scholarly journals Effectiveness of poultry litter amendments on bacterial survival and Eimeria oocyst sporulation

2018 ◽  
pp. 1064-1073 ◽  
Author(s):  
Essam S. Soliman ◽  
Nahla H. Sallam ◽  
Eman M. Abouelhassan
Keyword(s):  
Escherichia Coli ◽  
Negative Correlation ◽  
Total Bacterial Count ◽  
Bacterial Survival ◽  
Positive Correlation ◽  
E Coli ◽  
Weak Positive Correlation ◽  
Moisture Percentage ◽  
Weak Negative Correlation

Aim: Broilers' optimum performance in response to their genetic potential depends on litter environment which is ideal for bacterial survival and coccidian oocyst sporulation. An in vitro evaluation was conducted for the effectiveness of superphosphate, meta-bisulfide, and charcoal litter amendments in minimizing Escherichia coli O157:H7 and Salmonella Typhimurium survival, Eimeria oocyst count, and sporulation. Materials and Methods: Three groups of 16 litter trays were prepared and inoculated with E. coli O157:H7, S. Typhimurium, and Eimeria non-sporulated oocyst. A set of four trays in each group was designed for each one of the chemical amendments. A total of 720 litter samples were collected and examined for bacterial counts, Eimeria oocyst count, and sporulation during the experimental period (35 days). Results: Litter moisture and pH revealed a highly significant (p<0.001) reduction in all treated litter trays compared to control. Total bacterial count (TBC), total Enterobacteriaceae count, and S. Typhimurium count showed a highly significant (p<0.001) reduction in meta-bisulfide-treated trays compared to other amendments and positive control. Meanwhile, Eimeria oocyst count and sporulation revealed a highly significant (p<0.001) reduction in superphosphate, meta-bisulfide, and charcoal-treated trays, respectively. Temperature revealed a highly significant (p<0.001) weak positive correlation with pH of all inoculated trays, a highly significant (p<0.001) weak negative correlation with moisture percentage of E. coli O157:H7 and S. Typhimurium inoculated trays, and a highly significant (p<0.001) weak negative correlation with TBC. Meanwhile, relative humidity revealed significant (p≤0.005) weak positive correlation with moisture percentage of E. coli O157:H7 inoculated trays. Conclusion: The study concluded that regular usage with periodical reapplication of litter amendments as meta-bisulfide or superphosphate in poultry farms is one of the indispensable managemental and preventive measures for minimizing bacterial survival and inhibiting Eimeria oocyst maturation and sporulation. Keywords: charcoal, Eimeria, Escherichia coli, litter, meta-bisulfide, Salmonella, superphosphate.

scholarly journals Relationship between Body Mass Index and Flexibility in Young Adults

2021 ◽  
pp. 119-126
Author(s):  
Ashwini Akash Gite ◽  
Neha Mukkamala ◽  
Lata Parmar
Keyword(s):  
Physical Activity ◽  
Young Adults ◽  
Negative Correlation ◽  
Knee Extension ◽  
Activity Levels ◽  
Low Back ◽  
Positive Correlation ◽  
Weak Positive Correlation ◽  
Weak Negative Correlation ◽  
Hamstring Flexibility

BACKGROUND: Flexibility is important for general health and fitness, athletic performance, injury prevention and rehabilitation. Sedentary behavior of students leads to decreased physical fitness levels which affects flexibility. There is little evidence regarding relationship between BMI and flexibility. Aim: To study the relationship between BMI and flexibility and BMI and physical activity in young adults. Study Design: Observational study Place and Duration Of Study: The study was carried out at College of Physiotherapy, Sumandeep Vidyapeeth, Vadodara from September 2019 to March 2020. Methods: Males and females between 18 to 30 years were included in the study. BMI, Sit and Reach Test for low back and hamstring flexibility, Active Knee Extension Test (AKET) for hamstrings and Global Physical Activity Questionnaire (GPAQ) for physical activity levels were assessed. Results: 570 participants with mean age of 20.23±2.07 years were included. Mean BMI was 21.83±4.81 kg/m2. Number of females were 462 and males were 108. There was no significant correlation between BMI and flexibility of low back (P=.247) and hamstring (Rt) (P=.668) (Lt) (P=.354). BMI and GPAQ showed a statistically significant association (P=0.02). There was a weak positive correlation which was statistically significant between GPAQ and SART (P =0.000) and a weak negative correlation which was statistically significant between GPAQ and Rt AKET (P=0.004). Conclusion:  There was no significant correlation found between BMI and flexibility. There was a statistically significant association between BMI and physical activity, a weak negative correlation between GPAQ and hamstring flexibility and weak positive correlation between GPAQ and SART.

