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Sample records for coli ef-tu mutants

  1. EF-Tu from the enacyloxin producing Frateuria W-315 strain: Structure/activity relationship and antibiotic resistance.

    PubMed

    Créchet, Jean-Bernard; Malosse, Christian; Hountondji, Codjo

    2016-08-01

    In this report, we have demonstrated that the poly(U)-dependent poly(Phe) synthesis activity of elongator factor Tu (EF-Tu) from the enacyloxin producing strain Frateuria sp. W-315 is inhibited by the antibiotic similarly to that of Escherichia coli EF-Tu. The inhibitory effect of enacyloxin observed in a purified system was the same as that obtained with an S30 extract from E. coli or Frateuria sp. W-315, respectively, suggesting that antibiotic resistance of enacyloxin producing Frateuria sp. W-315 is not due neither to EF-Tu nor to other components of the translation machinery but to a still unknown mechanism. The EF-Tu gene, as PCR amplified from Frateuria W-315 genomic DNA and sequenced represented an ORF of 1191 nucleotides corresponding to 396 amino acids. This protein is larger than the product of tufA from E. coli by only two amino acid residues. Alignment of the amino acid sequence of EF-Tu from E. coli with those of Frateuria and Ralstonia solanacearum indicates on average 80% identical amino acid residues and 9.7% conservative replacements between EF-Tu Frateuria and EF-Tu E. coli, on one hand, and 97% identity and 1.7% conservative replacement between EF-Tu Frateuria and EF-Tu Ralstonia solanacearum, on the other hand. These strong primary structure similarities between EF-Tu from different origins are consistent with the fact that this factor is essential for the translation process in all kingdoms of life. Comparison of the effects of antibiotics on EF-Tu Frateuria and EF-Tu E. coli revealed that enacyloxin, kirromycin and pulvomycin exert a stronger stimulation of the GDP dissociation rate on EF-Tu Frateuria, while the effects of the antibiotics on the GDP association rate were comparable for the two EF-Tu species. Different mutants of EF-Tu E. coli were constructed with the help of site directed mutagenesis by changing one or several residues of EF-Tu E. coli by the corresponding residues of EF-Tu Frateuria. The single A45K substitution did

  2. Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation.

    PubMed

    Chudaev, Maxim; Poruri, Kiran; Goldman, Emanuel; Jakubowski, Hieronim; Jain, Mohit Raja; Chen, Wei; Li, Hong; Tyagi, Sanjay; Mandecki, Wlodek

    2013-05-01

    Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.

  3. Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803.

    PubMed

    Yutthanasirikul, Rayakorn; Nagano, Takanori; Jimbo, Haruhiko; Hihara, Yukako; Kanamori, Takashi; Ueda, Takuya; Haruyama, Takamitsu; Konno, Hiroki; Yoshida, Keisuke; Hisabori, Toru; Nishiyama, Yoshitaka

    2016-03-11

    Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP- and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H2O2, whereas GDP-bound EF-Tu was resistant to H2O2. The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H2O2, confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

  4. Translation elongation factor EF-Tu modulates filament formation of actin-like MreB protein in vitro.

    PubMed

    Defeu Soufo, Hervé Joël; Reimold, Christian; Breddermann, Hannes; Mannherz, Hans G; Graumann, Peter L

    2015-04-24

    EF-Tu has been shown to interact with actin-like protein MreB and to affect its localization in Escherichia coli and in Bacillus subtilis cells. We have purified YFP-MreB in an active form, which forms filaments on glass slides in vitro and was active in dynamic light-scattering assays, polymerizing in milliseconds after addition of magnesium. Purified EF-Tu enhanced the amount of MreB filaments, as seen by sedimentation assays, the speed of filament formation and the length of MreB filaments in vitro. EF-Tu had the strongest impact on MreB filaments in a 1:1 ratio, and EF-Tu co-sedimented with MreB filaments, revealing a stoichiometric interaction between both proteins. This was supported by cross-linking assays where 1:1 species were well detectable. When expressed in E. coli cells, B. subtilis MreB formed filaments and induced the formation of co-localizing B. subtilis EF-Tu structures, indicating that MreB can direct the positioning of EF-Tu structures in a heterologous cell system. Fluorescence recovery after photobleaching analysis showed that MreB filaments have a higher turnover in B. subtilis cells than in E. coli cells, indicating different filament kinetics in homologous or heterologous cell systems. The data show that MreB can direct the localization of EF-Tu in vivo, which in turn positively affects the formation and dynamics of MreB filaments. Thus, EF-Tu is a modulator of the activity of a bacterial actin-like protein.

  5. EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways.

    PubMed

    Liu, Wei; Chen, Chunlai; Kavaliauskas, Darius; Knudsen, Charlotte R; Goldman, Yale E; Cooperman, Barry S

    2015-10-30

    The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and Pi release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.

  6. The 51-63 base pair of tRNA confers specificity for binding by EF-Tu.

    PubMed

    Sanderson, Lee E; Uhlenbeck, Olke C

    2007-06-01

    Elongation factor Tu (EF-Tu) exhibits significant specificity for the different elongator tRNA bodies in order to offset its variable affinity to the esterified amino acid. Three X-ray cocrystal structures reveal that while most of the contacts with the protein involve the phosphodiester backbone of tRNA, a single hydrogen bond is observed between the Glu390 and the amino group of a guanine in the 51-63 base pair in the T-stem of tRNA. Here we show that the Glu390Ala mutation of Thermus thermophilus EF-Tu selectively destabilizes binding of those tRNAs containing a guanine at either position 51 or 63 and that mutagenesis of the 51-63 base pair in several tRNAs modulates their binding affinities to EF-Tu. A comparison of Escherichia coli tRNA sequences suggests that this specificity mechanism is conserved across the bacterial domain. While this contact is an important specificity determinant, it is clear that others remain to be identified.

  7. Borrelia burgdorferi elongation factor EF-Tu is an immunogenic protein during Lyme borreliosis.

    PubMed

    Carrasco, Sebastian E; Yang, Youyun; Troxell, Bryan; Yang, Xiuli; Pal, Utpal; Yang, X Frank

    2015-09-02

    Borrelia burgdorferi, the etiological agent of Lyme disease, does not produce lipopolysaccharide but expresses a large number of lipoproteins on its cell surface. These outer membrane lipoproteins are highly immunogenic and have been used for serodiagnosis of Lyme disease. Recent studies have shown that highly conserved cytosolic proteins such as enolase and elongation factor Tu (EF-Tu) unexpectedly localized on the surface of bacteria including B. burgdorferi, and surface-localized enolase has shown to contribute to the enzootic cycle of B. burgdorferi. In this study, we studied the immunogenicity, surface localization, and function of B. burgdorferi EF-Tu. We found that EF-Tu is highly immunogenic in mice, and EF-Tu antibodies were readily detected in Lyme disease patients. On the other hand, active immunization studies showed that EF-Tu antibodies did not protect mice from infection when challenged with B. burgdorferi via either needle inoculation or tick bites. Borrelial mouse-tick cycle studies showed that EF-Tu antibodies also did not block B. burgdorferi migration and survival in ticks. Consistent with these findings, we found that EF-Tu primarily localizes in the protoplasmic cylinder of spirochetes and is not on the surface of B. burgdorferi. Taken together, our studies suggest that B. burgdorferi EF-Tu is not surfaced exposed, but it is highly immunogenic and is a potential serodiagnostic marker for Lyme borreliosis.

  8. Centers of motion associated with EF-Tu binding to the ribosome.

    PubMed

    Paci, Maxim; Fox, George E

    2016-05-03

    Structural centers of motion (pivot points) in the ribosome have recently been identified by measurement of conformational changes in rRNA resulting from EF-G GTP hydrolysis. This series of measurements is extended here to the ribosome's interactions with the cofactor EF-Tu. Four recent EF-Tu bound ribosome structures were compared to unbound structures. A total of 16 pivots were identified, of which 4 are unique to the EF-Tu interaction. Pivots in the GTPase associated center and the sarcin-ricin loop omitted previously, are found to be mobile in response to both EF-Tu and EF-G binding. Pivots in the intersubunit bridge rRNAs are found to be cofactor specific. Head swiveling motions in the small subunit are observed in the EF-Tu bound structures that were trapped post GTP hydrolysis. As in the case of pivots associated with EF-G, the additional pivots described here are associated with weak points in the rRNA structures such as non-canonical pairs and bulge loops. The combined set of pivots should be regarded as a minimal set. Only several states available to the ribosome have been presented in this work. Future, precise crystal structures in conjunction with experimental data will likely show additional functional pivoting elements in the rRNA.

  9. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens.

    PubMed

    Lee, Kyungwoo; Lillehoj, Hyun S; Li, Guangxing; Park, Myeong-Seon; Jang, Seung I; Jeong, Wooseog; Jeoung, Hye-Young; An, Dong-Jun; Lillehoj, Erik P

    2011-12-01

    Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.

  10. Heat tolerance and expression of protein synthesis elongation factors, EF-Tu and EF-1a, in spring wheat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein elongation factors, EF-Tu and EF-1a, have been implicated in cell response to heat stress. In spring wheat, EF-Tu displays chaperone activity and reduces thermal aggregation of Rubisco activase. Similarly, in mammalian cells, EF-1a displays chaperone-like activity and regulates the expressio...

  11. Two distinct EF-Tu epitopes induce immune responses in rice and Arabidopsis.

    PubMed

    Furukawa, Takehito; Inagaki, Hiroaki; Takai, Ryota; Hirai, Hiroyuki; Che, Fang-Sik

    2014-02-01

    Plants sense potential pathogens by recognizing conserved pathogen-associated molecular patterns (PAMPs) that cause PAMP-triggered immunity (PTI). We previously reported that rice recognizes flagellin from the rice-incompatible N1141 strain of Acidovorax avenae and subsequently induces immune responses. Cell extracts isolated from flagellin-deficient N1141 (Δfla1141) still induced PTI responses, suggesting that Δfla1141 possesses an additional PAMP distinct from flagellin. Here, we show that elongation factor Tu (EF-Tu), one of the most abundant bacterial proteins, acts as a PAMP in rice and causes several PTI responses. In Brassicaceae species, EF-Tu and an N-acetylated peptide comprising the first 18 amino acids of the N-terminus, termed elf18, are fully active as inducers of PTI responses. By contrast, elf18 did not cause any immune responses in rice, whereas an EF-Tu middle region comprising Lys176 to Gly225, termed EFa50, is fully active as a PAMP in rice. In the leaves of rice plants, EF-Tu induced H2O2 generation and callose deposition, and also triggered resistance to coinfection with pathogenic bacteria. Taken together, these data demonstrate that rice recognizes EFa50, which is distinct from elf18, and that this epitope induces PTI responses.

  12. The interface between Escherichia coli elongation factor Tu and aminoacyl-tRNA.

    PubMed

    Yikilmaz, Emine; Chapman, Stephen J; Schrader, Jared M; Uhlenbeck, Olke C

    2014-09-09

    Nineteen of the highly conserved residues of Escherichia coli (E. coli) Elongation factor Tu (EF-Tu) that form the binding interface with aa-tRNA were mutated to alanine to better understand how modifying the thermodynamic properties of EF-Tu-tRNA interaction can affect the decoding properties of the ribosome. Comparison of ΔΔG(o) values for binding EF-Tu to aa-tRNA show that the majority of the interface residues stabilize the ternary complex and their thermodynamic contribution can depend on the tRNA species that is used. Experiments with a very tight binding mutation of tRNA(Tyr) indicate that interface amino acids distant from the tRNA mutation can contribute to the specificity. For nearly all of the mutations, the values of ΔΔG(o) were identical to those previously determined at the orthologous positions of Thermus thermophilus (T. thermophilus) EF-Tu indicating that the thermodynamic properties of the interface were conserved between distantly related bacteria. Measurement of the rate of GTP hydrolysis on programmed ribosomes revealed that nearly all of the interface mutations were able to function in ribosomal decoding. The only interface mutation with greatly impaired GTPase activity was R223A which is the only one that also forms a direct contact with the ribosome. Finally, the ability of the EF-Tu interface mutants to destabilize the EF-Tu-aa-tRNA interaction on the ribosome after GTP hydrolysis were evaluated by their ability to suppress the hyperstable T1 tRNA(Tyr) variant where EF-Tu release is sufficiently slow to limit the rate of peptide bond formation (kpep) . In general, interface mutations that destabilize EF-Tu binding are also able to stimulate kpep of T1 tRNA(Tyr), suggesting that the thermodynamic properties of the EF-Tu-aa-tRNA interaction on the ribosome are quite similar to those found in the free ternary complex.

  13. An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue.

    PubMed

    Miller, Mickey R; Buskirk, Allen R

    2014-02-01

    In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon-anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB's affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA.

  14. An unusual mechanism for EF-Tu activation during tmRNA-mediated ribosome rescue

    PubMed Central

    Miller, Mickey R.; Buskirk, Allen R.

    2014-01-01

    In bacteria, ribosomes stalled on truncated mRNAs are rescued by transfer-messenger RNA (tmRNA) and its protein partner SmpB. Acting like tRNA, the aminoacyl-tmRNA/SmpB complex is delivered to the ribosomal A site by EF-Tu and accepts the transfer of the nascent polypeptide. Although SmpB binding within the decoding center is clearly critical for licensing tmRNA entry into the ribosome, it is not known how activation of EF-Tu occurs in the absence of a codon–anticodon interaction. A recent crystal structure revealed that SmpB residue His136 stacks on 16S rRNA nucleotide G530, a critical player in the canonical decoding mechanism. Here we use pre-steady-state kinetic methods to probe the role of this interaction in ribosome rescue. We find that although mutation of His136 does not reduce SmpB's affinity for the ribosomal A-site, it dramatically reduces the rate of GTP hydrolysis by EF-Tu. Surprisingly, the same mutation has little effect on the apparent rate of peptide-bond formation, suggesting that release of EF-Tu from the tmRNA/SmpB complex on the ribosome may occur prior to GTP hydrolysis. Consistent with this idea, we find that peptidyl transfer to tmRNA is relatively insensitive to the antibiotic kirromycin. Taken together, our studies provide a model for the initial stages of ribosomal rescue by tmRNA. PMID:24345396

  15. A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids.

    PubMed

    Ieong, Ka-Weng; Pavlov, Michael Y; Kwiatkowski, Marek; Ehrenberg, Måns; Forster, Anthony C

    2014-05-01

    There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNA(Ala)-based body (tRNA(AlaB)) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNA(PheB) body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNA(AlaB) body than from the tRNA(PheB) body. At ∼1 µM EF-Tu, tRNA(AlaB) conferred considerably faster incorporation kinetics than tRNA(PheB), especially in the case of the bulky bK. In contrast, the swap to the tRNA(AlaB) body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNA(AlaB) and tRNA(PheB) bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner.

  16. A tRNA body with high affinity for EF-Tu hastens ribosomal incorporation of unnatural amino acids

    PubMed Central

    Ieong, Ka-Weng; Pavlov, Michael Y.; Kwiatkowski, Marek; Ehrenberg, Måns; Forster, Anthony C.

    2014-01-01

    There is evidence that tRNA bodies have evolved to reduce differences between aminoacyl-tRNAs in their affinity to EF-Tu. Here, we study the kinetics of incorporation of L-amino acids (AAs) Phe, Ala allyl-glycine (aG), methyl-serine (mS), and biotinyl-lysine (bK) using a tRNAAla-based body (tRNAAlaB) with a high affinity for EF-Tu. Results are compared with previous data on the kinetics of incorporation of the same AAs using a tRNAPheB body with a comparatively low affinity for EF-Tu. All incorporations exhibited fast and slow phases, reflecting the equilibrium fraction of AA-tRNA in active ternary complex with EF-Tu:GTP before the incorporation reaction. Increasing the concentration of EF-Tu increased the amplitude of the fast phase and left its rate unaltered. This allowed estimation of the affinity of each AA-tRNA to EF-Tu:GTP during translation, showing about a 10-fold higher EF-Tu affinity for AA-tRNAs formed from the tRNAAlaB body than from the tRNAPheB body. At ∼1 µM EF-Tu, tRNAAlaB conferred considerably faster incorporation kinetics than tRNAPheB, especially in the case of the bulky bK. In contrast, the swap to the tRNAAlaB body did not increase the fast phase fraction of N-methyl-Phe incorporation, suggesting that the slow incorporation of N-methyl-Phe had a different cause than low EF-Tu:GTP affinity. The total time for AA-tRNA release from EF-Tu:GDP, accommodation, and peptidyl transfer on the ribosome was similar for the tRNAAlaB and tRNAPheB bodies. We conclude that a tRNA body with high EF-Tu affinity can greatly improve incorporation of unnatural AAs in a potentially generalizable manner. PMID:24671767

  17. Duplication of Drosophila melanogaster mitochondrial EF-Tu: pre-adaptation to T-arm truncation and exclusion of bulky aminoacyl residues.

    PubMed

    Sato, Aya; Suematsu, Takuma; Aihara, Koh-Ki; Kita, Kiyoshi; Suzuki, Tsutomu; Watanabe, Kimitsuna; Ohtsuki, Takashi; Watanabe, Yoh-Ichi

    2017-03-07

    Translation elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to ribosomes in protein synthesis. EF-Tu generally recognizes aminoacyl moieties and acceptor- and T-stems of aa-tRNAs. However, nematode mitochondrial (mt) tRNAs frequently lack all or part of the T-arm that is recognized by canonical EF-Tu. We previously reported that two distinct EF-Tu species, EF-Tu1 and EF-Tu2, respectively, recognize mt tRNAs lacking T-arms and D-arms in the mitochondria of the chromadorean nematode Caenorhabditis elegansC. elegans EF-Tu2 specifically recognizes the seryl moiety of serylated D-armless tRNAs. Mitochondria of the enoplean nematode Trichinella possess three structural types of tRNAs: T-armless tRNAs, D-armless tRNAs, and cloverleaf tRNAs with a short T-arm. Trichinella mt EF-Tu1 binds to all three types and EF-Tu2 binds only to D-armless Ser-tRNAs, showing an evolutionary intermediate state from canonical EF-Tu to chromadorean nematode (e.g. C. elegans) EF-Tu species. We report here that two EF-Tu species also participate in Drosophila melanogaster mitochondria. Both D. melanogaster EF-Tu1 and EF-Tu2 bound to cloverleaf and D-armless tRNAs. D. melanogaster EF-Tu1 has the ability to recognize T-armless tRNAs that do not evidently exist in D. melanogaster mitochondria, but do exist in related arthropod species. In addition, D. melanogaster EF-Tu2 preferentially bound to aa-tRNAs carrying small amino acids, but not to aa-tRNAs carrying bulky amino acids. These results suggest that the Drosophila mt translation system could be another intermediate state between the canonical and nematode mitochondria-type translation systems.

  18. Arabidopsis EF-Tu receptor enhances bacterial disease resistance in transgenic wheat.

    PubMed

    Schoonbeek, Henk-Jan; Wang, Hsi-Hua; Stefanato, Francesca L; Craze, Melanie; Bowden, Sarah; Wallington, Emma; Zipfel, Cyril; Ridout, Christopher J

    2015-04-01

    Perception of pathogen (or microbe)-associated molecular patterns (PAMPs/MAMPs) by pattern recognition receptors (PRRs) is a key component of plant innate immunity. The Arabidopsis PRR EF-Tu receptor (EFR) recognizes the bacterial PAMP elongation factor Tu (EF-Tu) and its derived peptide elf18. Previous work revealed that transgenic expression of AtEFR in Solanaceae confers elf18 responsiveness and broad-spectrum bacterial disease resistance. In this study, we developed a set of bioassays to study the activation of PAMP-triggered immunity (PTI) in wheat. We generated transgenic wheat (Triticum aestivum) plants expressing AtEFR driven by the constitutive rice actin promoter and tested their response to elf18. We show that transgenic expression of AtEFR in wheat confers recognition of elf18, as measured by the induction of immune marker genes and callose deposition. When challenged with the cereal bacterial pathogen Pseudomonas syringae pv. oryzae, transgenic EFR wheat lines had reduced lesion size and bacterial multiplication. These results demonstrate that AtEFR can be transferred successfully from dicot to monocot species, further revealing that immune signalling pathways are conserved across these distant phyla. As novel PRRs are identified, their transfer between plant families represents a useful strategy for enhancing resistance to pathogens in crops.

  19. Elongation Factor-Tu (EF-Tu) proteins structural stability and bioinformatics in ancestral gene reconstruction

    NASA Astrophysics Data System (ADS)

    Dehipawala, Sunil; Nguyen, A.; Tremberger, G.; Cheung, E.; Schneider, P.; Lieberman, D.; Holden, T.; Cheung, T.

    2013-09-01

    A paleo-experimental evolution report on elongation factor EF-Tu structural stability results has provided an opportunity to rewind the tape of life using the ancestral protein sequence reconstruction modeling approach; consistent with the book of life dogma in current biology and being an important component in the astrobiology community. Fractal dimension via the Higuchi fractal method and Shannon entropy of the DNA sequence classification could be used in a diagram that serves as a simple summary. Results from biomedical gene research provide examples on the diagram methodology. Comparisons between biomedical genes such as EEF2 (elongation factor 2 human, mouse, etc), WDR85 in epigenetics, HAR1 in human specificity, DLG1 in cognitive skill, and HLA-C in mosquito bite immunology with EF Tu DNA sequences have accounted for the reported circular dichroism thermo-stability data systematically; the results also infer a relatively less volatility geologic time period from 2 to 3 Gyr from adaptation viewpoint. Comparison to Thermotoga maritima MSB8 and Psychrobacter shows that Thermus thermophilus HB8 EF-Tu calibration sequence could be an outlier, consistent with free energy calculation by NUPACK. Diagram methodology allows computer simulation studies and HAR1 shows about 0.5% probability from chimp to human in terms of diagram location, and SNP simulation results such as amoebic meningoencephalitis NAF1 suggest correlation. Extensions to the studies of the translation and transcription elongation factor sequences in Megavirus Chiliensis, Megavirus Lba and Pandoravirus show that the studied Pandoravirus sequence could be an outlier with the highest fractal dimension and lowest entropy, as compared to chicken as a deviant in the DNMT3A DNA methylation gene sequences from zebrafish to human and to the less than one percent probability in computer simulation using the HAR1 0.5% probability as reference. The diagram methodology would be useful in ancestral gene

  20. Protein synthesis alongation factors EF-Tu and eEF1A: biosynthesis, functions and application in the improvement of heat tolerance in plants

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Protein synthesis elongation factors EF-Tu and eEF1A (EFs) represent a group of highly conserved and abundant GTPases with an important role in transporting the aminoacyl-tRNA complex to the A site of the ribosome during elongation phase of translation. EF-Tu proteins are located in bacteria and, du...

  1. Cloning, overexpression and purification of Bacillus subtilis elongation factor Tu in Escherichia coli.

    PubMed

    Kim, S I; Kim, H Y; Kwak, J H; Kwon, S H; Lee, S Y

    2000-02-29

    To establish the overexpression and one-step purification system of Bacillus subtilis elongation factor-Tu (EF-Tu), the EF-Tu gene was amplified with or without own ribosome binding site (rbs) by PCR and the only PCR product without rbs was subcloned successfully. For the expression of the EF-Tu gene cloned after PCR amplification, a constitutive expression system and inducible expression system with His6 tag at N-terminus or C-terminus, or glutathione-S-transferase (GST) fusion system were examined in E. coli and B. subtilis. Except GST fusion system in E. coli, however, all other trials were unsuccessful at the step of plasmid construction for the EF-Tu expression. The GST/EF-Tu fusion proteins were highly expressed by IPTG induction and obtained as both soluble and insoluble form. From the soluble GST/EF-Tu fusion protein, EF-Tu was obtained to near homogeneity by one-step purification with glutathione-sepharose affinity column chromatography followed by factor Xa treatment. The purified EF-Tu showed high GDP binding activity. These results indicate that the GST/EF-Tu fusion system is favorable to overexpression and purification of B. subtilis EF-Tu.

  2. Genetic studies of an Escherichia coli K-12 temperature-sensitive mutant defective in membrane protein synthesis.

    PubMed Central

    Sato, T; Ohki, M; Yura, T; Ito, K

    1979-01-01

    The mutant divE42(Ts) of Escherichia coli K-12, defective in the synthesis of membrane proteins and in the transcription of the lac operon at high temperature, has been further characterized. It was found that a mutation (divE42) located at about min 22 on the E. coli chromosome map is responsible for the Lac- phenotype and temperature-sensitive growth. The mutation could be contransduced with serC, pyrD, or pyrC by phage P1 at a frequency of 4, 16, or 0.5%, respectively, the gene order being serC-pyrD-ompA-sulA-divE-pyrC. Examination of temperature-independent revertants and Pyr+ transductants revealed that all the mutant phenotypes examined (deficiencies in the increase of activities of some membrane enzymes, expression of the lac operon, and synthesis of several other proteins) are due to a single mutation (divE42) which is recessive to the wild-type (divE+) allele. Protein synthesis in the mutant was also analyzed by dodecyl sulfate-polyacrylamide gel electrophoresis. Synthesis of a number of proteins, including membrane proteins, was found to decrease significantly, whereas that of an elongation factor, EF-Tu, increased upon transfer of a log-phase culture to high temperature (42 degrees C). These effects of temperature shift-up on protein synthesis were evident within 5 min under the conditions used. Images PMID:374381

  3. A conserved P-loop anchor limits the structural dynamics that mediate nucleotide dissociation in EF-Tu

    NASA Astrophysics Data System (ADS)

    Mercier, Evan; Girodat, Dylan; Wieden, Hans-Joachim

    2015-01-01

    The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu.GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C. We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

  4. How EF-Tu can contribute to efficient proofreading of aa-tRNA by the ribosome

    PubMed Central

    Noel, Jeffrey K.; Whitford, Paul C.

    2016-01-01

    It has long been recognized that the thermodynamics of mRNA–tRNA base pairing is insufficient to explain the high fidelity and efficiency of aminoacyl-tRNA (aa-tRNA) selection by the ribosome. To rationalize this apparent inconsistency, Hopfield proposed that the ribosome may improve accuracy by utilizing a multi-step kinetic proofreading mechanism. While biochemical, structural and single-molecule studies have provided a detailed characterization of aa-tRNA selection, there is a limited understanding of how the physical–chemical properties of the ribosome enable proofreading. To this end, we probe the role of EF-Tu during aa-tRNA accommodation (the proofreading step) through the use of energy landscape principles, molecular dynamics simulations and kinetic models. We find that the steric composition of EF-Tu can reduce the free-energy barrier associated with the first step of accommodation: elbow accommodation. We interpret this effect within an extended kinetic model of accommodation and show how EF-Tu can contribute to efficient and accurate proofreading. PMID:27796304

  5. How EF-Tu can contribute to efficient proofreading of aa-tRNA by the ribosome

    NASA Astrophysics Data System (ADS)

    Noel, Jeffrey K.; Whitford, Paul C.

    2016-10-01

    It has long been recognized that the thermodynamics of mRNA-tRNA base pairing is insufficient to explain the high fidelity and efficiency of aminoacyl-tRNA (aa-tRNA) selection by the ribosome. To rationalize this apparent inconsistency, Hopfield proposed that the ribosome may improve accuracy by utilizing a multi-step kinetic proofreading mechanism. While biochemical, structural and single-molecule studies have provided a detailed characterization of aa-tRNA selection, there is a limited understanding of how the physical-chemical properties of the ribosome enable proofreading. To this end, we probe the role of EF-Tu during aa-tRNA accommodation (the proofreading step) through the use of energy landscape principles, molecular dynamics simulations and kinetic models. We find that the steric composition of EF-Tu can reduce the free-energy barrier associated with the first step of accommodation: elbow accommodation. We interpret this effect within an extended kinetic model of accommodation and show how EF-Tu can contribute to efficient and accurate proofreading.

  6. A conserved P-loop anchor limits the structural dynamics that mediate nucleotide dissociation in EF-Tu.

    PubMed

    Mercier, Evan; Girodat, Dylan; Wieden, Hans-Joachim

    2015-01-08

    The phosphate-binding loop (P-loop) is a conserved sequence motif found in mononucleotide-binding proteins. Little is known about the structural dynamics of this region and its contribution to the observed nucleotide binding properties. Understanding the underlying design principles is of great interest for biomolecular engineering applications. We have used rapid-kinetics measurements in vitro and molecular dynamics (MD) simulations in silico to investigate the relationship between GTP-binding properties and P-loop structural dynamics in the universally conserved Elongation Factor (EF) Tu. Analysis of wild type EF-Tu and variants with substitutions at positions in or adjacent to the P-loop revealed a correlation between P-loop flexibility and the entropy of activation for GTP dissociation. The same variants demonstrate more backbone flexibility in two N-terminal amino acids of the P-loop during force-induced EF-Tu · GTP dissociation in Steered Molecular Dynamics simulations. Amino acids Gly18 and His19 are involved in stabilizing the P-loop backbone via interactions with the adjacent helix C. We propose that these P-loop/helix C interactions function as a conserved P-loop anchoring module and identify the presence of P-loop anchors within several GTPases and ATPases suggesting their evolutionary conservation.

  7. Structure of the Acinetobacter baumannii dithiol oxidase DsbA bound to elongation factor EF-Tu reveals a novel protein interaction site.

    PubMed

    Premkumar, Lakshmanane; Kurth, Fabian; Duprez, Wilko; Grøftehauge, Morten K; King, Gordon J; Halili, Maria A; Heras, Begoña; Martin, Jennifer L

    2014-07-18

    The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA.

  8. Structure of the Acinetobacter baumannii Dithiol Oxidase DsbA Bound to Elongation Factor EF-Tu Reveals a Novel Protein Interaction Site

    PubMed Central

    Premkumar, Lakshmanane; Kurth, Fabian; Duprez, Wilko; Grøftehauge, Morten K.; King, Gordon J.; Halili, Maria A.; Heras, Begoña; Martin, Jennifer L.

    2014-01-01

    The multidrug resistant bacterium Acinetobacter baumannii is a significant cause of nosocomial infection. Biofilm formation, that requires both disulfide bond forming and chaperone-usher pathways, is a major virulence trait in this bacterium. Our biochemical characterizations show that the periplasmic A. baumannii DsbA (AbDsbA) enzyme has an oxidizing redox potential and dithiol oxidase activity. We found an unexpected non-covalent interaction between AbDsbA and the highly conserved prokaryotic elongation factor, EF-Tu. EF-Tu is a cytoplasmic protein but has been localized extracellularly in many bacterial pathogens. The crystal structure of this complex revealed that the EF-Tu switch I region binds to the non-catalytic surface of AbDsbA. Although the physiological and pathological significance of a DsbA/EF-Tu association is unknown, peptides derived from the EF-Tu switch I region bound to AbDsbA with submicromolar affinity. We also identified a seven-residue DsbB-derived peptide that bound to AbDsbA with low micromolar affinity. Further characterization confirmed that the EF-Tu- and DsbB-derived peptides bind at two distinct sites. These data point to the possibility that the non-catalytic surface of DsbA is a potential substrate or regulatory protein interaction site. The two peptides identified in this work together with the newly characterized interaction site provide a novel starting point for inhibitor design targeting AbDsbA. PMID:24860094

  9. Identification and cloning of two immunogenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with...

  10. Effect of thiostrepton and 3'-terminal fragments of aminoacyl-tRNA on EF-Tu and ribosome-dependent GTP hydrolysis.

    PubMed

    Bhuta, P; Chládek, S

    1982-08-30

    The effect of the antibiotics thiostrepton and micrococcin on EF-Tu-catalyzed (ribosome-dependent) GTP hydrolysis in the presence of A-Phe, C-A-Phe, or C-C-A-Phe (related to the sequence of the 3'-terminus of aminoacyl-tRNA)(System I) or by methanol ('uncoupled GTPase', System II) was investigated. In System I, thiostrepton increases the binding affinities of the effectors to the EF-Tu.GTP.70 S ribosome complex, as well as the extent of the GTP hydrolysis, while the KmGTP is virtually unchanged. Similarly, in the uncoupled system (System II) and in the absence of effectors, thiostrepton significantly increases VmaxGTP, whereas KmGTP remains unaffected. Micrococcin is without any effect in both systems. The 'uncoupled GTPase' (in System II) is also strongly inhibited by C-A-Phe. The results indicate the crucial role of the EF-Tu site which binds the aminoacylated C-C-A terminus of aminoacyl-tRNA in promoting GTP hydrolysis. It follows that the binding of the model effectors (such as C-C-A-Phe) to that site is favorably influenced by the interaction of thiostrepton with the 50 S ribosomal subunit, whereas thiostrepton, per se, does not influence the affinity of EF-Tu for GTP.

  11. Analysis of transgenic wheat (Triticum aestivum L.) harboring a maize (Zea mays L.) gene for plastid EF-Tu: segregation pattern, expression and effects of the transgene.

    PubMed

    Fu, Jianming; Ristic, Zoran

    2010-06-01

    We previously reported that transgenic wheat (Triticum aestivum L.) carrying a maize (Zea mays L.) gene (Zmeftu1) for chloroplast protein synthesis elongation factor, EF-Tu, displays reduced thermal aggregation of leaf proteins, reduced injury to photosynthetic membranes (thylakoids), and enhanced rate of CO(2) fixation following exposure to heat stress (18 h at 45 degrees C) [Fu et al. in Plant Mol Biol 68:277-288, 2008]. In the current study, we investigated the segregation pattern and expression of the transgene Zmeftu1 and determined the grain yield of transgenic plants after exposure to a brief heat stress (18 h at 45 degrees C). We also assessed thermal aggregation of soluble leaf proteins in transgenic plants, testing the hypothesis that increased levels of EF-Tu will lead to a non-specific protection of leaf proteins against thermal aggregation. The transgenic wheat displayed a single-gene pattern of segregation of Zmeftu1. Zmeftu1 was expressed, and the transgenic plants synthesized and accumulated three anti-EF-Tu cross-reacting polypeptides of similar molecular mass but different pI, suggesting the possibility of posttranslational modification of this protein. The transgenic plants also showed better grain yield after exposure to heat stress compared with their non-transgenic counterparts. Soluble leaf proteins of various molecular masses displayed lower thermal aggregation in transgenic than in non-transgenic wheat. The results suggest that overexpression of chloroplast EF-Tu can be beneficial to wheat tolerance to heat stress. Moreover, the results also support the hypothesis that EF-Tu contributes to heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation in a non-specific manner.

  12. Elongation factor Tu D138N, a mutant with modified substrate specificity, as a tool to study energy consumption in protein biosynthesis.

    PubMed

    Weijland, A; Parlato, G; Parmeggiani, A

    1994-09-06

    Substitution Asp138-->Asn changes the substrate specificity of elongation factor (EF) Tu from GTP to XTP [Hwang & Miller (1987) J. Biol. Chem. 262, 13081-13085]. This mutated EF-Tu (EF-Tu D138N) was used to show that 2 XTP molecules are hydrolyzed for each elongation cycle [Weijland & Parmeggiani (1993) Science 259, 1311-1313]. Here we extend the study of the properties of this EF-Tu mutant and its function in the elongation process. In poly(U)-directed poly(phenylalanine) synthesis, the number of peptide chains synthesized using EF-Tu D138N.XTP was 30% higher than with EF-Tu wild type (wt).GTP. However, since in the former case the average peptide chain length was correspondingly reduced, the number of the residues incorporated turned out to be nearly the same in both systems. The K'd values of the XTP and XDP complexes of EF-Tu D138N were similar to those of the GTP and GDP complexes of EF-Tu wt. The extent of leucine misincorporation and the kirromycin effect were also comparable to those in the EF-Tu wt/GTP system. The hydrolysis of two XTP molecules, very likely as part of two EF-Tu D138N.XTP complexes, for each elongation cycle was found to be independent of (i) MgCl2 concentration, (ii) ribosome concentration, and (iii) temperature (5-40 degrees C). With rate-limiting amounts of XTP the K'm of its XTPase activity corresponded to the K'm for XTP of poly(phenylalanine) synthesis (0.3-0.6 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

  13. A SelB/EF-Tu/aIF2γ-like protein from Methanosarcina mazei in the GTP-bound form binds cysteinyl-tRNA(Cys.).

    PubMed

    Yanagisawa, Tatsuo; Ishii, Ryohei; Hikida, Yasushi; Fukunaga, Ryuya; Sengoku, Toru; Sekine, Shun-ichi; Yokoyama, Shigeyuki

    2015-03-01

    The putative translation elongation factor Mbar_A0971 from the methanogenic archaeon Methanosarcina barkeri was proposed to be the pyrrolysine-specific paralogue of EF-Tu ("EF-Pyl"). In the present study, the crystal structures of its homologue from Methanosarcina mazei (MM1309) were determined in the GMPPNP-bound, GDP-bound, and apo forms, by the single-wavelength anomalous dispersion phasing method. The three MM1309 structures are quite similar (r.m.s.d. < 0.1 Å). The three domains, corresponding to domains 1, 2, and 3 of EF-Tu/SelB/aIF2γ, are packed against one another to form a closed architecture. The MM1309 structures resemble those of bacterial/archaeal SelB, bacterial EF-Tu in the GTP-bound form, and archaeal initiation factor aIF2γ, in this order. The GMPPNP and GDP molecules are visible in their co-crystal structures. Isothermal titration calorimetry measurements of MM1309·GTP·Mg(2+), MM1309·GDP·Mg(2+), and MM1309·GMPPNP·Mg(2+) provided dissociation constants of 0.43, 26.2, and 222.2 μM, respectively. Therefore, the affinities of MM1309 for GTP and GDP are similar to those of SelB rather than those of EF-Tu. Furthermore, the switch I and II regions of MM1309 are involved in domain-domain interactions, rather than nucleotide binding. The putative binding pocket for the aminoacyl moiety on MM1309 is too small to accommodate the pyrrolysyl moiety, based on a comparison of the present MM1309 structures with that of the EF-Tu·GMPPNP·aminoacyl-tRNA ternary complex. A hydrolysis protection assay revealed that MM1309 binds cysteinyl (Cys)-tRNA(Cys) and protects the aminoacyl bond from non-enzymatic hydrolysis. Therefore, we propose that MM1309 functions as either a guardian protein that protects the Cys moiety from oxidation or an alternative translation factor for Cys-tRNA(Cys).

  14. Ethanol production using engineered mutant E. coli

    DOEpatents

    Ingram, Lonnie O.; Clark, David P.

    1991-01-01

    The subject invention concerns novel means and materials for producing ethanol as a fermentation product. Mutant E. coli are transformed with a gene coding for pyruvate decarboxylase activity. The resulting system is capable of producing relatively large amounts of ethanol from a variety of biomass sources.

  15. Endonuclease IV (nfo) mutant of Escherichia coli.

    PubMed Central

    Cunningham, R P; Saporito, S M; Spitzer, S G; Weiss, B

    1986-01-01

    A cloned gene, designated nfo, caused overproduction of an EDTA-resistant endonuclease specific for apurinic-apyrimidinic sites in DNA. The sedimentation coefficient of the enzyme was similar to that of endonuclease IV. An insertion mutation was constructed in vitro and transferred from a plasmid to the Escherichia coli chromosome. nfo mutants had an increased sensitivity to the alkylating agents methyl methanesulfonate and mitomycin C and to the oxidants tert-butyl hydroperoxide and bleomycin. The nfo mutation enhanced the killing of xth (exonuclease III) mutants by methyl methanesulfonate, H2O2, tert-butyl hydroperoxide, and gamma rays, and it enhanced their mutability by methyl methanesulfonate. It also increased the temperature sensitivity of an xth dut (dUTPase) mutant that is defective in the repair of uracil-containing DNA. These results are consistent with earlier findings that endonuclease IV and exonuclease III both cleave DNA 5' to an apurinic-apyrimidinic site and that exonuclease III is more active. However, nfo mutants were more sensitive to tert-butyl hydroperoxide and to bleomycin than were xth mutants, suggesting that endonuclease IV might recognize some lesions that exonuclease III does not. The mutants displayed no marked increase in sensitivity to 254-nm UV radiation, and the addition of an nth (endonuclease III) mutation to nfo or nfo xth mutants did not significantly increase their sensitivity to any of the agents tested. Images PMID:2430946

  16. Rejection of tmRNA·SmpB after GTP hydrolysis by EF-Tu on ribosomes stalled on intact mRNA.

    PubMed

    Kurita, Daisuke; Miller, Mickey R; Muto, Akira; Buskirk, Allen R; Himeno, Hyouta

    2014-11-01

    Messenger RNAs lacking a stop codon trap ribosomes at their 3' ends, depleting the pool of ribosomes available for protein synthesis. In bacteria, a remarkable quality control system rescues and recycles stalled ribosomes in a process known as trans-translation. Acting as a tRNA, transfer-messenger RNA (tmRNA) is aminoacylated, delivered by EF-Tu to the ribosomal A site, and accepts the nascent polypeptide. Translation then resumes on a reading frame within tmRNA, encoding a short peptide tag that targets the nascent peptide for degradation by proteases. One unsolved issue in trans-translation is how tmRNA and its protein partner SmpB preferentially recognize stalled ribosomes and not actively translating ones. Here, we examine the effect of the length of the 3' extension of mRNA on each step of trans-translation by pre-steady-state kinetic methods and fluorescence polarization binding assays. Unexpectedly, EF-Tu activation and GTP hydrolysis occur rapidly regardless of the length of the mRNA, although the peptidyl transfer to tmRNA decreases as the mRNA 3' extension increases and the tmRNA·SmpB binds less tightly to the ribosome with an mRNA having a long 3' extension. From these results, we conclude that the tmRNA·SmpB complex dissociates during accommodation due to competition between the downstream mRNA and the C-terminal tail for the mRNA channel. Rejection of the tmRNA·SmpB complex during accommodation is reminiscent of the rejection of near-cognate tRNA from the ribosome in canonical translation.

  17. Expression of the mucus adhesion genes Mub and MapA, adhesion-like factor EF-Tu and bacteriocin gene plaA of Lactobacillus plantarum 423, monitored with real-time PCR.

    PubMed

    Ramiah, K; van Reenen, C A; Dicks, L M T

    2007-05-30

    Expression of the mucus adhesion genes Mub and MapA, adhesion-like factor EF-Tu and bacteriocin gene plaA by Lactobacillus plantarum 423, grown in the presence of bile, pancreatin and at low pH, was studied by real-time PCR. Mub, MapA and EF-Tu were up-regulated in the presence of mucus, proportional to increasing concentrations. Expression of MapA was up-regulated in the presence of 3.0 g/l bile and 3.0 g/l pancreatin at pH 6.5. Similar results were recorded in the presence of 10.0 g/l bile and 10.0 g/l pancreatin at pH 6.5. Expression of Mub was down-regulated in the presence of bile and pancreatin, whilst the expression of EF-Tu and plaA remained unchanged. Expression of Mub and MapA remained unchanged at pH 4.0, whilst expression of EF-Tu and plaA were up-regulated. Expression of MapA was down-regulated in the presence of 1.0 g/l l-cysteine HCl, suggesting that the gene is regulated by transcription attenuation that involves cysteine.

  18. New types of Escherichia coli recombination-deficient mutants.

    PubMed

    Freifelder, D

    1976-11-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency.

  19. New types of Escherichia coli recombination-deficient mutants.

    PubMed Central

    Freifelder, D

    1976-01-01

    A set of Escherichia coli mutants deficient in intramolecular recombination and different from those previously found is described. All have temperature-sensitive lethal mutations. The mutants have been characterized with respect to the following properties: the Pap phenotype, deoxyribonucleic acid synthesis, sensitivity to ultraviolet light, ability to support the growth of phage lambda, filament formation, and mutation frequency. PMID:789362

  20. Division pattern of a round mutant of Escherichia coli.

    PubMed Central

    Cooper, S

    1997-01-01

    A round mutant of Escherichia coli, when grown in Methocel medium, forms chains of cells and does not form tetrads. This implies that successive division planes of the round mutant are parallel rather than perpendicular. These results differ from a previous proposal that division planes in this round mutant are perpendicular to the prior division plane (W. D. Donachie, S. Addinall, and K. Begg, Bioessays 17:569-576, 1995). PMID:9287016

  1. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2001-09-25

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  2. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, M.; Millard, C.S.; Stols, L.

    1998-06-23

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria. 2 figs.

  3. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    2002-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which has been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  4. Mutant E. coli strain with increased succinic acid production

    DOEpatents

    Donnelly, Mark; Millard, Cynthia S.; Stols, Lucy

    1998-01-01

    A method for isolating succinic acid producing bacteria is provided comprising increasing the biomass of an organism which lacks the ability to catabolize pyruvate, and then subjecting the biomass to glucose-rich medium in an anaerobic environment to enable pyruvate-catabolizing mutants to grow. The invention also provides for a mutant that produces high amounts of succinic acid, which as been derived from a parent which lacked the genes for pyruvate formate lyase and lactate dehydrogenase, and which belongs to the E.coli Group of Bacteria.

  5. Construction and characterization of avian Escherichia coli cya crp mutants.

    PubMed

    Peighambari, S M; Gyles, C L

    1998-01-01

    We constructed delta cya delta crp mutants of two avian septicemic Escherichia coli strains and evaluated their attenuation in virulence. The P1 phage was used to transfer cya::Tn10 from an E. coli K-12 strain into virulent avian O78 and O2 E. coli isolates. Tetracycline-resistant transductants were plated on Bochner-Maloy Medium, and tetracycline-sensitive colonies were selected, then tested by polymerase chain reaction to confirm that they had deletions of the cya gene. Deletions of crp were created by the same technique in isolates with deletions in cya. The delta cya and delta cya delta crp derivatives had slower growth rates, smaller colonies, and impaired fermentation of carbohydrates compared with their wild parents, and they did not revert. Attenuation of the mutant strains was evaluated by subcutaneous (s.c.) inoculation of day-old chicks and by intratracheal (i.t.) inoculation of 9-day-old chicks previously inoculated intranasally with infectious bronchitis virus. For the wild O78 strain and its delta cya and delta cya delta crp derivatives, the percentages of chicks that died within 6 days of s.c. injection of approximately 5 x 10(7) organisms were 100, 60, and 0, respectively. The corresponding percentages for wild-type O2 and its delta cya and delta cya delta crp mutants were 100, 70, and 20 at a dose of approximately 2 x 10(5) organisms. Following i.t. inoculation, group scores based on pathologic and bacteriologic findings were 51%, 15%, and 9% for wild, delta cya, and delta crp O78 strains (inoculum approximately 2 x 10(7) organisms) and 98%, 31%, and 11%, respectively, for the corresponding O2 strains (inoculum approximately 4 x 10(6) organisms). This study demonstrated reduced virulence and stability of the double mutant, which may useful as a live attenuated vaccine against poultry colibacillosis.

  6. Proton translocation in cytochrome-deficient mutants of Escherichia coli.

    PubMed Central

    Brookman, J J; Downie, J A; Gibson, F; Cox, G B; Rosenberg, H

    1979-01-01

    Cytochrome-deficient cells of a strain of Escherichia coli lacking 5-amino-levulinate synthetase have been used to study proton translocation associated with the reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase region of the electron transport chain. Menadione was used as electron acceptor, and mannitol was used as the substrate for the generation of intracellular NADH. The effects of iron deficiency on NADH- and D-lactate-menadione reductase activities were studied in iron-deficient cells of a mutant strain unable to synthesize the iron chelator enterochelin; both activities were reduced. The NADH- menadione reductase activity in cytochrome-deficient cells was associated with proton translocation and could be coupled to the uptake of proline. However proton translocation associated with the NADH-menadione reductase activity was prevented by a mutation in an unc gene. It was concluded that there is no proton translocation associated with the NADH-dehydrogenase region of the electron transport chain in E. coli and that the proton translocation obtained with mannitol as substrate is due to the activity of membrane-bound adenosine triphosphatase. PMID:154508

  7. Pleiotropic aspartate taxis and serine taxis mutants of Escherichia coli.

    PubMed

    Reader, R W; Tso, W W; Springer, M S; Goy, M F; Adler, J

    1979-04-01

    Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tar mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.

  8. Improved penicillin amidase production using a genetically engineered mutant of escherichia coli ATCC 11105

    SciTech Connect

    Robas, N.; Zouheiry, H.; Branlant, G.; Branlant, C. )

    1993-01-05

    Penicillin G amidase (PGA) is a key enzyme for the industrial production of penicillin G derivatives used in therapeutics. Escherichia coli ATCC 11105 is the more commonly used strain for PGA production. To improve enzyme yield, the authors constructed various recombinant E. coli HB 101 and ATCC 11105 strains. For each strain, PGA production was determined for various concentrations of glucose and phenylacetic acid (PAA) in the medium. The E. coli strain, G271, was identified as the best performer (800 U NIPAB/L). This strain was obtained as follows: an E. coli ATCC 11105 mutant (E. coli G133) was first selected based on a low negative effect of glucose on PGA production. This mutant was then transformed with a pBR322 derivative containing the PGA gene. Various experiments were made to try to understand the reason for the high productivity of E. coli G271. The host strain, E. coli G133, was found to be mutated in one (or more) gene(s) whose product(s) act(s) in trans on the PGA gene expression. Its growth is not inhibited by high glucose concentration in the medium. Interestingly, whereas glucose still exerts some negative effect on the PGA production by E. coli G133, PGA production by its transformant (E. coli G271) is stimulated by glucose. The reason for this stimulation is discussed. Transformation of E. coli G133 with a pBR322 derivative containing the HindIII fragment of the PGA gene, showed that the performance of E. coli G271 depends both upon the host strain properties and the plasmid structure. Study of the production by the less efficient E. coli HB101 derivatives brought some light on the mechanism of regulation of the PGA gene.

  9. Characterization of Escherichia coli d-Cycloserine Transport and Resistant Mutants

    PubMed Central

    Baisa, Gary; Stabo, Nicholas J.

    2013-01-01

    d-Cycloserine (DCS) is a broad-spectrum antibiotic that inhibits d-alanine ligase and alanine racemase activity. When Escherichia coli K-12 or CFT073 is grown in minimal glucose or glycerol medium, CycA transports DCS into the cell. E. coli K-12 cycA and CFT073 cycA mutant strains display increased DCS resistance when grown in minimal medium. However, the cycA mutants exhibit no change in DCS sensitivity compared to their parental strains when grown in LB (CFT073 and K-12) or human urine (CFT073 only). These data suggest that cycA does not participate in DCS sensitivity when strains are grown in a non-minimal medium. The small RNA GvcB acts as a negative regulator of E. coli K-12 cycA expression when grown in LB. Three E. coli K-12 gcvB mutant strains failed to demonstrate a change in DCS sensitivity when grown in LB. This further suggests a limited role for cycA in DCS sensitivity. To aid in the identification of E. coli genes involved in DCS sensitivity when grown on complex media, the Keio K-12 mutant collection was screened for DCS-resistant strains. dadA, pnp, ubiE, ubiF, ubiG, ubiH, and ubiX mutant strains showed elevated DCS resistance. The phenotypes associated with these mutants were used to further define three previously characterized E. coli DCS-resistant strains (χ316, χ444, and χ453) isolated by Curtiss and colleagues (R. Curtiss, III, L. J. Charamella, C. M. Berg, and P. E. Harris, J. Bacteriol. 90:1238–1250, 1965). A dadA mutation was identified in both χ444 and χ453. In addition, results are presented that indicate for the first time that DCS can antagonize d-amino acid dehydrogenase (DadA) activity. PMID:23316042

  10. Selection for Spontaneous "Escherichia coli" Streptomycin Mutants Using Basic Fuchsin.

    ERIC Educational Resources Information Center

    Walkosz, Ronald

    1991-01-01

    An exercise that uses a common bacterium, E. coli, in great numbers, to detect a demonstrable change in the ability of some cells to become resistant to the common antibiotic streptomycin is presented. The procedure for preparing and pouring the gradient antibiotic plates is provided. The advantages of using the Basic Fuchsin in the agar are…

  11. Novel E. coli mutants deficient in biosynthesis of 5-methylaminomethyl-2-thiouridine.

    PubMed Central

    Elseviers, D; Petrullo, L A; Gallagher, P J

    1984-01-01

    Novel E. coli mutants deficient in biosynthesis of 5- methylaminomethyl -2-thiouridine were isolated based on a phenotype of reduced readthrough at UAG codons. They define 2 new loci trmE and trmF , near 83' on the E. coli map. These mutants are different from strains carrying trmC mutations, which are known to confer a methylation deficiency in biosynthesis of 5- methylaminomethyl -2-thiouridine. tRNA from mutants carrying trmE or trmF mutations was shown to carry 2-thiouridine instead of 5- methylaminomethyl -2-thiouridine. This deficiency affects the triplet binding properties of the mutant tRNA. Our results suggest that the 5- methylaminomethyl group stabilizes the basepairing of this modified nucleotide with G, most likely through direct interaction with the ribosomal binding site(s). Images PMID:6427754

  12. Mutant Strains of Escherichia coli K-12 Unable to Form Ubiquinone

    PubMed Central

    Cox, G. B.; Gibson, F.; Pittard, James

    1968-01-01

    A strain of Escherichia coli was isolated which was unable to form ubiquinone. This mutant was obtained by selecting strains unable to grow on malate as sole source of carbon. Such strains were further screened by examination of the quinone content of cells grown on a glucose medium. A mutant unable to form vitamin K was also isolated by this procedure. A genetic analysis of the ubiquinoneless strain showed that it possessed two mutations affecting ubiquinone biosynthesis. Images PMID:4870277

  13. An Escherichia coli asr mutant has decreased fitness during colonization in a mouse model.

    PubMed

    Armalyte, Julija; Seputiene, Vaida; Melefors, Ojar; Suziedeliene, Edita

    2008-01-01

    The Escherichia coli asr gene, like its homologues in other enterobacteria, is strongly induced by low external pH. The E. coli asr mutant shows weakened ability to adapt to acidic pH. This suggests that the asr gene product is important for enterobacterial species, both commensal and pathogenic, in overcoming acid stress in the stomach and subsequently colonizing the intestine. We examined the relative fitness of an E. coli asr mutant compared to a wild type, by feeding both strains simultaneously to mice and letting them colonize the intestine. Analysis of the bacteria after passage through the intestine showed up to five orders of magnitude less asr mutant than wild type. Transcomplementation of the asr gene on a plasmid partially restored the number of mutants. Similar competition in liquid media demonstrated that the asr mutant has reduced viability during long-term incubation in rich media, but is as fit as the wild type when bacteria are challenged in minimal medium. Competition carried out under different pH conditions proved that pH of the media was not the main determinant leading to the decreased fitness of the asr mutant. This suggests that the asr gene product is important for adaptation to stress conditions other than acidity, including long periods of starvation.

  14. Activity of KB-5246 against outer membrane mutants of Escherichia coli and Salmonella typhimurium.

    PubMed Central

    Kotera, Y; Inoue, M; Mitsuhashi, S

    1990-01-01

    The inhibitory activity of KB-5246 against Escherichia coli DNA gyrase and the antibacterial activity and apparent uptake in E. coli and Salmonella typhimurium outer membrane mutants of KB-5246 were measured. The 50% inhibitory concentrations of KB-5246, ciprofloxacin, oflaxacin, and norfloxacin for E. coli KL-16 DNA gyrase were 0.72, 0.62, 0.84, and 1.16 micrograms/ml, respectively. The activity of KB-5246 was twofold lower against an OmpF-deficient mutant and twofold higher against a mutant which produced OmpF constitutively than against the parent with osmoregulated OmpF production. KB-5246 had twofold-higher activity against a deep rough mutant of S. typhimurium than against the parent. The apparent uptake of KB-5246 in the OmpF-deficient mutant was decreased and its uptake in the deep rough mutant was increased when compared with those in the parents. These results suggest that KB-5246 is taken up by porin and nonporin pathways and has strong inhibitory activity against DNA gyrase, resulting in potent antibacterial activity. PMID:2167038

  15. Persistence of Escherichia coli O157:H7 and its mutants in soils.

    PubMed

    Ma, Jincai; Ibekwe, A Mark; Yi, Xuan; Wang, Haizhen; Yamazaki, Akihiro; Crowley, David E; Yang, Ching-Hong

    2011-01-01

    The persistence of Shiga toxin-producing E. coli O157:H7 in the environment poses a serious threat to public health. However, the role of Shiga toxins and other virulence factors in the survival of E. coli O157:H7 is poorly defined. The aim of this study was to determine if the virulence factors, stx₁, stx₂, stx₁₋₂, and eae in E. coli O157:H7 EDL933 play any significant role in the growth of this pathogen in rich media and in soils. Isogenic deletion mutants that were missing one of four virulence factors, stx₁, stx₂, stx₁₋₂, and eae in E. coli O157:H7 EDL933 were constructed, and their growth in rich media and survival in soils with distinct texture and chemistry were characterized. The survival data were successfully analyzed using Double Weibull model, and the modeling parameters of the mutant strains were not significantly different from those of the wild type. The calculated T(d) (time needed to reach the detection limit, 100 CFU/g soil) for loamy sand, sandy loam, and silty clay was 32, 80, and 110 days, respectively. It was also found that T(d) was positively correlated with soil structure (e.g. clay content), and soil chemistry (e.g. total nitrogen, total carbon, and water extractable organic carbon). The results of this study showed that the possession of Shiga toxins and intimin in E. coli O157:H7 might not play any important role in its survival in soils. The double deletion mutant of E. coli O157:H7 (stx₁⁻stx₂⁻) may be a good substitute to use for the investigation of transport, fate, and survival of E. coli O157:H7 in the environment where the use of pathogenic strains are prohibited by law since the mutants showed the same characteristics in both culture media and environmental samples.

  16. Persistence of Escherichia coli O157:H7 and Its Mutants in Soils

    PubMed Central

    Ma, Jincai; Ibekwe, A. Mark; Yi, Xuan; Wang, Haizhen; Yamazaki, Akihiro; Crowley, David E.; Yang, Ching-Hong

    2011-01-01

    The persistence of Shiga toxin-producing E. coli O157:H7 in the environment poses a serious threat to public health. However, the role of Shiga toxins and other virulence factors in the survival of E. coli O157:H7 is poorly defined. The aim of this study was to determine if the virulence factors, stx1, stx2, stx1–2, and eae in E. coli O157:H7 EDL933 play any significant role in the growth of this pathogen in rich media and in soils. Isogenic deletion mutants that were missing one of four virulence factors, stx1, stx2, stx1–2, and eae in E. coli O157:H7 EDL933 were constructed, and their growth in rich media and survival in soils with distinct texture and chemistry were characterized. The survival data were successfully analyzed using Double Weibull model, and the modeling parameters of the mutant strains were not significantly different from those of the wild type. The calculated Td (time needed to reach the detection limit, 100 CFU/g soil) for loamy sand, sandy loam, and silty clay was 32, 80, and 110 days, respectively. It was also found that Td was positively correlated with soil structure (e.g. clay content), and soil chemistry (e.g. total nitrogen, total carbon, and water extractable organic carbon). The results of this study showed that the possession of Shiga toxins and intimin in E. coli O157:H7 might not play any important role in its survival in soils. The double deletion mutant of E. coli O157:H7 (stx1−stx2−) may be a good substitute to use for the investigation of transport, fate, and survival of E. coli O157:H7 in the environment where the use of pathogenic strains are prohibited by law since the mutants showed the same characteristics in both culture media and environmental samples. PMID:21826238

  17. Characterization of an Escherichia coli K12 mutant that is sensitive to chlorate when grown aerobically.

    PubMed Central

    Giordano, G; Grillet, L; Rosset, R; Dou, J H; Azoulay, E; Haddock, B A

    1978-01-01

    Escherichia coli can normally grow aerobically in the presence of chlorate; however, mutants can be isolated that can no longer grow under these conditions. We present here the biochemical characterization of one such mutant and show that the primary genetic lesion occurs in the ubiquinone-8-biosynthetic pathway. As a consequence of this, under aerobic growth conditions the mutant is apparently unable to synthesize formate dehydrogenase, but can synthesize a Benzyl Viologen-dependent nitrate reductase activity. The nature of this activity is discussed. PMID:369552

  18. Isolation and cloning of a Azospirillum lipoferum locus that complements Escherichia coli proU mutant.

    PubMed

    Tripathi, A K; Mishra, B M

    1998-05-15

    Glycine betaine relieved sodium chloride-mediated inhibition of growth in Azospirillum lipoferum ATCC 29708. 35S-methionine labelling of proteins after salinity up-shock revealed strong induction of a 30 kDa protein which cross-reacted with the anti-glycine betaine binding protein antibody from Escherichia coli. This suggested that A. lipoferum had a salinity-induced ProU-like high-affinity glycine betaine transport system. A genomic library of A. lipoferum ATCC 29708 was screened for the proU-like gene by complementation of a proU mutant of E. coli. Four recombinant cosmids, capable of restoring growth of the proU mutant on plates containing 600 mM NaCl and 1 mM glycine betaine were selected. Selected recombinant cosmids hybridized with a proU gene probe from E. coli. Complementation of E. coli proU mutant with the A. lipoferum genomic DNA was evident by the ability of proU mutant (containing selected recombinant cosmids) to grow on minimal medium supplemented with 600 mM NaCl and 1 mM glycine betaine.

  19. Ferritin Mutants of Escherichia coli Are Iron Deficient and Growth Impaired, and fur Mutants are Iron Deficient

    PubMed Central

    Abdul-Tehrani, Hossein; Hudson, Aaron J.; Chang, Yung-Sheng; Timms, Andrew R.; Hawkins, Chris; Williams, John M.; Harrison, Pauline M.; Guest, John R.; Andrews, Simon C.

    1999-01-01

    Escherichia coli contains at least two iron storage proteins, a ferritin (FtnA) and a bacterioferritin (Bfr). To investigate their specific functions, the corresponding genes (ftnA and bfr) were inactivated by replacing the chromosomal ftnA and bfr genes with disrupted derivatives containing antibiotic resistance cassettes in place of internal segments of the corresponding coding regions. Single mutants (ftnA::spc and bfr::kan) and a double mutant (ftnA::spc bfr::kan) were generated and confirmed by Western and Southern blot analyses. The iron contents of the parental strain (W3110) and the bfr mutant increased by 1.5- to 2-fold during the transition from logarithmic to stationary phase in iron-rich media, whereas the iron contents of the ftnA and ftnA bfr mutants remained unchanged. The ftnA and ftnA bfr mutants were growth impaired in iron-deficient media, but this was apparent only after the mutant and parental strains had been precultured in iron-rich media. Surprisingly, ferric iron uptake regulation (fur) mutants also had very low iron contents (2.5-fold less iron than Fur+ strains) despite constitutive expression of the iron acquisition systems. The iron deficiencies of the ftnA and fur mutants were confirmed by Mössbauer spectroscopy, which further showed that the low iron contents of ftnA mutants are due to a lack of magnetically ordered ferric iron clusters likely to correspond to FtnA iron cores. In combination with the fur mutation, ftnA and bfr mutations produced an enhanced sensitivity to hydroperoxides, presumably due to an increase in production of “reactive ferrous iron.” It is concluded that FtnA acts as an iron store accommodating up to 50% of the cellular iron during postexponential growth in iron-rich media and providing a source of iron that partially compensates for iron deficiency during iron-restricted growth. In addition to repressing the iron acquisition systems, Fur appears to regulate the demand for iron, probably by controlling

  20. Colonization of gnotobiotic piglets by a luxS mutant strain of Escherichia coli O157:H7.

    PubMed

    Jordan, Dianna M; Sperandio, Vanessa; Kaper, James B; Dean-Nystrom, Evelyn A; Moon, Harley W

    2005-02-01

    Gnotobiotic piglets inoculated with Escherichia coli O157:H7, its luxS mutant derivative, or nonpathogenic E. coli were evaluated for attaching and effacing lesions. Although no differences in clinical symptoms were seen between pigs inoculated with the parent and those inoculated with the luxS mutant, the luxS mutant-inoculated pigs had a lower frequency of attaching and effacing lesions in the spiral colon than parent strain-inoculated pigs.

  1. The use of suicide substrates to select mutants of Escherichia coli lacking enzymes of alcohol fermentation.

    PubMed

    Cunningham, P R; Clark, D P

    1986-12-01

    Mutants of Escherichia coli resistant to chloroethanol or to chloroacetaldehyde were selected. Such mutants were found to lack the fermentative coenzyme A (CoA) linked acetaldehyde dehydrogenase activity. Most also lacked the associated fermentative enzyme alcohol dehydrogenase. Both types of mutants, those lacking acetaldehyde dehydrogenase alone or lacking both enzymes, mapped close to the regulatory adhC gene at 27 min on the E. coli genetic map. The previously described acd mutants which lack acetaldehyde dehydrogenase and which map at 63 min were shown to be pleiotropic, affecting respiration and growth on a variety of substrates. It therefore seems likely that the structural genes for both the acetaldehyde and alcohol dehydrogenases lie in the adhCE operon. This interpretation was confirmed by the isolation of temperature sensitive chloracetaldehyde-resistant mutants, some of which produced thermolabile acetaldehyde dehydrogenase and alcohol dehydrogenase and were also found to map at the adh locus. Reversion analysis indicated that mutants lacking one or both enzymes carried single mutations. The gene order in the adh region was determined by three point crosses to be trp-zch::Tn10-adh-galU-bglY-tyrT-chlC.

  2. Chemotactic properties of Escherichia coli mutants having abnormal Ca2+ content.

    PubMed Central

    Tisa, L S; Adler, J

    1995-01-01

    The calA, calC, and calD mutants of Escherichia coli are known to be sensitive to Ca2+ (R. N. Brey and B. P. Rosen, J. Bacteriol. 139:824-834, 1979). In the absence of any added stimuli for chemotaxis, both the calC and the calD mutants swam with a tumbly bias. Both the calC and the calD mutants were defective in chemotaxis as measured by computer analysis, use of swarm plates, and capillary assays. The calA mutant was only slightly defective in motility and only slightly impaired in chemotaxis. Chemotactically wild-type cells had an intra-cellular free-Ca2+ level of about 105 nM. The intracellular free-Ca2+ levels of the mutants, as determined by use of the fluorescent Ca2+ indicator dye fura-2 or fluo-3, were about 90, about 1,130, and about 410 nM for calA, calC, and calD, respectively. Lowering the intracellular free-Ca2+ levels in wild-type cells and in the tumbly cal mutants by use of Ca2+ chelators promoted running (smooth swimming). Overexpression of CheZ (which causes dephosphorylation of CheY-phosphate) in the wild type and in the tumbly cal mutants decreased the level of tumbliness (which is caused by CheY-phosphate). The calA mutant was 4- to 10-fold more resistant than the wild type to the inhibitory effect of omega-conotoxin on chemotaxis. omega-Conotoxin had no effect on Ca2+ extrusion by wild-type E. coli; that result suggests that omega-conotoxin affects Ca2+ transport at the point of entry instead of exit. PMID:8522517

  3. Mutant DnaAs of Escherichia coli that are refractory to negative control

    PubMed Central

    Chodavarapu, Sundari; Felczak, Magdalena M.; Simmons, Lyle A.; Murillo, Alec; Kaguni, Jon M.

    2013-01-01

    DnaA is the initiator of DNA replication in bacteria. A mutant DnaA named DnaAcos is unusual because it is refractory to negative regulation. We developed a genetic method to isolate other mutant DnaAs that circumvent regulation to extend our understanding of mechanisms that control replication initiation. Like DnaAcos, one mutant bearing a tyrosine substitution for histidine 202 (H202Y) withstands the regulation exerted by datA, hda and dnaN (β clamp), and both DnaAcos and H202Y resist inhibition by the Hda-β clamp complex in vitro. Other mutant DnaAs carrying G79D, E244K, V303M or E445K substitutions are either only partially sensitive or refractory to inhibition by the Hda-β clamp complex in vitro but are responsive to hda expression in vivo. All mutant DnaAs remain able to interact directly with Hda. Of interest, both DnaAcos and DnaAE244K bind more avidly to Hda. These mutants, by sequestrating Hda, may limit its availability to regulate other DnaA molecules, which remain active to induce extra rounds of DNA replication. Other evidence suggests that a mutant bearing a V292M substitution hyperinitiates by escaping the effect of an unknown regulatory factor. Together, our results provide new insight into the mechanisms that regulate replication initiation in Escherichia coli. PMID:23990329

  4. Expression of the cloned Escherichia coli O9 rfb gene in various mutant strains of Salmonella typhimurium.

    PubMed Central

    Sugiyama, T; Kido, N; Komatsu, T; Ohta, M; Kato, N

    1991-01-01

    To investigate the effect of chromosomal mutation on the synthesis of rfe-dependent Escherichia coli O9 lipopolysaccharide (LPS), the cloned E. coli O9 rfb gene was introduced into Salmonella typhimurium strains defective in various genes involved in the synthesis of LPS. When E. coli O9 rfb was introduced into S. typhimurium strains possessing defects in rfb or rfc, they synthesized E. coli O9 LPS on their cell surfaces. The rfe-defective mutant of S. typhimurium synthesized only very small amounts of E. coli O9 LPS after the introduction of E. coli O9 rfb. These results confirmed the widely accepted idea that the biosynthesis of E. coli O9-specific polysaccharide does not require rfc but requires rfe. By using an rfbT mutant of the E. coli O9 rfb gene, the mechanism of transfer of the synthesized E. coli O9-specific polysaccharide from antigen carrier lipid to the R-core of S. typhimurium was investigated. The rfbT mutant of the E. coli O9 rfb gene failed to direct the synthesis of E. coli O9 LPS in the rfc mutant strain of S. typhimurium, in which rfaL and rfbT functions are intact, but directed the synthesis of the precursor. Because the intact E. coli O9 rfb gene directed the synthesis of E. coli O9 LPS in the same strain, it was suggested that the rfaL product of S. typhimurium and rfbT product of E. coli O9 cooperate to synthesize E. coli O9 LPS in S. typhimurium. Images PMID:1987133

  5. Physiology and pathogenicity of cpdB deleted mutant of avian pathogenic Escherichia coli.

    PubMed

    Liu, Huifang; Chen, Liping; Si, Wei; Wang, Chunlai; Zhu, Fangna; Li, Guangxing; Liu, Siguo

    2017-04-01

    Avian colibacillosis is one of the most common infectious diseases caused partially or entirely by avian pathogenic Escherichia coli (APEC) in birds. In addition to spontaneous infection, APEC can also cause secondary infections that result in greater severity of illness and greater losses to the poultry industry. In order to assess the role of 2', 3'-cyclic phosphodiesterase (cpdB) in APEC on disease physiology and pathogenicity, an avian pathogenic Escherichia coli-34 (APEC-34) cpdB mutant was obtained using the Red system. The cpdB mutant grew at a slower rate than the natural strain APEC-34. Scanning electron microscopy (SEM) indicated that the bacteria of the cpdB mutant were significantly longer than the bacteria observed in the natural strain (P<0.01), and that the width of the cpdB mutant was significantly smaller than its natural counterpart (P<0.01). In order to evaluate the role of cpdB in APEC in the colonization of internal organs (lung, liver and spleen) in poultry, seven-day-old SPF chicks were infected with 10(9)CFU/chick of the cpdB mutant or the natural strain. No colonizations of cpdB mutants were observed in the internal organs 10days following the infection, though numerous natural strains were observed at 20days following infection. Additionally, the relative expression of division protein ftsZ, outer membrane protein A ompA, ferric uptake regulator fur and tryptophanase tnaA genes in the mutant strain were all significantly lower than in the natural strain (P<0.05 or P<0.01). These results suggested that cpdB is involved in the long-term colonization of APEC in the internal organs of the test subjects. The deletion of the cpdB gene also significantly affected the APEC growth and morphology.

  6. Relative activities and stabilities of mutant Escherichia coli tryptophan synthase alpha subunits.

    PubMed Central

    Lim, W K; Shin, H J; Milton, D L; Hardman, J K

    1991-01-01

    In vitro mutagenesis of the Escherichia coli trpA gene has yielded 66 mutant tryptophan synthase alpha subunits containing single amino acid substitutions at 49 different residue sites and 29 double and triple amino acid substitutions at 16 additional sites, all within the first 121 residues of the protein. The 66 singly altered mutant alpha subunits encoded from overexpression vectors have been examined for their ability to support growth in trpA mutant host strains and for their enzymatic and stability properties in crude extracts. With the exception of mutant alpha subunits altered at catalytic residue sites Glu-49 and Asp-60, all support growth; this includes those (48 of 66) that have no enzymatic defects and those (18 of 66) that do. The majority of the enzymatically defective mutant alpha subunits have decreased capacities for substrate (indole-3-glycerol phosphate) utilization, typical of the early trpA missense mutants isolated by in vivo selection methods. These defects vary in severity from complete loss of activity for mutant alpha subunits altered at residue positions 49 and 60 to those, altered elsewhere, that are partially (up to 40 to 50%) defective. The complete inactivation of the proteins altered at the two catalytic residue sites suggest that, as found via in vitro site-specific mutagenesis of the Salmonella typhimurium tryptophan synthetase alpha subunit, both residues probably also participate in a push-pull general acid-base catalysis of indole-3-glycerol phosphate breakdown for the E. coli enzyme as well. Other classes of mutant alpha subunits include some novel types that are defective in their functional interaction with the other tryptophan synthetase component, the beta 2 subunit. Also among the mutant alpha subunits, 19 were found altered at one or another of the 34 conserved residue sites in this portion of the alpha polypeptide sequence; surprisingly, 10 of these have wild-type enzymatic activity, and 16 of these can satisfy growth

  7. The Proteomic Response to Mutants of the Escherichia coli RNA Degradosome

    DTIC Science & Technology

    2013-01-01

    pathway of RNA degradation. REPORT DOCUMENTATION PAGE (SF298) (Continuation Sheet) Continuation for Block 13 ARO Report Number The proteomic response...of the bacterial proteome and provide the first large-scale proteomic description of the response to perturbation of this major pathway of RNA...truncation mutant11 (rightmost column). Significant function enrichments (adjusted P-value o 0.01, compared to entire E. coli genome) are indicated

  8. Dynamics of SOS-Response in UVR-Mutants of Escherichia Coli Cells under Ultraviolet Irradiation

    NASA Astrophysics Data System (ADS)

    Tuchina, M. A.; Parkhomenko, A. Yu.; Belov, O. V.; Bugai, A. N.

    2010-01-01

    A mathematical model of the genetic regulatory system of the SOS-response induced by ultraviolet radiation in excision repair-deficient mutants of E. coli bacterial cells is developed. On the basis of the model, the dynamics of the SOS system regulatory proteins is analyzed. The influence of excision repair on the induction of the key gene products during the SOS-response is studied.

