scholarly journals Phosphorus Release and Regeneration Following Laboratory Lysis of Bacterial Cells

2021 ◽  
Vol 12 ◽  
Author(s):  
Aric H. Mine ◽  
Maureen L. Coleman ◽  
Albert S. Colman
Keyword(s):  
Escherichia Coli ◽  
Organic Phosphorus ◽  
Cell Lysis ◽  
Phosphorus Release ◽  
Bacterial Cells ◽  
Nutrient Regeneration ◽  
Cell Components ◽  
K 12 ◽  
The Impact ◽  
Apase Activity

The availability of phosphorus limits primary production in large regions of the oceans, and marine microbes use a variety of strategies to overcome this limitation. One strategy is the production of alkaline phosphatase (APase), which allows hydrolysis of larger dissolved organic phosphorus (DOP) compounds in the periplasm or at the cell surface for transport of orthophosphate into the cell. Cell lysis, driven by grazing and viral infection, releases phosphorus-containing cell components, along with active enzymes that could persist after lysis. The importance of this continued enzymatic activity for orthophosphate regeneration is unknown. We used three model bacteria – Escherichia coli K-12 MG1655, Synechococcus sp. WH7803, and Prochlorococcus sp. MED4 – to assess the impact of continued APase activity after cell lysis, via lysozyme treatment, on orthophosphate regeneration. Direct release of orthophosphate scaled with cell size and was reduced under phosphate-starved conditions where APase activity continued for days after lysis. All lysate incubations showed post-lysis orthophosphate generation suggesting phosphatases other than APase maintain activity. Rates of DOP hydrolysis and orthophosphate remineralization varied post-lysis among strains. Escherichia coli K-12 MG1655 rates of remineralization were 0.6 and 1.2 amol cell–1hr–1 under deplete and replete conditions; Synechococcus WH7803 lysates ranged from 0.04 up to 0.3 amol cell–1hr–1 during phosphorus deplete and replete conditions, respectively, while in Prochlorococcus MED4 lysates, rates were stable at 0.001 amol cell–1hr–1 in both conditions. The range of rates of hydrolysis and regeneration underscores the taxonomic and biochemical variability in the process of nutrient regeneration and further highlights the complexity of quantitatively resolving the major fluxes within the microbial loop.

scholarly journals Role of Escherichia coli O157:H7 Virulence Factors in Colonization at the Bovine Terminal Rectal Mucosa

2006 ◽  
Vol 74 (8) ◽  
pp. 4685-4693 ◽  
Author(s):  
Haiqing Sheng ◽  
Ji Youn Lim ◽  
Hannah J. Knecht ◽  
Jie Li ◽  
Carolyn J. Hovde
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Clinical Manifestations ◽  
Rectal Mucosa ◽  
Wild Type ◽  
E Coli ◽  
Healthy Cattle ◽  
Life Threatening ◽  
K 12 ◽  
The Impact

ABSTRACT The human pathogen Escherichia coli O157:H7 causes hemorrhagic colitis and life-threatening sequelae and transiently colonizes healthy cattle at the terminal rectal mucosa. This study analyzed virulence factors important for the clinical manifestations of human E. coli O157:H7 infection for their contribution to the persistence of E. coli in cattle. The colonizing ability of E. coli O157:H7 was compared with those of nonpathogenic E. coli K-12 and isogenic deletion mutants missing Shiga toxin (Stx), the adhesin intimin, its receptor Tir, hemolysin, or the ∼92-kb pO157. Fully ruminant steers received a single rectal application of one E. coli strain so that effects of mucosal attachment and survival at the terminal rectum could be measured without the impact of bacterial passage through the entire gastrointestinal tract. Colonization was monitored by sensitive recto-anal junction mucosal swab culture. Nonpathogenic E. coli K-12 did not colonize as well as E. coli O157:H7 at the bovine terminal rectal mucosa. The E. coli O157:H7 best able to persist had intimin, Tir, and the pO157. Strains missing even one of these factors were recovered in lower numbers and were cleared faster than the wild type. In contrast, E. coli O157:H7 strains that were missing Stx or hemolysin colonized like the wild type. For these three strains, the number of bacteria increased between days 1 and 4 postapplication and then decreased slowly. In contrast, the numbers of noncolonizing strains (K-12, Δtir, and Δeae) decreased from the day of application. These patterns consistently predicted long-term colonization or clearance of the bacteria from the bovine terminal rectal mucosa.

