scholarly journals Knockout of a labeled strain of Escherichia coli in the mouse gut using native phages

2020 ◽  
Author(s):  
Li Ping ◽  
Chen Jingchao ◽  
Zhiyu Zhang ◽  
Li Yi ◽  
Liu Lei ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Specificity ◽  
Fluorescent Protein ◽  
Bacterial Species ◽  
Sequencing Analysis ◽  
In Vitro Test ◽  
The Impact ◽  
Phage Treatment

Abstract Background: There is a lack of methodological investigation of the in situ functions of a bacterial species in microecosystems such as the animal gut, although the microbiome has become a focus in the microbiology field worldwide. Results: We used native mixed phages containing Escherichia phages T1 and T4 as a microbial editing tool for eliminating Escherichia coli MG1655 labeled with green fluorescent protein in the mouse gut. The phages possessed rigorous host specificity at both the genus and species levels, resulting in an 8.8-log10 decrease in the titer of viable bacteria after 12 h of phage treatment in an in vitro test. In vivo, they knocked out strain MG1655 not only at concentrations of 10 6 -10 8 CFU g -1 colonizing the mouse gut but also even in mice fed with feedstuff containing the bacterium. In addition, the impact of phage treatment on the microbial community structure of the mouse gut was not significantly ( p ＞0.05) based on a 16S rRNA amplicon gene sequencing analysis, although the richness of some bacteria changed significantly. Conclusions: We provide a feasible microbial editing technique for the animal gut. Native phages with strict host specificity can effectively knock out a target bacterium by single or continuous gastric perfusion, with limited perturbation of microbial diversity, which is beneficial for studies of the function of a specific bacterial species colonizing a complicated microecosystem.

scholarly journals Optimization of Protoplast Isolation and Transformation for a Pilot Study of Genome Editing in Peanut by Targeting the Allergen Gene Ara h 2

2022 ◽  
Vol 23 (2) ◽  
pp. 837
Author(s):  
Sudip Biswas ◽  
Nancy J. Wahl ◽  
Michael J. Thomson ◽  
John M. Cason ◽  
Bill F. McCutchen ◽  
...  
Keyword(s):  
Genome Editing ◽  
New Technologies ◽  
Fluorescent Protein ◽  
Peanut Butter ◽  
Processing System ◽  
Protoplast Isolation ◽  
Sequencing Analysis ◽  
Ara H 2

The cultivated peanut (Arachis hypogaea L.) is a legume consumed worldwide in the form of oil, nuts, peanut butter, and candy. Improving peanut production and nutrition will require new technologies to enable novel trait development. Clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR–Cas9) is a powerful and versatile genome-editing tool for introducing genetic changes for studying gene expression and improving crops, including peanuts. An efficient in vivo transient CRISPR–Cas9- editing system using protoplasts as a testbed could be a versatile platform to optimize this technology. In this study, multiplex CRISPR–Cas9 genome editing was performed in peanut protoplasts to disrupt a major allergen gene with the help of an endogenous tRNA-processing system. In this process, we successfully optimized protoplast isolation and transformation with green fluorescent protein (GFP) plasmid, designed two sgRNAs for an allergen gene, Ara h 2, and tested their efficiency by in vitro digestion with Cas9. Finally, through deep-sequencing analysis, several edits were identified in our target gene after PEG-mediated transformation in protoplasts with a Cas9 and sgRNA-containing vector. These findings demonstrated that a polyethylene glycol (PEG)-mediated protoplast transformation system can serve as a rapid and effective tool for transient expression assays and sgRNA validation in peanut.

scholarly journals Lysine Acetylation Regulates Alanyl-tRNA Synthetase Activity in Escherichia coli

Genes ◽  
2018 ◽  
Vol 9 (10) ◽  
pp. 473 ◽  
Author(s):  
Takuya Umehara ◽  
Saori Kosono ◽  
Dieter Söll ◽  
Koji Tamura
Keyword(s):  
Escherichia Coli ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Lysine Acetylation ◽  
Trna Synthetases ◽  
E Coli ◽  
Domains Of Life ◽  
The Impact

Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.

scholarly journals C-terminal eYFP fusion impairs Escherichia coli MinE function

Open Biology ◽  
2020 ◽  
Vol 10 (5) ◽  
pp. 200010
Author(s):  
Navaneethan Palanisamy ◽  
Mehmet Ali Öztürk ◽  
Emir Bora Akmeriç ◽  
Barbara Di Ventura
Keyword(s):  
Escherichia Coli ◽  
In Silico ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Yellow Fluorescent Protein ◽  
Time Lapse ◽  
Enhanced Yellow Fluorescent Protein ◽  
Min Proteins