  9. A Comparative Proteome Analysis of Escherichia coli ΔrelA Mutant Cells

    PubMed Central

    Carneiro, Sónia; Villas-Bôas, Silas; Ferreira, Eugénio C.; Rocha, Isabel

    2016-01-01

    The bacterial RelA-dependent stringent response exerts a strong influence over various processes. In this work, the impact of the relA gene mutation in Escherichia coli cells was evaluated by a quantitative proteomics analysis, employing stable-isotope labeling and high-resolution mass spectrometry. Chemostat cultures of E. coli W3110 and ΔrelA mutant strains were performed at two dilution rates (0.1 and 0.2 h−1) to assess the influence of the relA gene mutation in steady-state protein levels. A total of 121 proteins showed significant alterations in their abundance when comparing the proteome of mutant to wild-type cells. The relA gene mutation induced changes on key cellular processes, including the amino acids and nucleotide biosynthesis, the lipid metabolism, transport activities, transcription and translation processes, and responses to stress. Furthermore, some of those changes were more pronounced under specific growth conditions, as the most significant differences in protein ratios were observed at one of the dilution rates. An effect of the relA gene mutation in the acetate overflow was also observed, which confers interesting characteristics to this mutant strain that could be useful in the production of recombinant proteins. Overall, these results provide a valuable insight into the E. coli stringent response under defined steady-state conditions, suggesting that this stress response might influence multiple metabolic processes like the acetate overflow or the catabolite repression. PMID:27833909

  10. RESISTANCE AND CROSS RESISTANCE OF ESCHERICHIA COLI S MUTANTS TO THE RADIOMIMETIC AGENT NITROFURAZONE

    PubMed Central

    Woody-Karrer, Pearl; Greenberg, Joseph

    1963-01-01

    Woody-Karrer, Pearl (Palo Alto Medical Research Foundation, Palo Alto, Calif.) and Joseph Greenberg. Resistance and cross resistance of Escherichia coli S mutants to the radiomimetic agent nitrofurazone. J. Bacteriol. 85:1208–1216. 1963.—Cross-resistance relationships are described for 73 mutants of Escherichia coli strain S selected in one step for resistance to nitrofurazone. The test agents included ultraviolet radiation, five radiomimetic compounds, and penicillin; 12 different types of mutants could be selected. Two of these were chemoresistant, three were identical to radioresistant types previously isolated by use of other radiomimetic agents, and seven represented previously unobserved radioresistant types. The majority of radioresistant strains did not respond to plating-medium reactivation after ultraviolet radiation, despite the ultraviolet radiation responses of several minority representative strains. The data presented indicate that radioresistance in E. coli S does not involve resistance to most toxic agents; on the other hand, cross resistance to radiomimetic compounds is not restricted to alkylating agents. PMID:14047210

  11. Comparative mutant prevention concentration and antibacterial activity of fluoroquinolones against Escherichia coli in diarrheic buffalo calves.

    PubMed

    Beri, Supriya; Sidhu, Pritam K; Kaur, Gurpreet; Chandra, Mudit; Rampal, Satyavan

    2015-10-01

    Owing to emerging threat of antimicrobial resistance, mutant prevention concentration (MPC) is considered as an important parameter to evaluate the antimicrobials for their capacity to restrict/allow the emergence of resistant mutants. Therefore, MPCs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin, and norfloxacin were determined against Escherichia coli isolates of diarrheic buffalo calves. The minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) were also established. The MICs of ciprofloxacin, enrofloxacin, levofloxacin, moxifloxacin and norfloxacin were 0·009, 0·022, 0·024, 0·028, and 0·036 μg/ml, respectively. The MBCs obtained were very close to the MICs of respective drugs that suggested a bactericidal mode of action of antimicrobials. The MPCs (μg/ml) of ciprofloxacin (4·2×MIC), moxifloxacin (4·8×MIC), and norfloxacin (5·1×MIC) were approximately equal but slightly lower than enrofloxacin (7·6×MIC) and levofloxacin (8·5×MIC) against clinical isolates of E. coli. The MPC data suggested that enrofloxacin has the potential for restricting the selection of E. coli mutants during treatment at appropriate dosing.

  12. Functional interaction between bases C1049 in domain II and G2751 in domain VI of 23S rRNA in Escherichia coli ribosomes

    PubMed Central

    Miyoshi, Tomohiro; Uchiumi, Toshio

    2008-01-01

    The factor-binding center within the Escherichia coli ribosome is comprised of two discrete domains of 23S rRNA: the GTPase-associated region (GAR) in domain II and the sarcin–ricin loop in domain VI. These two regions appear to collaborate in the factor-dependent events that occur during protein synthesis. Current X-ray crystallography of the ribosome shows an interaction between C1049 in the GAR and G2751 in domain VI. We have confirmed this interaction by site-directed mutagenesis and chemical probing. Disruption of this base pair affected not only the chemical modification of some bases in domains II and VI and in helix H89 of domain V, but also ribosome function dependent on both EF-G and EF-Tu. Mutant ribosomes carrying the C1049 to G substitution, which show enhancement of chemical modification at G2751, were used to probe the interactions between the regions around 1049 and 2751. Binding of EF-G-GDP-fusidic acid, but not EF-G-GMP-PNP, to the ribosome protected G2751 from modification. The G2751 protection was also observed after tRNA binding to the ribosomal P and E sites. The results suggest that the interactions between the bases around 1049 and 2751 alter during different stages of the translation process. PMID:18252772

  13. Escherichia coli mar and acrAB mutants display no tolerance to simple alcohols.

    PubMed

    Ankarloo, Jonas; Wikman, Susanne; Nicholls, Ian A

    2010-03-31

    The inducible Mar phenotype of Escherichia coli is associated with increased tolerance to multiple hydrophobic antibiotics as well as some highly hydrophobic organic solvents such as cyclohexane, mediated mainly through the AcrAB/TolC efflux system. The influence of water miscible alcohols ethanol and 1-propanol on a Mar constitutive mutant and a mar deletion mutant of E. coli K-12, as well as the corresponding strains carrying the additional acrAB deletion, was investigated. In contrast to hydrophobic solvents, all strains were killed in exponential phase by 1-propanol and ethanol at rates comparable to the parent strain. Thus, the Mar phenotype does not protect E. coli from killing by these more polar solvents. Surprisingly, AcrAB does not contribute to an increased alcohol tolerance. In addition, sodium salicylate, at concentrations known to induce the mar operon, was unable to increase 1-propanol or ethanol tolerance. Rather, the toxicity of both solvents was increased in the presence of sodium salicylate. Collectively, the results imply that the resilience of E. coli to water miscible alcohols, in contrast to more hydrophobic solvents, does not depend upon the AcrAB/TolC efflux system, and suggests a lower limit for substrate molecular size and functionality. Implications for the application of microbiological systems in environments containing high contents of water miscible organic solvents, e.g., phage display screening, are discussed.

  14. Isolation and characterization of Escherichia coli mutants defective for phenylpropionate degradation.

    PubMed Central

    Burlingame, R P; Wyman, L; Chapman, P J

    1986-01-01

    Mutants of Escherichia coli defective in catabolism of 3-phenylpropionate, 3-(3-hydroxyphenyl)propionate, or both were isolated after mutagenesis with ethylmethane sulfonate. Nine phenotypically distinct classes of mutants were identified, including strains lacking each of the first five enzyme activities for the degradation of these compounds and mutants pleiotropically negative for some of these activities. Characterization of these mutants was greatly facilitated by the use of indicator media in which accumulation of 3-(2,3-dihydroxyphenyl)propionate or 2-hydroxy-6-ketononadienedioic acid led to the formation of dark red or bright yellow colors, respectively, in the medium. Assays with wild-type and mutant strains indicated that 3-phenylpropionate (or its dihydrodiol), but none of the hydroxylated derivatives tested, induced the synthesis of enzymes for its conversion to 3-(2,3-dihydroxyphenyl)propionate. The remaining enzymes were induced by the 2- or 3-hydroxy or 2,3-dihydroxy derivatives of 3-phenylpropionate, with the 2-hydroxy compound acting as an apparent gratuitous inducer. Metabolism to nonaromatic intermediates appeared to be unnecessary for full induction of any pathway enzyme. One unusual class of mutants, in which 2-keto-4-pentenoate hydratase appeared to be uninducible, indicated a level of control not previously shown in meta-fission catabolic pathways. PMID:3531186

  15. Minicell-forming mutants of Escherichia coli: production of minicells and anucleate rods.

    PubMed Central

    Jaffé, A; D'Ari, R; Hiraga, S

    1988-01-01

    The Escherichia coli minB mutant originally isolated is known to septate at cell poles to form spherical anucleate minicells. Three new minicell-producing mutants were isolated during a screening by autoradiography for chromosome partition mutants giving rise spontaneously to normal-sized anucleate cells. These min mutants were affected close to or in the minB locus. Autoradiography analysis as well as fluorescent staining of DNA showed that in addition to minicells, these strains and the original minB mutant also spontaneously produced anucleate rods of normal size and had an abnormal DNA distribution in filaments. These aberrations were not associated with spontaneous induction of the SOS response. Inhibition of DNA synthesis in these mutants gave rise to anucleate cells whose size was longer than unit cell length, suggesting that the min defect allows septation to take place at normally forbidden sites not only at cell poles but also far from poles. Abnormal DNA distribution and production of anucleate rods suggest that the Min product(s) could be involved in DNA distribution. Images PMID:2838458

  16. Biochemical characterization of the chlB mutant of E. coli

    SciTech Connect

    Johnson, J.L.; Indermauer, L.W.; Rajagopalan, K.V. )

    1991-03-11

    The chlorate resistant mutants of E. coli exhibit a pleiotropic loss of the activities of several molybdoenzymes suggestive of defective molybdenum cofactor synthesis. Indeed, mutants at the chlA and chlE loci have been shown to be deficient in molybdenum cofactor. ChlB mutants, on the other hand, contain high levels of molybdenum cofactor as measured by conversion to the Form A derivative and by reconstitution of the nitrate reductase in the high molecular weight fraction of extracts of the Neurospora crassa nit-1 mutant. The recent discovery that the molybdenum cofactors of E. coli nitrate reductase and formate dehydrogenase contain molybdopterin guanine dinucleotide (MGD) rather than the simpler molybdopterin (MPT) raised the possibility that the chlB locus could be essential for the biosynthesis of MGD from MPT. To test this, conditions were devised for conversion of MGD to a fluorescent, stable derivative, Form A-GMP, and the absorption, fluorescence and chromatographic properties of Form A-GMP were established. Both Form A, arising from MPT, and Form A-GMP arising from MGD, were quantitated in extracts of wild type and chlB cells. Wild type cells were found to contain both Form A and Form A-GMP. In contrast, chlB cells contained elevated levels of Form A but no Form A-GMP. These results suggest that the chlB gene product is essential for the conversion of MPT to MGD.

  17. Genetic and biochemical characterization of periplasmic-leaky mutants of Escherichia coli K-12.

    PubMed Central

    Lazzaroni, J C; Portalier, R C

    1981-01-01

    Periplasmic-leaky mutants of Escherichia coli K-12 were isolated after nitrosoguanidine-induced mutagenesis. They released periplasmic enzymes into the extracellular medium. Excretion of alkaline phosphatase, which started immediately in the early exponential phase of growth, could reach up to 90% of the total enzyme production in the stationary phase. Leaky mutants were sensitive to ethylenediaminetetraacetic acid, cholic acid, and the antibiotics rifampin, chloramphenicol, mitomycin C, and ampicillin. Furthermore, they were resistant to colicin E1 and partially resistant to phage TuLa. Their genetic characterization showed that the lky mutations mapped between the suc and gal markers, near or in the tolPAB locus. A biochemical analysis of cell envelope components showed that periplasmic-leaky mutants contained reduced amounts of major outer membrane protein OmpF and increased amounts of a 16,000-dalton outer membrane protein. Images PMID:7009581

  18. Phage Genetic Sites Involved in λ Growth Inhibition by the Escherichia Coli Rap Mutant

    PubMed Central

    Guzman, P.; Guarneros, G.

    1989-01-01

    The rap mutation of Escherichia coli prevents the growth of bacteriophage λ. We have isolated phage mutants that compensate for the host deficiency. The mutations, named bar, were genetically located to three different loci of the λ genome: barI in the attP site, barII in the cIII ea10 region, and barIII within or very near the imm434 region. The level of λ leftward transcription correlates with rap exclusion. Phage λ mutants partially defective in the pL promoter or in pL-transcript antitermination showed a Bar(-) phenotype. Conversely, mutants constitutive for transcription from the pI or pL promoters were excluded more stringently by rap bacteria. We conclude that rap exclusion depends on the magnitude of transcription through the wild type bar loci in the phage genome. PMID:2523838

  19. Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

    PubMed Central

    Noguera, Martín E.; Vazquez, Diego S.; Ferrer-Sueta, Gerardo; Agudelo, William A.; Howard, Eduardo; Rasia, Rodolfo M.; Manta, Bruno; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

    2017-01-01

    Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity. PMID:28181556

  20. Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants.

    PubMed

    Noguera, Martín E; Vazquez, Diego S; Ferrer-Sueta, Gerardo; Agudelo, William A; Howard, Eduardo; Rasia, Rodolfo M; Manta, Bruno; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

    2017-02-09

    Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

  1. Structural variability of E. coli thioredoxin captured in the crystal structures of single-point mutants

    NASA Astrophysics Data System (ADS)

    Noguera, Martín E.; Vazquez, Diego S.; Ferrer-Sueta, Gerardo; Agudelo, William A.; Howard, Eduardo; Rasia, Rodolfo M.; Manta, Bruno; Cousido-Siah, Alexandra; Mitschler, André; Podjarny, Alberto; Santos, Javier

    2017-02-01

    Thioredoxin is a ubiquitous small protein that catalyzes redox reactions of protein thiols. Additionally, thioredoxin from E. coli (EcTRX) is a widely-used model for structure-function studies. In a previous paper, we characterized several single-point mutants of the C-terminal helix (CTH) that alter global stability of EcTRX. However, spectroscopic signatures and enzymatic activity for some of these mutants were found essentially unaffected. A comprehensive structural characterization at the atomic level of these near-invariant mutants can provide detailed information about structural variability of EcTRX. We address this point through the determination of the crystal structures of four point-mutants, whose mutations occurs within or near the CTH, namely L94A, E101G, N106A and L107A. These structures are mostly unaffected compared with the wild-type variant. Notably, the E101G mutant presents a large region with two alternative traces for the backbone of the same chain. It represents a significant shift in backbone positions. Enzymatic activity measurements and conformational dynamics studies monitored by NMR and molecular dynamic simulations show that E101G mutation results in a small effect in the structural features of the protein. We hypothesize that these alternative conformations represent samples of the native-state ensemble of EcTRX, specifically the magnitude and location of conformational heterogeneity.

  2. Efficient hammerhead ribozyme and antisense RNA targeting in a slow ribosome Escherichia coli mutant.

    PubMed

    Chen, H; Ferbeyre, G; Cedergren, R

    1997-05-01

    We have evaluated inhibition of the plasmid-born chloramphenicol acetyl transferase gene (CAT) by the hammerhead ribozyme and antisense RNA in Escherichia coli where the translation and transcription rates have been modified. Whereas neither antisense nor the hammerhead had an inhibitory effect on CAT activity in wild-type E. coli, both reduced the level of the messenger RNA and the activity of the CAT gene by almost 60% in a slow ribosome mutant. Streptomycin, which increases the speed of translation in this mutant strain, restored full CAT activity. The level of CAT activity expressed from a T7 RNA polymerase promoter was not affected by the presence of either antisense RNA or the hammerhead ribozyme. When the target gene was expressed from a chromosomal locus in wild-type E. coli, both antisense RNA and the hammerhead ribozyme showed some inhibitory activity, but the level of inhibition was significantly increased in the slow ribosome strain. This bacterial system offers a unique entry to the study of cellular factors which mediate the activity of ribozymes in vivo.

  3. Adhesion of Type 1-Fimbriated Escherichia coli to Abiotic Surfaces Leads to Altered Composition of Outer Membrane Proteins

    PubMed Central

    Otto, Karen; Norbeck, Joakim; Larsson, Thomas; Karlsson, Karl-Anders; Hermansson, Malte

    2001-01-01

    Phenotypic differences between planktonic bacteria and those attached to abiotic surfaces exist, but the mechanisms involved in the adhesion response of bacteria are not well understood. By the use of two-dimensional (2D) polyacrylamide gel electrophoresis, we have demonstrated that attachment of Escherichia coli to abiotic surfaces leads to alteration in the composition of outer membrane proteins. A major decrease in the abundance of resolved proteins was observed during adhesion of type 1-fimbriated E. coli strains, which was at least partly caused by proteolysis. Moreover, a study of fimbriated and nonfimbriated mutants revealed that these changes were due mainly to type 1 fimbria-mediated surface contact and that only a few changes occurred in the outer membranes of nonfimbriated mutant strains. Protein synthesis and proteolytic degradation were involved to different extents in adhesion of fimbriated and nonfimbriated cells. While protein synthesis appeared to affect adhesion of only the nonfimbriated strain, proteolytic activity mostly seemed to contribute to adhesion of the fimbriated strain. Using matrix-assisted laser desorption ionization–time of flight mass spectrometry, six of the proteins resolved by 2D analysis were identified as BtuB, EF-Tu, OmpA, OmpX, Slp, and TolC. While the first two proteins were unaffected by adhesion, the levels of the last four were moderately to strongly reduced. Based on the present results, it may be suggested that physical interactions between type 1 fimbriae and the surface are part of a surface-sensing mechanism in which protein turnover may contribute to the observed change in composition of outer membrane proteins. This change alters the surface characteristics of the cell envelope and may thus influence adhesion. PMID:11274103

  4. Complementation of an Escherichia coli pyrF mutant with DNA from Desulfovibrio vulgaris

    SciTech Connect

    Li, C.; Peck, H.D. Jr.; Przybyla, A.E.

    1986-02-01

    A PyrF/sup -/ mutant of Escherichia coli (SK1108, pyrF::Tn5 Kan/sup r/) was complemented with the Desulfovibrio vulgaris (Hildenborough) structural gene for orotidine-5'-phosphate decarboxylase. Either orientation of a 1.6-kilobase-pair D. vulgaris DNA fragment (pLP3B or pLP3A) complemented the PyrF/sup -/ strain suggesting that the D. vulgaris pyrF promoter was functional. The apparent product of the D. vulgaris pyrF gene was a single 26-kilodalton polypeptide. These results demonstrate the utility of E. coli cloning systems in studying metabolic and energetic pathways in sulfate-reducing bacteria.

  5. A novel fermentation pathway in an Escherichia coli mutant producing succinic acid, acetic acid, and ethanol.

    SciTech Connect

    Donnelly, M. I.; Millard, C. S.; Clark, D. P.; Chen, M. J.; Rathke, J. W.; Southern Illinois Univ.

    1998-04-01

    Escherichia coli strain NZN111, which is unable to grow fermentatively because of insertional inactivation of the genes encoding pyruvate: formate lyase and the fermentative lactate dehydrogenase, gave rise spontaneously to a chromosomal mutation that restored its ability to ferment glucose. The mutant strain, named AFP111, fermented glucose more slowly than did its wild-type ancestor, strain W1485, and generated a very different spectrum of products. AFP111 produced succinic acid, acetic acid, and ethanol in proportions of approx 2:1:1. Calculations of carbon and electron balances accounted fully for the observed products; 1 mol of glucose was converted to 1 mol of succinic acid and 0.5 mol each of acetic acid and ethanol. The data support the emergence in E.coli of a novel succinic acid:acetic acid:ethanol fermentation pathway.

  6. [The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants].

    PubMed

    Doroshenko, V G; Lobanov, A O; Fedorina, E A

    2013-01-01

    Strain MG 1655+hisGr hisL'-Delta, purR, which produces histidine with a weight yield of approximately 12% from glucose, was constructed through directed chromosomal modifications of the laboratory Escherichia coli strain MG 1655+, which has a known genome sequence. A feedback-resistant ATP-phosphoribosyl transferase encoded by the mutant hisGr (E271 K) was the main determinant of histidine production. A further increase in histidine production was achieved by the expression enhance of a mutant his operon containing hisGr through the deleting attenuator region (hisL'-Delta). An increase in the expression of the wildtype his operon did not result in histidine accumulation. Deletion of the transcriptional regulator gene purR increased the biomass produced and maintained the level of histidine production per cell under the fermentation conditions used.

  7. Measuring cell wall elasticity on enteroaggregative Escherichia coli wild type and dispersin mutant by AFM

    SciTech Connect

    Beckmann, Melissa; Venkataraman, Sankar; Doktycz, Mitchel John; Nataro, James P; Sullivan, Claretta J; Morrell-Falvey, Jennifer L; Allison, David P

    2006-07-01

    Enteroaggregative Escherichia coli (EAEC) is pathogenic and produces severe diarrhea in humans. A mutant of EAEC that does not produce dispersin, a cell surface protein, is not pathogenic. It has been proposed that dispersin imparts a positive charge to the bacterial cell surface allowing the bacteria to colonize on the negatively charged intestinal mucosa. However, physical properties of the bacterial cell surface, such as rigidity, may be influenced by the presence of dispersin and may contribute to pathogenicity. Using the system developed in our laboratory for mounting and imaging bacterial cells by atomic force microscopy (AFM), in liquid, on gelatin coated mica surfaces, studies were initiated to measure cell surface elasticity. This was carried out in both wild type EAEC, that produces dispersin, and the mutant that does not produce dispersin. This was accomplished using AFM force-distance (FD) spectroscopy on the wild type and mutant grown in liquid or on solid medium. Images in liquid and in air of both the wild-type and mutant grown in liquid and on solid media are presented. This work represents an initial step in efforts to understand the pathogenic role of the dispersin protein in the wild-type bacteria.

  8. Elucidating the genetic basis for Escherichia coli defense against silver toxicity using mutant arrays.

    PubMed

    Xiu, Zongming; Liu, Yuanyuan; Mathieu, Jacques; Wang, Jing; Zhu, Dongqiang; Alvarez, Pedro J J

    2014-05-01

    Bacterial adaptation and defense mechanisms against silver are poorly understood at the genetic level. A library of Escherichia coli gene-deletion mutants was used to show that clones lacking sodB (coding for oxidative stress protection), lon (protein damage repair), or cusR (metal efflux pump) are quite sensitive to silver (with 7.3 ± 9.1%, 5.3 ± 1.8%, and 0.4 ± 0.1% of cells surviving, respectively, compared with 90.1 ± 5.4% survival for wild-type E. coli, after 6-h exposure to 8 mg/L AgNO(3)), suggesting the importance of the coded functions as defense mechanisms. Mutants lacking pgaB or wcaD, which code for production of extracellular polymeric substances (EPS), also showed significant (p < 0.05) sensitivity to silver exposure (23.4 ± 16.2% and 23.1 ± 32.6% survival, respectively). Transmission electron microscopy (TEM) with scanning TEM/energy-dispersive X-ray spectroscopy analysis showed accumulation of silver nanoparticles within EPS, suggesting that EPS serve as a protective barrier that also immobilizes dissolved silver as silver nanoparticles.

  9. Recombinant levels of Escherichia coli K-12 mutants deficient in various replication, recombination, or repair genes.

    PubMed Central

    Zieg, J; Maples, V F; Kushner, S R

    1978-01-01

    Escherichia coli strains containing mutations in lexA, rep, uvrA, uvrD, uvrE, lig, polA, dam, or xthA were constructed and tested for conjugation and transduction proficiencies and ability to form Lac+ recombinants in an assay system utilizing a nontandem duplication of two partially deleted lactose operons (lacMS286phi80dIIlacBK1). lexA and rep mutants were as deficient (20% of wild type) as recB and recC strains in their ability to produce Lac+ progeny. All the other strains exhibited increased frequencies of Lac+ recombinant formation, compared with wild type, ranging from 2- to 13-fold. Some strains showed markedly increased conjugation proficiency (dam uvrD) compared to wild type, while others appeared deficient (polA107). Some differences in transduction proficiency were also observed. Analysis of the Lac+ recombinants formed by the various mutants indicated that they were identical to the recombinants formed by a wild-type strain. The results indicate that genetic recombination in E. coli is a highly regulated process involving multiple gene products. PMID:350859

  10. Heterologous Expression of Der Homologs in an Escherichia coli der Mutant and Their Functional Complementation

    PubMed Central

    Choi, Eunsil; Kang, Nalae; Jeon, Young; Pai, Hyun-Sook

    2016-01-01

    clarified. In this study, we used five Der homologs from gammaproteobacteria, pathogenic bacteria, and an extremophile to elucidate their conserved function in 50S ribosomal subunit biogenesis. Among them, Klebsiella pneumoniae and Salmonella enterica serovar Typhimurium Der homologs implicated the participation of Der in ribosome assembly in E. coli. Our results show that the linker and C-terminal regions of Der homologs are correlated with its functional complementation in E. coli der mutants, suggesting that they are involved in species-specific recognition or interaction with 50S ribosomal subunits. PMID:27297882

  11. An Escherichia coli mutant resistant to phleomycin, bleomycin, and heat inactivation is defective in ubiquinone synthesis.

    PubMed Central

    Collis, C M; Grigg, G W

    1989-01-01

    A mutant of Escherichia coli, selected for resistance to the antibiotic and antitumor agent phleomycin, has been characterized, and the phleomycin resistance determinant has been identified. The mutant is equally resistant to bleomycins. The resistance to phleomycin is strongly dependent on the nature of the C-terminal amine of the drug, with the greatest resistance being shown to phleomycins and bleomycins with the most basic terminal amines. The mutation also confers resistance to the lethal effects of heating at 52 degrees C. Other characteristics of the phleomycin-resistant strain include a slow growth rate, an inability to grow on succinate as the sole carbon source (Suc- phenotype), cross resistance to aminoglycoside antibiotics, and a slight sensitivity to hydrogen peroxide, methyl methanesulfonate, and gamma-irradiation. Some of these characteristics, together with mapping data, suggested that the phleomycin resistance and Suc- determinant probably lies within the ubiF gene coding for an enzyme effecting a step in the biosynthesis of ubiquinone. The phenotypes of known mutants defective in this and other steps of the ubiquinone pathway were found to be closely similar to those of the original phleomycin-resistant strain. PMID:2475481

  12. Metabolic engineering of Escherichia coli: construction and characterization of a gltA (citrate synthase) knockout mutant.

    PubMed

    Vandedrinck, S; Deschamps, G; Sablon, E; Vandamme, E J

    2001-01-01

    E. coli is one of the most important host organisms for recombinant protein production. However, growth and recombinant protein production can be limited by acetate accumulation during high-cell-density fermentations. Some of the strategies used to overcome this problem are based on the alteration of the genotype of the host. This paper discusses the construction and characterization of an E. coli gltA- knockout mutant. The knockout of the gene was confirmed by the loss of citrate synthase activity in an enzyme assay. Also the growth rate of the mutant on Luria Broth and Luria Broth + acetate was reduced.

  13. Draft Genome Sequence of Escherichia coli O157:H7 ATCC 35150 and a Nalidixic Acid-Resistant Mutant Derivative

    PubMed Central

    Markell, James A.; Koziol, Adam G.

    2015-01-01

    Shiga toxin-producing Escherichia coli strains, occasionally isolated from food, are of public health importance. Here, we report on the 5.30-Mbp draft genome sequence of E. coli O157:H7 EDL931 (strain ATCC 35150) and the 5.32-Mbp draft genome sequence of a nalidixic acid-resistant mutant derivative used as a distinguishable control strain in food-testing laboratories. PMID:26205873

  14. Enhanced Biofilm Formation by Escherichia coli LPS Mutants Defective in Hep Biosynthesis

    PubMed Central

    Nakao, Ryoma; Ramstedt, Madeleine; Wai, Sun Nyunt; Uhlin, Bernt Eric

    2012-01-01

    Lipopolysaccharide (LPS) is the major component of the surface of Gram-negative bacteria and its polysaccharide portion is situated at the outermost region. We investigated the relationship between the polysaccharide portion of LPS and biofilm formation using a series of Escherichia coli mutants defective in genes earlier shown to affect the LPS sugar compositions. Biofilm formation by a deep rough LPS mutant, the hldE strain, was strongly enhanced in comparison with the parental strain and other LPS mutants. The hldE strain also showed a phenotype of increased auto-aggregation and stronger cell surface hydrophobicity compared to the wild-type. Similar results were obtained with another deep rough LPS mutant, the waaC strain whose LPS showed same molecular mass as that of the hldE strain. Confocal laser scanning microscopy (CLSM) analysis and biofilm formation assay using DNase I revealed that biofilm formation by the hldE strain was dependent on extracellular DNA. Furthermore, a loss of flagella and an increase in amount of outer membrane vesicles in case of the hldE strain were also observed by transmission electron microscopy and atomic force microscopy, respectively. In addition, we demonstrated that a mutation in the hldE locus, which alters the LPS structure, caused changes in both expression and properties of several surface bacterial factors involved in biofilm formation and virulence. We suggest that the implication of these results should be considered in the context of biofilm formation on abiotic surfaces, which is frequently associated with nosocominal infections such as the catheter-associated infections. PMID:23284671

  15. Isolation and characterization of the E. coli membrane protein production strain Mutant56(DE3)

    PubMed Central

    Baumgarten, Thomas; Schlegel, Susan; Wagner, Samuel; Löw, Mirjam; Eriksson, Jonas; Bonde, Ida; Herrgård, Markus J.; Heipieper, Hermann J.; Nørholm, Morten H. H.; Slotboom, Dirk Jan; de Gier, Jan-Willem

    2017-01-01

    Membrane protein production is usually toxic to E. coli. However, using genetic screens strains can be isolated in which the toxicity of membrane protein production is reduced, thereby improving production yields. Best known examples are the C41(DE3) and C43(DE3) strains, which are both derived from the T7 RNA polymerase (P)-based BL21(DE3) protein production strain. In C41(DE3) and C43(DE3) mutations lowering t7rnap expression levels result in strongly reduced T7 RNAP accumulation levels. As a consequence membrane protein production stress is alleviated in the C41(DE3) and C43(DE3) strains, thereby increasing membrane protein yields. Here, we isolated Mutant56(DE3) from BL21(DE3) using a genetic screen designed to isolate BL21(DE3)-derived strains with mutations alleviating membrane protein production stress other than the ones in C41(DE3) and C43(DE3). The defining mutation of Mutant56(DE3) changes one amino acid in its T7 RNAP, which weakens the binding of the T7 RNAP to the T7 promoter governing target gene expression rather than lowering T7 RNAP levels. For most membrane proteins tested yields in Mutant56(DE3) were considerably higher than in C41(DE3) and C43(DE3). Thus, the isolation of Mutant56(DE3) shows that the evolution of BL21(DE3) can be promoted towards further enhanced membrane protein production. PMID:28338018

  16. Escherichia coli pleiotropic mutant that reduces amounts of several periplasmic and outer membrane proteins.

    PubMed Central

    Wanner, B L; Sarthy, A; Beckwith, J

    1979-01-01

    We have isolated a mutant of Escherichia coli K-12 that is reduced from 6- to 10-fold in the amount of alkaline phosphatase found in the periplasmic space. The reduced synthesis is not due to effects at the level of transcription regulation of the phoA gene, the structural gene for the enzyme. In addition, the mutation (termed perA) responsible for this phenotype results in reduced amounts of possibly six or more other periplasmic proteins and at least three outer membrane proteins. One of the outer membrane proteins affected is protein IA (D. L. Diedrich, A. O. Summers, and C. A. Schnaitman, J. Bacteriol. 131:598-607, 1977). Although other possibilities exist, one explanation for the phenotype of the perA mutation is that it affects the cell's secretory apparatus. Images PMID:387722

  17. Isolating tryptophan regulatory mutants in Escherichia coli by using a trp-lac fusion strain.

    PubMed

    Reznikoff, W S; Thornton, K P

    1972-02-01

    A trp-lac fusion strain of Escherichia coli in which the lac structural genes are part of the tryptophan operon has been used to isolate trp regulatory mutants. This was accomplished by isolating lac(+) colonies on either lactose-minimal agar or lactose-MacConkey indicator agar. Seventy-seven of 78 lac(+) isolates contained mutations which mapped near the ara locus and most of these isolates were found to be 5-methyltryptophan-resistant after introduction of an F-trp episome. The lac(+) phenotypes of these 77 isolates were therefore probably the result of trpR(-) mutations. The one remaining isolate carried a mutation which was not part of the trp regulatory system.

  18. Selective targeting of mutant adenomatous polyposis coli (APC) in colorectal cancer.

    PubMed

    Zhang, Lu; Theodoropoulos, Panayotis C; Eskiocak, Ugur; Wang, Wentian; Moon, Young-Ah; Posner, Bruce; Williams, Noelle S; Wright, Woodring E; Kim, Sang Bum; Nijhawan, Deepak; De Brabander, Jef K; Shay, Jerry W

    2016-10-19

    Mutations in the adenomatous polyposis coli (APC) gene are common in colorectal cancer (CRC), and more than 90% of those mutations generate stable truncated gene products. We describe a chemical screen using normal human colonic epithelial cells (HCECs) and a series of oncogenically progressed HCECs containing a truncated APC protein. With this screen, we identified a small molecule, TASIN-1 (truncated APC selective inhibitor-1), that specifically kills cells with APC truncations but spares normal and cancer cells with wild-type APC. TASIN-1 exerts its cytotoxic effects through inhibition of cholesterol biosynthesis. In vivo administration of TASIN-1 inhibits tumor growth of CRC cells with truncated APC but not APC wild-type CRC cells in xenograft models and in a genetically engineered CRC mouse model with minimal toxicity. TASIN-1 represents a potential therapeutic strategy for prevention and intervention in CRC with mutant APC.

  19. Escherichia coli mutants deficient in the production of alkaline phosphatase isozymes.

    PubMed Central

    Nakata, A; Yamaguchi, M; Izutani, K; Amemura, M

    1978-01-01

    Escherichia coli K-12 mutants showing an altered isozyme pattern of alkaline phosphatase were isolated. Whereas wild-type strains synthesized all three isozymes in a synthetic medium supplemented with Casamino Acids or arginine but synthesized only isozyme 3 in a medium without supplement, the mutant strains synthesized isozyme 1 and a small amount (if any) of isozyme 2, but no isozyme 3, under all growth conditions. The mutation responsible for the altered isozyme pattern, designated iap, was mapped by P1 transduction in the interval between cysC and srl (at about 58.5 min on the E. coli genetic map). It was cotransducible with cysC and srl at frequencies of 0.54 and 0.08, respectively. The order of the genes in this region was srl-iap-cysC-argA-thyA-lysA. Three more independent mutations were also mapped in the same locus. We purified isozymes 1' and 3' from iap and iap+ strains and analyzed the sequences of four amino acids from the amino terminus of each polypeptide. They were Arg-Thr-Pro-Glu (or Gln) in isozyme 1' and Thr-Pro-Glu (or gln)-Met in isozyme 3', which were identical with those of corresponding isozymes produced by the wild-type phoA+ strain (P.M. Kelley, P.A. Neumann, K. Schriefer, F. Cancedda, M.J. Schlesinger, and R.A. Bradshaw, Biochemistry 12:3499-3503, 1973; M.J. Schlesinger, W. Bloch, and P.M. Kelley, p. 333-342, in Isozymes, Academic Press Inc., 1975). These results indicate that the different mobilities of isozymes 1, 2, and 3 are determined by the presence or absence of amino-terminal arginine residues in polypeptides. Images PMID:348683

  20. Fluorescent Trimethoprim Conjugate Probes To Assess Drug Accumulation in Wild Type and Mutant Escherichia coli

    PubMed Central

    2016-01-01

    Reduced susceptibility to antimicrobials in Gram-negative bacteria may result from multiple resistance mechanisms, including increased efflux pump activity or reduced porin protein expression. Up-regulation of the efflux pump system is closely associated with multidrug resistance (MDR). To help investigate the role of efflux pumps on compound accumulation, a fluorescence-based assay was developed using fluorescent derivatives of trimethoprim (TMP), a broad-spectrum synthetic antibiotic that inhibits an intracellular target, dihydrofolate reductase (DHFR). Novel fluorescent TMP probes inhibited eDHFR activity with comparable potency to TMP, but did not kill or inhibit growth of wild type Escherichia coli. However, bactericidal activity was observed against an efflux pump deficient E. coli mutant strain (ΔtolC). A simple and quick fluorescence assay was developed to measure cellular accumulation of the TMP probe using either fluorescence spectroscopy or flow cytometry, with validation by LC-MS/MS. This fluorescence assay may provide a simple method to assess efflux pump activity with standard laboratory equipment. PMID:27737551

  1. Functional and immunochemical characterization of a mutant of Escherichia coli energy uncoupled for lactose transport

    SciTech Connect

    Herzlinger, D.; Carrasco, N.; Kaback, H.R.

    1985-01-01

    Right-side-out cytoplasmic membrane vesicles from Escherichia coli ML 308-22, a mutant ''uncoupled'' for beta-galactoside/H/sup +/ symport are specifically defective in the ability to catalyze accumulation of methyl 1-thio-beta-D-galactopyranoside (TMG) in the presence of an H/sup +/ electrochemical gradient (interior negative and alkaline). Furthermore, the rate of carrier-mediated efflux under nonenergized conditions is slow and unaffected by ambient pH from pH 5.5 to 7.5, and TMG-induced H/sup +/ influx is only about 15% of that observed in vesicles containing wild-type lac permease (ML 308-225). Alternatively, ML 308-22 vesicles bind p-nitrophenyl alpha-D-galactopyranoside and monoclonal antibody 4B1 to the same extent as ML 308-225 vesicles and catalyze facilitated diffusion and equilibrium exchange as well as ML 308-225 vesicles. When entrance counterflow is studied with external substrate at saturating and subsaturating concentrations, it is apparent that the mutation simulates the effects of deuterium oxide. That is, the mutation has no effect on the rate or extent of counterflow when external substrate is saturating but stimulates the efficiency of counterflow when external substrate is below the apparent K/sub m/. Moreover, although replacement of protium with deuterium stimulates counterflow in ML 308-225 vesicles when external substrate is subsaturating, the isotope has no effect on the mutant vesicles under the same conditions.

  2. Mutant DnaK chaperones cause ribosome assembly defects in Escherichia coli.

    PubMed Central

    Alix, J H; Guérin, M F

    1993-01-01

    To determine whether the biogenesis of ribosomes in Escherichia coli is the result of the self-assembly of their different constituents or involves the participation of additional factors, we have studied the influence of a chaperone, the product of the gene dnaK, on ribosome assembly in vivo. Using three thermosensitive (ts) mutants carrying the mutations dnaK756-ts, dnaK25-ts, and dnaK103-ts, we have observed the accumulation at nonpermissive temperature (45 degrees C) of ribosomal particles with different sedimentation constants--namely, 45S, 35S, and 25S along with the normal 30S and 50S ribosomal subunits. This is the result of a defect not in thermostability but in ribosome assembly at the nonpermissive temperature. These abnormal ribosomal particles are rescued if the mutant cells are returned to 30 degrees C. Thus, the product of the dnaK gene is implicated in ribosome biogenesis at high temperature. PMID:8105482

  3. Lethal action of quinolones against a temperature-sensitive dnaB replication mutant of Escherichia coli.

    PubMed

    Zhao, Xilin; Malik, Muhammad; Chan, Nymph; Drlica-Wagner, Alex; Wang, Jian-Ying; Li, Xinying; Drlica, Karl

    2006-01-01

    Inhibition of DNA replication in an Escherichia coli dnaB-22 mutant failed to block quinolone-mediated lethality. Inhibition of protein synthesis by chloramphenicol inhibited nalidixic acid lethality and, to a lesser extent, ciprofloxacin lethality in both dnaB-22 and wild-type cells. Thus, major features of quinolone-mediated lethality do not depend on ongoing replication.

  4. [Repression of the enzyme inducible syntheses in Escherichia coli K12 mutant with a deleted ptsH gene].

    PubMed

    Gershanovich, V N; Il'ina, T S; Rusina, O Iu; Iurovitskaia, N V; Bol'shakova, T N

    1977-01-01

    The genome of lambda phage with thermosensitive repressor was integrated into the pts region of the E. coli chromosome. Such a lysogenic culture behaves as a pts mutant at 30 degrees. Heating of cells of this strain leads to the induction of lambda prophage and formation of deletions in the pts region. A mutant with a deletion covering ptsH gene was isolated after prophage induction. The deletion nature of pts mutation was confirmed in genetic and biochemical experiments. It was shown that the deletion is small and does not involve ptsI and lig genes. The isolated deltaptsH mutant possesses all characteristics of pts mutants: pleiotropic impairment of transport and utilization of a number of carbohydrates, repression of the enzyme inducible synthesis and resistance to catabolite repression with glucose. These data (together with earlier ones) allow us to conclude that the phosphorylated form of HPr is involved (in direct of indirect manner/ in activation of DNA transcription.

  5. Reduced hydroperoxidase (HPI and HPII) activity in the Deltafur mutant contributes to increased sensitivity to UVA radiation in Escherichia coli.

    PubMed

    Hoerter, James D; Arnold, Alan A; Ward, Christopher S; Sauer, Michael; Johnson, Steve; Fleming, Todd; Eisenstark, Abraham

    2005-05-13

    In Escherichia coli, Deltafur (ferric uptake regulator) mutants are hypersensitive to various oxidative agents, including UVA radiation (400-315 nm). Studies suggest that UVA radiation mediates its biological effects on bacteria via oxidative mechanisms that lead to reactive oxygen species, including the superoxide anion radical (O2.-), hydroxyl radical (HO.), hydrogen peroxide (H2O2) and singlet oxygen (1O2). There is accumulating evidence that Fur may play an important role in the defense against UVA radiation. In addition to regulating almost all genes directly involved in iron acquisition, Fur also regulates the expression of manganese and iron superoxide dismutase (MnSOD, FeSOD), key enzymes in the defense against oxygen toxicity in E. coli. In Deltafur mutants, there is a complete absence of FeSOD. Previous results suggest that the native iron chelating agent, enterobactin, which exists in increased levels in Deltafur mutants, is an endogenous chromophore for UVA, releasing Fe2+ into the cytoplasm to catalyze the production of highly reactive hydroxyl radicals. We now report that the hypersensitivity of Deltafur mutants to UVA irradiation is associated with reduced hydroperoxidase I (HPI) and hydroperoxidase II (HPII) activity, and is associated with a decrease in the transcription of katE and katG genes. The observed decrease in HPII activity in Deltafur mutants is also associated with reduced rpoS gene transcription. This study provides additional evidence that the Fur gene product, in addition to its known regulatory effect on the expression of SOD and iron uptake mechanisms, also regulates HPI and HPII activity levels in E. coli. An H2O2-inducible antioxidant defense system leading to an increase in HPI activity, is unaltered in Deltafur mutants.

  6. Functional Analysis of the Signal Recognition Particle in Escherichia coli by Characterization of a Temperature-Sensitive ffh Mutant

    PubMed Central

    Park, Sei-Kyoung; Jiang, Fenglei; Dalbey, Ross E.; Phillips, Gregory J.

    2002-01-01

    The Ffh protein of Escherichia coli is a 48-kDa polypeptide that is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP). Efforts to understand the function of Ffh in bacteria have depended largely on the use of E. coli strains that allow depletion of the wild-type gene product. As an alternative approach to studying Ffh, a temperature-sensitive ffh mutant was isolated. The ffh-10(Ts) mutation results in two amino acid changes in conserved regions of the Ffh protein, and characterization of the mutant revealed that the cells rapidly lose viability at the nonpermissive temperature of 42°C as well as show reduced growth at the permissive temperature of 30°C. While the ffh mutant is defective in insertion of inner membrane proteins, the export of proteins with cleavable signal sequences is not impaired. The mutant also shows elevated expression of heat shock proteins and accumulates insoluble proteins, especially at 42°C. It was further observed that the temperature sensitivity of the ffh mutant was suppressed by overproduction of 4.5S RNA, the RNA component of the bacterial SRP, by stabilizing the thermolabile protein. Collectively, these results are consistent with a model in which Ffh is required only for localization of proteins integral to the cytoplasmic membrane and suggest new genetic approaches to the study of how the structure of the SRP contributes to its function. PMID:11976293

  7. Arabinose-leucine deletion mutants of Escherichia coli B-r.

    PubMed

    Kessler, D P; Englesberg, E

    1969-06-01

    The control of ara gene expression was studied in mutants of Escherichia coli B/r containing deletions which fused the l-arabinose gene complex with the leucine operon (the normal gene order being araDABIOC...leuDCBAO). Complementation experiments with stable merodiploids showed that expression of ara genes cis to araC-leu deletions was controlled by the trans-acting product of the araC gene. Expression of ara genes cis to araB-leu deletions was under leucine control. These studies confirm the existence of a region between genes araC and araB essential for normal activator controlled expression of the ara structural genes. One deletion was characterized as an araO-leu deletion. Its effect on ara gene expression was unique in that ara genes were susceptible to potential regulation by both l-arabinose and leucine. These experiments suggest that two different species of messenger ribonucleic acid (mRNA) may be produced for the ara-leu region as a result of this deletion. One, under l-arabinose-activator control, is initiated in the l-arabinose region; the other, under leucine control, is initiated in the leucine region. The latter indicates that araI can be transcribed. Whether araI is transcribed in the former instance (mRNA made under activator control) remains to be established.

  8. Arabinose-Leucine Deletion Mutants of Escherichia coli B/r

    PubMed Central

    Kessler, Donald P.; Englesberg, Ellis

    1969-01-01

    The control of ara gene expression was studied in mutants of Escherichia coli B/r containing deletions which fused the l-arabinose gene complex with the leucine operon (the normal gene order being araDABIOC...leuDCBAO). Complementation experiments with stable merodiploids showed that expression of ara genes cis to araC-leu deletions was controlled by the trans-acting product of the araC gene. Expression of ara genes cis to araB-leu deletions was under leucine control. These studies confirm the existence of a region between genes araC and araB essential for normal activator controlled expression of the ara structural genes. One deletion was characterized as an araO-leu deletion. Its effect on ara gene expression was unique in that ara genes were susceptible to potential regulation by both l-arabinose and leucine. These experiments suggest that two different species of messenger ribonucleic acid (mRNA) may be produced for the ara-leu region as a result of this deletion. One, under l-arabinose-activator control, is initiated in the l-arabinose region; the other, under leucine control, is initiated in the leucine region. The latter indicates that araI can be transcribed. Whether araI is transcribed in the former instance (mRNA made under activator control) remains to be established. PMID:4892369

  9. Chromosomal Fragmentation in "Escherichia Coli": Its Absence in "mutT" Mutants and Its Mechanisms in "seqA" Mutants

    ERIC Educational Resources Information Center

    Rotman, Ella Rose

    2009-01-01

    Chromosomal fragmentation in "Escherichia coli" is a lethal event for the cell unless mended by the recombinational repair proteins RecA, RecBCD, and RuvABC. Certain mutations exacerbate problems that cause the cell to be dependent on the recombinational repair proteins for viability. We tested whether the absence of the MutT protein caused…

  10. Functional complementation of Leishmania (Leishmania) amazonensis AP endonuclease gene (lamap) in Escherichia coli mutant strains challenged with DNA damage agents

    PubMed Central

    Verissimo-Villela, Erika; Kitahara-Oliveira, Milene Yoko; dos Reis, Ana Beatriz de Bragança; Albano, Rodolpho Mattos; Da-Cruz, Alda Maria; Bello, Alexandre Ribeiro

    2016-01-01

    During its life cycle Leishmania spp. face several stress conditions that can cause DNA damages. Base Excision Repair plays an important role in DNA maintenance and it is one of the most conserved mechanisms in all living organisms. DNA repair in trypanosomatids has been reported only for Old World Leishmania species. Here the AP endonuclease from Leishmania (L.) amazonensis was cloned, expressed in Escherichia coli mutants defective on the DNA repair machinery, that were submitted to different stress conditions, showing ability to survive in comparison to the triple null mutant parental strain BW535. Phylogenetic and multiple sequence analyses also confirmed that LAMAP belongs to the AP endonuclease class of proteins. PMID:27223868

  11. Assembly of LamB and OmpF in deep rough lipopolysaccharide mutants of Escherichia coli K-12.

    PubMed Central

    Laird, M W; Kloser, A W; Misra, R

    1994-01-01

    Assembly of the OmpF and LamB proteins was kinetically retarded in deep rough lipopolysaccharide mutants of Escherichia coli K-12. OmpF assembly was affected at the step of conversion of metastable trimers to stable trimers, whereas LamB assembly was influenced both at the monomer-to-metastable trimer and metastable-to-stable trimer steps. These assembly defects were reversed in the presence of the sfaA1 and sfaB3 suppressor alleles, which were isolated by using ompF assembly mutants. Images PMID:8157594

  12. Multiplex growth rate phenotyping of synthetic mutants in selection to engineer glucose and xylose co-utilization in Escherichia coli.

    PubMed

    Groot, Joost; Cepress-Mclean, Sidney C; Robbins-Pianka, Adam; Knight, Rob; Gill, Ryan T

    2017-04-01

    Engineering the simultaneous consumption of glucose and xylose sugars is critical to enable the sustainable production of biofuels from lignocellulosic biomass. In most major industrial microorganisms glucose completely inhibits the uptake of xylose, limiting efficient sugar mixture conversion. In E. coli removal of the major glucose transporter PTS allows for glucose and xylose co-consumption but only after prolonged adaptation, which is an effective process but hard to control and prone to co-evolving undesired traits. Here we synthetically engineer mutants to target sugar co-consumption properties; we subject a PTS(-) mutant to a short adaptive step and subsequently either delete or overexpress key genes previously suggested to affect sugar consumption. Screening the co-consumption properties of these mutants individually is very laborious. We show we can evaluate sugar co-consumption properties in parallel by culturing the mutants in selection and applying a novel approach that computes mutant growth rates in selection using chromosomal barcode counts obtained from Next-Generation Sequencing. We validate this multiplex growth rate phenotyping approach with individual mutant pure cultures, identify new instances of mutants cross-feeding on metabolic byproducts, and, importantly, find that the rates of glucose and xylose co-consumption can be tuned by altering glucokinase expression in our PTS(-) background. Biotechnol. Bioeng. 2017;114: 885-893. © 2016 Wiley Periodicals, Inc.

  13. Characterization of FNR* mutant proteins indicates two distinct mechanisms for altering oxygen regulation of the Escherichia coli transcription factor FNR.

    PubMed Central

    Bates, D M; Lazazzera, B A; Kiley, P J

    1995-01-01

    In order to gain insight into the mechanism by which the Escherichia coli transcription factor FNR* is activated in response to anaerobiosis, we have analyzed FNR mutant proteins which, unlike the wild-type protein, stimulate gene expression in the presence of oxygen in vivo. Cell extracts containing seven different FNR* mutant proteins were tested in vitro for the ability to bind to the FNR consensus DNA site in a gel retardation assay under aerobic conditions. At the concentration of protein tested, only extracts which contained FNR* mutant proteins with amino acid substitutions at position 154 showed significant DNA binding. The three position-154 FNR* mutant proteins could be further distinguished from the other mutant proteins by analysis of the in vivo phenotypes of FNR* proteins containing amino acid substitutions at either of two essential cysteine residues. In the presence of oxygen, FNR* mutant proteins with amino acid substitutions at position 154 were the least affected when either Cys-23 or Cys-122 was substituted for Ser. On the basis of these in vivo and in vitro analyses, FNR* mutant proteins appear to segregate into at least two classes. Thus, it appears that each class of FNR* substitutions alters the normal pathway of FNR activation in response to oxygen deprivation by a different mechanism. PMID:7608069

  14. L-Arabinose-sensitive, L-ribulose 5-phosphate 4-epimerase-deficient mutants of Escherichia coli.

    PubMed

    ENGLESBERG, E; ANDERSON, R L; WEINBERG, R; LEE, N; HOFFEE, P; HUTTENHAUER, G; BOYER, H

    1962-07-01

    Englesberg, E. (University of Pittsburgh, Pittsburgh, Pa.), R L. Anderson, R. Weinberg, N. Lee, P. Hoffee, G. Huttenhauer, and H. Boyer. l-Arabinose-sensitive, l-ribulose 5-phosphate 4-epimerase-deficient mutants of Escherichia coli. J. Bacteriol. 84:137-146. 1962-l-Arabinose-negative mutants of Escherichia coli B/r, ara-53 and ara-139, are deficient in the enzyme l-ribulose 5-phosphate 4-epimerase; ara-53, further analyzed, accumulates large quantities of l-ribulose 5-phosphate when incubated with l-arabinose. The mutant sites are closely linked to the left of the previously ordered l-arabinose mutant sites, and probably represent the structural gene for l-ribulose 5-phosphate 4-epimerase (gene D) in the l-arabinose operon. The inducible levels of l-arabinose isomerase and l-ribulose 5-phosphate 4-epimerase vary correspondingly as a result of mutation in the structural gene for l-ribulokinase (gene B), further substantiating the dual structural and regulatory function of this gene locus. Ara-53 and ara-139 are strongly inhibited by l-arabinose and give rise to l-arabinose-resistant mutants. The one resistant mutant analyzed still lacks the 4-epimerase but is deficient in l-ribulokinase and has increased l-arabinose isomerase activity, a characteristic of a type of mutation in the B gene. It is proposed that accumulation of l-ribulose 5-phosphate is responsible for the inhibition, and that mutation to resistance will involve mutation in the A, B, C, permease, or repressor genes, thus providing a direct method for isolating these types of l-arabinose-negative mutants. Glucose prevents and cures the l-arabinose inhibition.

  15. Synthesis of outer membrane proteins in cpxA cpxB mutants of Escherichia coli K-12.

    PubMed Central

    McEwen, J; Sambucetti, L; Silverman, P M

    1983-01-01

    Two major proteins, the murein lipoprotein and the OmpF matrix porin, are deficient in the outer membrane of cpxA cpxB mutants of Escherichia coli K-12. We present evidence that the cpx mutations prevent or retard the translocation of these proteins to the outer membrane. The mutations had no effect on the rate of lipoprotein synthesis. Mutant cells labeled for 5 min with radioactive arginine accumulated as much lipoprotein as otherwise isogenic cpxA+ cpxB+ cells. This lipoprotein accumulated as such; no material synthesized in mutant cells and reactive with antilipoprotein antibodies had the electrophoretic mobility of prolipoprotein. Hence, the initial stages of prolipoprotein insertion into the inner membrane leading to its cleavage to lipoprotein appeared normal. However, after a long labeling interval, mutant cells were deficient in free lipoprotein and lacked lipoprotein covalently bound to peptidoglycan, suggesting that little if any of the lipoprotein synthesized in mutant cells reaches the outer membrane. Immunoreactive OmpF protein could also be detected in extracts of mutant cells labeled for 5 min, but the amount that accumulated was severalfold less in mutant cells than in cpxA+ cpxB+ cells. Analysis of beta-galactosidase synthesis from ompF-lacZ fusion genes showed this difference to be the result of a reduced rate of ompF transcription in mutant cells. Even so, little or none of the ompF protein synthesized in mutant cells was incorporated into the outer membrane. Images PMID:6339479

  16. Properties of adenyl cyclase and cyclic adenosine 3',5'-monophosphate receptor protein-deficient mutants of Escherichia coli.

    PubMed Central

    Kumar, S

    1976-01-01

    Several spontaneous cya and crp mutants of Escherichia coli have been selected as clones simultaneously resistant to phage lambda and nalidixic acid and characterized. Both cya and crp mutants have been found to grow as cocci with increased doubling times. They have increased resistance to some mutagens (methylmethanesulfonate, ultraviolet light, gamma rays), antibiotics (nalidixic acid, ampicillin), phages (lambda, T6), sublethal heat and hypotonic shock, and decreased resistance to neutral detergents (sodium dodecyl sulfate, sodium deoxycholate), a protein synthesis inhibitor (streptomycin), and a respiratory inhibitor (sodium azide). The nature of changes in cell parameters indicate fundamental alterations in the envelope structure of the cya and crp mutant cells. The new cya and crp mutants have been found to be multiply carbohydrate negative and nonmotile in conformity with similar previously isolated mutants. Studies of revertants and phi80 cya+ and phi80 cya transductants indicated that the pleiotropic phenotype is related to a single mutational event at the cya or the crp locus in the mutants. Images PMID:173710

  17. Characterization of mutant histidine-containing proteins of the phosphoenolpyruvate:sugar phosphotransferase system of Escherichia coli and Salmonella typhimurium

    SciTech Connect

    Waygood, E.B.; Reiche, B.; Hengstenberg, W.; Lee, J.S.

    1987-06-01

    Histidine-containing phosphocarrier protein (HPr) is common to all of the phosphoenolpyruvate:sugar phosphotransferase systems (PTS) in Escherichia coli and Salmonella typhimurium, except the fructose-specific PTS. Strains which lack HPr activity (ptsH) have been characterized in the past, and it has proved difficult to delineate between tight and leaky mutants. In this study four different parameters of ptsH strains were measured: in vitro sugar phosphorylation activity of the mutant HPr; detection of /sup 32/P-labeled P-HPr; ability of monoclonal antibodies to bind mutant HPr; and sensitivity of ptsH strains to fosfomycin. Tight ptsH strains could be defined; they were fosfomycin resistant and produced no HPr protein or completely inactive mutant HPr. All leaky ptsH strains were fosfomycin sensitive, Usually produced normal amounts of mutant HPr protein, and had low but measurable activity, and HPr was detectable as a phosphoprotein. This indicates that the regulatory functions of the PTS require a very low level of HPr activity (about 1%). The antibodies used to detect mutant HPr in crude extracts were two monoclonal immunoglobulin G antibodies Jel42 and Jel44. Both antibodies, which have different pIs, inhibited PTS sugar phosphorylation assays, but the antibody-JPr complex could still be phosphorylated by enzyme I. Preliminary evidence suggests that the antibodies bind to two different epitopes which are in part located in a ..beta..-sheet structure.

  18. A mutant of Escherichia coli showing constitutive expression of the lysogenic induction and error-prone DNA repair pathways.

    PubMed Central

    Mount, D W

    1977-01-01

    A mutant of E. coli (designated the STS mutant) has been isolated in which the phage induction and error-prone DNA repair pathways appear to be expressed constitutively without the cells having received an inducing signal. Phage lambda was not able to lysogenize this mutant, whereas a noninducible mutant of lambda, lambdacIind-, known to synthesize a repressor that is insensitive to the induction mechanism, lysogenized it normally. This result suggested that normal phage repressor was synthesized in the STS mutant but was then inactivated by the induction mechanism. The STS strain also had mutator characteristics, and showed spontaneous, error-prone repair of UV-damaged phage lambda. Derived from a lexA tif sfiA parent strain, the STS mutant carried an additional mutation spr at the lexA locus that resulted in a high level of expression of the induction pathways. The properties of this and related strains provide additional evidence that induction of phage and induction of error-prone DNA repair occur by a similar mechanism, and further suggest a model for the regulation of these pathways. Images PMID:319458

  19. Using student-generated UV-induced Escherichia coli mutants in a directed inquiry undergraduate genetics laboratory.

    PubMed

    Healy, Frank G; Livingstone, Kevin D

    2010-09-01

    We report a thematic sequence of directed inquiry-based labs taking students from bacterial mutagenesis and phenotypic identification of their own self-created mutant, through identification of mutated genes by biochemical testing, to verification of mutant alleles by complementation, and finally to mutant allele characterization by DNA sequence analysis. The lab utilizes UV mutagenesis with wild-type Escherichia coli and a UV-sensitive isogenic derivative optimized for undergraduate use. The labs take advantage of the simplicity of E. coli in a realistic genetic investigation using safe UV irradiation methods for creation and characterization of novel mutants. Assessment data collected over three offerings of the course suggest that the labs, which combine original investigation in a scientifically realistic intellectual environment with learned techniques and concepts, were instrumental in improving students' learning in a number of areas. These include the development of critical thinking skills and understanding of concepts and methods. Student responses also suggest the labs were helpful in improving students' understanding of the scientific process as a rational series of experimental investigations and awareness of the interdisciplinary nature of scientific inquiry.

  20. A mutant crp allele that differentially activates the operons of the fuc regulon in Escherichia coli.

    PubMed

    Zhu, Y; Lin, E C

    1988-05-01

    L-Fucose is used by Escherichia coli through an inducible pathway mediated by a fucP-encoded permease, a fucI-encoded isomerase, a fucK-encoded kinase, and a fucA-encoded aldolase. The adolase catalyzes the formation of dihydroxyacetone phosphate and L-lactaldehyde. Anaerobically, lactaldehyde is converted by a fucO-encoded oxidoreductase to L-1,2-propanediol, which is excreted. The fuc genes belong to a regulon comprising four linked operons: fucO, fucA, fucPIK, and fucR. The positive regulator encoded by fucR responds to fuculose 1-phosphate as the effector. Mutants serially selected for aerobic growth on propanediol became constitutive in fucO and fucA [fucO(Con) fucA(Con)], but noninducible in fucPIK [fucPIK(Non)]. An external suppressor mutation that restored growth on fucose caused constitutive expression of fucPIK. Results from this study indicate that this suppressor mutation occurred in crp, which encodes the cyclic AMP-binding (or receptor) protein. When the suppressor allele (crp-201) was transduced into wild-type strains, the recipient became fucose negative and fucose sensitive (with glycerol as the carbon and energy source) because of impaired expression of fucA. The fucPIK operon became hyperinducible. The growth rate on maltose was significantly reduced, but growth on L-rhamnose, D-galactose, L-arabinose, glycerol, or glycerol 3-phosphate was close to normal. Lysogenization of fuc+ crp-201 cells by a lambda bacteriophage bearing crp+ restored normal growth ability on fucose. In contrast, lysogenization of [fucO(Con)fucA(Con)fucPIK(Non)crp-201] cells by the same phage retarded their growth on fucose.

  1. Effect of Spermidine Analogues on Cell Growth of Escherichia coli Polyamine Requiring Mutant MA261

    PubMed Central

    Yoshida, Taketo; Sakamoto, Akihiko; Terui, Yusuke; Takao, Koichi; Sugita, Yoshiaki; Yamamoto, Kaneyoshi; Ishihama, Akira; Igarashi, Kazuei; Kashiwagi, Keiko

    2016-01-01

    The effects of spermidine analogues [norspermidine (NSPD, 33), spermidine (SPD, 34), homospermidine (HSPD, 44) and aminopropylcadaverine (APCAD, 35)] on cell growth were studied using Escherichia coli polyamine-requiring mutant MA261. Cell growth was compared at 32°C, 37°C, and 42°C. All four analogues were taken up mainly by the PotABCD spermidine-preferential uptake system. The degree of stimulation of cell growth at 32°C and 37°C was NSPD ≥ SPD ≥ HSPD > APCAD, and SPD ≥ HSPD ≥ NSPD > APCAD, respectively. However, at 42°C, it was HSPD » SPD > NSPD > APCAD. One reason for this is HSPD was taken up effectively compared with other triamines. In addition, since natural polyamines (triamines and teteraamines) interact mainly with RNA, and the structure of RNA is more flexible at higher temperatures, HSPD probably stabilized RNA more tightly at 42°C. We have thus far found that 20 kinds of protein syntheses are stimulated by polyamines at the translational level. Among them, synthesis of OppA, RpoE and StpA was more strongly stimulated by HSPD at 42°C than at 37°C. Stabilization of the initiation region of oppA and rpoE mRNA was tighter by HSPD at 42°C than 37°C determined by circular dichroism (CD). The degree of polyamine stimulation of OppA, RpoE and StpA synthesis by NSPD, SPD and APCAD was smaller than that by HSPD at 42°C. Thus, the degree of stimulation of cell growth by spermidine analogues at the different temperatures is dependent on the stimulation of protein synthesis by some components of the polyamine modulon. PMID:27434546

  2. Mutations Affecting Potassium Import Restore the Viability of the Escherichia coli DNA Polymerase III holD Mutant

    PubMed Central

    Durand, Adeline

    2016-01-01

    Mutants lacking the ψ (HolD) subunit of the Escherichia coli DNA Polymerase III holoenzyme (Pol III HE) have poor viability, but a residual growth allows the isolation of spontaneous suppressor mutations that restore ΔholD mutant viability. Here we describe the isolation and characterization of two suppressor mutations in the trkA and trkE genes, involved in the main E. coli potassium import system. Viability of ΔholD trk mutants is abolished on media with low or high K+ concentrations, where alternative K+ import systems are activated, and is restored on low K+ concentrations by the inactivation of the alternative Kdp system. These findings show that the ΔholD mutant is rescued by a decrease in K+ import. The effect of trk inactivation is additive with the previously identified ΔholD suppressor mutation lexAind that blocks the SOS response indicating an SOS-independent mechanism of suppression. Accordingly, although lagging-strand synthesis is still perturbed in holD trkA mutants, the trkA mutation allows HolD-less Pol III HE to resist increased levels of the SOS-induced bypass polymerase DinB. trk inactivation is also partially additive with an ssb gene duplication, proposed to stabilize HolD-less Pol III HE by a modification of the single-stranded DNA binding protein (SSB) binding mode. We propose that lowering the intracellular K+ concentration stabilizes HolD-less Pol III HE on DNA by increasing electrostatic interactions between Pol III HE subunits, or between Pol III and DNA, directly or through a modification of the SSB binding mode; these three modes of action are not exclusive and could be additive. To our knowledge, the holD mutant provides the first example of an essential protein-DNA interaction that strongly depends on K+ import in vivo. PMID:27280472

  3. Rapid site-specific DNA inversion in Escherichia coli mutants lacking the histonelike protein H-NS.

    PubMed Central

    Kawula, T H; Orndorff, P E

    1991-01-01

    Escherichia coli pilG mutants are thought to have a dramatically higher DNA inversion rate as measured by the site-specific DNA inversion of the type 1 pili pilA promoter. DNA sequence of the pilG gene confirmed its identity to the gene encoding the bacterial histonelike protein H-NS. Unlike other histonelike protein complexes that enhance site-specific DNA recombination, the H-NS protein inhibited this process. This inhibition was indicated by the increased inversion rate of the pilA promoter region effected by two different mutant pilG alleles. One of these alleles, pilG1, conferred a mutant phenotype only at low temperature attributable to a T-to-G transversion in the -35 sequence of the pilG promoter. The other allele, pilG2-tetR, was an insertion mutation in the pilG coding region that conferred the mutant phenotype independent of temperature. We measured an approximately 100-fold-increased pilA promoter inversion rate in the mutant by exploiting the temperature-dependent expression of pilG1 and using a novel rapid-population-sampling method. Contrary to one current view on how the H-NS protein might act to increase DNA inversion rate, we found no evidence to support the hypothesis that DNA supercoiling affected pilA promoter inversion. Images PMID:1648076

  4. Reduced LPS phosphorylation in Escherichia coli lowers the elevated ori/ter ratio in seqA mutants

    PubMed Central

    Rotman, Ella; Bratcher, Preston; Kuzminov, Andrei

    2009-01-01

    Summary The seqA defect in E. coli increases the ori/ter ratio and causes chromosomal fragmentation, making seqA mutants dependent on recombinational repair (the seqA recA co-lethality). To understand the nature of this chromosomal fragmentation, we characterized ΔseqA mutants and isolated suppressors of the ΔseqA recA lethality. We demonstrate that our ΔseqA alleles have normal function of the downstream pgm gene and normal ratios of the major phospholipids in the membranes, but increased surface lipopolysaccharide (LPS) phosphorylation. The predominant class of ΔseqA recA suppressors disrupts the rfaQGP genes, reducing phosphorylation of the inner core region of LPS. The rfaQGP suppressors also reduce the elevated ori/ter ratio of the ΔseqA mutants, but, unexpectedly, the suppressed mutants still exhibit the high levels of chromosomal fragmentation and SOS induction, characteristic of the ΔseqA mutants. We also found that co-lethality of rfaP with defects in the production of acidic phospholipids is suppressed by alternative initiation of chromosomal replication, suggesting that LPS phosphorylation stimulates replication initiation. The rfaQGP suppression of the seqA recA lethality provides genetic support for the surprising physical evidence that the oriC DNA forms complexes with the outer membrane. PMID:19432803

  5. Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

    PubMed Central

    Zengel, J M; Young, R; Dennis, P P; Nomura, M

    1977-01-01

    Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome. PMID:321423

  6. Role of ribosomal protein S12 in peptide chain elongation: analysis of pleiotropic, streptomycin-resistant mutants of Escherichia coli.

    PubMed

    Zengel, J M; Young, R; Dennis, P P; Nomura, M

    1977-03-01

    Some of the spontaneous streptomycin-resistant mutants of Escherichia coli strain C600 exhibit pleiotropic effects in addition to the antibiotic resistance. These effects include decreased growth rates, reduced levels of certain enzymes, and poor support of bacteriophage growth. One of these mutants, strain SM3, was studied further. We have examined the question of whether the reduced growth rate of the mutant SM3 is related to the reduction in relative amounts of ribosomes or to the reduction in the efficiency of ribosomes in protein synthesis. Measurements of alpha, the differential synthesis rate of ribosomal protein, revealed that the protein synthesis effeciency of ribosomes from the mutant strain SM3 was reduced about twofold relative to that of the parent strain C600. Measurements of the induction lag for beta-galactosidase and of the synthesis time of several different molecular-weight classes of proteins indicated that the mutation resulted in a marked reduction in the peptide chain growth rate. This reduction in the chain growth rate probably accounted for most of the observed reduction in the growth rate of the mutant strain. These experimental results show that the strA gene product, the S12 protein of the 30S subunit, is involved in some aspect of protein chain elongation. Presumably this involvement occurs during the messenger ribonucleic acid-directed binding of transfer ribonucleic acid to the ribosome.

  7. ssb Gene Duplication Restores the Viability of ΔholC and ΔholD Escherichia coli Mutants

    PubMed Central

    Duigou, Stéphane; Silvain, Maud; Viguera, Enrique; Michel, Bénédicte

    2014-01-01

    The HolC-HolD (χψ) complex is part of the DNA polymerase III holoenzyme (Pol III HE) clamp-loader. Several lines of evidence indicate that both leading- and lagging-strand synthesis are affected in the absence of this complex. The Escherichia coli ΔholD mutant grows poorly and suppressor mutations that restore growth appear spontaneously. Here we show that duplication of the ssb gene, encoding the single-stranded DNA binding protein (SSB), restores ΔholD mutant growth at all temperatures on both minimal and rich medium. RecFOR-dependent SOS induction, previously shown to occur in the ΔholD mutant, is unaffected by ssb gene duplication, suggesting that lagging-strand synthesis remains perturbed. The C-terminal SSB disordered tail, which interacts with several E. coli repair, recombination and replication proteins, must be intact in both copies of the gene in order to restore normal growth. This suggests that SSB-mediated ΔholD suppression involves interaction with one or more partner proteins. ssb gene duplication also suppresses ΔholC single mutant and ΔholC ΔholD double mutant growth defects, indicating that it bypasses the need for the entire χψ complex. We propose that doubling the amount of SSB stabilizes HolCD-less Pol III HE DNA binding through interactions between SSB and a replisome component, possibly DnaE. Given that SSB binds DNA in vitro via different binding modes depending on experimental conditions, including SSB protein concentration and SSB interactions with partner proteins, our results support the idea that controlling the balance between SSB binding modes is critical for DNA Pol III HE stability in vivo, with important implications for DNA replication and genome stability. PMID:25329071

  8. Polymorphic Variation in Susceptibility and Metabolism of Triclosan-Resistant Mutants of Escherichia coli and Klebsiella pneumoniae Clinical Strains Obtained after Exposure to Biocides and Antibiotics

    PubMed Central

    Curiao, Tânia; Marchi, Emmanuela; Viti, Carlo; Oggioni, Marco R.; Baquero, Fernando; Martinez, José Luis

    2015-01-01

    Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRIr) and triclosan-hypersusceptible (TRIhs) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRIr mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRIr mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRIr mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRIr mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRIr mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive traits. PMID

  9. Study the Expression of marA Gene in Ciprofloxacin and Tetracycline Resistant Mutants of Esherichia coli.

    PubMed

    Pourahmad Jaktaji, Razieh; Ebadi, Rayhaneh

    2013-01-01

    MarA activates two membrane dependent mechanisms of resistance to different antibiotics, such as ciprofloxacin and tetracycline, including promotion of outflux and inhibition of influx of antibiotics. Thus, MarA causes multiple antibiotic resistance phenotype. The activation of these mechanisms needs overexpression of marA. This could happen through mutation in marR. Thus, the aim of this study was to measure marA expression in ciprofloxacin resistant E. coli gyrA mutants and clones with or without marR mutation. For this purpose, real time PCR was used to measure relative expression of marA in above mutants and clones. Results showed that two clones, C14 and C17 overexpressed marA. It is concluded that the level of marA expression is important for activation of above mechanisms.

  10. Penicillin-binding protein 2 is essential in wild-type Escherichia coli but not in lov or cya mutants.

    PubMed Central

    Ogura, T; Bouloc, P; Niki, H; D'Ari, R; Hiraga, S; Jaffé, A

    1989-01-01

    Penicillin-binding protein 2 (PBP2), target of the beta-lactam mecillinam, is required for rod morphology and cell wall elongation in Escherichia coli. A new temperature-sensitive PBP2 allele and an in vitro-constructed insertion deletion allele were shown to be lethal in wild-type strains, establishing that the activity of this protein is essential. Mutations in the lov or cya genes, conferring mecillinam resistance, compensated for the deleterious effect of the absence of PBP2. The resulting double mutants grew as spheres. In a cya mutant lacking PBP2, the restoration of a Cya+ phenotype by addition of cyclic AMP caused lethality and a block in cell division. These results show that in wild-type cells, PBP2 is essential for growth and division. PMID:2656638

  11. H-NS and RpoS regulate emergence of Lac Ara+ mutants of Escherichia coli MCS2.

    PubMed

    Gómez-Gómez, J M; Blázquez, J; Baquero, F; Martínez, J L

    1997-07-01

    Two master growth-phase regulatory proteins, H-NS and sigmaS, are involved in the formation of araB-lacZ fusion clones of Escherichia coli MCS2. The stationary-phase sigma factor RpoS is strictly required for the appearance of such mutants, whereas the histone-like protein H-NS represses their emergence. Our results support the idea that genetic changes leading to adaptive mutation in this model system are regulated by physiological signal transduction networks.