scholarly journals Korelasi Hemoglobin A1c dengan Hemoglobin dan Laju Filtrasi Glomerulus Penderita Diabetes dengan dan tanpa Komplikasi Gagal Ginjal Kronik di Bogor

2020 ◽  
Vol 30 (4) ◽  
Author(s):  
Srilaning Driyah ◽  
Julianty Pradono
Keyword(s):  
Blood Sugar ◽  
Negative Correlation ◽  
Hemoglobin A1c ◽  
Complete Data ◽  
Bivariate Analysis ◽  
Strong Positive Correlation ◽  
Data Set ◽  
Positive Correlation ◽  
Weak Positive Correlation ◽  
Weak Negative Correlation

Diabetes mellitus type 2 (T2DM) is a chronic disease and can cause complications, one of which is decreased kidney function. Anemia is a complication of T2DM, especially if it is accompanied by renal disorders. The aim of this study was to show the relationship between HbA1c and hemoglobin (Hb), hematocrit (HCT), creatinine, and glomerular filtration rate (GFR) in T2DM respondents with and without complications of chronic renal failure (CRF). This study used a subset of the Non-Communicable Diseases cohort data set by the Center for Public Health Efforts in Central Bogor sub-district, Bogor City. The research design was an analytic observational study. Respondents were all T2DM with complete data as much as 303 people. The respondents diagnosed based on the results of previous blood sugar tests. The inclusion criteria were people with T2DM who had complete data (HBA1c, Hb, HCT, and creatinine). Bivariate analysis between the dependent variable (T2DM with or without CRF) and the independent variable (HbA1c, Hb, HCT, creatinine, and LFG) used the Spearman correlation. The results showed a strong positive correlation between HbA1c and Hb (r = 0,66, p<0,05) and HCT (r = 0,67, p<0,05)in T2DM respondents with CRF, but there is no correlation between HbA1c and creatinine and LFG. In T2DM without CRF there is a weak positive correlation between HbA1c and Hb (r = 0,26, p<0,05) and HCT (r = 0,21, p<0,05), a negative correlation between HbA1c and creatinine (r = -0,29, p<0,05), and there is a weak positive correlation between HbA1c and LFG (r = 0,24, p<0,05 ). The conclusion is that controlling blood sugar by examining HbA1c levels shows a strong positive correlation with Hb levels and HCT in T2DM with CRF and a weak negative correlation with LFG in T2DM without CRF. This difference is not in accordance with the existing theory Abstrak  Diabetes melitus tipe 2 (DMT2) merupakan penyakit kronik dan dapat menimbulkan komplikasi, salah satunya adalah penurunan fungsi ginjal. Anemia merupakan komplikasi DMT2 khususnya jika disertai gangguan renal. Tujuan penelitian ini menunjukkan hubungan hemoglobin A1c (HbA1c) dengan hemoglobin (Hb), hematokrit (HCT), kreatinin, dan laju filtrasi glomerulus (LFG) pada responden DMT2 dengan dan tanpa komplikasi gagal ginjal kronik (GGK). Penelitian ini menggunakan subset data kohor penyakit tidak menular (PTM) yang dilakukan oleh Puslitbang Upaya Kesehatan Masyarakat di Kecamatan Bogor Tengah, Kota Bogor. Desain penelitian adalah studi observasional analitik. Responden adalah semua penderita DMT2 dengan data lengkap sebanyak 303 orang yang didiagnosis berdasarkan hasil pemeriksaan gula darah sebelumnya. Kriteria inklusi adalah penderita DMT2 yang memiliki data lengkap (HbA1c, Hb, HCT, dan kreatinin). Analisis bivariat antara variabel dependen (DMT2 dengan atau tanpa GGK) dengan variabel independen (HbA1c, Hb, HCT, kreatinin dan LFG) mengunakan korelasi Spearman. Hasil penelitian menunjukkan pada responden DMT2 dengan GGK terdapat korelasi positif yang kuat antara HbA1c dengan Hb (r = 0,66, p<0,05) dan HCT (r = 0,67, p<.0,05). Sedangkan HbA1c dengan kreatin dan LFG tidak terdapat korelasi. Pada DMT2 tanpa GGK terdapat korelasi positif lemah antara HbA1c dengan Hb (r = 0,26, p<0,05 ) dan HCT (r = 0,21, p<0,05), terjadi korelasi negatif antara HbA1c dengan kreatinin sebesar (r = -0,29, p<0,05), dan terdapat korelasi positif lemah antara HbA1c dengan LFG ( r = 0,24, p<0,05 ). Simpulan yang dapat diambil adalah pengontrolan gula darah dengan pemeriksaan kadar HbA1c menunjukkan korelasi positif yang kuat dengan kadar Hb dan HCT pada DMT2 dengan GGK dan korelasi negatif lemah dengan LFG pada DMT2 tanpa GGK. Perbedaan tersebut belum sesuai teori yang ada.

