scholarly journals Fine-Tuning of the Fatty Acid Pathway by Synthetic Antisense RNA for Enhanced (2S)-Naringenin Production from l-Tyrosine in Escherichia coli

2014 ◽  
Vol 80 (23) ◽  
pp. 7283-7292 ◽  
Author(s):  
Junjun Wu ◽  
Oliver Yu ◽  
Guocheng Du ◽  
Jingwen Zhou ◽  
Jian Chen
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Antisense Rna ◽  
Fine Tuning ◽  
Content Type ◽  
Endogenous Gene ◽  
Malonyl Coa ◽  
Acid Pathway ◽  
And Function ◽  
Fatty Acid Pathway

ABSTRACTMalonyl coenzyme A (malonyl-CoA) is an important precursor for the synthesis of natural products, such as polyketides and flavonoids. The majority of this cofactor often is consumed for producing fatty acids and phospholipids, leaving only a small amount of cellular malonyl-CoA available for producing the target compound. The tuning of malonyl-CoA into heterologous pathways yields significant phenotypic effects, such as growth retardation and even cell death. In this study, fine-tuning of the fatty acid pathway inEscherichia coliwith antisense RNA (asRNA) to balance the demands on malonyl-CoA for target-product synthesis and cell health was proposed. To establish an efficient asRNA system, the relationship between sequence and function for asRNA was explored. It was demonstrated that the gene-silencing effect of asRNA could be tuned by directing asRNA to different positions in the 5′-UTR (untranslated region) of the target gene. Based on this principle, the activity of asRNA was quantitatively tailored to balance the need for malonyl-CoA in cell growth and the production of the main flavonoid precursor, (2S)-naringenin. Appropriate inhibitory efficiency of the anti-fabB/fabFasRNA improved the production titer by 431% (391 mg/liter). Therefore, the strategy presented in this study provided a useful tool for the fine-tuning of endogenous gene expression in bacteria.

The Classical, Yet Controversial, First Enzyme of Lipid Synthesis: Escherichia coli Acetyl-CoA Carboxylase

2021 ◽  
Author(s):  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Building Block ◽  
Lipid Synthesis ◽  
Acid Synthesis ◽  
Acetyl Coa Carboxylase ◽  
Acetyl Coa ◽  
Content Type ◽  
Malonyl Coa

SUMMARY Escherichia coli acetyl-CoA carboxylase (ACC), the enzyme responsible for synthesis of the malonyl-CoA, the building block of fatty acid synthesis, is the paradigm bacterial ACC. Many reports on the structures and stoichiometry of the four subunits comprising the active enzyme as well as on regulation of ACC activity and expression have appeared in the almost 20 years since this subject was last reviewed. This review seeks to update and expand on these reports.

scholarly journals One Secret of Longevity: The Fatty Acid Pathway

PLoS Biology ◽  
2011 ◽  
Vol 9 (3) ◽  
pp. e1001030
Author(s):  
Richard Robinson
Keyword(s):  
Fatty Acid ◽  
Acid Pathway ◽  
Fatty Acid Pathway

scholarly journals Identification and Characterization of Genes Required for 5-Hydroxyuridine Synthesis in Bacillus subtilis and Escherichia coli tRNA

2019 ◽  
Vol 201 (20) ◽  
Author(s):  
Charles T. Lauhon
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Genomic Context ◽  
Similar Reduction ◽  
Content Type ◽  
E Coli ◽  
Wobble Position ◽  
Fertile Ground ◽  
And Function