  12. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed Central

    DeWyngaert, M A; Hinkle, D C

    1980-01-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein. Images PMID:6997508

  13. Characterization of the defects in bacteriophage T7 DNA synthesis during growth in the Escherichia coli mutant tsnB.

    PubMed

    DeWyngaert, M A; Hinkle, D C

    1980-02-01

    The Escherichia coli mutant tsnB (M. Chamberlin, J. Virol. 14:509-516, 1974) is unable to support the growth of bacteriophage T7, although all classes of phage proteins are produced and the host is killed by the infection. During growth in this mutant host, the rate of phage DNA synthesis is reduced and the DNA is not packaged into stable, phagelike particles. The replicating DNA forms concatemers but the very large replicative intermediates (approximately 440S) identified by Paetkau et al. (J. Virol. 22:130-141, 1977) are not detected in T7+-infected tsnB cells. These large structures are formed in tsnB cells infected with a T7 gene 3 (endonuclease) mutant, where normal processing of the large intermediates into shorter concatemers is blocked. At later times during infection of tsnB cells, the replicating DNA accumulates in molecules about 30% shorter than unit length. Analysis of this DNA with a restriction endonuclease indicates that it is missing sequences from the ends (particularly the left end) of the genome. The loss of these specific sequences does not occur during infections with T7 gene 10 (head protein) or gene 19 (maturation protein) mutants. This suggests that the processing of concatemers into unit-length DNA molecules may occur normally in T7 -infected tsnB cells and that the shortened DNA arises from exonucleolytic degradation of the mature DNA molecules. These results are discussed in relation to our recent observation (M. A. DeWyngaert and D. C. Hinkle, J. Biol. Chem. 254:11247-11253, 1979) that E. coli tsnB produces an altered RNA polymerase which is resistance to inhibition by the T7 gene 2 protein.

  14. The sub-optimal phenotypes of double-knockout mutants of Escherichia coli depend on the order of gene deletions.†

    PubMed Central

    Gawand, Pratish; Abukar, Fatumina Said; Venayak, Naveen; Partow, Siavash; Motter, Adilson E.; Mahadevan, Radhakrishnan

    2016-01-01

    Metabolic networks are characterized by multiple redundant reactions that do not have a clear biological function. The redundancies in the metabolic networks are implicated in adaptation to random mutations and survival under different environmental conditions. Reactions that are not active under wild-type growth conditions, but get transiently activated after a mutation event such as gene deletion are known as latent reactions. Characterization of multiple-gene knockout mutants can identify the physiological roles of latent reactions. In this study, we characterized double-gene deletion mutants of E. coli with an aim to investigate the sub-optimal physiology of the mutants and the plausible roles of latent reactions. Specifically, we investigated the effects of deletion of the glyoxylate-shunt gene aceA (encoding a latent reaction enzyme, isocitrate lyase) on the growth characteristics of the mutant E. coli Δpgi. The deletion of aceA reduced the growth rate of E. coli Δpgi, indicating that the activation of the glyoxylate shunt plays an important role in adaptation of the mutant E. coli Δpgi. We also investigated the effect of the order of the gene deletions on the growth rates and substrate uptake rates of the double-gene deletion mutants. The results indicate that the order in which genes are deleted determines the phenotype of the mutants during the sub-optimal growth phase. To elucidate the mechanism behind the difference between the observed phenotypes, we carried out transcriptomic analysis and constraint-based modeling of the mutants. Transcriptomic analysis showed differential expression of the gene aceK (encoding the protein isocitrate dehydrogenase kinase) involved in controlling the isocitrate flux through the TCA cycle and the glyoxylate shunt. Higher acetate production in the E. coli ΔaceA1 Δpgi2 mutant was consistent with the increased aceK expression, which limits the TCA cycle flux and causes acetate production via overflow metabolism. PMID

  15. Isolation of catalase-deficient Escherichia coli mutants and genetic mapping of katE, a locus that affects catalase activity.

    PubMed Central

    Loewen, P C

    1984-01-01

    A number of catalase-deficient mutants of Escherichia coli which exhibit no assayable catalase activity were isolated. The only physiological difference between the catalase mutants and their parents was a 50- to 60-fold greater sensitivity to killing by hydrogen peroxide. For comparison, mutations in the xthA and recA genes of the same strains increased the sensitivity of the mutants to hydrogen peroxide by seven- and fivefold, respectively, showing that catalase was the primary defense against hydrogen peroxide. One class of mutants named katE was localized between pfkB and xthA at 37.8 min on the E. coli genome. A second class of catalase mutants was found which did not map in this region. PMID:6319370

  16. [Protective action of reactivating factor of Luteococcus japonicus subsp. casei toward cells of Escherichia coli reparation mutants inactivated with UV-light].

    PubMed

    Vorob'eva, L I; Fedotova, A V; Khodzhaev, E Iu

    2010-01-01

    Reactivating factor (RF) from Luteococcus japonicus subsp. casei had a protective action on UV-irradiated cells of Escherichia coli AB1157 with a native reparation system and on cells of isogenic reparation mutants of E. coli UvrA-, RecA-, and PolA-: the effect resulted in multifold increase of survivability. Defense action of L. casei exometabolite is not connected with stimulating reparation systems in E. coli, and, probably, it is mediated by involvement of the exometabolite in the mechanism of cell division. RF did not provoke the reactivation of E. coli cells inactivated by UV-light.

  17. Isolation and characterization of an Escherichia coli mutant lacking cytochrome d terminal oxidase.

    PubMed Central

    Green, G N; Gennis, R B

    1983-01-01

    A screening procedure was devised which permitted the isolation of a cytochrome d-deficient mutant by its failure to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Cytochrome a1 and probably cytochrome b558 were also missing in the mutant. Growth and oxygen uptake rates were similar for both parent and mutant strains. However, the strain lacking cytochrome d had an increased sensitivity to cyanide, indicating that cytochrome d confers some resistance to this respiratory inhibitor. The gene responsible for these phenotypes has been named cyd and maps between tolA and sucB. PMID:6304009

  18. Microbial biosensor array with transport mutants of Escherichia coli K12 for the simultaneous determination of mono-and disaccharides.

    PubMed

    Held, Michael; Schuhmann, Wolfgang; Jahreis, Knut; Schmidt, Hanns-Ludwig

    2002-12-01

    An automated flow-injection system with an integrated biosensor array using bacterial cells for the selective and simultaneous determination various mono- and disaccharides is described. The selectivity of the individually addressable sensors of the array was achieved by the combination of the metabolic response, measured as the O(2) consumption, of bacterial mutants of Escherichia coli K12 lacking different transport systems for individual carbohydrates. Kappa-carrageenan was used as immobilization matrix for entrapment of the bacterial cells in front of 6 individually addressable working electrodes of a screen-printed sensor array. The local consumption of molecular oxygen caused by the metabolic activity of the immobilized cells was amperometrically determined at the underlying screen-printed gold electrodes at a working potential of -600 mV vs. Ag/AgCl. Addition of mono- or disaccharides for which functional transport systems exist in the used transport mutant strains of E. coli K12 leads to an enhanced metabolic activity of the immobilized bacterial cells and to a concomitant depletion of oxygen at the electrode. Parallel determination of fructose, glucose, and sucrose was performed demonstrating the high selectivity of the proposed analytical system.

  19. Transition of deletion mutants of the composite resistance plasmid NR1 in Escherichia coli and Salmonella typhimurium.

    PubMed Central

    Huffman, G A; Rownd, R H

    1984-01-01

    Derivatives of the composite R plasmid NR1 from which a portion of the resistance determinants (r-determinants) component had been deleted were found to undergo amplification of the remaining r-determinants region in Escherichia coli and Salmonella typhimurium. The wild-type NR1 plasmid does not amplify in these genera, although all of these plasmids undergo amplification in Proteus mirabilis. The deletion mutants retained the mercuric ion resistance operon (mer) but conferred a much lower level of sulfonamide resistance than NR1. The remaining r-determinants region, which is bounded by direct repeats of the insertion element IS1, formed multiple tandem duplications in E. coli, S. typhimurium, and P. mirabilis after subculturing the host cells in medium containing high concentrations of sulfonamide. Gene amplification was characterized by restriction endonuclease analysis, analytical buoyant density centrifugation, DNA-DNA hybridization, and sedimentation in sucrose gradients. The tandem repeats remained attached to the resistance transfer factor component of the plasmid in at least part of the plasmid population; autonomous tandem repeats of r-determinants were probably also present. Amplification did not occur in host recA mutants. Amplified strains subcultured in drug-free medium lost the amplified r-determinants. By using a strain temperature sensitive for the recA gene, it was possible to obtain gene amplification at the permissive temperature. Loss of r-determinants took place at the permissive temperature, but not at the nonpermissive temperature. The termini of the deletions of several independent mutants which conferred low sulfonamide resistance were found to be located within the adjacent streptomycin-spectinomycin resistance gene. Images PMID:6086573

  20. Characterization of an Escherichia coli mutant (radB101) sensitive to. gamma. and uv radiation, and methyl methanesulfonate

    SciTech Connect

    Sargentini, N.J.; Smith, K.C.

    1983-03-01

    After N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis of Escherichia coli K-12 (xthA14), an X-ray-sensitive mutant was isolated. This sensitivity is due to a mutation, radB101, which is located at 56.5 min on the E.coli K-12 linkage map. The radB101 mutation sensitized wild-type cells to ..gamma.. and uv radiation, and to methyl methanesulfonate. When known DNA repair-deficient mutants were ranked for their ..gamma..-radiation sensitivity relative to their uv-radiation sensitivity, their order was (starting with the most selectively ..gamma..-radiation-sensitive strain): recB21, radB101, wild type, polA1, recF143, lexA101, recA56, uvrD3, and uvrA6. The radB mutant was normal for ..gamma..- and uv-radiation mutagenesis, it showed only a slight enhancement of ..gamma..- and uv-radiation-induced DNA degradation, and it was approx. 60% deficient in recombination ability. The radB gene is suggested to play a role in the recA gene-dependent (Type III) repair of DNA single-strand breaks after ..gamma.. irradiation and in postreplication repair after uv irradiation for the following reasons: the radB strain was normal for the host-cell reactivation of ..gamma..- and uv-irradiated bacteriophage lambda; the radB mutation did not sensitize a recA strain, but did sensitize a polA strain to ..gamma.. and uv radiation; the radB mutation sensitized a uvrB strain to uv radiation.

  1. Complementation of growth defect in an ampC deletion mutant of Escherichia coli.

    PubMed

    Bishop, R E; Weiner, J H

    1993-12-15

    beta-Lactamase genes of class-A (Rtem) and class-C (ampC) were placed under control of an inducible tac-promoter and expressed in Escherichia coli. Expression of RTEM had no observable effect on the growth properties of E. coli strains HB101 (ampC+) or MI1443 (delta ampC). E. coli MI1443 exhibited a decline in growth rate at mid-exponential phase which could be delayed by expression of AmpC at early-exponential phase. AmpC expression otherwise inhibited growth, particularly during the transition into exponential phase where growth was prevented altogether. We suggest that the AmpC beta-lactamase, but not RTEM, may have an additional cellular function as a peptidoglycan hydrolase.

  2. Colonization of porcine small intestine by Escherichia coli: ileal colonization and adhesion by pig enteropathogens that lack K88 antigen and by some acapsular mutants.

    PubMed

    Nagy, B; Moon, H W; Isaacson, R E

    1976-04-01

    Seven K88-negative porcine enteropathogenic Escherichia coli, representing three different serogroups, caused severe diarrhea and characteristically colonized the ileum, but not the jejunum, of intragastrically exposed newborn pigs. Bacterial counts of intestinal contents and wall, fluorescence, and scanning electron microscopy all suggested that these strains colonized the ileum by adhesion to the villous epithelium. However, in ligated intestinal loops, these enteropathogenic E. coli strains adhered to jejunal epithelium as well as to ileal epithelium. Acapsular (K-) mutants, derived from one of the principal strains, retained their colonizing and adhesive abilities, whereas K- mutants from three other enteropathogenic E. coli strains did not. It is suggested that: (i) these K88-negative enteropathogenic E. coli colonize the ileum by adhesion, and (ii) the adhesion of some K-88-negative strains is mediated by surface factors other than, or in addition to, the polysaccharide K antigen.

  3. Induction of the SOS response by hydrogen peroxide in various Escherichia coli mutants with altered protection against oxidative DNA damage.

    PubMed Central

    Goerlich, O; Quillardet, P; Hofnung, M

    1989-01-01

    The induction of the SOS response by H2O2 was measured in Escherichia coli by means of a sfiA::lacZ operon fusion. The effects of mutations in genes involved in DNA repair or DNA metabolism on the SOS response were investigated. We found that in an uvrA mutant, H2O2 induced the SOS response at lower concentrations than in the uvr+ parent strain, indicating that some lesions induced by H2O2 may be repaired by the uvrABC-dependent excision repair system. A nth mutation, yielding deficiency in thymine glycol DNA glycosylase, had no detectable effect on SOS induction, indicating that thymine glycol, a DNA lesion expected to be induced by H2O2, does not participate detectably in the induction of the SOS response by this chemical under our conditions. H2O2 still induced the SOS response in a dnaC(Ts) uvrA double mutant under conditions in which no DNA replication proceeds, suggesting that this chemical induces DNA strand breaks. Induction of the SOS response by H2O2 was also assayed in various mutants affected in genes suspected to be important for protection against oxidative stress. Mutations in the catalase genes, katE and katG, had only minor effects. However, in an oxyR deletion mutant, in which the adaptative response to H2O2 does not occur, SOS induction occurred at much lower H2O2 concentrations than in the oxyR+ parent strain. These results indicate that some enzymes regulated by the oxyR gene are, under our conditions, more important than catalase for protection against the H2O2-induced DNA damages which trigger the SOS response. PMID:2681154

  4. Evidence that recBC-dependent degradation of duplex DNA in Escherichia coli recD mutants involves DNA unwinding.

    PubMed Central

    Rinken, R; Thomas, B; Wackernagel, W

    1992-01-01

    Infection of Escherichia coli with phage T4 gene 2am was used to transport 3H-labeled linear duplex DNA into cells to follow its degradation in relation to the cellular genotype. In wild-type cells, 49% of the DNA was made acid soluble within 60 min; in recB or recC cells, only about 5% of the DNA was made acid soluble. Remarkably, in recD cells about 25% of the DNA was rendered acid soluble. The DNA degradation in recD cells depended on intact recB and recC genes. The degradation in recD cells was largely decreased by mutations in recJ (which eliminates the 5' single-strand-specific exonuclease coded by this gene) or xonA (which abolishes the 3' single-strand-specific exonuclease I). In a recD recJ xonA triple mutant, the degradation of linear duplex DNA was roughly at the level of a recB mutant. Results similar to those with the set of recD strains were also obtained with a recC++ mutant (in which the RecD protein is intact but does not function) and its recJ, xonA, and recJ xonA derivatives. The observations provide evidence for a recBC-dependent DNA-unwinding activity that renders unwound DNA susceptible to exonucleolytic degradation. It is proposed that the DNA-unwinding activity causes the efficient recombination, DNA repair, and SOS induction (after application of nalidixic acid) in recD mutants. The RecBC helicase indirectly detected here may have a central function in Chi-dependent recombination and in the recombinational repair of double-strand breaks by the RecBCD pathway. PMID:1322885

  5. Genetic requirements for high constitutive SOS expression in recA730 mutants of Escherichia coli.

    PubMed

    Vlašić, Ignacija; Šimatović, Ana; Brčić-Kostić, Krunoslav

    2011-09-01

    The RecA protein in its functional state is in complex with single-stranded DNA, i.e., in the form of a RecA filament. In SOS induction, the RecA filament functions as a coprotease, enabling the autodigestion of the LexA repressor. The RecA filament can be formed by different mechanisms, but all of them require three enzymatic activities essential for the processing of DNA double-stranded ends. These are helicase, 5'-3' exonuclease, and RecA loading onto single-stranded DNA (ssDNA). In some mutants, the SOS response can be expressed constitutively during the process of normal DNA metabolism. The RecA730 mutant protein is able to form the RecA filament without the help of RecBCD and RecFOR mediators since it better competes with the single-strand binding (SSB) protein for ssDNA. As a consequence, the recA730 mutants show high constitutive SOS expression. In the study described in this paper, we studied the genetic requirements for constitutive SOS expression in recA730 mutants. Using a β-galactosidase assay, we showed that the constitutive SOS response in recA730 mutants exhibits different requirements in different backgrounds. In a wild-type background, the constitutive SOS response is partially dependent on RecBCD function. In a recB1080 background (the recB1080 mutation retains only helicase), constitutive SOS expression is partially dependent on RecBCD helicase function and is strongly dependent on RecJ nuclease. Finally, in a recB-null background, the constitutive SOS expression of the recA730 mutant is dependent on the RecJ nuclease. Our results emphasize the importance of the 5'-3' exonuclease for high constitutive SOS expression in recA730 mutants and show that RecBCD function can further enhance the excellent intrinsic abilities of the RecA730 protein in vivo.

  6. Escherichia coli endonuclease VIII: cloning, sequencing, and overexpression of the nei structural gene and characterization of nei and nei nth mutants.

    PubMed Central

    Jiang, D; Hatahet, Z; Blaisdell, J O; Melamede, R J; Wallace, S S

    1997-01-01

    Escherichia coli possesses two DNA glycosylase/apurinic lyase activities with overlapping substrate specificities, endonuclease III and endonuclease VIII, that recognize and remove oxidized pyrimidines from DNA. Endonuclease III is encoded by the nth gene. Endonuclease VIII has now been purified to apparent homogeneity, and the gene, nei, has been cloned by using reverse genetics. The gene nei is located at 16 min on the E. coli chromosome and encodes a 263-amino-acid protein which shows significant homology in the N-terminal and C-terminal regions to five bacterial Fpg proteins. A nei partial deletion replacement mutant was constructed, and deletion of nei was confirmed by genomic PCR, activity analysis, and Western blot analysis. nth nei double mutants were hypersensitive to ionizing radiation and hydrogen peroxide but not as sensitive as mutants devoid of base excision repair (xth nfo). Single nth mutants exhibited wild-type sensitivity to X rays, while nei mutants were consistently slightly more sensitive than the wild type. Double mutants lacking both endonucleases III and VIII exhibited a strong spontaneous mutator phenotype (about 20-fold) as determined by a rifampin forward mutation assay. In contrast to nth mutants, which showed a weak mutator phenotype, nei single mutants behaved as the wild type. PMID:9171429

  7. Regulation of ribosomal protein synthesis in an Escherichia coli mutant missing ribosomal protein L1.

    PubMed Central

    Jinks-Robertson, S; Nomura, M

    1981-01-01

    In an Escherichia coli B strain missing ribosomal protein L1, the synthesis rate of L11 is 50% greater than that of other ribosomal proteins. This finding is in agreement with the previous conclusion that L1 regulates synthesis of itself and L11 and indicates that this regulation is important for maintaining the balanced synthesis of ribosomal proteins under physiological conditions. PMID:7009590

  8. Bacteriophage Mu-1-induced permeability mutants in Escherichia coli K-12.

    PubMed Central

    Aline, R F; Reznikoff, W S

    1975-01-01

    Apparent permeability mutations were produced in Escherichia coli K-12 by bacteriophage mu-1 mutagenesis. They are pleiotropic mutations showing sensitivity to a number of detergents and unrelated antibiotics, and presumably they affect cell wall or membrane biosynthesis. One of the mutations was genetically mapped at a site in or near the acrA and mtc loci at approximately 10.5 min on the Taylor and Trotter map (1972). PMID:1100615

  9. Escherichia coli B/r leuK mutant lacking pseudouridine synthase I activity.

    PubMed Central

    Searles, L L; Jones, J W; Fournier, M J; Grambow, N; Tyler, B; Calvo, J M

    1986-01-01

    Escherichia coli B/r strain EB146 containing mutation leuK16 has elevated levels of enzymes involved in the synthesis of leucine, valine, isoleucine, histidine, and tryptophan (Brown et al., J. Bacteriol. 135:542-550, 1978). We show here that strain EB146 (leuK16) has properties that are similar to those of E. coli and Salmonella typhimurium hisT strains. In tRNA1Leu from both hisT and leuK strains, positions 39 and 41 are uridine residues rather than pseudouridine residues. Furthermore, in tRNA3Leu and tRNA4Leu from a leuK strain, uridine residues at positions 39 and 40, respectively, are unmodified. Pseudouridine synthase I activity is missing in extracts of strain EB146 (leuK16), and extracts of strain EB146 (leuK16) and of a hisT strain do not complement one another in vitro. Four phenotypes of strain EB146 (leuK16), leucine excretion, wrinkled colony morphology, and elevated levels of leu and his enzymes, are complemented by a plasmid having a 1.65-kilobase DNA fragment containing the E. coli K-12 hisT locus. These results indicate that either leuK codes for pseudouridine synthase I (and is thus a hisT locus in reality) or, less likely, it codes for a product that affects the synthesis or activity of pseudouridine synthase I. Images PMID:3514581

  10. [Heavy metal cation-induced increase in the antimicrobial activity of gramicidin S. Increased sensitivity of metal-resistant mutants of Escherichia coli B to the antibiotic].

    PubMed

    Kuzovnikova, T A; Fedorov, Iu I

    1990-04-01

    Gramicidin S response of metal resistant mutants of E. coli B and the effect of concentrations of Cu2+, Ag+, Co2+ and Cd2+ on the growth and sensitivity of E. coli B to cationic antibiotics, i.e. gramicidin S2+ and streptomycin2+, were studied. It was shown that the metal-cumulating mutants of E. coli B with two different mechanisms of cross resistance to Cu2+, Cd2+ and Ag+ had higher sensitivity to gramicidin S than the initial wild type strain of E. coli B. It was found that in the threshold or higher doses the salts of Cu, Ag, Co and Cd increased the gramicidin S antimicrobial action on actively metabolizing cells of E. coli B. Analysis of the experimental data as well as the literature ones suggested that the synergic action of gramicidin S and the heavy metals stemmed from an increase in the cationic conductivity of the cytoplasma membrane modified by the metals in the threshold doses which induced an increase in the transport and accumulation of the cations in the bacterial cells by the electric field gradient (with the negative sign inside). Withdrawal of Ca2+ and Mg2+ from the E. coli outer structures into the cytoplasm impaired the barrier properties of the outer membrane and promoted binding of the gramicidin S cations to the liberated anionic groups of the E. coli outer structures and potentiation of the gramicidin S antimicrobial activity as was shown in our experiments.

  11. Selection of multiple-antibiotic-resistant (mar) mutants of Escherichia coli by using the disinfectant pine oil: roles of the mar and acrAB loci.

    PubMed Central

    Moken, M C; McMurry, L M; Levy, S B

    1997-01-01

    Mutants of Escherichia coli selected for resistance to the disinfectant pine oil or to a household product containing pine oil also showed resistance to multiple antibiotics (tetracycline, ampicillin, chloramphenicol, and nalidixic acid) and overexpressed the marA gene. Likewise, antibiotic-selected Mar mutants, which also overexpress marA, were resistant to pine oil. Deletion of the mar or acrAB locus, the latter encoding a multidrug efflux pump positively regulated in part by MarA, increased the susceptibility of wild-type and mutant strains to pine oil. PMID:9420057

  12. The effect of catalase on recovery of heat-injured DNA-repair mutants of Escherichia coli.

    PubMed

    Mackey, B M; Seymour, D A

    1987-06-01

    The apparent sensitivity of Escherichia coli K12 to mild heat was increased by recA (def), recB and polA, but not by uvrA, uvrB or recF mutations. However, addition of catalase to the rich plating medium used to assess viability restored counts of heat-injured recA, recB and polA strains to wild-type levels. E. coli p3478 polA was sensitized by heat to a concentration of hydrogen peroxide similar to that measured in autoclaved recovery medium. The apparent heat sensitivity of DNA-repair mutants is thus due to heat-induced sensitivity to the low levels of peroxide present in rich recovery media. It is proposed that DNA damage in heated cells could occur indirectly by an oxidative mechanism. The increased peroxide sensitivity of heat-injured cells was not due to a decrease in total catalase activity but may be related specifically to inactivation of the inducible catalase/peroxidase (HPI).

  13. Functional citric acid cycle in an arcA mutant of Escherichia coli during growth with nitrate under anoxic conditions.

    PubMed

    Prohl, C; Wackwitz, B; Vlad, D; Unden, G

    1998-07-01

    The operation of the citric acid cycle of Escherichia coli during nitrate respiration (anoxic conditions) was studied by measuring end products and enzyme activities. Excretion of products other than CO2, such as acetate or ethanol, was taken as an indication for a non-functional cycle. From glycerol, approximately 0.3 mol acetate was produced; the residual portion was completely oxidized, indicating the presence of a partially active citric acid cycle. In an arcA mutant devoid of the transcriptional regulator ArcA, glycerol was completely oxidized with nitrate as an electron acceptor, demonstrating derepression and function of the complete pathway. Glucose, on the other hand, was excreted mostly as acetate by the wild-type and by the arcA mutant. During growth on glucose, but not on glycerol, activities of succinate dehydrogenase and of 2-oxoglutarate dehydrogenase were missing nearly completely. Thus, the previously described strong repression of the citric acid cycle during nitrate respiration occurs only during growth on glucose and is the effect of anaerobic and, more important, of glucose repression. In Pseudomonas fluorescens (but not Pseudomonas stutzeri), a similar decrease of citric acid cycle function during anaerobic growth with nitrate was found, indicating a broad distribution of this regulatory principle.

  14. A genome-scale Escherichia coli kinetic metabolic model k-ecoli457 satisfying flux data for multiple mutant strains

    PubMed Central

    Khodayari, Ali; Maranas, Costas D.

    2016-01-01

    Kinetic models of metabolism at a genome scale that faithfully recapitulate the effect of multiple genetic interventions would be transformative in our ability to reliably design novel overproducing microbial strains. Here, we introduce k-ecoli457, a genome-scale kinetic model of Escherichia coli metabolism that satisfies fluxomic data for wild-type and 25 mutant strains under different substrates and growth conditions. The k-ecoli457 model contains 457 model reactions, 337 metabolites and 295 substrate-level regulatory interactions. Parameterization is carried out using a genetic algorithm by simultaneously imposing all available fluxomic data (about 30 measured fluxes per mutant). The Pearson correlation coefficient between experimental data and predicted product yields for 320 engineered strains spanning 24 product metabolites is 0.84. This is substantially higher than that using flux balance analysis, minimization of metabolic adjustment or maximization of product yield exhibiting systematic errors with correlation coefficients of, respectively, 0.18, 0.37 and 0.47 (k-ecoli457 is available for download at http://www.maranasgroup.com). PMID:27996047

  15. Efficient production of mutant phytase (phyA-7) derived from Selenomonas ruminantium using recombinant Escherichia coli in pilot scale.

    PubMed

    Chi-Wei Lan, John; Chang, Chih-Kai; Wu, Ho-Shing

    2014-09-01

    A mutant gene of rumen phytase (phyA-7) was cloned into pET23b(+) vector and expressed in the Escherichia coli BL21 under the control of the T7 promoter. The study of fermentation conditions includes the temperature impacts of mutant phytase expression, the effect of carbon supplements over induction stage, the inferences of acetic acid accumulation upon enzyme expression and the comparison of one-stage and two-stage operations in batch mode. The maximum value of phytase activity was reached 107.0 U mL(-1) at induction temperature of 30°C. Yeast extract supplement demonstrated a significant increase on both protein concentration and phytase activity. The acetic acid (2 g L(-1)) presented in the modified synthetic medium demonstrated a significant decrease on expressed phytase activity. A two-stage batch operation enhanced the level of phytase activity from 306 to 1204 U mL(-1) in the 20 L of fermentation scale. An overall 3.7-fold improvement in phytase yield (35,375.72-1,31,617.50 U g(-1) DCW) was achieved in the two-stage operation.

  16. Polymorphic variation in susceptibility and metabolism of triclosan-resistant mutants of Escherichia coli and Klebsiella pneumoniae clinical strains obtained after exposure to biocides and antibiotics.

    PubMed

    Curiao, Tânia; Marchi, Emmanuela; Viti, Carlo; Oggioni, Marco R; Baquero, Fernando; Martinez, José Luis; Coque, Teresa M

    2015-01-01

    Exposure to biocides may result in cross-resistance to other antimicrobials. Changes in biocide and antibiotic susceptibilities, metabolism, and fitness costs were studied here in biocide-selected Escherichia coli and Klebsiella pneumoniae mutants. E. coli and K. pneumoniae mutants with various degrees of triclosan susceptibility were obtained after exposure to triclosan (TRI), benzalkonium chloride (BKC), chlorhexidine (CHX) or sodium hypochlorite (SHC), and ampicillin or ciprofloxacin. Alterations in antimicrobial susceptibility and metabolism in mutants were tested using Phenotype MicroArrays. The expression of AcrAB pump and global regulators (SoxR, MarA, and RamA) was measured by quantitative reverse transcription-PCR (qRT-PCR), and the central part of the fabI gene was sequenced. The fitness costs of resistance were assessed by a comparison of relative growth rates. Triclosan-resistant (TRI(r)) and triclosan-hypersusceptible (TRI(hs)) mutants of E. coli and K. pneumoniae were obtained after selection with biocides and/or antibiotics. E. coli TRI(r) mutants, including those with mutations in the fabI gene or in the expression of acrB, acrF, and marA, exhibited changes in susceptibility to TRI, CHX, and antibiotics. TRI(r) mutants for which the TRI MIC was high presented improved metabolism of carboxylic acids, amino acids, and carbohydrates. In TRI(r) mutants, resistance to one antimicrobial provoked hypersusceptibility to another one(s). TRI(r) mutants had fitness costs, particularly marA-overexpressing (E. coli) or ramA-overexpressing (K. pneumoniae) mutants. TRI, BKC, and CIP exposure frequently yielded TRI(r) mutants exhibiting alterations in AraC-like global regulators (MarA, SoxR, and RamA), AcrAB-TolC, and/or FabI, and influencing antimicrobial susceptibility, fitness, and metabolism. These various phenotypes suggest a trade-off of different selective processes shaping the evolution toward antibiotic/biocide resistance and influencing other adaptive

  17. Modeling mutant distribution in a stressed Escherichia coli bacteria population using experimental data

    NASA Astrophysics Data System (ADS)

    Bazzani, Armando; Fani, Renato; Freguglia, Paolo

    2014-01-01

    In this paper we propose a statistical physics approach to experimental results on bacterial mutations (Escherichia coli). We get scaling laws that describe some generic traits and suggest some features of the underlying dynamical structure for the considered evolution process. Our main assumption is that the evolution dynamics could be visualized as a random walk on a fitness landscape whose topological structure is analogous to the structure of energy landscape potentials used in Physics and Chemistry. Then we relate the generic distribution of local minima attraction basins to the number of bacterial mutations and we discuss the comparison with experimental results.

  18. Increased Hydrolysis of Oximino-β-Lactams by CMY-107, a Tyr199Cys Mutant Form of CMY-2 Produced by Escherichia coli

    PubMed Central

    Vetouli, E. E.; Bozavoutoglou, E.; Lebessi, E.; Tzelepi, E.; Tzouvelekis, L. S.

    2015-01-01

    The cephalosporinase CMY-107, a Tyr199Cys mutant form of CMY-2 encoded by an IncI self-transferable plasmid carried by an Escherichia coli clinical strain, was characterized. The enzyme hydrolyzed oximino-cephalosporins and aztreonam more efficiently than CMY-2 did. PMID:26438499

  19. Validation of the mutant selection window hypothesis with fosfomycin against Escherichia coli and Pseudomonas aeruginosa: an in vitro and in vivo comparative study.

    PubMed

    Pan, Ai-Jun; Mei, Qing; Ye, Ying; Li, Hong-Ru; Liu, Bao; Li, Jia-Bin

    2017-02-01

    The purpose of this study was to validate the mutant selection window (MSW) hypothesis in vitro and in vivo with Escherichia coli and Pseudomonas aeruginosa exposed to fosfomycin. Two standard strains of Gram-negative bacteria, those are E. coli ATCC 25922 and P. aeruginosa ATCC 27853, were exposed to fosfomycin at concentrations below MIC, between the MIC and the mutant prevention concentration (MPC), and above the MPC in Luria-Bertani broth and in a tissue-cage infection model, respectively. With the in vitro time-kill studies, there were bacterial re-growth and emergence of resistance thereafter for both strains at antibiotic concentrations of × 4, × 8 and × 16 MIC. In our animal model, the loss in susceptibility of P. aeruginosa at fosfomycin concentrations fluctuated between the lower and upper boundaries of the MSW. In contrast, the emergence of resistant mutants of E. coli was not observed in vivo, regardless of fosfomycin dosage. Interestingly, the in vitro-isolated resistant mutants of E. coli showed a decreased growth rate compared with the susceptible parental strains, whereas no fitness cost in P. aeruginosa was observed. The emergence of antibiotic resistance is shaped by several factors. MSW theory may not apply to all antimicrobial-pathogen combinations. Before it can be used as a framework for the design of antimicrobial therapy, the existence of the window must be demonstrated not only in vitro but also in vivo.

  20. Physiological characterization of an Escherichia coli mutant altered in the structure of murein lipoprotein.

    PubMed Central

    Yem, D W; Wu, H C

    1978-01-01

    Studies using isogenic transductant strains mlpA+ and mlpA as well as reversion analysis suggested that the physiological consequences of a structural gene mutation in murein lipoprotein include (i) increased sensitivity toward chelating agents ethylenediaminetetraacetic acid and ethyleneglycol-bis (beta-aminoethyl ether)-N,N-tetraacetic acid, (ii) leakage of periplasmic enzyme ribonuclease, (iii) weakened association between the outer membrane and the rigid layer accentuated by Mg2+ starvation, resulting in the formation of outer membrane blebs, and (iv) decreased growth rate in media of low ionic strength or low osmolarity. It is suggested that the bound form of lipoprotein plays an important role in the maintenance of the structural integrity of the outer membrane of the Escherichia coli cell envelope. Other outer membrane components may also contribute to the anchorage of outer membrane to the rigid layer, probably through ionic interactions with divalent cations. Using the phenotype of ribonuclease leakage as an unselected marker in a three-factor cross with P1 transduction, we were able to establish the gene order of man mlpA aroD pps on the E. coli chromosome. Images PMID:417067

  1. Complexes of mutants of Escherichia coli aminopeptidase P and the tripeptide substrate ValProLeu

    SciTech Connect

    Graham, Stephen C.; Guss, J. Mitchell

    2008-09-17

    Aminopeptidase P (APPro) is a manganese-containing enzyme that catalyses the hydrolysis of the N-terminal residue of a polypeptide if the second residue is proline. Structures of APPro mutants with reduced or negligible activity have been determined in complex with the tripeptide substrate ValProLeu. In the complex of Glu383Ala APPro with ValProLeu one of the two metal sites is only partly occupied, indicating an essential role for Glu383 in metal binding in the presence of substrate. His361Ala APPro clearly possesses residual activity as the ValProLeu substrate has been cleaved in the crystals; difference electron density consistent with bound ProLeu dipeptide and a disordered Val amino acid is present at the active site. Contrary to previous suggestions, the His243Ala mutant is capable of binding substrate. The structure of the His243Ala APPro complex with ValProLeu shows that the peptide interacts with one of the active-site metal atoms via its terminal amino group. The implications of these complexes for the roles of the respective residues in APPro catalysis are discussed.

  2. The Genetic Dependence of Recombination in Recd Mutants of Escherichia Coli

    PubMed Central

    Lovett, S. T.; Luisi-DeLuca, C.; Kolodner, R. D.

    1988-01-01

    RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunbits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD(+) strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations. PMID:3065139

  3. Genetic dependence of recombination in recD mutants of Escherichia coli

    SciTech Connect

    Lovett, S.T.; Luisi-DeLuca, C.; Kolodner, R.D.

    1988-09-01

    RecBCD enzyme has multiple activities including helicase, exonuclease and endonuclease activities. Mutations in the genes recB or recC, encoding two subunits of the enzyme, reduce the frequency of many types of recombinational events. Mutations in recD, encoding the third subunit, do not reduce recombination even though most of the activities of the RecBCD enzyme are severely reduced. In this study, the genetic dependence of different types of recombination in recD mutants has been investigated. The effects of mutations in genes in the RecBCD pathway (recA and recC) as well as the genes specific for the RecF pathway (recF, recJ, recN, recO, recQ, ruv and lexA) were tested on conjugational, transductional and plasmid recombination, and on UV survival. recD mutants were hyper-recombinogenic for all the monitored recombination events, especially those involving plasmids, and all recombination events in recD strains required recA and recC. In addition, unlike recD+ strains, chromosomal recombination events and the repair of UV damage to DNA in recD strains were dependent on one RecF pathway gene, recJ. Only a subset of the tested recombination events were affected by ruv, recN, recQ, recO and lexA mutations.

  4. Mutagenesis in the lacI gene target of E. coli: improved analysis for lacI(d) and lacO mutants.

    PubMed

    Swerdlow, Sarah J; Schaaper, Roel M

    2014-12-01

    The lacI gene of Escherichia coli has been a highly useful target for studies of mutagenesis, particularly for analysis of the specificity (spectrum) of mutations generated under a variety of conditions and in various genetic backgrounds. The gene encodes the repressor of the lac operon, and lacI-defective mutants displaying constitutive expression of the operon are readily selected. DNA sequencing of the lacI mutants has often been confined to the N-terminal region of the protein, as it presents a conveniently short target with a high density of detectably mutable sites. Mutants in this region are easily selected due to their dominance in a genetic complementation test (lacI(d) mutants). A potential complication in these studies is that constitutive expression of lac may also arise due to mutations in the lac operator (lacO mutants). Under some conditions, for example when analyzing spontaneous mutations, lacO mutants can comprise a very high fraction of the constitutive mutants due to a strong base-substitution hotspot in the lac operator. Such mutational hot spots diminish the return of the sequencing effort and do not yield significant new information. For this reason, a procedure to eliminate the lacO mutants prior to DNA sequencing is desirable. Here, we report a simple method that allows screening out of lacO mutants. This method is based on the lack of resistance of lacO mutants to kanamycin under conditions when the kan gene is expressed from a plasmid under control of the lac promoter-operator (lacPO). We show data validating the new approach with sets of known lacI(d) and lacO mutants, and further apply it to the generation of a new collection of spontaneous mutations, where lacO mutants have historically been a significant contributor.

  5. Dual Roles of Capsular Extracellular Polymeric Substances in Photocatalytic Inactivation of Escherichia coli: Comparison of E. coli BW25113 and Isogenic Mutants

    PubMed Central

    Huang, Guocheng; Xia, Dehua; Ng, Tsz Wai; Yip, Ho Yin; Li, Guiying; Zhao, Huijun

    2015-01-01

    The dual roles of capsular extracellular polymeric substances (EPS) in the photocatalytic inactivation of bacteria were demonstrated in a TiO2-UVA system, by comparing wild-type Escherichia coli strain BW25113 and isogenic mutants with upregulated and downregulated production of capsular EPS. In a partition system in which direct contact between bacterial cells and TiO2 particles was inhibited, an increase in the amount of EPS was associated with increased bacterial resistance to photocatalytic inactivation. In contrast, when bacterial cells were in direct contact with TiO2 particles, an increase in the amount of capsular EPS decreased cell viability during photocatalytic treatment. Taken together, these results suggest that although capsular EPS can protect bacterial cells by consuming photogenerated reactive species, it also facilitates photocatalytic inactivation of bacteria by promoting the adhesion of TiO2 particles to the cell surface. Fluorescence microscopy and scanning electron microscopy analyses further confirmed that high capsular EPS density led to more TiO2 particles attaching to cells and forming bacterium-TiO2 aggregates. Calculations of interaction energy, represented by extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) potential, suggested that the presence of capsular EPS enhances the attachment of TiO2 particles to bacterial cells via acid-base interactions. Consideration of these mechanisms is critical for understanding bacterium-nanoparticle interactions and the photocatalytic inactivation of bacteria. PMID:26002903

  6. Cloning, sequencing, and expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in E. coli.

    PubMed

    Pratush, Amit; Seth, Amit; Bhalla, T C

    2012-10-01

    The NHase encoding gene of mutant 4D was isolated by PCR amplification. The NHase gene of mutant 4D was successfully cloned and expressed in Escherichia coli by using Ek/LIC Duet cloning kits (Novagen). For the active expression of the NHase gene, the co-expression of small cobalt transporter gene (P-protein gene) has also been co-expressed with NHase gene E. coli. The nucleotide sequence of this NHase gene revealed high homology with the H-NHase of Rhodococcus rhodochrous J1. The recombinant E. coli cells showed higher NHase activity (5.9 U/mg dcw) as compared to the wild (4.1 U/mg dcw) whereas it is less than the mutant strain (8.4 U/mg dcw). Addition of cobalt ion in Luria-Bertani medium is needed up to a very small concentration (0.4 mM) for NHase activity. The recombinant E. coli exhibited maximum NHase activity at 6 h of incubation and was purified with a yield of 56 % with specific activity of 37.1 U/mg protein.

  7. Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival

    PubMed Central

    Kuban, Wojciech; Vaisman, Alexandra; McDonald, John P.; Karata, Kiyonobu; Yang, Wei; Goodman, Myron F.; Woodgate, Roger

    2012-01-01

    Escherichia coli polymerase V (pol V/UmuD'2C) is a low-fidelity DNA polymerase that has recently been shown to avidly incorporate ribonucleotides (rNTPs) into undamaged DNA. The fidelity and sugar selectivity of pol V can be modified by missense mutations around the “steric gate” of UmuC. Here, we analyze the ability of three steric gate mutants of UmuC to facilitate translesion DNA synthesis (TLS) of a cyclobutane pyrimidine dimer (CPD) in vitro, and to promote UV-induced mutagenesis and cell survival in vivo. The pol V (UmuC_F10L) mutant discriminates against rNTP and incorrect dNTP incorporation much better than wild-type pol V and although exhibiting a reduced ability to bypass a CPD in vitro, does so with high-fidelity and consequently produces minimal UV-induced mutagenesis in vivo. In contrast, pol V (UmuC_Y11A) readily misincorporates both rNTPs and dNTPs during efficient TLS of the CPD in vitro. However, cells expressing umuD'C (Y11A) were considerably more UV-sensitive and exhibited lower levels of UV-induced mutagenesis than cells expressing wild-type umuD'C or umuD'C (Y11F). We propose that the increased UV-sensitivity and reduced UV-mutability of umuD'C (Y11A) is due to excessive incorporation of rNTPs during TLS that are subsequently targeted for repair, rather than an inability to traverse UV-induced lesions. PMID:22784977

  8. A double, long polar fimbria mutant of Escherichia coli O157:H7 expresses Curli and exhibits reduced in vivo colonization.

    PubMed

    Lloyd, Sonja J; Ritchie, Jennifer M; Rojas-Lopez, Maricarmen; Blumentritt, Carla A; Popov, Vsevolod L; Greenwich, Jennifer L; Waldor, Matthew K; Torres, Alfredo G

    2012-03-01

    Escherichia coli O157:H7 causes food and waterborne enteric infections that can result in hemorrhagic colitis and life-threatening hemolytic uremic syndrome. Intimate adherence of the bacteria to intestinal epithelial cells is mediated by intimin, but E. coli O157:H7 also possess several other putative adhesins, including curli and two operons that encode long polar fimbriae (Lpf). To assess the importance of Lpf for intestinal colonization, we performed competition experiments between E. coli O157:H7 and an isogenic ΔlpfA1 ΔlpfA2 double mutant in the infant rabbit model. The mutant was outcompeted in the ileum, cecum, and midcolon, suggesting that Lpf contributes to intestinal colonization. In contrast, the ΔlpfA1 ΔlpfA2 mutant showed increased adherence to colonic epithelial cells in vitro. Transmission electron microscopy revealed curli-like structures on the surface of the ΔlpfA1 ΔlpfA2 mutant, and the presence of curli was confirmed by Congo red binding, immunogold-labeling electron microscopy, immunoblotting, and quantitative real-time reverse transcription-PCR (qRT-PCR) measuring csgA expression. However, deletion of csgA, which encodes the major curli subunit, does not appear to affect intestinal colonization. In addition to suggesting that Lpf can contribute to EHEC intestinal colonization, our observations indicate that the regulatory pathways governing the expression of Lpf and curli are interdependent.

  9. Genetic analysis of the Rhizobium meliloti bacA gene: functional interchangeability with the Escherichia coli sbmA gene and phenotypes of mutants.

    PubMed

    Ichige, A; Walker, G C

    1997-01-01

    The Rhizobium meliloti bacA gene encodes a function that is essential for bacterial differentiation into bacteroids within plant cells in the symbiosis between R. meliloti and alfalfa. An Escherichia coli homolog of BacA, SbmA, is implicated in the uptake of microcin B17, microcin J25 (formerly microcin 25), and bleomycin. When expressed in E. coli with the lacZ promoter, the R. meliloti bacA gene was found to suppress all the known defects of E. coli sbmA mutants, namely, increased resistance to microcin B17, microcin J25, and bleomycin, demonstrating the functional similarity between the two proteins. The R. meliloti bacA386::Tn(pho)A mutant, as well as a newly constructed bacA deletion mutant, was found to show increased resistance to bleomycin. However, it also showed increased resistance to certain aminoglycosides and increased sensitivity to ethanol and detergents, suggesting that the loss of bacA function causes some defect in membrane integrity. The E. coli sbmA gene suppressed all these bacA mutant phenotypes as well as the Fix- phenotype when placed under control of the bacA promoter. Taken together, these results strongly suggest that the BacA and SbmA proteins are functionally similar and thus provide support for our previous hypothesis that BacA may be required for uptake of some compound that plays an important role in bacteroid development. However, the additional phenotypes of bacA mutants identified in this study suggest the alternative possibility that BacA may be needed for membrane integrity, which is likely to be critically important during the early stages of bacterial differentiation within plant cells.

  10. Pathogenicity of an Escherichia coli O115:K"V165" mutant negative for F165(1) fimbriae in septicemia of gnotobiotic pigs.

    PubMed Central

    Ngeleka, M; Jacques, M; Martineau-Doizé, B; Daigle, F; Harel, J; Fairbrother, J M

    1993-01-01

    To evaluate the role of the F165(1) fimbrial system in the pathogenesis of septicemia, 2-day-old germfree pigs were inoculated intragastrically with Escherichia coli O115:K"V165":F165 wild-type strain 5131, its F165(1)-negative TnphoA mutant M48, or E. coli O115:K(-):F165(-) wild-type strain 862B. Pigs were sacrificed at different times (3, 6, 12, 24, 48, and 96 h) postinfection (p.i.). Pigs inoculated with strain 5131 developed clinical signs (anorexia, lameness, reluctance to move, or lack of motor coordination) and were moribund within 48 h p.i., and, at necropsy, infecting bacteria were isolated in various extraintestinal organs. Strain 5131 was isolated as early as 6 h p.i. from the blood of inoculated pigs. Pigs inoculated with mutant M48 developed only mild clinical signs at 96 h p.i. Mutant M48 colonized extraintestinal organs of pigs but to a lesser extent than the parent strain did. In contrast to the parent strain, this mutant was not isolated in the blood of inoculated pigs. Pigs inoculated with strain 862B remained normal during the experiment. All of the strains colonized the mucus layer of the intestine, but no histological changes of intestinal mucosa were observed by either light or electron microscopy. The parent strain, but not the mutant M48, expressed F165(1) in vivo. In a competitive study in which the parent strain and its afimbrial mutant were inoculated simultaneously, clinical signs of septicemia developed 24 h after inoculation, and only the parent strain 5131 was isolated from the blood of inoculated pigs. Our results suggest that the F165(1) fimbrial system of E. coli O115:K"V165" strains may play an important role in the ability of the bacteria to survive in the blood and spread systemically through the porcine host. Images PMID:8094383

  11. Novel Escherichia coli RF1 mutants with decreased translation termination activity and increased sensitivity to the cytotoxic effect of the bacterial toxins Kid and RelE.

    PubMed

    Diago-Navarro, Elizabeth; Mora, Liliana; Buckingham, Richard H; Díaz-Orejas, Ramón; Lemonnier, Marc

    2009-01-01

    Novel mutations in prfA, the gene for the polypeptide release factor RF1 of Escherichia coli, were isolated using a positive genetic screen based on the parD (kis, kid) toxin-antitoxin system. This original approach allowed the direct selection of mutants with altered translational termination efficiency at UAG codons. The isolated prfA mutants displayed a approximately 10-fold decrease in UAG termination efficiency with no significant changes in RF1 stability in vivo. All three mutations, G121S, G301S and R303H, were situated close to the nonsense codon recognition site in RF1:ribosome complexes. The prfA mutants displayed increased sensitivity to the RelE toxin encoded by the relBE system of E. coli, thus providing in vivo support for the functional interaction between RF1 and RelE. The prfA mutants also showed increased sensitivity to the Kid toxin. Since this toxin can cleave RNA in a ribosome-independent manner, this result was not anticipated and provided first evidence for the involvement of RF1 in the pathway of Kid toxicity. The sensitivity of the prfA mutants to RelE and Kid was restored to normal levels upon overproduction of the wild-type RF1 protein. We discuss these results and their utility for the design of novel antibacterial strategies in the light of the recently reported structure of ribosome-bound RF1.

  12. Crystallization and preliminary X-ray diffraction analysis of D53H mutant Escherichia coli cAMP receptor protein.

    PubMed

    Huang, Jing; Wu, Tong; Guo, Zheng; Lou, Tiantian; Yu, Shaoning; Gong, Weimin; Ji, Chaoneng

    2013-12-01

    The Escherichia coli cyclic AMP receptor protein (CRP) is a prokaryotic global transcription activator protein that controls the expression of many different genes. Wild-type CRP can bind to special DNA sequences in the presence of cAMP. The substitution of Asp53 by His results in the CRP* phenotype, which does not require exogenous cAMP. In the present study, the D53H CRP mutant was overexpressed, purified and crystallized. cAMP-free D53H CRP crystals were obtained and diffracted to a resolution of 2.9 Å. Based on the systematic absences of the crystals, the space group is likely to be P2(1)2(1)2(1), with unit-cell parameters a = 76.66, b = 152.14, c = 176.11 Å. The asymmetric unit was confirmed to contain four protein dimers, with a Matthews coefficient of 2.71 Å(3) Da(-1) and a solvent content of 54.68%.

  13. Stimulation of Ribonucleic Acid Synthesis by Chloramphenicol in a rel+ Aminoacyl-Transfer Ribonucleic Acid Synthetase Mutant of Escherichia coli

    PubMed Central

    Yegian, Charles D.; Vanderslice, Rebecca W.

    1971-01-01

    Escherichia coli strain 9D3 possesses a highly temperature-sensitive valyl-transfer ribonucleic acid (tRNA) synthetase (EC 6.1.1.9). Since 9D3 is a rel+ strain, it cannot carry out net RNA synthesis at high temperature. A 100-μg amount of chloramphenicol (CAP) per ml added in the absence of valine cannot stimulate RNA synthesis. Either 300 μg of CAP or 100 μg of CAP plus 50 μg of valine per ml, however, promotes nearly maximal RNA synthesis. These results can be understood as follows. (i) Valyl-tRNA is required for net RNA synthesis, (ii) the synthetase lesion is incomplete, (iii) the rate of mutant acylation of tRNAval at high temperature is valine-dependent, and (iv) the CAP concentration determines the rate of residual protein synthesis. Data are also presented which demonstrate that the rate of net RNA synthesis can greatly increase long after the addition of CAP, if the amount of valyl-tRNA increases. PMID:4942766

  14. Inducible Prophage Mutant of Escherichia coli Can Lyse New Host and the Key Sites of Receptor Recognition Identification

    PubMed Central

    Chen, Mianmian; Zhang, Lei; Xin, Sipei; Yao, Huochun; Lu, Chengping; Zhang, Wei

    2017-01-01

    The use of bacteriophages as therapeutic agents is hindered by their narrow and specific host range, and by a lack of the knowledge concerning the molecular mechanism of receptor recognition. Two P2-like coliphages, named P88 and pro147, were induced from Escherichia coli strains K88 and DE147, respectively. A comparison of the genomes of these two and other P2-like coliphages obtained from GenBank showed that the tail fiber protein genes, which are the key genes for receptor recognition in other myoviridae phages, showed more diversity than the conserved lysin, replicase, and terminase genes. Firstly, replacing hypervariable region 2 (HR2: amino acids 716–746) of the tail fiber protein of P88 with that of pro147 changed the host range of P88. Then, replacing six amino acids in HR2 with the corresponding residues from pro147 altered the host range only in these mutants with changes at position 730 (leucine) and 744 (glutamic acid). Thus, we predicted that these amino acids are vital to establish the host range of P88. This study provided a vector of lysogenic bacteria that could be used to change or expand the phage host range of P88. These results illustrated that, in P2-like phage P88, the tail fiber protein determined the receptor recognition. Amino acids 716–746 and the amino acids at positions 730 and 744 were important for receptor recognition. PMID:28203234

  15. Laboratory-Evolved Mutants of an Exogenous Global Regulator, IrrE from Deinococcus radiodurans, Enhance Stress Tolerances of Escherichia coli

    PubMed Central

    Chen, Tingjian; Wang, Jianqing; Yang, Rong; Li, Jicong; Lin, Min; Lin, Zhanglin

    2011-01-01

    Background The tolerance of cells toward different stresses is very important for industrial strains of microbes, but difficult to improve by the manipulation of single genes. Traditional methods for enhancing cellular tolerances are inefficient and time-consuming. Recently, approaches employing global transcriptional or translational engineering methods have been increasingly explored. We found that an exogenous global regulator, irrE from an extremely radiation-resistant bacterium, Deinococcus radiodurans, has the potential to act as a global regulator in Escherichia coli, and that laboratory-evolution might be applied to alter this regulator to elicit different phenotypes for E. coli. Methodology/Principal Findings To extend the methodology for strain improvement and to obtain higher tolerances toward different stresses, we here describe an approach of engineering irrE gene in E. coli. An irrE library was constructed by randomly mutating the gene, and this library was then selected for tolerance to ethanol, butanol and acetate stresses. Several mutants showing significant tolerances were obtained and characterized. The tolerances of E. coli cells containing these mutants were enhanced 2 to 50-fold, based on cell growth tests using different concentrations of alcohols or acetate, and enhanced 10 to 100-fold based on ethanol or butanol shock experiments. Intracellular reactive oxygen species (ROS) assays showed that intracellular ROS levels were sharply reduced for cells containing the irrE mutants. Sequence analysis of the mutants revealed that the mutations distribute cross all three domains of the protein. Conclusions To our knowledge, this is the first time that an exogenous global regulator has been artificially evolved to suit its new host. The successes suggest the possibility of improving tolerances of industrial strains by introducing and engineering exogenous global regulators, such as those from extremophiles. This new approach can be applied alone or

  16. Genetic and biochemical analysis of gonococcal IgA1 protease: cloning in Escherichia coli and construction of mutants of gonococci that fail to produce the activity.

    PubMed Central

    Koomey, J M; Gill, R E; Falkow, S

    1982-01-01

    The biological significance of bacterial extracellular proteases that specifically cleave human IgA1 is unknown. We have prepared a gene bank of gonococcal chromosomal DNA in Escherichia coli K-12 using a cosmid cloning system. Among these clones, we have identified and characterized an E. coli strain that elaborates an extracellular endopeptidase that is indistinguishable from gonococcal IgA1 protease in its substrate specificity and action on human IgA1. Analysis of recombinant plasmids and examination of plasmid-specific peptides in minicells have shown that the IgA1 protease activity in E. coli is associated with expression of a Mr 140,000 peptide. We have isolated IgA1 protease-deficient mutants of Neisseria gonorrhoeae by reintroduction of physically defined deletions of the cloned gene into the gonococcal chromosome by transformation. Images PMID:6818556

  17. Functional characterization of Escherichia coli GlpG and additional rhomboid proteins using an aarA mutant of Providencia stuartii.

    PubMed

    Clemmer, Katy M; Sturgill, Gwen M; Veenstra, Alexander; Rather, Philip N

    2006-05-01

    The Providencia stuartii AarA protein is a member of the rhomboid family of intramembrane serine proteases and required for the production of an extracellular signaling molecule that regulates cellular functions including peptidoglycan acetylation, methionine transport, and cysteine biosynthesis. Additional aarA-dependent phenotypes include (i) loss of an extracellular yellow pigment, (ii) inability to grow on MacConkey agar, and (iii) abnormal cell division. Since these phenotypes are easily assayed, the P. stuartii aarA mutant serves as a useful host system to investigate rhomboid function. The Escherichia coli GlpG protein was shown to be functionally similar to AarA and rescued the above aarA-dependent phenotypes in P. stuartii. GlpG proteins containing single alanine substitutions at the highly conserved catalytic triad of asparagine (N154A), serine (S201A), or histidine (H254A) residues were nonfunctional. The P. stuartii aarA mutant was also used as a biosensor to demonstrate that proteins from a variety of diverse sources exhibited rhomboid activity. In an effort to further investigate the role of a rhomboid protein in cell physiology, a glpG mutant of E. coli was constructed. In phenotype microarray experiments, the glpG mutant exhibited a slight increase in resistance to the beta-lactam antibiotic cefotaxime.

  18. Conversion of bacteriophage G4 single-stranded viral DNA to double-stranded replicative form in dna mutants of Escherichia coli.

    PubMed

    Kodaira, K I; Taketo, A

    1977-05-17

    Host functions involved in synthesis of parental replicative form of bacteriophage G4 were investigated using various replication mutants of Escheria coli. In dna+ bacteria, conversion of single-stranded viral DNA to replicative form DNA was insensitive to 200 microng/ml of rifampicin or 25 microng/ml of chloramphenicol. At high temperature, synthesis of parental replicative form was unaffected in mutants thermosensitive for dnaA, dnaB, dnaC(D), dnaE or dnaH. In dnaG or dnaZ mutants, however, parental replicative from DNA synthesis was clearly thermosensitive at 43 degrees C. Although the host rep product was essential for viral multiplication, the conversion of single stranded to replicative form was independent of the rep function.

  19. Effect of moderate salinity stress treatment on the stimulation of proline uptake and growth in Escherichia coli CSH4 and its mutants under high salinity.

    PubMed

    Nagata, Shinichi; Wang, Yaoqiang; Zhang, Hongyan; Sasaki, Hideaki; Oshima, Akinobu; Ishida, Akio

    2009-09-01

    Activity of proline uptake in Escherichia coli CSH4 was inhibited in the presence of 1 M NaCl, while it was recovered if the cells were incubated at 30 degrees C for 1 h in a moderate salinity stress (MSS) solution which consists of Davis minimal medium with 5 mM proline and 0.5 M NaCl. Then, an attempt was made to examine whether MSS treatment is also effective on the activity restoration of proline uptake and growth under high salinity for E. coli CSH4 mutants with different combinations of proP, putA, putP, and proU which are related to the transport and metabolization of proline. After MSS treatment, proline uptake was vigorously occurred for the mutants with proline transporter gene proP but not for its deficient ones. For the expression of proline uptake activities of these mutant strains after MSS treatment, PO(4)(3-) in MSS solution is more important than K(+). No growth of strain CSH4 and its mutants without MSS treatment was observed, when cultured in high osmotic medium G (0.8 M NaCl) consisting of 1 mM glycine betaine and Davis minimal medium without potassium phosphate supplemented. After MSS treatment, however, mutant strains lacking proP showed sufficient growth in medium G. Cell growth of proP(+) strains was recognized if MSS treatment was performed in the absence of proline. In conclusion, growth of mutant strains under high-salinity medium G depended on their amount of proline accumulated during MSS treatment, in which K(+) and PO(4)(3-) might play a key role to guarantee their sufficient growth.

  20. Deletion of luxS further attenuates the virulence of the avian pathogenic Escherichia coli aroA mutant.

    PubMed

    Han, Xiangan; Bai, Hao; Tu, Jian; Yang, Lijun; Xu, Da; Wang, Shaohui; Qi, Kezong; Fan, Guobo; Zhang, Yuxi; Zuo, Jiakun; Tian, Mingxing; Ding, Chan; Yu, Shengqing

    2015-11-01

    In this study, an aroA-deletion avian pathogenic Escherichia coli (APEC) mutant (strain DE17ΔaroA) and aroA and luxS double deletion APEC mutant (strain DE17ΔluxSΔaroA) were constructed from the APEC DE17 strain. The results showed that as compared to DE17ΔaroA, the virulence of DE17ΔluxSΔaroA was further attenuated by 200- and 31.7-fold, respectively, in ducklings based on the 50% lethal dose. The adherence and invasion abilities of DE17ΔluxSΔaroA and DE17ΔaroA were reduced by 36.5%/42.5% and 25.8%/29.3%, respectively, as compared to the wild-type strain DE17 (p < 0.05 and 0.01, respectively). Furthermore, in vivo studies showed that the bacterial loads of DE17ΔluxSΔaroA were reduced by 8400- and 11,333-fold in the spleen and blood of infected birds, respectively, while those of DE17ΔaroA were reduced by 743- and 1000-fold, respectively, as compared to the wild-type strain DE17. Histopathological analysis showed both that the mutants were associated with reduced pathological changes in the liver, spleen, and kidney of ducklings, and changes in DE17ΔluxSΔaroA-infected ducklings were reduced to a greater degree than those infected with DE17ΔaroA. Real-time polymerase chain reaction analysis further demonstrated that the mRNA levels of virulence-related genes (i.e., tsh, ompA, vat, iucD, pfs, fyuA, and fimC) were significantly decreased in DE17ΔaroA, especially in DE17ΔluxSΔaroA, as compared to DE17 (p < 0.05). In addition, the deletion of aroA or the double deletion of aroA and luxS reduced bacterial motility. To evaluate the potential use of DE17ΔluxSΔaroA as a vaccine candidate, 50 7-day-old ducklings were divided randomly into five groups of ten each for the experiment. The results showed that the ducklings immunized with inactivated DE17, DE17ΔluxS, DE17ΔaroA, and DE17ΔluxSΔaroA were 70.0%, 70.0%, 70.0, and 80.0% protected, respectively, after challenge with strain APEC DE17. The results of this study suggest that the double deletion of

  1. Modulation of Escherichia coli Adenylyl Cyclase Activity by Catalytic-Site Mutants of Protein IIAGlc of the Phosphoenolpyruvate:Sugar Phosphotransferase System

    PubMed Central

    Reddy, Prasad; Kamireddi, Madhavi

    1998-01-01

    It is demonstrated here that in Escherichia coli, the phosphorylated form of the glucose-specific phosphocarrier protein IIAGlc of the phosphoenolpyruvate:sugar phosphotransferase system is an activator of adenylyl cyclase and that unphosphorylated IIAGlc has no effect on the basal activity of adenylyl cyclase. To elucidate the specific role of IIAGlc phosphorylation in the regulation of adenylyl cyclase activity, both the phosphorylatable histidine (H90) and the interactive histidine (H75) of IIAGlc were mutated by site-directed mutagenesis to glutamine and glutamate. Wild-type IIAGlc and the H75Q mutant, in which the histidine in position 75 has been replaced by glutamine, were phosphorylated by the phosphohistidine-containing phosphocarrier protein (HPr∼P) and were equally potent activators of adenylyl cyclase. Neither the H90Q nor the H90E mutant of IIAGlc was phosphorylated by HPr∼P, and both failed to activate adenylyl cyclase. Furthermore, replacement of H75 by glutamate inhibited the appearance of a steady-state level of phosphorylation of H90 of this mutant protein by HPr∼P, yet the H75E mutant of IIAGlc was a partial activator of adenylyl cyclase. The H75E H90A double mutant, which cannot be phosphorylated, did not activate adenylyl cyclase. This suggests that the H75E mutant was transiently phosphorylated by HPr∼P but the steady-state level of the phosphorylated form of the mutant protein was decreased due to the repulsive forces of the negatively charged glutamate at position 75 in the catalytic pocket. These results are discussed in the context of the proximity of H75 and H90 in the IIAGlc structure and the disposition of the negative charge in the modeled glutamate mutants. PMID:9457881

  2. Isolation of an Escherichia coli K-12 mutant strain able to form biofilms on inert surfaces: involvement of a new ompR allele that increases curli expression.

    PubMed

    Vidal, O; Longin, R; Prigent-Combaret, C; Dorel, C; Hooreman, M; Lejeune, P

    1998-05-01

    Classical laboratory strains of Escherichia coli do not spontaneously colonize inert surfaces. However, when maintained in continuous culture for evolution studies or industrial processes, these strains usually generate adherent mutants which form a thick biofilm, visible with the naked eye, on the wall of the culture apparatus. Such a mutant was isolated to identify the genes and morphological structures involved in biofilm formation in the very well characterized E. coli K-12 context. This mutant acquired the ability to colonize hydrophilic (glass) and hydrophobic (polystyrene) surfaces and to form aggregation clumps. A single point mutation, resulting in the replacement of a leucine by an arginine residue at position 43 in the regulatory protein OmpR, was responsible for this phenotype. Observations by electron microscopy revealed the presence at the surfaces of the mutant bacteria of fibrillar structures looking like the particular fimbriae described by the Olsén group and designated curli (A. Olsén, A. Jonsson, and S. Normark, Nature 338:652-655, 1989). The production of curli (visualized by Congo red binding) and the expression of the csgA gene encoding curlin synthesis (monitored by coupling a reporter gene to its promoter) were significantly increased in the presence of the ompR allele described in this work. Transduction of knockout mutations in either csgA or ompR caused the loss of the adherence properties of several biofilm-forming E. coli strains, including all those which were isolated in this work from the wall of a continuous culture apparatus and two clinical strains isolated from patients with catheter-related infections. These results indicate that curli are morphological structures of major importance for inert surface colonization and biofilm formation and demonstrate that their synthesis is under the control of the EnvZ-OmpR two-component regulatory system.

  3. The alternative sigma factor sigma28 of Legionella pneumophila restores flagellation and motility to an Escherichia coli fliA mutant.

    PubMed Central

    Heuner, K; Hacker, J; Brand, B C

    1997-01-01

    Gene expression in Legionella pneumophila, the etiological agent of Legionnaires' disease, can be controlled by alternative forms of RNA polymerase programmed by distinct sigma factors. To understand the regulation of L. pneumophila flagellin expression, we cloned the sigma factor (FliA) of RNA polymerase responsible for the transcription of the flagellin gene, flaA. FliA is a member of the sigma28 class of alternative sigma factors identified in several bacterial genera. The gene fliA has been isolated from an expression library of L. pneumophila isolate Corby in Escherichia coli K-12. This library was transformed into a fliA mutant of E. coli K-12 containing a plasmid carrying the L. pneumophila-specific flaA promoter fused to the reporter gene luxAB. Screening the obtained transformants for luciferase activity, we isolated the major part of the fliA gene on a 1.64-kb fragment. This fragment was sequenced and used for reverse PCR in order to recover the complete fliA gene. The resulting 1.03-kb fragment was shown to contain the entire fliA gene. L. pneumophila FliA has 55 and 43% amino acid identity with the homologous sequences of Pseudomonas aeruginosa and E. coli. Furthermore, the L. pneumophila fliA gene was able to restore the flagellation and the motility defect of an E. coli fliA mutant. This result suggests that the L. pneumophila sigma28 protein can bind to the E. coli core RNA polymerase to direct transcription initiation from the flaA-specific promoter. PMID:8981975

  4. Dominance relationships among mutant alleles of regulatory gene araC in the Escherichia coli B/R L-arabinose operon.

    PubMed

    Sheppard, D E

    1986-11-01

    The araBAD operon of Escherichia coli B/r is positively and negatively regulated by the araC+ regulatory protein. Mutations in gene araC can result in a variety of different regulatory phenotypes: araC null mutants (those carrying a null allele exhibiting no repressor or activator activity) are unable to achieve operon induction; araC-constitutive (araCc) mutants are partially constitutive, inducible by D-fucose, and resistant to catabolite repression; araCh mutants are hypersensitive to catabolite repression; and araCi mutants are resistant to catabolite repression. Various mutant alleles of gene araC were cloned into a derivative of plasmid pBR322 by in vivo recombination. Various heterozygous araC allelic combinations were constructed by transformation. Analysis of isomerase (araA) specific activity levels under various growth conditions indicated the following dominance relationships with regard to sensitivity to catabolite repression: araCh greater than araC+ greater than (araCc and araCi) greater than araC. It was concluded that the araCh protein may form a repressor complex that is refractory to removal by cyclic AMP receptor protein-cyclic AMP complex. This was interpreted in terms of the known nucleoprotein interactions between ara regulatory proteins and ara regulatory DNA.

  5. Analysis of mRNA decay and rRNA processing in Escherichia coli multiple mutants carrying a deletion in RNase III.

    PubMed

    Babitzke, P; Granger, L; Olszewski, J; Kushner, S R

    1993-01-01

    RNase III is an endonuclease involved in processing both rRNA and certain mRNAs. To help determine whether RNase III (rnc) is required for general mRNA turnover in Escherichia coli, we have created a deletion-insertion mutation (delta rnc-38) in the structural gene. In addition, a series of multiple mutant strains containing deficiencies in RNase II (rnb-500), polynucleotide phosphorylase (pnp-7 or pnp-200), RNase E (rne-1 or rne-3071), and RNase III (delta rnc-38) were constructed. The delta rnc-38 single mutant was viable and led to the accumulation of 30S rRNA precursors, as has been previously observed with the rnc-105 allele (P. Gegenheimer, N. Watson, and D. Apirion, J. Biol. Chem. 252:3064-3073, 1977). In the multiple mutant strains, the presence of the delta rnc-38 allele resulted in the more rapid decay of pulse-labeled RNA but did not suppress conditional lethality, suggesting that the lethality associated with altered mRNA turnover may be due to the stabilization of specific mRNAs. In addition, these results indicate that RNase III is probably not required for general mRNA decay. Of particular interest was the observation that the delta rnc-38 rne-1 double mutant did not accumulate 30S rRNA precursors at 30 degrees C, while the delta rnc-38 rne-3071 double mutant did. Possible explanations of these results are discussed.

  6. Towards a vaccine for attaching/effacing Escherichia coli: a LEE encoded regulator (ler) mutant of rabbit enteropathogenic Escherichia coli is attenuated, immunogenic, and protects rabbits from lethal challenge with the wild-type virulent strain.

    PubMed

    Zhu, Chengru; Feng, Shuzhang; Thate, Timothy E; Kaper, James B; Boedeker, Edgar C

    2006-05-01

    The ler (LEE encoded regulator) gene product is a central regulator for the genes encoded on the locus of enterocyte effacement (LEE) pathogenicity island of attaching/effacing (A/E) pathogens, including human enteropathogenic E. coli (EPEC) and enterohemorrhagic E. coli (EHEC) as well as animal isolates. Although an in vivo role for Ler in bacterial virulence has not been documented, we hypothesized that a Ler deletion mutant should be attenuated for virulence but might retain immunogenicity. The goals of this study were to genetically characterize ler of a rabbit EPEC (rEPEC) strain (O103:H2), to examine the effect of ler on in vivo virulence, and to determine if intragastric inoculation of an attenuated rEPEC ler mutant was immunogenic and could protect rabbits against subsequent challenge with the wild-type virulent parent strain. The predicted ler gene product of rEPEC strain O103:H2 shares high homology (over 95% amino acid identity) with the Lers of another rEPEC strain RDEC-1 (O15:H-) and human EPEC and EHEC. A defined internal ler deletion mutant of rEPEC O103:H2 showed reduced production of secreted proteins. Although orogastric inoculation of rabbits with the virulent parent O103:H2 strain induced severe diarrhea, significant weight loss and early mortality with adherent mucosal bacteria found at sacrifice, the isogeneic ler mutant strain was well tolerated. Animals gained weight and showed no clinical signs of disease. Examination of histological sections of intestinal segments revealed the absence of mucosal bacterial adherence. This result demonstrates an essential role for Ler in in vivo pathogenicity of A/E E. coli. Single dose orogastric immunization with the rEPEC ler mutant induced serum IgG antibody to whole bacteria (but not to intimin). Immunized animals were protected against enteric infection with the WT virulent parent strain exhibiting normal weight gain, absence of diarrhea and absence of mucosally adherent bacteria at sacrifice. Such

  7. A systematic analysis of TCA Escherichia coli mutants reveals suitable genetic backgrounds for enhanced hydrogen and ethanol production using glycerol as main carbon source.

    PubMed

    Valle, Antonio; Cabrera, Gema; Muhamadali, Howbeer; Trivedi, Drupad K; Ratray, Nicholas J W; Goodacre, Royston; Cantero, Domingo; Bolivar, Jorge

    2015-09-01

    Biodiesel has emerged as an environmentally friendly alternative to fossil fuels; however, the low price of glycerol feed-stocks generated from the biodiesel industry has become a burden to this industry. A feasible alternative is the microbial biotransformation of waste glycerol to hydrogen and ethanol. Escherichia coli, a microorganism commonly used for metabolic engineering, is able to biotransform glycerol into these products. Nevertheless, the wild type strain yields can be improved by rewiring the carbon flux to the desired products by genetic engineering. Due to the importance of the central carbon metabolism in hydrogen and ethanol synthesis, E. coli single null mutant strains for enzymes of the TCA cycle and other related reactions were studied in this work. These strains were grown anaerobically in a glycerol-based medium and the concentrations of ethanol, glycerol, succinate and hydrogen were analysed by HPLC and GC. It was found that the reductive branch is the more relevant pathway for the aim of this work, with malate playing a central role. It was also found that the putative C4-transporter dcuD mutant improved the target product yields. These results will contribute to reveal novel metabolic engineering strategies for improving hydrogen and ethanol production by E. coli.

  8. The lux genes of the luminous bacterial symbiont, Photobacterium leiognathi, of the ponyfish. Nucleotide sequence, difference in gene organization, and high expression in mutant Escherichia coli.

    PubMed

    Lee, C Y; Szittner, R B; Meighen, E A

    1991-10-01

    The lux genes required for light expression in the luminescent bacterium Photobacterium leiognathi (ATCC 25521) have been cloned and expressed in Escherichia coli and their organization and nucleotide sequence determined. Transformation of a recombinant 9.5-kbp chromosomal DNA fragment of P. leiognathi into an E. coli mutant (43R) gave luminescent colonies that were as bright as those of the parental strain. Moreover, expression of the lux genes in the mutant E. coli was strong enough so that not only were high levels of luciferase detected in crude extracts, but the fatty-acid reductase activity responsible for synthesis of the aldehyde substrate for the luminescent reaction could readily be measured. Determination of the 7.3-kbp nucleotide sequence of P. leiognathi DNA, including the genes for luciferase (luxAB) and fatty-acid reductase (luxCDE) as well as a new lux gene (luxG) found recently in luminescent Vibrio species, showed that the order of the lux genes was luxCDABEG. Moreover, luxF, a gene homologous to luxB and located between luxB and luxE in Photobacterium but not Vibrio strains, was absent. In spite of this different lux gene organization, an intergenic stem-loop structure between luxB and luxE was discovered to be highly conserved in other Photobacterium species after luxF.