scholarly journals Role of Shiga Toxins in Cytotoxicity and Immunomodulatory Effects of Escherichia coli O157:H7 during Host-Bacterial Interactions in vitro

Toxins ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 48 ◽  
Author(s):  
Bruballa ◽  
Shiromizu ◽  
Bernal ◽  
Pineda ◽  
Sabbione ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Time Course ◽  
Cell Damage ◽  
Enterohemorrhagic Escherichia Coli ◽  
Host Cells ◽  
Epithelial Cell Line ◽  
Bacterial Survival ◽  
E Coli ◽  
Clinical Conditions

Enterohemorrhagic Escherichia coli (EHEC) strains are food-borne pathogens that can cause different clinical conditions. Shiga toxin 2a and/or 2c (Stx2)-producing E. coli O157:H7 is the serotype most frequently associated with severe human disease. In this work we analyzed the hypothesis that host cells participate in Stx2 production, cell damage, and inflammation during EHEC infection. With this aim, macrophage-differentiated THP-1 cells and the intestinal epithelial cell line HCT-8 were incubated with E. coli O157:H7. A time course analysis of cellular and bacterial survival, Stx2 production, stx2 transcription, and cytokine secretion were analyzed in both human cell lines. We demonstrated that macrophages are able to internalize and kill EHEC. Simultaneously, Stx2 produced by internalized bacteria played a major role in macrophage death. In contrast, HCT-8 cells were completely resistant to EHEC infection. Besides, macrophages and HCT-8 infected cells produce IL-1β and IL-8 inflammatory cytokines, respectively. At the same time, bacterial stx2-specific transcripts were detected only in macrophages after EHEC infection. The interplay between bacteria and host cells led to Stx production, triggering of inflammatory response and cell damage, all of which could contribute to a severe outcome after EHEC infections.

scholarly journals Genome rearrangements induce biofilm formation in Escherichia coli C – an old model organism with a new application in biofilm research

BMC Genomics ◽  
2019 ◽  
Vol 20 (1) ◽  
Author(s):  
Jarosław E. Król ◽  
Donald C. Hall ◽  
Sergey Balashov ◽  
Steven Pastor ◽  
Justin Sibert ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Insertion Sequence ◽  
Genomic Sequence ◽  
Model Organism ◽  
Start Codon ◽  
Bacterial Survival ◽  
E Coli ◽  
Full Genomic Sequence