scholarly journals The Impact of ackA, pta, and ackA-pta Mutations on Growth, Gene Expression and Protein Acetylation in Escherichia coli K-12

2020 ◽  
Vol 11 ◽  
Author(s):  
Andrea Schütze ◽  
Dirk Benndorf ◽  
Sebastian Püttker ◽  
Fabian Kohrs ◽  
Katja Bettenbrock
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Protein Acetylation ◽  
K 12 ◽  
The Impact

scholarly journals Location and dynamics of an active promoter in Escherichia coli K-12

2011 ◽  
Vol 441 (1) ◽  
pp. 481-485 ◽  
Author(s):  
María-Antonia Sánchez-Romero ◽  
David J. Lee ◽  
Eugenio Sánchez-Morán ◽  
Stephen J. W. Busby
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Lac Repressor ◽  
Fluorescence Signal ◽  
Bacterial Cells ◽  
Protein Fusion ◽  
Lac Operator ◽  
Low Copy Number ◽  
Green Fluorescent ◽  
K 12

In the present paper, we report that transcription affects the location of a DNA target in Escherichia coli K-12. A strain whose chromosome had been engineered to encode a lac repressor–GFP (green fluorescent protein) fusion was used as a host for a low copy number plasmid that carries an array of five lac operator sites. Individual cells of this strain exhibited a diffuse fluorescence signal, suggesting that the plasmid is distributed throughout the cell cytoplasm. However, a derivative of this plasmid carrying a cloned constitutive promoter is targeted to a location at the edge of the nucleoid towards the pole of the host cell. We conclude that transcription from the cloned promoter is driving the location of the plasmid and that specific locations in bacterial cells may favour gene expression.

scholarly journals Model System To Evaluate the Effect of ampD Mutations on AmpC-Mediated β-Lactam Resistance

2006 ◽  
Vol 50 (6) ◽  
pp. 2030-2037 ◽  
Author(s):  
Amber J. Schmidtke ◽  
Nancy D. Hanson
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Structural Gene ◽  
Model System ◽  
Alternative Mechanism ◽  
Citrobacter Freundii ◽  
E Coli ◽  
K 12 ◽  
Carboxy Terminal ◽  
The Impact

ABSTRACT Mutations within the structural gene of ampD can lead to AmpC overproduction and increases in β-lactam MICs in organisms with an inducible ampC. However, identification of mutations alone cannot predict the impact that those mutations have on AmpD function. Therefore, a model system was designed to determine the effect of ampD mutations on ceftazidime MICs using an AmpD− mutant Escherichia coli strain which produced an inducible plasmid-encoded AmpC. ampD genes were amplified by PCR from strains of E. coli, Citrobacter freundii, and Pseudomonas aeruginosa. Also, carboxy-terminal truncations of C. freundii ampD genes were constructed representing deletions of 10, 21, or 25 codons. Amplified ampD products were cloned into pACYC184 containing inducible bla ACT-1-ampR. Plasmids were transformed into E. coli strains JRG582 (AmpD−) and K-12 259 (AmpD+). The strains were evaluated for a derepressed phenotype using ceftazidime MICs. Some mutated ampD genes, including the ampD gene of a derepressed C. freundii isolate, resulted in substantial decreases in ceftazidime MICs (from >256 μg/ml to 12 to 24 μg/ml) for the AmpD− strain, indicating no role for these mutations in derepressed phenotypes. However, ampD truncation products and ampD from a partially derepressed P. aeruginosa strain resulted in ceftazidime MICs of >256 μg/ml, indicating a role for these gene modifications in derepressed phenotypes. The use of this model system indicated that alternative mechanisms were involved in the derepressed phenotype observed in strains of C. freundii and P. aeruginosa. The alternative mechanism involved in the derepressed phenotype of the C. freundii isolate was downregulation of ampD transcription.

