scholarly journals Efficient Production of Active Polyhydroxyalkanoate Synthase in Escherichia coli by Coexpression of Molecular Chaperones

2013 ◽  
Vol 79 (6) ◽  
pp. 1948-1955 ◽  
Author(s):  
Nicholas M. Thomson ◽  
Azusa Saika ◽  
Kazunori Ushimaru ◽  
Smith Sangiambut ◽  
Takeharu Tsuge ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Molecular Chaperones ◽  
Specific Activity ◽  
Soluble Fraction ◽  
Efficient Production ◽  
Type I ◽  
Content Type ◽  
Molecular Weights ◽  
Polyhydroxyalkanoate Synthase ◽  
Potential Use

ABSTRACTThe type I polyhydroxyalkanoate synthase fromCupriavidus necatorwas heterologously expressed inEscherichia coliwith simultaneous overexpression of chaperone proteins. Compared to expression of synthase alone (14.55 mg liter−1), coexpression with chaperones resulted in the production of larger total quantities of enzyme, including a larger proportion in the soluble fraction. The largest increase was seen when the GroEL/GroES system was coexpressed, resulting in approximately 6-fold-greater enzyme yields (82.37 mg liter−1) than in the absence of coexpressed chaperones. The specific activity of the purified enzyme was unaffected by coexpression with chaperones. Therefore, the increase in yield was attributed to an enhanced soluble fraction of synthase. Chaperones were also coexpressed with a polyhydroxyalkanoate production operon, resulting in the production of polymers with generally reduced molecular weights. This suggests a potential use for chaperones to control the physical properties of the polymer.

scholarly journals Characterization of the Highly Active Polyhydroxyalkanoate Synthase of Chromobacterium sp. Strain USM2

2011 ◽  
Vol 77 (9) ◽  
pp. 2926-2933 ◽  
Author(s):  
Kesaven Bhubalan ◽  
Jo-Ann Chuah ◽  
Fumi Shozui ◽  
Christopher J. Brigham ◽  
Seiichi Taguchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Weight Percent ◽  
Pha Synthase ◽  
Content Type ◽  
Highly Active ◽  
Key Enzyme ◽  
Polyhydroxyalkanoate Synthase

ABSTRACTThe synthesis of bacterial polyhydroxyalkanoates (PHA) is very much dependent on the expression and activity of a key enzyme, PHA synthase (PhaC). Many efforts are being pursued to enhance the activity and broaden the substrate specificity of PhaC. Here, we report the identification of a highly active wild-type PhaC belonging to the recently isolatedChromobacteriumsp. USM2 (PhaCCs). PhaCCsshowed the ability to utilize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), and 3-hydroxyhexanoate (3HHx) monomers in PHA biosynthesis. Anin vitroassay of recombinant PhaCCsexpressed inEscherichia colishowed that its polymerization of 3-hydroxybutyryl-coenzyme A activity was nearly 8-fold higher (2,462 ± 80 U/g) than that of the synthase from the model strainC. necator(307 ± 24 U/g). Specific activity using a Strep2-tagged, purified PhaCCswas 238 ± 98 U/mg, almost 5-fold higher than findings of previous studies using purified PhaC fromC. necator. Efficient poly(3-hydroxybutyrate) [P(3HB)] accumulation inEscherichia coliexpressing PhaCCsof up to 76 ± 2 weight percent was observed within 24 h of cultivation. To date, this is the highest activity reported for a purified PHA synthase. PhaCCsis a naturally occurring, highly active PHA synthase with superior polymerizing ability.

scholarly journals Influence of Type I Fimbriae and Fluid Shear Stress on Bacterial Behavior and Multicellular Architecture of Early Escherichia coli Biofilms at Single-Cell Resolution

2018 ◽  
Vol 84 (6) ◽  
Author(s):  
Liyun Wang ◽  
Robert Keatch ◽  
Qi Zhao ◽  
John A. Wright ◽  
Clare E. Bryant ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Shear Stress ◽  
Single Cell ◽  
Biofilm Formation ◽  
Fluid Shear Stress ◽  
Cell Membrane Permeability ◽  
Type I ◽  
Fluid Shear ◽  
Content Type ◽  
Type I Fimbriae