Abstract Background Escherichia coli C forms more robust biofilms than other laboratory strains. Biofilm formation and cell aggregation under a high shear force depend on temperature and salt concentrations. It is the last of five E. coli strains (C, K12, B, W, Crooks) designated as safe for laboratory purposes whose genome has not been sequenced. Results Here we present the complete genomic sequence of this strain in which we utilized both long-read PacBio-based sequencing and high resolution optical mapping to confirm a large inversion in comparison to the other laboratory strains. Notably, DNA sequence comparison revealed the absence of several genes thought to be involved in biofilm formation, including antigen 43, waaSBOJYZUL for lipopolysaccharide (LPS) synthesis, and cpsB for curli synthesis. The first main difference we identified that likely affects biofilm formation is the presence of an IS3-like insertion sequence in front of the carbon storage regulator csrA gene. This insertion is located 86 bp upstream of the csrA start codon inside the − 35 region of P4 promoter and blocks the transcription from the sigma32 and sigma70 promoters P1-P3 located further upstream. The second is the presence of an IS5/IS1182 in front of the csgD gene. And finally, E. coli C encodes an additional sigma70 subunit driven by the same IS3-like insertion sequence. Promoter analyses using GFP gene fusions provided insights into understanding this regulatory pathway in E. coli. Conclusions Biofilms are crucial for bacterial survival, adaptation, and dissemination in natural, industrial, and medical environments. Most laboratory strains of E. coli grown for decades in vitro have evolved and lost their ability to form biofilm, while environmental isolates that can cause infections and diseases are not safe to work with. Here, we show that the historic laboratory strain of E. coli C produces a robust biofilm and can be used as a model organism for multicellular bacterial research. Furthermore, we ascertained the full genomic sequence of this classic strain, which provides for a base level of characterization and makes it useful for many biofilm-based applications.

scholarly journals Clinical profile and outcomes of patients with acute pancreatitis and correlation with severity index from a tertiary care centre in South India-retrospective analysis

2021 ◽  
Vol 8 (6) ◽  
pp. 814
Author(s):  
Sultan Nawahir ◽  
Babu Kumar S. ◽  
Bala Kasi Naik ◽  
Ealai Athmarao Parthasarathy
Keyword(s):  
Acute Pancreatitis ◽  
Negative Correlation ◽  
Clinical Profile ◽  
Systemic Hypertension ◽  
Serum Lipase ◽  
Positive Correlation ◽  
Weak Positive Correlation ◽  
Study Population ◽  
Weak Negative Correlation ◽  
The Mean

Background: Acute pancreatitis is an inflammatory condition of the pancreas having a varied clinical presentation. It is one of the commonest causes of abdominal pain requiring hospital admission. This retrospective study aims to describe the clinical profile and outcomes of patients with acute pancreatitis and its correlation with severity index.Methods: All patients admitted with a diagnosis of acute pancreatitis to medical gastroenterology department between January 2018 to December 2020 were included in this retrospective study.Results: A total of 61 subjects were included in the final analysis. The mean age was 41.64, the ranged between 19 to 88 years. Among the study population, 53 (86.89%) were male and 8 (13.11%) were female. Among the people 43 (70.49%) were alcoholics, 22 (36.07%) were smokers, 11(18.03%) had systemic hypertension and 5 (8.20%) participants had type 2 diabetic mellitus. The mean CTSI was 2.15±2.82, the mean Balthazar was 3.43±2.4 and the mean procalcitonin was 2.52±9.28. The mean Lipase was 7822. There was a weak positive correlation between CTSI and CRP (rs value: 0.147, p=0.260). There was a weak negative correlation between Balthazar and CRP (rs value:-0.067, p=0.606).Conclusions: Among the study population, 87% of the patients were males. 70% of the population were alcoholics. Mean serum lipase was 7822. There was a weak positive correlation between CTSI and CRP (rs value: 0.147, p=0.260). There was a weak negative correlation between Balthazar and CRP (rs value:-0.067, p=0.606).