  9. Annexin-like protein from Arabidopsis thaliana rescues delta oxyR mutant of Escherichia coli from H2O2 stress.

    PubMed Central

    Gidrol, X; Sabelli, P A; Fern, Y S; Kush, A K

    1996-01-01

    Reactive oxygen species are common causes of cellular damages in all aerobic organisms. In Escherichia coli, the oxyR gene product is a positive regulator of the oxyR regulon that is induced in response to H2O2 stress. To identify genes involved in counteracting oxidative stress in plants, we transformed a delta oxyR mutant of E. coli with an Arabidopsis thaliana cDNA library and selected for clones that restored the ability of the delta oxyR mutant to grow in the presence of H2O2. Using this approach, we isolated a cDNA that has strong homology with the annexin super-gene family. The complemented mutant showed higher catalase activity. mRNA expression of the annexin gene in A. thaliana was higher in roots as compared with other organs and was also increased when the plants were exposed to H2O2 stress or salicylic acid. Based on the results presented in this study, we propose a novel physiological role for annexin in counteracting H2O2 stress. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 7 Fig. 8 PMID:8855345

  10. A mutant pyruvate dehydrogenase E1 subunit allows survival of Escherichia coli strains defective in 1-deoxy-D-xylulose 5-phosphate synthase.

    PubMed

    Sauret-Güeto, Susanna; Urós, Eva María; Ibáñez, Ester; Boronat, Albert; Rodríguez-Concepción, Manuel

    2006-02-06

    The 2-C-methyl-D-erythritol 4-phosphate pathway has been proposed as a promising target to develop new antimicrobial agents. However, spontaneous mutations in Escherichia coli were observed to rescue the otherwise lethal loss of the first two enzymes of the pathway, 1-deoxy-D-xylulose 5-phosphate (DXP) synthase (DXS) and DXP reductoisomerase (DXR), with a relatively high frequency. A mutation in the gene encoding the E1 subunit of the pyruvate dehydrogenase complex was shown to be sufficient to rescue the lack of DXS but not DXR in vivo, suggesting that the mutant enzyme likely allows the synthesis of DXP or an alternative substrate for DXR.

  11. Recombinant soluble human tissue factor secreted by Saccharomyces cerevisiae and refolded from Escherichia coli inclusion bodies: glycosylation of mutants, activity and physical characterization.

    PubMed Central

    Stone, M J; Ruf, W; Miles, D J; Edgington, T S; Wright, P E

    1995-01-01

    Tissue factor (TF) is the cell-surface transmembrane receptor that initiates both the extrinsic and intrinsic blood coagulation cascades. The abilities of TF to associate with Factor VIIa and Factor X in a ternary complex and to enable proteolytic activation of Factor X by Factor VIIa reside in the extracellular domain of TF. We describe the expression of the surface domain of TF (truncated TF, tTF) in both Saccharomyces cerevisiae and Escherichia coli and the biochemical and physical characterization of the recombinant proteins. Wild-type tTF and several glycosylation-site mutants were secreted efficiently by S. cerevisiae under the control of the yeast prepro-alpha-signal sequence; the T13A,N137D double mutant was the most homogeneous variant expressed in milligram quantities. Wild-type tTF was expressed in a non-native state in E. coli inclusion bodies as a fusion protein with a poly(His) leader. The fusion protein could be fully renatured and the leader removed by proteolysis with thrombin; the correct molecular mass (24,729 Da) of the purified protein was confirmed by electrospray mass spectrometry. Recombinant tTFs from yeast, E. coli and Chinese hamster ovary cells were identical in their abilities to bind Factor VIIa, to enhance the catalytic activity of Factor VIIa and to enhance the proteolytic activation of Factor X by Factor VIIa. Furthermore, CD, fluorescence emission and NMR spectra of the yeast and E. coli proteins indicated that these proteins are essentially identical structurally. Images Figure 1 Figure 2 Figure 3 PMID:7654202

  12. Feedback inhibition of chorismate mutase/prephenate dehydrogenase (TyrA) of Escherichia coli: generation and characterization of tyrosine-insensitive mutants.

    PubMed

    Lütke-Eversloh, Tina; Stephanopoulos, Gregory

    2005-11-01

    In order to get insights into the feedback regulation by tyrosine of the Escherichia coli chorismate mutase/prephenate dehydrogenase (CM/PDH), which is encoded by the tyrA gene, feedback-inhibition-resistant (fbr) mutants were generated by error-prone PCR. The tyrA(fbr) mutants were selected by virtue of their resistance toward m-fluoro-D,L-tyrosine, and seven representatives were characterized on the biochemical as well as on the molecular level. The PDH activities of the purified His6-tagged TyrA proteins exhibited up to 35% of the enzyme activity of TyrA(WT), but tyrosine did not inhibit the mutant PDH activities. On the other hand, CM activities of the TyrA(fbr) mutants were similar to those of the TyrA(WT) protein. Analyses of the DNA sequences of the tyrA genes revealed that tyrA(fbr) contained amino acid substitutions either at Tyr263 or at residues 354 to 357, indicating that these two sites are involved in the feedback inhibition by tyrosine.

  13. Suppression by enhanced RpoE activity of the temperature-sensitive phenotype of a degP ssrA double mutant in Escherichia coli.

    PubMed

    Ono, Katsuhiko; Kutsukake, Kazuhiro; Abo, Tatsuhiko

    2009-02-01

    SsrA is a small RNA playing a crucial role in trans-translation, which leads to rescue of stalled ribosomes on or at the end of mRNA and addition of the degradation tag to a growing polypeptide. The lack of SsrA has been shown to enhance the temperature-sensitive (ts) phenotype of an E. coli strain defective in the degP gene, which encodes one of the periplasmic proteases. This severe ts phenotype was relieved only partially by an SsrADD variant, which can lead to ribosome rescue but adds a protease-resistant tag instead of the degradation tag, suggesting that accumulation of polypeptides programmed by truncated mRNAs is responsible for growth defect of the ssrA degP mutant. Expression of an S210A-mutant DegP protein, which lacks the protease activity but retains the chaperone activity, could relieve the ts phenotype of the double mutant, suggesting that the chaperone activity but not the protease activity of DegP is required for growth of the ssrA-deficient cells at high temperature. Overexpression of the rpoE gene, which encodes sigmaE responsible for the expression of factors involved in extracellular stress response, also suppressed the ts phenotype of the ssrA degP mutant. This suggests that the stress-responsing pathway(s) may be involved in the enhancement of ts phenotype of degP mutant in the absence of SsrA.

  14. Unveiling the photoelectrocatalytic inactivation mechanism of Escherichia coli: Convincing evidence from responses of parent and anti-oxidation single gene knockout mutants.

    PubMed

    Sun, Hongwei; Li, Guiying; An, Taicheng; Zhao, Huijun; Wong, Po Keung

    2016-01-01

    This study investigated photoelectrocatalytic (PEC) inactivation mechanism of bacteria using parental Escherichia coli (E. coli) BW25113 and its isogenic mutants deficient in catalase HPI (katG(-), JW3914-1) and Mn-SOD (sodA(-), JW3879-1). BW25113 in the mid-log phase was less susceptible to PEC inactivation than those in early-log and stationary phases, consistent with the peak activities of catalase and superoxide dismutase (SOD) at mid-log phase (30.6 and 13.0 Unit/ml/OD600). For different strains all in mid-log phase, PEC inactivation efficiency followed the order katG(-) > sodA(-) > BW25113, with the duration of 60, 60 and 90 min for complete inactivation of ∼2 × 10(7) CFU mL(-1) bacteria, respectively. Correspondingly, catalase and SOD levels of BW25113 were also higher than the mutants by 5.9 and 11.7 Unit/mL/OD600, respectively. Reactive oxygen species (ROSs) concentrations in PEC systems revealed that the inactivation performance coincided with H2O2 levels, rather than OH. Moreover, pre-incubation with H2O2 elevated catalase activities and PEC inactivation resistance of BW25113 were positively correlated. The above results indicated that H2O2 was the dominant PEC generated bactericide, and anti-oxidative enzymes especially catalase contributed greatly to the bacterial PEC resistance capacity. Further tests revealed that PEC treatment raised the intracellular ROSs concentration by more than 3 times, due to the permeated H2O2 and its intracellular derivative, OH. However, oxidative stress response of E. coli, such as increased catalase or SOD were not observed, perhaps because the ROSs overwhelmed the bacterial protective capacity. The accumulated ROSs subsequently caused oxidative damages to E. coli cells, including membrane damage, K(+) leakage, and protein oxidation. Compared with BW25113, the mutants experienced damages earlier and at higher levels, confirming the essential roles of catalase and SOD in the bacterial PEC resistance.

  15. Heterologous expression in Escherichia coli of native and mutant forms of the major intrinsic protein of rat eye lens (MIP26).

    PubMed Central

    Dilsiz, N; Crabbe, M J

    1995-01-01

    The complete cDNA of rat eye lens major intrinsic protein (MIP26) was sequenced using the dideoxy chain termination method. The sequence displayed 89% nucleotide identity and 95% identity at the amino acid level with bovine MIP26 [Gorin, Yancey, Cline, Revel and Horwitz (1984) Cell, 39, 49-54]. Both native and mutant cDNAs coding for rat MIP26 were amplified by PCR and subcloned into the pPOW expression vector for expression of Escherichia coli. A membrane signal peptide (PelB) was used for secretion of MIP26 into the cytoplasmic membrane. A hydrophilic octapeptide tail (FLAG) was fused to either the N- or C-terminus of MIP26 to aid monoclonal antibody-mediated identification and purification. Heterologously expressed MIP26 was identified by using a monoclonal antibody corresponding to the FLAG peptide located at the termini of MIP26. Immunofluorescently labelled monoclonal antibody was used to determine the localization of MIP26 in the cytoplasmic membrane. The majority of the protein was integrated into cell plasma membrane. MIP26 was extracted with n-octyl beta-D-glucopyranoside and then purified on an affinity gel column. Rat MIP26 cDNA contains an -Asn-Gly- sequence at the C-terminus, which has been shown in other proteins to be particularly susceptible to spontaneous deamidation [Takemoto and Emmons (1991) Curr. Eye Res. 10, 863-869]. We therefore modified the MIP26 molecule using a site-directed mutagenesis method to generate a mutant MIP26 at the appropriate asparagine residue (Asn244-->Asp) near the C-terminus. The mutation was confirmed by DNA sequencing. The mutant MIP26 protein was also expressed in E. coli and incorporated predominantly into the cytoplasmic membrane. Images Figure 5 Figure 6 Figure 7 PMID:7848273

  16. Functional Genomics Via Metabolic Footprinting: Monitoring Metabolite Secretion by Escherichia Coli Tryptophan Metabolism Mutants Using FT–IR and Direct Injection Electrospray Mass Spectrometry

    PubMed Central

    Kaderbhai, Naheed N.; Broadhurst, David I.; Ellis, David I.; Goodacre, Royston

    2003-01-01

    We sought to test the hypothesis that mutant bacterial strains could be discriminated from each other on the basis of the metabolites they secrete into the medium (their ‘metabolic footprint’), using two methods of ‘global’ metabolite analysis (FT–IR and direct injection electrospray mass spectrometry). The biological system used was based on a published study of Escherichia coli tryptophan mutants that had been analysed and discriminated by Yanofsky and colleagues using transcriptome analysis. Wild-type strains supplemented with tryptophan or analogues could be discriminated from controls using FT–IR of 24 h broths, as could each of the mutant strains in both minimal and supplemented media. Direct injection electrospray mass spectrometry with unit mass resolution could also be used to discriminate the strains from each other, and had the advantage that the discrimination required the use of just two or three masses in each case. These were determined via a genetic algorithm. Both methods are rapid, reagentless, reproducible and cheap, and might beneficially be extended to the analysis of gene knockout libraries. PMID:18629082

  17. Enhanced expression of tandem multimers of the antimicrobial peptide buforin II in Escherichia coli by the DEAD-box protein and trxB mutant.

    PubMed

    Lee, J H; Kim, M S; Cho, J H; Kim, S C

    2002-05-01

    The tandem multimeric expression of various peptides has been explored by many researchers. However, expression levels have usually not been proportional to the degree of multimerization. To increase the expression level in Escherichia coli of tandem multimers of a cationic antimicrobial peptide, buforin II, fused to an anionic peptide, we studied the effect of the DEAD-box protein and the trxB mutant on the expression of tandem multimers. An expression vector with a tac promoter was more effective in directing multimeric expression than one with a T7 promoter. The expression level of large multimers was substantially increased with the tac promoter, possibly through stabilization of long transcripts by synchronization of transcription and translation. Coexpression of the DEAD-box protein, an RNA-binding protein, with the T7 expression system increased the expression level of multimers, especially large multimers, due to protection of the long RNA transcripts. In addition, the use of the trxB mutant also enhanced the expression level of tandem multimers, which contain two cysteine residues at both ends of the monomeric unit. It seems that disulfide bonds formed in the multimers in the trxB mutant might help efficient charge neutralization for inclusion body formation of the multimers, resulting in enhancement of expression. Our results show that the expression of multimers can be improved through the stabilization of the long transcripts by the DEAD-box protein or the expression, under an oxidizing environment, of the trxB mutant in which covalent cross-links through disulfide bonds facilitate inclusion body formation of the multimeric fusion peptide.

  18. Production of succinic acid through overexpression of NAD{sup +}-dependent malic enzyme in an Escherichia coli mutant

    SciTech Connect

    Stols, L.; Donnelly, M.I.

    1997-07-01

    NAD{sup +}-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene. The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions. The K{sub m} values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction). When malic enzyme was induced under appropriate culture conditions in a strain of E. coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism of glucose was restored. Succinic acid was the major fermentation product formed. When this fermentation was performed in the presence of hydrogen, the yield of succinic acid increased. The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks. 27 refs., 5 figs., 4 tabs.

  19. Correlation Between the Rate of Ribonucleic Acid Synthesis and the Level of Valyl Transfer Ribonucleic Acid in Mutants of Escherichia coli

    PubMed Central

    Kaplan, Sam

    1969-01-01

    By use of a mutant of Escherichia coli with a partially thermolabile transfer ribonucleic acid (tRNA) synthase, it was possible to regulate the rate of RNA synthesis over a 10-fold range. The addition of chloramphenicol to cultures kept at the nonpermissive temperature stimulated RNA synthesis. The longer the culture was kept at the nonpermissive temperature prior to addition of chloramphenicol, the lower was the resulting rate of RNA synthesis. The decrease in the rate of incorporation of labeled uracil into RNA was correlated with the decrease in the level of valyl tRNA. Additional experiments provided evidence which may be interpreted as indicating that valyl tRNA does not, by itself, react with the RNA-forming system. PMID:4891259

  20. recA730-dependent suppression of recombination deficiency in RecA loading mutants of Escherichia coli.

    PubMed

    Vlašić, Ignacija; Simatović, Ana; Brčić-Kostić, Krunoslav

    2011-04-01

    Homologous recombination is an essential process in double-strand break repair. The main requirement for recombination is formation of a RecA filament. Double-strand breaks can be processed into a RecA filament by the action of three enzymatic activities: helicase, 5'-3' exonuclease and RecA loading onto ssDNA. These activities are provided by the RecBCD enzyme in wild type cells or by the RecF pathway gene products in recBC sbcBC(D) cells. In the recBD1080A mutant (recB∗ mutant), the recombination machineries of RecBCD and RecF pathways are interchangeable and include RecB∗CD enzyme (helicase), RecJ (5'-3' exonuclease) and RecFOR (RecA loading). The mutant RecA730 protein is able to produce a RecA filament without the help of RecFOR mediators, since it more efficiently competes with SSB protein for ssDNA than the normal RecA protein. It was previously shown that the recA730 mutation suppresses UV sensitivity in a uvrA recFOR genetic background. We tested whether the recA730 mutation can suppress recombination and DNA repair deficiency in a recB∗ mutant and its derivatives. We show that the recA730 mutation suppresses recombination deficiency in a recB∗ recFOR background, where the defect is at the level of RecA loading, but not in the recB∗ recJ background where the defect is at the level of nuclease activity.

  1. Regulatory properties of araC(c) mutants in the L-arabinose operon of escherichia coliB/r.

    PubMed

    MacInnes, K R; Sheppard, D E; Falgout, B

    1978-01-01

    Merodiploids containing a high-constitutive and a low-constitutive araC(c) allele were assayed for constitutive expression of the ara operon. Low-constitutive araC(c) alleles either were unable to repress the constitutive rate of ara operon expression exhibited by by high-constitutive araC(c) alleles or achieved a partial repression of the high-constitutive rate of operon expression. Either mutation to a low-constitutive araC(c) mutant resulted in a partial or complete loss of repressor function, or subunit mixing between the two araC(c) mutant proteins resulted in a partial or complete dominance of the high-constitutive araC(c) allele. Five of the six araC(c) alleles tested allowed a partial induction of the ara operon in cya crp background. In general, a higher level of ara operon induction was achieved in the cya crp background by high araC(c) alleles than by low araC(c) alleles. Furthermore, several araC(c) mutants exhibited decreased sensitivity to catabolite repression, particularly in the presence of inducer. The results suggest a model in which certain araC(c) gene products can achieve ara operon induction in the presence of either arabinose (inducer) or catabolite activator protein-cyclic adenosine monophosphate, whereas the wild-type araC gene product requires the presence of both of these factors for operon expression.

  2. Interaction between mutant alleles of araC of the Escherichia coli B/r L-arabinose operon.

    PubMed

    Sheppard, D E; Eleuterio, M; Falgout, B

    1979-09-01

    Strains were constructed that contain mutational alterations affecting two distinct functional domains within the araC gene protein. The araCi (catabolite repression insensitivity) and araCh (catabolite repression hypersensitivity) mutations were used to alter the catabolite repression sensitivity domain, and mutation to D-fucose resistance was used to alter the inducer binding domain. araCh, D-fucose-resistant double mutants never exhibited constitutive ara operon expression, whereas all of the araCi, D-fucose-resistant double mutants did exhibit constitutivity. When L-arabinose was used as an inducer, most of the double mutants exhibited the sensitivity to catabolite repression associated with the araCi or araCh mutation. However, when D-fucose was used as an inducer, changes in sensitivity to catabolite repression were observed that were attributed to interactions between the two protein domains. The roles of catabolite activator protein and araC gene protein in the induction of the araBAD operon were discussed.

  3. Chemical rescue of Asp237-->Ala and Lys358-->Ala mutants in the lactose permease of Escherichia coli.

    PubMed

    Frillingos, S; Kaback, H R

    1996-10-15

    Asp237 (helix VII) and Lys358 (helix XI) form a salt bridge in the lactose permease, and neutral replacement of either residue inactivates. Remarkably, noncovalent neutralization of the unpaired Asp or Lys residue, respectively, with n-alkylsulfonates or n-alkylamines of appropriate size restores active transport to high levels in the mutants. Saturation with respect to the concentration of the alkylamines and different size preferences suggest that the alkylamines bind sterically at position 358. Rescue of Asp237-->Ala by alkylsulfonates is apparently more indiscriminate, since methane-, ethane-, or propane-sulfonate have comparable effects. Sodium and chloride, respectively, are also effective in rescuing the Lys358-->Ala and Asp237-->Ala mutants, while various other compounds are ineffective. In marked contrast to Asp237-->Ala or Lys358-->Ala permease, alkylsulfonates or alkylamines have no effect whatsoever on the activity of mutants with neutral replacements for Asp240, Glu269, Arg302, Lys319, His322, or Glu325. The results support the conclusion that neutral replacement of one member of the charge pair between Asp237 and Lys358 leads to inactivation because of an unpaired charge in the low dielectric of the membrane. In addition, the findings are consistent with the idea that interactions between Arg302 and Glu325, His 322 and Glu269, and Asp240 and Lys319 play important roles in the mechanism of the permease, which is not the case for either Asp237 or Lys358 or the salt bridge between the two residues.

  4. Herpes simplex virus thymidine kinase activity of thymidine kinase-deficient Escherichia coli K-12 mutant transformed by hybrid plasmids.

    PubMed

    Kit, S; Otsuka, H; Qavi, H; Hazen, M

    1981-01-01

    A hybrid plasmid (pAGO) that contains the herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) gene in the form of a 2-kilobase-pair (kbp) Pvu II fragment inserted at the Pvu II site of plasmid pBR322 was used to transform TK- Escherichia coli K-12 strain KY895. pAGO-transformed KY895 cells exhibited partially restored ability to incorporate [3H]dThd into DNA and an HSv-1-specific TK activity. Bacteria cured of plasmid pAGO (or transformed by plasmid pBR322) did not show enhanced incorporation of [3H]dThd into DNA or HSV-1 TK activity. Plasmid pMH1A was derived from pAGO by deletion of 2067 bp of DNA sequence from pBR322 and 105 bp from the HSV-1 TK gene. E. coli K-12 strain KY895 cells transformed by pMH1A did not show enhanced incorporation of [3H]dThd into bacterial DNA, although pMH1A DNA isolated from transformed KY895 cells, like pAGO DNA, did transform TK- mouse fibroblast [LM(TK-)] cells to the TK+ phenotype. The expression of HSV-1 TK activity by E. coli K-12 suggests that intervening sequences may be absent from the coding region of HSV-1 tk or that the coding region of the gene possesses short intervening sequences which do not disrupt the translational reading frame.

  5. Enhanced generation of A:T-->T:A transversions in a recA730 lexA51(Def) mutant of Escherichia coli.

    PubMed

    Watanabe-Akanuma, M; Woodgate, R; Ohta, T

    1997-01-03

    RecA730 belongs to a class of mutant RecA protein that is often referred to as RecA*, since it is constitutively activated for coprotease functions in the absence of exogenous DNA-damage. Escherichia coli strains carrying recA730 (or other recA* alleles) exhibit dramatic increases in SOS-dependent spontaneous mutator activity. We have analyzed the specificity of this mutator phenotype by employing F'-plasmids carrying a set of mutant lacZ genes that can individually detect two types of transitions, four types of transversions, and four kinds of specific frameshift events. Analysis revealed that most of the spontaneous mutagenesis in a recA730 lexA51(Def) strain (which expresses derepressed levels of all LexA-regulated proteins) can be attributed to a specific increase in A:T-->T:A, A:T-->C:G and G:C-->T:A transversions, with the A:T-->T:A transversions occurring most frequently. These transversion events were completely abolished in a delta umuDC strain, indicating that the functionally active UmuD'C proteins are normally required for their generation. The spectrum obtained was similar to that of strains with a defect in the epsilon (3'-->5' proofreading) subunit of DNA polymerase III. Such an observation raises the possibility that the wild-type epsilon protein is in activated in strains expressing the RecA730 and UmuD'C proteins.

  6. Evolved osmotolerant Escherichia coli mutants frequently exhibit defective N-acetylglucosamine catabolism and point mutations in cell shape-regulating protein MreB.

    PubMed

    Winkler, James D; Garcia, Carlos; Olson, Michelle; Callaway, Emily; Kao, Katy C

    2014-06-01

    Biocatalyst robustness toward stresses imposed during fermentation is important for efficient bio-based production. Osmotic stress, imposed by high osmolyte concentrations or dense populations, can significantly impact growth and productivity. In order to better understand the osmotic stress tolerance phenotype, we evolved sexual (capable of in situ DNA exchange) and asexual Escherichia coli strains under sodium chloride (NaCl) stress. All isolates had significantly improved growth under selection and could grow in up to 0.80 M (47 g/liter) NaCl, a concentration that completely inhibits the growth of the unevolved parental strains. Whole genome resequencing revealed frequent mutations in genes controlling N-acetylglucosamine catabolism (nagC, nagA), cell shape (mrdA, mreB), osmoprotectant uptake (proV), and motility (fimA). Possible epistatic interactions between nagC, nagA, fimA, and proV deletions were also detected when reconstructed as defined mutations. Biofilm formation under osmotic stress was found to be decreased in most mutant isolates, coupled with perturbations in indole secretion. Transcriptional analysis also revealed significant changes in ompACGL porin expression and increased transcription of sulfonate uptake systems in the evolved mutants. These findings expand our current knowledge of the osmotic stress phenotype and will be useful for the rational engineering of osmotic tolerance into industrial strains in the future.

  7. Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant.

    PubMed

    Arvizu-Flores, Aldo A; Sugich-Miranda, Rocio; Arreola, Rodrigo; Garcia-Orozco, Karina D; Velazquez-Contreras, Enrique F; Montfort, William R; Maley, Frank; Sotelo-Mundo, Rogerio R

    2008-01-01

    Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH(2)THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The k(cat) of the K48Q mutant was 430-fold lower than wild-type TS in activity, while the K(m) for the (R)-stereoisomer of CH(2)THF was 300 microM, about 30-fold larger than K(m) from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideazafolate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutant, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild-type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH(2)THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group.

  8. Double helicase II (uvrD)-helicase IV (helD) deletion mutants are defective in the recombination pathways of Escherichia coli.

    PubMed Central

    Mendonca, V M; Kaiser-Rogers, K; Matson, S W

    1993-01-01

    The Escherichia coli helD (encoding helicase IV) and uvrD (encoding helicase II) genes have been deleted, independently and in combination, from the chromosome and replaced with genes encoding antibiotic resistance. Each deletion was verified by Southern blots, and the location of each deletion was confirmed by P1-mediated transduction. Cell strains containing the single and double deletions were viable, indicating that helicases II and IV are not essential for viability. Cell strains lacking helicase IV (delta helD) exhibited no increase in sensitivity to UV irradiation but were slightly more resistant to methyl methanesulfonate (MMS) than the isogenic wild-type cell strain. As expected, cell strains containing the helicase II deletion (delta uvrD) were sensitive to both UV irradiation and MMS. The introduction of the helicase IV deletion into a delta uvrD background had essentially no effect on the UV and MMS sensitivity of the cell strains analyzed. The double deletions, however, conferred a Rec- mutant phenotype for conjugational and transductional recombination in both recBC sbcB(C) and recBC sbcA backgrounds. The Rec- mutant phenotype was more profound in the recBC sbcB(C) background than in the recBC sbcA background. The recombination-deficient phenotype indicates the direct involvement of helicase II and/or helicase IV in the RecF pathway [recBC sbcB(C) background] and RecE pathway (recBC sbcA background) of recombination. The modest decrease in the recombination frequency observed in single-deletion mutants in the recBC sbcB(C) background suggests that either helicase is sufficient. In addition, helicase IV has been overexpressed in a tightly regulated system. The data suggest that even modest overexpression of helicase IV is lethal to the cell. Images PMID:8335623

  9. Membrane Vesicles Released by a hypervesiculating Escherichia coli Nissle 1917 tolR Mutant Are Highly Heterogeneous and Show Reduced Capacity for Epithelial Cell Interaction and Entry

    PubMed Central

    Pérez-Cruz, Carla; Cañas, María-Alexandra; Giménez, Rosa; Badia, Josefa; Mercade, Elena; Aguilera, Laura

    2016-01-01

    Membrane vesicles (MVs) produced by Gram-negative bacteria are being explored for novel clinical applications due to their ability to deliver active molecules to distant host cells, where they can exert immunomodulatory properties. MVs released by the probiotic Escherichia coli Nissle 1917 (EcN) are good candidates for testing such applications. However, a drawback for such studies is the low level of MV isolation from in vitro culture supernatants, which may be overcome by the use of mutants in cell envelope proteins that yield a hypervesiculation phenotype. Here, we confirm that a tolR mutation in EcN increases MV production, as determined by protein, LPS and fluorescent lipid measurements. Transmission electron microscopy (TEM) of negatively stained MVs did not reveal significant differences with wild type EcN MVs. Conversely, TEM observation after high-pressure freezing followed by freeze substitution of bacterial samples, together with cryo-TEM observation of plunge-frozen hydrated isolated MVs showed considerable structural heterogeneity in the EcN tolR samples. In addition to common one-bilayer vesicles (OMVs) and the recently described double-bilayer vesicles (O-IMVs), other types of MVs were observed. Time-course experiments of MV uptake in Caco-2 cells using rhodamine- and DiO-labelled MVs evidenced that EcN tolR MVs displayed reduced internalization levels compared to the wild-type MVs. The low number of intracellular MVs was due to a lower cell binding capacity of the tolR-derived MVs, rather than a different entry pathway or mechanism. These findings indicate that heterogeneity of MVs from tolR mutants may have a major impact on vesicle functionality, and point to the need for conducting a detailed structural analysis when MVs from hypervesiculating mutants are to be used for biotechnological applications. PMID:28036403

  10. Ophthalmic acid accumulation in an Escherichia coli mutant lacking the conserved pyridoxal 5'-phosphate-binding protein YggS.

    PubMed

    Ito, Tomokazu; Yamauchi, Ayako; Hemmi, Hisashi; Yoshimura, Tohru

    2016-12-01

    Escherichia coli YggS is a highly conserved pyridoxal 5'-phosphate (PLP)-binding protein whose biochemical function is currently unknown. A previous study with a yggS-deficient E. coli strain (ΔyggS) demonstrated that YggS controls l-Ile- and l-Val-metabolism by modulating 2-ketobutyrate (2-KB), l-2-aminobutyrate (l-2-AB), and/or coenzyme A (CoA) availability in a PLP-dependent fashion. In this study, we found that ΔyggS accumulates an unknown metabolite as judged by amino acid analyses. LC/MS and MS/MS analyses of the compound with propyl chloroformate derivatization, and co-chromatography analysis identified this compound as γ-l-glutamyl-l-2-aminobutyryl-glycine (ophthalmic acid), a glutathione (GSH) analogue in which the l-Cys moiety is replaced by l-2-AB. We also determine the metabolic consequence of the yggS mutation. Absence of YggS initially increases l-2-AB availability, and then causes ophthalmic acid accumulation and CoA limitation in the cell. The expression of a γ-glutamylcysteine synthetase and a glutathione synthetase in a ΔyggS background causes high-level accumulation of ophthalmic acid in the cells (∼1.2 nmol/mg cells) in a minimal synthetic medium. This opens the possibility of a first fermentative production of ophthalmic acid.

  11. Suppression of Recj Exonuclease Mutants of Escherichia Coli by Alterations in DNA Helicases II (Uvrd) and IV (Held)

    PubMed Central

    Lovett, S. T.; Sutera-Jr., V. A.

    1995-01-01

    The recJ gene encodes a single-strand DNA-specific exonuclease involved in homologous recombination. We have isolated a pseudorevertant strain in which recJ mutant phenotypes were alleviated. Suppression of recJ was due to at least three mutations, two of which we have identified as alterations in DNA helicase genes. A recessive amber mutation, ``uvrD517(am),'' at codon 503 of the gene encoding helicase II was sufficient to suppress recJ partially. The uvrD517(am) mutation does not eliminate uvrD function because it affects UV survival only weakly; moreover, a uvrD insertion mutation could not replace uvrD517(am) as a suppressor. However, suppression may result from differential loss of uvrD function: mutation rate in a uvrD517(am) derivative was greatly elevated, equal to that in a uvrD insertion mutant. The second cosuppressor mutation is an allele of the helD gene, encoding DNA helicase IV, and could be replaced by insertion mutations in helD. The identity of the third cosuppressor ``srjD'' is not known. Strains carrying the three cosuppressor mutations exhibited hyperrecombinational phenotypes including elevated excision of repeated sequences. To explain recJ suppression, we propose that loss of antirecombinational helicase activity by the suppressor mutations stabilizes recombinational intermediates formed in the absence of recJ. PMID:7635292

  12. Spectrophotometric and kinetic studies on the interaction of antibiotic X5108, the N-methylated derivative of kirromycin, with elongation factor Tu from Escherichia coli.

    PubMed

    Eccleston, J F

    1981-04-10

    The absorption spectrum of antibiotic X5108, the N-methylated derivative of kirromycin, has been found to be decreased in intensity on binding to elongation factor (EF)-Tu . GDP, EF-Tu . GTP, and nucleotide-free EF-Tu. This has allowed the binding of X5108 to be studied directly. In agreement with previous studies, a 1:1 stoichiometry is observed, with a dissociation constant of less than 1 microM. Identical results were obtained with all three EF-Tu species. The absorption spectrum of X5108 in increasing concentrations of isopropyl alcohol first intensifies and then decreases, 80% isopropyl alcohol giving the same spectrum as that of X5108 bound to EF-Tu. This result is interpreted as showing that the chromophoric moiety of X5108 is bound in a highly hydrophobic environment on EF-Tu. The rate of binding of X5108 to EF-Tu . GDP was measured using a stopped flow spectrophotometer. This rate was proportional to the concentration of X5108, giving a second order binding rate constant of 4.8 X 10(3) M-1 s-1. Since this is several orders of magnitude too slow for a diffusion-controlled reaction, the results are interpreted based on a two-step binding process. A half-time of about 10 min is calculated for the dissociation of X5108 from EF-Tu . GDP. The fact that X5108 bound to EF-Tu is not in rapid equilibrium with X5108 free in solution needs to be considered in studies on the effect of X5108 and kirromycin on partial reactions of protein biosynthesis.

  13. Expression of Ascaris suum malic enzyme in a mutant Escherichia coli allows production of succinic acid from glucose

    SciTech Connect

    Stols, L.; Donnelly, M.I.; Kulkarni, G.; Harris, B.G.

    1997-12-31

    The malic enzyme gene of Ascaris suum was cloned into the vector pTRC99a in two forms encoding alternative amino-termini. The resulting plasmids, pMEA1 and pMEA2, were introduced into Escherichia coli NZN111, a strain that is unable to grow fermentatively because of inactivation of the genes encoding pyruvate dissimilation. Induction of pMEA1, which encodes the native animoterminus, gave better overexpression of malic enzyme, approx 12-fold compared to uninduced cells. Under the appropriate culture conditions, expression of malic enzyme allowed the fermentative dissimilation of glucose by NZN111. The major fermentation product formed in induced cultures was succinic acid.

  14. Amplified UvrA protein can ameliorate the ultraviolet sensitivity of an Escherichia coli recA mutant.

    PubMed

    Kiyosawa, K; Tanaka, M; Matsunaga, T; Nikaido, O; Yamamoto, K

    2001-12-19

    When a recA strain of Escherichia coli was transformed with the multicopy plasmid pSF11 carrying the uvrA gene of E. coli, its extreme ultraviolet (UV) sensitivity was decreased. The sensitivity of the lexA1 (Ind(-)) strain to UV was also decreased by pSF11. The recA cells expressing Neurospora crassa UV damage endonuclease (UVDE), encoding UV-endonuclease, show UV resistance. On the other hand, only partial amelioration of UV sensitivity of the recA strain was observed in the presence of the plasmid pNP10 carrying the uvrB gene. Host cell reactivation of UV-irradiated lambda phage in recA cells with pSF11 was as efficient as that in wild-type cells. Using an antibody to detect cyclobutane pyrimidine dimers, we found that UV-irradiated recA cells removed dimers from their DNA more rapidly if they carried pSF11 than if they carried a vacant control plasmid. Using anti-UvrA antibody, we observed that the expression level of UvrA protein was about 20-fold higher in the recA strain with pSF11 than in the recA strain without pSF11. Our results were consistent with the idea that constitutive level of UvrA protein in the recA cells results in constitutive levels of active UvrABC nuclease which is not enough to operate full nucleotide excision repair (NER), thus leading to extreme UV sensitivity.

  15. Uracil uptake in Escherichia coli K-12: isolation of uraA mutants and cloning of the gene.

    PubMed Central

    Andersen, P S; Frees, D; Fast, R; Mygind, B

    1995-01-01

    Mutants defective in utilization of uracil at low concentrations have been isolated and characterized. The mutations in question (uraA) map close to the upp gene encoding uracil phosphoribosyltransferase. By complementation analysis, a plasmid that complements the uraA mutation has been isolated. The uraA gene was shown to be the second gene in a bicistronic operon with upp as the promoter proximal gene. The nucleotide sequence of the gene was determined, and the gene encodes a hydrophobic membrane protein with a calculated Mr of 45,030. The UraA protein has been identified in sodium dodecyl sulfate-polyacrylamide gels in the membrane fraction of minicells harboring the uraA plasmids. PMID:7721693

  16. A spontaneous Escherichia coli K12 mutant which inhibits the excision-reintegration process of Mu gem2ts.

    PubMed

    Di Lallo, G; Fabozzi, G; Ghelardini, P; Paolozzi, L

    1997-09-01

    Escherichia coli K12 strains lysogenic for Mu gem2ts with the prophage inserted in a target gene (i.e., lacZ::Mu gem2ts lysogenic strains) revert to Lac+ by prophage precise excision with a relatively high frequency (about 1 x 10(-6)). The revertants obtained are still lysogens with the prophage inserted elsewhere in the bacterial chromosome. We have observed that, with the time of storage in stabs, bacterial cultures lysogenic for Mu gem2ts lose the ability to excise the prophage. The mutation responsible for this effect was co-transducible with the gyrB gene. After the removal of the prophage by P1 vir transduction from these strains, one randomly chosen clone, R3538, was further analyzed. It shows an increment of DNA supercoiling of plasmid pAT153, used as a reporter, and a reduced beta-galactosidase activity. On the other hand, R3538 is totally permissive to both lytic and lysogenic cycles of bacteriophage Mu.

  17. Escherichia coli deletion mutants illuminate trade-offs between growth rate and flux through a foreign anabolic pathway.

    PubMed

    Falls, Kelly C; Williams, Aimee L; Bryksin, Anton V; Matsumura, Ichiro

    2014-01-01

    Metabolic engineers strive to improve the production yields of microbial fermentations, sometimes by mutating the genomes of production strains. Some mutations are detrimental to the health of the organism, so a quantitative and mechanistic understanding of the trade-offs could inform better designs. We employed the bacterial luciferase operon (luxABCDE), which uses ubiquitous energetic cofactors (NADPH, ATP, FMNH2, acetyl-CoA) from the host cell, as a proxy for a novel anabolic pathway. The strains in the Escherichia coli Keio collection, each of which contains a single deletion of a non-essential gene, represent mutational choices that an engineer might make to optimize fermentation yields. The Keio strains and the parental BW25113 strain were transformed with a luxABCDE expression vector. Each transformant was propagated in defined M9 medium at 37 °C for 48 hours; the cell density (optical density at 600 nanometers, OD600) and luminescence were measured every 30 minutes. The trade-offs were visualized by plotting the maximum growth rate and luminescence/OD600 of each transformant across a "production possibility frontier". Our results show that some loss-of-function mutations enhance growth in vitro or light production, but that improvement in one trait generally comes at the expense of the other.

  18. Identification of the enzymatic basis for. delta. -aminolevulinic acid auxotrophy in a hemA mutant of escherichia coli

    SciTech Connect

    Avissar, Y.J.; Beale, S.I. )

    1989-06-01

    The hemA mutation of Escherichia coli K-12 confers a requirement for {delta}-aminolevulinic acid (ALA). Cell extract prepared from the hemA strain SASX41B was incapable of producing ALA from either glutamate or glutamyl-tRNA, whereas extract of the hem{sup +} strain HB101 formed colorimetrically detectable amounts of ALA and transferred label from 1-({sup 14}C)glutamate and 3,4-({sup 3}H)glutamyl-tRNA to ALA. Extracts of both strains converted glutamate-1-semialdehyde to ALA and were capable of aminoacylating tRNA{sup Glu}. Glutamyl-tRNA formed by extracts of both strains could be converted to ALA by the extract of hem{sup +} cells. The extract of hemA cells did not convert glutamyl-tRNA formed by either strain to ALA. However, the hemA cell extract, when supplemented in vitro with glutamyl-tRNA dehydrogenase isolated from Chlorella vulgaris cells, formed about as much ALA as did the unsupplemented hem{sup +} cell extract. We conclude from these observations that the enzyme activity that is lacking in the ALA auxotrophic strain carrying the hemA mutation is that of glutamyl-tRNA dehydrogenase.

  19. Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures.

    PubMed

    Yoshimune, Kazuaki; Galkin, Andrey; Kulakova, Ljudmila; Yoshimura, Tohru; Esaki, Nobuyoshi

    2005-04-01

    Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0 degrees C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24 degrees C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43 degrees C nor lambda phage propagation at an even lower temperature, 30 degrees C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15 degrees C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.

  20. Role of an invariant lysine residue in folate binding on Escherichia coli thymidylate synthase: calorimetric and crystallographic analysis of the K48Q mutant

    PubMed Central

    Arvizu-Flores, Aldo A.; Sugich-Miranda, Rocio; Arreola, Rodrigo; Garcia-Orozco, Karina D.; Velazquez-Contreras, Enrique F.; Montfort, William R.; Maley, Frank; Sotelo-Mundo, Rogerio R.

    2008-01-01

    Thymidylate synthase (TS) catalyzes the reductive methylation of deoxyuridine monophosphate (dUMP) using methylene tetrahydrofolate (CH2THF) as cofactor, the glutamate tail of which forms a water-mediated hydrogen-bond with an invariant lysine residue of this enzyme. To understand the role of this interaction, we studied the K48Q mutant of Escherichia coli TS using structural and biophysical methods. The kcat of the K48Q mutant was 430 fold lower than wild-type TS in activity, while the the Km for the (R)-stereoisomer of CH2THF was 300 µM, about 30 fold larger than Km from the wild-type TS. Affinity constants were determined using isothermal titration calorimetry, which showed that binding was reduced by one order of magnitude for folate-like TS inhibitors, such as propargyl-dideaza folate (PDDF) or compounds that distort the TS active site like BW1843U89 (U89). The crystal structure of the K48Q-dUMP complex revealed that dUMP binding is not impaired in the mutamt, and that U89 in a ternary complex of K48Q-nucleotide-U89 was bound in the active site with subtle differences relative to comparable wild type complexes. PDDF failed to form ternary complexes with K48Q and dUMP. Thermodynamic data correlated with the structural determinations, since PDDF binding was dominated by enthalpic effects while U89 had an important entropic component. In conclusion, K48 is critical for catalysis since it leads to a productive CH2THF binding, while mutation at this residue does not affect much the binding of inhibitors that do not make contact with this group. PMID:18403248

  1. NADPH-dependent reductive biotransformation with Escherichia coli and its pfkA deletion mutant: influence on global gene expression and role of oxygen supply.

    PubMed

    Siedler, Solvej; Bringer, Stephanie; Polen, Tino; Bott, Michael

    2014-10-01

    An Escherichia coli ΔpfkA mutant lacking the major phosphofructokinase possesses a partially cyclized pentose phosphate pathway leading to an increased NADPH per glucose ratio. This effect decreases the amount of glucose required for NADPH regeneration in reductive biotransformations, such as the conversion of methyl acetoacetate (MAA) to (R)-methyl 3-hydroxybutyrate (MHB) by an alcohol dehydrogenase from Lactobacillus brevis. Here, global transcriptional analyses were performed to study regulatory responses during reductive biotransformation. DNA microarray analysis revealed amongst other things increased expression of soxS, supporting previous results indicating that a high NADPH demand contributes to the activation of SoxR, the transcriptional activator of soxS. Furthermore, several target genes of the ArcAB two-component system showed a lower mRNA level in the reference strain than in the ΔpfkA mutant, pointing to an increased QH2 /Q ratio in the reference strain. This prompted us to analyze yields and productivities of MAA reduction to MHB under different oxygen regimes in a bioreactor. Under anaerobic conditions, the specific MHB production rates of both strains were comparable (7.4 ± 0.2 mmolMHB  h(-1)  gcdw (-1) ) and lower than under conditions of 15% dissolved oxygen, where those of the reference strain (12.8 mmol h(-1)  gcdw (-1) ) and of the ΔpfkA mutant (11.0 mmol h(-1)  gcdw (-1) ) were 73% and 49% higher. While the oxygen transfer rate (OTR) of the reference strain increased after the addition of MAA, presumably due to the oxidation of the acetate accumulated before MAA addition, the OTR of the ΔpfkA strain strongly decreased, indicating a very low respiration rate despite sufficient oxygen supply. The latter effect can likely be attributed to a restricted conversion of NADPH into NADH via the soluble transhydrogenase SthA, as the enzyme is outcompeted in the presence of MAA by the recombinant NADPH-dependent alcohol

  2. Differential survival of Escherichia coli uvrA, uvrB, and uvrC mutants to psoralen plus UV-A (PUVA): Evidence for uncoupled action of nucleotide excision repair to process DNA adducts.

    PubMed

    Lage, Claudia; Gonçalves, Silvia R F; Souza, Luciana L; de Pádula, Marcelo; Leitão, Alvaro C

    2010-01-21

    The nucleotide excision repair mechanism (NER) of Escherichia coli is responsible for the recognition and elimination of more than twenty different DNA lesions. Herein, we evaluated the in vivo role of NER in the repair of DNA adducts generated by psoralens (mono- or bi-functional) and UV-A light (PUVA) in E. coli. Cultures of wild-type E. coli K12 and mutants for uvrA, uvrB, uvrC or uvrAC genes were treated with PUVA and cell survival was determined. In parallel, kinetics of DNA repair was also evaluated by the comparison of DNA sedimentation profiles in all the strains after PUVA treatment. The uvrB mutant was more sensitive to PUVA treatment than all the other uvr mutant strains. Wild-type strain, and uvrA and uvrC mutants were able to repair PUVA-induced lesions, as seen by DNA sedimentation profiles, while the uvrB mutant was unable to repair the lesions. In addition, a quadruple fpg nth xth nfo mutant was unable to nick PUVA-treated DNA when the crude cell-free extract was used to perform plasmid nicking. These data suggest that DNA repair of PUVA-induced lesions may require base excision repair functions, despite proficient UvrABC activity. These results point to a specific role for UvrB protein in the repair of psoralen adducts, which appear to be independent of UvrA or UvrC proteins, as described for the classical UvrABC endonuclease mechanism.

  3. A Bacillus subtilis dnaG mutant harbours a mutation in a gene homologous to the dnaN gene of Escherichia coli.

    PubMed

    Ogasawara, N; Moriya, S; Mazza, G; Yoshikawa, H

    1986-01-01

    A dnaG mutation of Bacillus subtilis, dnaG5, was found to be linked closely to recF. We have reported previously that two putative dna genes, 'dnaA' and 'dnaN', highly homologous to Escherichia coli's dnaA and dnaN, respectively, were located adjacent to recF [Ogasawara et al., EMBO J., 4 (1985) 3345-3350]. Transformation by various fragments cloned from the 'dnaA'-recF region of the wild-type cell revealed that a 532-bp AluI fragment containing 5'-portion of the 'dnaN' gene could transform the dnaG5 mutation. The nucleotide (nt) sequence of the same fragment cloned from the mutant cell shows a single nt change in the ORF of 'dnaN' which in turn causes a single amino acid alteration from Gly to Arg. The 'dnaN' gene is now proven to be a dna gene, mutations in which result in instant arrest of chromosomal replication.

  4. D-Fucose as a gratuitous inducer of the L-arabinose operon in strains of Escherichia coli B-r mutant in gene araC.

    PubMed

    Beverin, S; Sheppard, D E; Park, S S

    1971-07-01

    d-Fucose, a nonmetabolizable analogue of l-arabinose, prevents growth of Escherichia coli B/r on a mineral salts medium plus l-arabinose by inhibiting induction of the l-arabinose operon. Mutations giving rise to d-fucose resistance map in gene araC and result in constitutive expression of the l-arabinose operon. Most of these mutations also permit d-fucose to serve as a gratuitous inducer. It is concluded that d-fucose-resistant mutants produce an araC gene product with an altered inducer specificity. Addition of l-arabinose to cells induced with the gratuitous inducer, d-fucose, resulted in severe transient repression of operon expression followed by permanent catabolite repression. Transient repression but no permanent catabolite repression was obtained when cells unable to metabolize l-arabinose were employed. It is concluded that transport of l-arabinose alone is sufficient to achieve transient repression of its own operon, but that metabolism of l-arabinose must occur to achieve permanent catabolite repression of the l-arabinose operon. This general effect has been termed "self-catabolite repression."

  5. Kinetics and crystal structure of a mutant Escherichia coli alkaline phosphatase (Asp-369-->Asn): a mechanism involving one zinc per active site.

    PubMed

    Tibbitts, T T; Xu, X; Kantrowitz, E R

    1994-11-01

    Using site-directed mutagenesis, an aspartate side chain involved in binding metal ions in the active site of Escherichia coli alkaline phosphatase (Asp-369) was replaced, alternately, by asparagine (D369N) and by alanine (D369A). The purified mutant enzymes showed reduced turnover rates (kcat) and increased Michaelis constants (Km). The kcat for the D369A enzyme was 5,000-fold lower than the value for the wild-type enzyme. The D369N enzyme required Zn2+ in millimolar concentrations to become fully active; even under these conditions the kcat measured for hydrolysis of p-nitrophenol phosphate was 2 orders of magnitude lower than for the wild-type enzyme. Thus the kcat/Km ratios showed that catalysis is 50 times less efficient when the carboxylate side chain of Asp-369 is replaced by the corresponding amide; and activity is reduced to near nonenzymic levels when the carboxylate is replaced by a methyl group. The crystal structure of D369N, solved to 2.5 A resolution with an R-factor of 0.189, showed vacancies at 2 of the 3 metal binding sites. On the basis of the kinetic results and the refined X-ray coordinates, a reaction mechanism is proposed for phosphate ester hydrolysis by the D369N enzyme involving only 1 metal with the possible assistance of a histidine side chain.

  6. Antimicrobial peptides at work: interaction of myxinidin and its mutant WMR with lipid bilayers mimicking the P. aeruginosa and E. coli membranes

    PubMed Central

    Lombardi, Lucia; Stellato, Marco Ignazio; Oliva, Rosario; Falanga, Annarita; Galdiero, Massimiliano; Petraccone, Luigi; D’Errico, Geradino; De Santis, Augusta; Galdiero, Stefania; Del Vecchio, Pompea

    2017-01-01

    Antimicrobial peptides are promising candidates as future therapeutics in order to face the problem of antibiotic resistance caused by pathogenic bacteria. Myxinidin is a peptide derived from the hagfish mucus displaying activity against a broad range of bacteria. We have focused our studies on the physico-chemical characterization of the interaction of myxinidin and its mutant WMR, which contains a tryptophan residue at the N-terminus and four additional positive charges, with two model biological membranes (DOPE/DOPG 80/20 and DOPE/DOPG/CL 65/23/12), mimicking respectively Escherichia coli and Pseudomonas aeruginosa membrane bilayers. All our results have coherently shown that, although both myxinidin and WMR interact with the two membranes, their effect on membrane microstructure and stability are different. We further have shown that the presence of cardiolipin plays a key role in the WMR-membrane interaction. Particularly, WMR drastically perturbs the DOPE/DOPG/CL membrane stability inducing a segregation of anionic lipids. On the contrary, myxinidin is not able to significantly perturb the DOPE/DOPG/CL bilayer whereas interacts better with the DOPE/DOPG bilayer causing a significant perturbing effect of the lipid acyl chains. These findings are fully consistent with the reported greater antimicrobial activity of WMR against P. aeruginosa compared with myxinidin. PMID:28294185

  7. A Novel Method for Efficient Preparation of Mucosal Adjuvant Escherichia coli Heat-Labile Enterotoxin Mutant (LTm) by Artificially Assisted Self-Assembly In Vitro.

    PubMed

    Liu, Di; Zhang, Na; Zheng, Wenyun; Guo, Hua; Wang, Xiaoli; Wang, Tianwen; Wang, Ping; Ma, Xingyuan

    2016-04-01

    As well-known powerful mucosal adjuvant proteins, Escherichia coli heat-labile enterotoxin (LT) and its non-toxic or low-toxic mutants (LTm) are capable of promoting strong mucosal immune responses to co-administered antigens in various types of vaccines. However, due to the complex composition and special structure, the yield of LTm directly from the recombinant genetic engineering strains is quite low. Here, we put forward a novel method to prepare LTm protein which designed, expressed, and purified three kinds of component subunits respectively and assembled them into a hexamer structure in vitro by two combination modes. In addition, by simulated in vivo environment of polymer protein assembly, the factors of the protein solution system which include environment temperature, pH, ionic strength of the solution, and ratio between each subunit were taken into consideration. Finally, we confirmed the optimal conditions of two assembly strategies and prepared the hexamer holotoxin in vitro. These results are not only an important significance in promoting large-scale preparation of the mucosal adjuvant LTm but also an enlightening to produce other multi-subunit proteins.

  8. Antimicrobial peptides at work: interaction of myxinidin and its mutant WMR with lipid bilayers mimicking the P. aeruginosa and E. coli membranes

    NASA Astrophysics Data System (ADS)

    Lombardi, Lucia; Stellato, Marco Ignazio; Oliva, Rosario; Falanga, Annarita; Galdiero, Massimiliano; Petraccone, Luigi; D’Errico, Geradino; de Santis, Augusta; Galdiero, Stefania; Del Vecchio, Pompea

    2017-03-01

    Antimicrobial peptides are promising candidates as future therapeutics in order to face the problem of antibiotic resistance caused by pathogenic bacteria. Myxinidin is a peptide derived from the hagfish mucus displaying activity against a broad range of bacteria. We have focused our studies on the physico-chemical characterization of the interaction of myxinidin and its mutant WMR, which contains a tryptophan residue at the N-terminus and four additional positive charges, with two model biological membranes (DOPE/DOPG 80/20 and DOPE/DOPG/CL 65/23/12), mimicking respectively Escherichia coli and Pseudomonas aeruginosa membrane bilayers. All our results have coherently shown that, although both myxinidin and WMR interact with the two membranes, their effect on membrane microstructure and stability are different. We further have shown that the presence of cardiolipin plays a key role in the WMR-membrane interaction. Particularly, WMR drastically perturbs the DOPE/DOPG/CL membrane stability inducing a segregation of anionic lipids. On the contrary, myxinidin is not able to significantly perturb the DOPE/DOPG/CL bilayer whereas interacts better with the DOPE/DOPG bilayer causing a significant perturbing effect of the lipid acyl chains. These findings are fully consistent with the reported greater antimicrobial activity of WMR against P. aeruginosa compared with myxinidin.

  9. Hyperadherence of an hha mutant of Escherichia coli O157:H7 is correlated with enhanced expression of LEE-encoded adherence genes.

    PubMed

    Sharma, Vijay K; Carlson, Steven A; Casey, Thomas A

    2005-02-01

    Enterohemorrhagic Escherichia coli (EHEC) O157:H7 virulence factors, specifically those conferring intimate adherence to and formation of attaching and effacing lesions (A/E) on host cells, are encoded by a horizontally acquired locus of enterocyte effacement (LEE). Expression of several LEE-encoded genes, which are organized into operons LEE1 through LEE5, is under the positive regulation of ler, the first gene in the LEE1 operon. We have recently demonstrated that EHEC O157:H7 lacking hha exhibited greater than a 10-fold increase in ler expression and that the repression of ler results from the binding of Hha to the ler promoter. In this report, we show that an hha mutant of EHEC O157:H7 exhibited increased adherence to Hep-2 cells, had increased transcriptional activities of LEE1, LEE2, LEE3, and LEE5 as determined by reverse transcriptase-polymerase chain reaction assays, and expressed LEE5::lac transcriptional fusion at levels that were several-fold higher than that expressed by the parental hha+ strain. These results demonstrate that hha is an important regulatory component of the cascade that governs the expression of LEE operons and the resulting ability of EHEC O157:H7 to intimately adhere to host cells.

  10. Impact of membrane-associated hydrogenases on the F₀F₁-ATPase in Escherichia coli during glycerol and mixed carbon fermentation: ATPase activity and its inhibition by N,N'-dicyclohexylcarbodiimide in the mutants lacking hydrogenases.

    PubMed

    Blbulyan, Syuzanna; Trchounian, Armen

    2015-08-01

    Escherichia coli is able to ferment glycerol and to produce molecular hydrogen (H2) by four membrane-associated hydrogenases (Hyd) changing activity in response to different conditions. In this study, overall ATPase activity of glycerol alone and mixed carbon sources (glucose and glycerol) fermented E. coli wild type and different Hyd mutants and its inhibition by N,N'-dicyclohexylcarbodiimide (DCCD) were first investigated. ATPase activity was higher in glycerol fermented wild type cells at pH 7.5 compared to pH 6.5 and pH 5.5; DCCD inhibited markedly ATPase activity at pH 7.5. The ATPase activity at pH 7.5, compared with wild type, was lower in selC and less in hypF single mutants, suppressed in hyaB hybC selC triple mutant. Moreover, total ATPase activity of mixed carbon fermented wild type cells was maximal at pH 7.5 and lowered at pH 5.5. The ATPase activities of hypF and hyaB hybC selC mutants were higher at pH 5.5, compared with wild type; DCCD inhibited markedly ATPase activity of hypF mutant. These results demonstrate that in E. coli during glycerol fermentation the membrane proton-translocating FOF1-ATPase has major input in overall ATPase activity and alkaline pH is more optimal for the FOF1-ATPase operation. Hyd-1 and Hyd-2 are required for the FOF1-ATPase activity upon anaerobic fermentation of glycerol. The impact of Hyd-1 and Hyd-2 on the FOF1-ATPase is more obvious during mixed carbon fermentation at slightly acidic pH.

  11. Synthetic lipid A with endotoxic and related biological activities comparable to those of a natural lipid A from an Escherichia coli re-mutant.

    PubMed Central

    Kotani, S; Takada, H; Tsujimoto, M; Ogawa, T; Takahashi, I; Ikeda, T; Otsuka, K; Shimauchi, H; Kasai, N; Mashimo, J

    1985-01-01

    A synthetic compound (506), beta (1-6) D-glucosamine disaccharide 1,4'-bisphosphate, which is acylated at 2'-amino and 3'-hydroxyl groups with (R)-3-dodecanoyloxytetradecanoyl and (R)-3-tetradecanoyloxytetradecanoyl groups, respectively, and has (R)-3-hydroxytetradecanoyl groups at 2-amino and 3-hydroxyl groups, exhibited full endotoxic activities identical to or sometimes stronger than those of a reference lipid A from an Escherichia coli Re-mutant (strain F515). Endotoxic activities tested include pyrogenicity and leukopenia-inducing activity in rabbits, body weight-decreasing toxicity in normal mice, lethal toxicity in galactosamine-sensitized mice and chicken embryos, and the preparation and provocation of the local Shwartzman reaction in rabbits. Compound 406, a synthetic counterpart of a biosynthetic precursor of lipid A molecule, showed by contrast only weak activities in all of the above assay systems except for the lethality in galactosamine-loaded mice. This finding strongly suggests that the presence of acyloxyacyl groups at the C-2' and C-3' positions of the disaccharide backbone is one of the most important determinant structures of the lipid A molecule for exhibition of strong biological activities characteristic of lipopolysaccharide and its lipid A moiety. The activities of the corresponding 4'-monophosphate (compound 504) and 1-monophosphate (505) analogs were considerably less than those of the parent molecule 506 and the reference F515 lipid A. Regarding other biological activities, not only compound 506 but also compounds 504, 505, and 406 showed definite activities, sometimes comparable to those of F515 lipid A and other reference natural products. These are the activation of Tachypleus tridentatus amoebocyte clotting enzyme cascade and human complement via the classical pathway, mitogenic and polyclonal B-cell activation of murine splenocytes, stimulation of peritoneal macrophages in a guinea pig, enhancement of migration of human blood

  12. Cloning of a chicken liver cDNA encoding 5-aminoimidazole ribonucleotide carboxylase and 5-aminoimidazole-4-N-succinocarboxamide ribonucleotide synthetase by functional complementation of Escherichia coli pur mutants.

    PubMed Central

    Chen, Z D; Dixon, J E; Zalkin, H

    1990-01-01

    We have used functional complementation of Escherichia coli pur mutants to clone avian cDNA encoding 5-aminoimidazole ribonucleotide (AIR) carboxylase-5-aminoimidazole-4-N-succinocarboxamide ribonucleotide (SAICAR) synthetase, the bifunctional enzyme catalyzing steps 6 and 7 in the pathway for de novo purine nucleotide synthesis. Mutational analyses have been used to establish the structure-function relationship: NH2-SAICAR synthetase-AIR carboxylase-COOH. The amino acid sequence of the SAICAR synthetase domain is homologous to that of bacterial purC-encoded enzymes, and the sequence of the following AIR carboxylase domain is homologous to that of bacterial purE-encoded enzymes. In E. coli, AIR carboxylase is the product of genes purEK with the purK subunit postulated to have a role in CO2 binding. The avian enzyme lacks sequences corresponding to purK yet functions in E. coli. Functional complementation of E. coli pur mutants can be used to clone additional avian cDNAs for de novo purine nucleotide synthesis. Images PMID:1691501

  13. Characterization of the membrane quinoprotein glucose dehydrogenase from Escherichia coli and characterization of a site-directed mutant in which histidine-262 has been changed to tyrosine.

    PubMed Central

    Cozier, G E; Salleh, R A; Anthony, C

    1999-01-01

    The requirements for substrate binding in the quinoprotein glucose dehydrogenase (GDH) in the membranes of Escherichia coli are described, together with the changes in activity in a site-directed mutant in which His262 has been altered to a tyrosine residue (H262Y-GDH). The differences in catalytic efficiency between substrates are mainly related to differences in their affinity for the enzyme. Remarkably, it appears that, if a hexose is able to bind in the active site, then it is also oxidized, whereas some pentoses are able to bind (and act as competitive inhibitors), but are not substrates. The activation energies for the oxidation of hexoses and pentoses are almost identical. In a previously published model of the enzyme, His262 is at the entrance to the active site and appears to be important in holding the prosthetic group pyrroloquinoline quinone (PQQ) in place, and it has been suggested that it might play a role in electron transfer from the reduced PQQ to the ubiquinone in the membrane. The H262Y-GDH has a greatly diminished catalytic efficiency for all substrates, which is mainly due to a marked decrease in their affinities for the enzyme, but the rate of electron transfer to oxygen is unaffected. During the processing of the PQQ into the apoenzyme to give active enzyme, its affinity is markedly dependent on the pH, four groups with pK values between pH7 and pH8 being involved. Identical results were obtained with H262Y-GDH, showing that His262 it is not directly involved in this process. PMID:10359647

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

    PubMed Central

    Lee, Pyung-Gang; Kim, Joonwon; Kim, Eun-Jung; Jung, EunOk; Pandey, Bishnu Prasad

    2016-01-01

    (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 from Slackia isoflavoniconvertens into Escherichia coli BL21(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 of trans-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%. PMID:26801575

  15. Altered Antibiotic Transport in OmpC Mutants Isolated from a Series of Clinical Strains of Multi-Drug Resistant E. coli

    PubMed Central

    Ceccarelli, Matteo; Mach, Tivadar; Beis, Konstantinos; Low, Alison S.; Bamford, Victoria A.; Booth, Ian R.; Bayley, Hagan; Naismith, James H.

    2011-01-01

    Antibiotic-resistant bacteria, particularly Gram negative species, present significant health care challenges. The permeation of antibiotics through the outer membrane is largely effected by the porin superfamily, changes in which contribute to antibiotic resistance. A series of antibiotic resistant E. coli isolates were obtained from a patient during serial treatment with various antibiotics. The sequence of OmpC changed at three positions during treatment giving rise to a total of four OmpC variants (denoted OmpC20, OmpC26, OmpC28 and OmpC33, in which OmpC20 was derived from the first clinical isolate). We demonstrate that expression of the OmpC K12 porin in the clinical isolates lowers the MIC, consistent with modified porin function contributing to drug resistance. By a range of assays we have established that the three mutations that occur between OmpC20 and OmpC33 modify transport of both small molecules and antibiotics across the outer membrane. This results in the modulation of resistance to antibiotics, particularly cefotaxime. Small ion unitary conductance measurements of the isolated porins do not show significant differences between isolates. Thus, resistance does not appear to arise from major changes in pore size. Crystal structures of all four OmpC clinical mutants and molecular dynamics simulations also show that the pore size is essentially unchanged. Molecular dynamics simulations suggest that perturbation of the transverse electrostatic field at the constriction zone reduces cefotaxime passage through the pore, consistent with laboratory and clinical data. This subtle modification of the transverse electric field is a very different source of resistance than occlusion of the pore or wholesale destruction of the transverse field and points to a new mechanism by which porins may modulate antibiotic passage through the outer membrane. PMID:22053181

  16. A dnaN Plasmid Shuffle Strain for Rapid In Vivo Analysis of Mutant Escherichia coli β Clamps Provides Insight Into the Role of Clamp in umuDC-Mediated Cold Sensitivity

    PubMed Central

    Babu, Vignesh M. P.; Sutton, Mark D.

    2014-01-01

    The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C. PMID:24896652

  17. A dnaN plasmid shuffle strain for rapid in vivo analysis of mutant Escherichia coli β clamps provides insight into the role of clamp in umuDC-mediated cold sensitivity.

    PubMed

    Babu, Vignesh M P; Sutton, Mark D

    2014-01-01

    The E. coli umuDC gene products participate in two temporally distinct roles: UmuD2C acts in a DNA damage checkpoint control, while UmuD'2C, also known as DNA polymerase V (Pol V), catalyzes replication past DNA lesions via a process termed translesion DNA synthesis. These different roles of the umuDC gene products are managed in part by the dnaN-encoded β sliding clamp protein. Co-overexpression of the β clamp and Pol V severely blocked E. coli growth at 30°C. We previously used a genetic assay that was independent of the ability of β clamp to support E. coli viability to isolate 8 mutant clamp proteins (βQ61K, βS107L, βD150N, βG157S, βV170M, βE202K, βM204K and βP363S) that failed to block growth at 30°C when co-overexpressed with Pol V. It was unknown whether these mutant clamps were capable of supporting E. coli viability and normal umuDC functions in vivo. The goals of this study were to answer these questions. To this end, we developed a novel dnaN plasmid shuffle assay. Using this assay, βD150N and βP363S were unable to support E. coli viability. The remaining 6 mutant clamps, each of which supported viability, were indistinguishable from β+ with respect to umuDC functions in vivo. In light of these findings, we analyzed phenotypes of strains overexpressing either β clamp or Pol V alone. The strain overexpressing β+, but not those expressing mutant β clamps, displayed slowed growth irrespective of the incubation temperature. Moreover, growth of the Pol V-expressing strain was modestly slowed at 30°, but not 42°C. Taken together, these results suggest the mutant clamps were identified due to their inability to slow growth rather than an inability to interact with Pol V. They further suggest that cold sensitivity is due, at least in part, to the combination of their individual effects on growth at 30°C.

  18. Genome-wide screen identifies Escherichia coli TCA cycle-related mutants with extended chronological lifespan dependent on acetate metabolism and the hypoxia-inducible transcription factor ArcA

    PubMed Central

    Gonidakis, Stavros; Finkel, Steven E.; Longo, Valter D.

    2010-01-01

    Summary Single-gene mutants with extended lifespan have been described in several model organisms. We performed a genome-wide screen for long-lived mutants in Escherichia coli which revealed strains lacking TCA cycle-related genes that exhibit longer stationary phase survival and increased resistance to heat stress compared to wild-type. Extended lifespan in the sdhA mutant, lacking subunit A of succinate dehydrogenase, is associated with reduced production of superoxide and increased stress resistance. On the other hand, the longer lifespan of the lipoic acid synthase mutant (lipA) is associated with reduced oxygen consumption and requires the acetate-producing enzyme pyruvate oxidase, as well as acetyl-CoA synthetase, the enzyme that converts extracellular acetate to acetyl-CoA. The hypoxia-inducible transcription factor ArcA, acting independently of acetate metabolism, is also required for maximum lifespan extension in the lipA and lpdA mutants, indicating that these mutations promote entry into a mode normally associated with a low-oxygen environment. Since analogous changes from respiration to fermentation have been observed in long-lived Saccharomyces cerevisiae and Caenorhabditis elegans strains, such metabolic alterations may represent an evolutionarily conserved strategy to extend lifespan. PMID:20707865

  19. Heterologous expression of the human Phosphoenol Pyruvate Carboxykinase (hPEPCK-M) improves hydrogen and ethanol synthesis in the Escherichia coli dcuD mutant when grown in a glycerol-based medium.

    PubMed

    Valle, Antonio; Cabrera, Gema; Cantero, Domingo; Bolivar, Jorge

    2017-03-25

    The production of biodiesel has emerged as an alternative to fossil fuels. However, this industry generates glycerol as a by-product in such large quantities that it has become an environmental problem. The biotransformation of this excess glycerol into other renewable bio-energy sources, like H2 and ethanol, by microorganisms such as Escherichia coli is an interesting possibility that warrants investigation. In this work we hypothesized that the conversion of oxaloacetate (OAA) to phosphoenolpyruvate (PEP) could be improved by a controlled expression of the human mitochondrial GTP-dependent PEP carboxykinase. This heterologous expression was tested in several E. coli mutant backgrounds with increased availability of C4 intermediates. It was found that this metabolic rewiring improved the synthesis of the target products in several mutants, with the dcuD mutant being the most suitable background for hydrogen and ethanol specific productions and glycerol consumption. These factors increased by 2.46, 1.73 and 1.95 times, respectively, when compared to those obtained for the wild-type strain.

  20. Elongation factors in protein synthesis.

    PubMed

    Kraal, B; Bosch, L; Mesters, J R; de Graaf, J M; Woudt, L P; Vijgenboom, E; Heinstra, P W; Zeef, L A; Boon, C

    1993-01-01

    Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

  1. A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome.

    PubMed Central

    Tubulekas, I; Hughes, D

    1993-01-01

    Elongation factor Tu (EF-Tu).GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tuf gene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EF-Tu.GTP.aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed. Images PMID:8416899

  2. Two proofreading steps amplify the accuracy of genetic code translation.

    PubMed

    Ieong, Ka-Weng; Uzun, Ülkü; Selmer, Maria; Ehrenberg, Måns

    2016-11-29

    Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aa-tRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EF-Tu-independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information.

  3. Location of functional regions of the Escherichia coli RecA protein by DNA sequence analysis of RecA protease-constitutive mutants.

    PubMed Central

    Wang, W B; Tessman, E S

    1986-01-01

    In previous work (E. S. Tessman and P. K. Peterson, J. Bacteriol. 163:677-687 and 688-695, 1985), we isolated many novel protease-constitutive (Prtc) recA mutants, i.e., mutants in which the RecA protein was always in the protease state without the usual need for DNA damage to activate it. Most Prtc mutants were recombinase positive and were designated Prtc Rec+; only a few Prtc mutants were recombinase negative, and those were designated Prtc Rec-. We report changes in DNA sequence of the recA gene for several of these mutants. The mutational changes clustered at three regions on the linear RecA polypeptide. Region 1 includes amino acid residues 25 through 39, region 2 includes amino acid residues 157 through 184, and region 3 includes amino acid residues 298 through 301. The in vivo response of these Prtc mutants to different effectors suggests that the RecA effector-binding sites have been altered. In particular we propose that the mutations may define single-stranded DNA- and nucleoside triphosphate-binding domains of RecA, that polypeptide regions 1 and 3 comprise part of the single-stranded DNA-binding domain, and that polypeptide regions 2 and 3 comprise part of the nucleoside triphosphate-binding domain. The overlapping of single-stranded DNA- and nucleoside triphosphate-binding domains in region 3 can explain previously known complex allosteric effects. Each of four Prtc Rec- mutants sequenced was found to contain a single amino acid change, showing that the change of just one amino acid can affect both the protease and recombinase activities and indicating that the functional domains for these two activities of RecA overlap. A recA promoter-down mutation was isolated by its ability to suppress the RecA protease activity of one of our strong Prtc mutants. PMID:3536864

  4. Lack of discrimination against non-proteinogenic amino acid norvaline by elongation factor Tu from Escherichia coli.

    PubMed

    Cvetesic, Nevena; Akmacic, Irena; Gruic-Sovulj, Ita

    2013-01-01

    The GTP-bound form of elongation factor Tu (EF-Tu) brings aminoacylated tRNAs (aa-tRNA) to the A-site of the ribosome. EF-Tu binds all cognate elongator aa-tRNAs with highly similar affinities, and its weaker or tighter binding of misacylated tRNAs may discourage their participation in translation. Norvaline (Nva) is a non-proteinogenic amino acid that is activated and transferred to tRNA(Leu) by leucyl-tRNA synthetase (LeuRS). No notable accumulation of Nva-tRNA(Leu) has been observed in vitro, because of the efficient post-transfer hydrolytic editing activity of LeuRS. However, incorporation of norvaline into proteins in place of leucine does occur under certain conditions in vivo. Here we show that EF-Tu binds Nva-tRNA(Leu) and Leu-tRNA(Leu) with similar affinities, and that Nva-tRNA(Leu) and Leu-tRNA(Leu) dissociate from EF-Tu at comparable rates. The inability of EF-Tu to discriminate against norvaline may have driven evolution of highly efficient LeuRS editing as the main quality control mechanism against misincorporation of norvaline into proteins.

  5. Problem-Solving Test: Tryptophan Operon Mutants

    ERIC Educational Resources Information Center

    Szeberenyi, Jozsef

    2010-01-01

    This paper presents a problem-solving test that deals with the regulation of the "trp" operon of "Escherichia coli." Two mutants of this operon are described: in mutant A, the operator region of the operon carries a point mutation so that it is unable to carry out its function; mutant B expresses a "trp" repressor protein unable to bind…

  6. Reconstitution of mammalian excision repair activity with mutant cell-free extracts and XPAC and ERCC1 proteins expressed in Escherichia coli.

    PubMed Central

    Park, C H; Sancar, A

    1993-01-01

    Nucleotide excision repair in humans involves the coordinated actions of 8-10 proteins. To understand the roles of each of these proteins in excision it is necessary to develop an in vitro excision repair system reconstituted entirely from purified proteins. Towards this goal we have expressed in E. coli two of the 8 genes known to be essential for the excision reaction. XPAC and ERCC1 were expressed as fusion proteins with the Escherichia coli maltose binding protein (MBP) and purified to > 80% homogeneity by affinity chromatography. The purified proteins either as fusions or after cleavage from the MBP were able to complement the CFE of cells with mutations in the corresponding genes in an excision assay with thymine dimer containing substrate. Images PMID:8255764

  7. Growth of wildtype and mutant E. coli strains in minimal media for optimal production of nucleic acids for preparing labeled nucleotides.