ABSTRACT Biofilm formation on abiotic surfaces in the food and medical industry can cause severe contamination and infection, yet how biological and physical factors determine the cellular architecture of early biofilms and the bacterial behavior of the constituent cells remains largely unknown. In this study, we examined the specific role of type I fimbriae in nascent stages of biofilm formation and the response of microcolonies to environmental flow shear at the single-cell resolution. The results show that type I fimbriae are not required for reversible adhesion from plankton, but they are critical for the irreversible adhesion of Escherichia coli strain MG1655 cells that form biofilms on polyethylene terephthalate (PET) surfaces. Besides establishing firm cell surface contact, the irreversible adhesion seems necessary to initiate the proliferation of E. coli on the surface. After the application of shear stress, bacterial retention is dominated by the three-dimensional architecture of colonies, independent of the population size, and the multilayered structure could protect the embedded cells from being insulted by fluid shear, while the cell membrane permeability mainly depends on the biofilm population size and the duration of the shear stress. IMPORTANCE Bacterial biofilms could lead to severe contamination problems in medical devices and food processing equipment. However, biofilms are usually studied at a rough macroscopic level; thus, little is known about how individual bacterium behavior within biofilms and the multicellular architecture are influenced by bacterial appendages (e.g., pili/fimbriae) and environmental factors during early biofilm formation. We applied confocal laser scanning microscopy (CLSM) to visualize Escherichia coli microcolonies at a single-cell resolution. Our findings suggest that type I fimbriae are vital to the initiation of bacterial proliferation on surfaces. We also found that the fluid shear stress affects the biofilm architecture and cell membrane permeability of the constituent bacteria in a different way: the onset of the biofilm is linked with the three-dimensional morphology, while membranes are regulated by the overall population of microcolonies.

scholarly journals Histone-like Nucleoid-Structuring Protein (H-NS) Paralogue StpA Activates the Type I-E CRISPR-Cas System against Natural Transformation in Escherichia coli

2020 ◽  
Vol 86 (14) ◽  
Author(s):  
Dongchang Sun ◽  
Xudan Mao ◽  
Mingyue Fei ◽  
Ziyan Chen ◽  
Tingheng Zhu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Natural Transformation ◽  
Inducible Promoter ◽  
Regulation Mechanism ◽  
Type I ◽  
Content Type ◽  
E Coli ◽  
Double Stranded Dna ◽  
Dna Binding Site

ABSTRACT Working mechanisms of CRISPR-Cas systems have been intensively studied. However, far less is known about how they are regulated. The histone-like nucleoid-structuring protein H-NS binds the promoter of cas genes (Pcas) and suppresses the type I-E CRISPR-Cas system in Escherichia coli. Although the H-NS paralogue StpA also binds Pcas, its role in regulating the CRISPR-Cas system remains unidentified. Our previous work established that E. coli is able to take up double-stranded DNA during natural transformation. Here, we investigated the function of StpA in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. We first documented that although the activated type I-E CRISPR-Cas system, due to hns deletion, interfered with CRISPR-Cas-targeted plasmid transfer, stpA inactivation restored the level of natural transformation. Second, we showed that inactivating stpA reduced the transcriptional activity of Pcas. Third, by comparing transcriptional activities of the intact Pcas and the Pcas with a disrupted H-NS binding site in the hns and hns stpA null deletion mutants, we demonstrated that StpA activated transcription of cas genes by binding to the same site as H-NS in Pcas. Fourth, by expressing StpA with an arabinose-inducible promoter, we confirmed that StpA expressed at a low level stimulated the activity of Pcas. Finally, by quantifying the level of mature CRISPR RNA (crRNA), we demonstrated that StpA was able to promote the amount of crRNA. Taken together, our work establishes that StpA serves as a transcriptional activator in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. IMPORTANCE StpA is normally considered a molecular backup of the nucleoid-structuring protein H-NS, which was reported as a transcriptional repressor of the type I-E CRISPR-Cas system in Escherichia coli. However, the role of StpA in regulating the type I-E CRISPR-Cas system remains elusive. Our previous work uncovered a new route for double-stranded DNA (dsDNA) entry during natural transformation of E. coli. In this study, we show that StpA plays a role opposite to that of its paralogue H-NS in regulating the type I-E CRISPR-Cas system against natural transformation of E. coli. Our work not only expands our knowledge on CRISPR-Cas-mediated adaptive immunity against extracellular nucleic acids but also sheds new light on understanding the complex regulation mechanism of the CRISPR-Cas system. Moreover, the finding that paralogues StpA and H-NS share a DNA binding site but play opposite roles in transcriptional regulation indicates that higher-order compaction of bacterial chromatin by histone-like proteins could switch prokaryotic transcriptional modes.

