scholarly journals Localization of Cell Division Protein FtsK to theEscherichia coli Septum and Identification of a Potential N-Terminal Targeting Domain

1998 ◽  
Vol 180 (5) ◽  
pp. 1296-1304 ◽  
Author(s):  
Xuan-chuan Yu ◽  
Anthony H. Tran ◽  
Qin Sun ◽  
William Margolin
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Green Fluorescent Protein ◽  
Late Stage ◽  
Fluorescent Protein ◽  
Mutant Protein ◽  
E Coli ◽  
Cell Division Protein ◽  
Green Fluorescent

ABSTRACT Escherichia coli cell division protein FtsK is a homolog of Bacillus subtilis SpoIIIE and appears to act late in the septation process. To determine whether FtsK localizes to the septum, we fused three N-terminal segments of FtsK to green fluorescent protein (GFP) and expressed them in E. colicells. All three segments were sufficient to target GFP to the septum, suggesting that as little as the first 15% of the protein is a septum-targeting domain. Localized fluorescence was detectable only in cells containing a visible midcell constriction, suggesting that FtsK targeting normally occurs only at a late stage of septation. The largest two FtsK-GFP fusions were able at least partially to complement the ftsK44 mutation in trans, suggesting that the N- and C-terminal domains are functionally separable. However, overproduction of FtsK-GFP resulted in a late-septation phenotype similar to that of ftsK44, with fluorescent dots localized at the blocked septa, suggesting that high levels of the N-terminal domain may still localize but also inhibit FtsK activity. Interestingly, under these conditions fluorescence was also sometimes localized as bands at potential division sites, suggesting that FtsK-GFP is capable of targeting very early. In addition, FtsK-GFP localized to potential division sites in cephalexin-induced andftsI mutant filaments, further supporting the idea that FtsK-GFP can target early, perhaps by recognizing FtsZ directly. This hypothesis was supported by the failure of FtsK-GFP to localize inftsZ mutant filaments. In ftsK44 mutant filaments, FtsA and FtsZ were usually localized to potential division sites between the blocked septa. When the ftsK44 mutation was incorporated into the FtsK-GFP fusions, localization to midcell ranged between very weak and undetectable, suggesting that the FtsK44 mutant protein is defective in targeting the septum.

scholarly journals Artificial Septal Targeting of Bacillus subtilis Cell Division Proteins in Escherichia coli: an Interspecies Approach to the Study of Protein-Protein Interactions in Multiprotein Complexes

2008 ◽  
Vol 190 (18) ◽  
pp. 6048-6059 ◽  
Author(s):  
Carine Robichon ◽  
Glenn F. King ◽  
Nathan W. Goehring ◽  
Jon Beckwith
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Protein Interactions ◽  
Fluorescent Protein ◽  
Bacterial Cell Division ◽  
Protein Protein Interactions ◽  
Positive Interaction ◽  
E Coli ◽  
Multiprotein Complexes

ABSTRACT Bacterial cell division is mediated by a set of proteins that assemble to form a large multiprotein complex called the divisome. Recent studies in Bacillus subtilis and Escherichia coli indicate that cell division proteins are involved in multiple cooperative binding interactions, thus presenting a technical challenge to the analysis of these interactions. We report here the use of an E. coli artificial septal targeting system for examining the interactions between the B. subtilis cell division proteins DivIB, FtsL, DivIC, and PBP 2B. This technique involves the fusion of one of the proteins (the “bait”) to ZapA, an E. coli protein targeted to mid-cell, and the fusion of a second potentially interacting partner (the “prey”) to green fluorescent protein (GFP). A positive interaction between two test proteins in E. coli leads to septal localization of the GFP fusion construct, which can be detected by fluorescence microscopy. Using this system, we present evidence for two sets of strong protein-protein interactions between B. subtilis divisomal proteins in E. coli, namely, DivIC with FtsL and DivIB with PBP 2B, that are independent of other B. subtilis cell division proteins and that do not disturb the cytokinesis process in the host cell. Our studies based on the coexpression of three or four of these B. subtilis cell division proteins suggest that interactions among these four proteins are not strong enough to allow the formation of a stable four-protein complex in E. coli in contrast to previous suggestions. Finally, our results demonstrate that E. coli artificial septal targeting is an efficient and alternative approach for detecting and characterizing stable protein-protein interactions within multiprotein complexes from other microorganisms. A salient feature of our approach is that it probably only detects the strongest interactions, thus giving an indication of whether some interactions suggested by other techniques may either be considerably weaker or due to false positives.

