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 Green Fluorescent Chimeras Indicate Nonpolar Localization of Pullulanase Secreton Components PulL and PulM

2006 ◽  
Vol 188 (8) ◽  
pp. 2928-2935 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Olivera Francetic ◽  
Anthony P. Pugsley
Keyword(s):  
Fluorescence Microscopy ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Klebsiella Oxytoca ◽  
Subcellular Location ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Native Proteins ◽  
Type Ii Secretion System ◽  
Green Fluorescent

ABSTRACT The Klebsiella oxytoca pullulanase secreton (type II secretion system) components PulM and PulL were tagged at their N termini with green fluorescent protein (GFP), and their subcellular location was examined by fluorescence microscopy and fractionation. When produced at moderate levels without other secreton components in Escherichia coli, both chimeras were envelope associated, as are the native proteins. Fluorescent GFP-PulM was evenly distributed over the cell envelope, with occasional brighter foci. Under the same conditions, GFP-PulL was barely detectable in the envelope by fluorescence microscopy. When produced together with all other secreton components, GFP-PulL exhibited circumferential fluorescence, with numerous brighter patches. The envelope-associated fluorescence of GFP-PulL was almost completely abolished when native PulL was also produced, suggesting that the chimera cannot compete with PulL for association with other secreton components. The patches of GFP-PulL might represent functional secretons, since GFP-PulM also appeared in similar patches. GFP-PulM and GFP-PulL both appeared in spherical polar foci when made at high levels. In K. oxytoca, GFP-PulM was evenly distributed over the cell envelope, with few patches, whereas GFP-PulL showed only weak envelope-associated fluorescence. These data suggest that, in contrast to their Vibrio cholerae Eps secreton counterparts (M. Scott, Z. Dossani, and M. Sandkvist, Proc. Natl. Acad. Sci. USA 98:13978-13983, 2001), PulM and PulL do not localize specifically to the cell poles and that the Pul secreton is distributed over the cell surface.

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.

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.

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.

scholarly journals A Subassembly of R27-Encoded Transfer Proteins Is Dependent on TrhC Nucleoside Triphosphate-Binding Motifs for Function but Not Formation

2004 ◽  
Vol 186 (6) ◽  
pp. 1606-1613 ◽  
Author(s):  
Matthew W. Gilmour ◽  
Diane E. Taylor
Keyword(s):  
Fluorescent Protein ◽  
Experimental System ◽  
Nucleoside Triphosphate ◽  
Conjugative Transfer ◽  
Bacterial Cells ◽  
Protein Fusion ◽  
Homologous Proteins ◽  
Focus Formation ◽  
Binding Motifs ◽  
Green Fluorescent

ABSTRACT The transfer of plasmid DNA molecules between bacterial cells is achieved by a large array of conjugative transfer proteins which assemble into both cytoplasmic and membrane-associated complexes. TrhC is a membrane-associated protein that is required for the transfer of the IncHI1 resistance plasmid R27. Homologous proteins are encoded in all known conjugative systems, and each contains characteristic nucleoside triphosphate (NTP)-binding domains. An assembly of R27-encoded proteins was previously visualized by use of a TrhC-green fluorescent protein fusion, which appeared as discrete membrane-associated fluorescent foci. We have utilized this experimental system to determine the requirements for assembly of this TrhC-associated protein complex, and we found that 12 of the other 18 R27 transfer proteins are required for focus formation. An individual focus possibly represents a subassembly comprised of some or all of these transfer proteins. These data support the notion that the transfer apparatus is a multicomponent structure. In contrast, substitutions and deletions within TrhC NTP-binding motifs had minor effects on focus formation, but these mutations did affect plasmid transfer and bacteriophage susceptibility. These results indicate that TrhC requires intact NTP-binding motifs to function during conjugative transfer but that these motifs are not essential for the assembly of TrhC into a complex with other transfer proteins.

scholarly journals Natural Competence Rates Are Variable Among Xylella fastidiosa Strains and Homologous Recombination Occurs In Vitro Between Subspecies fastidiosa and multiplex

