeri cells was detected. These findings suggested that dominance results from the ability of V. fischeri either to accumulate or to be retained more effectively within the mucus. Viability of the V. fischeri cells was required for both the formation of tight aggregates and their dominance in the mucus. Neither of the V. fischeri quorum-sensing compounds accumulated in the aggregations, which suggested that the effects of these small signal molecules are not critical to V. fischeri dominance. Taken together, these data provide evidence that the specificity of the squid-vibrio symbiosis begins early in the interaction, in the mucus where the symbionts aggregate outside of the light organ.

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