A Novel Approach To Investigate the Uptake and Internalization of Escherichia coli O157:H7 in Spinach Cultivated in Soil and Hydroponic Medium†

2009 ◽  
Vol 72 (7) ◽  
pp. 1513-1520 ◽  
Author(s):  
MANAN SHARMA ◽  
DAVID T. INGRAM ◽  
JITENDRA R. PATEL ◽  
PATRICIA D. MILLNER ◽  
XIAOLIN WANG ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Plasmid Vector ◽  
Green Fluorescent Protein Gene ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
Novel Approach ◽  
Efficient Uptake ◽  
Green Fluorescent

Internalization of Escherichia coli O157:H7 into spinach plants through root uptake is a potential route of contamination. ATn7-based plasmid vector was used to insert a green fluorescent protein gene into the attTn7 site in the E. coli chromosome. Three green fluorescent protein–labeled E. coli inocula were used: produce outbreak O157:H7 strains RM4407 and RM5279 (inoculum 1), ground beef outbreak O157:H7 strain 86-24h11 (inoculum 2), and commensal strain HS (inoculum 3). These strains were cultivated in fecal slurries and applied at ca. 103 or 107 CFU/g to pasteurized soils in which baby spinach seedlings were planted. No E. coli was recovered by spiral plating from surface-sanitized internal tissues of spinach plants on days 0, 7, 14, 21, and 28. Inoculum 1 survived at significantly higher populations (P < 0.05) in the soil than did inoculum 3 after 14, 21, and 28 days, indicating that produce outbreak strains of E. coli O157:H7 may be less physiologically stressed in soils than are nonpathogenic E. coli isolates. Inoculum 2 applied at ca. 107 CFU/ml to hydroponic medium was consistently recovered by spiral plating from the shoot tissues of spinach plants after 14 days (3.73 log CFU per shoot) and 21 days (4.35 log CFU per shoot). Fluorescent E. coli cells were microscopically observed in root tissues in 23 (21%) of 108 spinach plants grown in inoculated soils. No internalized E. coli was microscopically observed in shoot tissue of plants grown in inoculated soil. These studies do not provide evidence for efficient uptake of E. coli O157:H7 from soil to internal plant tissue.

scholarly journals Inhibition of Salmonella Binding to Porcine Intestinal Cells by a Wood-Derived Prebiotic

2020 ◽  
Vol 8 (7) ◽  
pp. 1051 ◽  
Author(s):  
Aleksandar Božić ◽  
Robin C. Anderson ◽  
Tawni L. Crippen ◽  
Christina L. Swaggerty ◽  
Michael E. Hume ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Saccharomyces Boulardii ◽  
Binding Activity ◽  
Intestinal Cells ◽  
E Coli ◽  
Enteric Infections ◽  
Green Fluorescent

Numerous Salmonella enterica serovars can cause disease and contamination of animal-produced foods. Oligosaccharide-rich products capable of blocking pathogen adherence to intestinal mucosa are attractive alternatives to antibiotics as these have potential to prevent enteric infections. Presently, a wood-derived prebiotic composed mainly of glucose-galactose-mannose-xylose oligomers was found to inhibit mannose-sensitive binding of select Salmonella Typhimurium and Escherichia coli strains when reacted with Saccharomyces boulardii. Tests for the ability of the prebiotic to prevent binding of a green fluorescent protein (GFP)-labeled S. Typhimurium to intestinal porcine epithelial cells (IPEC-J2) cultured in vitro revealed that prebiotic-exposed GFP-labeled S. Typhimurium bound > 30% fewer individual IPEC-J2 cells than did GFP-labeled S. Typhimurium having no prebiotic exposure. Quantitatively, 90% fewer prebiotic-exposed GFP-labeled S. Typhimurium cells were bound per individual IPEC-J2 cell compared to non-prebiotic exposed GFP-labeled S. Typhimurium. Comparison of invasiveness of S. Typhimurium DT104 against IPEC-J2 cells revealed greater than a 90% decrease in intracellular recovery of prebiotic-exposed S. Typhimurium DT104 compared to non-exposed controls (averaging 4.4 ± 0.2 log10 CFU/well). These results suggest compounds within the wood-derived prebiotic bound to E. coli and S. Typhimurium-produced adhesions and in the case of S. Typhimurium, this adhesion-binding activity inhibited the binding and invasion of IPEC-J2 cells.

