scholarly journals Escherichia coli YqjA, a Member of the Conserved DedA/Tvp38 Membrane Protein Family, Is a Putative Osmosensing Transporter Required for Growth at Alkaline pH

2015 ◽  
Vol 197 (14) ◽  
pp. 2292-2300 ◽  
Author(s):  
Sujeet Kumar ◽  
William T. Doerrler
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Amino Acid Identity ◽  
Protein Family ◽  
Cytoplasmic Ph ◽  
Alkaline Ph ◽  
Ph Homeostasis ◽  
Content Type ◽  
E Coli ◽  
Alkaline Conditions

ABSTRACTThe ability to persist and grow under alkaline conditions is an important characteristic of many bacteria. In order to survive at alkaline pH,Escherichia colimust maintain a stable cytoplasmic pH of about 7.6. Membrane cation/proton antiporters play a major role in alkaline pH homeostasis by catalyzing active inward proton transport. The DedA/Tvp38 family is a highly conserved membrane protein family of unknown function present in most sequenced genomes. YqjA and YghB are members of theE. coliDedA family with 62% amino acid identity and partially redundant functions. We have shown thatE. coliwith ΔyqjAand ΔyghBmutations cannot properly maintain the proton motive force (PMF) and is compromised in PMF-dependent drug efflux and other PMF-dependent functions. Furthermore, the functions of YqjA and YghB are dependent upon membrane-embedded acidic amino acids, a hallmark of several families of proton-dependent transporters. Here, we show that the ΔyqjAmutant (but not ΔyghB) cannot grow under alkaline conditions (ranging from pH 8.5 to 9.5), unlike the parentE. coli. Overexpression ofyqjArestores growth at alkaline pH, but only when more than ∼100 mM sodium or potassium is present in the growth medium. Increasing the osmotic pressure by the addition of sucrose enhances the ability of YqjA to support growth under alkaline conditions in the presence of low salt concentrations, consistent with YqjA functioning as an osmosensor. We suggest that YqjA possesses proton-dependent transport activity that is stimulated by osmolarity and that it plays a significant role in the survival ofE. coliat alkaline pH.IMPORTANCEThe ability to survive under alkaline conditions is important for many species of bacteria.Escherichia colican grow at pH 5.5 to 9.5 while maintaining a constant cytoplasmic pH of about 7.6. Under alkaline conditions, bacteria rely upon proton-dependent transporters to maintain a constant cytoplasmic pH. The DedA/Tvp38 protein family is a highly conserved but poorly characterized family of membrane proteins. Here, we show that the DedA/Tvp38 protein YqjA is critical forE. colito survive at pH 8.5 to 9.5. YqjA requires sodium and potassium for this function. At low cation concentrations, osmolytes, including sucrose, can facilitate rescue ofE. coligrowth by YqjA at high pH. These data are consistent with YqjA functioning as an osmosensing cation-dependent proton transporter.

scholarly journals Tail-Anchored Inner Membrane Protein ElaB Increases Resistance to Stress While Reducing Persistence in Escherichia coli

2017 ◽  
Vol 199 (9) ◽  
Author(s):  
Yunxue Guo ◽  
Xiaoxiao Liu ◽  
Baiyuan Li ◽  
Jianyun Yao ◽  
Thomas K. Wood ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Escherichia Coli ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Stationary Phase ◽  
Transmembrane Domain ◽  
Inner Membrane ◽  
Content Type ◽  
E Coli ◽  
Inner Membrane Protein

ABSTRACT Host-associated bacteria, such as Escherichia coli, often encounter various host-related stresses, such as nutritional deprivation, oxidative stress, and temperature shifts. There is growing interest in searching for small endogenous proteins that mediate stress responses. Here, we characterized the small C-tail-anchored inner membrane protein ElaB in E. coli. ElaB belongs to a class of tail-anchored inner membrane proteins with a C-terminal transmembrane domain but lacking an N-terminal signal sequence for membrane targeting. Proteins from this family have been shown to play vital roles, such as in membrane trafficking and apoptosis, in eukaryotes; however, their role in prokaryotes is largely unexplored. Here, we found that the transcription of elaB is induced in the stationary phase in E. coli and stationary-phase sigma factor RpoS regulates elaB transcription by binding to the promoter of elaB. Moreover, ElaB protects cells against oxidative stress and heat shock stress. However, unlike membrane peptide toxins TisB and GhoT, ElaB does not lead to cell death, and the deletion of elaB greatly increases persister cell formation. Therefore, we demonstrate that disruption of C-tail-anchored inner membrane proteins can reduce stress resistance; it can also lead to deleterious effects, such as increased persistence, in E. coli. IMPORTANCE Escherichia coli synthesizes dozens of poorly understood small membrane proteins containing a predicted transmembrane domain. In this study, we characterized the function of the C-tail-anchored inner membrane protein ElaB in E. coli. ElaB increases resistance to oxidative stress and heat stress, while inactivation of ElaB leads to high persister cell formation. We also demonstrated that the transcription of elaB is under the direct regulation of stationary-phase sigma factor RpoS. Thus, our study reveals that small inner membrane proteins may have important cellular roles during the stress response.