    PubMed

    Thakur, Chandar S; Brown, Margaret E; Sama, Jacob N; Jackson, Melantha E; Dayie, T Kwaku

    2010-10-01

    Since RNAs lie at the center of most cellular processes, there is a need for synthesizing large amounts of RNAs made from stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (NMR) spectroscopy. A particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria grown in defined minimal media supplemented with 15NH4Cl and various carbon sources. Given the high cost of carbon precursors required for labeling nucleic acids for NMR studies, it becomes important to evaluate the optimal growth for commonly used strains under standard minimal media conditions. Such information is lacking. In this study, we characterize the growth for Escherichia coli strains K12, K10zwf, and DL323 in three minimal media with isotopic-labeled carbon sources of acetate, glycerol, and glycerol combined with formate. Of the three media, the LeMaster-Richards and the Studier media outperform the commonly used M9 media and both support optimal growth of E. coli for the production of nucleotides. However, the growth of all three E. coli strains in acetate is reduced almost twofold compared to growth in glycerol. Analysis of the metabolic pathway and previous gene array studies help to explain this differential growth in glycerol and acetate. These studies should benefit efforts to make selective 13C-15N isotopic-labeled nucleotides for synthesizing biologically important RNAs.

  8. Cadmium resistance mechanism in Escherichia coli P4 and its potential use to bioremediate environmental cadmium.

    PubMed

    Khan, Zaman; Nisar, Muhammad Atif; Hussain, Syed Zajif; Arshad, Muhammad Nauman; Rehman, Abdul

    2015-12-01

    A cadmium-resistant bacterium was isolated from industrial wastewater and identified as Escherichia coli (dubbed as P4) on the basis of morphological, biochemical tests and 16S rRNA ribotyping. It showed optimum growth at 30 °C and pH 7. E. coli P4 found to resist Cd(+2) (10.6 mM) as well as Zn(+2) (4.4 mM), Pb(+2) (17 mM), Cu(+2) (3.5 mM), Cr(+6) (4.4 mM), As(+2) (10.6 mM), and Hg(+2) (0.53 mM). It could remove 18.8, 37, and 56 % Cd(+2) from aqueous medium after 48, 96, and 144 h, respectively. Fourier transform infrared spectroscopy (FTIR), scanning electron microscope (SEM), and Energy-dispersive X-ray (EDX) analysis also confirmed the biosorption of Cd(+2) by E. coli P4. However, temperature and pH were found to be the most critical factors in biosorption of Cd(+2) by E. coli P4. Cd(+2) stress altered E. coli P4 cell physiology analyzed by measuring glutathione (GSH) and non-protein thiol (cysteine) levels which were increased up to 130 and 48 %, respectively. Quantitative real-time polymerase chain reaction (qRT-PCR) showed alteration in the expression levels of ftsZ, mutS, clpB, ef-tu, and dnaK genes in the presence of Cd(+2). Total protein profiles of E. coli P4 in the absence and presence of Cd(+2) were compared by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which showed remarkable difference in the banding pattern. czcB gene, a component of czcCBA operon, was amplified from genomic DNA which suggested the chromosomal-borne Cd(+2) resistance in E. coli P4. Furthermore, it harbors smtAB gene which plays a significant role in Cd(+2) resistance.

  9. Growth of wildtype and mutant E. coli strains in minimal media for optimal production of nucleic acids for preparing labeled nucleotides

    PubMed Central

    Thakur, Chandar S.; Brown, Margaret E.; Sama, Jacob N.; Jackson, Melantha E.

    2010-01-01

    Since RNAs lie at the center of most cellular processes, there is a need for synthesizing large amounts of RNAs made from stable isotope-labeled nucleotides to advance the study of their structure and dynamics by nuclear magnetic resonance (NMR) spectroscopy. A particularly effective means of obtaining labeled nucleotides is to harvest these nucleotides from bacteria grown in defined minimal media supplemented with 15NH4Cl and various carbon sources. Given the high cost of carbon precursors required for labeling nucleic acids for NMR studies, it becomes important to evaluate the optimal growth for commonly used strains under standard minimal media conditions. Such information is lacking. In this study, we characterize the growth for Escherichia coli strains K12, K10zwf, and DL323 in three minimal media with isotopic-labeled carbon sources of acetate, glycerol, and glycerol combined with formate. Of the three media, the LeMaster-Richards and the Studier media outperform the commonly used M9 media and both support optimal growth of E. coli for the production of nucleotides. However, the growth of all three E. coli strains in acetate is reduced almost twofold compared to growth in glycerol. Analysis of the metabolic pathway and previous gene array studies help to explain this differential growth in glycerol and acetate. These studies should benefit efforts to make selective 13C-15N isotopic-labeled nucleotides for synthesizing biologically important RNAs. Electronic supplementary material The online version of this article (doi:10.1007/s00253-010-2813-y) contains supplementary material, which is available to authorized users. PMID:20730533

  10. Characterisation of mutant alleles of the cell division protein FtsA, a regulator and structural component of the Escherichia coli septator.

    PubMed

    Sánchez, M; Dopazo, A; Pla, J; Robinson, A C; Vicente, M

    1994-01-01

    Two alleles of ftsA, a gene that encodes an essential cell division protein in Escherichia coli, have-been mapped at the nucleotide level. The mutations are located inside domains that are conserved in an ATP-binding protein family. The ftsA2 mutation lies in the adenine-binding domain, and the ftsA3 in the ribose-binding domain. The defect in ampicillin binding to PBP3 described for allele ftsA3 is allele-specific. This supports the hypothesis of the existence of different domains in FtsA having different functions.

  11. An excretory function for the Escherichia coli outer membrane pore TolC: upregulation of marA and soxS transcription and Rob activity due to metabolites accumulated in tolC mutants.

    PubMed

    Rosner, Judah L; Martin, Robert G

    2009-08-01

    Efflux pumps function to rid bacteria of xenobiotics, including antibiotics, bile salts, and organic solvents. TolC, which forms an outer membrane channel, is an essential component of several efflux pumps in Escherichia coli. We asked whether TolC has a role during growth in the absence of xenobiotics. Because tolC transcription is activated by three paralogous activators, MarA, SoxS, and Rob, we examined the regulation of these activators in tolC mutants. Using transcriptional fusions, we detected significant upregulation of marRAB and soxS transcription and Rob protein activity in tolC mutants. Three mechanisms could be distinguished: (i) activation of marRAB transcription was independent of marRAB, soxR, and rob functions; (ii) activation of soxS transcription required SoxR, a sensor of oxidants; and (iii) Rob protein was activated posttranscriptionally. This mechanism is similar to the mechanisms of upregulation of marRAB, soxS, and Rob by treatment with certain phenolics, superoxides, and bile salts, respectively. The transcription of other marA/soxS/rob regulon promoters, including tolC itself, was also elevated in tolC mutants. We propose that TolC is involved in the efflux of certain cellular metabolites, not only xenobiotics. As these metabolites accumulate during growth, they trigger the upregulation of MarA, SoxS, and Rob, which in turn upregulate tolC and help rid the bacteria of these metabolites, thereby restoring homeostasis.

  12. Ribosome-induced changes in elongation factor Tu conformation control GTP hydrolysis

    PubMed Central

    Villa, Elizabeth; Sengupta, Jayati; Trabuco, Leonardo G.; LeBarron, Jamie; Baxter, William T.; Shaikh, Tanvir R.; Grassucci, Robert A.; Nissen, Poul; Ehrenberg, Måns; Schulten, Klaus; Frank, Joachim

    2009-01-01

    In translation, elongation factor Tu (EF-Tu) molecules deliver aminoacyl-tRNAs to the mRNA-programmed ribosome. The GTPase activity of EF-Tu is triggered by ribosome-induced conformational changes of the factor that play a pivotal role in the selection of the cognate aminoacyl-tRNAs. We present a 6.7-Å cryo-electron microscopy map of the aminoacyl-tRNA·EF-Tu·GDP·kirromycin-bound Escherichia coli ribosome, together with an atomic model of the complex obtained through molecular dynamics flexible fitting. The model reveals the conformational changes in the conserved GTPase switch regions of EF-Tu that trigger hydrolysis of GTP, along with key interactions, including those between the sarcin-ricin loop and the P loop of EF-Tu, and between the effector loop of EF-Tu and a conserved region of the 16S rRNA. Our data suggest that GTP hydrolysis on EF-Tu is controlled through a hydrophobic gate mechanism. PMID:19122150

  13. Two proofreading steps amplify the accuracy of genetic code translation

    PubMed Central

    Uzun, Ülkü; Selmer, Maria; Ehrenberg, Måns

    2016-01-01

    Aminoacyl-tRNAs (aa-tRNAs) are selected by the messenger RNA programmed ribosome in ternary complex with elongation factor Tu (EF-Tu) and GTP and then, again, in a proofreading step after GTP hydrolysis on EF-Tu. We use tRNA mutants with different affinities for EF-Tu to demonstrate that proofreading of aa-tRNAs occurs in two consecutive steps. First, aa-tRNAs in ternary complex with EF-Tu·GDP are selected in a step where the accuracy increases linearly with increasing aa-tRNA affinity to EF-Tu. Then, following dissociation of EF-Tu·GDP from the ribosome, the accuracy is further increased in a second and apparently EF-Tu−independent step. Our findings identify the molecular basis of proofreading in bacteria, highlight the pivotal role of EF-Tu for fast and accurate protein synthesis, and illustrate the importance of multistep substrate selection in intracellular processing of genetic information. PMID:27837019

  14. Decreased susceptibility to 4'-deoxy-6'-N-methylamikacin (BB-K311) conferred by a mutant plasmid in Escherichia coli.

    PubMed

    Perlin, M H; Lerner, S A

    1982-07-01

    Escherichia coli MP6 contains a plasmid that encodes aminoglycoside 3'-phosphotransferase II, which phosphorylates kanamycin and confers high-level kanamycin resistance, Amikacin is a minor substrate of this enzyme, but MP6 is susceptible to amikacin. Strain MP10 has a spontaneous mutation in the plasmid of MP6 that increases the aminoglycoside 3'-phosphotransferase II activity not only against kanamycin but also against amikacin. This mutation is also responsible for the appearance of resistance to amikacin in MP10. Resistance to 4'-deoxy-6'-N-methylamikacin (BB-K311) by enzymatic modification has not been reported previously. As with amikacin, MP6 was susceptible to BB-K311 and its aminoglycoside 3'-phosphotransferase II did not phosphorylate this amikacin derivative appreciably. We found that the plasmid-borne mutation in MP10, however, localized by being cloned with a 3.7-megadalton HindIII fragment containing the aminoglycoside 3'-phosphotransferase II gene, resulted in increased phosphorylation of BB-K311 and resistance to it. Thus, the mutation distinguishing MP6 and MP10 has increased the activity of an existing aminoglycoside-modifying enzyme and produced new bacterial resistance to two previously minor substrates of the enzyme.

  15. Kinetic analysis of Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase, wild type and mutants, reveals roles of active site amino acids.

    PubMed

    Richard, Stéphane B; Lillo, Antonietta M; Tetzlaff, Charles N; Bowman, Marianne E; Noel, Joseph P; Cane, David E

    2004-09-28

    Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP or IspD) catalyzes the conversion of 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytidine triphosphate (CTP) to 4-diphosphocytidyl-2-C-methylerythritol (CDPME). Pulse chase experiments established that the reaction involves an ordered sequential mechanism with mandatory initial binding of CTP. On the basis of analysis of the previously reported crystal structures of apo-YgbP as well as YgbP complexed with both CTP.Mg(2+) and CDPME.Mg(2+) [Richard, S. B., Bowman, M. E., Kwiatkowski, W., Kang, I., Chow, C., Lillo, A. M., Cane, D. E., and Noel, J. P. (2001) Nat. Struct. Biol. 8, 641-648], a group of active site residues were selected for site-directed mutagenesis and steady-state kinetic analysis. Both Lys27 and Lys213 were shown to be essential to catalytic activity, consistent with their proposed role in stabilization of a pentacoordinate phosphate transition state resulting from in-line attack of the MEP phosphate on the alpha-phosphate of CTP. In addition, Thr140, Arg109, Asp106, and Thr165 were all shown to play critical roles in the binding and proper orientation of the MEP substrate.

  16. Expression of escherichia coli otsA in a Saccharomyces cerevisiae tps1 mutant restores trehalose 6-phosphate levels and partly restores growth and fermentation with glucose and control of glucose influx into glycolysis.

    PubMed

    Bonini, B M; Van Vaeck, C; Larsson, C; Gustafsson, L; Ma, P; Winderickx, J; Van Dijck, P; Thevelein, J M

    2000-08-15

    The TPS1 gene, encoding trehalose-6-phosphate synthase (TPS), exerts an essential control on the influx of glucose into glycolysis in the yeast Saccharomyces cerevisiae. The deletion of TPS1 causes an inability to grow on glucose because of a hyperaccumulation of sugar phosphates and depletion of ATP and phosphate. We show that expression of the Escherichia coli homologue, otsA, in a yeast tps1 mutant results in high TPS activity. Although the trehalose 6-phosphate (Tre6P) level during exponential growth on glucose was at least as high as in a wild-type yeast strain, growth on glucose was only partly restored and the lag phase was much longer. Measurement of the glycolytic metabolites immediately after the addition of glucose showed that in spite of a normal Tre6P accumulation there was still a partial hyperaccumulation of sugar phosphates. Strong elevation of the Tre6P level by the additional deletion of the TPS2 gene, which encodes Tre6P phosphatase, was not able to cause a strong decrease in the sugar phosphate levels in comparison with the wild-type strain. In addition, in chemostat experiments the short-term response to a glucose pulse was delayed, but normal metabolism was regained over a longer period. These results show that Tre6P synthesis from a heterologous TPS enzyme can to some extent restore the control of glucose influx into glycolysis and growth on glucose in yeast. However, they also indicate that the yeast TPS enzyme, as opposed to the E. coli otsA gene product, is able to increase the efficiency of the Tre6P control on glucose influx into yeast glycolysis.

  17. Electron injection through a specific pathway determines the outcome of oxygen activation at the diiron cluster in the F208Y mutant of Escherichia coli ribonucleotide reductase protein R2.

    PubMed

    Parkin, S E; Chen, S; Ley, B A; Mangravite, L; Edmondson, D E; Huynh, B H; Bollinger, J M

    1998-01-27

    Protein R2 of ribonucleotide reductase from Escherichia coli contains a dinuclear iron cluster, which reductively activates O2 to produce the enzyme's functionally essential tyrosyl radical by one-electron oxidation of residue Y122. A key step in this reaction is the rapid injection of a single electron from an exogenous reductant (Fe2+ or ascorbate) during formation of the radical-generating intermediate, cluster X, from the diiron(II) cluster and O2. As this step leaves only one of the two oxidizing equivalents of the initial diiron(II)-O2 adduct, it commits the reaction to a one-electron oxidation outcome and precludes possible two-electron alternatives (as occur in the related diiron bacterial alkane hydroxylases and fatty acyl desaturases). In the F208Y site-directed mutant of R2, Y208 is hydroxylated (a two-electron oxidation) in preference to the normal reaction [Aberg, A., Ormö, M., Nordlund, P., & Sjöberg, B. M. (1993) Biochemistry 32, 9845-9850], implying that this substitution blocks electron injection or (more likely) introduces an endogenous reductant (Y208) that effectively competes. Here we demonstrate that O2 activation in the F208Y mutant of R2 partitions between these two-electron (Y208 hydroxylation) and one-electron (Y122 radical production) outcomes and that the latter becomes predominant under conditions which favor electron injection (namely, high concentration of the reductant ascorbate). Moreover, we show that the sensitivity of the partition ratio to ascorbate concentration is strictly dependent on the integrity of a hydrogen-bond network involving the near surface residue W48: when this residue is substituted with F, Y208 hydroxylation predominates irrespective of ascorbate concentration. These data suggest that the hydrogen-bond network involving W48 is a specific electron-transfer pathway between the cofactor site and the protein surface.

  18. RecQ helicase acts before RuvABC, RecG and XerC proteins during recombination in recBCD sbcBC mutants of Escherichia coli.

    PubMed

    Buljubašić, Maja; Zahradka, Davor; Zahradka, Ksenija

    2013-12-01

    The RecQ helicase is required by the RecF recombination pathway that is operative in recBC(D) sbcB sbcC(D) mutants of Escherichia coli. Genetic data suggest that RecQ participates in resection of DNA ends during initiation of recombination. In vitro, RecQ can unwind a variety of DNA substrates, including recombination intermediates such as D-loops and Holliday junctions. However, its potential role in processing of recombination intermediates during the late stage of the RecF pathway has not been genetically tested. Here we studied the effect of a recQ mutation on transductional recombination and DNA repair after γ-irradiation in ΔrecBCD ΔsbcB sbcC strains deficient for RuvABC, RecG and XerC proteins. RuvABC and RecG proteins process recombination intermediates in the late stage of recombination, whereas XerC is required to resolve chromosome dimers formed upon recombination. Our results do not reveal any substantial synergistic effect between the recQ mutation, on one hand, and ruvABC, recG and xerC mutations on the other. In addition, the recQ mutation suppresses chromosome segregation defects in γ-irradiated ruvABC recG and xerC mutants. These results suggest that RecQ acts upstream of RuvABC, RecG and XerC proteins, a finding that is compatible with its primary role in initiation of the RecF recombination pathway.

  19. E. Coli

    MedlinePlus

    ... CDC Cancel Submit Search The CDC E.coli (Escherichia coli) Note: Javascript is disabled or is not supported ... Brand SoyNut Butter Read the Latest E. coli (Escherichia coli) General Information Protect Yourself Learn about E. coli ...

  20. Structural analysis and mutant growth properties reveal distinctive enzymatic and cellular roles for the three major L-alanine transaminases of Escherichia coli.

    PubMed

    Peña-Soler, Esther; Fernandez, Francisco J; López-Estepa, Miguel; Garces, Fernando; Richardson, Andrew J; Quintana, Juan F; Rudd, Kenneth E; Coll, Miquel; Vega, M Cristina

    2014-01-01

    In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.

  1. Efficient biosynthesis of rare natural product scopolamine using E. coli cells expressing a S14P/K97A mutant of hyoscyamine 6β-hydroxylase AaH6H.

    PubMed

    Cao, Yue-De; He, Yu-Cai; Li, Hao; Kai, Guo-Yin; Xu, Jian-He; Yu, Hui-Lei

    2015-10-10

    Hyoscyamine 6β-hydroxylase (H6H, EC 1.14.11.11), an α-ketoglutarate dependent dioxygenase catalyzes the hydroxylation of (-)-hyoscyamine and the subsequent epoxidation of 6β-hydroxyhyoscyamine to form scopolamine, a valuable natural alkaloid. In this study, random mutagenesis and site-directed saturation mutagenesis were used to enhance the hydroxylation activity of H6H from Anisodus acutangulus (AaH6H). A double mutant, AaH6HM1 (S14P/K97A), showed a 3.4-fold improved hydroxylation activity compared with the wild-type enzyme, and the in vivo epoxidation activity was also improved by 2.3 times. After 34h cultivation of Escherichia coli cells harboring Aah6hm1 in a 5-L bioreactor with a working volume of 3L, scopolamine was produced via a single-enzyme-mediated two-step transformation from 500mgL(-1) (-)-hyoscyamine in 97% conversion, and 1.068g of the product were isolated, corresponding to a space-time yield of 251mgL(-1)d(-1). This study shows that the protein engineering of some key enzymes is a promising and effective way for improving the production of rare natural products such as scopolamine.

  2. The presence of the region on pBR322 that encodes resistance to tetracycline is responsible for high levels of plasmid DNA knotting in Escherichia coli DNA topoisomerase I deletion mutant.

    PubMed Central

    Shishido, K; Ishii, S; Komiyama, N

    1989-01-01

    Plasmid pBR322 DNA isolated from Escherichia coli DNA topoisomerase I deletion mutant DM800 is estimated to contain about 10% of the knotted forms (Shishido et al., 1987). These knotted DNA species were shown to have the same primary structure as usual, unknotted pBR322 DNA. Analysis of the knotting level of deletion, insertion and sequence-rearranged derivatives of pBR322 in DM800 showed that the presence of the region on pBR322 encoding resistance to tetracycline (tet) is required for high levels of plasmid knotting. When the entire tet region is present in a native orientation, the level of knotting is highest. Inactivating the tet promoter is manifested by a middle level of knotting. For deletion derivatives lacking various portions of the tet region, the level of knotting ranges from lowest to high depending on the site and length of the tet gene remaining. Inverting the orientation of tet region on the pBR322 genome results in a middle level of knotting. Deleting the ampicillin-resistance (bla)gene outside of its second promoter does not affect the level of knotting, if the entire tet gene remains. A possible mechanism of regulation of plasmid knotting is discussed. Images PMID:2557587

  3. Mutant fatty acid desaturase

    DOEpatents

    Shanklin, John; Cahoon, Edgar B.

    2004-02-03

    The present invention relates to a method for producing mutants of a fatty acid desaturase having a substantially increased activity towards fatty acid substrates with chains containing fewer than 18 carbons relative to an unmutagenized precursor desaturase having an 18 carbon atom chain length substrate specificity. The method involves inducing one or more mutations in the nucleic acid sequence encoding the precursor desaturase, transforming the mutated sequence into an unsaturated fatty acid auxotroph cell such as MH13 E. coli, culturing the cells in the absence of supplemental unsaturated fatty acids, thereby selecting for recipient cells which have received and which express a mutant fatty acid desaturase with an elevated specificity for fatty acid substrates having chain lengths of less than 18 carbon atoms. A variety of mutants having 16 or fewer carbon atom chain length substrate specificities are produced by this method. Mutant desaturases produced by this method can be introduced via expression vectors into prokaryotic and eukaryotic cells and can also be used in the production of transgenic plants which may be used to produce specific fatty acid products.

  4. Protein Synthesis in E. coli: Dependence of Codon-Specific Elongation on tRNA Concentration and Codon Usage.

    PubMed

    Rudorf, Sophia; Lipowsky, Reinhard

    2015-01-01

    To synthesize a protein, a ribosome moves along a messenger RNA (mRNA), reads it codon by codon, and takes up the corresponding ternary complexes which consist of aminoacylated transfer RNAs (aa-tRNAs), elongation factor Tu (EF-Tu), and GTP. During this process of translation elongation, the ribosome proceeds with a codon-specific rate. Here, we present a general theoretical framework to calculate codon-specific elongation rates and error frequencies based on tRNA concentrations and codon usages. Our theory takes three important aspects of in-vivo translation elongation into account. First, non-cognate, near-cognate and cognate ternary complexes compete for the binding sites on the ribosomes. Second, the corresponding binding rates are determined by the concentrations of free ternary complexes, which must be distinguished from the total tRNA concentrations as measured in vivo. Third, for each tRNA species, the difference between total tRNA and ternary complex concentration depends on the codon usages of the corresponding cognate and near-cognate codons. Furthermore, we apply our theory to two alternative pathways for tRNA release from the ribosomal E site and show how the mechanism of tRNA release influences the concentrations of free ternary complexes and thus the codon-specific elongation rates. Using a recently introduced method to determine kinetic rates of in-vivo translation from in-vitro data, we compute elongation rates for all codons in Escherichia coli. We show that for some tRNA species only a few tRNA molecules are part of ternary complexes and, thus, available for the translating ribosomes. In addition, we find that codon-specific elongation rates strongly depend on the overall codon usage in the cell, which could be altered experimentally by overexpression of individual genes.

  5. Thiophene metabolism by E. coli

    SciTech Connect

    Clark, D.P.

    1990-01-01

    The objective of this project is to investigate the mechanism of degradation of sulfur containing heterocyclic molecules by mutants of Escherichia coli K-12. We previously isolated multiple mutants of E. coli which were selected for improved oxidation of furan and thiophene derivatives. We have focused on the thdA mutation in our subsequent research as it appears to be of central importance in thiophene oxidation. We hope that analysis of the thd gene of E. coli will lead to improvement of our thiophene metabolizing bacterial strains.

  6. Mechanism of activation of elongation factor Tu by ribosome: catalytic histidine activates GTP by protonation.

    PubMed

    Aleksandrov, Alexey; Field, Martin

    2013-09-01

    Elongation factor Tu (EF-Tu) is central to prokaryotic protein synthesis as it has the role of delivering amino-acylated tRNAs to the ribosome. Release of EF-Tu, after correct binding of the EF-Tu:aa-tRNA complex to the ribosome, is initiated by GTP hydrolysis. This reaction, whose mechanism is uncertain, is catalyzed by EF-Tu, but requires activation by the ribosome. There have been a number of mechanistic proposals, including those spurred by a recent X-ray crystallographic analysis of a ribosome:EF-Tu:aa-tRNA:GTP-analog complex. In this work, we have investigated these and alternative hypotheses, using high-level quantum chemical/molecular mechanical simulations for the wild-type protein and its His85Gln mutant. For both proteins, we find previously unsuggested mechanisms as being preferred, in which residue 85, either His or Gln, directly assists in the reaction. Analysis shows that the RNA has a minor catalytic effect in the wild-type reaction, but plays a significant role in the mutant by greatly stabilizing the reaction's transition state. Given the similarity between EF-Tu and other members of the translational G-protein family, it is likely that these mechanisms of ribosome-activated GTP hydrolysis are pertinent to all of these proteins.

  7. E. Coli

    MedlinePlus

    ... Emergency Room? What Happens in the Operating Room? E. Coli KidsHealth > For Kids > E. Coli A A A What's in this article? What ... Doctor Do? What Can Kids Do? en español E. coli What Is It? E. coli is a common ...

  8. Conserved discrimination against misacylated tRNAs by two mesophilic elongation factor Tu orthologs.

    PubMed

    Cathopoulis, Terry J T; Chuawong, Pitak; Hendrickson, Tamara L

    2008-07-22

    Elongation factor Tu (EF-Tu) binds and loads elongating aminoacyl-tRNAs (aa-tRNAs) onto the ribosome for protein biosynthesis. Many bacteria biosynthesize Gln-tRNA (Gln) and Asn-tRNA (Asn) by an indirect, two-step pathway that relies on the misacylated tRNAs Glu-tRNA (Gln) and Asp-tRNA (Asn) as intermediates. Previous thermodynamic and experimental analyses have demonstrated that Thermus thermophilus EF-Tu does not bind Asp-tRNA (Asn) and predicted a similar discriminatory response against Glu-tRNA (Gln) [Asahara, H., and Uhlenbeck, O. (2005) Biochemistry 46, 6194-6200; Roy, H., et al. (2007) Nucleic Acids Res. 35, 3420-3430]. By discriminating against these misacylated tRNAS, EF-Tu plays a direct role in preventing misincorporation of aspartate and glutamate into proteins at asparagine and glutamine codons. Here we report the characterization of two different mesophilic EF-Tu orthologs, one from Escherichia coli, a bacterium that does not utilize either Glu-tRNA (Gln) or Asp-tRNA (Asn), and the second from Helicobacter pylori, an organism in which both misacylated tRNAs are essential. Both EF-Tu orthologs discriminate against these misacylated tRNAs, confirming the prediction that Glu-tRNA (Gln), like Asp-tRNA (Asn), will not form a complex with EF-Tu. These results also demonstrate that the capacity of EF-Tu to discriminate against both of these aminoacyl-tRNAs is conserved even in bacteria like E. coli that do not generate either misacylated tRNA.

  9. Growth phase-dependent transcription of the Streptomyces ramocissimus tuf1 gene occurs from two promoters.

    PubMed Central

    Tieleman, L N; van Wezel, G P; Bibb, M J; Kraal, B

    1997-01-01

    The str operon of Streptomyces ramocissimus contains the genes for ribosomal proteins S12 (rpsL) and S7 (rpsG) and for the polypeptide chain elongation factors G (EF-G) (fus) and Tu (EF-Tu) (tuf). This kirromycin producer contains three tuf or tuf-like genes; tuf1 encodes the regular EF-Tu and is located immediately downstream of fus. In vivo and in vitro transcription analysis revealed a transcription start site directly upstream of S. ramocissimus tuf1, in addition to the operon promoter rpsLp. Transcription from these promoters appeared to be growth phase dependent, diminishing drastically upon entry into stationary phase and at the onset of production of the EF-Tu-targeted antibiotic kirromycin. In surface-grown cultures, a second round of tuf1 transcription, coinciding with aerial mycelium formation and kirromycin production, was observed. The tuf1-specific promoter (tuf1p) was located in the intercistronic region between fus and tuf1 by high-resolution S1 mapping, in vitro transcription, and in vivo promoter probing. During logarithmic growth, the tuf1p and rpsLp transcripts are present at comparable levels. In contrast to Escherichia coli, which has two almost identical tuf genes, the gram-positive S. ramocissimus contains only tuf1 for its regular EF-Tu. High levels of EF-Tu may therefore be achieved by the compensatory activity of tuf1p. PMID:9171408

  10. E. Coli

    MedlinePlus

    ... of Your Teeth El cuidado de los dientes Video: Getting an X-ray E. Coli KidsHealth > For Kids > E. Coli Print A A A What's in ... recalls affecting contaminated vegetables or other products. But kids can ... inside. Don't swallow lake, ocean, or pool water. If the water contains ...

  11. The characterization of Mycoplasma synoviae EF-Tu protein and proteins involved in hemadherence and their N-terminal amino acid sequences.

    PubMed

    Bencina, D; Narat, M; Dovc, P; Drobnic-Valic, M; Habe, F; Kleven, S H

    1999-04-01

    An abundant cytoplasmic 43-kDa protein from Mycoplasma synoviae, a major pathogen from poultry, was identified as elongation factor Tu. The N-terminal amino acid sequence (AKLDFDRSKEHVNVGTIGHV) has 90% identity with the sequence of the Mycoplasma hominis elongation factor Tu protein. Monoclonal antibodies reacting with the M. synoviae elongation factor Tu protein also reacted with 43-kDa proteins from the avian Mycoplasma species Mycoplasma gallinarum, Mycoplasma gallinaceum, Mycoplasma pullorum, Mycoplasma cloacale, Mycoplasma iners and Mycoplasma meleagridis, but not with the proteins from Mycoplasma gallisepticum, Mycoplasma imitans or Mycoplasma iowae. In addition, two groups of phase variable integral membrane proteins, pMSA and pMSB, associated with hemadherence and pathogenicity of M. synoviae strains AAY-4 and ULB925 were identified. The cleavage of a larger hemagglutinating protein encoded by a gene homologous to the vlhA gene of M. synoviae generates pMSB1 and pMSA1 proteins defined by mAb 125 and by hemagglutination inhibiting mAb 3E10, respectively. The N-terminal amino acid sequences of pMSA proteins (SENKLI ... and SENETQ ...) probably indicate the cleavage site of the M. synoviae strain ULB 925 hemagglutinin.

  12. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related diseases such as gangrenous dermatitis (GD) and necrotic enteritis (NE) are increasingly emerging as major diseases in recent years with high economic loss around the world. In this report, we characterized two immunogenic Clostridium perfringens (CP) proteins (e.g., elongation f...

  13. Cleavage of the sarcin–ricin loop of 23S rRNA differentially affects EF-G and EF-Tu binding

    PubMed Central

    García-Ortega, Lucía; Álvarez-García, Elisa; Gavilanes, José G.; Martínez-del-Pozo, Álvaro; Joseph, Simpson

    2010-01-01

    Ribotoxins are potent inhibitors of protein biosynthesis and inactivate ribosomes from a variety of organisms. The ribotoxin α-sarcin cleaves the large 23S ribosomal RNA (rRNA) at the universally conserved sarcin–ricin loop (SRL) leading to complete inactivation of the ribosome and cellular death. The SRL interacts with translation factors that hydrolyze GTP, and it is important for their binding to the ribosome, but its precise role is not yet understood. We studied the effect of α-sarcin on defined steps of translation by the bacterial ribosome. α-Sarcin-treated ribosomes showed no defects in mRNA and tRNA binding, peptide-bond formation and sparsomycin-dependent translocation. Cleavage of SRL slightly affected binding of elongation factor Tu ternary complex (EF-Tu•GTP•tRNA) to the ribosome. In contrast, the activity of elongation factor G (EF-G) was strongly impaired in α-sarcin-treated ribosomes. Importantly, cleavage of SRL inhibited EF-G binding, and consequently GTP hydrolysis and mRNA–tRNA translocation. These results suggest that the SRL is more critical in EF-G than ternary complex binding to the ribosome implicating different requirements in this region of the ribosome during protein elongation. PMID:20215430

  14. Isolation and characterization of unusual gin mutants.

    PubMed Central

    Klippel, A; Cloppenborg, K; Kahmann, R

    1988-01-01

    Site-specific inversion of the G segment in phage Mu DNA is promoted by two proteins, the DNA invertase Gin and the host factor FIS. Recombination occurs if the recombination sites (IR) are arranged as inverted repeats and a recombinational enhancer sequence is present in cis. Intermolecular reactions as well as deletions between direct repeats of the IRs rarely occur. Making use of a fis- mutant of Escherichia coli we have devised a scheme to isolate gin mutants that have a FIS independent phenotype. This mutant phenotype is caused by single amino acid changes at five different positions of gin. The mutant proteins display a whole set of new properties in vivo: they promote inversions, deletions and intermolecular recombination in an enhancer- and FIS-independent manner. The mutants differ in recombination activity. The most active mutant protein was analysed in vitro. The loss of site orientation specificity was accompanied with the ability to recombine even linear substrates. We discuss these results in connection with the role of the enhancer and FIS protein in the wild-type situation. Images PMID:2974801

  15. E. Coli Infection

    MedlinePlus

    ... is E. coli?E. coli is short for Escherichia coli -- bacteria (germs) that cause severe cramps and diarrhea. ... staff Tags: bacterial endotoxin, bloody diarrhea, enterohemorrhagic infection, Escherichia coli, food-borne illness, gastroenteritis, hemorrhagic colitis, HUS, thrombotic ...

  16. Isolation and characterization of transcription fidelity mutants.

    PubMed

    Strathern, Jeffrey N; Jin, Ding Jun; Court, Donald L; Kashlev, Mikhail

    2012-07-01

    Accurate transcription is an essential step in maintaining genetic information. Error-prone transcription has been proposed to contribute to cancer, aging, adaptive mutagenesis, and mutagenic evolution of retroviruses and retrotransposons. The mechanisms controlling transcription fidelity and the biological consequences of transcription errors are poorly understood. Because of the transient nature of mRNAs and the lack of reliable experimental systems, the identification and characterization of defects that increase transcription errors have been particularly challenging. In this review we describe novel genetic screens for the isolation of fidelity mutants in both Saccharomyces cerevisiae and Escherichia coli RNA polymerases. We obtained and characterized two distinct classes of mutants altering NTP misincorporation and transcription slippage both in vivo and in vitro. Our study not only validates the genetic schemes for the isolation of RNA polymerase mutants that alter fidelity, but also sheds light on the mechanism of transcription accuracy. This article is part of a Special Issue entitled: Chromatin in time and space.

  17. CO-Releasing Molecules Have Nonheme Targets in Bacteria: Transcriptomic, Mathematical Modeling and Biochemical Analyses of CORM-3 [Ru(CO)3Cl(glycinate)] Actions on a Heme-Deficient Mutant of Escherichia coli

    PubMed Central

    Wilson, Jayne Louise; Wareham, Lauren K.; McLean, Samantha; Begg, Ronald; Greaves, Sarah; Mann, Brian E.; Sanguinetti, Guido

    2015-01-01

    Abstract Aims: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy. Hemes are generally considered the prime targets of CO and CORMs, so we tested this hypothesis using heme-deficient bacteria, applying cellular, transcriptomic, and biochemical tools. Results: CORM-3 [Ru(CO)3Cl(glycinate)] readily penetrated Escherichia coli hemA bacteria and was inhibitory to these and Lactococcus lactis, even though they lack all detectable hemes. Transcriptomic analyses, coupled with mathematical modeling of transcription factor activities, revealed that the response to CORM-3 in hemA bacteria is multifaceted but characterized by markedly elevated expression of iron acquisition and utilization mechanisms, global stress responses, and zinc management processes. Cell membranes are disturbed by CORM-3. Innovation: This work has demonstrated for the first time that CORM-3 (and to a lesser extent its inactivated counterpart) has multiple cellular targets other than hemes. A full understanding of the actions of CORMs is vital to understand their toxic effects. Conclusion: This work has furthered our understanding of the key targets of CORM-3 in bacteria and raises the possibility that the widely reported antimicrobial effects cannot be attributed to classical biochemical targets of CO. This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy. Antioxid. Redox Signal. 23, 148–162. PMID:25811604

  18. Intimin facilitates colonization by Escherichia coli O157:H7 in adult ruminants.

    PubMed

    Cornick, Nancy A; Booher, Sheridan L; Moon, Harley W

    2002-05-01

    We compared the magnitude and duration of fecal shedding of wild-type Escherichia coli O157:H7 to that of an isogenic intimin mutant in young adult cattle and sheep. In both ruminant species, wild-type E. coli O157:H7 was shed in greater numbers and for a longer duration than was the intimin mutant.

  19. Multicopy suppressors of prc mutant Escherichia coli include two HtrA (DegP) protease homologs (HhoAB), DksA, and a truncated R1pA.

    PubMed Central

    Bass, S; Gu, Q; Christen, A

    1996-01-01

    We have isolated three multicopy suppressors of the conditional lethal phenotype of a prc (tsp) null strain of Escherichia coli. One of these suppressors included two novel putative protease genes in tandem that map to 3400 kb or 72.5 centisomes on the chromosome. We propose the names hhoA and hhoB, for htrA homolog, to denote that these genes encode proteins that are 58 and 35% identical, respectively, to the HtrA (DegP) serine protease and 36% identical to each other. The HhoA and HhoB proteins are predicted to be 455 and 355 amino acids, respectively, in length. The mature HhoA protein is periplasmic in location, and amino-terminal sequencing shows that it arises following cleavage of a 27-amino-acid signal peptide. Searches of the protein and DNA databases reveal a rapidly growing family of homologous genes in a variety of other bacteria, including several which are required for virulence in their host. Deletion of the hhoAB genes shows that they are not required for viability at high temperatures like the homologous htrA but grow more slowly than wild-type strains. A second multicopy prc suppressor is the dksA (dnaK suppressor) gene, which is also a multicopy suppressor of defects in the heat shock genes dnaK, dnaJ, and grpE. The dksA gene was independently isolated as a multicopy suppressor of a mukB mutation, which is required for chromosomal partitioning. A third dosage-dependent prc suppressor includes a truncated rare lipoprotein A (rlpA) gene. PMID:8576052

  20. Dialogue between E. coli free radical pathways and the mitochondria of C. elegans.

    PubMed

    Govindan, J Amaranath; Jayamani, Elamparithi; Zhang, Xinrui; Mylonakis, Eleftherios; Ruvkun, Gary

    2015-10-06

    The microbial world presents a complex palette of opportunities and dangers to animals, which have developed surveillance and response strategies to hints of microbial intent. We show here that the mitochondrial homeostatic response pathway of the nematode Caenorhabditis elegans responds to Escherichia coli mutations that activate free radical detoxification pathways. Activation of C. elegans mitochondrial responses could be suppressed by additional mutations in E. coli, suggesting that C. elegans responds to products of E. coli to anticipate challenges to its mitochondrion. Out of 50 C. elegans gene inactivations known to mediate mitochondrial defense, we found that 7 genes were required for C. elegans response to a free radical producing E. coli mutant, including the bZip transcription factor atfs-1 (activating transcription factor associated with stress). An atfs-1 loss-of-function mutant was partially resistant to the effects of free radical-producing E. coli mutant, but a constitutively active atfs-1 mutant growing on wild-type E. coli inappropriately activated the pattern of mitochondrial responses normally induced by an E. coli free radical pathway mutant. Carbonylated proteins from free radical-producing E. coli mutant may directly activate the ATFS-1/bZIP transcription factor to induce mitochondrial stress response: feeding C. elegans with H2O2-treated E. coli induces the mitochondrial unfolded protein response, and inhibition of a gut peptide transporter partially suppressed C. elegans response to free radical damaged E. coli.

  1. Dialogue between E. coli free radical pathways and the mitochondria of C. elegans

    PubMed Central

    Govindan, J. Amaranath; Jayamani, Elamparithi; Zhang, Xinrui; Mylonakis, Eleftherios; Ruvkun, Gary

    2015-01-01

    The microbial world presents a complex palette of opportunities and dangers to animals, which have developed surveillance and response strategies to hints of microbial intent. We show here that the mitochondrial homeostatic response pathway of the nematode Caenorhabditis elegans responds to Escherichia coli mutations that activate free radical detoxification pathways. Activation of C. elegans mitochondrial responses could be suppressed by additional mutations in E. coli, suggesting that C. elegans responds to products of E. coli to anticipate challenges to its mitochondrion. Out of 50 C. elegans gene inactivations known to mediate mitochondrial defense, we found that 7 genes were required for C. elegans response to a free radical producing E. coli mutant, including the bZip transcription factor atfs-1 (activating transcription factor associated with stress). An atfs-1 loss-of-function mutant was partially resistant to the effects of free radical-producing E. coli mutant, but a constitutively active atfs-1 mutant growing on wild-type E. coli inappropriately activated the pattern of mitochondrial responses normally induced by an E. coli free radical pathway mutant. Carbonylated proteins from free radical-producing E. coli mutant may directly activate the ATFS-1/bZIP transcription factor to induce mitochondrial stress response: feeding C. elegans with H2O2-treated E. coli induces the mitochondrial unfolded protein response, and inhibition of a gut peptide transporter partially suppressed C. elegans response to free radical damaged E. coli. PMID:26392561

  2. Repair-defective mutants of Alteromonas espejiana, the host for bacteriophage PM2

    SciTech Connect

    Zerler, B.R.; Wallace, S.S.

    1984-02-01

    The in vivo repair processes of Alteromonas espejiana, the host for bacteriophage PM2, were characterized, and UV- and methyl methanesulfonate (MMS)-sensitive mutants were isolated. Wild-type A. espejiana cells were capable of photoreactivation, excision, recombination, and inducible repair. There was no detecttable pyrimidine dimer-DNA N-glycosylase activity, and pyrimidine dimer removal appeared to occur by a pathway analogous to the Escherichia coli Uvr pathway. The UV- and MMS-sensitive mutants of A. espejiana included three groups, each containing at least one mutation involved with excision, recombination, or inducible repair. One group that was UV sensitive but not sensitive to MMS or X rays showed a decreased ability to excise pyrimidine dimers. Mutants in this group were also sensitive to psoralen plus near-UV light and were phenotypically analogous to the E. coli uvr mutants. A second group was UV and MMS sensitive but not sensitive to X rays and appeared to contain mutations in a gene(s) involved in recombination repair. These recombination-deficient mutants differed from the E. coli rec mutants, which are MMS and X-ray sensitive. The third group of A. espejiana mutants was sensitive to UV, MMS, and X rays. These mutants were recombination deficient, lacked inducible repair, and were phenotypically similar to E. coli recA mutants.

  3. Saccharomyces cerevisiae aldolase mutants.

    PubMed Central

    Lobo, Z

    1984-01-01

    Six mutants lacking the glycolytic enzyme fructose 1,6-bisphosphate aldolase have been isolated in the yeast Saccharomyces cerevisiae by inositol starvation. The mutants grown on gluconeogenic substrates, such as glycerol or alcohol, and show growth inhibition by glucose and related sugars. The mutations are recessive, segregate as one gene in crosses, and fall in a single complementation group. All of the mutants synthesize an antigen cross-reacting to the antibody raised against yeast aldolase. The aldolase activity in various mutant alleles measured as fructose 1,6-bisphosphate cleavage is between 1 to 2% and as condensation of triose phosphates to fructose 1,6-bisphosphate is 2 to 5% that of the wild-type. The mutants accumulate fructose 1,6-bisphosphate from glucose during glycolysis and dihydroxyacetone phosphate during gluconeogenesis. This suggests that the aldolase activity is absent in vivo. PMID:6384192

  4. E. Coli and Pregnancy

    MedlinePlus

    ... care provider. What is E. coli? E. coli (Escherichia coli) is a bacterium that lives in your colon ( ... 10):1411-1413. Jones B, et al. 2004. Escherichia coli: a growing problem in early onset neonatal sepsis. ...

  5. Escherichia Coli

    ERIC Educational Resources Information Center

    Goodsell, David S.

    2009-01-01

    Diverse biological data may be used to create illustrations of molecules in their cellular context. I describe the scientific results that support a recent textbook illustration of an "Escherichia coli cell". The image magnifies a portion of the bacterium at one million times, showing the location and form of individual macromolecules. Results…

  6. Comprehensive transposon mutant library of Pseudomonas aeruginosa

    PubMed Central

    Jacobs, Michael A.; Alwood, Ashley; Thaipisuttikul, Iyarit; Spencer, David; Haugen, Eric; Ernst, Stephen; Will, Oliver; Kaul, Rajinder; Raymond, Christopher; Levy, Ruth; Chun-Rong, Liu; Guenthner, Donald; Bovee, Donald; Olson, Maynard V.; Manoil, Colin

    2003-01-01

    We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering. PMID:14617778

  7. Comprehensive transposon mutant library of Pseudomonas aeruginosa.

    PubMed

    Jacobs, Michael A; Alwood, Ashley; Thaipisuttikul, Iyarit; Spencer, David; Haugen, Eric; Ernst, Stephen; Will, Oliver; Kaul, Rajinder; Raymond, Christopher; Levy, Ruth; Chun-Rong, Liu; Guenthner, Donald; Bovee, Donald; Olson, Maynard V; Manoil, Colin

    2003-11-25

    We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300-400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.

  8. Thymidine kinase mutants obtained by random sequence selection.

    PubMed

    Munir, K M; French, D C; Loeb, L A

    1993-05-01

    Knowledge of the catalytic properties and structural information regarding the amino acid residues that comprise the active site of an enzyme allows one, in principle, to use site-specific mutagenesis to construct genes that encode enzymes with altered functions. However, such information about most enzymes is not known and the effects of specific amino acid substitutions are not generally predictable. An alternative approach is to substitute random nucleotides for key codons in a gene and to use genetic selection to identify new and interesting enzyme variants. We describe here the construction, selection, and characterization of herpes simplex virus type 1 thymidine kinase mutants either with different catalytic properties or with enhanced thermostability. From a library containing 2 x 10(6) plasmid-encoded herpes thymidine kinase genes, each with a different nucleotide sequence at the putative nucleoside binding site, we obtained 1540 active mutants. Using this library and one previously constructed, we identified by secondary selection Escherichia coli harboring thymidine kinase mutant clones that were unable to grow in the presence of concentrations of 3'-azido-3'-deoxythymidine (AZT) that permits colony formation by E. coli harboring the wild-type plasmid. Two of the mutant enzymes exhibited a reduced Km for AZT, one of which displayed a higher catalytic efficiency for AZT over thymidine relative to that of the wild type. We also identified one mutant with enhanced thermostability. These mutants may have clinical potential as the promise of gene therapy is increasingly becoming a reality.

  9. Flagella from F18+Escherichia coli play a role in adhesion to pig epithelial cell lines.

    PubMed

    Duan, Qiangde; Zhou, Mingxu; Zhu, Xiaofang; Yang, Yang; Zhu, Jun; Bao, Wenbin; Wu, Shenglong; Ruan, Xiaosai; Zhang, Weiping; Zhu, Guoqiang

    2013-02-01

    F18 fimbriae and toxins produced by F18 fimbriae-carrying Escherichia coli (E. coli) strains are known virulence factors responsible for post-weaning diarrhea (PWD) and edema disease (ED). In this study, we showed that fliC isogenic mutants constructed in two reference wild-type F18 fimbriae (F18+) E. coli were markedly impaired in adherence in vitro cell models (p < 0.05). Flagella purified from F18+E. coli could directly bind to cultured piglet epithelial cells and block adherence of F18+E. coli to cells when pre-incubated. In addition, the F18+E. coli fliC deletion mutants up-regulated the expression of type I fimbriae produced by F18+E. coli strains. These results demonstrated that expression of flagella is essential for the adherence of F18+E. coli in vitro.

  10. PDGFRA-mutant syndrome.

    PubMed

    Ricci, Riccardo; Martini, Maurizio; Cenci, Tonia; Carbone, Arnaldo; Lanza, Paola; Biondi, Alberto; Rindi, Guido; Cassano, Alessandra; Larghi, Alberto; Persiani, Roberto; Larocca, Luigi M

    2015-07-01

    Germline PDGFRA mutations cause multiple heterogeneous gastrointestinal mesenchymal tumors. In its familial form this disease, which was formerly termed intestinal neurofibromatosis/neurofibromatosis 3b (INF/NF3b), has been included among familial gastrointestinal stromal tumors (GISTs) because of its genotype, described when GIST was the only known PDGFRA-mutant gastrointestinal tumor. Shortly afterwards, however, inflammatory fibroid polyps also revealed PDGFRA mutations. Subsequently, gastrointestinal CD34+ 'fibrous tumors' of uncertain classification were described in a germline PDGFRA-mutant context. Our aim was to characterize the syndrome produced by germline PDGFRA mutations and establish diagnostic criteria and management strategies for this hitherto puzzling disease. We studied a kindred displaying multiple gastrointestinal mesenchymal tumors, comparing it with published families/individuals with possible analogous conditions. We identified a novel inherited PDGFRA mutation (P653L), constituting the third reported example of familial PDGFRA mutation. In adult mutants we detected inflammatory fibroid polyps, gastric GISTs and gastrointestinal fibrous tumors of uncertain nosology. We demonstrate that the syndrome formerly defined as INF/NF3b (exemplified by the family reported herein) is simplistically considered a form of familial GIST, because inflammatory fibroid polyps often prevail. Fibrous tumors appear variants of inflammatory fibroid polyps. 'INF/NF3b' and 'familial GIST' are misleading terms which we propose changing to 'PDGFRA-mutant syndrome'. In this condition, unlike KIT-dependent familial GIST syndromes, if present, GISTs are stomach-restricted and diffuse Cajal cell hyperplasia is not observed. This restriction of GISTs to the stomach in PDGFRA-mutant syndrome: (i) focuses oncological concern on gastric masses, as inflammatory fibroid polyps are benign; (ii) supports a selective role of gastric environment for PDGFRA mutations to elicit GISTs

  11. AraC regulatory protein mutants with altered effector specificity.

    PubMed

    Tang, Shuang-Yan; Fazelinia, Hossein; Cirino, Patrick C

    2008-04-16

    The AraC regulatory protein of the Escherichia coli ara operon has been engineered to activate transcription in response to D-arabinose and not in response to its native effector L-arabinose. Two different AraC mutant libraries, each with four randomized binding pocket residues, were subjected to FACS-mediated dual screening using a GFP reporter. Both libraries yielded mutants with the desired switch in effector specificity, and one mutant we describe maintains tight repression in the absence of effector. The presence of 100 mM L-arabinose does not influence the response of the reported mutants to D-arabinose, and the mutants are not induced by other sugars tested (D-xylose, D-fucose, D-lyxose). Co-expression of the FucP transporter in E. coli enabled induction by D-arabinose in the 0.1 mM range. Our results demonstrate the power of dual screening for altering AraC inducer specificity and represent steps toward the design of customized in vivo molecular reporters and gene switches for metabolic engineering.

  12. Proton suicide: general method for direct selection of sugar transport- and fermentation-defective mutants

    SciTech Connect

    Winkelman, J.W.; Clark, D.P.

    1984-11-01

    A positive selection procedure was devised for bacterial mutants incapable of producing acid from sugars by fermentation. The method relied on the production of elemental bromine from a mixture of bromide and bromate under acidic conditions. When wild-type Escherichia coli cells were plated on media containing a fermentable sugar and an equimolar mixture of bromide and bromate, most of the cells were killed but a variety of mutants unable to produce acid from the sugar survived. Among these mutants were those defective in (i) sugar uptake, (ii) the glycolytic pathway, and (iii) the excretion. There were also novel mutants with some presumed regulatory defects affecting fermentation.

  13. Engineering the elongation factor Tu for efficient selenoprotein synthesis.

    PubMed

    Haruna, Ken-ichi; Alkazemi, Muhammad H; Liu, Yuchen; Söll, Dieter; Englert, Markus

    2014-09-01

    Selenocysteine (Sec) is naturally co-translationally incorporated into proteins by recoding the UGA opal codon with a specialized elongation factor (SelB in bacteria) and an RNA structural signal (SECIS element). We have recently developed a SECIS-free selenoprotein synthesis system that site-specifically--using the UAG amber codon--inserts Sec depending on the elongation factor Tu (EF-Tu). Here, we describe the engineering of EF-Tu for improved selenoprotein synthesis. A Sec-specific selection system was established by expression of human protein O(6)-alkylguanine-DNA alkyltransferase (hAGT), in which the active site cysteine codon has been replaced by the UAG amber codon. The formed hAGT selenoprotein repairs the DNA damage caused by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine, and thereby enables Escherichia coli to grow in the presence of this mutagen. An EF-Tu library was created in which codons specifying the amino acid binding pocket were randomized. Selection was carried out for enhanced Sec incorporation into hAGT; the resulting EF-Tu variants contained highly conserved amino acid changes within members of the library. The improved UTu-system with EF-Sel1 raises the efficiency of UAG-specific Sec incorporation to >90%, and also doubles the yield of selenoprotein production.

  14. Secretion of Alpha-Hemolysin by Escherichia coli Disrupts Tight Junctions in Ulcerative Colitis Patients

    PubMed Central

    Mirsepasi-Lauridsen, Hengameh Chloé; Du, Zhengyu; Struve, Carsten; Charbon, Godefroid; Karczewski, Jurgen; Krogfelt, Karen Angeliki; Petersen, Andreas Munk; Wells, Jerry M

    2016-01-01

    Objectives: The potential of Escherichia coli (E. coli) isolated from inflammatory bowel disease (IBD) patients to damage the integrity of the intestinal epithelium was investigated. Methods: E. coli strains isolated from patients with ulcerative colitis (UC) and healthy controls were tested for virulence capacity by molecular techniques and cytotoxic assays and transepithelial electric resistance (TER). E. coli isolate p19A was selected, and deletion mutants were created for alpha-hemolysin (α-hemolysin) (hly) clusters and cytotoxic necrotizing factor type 1 (cnf1). Probiotic E. coli Nissle and pathogenic E. coli LF82 were used as controls. Results: E. coli strains from patients with active UC completely disrupted epithelial cell tight junctions shortly after inoculation. These strains belong to phylogenetic group B2 and are all α-hemolysin positive. In contrast, probiotic E. coli Nissle, pathogenic E. coli LF82, four E. coli from patients with inactive UC and three E. coli strains from healthy controls did not disrupt tight junctions. E. coli p19A WT as well as cnf1, and single loci of hly mutants from cluster I and II were all able to damage Caco-2 (Heterogeneous human epithelial colorectal adenocarcinoma) cell tight junctions. However, this phenotype was lost in a mutant with knockout (Δ) of both hly loci (P<0.001). Conclusions: UC-associated E. coli producing α-hemolysin can cause rapid loss of tight junction integrity in differentiated Caco-2 cell monolayers. This effect was abolished in a mutant unable to express α-hemolysin. These results suggest that high Hly expression may be a mechanism by which specific strains of E. coli pathobionts can contribute to epithelial barrier dysfunction and pathophysiology of disease in IBD. PMID:26938480

  15. [Escherichia coli R live vaccine Suicolplex "Dessau"].

    PubMed

    Michael-Meese, M; Klie, H; Schöll, W

    1980-01-01

    Immunisation of pregnant sows prior to parturition has long proved to be a good method to forestall coli dysentery in piglets before weaning. Inactivated vaccines of the pathogenetically important E. coli serogroups with and without adjuvant so far were primarily used at international level. A vaccine of that kind has become available in the GDR more than eight years ago. Its name is Coliporc "Dessau". A live vaccine has been developed from two R-mutants at the authors' institute. The effectiveness of that live vaccine on laboratory animals and in field experiments is reported in this paper together with possibilities of differential diagnosis to distinguish wild strains from the mutants. The live vaccine was commercially registered under the name of Suicolpex "Dessau", in spring 1976.

  16. Low Ubiquinone Content in Escherichia coli Causes Thiol Hypersensitivity

    PubMed Central

    Zeng, H.; Snavely, I.; Zamorano, P.; Javor, G. T.

    1998-01-01

    Thiol hypersensitivity in a mutant of Escherichia coli (IS16) was reversed by complementation with a plasmid that carried the ubiX gene. The mutant had low ubiquinone content. Complementation elevated the ubiquinone level and eliminated thiol hypersensitivity. Analysis of chromosomal ubiX genes indicated that both parent and mutant strains were ubiX mutants. The low ubiquinone content of IS16 was possibly caused by a ubiD ubiX genotype. A ubiA mutant also exhibited thiol hypersensitivity. Neither IS16 nor the ubiA mutant strain could produce alkaline phosphatase (in contrast to their parent strains) after 2 h of induction, thus showing Dsb− phenotypes. The phenomena of thiol hypersensitivity and low ubiquinone content may be linked by their connections to the periplasmic disulfide bond redox machinery. PMID:9658014

  17. Escherichia coli exports cyclic AMP via TolC.

    PubMed

    Hantke, Klaus; Winkler, Karin; Schultz, Joachim E

    2011-03-01

    In Escherichia coli more than 180 genes are regulated by the cyclic AMP (cAMP)-cAMP receptor protein (CRP) complex. However, more than 90% of cAMP that is made by intracellular adenylyl cyclases is found in the culture medium. How is cAMP exported from E. coli? In a tolC mutant, 0.03 mM IPTG (isopropyl-β-d-thiogalactopyranoside) was sufficient to induce β-galactosidase compared to 0.1 mM IPTG in the parent strain. In a cya mutant unable to produce cAMP about 1 mM extracellular cAMP was required to induce β-galactosidase, whereas in a cya tolC mutant 0.1 mM cAMP was sufficient. When cAMP in E. coli cya was generated intracellularly by a recombinant, weakly active adenylyl cyclase from Corynebacterium glutamicum, the critical level of cAMP necessary for induction of maltose degradation was only achieved in a tolC mutant and not in the parent strain. Deletion of a putative cAMP phosphodiesterase of E. coli, CpdA, resulted in a slightly similar, yet more diffuse phenotype. The data demonstrate that export of cAMP via TolC is a most efficient way of E. coli to lower high concentrations of cAMP in the cell and maintain its sensitivity in changing metabolic environments.

  18. Increasing the Oxidative Stress Response Allows Escherichia coli To Overcome Inhibitory Effects of Condensed Tannins

    PubMed Central

    Smith, Alexandra H.; Imlay, James A.; Mackie, Roderick I.

    2003-01-01

    Tannins are plant-derived polyphenols with antimicrobial effects. The mechanism of tannin toxicity towards Escherichia coli was determined by using an extract from Acacia mearnsii (Black wattle) as a source of condensed tannins (proanthocyanidins). E. coli growth was inhibited by tannins only when tannins were exposed to oxygen. Tannins auto-oxidize, and substantial hydrogen peroxide was generated when they were added to aerobic media. The addition of exogenous catalase permitted growth in tannin medium. E. coli mutants that lacked HPI, the major catalase, were especially sensitive to tannins, while oxyR mutants that constitutively overexpress antioxidant enzymes were resistant. A tannin-resistant mutant was isolated in which a promoter-region point mutation increased the level of HPI by 10-fold. Our results indicate that wattle condensed tannins are toxic to E. coli in aerobic medium primarily because they generate H2O2. The oxidative stress response helps E. coli strains to overcome their inhibitory effect. PMID:12788743

  19. Fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni.

    PubMed

    Zeitouni, Salman; Collin, Olivier; Andraud, Mathieu; Ermel, Gwennola; Kempf, Isabelle

    2012-04-01

    The aim of this study was to investigate the fitness of macrolide resistant Campylobacter coli and Campylobacter jejuni. The in vitro growth, the survival on food matrix, and the in vivo colonization of C. jejuni and C. coli susceptible isolates and their isogenic resistant mutants were studied. In vitro experiments demonstrated that macrolide resistance imposed a fitness cost when the susceptible strains and their isogenic resistant mutants were cultured in competition. When inoculated in food matrix, the resistant C. jejuni mutant was no longer detectable after 3 to 5 days but the susceptible strain remained detectable for over 18 days. No difference in survival in food matrix was observed between susceptible and resistant C. coli. When inoculated in vivo in chickens, the macrolide susceptible and resistant C. coli displayed similar levels of colonization, both in separated inoculations and during competitive assays. Strikingly, when mono-inoculated or co-inoculated into chickens, macrolide susceptible C. jejuni outcompeted the macrolide resistant population. However, a spontaneous mutant that evolved in vivo showed a colonization capacity similar to the susceptible strain. Our findings demonstrate the effect of macrolide resistance on the fitness of Campylobacter but suggest that evolved mutants may be as fit as susceptible strains.

  20. Identification of ribosomal protein S7 as a repressor of translation within the str operon of E. coli.

    PubMed

    Dean, D; Yates, J L; Nomura, M

    1981-05-01

    A DNA-directed in vitro protein-synthesizing system was used to demonstrate that r protein S7 has the capacity to inhibit the translation of mRNA for the second and third gene products of the str operon (S7 and EF-G) but not for the first gene product (S12). Translation of mRNA of the last gene product in the operon (EF-Tu) is also probably not inhibited by S7. In addition, we localized the target site for S7 repressor action on the polycistronic str mRNA by examining the repressor activity of S7 in vitro using various template DNAs that contain the gene. The target site was found not to include a promoter-proximal portion of the mRNA for S12. To test for regulatory properties of S7 in vivo, we inserted the S7 gene into a plasmid vector containing the ara regulatory elements such that S7 synthesis was placed under ara control. A specific increase in S7 synthesis caused by stimulation in transcription originating from the arabinose promoter decreased the synthetic rate for EF-G but had no effect on S12 or EF-Tu synthesis.

  1. RECOMBINATIONS OF MUTANT PHAGES OF BACILLUS MEGATHERIUM 899A

    PubMed Central

    Murphy, James S.

    1953-01-01

    A group of mutant phages stemming from the virus of B. megatherium 899a (lysogenic), growing on a sensitive B. megatherium strain (KM), have been studied with respect to their recombination reactions. All these mutants and many of their recombinations can be recognized by a characteristic plaque morphology. A similar group of phages have been isolated directly from a culture of B. megatherium 899a in this laboratory. Previous work has shown that when two different plaque mutant phages both infect essentially all the bacteria in a culture, a characteristic per cent of recombinants is produced. This percentage depends on the two recombinants used, each pair having its own value. Hershey and coworkers (2–5) have demonstrated with coli-phage T2, that the percentages of recombination found can be handled mathematically and that they demonstrate the existence of a relationship between the mutations entirely comparable to crossover percentages as used in gene locus maps in genetics. This has been found to hold true for the phages studied in the present work. Only one "linkage group" has been detected and all the mutants studied showed low percentages of recombination (0.8 to 7.6). B. megatherium 899a phage and some of its mutants have been examined with an electron microscope and no differences have been detected between the different mutant strains. PMID:13109115

  2. The zebrafish early arrest mutants.

    PubMed

    Kane, D A; Maischein, H M; Brand, M; van Eeden, F J; Furutani-Seiki, M; Granato, M; Haffter, P; Hammerschmidt, M; Heisenberg, C P; Jiang, Y J; Kelsh, R N; Mullins, M C; Odenthal, J; Warga, R M; Nüsslein-Volhard, C

    1996-12-01

    This report describes mutants of the zebrafish having phenotypes causing a general arrest in early morphogenesis. These mutants identify a group of loci making up about 20% of the loci identified by mutants with visible morphological phenotypes within the first day of development. There are 12 Class I mutants, which fall into 5 complementation groups and have cells that lyse before morphological defects are observed. Mutants at three loci, speed bump, ogre and zombie, display abnormal nuclei. The 8 Class II mutants, which fall into 6 complementation groups, arrest development before cell lysis is observed. These mutants seemingly stop development in the late segmentation stages, and maintain a body shape similar to a 20 hour embryo. Mutations in speed bump, ogre, zombie, specter, poltergeist and troll were tested for cell lethality by transplanting mutant cells into wild-type hosts. With poltergeist, transplanted mutant cells all survive. The remainder of the mutants tested were autonomously but conditionally lethal: mutant cells, most of which lyse, sometimes survive to become notochord, muscles, or, in rare cases, large neurons, all cell types which become postmitotic in the gastrula. Some of the genes of the early arrest group may be necessary for progression though the cell cycle; if so, the survival of early differentiating cells may be based on having their terminal mitosis before the zygotic requirement for these genes.

  3. Diarrheagenic Escherichia coli

    PubMed Central

    Nataro, James P.; Kaper, James B.

    1998-01-01

    Escherichia coli is the predominant nonpathogenic facultative flora of the human intestine. Some E. coli strains, however, have developed the ability to cause disease of the gastrointestinal, urinary, or central nervous system in even the most robust human hosts. Diarrheagenic strains of E. coli can be divided into at least six different categories with corresponding distinct pathogenic schemes. Taken together, these organisms probably represent the most common cause of pediatric diarrhea worldwide. Several distinct clinical syndromes accompany infection with diarrheagenic E. coli categories, including traveler’s diarrhea (enterotoxigenic E. coli), hemorrhagic colitis and hemolytic-uremic syndrome (enterohemorrhagic E. coli), persistent diarrhea (enteroaggregative E. coli), and watery diarrhea of infants (enteropathogenic E. coli). This review discusses the current level of understanding of the pathogenesis of the diarrheagenic E. coli strains and describes how their pathogenic schemes underlie the clinical manifestations, diagnostic approach, and epidemiologic investigation of these important pathogens. PMID:9457432

  4. Mechanism of Escherichia coli Resistance to Pyrrhocoricin

    PubMed Central

    Narayanan, Shalini; Modak, Joyanta K.; Ryan, Catherine S.; Garcia-Bustos, Jose; Davies, John K.

    2014-01-01

    Due to their lack of toxicity to mammalian cells and good serum stability, proline-rich antimicrobial peptides (PR-AMPs) have been proposed as promising candidates for the treatment of infections caused by antimicrobial-resistant bacterial pathogens. It has been hypothesized that these peptides act on multiple targets within bacterial cells, and therefore the likelihood of the emergence of resistance was considered to be low. Here, we show that spontaneous Escherichia coli mutants resistant to pyrrhocoricin arise at a frequency of approximately 6 × 10−7. Multiple independently derived mutants all contained a deletion in a nonessential gene that encodes the putative peptide uptake permease SbmA. Sensitivity could be restored to the mutants by complementation with an intact copy of the sbmA gene. These findings question the viability of the development of insect PR-AMPs as antimicrobials. PMID:24590485

  5. Fimbriae and lipopolysaccharides are necessary for co-aggregation between Lactobacilli and Escherichia coli.

    PubMed

    Mizuno, Kouhei; Furukawa, Soichi; Usui, Yumi; Ishiba, Madoka; Ogihara, Hirokazu; Morinaga, Yasushi

    2014-01-01

    Cells of Lactobacilli co-aggregated with Escherichia coli K-12 cells to form co-aggregates under mixed-culture conditions at 37 °C for 24 h. Co-aggregation was inhibited by sodium dodecyl sulfate but not by protease. E. coli deletion mutants of fimbriae formation and lipopolysaccharide (LPS) formation did not co-aggregate with Lactobacilli. These results showed that fimbriae and LPS are necessary for co-aggregation between Lactobacilli and E. coli.

  6. Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity.

    PubMed

    Lassila, Jonathan Kyle; Keeffe, Jennifer R; Oelschlaeger, Peter; Mayo, Stephen L

    2005-04-01

    Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency.

  7. ECB deacylase mutants

    DOEpatents

    Arnold, Frances H.; Shao, Zhixin; Zhao, Huimin; Giver, Lorraine J.

    2002-01-01

    A method for in vitro mutagenesis and recombination of polynucleotide sequences based on polymerase-catalyzed extension of primer oligonucleotides is disclosed. The method involves priming template polynucleotide(s) with random-sequences or defined-sequence primers to generate a pool of short DNA fragments with a low level of point mutations. The DNA fragments are subjected to denaturization followed by annealing and further enzyme-catalyzed DNA polymerization. This procedure is repeated a sufficient number of times to produce full-length genes which comprise mutants of the original template polynucleotides. These genes can be further amplified by the polymerase chain reaction and cloned into a vector for expression of the encoded proteins.

  8. E. Coli Infections

    MedlinePlus

    ... adults with weak immune systems. You can get E. coli infections by eating foods containing the bacteria. Symptoms of ... pool contaminated with human waste. Most cases of E. coli infection get better without treatment in 5 to 10 ...

  9. The 503nm pigment of Escherichia coli

    PubMed Central

    Kamitakahara, Joyce R.; Polglase, W. J.

    1970-01-01

    The yield of cell protein was one-third less for streptomycin-dependent Escherichia coli B than for the wild-type parent strain when both were grown aerobically on a medium with limiting glucose, but anaerobically the yield of protein was similar for both strains. The transient pigment absorbing at 503nm that is known to be present in E. coli and other organisms was not detectable in streptomycin-dependent mutants nor in a non-dependent (energy-deficient) revertant. When wild-type E. coli B was grown on limiting glucose–salts medium containing 2,4 dinitrophenol, the yield of cell protein was decreased and formation of the 503nm pigment was inhibited. Fumarase, aconitase and glucose 6-phosphate dehydrogenase were de-repressed in E. coli B cells grown with excess of glucose in a medium containing 2,4-dinitrophenol. In air-oxidized, wild-type E. coli B cells, the 503nm pigment appeared before reduced cytochromes when gluconate was the substrate but failed to appear when succinate was the substrate. The results provide evidence for a role of the 503nm pigment in aerobic energy metabolism, possibly as an electron acceptor from NADPH. PMID:4395501

  10. Highly ordered crystals of channel-forming membrane proteins, of nucleoside-monophosphate kinases, of FAD-containing oxidoreductases and of sugar-processing enzymes and their mutants

    NASA Astrophysics Data System (ADS)

    Schulz, G. E.; Dreyer, M.; Klein, C.; Kreusch, A.; Mittl, P.; Mu¨ller, C. W.; Mu¨ller-Dieckmann, J.; Muller, Y. A.; Proba, K.; Schlauderer, G.; Spu¨rgin, P.; Stehle, T.; Weiss, M. S.

    1992-08-01

    Preparation and crystallization procedures as well as crystal properties are reported for 12 proteins plus numerous site-directed mutants. The proteins are: the integral membrane protein porin from Rhodobacter capsulatus which diffracts to at least 1.8A˚resolution, porin from Rhodopseudomonas blastica which diffracts to at least 2.0A˚resolution, adenylate kinase from yeast and mutants, adenylate kinase from Escherichia coli and mutants, bovine liver mitochondrial adenylate kinase, guanylate kinase from yeast, uridylate kinase from yeast, glutathione reductase from E. coli and mutants, NADH peroxidase from Streptococcus faecalis containing a sulfenic acid as redox-center, pyruvate oxidase from Lactobacillus plantarum containing FAD and TPP, cyclodextrin glycosyltransferase from Bacillus circulans and mutants, and a fuculose aldolase from E. coli.

  11. F'-plasmid transfer from Escherichia coli to Pseudomonas fluorescens.

    PubMed Central

    Mergeay, M; Gerits, J

    1978-01-01

    Various F' plasmids of Escherichia coli K-12 could be transferred into mutants of the soil strain 6.2, classified herein as a Pseudomonas fluorescens biotype IV. This strain was previously found to receive Flac plasmid (N. Datta and R.W. Hedges, J. Gen Microbiol. 70:453-460, 1972). ilv, leu, met, arg, and his auxotrophs were complemented by plasmids carrying isofunctional genes; trp mutants were not complemented or were very poorly complemented. The frequency of transfer was 10(-5). Subsequent transfer into other P. fluorescens recipients was of the same order of magnitude. Some transconjugants were unable to act as donors, and these did not lose the received information if subcultured on nonselective media. Use of F' plasmids helped to discriminate metabolic blocks in P. fluorescens. In particular, metA, metB, and argH mutants were so distinguished. In addition, F131 plasmid carrying the his operon and a supD mutation could partially relieve the auxotrophy of thr, ilv, and metA13 mutants, suggesting functional expression of E. coli tRNA in P. fluorescens. In P. fluorescens metA Rifr mutants carrying the F110 plasmid, which carried the E. coli metA gene and the E. coli rifs allele, sensitivity to rifampin was found to be dominant at least temporarily over resistance. This suggests interaction of E. coli and P. fluorescens subunits of RNA polymerase. his mutations were also complemented by composite P plasmids containing the his-nif region of Klebsiella pneumoniae (plasmids FN68 and RP41). nif expression could be detected by acetylene reduction in some his+ transconjugants. The frequency of transfer of these P plasmids was 5 X 10(-4). PMID:97267

  12. Fitness cost of fluoroquinolone resistance in Campylobacter coli and Campylobacter jejuni.

    PubMed

    Zeitouni, Salman; Kempf, Isabelle

    2011-06-01

    In this study, the fitness cost of fluoroquinolone resistance was evaluated in vitro, on food matrices, and in vivo, using Campylobacter coli and Campylobacter jejuni in vitro selected mutants. In vitro, the growth rate of the susceptible (wild type) and resistant (mutant) strains did not differ when cultured separately. However, by conducting sequential passages of mixed cultures, the ratio of the resistant mutant to the susceptible strain decreased for C. coli but not for C. jejuni. When the wild type and the mutant were co-inoculated on food matrices, mutants were no longer detectable 3 to 5 days after artificial contamination, but the wild-type strains remained detectable for over 13 days. In mono-inoculated animals, no difference was observed between wild-type and mutant fecal titers. When co-inoculated into chickens, the susceptible strain outcompeted the resistant mutant for C. coli and for C. jejuni. However, for C. coli, if the resistant strain was already present in animals, it could persist at high titers in the digestive tract even in the presence of the wild-type strain. Together, these findings suggest that, depending on strain and study conditions, fluoroquinolone resistance can impose a fitness cost on Campylobacter.

  13. Measuring Escherichia coli Gene Expression during Human Urinary Tract Infections

    PubMed Central

    Mobley, Harry L. T.