scholarly journals Spread from the Sink to the Patient: In Situ Study Using Green Fluorescent Protein (GFP)-Expressing Escherichia coli To Model Bacterial Dispersion from Hand-Washing Sink-Trap Reservoirs

2017 ◽  
Vol 83 (8) ◽  
Author(s):  
Shireen Kotay ◽  
Weidong Chai ◽  
William Guilford ◽  
Katie Barry ◽  
Amy J. Mathers
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Hand Washing ◽  
Resistant Bacteria ◽  
Droplet Dispersion ◽  
Content Type ◽  
E Coli ◽  
Mode Of Transmission ◽  
Green Fluorescent

ABSTRACT There have been an increasing number of reports implicating Gammaproteobacteria as often carrying genes of drug resistance from colonized sink traps to vulnerable hospitalized patients. However, the mechanism of transmission from the wastewater of the sink P-trap to patients remains poorly understood. Herein we report the use of a designated hand-washing sink lab gallery to model dispersion of green fluorescent protein (GFP)-expressing Escherichia coli from sink wastewater to the surrounding environment. We found no dispersion of GFP-expressing E. coli directly from the P-trap to the sink basin or surrounding countertop with coincident water flow from a faucet. However, when the GFP-expressing E. coli cells were allowed to mature in the P-trap under conditions similar to those in a hospital environment, a GFP-expressing E. coli-containing putative biofilm extended upward over 7 days to reach the strainer. This subsequently resulted in droplet dispersion to the surrounding areas (<30 in.) during faucet operation. We also demonstrated that P-trap colonization could occur by retrograde transmission along a common pipe. We postulate that the organisms mobilize up to the strainer from the P-trap, resulting in droplet dispersion rather than dispersion directly from the P-trap. This work helps to further define the mode of transmission of bacteria from a P-trap reservoir to a vulnerable hospitalized patient. IMPORTANCE Many recent reports demonstrate that sink drain pipes become colonized with highly consequential multidrug-resistant bacteria, which then results in hospital-acquired infections. However, the mechanism of dispersal of bacteria from the sink to patients has not been fully elucidated. Through establishment of a unique sink gallery, this work found that a staged mode of transmission involving biofilm growth from the lower pipe to the sink strainer and subsequent splatter to the bowl and surrounding area occurs rather than splatter directly from the water in the lower pipe. We have also demonstrated that bacterial transmission can occur via connections in wastewater plumbing to neighboring sinks. This work helps to more clearly define the mechanism and risk of transmission from a wastewater source to hospitalized patients in a world with increasingly antibiotic-resistant bacteria that can thrive in wastewater environments and cause infections in vulnerable patients.

scholarly journals PVv3, a New Shuttle Vector for Gene Expression in Vibrio vulnificus

2013 ◽  
Vol 80 (4) ◽  
pp. 1477-1481 ◽  
Author(s):  
Karina Klevanskaa ◽  
Nadja Bier ◽  
Kerstin Stingl ◽  
Eckhard Strauch ◽  
Stefan Hertwig
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Vibrio Parahaemolyticus ◽  
Fluorescent Protein ◽  
Vibrio Vulnificus ◽  
Shuttle Vector ◽  
Content Type ◽  
E Coli ◽  
Green Fluorescent

ABSTRACTAn efficient electroporation procedure forVibrio vulnificuswas designed using the new cloning vector pVv3 (3,107 bp). Transformation efficiencies up to 2 × 106transformants per μg DNA were achieved. The vector stably replicated in bothV. vulnificusandEscherichia coliand was also successfully introduced intoVibrio parahaemolyticusandVibrio cholerae. To demonstrate the suitability of the vector for molecular cloning, the green fluorescent protein (GFP) gene and thevvhBAhemolysin operon were inserted into the vector and functionally expressed inVibrioandE. coli.