scholarly journals The EcoKI Type I Restriction-Modification System in Escherichia coli Affects but Is Not an Absolute Barrier for Conjugation

2014 ◽  
Vol 197 (2) ◽  
pp. 337-342 ◽  
Author(s):  
Louise Roer ◽  
Frank M. Aarestrup ◽  
Henrik Hasman
Keyword(s):  
Escherichia Coli ◽  
Rapid Evolution ◽  
Type I ◽  
Modification System ◽  
Major Barrier ◽  
Exogenous Dna ◽  
Content Type ◽  
Restriction Modification System ◽  
K 12 ◽  
Restriction Modification

The rapid evolution of bacteria is crucial to their survival and is caused by exchange, transfer, and uptake of DNA, among other things. Conjugation is one of the main mechanisms by which bacteria share their DNA, and it is thought to be controlled by varied bacterial immune systems. Contradictory results about restriction-modification systems based on phenotypic studies have been presented as reasons for a barrier to conjugation with and other means of uptake of exogenous DNA. In this study, we show that inactivation of the R.EcoKI restriction enzyme in strainEscherichia coliK-12 strain MG1655 increases the conjugational transfer of plasmid pOLA52, which carriers two EcoKI recognition sites. Interestingly, the results were not absolute, and uptake of unmethylated pOLA52 was still observed in the wild-type strain (with an intacthsdRgene) but at a reduction of 85% compared to the uptake of the mutant recipient with a disruptedhsdRgene. This leads to the conclusion that EcoKI restriction-modification affects the uptake of DNA by conjugation but is not a major barrier to plasmid transfer.

scholarly journals Fur Represses Adhesion to, Invasion of, and Intracellular Bacterial Community Formation within Bladder Epithelial Cells and Motility in Uropathogenic Escherichia coli

2016 ◽  
Vol 84 (11) ◽  
pp. 3220-3231 ◽  
Author(s):  
Kumiko Kurabayashi ◽  
Tomohiro Agata ◽  
Hirofumi Asano ◽  
Haruyoshi Tomita ◽  
Hidetada Hirakawa
Keyword(s):  
Escherichia Coli ◽  
Epithelial Cells ◽  
Antimicrobial Agents ◽  
Host Cells ◽  
Type I ◽  
Wild Type ◽  
Content Type ◽  
Type I Fimbriae ◽  
Tract Infections

Uropathogenic Escherichia coli (UPEC) is a major pathogen that causes urinary tract infections (UTIs). This bacterium adheres to and invades the host cells in the bladder, where it forms biofilm-like polymicrobial structures termed intracellular bacterial communities (IBCs) that protect UPEC from antimicrobial agents and the host immune systems. Using genetic screening, we found that deletion of the fur gene, which encodes an iron-binding transcriptional repressor for iron uptake systems, elevated the expression of type I fimbriae and motility when UPEC was grown under iron-rich conditions, and it led to an increased number of UPEC cells adhering to and internalized in bladder epithelial cells. Consequently, the IBC colonies that the fur mutant formed in host cells were denser and larger than those formed by the wild-type parent strain. Fur is inactivated under iron-restricted conditions. When iron was depleted from the bacterial cultures, wild-type UPEC adhesion, invasion, and motility increased, similar to the case with the fur mutant. The purified Fur protein bound to regions upstream of fimA and flhD , which encode type I fimbriae and an activator of flagellar expression that contributes to motility, respectively. These results suggest that Fur is a repressor of fimA and flhD and that its repression is abolished under iron-depleted conditions. Based on our in vitro experiments, we conclude that UPEC adhesion, invasion, IBC formation, and motility are suppressed by Fur under iron-rich conditions but derepressed under iron-restricted conditions, such as in patients with UTIs.