The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo , in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.

scholarly journals Adaptation in a Mouse Colony Monoassociated with Escherichia coli K-12 for More than 1,000 Days

2010 ◽  
Vol 76 (14) ◽  
pp. 4655-4663 ◽  
Author(s):  
Sean M. Lee ◽  
Aaron Wyse ◽  
Aaron Lesher ◽  
Mary Lou Everett ◽  
Linda Lou ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rates ◽  
Bacterial Species ◽  
Intestinal Flora ◽  
Average Density ◽  
Host Bacterium ◽  
E Coli ◽  
K 12

ABSTRACT Although mice associated with a single bacterial species have been used to provide a simple model for analysis of host-bacteria relationships, bacteria have been shown to display adaptability when grown in a variety of novel environments. In this study, changes associated with the host-bacterium relationship in mice monoassociated with Escherichia coli K-12 over a period of 1,031 days were evaluated. After 80 days, phenotypic diversification of E. coli was observed, with the colonizing bacteria having a broader distribution of growth rates in the laboratory than the parent E. coli. After 1,031 days, which included three generations of mice and an estimated 20,000 generations of E. coli, the initially homogeneous bacteria colonizing the mice had evolved to have widely different growth rates on agar, a potential decrease in tendency for spontaneous lysis in vivo, and an increased tendency for spontaneous lysis in vitro. Importantly, mice at the end of the experiment were colonized at an average density of bacteria that was more than 3-fold greater than mice colonized on day 80. Evaluation of selected isolates on day 1,031 revealed unique restriction endonuclease patterns and differences between isolates in expression of more than 10% of the proteins identified by two-dimensional electrophoresis, suggesting complex changes underlying the evolution of diversity during the experiment. These results suggest that monoassociated mice might be used as a tool for characterizing niches occupied by the intestinal flora and potentially as a method of targeting the evolution of bacteria for applications in biotechnology.

scholarly journals Visualization of Periplasmic and Cytoplasmic Proteins with a Self-Labeling Protein Tag

2016 ◽  
Vol 198 (7) ◽  
pp. 1035-1043 ◽  
Author(s):  
Na Ke ◽  
Dirk Landgraf ◽  
Johan Paulsson ◽  
Mehmet Berkmen
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Living Cells ◽  
Functional Protein ◽  
Content Type ◽  
Additional Tool

ABSTRACTThe use of fluorescent and luminescent proteins in visualizing proteins has become a powerful tool in understanding molecular and cellular processes within living organisms. This success has resulted in an ever-increasing demand for new and more versatile protein-labeling tools that permit light-based detection of proteins within living cells. In this report, we present data supporting the use of the self-labeling HaloTag protein as a light-emitting reporter for protein fusions within the model prokaryoteEscherichia coli. We show that functional protein fusions of the HaloTag can be detected bothin vivoandin vitrowhen expressed within the cytoplasmic or periplasmic compartments ofE. coli. The capacity to visually detect proteins localized in various prokaryotic compartments expands today's molecular biologist toolbox and paves the path to new applications.IMPORTANCEVisualizing proteins microscopically within living cells is important for understanding both the biology of cells and the role of proteins within living cells. Currently, the most common tool is green fluorescent protein (GFP). However, fluorescent proteins such as GFP have many limitations; therefore, the field of molecular biology is always in need of new tools to visualize proteins. In this paper, we demonstrate, for the first time, the use of HaloTag to visualize proteins in two different compartments within the model prokaryoteEscherichia coli. The use of HaloTag as an additional tool to visualize proteins within prokaryotes increases our capacity to ask about and understand the role of proteins within living cells.

scholarly journals Using Unique ORFan Genes as Strain-specific Identifiers for Escherichia Coli

2021 ◽  
Author(s):  
Marta Ferrandis-Vila ◽  
Sumeet K. Tiwari ◽  
Svenja Mamerow ◽  
Torsten Semmler ◽  
Christian Menge ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Species ◽  
Strain Identification ◽  
Specific Primers ◽  
Faecal Material ◽  
Specific Strain ◽  
Selection Step ◽  
Multiple Strains

