Tinkering with Osmotically Controlled Transcription Allows Enhanced Production and Excretion of Ectoine and Hydroxyectoine from a Microbial Cell Factory

ABSTRACTEctoine and hydroxyectoine are widely synthesized by members of theBacteriaand a few members of theArchaeaas potent osmostress protectants. We have studied the salient features of the osmostress-responsive promoter directing the transcription of the ectoine/hydroxyectoine biosynthetic gene cluster from the plant-root-associated bacteriumPseudomonas stutzeriby transferring it intoEscherichia coli, an enterobacterium that does not produce ectoines naturally. Usingect-lacZreporter fusions, we found that the heterologousectpromoter reacted with exquisite sensitivity in its transcriptional profile to graded increases in sustained high salinity, responded to a true osmotic signal, and required the buildup of an osmotically effective gradient across the cytoplasmic membrane for its induction. The involvement of the −10, −35, and spacer regions of the sigma-70-typeectpromoter in setting promoter strength and response to osmotic stress was assessed through site-directed mutagenesis. Moderate changes in theectpromoter sequence that increase its resemblance to housekeeping sigma-70-type promoters ofE. coliafforded substantially enhanced expression, both in the absence and in the presence of osmotic stress. Building on this set ofectpromoter mutants, we engineered anE. colichassis strain for the heterologous production of ectoines. This synthetic cell factory lacks the genes for the osmostress-responsive synthesis of trehalose and the compatible solute importers ProP and ProU, and it continuously excretes ectoines into the growth medium. By combining appropriate host strains and different plasmid variants, excretion of ectoine, hydroxyectoine, or a mixture of both compounds was achieved under mild osmotic stress conditions.IMPORTANCEEctoines are compatible solutes, organic osmolytes that are used by microorganisms to fend off the negative consequences of high environmental osmolarity on cellular physiology. An understanding of the salient features of osmostress-responsive promoters directing the expression of the ectoine/hydroxyectoine biosynthetic gene clusters is lacking. We exploited theectpromoter from an ectoine/hydroxyectoine-producing soil bacterium for such a study by transferring it into a surrogate bacterial host. Despite the fact thatE. colidoes not synthesize ectoines naturally, theectpromoter retained its exquisitely sensitive osmotic control, indicating that osmoregulation ofecttranscription is an inherent feature of the promoter and its flanking sequences. These sequences were narrowed to a 116-bp DNA fragment. Ectoines have interesting commercial applications. Building on data from a site-directed mutagenesis study of theectpromoter, we designed a synthetic cell factory that secretes ectoine, hydroxyectoine, or a mixture of both compounds into the growth medium.

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Hydrolysis Spectrum Extension of CMY-2-Like β-Lactamases Resulting from Structural Alteration in the Y-X-N Loop

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05630-11 ◽

2012 ◽

Vol 56 (3) ◽

pp. 1151-1156 ◽

Cited By ~ 4

Author(s):

Sandrine Dahyot ◽

Hedi Mammeri

Keyword(s):

Hot Spot ◽

Catalytic Efficiency ◽

Site Directed Mutagenesis ◽

Hydrolysis Rate ◽

Directed Mutagenesis ◽

Content Type ◽

E Coli ◽

Class A ◽

Extended Spectrum ◽

Cloning Experiment

ABSTRACTTheCitrobacter freundiiisolate CHA, which was responsible for postoperative peritonitis after 10 days of cefepime therapy, displayed a phenotype of resistance consistent with extended-spectrum AmpC (ESAC) β-lactamase. The chromosome-borneblaAmpC-CHAgene was amplified and sequenced, revealing five amino acid substitutions, I125V, R148H, Q196H, V305A, and V348A, in the product compared to the sequence of native AmpC. A cloning experiment yielded theEscherichia coliTOP10(pAmpC-CHA) strain, which was resistant to all extended-spectrum cephalosporins (ESCs), including cefepime. To ascertain whether the R148H substitution accounted for the hydrolysis spectrum extension, it was reverted by site-directed mutagenesis. The resultingE. coliTOP10(pAmpC-CHA-H148R) strain was fully susceptible to cefepime, thus confirming that the Arg-148 replacement was mandatory for substrate profile enlargement. To further characterize the phenotypical and biochemical effects induced by the R148H change, it was introduced by site-directed mutagenesis into the CMY-2 β-lactamase, which is structurally related to the chromosome-borne cephalosporinase ofC. freundii. The CMY-2-R148H variant conferred increased MICs of ESCs, whereas those of carbapenems were unchanged even in a porin-deficientE. colistrain. Moreover, it exhibited increased catalytic efficiency (kcat/Km) toward ceftazidime (100-fold) due to an enhanced hydrolysis rate (kcat), whereas the enzymatic parameters toward imipenem were unchanged. The structural analysis of the AmpC variant showed that the R148H replacement occurred in the loop containing the Y-X-N motif, which is the counterpart of the SDN loop in class A β-lactamases. This study shows that the Y-X-N loop is a novel hot spot for mutations accounting for hydrolysis spectrum extension in CMY-2-type enzymes.