scholarly journals NlpI Facilitates Deposition of C4bp on Escherichia coli by Blocking Classical Complement-Mediated Killing, Which Results in High-Level Bacteremia

2012 ◽  
Vol 80 (10) ◽  
pp. 3669-3678 ◽  
Author(s):  
Yu-ting Tseng ◽  
Shainn-Wei Wang ◽  
Kwang Sik Kim ◽  
Ying-Hsiang Wang ◽  
Yufeng Yao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Neonatal Meningitis ◽  
Content Type ◽  
E Coli ◽  
Classical Complement Pathway ◽  
High Level ◽  
Classical Complement

ABSTRACTNeonatal meningitisEscherichia coli(NMEC) is the most common Gram-negative organism that is associated with neonatal meningitis, which usually develops as a result of hematogenous spread of the bacteria. There are two key pathogenesis processes for NMEC to penetrate into the brain, the essential step for the development ofE. colimeningitis: a high-level bacteremia and traversal of the blood-brain barrier (BBB). Our previous study has shown that the bacterial outer membrane protein NlpI contributes to NMEC binding to and invasion of brain microvascular endothelial cells, the major component cells of the BBB, suggesting a role for NlpI in NMEC crossing of the BBB. In this study, we showed that NlpI is involved in inducing a high level of bacteremia. In addition, NlpI contributed to the recruitment of the complement regulator C4bp to the surface of NMEC to evade serum killing, which is mediated by the classical complement pathway. NlpI may be involved in the interaction between C4bp and OmpA, which is an outer membrane protein that directly interacts with C4bp on the bacterial surface. The involvement of NlpI in two key pathogenesis processes of NMEC meningitis may make this bacterial factor a potential target for prevention and therapy ofE. colimeningitis.

scholarly journals Growth Inhibition by External Potassium of Escherichia coli Lacking PtsN (EIIANtr) Is Caused by Potassium Limitation Mediated by YcgO

2016 ◽  
Vol 198 (13) ◽  
pp. 1868-1882 ◽  
Author(s):  
Ravish Sharma ◽  
Tomohiro Shimada ◽  
Vinod K. Mishra ◽  
Suchitra Upreti ◽  
Abhijit A. Sardesai
Keyword(s):  
Escherichia Coli ◽  
Membrane Protein ◽  
Phosphorylation Site ◽  
Phosphotransferase System ◽  
Inner Membrane ◽  
Content Type ◽  
E Coli ◽  
External Potassium ◽  
Inner Membrane Protein ◽  
External K