Abstract BackgroundBacterial identification at the strain level is a much-needed, but arduous and challenging task. This study aimed to develop a method for identifying and differentiating individual strains among multiple strains of the same bacterial species. The set used for testing the method consisted of 17 Escherichia coli strains picked from a collection of strains isolated in Germany, Spain, the United Kingdom and Vietnam from humans, cattle, swine, wild boars, and chickens. We targeted unique or rare ORFan genes to address the problem of selective and specific strain identification. These ORFan genes, exclusive to each strain, served as templates for developing strain-specific primers.ResultsMost of the strains to be deployed experimentally (14 out of 17) possessed unique ORFan genes that were used to develop strain-specific primers. The remaining three strains were identified by combining a PCR for a rare gene with a selection step for isolating the experimental strains. Multiplex PCR allowed the successful identification of the strains both in vitro in spiked faecal material in addition to in vivo after experimental infections of pigs and recovery of bacteria from faecal material. In addition, primers for qPCR were also developed and quantitative readout from faecal samples after experimental infection was also possible.ConclusionsThe method described in this manuscript using strain-specific unique genes to identify single strains in a mixture of strains proved itself efficient and reliable in detecting and following individual strains both in vitro and in vivo, representing a fast and inexpensive alternative to more costly methods.

scholarly journals Impact of thrombocytes, on bacterial growth and antimicrobial activity of selected antibiotics

2019 ◽  
Vol 39 (3) ◽  
pp. 593-597 ◽  
Author(s):  
Alina Karoline Nussbaumer-Pröll ◽  
Sabine Eberl ◽  
Birgit Reiter ◽  
Thomas Stimpfl ◽  
Walter Jäger ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacterial Growth ◽  
Limiting Factor ◽  
Growth Media ◽  
Healthy Human ◽  
Bacterial Killing ◽  
Bacteria Growth ◽  
The Impact

AbstractIn vitro pharmacodynamic models are used to optimize in vivo dosing regimens in antimicrobial drug development. One limiting factor of such models is the lack of host factors such as corpuscular blood components as erythrocytes which have already been shown to impact activity of antibiotics and/or growth of the pathogen. However, the impact of thrombocytes has not previously been investigated. We set out to investigate if the addition of thrombocytes (set to physiological concentrations in blood of healthy human, i.e., 5 × 105 thrombocytes/μL standard growth media Mueller Hinton Broth, MHB) has an influence on bacterial growth and on the efficacy of antibiotics against Gram+ and Gram− bacteria. Growth assays and time-killing-curves (TKC) were performed with ATCC-strains of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa in triplicate over 24 h. The same approach was followed for 5 clinical isolates of Escherichia coli. Meropenem, ciprofloxacin, and tigecycline were tested as representatives of broad-spectrum antibiotics, and concentrations several-fold above and below the minimal inhibitory concentration (MIC) were simulated. No significant impact of thrombocytes was found on bacterial growth or antimicrobial stability for the investigated agents. Bacteria reduced thrombocyte content to different degree, indicating direct interaction of pathogens and thrombocytes. Impact on bacterial killing was observed but was not fully reproducible when thrombocytes from different donors where used. While interaction of bacteria and thrombocytes was evident in the present study, interaction between antibiotic activity and thrombocytes seems unlikely. Whether variability was caused by different thrombocyte concentrates needs further investigation.

scholarly journals Emergent Bacteria in Cystic Fibrosis:In VitroBiofilm Formation and Resilience under Variable Oxygen Conditions

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Susana P. Lopes ◽  
Nuno F. Azevedo ◽  
Maria O. Pereira
Keyword(s):  
Bacterial Species ◽  
Low Oxygen ◽  
Antibiotic Susceptibilities ◽  
Oxygen Conditions ◽  
Mode Of Life ◽  
Lung Infections ◽  
Biofilm Development ◽  
The Impact

Concurrent to conventional bacterial pathogens, unusual microbes are emerging from cystic fibrosis (CF) airways. Nonetheless, little is known about the contribution of these newly microbes to the resilience of CF-associated biofilms, particularly under variable-oxygen concentrations that are known to occurin vivoin the mucus of CF patients. Two CF-emergent bacterial species,Inquilinus limosusandDolosigranulum pigrum, and the major pathogenPseudomonas aeruginosawere studied in terms of biofilm development and antibiotic susceptibilities underin vitroatmospheres with different oxygen availabilities. All species were able to developin vitrobiofilms under different oxygen-available environments, withD. pigrumaccumulating high amounts of biomass and respiratory activities. When established, biofilms were of difficult eradication, with antibiotics losing their effectiveness in comparison with the corresponding planktonic populations. Surprisingly, biofilms of each emergent organism displayed multidrug resistance under aerobic environments, enduring even in low-oxygen atmospheres. This study suggests a potential prospect on the impact of nonconventional organismsI. limosusandD. pigrumon CF lung infections, demonstrating capacity to adapt to biofilm mode of life under restricted-oxygen atmospheres resembling CF airways, which may ultimately endanger the efficacy of currently used antibiotic regimens.