2017 ◽  
Vol 30 (7) ◽  
pp. 589-600 ◽  
Author(s):  
Prem P. Kandel ◽  
Rodrigo P. P. Almeida ◽  
Paul A. Cobine ◽  
Leonardo De La Fuente
Keyword(s):  
Homologous Recombination ◽  
Fluorescent Protein ◽  
Xylella Fastidiosa ◽  
Dna Recombination ◽  
Bacterial Cells ◽  
Disease Emergence ◽  
Natural Competence ◽  
In Silico Studies ◽  
Green Fluorescent

Xylella fastidiosa, an etiological agent of emerging crop diseases around the world, is naturally competent for the uptake of DNA from the environment that is incorporated into its genome by homologous recombination. Homologous recombination between subspecies of X. fastidiosa was inferred by in silico studies and was hypothesized to cause disease emergence. However, no experimental data are available on the degree to which X. fastidiosa strains are capable of competence and whether recombination can be experimentally demonstrated between subspecies. Here, using X. fastidiosa strains from different subspecies, natural competence in 11 of 13 strains was confirmed with plasmids containing antibiotic markers flanked by homologous regions and, in three of five strains, with dead bacterial cells used as source of donor DNA. Recombination frequency differed among strains and was correlated to growth rate and twitching motility. Moreover, intersubspecific recombination occurred readily between strains of subsp. fastidiosa and multiplex, as demonstrated by movement of antibiotic resistance and green fluorescent protein from donor to recipient cells and confirmed by DNA sequencing of the flanking arms of recombinant strains. Results demonstrate that natural competence is widespread among X. fastidiosa strains and could have an impact in pathogen adaptation and disease development.

scholarly journals Predictive and Interpretive Simulation of Green Fluorescent Protein Expression in Reporter Bacteria

2001 ◽  
Vol 183 (23) ◽  
pp. 6752-6762 ◽  
Author(s):  
Johan H. J. Leveau ◽  
Steven E. Lindow
Keyword(s):  
Growth Rate ◽  
Green Fluorescent Protein ◽  
Green Fluorescent Protein Expression ◽  
Fluorescent Protein ◽  
Proteolytic Degradation ◽  
Bacterial Cells ◽  
Prior Testing ◽  
Gfp Fluorescence ◽  
Green Fluorescent ◽  
Best Fit

ABSTRACT We have formulated a numerical model that simulates the accumulation of green fluorescent protein (GFP) in bacterial cells from a generic promoter-gfp fusion. The model takes into account the activity of the promoter, the time it takes GFP to mature into its fluorescent form, the susceptibility of GFP to proteolytic degradation, and the growth rate of the bacteria. From the model, we derived a simple formula with which promoter activity can be inferred easily and quantitatively from actual measurements of GFP fluorescence in growing bacterial cultures. To test the usefulness of the formula, we determined the activity of the LacI-repressible promoter P A1/O4/O3 in response to increasing concentrations of the inducer IPTG (isopropyl-β-d-thiogalactopyranoside) and were able to predict cooperativity between the LacI repressors on each of the two operator sites within P A1/O4/O3 . Aided by the model, we also quantified the proteolytic degradation of GFP[AAV], GFP[ASV], and GFP[LVA], which are popular variants of GFP with reduced stability in bacteria. Best described by Michaelis-Menten kinetics, the rate at which these variants were degraded was a function of the activity of the promoter that drives their synthesis: a weak promoter yielded proportionally less GFP fluorescence than a strong one. The degree of disproportionality is species dependent: the effect was more pronounced in Erwinia herbicola than in Escherichia coli. This phenomenon has important implications for the interpretation of fluorescence from bacterial reporters based on these GFP variants. The model furthermore predicted a significant effect of growth rate on the GFP content of individual bacteria, which if not accounted for might lead to misinterpretation of GFP data. In practice, our model will be helpful for prior testing of different combinations of promoter-gfpfusions that best fit the application of a particular bacterial reporter strain, and also for the interpretation of actual GFP fluorescence data that are obtained with that reporter.