scholarly journals Influence of Environmental and Anthropogenic Factors on Microbial Ecology and Sanitary Threat in the Final Stretch of the Brda River

Water ◽  
2019 ◽  
Vol 11 (5) ◽  
pp. 922 ◽  
Author(s):  
Łukasz Kubera ◽  
Marta Małecka-Adamowicz ◽  
Emilia Jankowiak ◽  
Ewa Dembowska ◽  
Piotr Perliński ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Urban Areas ◽  
Sampling Site ◽  
Environmental Parameters ◽  
Diffusion Method ◽  
Anthropogenic Factors ◽  
Bacterial Cells ◽  
Disk Diffusion Method ◽  
Spatio Temporal ◽  
The Impact

Ecology of aquatic microorganisms depends on a number of environmental parameters. The additional influence of anthropogenic factors is connected with sanitary risk, particularly in urban areas. The study was aimed at assessing the impact of physicochemical and biological parameters on the abundance and activity of bacterioplankton under different spatio-temporal conditions in the urbanized section of the Brda River. The evaluation of sanitary pollution of water was accompanied by the assessment of antibiotic resistance of isolated faecal strains determined using the disk diffusion method. The results indicate that the location of sampling sites significantly affected by the distribution of faecal Escherichia coli and enterococci in the studied part of the river. On the other hand, there were no significant seasonal changes in respiratory activity or abundance of planktonic bacteria. In addition, the abundance of bacterioplankton was not correlated with all measured physico-chemical parameters, though it was correlated with the organic carbon oxidation rate. Depending on the sampling site, bacterial cells with damaged membranes constituted between 8% and 20% of the population. Antibiograms showed the absence of multi-drug resistant strains. Enterococci exhibited the highest resistance to imipenem (45%), while Escherichia coli, to cefoxitin (31%).

scholarly journals Phenotype inference in an Escherichia coli strain panel

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Marco Galardini ◽  
Alexandra Koumoutsi ◽  
Lucia Herrera-Dominguez ◽  
Juan Antonio Cordero Varela ◽  
Anja Telzerow ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Association Studies ◽  
Growth Conditions ◽  
Coli Strain ◽  
Genome Wide Association Studies ◽  
Loss Of Function ◽  
Promising Alternative ◽  
Genetic Interventions ◽  
K 12 ◽  
The Impact

Understanding how genetic variation contributes to phenotypic differences is a fundamental question in biology. Combining high-throughput gene function assays with mechanistic models of the impact of genetic variants is a promising alternative to genome-wide association studies. Here we have assembled a large panel of 696 Escherichia coli strains, which we have genotyped and measured their phenotypic profile across 214 growth conditions. We integrated variant effect predictors to derive gene-level probabilities of loss of function for every gene across all strains. Finally, we combined these probabilities with information on conditional gene essentiality in the reference K-12 strain to compute the growth defects of each strain. Not only could we reliably predict these defects in up to 38% of tested conditions, but we could also directly identify the causal variants that were validated through complementation assays. Our work demonstrates the power of forward predictive models and the possibility of precision genetic interventions.

scholarly journals The Global Regulatory hns Gene Negatively Affects Adhesion to Solid Surfaces by Anaerobically Grown Escherichia coli by Modulating Expression of Flagellar Genes and Lipopolysaccharide Production

2002 ◽  
Vol 184 (6) ◽  
pp. 1522-1529 ◽  
Author(s):  
Paolo Landini ◽  
Alexander J. B. Zehnder
Keyword(s):  
Escherichia Coli ◽  
Sharp Decrease ◽  
Luria Broth ◽  
Bacterial Cells ◽  
Gene Encoding ◽  
Aerobic Conditions ◽  
E Coli ◽  
Hydrophilic Surfaces ◽  
K 12 ◽  
Initial Binding