ABSTRACT In bacteria, tRNAs that decode 4-fold degenerate family codons and have uridine at position 34 of the anticodon are typically modified with either 5-methoxyuridine (mo5U) or 5-methoxycarbonylmethoxyuridine (mcmo5U). These modifications are critical for extended recognition of some codons at the wobble position. Whereas the alkylation steps of these modifications have been described, genes required for the hydroxylation of U34 to give 5-hydroxyuridine (ho5U) remain unknown. Here, a number of genes in Escherichia coli and Bacillus subtilis are identified that are required for wild-type (wt) levels of ho5U. The yrrMNO operon is identified in B. subtilis as important for the biosynthesis of ho5U. Both yrrN and yrrO are homologs to peptidase U32 family genes, which includes the rlhA gene required for ho5C synthesis in E. coli. Deletion of either yrrN or yrrO, or both, gives a 50% reduction in mo5U tRNA levels. In E. coli, yegQ was found to be the only one of four peptidase U32 genes involved in ho5U synthesis. Interestingly, this mutant shows the same 50% reduction in (m)cmo5U as that observed for mo5U in the B. subtilis mutants. By analyzing the genomic context of yegQ homologs, the ferredoxin YfhL is shown to be required for ho5U synthesis in E. coli to the same extent as yegQ. Additional genes required for Fe-S biosynthesis and biosynthesis of prephenate give the same 50% reduction in modification. Together, these data suggest that ho5U biosynthesis in bacteria is similar to that of ho5C, but additional genes and substrates are required for complete modification. IMPORTANCE Modified nucleotides in tRNA serve to optimize both its structure and function for accurate translation of the genetic code. The biosynthesis of these modifications has been fertile ground for uncovering unique biochemistry and metabolism in cells. In this work, genes that are required for a novel anaerobic hydroxylation of uridine at the wobble position of some tRNAs are identified in both Bacillus subtilis and Escherichia coli. These genes code for Fe-S cluster proteins, and their deletion reduces the levels of the hydroxyuridine by 50% in both organisms. Additional genes required for Fe-S cluster and prephenate biosynthesis and a previously described ferredoxin gene all display a similar reduction in hydroxyuridine levels, suggesting that still other genes are required for the modification.

scholarly journals The Influence of Repressor DNA Binding Site Architecture on Transcriptional Control

mBio ◽  
2014 ◽  
Vol 5 (5) ◽  
Author(s):  
Dan M. Park ◽  
Patricia J. Kiley
Keyword(s):  
Escherichia Coli ◽  
Dna Binding ◽  
Binding Site ◽  
Information Content ◽  
Fine Tuning ◽  
Carbon Oxidation ◽  
Content Type ◽  
Architectural Features ◽  
Recognition Elements ◽  
Insight Into

ABSTRACTHow the architecture of DNA binding sites dictates the extent of repression of promoters is not well understood. Here, we addressed the importance of the number and information content of the three direct repeats (DRs) in the binding and repression of theicdApromoter by the phosphorylated form of the globalEscherichia colirepressor ArcA (ArcA-P). We show that decreasing the information content of the two sites with the highest information (DR1 and DR2) eliminated ArcA binding to all three DRs and ArcA repression oficdA. Unexpectedly, we also found that DR3 occupancy functions principally in repression, since mutation of this low-information-content site both eliminated DNA binding to DR3 and significantly weakenedicdArepression, despite the fact that binding to DR1 and DR2 was intact. In addition, increasing the information content of any one of the three DRs or addition of a fourth DR increased ArcA-dependent repression but perturbed signal-dependent regulation of repression. Thus, our data show that the information content and number of DR elements are critical architectural features for maintaining a balance between high-affinity binding and signal-dependent regulation oficdApromoter function in response to changes in ArcA-P levels. Optimization of such architectural features may be a common strategy to either dampen or enhance the sensitivity of DNA binding among the members of the large OmpR/PhoB family of regulators as well as other transcription factors.IMPORTANCEInEscherichia coli, the response regulator ArcA maintains homeostasis of redox carriers under O2-limiting conditions through a comprehensive repression of carbon oxidation pathways that require aerobic respiration to recycle redox carriers. Although a binding site architecture comprised of a variable number of sequence recognition elements has been identified within the promoter regions of ArcA-repressed operons, it is unclear how this variable architecture dictates transcriptional regulation. By dissecting the role of multiple sequence elements within theicdApromoter, we provide insight into the design principles that allow ArcA to repress transcription within diverse promoter contexts. Our data suggest that the arrangement of recognition elements is tailored to achieve sufficient repression of a given promoter while maintaining appropriate signal-dependent regulation of repression, providing insight into how diverse binding site architectures link changes in O2with the fine-tuning of carbon oxidation pathway levels.