scholarly journals P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

2016 ◽  
Vol 82 (7) ◽  
pp. 1992-2002 ◽  
Author(s):  
Pyung-Gang Lee ◽  
Joonwon Kim ◽  
Eun-Jung Kim ◽  
EunOk Jung ◽  
Bishnu Prasad Pandey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Reaction System ◽  
Gut Bacteria ◽  
Site Directed Mutagenesis ◽  
Anaerobic Growth ◽  
Efficient Production ◽  
Cell Reaction ◽  
Whole Cell ◽  
Content Type ◽  
Postmenopausal Symptoms

ABSTRACT(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis fromSlackia isoflavoniconvertensintoEscherichia coliBL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 μM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis oftrans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

scholarly journals Lineage-Specific Methyltransferases Define the Methylome of the Globally Disseminated Escherichia coli ST131 Clone

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Brian M. Forde ◽  
Minh-Duy Phan ◽  
Jayde A. Gawthorne ◽  
Melinda M. Ashcroft ◽  
Mitchell Stanton-Cook ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Methylation ◽  
Real Time ◽  
Single Molecule ◽  
Mobile Genetic Elements ◽  
Sequence Type ◽  
Type I ◽  
Smrt Sequencing ◽  
Content Type ◽  
E Coli

ABSTRACTEscherichia colisequence type 131 (ST131) is a clone of uropathogenicE. colithat has emerged rapidly and disseminated globally in both clinical and community settings. Members of the ST131 lineage from across the globe have been comprehensively characterized in terms of antibiotic resistance, virulence potential, and pathogenicity, but to date nothing is known about the methylome of these important human pathogens. Here we used single-molecule real-time (SMRT) PacBio sequencing to determine the methylome ofE. coliEC958, the most-well-characterized completely sequenced ST131 strain. Our analysis of 52,081 methylated adenines in the genome of EC958 discovered threem6A methylation motifs that have not been described previously. Subsequent SMRT sequencing of isogenic knockout mutants identified the two type I methyltransferases (MTases) and one type IIG MTase responsible form6A methylation of novel recognition sites. Although both type I sites were rare, the type IIG sites accounted for more than 12% of all methylated adenines in EC958. Analysis of the distribution of MTase genes across 95 ST131 genomes revealed their prevalence is highly conserved within the ST131 lineage, with most variation due to the presence or absence of mobile genetic elements on which individual MTase genes are located.IMPORTANCEDNA modification plays a crucial role in bacterial regulation. Despite several examples demonstrating the role of methyltransferase (MTase) enzymes in bacterial virulence, investigation of this phenomenon on a whole-genome scale has remained elusive until now. Here we used single-molecule real-time (SMRT) sequencing to determine the first complete methylome of a strain from the multidrug-resistantE. colisequence type 131 (ST131) lineage. By interrogating the methylome computationally and with further SMRT sequencing of isogenic mutants representing previously uncharacterized MTase genes, we defined the target sequences of three novel ST131-specific MTases and determined the genomic distribution of all MTase target sequences. Using a large collection of 95 previously sequenced ST131 genomes, we identified mobile genetic elements as a major factor driving diversity in DNA methylation patterns. Overall, our analysis highlights the potential for DNA methylation to dramatically influence gene regulation at the transcriptional level within a well-definedE. coliclone.

scholarly journals Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

2015 ◽  
Vol 81 (15) ◽  
pp. 5103-5114 ◽  
Author(s):  
Michael E. Pyne ◽  
Murray Moo-Young ◽  
Duane A. Chung ◽  
C. Perry Chou
Keyword(s):  
Escherichia Coli ◽  
Gene Replacement ◽  
Efficient Production ◽  
Chromosomal Gene ◽  
Content Type ◽  
Pcr Screening ◽  
Colony Pcr ◽  
Marker Recycling ◽  
Dna Deletions ◽  
Pcr Products