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A genetically detoxified derivative of heat-labile Escherichia coli enterotoxin induces neutralizing antibodies against the A subunit.

Journal of Experimental Medicine ◽

10.1084/jem.180.6.2147 ◽

1994 ◽

Vol 180 (6) ◽

pp. 2147-2153 ◽

Cited By ~ 69

Author(s):

M Pizza ◽

M R Fontana ◽

M M Giuliani ◽

M Domenighini ◽

C Magagnoli ◽

...

Keyword(s):

Escherichia Coli ◽

Cholera Toxin ◽

Neutralizing Antibodies ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

E Coli ◽

Heat Labile ◽

A Subunit ◽

Adp Ribosyltransferase ◽

Derivatives Of

Escherichia coli enterotoxin (LT) and the homologous cholera toxin (CT) are A-B toxins that cause travelers' diarrhea and cholera, respectively. So far, experimental live and killed vaccines against these diseases have been developed using only the nontoxic B portion of these toxins. The enzymatically active A subunit has not been used because it is responsible for the toxicity and it is reported to induce a negligible titer of toxin neutralizing antibodies. We used site-directed mutagenesis to inactivate the ADP-ribosyltransferase activity of the A subunit and obtained nontoxic derivatives of LT that elicited a good titer of neutralizing antibodies recognizing the A subunit. These LT mutants and equivalent mutants of CT may be used to improve live and killed vaccines against cholera and enterotoxinogenic E. coli.

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Site-Directed Mutagenesis of the Heterotrimeric Killer Toxin Zymocin Identifies Residues Required for Early Steps in Toxin Action

Applied and Environmental Microbiology ◽

10.1128/aem.02197-14 ◽

2014 ◽

Vol 80 (20) ◽

pp. 6549-6559 ◽

Cited By ~ 6

Author(s):

Sabrina Wemhoff ◽

Roland Klassen ◽

Friedhelm Meinhardt

Keyword(s):

Kluyveromyces Lactis ◽

Killer Toxin ◽

Cysteine Residue ◽

Site Directed Mutagenesis ◽

Target Cells ◽

Directed Mutagenesis ◽

Content Type ◽

Protein Toxin ◽

Assisted Delivery

ABSTRACTZymocin is aKluyveromyces lactisprotein toxin composed of αβγ subunits encoded by the cytoplasmic virus-like element k1 and functions by αβ-assisted delivery of the anticodon nuclease (ACNase) γ into target cells. The toxin binds to cells' chitin and exhibits chitinase activityin vitrothat might be important during γ import.Saccharomyces cerevisiaestrains carrying k1-derived hybrid elements deficient in either αβ (k1ORF2) or γ (k1ORF4) were generated. Loss of either gene abrogates toxicity, and unexpectedly, Orf2 secretion depends on Orf4 cosecretion. Functional zymocin assembly can be restored by nuclear expression of k1ORF2 or k1ORF4, providing an opportunity to conduct site-directed mutagenesis of holozymocin. Complementation required active site residues of α's chitinase domain and the sole cysteine residue of β (Cys250). Since βγ are reportedly disulfide linked, the requirement for the conserved γ C231 was probed. Toxicity of intracellularly expressed γ C231A indicated no major defect in ACNase activity, while complementation of k1ΔORF4 by γ C231A was lost, consistent with a role of β C250 and γ C231 in zymocin assembly. To test the capability of αβ to carry alternative cargos, the heterologous ACNase fromPichia acaciae(P. acaciaeOrf2 [PaOrf2]) was expressed, along with its immunity gene, in k1ΔORF4. While efficient secretion of PaOrf2 was detected, suppression of the k1ΔORF4-derived k1Orf2 secretion defect was not observed. Thus, the dependency of k1Orf2 on k1Orf4 cosecretion needs to be overcome prior to studying αβ's capability to deliver other cargo proteins into target cells.