scholarly journals The Impact of Plasma Membrane Lipid Composition on Flagellum-Mediated Adhesion of Enterohemorrhagic Escherichia coli

mSphere ◽  
2020 ◽  
Vol 5 (5) ◽  
Author(s):  
Hélène Cazzola ◽  
Laurine Lemaire ◽  
Sébastien Acket ◽  
Elise Prost ◽  
Luminita Duma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Plasma Membrane ◽  
Fatty Acid ◽  
Linolenic Acid ◽  
Lipid Rafts ◽  
Bacterial Adhesion ◽  
Human Pathogen ◽  
Content Type ◽  
E Coli ◽  
The Impact

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a major cause of foodborne gastrointestinal illness. The adhesion of EHEC to host tissues is the first step enabling bacterial colonization. Adhesins such as fimbriae and flagella mediate this process. Here, we studied the interaction of the bacterial flagellum with the host cell’s plasma membrane using giant unilamellar vesicles (GUVs) as a biologically relevant model. Cultured cell lines contain many different molecular components, including proteins and glycoproteins. In contrast, with GUVs, we can characterize the bacterial mode of interaction solely with a defined lipid part of the cell membrane. Bacterial adhesion on GUVs was dependent on the presence of the flagellar filament and its motility. By testing different phospholipid head groups, the nature of the fatty acid chains, or the liposome curvature, we found that lipid packing is a key parameter to enable bacterial adhesion. Using HT-29 cells grown in the presence of polyunsaturated fatty acid (α-linolenic acid) or saturated fatty acid (palmitic acid), we found that α-linolenic acid reduced adhesion of wild-type EHEC but not of a nonflagellated mutant. Finally, our results reveal that the presence of flagella is advantageous for the bacteria to bind to lipid rafts. We speculate that polyunsaturated fatty acids prevent flagellar adhesion on membrane bilayers and play a clear role for optimal host colonization. Flagellum-mediated adhesion to plasma membranes has broad implications for host-pathogen interactions. IMPORTANCE Bacterial adhesion is a crucial step to allow bacteria to colonize their hosts, invade tissues, and form biofilm. Enterohemorrhagic Escherichia coli O157:H7 is a human pathogen and the causative agent of diarrhea and hemorrhagic colitis. Here, we use biomimetic membrane models and cell lines to decipher the impact of lipid content of the plasma membrane on enterohemorrhagic E. coli flagellum-mediated adhesion. Our findings provide evidence that polyunsaturated fatty acid (α-linolenic acid) inhibits E. coli flagellar adhesion to the plasma membrane in a mechanism separate from its antimicrobial and anti-inflammatory functions. In addition, we confirm that cholesterol-enriched lipid microdomains, often called lipid rafts, are important in bacterial adhesion. These findings demonstrate that plasma membrane adhesion via bacterial flagella play a significant role for an important human pathogen. This mechanism represents a promising target for the development of novel antiadhesion therapies.

scholarly journals Mutational Analysis of Residues in PriA and PriC Affecting Their Ability To Interact with SSB in Escherichia coli K-12

2020 ◽  
Vol 202 (23) ◽  
Author(s):  
Anastasiia N. Klimova ◽  
Steven J. Sandler
Keyword(s):  
Escherichia Coli ◽  
Mutational Analysis ◽  
Wild Type ◽  
Content Type ◽  
Growth Defects ◽  
C Terminus ◽  
K 12 ◽  
And Function