Transformation of Escherichia coli K-12 with a High-Copy Plasmid Encoding the Green Fluorescent Protein Reduces Growth: Implications for Predictive Microbiology†

2006 ◽  
Vol 69 (2) ◽  
pp. 276-281 ◽  
Author(s):  
T. P. OSCAR ◽  
K. DULAL ◽  
D. BOUCAUD
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Predictive Models ◽  
Fluorescent Protein ◽  
Inducible Promoter ◽  
E Coli ◽  
Contaminated Food ◽  
Green Fluorescent ◽  
K 12 ◽  
High Copy Plasmid

The green fluorescent protein (GFP) of the jellyfish Aequorea victoria has been widely used as a biomarker and has potential for use in developing predictive models for growth of pathogens on naturally contaminated food. However, constitutive production of GFP can reduce growth of transformed strains. Consequently, a high-copy plasmid with gfp under the control of a tetracycline-inducible promoter (pTGP) was constructed. The plasmid was first introduced into a tetracycline-resistant strain of Escherichia coli K-12 to propagate it for subsequent transformation of tetracycline-resistant strains of Salmonella. In contrast to transformed E. coli K-12, which only fluoresced in response to tetracycline, transformed Salmonella fluoresced maximally without tetracycline induction of gfp. Although pTGP did not function as intended in Salmonella, growth of parent and GFP E. coli K-12 was compared to test the hypothesis that induction of GFP production reduced growth. Although GFP production was not induced during growth on sterile chicken in the absence of tetracycline, maximum specific growth rate (μmax) of GFP E. coli K-12 was reduced 40 to 50% (P &lt; 0.05) at 10, 25, and 40°C compared with the parent strain. When growth of parent and GFP strains of E. coli K-12 was compared in sterile broth at 40°C, μmax and maximum population density of the GFP strain were reduced (P &lt; 0.05) to the same extent (50 to 60%) in the absence and presence of tetracycline. These results indicated that transformation reduced growth of E. coli K-12 independent of gfp induction. Thus, use of a low-copy plasmid or insertion of gfp into the chromosome may be required to construct valid strains for development of predictive models for growth of pathogens on naturally contaminated food.

scholarly journals Escherichia coli Division Inhibitor MinCD Blocks Septation by Preventing Z-Ring Formation

2001 ◽  
Vol 183 (22) ◽  
pp. 6630-6635 ◽  
Author(s):  
Sebastien Pichoff ◽  
Joe Lutkenhaus
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Direct Interaction ◽  
Ring Formation ◽  
Ring Assembly ◽  
Z Ring ◽  
Alternative Fixation ◽  
Green Fluorescent

ABSTRACT The min system spatially regulates division through the topological regulation of MinCD, an inhibitor of cell division. MinCD was previously shown to inhibit division by preventing assembly of the Z ring (E. Bi and J. Lutkenhaus, J. Bacteriol. 175:1118–1125, 1993); however, this was questioned in a recent report (S. S. Justice, J. Garcia-Lara, and L. I. Rothfield, Mol. Microbiol. 37:410–423, 2000) which indicated that MinCD acted after Z-ring formation and prevented the recruitment of FtsA to the Z ring. This discrepancy was due in part to alternative fixation conditions. We have therefore reinvestigated the action of MinCD and avoided fixation by using green fluorescent protein (GFP) fusions to division proteins. MinCD prevented the localization of both FtsZ-GFP and ZipA-GFP, consistent with it preventing Z-ring assembly. Consistent with a direct interaction between FtsZ and the MinCD inhibitor, we find that increased FtsZ, but not FtsA, suppresses MinCD-induced lethality. Furthermore, strains carrying various alleles offtsZ, selected on the basis of resistance to the inhibitor SulA, displayed variable resistance to MinCD. These results are consistent with FtsZ as the target of MinCD and confirm that this inhibitor prevents Z-ring assembly.