ABSTRACTThe absence of PtsN, the terminal phosphoacceptor of the phosphotransferase system comprising PtsP-PtsO-PtsN, inEscherichia coliconfers a potassium-sensitive (Ks) phenotype as the external K+concentration ([K+]e) is increased above 5 mM. A growth-inhibitory increase in intracellular K+content, resulting from hyperactivated Trk-mediated K+uptake, is thought to cause this Ks. We provide evidence that the Ksof the ΔptsNmutant is associated with K+limitation. Accordingly, the moderate Ksdisplayed by the ΔptsNmutant was exacerbated in the absence of the Trk and Kup K+uptake transporters and was associated with reduced cellular K+content. Conversely, overproduction of multiple K+uptake proteins suppressed the Ks. Expression of PtsN variants bearing the H73A, H73D, and H73E substitutions of the phosphorylation site histidine of PtsN complemented the Ks. Absence of the predicted inner membrane protein YcgO (also called CvrA) suppressed the Ks, which was correlated with elevated cellular K+content in the ΔptsNmutant, but the ΔptsNmutation did not alter YcgO levels. Heterologous overexpression ofycgOalso led to Ksthat was associated with reduced cellular K+content, exacerbated by the absence of Trk and Kup and alleviated by overproduction of Kup. Our findings are compatible with a model that postulates that Ksin the ΔptsNmutant occurs due to K+limitation resulting from activation of K+efflux mediated by YcgO, which may be additionally stimulated by [K+]e, implicating a role for PtsN (possibly its dephosphorylated form) as an inhibitor of YcgO activity.IMPORTANCEThis study examines the physiological link between the phosphotransferase system comprising PtsP-PtsO-PtsN and K+ion metabolism inE. coli. Studies on the physiological defect that renders anE. colimutant lacking PtsN to be growth inhibited by external K+indicate that growth impairment results from cellular K+limitation that is mediated by YcgO, a predicted inner membrane protein. Additional observations suggest that dephospho-PtsN may inhibit and external K+may stimulate K+limitation mediated by YcgO. It is speculated that YcgO-mediated K+limitation may be an output of a response to certain stresses, which by modulating the phosphotransfer capacity of the PtsP-PtsO-PtsN phosphorelay leads to growth cessation and stress tolerance.

scholarly journals Identification of FosA8, a Plasmid-Encoded Fosfomycin Resistance Determinant from Escherichia coli, and Its Origin in Leclercia adecarboxylata

2019 ◽  
Vol 63 (11) ◽  
Author(s):  
Laurent Poirel ◽  
Xavier Vuillemin ◽  
Nicolas Kieffer ◽  
Linda Mueller ◽  
Marie-Christine Descombes ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid Identity ◽  
Natural Resistance ◽  
Whole Genome ◽  
Content Type ◽  
Acid Identity ◽  
E Coli ◽  
Fosfomycin Resistance ◽  
High Level ◽  
Level Resistance

ABSTRACT A plasmid-located fosfomycin resistance gene, fosA8, was identified from a CTX-M-15-producing Escherichia coli isolate recovered from urine. Identification of this gene was obtained by whole-genome sequencing. It encoded FosA8, which shares 79% and 78% amino acid identity with the most closely related FosA2 and FosA1 enzymes, respectively. The fosA8 gene was located on a transferable 50-kb plasmid of IncN type encoding high-level resistance to fosfomycin. In silico analysis and cloning experiments identified fosA8 analogues (99% identity) in the genome of Leclercia decarboxylata, which is an enterobacterial species with natural resistance to fosfomycin. This finding adds L. decarboxylata to the list of enterobacterial species that are a reservoir of fosA-like genes which have been captured from the chromosome of a progenitor and are then acquired by E. coli.

scholarly journals Escherichia coli SRP, Its Protein Subunit Ffh, and the Ffh M Domain Are Able To Selectively Limit Membrane Protein Expression When Overexpressed

mBio ◽  
2010 ◽  
Vol 1 (2) ◽  
Author(s):  
Ido Yosef ◽  
Elena S. Bochkareva ◽  
Eitan Bibi
Keyword(s):  
Escherichia Coli ◽  
Quality Control ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Protein Translation ◽  
Mrna Levels ◽  
Content Type ◽  
E Coli ◽  
Quality Control Process

ABSTRACT The Escherichia coli signal recognition particle (SRP) system plays an important role in membrane protein biogenesis. Previous studies have suggested indirectly that in addition to its role during the targeting of ribosomes translating membrane proteins to translocons, the SRP might also have a quality control role in preventing premature synthesis of membrane proteins in the cytoplasm. This proposal was studied here using cells simultaneously overexpressing various membrane proteins and either SRP, the SRP protein Ffh, its 4.5S RNA, or the Ffh M domain. The results show that SRP, Ffh, and the M domain are all able to selectively inhibit the expression of membrane proteins. We observed no apparent changes in the steady-state mRNA levels or membrane protein stability, suggesting that inhibition may occur at the level of translation, possibly through the interaction between Ffh and ribosome-hydrophobic nascent chain complexes. Since E. coli SRP does not have a eukaryote-like translation arrest domain, we discuss other possible mechanisms by which this SRP might regulate membrane protein translation when overexpressed. IMPORTANCE The eukaryotic SRP slows down translation of SRP substrates by cytoplasmic ribosomes. This activity is important for preventing premature synthesis of secretory and membrane proteins in the cytoplasm. It is likely that an analogous quality control step would be required in all living cells. However, on the basis of its composition and domain structure and limited in vitro studies, it is believed that the E. coli SRP is unable to regulate ribosomes translating membrane proteins. Nevertheless, several in vivo studies have suggested otherwise. To address this issue further in vivo, we utilized unbalanced conditions under which E. coli simultaneously overexpresses SRP and each of several membrane or cytosolic proteins. Surprisingly, our results clearly show that the E. coli SRP is capable of regulating membrane protein synthesis and demonstrate that the M domain of Ffh mediates this activity. These results thus open the way for mechanistic characterization of this quality control process in bacteria.