ABSTRACT The initial binding of bacterial cells to a solid surface is a critical and essential step in biofilm formation. In this report we show that stationary-phase cultures of Escherichia coli W3100 (a K-12 strain) can efficiently attach to sand columns when they are grown in Luria broth medium at 28°C in fully aerobic conditions. In contrast, growth in oxygen-limited conditions results in a sharp decrease in adhesion to hydrophilic substrates. We show that the production of lipopolysaccharide (LPS) and of flagella, as well as the transcription of the fliC gene, encoding the major flagellar subunit, increases under oxygen-limited conditions. Inactivation of the global regulatory hns gene counteracts increased production of LPS and flagella in response to anoxia and allows E. coli W3100 to attach to sand columns even when it is grown under oxygen-limited conditions. We propose that increased production of the FliC protein and of LPS in response to oxygen limitation results in the loss of the ability of E. coli W3100 to adhere to hydrophilic surfaces. Indeed, overexpression of the fliC gene results in a decreased adhesion to sand even when W3100 is grown in fully aerobic conditions. Our observations strongly suggest that anoxia is a negative environmental signal for adhesion in E. coli.

scholarly journals Unexpected Phytostimulatory Behavior for Escherichia coli and Agrobacterium tumefaciens Model Strains

2013 ◽  
Vol 26 (5) ◽  
pp. 495-502 ◽  
Author(s):  
Vincent Walker ◽  
Maxime Bruto ◽  
Floriant Bellvert ◽  
René Bally ◽  
Daniel Muller ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Nitrite Reductase ◽  
Azospirillum Brasilense ◽  
High Performance ◽  
Positive Control ◽  
Shoot Biomass ◽  
E Coli ◽  
K 12 ◽  
The Impact

Plant-beneficial effects of bacteria are often underestimated, especially for well-studied strains associated with pathogenicity or originating from other environments. We assessed the impact of seed inoculation with the emblematic bacterial models Agrobacterium tumefaciens C58 (plasmid-cured) or Escherichia coli K-12 on maize seedlings in nonsterile soil. Compared with the noninoculated control, root biomass (with A. tumefaciens or E. coli) and shoot biomass (with A. tumefaciens) were enhanced at 10 days for ‘PR37Y15’ but not ‘DK315’, as found with the phytostimulator Azospirillum brasilense UAP-154 (positive control). In roots as well as in shoots, Agrobacterium tumefaciens and E. coli triggered similar (in PR37Y15) or different (in DK315) changes in the high-performance liquid chromatography profiles of secondary metabolites (especially benzoxazinoids), distinct from those of Azospirillum brasilense UAP-154. Genome sequence analysis revealed homologs of nitrite reductase genes nirK and nirBD and siderophore synthesis genes for Agrobacterium tumefaciens, as well as homologs of nitrite reductase genes nirBD and phosphatase genes phoA and appA in E. coli, whose contribution to phytostimulation will require experimental assessment. In conclusion, the two emblematic bacterial models had a systemic impact on maize secondary metabolism and resulted in unexpected phytostimulation of seedlings in the Azospirillum sp.-responsive cultivar.

scholarly journals Impact of Nutritional Factors on the Proteome of Intestinal Escherichia coli: Induction of OxyR-Dependent Proteins AhpF and Dps by a Lactose-Rich Diet

2012 ◽  
Vol 78 (10) ◽  
pp. 3580-3591 ◽  
Author(s):  
Monique Rothe ◽  
Carl Alpert ◽  
Wolfram Engst ◽  
Stephanie Musiol ◽  
Gunnar Loh ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Osmotic Stress ◽  
Stress Responses ◽  
Luciferase Reporter ◽  
Nutritional Factors ◽  
Content Type ◽  
E Coli ◽  
K 12 ◽  
The Impact