    2016-01-01

    Extraintestinal Escherichia coli (E. coli) evolved by acquisition of pathogenicity islands, phage, plasmids, and DNA segments by horizontal gene transfer. Strains are heterogeneous but virulent uropathogenic isolates more often have specific fimbriae, toxins, and iron receptors than commensal strains. One may ask whether it is the virulence factors alone that are required to establish infection. While these virulence factors clearly contribute strongly to pathogenesis, bacteria must survive by metabolizing nutrients available to them. By constructing mutants in all major metabolic pathways and co-challenging mice transurethrally with each mutant and the wild type strain, we identified which major metabolic pathways are required to infect the urinary tract. We must also ask what else is E. coli doing in vivo? To answer this question, we examined the transcriptome of E. coli CFT073 in the murine model of urinary tract infection (UTI) as well as for E. coli strains collected and analyzed directly from the urine of patients attending either a urology clinic or a university health clinic for symptoms of UTI. Using microarrays and RNA-seq, we measured in vivo gene expression for these uropathogenic E. coli strains, identifying genes upregulated during murine and human UTI. Our findings allow us to propose a new definition of bacterial virulence. PMID:26784237

  14. Capsule reduces adherence of enterotoxigenic Escherichia coli to isolated intestinal epithelial cells of pigs.

    PubMed Central

    Runnels, P L; Moon, H W

    1984-01-01

    Previous reports have demonstrated that heat-stable (A-type) capsule on piliated enterotoxigenic Escherichia coli enhances colonization of enterotoxigenic E. coli in the small intestine and enhances virulence of enterotoxigenic E. coli. In this report, four encapsulated enterotoxigenic E. coli strains and one encapsulated nonenterotoxigenic strain of E. coli and their nonencapsulated mutants were tested for adhesion to isolated intestinal epithelial cells or brush borders from neonatal pigs. The enterotoxigenic E. coli also expressed the K99 pilus antigen. The nonencapsulated mutants of the four enterotoxigenic E. coli adhered in higher numbers than did the encapsulated parental strains. Both the encapsulated and nonencapsulated forms of enterotoxigenic E. coli 431 grown at 18 degrees C (K99 production suppressed) adhered poorly to the isolated cells. The nonenterotoxigenic E. coli 1793 which does not express K99 antigen also adhered poorly in both encapsulated and nonencapsulated forms. Fab fragments of anticapsular immunoglobulin G failed to block the effect of capsule on adherence of strain 431. The results indicated that K99 was the principal mediator of in vitro adhesion of the enterotoxigenic E. coli strains and that capsule impedes the in vitro adhesion. They also suggested that the capsular enhancement of colonization by such strains in vivo probably is by some mechanism other than enhanced adhesion to epithelium. PMID:6147310

  15. Capsule reduces adherence of enterotoxigenic Escherichia coli to isolated intestinal epithelial cells of pigs.

    PubMed

    Runnels, P L; Moon, H W

    1984-09-01

    Previous reports have demonstrated that heat-stable (A-type) capsule on piliated enterotoxigenic Escherichia coli enhances colonization of enterotoxigenic E. coli in the small intestine and enhances virulence of enterotoxigenic E. coli. In this report, four encapsulated enterotoxigenic E. coli strains and one encapsulated nonenterotoxigenic strain of E. coli and their nonencapsulated mutants were tested for adhesion to isolated intestinal epithelial cells or brush borders from neonatal pigs. The enterotoxigenic E. coli also expressed the K99 pilus antigen. The nonencapsulated mutants of the four enterotoxigenic E. coli adhered in higher numbers than did the encapsulated parental strains. Both the encapsulated and nonencapsulated forms of enterotoxigenic E. coli 431 grown at 18 degrees C (K99 production suppressed) adhered poorly to the isolated cells. The nonenterotoxigenic E. coli 1793 which does not express K99 antigen also adhered poorly in both encapsulated and nonencapsulated forms. Fab fragments of anticapsular immunoglobulin G failed to block the effect of capsule on adherence of strain 431. The results indicated that K99 was the principal mediator of in vitro adhesion of the enterotoxigenic E. coli strains and that capsule impedes the in vitro adhesion. They also suggested that the capsular enhancement of colonization by such strains in vivo probably is by some mechanism other than enhanced adhesion to epithelium.

  16. Fungal β-1,3-glucan increases ofloxacin tolerance of Escherichia coli in a polymicrobial E. coli/Candida albicans biofilm.

    PubMed

    De Brucker, Katrijn; Tan, Yulong; Vints, Katlijn; De Cremer, Kaat; Braem, Annabel; Verstraeten, Natalie; Michiels, Jan; Vleugels, Jef; Cammue, Bruno P A; Thevissen, Karin

    2015-01-01

    In the past, biofilm-related research has focused mainly on axenic biofilms. However, in nature, biofilms are often composed of multiple species, and the resulting polymicrobial interactions influence industrially and clinically relevant outcomes such as performance and drug resistance. In this study, we show that Escherichia coli does not affect Candida albicans tolerance to amphotericin or caspofungin in an E. coli/C. albicans biofilm. In contrast, ofloxacin tolerance of E. coli is significantly increased in a polymicrobial E. coli/C. albicans biofilm compared to its tolerance in an axenic E. coli biofilm. The increased ofloxacin tolerance of E. coli is mainly biofilm specific, as ofloxacin tolerance of E. coli is less pronounced in polymicrobial E. coli/C. albicans planktonic cultures. Moreover, we found that ofloxacin tolerance of E. coli decreased significantly when E. coli/C. albicans biofilms were treated with matrix-degrading enzymes such as the β-1,3-glucan-degrading enzyme lyticase. In line with a role for β-1,3-glucan in mediating ofloxacin tolerance of E. coli in a biofilm, we found that ofloxacin tolerance of E. coli increased even more in E. coli/C. albicans biofilms consisting of a high-β-1,3-glucan-producing C. albicans mutant. In addition, exogenous addition of laminarin, a polysaccharide composed mainly of poly-β-1,3-glucan, to an E. coli biofilm also resulted in increased ofloxacin tolerance. All these data indicate that β-1,3-glucan from C. albicans increases ofloxacin tolerance of E. coli in an E. coli/C. albicans biofilm.

  17. Genetic mapping of nth, a gene affecting endonuclease III (thymine glycol-DNA glycosylase) in Escherichia coli K-12.

    PubMed Central

    Weiss, B; Cunningham, R P

    1985-01-01

    The nth gene of Escherichia coli affects the production of endonuclease III, a glycosylase-endonuclease that attacks DNA damaged by oxidizing agents or by ionizing radiation. An nth insertion mutant and a deletion mutant were studied. nth is located between add and tyrS on the linkage map of E. coli K-12 and was 97% linked to tyrS in a transduction with phage P1. PMID:3886628

  18. Influence of cyclopropane fatty acids on heat, high pressure, acid and oxidative resistance in Escherichia coli.

    PubMed

    Chen, Yuan Yao; Gänzle, Michael G

    2016-04-02

    Heat and high pressure resistant strains of Escherichia coli are a challenge to food safety. This study investigated effects of cyclopropane fatty acids (CFAs) on stress tolerance in the heat- and pressure-resistant strain E. coli AW1.7 and the sensitive strain E. coli MG1655. The role of CFAs was explored by disruption of cfa coding for CFA synthase with an in-frame, unmarked deletion method. Both wild-type strains consumed all the unsaturated fatty acids (C16:1 and C18:1) that were mostly converted to CFAs and a low proportion to saturated fatty acid (C16:0). Moreover, E. coli AW1.7 contained a higher proportion of membrane C19:0 cyclopropane fatty acid than E. coli MG1655 (P<0.05). The Δcfa mutant strains did not produce CFAs, and the corresponding substrates C16:1 and C18:1 accumulated in membrane lipids. The deletion of cfa did not alter resistance to H2O2 but increased the lethality of heat, high pressure and acid treatments in E. coli AW1.7, and E. coli MG1655. E. coli AW1.7 and its Δcfa mutant were more resistant to pressure and heat but less resistant to acid stress than E. coli MG1655. Heat resistance of wild-type strains and their Δcfa mutant was also assessed in beef patties grilled to an internal temperature of 71 °C. After treatment, cell counts of wild type strains were higher than those of the Δcfa mutant strains. In conclusion, CFA synthesis in E. coli increases heat, high pressure and acid resistance, and increases heat resistance in food. This knowledge on mechanisms of stress resistance will facilitate the design of intervention methods for improved pathogen control in food production.

  19. An Escherichia coli gene required for bacteriophage P2-lambda interference.

    PubMed Central

    Ghisotti, D; Zangrossi, S; Sironi, G

    1983-01-01

    The gene old of bacteriophage P2 is known to (i) cause interference with phage lambda growth; (ii) kill recB- mutants of Escherichia coli after P2 infection; and (iii) determine increased sensitivity of P2 lysogenic cells to X-ray irradiation. In all of these phenomena, inhibition of protein synthesis occurs. We have isolated bacterial mutants, named pin (P2 interference), able to suppress all of the above-mentioned phenomena caused by the old+ gene product and the concurrent protein synthesis inhibition. Pin mutations are recessive, map at 12 min on the E. coli map, and identify a new gene. Satellite bacteriophage P4 does not plate on pin-3 mutant strains and causes cell lethality and protein synthesis inhibition in such mutants. P4 mutants able to grow on pin-3 strains have been isolated. PMID:6355505

  20. (Regulation of alcohol fermentation by Escherichia coli). Progress report

    SciTech Connect

    Clark, D.P.

    1985-01-01

    Constitutive adhC mutants were used as a starting point for the isolation of further mutants, some of which are defective in alcohol dehydrogenase (ADH) and/or acetaldehyde dehydrogenase (ACDH) activities and some of which are regulatory and express elevated enzyme levels. The structural mutants map close to the adhC gene, suggesting the existence of an anaerobically controlled operon responsible for the conversion of acetyl-CoA to ethanol. Purification of the two enzyme activities indicates that both copurify as a complex of approximately 200,000 daltons. Although confirmation is required, both enzyme activities appear to be functions of a single polypeptide of MW 100,000 daltons. This interpretation is consistent with genetic data which show that most mutants selected directly for loss of either enzyme have also lost the other activity. Temperature sensitive mutants in which both enzymes are thermolabile also support the idea of a single polypeptide for the two activities. Regulatory mutants located away from the adhC locus have been isolated, and result in two to tenfold elevation of both ADH and ACDH. These mutants are in process of further characterization. Study of adh regulation by means of gene fusions has been slowed by technical problems, however we have devised a direct method for the selection of mutants unable to excrete acidic fermentation products and have accumulated a variety of anaerobically regulated gene fusions which have allowed us to estimate that anaerobiosis in E. coli requires the induction of around 50 genes.

  1. Use of Electroporation To Generate a Thiobacillus neapolitanus Carboxysome Mutant

    PubMed Central

    English, R. S.; Jin, S.; Shively, J. M.

    1995-01-01

    Two cloning vectors designed for use in Escherichia coli and the thiobacilli were constructed by combining a Thiobacillus intermedius plasmid replicon with a multicloning site, lacZ(prm1), and either a kanamycin or a streptomycin resistance gene. Conditions necessary for the introduction of DNA into T. intermedius and T. neapolitanus via electroporation were examined and optimized. By using optimal electroporation conditions, the gene encoding a carboxysome shell protein, csoS1A, was insertionally inactivated in T. neapolitanus. The mutant showed a reduced number of carboxysomes and an increased level of CO(inf2) necessary for growth. PMID:16535117

  2. Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation.

    PubMed Central

    Gupta, S; Clark, D P

    1989-01-01

    Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed in terms of redox balance. PMID:2661531

  3. Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation.

    PubMed

    Gupta, S; Clark, D P

    1989-07-01

    Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed in terms of redox balance.

  4. Escherichia coli derivatives lacking both alcohol dehydrogenase and phosphotransacetylase grow anaerobically by lactate fermentation

    SciTech Connect

    Gupta, S.; Clark, D.P. )

    1989-07-01

    Escherichia coli mutants lacking alcohol dehydrogenase (adh mutants) cannot synthesize the fermentation product ethanol and are unable to grow anaerobically on glucose and other hexoses. Similarly, phosphotransacetylase-negative mutants (pta mutants) neither excrete acetate nor grow anaerobically. However, when a strain carrying an adh deletion was selected for anaerobic growth on glucose, spontaneous pta mutants were isolated. Strains carrying both adh and pta mutations were observed by in vivo nuclear magnetic resonance and shown to produce lactic acid as the major fermentation product. Various combinations of adh pta double mutants regained the ability to grow anaerobically on hexoses, by what amounts to a homolactic fermentation. Unlike wild-type strains, such adh pta double mutants were unable to grow anaerobically on sorbitol or on glucuronic acid. The growth properties of strains carrying various mutations affecting the enzymes of fermentation are discussed terms of redox balance.

  5. Subunit interface mutants of rabbit muscle aldolase form active dimers.

    PubMed Central

    Beernink, P. T.; Tolan, D. R.

    1994-01-01

    We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability. PMID:7833800

  6. Escherichia coli biofilms

    PubMed Central

    Beloin, Christophe; Roux, Agnès; Ghigo, Jean-Marc

    2008-01-01

    Escherichia coli is a predominant species among facultative anaerobic bacteria of the gastrointestinal tract. Both its frequent community lifestyle and the availability of a wide array of genetic tools contributed to establish E. coli as a relevant model organism for the study of surface colonization. Several key factors, including different extracellular appendages, are implicated in E. coli surface colonization and their expression and activity are finely regulated, both in space and time, to ensure productive events leading to mature biofilm formation. This chapter will present known molecular mechanisms underlying biofilm development in both commensal and pathogenic E. coli. PMID:18453280

  7. Bacteriophage PhiX174's Ecological Niche and the Flexibility of Its Escherichia coli Lipopolysaccharide Receptor▿

    PubMed Central

    Michel, Alix; Clermont, Olivier; Denamur, Erick; Tenaillon, Olivier

    2010-01-01

    To determine bacteriophage PhiX174's ecological niche, 783 Escherichia coli isolates were screened for susceptibility. Sensitive strains are diverse regarding their phylogenies and core lipopolysaccharides (LPS), but all have rough phenotypes. Further analysis of E. coli K-12 LPS mutants revealed that PhiX174 can use a wide diversity of LPS structures to initiate its infectious process. PMID:20833781

  8. Cross-species complementation of the indispensable Escherichia coli era gene highlights amino acid regions essential for activity.

    PubMed Central

    Pillutla, R C; Sharer, J D; Gulati, P S; Wu, E; Yamashita, Y; Lerner, C G; Inouye, M; March, P E

    1995-01-01

    Era is an essential GTP binding protein in Escherichia coli. Two homologs of this protein, Sgp from Streptococcus mutans and Era from Coxiella burnetii, can substitute for the essential function of Era in E. coli. Site-specific and randomly generated Era mutants which may indicate regions of the protein that are of functional importance are described. PMID:7721709

  9. A second DNA methyltransferase repair enzyme in Escherichia coli.

    PubMed Central

    Rebeck, G W; Coons, S; Carroll, P; Samson, L

    1988-01-01

    The Escherichia coli ada-alkB operon encodes a 39-kDa protein (Ada) that is a DNA-repair methyltransferase and a 27-kDa protein (AlkB) of unknown function. By DNA blot hybridization analysis we show that the alkylation-sensitive E. coli mutant BS23 [Sedgwick, B. & Lindahl, T. (1982) J. Mol. Biol. 154, 169-175] is a deletion mutant lacking the entire ada-alkB operon. Despite the absence of the ada gene and its product, the cells contain detectable levels of a DNA-repair methyltransferase activity. We conclude that the methyltransferase in BS23 cells is the product of a gene other than ada. A similar activity was detected in extracts of an ada-10::Tn10 insertion mutant of E. coli AB1157. This DNA methyltransferase has a molecular mass of about 19 kDa and transfers the methyl groups from O6-methylguanine and O4-methylthymine in DNA, but not those from methyl phosphotriester lesions. This enzyme was not induced by low doses of alkylating agent and is expressed at low levels in ada+ and a number of ada- E. coli strains. Images PMID:3283737

  10. Mutations in Escherichia coli that effect sensitivity to oxygen

    SciTech Connect

    Jamison, C.S.; Adler, H.I.

    1987-11-01

    Fifteen oxygen-sensitive (Oxy/sup s/) mutants of Escherichia coli were isolated after exposure to UV light. The mutants did not form macroscopic colonies when plated aerobically. They did form macroscopic colonies anaerobically. Oxygen, introduced during log phase, inhibited the growth of liquid cultures. The degree of inhibition was used to separate the mutants into three classes. Class I mutants did not grow after exposure to oxygen. Class II mutants were able to grow, but at a reduced rate and to a reduced final titer, when compared with the wild-type parent. Class III mutants formed filaments in response to oxygen. Genetic experiments indicated that the mutations map to six different chromosomal regions. The results of enzymatic assays indicated that 7 of the 10 class I mutants have low levels of catalase, peroxidase, superoxide dismutase, and respiratory enzymes when compared with the wild-type parent. Mutations in five of the seven class I mutants which have the low enzyme activities mapped within the region 8 to 13.5 min. P1 transduction data indicated that mutations in three of these five mutants, Oxy/sup s/-6, Oxy/sup s/-14, and Oxy/sup s/-17, mapped to 8.4 min. The correlation of low enzyme levels and mapping data suggest that a single gene may regulate several enzymes in response to oxygen. The remaining three class I mutants had wild-type levels of catalase, peroxidase, and superoxide dismutase, but decreased respiratory activity. The class II and III mutants had enzyme activities similar to those of the wild-type parent.

  11. Escherichia coli genes affecting recipient ability in plasmid conjugation: Are there any?

    PubMed Central

    Pérez-Mendoza, Daniel; de la Cruz, Fernando

    2009-01-01

    Background How does the recipient cell contribute to bacterial conjugation? To answer this question we systematically analyzed the individual contribution of each Escherichia coli gene in matings using plasmid R388 as a conjugative plasmid. We used an automated conjugation assay and two sets of E. coli mutant collections: the Keio collection (3,908 E. coli single-gene deletion mutants) and a collection of 20,000 random mini-Tn10::Km insertion mutants in E. coli strain DH5α. The combined use of both collections assured that we screened > 99% of the E. coli non-essential genes in our survey. Results Results indicate that no non-essential recipient E. coli genes exist that play an essential role in conjugation. Mutations in the lipopolysaccharide (LPS) synthesis pathway had a modest effect on R388 plasmid transfer (6 – 32% of wild type). The same mutations showed a drastic inhibition effect on F-plasmid transfer, but only in liquid matings, suggesting that previously isolated conjugation-defective mutants do in fact impair mating pair formation in liquid mating, but not conjugative DNA processing or transport per se. Conclusion We conclude from our genome-wide screen that recipient bacterial cells cannot avoid being used as recipients in bacterial conjugation. This is relevant as an indication of the problems in curbing the dissemination of antibiotic resistance and suggests that conjugation acts as a pure drilling machine, with little regard to the constitution of the recipient cell. PMID:19203375

  12. Active-site mutants of beta-lactamase: use of an inactive double mutant to study requirements for catalysis.

    PubMed

    Dalbadie-McFarland, G; Neitzel, J J; Richards, J H

    1986-01-28

    We have studied the catalytic activity and some other properties of mutants of Escherichia coli plasmid-encoded RTEM beta-lactamase (EC 3.5.2.6) with all combinations of serine and threonine residues at the active-site positions 70 and 71. (All natural beta-lactamases have conserved serine-70 and threonine-71.) From the inactive double mutant Ser-70----Thr, Thr-71----Ser [Dalbadie-McFarland, G., Cohen, L. W., Riggs, A. D., Morin, C., Itakura, K., & Richards, J. H. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 6409-6413], an active revertant, Thr-71----Ser (i.e., residue 70 in the double mutant had changed from threonine to the serine conserved at position 70 in the wild-type enzyme), was isolated by an approach that allows identification of active revertants in the absence of a background of wild-type enzyme. This mutant (Thr-71----Ser) has about 15% of the catalytic activity of wild-type beta-lactamase. The other possible mutant involving serine and threonine residues at positions 70 and 71 (Ser-70----Thr) shows no catalytic activity. The primary nucleophiles of a serine or a cysteine residue [Sigal, I. S., Harwood, B. G., & Arentzen, R. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7157-7160] at position 70 thus seem essential for enzymatic activity. Compared to wild-type enzyme, all three mutants show significantly reduced resistance to proteolysis; for the active revertant (Thr-71----Ser), we have also observed reduced thermal stability and reduced resistance to denaturation by urea.

  13. Anaerobic respiration of Escherichia coli in the mouse intestine.

    PubMed

    Jones, Shari A; Gibson, Terri; Maltby, Rosalie C; Chowdhury, Fatema Z; Stewart, Valley; Cohen, Paul S; Conway, Tyrrell

    2011-10-01

    The intestine is inhabited by a large microbial community consisting primarily of anaerobes and, to a lesser extent, facultative anaerobes, such as Escherichia coli, which we have shown requires aerobic respiration to compete successfully in the mouse intestine (S. A. Jones et al., Infect. Immun. 75:4891-4899, 2007). If facultative anaerobes efficiently lower oxygen availability in the intestine, then their sustained growth must also depend on anaerobic metabolism. In support of this idea, mutants lacking nitrate reductase or fumarate reductase have extreme colonization defects. Here, we further explore the role of anaerobic respiration in colonization using the streptomycin-treated mouse model. We found that respiratory electron flow is primarily via the naphthoquinones, which pass electrons to cytochrome bd oxidase and the anaerobic terminal reductases. We found that E. coli uses nitrate and fumarate in the intestine, but not nitrite, dimethyl sulfoxide, or trimethylamine N-oxide. Competitive colonizations revealed that cytochrome bd oxidase is more advantageous than nitrate reductase or fumarate reductase. Strains lacking nitrate reductase outcompeted fumarate reductase mutants once the nitrate concentration in cecal mucus reached submillimolar levels, indicating that fumarate is the more important anaerobic electron acceptor in the intestine because nitrate is limiting. Since nitrate is highest in the absence of E. coli, we conclude that E. coli is the only bacterium in the streptomycin-treated mouse large intestine that respires nitrate. Lastly, we demonstrated that a mutant lacking the NarXL regulator (activator of the NarG system), but not a mutant lacking the NarP-NarQ regulator, has a colonization defect, consistent with the advantage provided by NarG. The emerging picture is one in which gene regulation is tuned to balance expression of the terminal reductases that E. coli uses to maximize its competitiveness and achieve the highest possible population in

  14. Slugs: Potential Novel Vectors of Escherichia coli O157

    PubMed Central

    Sproston, Emma L.; Macrae, M.; Ogden, Iain D.; Wilson, Michael J.; Strachan, Norval J. C.

    2006-01-01

    Field and laboratory studies were performed to determine whether slugs could act as novel vectors for pathogen (e.g., Escherichia coli O157) transfer from animal feces to salad vegetables. Escherichia coli O157 was isolated from 0.21% of field slugs from an Aberdeenshire sheep farm. These isolates carried the verocytotoxin genes (vt1 and vt2) and the attaching and effacing gene (eae), suggesting that they are potentially pathogenic to humans. Strain typing using multilocus variable number tandem repeats analysis showed that slug and sheep isolates were indistinguishable. Laboratory experiments using an E. coli mutant resistant to nalidixic acid showed that the ubiquitous slug species Deroceras reticulatum could carry viable E. coli on its external surface for up to 14 days. Slugs that had been fed E. coli shed viable bacteria in their feces with numbers showing a short but statistically significant linear log decline. Further, it was found that E. coli persisted for up to 3 weeks in excreted slug feces, and hence, we conclude that slugs have the potential to act as novel vectors of E. coli O157. PMID:16391036

  15. fur (-) mutation increases the survival time of Escherichia coli under photooxidative stress in aquatic environments.

    PubMed

    Darcan, C; Aydin, Ebru

    2012-09-01

    We investigated the survival of the wild type Escherichia coli (QC771) and fur- mutant strain (QC1732) under photooxidative stress in different water sources. The survival of fur- mutant and wild type E. coli was seen as a significant decrease in the visible light samples in the presence of methylene blue (MB). The fur-E. coli strain lived longer than the wild type E. coli strain on exposure to MB and visible light, which generates singlet oxygen, in both lake water (48-h) and pure water (16-h). It is interesting to note that the survival of both wild type and the fur- mutant strain was more protected at 24 °C than at other temperatures. The Fur protein does not have any relation to the entry of E. coli into the viable but nonculturable state (VBNC) under photooxidative stress. This is the first study which shows that fur- mutation increases the resistance of E. coli to photooxidative stress in aquatic environments, and the Fur protein does not have any relation to the entry of E. coli into the VBNC state.

  16. PATHOGENIC ESCHERICHIA COLI

    EPA Science Inventory

    Escherichia coli is a bacterial species which inhabits the gastrointestinal tract of man and warm-blooded animals. Because of the ubiquity of this bacterium in the intestinal flora, it serves as an important indicator organism of fecal contamination. E. coli, aside from serving a...

  17. Pathogenic Escherichia coli

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Escherichia coli, a member of the Enterobacteriaceae family, is a part of the normal flora of the intestinal tract of humans and a variety of animals. E. coli strains are classified on the basis of antigenic differences in two surface components (serotyping), the somatic antigen (O) of the lipopoly...

  18. Diarrheagenic Escherichia coli.

    PubMed

    Gomes, Tânia A T; Elias, Waldir P; Scaletsky, Isabel C A; Guth, Beatriz E C; Rodrigues, Juliana F; Piazza, Roxane M F; Ferreira, Luís C S; Martinez, Marina B

    2016-12-01

    Most Escherichia coli strains live harmlessly in the intestines and rarely cause disease in healthy individuals. Nonetheless, a number of pathogenic strains can cause diarrhea or extraintestinal diseases both in healthy and immunocompromised individuals. Diarrheal illnesses are a severe public health problem and a major cause of morbidity and mortality in infants and young children, especially in developing countries. E. coli strains that cause diarrhea have evolved by acquiring, through horizontal gene transfer, a particular set of characteristics that have successfully persisted in the host. According to the group of virulence determinants acquired, specific combinations were formed determining the currently known E. coli pathotypes, which are collectively known as diarrheagenic E. coli. In this review, we have gathered information on current definitions, serotypes, lineages, virulence mechanisms, epidemiology, and diagnosis of the major diarrheagenic E. coli pathotypes.

  19. Indole production provides limited benefit to Escherichia coli during co-culture with Enterococcus faecalis.

    PubMed

    Pringle, Shelly L; Palmer, Kelli L; McLean, Robert J C

    2017-01-01

    Escherichia coli lives in the gastrointestinal tract and elsewhere, where it coexists within a mixed population. Indole production enables E. coli to grow with other gram-negative bacteria as indole inhibits N-acyl-homoserine lactone (AHL) quorum regulation. We investigated whether E. coli indole production enhanced competition with gram-positive Enterococcus faecalis, wherein quorum signaling is mediated by small peptides. During planktonic co-culture with E. faecalis, the fitness and population density of E. coli tnaA mutants (unable to produce indole) equaled or surpassed that of E. coli wt. During biofilm growth, the fitness of both populations of E. coli stabilized around 100 %, whereas the fitness of E. faecalis declined over time to 85-90 %, suggesting that biofilm and planktonic populations have different competition strategies. Media supplementation with indole removed the competitive advantage of E. coli tnaA in planktonic populations but enhanced it in biofilm populations. E. coli wt and tnaA showed similar growth in Luria-Bertani (LB) broth. However, E. coli growth was inhibited in the presence of filter-sterilized spent LB from E. faecalis, with inhibition being enhanced by indole. Similarly, there was also an inhibition of E. faecalis growth by proteinaceous components (likely bacteriocins) from spent culture media from both E. coli strains. We conclude that E. coli indole production is not a universal competition strategy, but rather works against gram-negative, AHL-producing bacteria.

  20. Interactions between chemotaxis genes and flagellar genes in Escherichia coli.

    PubMed Central

    Parkinson, J S; Parker, S R; Talbert, P B; Houts, S E

    1983-01-01

    Escherichia coli mutants defective in cheY and cheZ function are motile but generally nonchemotactic; cheY mutants have an extreme counterclockwise bias in flagellar rotation, whereas cheZ mutants have a clockwise rotational bias. Chemotactic pseudorevertants of cheY and cheZ mutants were isolated on semisolid agar and examined for second-site suppressors in other chemotaxis-related loci. Approximately 15% of the cheZ revertants and over 95% of the cheY revertants contained compensatory mutations in the flaA or flaB locus. When transferred to an otherwise wild-type background, most of these suppressor mutations resulted in a generally nonchemotactic phenotype: suppressors of cheY caused a clockwise rotational bias; suppressors of cheZ produced a counterclockwise rotational bias. Chemotactic double mutants containing a che and a fla mutation invariably exhibited flagellar rotation patterns in between the opposing extremes characteristic of the component mutations. This additive effect on flagellar rotation resulted in essentially wild-type swimming behavior and is probably the major basis of suppressor action. However, suppression effects were also allele specific, suggesting that the cheY and cheZ gene products interact directly with the flaA and flaB products. These interactions may be instrumental in establishing the unstimulated swimming pattern of E. coli. Images PMID:6305913

  1. Role of glycoside hydrolase genes in sinigrin degradation by E. coli O157:H7.

    PubMed

    Cordeiro, Roniele P; Doria, Juan H; Zhanel, George G; Sparling, Richard; Holley, Richard A

    2015-07-16

    This work examined Escherichia coli O157:H7 strain 02-0304 for putative genes responsible for sinigrin hydrolysis. Sinigrin is a glucosinolate present in Oriental mustard (Brassica juncea), and its hydrolysis is mediated in plants by the enzyme myrosinase. Sinigrin hydrolysis by plant or bacterial myrosinase yields allyl isothiocyanate (AITC) which is bactericidal. In silico analysis using public databases found sequence similarity between plant myrosinase and enzymes encoded by genes from β-glucosidase families in E. coli O157:H7. Specifically, 6-phospho-β-glucosidase encoded by the genes bglA and ascB (family 1), and chbF (family 4) present in E. coli O157:H7 showed the highest similarity. Polymerase chain reaction (PCR) confirmed the presence of bglA, ascB, and chbF in the clinical E. coli strain tested. Disruption of these genes in wild-type E. coli O157:H7 strain 02-0304 using lambda-red replacement created single and double mutants. The relative importance of each gene in the hydrolysis of sinigrin by E. coli O157:H7 was also assessed by comparing gene expression and sinigrin degradation rates among the E. coli O157:H7 wild-type strain and its mutants. The results suggested that the genes bglA and ascB play a substantial role in the degradation of sinigrin by E. coli O157:H7 strain 02-0304.

  2. Inversions between ribosomal RNA genes of Escherichia coli.

    PubMed Central

    Hill, C W; Harnish, B W

    1981-01-01

    It might be anticipated that the presence of redundant but oppositely oriented sequences in a chromosome could allow inversion of the intervening material through homologous recombination. For example, the ribosomal RNA gene rrnD of Escherichia coli has the opposite orientation fro rrnB and rrnE and is separated from these genes by roughly 20% of the chromosome. Starting with a derivative of Cavalli Hfr, we have constructed mutants that have an inversion of the segment between rrnD and either rrnB or rrnE. These mutants are generally quite viable but do exhibit a slight reduction in growth rate relative to the parental strain. A major line of laboratory E. coli, W3110 and its derivatives, also has an inversion between rrnD and rrnE, probably created directly by a recombinational event between these highly homologous genes. Images PMID:6273909

  3. Thiolases of Escherichia coli: purification and chain length specificities.

    PubMed Central

    Feigenbaum, J; Schulz, H

    1975-01-01

    The presence of only one thiolase (EC 2.3.1.9) in wild-type Escherichia coli induced for enzymes of beta oxidation was demonstrated. A different thiolase was shown to be present in a mutant constitutive for the enzymes of butyrate degradation. The two thiolases were purified to near homogeneity by a simple two-step procedure and were found to be associated with different proteins as shown by gel electrophoresis. The thiolase isolated from induced wild-type Escherichia coli cell was active on beta-ketoacyl-coenzyme A derivatives containing 4 to 16 carbons, but exhibited optimal activity with medium-chain substrates. In contrast, the thiolase isolated from the constitutive mutant was shown to be specific for acetoacetyl-coenzyme A. PMID:236278

  4. TRYPTOPHANASE-TRYPTOPHAN SYNTHETASE SYSTEMS IN ESCHERICHIA COLI III.

    PubMed Central

    Freundlich, Martin; Lichstein, Herman C.

    1962-01-01

    Freundlich, Martin (University of Minnesota, Minneapolis) and Herman C. Lichstein. Tryptophanase-tryptophan synthetase systems in Escherichia coli. III. Requirements for enzyme synthesis. J. Bacteriol. 84:996–1006. 1962.—The requirements for the formation of tryptophanase and tryptophan synthetase in Escherichia coli during repression release were studied. The kinetics of the formation of tryptophan synthetase differed in the two strains examined; this was attributed to differences in the endogenous level of tryptophan in the bacterial cells. The formation of both enzymes was inhibited by chloramphenicol, and by the absence of arginine in an arginine-requiring mutant. These results are indicative of a requirement for protein synthesis for enzyme formation. Requirements for nucleic acid synthesis were examined by use of a uracil- and thymine-requiring mutant, and with purine and pyrimidine analogues. The results obtained suggest that some type of ribonucleic acid synthesis was necessary for the formation of tryptophanase and tryptophan synthetase. PMID:13959620

  5. Web application for genetic modification flux with database to estimate metabolic fluxes of genetic mutants.

    PubMed

    Mohd Ali, Noorlin; Tsuboi, Ryo; Matsumoto, Yuta; Koishi, Daisuke; Inoue, Kentaro; Maeda, Kazuhiro; Kurata, Hiroyuki

    2016-07-01

    Computational analysis of metabolic fluxes is essential in understanding the structure and function of a metabolic network and in rationally designing genetically modified mutants for an engineering purpose. We had presented the genetic modification flux (GMF) that predicts the flux distribution of a broad range of genetically modified mutants. To enhance the feasibility and usability of GMF, we have developed a web application with a metabolic network database to predict a flux distribution of genetically modified mutants. One hundred and twelve data sets of Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Chinese hamster ovary were registered as standard models.

  6. A beta-turn rich barley seed protein is correctly folded in Escherichia coli.

    PubMed

    Tamas, L; Greenfield, J; Halford, N G; Tatham, A S; Shewry, P R

    1994-08-01

    Wild-type and cysteine-containing mutant C hordeins from barley were expressed in Escherichia coli at high levels (> or = 30mg/liter). N-terminal sequence analysis, SDS-PAGE, RP-HPLC, cd spectroscopy, and small angle X-ray scattering demonstrated that their physicochemical properties were similar to those of C hordeins isolated from barley grain. This indicates that the expressed proteins were correctly folded. The cysteine-containing mutant showed evidence of polymer formation in E. coli, nonreduced preparations of the protein showing the presence of polymers that were replaced by a single protein when a reducing agent was added.

  7. Cloning and expression of the Thiobacillus ferrooxidans glutamine synthetase gene in Escherichia coli

    SciTech Connect

    Barros, M.E.C.; Rawlings, D.E.; Woods, D.R.

    1985-12-01

    The glutamine synthetase (GS) gene glnA of Thiobacillus ferrooxidans was cloned on recombinant plasmid pMEB100 which enabled Escherichia coli glnA deletion mutants to utilize (NH/sub 4/)/sub 2/SO/sub 4/ as the sole source of nitrogen. High levels of GS-specific activity were obtained in the E. coli glnA deletion mutants containing the T. ferrooxidans GS gene. The cloned T. ferrooxidans DNA fragment containing the glnA gene activated histidase activity in an E. coli glnA glnL glnG deletion mutant containing the Klebsiella aerogenes hut operon. Plasmid pMEB100 also enabled the E. coli glnA glnL glnG deletion mutant to utilize arginine or low levels of glutamine as the sole source of nitrogen. There was no detectable DNA homology between the T. ferrooxidans glnA gene and the E. coli glnA gene.

  8. High-throughput, quantitative analyses of genetic interactions in E. coli.

    PubMed

    Typas, Athanasios; Nichols, Robert J; Siegele, Deborah A; Shales, Michael; Collins, Sean R; Lim, Bentley; Braberg, Hannes; Yamamoto, Natsuko; Takeuchi, Rikiya; Wanner, Barry L; Mori, Hirotada; Weissman, Jonathan S; Krogan, Nevan J; Gross, Carol A

    2008-09-01

    Large-scale genetic interaction studies provide the basis for defining gene function and pathway architecture. Recent advances in the ability to generate double mutants en masse in Saccharomyces cerevisiae have dramatically accelerated the acquisition of genetic interaction information and the biological inferences that follow. Here we describe a method based on F factor-driven conjugation, which allows for high-throughput generation of double mutants in Escherichia coli. This method, termed genetic interaction analysis technology for E. coli (GIANT-coli), permits us to systematically generate and array double-mutant cells on solid media in high-density arrays. We show that colony size provides a robust and quantitative output of cellular fitness and that GIANT-coli can recapitulate known synthetic interactions and identify previously unidentified negative (synthetic sickness or lethality) and positive (suppressive or epistatic) relationships. Finally, we describe a complementary strategy for genome-wide suppressor-mutant identification. Together, these methods permit rapid, large-scale genetic interaction studies in E. coli.

  9. Stereochemistry of phospho group transfer catalyzed by a mutant alkaline phosphatase

    SciTech Connect

    Butler-Ransohoff, J.E.; Kendall, D.A.; Freeman, S.; Knowles, J.R.; Kaiser, E.T.

    1988-06-28

    The stereochemical course of the phospho group transfer catalyzed by mutant (S102C) alkaline phosphatase from Escherichia coli was investigated by using /sup 31/P nuclear magnetic resonance spectroscopy. Transphosphorylation from 4-nitrophenyl (R/sub P/)-/sup 17/O, /sup 16/O, /sup 18/O)phosphate to (S)-propane-1,2-diol occurs with overall retention of configuration at phosphorus. This result is consistent with the view that the hydrolysis of substrates by this mutant enzyme proceeds by way of a covalent phosphoenzyme intermediate in the same manner as the wild-type alkaline phosphatase.

  10. Mutant power: using mutant allele collections for yeast functional genomics.

    PubMed

    Norman, Kaitlyn L; Kumar, Anuj

    2016-03-01

    The budding yeast has long served as a model eukaryote for the functional genomic analysis of highly conserved signaling pathways, cellular processes and mechanisms underlying human disease. The collection of reagents available for genomics in yeast is extensive, encompassing a growing diversity of mutant collections beyond gene deletion sets in the standard wild-type S288C genetic background. We review here three main types of mutant allele collections: transposon mutagen collections, essential gene collections and overexpression libraries. Each collection provides unique and identifiable alleles that can be utilized in genome-wide, high-throughput studies. These genomic reagents are particularly informative in identifying synthetic phenotypes and functions associated with essential genes, including those modeled most effectively in complex genetic backgrounds. Several examples of genomic studies in filamentous/pseudohyphal backgrounds are provided here to illustrate this point. Additionally, the limitations of each approach are examined. Collectively, these mutant allele collections in Saccharomyces cerevisiae and the related pathogenic yeast Candida albicans promise insights toward an advanced understanding of eukaryotic molecular and cellular biology.

  11. ESCHERICHIA COLI Gene Induction by Alkylation Treatment

    PubMed Central

    Volkert, Michael R.; Nguyen, Dinh C.; Beard, K. Christopher

    1986-01-01

    Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased β-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes. PMID:3080354

  12. Escherichia coli gene induction by alkylation treatment.

    PubMed

    Volkert, M R; Nguyen, D C; Beard, K C

    1986-01-01

    Searches for alkylation-inducible (aid) genes of Escherichia coli have been conducted by screening random fusions of the Mu-dl(ApR lac) phage for fusions showing increased beta-galactosidase activity after treatment with methylating agents, but not after treatments with UV-irradiation. In this report we describe gene fusions that are specifically induced by alkylation treatments. Nine new mutants are described, and their properties are compared with the five mutants described previously. The total of 14 fusion mutants map at five distinct genetic loci. They can be further subdivided on the basis of their induction by methyl methanesulfonate (MMS) and N-methyl-N' -nitro-N-nitrosoguanidine (MNNG). alkA, aidB and aidD are induced by both agents and appear to be regulated by ada. Neither aidC nor aidI is regulated by ada. Moreover, since aidC is induced only by MNNG and aidI is induced only by MMS, these two genes are likely to be individually regulated. Thus, there appear to be at least three different regulatory mechanisms controlling aid genes.

  13. Colonization of porcine small intestine by Escherichia coli: colonization and adhesion factors of pig enteropathogens that lack K88.

    PubMed

    Isaacson, R E; Nagy, B; Moon, H W

    1977-04-01

    The colonizing and adhesive attributes of enterotoxigenic acapsular and/or nonpiliated mutants from K88-negative enteropathogenic Escherichia coli strains were compared with their capsulated and piliated parents (parents were piliated when grown in vitro and in vivo). Acapsular, nonpiliated mutants from three different colonizing strains of enteropathogenic E. coli lost their ability to colonize the ileum of newborn pigs. Acapsular, piliated and capsular, nonpiliated mutants were derived from one of the parental strains (987), and both mutants lacked the ability to colonize the ileum of pigs. The only mutants available from a fourth strain (431) were acapsular and piliated, and they colonized as well as their parents. These data indicate that both capsule and pili are involved in colonization by strain 987. In contrast, capsule is not required for colonization by strain 431, but pili may be.

  14. Curli fimbria: an Escherichia coli adhesin associated with human cystitis.

    PubMed

    Cordeiro, Melina Aparecida; Werle, Catierine Hirsch; Milanez, Guilherme Paier; Yano, Tomomasa

    2016-01-01

    Escherichia coli is the major causative agent of human cystitis. In this study, a preliminary molecular analysis carried out by PCR (polymerase chain reaction) demonstrated that 100% of 31 E. coli strains isolated from patients with recurrent UTIs (urinary tract infections) showed the presence of the curli fimbria gene (csgA). Curli fimbria is known to be associated with bacterial biofilm formation but not with the adhesion of human cystitis-associated E. coli. Therefore, this work aimed to study how curli fimbria is associated with uropathogenic E. coli (UPEC) as an adhesion factor. For this purpose, the csgA gene was deleted from strain UPEC-4, which carries three adhesion factor genes (csgA, fimH and ompA). The wild-type UPEC-4 strain and its mutant (ΔcsgA) were analyzed for their adhesion ability over HTB-9 (human bladder carcinoma), Vero (kidney cells of African green monkey) and HUVEC (human umbilical vein) cells in the presence of α-d-mannose. All the wild-type UPEC strains tested (100%) were able to adhere to all three cell types, while the UPEC-4 ΔcsgA mutant lost its adherence to HTB-9 but continued to adhere to the HUVEC and Vero cells. The results suggest that curli fimbria has an important role in the adhesion processes associated with human UPEC-induced cystitis.

  15. A genetic system for isolation and characterization of TaqI restriction endonuclease mutants.

    PubMed

    Barany, F

    1987-01-01

    The gene encoding TaqI restriction endonuclease has been subcloned downstream from an inducible phoA promoter. Certain strains of Escherichia coli remain viable when endonuclease is expressed, even in the absence of (protective) methylation. Infecting lambda phage DNA is not restricted in vivo. One E. coli strain, MM294, exhibited a temperature-sensitive phenotype when TaqI endonuclease was induced. This allowed for design of an in vivo plate assay for identification of specially constructed two-codon insertion mutants in the endonuclease gene. These mutants exhibited a wide range of in vitro activities, including wild-type activity, greater activity in low-salt buffer, and sequence-specific nicking activity.

  16. A (p)ppGpp-null mutant of Haemophilus ducreyi is partially attenuated in humans due to multiple conflicting phenotypes.

    PubMed

    Holley, Concerta; Gangaiah, Dharanesh; Li, Wei; Fortney, Kate R; Janowicz, Diane M; Ellinger, Sheila; Zwickl, Beth; Katz, Barry P; Spinola, Stanley M

    2014-08-01

    (p)ppGpp responds to nutrient limitation through a global change in gene regulation patterns to increase survival. The stringent response has been implicated in the virulence of several pathogenic bacterial species. Haemophilus ducreyi, the causative agent of chancroid, has homologs of both relA and spoT, which primarily synthesize and hydrolyze (p)ppGpp in Escherichia coli. We constructed relA and relA spoT deletion mutants to assess the contribution of (p)ppGpp to H. ducreyi pathogenesis. Both the relA single mutant and the relA spoT double mutant failed to synthesize (p)ppGpp, suggesting that relA is the primary synthetase of (p)ppGpp in H. ducreyi. Compared to the parent strain, the double mutant was partially attenuated for pustule formation in human volunteers. The double mutant had several phenotypes that favored attenuation, including increased sensitivity to oxidative stress. The increased sensitivity to oxidative stress could be complemented in trans. However, the double mutant also exhibited phenotypes that favored virulence. When grown to the mid-log phase, the double mutant was significantly more resistant than its parent to being taken up by human macrophages and exhibited increased transcription of lspB, which is involved in resistance to phagocytosis. Additionally, compared to the parent, the double mutant also exhibited prolonged survival in the stationary phase. In E. coli, overexpression of DksA compensates for the loss of (p)ppGpp; the H. ducreyi double mutant expressed higher transcript levels of dksA than the parent strain. These data suggest that the partial attenuation of the double mutant is likely the net result of multiple conflicting phenotypes.

  17. A structural view of the dissociation of Escherichia coli tryptophanase.

    PubMed

    Green, Keren; Qasim, Nasrin; Gdaelvsky, Garik; Kogan, Anna; Goldgur, Yehuda; Parola, Abraham H; Lotan, Ofra; Almog, Orna

    2015-12-01

    Tryptophanase (Trpase) is a pyridoxal 5'-phosphate (PLP)-dependent homotetrameric enzyme which catalyzes the degradation of L-tryptophan. Trpase is also known for its cold lability, which is a reversible loss of activity at low temperature (2°C) that is associated with the dissociation of the tetramer. Escherichia coli Trpase dissociates into dimers, while Proteus vulgaris Trpase dissociates into monomers. As such, this enzyme is an appropriate model to study the protein-protein interactions and quaternary structure of proteins. The aim of the present study was to understand the differences in the mode of dissociation between the E. coli and P. vulgaris Trpases. In particular, the effect of mutations along the molecular axes of homotetrameric Trpase on its dissociation was studied. To answer this question, two groups of mutants of the E. coli enzyme were created to resemble the amino-acid sequence of P. vulgaris Trpase. In one group, residues 15 and 59 that are located along the molecular axis R (also termed the noncatalytic axis) were mutated. The second group included a mutation at position 298, located along the molecular axis Q (also termed the catalytic axis). Replacing amino-acid residues along the R axis resulted in dissociation of the tetramers into monomers, similar to the P. vulgaris Trpase, while replacing amino-acid residues along the Q axis resulted in dissociation into dimers only. The crystal structure of the V59M mutant of E. coli Trpase was also determined in its apo form and was found to be similar to that of the wild type. This study suggests that in E. coli Trpase hydrophobic interactions along the R axis hold the two monomers together more strongly, preventing the dissociation of the dimers into monomers. Mutation of position 298 along the Q axis to a charged residue resulted in tetramers that are less susceptible to dissociation. Thus, the results indicate that dissociation of E. coli Trpase into dimers occurs along the molecular Q axis.

  18. Gene expression in E. coli after treatment with streptozotocin.

    PubMed

    Fram, R J; Marinus, M G; Volkert, M R

    1988-03-01

    Gene induction by the methylating agents streptozotocin (STZ), N-methyl-N-nitrosourea (MNU), and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was evaluated in E. coli fusion mutants. These mutants have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents and were previously selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methyl methanesulfonate or MNNG. The results demonstrate that STZ differs from MNNG and MNU in failing to induce aidC expression. Further, expression of aidC after exposure to MNU and MNNG occurs only in nonaerated cultures; aeration blocks the induction. Induction of aidD, alkA, aidB, and sfiA expression occurs with all 3 agents although at markedly lower concentrations of MNNG and STZ compared to MNU. alkA and to a lesser extent aidD mutants of E. coli strains were more sensitive to these agents, while no differences were evident between wild-type and aidB or aidC fusion mutants.

  19. Synthesis and processing of Escherichia coli TEM-beta-lactamase and Bacillus licheniformis alpha-amylase in E. coli: the role of signal peptidase I.

    PubMed

    van Dijl, J M; Smith, H; Bron, S; Venema, G

    1988-09-01

    A mutant of Escherichia coli, in which signal peptidase I synthesis can be regulated, was constructed. The mutant was used to study the effects of signal peptidase I limitation on the synthesis and efficiency of processing of two proteins: the periplasmic E. coli TEM-beta-lactamase and Bacillus licheniformis alpha-amylase, which also accumulates in the periplasm of E. coli. Signal peptidase I limitation resulted in reduced rates of processing of pre-beta-lactamase and in strong inhibition of synthesis of alpha-amylase. The data suggest that beta-lactamase is processed post-translationally and that an intimate relationship exists between the synthesis and processing of alpha-amylase.

  20. Transfer of Plasmids to an Antibiotic-Sensitive Mutant of Zymomonas mobilis†

    PubMed Central

    Buchholz, Steven E.; Eveleigh, Douglas E.

    1986-01-01

    Wild-type strains of Zymomonas mobilis exhibit multiple antibiotic resistance and thus restrict the use of many broad-host-range plasmids in them as cloning vehicles. Antibiotic-sensitive mutants of Z. mobilis were isolated and used as hosts for the conjugal transfer of broad-host-range plasmids from Escherichia coli. Such antibiotic-sensitive strains can facilitate the application of broad-host-range plasmids to the study of Z. mobilis. Images PMID:16347136

  1. Molecular Determinants of Mutant Phenotypes, Inferred from Saturation Mutagenesis Data

    PubMed Central

    Tripathi, Arti; Gupta, Kritika; Khare, Shruti; Jain, Pankaj C.; Patel, Siddharth; Kumar, Prasanth; Pulianmackal, Ajai J.; Aghera, Nilesh; Varadarajan, Raghavan

    2016-01-01

    Understanding how mutations affect protein activity and organismal fitness is a major challenge. We used saturation mutagenesis combined with deep sequencing to determine mutational sensitivity scores for 1,664 single-site mutants of the 101 residue Escherichia coli cytotoxin, CcdB at seven different expression levels. Active-site residues could be distinguished from buried ones, based on their differential tolerance to aliphatic and charged amino acid substitutions. At nonactive-site positions, the average mutational tolerance correlated better with depth from the protein surface than with accessibility. Remarkably, similar results were observed for two other small proteins, PDZ domain (PSD95pdz3) and IgG-binding domain of protein G (GB1). Mutational sensitivity data obtained with CcdB were used to derive a procedure for predicting functional effects of mutations. Results compared favorably with those of two widely used computational predictors. In vitro characterization of 80 single, nonactive-site mutants of CcdB showed that activity in vivo correlates moderately with thermal stability and solubility. The inability to refold reversibly, as well as a decreased folding rate in vitro, is associated with decreased activity in vivo. Upon probing the effect of modulating expression of various proteases and chaperones on mutant phenotypes, most deleterious mutants showed an increased in vivo activity and solubility only upon over-expression of either Trigger factor or SecB ATP-independent chaperones. Collectively, these data suggest that folding kinetics rather than protein stability is the primary determinant of activity in vivo. This study enhances our understanding of how mutations affect phenotype, as well as the ability to predict fitness effects of point mutations. PMID:27563054

  2. In silico design of anaerobic growth-coupled product formation in Escherichia coli: experimental validation using a simple polyol, glycerol.

    PubMed

    Balagurunathan, Balaji; Jain, Vishist Kumar; Tear, Crystal Jing Ying; Lim, Chan Yuen; Zhao, Hua

    2017-03-01

    Integrated approaches using in silico model-based design and advanced genetic tools have enabled efficient production of fuels, chemicals and functional ingredients using microbial cell factories. In this study, using a recently developed genome-scale metabolic model for Escherichia coli iJO1366, a mutant strain has been designed in silico for the anaerobic growth-coupled production of a simple polyol, glycerol. Computational complexity was significantly reduced by systematically reducing the target reactions used for knockout simulations. One promising penta knockout E. coli mutant (E. coli ΔadhE ΔldhA ΔfrdC ΔtpiA ΔmgsA) was selected from simulation study and was constructed experimentally by sequentially deleting five genes. The penta mutant E. coli bearing the Saccharomyces cerevisiae glycerol production pathway was able to grow anaerobically and produce glycerol as the major metabolite with up to 90% of theoretical yield along with stoichiometric quantities of acetate and formate. Using the penta mutant E. coli strain we have demonstrated that the ATP formation from the acetate pathway was essential for growth under anaerobic conditions. The general workflow developed can be easily applied to anaerobic production of other platform chemicals using E. coli as the cell factory.

  3. Transcriptional and functional responses of Escherichia coli O157:H7 growing in the lettuce rhizoplane.

    PubMed

    Hou, Zhe; Fink, Ryan C; Sugawara, Masayuki; Diez-Gonzalez, Francisco; Sadowsky, Michael J

    2013-09-01

    Lettuce and spinach are increasingly implicated in foodborne illness outbreaks due to contamination by Escherichia coli O157:H7. While this bacterium has been shown to colonize and survive on lettuce leaf surfaces, little is known about its interaction with the roots of growing lettuce plants. In these studies, a microarray analyses, mutant construction and confocal microscopy were used to gain an understanding of structure and function of bacterial genes involved in the colonization and growth of E. coli O157:H7 on lettuce roots. After three days of interaction with lettuce roots, 94 and 109 E. coli O157:H7 genes were significantly up- and down-regulated at least 1.5 fold, respectively. While genes involved in biofilm modulation (ycfR and ybiM) were significantly up-regulated, 40 of 109 (37%) of genes involved in protein synthesis were significantly repressed. E. coli O157:H7 was 2 logs less efficient in lettuce root colonization than was E. coli K12. We also unambiguously showed that a ΔycfR mutant of E. coli O157:H7 was unable to attach to or colonize lettuce roots. Taken together these results indicate that bacterial genes involved in attachment and biofilm formation are likely important for contamination of lettuce plants with Shiga toxin-producing E. coli strains.

  4. Lytic and lysogenic infection of diverse Escherichia coli and Shigella strains with a verocytotoxigenic bacteriophage.

    PubMed

    James, C E; Stanley, K N; Allison, H E; Flint, H J; Stewart, C S; Sharp, R J; Saunders, J R; McCarthy, A J

    2001-09-01

    A verocytotoxigenic bacteriophage isolated from a strain of enterohemorrhagic Escherichia coli O157, into which a kanamycin resistance gene (aph3) had been inserted to inactivate the verocytotoxin gene (vt2), was used to infect Enterobacteriaceae strains. A number of Shigella and E. coli strains were susceptible to lysogenic infection, and a smooth E. coli isolate (O107) was also susceptible to lytic infection. The lysogenized strains included different smooth E. coli serotypes of both human and animal origin, indicating that this bacteriophage has a substantial capacity to disseminate verocytotoxin genes. A novel indirect plaque assay utilizing an E. coli recA441 mutant in which phage-infected cells can enter only the lytic cycle, enabling detection of all infective phage, was developed.

  5. Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase

    PubMed Central

    Franke, Kamila B.; Bukau, Bernd; Mogk, Axel

    2017-01-01

    The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings. PMID:28275610

  6. Mutant Analysis Reveals Allosteric Regulation of ClpB Disaggregase.

    PubMed

    Franke, Kamila B; Bukau, Bernd; Mogk, Axel

    2017-01-01

    The members of the hexameric AAA+ disaggregase of E. coli and S. cerevisiae, ClpB, and Hsp104, cooperate with the Hsp70 chaperone system in the solubilization of aggregated proteins. Aggregate solubilization relies on a substrate threading activity of ClpB/Hsp104 fueled by ATP hydrolysis in both ATPase rings (AAA-1, AAA-2). ClpB/Hsp104 ATPase activity is controlled by the M-domains, which associate to the AAA-1 ring to downregulate ATP hydrolysis. Keeping M-domains displaced from the AAA-1 ring by association with Hsp70 increases ATPase activity due to enhanced communication between protomers. This communication involves conserved arginine fingers. The control of ClpB/Hsp104 activity is crucial, as hyperactive mutants with permanently dissociated M-domains exhibit cellular toxicity. Here, we analyzed AAA-1 inter-ring communication in relation to the M-domain mediated ATPase regulation, by subjecting a conserved residue of the AAA-1 domain subunit interface of ClpB (A328) to mutational analysis. While all A328X mutants have reduced disaggregation activities, their ATPase activities strongly differed. ClpB-A328I/L mutants have reduced ATPase activity and when combined with the hyperactive ClpB-K476C M-domain mutation, suppress cellular toxicity. This underlines that ClpB ATPase activation by M-domain dissociation relies on increased subunit communication. The ClpB-A328V mutant in contrast has very high ATPase activity and exhibits cellular toxicity on its own, qualifying it as novel hyperactive ClpB mutant. ClpB-A328V hyperactivity is however, different from that of M-domain mutants as M-domains stay associated with the AAA-1 ring. The high ATPase activity of ClpB-A328V primarily relies on the AAA-2 ring and correlates with distinct conformational changes in the AAA-2 catalytic site. These findings characterize the subunit interface residue A328 as crucial regulatory element to control ATP hydrolysis in both AAA rings.

  7. Stringency of substrate specificity of Escherichia coli malate dehydrogenase.

    SciTech Connect

    Boernke, W. E.; Millard, C. S.; Stevens, P. W.; Kakar, S. N.; Stevens, F. J.; Donnelly, M. I.; Nebraska Wesleyan Univ.

    1995-09-10

    Malate dehydrogenase and lactate dehydrogenase are members of the structurally and functionally homologous family of 2-ketoacid dehydrogenases. Both enzymes display high specificity for their respective keto substrates, oxaloacetate and pyruvate. Closer analysis of their specificity, however, reveals that the specificity of malate dehydrogenase is much stricter and less malleable than that of lactate dehydrogenase. Site-specific mutagenesis of the two enzymes in an attempt to reverse their specificity has met with contrary results. Conversion of a specific active-site glutamine to arginine in lactate dehydrogenase from Bacillus stearothermophilus generated an enzyme that displayed activity toward oxaloacetate equal to that of the native enzyme toward pyruvate (H. M. Wilks et al. (1988) Science 242, 1541-1544). We have constructed a series of mutants in the mobile, active site loop of the Escherichia coli malate dehydrogenase that incorporate the complementary change, conversion of arginine 81 to glutamine, to evaluate the role of charge distribution and conformational flexibility within this loop in defining the substrate specificity of these enzymes. Mutants incorporating the change R81Q all had reversed specificity, displaying much higher activity toward pyruvate than to the natural substrate, oxaloacetate. In contrast to the mutated lactate dehydrogenase, these reversed-specificity mutants were much less active than the native enzyme. Secondary mutations within the loop of the E. coli enzyme (A80N, A80P, A80P/M85E/D86T) had either no or only moderately beneficial effects on the activity of the mutant enzyme toward pyruvate. The mutation A80P, which can be expected to reduce the overall flexibility of the loop, modestly improved activity toward pyruvate. The possible physiological relevance of the stringent specificity of malate dehydrogenase was investigated. In normal strains of E. coli, fermentative metabolism was not affected by expression of the mutant

  8. Activation of contact-dependent antibacterial tRNase toxins by translation elongation factors.

    PubMed

    Jones, Allison M; Garza-Sánchez, Fernando; So, Jaime; Hayes, Christopher S; Low, David A

    2017-03-07

    Contact-dependent growth inhibition (CDI) is a mechanism by which bacteria exchange toxins via direct cell-to-cell contact. CDI systems are distributed widely among Gram-negative pathogens and are thought to mediate interstrain competition. Here, we describe tsf mutations that alter the coiled-coil domain of elongation factor Ts (EF-Ts) and confer resistance to the CdiA-CT(EC869) tRNase toxin from enterohemorrhagic Escherichia coli EC869. Although EF-Ts is required for toxicity in vivo, our results indicate that it is dispensable for tRNase activity in vitro. We find that CdiA-CT(EC869) binds to elongation factor Tu (EF-Tu) with high affinity and this interaction is critical for nuclease activity. Moreover, in vitro tRNase activity is GTP-dependent, suggesting that CdiA-CT(EC869) only cleaves tRNA in the context of translationally active GTP·EF-Tu·tRNA ternary complexes. We propose that EF-Ts promotes the formation of GTP·EF-Tu·tRNA ternary complexes, thereby accelerating substrate turnover for rapid depletion of target-cell tRNA.

  9. Rapid Evolution of Citrate Utilization by Escherichia coli by Direct Selection Requires citT and dctA

    PubMed Central

    Van Hofwegen, Dustin J.; Hovde, Carolyn J.

    2016-01-01

    ABSTRACT The isolation of aerobic citrate-utilizing Escherichia coli (Cit+) in long-term evolution experiments (LTEE) has been termed a rare, innovative, presumptive speciation event. We hypothesized that direct selection would rapidly yield the same class of E. coli Cit+ mutants and follow the same genetic trajectory: potentiation, actualization, and refinement. This hypothesis was tested with wild-type E. coli strain B and with K-12 and three K-12 derivatives: an E. coli ΔrpoS::kan mutant (impaired for stationary-phase survival), an E. coli ΔcitT::kan mutant (deleted for the anaerobic citrate/succinate antiporter), and an E. coli ΔdctA::kan mutant (deleted for the aerobic succinate transporter). E. coli underwent adaptation to aerobic citrate metabolism that was readily and repeatedly achieved using minimal medium supplemented with citrate (M9C), M9C with 0.005% glycerol, or M9C with 0.0025% glucose. Forty-six independent E. coli Cit+ mutants were isolated from all E. coli derivatives except the E. coli ΔcitT::kan mutant. Potentiation/actualization mutations occurred within as few as 12 generations, and refinement mutations occurred within 100 generations. Citrate utilization was confirmed using Simmons, Christensen, and LeMaster Richards citrate media and quantified by mass spectrometry. E. coli Cit+ mutants grew in clumps and in long incompletely divided chains, a phenotype that was reversible in rich media. Genomic DNA sequencing of four E. coli Cit+ mutants revealed the required sequence of mutational events leading to a refined Cit+ mutant. These events showed amplified citT and dctA loci followed by DNA rearrangements consistent with promoter capture events for citT. These mutations were equivalent to the amplification and promoter capture CitT-activating mutations identified in the LTEE. IMPORTANCE E. coli cannot use citrate aerobically. Long-term evolution experiments (LTEE) performed by Blount et al. (Z. D. Blount, J. E. Barrick, C. J. Davidson, and

  10. Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation

    PubMed Central

    Scotti, John S.; Leung, Ivanhoe K. H.; Ge, Wei; Bentley, Michael A.; Paps, Jordi; Kramer, Holger B.; Lee, Joongoo; Aik, WeiShen; Choi, Hwanho; Paulsen, Steinar M.; Bowman, Lesley A. H.; Loik, Nikita D.; Horita, Shoichiro; Ho, Chia-hua; Kershaw, Nadia J.; Tang, Christoph M.; Claridge, Timothy D. W.; Preston, Gail M.; McDonough, Michael A.; Schofield, Christopher J.

    2014-01-01

    The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins. PMID:25197067

  11. Human oxygen sensing may have origins in prokaryotic elongation factor Tu prolyl-hydroxylation.

    PubMed

    Scotti, John S; Leung, Ivanhoe K H; Ge, Wei; Bentley, Michael A; Paps, Jordi; Kramer, Holger B; Lee, Joongoo; Aik, WeiShen; Choi, Hwanho; Paulsen, Steinar M; Bowman, Lesley A H; Loik, Nikita D; Horita, Shoichiro; Ho, Chia-hua; Kershaw, Nadia J; Tang, Christoph M; Claridge, Timothy D W; Preston, Gail M; McDonough, Michael A; Schofield, Christopher J

    2014-09-16

    The roles of 2-oxoglutarate (2OG)-dependent prolyl-hydroxylases in eukaryotes include collagen stabilization, hypoxia sensing, and translational regulation. The hypoxia-inducible factor (HIF) sensing system is conserved in animals, but not in other organisms. However, bioinformatics imply that 2OG-dependent prolyl-hydroxylases (PHDs) homologous to those acting as sensing components for the HIF system in animals occur in prokaryotes. We report cellular, biochemical, and crystallographic analyses revealing that Pseudomonas prolyl-hydroxylase domain containing protein (PPHD) contain a 2OG oxygenase related in structure and function to the animal PHDs. A Pseudomonas aeruginosa PPHD knockout mutant displays impaired growth in the presence of iron chelators and increased production of the virulence factor pyocyanin. We identify elongation factor Tu (EF-Tu) as a PPHD substrate, which undergoes prolyl-4-hydroxylation on its switch I loop. A crystal structure of PPHD reveals striking similarity to human PHD2 and a Chlamydomonas reinhardtii prolyl-4-hydroxylase. A crystal structure of PPHD complexed with intact EF-Tu reveals that major conformational changes occur in both PPHD and EF-Tu, including a >20-Å movement of the EF-Tu switch I loop. Comparison of the PPHD structures with those of HIF and collagen PHDs reveals conservation in substrate recognition despite diverse biological roles and origins. The observed changes will be useful in designing new types of 2OG oxygenase inhibitors based on various conformational states, rather than active site iron chelators, which make up most reported 2OG oxygenase inhibitors. Structurally informed phylogenetic analyses suggest that the role of prolyl-hydroxylation in human hypoxia sensing has ancient origins.

  12. A genetic system to identify DNA polymerase β mutator mutants

    PubMed Central

    Washington, Stacy L.; Yoon, Margaret S.; Chagovetz, Alexander M.; Li, Shu-Xia; Clairmont, Caroline A.; Preston, Bradley D.; Eckert, Kristin A.; Sweasy, Joann B.

    1997-01-01

    DNA polymerase β (pol β) is a 39-kDa protein that functions in DNA repair processes in mammalian cells. As a first step toward understanding mechanisms of polymerase fidelity, we developed a genetic method to identify mammalian pol β mutator mutants. This screen takes advantage of a microbial genetics assay and the ability of rat pol β to substitute for Escherichia coli DNA polymerase I in DNA replication in vivo. Using this screen, we identified 13 candidate pol β mutator mutants. Three of the candidate mutator mutants were further characterized in vivo and shown to confer an increased spontaneous mutation frequency over that of wild-type pol β to our bacterial strain. Purification and subsequent analysis of one of our putative mutator proteins, the pol β-14 protein, showed that it possesses intrinsic mutator activity in four different assays that measure the fidelity of DNA synthesis. Therefore, residue 265, which is altered in pol β-14 and another of our mutant proteins, pol β-166, is probably critical for accurate DNA synthesis by pol β. Thus, our genetic method of screening for pol β mutator mutants is useful in identifying active mammalian DNA polymerase mutants that encode enzymes that catalyze DNA synthesis with altered fidelity compared with the wild-type pol β enzyme. PMID:9037051

  13. A Mutant Library Approach to Identify Improved Meningococcal Factor H Binding Protein Vaccine Antigens

    PubMed Central

    Konar, Monica; Rossi, Raffaella; Walter, Helen; Pajon, Rolando; Beernink, Peter T.

    2015-01-01

    Factor H binding protein (FHbp) is a virulence factor used by meningococci to evade the host complement system. FHbp elicits bactericidal antibodies in humans and is part of two recently licensed vaccines. Using human complement Factor H (FH) transgenic mice, we previously showed that binding of FH decreased the protective antibody responses to FHbp vaccination. Therefore, in the present study we devised a library-based method to identify mutant FHbp antigens with very low binding of FH. Using an FHbp sequence variant in one of the two licensed vaccines, we displayed an error-prone PCR mutant FHbp library on the surface of Escherichia coli. We used fluorescence-activated cell sorting to isolate FHbp mutants with very low binding of human FH and preserved binding of control anti-FHbp monoclonal antibodies. We sequenced the gene encoding FHbp from selected clones and introduced the mutations into a soluble FHbp construct. Using this approach, we identified several new mutant FHbp vaccine antigens that had very low binding of FH as measured by ELISA and surface plasmon resonance. The new mutant FHbp antigens elicited protective antibody responses in human FH transgenic mice that were up to 20-fold higher than those elicited by the wild-type FHbp antigen. This approach offers the potential to discover mutant antigens that might not be predictable even with protein structural information and potentially can be applied to other microbial vaccine antigens that bind host proteins. PMID:26057742

  14. A genome-wide screen of bacterial mutants that enhance dauer formation in C. elegans

    PubMed Central

    Khanna, Amit; Kumar, Jitendra; Vargas, Misha A.; Barrett, LaKisha; Katewa, Subhash; Li, Patrick; McCloskey, Tom; Sharma, Amit; Naudé, Nicole; Nelson, Christopher; Brem, Rachel; Killilea, David W.; Mooney, Sean D.; Gill, Matthew; Kapahi, Pankaj

    2016-01-01

    Molecular pathways involved in dauer formation, an alternate larval stage that allows Caenorhabditis elegans to survive adverse environmental conditions during development, also modulate longevity and metabolism. The decision to proceed with reproductive development or undergo diapause depends on food abundance, population density, and temperature. In recent years, the chemical identities of pheromone signals that modulate dauer entry have been characterized. However, signals derived from bacteria, the major source of nutrients for C. elegans, remain poorly characterized. To systematically identify bacterial components that influence dauer formation and aging in C. elegans, we utilized the individual gene deletion mutants in E. coli (K12). We identified 56 diverse E. coli deletion mutants that enhance dauer formation in an insulin-like receptor mutant (daf-2) background. We describe the mechanism of action of a bacterial mutant cyaA, that is defective in the production of cyclic AMP, which extends lifespan and enhances dauer formation through the modulation of TGF-β (daf-7) signaling in C. elegans. Our results demonstrate the importance of bacterial components in influencing developmental decisions and lifespan in C. elegans. Furthermore, we demonstrate that C. elegans is a useful model to study bacterial-host interactions. PMID:27958277

  15. An episomal expression vector for screening mutant gene libraries in Pichia pastoris.

    PubMed

    Lee, Charles C; Williams, Tina G; Wong, Dominic W S; Robertson, George H

    2005-07-01

    Screening mutant gene libraries for isolating improved enzyme variants is a powerful technique that benefits from effective and reliable biological expression systems. Pichia pastoris is a very useful organism to express proteins that are inactive in other hosts such as Escherichia coli and Saccharomyces cerevisiae. However, most P. pastoris expression plasmids are designed to integrate into the host chromosome and hence are not as amenable to high-throughput screening projects. We have designed a P. pastoris expression vector, pBGP1, incorporating an autonomous replication sequence that allows the plasmid to exist as an episomal element. This vector contains the alpha-factor signal sequence to direct secretion of the mutant enzymes. Expression of the genes is driven by the constitutive GAP promoter, thus eliminating the need for timed or cell density-specific inductions. The pBGP1 plasmid was used to screen a xylanase gene library to isolate higher activity mutants.

  16. Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond.

    PubMed

    Schultz, S C; Dalbadie-McFarland, G; Neitzel, J J; Richards, J H

    1987-01-01

    Uniquely among class A beta-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123. To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 beta-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77----Ser mutation. Both the wild-type enzyme and the single mutant Cys 77----Ser confer the same high levels of resistance to ampicillin in vivo to Escherichia coli; at 30 degrees C the specific activity of purified Cys 77----Ser mutant is also the same as that of the wild-type enzyme. Also, neither wild-type enzyme nor the Cys 77----Ser mutant is inactivated by brief exposure to p-hydroxymercuribenzoate. However, above 40 degrees C the mutant enzyme is less stable than wild-type enzyme. After introduction of the Cys 77----Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37 degrees C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30 degrees C. The use of electrophoretic blots stained with antibodies against beta-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E. coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Differential regulation by cyclic AMP of starvation protein synthesis in Escherichia coli.

    PubMed

    Schultz, J E; Latter, G I; Matin, A

    1988-09-01

    Of the 30 carbon starvation proteins whose induction has been previously shown to be important for starvation survival of Escherichia coli, two-thirds were not induced in cya or crp deletion mutants of E. coli at the onset of carbon starvation. The rest were induced, although not necessarily with the same temporal pattern as exhibited in the wild type. The starvation proteins that were homologous to previously identified heat shock proteins belonged to the latter class and were hyperinduced in delta cya or delta crp mutants during starvation. Most of the cyclic AMP-dependent proteins were synthesized in the delta cya mutant if exogenous cyclic AMP was added at the onset of starvation. Furthermore, beta-galactosidase induction of several carbon starvation response gene fusions occurred only in a cya+ genetic background. Thus, two-thirds of the carbon starvation proteins of E. coli require cyclic AMP and its receptor protein for induction; the rest do not. The former class evidently has no role in starvation survival, since delta cya or delta crp mutants of either E. coli or Salmonella typhimurium survived starvation as well as their wild-type parents did. The latter class, therefore, is likely to have a direct role in starvation survival. This possibility is strengthened by the finding that nearly all of the cya- and crp-independent proteins were also induced during nitrogen starvation and, as shown previously, during phosphate starvation. Proteins whose synthesis is independent of cya- and crp control are referred to as Pex (postexponential).

  18. Mutations That Enhance the Ciprofloxacin Resistance of Escherichia coli with qnrA1.

    PubMed

    Vinué, Laura; Corcoran, Marian A; Hooper, David C; Jacoby, George A

    2015-12-28

    Plasmid-mediated qnr genes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. In Escherichia coli strain J53 without qnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations in gyrA were absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) with qnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations in marR and soxR associated with increased expression of marA and soxS and, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of the ydhE pump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations in rfaD and rfaE in the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression of qnrA that persisted when pMG252 from these strains was outcrossed. gyrA mutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected from E. coli J53 with aac(6')-Ib-cr or qepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from an E. coli strain carrying qnrA1, aac(6')-Ib-cr, or qepA because these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those in gyrA to predominate.

  19. Mutations That Enhance the Ciprofloxacin Resistance of Escherichia coli with qnrA1

    PubMed Central

    Vinué, Laura; Corcoran, Marian A.; Hooper, David C.