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Genome-Wide Screening Identifies Six Genes That Are Associated with Susceptibility to Escherichia coli Microcin PDI

Applied and Environmental Microbiology ◽

10.1128/aem.01704-15 ◽

2015 ◽

Vol 81 (20) ◽

pp. 6953-6963 ◽

Cited By ~ 5

Author(s):

Zhe Zhao ◽

Lauren J. Eberhart ◽

Lisa H. Orfe ◽

Shao-Yeh Lu ◽

Thomas E. Besser ◽

...

Keyword(s):

Escherichia Coli ◽

Atp Synthase ◽

Disulfide Bonds ◽

Gene Knockout ◽

Single Gene ◽

Site Directed Mutagenesis ◽

Content Type ◽

E Coli ◽

Synthase Inhibitor ◽

Loss Of Susceptibility

ABSTRACTThe microcin PDI inhibits a diverse group of pathogenicEscherichia colistrains. Coculture of a single-gene knockout library (BW25113;n= 3,985 mutants) against a microcin PDI-producing strain (E. coli25) identified six mutants that were not susceptible (ΔatpA, ΔatpF, ΔdsbA, ΔdsbB, ΔompF, and ΔompR). Complementation of these genes restored susceptibility in all cases, and the loss of susceptibility was confirmed through independent gene knockouts inE. coliO157:H7 Sakai. Heterologous expression ofE. coliompFconferred susceptibility toSalmonella entericaandYersinia enterocoliticastrains that are normally unaffected by microcin PDI. The expression of chimeric OmpF and site-directed mutagenesis revealed that the K47G48N49region within the first extracellular loop ofE. coliOmpF is a putative binding site for microcin PDI. OmpR is a transcriptional regulator forompF, and consequently loss of susceptibility by the ΔompRstrain most likely is related to this function. Deletion of AtpA and AtpF, as well as AtpE and AtpH (missed in the original library screen), resulted in the loss of susceptibility to microcin PDI and the loss of ATP synthase function. Coculture of a susceptible strain in the presence of an ATP synthase inhibitor resulted in a loss of susceptibility, confirming that a functional ATP synthase complex is required for microcin PDI activity. Intransexpression ofompFin the ΔdsbAand ΔdsbBstrains did not restore a susceptible phenotype, indicating that these proteins are probably involved with the formation of disulfide bonds for OmpF or microcin PDI.

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Determination of the Catalytic Base in Family 48 Glycosyl Hydrolases

Applied and Environmental Microbiology ◽

10.1128/aem.05532-11 ◽

2011 ◽

Vol 77 (17) ◽

pp. 6274-6276 ◽

Cited By ~ 14

Author(s):

Maxim Kostylev ◽

David B. Wilson

Keyword(s):

Aspartic Acid ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Thermobifida Fusca ◽

Glycosyl Hydrolases ◽

Content Type ◽

Modeling Data ◽

Partial Activity

ABSTRACTThe catalytic base in family 48 glycosyl hydrolases has not been previously established experimentally. Based on structural and modeling data published to date, we used site-directed mutagenesis and azide rescue activity assays to show definitively that the catalytic base inThermobifida fuscaCel48A is aspartic acid 225. Of the tested mutants, only Cel48A with the D225E mutation retained partial activity on soluble and insoluble substrates. In azide rescue experiments, only the D225G mutation, in the smallest residue tested, showed an increase in activity with added azide.

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E. coli F1 -ATPase: Site-directed mutagenesis of the β-subunit

FEBS Letters ◽

10.1016/0014-5793(88)80397-1 ◽

1988 ◽

Vol 232 (1) ◽

pp. 111-114 ◽

Cited By ~ 21

Author(s):

Derek Parsonage ◽

Susan Wilke-Mounts ◽

Alan E. Senior

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Β Subunit ◽

E Coli ◽

F1 Atpase

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Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

Applied and Environmental Microbiology ◽

10.1128/aem.01628-18 ◽

2018 ◽

Vol 84 (23) ◽

Cited By ~ 2

Author(s):

Efstratios Nikolaivits ◽

Maria Dimarogona ◽

Ioanna Karagiannaki ◽

Angelina Chalima ◽

Ayelet Fishman ◽

...

Keyword(s):

Substrate Specificity ◽

Fruits And Vegetables ◽

Homology Model ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Gene Encoding ◽

Content Type ◽

Polyphenol Oxidases ◽

Increased Activity ◽

Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

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Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

10.21248/gups.61063 ◽

2021 ◽

Author(s):

◽

Madeleine Huber

Keyword(s):

Escherichia Coli ◽

De Novo ◽

Stop Codon ◽

Haloferax Volcanii ◽

Site Directed Mutagenesis ◽

Start Codon ◽

Directed Mutagenesis ◽

Ribosome Display ◽

E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

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