Substituted 4-Formyl-2H-chromen-2-ones: Their Reaction with N-(2,3,4,6-Tetra-Oacetyl- β-D-galactopyranosyl)thiosemicarbazide, Antibacterial and Antifungal Activity of Their Thiosemicarbazone Products

2020 ◽  
Vol 24 (19) ◽  
pp. 2272-2282
Author(s):  
Vu Ngoc Toan ◽  
Nguyen Minh Tri ◽  
Nguyen Dinh Thanh
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Candida Albicans ◽  
Selenium Dioxide ◽  
Antibacterial And Antifungal Activity ◽  
E Coli ◽  
Antibacterial And Antifungal Activities ◽  
Alkyl Ethers ◽  
Antibacterial And Antifungal

Several 6- and 7-alkoxy-2-oxo-2H-chromene-4-carbaldehydes were prepared from corresponding alkyl ethers of 6- and 7-hydroxy-4-methyl-2-oxo-2H-chromen-2-ones by oxidation using selenium dioxide. 6- and 7-Alkoxy-4-methyl-2H-chromenes were obtained with yields of 57-85%. Corresponding 4-carbaldehyde derivatives were prepared with yields of 41-67%. Thiosemicarbazones of these aldehydes with D-galactose moiety were synthesized by reaction of these aldehydes with N-(2,3,4,6-tetra-O-acetyl-β-Dgalactopyranosyl) thiosemicarbazide with yields of 62-74%. These thiosemicarbazones were screened for their antibacterial and antifungal activities in vitro against bacteria, such as Staphylococcus aureus, Escherichia coli, and fungi, such as Aspergillus niger, Candida albicans. Several compounds exhibited strong inhibitory activity with MIC values of 0.78- 1.56 μM, including 8a (against S. aureus, E. coli, and C. albicans), 8d (against E. coli and A. niger), 9a (against S. aureus), and 9c (against S. aureus and C. albicans).

scholarly journals In vitro activity of antimicrobial peptide CDP-B11 alone and in combination with colistin against colistin-resistant and multidrug-resistant Escherichia coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kaitlin S. Witherell ◽  
Jason Price ◽  
Ashok D. Bandaranayake ◽  
James Olson ◽  
Douglas R. Call
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Peptide ◽  
Membrane Integrity ◽  
Antibiotic Resistance Genes ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Multidrug Resistant Bacteria ◽  
E Coli ◽  
Minimal Media

AbstractMultidrug-resistant bacteria are a growing global concern, and with increasingly prevalent resistance to last line antibiotics such as colistin, it is imperative that alternative treatment options are identified. Herein we investigated the mechanism of action of a novel antimicrobial peptide (CDP-B11) and its effectiveness against multidrug-resistant bacteria including Escherichia coli #0346, which harbors multiple antibiotic-resistance genes, including mobilized colistin resistance gene (mcr-1). Bacterial membrane potential and membrane integrity assays, measured by flow cytometry, were used to test membrane disruption. Bacterial growth inhibition assays and time to kill assays measured the effectiveness of CDP-B11 alone and in combination with colistin against E. coli #0346 and other bacteria. Hemolysis assays were used to quantify the hemolytic effects of CDP-B11 alone and in combination with colistin. Findings show CDP-B11 disrupts the outer membrane of E. coli #0346. CDP-B11 with colistin inhibits the growth of E. coli #0346 at ≥ 10× lower colistin concentrations compared to colistin alone in Mueller–Hinton media and M9 media. Growth is significantly inhibited in other clinically relevant strains, such as Acinetobacter baumannii, Pseudomonas aeruginosa, and Klebsiella pneumoniae. In rich media and minimal media, the drug combination kills bacteria at a lower colistin concentration (1.25 μg/mL) compared to colistin alone (2.5 μg/mL). In minimal media, the combination is bactericidal with killing accelerated by up to 2 h compared to colistin alone. Importantly, no significant red blood hemolysis is evident for CDP-B11 alone or in combination with colistin. The characteristics of CDP-B11 presented here indicate that it can be used as a potential monotherapy or as combination therapy with colistin for the treatment of multidrug-resistant infections, including colistin-resistant infections.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals A Rhodobacter sphaeroides Protein Mechanistically Similar to Escherichia coli DksA Regulates Photosynthetic Growth

mBio ◽  
2014 ◽  
Vol 5 (3) ◽  
Author(s):  
Christopher W. Lennon ◽  
Kimberly C. Lemmer ◽  
Jessica L. Irons ◽  
Max I. Sellman ◽  
Timothy J. Donohue ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structure Function ◽  
Rhodobacter Sphaeroides ◽  
Fatty Acid Content ◽  
Regulatory Protein ◽  
Growth Conditions ◽  
Globular Domain ◽  
Content Type ◽  
E Coli