scholarly journals mcr-9, an Inducible Gene Encoding an Acquired Phosphoethanolamine Transferase in Escherichia coli, and Its Origin

2019 ◽  
Vol 63 (9) ◽  
Author(s):  
Nicolas Kieffer ◽  
Guilhem Royer ◽  
Jean-Winoc Decousser ◽  
Anne-Sophie Bourrel ◽  
Mattia Palmieri ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Lipid A ◽  
Acquired Resistance ◽  
Regulatory System ◽  
Amino Acid Identity ◽  
Gene Encoding ◽  
Content Type ◽  
E Coli ◽  
K 12 ◽  
A Minor

ABSTRACT The plasmid-located mcr-9 gene, encoding a putative phosphoethanolamine transferase, was identified in a colistin-resistant human fecal Escherichia coli strain belonging to a very rare phylogroup, the D-ST69-O15:H6 clone. This MCR-9 protein shares 33% to 65% identity with the other plasmid-encoded MCR-type enzymes identified (MCR-1 to -8) that have been found as sources of acquired resistance to polymyxins in Enterobacteriaceae. Analysis of the lipopolysaccharide of the MCR-9-producing isolate revealed a function similar to that of MCR-1 by adding a phosphoethanolamine group to lipid A and subsequently modifying the structure of the lipopolysaccharide. However, a minor impact on susceptibility to polymyxins was noticed once the mcr-9 gene was cloned and produced in an E. coli K-12-derived strain. Nevertheless, we showed here that subinhibitory concentrations of colistin induced the expression of the mcr-9 gene, leading to increased MIC levels. This inducible expression was mediated by a two-component regulatory system encoded by the qseC and qseB genes located downstream of mcr-9. Genetic analysis showed that the mcr-9 gene was carried by an IncHI2 plasmid. In silico analysis revealed that the plasmid-encoded MCR-9 shared significant amino acid identity (ca. 80%) with the chromosomally encoded MCR-like proteins from Buttiauxella spp. In particular, Buttiauxella gaviniae was found to harbor a gene encoding MCR-BG, sharing 84% identity with MCR-9. That gene was neither expressed nor inducible in its original host, which was fully susceptible to polymyxins. This work showed that mcr genes may circulate silently and remain undetected unless induced by colistin.

scholarly journals The Lysine 299 Residue Endows the Multisubunit Mrp1 Antiporter with Dominant Roles in Na+Resistance and pH Homeostasis inCorynebacterium glutamicum

2018 ◽  
Vol 84 (10) ◽  
Author(s):  
Ning Xu ◽  
Yingying Zheng ◽  
Xiaochen Wang ◽  
Terry A. Krulwich ◽  
Yanhe Ma ◽  
...  
Keyword(s):  
Corynebacterium Glutamicum ◽  
Expression Patterns ◽  
Alkaline Stress ◽  
Alkaline Ph ◽  
Ph Homeostasis ◽  
Conserved Residues ◽  
Content Type ◽  
Ion Translocation ◽  
Alkaline Tolerance ◽  
Alkaline Conditions