scholarly journals Arginine Improves pH Homeostasis via Metabolism and Microbiome Modulation

2017 ◽  
Vol 96 (8) ◽  
pp. 924-930 ◽  
Author(s):  
M. Agnello ◽  
L. Cen ◽  
N.C. Tran ◽  
W. Shi ◽  
J.S. McLean ◽  
...  
Keyword(s):  
Microbial Community ◽  
Bacterial Species ◽  
Oral Microbiome ◽  
Ecological Effect ◽  
Sequencing Analysis ◽  
Ph Homeostasis ◽  
16S Sequencing ◽  
Plaque Ph

Dental caries can be described as a dysbiosis of the oral microbial community, in which acidogenic, aciduric, and acid-adapted bacterial species promote a pathogenic environment, leading to demineralization. Alkali generation by oral microbes, specifically via arginine catabolic pathways, is an essential factor in maintaining plaque pH homeostasis. There is evidence that the use of arginine in dentifrices helps protect against caries. The aim of the current study was to investigate the mechanistic and ecological effect of arginine treatment on the oral microbiome and its regulation of pH dynamics, using an in vitro multispecies oral biofilm model that was previously shown to be highly reflective of the in vivo oral microbiome. Pooled saliva from 6 healthy subjects was used to generate overnight biofilms, reflecting early stages of biofilm maturation. First, we investigated the uptake of arginine by the cells of the biofilm as well as the metabolites generated. We next explored the effect of arginine on pH dynamics by pretreating biofilms with 75 mM arginine, followed by the addition of sucrose (15 mM) after 0, 6, 20, or 48 h. pH was measured at each time point and biofilms were collected for 16S sequencing and targeted arginine quantification, and supernatants were prepared for metabolomic analysis. Treatment with only sucrose led to a sustained pH drop from 7 to 4.5, while biofilms treated with sucrose after 6, 20, or 48 h of preincubation with arginine exhibited a recovery to higher pH. Arginine was detected within the cells of the biofilms, indicating active uptake, and arginine catabolites citrulline, ornithine, and putrescine were detected in supernatants, indicating active metabolism. Sequencing analysis revealed a shift in the microbial community structure in arginine-treated biofilms as well as increased species diversity. Overall, we show that arginine improved pH homeostasis through a remodeling of the oral microbial community.

scholarly journals Assembly Dynamics of FtsZ Rings in Bacillus subtilis and Escherichia coli and Effects of FtsZ-Regulating Proteins

2004 ◽  
Vol 186 (17) ◽  
pp. 5775-5781 ◽  
Author(s):  
David E. Anderson ◽  
Frederico J. Gueiros-Filho ◽  
Harold P. Erickson
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Fluorescence Recovery ◽  
Half Time ◽  
Experimental Conditions ◽  
Z Ring

ABSTRACT FtsZ is the major cytoskeletal component of the bacterial cell division machinery. It forms a ring-shaped structure (the Z ring) that constricts as the bacterium divides. Previous in vivo experiments with green fluorescent protein-labeled FtsZ and fluorescence recovery after photobleaching have shown that the Escherichia coli Z ring is extremely dynamic, continually remodeling itself with a half time of 30 s, similar to microtubules in the mitotic spindle. In the present work, under different experimental conditions, we have found that the half time for fluorescence recovery of E. coli Z rings is even shorter (∼9 s). As before, the turnover appears to be coupled to GTP hydrolysis, since the mutant FtsZ84 protein, with reduced GTPase in vitro, showed an ∼3-fold longer half time. We have also extended the studies to Bacillus subtilis and found that this species exhibits equally rapid dynamics of the Z ring (half time, ∼8 s). Interestingly, null mutations of the FtsZ-regulating proteins ZapA, EzrA, and MinCD had only modest effects on the assembly dynamics. This suggests that these proteins do not directly regulate FtsZ subunit exchange in and out of polymers. In B. subtilis, only 30 to 35% of the FtsZ protein was in the Z ring, from which we conclude that a Z ring only 2 or 3 protofilaments thick can function for cell division.

Sign in / Sign up

Export Citation Format

Share Document