ABSTRACT Escherichia coli PriA and PriC recognize abandoned replication forks and direct reloading of the DnaB replicative helicase onto the lagging-strand template coated with single-stranded DNA-binding protein (SSB). Both PriA and PriC have been shown by biochemical and structural studies to physically interact with the C terminus of SSB. In vitro, these interactions trigger remodeling of the SSB on ssDNA. priA341(R697A) and priC351(R155A) negated the SSB remodeling reaction in vitro. Plasmid-carried priC351(R155A) did not complement priC303::kan, and priA341(R697A) has not yet been tested for complementation. Here, we further studied the SSB-binding pockets of PriA and PriC by placing priA341(R697A), priA344(R697E), priA345(Q701E), and priC351(R155A) on the chromosome and characterizing the mutant strains. All three priA mutants behaved like the wild type. In a ΔpriB strain, the mutations caused modest increases in SOS expression, cell size, and defects in nucleoid partitioning (Par−). Overproduction of SSB partially suppressed these phenotypes for priA341(R697A) and priA344(R697E). The priC351(R155A) mutant behaved as expected: there was no phenotype in a single mutant, and there were severe growth defects when this mutation was combined with ΔpriB. Analysis of the priBC mutant revealed two populations of cells: those with wild-type phenotypes and those that were extremely filamentous and Par− and had high SOS expression. We conclude that in vivo, priC351(R155A) identified an essential residue and function for PriC, that PriA R697 and Q701 are important only in the absence of PriB, and that this region of the protein may have a complicated relationship with SSB. IMPORTANCE Escherichia coli PriA and PriC recruit the replication machinery to a collapsed replication fork after it is repaired and needs to be restarted. In vitro studies suggest that the C terminus of SSB interacts with certain residues in PriA and PriC to recruit those proteins to the repaired fork, where they help remodel it for restart. Here, we placed those mutations on the chromosome and tested the effect of mutating these residues in vivo. The priC mutation completely abolished function. The priA mutations had no effect by themselves. They did, however, display modest phenotypes in a priB-null strain. These phenotypes were partially suppressed by SSB overproduction. These studies give us further insight into the reactions needed for replication restart.

scholarly journals Validation of Putative Apicoplast-Targeting Drugs Using a Chemical Supplementation Assay in Cultured Human Malaria Parasites

2017 ◽  
Vol 62 (1) ◽  
Author(s):  
Taher Uddin ◽  
Geoffrey Ian McFadden ◽  
Christopher Dean Goodman
Keyword(s):  
Fatty Acid ◽  
Posttranslational Modification ◽  
Drug Target ◽  
Acid Synthesis ◽  
Blood Stage ◽  
Parasite Life Cycle ◽  
Malaria Parasites ◽  
Acid Pathway ◽  
Synthase Inhibitor ◽  
Fatty Acid Pathway

ABSTRACTMalaria parasites contain a relict plastid, the apicoplast, which is considered an excellent drug target due to its bacterial-like ancestry. Numerous parasiticidals have been proposed to target the apicoplast, but few have had their actual targets substantiated. Isopentenyl pyrophosphate (IPP) production is the sole required function of the apicoplast in the blood stage of the parasite life cycle, and IPP supplementation rescues parasites from apicoplast-perturbing drugs. Hence, any drug that kills parasites when IPP is supplied in culture must have a nonapicoplast target. Here, we use IPP supplementation to discriminate whether 23 purported apicoplast-targeting drugs are on- or off-target. We demonstrate that a prokaryotic DNA replication inhibitor (ciprofloxacin), several prokaryotic translation inhibitors (chloramphenicol, doxycycline, tetracycline, clindamycin, azithromycin, erythromycin, and clarithromycin), a tRNA synthase inhibitor (mupirocin), and two IPP synthesis pathway inhibitors (fosmidomycin and FR900098) have apicoplast targets. Intriguingly, fosmidomycin and FR900098 leave the apicoplast intact, whereas the others eventually result in apicoplast loss. Actinonin, an inhibitor of bacterial posttranslational modification, does not produce a typical delayed-death response but is rescued with IPP, thereby confirming its apicoplast target. Parasites treated with putative apicoplast fatty acid pathway inhibitors could not be rescued, demonstrating that these drugs have their primary targets outside the apicoplast, which agrees with the dispensability of the apicoplast fatty acid synthesis pathways in the blood stage of malaria parasites. IPP supplementation provides a simple test of whether a compound has a target in the apicoplast and can be used to screen novel compounds for mode of action.

scholarly journals Application of AlkBGT and AlkL from Pseudomonas putida GPo1 for Selective Alkyl Ester ω-Oxyfunctionalization in Escherichia coli

2016 ◽  
Vol 82 (13) ◽  
pp. 3801-3807 ◽  
Author(s):  
Youri M. van Nuland ◽  
Gerrit Eggink ◽  
Ruud A. Weusthuis
Keyword(s):  
Escherichia Coli ◽  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Esters ◽  
Reaction Conditions ◽  
Oxidation Activity ◽  
Content Type ◽  
Whole Cells ◽  
Butyl Esters ◽  
Acid Esters