ABSTRACTCorynebacterium glutamicumis generally regarded as a moderately salt- and alkali-tolerant industrial organism. However, relatively little is known about the molecular mechanisms underlying these specific adaptations. Here, we found that the Mrp1 antiporter played crucial roles in conferring both environmental Na+resistance and alkali tolerance whereas the Mrp2 antiporter was necessary in coping with high-KCl stress at alkaline pH. Furthermore, the Δmrp1Δmrp2double mutant showed the most-severe growth retardation and failed to grow under high-salt or alkaline conditions. Consistent with growth properties, the Na+/H+antiporters ofC. glutamicumwere differentially expressed in response to specific salt or alkaline stress, and an alkaline stimulus particularly induced transcript levels of the Mrp-type antiporters. When the major Mrp1 antiporter was overwhelmed,C. glutamicummight employ alternative coordinate strategies to regulate antiport activities. Site-directed mutagenesis demonstrated that several conserved residues were required for optimal Na+resistance, such as Mrp1A K299, Mrp1C I76, Mrp1A H230, and Mrp1D E136. Moreover, the chromosomal replacement of lysine 299 in the Mrp1A subunit resulted in a higher intracellular Na+level and a more alkaline intracellular pH value, thereby causing a remarkable growth attenuation. Homology modeling of the Mrp1 subcomplex suggested two possible ion translocation pathways, and lysine 299 might exert its effect by affecting the stability and flexibility of the cytoplasm-facing channel in the Mrp1A subunit. Overall, these findings will provide new clues to the understanding of salt-alkali adaptation duringC. glutamicumstress acclimatization.IMPORTANCEThe capacity to adapt to harsh environments is crucial for bacterial survival and product yields, including industrially usefulCorynebacterium glutamicum. AlthoughC. glutamicumexhibits a marked resistance to salt-alkaline stress, the possible mechanism for these adaptations is still unclear. Here, we present the physiological functions and expression patterns ofC. glutamicumputative Na+/H+antiporters and conserved residues of Mrp1 subunits, which respond to different salt and alkaline stresses. We found that the Mrp-type antiporters, particularly the Mrp1 antiporter, played a predominant role in maintaining intracellular nontoxic Na+levels and alkaline pH homeostasis. Loss of the major Mrp1 antiporter had a profound effect on gene expression of other antiporters under salt or alkaline conditions. The lysine 299 residue may play its essential roles in conferring salt and alkaline tolerance by affecting the ion translocation channel of the Mrp1A subunit. These findings will contribute to a better understanding of Na+/H+antiporters in sodium antiport and pH regulation.

scholarly journals Outer Membrane Protein AlkL Boosts Biocatalytic Oxyfunctionalization of Hydrophobic Substrates in Escherichia coli

2012 ◽  
Vol 78 (16) ◽  
pp. 5724-5733 ◽  
Author(s):  
Mattijs K. Julsing ◽  
Manfred Schrewe ◽  
Sjef Cornelissen ◽  
Inna Hermann ◽  
Andreas Schmid ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Methyl Ester ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Acid Methyl Ester ◽  
Dodecanoic Acid ◽  
Content Type ◽  
E Coli ◽  
Acid Methyl

ABSTRACTThe outer membrane of microbial cells forms an effective barrier for hydrophobic compounds, potentially causing an uptake limitation for hydrophobic substrates. Low bioconversion activities (1.9 U gcdw−1) have been observed for the ω-oxyfunctionalization of dodecanoic acid methyl ester by recombinantEscherichia colicontaining the alkane monooxygenase AlkBGT ofPseudomonas putidaGPo1. Using fatty acid methyl ester oxygenation as the model reaction, this study investigated strategies to improve bacterial uptake of hydrophobic substrates. Admixture of surfactants and cosolvents to improve substrate solubilization did not result in increased oxygenation rates. Addition of EDTA increased the initial dodecanoic acid methyl ester oxygenation activity 2.8-fold. The use of recombinantPseudomonas fluorescensCHA0 instead ofE. coliresulted in a similar activity increase. However, substrate mass transfer into cells was still found to be limiting. Remarkably, the coexpression of thealkLgene ofP. putidaGPo1 encoding an outer membrane protein with so-far-unknown function increased the dodecanoic acid methyl ester oxygenation activity of recombinantE. coli28-fold. In a two-liquid-phase bioreactor setup, a 62-fold increase to a maximal activity of 87 U gcdw−1was achieved, enabling the accumulation of high titers of terminally oxyfunctionalized products. Coexpression ofalkLalso increased oxygenation activities toward the natural AlkBGT substrates octane and nonane, showing for the first time clear evidence for a prominent role of AlkL in alkane degradation. This study demonstrates that AlkL is an efficient tool to boost productivities of whole-cell biotransformations involving hydrophobic aliphatic substrates and thus has potential for broad applicability.

scholarly journals Cytoplasmic pH Response to Acid Stress in Individual Cells of Escherichia coli and Bacillus subtilis Observed by Fluorescence Ratio Imaging Microscopy