Thermal Inactivation of Escherichia coli O157:H7 in Cow Manure Compost

2003 ◽  
Vol 66 (10) ◽  
pp. 1771-1777 ◽  
Author(s):  
XIUPING JIANG ◽  
JENNIE MORGAN ◽  
MICHAEL P. DOYLE
Keyword(s):  
Escherichia Coli ◽  
Green Fluorescent Protein ◽  
Thermal Inactivation ◽  
Fluorescent Protein ◽  
Escherichia Coli O157 ◽  
Cow Manure ◽  
E Coli ◽  
Manure Compost ◽  
D Values ◽  
Green Fluorescent

Rates of inactivation of a five-strain mixture of green fluorescent protein–labeled Escherichia coli O157:H7 in autoclaved and unautoclaved commercial cow manure compost with a moisture content of ca. 38% were determined at temperatures of 50, 55, 60, 65, and 70°C. Trypticase soy agar with ampicillin was determined to be the best medium for the enumeration of heat-injured and uninjured cells of green fluorescent protein–labeled E. coli O157:H7. The results obtained in this study revealed that in autoclaved compost, E. coli O157:H7 reductions of ca. 4 log CFU/g occurred within 8 h, 3 h, 15 min, 2 min, and &lt;1 min at 50, 55, 60, 65, and 70°C, respectively. At 65 and 70°C, considerably less time was required to kill the pathogen in unautoclaved compost than in autoclaved compost. Decimal reduction times (D-values) for autoclaved compost at 50, 55, 60, 65, and 70°C were 137, 50.3, 4.1, 1.8, and 0.93 min, respectively, and D-values for unautoclaved compost at 50, 55, and 60°C were 135, 35.4, and 3.9 min, respectively. Considerable tailing was observed for inactivation curves, especially at 60, 65, and 70°C. These results are useful for identifying composting conditions that will reduce the risk of the transmission of E. coli O157:H7 to foods produced in the presence of animal fecal waste.

scholarly journals Phosphatidylethanolamine Domains and Localization of Phospholipid Synthases in Bacillus subtilis Membranes

2005 ◽  
Vol 187 (6) ◽  
pp. 2163-2174 ◽  
Author(s):  
Ayako Nishibori ◽  
Jin Kusaka ◽  
Hiroshi Hara ◽  
Masato Umeda ◽  
Kouji Matsumoto
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Green Fluorescent Protein ◽  
Acridine Orange ◽  
Fluorescent Protein ◽  
Cyclic Peptide ◽  
Peptide Probe ◽  
Phosphatidylserine Synthase ◽  
Green Fluorescent ◽  
Mutant Cells

ABSTRACT Application of the cardiolipin (CL)-specific fluorescent dye 10-N-nonyl-acridine orange has recently revealed CL-rich domains in the septal regions and at the poles of the Bacillus subtilis membrane (F. Kawai, M. Shoda, R. Harashima, Y. Sadaie, H. Hara, and K. Matsumoto, J. Bacteriol. 186:1475-1483, 2004). This finding prompted us to examine the localization of another phospholipid, phosphatidylethanolamine (PE), with the cyclic peptide probe, Ro09-0198 (Ro), that binds specifically to PE. Treatment with biotinylated Ro followed by tetramethyl rhodamine-conjugated streptavidin revealed that PE is localized in the septal membranes of vegetative cells and in the membranes of the polar septum and the engulfment membranes of sporulating cells. When the mutant cells of the strains SDB01 (psd1::neo) and SDB02 (pssA10::spc), which both lack PE, were examined under the same conditions, no fluorescence was observed. The localization of the fluorescence thus evidently reflected the localization of PE-rich domains in the septal membranes. Similar PE-rich domains were observed in the septal regions of the cells of many Bacillus species. In Escherichia coli cells, however, no PE-rich domains were found. Green fluorescent protein fusions to the enzymes that catalyze the committed steps in PE synthesis, phosphatidylserine synthase, and in CL synthesis, CL synthase and phosphatidylglycerophosphate synthase, were localized mainly in the septal membranes in B. subtilis cells. The majority of the lipid synthases were also localized in the septal membranes; this includes 1-acyl-glycerol-3-phosphate acyltransferase, CDP-diacylglycerol synthase, phosphatidylserine decarboxylase, diacylglycerol kinase, glucolipid synthase, and lysylphosphatidylglycerol synthase. These results suggest that phospholipids are produced mostly in the septal membranes and that CL and PE are kept from diffusing out to lateral ones.

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