ABSTRACTThe enzyme system AlkBGT fromPseudomonas putidaGPo1 can efficiently ω-functionalize fatty acid methyl esters. Outer membrane protein AlkL boosts this ω-functionalization. In this report, it is shown that whole cells ofEscherichia coliexpressing the AlkBGT system can also ω-oxidize ethyl nonanoate (NAEE). Coexpression of AlkBGT and AlkL resulted in 1.7-fold-higher ω-oxidation activity on NAEE. With this strain, initial activity on NAEE was 70 U/g (dry weight) of cells (gcdw), 67% of the initial activity on methyl nonanoate. In time-lapse conversions with 5 mM NAEE the main product was 9-hydroxy NAEE (3.6 mM), but also 9-oxo NAEE (0.1 mM) and 9-carboxy NAEE (0.6 mM) were formed. AlkBGT also ω-oxidized ethyl, propyl, and butyl esters of fatty acids ranging from C6to C10. Increasing the length of the alkyl chain improved the ω-oxidation activity of AlkBGT on esters of C6and C7fatty acids. From these esters, application of butyl hexanoate resulted in the highest ω-oxidation activity, 82 U/gcdw. Coexpression of AlkL only had a positive effect on ω-functionalization of substrates with a total length of C11or longer. These findings indicate that AlkBGT(L) can be applied as a biocatalyst for ω-functionalization of ethyl, propyl, and butyl esters of medium-chain fatty acids.IMPORTANCEFatty acid esters are promising renewable starting materials for the production of ω-hydroxy fatty acid esters (ω-HFAEs). ω-HFAEs can be used to produce sustainable polymers. Chemical conversion of the fatty acid esters to ω-HFAEs is challenging, as it generates by-products and needs harsh reaction conditions. Biocatalytic production is a promising alternative. In this study, biocatalytic conversion of fatty acid esters toward ω-HFAEs was investigated using whole cells. This was achieved with recombinantEscherichia colicells that produce the AlkBGT enzymes. These enzymes can produce ω-HFAEs from a wide variety of fatty acid esters. Medium-chain-length acids (C6to C10) esterified with ethanol, propanol, or butanol were applied. This is a promising production platform for polymer building blocks that uses renewable substrates and mild reaction conditions.

scholarly journals BfpI, BfpJ, and BfpK Minor Pilins Are Important for the Function and Biogenesis of Bundle-Forming Pili Expressed by Enteropathogenic Escherichia coli

2015 ◽  
Vol 198 (5) ◽  
pp. 846-856 ◽  
Author(s):  
Claudia F. Martinez de la Peña ◽  
Leon De Masi ◽  
Shahista Nisa ◽  
George Mulvey ◽  
Jesse Tong ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Structural Integrity ◽  
Vaccine Development ◽  
Intestinal Epithelial ◽  
Adhesive Properties ◽  
Content Type ◽  
Structural And Functional Properties ◽  
Structural Subunit ◽  
Typical Epec ◽  
And Function

ABSTRACTEnteropathogenicEscherichia coli(EPEC) remains a significant cause of infant diarrheal illness and associated morbidity and mortality in developing countries. EPEC strains are characterized by their ability to colonize the small intestines of their hosts by a multistep program involving initial loose attachment to intestinal epithelial cells followed by an intimate adhesion phase. The initial loose interaction of typical EPEC with host intestinal cells is mediated by bundle-forming pili (BFP). BFP are type 4b pili (T4bP) based on structural and functional properties shared with T4bP expressed by other bacteria. The major structural subunit of BFP is called bundlin, a T4b pilin expressed from thebfpAgene in the BFP operon, which contains three additional genes that encode the pilin-like proteins BfpI, BfpJ, and BfpK. In this study, we show that, in the absence of the BFP retraction ATPase (BfpF), BfpI, BfpJ, and BfpK are dispensable for BFP biogenesis. We also demonstrate that these three minor pilins are incorporated along with bundlin into the BFP filament and contribute to its structural integrity and host cell adhesive properties. The results confirm that previous findings in T4aP systems can be extended to a model T4bP such as BFP.IMPORTANCEBundle-forming pili contribute to the host colonization strategy of enteropathogenicEscherichia coli. The studies described here investigate the role for three minor pilin subunits in the structure and function of BFP in EPEC. The studies also suggest that these subunits could be antigens for vaccine development.

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