    2015-01-01

    Plasmid-mediated qnr genes provide only a modest decrease in quinolone susceptibility but facilitate the selection of higher-level resistance. In Escherichia coli strain J53 without qnr, ciprofloxacin resistance often involves mutations in the GyrA subunit of DNA gyrase. Mutations in gyrA were absent, however, when 43 mutants with decreased ciprofloxacin susceptibility were selected from J53(pMG252) with qnrA1. Instead, in 13 mutants, individual and whole-genome sequencing identified mutations in marR and soxR associated with increased expression of marA and soxS and, through them, increased expression of the AcrAB pump, which effluxes quinolones. Nine mutants had increased expression of the MdtE efflux pump, and six demonstrated increased expression of the ydhE pump gene. Many efflux mutants also had increased resistance to novobiocin, another pump substrate, but other mutants were novobiocin hypersusceptible. Mutations in rfaD and rfaE in the pathway for inner core lipopolysaccharide (LPS) biosynthesis were identified in five such strains. Many of the pump and LPS mutants had decreased expression of OmpF, the major porin channel for ciprofloxacin entry. Three mutants had increased expression of qnrA that persisted when pMG252 from these strains was outcrossed. gyrA mutations were also rare when mutants with decreased ciprofloxacin susceptibility were selected from E. coli J53 with aac(6′)-Ib-cr or qepA. We suggest that multiple genes conferring low-level resistance contribute to enhanced ciprofloxacin resistance selected from an E. coli strain carrying qnrA1, aac(6′)-Ib-cr, or qepA because these determinants decrease the effective ciprofloxacin concentration and allow more common but lower-resistance mutations than those in gyrA to predominate. PMID:26711751

  20. Substitutions in Bacteriophage T4 AsiA and Escherichia coli ς70 That Suppress T4 motA Activation Mutations

    PubMed Central

    Cicero, Marco P.; Sharp, Meghan M.; Gross, Carol A.; Kreuzer, Kenneth N.

    2001-01-01

    Bacteriophage T4 middle-mode transcription requires two phage-encoded proteins, the MotA transcription factor and AsiA coactivator, along with Escherichia coli RNA polymerase holoenzyme containing the ς70 subunit. A motA positive control (pc) mutant, motA-pc1, was used to select for suppressor mutations that alter other proteins in the transcription complex. Separate genetic selections isolated two AsiA mutants (S22F and Q51E) and five ς70 mutants (Y571C, Y571H, D570N, L595P, and S604P). All seven suppressor mutants gave partial suppressor phenotypes in vivo as judged by plaque morphology and burst size measurements. The S22F mutant AsiA protein and glutathione S-transferase fusions of the five mutant ς70 proteins were purified. All of these mutant proteins allowed normal levels of in vitro transcription when tested with wild-type MotA protein, but they failed to suppress the mutant MotA-pc1 protein in the same assay. The ς70 substitutions affected the 4.2 region, which binds the −35 sequence of E. coli promoters. In the presence of E. coli RNA polymerase without T4 proteins, the L595P and S604P substitutions greatly decreased transcription from standard E. coli promoters. This defect could not be explained solely by a disruption in −35 recognition since similar results were obtained with extended −10 promoters. The generalized transcriptional defect of these two mutants correlated with a defect in binding to core RNA polymerase, as judged by immunoprecipitation analysis. The L595P mutant, which was the most defective for in vitro transcription, failed to support E. coli growth. PMID:11244069

  1. Molecular analysis of cytolysin A (ClyA) in pathogenic Escherichia coli strains.

    PubMed

    Ludwig, Albrecht; von Rhein, Christine; Bauer, Susanne; Hüttinger, Christian; Goebel, Werner

    2004-08-01

    Cytolysin A (ClyA) of Escherichia coli is a pore-forming hemolytic protein encoded by the clyA (hlyE, sheA) gene that was first identified in E. coli K-12. In this study we examined various clinical E. coli isolates with regard to the presence and integrity of clyA. PCR and DNA sequence analyses demonstrated that 19 of 23 tested Shiga toxin-producing E. coli (STEC) strains, all 7 tested enteroinvasive E. coli (EIEC) strains, 6 of 8 enteroaggregative E. coli (EAEC) strains, and 4 of 7 tested enterotoxigenic E. coli (ETEC) strains possess a complete clyA gene. The remaining STEC, EAEC, and ETEC strains and 9 of the 17 tested enteropathogenic E. coli (EPEC) strains were shown to harbor mutant clyA derivatives containing 1-bp frameshift mutations that cause premature termination of the coding sequence. The other eight EPEC strains and all tested uropathogenic and new-born meningitis-associated E. coli strains (n = 14 and 3, respectively) carried only nonfunctional clyA fragments due to the deletion of two sequences of 493 bp and 204 or 217 bp at the clyA locus. Expression of clyA from clinical E. coli isolates proved to be positively controlled by the transcriptional regulator SlyA. Several tested E. coli strains harboring a functional clyA gene produced basal amounts of ClyA when grown under standard laboratory conditions, but most of them showed a clyA-dependent hemolytic phenotype only when SlyA was overexpressed. The presented data indicate that cytolysin A can play a role only for some of the pathogenic E. coli strains.

  2. Enhanced oxidative damage by the familial amyotrophic lateral sclerosis-associated Cu,Zn-superoxide dismutase mutants.

    PubMed

    Kang, J H; Eum, W S

    2000-12-15

    Some cases of familial amyotrophic lateral sclerosis (FALS), a degenerative disorder of motor neurons, is associated with mutation in the Cu,Zn-superoxide dismutase (SOD) gene SOD1. The purified FALS mutant and wild-type Cu,Zn-SODs expressed in Escherichia coli cells have identical dismutation activity whereas the hydroxyl radical formation of FALS mutants was enhanced relative to that of the wild-type enzyme. These higher free radical-generating activities of mutants facilitated the release of copper ions from their own molecules. The reaction of the mutants with hydrogen peroxide enhanced DNA strand breaks and lipid peroxidation. The results suggested that the enhanced oxidative damage of macromolecules is mediated in the Cu,Zn-SOD mutants and hydrogen peroxide system via the generation of hydroxyl radicals by a combination of the higher free radical-generating activities of mutants and a Fenton-like reaction of copper ions released from oxidatively damaged Cu,Zn-SODs. Carnosine has been proposed to act as antioxidant in vivo. We investigated whether carnosine could protect the oxidative damage induced by FALS mutants. Carnosine effectively inhibited the DNA cleavage and lipid peroxidation. These results suggest that the higher free radical-generating function of FALS mutants can lead to increased oxidative damage of macromolecules which further implicates free radical-mediated motor neuronal injury in the pathogenesis of FALS and carnosine may be explored as potential therapeutic agents for FALS patients.

  3. Thermosensitive omsA mutation of Escherichia coli that causes thermoregulated release of periplasmic proteins.

    PubMed

    Tsuruoka, T; Ito, M; Tomioka, S; Hirata, A; Matsuhashi, M

    1988-11-01

    A mutant of Escherichia coli with a thermosensitive defect, possibly in the outer membrane (omsA mutant), was isolated from E. coli K-12 by mutagenization and selection for thermosensitivity and beta-lactam supersensitivity of growth. The mutant also showed very high sensitivity to other antibiotics, such as macarbomycin, midecamycin, rifampin, and bacitracin. The mutation was recessive to the wild type and was mapped at about 4 min on the E. coli chromosome between fhuA and metD. The mutation caused rapid release into the medium of periplasmic enzymes such as RTEM penicillinase but practically no cytoplasmic enzyme when cells grown at 30 degrees C were transferred to 37 or 42 degrees C. Electron microscopic observations showed many large double-layered vesicles attached to the surface of cells incubated at 42 degrees C. We conclude that the mutant had a mutation that caused a temperature-dependent defect in the outer membrane structure or its assembly (named an oms mutation). The omsA mutant may be useful for production of periplasmic proteins, which it releases into the culture medium on shift up of temperature.

  4. Escherichia coli Unsaturated Fatty Acid Synthesis

    PubMed Central

    Feng, Youjun; Cronan, John E.

    2009-01-01

    Although the unsaturated fatty acid (UFA) synthetic pathway of Escherichia coli is the prototype of such pathways, several unresolved issues have accumulated over the years. The key players are the fabA and fabB genes. Earlier studies of fabA transcription showed that the gene was transcribed from two promoters, with one being positively regulated by the FadR protein. The other weaker promoter (which could not be mapped with the technology then available) was considered constitutive because its function was independent of FadR. However, the FabR negative regulator was recently shown to represses fabA transcription. We report that the weak promoter overlaps the FadR-dependent promoter and is regulated by FabR. This promoter is strictly conserved in all E. coli and Salmonella enterica genomes sequenced to date and is thought to provide insurance against inappropriate regulation of fabA transcription by exogenous saturated fatty acids. Also, the fabAup promoter, a mutant promoter previously isolated by selection for increased FabA activity, was shown to be a promoter created de novo by a four-base deletion within the gene located immediately upstream of fabA. Demonstration of the key UFA synthetic reaction catalyzed by FabB has been elusive, although it was known to catalyze an elongation reaction. Strains lacking FabB are UFA auxotrophs indicating that the enzyme catalyzes an essential step in UFA synthesis. Using thioesterases specific for hydrolysis of short chain acyl-ACPs, the intermediates of the UFA synthetic pathway have been followed in vivo for the first time. These experiments showed that a fabB mutant strain accumulated less cis-5-dodecenoic acid than the parental wild-type strain. These data indicate that the key reaction in UFA synthesis catalyzed by FabB is elongation of the cis-3-decenoyl-ACP produced by FabA. PMID:19679654

  5. How much territory can a single E. coli cell control?

    PubMed Central

    El-Hajj, Ziad W.; Newman, Elaine B.

    2015-01-01

    Bacteria have been traditionally classified in terms of size and shape and are best known for their very small size. Escherichia coli cells in particular are small rods, each 1–2 μ. However, the size varies with the medium, and faster growing cells are larger because they must have more ribosomes to make more protoplasm per unit time, and ribosomes take up space. Indeed, Maaløe’s experiments on how E. coli establishes its size began with shifts between rich and poor media. Recently much larger bacteria have been described, including Epulopiscium fishelsoni at 700 μm and Thiomargarita namibiensis at 750 μm. These are not only much longer than E. coli cells but also much wider, necessitating considerable intracellular organization. Epulopiscium cells for instance, at 80 μm wide, enclose a large enough volume of cytoplasm to present it with major transport problems. This review surveys E. coli cells much longer than those which grow in nature and in usual lab cultures. These include cells mutated in a single gene (metK) which are 2–4 × longer than their non-mutated parent. This metK mutant stops dividing when slowly starved of S-adenosylmethionine but continues to elongate to 50 μm and more. FtsZ mutants have been routinely isolated as long cells which form during growth at 42°C. The SOS response is a well-characterized regulatory network that is activated in response to DNA damage and also results in cell elongation. Our champion elongated E. coli is a metK strain with a further, as yet unidentified mutation, which reaches 750 μm with no internal divisions and no increase in width. PMID:25954251

  6. Selection of chemotaxis mutants of Dictyostelium discoideum

    PubMed Central

    1987-01-01

    A method has been developed for the efficient selection of chemotaxis mutants of Dictyostelium discoideum. Mutants defective in the chemotactic response to folate could be enriched up to 30-fold in one round of selection using a chamber in which a compartment that contained the chemoattractant was separated by a sandwich of four nitrocellulose filters from a compartment that contained buffer. Mutagenized cells were placed in the center of the filter layer and exposed to the attractant gradient built up between the compartments for a period of 3-4 h. While wild-type cells moved through the filters in a wave towards the compartment that contained attractant, mutant cells remained in the filter to which they were applied. After several repetitions of the selection procedure, mutants defective in chemotaxis made up 10% of the total cell population retained in that filter. Mutants exhibiting three types of alterations were collected: motility mutants with either reduced speed of movement, or altered rates of turning; a single mutant defective in production of the attractant- degrading enzyme, folate deaminase; and mutants with normal motility but reduced chemotactic responsiveness. One mutant showed drastically reduced sensitivity in folate-induced cGMP production. Morphogenetic alterations of mutants defective in folate chemotaxis are described. PMID:3793759

  7. Superoxide dismutase and the resistance of Escherichia coli to phagocytic killing by human neutrophils.

    PubMed Central

    Papp-Szabò, E; Sutherland, C L; Josephy, P D

    1993-01-01

    Transformation of Escherichia coli K-12-derived strains with a plasmid carrying the genetic determinants for synthesis of lipopolysaccharide O antigen by Shigella dysenteriae allows the construction of phenotypically smooth derivatives. We show that such E. coli K-12 derivatives are highly resistant to killing by human serum. Isogenic wild-type and sodB mutant (Fe superoxide dismutase-deficient) strains were constructed. The results of experiments on phagocytic killing of these strains by human neutrophils are reported. We observed no difference between the sensitivities of wild-type and sodB mutant strains to phagocytic killing, in contrast to the results reported by other researchers who used species other than E. coli or strains other than K-12. Images PMID:8454348

  8. Impact of metal ion homeostasis of genetically modified Escherichia coli Nissle 1917 and K12 (W3110) strains on colonization properties in the murine intestinal tract.

    PubMed

    Kupz, Andreas; Fischer, André; Nies, Dietrich H; Grass, Gregor; Göbel, Ulf B; Bereswill, Stefan; Heimesaat, Markus M

    2013-09-01

    Metal ions are integral parts of pro- as well as eukaryotic cell homeostasis. Escherichia coli proved a valuable in vitro model organism to elucidate essential mechanisms involved in uptake, storage, and export of metal ions. Given that E. coli Nissle 1917 is able to overcome murine colonization resistance, we generated several E. coli Nissle 1917 mutants with defects in zinc, iron, copper, nickel, manganese homeostasis and performed a comprehensive survey of the impact of metal ion transport and homeostasis for E. coli colonization capacities within the murine intestinal tract. Seven days following peroral infection of conventional mice with E. coli Nissle 1917 strains exhibiting defined defects in zinc or iron uptake, the respective mutant and parental strains could be cultured at comparable, but low levels from the colonic lumen. We next reassociated gnotobiotic mice in which the microbiota responsible for colonization resistance was abrogated by broad-spectrum antibiotics with six different E. coli K12 (W3110) mutants. Seven days following peroral challenge, each mutant and parental strain stably colonized duodenum, ileum, and colon at comparable levels. Taken together, defects in zinc, iron, copper, nickel, and manganese homeostasis do not compromise colonization capacities of E. coli in the murine intestinal tract.

  9. The Escherichia coli argW-dsdCXA Genetic Island Is Highly Variable, and E. coli K1 Strains Commonly Possess Two Copies of dsdCXA

    PubMed Central

    Moritz, Rebecca L.; Welch, Rodney A.

    2006-01-01

    The genome sequences of Escherichia coli pathotypes reveal extensive genetic variability in the argW-dsdCXA island. Interestingly, the archetype E. coli K1 neonatal meningitis strain, strain RS218, has two copies of the dsdCXA genes for d-serine utilization at the argW and leuX islands. Because the human brain contains d-serine, an epidemiological study emphasizing K1 isolates surveyed the dsdCXA copy number and function. Forty of 41 (97.5%) independent E. coli K1 isolates could utilize d-serine. Southern blot hybridization revealed physical variability within the argW-dsdC region, even among 22 E. coli O18:K1:H7 isolates. In addition, 30 of 41 K1 strains, including 21 of 22 O18:K1:H7 isolates, had two dsdCXA loci. Mutational analysis indicated that each of the dsdA genes is functional in a rifampin-resistant mutant of RS218, mutant E44. The high percentage of K1 strains that can use d-serine is in striking contrast to our previous observation that only 4 of 74 (5%) isolates in the diarrheagenic E. coli (DEC) collection have this activity. The genome sequence of diarrheagenic E. coli isolates indicates that the csrRAKB genes for sucrose utilization are often substituted for dsdC and a portion of dsdX present at the argW-dsdCXA island of extraintestinal isolates. Among DEC isolates there is a reciprocal pattern of sucrose fermentation versus d-serine utilization. The ability to use d-serine is a trait strongly selected for among E. coli K1 strains, which have the ability to infect a wide range of extraintestinal sites. Conversely, diarrheagenic E. coli pathotypes appear to have substituted sucrose for d-serine as a potential nutrient. PMID:17088369

  10. In vitro and in vivo activities of T4 endonuclease V mutants altered in the C-terminal aromatic region

    SciTech Connect

    Ishida, M.; Kanamori, Y.; Hori, N.; Inaoka, T.; Ohtsuka, E. )

    1990-04-24

    Genes encoding mutants of the thymine photodimer repair enzyme from bacteriophage T4 (T4 endonuclease V) having an amino acid substitution (T127M, W128A, W128S, Y129A, K130L, Y131A, Y132A) were constructed by use of a previously obtained synthetic gene and expressed in Escherichia coli under the control of the E. coli tryptophan promoter. An in vitro assay of partially fractionated mutant proteins for glycosylase activity was performed with chemically synthesized substrates containing a thymine photodimer. T127M and K130L showed almost the same activity as the wild-type protein. Although W128S, Y131A, and Y132A were slightly active, W128A and Y129A lost activity. The results indicated that the aromatic amino acids around position 130 may be important for the glycosylase activity. Mutant T127M was purified, and the Km value was found to be of the same order as that of the wild type (10(-8) M). In vivo activities for all mutants were characterized with UV-sensitive E. coli. The results showed that substitution of Thr-127 with Met or Lys-130 with Leu did not have an effect on the survival of the bacteria but substitution of aromatic amino acids (128-132) had various effects on survival.

  11. A retrospective: Use of Escherichia coli as a vehicle to study phospholipid synthesis and function

    PubMed Central

    Dowhan, William

    2012-01-01

    Although the study of individual phospholipids and their synthesis began in the 1920’s first in plants and then mammals, it was not until the early 1960’s that Eugene Kennedy using Escherichia coli initiated studies of bacterial phospholipid metabolism. With the base of information already available from studies of mammalian tissue, the basic blueprint of phospholipid biosynthesis in E. coli was worked out by the late 1960’s. In 1970’s and 1980’s most of the enzymes responsible for phospholipid biosynthesis were purified and many of the genes encoding these enzymes were identified. By the late 1990’s conditional and null mutants were available along with clones of the genes for every step of phospholipid biosynthesis. Most of these genes had been sequenced before the complete E. coli genome sequence was available. Strains of E. coli were developed in which phospholipid composition could be changed in a systematic manner while maintaining cell viability. Null mutants, strains in which phospholipid metabolism was artificially regulated, and strains synthesizing foreign lipids not found in E. coli have been used to this day to define specific roles for individual phospholipid. This review will trace the findings that have led to the development of E. coli as an excellent model system to study mechanisms underlying the synthesis and function of phospholipids that are widely applicable to other prokaryotic and eukaryotic systems. PMID:22925633

  12. Targeting ESR1-Mutant Breast Cancer

    DTIC Science & Technology

    2015-09-01

    Award Number: W81XWH-14-1-0360 TITLE: Targeting ESR1- Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Geoffrey L. Greene, Ph.D. CONTRACTING...ADDRESS. 1. REPORT DATE September 2015 2. REPORT TYPE Annual 3. DATES COVERED 1 Sep 2014 - 31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1- Mutant ...approved hormonal therapies and that more potent, selective estrogen receptor degraders (SERDs) will enable complete inhibition of mutant ER signaling and

  13. Targeting ESR1-Mutant Breast Cancer

    DTIC Science & Technology

    2015-09-01

    AWARD NUMBER: W81XWH-14-1-0359 TITLE: Targeting ESR1- Mutant Breast Cancer PRINCIPAL INVESTIGATOR: Dr. Sarat Chandarlapaty CONTRACTING...31 Aug 2015 4. TITLE AND SUBTITLE Targeting ESR1- Mutant Breast Cancer 5a. CONTRACT NUMBER 5b. GRANT NUMBER W81XWH-14-1-0359 5c. PROGRAM ELEMENT...current FDA approved hormonal therapies and that more potent, selective estrogen receptor degraders (SERDs) will enable complete inhibition of mutant

  14. Bacterial Resistance Studies Using In Vitro Dynamic Models: the Predictive Power of the Mutant Prevention and Minimum Inhibitory Antibiotic Concentrations

    PubMed Central

    Strukova, Elena N.; Shlykova, Darya S.; Portnoy, Yury A.; Kozyreva, Varvara K.; Edelstein, Mikhail V.; Dovzhenko, Svetlana A.; Kobrin, Mikhail B.; Zinner, Stephen H.

    2013-01-01

    In light of the concept of the mutant selection window, i.e., the range between the MIC and the mutant prevention concentration (MPC), MPC-related pharmacokinetic indices should be more predictive of bacterial resistance than the respective MIC-related indices. However, experimental evidence of this hypothesis remains limited and contradictory. To examine the predictive power of the ratios of the area under the curve (AUC24) to the MPC and the MIC, the selection of ciprofloxacin-resistant mutants of four Escherichia coli strains with different MPC/MIC ratios was studied. Each organism was exposed to twice-daily ciprofloxacin for 3 days at AUC24/MIC ratios that provide peak antibiotic concentrations close to the MIC, between the MIC and the MPC, and above the MPC. Resistant E. coli was intensively enriched at AUC24/MPCs from 1 to 10 h (AUC24/MIC from 60 to 360 h) but not at the lower or higher AUC24/MPC and AUC24/MIC ratios. AUC24/MPC and AUC24/MIC relationships of the areas under the time courses of ciprofloxacin-resistant E. coli (AUBCM) were bell-shaped. A Gaussian-like function fits the AUBCM-AUC24/MPC and AUBCM-AUC24/MIC data combined for all organisms (r2 = 0.69 and 0.86, respectively). The predicted anti-mutant AUC24/MPC ratio was 58 ± 35 h, and the respective AUC24/MIC ratio was 1,080 ± 416 h. Although AUC24/MPC was less predictive of strain-independent E. coli resistance than AUC24/MIC, the established anti-mutant AUC24/MPC ratio was closer to values reported for Staphylococcus aureus (60 to 69 h) than the respective AUC24/MIC ratio (1,080 versus 200 to 240 h). This implies that AUC24/MPC might be a better interspecies predictor of bacterial resistance than AUC24/MIC. PMID:23896481

  15. Electrophysiological study of Drosophila rhodopsin mutants

    PubMed Central

    1986-01-01

    Electrophysiological investigations were carried out on several independently isolated mutants of the ninaE gene, which encodes opsin in R1-6 photoreceptors, and a mutant of the ninaD gene, which is probably important in the formation of the rhodopsin chromophore. In these mutants, the rhodopsin content in R1-6 photoreceptors is reduced by 10(2)-10(6)-fold. Light-induced bumps recorded from even the most severely affected mutants are physiologically normal. Moreover, a detailed noise analysis shows that photoreceptor responses of both a ninaE mutant and a ninaD mutant follow the adapting bump model. Since any extensive rhodopsin-rhodopsin interactions are not likely in these mutants, the above results suggest that such interactions are not needed for the generation and adaptation of light-induced bumps. Mutant bumps are strikingly larger in amplitude than wild-type bumps. This difference is observed both in ninaD and ninaE mutants, which suggests that it is due to severe depletion of rhodopsin content, rather than to any specific alterations in the opsin protein. Lowering or buffering the intracellular calcium concentration by EGTA injection mimics the effects of the mutations on the bump amplitude, but, unlike the mutations, it also affects the latency and kinetics of light responses. PMID:3097245

  16. Identifying representative drug resistant mutants of HIV

    PubMed Central

    2015-01-01

    Background Drug resistance is one of the most important causes for failure of anti-AIDS treatment. During therapy, multiple mutations accumulate in the HIV genome, eventually rendering the drugs ineffective in blocking replication of the mutant virus. The huge number of possible mutants precludes experimental analysis to explore the molecular mechanisms of resistance and develop improved antiviral drugs. Results In order to solve this problem, we have developed a new algorithm to reveal the most representative mutants from the whole drug resistant mutant database based on our newly proposed unified protein sequence and 3D structure encoding method. Mean shift clustering and multiple regression analysis were applied on genotype-resistance data for mutants of HIV protease and reverse transcriptase. This approach successfully chooses less than 100 mutants with the highest resistance to each drug out of about 10K in the whole database. When considering high level resistance to multiple drugs, the numbers reduce to one or two representative mutants. Conclusion This approach for predicting the most representative mutants for each drug has major importance for experimental verification since the results provide a small number of representative sequences, which will be amenable for in vitro testing and characterization of the expressed mutant proteins. PMID:26678327

  17. Molecular analysis of the Deinococcus radiodurans recA locus and identification of a mutation site in a DNA repair-deficient mutant, rec30.

    PubMed

    Narumi, I; Satoh, K; Kikuchi, M; Funayama, T; Kitayama, S; Yanagisawa, T; Watanabe, H; Yamamoto, K

    1999-12-07

    Deinococcus radiodurans strain rec30, which is a DNA damage repair-deficient mutant, has been estimated to be defective in the deinococcal recA gene. To identify the mutation site of strain rec30 and obtain information about the region flanking the gene, a 4.4-kb fragment carrying the wild-type recA gene was sequenced. It was revealed that the recA locus forms a polycistronic operon with the preceding cistrons (orf105a and orf105b). Predicted amino acid sequences of orf105a and orf105b showed substantial similarity to the competence-damage inducible protein (cinA gene product) from Streptococcus pneumoniae and the 2'-5' RNA ligase from Escherichia coli, respectively. By analyzing polymerase chain reaction (PCR) fragments derived from the genomic DNA of strain rec30, the mutation site in the strain was identified as a single G:C to A:T transition which causes an amino acid substitution at position 224 (Gly to Ser) of the deinococcal RecA protein. Furthermore, we succeeded in expressing both the wild-type and mutant recA genes of D. radiodurans in E. coli without any obvious toxicity or death. The gamma-ray resistance of an E. coli recA1 strain was fully restored by the expression of the wild-type recA gene of D. radiodurans that was cloned in an E. coli vector plasmid. This result is consistent with evidence that RecA proteins from many bacterial species can functionally complement E. coli recA mutants. In contrast with the wild-type gene, the mutant recA gene derived from strain rec30 did not complement E. coli recA1, suggesting that the mutant RecA protein lacks functional activity for recombinational repair.

  18. In vitro selection of resistance to pradofloxacin and ciprofloxacin in canine uropathogenic Escherichia coli isolates.

    PubMed

    Liu, Xiaoqiang; Lazzaroni, Caterina; Aly, Sherine A; Thungrat, Kamoltip; Boothe, Dawn M

    2014-12-05

    This study explored and compared the mechanisms and selective concentration of resistance between a 3rd (pradofloxacin) and 2nd (ciprofloxacin) generation fluoroquinolone. Pradofloxacin- and ciprofloxacin-resistant mutants were selected by stepwise exposure of Escherichia coli (E. coli) to escalating concentrations of pradofloxacin and ciprofloxacin. The sequence of the quinolone resistance determining region (QRDR) and the transcriptional regulator soxS were analyzed, and efflux pump AcrAB-TolC activity was measured by quantitative real-time reverse transcription-PCR (qRT-PCR). First-step mutants reduced the fluoroquinolone sensitivity and one mutant bore a single substitution in gyrA. Four of six second-step mutants expressed ciprofloxacin resistance, and displayed additional mutations in gyrA and/or parC, while these mutants retained susceptibility to pradofloxacin. All the third-step mutants were fluoroquinolone resistant, and each expressed multidrug resistance (MDR) phenotypes. Further, they displayed resistance to all antibacterials tested except cefotaxime, ceftazidime and meropenem. The number of mutations in QRDR of gyrA and parC correlated with fluoroquinolone MICs. Mutations in parC were not common in pradofloxacin-associated mutants. Moreover, one second- and one third-step ciprofloxacin-associated mutants bore both mutations at position 12 (Ala12Ser) and 78 (Met78Leu) in the soxS gene, yet no mutations in the soxS gene were detected in the pradofloxacin-selected mutants. Altogether, these results demonstrated that resistance emerged relatively more rapidly in 2nd compared to 3rd generation fluoroquinolones. Point mutations in gyrA were a key mechanism of resistance to pradofloxacin, and overexpression of efflux pump gene acrB played a potential role in the emergence of MDR phenotypes identified in this study.

  19. Mutants of staphylococcal toxic shock syndrome toxin 1: mitogenicity and recognition by a neutralizing monoclonal antibody.

    PubMed Central

    Blanco, L; Choi, E M; Connolly, K; Thompson, M R; Bonventre, P F

    1990-01-01

    Toxic shock syndrome toxin 1 (TSST-1), a 22-kilodalton protein made by strains of Staphylococcus aureus harboring the chromosomal toxin gene, may elicit toxic shock syndrome in humans. In vitro, TSST-1 induces T cells to proliferate and macrophages to secrete interleukin-1. To conduct a structure-function analysis, point mutations on the TSST-1 gene were generated by site-directed mutagenesis to identify amino acids critical for activity of the toxin. Specific tyrosine and histidine residues were replaced by alanines. Wild-type and mutant TSST-1 gene constructs were expressed in Escherichia coli, and the products were tested for their mitogenic potential and reactivity with a TSST-1 neutralizing monoclonal antibody (MAb 8-5-7). Four of the mutants were similar to the wild type; i.e., the mutant toxins stimulated murine T cells and reacted with MAb 8-5-7 equally as well as the wild type. Two mutants exhibited a decrease in mitogenic activity, but one of these retained the capacity to bind with MAb 8-5-7 while the other was no longer recognized by the same antibody. One double mutant demonstrated minimal mitogenic activity and did not react in enzyme-linked immunosorbent and immunoblot assays with MAb 8-5-7. The data show that specific residues near the carboxy terminus of TSST-1 are essential for mitogenic activity and in forming the epitope recognized by neutralizing MAb 8-5-7. Images PMID:1696937

  20. Viable transmembrane region mutants of bacteriophage M13 coat protein prepared by site-directed mutagenesis.

    PubMed

    Li, Z; Deber, C M

    1991-10-31

    Bacteriophage M13 coat protein - a 50-residue protein located at the E. coli host membrane during phage reproduction - is subjected to cytoplasmic, membrane-bound, and DNA-interactive environments during the phage life cycle. In research to examine the specific features of primary/secondary structure in the effective transmembrane (TM) region of the protein (residues 21-39: YIGYAWAMVVVIVGATIGI) which modulate its capacity to respond conformationally to the progressive influences of these varying environments, we have prepared over two dozen viable mutant phages with alterations in their coat protein TM regions. Mutants were obtained through use of site-directed mutagenesis techniques in combination with three "randomized" oligonucleotides which spanned the TM region. No subcloning was required. Among mutations observed were those in which each of the four TM Val residues was changed to Ala, and several with increased Ser or Thr content, including one double Ser mutant (G23S-A25S). Polar substitutions arising at Gly23 and Tyr24-including G23D, Y24H, Y24D and Y24N-suggested that this local segment resides external to the host membrane. Milligram quantities of mutant coat proteins are obtained by growing M13 mutant phages in liter preparations, with isotopic (e.g., 13C) labelling at desired sites, for subsequent characterization and conformational analysis in membrane-mimetic media.

  1. Production of hydroxycinnamoyl-shikimates and chlorogenic acid in Escherichia coli: production of hydroxycinnamic acid conjugates

    PubMed Central

    2013-01-01

    Background Hydroxycinnamates (HCs) are mainly produced in plants. Caffeic acid (CA), p-coumaric acid (PA), ferulic acid (FA) and sinapic acid (SA) are members of the HC family. The consumption of HC by human might prevent cardiovascular disease and some types of cancer. The solubility of HCs is increased through thioester conjugation to various compounds such as quinic acid, shikimic acid, malic acid, anthranilic acid, and glycerol. Although hydroxycinnamate conjugates can be obtained from diverse plant sources such as coffee, tomato, potato, apple, and sweet potato, some parts of the world have limited availability to these compounds. Thus, there is growing interest in producing HC conjugates as nutraceutical supplements. Results Hydroxycinnamoyl transferases (HCTs) including hydroxycinnamate-CoA shikimate transferase (HST) and hydroxycinnamate-CoA quinate transferase (HQT) were co-expressed with 4-coumarateCoA:ligase (4CL) in Escherichia coli cultured in media supplemented with HCs. Two hydroxycinnamoyl conjugates, p-coumaroyl shikimates and chlorogenic acid, were thereby synthesized. Total 29.1 mg/L of four different p-coumaroyl shikimates (3-p-coumaroyl shikimate, 4-p-coumaroyl shikimate, 3,4-di-p-coumaroyl shikimate, 3,5-di-p-coumaroyl shikimate, and 4,5-di-p-coumaroyl shikimate) was obtained and 16 mg/L of chlorogenic acid was synthesized in the wild type E. coli strain. To increase the concentration of endogenous acceptor substrates such as shikimate and quinate, the shikimate pathway in E. coli was engineered. A E. coli aroL and aroK gene were mutated and the resulting mutants were used for the production of p-coumaroyl shikimate. An E. coli aroD mutant was used for the production of chlorogenic acid. We also optimized the vector and cell concentration optimization. Conclusions To produce p-coumaroyl-shikimates and chlorogenic acid in E. coli, several E. coli mutants (an aroD mutant for chlorogenic acid production; an aroL, aroK, and aroKL mutant for p

  2. Mutant calreticulin requires both its mutant C-terminus and the thrombopoietin receptor for oncogenic transformation

    PubMed Central

    Elf, Shannon; Abdelfattah, Nouran S.; Chen, Edwin; Perales-Patón, Javier; Rosen, Emily A.; Ko, Amy; Peisker, Fabian; Florescu, Natalie; Giannini, Silvia; Wolach, Ofir; Morgan, Elizabeth A.; Tothova, Zuzana; Losman, Julie-Aurore; Schneider, Rebekka K.; Al-Shahrour, Fatima; Mullally, Ann

    2016-01-01

    Somatic mutations in calreticulin (CALR) are present in approximately 40% of patients with myeloproliferative neoplasms (MPN) but the mechanism by which mutant CALR is oncogenic remains unclear. Here, we demonstrate that expression of mutant CALR alone is sufficient to engender MPN in mice and recapitulates the disease phenotype of CALR-mutant MPN patients. We further show that the thrombopoietin receptor, MPL is required for mutant CALR-driven transformation through JAK-STAT pathway activation, thus rendering mutant CALR-transformed hematopoietic cells sensitive to JAK2 inhibition. Finally, we demonstrate that the oncogenicity of mutant CALR is dependent on the positive electrostatic charge of the C-terminus of the mutant protein, which is necessary for physical interaction between mutant CALR and MPL. Together, our findings elucidate a novel paradigm of cancer pathogenesis and reveal how CALR mutations induce MPN. PMID:26951227

  3. Elevated Mutagenesis Does Not Explain the Increased Frequency of Antibiotic Resistant Mutants in Starved Aging Colonies

    PubMed Central

    Katz, Sophia; Hershberg, Ruth

    2013-01-01

    The frequency of mutants resistant to the antibiotic rifampicin has been shown to increase in aging (starved), compared to young colonies of Eschierchia coli. These increases in resistance frequency occur in the absence of any antibiotic exposure, and similar increases have also been observed in response to additional growth limiting conditions. Understanding the causes of such increases in the frequency of resistance is important for understanding the dynamics of antibiotic resistance emergence and spread. Increased frequency of rifampicin resistant mutants in aging colonies is cited widely as evidence of stress-induced mutagenesis (SIM), a mechanism thought to allow bacteria to increase mutation rates upon exposure to growth-limiting stresses. At the same time it has been demonstrated that some rifampicin resistant mutants are relatively fitter in aging compared to young colonies, indicating that natural selection may also contribute to increased frequency of rifampicin resistance in aging colonies. Here, we demonstrate that the frequency of mutants resistant to both rifampicin and an additional antibiotic (nalidixic-acid) significantly increases in aging compared to young colonies of a lab strain of Escherichia coli. We then use whole genome sequencing to demonstrate conclusively that SIM cannot explain the observed magnitude of increased frequency of resistance to these two antibiotics. We further demonstrate that, as was previously shown for rifampicin resistance mutations, mutations conferring nalidixic acid resistance can also increase fitness in aging compared to young colonies. Our results show that increases in the frequency of antibiotic resistant mutants in aging colonies cannot be seen as evidence of SIM. Furthermore, they demonstrate that natural selection likely contributes to increases in the frequency of certain antibiotic resistance mutations, even when no selection is exerted due to the presence of antibiotics. PMID:24244205

  4. Alanine-scanning mutagenesis reveals a cytosine deaminase mutant with altered substrate preference.

    PubMed

    Mahan, Sheri D; Ireton, Greg C; Stoddard, Barry L; Black, Margaret E

    2004-07-20

    Suicide gene therapy of cancer is a method whereby cancerous tumors can be selectively eradicated while sparing damage to normal tissue. This is accomplished by delivering a gene, encoding an enzyme capable of specifically converting a nontoxic prodrug into a cytotoxin, to cancer cells followed by prodrug administration. The Escherichia coli gene, codA, encodes cytosine deaminase and is introduced into cancer cells followed by administration of the prodrug 5-fluorocytosine (5-FC). Cytosine deaminase converts 5-FC into cytotoxic 5-fluorouracil, which leads to tumor-cell eradication. One limitation of this enzyme/prodrug combination is that 5-FC is a poor substrate for bacterial cytosine deaminase. The crystal structure of bacterial cytosine deaminase (bCD) reveals that a loop structure in the active site pocket of wild-type bCD comprising residues 310-320 undergoes a conformational change upon cytosine binding, making several contacts to the pyrimidine ring. Alanine-scanning mutagenesis was used to investigate the structure-function relationship of amino acid residues within this region, especially with regard to substrate specificity. Using an E. coli genetic complementation system, seven active mutants were identified (F310A, G311A, H312A, D314A, V315A, F316A, and P318A). Further characterization of these mutants reveals that mutant F316A is 14-fold more efficient than the wild-type at deaminating cytosine to uracil. The mutant D314A enzyme demonstrates a dramatic decrease in cytosine activity (17-fold) as well as a slight increase in activity toward 5-FC (2-fold), indicating that mutant D314A prefers the prodrug over cytosine by almost 20-fold, suggesting that it may be a superior suicide gene.

  5. Monomeric yeast PCNA mutants are defective in interacting with and stimulating the ATPase activity of RFC.

    PubMed

    Ionescu, Costin N; Shea, Kathleen A; Mehra, Rajendra; Prundeanu, Lucia; McAlear, Michael A

    2002-10-29

    Yeast PCNA is a homo-trimeric, ring-shaped DNA polymerase accessory protein that can encircle duplex DNA. The integrity of this multimeric sliding DNA clamp is maintained through the protein-protein interactions at the interfaces of adjacent subunits. To investigate the importance of trimer stability for PCNA function, we introduced single amino acid substitutions at residues (A112T, S135F) that map to opposite ends of the monomeric protein. Recombinant wild-type and mutant PCNAs were purified from E. coli, and they were tested for their properties in vitro. Unlike the stable wild-type PCNA trimers, the mutant PCNA proteins behaved as monomers when diluted to low nanomolar concentrations. In contrast to what has been reported for a monomeric form of the beta clamp in E. coli, the monomeric PCNAs were compromised in their ability to interact with their associated clamp loader, replication factor C (RFC). Similarly, monomeric PCNAs were not effective in stimulating the ATPase activity of RFC. The mutant PCNAs were able to form mixed trimers with wild-type subunits, although these mixed trimers were unstable when loaded onto DNA. They were able to function as weak DNA polymerase delta processivity factors in vitro, and when the monomeric PCNA-41 (A112T, S135F double mutant) allele was introduced as the sole source of PCNA in vivo, the cells were viable and healthy. These pol30-41 mutants were, however, sensitive to UV irradiation and to the DNA damaging agent methylmethane sulfonate, implying that DNA repair pathways have a distinct requirement for stable DNA clamps.

  6. Enhancers of Conidiation Mutants in Aspergillus Nidulans

    PubMed Central

    Gems, D. H.; Clutterbuck, A. J.

    1994-01-01

    Mutants at a number of loci, designated sthenyo, have been isolated as enhancers of the oligoconidial mutations at the medA locus. Two loci have been mapped: sthA on linkage group I, and sthB on linkage group V. Two probable alleles have been identified at each locus but two further mutants were unlinked to either sthA or sthB. Neither sthA nor sthB mutants have conspicuous effects on morphology on their own, nor could the sthA1 sthB2 double mutant be distinguished from wild type. Mutants at both loci also interact with the temperature-sensitive brlA42 mutant at the permissive temperature to give a phenotype described as ``Abacoid.'' sthA1 also induces a slight modification of the phenotype of an abaA mutant. We conclude that sthenyo genes act mainly at the phialide stage of conidiation. We also describe the isolation of new medA mutants arising spontaneously as outgrowths on brlA42 colonies. PMID:8056325

  7. Regulation of Mutant p53 Protein Expression.

    PubMed

    Vijayakumaran, Reshma; Tan, Kah Hin; Miranda, Panimaya Jeffreena; Haupt, Sue; Haupt, Ygal

    2015-01-01

    For several decades, p53 has been detected in cancer biopsies by virtue of its high protein expression level which is considered indicative of mutation. Surprisingly, however, mouse genetic studies revealed that mutant p53 is inherently labile, similar to its wild type (wt) counterpart. Consistently, in response to stress conditions, both wt and mutant p53 accumulate in cells. While wt p53 returns to basal level following recovery from stress, mutant p53 remains stable. In part, this can be explained in mutant p53-expressing cells by the lack of an auto-regulatory loop with Mdm2 and other negative regulators, which are pivotal for wt p53 regulation. Further, additional protective mechanisms are acquired by mutant p53, largely mediated by the co-chaperones and their paralogs, the stress-induced heat shock proteins. Consequently, mutant p53 is accumulated in cancer cells in response to chronic stress and this accumulation is critical for its oncogenic gain of functions (GOF). Building on the extensive knowledge regarding wt p53, the regulation of mutant p53 is unraveling. In this review, we describe the current understanding on the major levels at which mutant p53 is regulated. These include the regulation of p53 protein levels by microRNA and by enzymes controlling p53 proteasomal degradation.

  8. A halotolerant mutant of Saccharomyces cerevisiae.

    PubMed Central

    Gaxiola, R; Corona, M; Zinker, S

    1996-01-01

    FRD, a nuclear and dominant spontaneous mutant of Saccharomyces cerevisiae capable of growing in up to 2 M NaCl, was isolated. Compared with parental cells, the mutant cells have a lower intracellular Na+/K+ ratio, shorter generation times in the presence of 1 M NaCl, and alterations in gene expression. PMID:8631691

  9. Phenotype Sequencing: Identifying the Genes That Cause a Phenotype Directly from Pooled Sequencing of Independent Mutants

    PubMed Central

    Harper, Marc A.; Chen, Zugen; Toy, Traci; Machado, Iara M. P.; Nelson, Stanley F.; Liao, James C.; Lee, Christopher J.

    2011-01-01

    Random mutagenesis and phenotype screening provide a powerful method for dissecting microbial functions, but their results can be laborious to analyze experimentally. Each mutant strain may contain 50–100 random mutations, necessitating extensive functional experiments to determine which one causes the selected phenotype. To solve this problem, we propose a “Phenotype Sequencing” approach in which genes causing the phenotype can be identified directly from sequencing of multiple independent mutants. We developed a new computational analysis method showing that 1. causal genes can be identified with high probability from even a modest number of mutant genomes; 2. costs can be cut many-fold compared with a conventional genome sequencing approach via an optimized strategy of library-pooling (multiple strains per library) and tag-pooling (multiple tagged libraries per sequencing lane). We have performed extensive validation experiments on a set of E. coli mutants with increased isobutanol biofuel tolerance. We generated a range of sequencing experiments varying from 3 to 32 mutant strains, with pooling on 1 to 3 sequencing lanes. Our statistical analysis of these data (4099 mutations from 32 mutant genomes) successfully identified 3 genes (acrB, marC, acrA) that have been independently validated as causing this experimental phenotype. It must be emphasized that our approach reduces mutant sequencing costs enormously. Whereas a conventional genome sequencing experiment would have cost $7,200 in reagents alone, our Phenotype Sequencing design yielded the same information value for only $1200. In fact, our smallest experiments reliably identified acrB and marC at a cost of only $110–$340. PMID:21364744

  10. Production of 3-O-xylosyl quercetin in Escherichia coli.

    PubMed

    Pandey, Ramesh Prasad; Malla, Sailesh; Simkhada, Dinesh; Kim, Byung-Gee; Sohng, Jae Kyung

    2013-03-01

    Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/∆pgi, E. coli BL21(DE3)/∆zwf, E. coli BL21(DE3)/∆pgi∆zwf, and E. coli BL21(DE3)/∆pgi∆zwf∆ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/∆pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/∆pgi∆zwf∆ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.

  11. Ultraviolet-Sensitive Mutator Strain of Escherichia coli K-12

    PubMed Central

    Siegel, Eli C.

    1973-01-01

    An ultraviolet (UV)-sensitive mutator gene, mutU, was identified in Escherichia coli K-12. The mutation mutU4 is very close to uvrD, between metE and ilv, on the E. coli chromosome. It was recessive as a mutator and as a UV-sensitive mutation. The frequency of reversion of trpA46 on an F episome was increased by mutU4 on the chromosome. The mutator gene did not increase mutation frequencies in virulent phages or in lytically grown phage λ. The mutU4 mutation predominantly induced transitional base changes. Mutator strains were normal for recombination and host-cell reactivation of UV-irradiated phage T1. They were normally resistant to methyl methanesulfonate and were slightly more sensitive to gamma irradiation than Mut+ strains. UV irradiation induced mutations in a mutU4 strain, and phage λ was UV-inducible. Double mutants containing mutU4 and recA, B, or C were extremely sensitive to UV irradiation; a mutU4 uvrA6 double mutant was only slightly more sensitive than a uvrA6 strain. The mutU4 uvrA6 and mutU4 recA, B, or C double mutants had mutation rates similar to that of a mutU4 strain. Two UV-sensitive mutators, mut-9 and mut-10, isolated by Liberfarb and Bryson in E. coli B/UV, were found to be co-transducible with ilv in the same general region as mutU4. PMID:4345920

  12. Growth and Division of Filamentous Forms of Escherichia coli.

    PubMed

    Adler, H I; Hardigree, A A

    1965-07-01

    Adler, Howard I. (Oak Ridge National Laboratory, Oak Ridge, Tenn.), and Alice A. Hardigree. Growth and division of filamentous forms of Escherichia coli. J. Bacteriol. 90:223-226. 1965.-Cells of certain mutant strains of Escherichia coli grow into long multinucleate filaments after exposure to radiation. Deoxyribonucleic acid, ribonucleic acid, and protein synthesis proceed, but cytokinesis does not occur. Cytokinesis (cross-septation) can be initiated by exposure of the filaments to pantoyl lactone or a temperature of 42 C. If growing filaments are treated with mitomycin C, nuclear division does not occur, and nuclear material is confined to the central region of the filament. Cytokinesis cannot be induced in mitomycin C-treated filaments by pantoyl lactone or treatment at 42 C.

  13. Investigation of E. coli Enterotoxins.

    DTIC Science & Technology

    1976-08-01

    It has been determined that representative culture filtrates from two different strains (H197 and 74-114) of enterotoxigenic E . coli contain at least...for E . coli entorotoxin (soluble) and that trypsin-activated insol ECT is more antigenic than unactivated insol ECT. In contrast, it was determined...that cholera (ga) toxoid, with or without adjuvant, stimulates antitoxin capable of neutralizing both cholera and E . coli enterotoxins. It has been

  14. Mutational analysis of the lac regulatory region: second-site changes that activate mutant promoters.

    PubMed Central

    Rothmel, R K; LeClerc, J E

    1989-01-01

    Second-site mutations that restored activity to severe lacP1 down-promoter mutants were isolated. This was accomplished by using a bacteriophage f1 vector containing a fusion of the mutant E. coli lac promoters with the structural gene for chloramphenicol acetyltransferase (CAT), so that a system was provided for selecting phage revertants (or pseudorevertants) that conferred resistance of phage-infected cells to chloramphenicol. Among the second-site changes that relieved defects in mutant lac promoters, the only one that restored lacP1 activity was a T----G substitution at position -14, a weakly conserved site in E. coli promoters. Three other sequence changes, G----A at -2, A----T at +1, and C----A at +10, activated nascent promoters in the lac regulatory region. The nascent promoters conformed to the consensus rule, that activity is gained by sequence changes toward homology with consensus sequences at the -35 and -10 regions of the promoter. However, the relative activities of some promoters cannot be explained solely by consideration of their conserved sequence elements. Images PMID:2660105

  15. Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium

    SciTech Connect

    Kramer, G.F.; Ames, B.N.

    1988-02-01

    Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm/sup 5/Se/sup 2/U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO/sub 3//sup 2 -/) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm/sup 5/Se/sup 2/U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm/sup 5/Se/sup 2/U in tRNA may be in codon-anticodon interactions.

  16. Occurrence of homologs of the Escherichia coli lytB gene in gram-negative bacterial species.

    PubMed

    Potter, S; Yang, X; Boulanger, M J; Ishiguro, E E

    1998-04-01

    The Escherichia coli LytB protein regulates the activity of guanosine 3',5'-bispyrophosphate synthetase I (RelA). A Southern blot analysis of chromosomal DNA with the E. coli lytB gene as a probe revealed the presence of lytB homologs in all of the gram-negative bacterial species examined but not in gram-positive species. The lytB homologs from Enterobacter aerogenes and Pseudomonas fluorescens complemented the E. coli lytB44 mutant allele.

  17. crl mutants of Saccharomyces cerevisiae resemble both mutants affecting general control of amino acid biosynthesis and omnipotent translational suppressor mutants.

    PubMed

    McCusker, J H; Haber, J E

    1988-06-01

    Cyocloheximide resistant lethal (crl) mutants of Saccharomyces cerevisiae, defining 22 unlinked complementation groups, are unable to grow at 37 degrees. They are also highly pleiotropic at their permissive temperature of 25 degrees. The mutants are all unable to arrest at the G1 stage of the cell cycle when grown to stationary phase or when starved for a single amino acid, though they do arrest at G1 when deprived of all nitrogen. The crl mutants are also hypersensitive to various amino acid analogs and to 3-aminotriazole. These mutants also "tighten" leaky auxotrophic mutations that permit wild-type cells to grow in the absence of the appropriate amino acid. All of these phenotypes are also exhibited by gcn mutants affecting general control of amino acid biosynthesis. In addition, the crl mutants are all hypersensitive to hygromycin B, an aminoglycoside antibiotic that stimulates translational misreading. The crl mutations also suppress one nonsense mutation which is phenotypically suppressed by hygromycin B. Many crl mutants are also osmotically sensitive. These are phenotypes which the crl mutations have in common with previously isolated omnipotent suppressors. We suggest that the the crl mutations all affect the fidelity of protein translation.

  18. Dihydroxyacetone production in an engineered Escherichia coli through expression of Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase.

    PubMed

    Jain, Vishist Kumar; Tear, Crystal Jing Ying; Lim, Chan Yuen

    2016-05-01

    Dihydroxyacetone (DHA) has several industrial applications such as a tanning agent in tanning lotions in the cosmetic industry; its production via microbial fermentation would present a more sustainable option for the future. Here we genetically engineered Escherichia coli (E. coli) for DHA production from glucose. Deletion of E. coli triose phosphate isomerase (tpiA) gene was carried out to accumulate dihydroxyacetone phosphate (DHAP), for use as the main intermediate or precursor for DHA production. The accumulated DHAP was then converted to DHA through the heterologous expression of Corynebacterium glutamicum DHAP dephosphorylase (cghdpA) gene. To conserve DHAP exclusively for DHA production we removed methylglyoxal synthase (mgsA) gene in the ΔtpiA strain. This drastically improved DHA production from 0.83g/l (0.06g DHA/g glucose) in the ΔtpiA strain bearing cghdpA to 5.84g/l (0.41g DHA/g glucose) in the ΔtpiAΔmgsA double mutant containing the same gene. To limit the conversion of intracellular DHA to glycerol, glycerol dehydrogenase (gldA) gene was further knocked out resulting in a ΔtpiAΔmgsAΔgldA triple mutant. This triple mutant expressing the cghdpA gene produced 6.60g/l of DHA at 87% of the maximum theoretical yield. In summary, we demonstrated an efficient system for DHA production in genetically engineered E. coli strain.

  19. Compilation and analysis of Escherichia coli promoter DNA sequences.

    PubMed Central

    Hawley, D K; McClure, W R

    1983-01-01

    The DNA sequence of 168 promoter regions (-50 to +10) for Escherichia coli RNA polymerase were compiled. The complete listing was divided into two groups depending upon whether or not the promoter had been defined by genetic (promoter mutations) or biochemical (5' end determination) criteria. A consensus promoter sequence based on homologies among 112 well-defined promoters was determined that was in substantial agreement with previous compilations. In addition, we have tabulated 98 promoter mutations. Nearly all of the altered base pairs in the mutants conform to the following general rule: down-mutations decrease homology and up-mutations increase homology to the consensus sequence. PMID:6344016

  20. Gene expression caused by alkylating agents and cis-diamminedichloroplatinum(II) in Escherichia coli.

    PubMed

    Fram, R J; Crockett, J; Volkert, M R

    1988-09-01

    Previous work has demonstrated heterogeneous effects of methylating agents on induction of DNA damage inducible genes in Escherichia coli. These studies employed E. coli mutants that have fusions of the lac operon to genes induced by treatment with sublethal levels of alkylating agents. These mutants were selected from random insertions of the Mu-dl (Apr lac) phage by screening for induction of beta-galactosidase activity in the presence of methylmethanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine. The current report extends these findings by analyzing gene expression caused by mechlorethamine, chloroethylnitrosoureas and cis-diamminedichloroplatinum(II) (cis-DDP). The results demonstrate heterogeneous effects by these agents on gene expression. While 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea induces alkA, other nitrosoureas, mechlorethamine, and cis-DDP do not cause expression of this gene. Further, while all nitrosoureas caused expression of aidC, mechlorethamine and cis-DDP did not. Lastly, cis-DDP caused marked expression of a sulA fusion mutant while not inducing any of the other E. coli fusion mutants.

  1. Evolutionary cheating in Escherichia coli stationary phase cultures.

    PubMed Central

    Vulic, M; Kolter, R

    2001-01-01

    Starved cultures of Escherichia coli are highly dynamic, undergoing frequent population shifts. The shifts result from the spread of mutants able to grow under conditions that impose growth arrest on the ancestral population. To analyze competitive interactions underlying this dynamic we measured the survival of a typical mutant and the wild type during such population shifts. Here we show that the survival advantage of the mutant at any given time during a takeover is inversely dependent on its frequency in the population, its growth adversely affects the survival of the wild type, and its ability to survive in stationary phase at fixation is lower than that of its ancestor. These mutants do not enter, or exit early, the nondividing stationary-phase state, cooperatively maintained by the wild type. Thus they end up overrepresented as compared to their initial frequency at the onset of the stationary phase, and subsequently they increase disproportionately their contribution in terms of progeny to the succeeding generation in the next growth cycle, which is a case of evolutionary cheating. If analyzed through the game theory framework, these results might be explained by the prisoner's dilemma type of conflict, which predicts that selfish defection is favored over cooperation. PMID:11404318

  2. Properties of bacteriophage T4 mutants defective in gene 30 (deoxyribonucleic acid ligase) and the rII gene.

    PubMed

    Karam, J D; Barker, B

    1971-02-01

    In Escherichia coli K-12 strains infected with phage T4 which is defective in gene 30 [deoxyribonucleic acid (DNA) ligase] and in the rII gene (product unknown), near normal levels of DNA and viable phage were produced. Growth of such T4 ligase-rII double mutants was less efficient in E. coli B strains which show the "rapidlysis" phenotype of rII mutations. In pulse-chase experiments coupled with temperature shifts and with inhibition of DNA synthesis, it was observed that DNA synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or inhibited by continued DNA synthesis. Mutations of the rII gene decreased but did not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivity to ultraviolet irradiation.

  3. Safranal and its analogs inhibit Escherichia coli ATP synthase and cell growth.

    PubMed

    Liu, Mason; Amini, Amon; Ahmad, Zulfiqar

    2017-02-01

    Safranal, a dominant component of saffron, is known to have antitumor, cytotoxic, and antibacterial properties. In this study, we examined safranal and its structural analogs-thymol, carvacrol, damascenone, cuminol, 2,6,6-trimethyl-2-cyclohexene-1,4-dione (TMCHD), 4-isopropylbenzyl bromide (IPBB), and 4-tert-butylphenol (TBP) induced inhibition of Escherichia coli membrane bound F1Fo ATP synthase. Safranal and its analogs inhibited wild-type enzyme to variable degrees. While safranal caused 100% inhibition of wild-type F1Fo ATP synthase, only about 50% inhibition occurred for αR283D mutant ATP synthase. Moreover, safranal, thymol, carvacrol, damascenone, cuminol, TMCHD, IPBB, and TBP all fully abrogated the growth of wild-type E. coli cells and had partial or no effect on the growth of null and mutant E. coli strains. Therefore, the antimicrobial properties of safranal, thymol, carvacrol, damascenone, cuminol, TMCHD, IPBB, and TBP can be linked to their binding and inhibition of ATP synthase. Total loss of growth in wild-type and partial or no growth loss in null or mutant E. coli strains demonstrates that ATP synthase is a molecular target for safranal and its structural analogs. Partial inhibition of the αArg-283 mutant enzyme establishes that αArg-283 residue is required in the polyphenol binding pocket of ATP synthase for the binding of safranal. Furthermore, partial growth loss for the null and mutant strains in the presence of inhibitors also suggests the role of other targets and residues in the process of inhibition.

  4. Phenotypic Diversity Caused by Differential RpoS Activity among Environmental Escherichia coli Isolates▿†

    PubMed Central

    Chiang, Sarah M.; Dong, Tao; Edge, Thomas A.; Schellhorn, Herb E.

    2011-01-01

    Enteric bacteria deposited into the environment by animal hosts are subject to diverse selective pressures. These pressures may act on phenotypic differences in bacterial populations and select adaptive mutations for survival in stress. As a model to study phenotypic diversity in environmental bacteria, we examined mutations of the stress response sigma factor, RpoS, in environmental Escherichia coli isolates. A total of 2,040 isolates from urban beaches and nearby fecal pollution sources on Lake Ontario (Canada) were screened for RpoS function by examining growth on succinate and catalase activity, two RpoS-dependent phenotypes. The rpoS sequence was determined for 45 isolates, including all candidate RpoS mutants, and of these, six isolates were confirmed as mutants with the complete loss of RpoS function. Similarly to laboratory strains, the RpoS expression of these environmental isolates was stationary phase dependent. However, the expression of RpoS regulon members KatE and AppA had differing levels of expression in several environmental isolates compared to those in laboratory strains. Furthermore, after plating rpoS+ isolates on succinate, RpoS mutants could be readily selected from environmental E. coli. Naturally isolated and succinate-selected RpoS mutants had lower generation times on poor carbon sources and lower stress resistance than their rpoS+ isogenic parental strains. These results show that RpoS mutants are present in the environment (with a frequency of 0.003 among isolates) and that, similarly to laboratory and pathogenic strains, growth on poor carbon sources selects for rpoS mutations in environmental E. coli. RpoS selection may be an important determinant of phenotypic diversification and, hence, the survival of E. coli in the environment. PMID:21948830

  5. Comparative Study on Different Expression Hosts for Alkaline Phytase Engineered in Escherichia coli.

    PubMed

    Chen, Weiwei; Yu, Hongwei; Ye, Lidan

    2016-07-01

    The application of alkaline phytase as a feed additive is restricted by the poor specific activity. Escherichia coli is a frequently used host for directed evolution of proteins including alkaline phytase towards improved activity. However, it is not suitable for production of food-grade products due to potential pathogenicity. To combine the advantages of different expression systems, mutants of the alkaline phytase originated from Bacillus subtilis 168 (phy168) were first generated via directed evolution in E. coli and then transformed to food-grade hosts B. subtilis and Pichia pastoris for secretory expression. In order to investigate the suitability of different expression systems, the phy168 mutants expressed in different hosts were characterized and compared in terms of specific activity, pH profile, pH stability, temperature profile, and thermostability. The specific activity of B. subtilis-expressed D24G/K70R/K111E/N121S mutant at pH 7.0 and 60 °C was 30.4 U/mg, obviously higher than those in P. pastoris (22.7 U/mg) and E. coli (19.7 U/mg). Moreover, after 10 min incubation at 80 °C, the B. subtilis-expressed D24G/K70R/K111E/N121S retained about 70 % of the activity at pH 7.0 and 37 °C, whereas the values were only about 25 and 50 % when expressed in P. pastoris and E. coli, respectively. These results suggested B. subtilis as an appropriate host for expression of phy168 mutants and that the strategy of creating mutants in one host and expressing them in another might be a new solution to industrial production of proteins with desired properties.

  6. ANIMAL ENTEROTOXIGENIC ESCHERICHIA COLI

    PubMed Central

    Dubreuil, J. Daniel; Isaacson, Richard E.; Schifferli, Dieter M.

    2016-01-01

    Enterotoxigenic Escherichia coli (ETEC) is the most common cause of E. coli diarrhea in farm animals. ETEC are characterized by the ability to produce two types of virulence factors; adhesins that promote binding to specific enterocyte receptors for intestinal colonization and enterotoxins responsible for fluid secretion. The best-characterized adhesins are expressed in the context of fimbriae, such as the F4 (also designated K88), F5 (K99), F6 (987P), F17 and F18 fimbriae. Once established in the animal small intestine, ETEC produces enterotoxin(s) that lead to diarrhea. The enterotoxins belong to two major classes; heat-labile toxin that consist of one active and five binding subunits (LT), and heat-stable toxins that are small polypeptides (STa, STb, and EAST1). This chapter describes the disease and pathogenesis of animal ETEC, the corresponding virulence genes and protein products of these bacteria, their regulation and targets in animal hosts, as well as mechanisms of action. Furthermore, vaccines, inhibitors, probiotics and the identification of potential new targets identified by genomics are presented in the context of animal ETEC. PMID:27735786

  7. Improved thermostability and acetic acid tolerance of Escherichia coli via directed evolution of homoserine o-succinyltransferase.

    PubMed

    Mordukhova, Elena A; Lee, Hee-Soon; Pan, Jae-Gu

    2008-12-01

    In Escherichia coli, growth is limited at elevated temperatures mainly because of the instability of a single enzyme, homoserine o-succinyltransferase (MetA), the first enzyme in the methionine biosynthesis pathway. The metA gene from the thermophile Geobacillus kaustophilus cloned into the E. coli chromosome was found to enhance the growth of the host strain at elevated temperature (44 degrees C), thus confirming the limited growth of E. coli due to MetA instability. In order to improve E. coli growth at higher temperatures, we used random mutagenesis to obtain a thermostable MetA(E. coli) protein. Sequencing of the thermotolerant mutant showed five amino acid substitutions: S61T, E213V, I229T, N267D, and N271K. An E. coli strain with the mutated metA gene chromosomally inserted showed accelerated growth over a temperature range of 34 to 44 degrees C. We used the site-directed metA mutants to identify two amino acid residues responsible for the sensitivity of MetA(E. coli) to both heat and acids. Replacement of isoleucine 229 with threonine and asparagine 267 with aspartic acid stabilized the protein. The thermostable MetA(E. coli) enzymes showed less aggregation in vivo at higher temperature, as well as upon acetic acid treatment. The data presented here are the first to show improved E. coli growth at higher temperatures solely due to MetA stabilization and provide new knowledge for designing E. coli strains that grow at higher temperatures, thus reducing the cooling cost of bioprocesses.

  8. Salmonella typhimurium mutants lacking NAD pyrophosphatase.

    PubMed Central

    Park, U E; Roth, J R; Olivera, B M

    1988-01-01

    NAD can serve as both a purine and a pyridine source for Salmonella typhimurium. Exogenous NAD is rapidly broken down into nicotinamide mononucleotide and AMP by an NAD pyrophosphatase, the first step in the pathway for the assimilation of exogenous NAD. We isolated and characterized mutants of S. typhimurium lacking NAD pyrophosphatase activity; such mutants were identified by their failure to use exogenous NAD as a purine source. These mutants carry mutations that map at a new locus, designated pnuE, between 86 and 87 min on the Salmonella chromosome. PMID:2841298

  9. Investigation of E. coli Enterotoxins.

    DTIC Science & Technology

    In the course of investigating E . coli enterotoxins, it was discovered that trypsin treatment of partially purified enterotoxin from strain H197 (078...loops) did exhibit elevated PF titers compared with uninoculated controls. These findings are consistent with the hypothesis that E . coli enterotoxins

  10. Binding and Cleavage of E. coli HUβ by the E. coli Lon Protease

    PubMed Central

    Liao, Jiahn-Haur; Lin, Yu-Ching; Hsu, Jowey; Lee, Alan Yueh-Luen; Chen, Tse-An; Hsu, Chun-Hua; Chir, Jiun-Ly; Hua, Kuo-Feng; Wu, Tzu-Hua; Hong, Li-Jenn; Yen, Pei-Wen; Chiou, Arthur; Wu, Shih-Hsiung

    2010-01-01

    Abstract The Escherichia coli Lon protease degrades the E. coli DNA-binding protein HUβ, but not the related protein HUα. Here we show that the Lon protease binds to both HUβ and HUα, but selectively degrades only HUβ in the presence of ATP. Mass spectrometry of HUβ peptide fragments revealed that region K18-G22 is the preferred cleavage site, followed in preference by L36-K37. The preferred cleavage site was further refined to A20-A21 by constructing and testing mutant proteins; Lon degraded HUβ-A20Q and HUβ-A20D more slowly than HUβ. We used optical tweezers to measure the rupture force between HU proteins and Lon; HUα, HUβ, and HUβ-A20D can bind to Lon, and in the presence of ATP, the rupture force between each of these proteins and Lon became weaker. Our results support a mechanism of Lon protease cleavage of HU proteins in at least three stages: binding of Lon with the HU protein (HUβ, HUα, or HUβ-A20D); hydrolysis of ATP by Lon to provide energy to loosen the binding to the HU protein and to allow an induced-fit conformational change; and specific cleavage of only HUβ. PMID:20085725

  11. Firefly luciferase as the reporter for transcriptional response to the environment in Escherichia coli.

    PubMed

    Ryo, Masashi; Oshikoshi, Yuta; Doi, Shosei; Motoki, Shogo; Niimi, Atsuko; Aoki, Setsuyuki

    2013-12-15

    We demonstrate that firefly luciferase is a good reporter in Escherichia coli for transcription dynamics in response to the environment. E. coli strains, carrying a fusion of the promoter of the ycgZ gene and the coding region of the luciferase gene, showed transient bioluminescence on receiving blue light. This response was compromised in mutants lacking known regulators in manners consistent with each regulator's function. We also show that relA, a gene encoding a (p)ppGpp synthetase, affects ycgZ dynamics when nullified. Moreover, two unstable luciferase variants showed improved response dynamics and should be useful to study quick changes of gene expression.

  12. Cloning of Bacteroides fragilis plasmid genes affecting metronidazole resistance and ultraviolet survival in Escherichia coli

    SciTech Connect

    Wehnert, G.U.; Abratt, V.R.; Goodman, H.J.; Woods, D.R. )

    1990-03-01

    Since reduced metronidazole causes DNA damage, resistance to metronidazole was used as a selection method for the cloning of Bacteroides fragilis genes affecting DNA repair mechanisms in Escherichia coli. Genes from B. fragilis Bf-2 were cloned on a recombinant plasmid pMT100 which made E. coli AB1157 and uvrA, B, and C mutant strains more resistant to metronidazole, but more sensitive to far uv irradiation under aerobic conditions. The loci affecting metronidazole resistance and uv sensitivity were linked and located on a 5-kb DNA fragment which originated from the small 6-kb cryptic plasmid pBFC1 present in B. fragilis Bf-2 cells.

  13. Lysis of Escherichia coli by Glycine Is Potentiated by Pyridoxine Starvation

    PubMed Central

    Dempsey, Walter B.

    1973-01-01

    Pyridoxineless mutants of Escherichia coli are lysed in a few hours when starved for pyridoxine in a glucose minimal medium containing glycine at 10 mM. The lysis is prevented equally well by l-alanine and by d-alanine when either is present at 0.1 mM. The lysis is potentiated by 0.5 mM l-methionine. The peculiar susceptibility of E. coli B to glycine-mediated lysis during starvation for pyridoxine suggests that the starvation reduces the availability of some normal antagonist of glycine, presumably alanine. PMID:4583221

  14. Activation by Gene Amplification of pitB, Encoding a Third Phosphate Transporter of Escherichia coli K-12

    PubMed Central

    Hoffer, Sally M.; Schoondermark, Paul; van Veen, Hendrik W.; Tommassen, Jan

    2001-01-01

    Two systems for the uptake of inorganic phosphate (Pi) in Escherichia coli, PitA and Pst, have been described. A revertant of a pitA pstS double mutant that could grow on Pi was isolated. We demonstrate that the expression of a new Pi transporter, PitB, is activated in this strain by a gene amplification event. PMID:11443103

  15. Solubilization and renaturation of overexpressed aggregates of mutant tryptophan synthase alpha-subunits.

    PubMed Central

    Lim, W K; Smith-Somerville, H E; Hardman, J K

    1989-01-01

    Certain Escherichia coli tryptophan synthase mutant alpha-subunits encoded from mutagenized trpA-containing plasmids were overexpressed as insoluble aggregates which were seen as large, intracellular inclusion bodies. The insoluble aggregates were solubilized to various degrees by several neutral, chaotropic salts. The order of effectiveness of these salts (KSCN, NaI greater than NaNO3, LiBr greater than CaCl2) followed that for the Hofmeister series. Optimum conditions for the use of KSCN resulted in a maximum 70 to 75% solubilization of the aggregate forms for all mutant alpha-subunits examined. Removal of KSCN by dialysis resulted in the recovery of biological activity and of certain characteristic structural properties. Such salts may be a useful alternative for other recombinant protein aggregates which resist complete renaturation by commonly used treatments with guanidine or urea. Images PMID:2547334

  16. Characterization of rag1 mutant zebrafish leukocytes

    PubMed Central

    Petrie-Hanson, Lora; Hohn, Claudia; Hanson, Larry

    2009-01-01

    Background Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants). Results Differential counts of peripheral blood leukocytes (PBL) showed that rag1 mutants had significantly decreased lymphocyte-like cell populations (34.7%) compared to wild-types (70.5%), and significantly increased granulocyte populations (52.7%) compared to wild-types (17.6%). Monocyte/macrophage populations were similar between mutants and wild-types, 12.6% and 11.3%, respectively. Differential leukocyte counts of rag1 mutant kidney hematopoietic tissue showed a significantly reduced lymphocyte-like cell population (8%), a significantly increased myelomonocyte population (57%), 34.8% precursor cells, and 0.2% thrombocytes, while wild-type hematopoietic kidney tissue showed 29.4% lymphocytes/lymphocyte-like cells, 36.4% myelomonocytes, 33.8% precursors and 0.5% thrombocytes. Flow cytometric analyses of kidney hematopoietic tissue revealed three leukocyte populations. Population A was monocytes and granulocytes and comprised 34.7% of the gated cells in rag1 mutants and 17.6% in wild-types. Population B consisted of hematopoietic precursors, and comprised 50% of the gated cells for rag1 mutants and 53% for wild-types. Population C consisted of lymphocytes and lymphocyte

  17. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity.

    PubMed

    Lippincott, B B; Lippincott, J A

    1966-10-01

    Lippincott, Barbara B. (Northwestern University, Evanston, Ill.), and James A. Lippincott. Characteristics of Agrobacterium tumefaciens auxotrophic mutant infectivity. J. Bacteriol. 92:937-945. 166.-Mutants of Agrobacterium tumefaciens auxotrophic for adenine, methionine, or asparagine are less infectious than the wild-type strain B6 from which they were derived and show increased infectivity on pinto bean leaves when the specific compounds required for growth of the mutants are added to the infected leaf. Reversion to a prototrophic form of nutrition is accompanied by increased infectivity. Tumors initiated by these auxotrophic mutants are shown to arise only at large wound sites where nutritional conditions may be less restricting. The data indicate that, after inoculation, the bacteria pass through a phase in which host-supplied nutrients are utilized for the production of one or more factors necessary for successful tumor initiation.

  18. Mutant IDH1 and thrombosis in gliomas.

    PubMed

    Unruh, Dusten; Schwarze, Steven R; Khoury, Laith; Thomas, Cheddhi; Wu, Meijing; Chen, Li; Chen, Rui; Liu, Yinxing; Schwartz, Margaret A; Amidei, Christina; Kumthekar, Priya; Benjamin, Carolina G; Song, Kristine; Dawson, Caleb; Rispoli, Joanne M; Fatterpekar, Girish; Golfinos, John G; Kondziolka, Douglas; Karajannis, Matthias; Pacione, Donato; Zagzag, David; McIntyre, Thomas; Snuderl, Matija; Horbinski, Craig

    2016-12-01

    Mutant isocitrate dehydrogenase 1 (IDH1) is common in gliomas, and produces D-2-hydroxyglutarate (D-2-HG). The full effects of IDH1 mutations on glioma biology and tumor microenvironment are unknown. We analyzed a discovery cohort of 169 World Health Organization (WHO) grade II-IV gliomas, followed by a validation cohort of 148 cases, for IDH1 mutations, intratumoral microthrombi, and venous thromboemboli (VTE). 430 gliomas from The Cancer Genome Atlas were analyzed for mRNAs associated with coagulation, and 95 gliomas in a tissue microarray were assessed for tissue factor (TF) protein. In vitro and in vivo assays evaluated platelet aggregation and clotting time in the presence of mutant IDH1 or D-2-HG. VTE occurred in 26-30 % of patients with wild-type IDH1 gliomas, but not in patients with mutant IDH1 gliomas (0 %). IDH1 mutation status was the most powerful predictive marker for VTE, independent of variables such as GBM diagnosis and prolonged hospital stay. Microthrombi were far less common within mutant IDH1 gliomas regardless of WHO grade (85-90 % in wild-type versus 2-6 % in mutant), and were an independent predictor of IDH1 wild-type status. Among all 35 coagulation-associated genes, F3 mRNA, encoding TF, showed the strongest inverse relationship with IDH1 mutations. Mutant IDH1 gliomas had F3 gene promoter hypermethylation, with lower TF protein expression. D-2-HG rapidly inhibited platelet aggregation and blood clotting via a novel calcium-dependent, methylation-independent mechanism. Mutant IDH1 glioma engraftment in mice significantly prolonged bleeding time. Our data suggest that mutant IDH1 has potent antithrombotic activity within gliomas and throughout the peripheral circulation. These findings have implications for the pathologic evaluation of gliomas, the effect of altered isocitrate metabolism on tumor microenvironment, and risk assessment of glioma patients for VTE.