2012 ◽  
Vol 78 (10) ◽  
pp. 3706-3714 ◽  
Author(s):  
Keith A. Martinez ◽  
Ryan D. Kitko ◽  
J. Patrick Mershon ◽  
Haley E. Adcox ◽  
Kotiba A. Malek ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Acid Stress ◽  
Adherent Cells ◽  
Cytoplasmic Ph ◽  
Fluorescence Ratio ◽  
Isolated Cells ◽  
Content Type ◽  
E Coli ◽  
Standard Curve

ABSTRACTThe ability ofEscherichia coliandBacillus subtilisto regulate their cytoplasmic pH is well studied in cell suspensions but is poorly understood in individual adherent cells and biofilms. We observed the cytoplasmic pH of individual cells using ratiometric pHluorin. A standard curve equating the fluorescence ratio with pH was obtained by perfusion at a range of external pH 5.0 to 9.0, with uncouplers that collapse the transmembrane pH difference. Adherent cells were acid stressed by switching the perfusion medium from pH 7.5 to pH 5.5. TheE. colicytoplasmic pH fell to a value that varied among individual cells (range of pH 6.2 to 6.8), but a majority of cells recovered (to pH 7.0 to 7.5) within 2 min. In anE. colibiofilm, cells shifted from pH 7.5 to pH 5.5 failed to recover cytoplasmic pH. Following a smaller shift (from pH 7.5 to pH 6.0), most biofilm cells recovered fully, although the pH decreased further than that of isolated adherent cells, and recovery took longer (7 min or longer). Some biofilm cells began to recover pH and then failed, a response not seen in isolated cells.B. subtiliscells were acid shifted from pH 7.5 to pH 6.0. InB. subtilis, unlike the case withE. coli, cytoplasmic pH showed no “overshoot” but fell to a level that was maintained. This level of cytoplasmic pH post-acid shift varied among individualB. subtiliscells (range of pH, 7.0 to 7.7). Overall, the cytoplasmic pHs of individual bacteria show important variation in the acid stress response, including novel responses in biofilms.

scholarly journals Isolation of a Gene Responsible for the Oxidation oftrans-Anethole topara-Anisaldehyde by Pseudomonas putida JYR-1 and Its Expression in Escherichia coli

2012 ◽  
Vol 78 (15) ◽  
pp. 5238-5246 ◽  
Author(s):  
Dongfei Han ◽  
Ji-Young Ryu ◽  
Robert A. Kanaly ◽  
Hor-Gil Hur
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Putida ◽  
Hypothetical Protein ◽  
Aldehyde Group ◽  
Agrobacterium Vitis ◽  
T7 Promoter ◽  
Content Type ◽  
E Coli ◽  
Cell Extracts ◽  
Isoeugenol Monooxygenase

ABSTRACTA plasmid, pTA163, inEscherichia colicontained an approximately 34-kb gene fragment fromPseudomonas putidaJYR-1 that included the genes responsible for the metabolism oftrans-anethole to protocatechuic acid. Three Tn5-disrupted open reading frame 10 (ORF 10) mutants of plasmid pTA163 lost their abilities to catalyzetrans-anethole. Heterologously expressed ORF 10 (1,047 nucleotides [nt]) under a T7 promoter inE. colicatalyzed oxidative cleavage of a propenyl group oftrans-anethole to an aldehyde group, resulting in the production ofpara-anisaldehyde, and this gene was designatedtao(trans-anetholeoxygenase). The deduced amino acid sequence of TAO had the highest identity (34%) to a hypothetical protein ofAgrobacterium vitisS4 and likely contained a flavin-binding site. Preferred incorporation of an oxygen molecule from water intop-anisaldehyde using18O-labeling experiments indicated stereo preference of TAO for hydrolysis of the epoxide group. Interestingly, unlike the narrow substrate range of isoeugenol monooxygenase fromPseudomonas putidaIE27 andPseudomonas nitroreducensJin1, TAO fromP. putidaJYR-1 catalyzed isoeugenol,O-methyl isoeugenol, and isosafrole, all of which contain the 2-propenyl functional group on the aromatic ring structure. Addition of NAD(P)H to the ultrafiltered cell extracts ofE. coli(pTA163) increased the activity of TAO. Due to the relaxed substrate range of TAO, it may be utilized for the production of various fragrance compounds from plant phenylpropanoids in the future.

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