ABSTRACTDksA is a global regulatory protein that, together with the alarmone ppGpp, is required for the “stringent response” to nutrient starvation in the gammaproteobacteriumEscherichia coliand for more moderate shifts between growth conditions. DksA modulates the expression of hundreds of genes, directly or indirectly. Mutants lacking a DksA homolog exhibit pleiotropic phenotypes in other gammaproteobacteria as well. Here we analyzed the DksA homolog RSP2654 in the more distantly relatedRhodobacter sphaeroides, an alphaproteobacterium. RSP2654 is 42% identical and similar in length toE. coliDksA but lacks the Zn finger motif of theE. coliDksA globular domain. Deletion of the RSP2654 gene results in defects in photosynthetic growth, impaired utilization of amino acids, and an increase in fatty acid content. RSP2654 complements the growth and regulatory defects of anE. colistrain lacking thedksAgene and modulates transcriptionin vitrowithE. coliRNA polymerase (RNAP) similarly toE. coliDksA. RSP2654 reduces RNAP-promoter complex stabilityin vitrowith RNAPs fromE. coliorR. sphaeroides, alone and synergistically with ppGpp, suggesting that even though it has limited sequence identity toE. coliDksA (DksAEc), it functions in a mechanistically similar manner. We therefore designate the RSP2654 protein DksARsp. Our work suggests that DksARsphas distinct and important physiological roles in alphaproteobacteria and will be useful for understanding structure-function relationships in DksA and the mechanism of synergy between DksA and ppGpp.IMPORTANCEThe role of DksA has been analyzed primarily in the gammaproteobacteria, in which it is best understood for its role in control of the synthesis of the translation apparatus and amino acid biosynthesis. Our work suggests that DksA plays distinct and important physiological roles in alphaproteobacteria, including the control of photosynthesis inRhodobacter sphaeroides. The study of DksARsp, should be useful for understanding structure-function relationships in the protein, including those that play a role in the little-understood synergy between DksA and ppGpp.

scholarly journals Enhancement of the Efficiency of Secretion of Heterologous Lipase in Escherichia coli by Directed Evolution of the ABC Transporter System

2005 ◽  
Vol 71 (7) ◽  
pp. 3468-3474 ◽  
Author(s):  
Gyeong Tae Eom ◽  
Jae Kwang Song ◽  
Jung Hoon Ahn ◽  
Yeon Soo Seo ◽  
Joon Shick Rhee
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Abc Transporter ◽  
Microtiter Plate ◽  
Single Amino Acid ◽  
Wild Type ◽  
E Coli ◽  
Single Amino Acid Change ◽  
Secretion Efficiency

ABSTRACT The ABC transporter (TliDEF) from Pseudomonas fluorescens SIK W1, which mediated the secretion of a thermostable lipase (TliA) into the extracellular space in Escherichia coli, was engineered using directed evolution (error-prone PCR) to improve its secretion efficiency. TliD mutants with increased secretion efficiency were identified by coexpressing the mutated tliD library with the wild-type tliA lipase in E. coli and by screening the library with a tributyrin-emulsified indicator plate assay and a microtiter plate-based assay. Four selected mutants from one round of error-prone PCR mutagenesis, T6, T8, T24, and T35, showed 3.2-, 2.6-, 2.9-, and 3.0-fold increases in the level of secretion of TliA lipase, respectively, but had almost the same level of expression of TliD in the membrane as the strain with the wild-type TliDEF transporter. These results indicated that the improved secretion of TliA lipase was mediated by the transporter mutations. Each mutant had a single amino acid change in the predicted cytoplasmic regions in the membrane domain of TliD, implying that the corresponding region of TliD was important for the improved and successful secretion of the target protein. We therefore concluded that the efficiency of secretion of a heterologous protein in E. coli can be enhanced by in vitro engineering of the ABC transporter.

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