scholarly journals Multicopy Plasmids Affect Replisome Positioning in Bacillus subtilis

2004 ◽  
Vol 186 (21) ◽  
pp. 7084-7090 ◽  
Author(s):  
Jue D. Wang ◽  
Megan E. Rokop ◽  
Melanie M. Barker ◽  
Nathaniel R. Hanson ◽  
Alan D. Grossman
Keyword(s):  
Bacillus Subtilis ◽  
Dna Polymerase ◽  
Fluorescent Protein ◽  
Subcellular Location ◽  
Multicopy Plasmid ◽  
Bacterial Cells ◽  
Initiation Of Replication ◽  
A Cell ◽  
A Subunit ◽  
Green Fluorescent

ABSTRACT The DNA replication machinery, various regions of the chromosome, and some plasmids occupy characteristic subcellular positions in bacterial cells. We visualized the location of a multicopy plasmid, pHP13, in living cells of Bacillus subtilis using an array of lac operators and LacI-green fluorescent protein (GFP). In the majority of cells, plasmids appeared to be highly mobile and randomly distributed. In a small fraction of cells, there appeared to be clusters of plasmids located predominantly at or near a cell pole. We also monitored the effects of the presence of multicopy plasmids on the position of DNA polymerase using a fusion of a subunit of DNA polymerase to GFP. Many of the plasmid-containing cells had extra foci of the replisome, and these were often found at uncharacteristic locations in the cell. Some of the replisome foci were dynamic and highly mobile, similar to what was observed for the plasmid. In contrast, replisome foci in plasmid-free cells were relatively stationary. Our results indicate that in B. subtilis, plasmid-associated replisomes are recruited to the subcellular position of the plasmid. Extending this notion to the chromosome, we postulated that the subcellular position of the chromosomally associated replisome is established by the subcellular location of oriC at the time of initiation of replication.

scholarly journals Methylotrophic Metabolism Is Advantageous for Methylobacterium extorquens during Colonization of Medicago truncatula under Competitive Conditions

2005 ◽  
Vol 71 (11) ◽  
pp. 7245-7252 ◽  
Author(s):  
Abdoulaye Sy ◽  
Antonius C. J. Timmers ◽  
Claudia Knief ◽  
Julia A. Vorholt
Keyword(s):  
Medicago Truncatula ◽  
Fluorescent Protein ◽  
Selective Advantage ◽  
Plant Colonization ◽  
Methylotrophic Bacteria ◽  
Bacterial Cells ◽  
Wild Type ◽  
Methylobacterium Extorquens ◽  
Model Legume ◽  
Green Fluorescent

ABSTRACT Facultative methylotrophic bacteria of the genus Methylobacterium are commonly found in association with plants. Inoculation experiments were performed to study the importance of methylotrophic metabolism for colonization of the model legume Medicago truncatula. Competition experiments with Methylobacterium extorquens wild-type strain AM1 and methylotrophy mutants revealed that the ability to use methanol as a carbon and energy source provides a selective advantage during colonization of M. truncatula. Differences in the fitness of mutants defective in different stages of methylotrophic metabolism were found; whereas approximately 25% of the mutant incapable of oxidizing methanol to formaldehyde (deficient in methanol dehydrogenase) was recovered, 10% or less of the mutants incapable of oxidizing formaldehyde to CO2 (defective in biosynthesis of the cofactor tetrahydromethanopterin) was recovered. Interestingly, impaired fitness of the mutant strains compared with the wild type was found on leaves and roots. Single-inoculation experiments showed, however, that mutants with defects in methylotrophy were capable of plant colonization at the wild-type level, indicating that methanol is not the only carbon source that is accessible to Methylobacterium while it is associated with plants. Fluorescence microscopy with a green fluorescent protein-labeled derivative of M. extorquens AM1 revealed that the majority of the bacterial cells on leaves were on the surface and that the cells were most abundant on the lower, abaxial side. However, bacterial cells were also found in the intercellular spaces inside the leaves, especially in the epidermal cell layer and immediately underneath this layer.

Sign in / Sign up

Export Citation Format

Share Document