ABSTRACTTo study the impact of nutritional factors on protein expression of intestinal bacteria, gnotobiotic mice monoassociated withEscherichia coliK-12 were fed three different diets: a diet rich in starch, a diet rich in nondigestible lactose, and a diet rich in casein. Two-dimensional gel electrophoresis and electrospray-tandem mass spectrometry were used to identify differentially expressed proteins of bacteria recovered from small intestine and cecum. Oxidative stress response proteins such as AhpF, Dps, and Fur, all of which belong to the oxyR regulon, were upregulated inE. coliisolates from mice fed the lactose-rich diet. Luciferase reporter gene assays demonstrated that osmotic stress caused by carbohydrates led to the expression ofahpCFanddps, which was not observed in anE. coliΔoxyRmutant. Growth ofahpCFandoxyRdeletion mutants was strongly impaired when nondigestible sucrose was present in the medium. The wild-type phenotype could be restored by complementation of the deletions with plasmids containing the corresponding genes and promoters. The results indicate that some OxyR-dependent proteins play a major role in the adaptation ofE. colito osmotic stress. We conclude that there is an overlap of osmotic and oxidative stress responses. Mice fed the lactose-rich diet possibly had a higher intestinal osmolality, leading to the upregulation of OxyR-dependent proteins, which enable intestinalE. colito better cope with diet-induced osmotic stress.

scholarly journals Novel perspective on a conventional technique: Impact of ultra-low temperature on bacterial viability and protein extraction

PLoS ONE ◽  
2021 ◽  
Vol 16 (5) ◽  
pp. e0251640
Author(s):  
Aditya Sarnaik ◽  
Apurv Mhatre ◽  
Muhammad Faisal ◽  
Dylan Smith ◽  
Ryan Davis ◽  
...  
Keyword(s):  
Low Temperature ◽  
Cell Viability ◽  
Bacterial Cell ◽  
Storage Time ◽  
Extraction Efficiency ◽  
Protein Extraction ◽  
Fold Increase ◽  
Cell Lysis ◽  
Bacterial Cells ◽  
The Impact

Ultra-low temperature (ULT) storage of microbial biomass is routinely practiced in biological laboratories. However, there is very little insight regarding the effects of biomass storage at ULT and the structure of the cell envelope, on cell viability. Eventually, these aspects influence bacterial cell lysis which is one of the critical steps for biomolecular extraction, especially protein extraction. Therefore, we studied the effects of ULT-storage (-80°C) on three different bacterial platforms: Escherichia coli, Bacillus subtilis and the cyanobacterium Synechocystis sp. PCC 6803. By using a propidium iodide assay and a modified MTT assay we determined the impact of ULT storage on cellular viability. Subsequently, the protein extraction efficiency was determined by analyzing the amount of protein released following the storage. The results successfully established that longer the ULT-storage time lower is the cell viability and larger is the protein extraction efficiency. Interestingly, E. coli and B. subtilis exhibited significant reduction in cell viability over Synechocystis 6803. This indicates that the cell membrane structure and composition may play a major role on cell viability in ULT storage. Interestingly, E. coli exhibited concomitant increase in cell lysis efficiency resulting in a 4.5-fold increase (from 109 μg/ml of protein on day 0 to 464 μg/ml of protein on day 2) in the extracted protein titer following ULT storage. Furthermore, our investigations confirmed that the protein function, tested through the extraction of fluorescent proteins from cells stored at ULT, remained unaltered. These results established the plausibility of using ULT storage to improve protein extraction efficiency. Towards this, the impact of shorter ULT storage time was investigated to make the strategy more time efficient to be adopted into protocols. Interestingly, E. coli transformants expressing mCherry yielded 2.7-fold increase (93 μg/mL to 254 μg/mL) after 10 mins, while 4-fold increase (380 μg/mL) after 120 mins of ULT storage in the extracted soluble protein. We thereby substantiate that: (1) the storage time of bacterial cells in -80°C affect cell viability and can alter protein extraction efficiency; and (2) exercising a simple ULT-storage prior to bacterial cell lysis can improve the desired protein yield without impacting its function.

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