ABSTRACTTo date, most genetic engineering approaches coupling the type IIStreptococcus pyogenesclustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements inEscherichia colican be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and “scar”-less.

scholarly journals Driving Forces Enable High-Titer Anaerobic 1-Butanol Synthesis in Escherichia coli

2011 ◽  
Vol 77 (9) ◽  
pp. 2905-2915 ◽  
Author(s):  
Claire R. Shen ◽  
Ethan I. Lan ◽  
Yasumasa Dekishima ◽  
Antonino Baez ◽  
Kwang Myung Cho ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Driving Forces ◽  
High Titer ◽  
Catalytic Efficiency ◽  
Anaerobic Growth ◽  
High Yield ◽  
Efficient Production ◽  
Acetyl Coa ◽  
Content Type ◽  
Irreversible Reaction

ABSTRACT1-Butanol, an important chemical feedstock and advanced biofuel, is produced byClostridiumspecies. Various efforts have been made to transfer the clostridial 1-butanol pathway into other microorganisms. However, in contrast to similar compounds, only limited titers of 1-butanol were attained. In this work, we constructed a modified clostridial 1-butanol pathway inEscherichia colito provide an irreversible reaction catalyzed bytrans-enoyl-coenzyme A (CoA) reductase (Ter) and created NADH and acetyl-CoA driving forces to direct the flux. We achieved high-titer (30 g/liter) and high-yield (70 to 88% of the theoretical) production of 1-butanol anaerobically, comparable to or exceeding the levels demonstrated by native producers. Without the NADH and acetyl-CoA driving forces, the Ter reaction alone only achieved about 1/10 the level of production. The engineered host platform also enables the selection of essential enzymes with better catalytic efficiency or expression by anaerobic growth rescue. These results demonstrate the importance of driving forces in the efficient production of nonnative products.

scholarly journals Reconstitution of Active Mycobacterial Binuclear Iron Monooxygenase Complex in Escherichia coli

2013 ◽  
Vol 79 (19) ◽  
pp. 6033-6039 ◽  
Author(s):  
Toshiki Furuya ◽  
Mika Hayashi ◽  
Kuniki Kino
Keyword(s):  
Escherichia Coli ◽  
Soluble Fraction ◽  
Sodium Dodecyl ◽  
Electrophoretic Analysis ◽  
Functional Expression ◽  
Content Type ◽  
E Coli ◽  
Efficient Expression ◽  
Coupling Protein ◽  
Binuclear Iron

ABSTRACTBacterial binuclear iron monooxygenases play numerous physiological roles in oxidative metabolism. Monooxygenases of this type found in actinomycetes also catalyze various useful reactions and have attracted much attention as oxidation biocatalysts. However, difficulties in expressing these multicomponent monooxygenases in heterologous hosts, particularly inEscherichia coli, have hampered the development of engineered oxidation biocatalysts. Here, we describe a strategy to functionally express the mycobacterial binuclear iron monooxygenase MimABCD inEscherichia coli. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis of themimABCDgene expression inE. colirevealed that the oxygenase components MimA and MimC were insoluble. Furthermore, although the reductase MimB was expressed at a low level in the soluble fraction ofE. colicells, a band corresponding to the coupling protein MimD was not evident. This situation rendered the transformedE. colicells inactive. We found that the following factors are important for functional expression of MimABCD inE. coli: coexpression of the specific chaperonin MimG, which caused MimA and MimC to be soluble inE. colicells, and the optimization of themimDnucleotide sequence, which led to efficient expression of this gene product. These two remedies enabled this multicomponent monooxygenase to be actively expressed inE. coli. The strategy described here should be generally applicable to theE. coliexpression of other actinomycetous binuclear iron monooxygenases and related enzymes and will accelerate the development of engineered oxidation biocatalysts for industrial processes.

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