  19. Mutations in rpoBC suppress the defects of a Sinorhizobium meliloti relA mutant.

    PubMed

    Wells, Derek H; Long, Sharon R

    2003-09-01

    The nitrogen-fixing symbiosis between Sinorhizobium meliloti and Medicago sativa requires complex physiological adaptation by both partners. One method by which bacteria coordinately control physiological adaptation is the stringent response, which is triggered by the presence of the nucleotide guanosine tetraphosphate (ppGpp). ppGpp, produced by the RelA enzyme, is thought to bind to and alter the ability of RNA polymerase (RNAP) to initiate and elongate transcription and affect the affinity of the core enzyme for various sigma factors. An S. meliloti relA mutant which cannot produce ppGpp was previously shown to be defective in the ability to form nodules. This mutant also overproduces a symbiotically necessary exopolysaccharide called succinoglycan. The work presented here encompasses the analysis of suppressor mutants, isolated from host plants, that suppress the symbiotic defects of the relA mutant. All suppressor mutations are extragenic and map to either rpoB or rpoC, which encode the beta and beta' subunits of RNAP. Phenotypic, structural, and gene expression analyses reveal that suppressor mutants can be divided into two classes; one is specific in its effect on stringent response-regulated genes and shares striking similarity with suppressor mutants of Escherichia coli strains that lack ppGpp, and another reduces transcription of all genes tested in comparison to that in the relA parent strain. Our findings indicate that the ability to successfully establish symbiosis is tightly coupled with the bacteria's ability to undergo global physiological adjustment via the stringent response.

  20. RecBCD Enzyme "Chi Recognition" Mutants Recognize Chi Recombination Hotspots in the Right DNA Context.

    PubMed

    Amundsen, Susan K; Sharp, Jake W; Smith, Gerald R

    2016-09-01

    RecBCD enzyme is a complex, three-subunit protein machine essential for the major pathway of DNA double-strand break repair and homologous recombination in Escherichia coli Upon encountering a Chi recombination-hotspot during DNA unwinding, RecBCD nicks DNA to produce a single-stranded DNA end onto which it loads RecA protein. Conformational changes that regulate RecBCD's helicase and nuclease activities are induced upon its interaction with Chi, defined historically as 5' GCTGGTGG 3'. Chi is thought to be recognized as single-stranded DNA passing through a tunnel in RecC. To define the Chi recognition-domain in RecC and thus the mechanism of the RecBCD-Chi interaction, we altered by random mutagenesis eight RecC amino acids lining the tunnel. We screened for loss of Chi activity with Chi at one site in bacteriophage λ. The 25 recC mutants analyzed thoroughly had undetectable or strongly reduced Chi-hotspot activity with previously reported Chi sites. Remarkably, most of these mutants had readily detectable, and some nearly wild-type, activity with Chi at newly generated Chi sites. Like wild-type RecBCD, these mutants had Chi activity that responded dramatically (up to fivefold, equivalent to Chi's hotspot activity) to nucleotide changes flanking 5' GCTGGTGG 3'. Thus, these and previously published RecC mutants thought to be Chi-recognition mutants are actually Chi context-dependence mutants. Our results fundamentally alter the view that Chi is a simple 8-bp sequence recognized by the RecC tunnel. We propose that Chi hotspots have dual nucleotide sequence interactions, with both the RecC tunnel and the RecB nuclease domain.

  1. Nucleotide sequence of the wild-type RAD4 gene of Saccharomyces cerevisiae and characterization of mutant rad4 alleles.

    PubMed Central

    Couto, L B; Friedberg, E C

    1989-01-01

    Shuttle plasmids carrying the wild-type RAD4 gene of Saccharomyces cerevisiae cannot be propagated in Escherichia coli (R. Fleer, W. Siede, and E. C. Friedberg, J. Bacteriol. 169:4884-4892, 1987). In order to determine the nucleotide sequence of the cloned gene, we used a plasmid carrying a mutant allele that allows plasmid propagation in E. coli. The wild-type sequence in the region of this mutation was determined from a second plasmid carrying a different mutant rad4 allele. We established the locations and characteristics of a number of spontaneously generated plasmid-borne RAD4 mutations that alleviate the toxicity of the wild-type gene in E. coli and of several mutagen-induced chromosomal mutations that inactivate the excision repair function of RAD4. These mutations are situated in very close proximity to each other, and all are expected to result in the expression of truncated polypeptides missing the carboxy-terminal one-third of the Rad4 polypeptide. This region of the gene may be important both for the toxic effect of the Rad4 protein in E. coli and for its role in DNA repair in S. cerevisiae. PMID:2649477

  2. Quantitative Analysis of Triple Mutant Genetic Interactions

    PubMed Central

    Braberg, Hannes; Alexander, Richard; Shales, Michael; Xu, Jiewei; Franks-Skiba, Kathleen E.; Wu, Qiuqin; Haber, James E.; Krogan, Nevan J.

    2014-01-01

    The quantitative analysis of genetic interactions between pairs of gene mutations has proven effective for characterizing cellular functions but can miss important interactions for functionally redundant genes. To address this limitation, we have developed an approach termed Triple Mutant Analysis (TMA). The procedure relies on a query strain that contains two deletions in a pair of redundant or otherwise related genes, that is crossed against a panel of candidate deletion strains to isolate triple mutants and measure their growth. A central feature of TMA is to interrogate mutants that are synthetically sick when two other genes are deleted but interact minimally with either single deletion. This approach has been valuable for discovering genes that restore critical functions when the principle actors are deleted. TMA has also uncovered double mutant combinations that produce severe defects because a third protein becomes deregulated and acts in a deleterious fashion, and it has revealed functional differences between proteins presumed to act together. The protocol is optimized for Singer ROTOR pinning robots, takes 3 weeks to complete, and measures interactions for up to 30 double mutants against a library of 1536 single mutants. PMID:25010907

  3. Mono and diterpene production in Escherichia coli.

    PubMed

    Reiling, K Kinkead; Yoshikuni, Yasuo; Martin, Vincent J J; Newman, Jack; Bohlmann, Jörg; Keasling, Jay D

    2004-07-20

    Mono- and diterpenoids are of great industrial and medical value as specialty chemicals and pharmaceuticals. Production of these compounds in microbial hosts, such as Escherichia coli, can be limited by intracellular levels of the polyprenyl diphosphate precursors, geranyl diphosphate (GPP), and geranylgeranyl diphosphate (GGPP). To alleviate this limitation, we constructed synthetic operons that express three key enzymes for biosynthesis of these precursors: (1). DXS,1-deoxy-d-xylulose-5-phosphate synthase; (2). IPPHp, IPP isomerase from Haematococcus pluvialis; and (3). one of two variants of IspA, FPP synthase that produces either GPP or GGPP. The reporter plasmids pAC-LYC and pACYC-IB, which encode enzymes that convert either FPP or GGPP, respectively, to the pigment lycopene, were used to demonstrate that at full induction, the operon encoding the wild-type FPP synthase and mutant GGPP synthase produced similar levels of lycopene. To synthesize di- or monoterpenes in E. coli using the GGPP and GPP encoding operons either a diterpene cyclase [casbene cyclase (Ricinus communis L) and ent-kaurene cyclase (Phaeosphaeria sp. L487)] or a monoterpene cyclase [3-carene cyclase (Picea abies)] was coexpressed with their respective precursor production operon. Analysis of culture extracts or headspace by gas chromatography-mass spectrometry confirmed the in vivo production of the diterpenes casbene, kaur-15-ene, and kaur-16-ene and the monoterpenes alpha-pinene, myrcene, sabinene, 3-carene, alpha-terpinene, limonene, beta-phellandrene, alpha-terpinene, and terpinolene. Construction and functional expression of GGPP and GPP operons provides an in vivo precursor platform host for the future engineering of di- and monoterpene cyclases and the overproduction of terpenes in bacteria.

  4. A Novel Putrescine Exporter SapBCDF of Escherichia coli.

    PubMed

    Sugiyama, Yuta; Nakamura, Atsuo; Matsumoto, Mitsuharu; Kanbe, Ayaka; Sakanaka, Mikiyasu; Higashi, Kyohei; Igarashi, Kazuei; Katayama, Takane; Suzuki, Hideyuki; Kurihara, Shin

    2016-12-16

    Recent research has suggested that polyamines (putrescine, spermidine, and spermine) in the intestinal tract impact the health of animals either negatively or positively. The concentration of polyamines in the intestinal tract results from the balance of uptake and export of the intestinal bacteria. However, the mechanism of polyamine export from bacterial cells to the intestinal lumen is still unclear. In Escherichia coli, PotE was previously identified as a transporter responsible for putrescine excretion in an acidic growth environment. We observed putrescine concentration in the culture supernatant was increased from 0 to 50 μm during growth of E. coli under neutral conditions. Screening for the unidentified putrescine exporter was performed using a gene knock-out collection of E. coli, and deletion of sapBCDF significantly decreased putrescine levels in the culture supernatant. Complementation of the deletion mutant with the sapBCDF genes restored putrescine levels in the culture supernatant. Additionally, the ΔsapBCDF strain did not facilitate uptake of putrescine from the culture supernatant. Quantification of stable isotope-labeled putrescine derived from stable isotope-labeled arginine supplemented in the medium revealed that SapBCDF exported putrescine from E. coli cells to the culture supernatant. It was previously reported that SapABCDF of Salmonella enterica sv. typhimurium and Haemophilus influenzae conferred resistance toantimicrobial peptides; however, the E. coli ΔsapBCDF strain did not affect resistance to antimicrobial peptide LL-37. These results strongly suggest that the natural function of the SapBCDF proteins is the export of putrescine.

  5. Genetic and biochemical analysis of peptide transport in Escherichia coli

    SciTech Connect

    Andrews, J.C.

    1986-01-01

    E. coli peptide transport mutants have been isolated based on their resistance to toxic tripeptides. These genetic defects were found to map in two distinct chromosomal locations. The transport systems which require expression of the trp-linked opp genes and the oppE gene(s) for activity were shown to have different substrate preferences. Growth of E. coli in medium containing leucine results in increased entry of exogenously supplied tripeptides into the bacterial cell. This leucine-mediated elevation of peptide transport required expression of the trp-linked opp operon and was accompanied by increased sensitivity to toxic tripeptides, by an enhanced capacity to utilize nutritional peptides, and by an increase in both the velocity and apparent steady-state level of L-(U-/sup 14/C)alanyl-L-alanyl-L-alanine accumulation for E. coli grown in leucine-containing medium relative to these parameters of peptide transport measured with bacteria grown in media lacking leucine. Direct measurement of opp operon expression by pulse-labeling experiments demonstrated that growth of E. coli in the presence of leucine resulted in increased synthesis of the oppA-encoded periplasmic binding protein. The transcriptional regulation of the trp-linked opp operon of E. coli was investigated using lambda placMu51-generated lac operon fusions. Synthesis of ..beta..-galactosidase by strains harboring oppA-lac, oppB-lac, and oppD-lac fusions occurred at a basal level when the fusion-containing strains were grown in minimal medium.

  6. Ribosomal protein alterations in thiostrepton- and Micrococcin-resistant mutants of Bacillus subtilis.

    PubMed

    Wienen, B; Ehrlich, R; Stöffler-Meilicke, M; Stöffler, G; Smith, I; Weiss, D; Vince, R; Pestka, S

    1979-08-25

    Ribosomal proteins of parental thiostrepton- and micrococcin-sensitive Bacillus subtilis cysA14 and thiostrepton-and micrococcin-resistant mutants were compared. Several electrophoretic and immunochemical techniques showed unambiguously that BS-L11 was not present on 50 S ribosomal subunits from the six thiostrepton-resistant mutants. Protein BS-L11 reappeared in all six revertants from thiostrepton resistance to thiostrepton sensitivity. No definitive protein alteration could be ascribed to the mutation from micrococcin sensitivity to resistance. It was also demonstrated that B. subtilis protein BS-L11 is homologous to Escherichia coli ribosomal protein L11. The finding that ribosomes from thiostrepton-resistant mutants do not contain protein L11 suggests that L11 not only is involved in binding of thiostrepton, but also, when mutationally altered, confers resistance to this antibiotic. Although the ribosomes of these strains do not contain protein L11, all thiostrepton-resistant mutants showed the same viability as the parental strain. Thus protein L11 cannot be obligatory for the structure and function of the ribosome.

  7. Isolation and characterization of a selenium metabolism mutant of Salmonella typhimurium.

    PubMed Central

    Kramer, G F; Ames, B N

    1988-01-01

    Selenium is a constituent in Escherichia coli of the anaerobic enzyme formate dehydrogenase in the form of selenocysteine. Selenium is also present in the tRNA of E. coli in the modified base 5-methylaminomethyl-2-selenouracil (mnm5Se2U). The pathways of bacterial selenium metabolism are largely uncharacterized, and it is unclear whether nonspecific reactions in the sulfur metabolic pathways may be involved. We demonstrated that sulfur metabolic pathway mutants retain a wild-type pattern of selenium incorporation, indicating that selenite (SeO32-) is metabolized entirely via selenium-specific pathways. To investigate the function of mnm5Se2U, we isolated a mutant which is unable to incorporate selenium into tRNA. This strain was obtained by isolating mutants lacking formate dehydrogenase activity and then screening for the inability to metabolize selenium. This phenotype is the result of a recessive mutation which appears to map in the general region of 21 min on the Salmonella typhimurium chromosome. A mutation in this gene, selA, thus has a pleiotropic effect of eliminating selenium incorporation into both protein and tRNA. The selA mutant appears to be blocked in a step of selenium metabolism after reduction, such as in the actual selenium insertion process. We showed that the absence of selenium incorporation into suppressor tRNA reduces the efficiency of suppression of nonsense codons in certain contexts and when wobble base pairing is required. Thus, one function of mnm5Se2U in tRNA may be in codon-anticodon interactions. Images PMID:2448290

  8. A recombinant hypoallergenic parvalbumin mutant for immunotherapy of IgE-mediated fish allergy.

    PubMed

    Swoboda, Ines; Bugajska-Schretter, Agnes; Linhart, Birgit; Verdino, Petra; Keller, Walter; Schulmeister, Ulrike; Sperr, Wolfgang R; Valent, Peter; Peltre, Gabriel; Quirce, Santiago; Douladiris, Nikolaos; Papadopoulos, Nikolaos G; Valenta, Rudolf; Spitzauer, Susanne

    2007-05-15

    IgE-mediated allergy to fish is a frequent cause of severe anaphylactic reactions. Parvalbumin, a small calcium-binding protein, is the major fish allergen. We have recently isolated a cDNA coding for carp parvalbumin, Cyp c 1, and expressed in Escherichia coli a recombinant Cyp c 1 molecule, which contained most IgE epitopes of saltwater and freshwater fish. In this study, we introduced mutations into the calcium-binding domains of carp parvalbumin by site-directed mutagenesis and produced in E. coli three parvalbumin mutants containing amino acid exchanges either in one (single mutants; Mut-CD and Mut-EF) or in both of the calcium-binding sites (double mutant; Mut-CD/EF). Circular dichroism analyses of the purified derivatives and the wild-type allergen showed that Mut-CD/EF exhibited the greatest reduction of overall protein fold. Dot blot assays and immunoblot inhibition experiments performed with sera from 21 fish-allergic patients showed that Mut-CD/EF had a 95% reduced IgE reactivity and represented the derivative with the least allergenic activity. The latter was confirmed by in vitro basophil histamine release assays and in vivo skin prick testing. The potential applicability for immunotherapy of Mut-CD/EF was demonstrated by the fact that mouse IgG Abs could be raised by immunization with the mutated molecule, which cross-reacted with parvalbumins from various fish species and inhibited the binding of fish-allergic patients' IgE to the wild-type allergen. Using the hypoallergenic carp parvalbumin mutant Mut-CD/EF, it may be possible to treat fish allergy by immunotherapy.

  9. Molecular evaluation of a spearmint mutant altered in the expression of limonene hydroxylases that direct essential oil monoterpene biosynthesis.

    PubMed

    Bertea, Cinzia; Schalk, Michel; Mau, Christopher J D; Karp, Frank; Wildung, Mark R; Croteau, Rodney

    2003-12-01

    Gamma irradiation of Scotch spearmint created a mutant line, 643-10-74, which has an altered essential oil reminiscent of peppermint because the monoterpene metabolites in the oil glands of the mutant are predominantly oxygenated at the C3 position of the p-menthane ring instead of the C6 position normally found in spearmint. The limonene hydroxylase genes responsible for directing the regiochemistry of oxygenation were cloned from Scotch spearmint and mutant 643 and expressed in Escherichia coli. The limonene bydroxylase from the wild-type parent hydroxylated the C6 position while the enzyme from the mutant oxygenated the C3 position. Comparison of the amino acid sequences with other limonene hydroxylases showed that the mutant enzyme was more closely related to the peppermint limonene-3-hydroxylases than to the spearmint limonene-6-hydroxylases. Because of the sequence differences between the Scotch spearmint and mutant 643 limonene hydroxylases, it is most likely that the mutation did not occur within the structural gene for limonene hydroxylase but rather at a regulatory site within the genome that controls the expression of one or the other regiospecific variants.

  10. Genome-wide screening of transcription factor deletion targets in Escherichia coli for enhanced production of lactate-based polyesters.

    PubMed

    Kadoya, Ryosuke; Kodama, Yu; Matsumoto, Ken'ichiro; Ooi, Toshihiko; Taguchi, Seiichi

    2017-05-01

    Engineered Escherichia coli is a useful platform for production of lactate (LA)-based polyester poly[LA-co-3-hydroxybutyrate (3HB)] from renewable sugars. Here we screened all non-lethal transcription factor deletions of E. coli for efficient production of the polymer. This approach aimed at drawing out the latent potential of the host for efficient polymer production via indirect positive effects. Among 252 mutants from Keio Collection tested, eight mutants (ΔpdhR, ΔcspG, ΔyneJ, ΔchbR, ΔyiaU, ΔcreB, ΔygfI and ΔnanK) accumulated greater amount of polymer (6.2-10.1 g/L) compared to the parent strain E. coli BW25113 (5.1 g/L). The mutants increased polymer production per cell (1.1-1.5-fold) without significant change in cell density. The yield of the polymer from glucose was also higher for the selected mutants (0.34-0.38 g/g) than the parent strain (0.27 g/g). Therefore, the deletions of transcription factors should channel the carbon flux towards polymer production. It should be noted that the screening employed in this study identified beneficial mutants without analyzing causal relationship between the mutation and the enhanced polymer production. This approach, therefore, should be applicable to broad range of fermentation productions.

  11. Staphylococcal enterotoxin type A internal deletion mutants: serological activity and induction of T-cell proliferation.

    PubMed Central

    Harris, T O; Hufnagle, W O; Betley, M J

    1993-01-01

    Previous findings indicate that the N-terminal region of staphylococcal enterotoxin type A (SEA) is required for its ability to induce T-cell proliferation. To better localize internal peptides of SEA that are important for induction of murine T-cell proliferation, SEA mutants that had internal deletions in their N-terminal third were constructed. A series of unique restriction enzyme sites were first engineered into sea; only one of these changes resulted in an amino acid substitution (the aspartic acid residue at position 60 of mature SEA was changed to a glycine [D60G]). Because the D60G substitution had no discernible effect on serological or biological activity, the sea allele encoding this mutant SEA was used to construct a panel of mutant SEAs lacking residues 3 to 17, 19 to 23, 24 to 28, 29 to 49, 50 to 55, 56 to 59, 61 to 73, 68 to 74, or 74 to 85. Recombinant plasmids with the desired mutations were constructed in Escherichia coli and transferred to Staphylococcus aureus. Staphylococcal culture supernatants containing the mutant SEAs were examined. Western immunoblot analysis with polyclonal anti-SEA antiserum revealed that each of the recombinant S. aureus strains produced a mutant SEA of the predicted size. All the mutant SEAs exhibited increased sensitivity to monkey stomach lavage fluid in vitro, which is consistent with these mutants having conformations unlike that of wild-type SEA or the SEA D60G mutant. In general, deletion of internal peptides had a deleterious effect on the ability to induce T-cell proliferation; only SEA mutants lacking either residues 3 to 17 or 56 to 59 consistently produced a statistically significant increase in the incorporation of [3H]thymidine. In the course of this work, two monoclonal antibodies that had different requirements for binding to SEA in Western blots were identified. The epitope for one monoclonal antibody was contained within residues 108 to 230 of mature SEA. Binding of the other monoclonal antibody to

  12. Novel CDC34 (UBC3) ubiquitin-conjugating enzyme mutants obtained by charge-to-alanine scanning mutagenesis.

    PubMed

    Pitluk, Z W; McDonough, M; Sangan, P; Gonda, D K

    1995-03-01

    CDC34 (UBC3) encodes a ubiquitin-conjugating (E2) enzyme required for transition from the G1 phase to the S phase of the budding yeast cell cycle. CDC34 consists of a 170-residue catalytic N-terminal domain onto which is appended an acidic C-terminal domain. A portable determinant of cell cycle function resides in the C-terminal domain, but determinants for specific function must reside in the N-terminal domain as well. We have explored the utility of "charge-to-alanine" scanning mutagenesis to identify novel N-terminal domain mutants of CDC34 that are enzymatically competent with respect to unfacilitated (E3-independent) ubiquitination but that nevertheless are defective with respect to its cell cycle function. Such mutants may reveal determinants of specific in vivo function, such as those required for interaction with substrates or trans-acting regulators of activity and substrate selectivity. Three of 18 "single-scan" mutants (in which small clusters of charged residues were mutated to alanine) were compromised with respect to in vivo function. One mutant (cdc34-109, 111, 113A) targeted a 12-residue segment of the Cdc34 protein not found in most other E2s and was unable to complement a cdc34 null mutant at low copy numbers but could complement a null mutant when overexpressed from an induced GAL1 promoter. Combining adjacent pairs of single-scan mutants to produce "double-scan" mutants yielded four additional mutants, two of which showed heat and cold sensitivity conditional defects. Most of the mutant proteins expressed in Escheria coli displayed unfacilitated (E3-independent) ubiquitin-conjugating activity, but two mutants differed from wild-type and other mutant Cdc34 proteins in the extent of multiubiquitination they catalyzed during an autoubiquitination reation-conjugating enzyme function and have identified additional mutant alleles of CDC34 that will be valuable in further genetic and biochemical studies of Cdc34-dependent ubiquitination.

  13. Transcriptional Responses of Escherichia coli K-12 and O157:H7 Associated with Lettuce Leaves

    PubMed Central

    Fink, Ryan C.; Black, Elaine P.; Hou, Zhe; Sugawara, Masayuki; Sadowsky, Michael J.

    2012-01-01

    An increasing number of outbreaks of gastroenteritis recently caused by Escherichia coli O157:H7 have been linked to the consumption of leafy green vegetables. Although it is known that E. coli survives and grows in the phyllosphere of lettuce plants, the molecular mechanisms by which this bacterium associates with plants are largely unknown. The goal of this study was to identify E. coli genes relevant to its interaction, survival, or attachment to lettuce leaf surfaces, comparing E. coli K-12, a model system, and E. coli O157:H7, a pathogen associated with a large number of outbreaks. Using microarrays, we found that upon interaction with intact leaves, 10.1% and 8.7% of the 3,798 shared genes were differentially expressed in K-12 and O157:H7, respectively, whereas 3.1% changed transcript levels in both. The largest group of genes downregulated consisted of those involved in energy metabolism, including tnaA (33-fold change), encoding a tryptophanase that converts tryptophan into indole. Genes involved in biofilm modulation (bhsA and ybiM) and curli production (csgA and csgB) were significantly upregulated in E. coli K-12 and O157:H7. Both csgA and bhsA (ycfR) mutants were impaired in the long-term colonization of the leaf surface, but only csgA mutants had diminished ability in short-term attachment experiments. Our data suggested that the interaction of E. coli K-12 and O157:H7 with undamaged lettuce leaves likely is initiated via attachment to the leaf surface using curli fibers, a downward shift in their metabolism, and the suppression of biofilm formation. PMID:22247152

  14. Saccharomyces cerevisiae RAD27 complements its Escherichia coli homolog in damage repair but not mutation avoidance.

    PubMed

    Ohnishi, Gaku; Daigaku, Yasukazu; Nagata, Yuki; Ihara, Makoto; Yamamoto, Kazuo

    2004-06-01

    In eukaryotes, the flap endonuclease of Rad27/Fen-1 is thought to play a critical role in lagging-strand DNA replication by removing ribonucleotides present at the 5' ends of Okazaki fragments, and in base excision repair by cleaving a 5' flap structure that may result during base excision repair. Saccharomyces cerevisiae rad27Delta mutants further display a repeat tract instability phenotype and a high rate of forward mutations to canavanine resistance that result from duplications of DNA sequence, indicating a role in mutation avoidance. Two conserved motifs in Rad27/Fen-1 show homology to the 5' --> 3' exonuclease domain of Escherichia coli DNA polymerase I. The strain defective in the 5' --> 3' exonuclease domain in DNA polymerase I shows essentially the same phenotype as the yeast rad27Delta strain. In this study, we expressed the yeast RAD27 gene in an E. coli strain lacking the 5' --> 3' exonuclease domain in DNA polymerase I in order to test whether eukaryotic RAD27/FEN-1 can complement the defect of its bacterial homolog. We found that the yeast Rad27 protein complements sensitivity to methyl methanesulfonate in an E. coli mutant. On the other hand, Rad27 protein did not reduce the high rate of spontaneous mutagenesis in the E. coli tonB gene which results from duplication of DNA. These results indicate that the yeast Rad27 and E. coli 5' --> 3' exonuclease act on the same substrate. We argue that the lack of mutation avoidance of yeast RAD27 in E. coli results from a lack of interaction between the yeast Rad27 protein and the E. coli replication clamp (beta-clamp).

  15. Characterization of the rec-1 gene of Haemophilus influenzae and behavior of the gene in Escherichia coli.

    PubMed Central

    Setlow, J K; Spikes, D; Griffin, K

    1988-01-01

    The rec-1 gene of Haemophilus influenzae was cloned into a shuttle vector that replicates in Escherichia coli as well as in H. influenzae. The plasmid, called pRec1, complemented the defects of a rec-1 mutant in repair of UV damage, transformation, and ability of prophage to be induced by UV radiation. Although UV resistance and recombination were caused by pRec1 in E. coli recA mutants, UV induction of lambda and UV mutagenesis were not. We suggest that the ability of the H. influenzae Rec-1 protein to cause cleavage of repressors but not the recombinase function differs from that of the E. coli RecA protein. PMID:3045079

  16. Deoxyribonucleic acid sequence of araBAD promoter mutants of Escherichia coli.

    PubMed

    Horwitz, A H; Morandi, C; Wilcox, G

    1980-05-01

    The controlling site region for the araBAD operon is defined, in part, by two classes of cis-acting constitutive mutations. The aralc mutations allow low-level constitutive expression of ara-BAD in the absence of the positive regulatory protein coded for by the araC gene, whereas the araXc mutations allow expression of araBAD in the absence of the cyclic adenosine monophosphate receptor protein. Six independently isolated aralc mutations and three independently isolated araXc mutations were cloned onto the plasmid pBR322 using in vitro recombinant deoxyribonucleic acid techniques and in vivo recombination between plasmid and chromosomal deoxyribonucleic acid. The location of these mutations was determined by deoxyribonucleic acid sequence analysis. All of the aralc mutations occurred at position -35 within the araBAD promoter (+1 = messenger ribonucleic acid start for araBAD) and resulted from an AT leads to GC transition. All of the araXc mutations occurred at position -10 within the araBAD promoter and resulted from a GC leads to AT transition. Models are presented to explain the mode of action of the aralc and araXc mutations.

  17. Identification of a Glyphosate-Resistant Mutant of Rice 5-Enolpyruvylshikimate 3-Phosphate Synthase Using a Directed Evolution Strategy1[W][OA

    PubMed Central

    Zhou, Min; Xu, Honglin; Wei, Xiaoli; Ye, Zhiqiang; Wei, Liping; Gong, Weimin; Wang, Yongqin; Zhu, Zhen

    2006-01-01

    5-Enolpyruvylshikimate 3-phosphate synthase (EPSPS) is a key enzyme in the shikimate pathway and is targeted by the wide-spectrum herbicide glyphosate. Here, we describe the use of a selection system based on directed evolution to select glyphosate-resistant mutants of EPSPS. Using this system, the rice (Oryza sativa) EPSPS gene, mutagenized by Error-Prone polymerase chain reaction, was introduced into an EPSPS-deficient Escherichia coli strain, AB2829, and transformants were selected on minimal medium by functional complementation. Three mutants with high glyphosate resistance were identified in three independent glyphosate selection experiments. Each mutant contained a C317→T transition within the EPSPS coding sequence, causing a change of proline-106 to leucine (P106L) in the protein sequence. Glyphosate resistance assays indicated a 3-fold increase in glyphosate resistance of E. coli expressing the P106L mutant. Affinity of the P106L mutant for glyphosate and phosphoenolpyruvate was decreased about 70-fold and 4.6-fold, respectively, compared to wild-type EPSPS. Analysis based on a kinetic model demonstrates that the P106L mutant has a high glyphosate resistance while retaining relatively high catalytic efficiency at low phosphoenolpyruvate concentrations. A mathematical model derived from the Michaelis-Menten equation was used to characterize the effect of expression level and selection conditions on kinetic (Ki and Km) variation of the mutants. This prediction suggests that the expression level is an important aspect of the selection system. Furthermore, glyphosate resistance of the P106L mutant was confirmed in transgenic tobacco (Nicotiana tabacum), demonstrating the potential for using the P106L mutant in transgenic crops. PMID:16361526

  18. Cyclic AMP and cell division in Escherichia coli.

    PubMed Central

    D'Ari, R; Jaffé, A; Bouloc, P; Robin, A

    1988-01-01

    We examined several aspects of cell division regulation in Escherichia coli which have been thought to be controlled by cyclic AMP (cAMP) and its receptor protein (CAP). Mutants lacking adenyl cyclase (cya) or CAP (crp) were rod shaped, not spherical, during exponential growth in LB broth or glucose-Casamino Acids medium, and lateral wall elongation was normal; in broth, stationary-phase cells became ovoid. Cell mass was smaller for the mutants than for the wild type, but it remained appropriate for their slower growth rate and thus probably does not reflect early (uncontrolled) septation. The slow growth did not seem to reflect a gross metabolic disorder, since the mutants gave a normal yield on limiting glucose; surprisingly, however, the cya mutant (unlike crp) was unable to grow anaerobically on glucose, suggesting a role for cAMP (but not for CAP) in the expression of some fermentation enzyme. Both cya and crp mutants are known to be resistant to mecillinam, an antibiotic which inhibits penicillin-binding protein 2 (involved in lateral wall elongation) and also affects septation. This resistance does not reflect a lack of PBP2. Furthermore, it was not simply the result of slow growth and small cell mass, since small wild-type cells growing in acetate remained sensitive. The cAMP-CAP complex may regulate the synthesis of some link between PBP2 and the septation apparatus. The ftsZ gene, coding for a cell division protein, was expressed at a higher level in the absence of cAMP, as measured with an ftsZ::lacZ fusion, but the amount of protein per cell, shown by others to be invariable over a 10-fold range of cell mass, was independent of cAMP, suggesting that ftsZ expression is not regulated by the cAMP-CAP complex. Images PMID:2826407

  19. Genome-Wide Assessment of Outer Membrane Vesicle Production in Escherichia coli

    PubMed Central

    Kulp, Adam J.; Sun, Bo; Ai, Teresa; Manning, Andrew J.; Orench-Rivera, Nichole

    2015-01-01

    The production of outer membrane vesicles by Gram-negative bacteria has been well documented; however, the mechanism behind the biogenesis of these vesicles remains unclear. Here a high-throughput experimental method and systems-scale analysis was conducted to determine vesiculation values for the whole genome knockout library of Escherichia coli mutant strains (Keio collection). The resultant dataset quantitatively recapitulates previously observed phenotypes and implicates nearly 150 new genes in the process of vesiculation. Gene functional and biochemical pathway analyses suggest that mutations that truncate outer membrane structures such as lipopolysaccharide and enterobacterial common antigen lead to hypervesiculation, whereas mutants in oxidative stress response pathways result in lower levels. This study expands and refines the current knowledge regarding the cellular pathways required for outer membrane vesiculation in E. coli. PMID:26406465

  20. Antisuppressor mutation in Escherichia coli defective in biosynthesis of 5-methylaminomethyl-2-thiouridine.

    PubMed Central

    Sullivan, M A; Cannon, J F; Webb, F H; Bock, R M

    1985-01-01

    Mutations in three Escherichia coli K-12 genes were isolated that reduce the efficiency of the lysine-inserting nonsense suppressor supL. These antisuppressor mutations asuD, asuE, and asuF map at 61.9, 25.3, and 76.3 min, respectively, on the E. coli chromosome. Biochemical and genetic analysis of the mutant strains revealed the reason for the antisuppressor phenotype for two of these genes. The activity of lysyl-tRNA synthetase was reduced in strains with asuD mutations. The modification of 5-methylaminomethyl-2-thiouridine, the wobble base of tRNALys, was impaired in asuE mutant strains, presumably at the 2-thiolation step. Images PMID:3881393

  1. Overexpression of Salmonella enterica serovar Typhi recA gene confers fluoroquinolone resistance in Escherichia coli DH5α

    PubMed Central

    Yassien, M.A.M.; Elfaky, M.A.

    2015-01-01

    A spontaneous fluoroquinolone-resistant mutant (STM1) was isolated from its parent Salmonella enterica serovar Typhi (S. Typhi) clinical isolate. Unlike its parent isolate, this mutant has selective resistance to fluoroquinolones without any change in its sensitivity to various other antibiotics. DNA gyrase assays revealed that the fluoroquinolone resistance phenotype of the STM1 mutant did not result from alteration of the fluoroquinolone sensitivity of the DNA gyrase isolated from it. To study the mechanism of fluoroquinolone resistance, a genomic library from the STM1 mutant was constructed in Escherichia coli DH5α and two recombinant plasmids were obtained. Only one of these plasmids (STM1-A) conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. The chromosomal insert from STM1-A, digested with EcoRI and HindIII restriction endonucleases, produced two DNA fragments and these were cloned separately into pUC19 thereby generating two new plasmids, STM1-A1 and STM1-A2. Only STM1-A1 conferred the selective fluoroquinolone resistance phenotype to E. coli DH5α. Sequence and subcloning analyses of STM1-A1 showed the presence of an intact RecA open reading frame. Unlike that of the wild-type E. coli DH5α, protein analysis of a crude STM1-A1 extract showed overexpression of a 40 kDa protein. Western blotting confirmed the 40 kDa protein band to be RecA. When a RecA PCR product was cloned into pGEM-T and introduced into E. coli DH5α, the STM1-A11 subclone retained fluoroquinolone resistance. These results suggest that overexpression of RecA causes selective fluoroquinolone resistance in E. coli DH5α. PMID:26375447

  2. Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli.

    PubMed Central

    Wanner, B L; Boline, J A

    1990-01-01

    The Escherichia coli phn (psiD) locus encodes genes for phosphonate (Pn) utilization, for phn (psiD) mutations abolish the ability to use as a sole P source a Pn with a substituted C-2 or unsubstituted hydrocarbon group such as 2-aminoethylphosphonate (AEPn) or methylphosphonate (MPn), respectively. Even though the E. coli K-12 phosphate starvation-inducible (psi) phn (psiD) gene(s) shows normal phosphate (Pi) control, Pn utilization is cryptic in E. coli K-12, as well as in several members of the E. coli reference (ECOR) collection which are closely related to K-12. For these bacteria, an activating mutation near the phn (psiD) gene is necessary for growth on a Pn as the sole P source. Most E. coli strains, including E. coli B, are naturally Phn+; a few E. coli strains are Phn- and are deleted for phn DNA sequences. The Phn+ phn(EcoB) DNA was molecularly cloned by using the mini-Mu in vivo cloning procedure and complementation of an E. coli K-12 delta phn mutant. The phn(EcoB) DNA hybridized to overlapping lambda clones in the E. coli K-12 gene library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) which contain the 93-min region, thus showing that the phn (psiD) locus was itself cloned and verifying our genetic data on its map location. The cryptic phn(EcoK) DNA has an additional 100 base pairs that is absent in the naturally Phn+ phn(EcoB) sequence. However, no gross structural change was detected in independent Phn+ phn(EcoK) mutants that have activating mutations near the phn locus. Images FIG. 2 PMID:2155195

  3. Mapping and molecular cloning of the phn (psiD) locus for phosphonate utilization in Escherichia coli.

    PubMed

    Wanner, B L; Boline, J A

    1990-03-01

    The Escherichia coli phn (psiD) locus encodes genes for phosphonate (Pn) utilization, for phn (psiD) mutations abolish the ability to use as a sole P source a Pn with a substituted C-2 or unsubstituted hydrocarbon group such as 2-aminoethylphosphonate (AEPn) or methylphosphonate (MPn), respectively. Even though the E. coli K-12 phosphate starvation-inducible (psi) phn (psiD) gene(s) shows normal phosphate (Pi) control, Pn utilization is cryptic in E. coli K-12, as well as in several members of the E. coli reference (ECOR) collection which are closely related to K-12. For these bacteria, an activating mutation near the phn (psiD) gene is necessary for growth on a Pn as the sole P source. Most E. coli strains, including E. coli B, are naturally Phn+; a few E. coli strains are Phn- and are deleted for phn DNA sequences. The Phn+ phn(EcoB) DNA was molecularly cloned by using the mini-Mu in vivo cloning procedure and complementation of an E. coli K-12 delta phn mutant. The phn(EcoB) DNA hybridized to overlapping lambda clones in the E. coli K-12 gene library (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) which contain the 93-min region, thus showing that the phn (psiD) locus was itself cloned and verifying our genetic data on its map location. The cryptic phn(EcoK) DNA has an additional 100 base pairs that is absent in the naturally Phn+ phn(EcoB) sequence. However, no gross structural change was detected in independent Phn+ phn(EcoK) mutants that have activating mutations near the phn locus.

  4. Folate Acts in E. coli to Accelerate C. elegans Aging Independently of Bacterial Biosynthesis.

    PubMed

    Virk, Bhupinder; Jia, Jie; Maynard, Claire A; Raimundo, Adelaide; Lefebvre, Jolien; Richards, Shane A; Chetina, Natalia; Liang, Yen; Helliwell, Noel; Cipinska, Marta; Weinkove, David

    2016-02-23

    Folates are cofactors for biosynthetic enzymes in all eukaryotic and prokaryotic cells. Animals cannot synthesize folate and must acquire it from their diet or microbiota. Previously, we showed that inhibiting E. coli folate synthesis increases C. elegans lifespan. Here, we show that restriction or supplementation of C. elegans folate does not influence lifespan. Thus, folate is required in E. coli to shorten worm lifespan. Bacterial proliferation in the intestine has been proposed as a mechanism for the life-shortening influence of E. coli. However, we found no correlation between C. elegans survival and bacterial growth in a screen of 1,000+ E. coli deletion mutants. Nine mutants increased worm lifespan robustly, suggesting specific gene regulation is required for the life-shortening activity of E. coli. Disrupting the biosynthetic folate cycle did not increase lifespan. Thus, folate acts through a growth-independent route in E. coli to accelerate animal aging.

  5. Phenol removal from refinery wastewater by mutant recombinant horseradish peroxidase.

    PubMed

    Asad, Sedigheh; Dabirmanesh, Bahareh; Khajeh, Khosro

    2014-01-01

    Application of mutated recombinant horseradish peroxidase (HRP) for phenol removal from refinery effluents is reported. Recombinant HRP produced in Escherichia coli suffers from the disadvantage of lacking glycosylation, which affects its catalytic efficiency and stability toward inactivating parameters such as increased temperature and enhanced amounts of hydrogen peroxide. In the present study, the previously reported variant (in which Asn268 was substituted with Asp, N268D) with improved stability characteristics and catalytic efficiency was used to remove phenol from a petroleum refinery effluent. The presence and removal of phenol was studied by high-performance liquid chromatography; the precipitated oxidized phenol was also observed and removed from the sample by centrifugation. Results showed that the N268D variant can remove 61%, 67%, and 81% of phenol from effluent in 1, 2, and 16 H, respectively. By exploiting the N268D mutant, removal of 50% phenol could be achieved in 42 Min, which was more than 22 times less than the treatment time required by native recombinant enzyme.

  6. Characterization of shrunken endosperm mutants in barley.

    PubMed

    Ma, Jian; Jiang, Qian-Tao; Wei, Long; Wang, Ji-Rui; Chen, Guo-Yue; Liu, Ya-Xi; Li, Wei; Wei, Yu-Ming; Liu, Chunji; Zheng, You-Liang

    2014-04-10

    Despite numerous studies on shrunken endosperm mutants caused by either maternal tissues (seg) or kernel per se (sex) in barley, the molecular mechanism for all of the eight seg mutants (seg1-seg8) and some sex mutants is yet to be uncovered. In this study, we determined the amylose content, characterized granule-binding proteins, analyzed the expression of key genes involved in starch synthesis, and examined starch granule structure of both normal (Bowman and Morex) and shrunken endosperm (seg1, seg3, seg4a, seg4b, seg5, seg6, seg7, and sex1) barley accessions. Our results showed that amylose contents of shrunken endosperm mutants ranged from 8.9% (seg4a) to 25.8% (seg1). SDS-PAGE analysis revealed that 87 kDa proteins corresponding to the starch branching enzyme II (SBEII) and starch synthase II (SSII) were not present in seg1, seg3, seg6, and seg7 mutants. Real-time quantitative PCR (RT-qPCR) analysis indicated that waxy expression levels of seg1, seg3, seg6, and seg7 mutants decreased in varying degrees to lower levels until 27 days after anthesis (DAA) after reaching the peak at 15-21 DAA, which differed from the pattern of normal barley accessions. Further characterization of waxy alleles revealed 7 non-synonymous single nucleotide polymorphisms (SNPs) in the coding sequences and 16 SNPs and 8 indels in the promoter sequences of the mutants. Results from starch granule by scanning electron microscopy (SEM) indicated that, in comparison with normal barley accessions, seg4a, seg4b, and sex1 had fewer starch granules per grain; seg3 and seg6 had less small B-type granules; some large A-type granules in seg7 had a hollow surface. These results improve our understanding about effects of seg and sex mutants on starch biosynthesis and granule structure during endosperm development and provide information for identification of key genes responsible for these shrunken endosperm mutants.

  7. Bacteriophage T5 gene A2 protein alters the outer membrane of Escherichia coli.

    PubMed Central

    Snyder, C E

    1984-01-01

    Evidence for changes in Escherichia coli envelope structure caused by the bacteriophage T5 gene A2 protein was obtained by the use of mutant bacteriophages, envelope fractionation procedures, electrophoretic analysis, and in vitro binding studies with purified gene A2 protein. The results suggested that the T5 gene A2 protein perturbs the host envelope as it functions to promote DNA transfer. Images PMID:6389511

  8. Cloning and expression in Escherichia coli of genes involved in the lysine pathway of Brevibacterium lactofermentum.

    PubMed Central

    Márquez, G; Sousa, J M; Sánchez, F

    1985-01-01

    The Brevibacterium lactofermentum genes which complement Escherichia coli lysA and asd-1 mutants were identified, respectively, as a 1.9-kilobase PstI-ClaI fragment and a 2.5-kilobase PstI fragment by cloning into pBR325. Southern blot transfers show hybridization to chromosomal fragments of identical size. The putative B. lactofermentum asd and lysA products are 44 and 48 kilodaltons, respectively. Images PMID:2864331

  9. De Novo Characterization of Genes That Contribute to High-Level Ciprofloxacin Resistance in Escherichia coli

    PubMed Central

    Tran, Thu; Ran, Qinghong; Ostrer, Lev

    2016-01-01

    Sensitization of resistant bacteria to existing antibiotics depends on the identification of candidate targets whose activities contribute to resistance. Using a transposon insertion library in an Escherichia coli mutant that was 2,000 times less susceptible to ciprofloxacin than its parent and the relative fitness scores, we identified 19 genes that contributed to the acquired ciprofloxacin resistance and mapped the shortest genetic path that increased the antibiotic susceptibility of the resistant bacteria back to a near wild-type level. PMID:27431218

  10. Multivalent Repression of Aspartic Semialdehyde Dehydrogenase in Escherichia coli K-12

    PubMed Central

    Boy, Emmanuelle; Patte, Jean-Claude

    1972-01-01

    Mutants of Escherichia coli in which the lysine-sensitive aspartokinase is feedback-resistant are described. In these strains, as well as in the wild type, aspartic semialdehyde dehydrogenase is subject to multivalent repression by lysine, threonine, and methionine. When these amino acids were added to a culture in minimal medium, the differential rate of synthesis of the enzyme dropped to zero and remained there for about one generation. PMID:4404058

  11. Metabolism of L-glyceraldehyde 3-phosphate in Escherichia coli

    SciTech Connect

    Kalyananda, M.K.G.S.

    1985-01-01

    E. coli is able to incorporate L-glyceraldehyde and L-glyceraldehyde 3-phosphate into phospholipids, L-(3-/sup 3/H)Glyceraldehyde was synthesized and the purity and the chemical identity of the product were checked by paper chromatography. L-(3-/sup 3/H)Glyceraldehyde 3-phosphate was synthesized from L-(3-/sup 3/H)glyceraldehyde in a reaction catalyzed by glycerokinase. E. coli extract contains a new enzyme activity which catalyzes an NADPH dependent reduction of L-glyceraldehyde 3-phosphate into sn-glycerol 3-phosphate. A procedure, specifically suitable for assaying the reductase activity in the crude extract, was developed. A more convenient spectrophotometric assay method was employed for the purified enzyme. At moderate concentrations sulfhydryl group inhibitors had no effect on the enzyme activity of L-GAP reductase. At 100..mu..M concentration Zn/sup +2/ inhibited the enzyme activity by about 30% while Mn/sup +2/ elevated the activity by about the same margin. Mg/sup +2/, Ca/sup +2/ and Fe/sup +2/ were without effect at this concentration. L-Glyceraldehyde 3-phosphate is known to be bactericidal at 1.25 ..mu..M concentration and the D-enantiomer is without effect. Furthermore, methylglyoxal is known to be bactericidal at or above 0.5 mM concentration. Strains of E. coli resistant to 1 mM methylglyoxal were isolated. The cell extract prepared from the mutant possessed increased capacity to transform methylglyoxal into D-lactate via a glutathione dependent reaction. These mutants were less sensitive to 2.5 mM DL-GAP suggesting that conversion of L-glyceraldehyde 3-phosphate into methylglyoxal may at least partly be responsible for the bactericidal activity of L-GAP.

  12. Phanerochaete mutants with enhanced ligninolytic activity

    SciTech Connect

    Kakar, S.N.; Perez, A.; Gonzales, J.

    1993-06-01

    In addition to lignin, the white rot fungus Phanerochaete chrysosporium has the ability to degrade a wide spectrum of recalcitrant organopollutants in soils and aqueous media. Although some of the organic compounds are degraded under nonligninolytic conditions, most are degraded under ligninolytic conditions with the involvement of the extracellular enzymes, lignin peroxidases, and manganese-dependent peroxidases, which are produced as secondary metabolites triggered by conditions of nutrient starvation (e.g., nitrogen limitation). The fungus and its enzymes can thus provide alternative technologies for bioremediation, biopulping, biobleaching, and other industrial applications. The efficiency and effectiveness of the fungus can be enhanced by increasing production and secretion of the important enzymes in large quantities and as primary metabolites under enriched conditions. One way this can be achieved is through isolation of mutants that are deregulated or are hyperproducers or supersecretors of key enzymes under enriched conditions. Through ultraviolet-light and gamma-rays mutagenesis we have isolated a variety of mutants, some of which produce key enzymes of the ligninolytic system under high-nitrogen growth conditions. One of the mutants produced 272 units (U) of lignin peroxidases enzyme activity per liter after nine days under high nitrogen. The mutant and the parent strains produced up to 54 U/L and 62 U/L, respectively, of the enzyme activity under low-nitrogen growth conditions during this period. In some experiments the mutant showed 281 U/L of enzyme activity under high nitrogen after 17 days.

  13. Computing border bases using mutant strategies

    NASA Astrophysics Data System (ADS)

    Ullah, E.; Abbas Khan, S.

    2014-01-01

    Border bases, a generalization of Gröbner bases, have actively been addressed during recent years due to their applicability to industrial problems. In cryptography and coding theory a useful application of border based is to solve zero-dimensional systems of polynomial equations over finite fields, which motivates us for developing optimizations of the algorithms that compute border bases. In 2006, Kehrein and Kreuzer formulated the Border Basis Algorithm (BBA), an algorithm which allows the computation of border bases that relate to a degree compatible term ordering. In 2007, J. Ding et al. introduced mutant strategies bases on finding special lower degree polynomials in the ideal. The mutant strategies aim to distinguish special lower degree polynomials (mutants) from the other polynomials and give them priority in the process of generating new polynomials in the ideal. In this paper we develop hybrid algorithms that use the ideas of J. Ding et al. involving the concept of mutants to optimize the Border Basis Algorithm for solving systems of polynomial equations over finite fields. In particular, we recall a version of the Border Basis Algorithm which is actually called the Improved Border Basis Algorithm and propose two hybrid algorithms, called MBBA and IMBBA. The new mutants variants provide us space efficiency as well as time efficiency. The efficiency of these newly developed hybrid algorithms is discussed using standard cryptographic examples.

  14. Recombinant expression of Streptococcus pneumoniae capsular polysaccharides in Escherichia coli

    PubMed Central

    Kay, Emily J.; Yates, Laura E.; Terra, Vanessa S.; Cuccui, Jon; Wren, Brendan W.

    2016-01-01

    Currently, Streptococcus pneumoniae is responsible for over 14 million cases of pneumonia worldwide annually, and over 1 million deaths, the majority of them children. The major determinant for pathogenesis is a polysaccharide capsule that is variable and is used to distinguish strains based on their serotype. The capsule forms the basis of the pneumococcal polysaccharide vaccine (PPV23) that contains purified capsular polysaccharide from 23 serotypes, and the pneumococcal conjugate vaccine (PCV13), containing 13 common serotypes conjugated to CRM197 (mutant diphtheria toxin). Purified capsule from S. pneumoniae is required for pneumococcal conjugate vaccine production, and costs can be prohibitively high, limiting accessibility of the vaccine in low-income countries. In this study, we demonstrate the recombinant expression of the capsule-encoding locus from four different serotypes of S. pneumoniae within Escherichia coli. Furthermore, we attempt to identify the minimum set of genes necessary to reliably and efficiently express these capsules heterologously. These E. coli strains could be used to produce a supply of S. pneumoniae serotype-specific capsules without the need to culture pathogenic bacteria. Additionally, these strains could be applied to synthetic glycobiological applications: recombinant vaccine production using E. coli outer membrane vesicles or coupling to proteins using protein glycan coupling technology. PMID:27110302

  15. Dissecting the Escherichia coli periplasmic chaperone network using differential proteomics

    PubMed Central

    Vertommen, Didier; Silhavy, Thomas J.; Collet, Jean-Francois

    2013-01-01

    β-barrel proteins, or outer membrane proteins (OMPs), perform many essential functions in Gram-negative bacteria, but questions remain about the mechanism by which they are assembled into the outer membrane (OM). In Escherichia coli, β-barrels are escorted across the periplasm by chaperones, most notably SurA and Skp. However, the contributions of these two chaperones to the assembly of the OM proteome remained unclear. We used differential proteomics to determine how the elimination of Skp and SurA affects the assembly of many OMPs. We have shown that removal of Skp has no impact on the levels of the 63 identified OM proteins. However, depletion of SurA in the skp strain has a marked impact on the OM proteome, diminishing the levels of almost all β-barrel proteins. Our results are consistent with a model in which SurA plays a primary chaperone role in E. coli. Furthermore, they suggest that while no OMPs prefer the Skp chaperone pathway in wild-type cells, most can use Skp efficiently when SurA is absent. Our data, which provide a unique glimpse into the protein content of the non-viable surA skp mutant, clarify the roles of the periplasmic chaperones in E. coli. PMID:22589188

  16. The Application of Molecular Modeling for Prediction of Substrate Specificity in Cytochrome P450 1A2 Mutants

    PubMed Central

    Tu, Youbin; Deshmukh, Rahul; Sivaneri, Meena; Szklarz, Grazyna D.

    2008-01-01

    Molecular dynamics (MD) simulations of 7-ethoxy and 7-methoxyresorufin bound in the active site of P450 1A2 wild type and various mutants were used to predict changes in substrate specificity of the mutants. A total of 26 multiple mutants representing all possible combinations of five key amino acid residues which are different between P450 1A1 and 1A2, were examined. The resorufin substrates were docked in the active site of each enzyme in the productive binding orientation and MD simulations were performed on the ES complex. Ensembles collected from MD trajectories were then scored based on geometric parameters relating substrate position with respect to the activated oxoheme cofactor. The results showed a high correlation between the previous experimental data on P450 1A2 wild type and single mutants with respect to the ratio between 7-ethoxyresorufin-O-deethylase (EROD) and 7-methoxyresorufin-O-demethylase (MROD) activities, and the equivalent in silico E/M scores. Moreover, this correlation served to establish linear regression models utilized to evaluate E/M scores of multiple P450 1A2 mutants. Seven mutants, all of them incorporating the L382V substitution, were predicted to shift specificity to that of P450 1A1. The predictions were then verified experimentally. The appropriate P450 1A2 multiple mutants were constructed by site-directed mutagenesis, expressed in E. coli, and assayed for EROD and MROD activities. Out of six mutants, five demonstrated increased EROD/MROD ratio confirming modeling predictions. PMID:18703643

  17. Zinc treatment is efficient against Escherichia coli α-haemolysin-induced intestinal leakage in mice

    PubMed Central

    Wiegand, Stephanie; Zakrzewski, Silke S.; Eichner, Miriam; Schulz, Emanuel; Günzel, Dorothee; Pieper, Robert; Rosenthal, Rita; Barmeyer, Christian; Bleich, André; Dobrindt, Ulrich; Schulzke, Jörg-Dieter; Bücker, Roland

    2017-01-01

    Zinc homoeostasis exerts protective effects in inflammatory intestinal diseases and zinc supplementation has been successfully used for treating infectious diarrhoea. This study aimed at a characterisation of zinc effects on focal leak induction by α-haemolysin (HlyA)-producing Escherichia coli (E. coli) as protective mechanism for colitis. We conducted in vivo experiments by oral challenge of gnotobiotic mice colonised with HlyA-expressing E. coli-536. Mice were either fed a defined normal or high zinc diet to analyse effects of zinc as a therapeutic regimen. HlyA-deficient E. coli-536 mutants were used as controls. Mice infected with HlyA-producing E. coli showed impaired barrier integrity when receiving normal zinc. High zinc supplementation in HlyA-producing E. coli-infected mice reduced epithelial dysfunction as indicated by ameliorated macromolecule permeability. Reduced size of focal leaks with diminished bacterial translocation was observed as inherent mechanisms of this zinc action. In human colon cell monolayers application of zinc rescued the HlyA-dependent decline in transepithelial electrical resistance via reduction of the calcium entry into HlyA-exposed cells. Calcium-dependent cell exfoliation was identified as mechanism for focal leak induction. In conclusion, zinc supplementation protects from HlyA-induced barrier dysfunction in vivo and in vitro, providing an explanation for the protective efficacy of zinc in intestinal disorders. PMID:28361997

  18. Uropathogenic virulence factor FimH facilitates binding of uteropathogenic Escherichia coli to canine endometrium.

    PubMed

    Krekeler, N; Marenda, M S; Browning, G F; Holden, K M; Charles, J A; Wright, P J

    2012-09-01

    Pyometra is a potentially life-threatening condition in bitches and is often caused by Escherichia coli infection. Both pathogenic and non-pathogenic E. coli strains commonly carry the genes for type 1 fimbriae that mediate bacterial adhesion onto host epithelium. To investigate whether the type 1 fimbrial adhesin, FimH, facilitates the binding of uropathogenic E. coli to canine endometrium, the fimH gene was insertionally inactivated in a pathogenic E. coli strain. The ability of E. coli to bind to canine endometrial epithelial cells was determined in vitro using canine uterine biopsies. Binding of the fimH mutant was only 0.3% of that of the wild type. Complementation of the mutation restored the phenotype to that of the parent. This study has developed an in vitro model that allows quantitative and qualitative assessment of bacterial binding to canine endometrium and has demonstrated that the fimH gene plays a role in adherence of pathogenic E. coli to canine endometrium.

  19. Mechanism of gallic acid biosynthesis in bacteria (Escherichia coli) and walnut (Juglans regia).

    PubMed

    Muir, Ryann M; Ibáñez, Ana M; Uratsu, Sandra L; Ingham, Elizabeth S; Leslie, Charles A; McGranahan, Gale H; Batra, Neelu; Goyal, Sham; Joseph, Jorly; Jemmis, Eluvathingal D; Dandekar, Abhaya M

    2011-04-01

    Gallic acid (GA), a key intermediate in the synthesis of plant hydrolysable tannins, is also a primary anti-inflammatory, cardio-protective agent found in wine, tea, and cocoa. In this publication, we reveal the identity of a gene and encoded protein essential for GA synthesis. Although it has long been recognized that plants, bacteria, and fungi synthesize and accumulate GA, the pathway leading to its synthesis was largely unknown. Here we provide evidence that shikimate dehydrogenase (SDH), a shikimate pathway enzyme essential for aromatic amino acid synthesis, is also required for GA production. Escherichia coli (E. coli) aroE mutants lacking a functional SDH can be complemented with the plant enzyme such that they grew on media lacking aromatic amino acids and produced GA in vitro. Transgenic Nicotiana tabacum lines expressing a Juglans regia SDH exhibited a 500% increase in GA accumulation. The J. regia and E. coli SDH was purified via overexpression in E. coli and used to measure substrate and cofactor kinetics, following reduction of NADP(+) to NADPH. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (RP-LC/ESI-MS) was used to quantify and validate GA production through dehydrogenation of 3-dehydroshikimate (3-DHS) by purified E. coli and J. regia SDH when shikimic acid (SA) or 3-DHS were used as substrates and NADP(+) as cofactor. Finally, we show that purified E. coli and J. regia SDH produced GA in vitro.

  20. Expression and mutation of soybean beta-amylase in Escherichia coli.

    PubMed

    Totsuka, A; Fukazawa, C

    1993-06-15

    The cDNA clones corresponding to soybean beta-amylase mRNA were isolated and sequenced. The cDNA contained an open-reading frame composed of 496 amino acids. The comparison of the amino acid sequence deduced from the cDNA with the N-terminal peptide sequence from mature enzyme proved that beta-amylase had no leader sequence. Employing the cDNA, the beta-amylase was directly synthesized in Escherichia coli by the expression vector pKK233-2 controlled by the tac promoter. The enzyme activity detected in E. coli lysate drastically increased with a lower cultivation temperature, and the total activity and specific activity of the enzyme in E. coli lysate cultured at 13 degrees C was 130-fold and 280-fold, respectively, the value at 37 degrees C. The enzyme produced in E. coli was purified by the affinity column chromatography of cyclomaltohexaose-immobilized Sepharose 6B. Employing the established expression and purification system of the enzyme, the functional ionizable groups in the active site were searched. His93, involving an imidazole, and Asp348, involving a carboxylate, in the highly conserved regions within the beta-amylases were replaced by Arg (H93R) and Ash (D348N) by site-directed mutagenesis, respectively. All beta-amylases, including the non-mutant and mutant beta-amylases, produced in E. coli exhibited lower Vmax values than that of beta-amylase isolated conventionally from soybean seeds. Especially the Vmax value of [H93R]beta-amylase was reduced drastically compared to that of the non-mutant; however, none of them lost their enzyme activities completely. Therefore, neither His93 nor Asp348 may participate in the catalytic reaction directly.

  1. Effects of Outer Membrane Protein TolC on the Transport of Escherichia coli within Saturated Quartz Sands

    PubMed Central

    Feriancikova, Lucia; Bardy, Sonia L.; Wang, Lixia; Li, Jin; Xu, Shangping

    2013-01-01

    The outer membrane protein (OMP) TolC is the cell surface component of several drug efflux pumps that are responsible for bacterial resistance against a variety of antibiotics. In this research, we investigated the effects of OMP TolC on E. coli transport within saturated sands through column experiments using a wide type E. coli K12 strain (with OMP TolC), as well as the corresponding transposon mutant (tolC∷kan) and the markerless deletion mutant (ΔtolC). Our results showed OMP TolC could significantly enhance the transport of E. coli when the ionic strength was 20 mM NaCl or higher. The deposition rate coefficients for the wild type E. coli strain (with OMP TolC) was usually >50% lower than those of the tolC-negative mutants. The measurements of contact angles using three probe liquids suggested that TolC altered the surface tension components of E. coli cells and lead to lower Hamaker constants for the cell-water-sand system. The interaction energy calculations using the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory suggested that the deposition of the E. coli cell primarily occurred at the secondary energy minimum. The depth of the secondary energy minimum increased with ionic strength, and was greater for the TolC-deletion strains under high ionic strength conditions. Overall, the transport behavior of three E. coli strain within saturated sands could be explained by the XDLVO calculations. Results from this research suggested that antibiotic resistant bacteria expressing OMP TolC could spread more widely within sandy aquifers. PMID:23627691

  2. Effects of outer membrane protein TolC on the transport of Escherichia coli within saturated quartz sands.

    PubMed

    Feriancikova, Lucia; Bardy, Sonia L; Wang, Lixia; Li, Jin; Xu, Shangping

    2013-06-04

    The outer membrane protein (OMP) TolC is the cell surface component of several drug efflux pumps that are responsible for bacterial resistance against a variety of antibiotics. In this research, we investigated the effects of OMP TolC on E. coli transport within saturated sands through column experiments using a wild-type E. coli K12 strain (with OMP TolC), as well as the corresponding transposon mutant (tolC::kan) and the markerless deletion mutant (ΔtolC). Our results showed OMP TolC could significantly enhance the transport of E. coli when the ionic strength was 20 mM NaCl or higher. The deposition rate coefficients for the wild-type E. coli strain (with OMP TolC) was usually >50% lower than those of the tolC-negative mutants. The measurements of contact angles using three probe liquids suggested that TolC altered the surface tension components of E. coli cells and lead to lower Hamaker constants for the cell-water-sand system. The interaction energy calculations using the extended Derjaguin-Landau-Verwey-Overbeek (XDLVO) theory suggested that the deposition of the E. coli cell primarily occurred at the secondary energy minimum. The depth of the secondary energy minimum increased with ionic strength, and was greater for the TolC-deletion strains under high ionic strength conditions. Overall, the transport behavior of three E. coli strains within saturated sands could be explained by the XDLVO calculations. Results from this research suggested that antibiotic resistant bacteria expressing OMP TolC could spread more widely within sandy aquifers.

  3. Regulation of alcohol fermentation by Escherichia coli

    SciTech Connect

    Clark, D.P.

    1986-03-01

    The purpose of this project is to elucidate the way in which the fermentative synthesis of ethanol is regulated in the facultative anaerobe Escherichia coli. Focus is on the two final steps in alcohol synthesis, which are catalyzed by alcohol dehydrogenase and acetaldehyde CoA dehydrogenase. We have isolated a series of mutations affecting the expression of these enzymes. Some of these mutations are in the structural genes for these enzymes; others affect the regulation of the adh operon. We have recently cloned the genes coding for these enzymes and are now studying the effect of multiple copies of the adh gene on fermentative growth and its regulation. A recently invented technique, proton suicide has allowed the selection of a variety of novel mutants affecting fermentation which are presently being characterized. We have isolated a comprehensive collection of operon fusions in which the lacZ structural gene is fused to promoters that are inactive aerobically but active anaerobically. Although these genes (like adh) are only expressed under anaerobic conditions, the level of induction varies from two-fold to nearly 100-fold. The nitrogen source, medium pH, nature of the buffer, presence of alternative electron acceptors (e.g., nitrate), and other factors exert a great effect on the expression of many of these genes. In the near future we will investigate control mechanisms common to the adh operon and other anaerobically regulated genes.

  4. Oligosaccharide Binding in Escherichia coli Glycogen Synthase

    SciTech Connect

    Sheng, Fang; Yep, Alejandra; Feng, Lei; Preiss, Jack; Geiger, James H.

    2010-11-17

    Glycogen/starch synthase elongates glucan chains and is the key enzyme in the synthesis of glycogen in bacteria and starch in plants. Cocrystallization of Escherichia coli wild-type glycogen synthase (GS) with substrate ADPGlc and the glucan acceptor mimic HEPPSO produced a closed form of GS and suggests that domain-domain closure accompanies glycogen synthesis. Cocrystallization of the inactive GS mutant E377A with substrate ADPGlc and oligosaccharide results in the first oligosaccharide-bound glycogen synthase structure. Four bound oligosaccharides are observed, one in the interdomain cleft (G6a) and three on the N-terminal domain surface (G6b, G6c, and G6d). Extending from the center of the enzyme to the interdomain cleft opening, G6a mostly interacts with the highly conserved N-terminal domain residues lining the cleft of GS. The surface-bound oligosaccharides G6c and G6d have less interaction with enzyme and exhibit a more curled, helixlike structural arrangement. The observation that oligosaccharides bind only to the N-terminal domain of GS suggests that glycogen in vivo probably binds to only one side of the enzyme to ensure unencumbered interdomain movement, which is required for efficient, continuous glucan-chain synthesis.

  5. Metabolic Engineering of Escherichia coli for Poly(3-hydroxybutyrate) Production under Microaerobic Condition.

    PubMed

    Wei, Xiao-Xing; Zheng, Wei-Tao; Hou, Xue; Liang, Jian; Li, Zheng-Jun

    2015-01-01

    The alcohol dehydrogenase promoter PadhE and mixed acid fermentation pathway deficient mutants of Escherichia coli were employed to produce poly(3-hydroxybutyrate) (P3HB) under microaerobic condition. The E. coli mutant with ackA-pta, poxB, ldhA, and adhE deletions accumulated 0.67 g/L P3HB, up to 78.84% of cell dry weight in tube cultivation. The deletion of pyruvate formate-lyase gene pflB drastically decreased P3HB production and P3HB content to 0.09 g/L and 24.44%, respectively. Overexpressing pflB via the plasmid in its knocked out mutant restored cell growth and P3HB accumulation, indicating the importance of the pyruvate formate-lyase in microaerobic carbon metabolism. The engineered E. coli BWapld (pWYC09) produced 5.00 g/L P3HB from 16.50 g/L glucose in 24 h batch fermentation, and P3HB production yield from glucose was 0.30 g/g, which reached up to 63% of maximal theoretical yield.

  6. Aromatic amino acid biosynthesis: regulation of shikimate kinase in Escherichia coli K-12.

    PubMed Central

    Ely, B; Pittard, J

    1979-01-01

    Starvation of cells of Escherichia coli K-12 for the aromatic amino acids results in an increased rate of synthesis of shikimate kinase activity. The two controlling amino acids are tyrosine and tryptophan, and starvation for both results in derepression. The product of the regulator gene tyrR also participates in this control, and shikimate kinase synthesis was depressed in tyrR mutants. Chromatography of cell extracts on diethylaminoethyl-Sephadex allowed partial separation of two shikimate kinase enzymes and demonstrated that only one of these subject to specific repression control involving tyrR. By contrast, chromatography of cell extracts with G-75 or G-200 columns revealed a singl-molecular-weight species of shikimate kinase activity with an apparent molecular weight of 20,000. The levels of shikimate kinase in a series of partial diploid strains indicated that aroL, the structural gene for the tyrR-controlled shikimate kinase enzyme, is located on the E. coli chromosome between the structural genes proC and purE. By means of localized mutagenesis, an aroL mutant of E. coli was isolated. The mutant was an aromatic prototroph and, by the criterion of column chromatography, appeared to have only a single functional species of shikimate kinase enzyme. PMID:222728

  7. DNA end resection controls the balance between homologous and illegitimate recombination in Escherichia coli.

    PubMed

    Ivanković, Siniša; Đermić, Damir

    2012-01-01

    Even a partial loss of function of human RecQ helicase analogs causes adverse effects such as a cancer-prone Werner, Bloom or Rothmund-Thompson syndrome, whereas a complete RecQ deficiency in Escherichia coli is not deleterious for a cell. We show that this puzzling difference is due to different mechanisms of DNA double strand break (DSB) resection in E. coli and humans. Coupled helicase and RecA loading activities of RecBCD enzyme, which is found exclusively in bacteria, are shown to be responsible for channeling recombinogenic 3' ending tails toward productive, homologous and away from nonproductive, aberrant recombination events. On the other hand, in recB1080/recB1067 mutants, lacking RecBCD's RecA loading activity while preserving its helicase activity, DSB resection is mechanistically more alike that in eukaryotes (by its uncoupling from a recombinase polymerization step), and remarkably, the role of RecQ also becomes akin of its eukaryotic counterparts in a way of promoting homologous and suppressing illegitimate recombination. The sickly phenotype of recB1080 recQ mutant was further exacerbated by inactivation of an exonuclease I, which degrades the unwound 3' tail. The respective recB1080 recQ xonA mutant showed poor viability, DNA repair and homologous recombination deficiency, and very increased illegitimate recombination. These findings demonstrate that the metabolism of the 3' ending overhang is a decisive factor in tuning the balance of homologous and illegitimate recombination in E. coli, thus highlighting the importance of regulating DSB resection for preserving genome integrity. recB mutants used in this study, showing pronounced RecQ helicase and exonuclease I dependence, make up a suitable model system for studying mechanisms of DSB resection in bacteria. Also, these mutants might be useful for investigating functions of the conserved RecQ helicase family members, and congruently serve as a simpler, more defined model system for human

  8. The role of oxidative stress genes and effect of pH on methylene blue sensitized photooxidation of Escherichia coli.

    PubMed

    İdil, Önder; Macit, İkbal; Kaygusuz, Özge; Darcan, Cihan

    2016-03-01

    In this study, the survival time of wild type E. coli W3110 and 11 mutants was analysed with a plate count method in methylene blue added or control groups under daylight fluoroscence illumination (4950 lux) at different pH values (5.0, 6.0, 7.0, and 8.0) in phosphate buffer. As a result, while the number of bacteria did not decrease under photooxidative stress at pH 5.0 and 6.0 during a 6-hour incubation, the wild type and all mutants decreased more than 2 log. at pH 8.0, and approximately one log. at pH 7.0. It was determined that a 2 log decrease in wild type E. coli takes 3.7 h according to t99 value at pH 8, these values were 2.39 h in the katE mutant, 2.64 h in the soxR mutant, 2.67 h in the oxyR mutant, 2.71 h in the sodB mutant, 3 h in the btuE mutant, 3.38 h in the zwf mutant and 3.40 h in the soxS mutant, respectively (p < 0.05). The roles of these genes were proved with complement tests. Finally, it is found that the effectiveness of photooxidative stress is in direct relation with pH, and the katE, soxR, oxyR, sodB, btuE, zwf, and soxS genes are important for the protection against this stress.

  9. Sleep restores behavioral plasticity to Drosophila mutants.

    PubMed

    Dissel, Stephane; Angadi, Veena; Kirszenblat, Leonie; Suzuki, Yasuko; Donlea, Jeff; Klose, Markus; Koch, Zachary; English, Denis; Winsky-Sommerer, Raphaelle; van Swinderen, Bruno; Shaw, Paul J

    2015-05-18

    Given the role that sleep plays in modulating plasticity, we hypothesized that increasing sleep would restore memory to canonical memory mutants without specifically rescuing the causal molecular lesion. Sleep was increased using three independent strategies: activating the dorsal fan-shaped body, increasing the expression of Fatty acid binding protein (dFabp), or by administering the GABA-A agonist 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridine-3-ol (THIP). Short-term memory (STM) or long-term memory (LTM) was evaluated in rutabaga (rut) and dunce (dnc) mutants using aversive phototaxic suppression and courtship conditioning. Each of the three independent strategies increased sleep and restored memory to rut and dnc mutants. Importantly, inducing sleep also reverses memory defects in a Drosophila model of Alzheimer's disease. Together, these data demonstrate that sleep plays a more fundamental role in modulating behavioral plasticity than previously appreciated and suggest that increasing sleep may benefit patients with certain neurological disorders.

  10. Membrane-bound beta-lactamase forms in Escherichia coli.

    PubMed

    Plückthun, A; Pfitzinger, I

    1988-10-05

    Frameshift pseudo-revertants of Escherichia coli RTEM beta-lactamase were obtained by a selection procedure, starting from frameshift mutants at the signal-processing site. These pseudo-revertant proteins, which have a totally altered COOH-terminal part of the signal sequence, are attached to the outer face of the inner membrane. The mutant proteins are enzymatically active in vitro and in vivo, and the membrane localization has no deleterious effect on cell growth. We conclude that initiation of transport across the membrane does not require the COOH-terminal part of the signal, but this part of the sequence determines whether the protein is released to the periplasm either with or without cleavage of the signal, or whether the protein remains anchored to the membrane. Mutants with two signals in series were used to show that a truncated signal is not refractory to transport per se. If neither signal contains a functional cleavage site, the protein is at least partially found on the outer face of the inner membrane. If both signals contain functional cleavage sites, both are removed and the protein is released to the periplasm. If only the first signal contains a cleavage site, a longer fusion protein is transported and released. The results presented here show that a pre-beta-lactamase-like protein can fold properly even as a membrane-bound species.

  11. Aminoacyl-tRNA analogues; synthesis, purification and properties of 3'-anthraniloyl oligoribonucleotides.

    PubMed

    Nawrot, B; Sprinzl, M

    1998-04-01

    Reaction of isatoic anhydride with adenosine, adenosine 5'-phosphate, oligoribonucleotides or with the E. coli tRNAVal led to attachment of an anthraniloyl residue at 2'- or 3'-OH groups of 3'-terminal ribose residue. No protection of the 5'-hydroxyl group or internal 2'-hydroxyl groups is required for this specific reaction. Anthraniloyl-tRNA which is an analogue of aminoacyl-tRNA forms a ternary complex with EF-Tu*GTP. The anthraniloyl-residue is used as a fluorescent reporter group to monitor interactions with proteins.

  12. Role of the host cell in bacteriophage T4 development. II. Characterization of host mutants that have pleiotropic effects on T4 growth.

    PubMed Central

    Stitt, B L; Revel, H R; Lielausis, I; Wood, W B

    1980-01-01

    Mutant host-defective Escherichi coli that fail to propagate bacteriophage T4 and have a pleiotropic effect on T4 development have been isolated and characterized. In phage-infected mutant cells, specific early phage proteins are absent or reduced in amount, phage DNA synthesis is depressed by about 50%, specific structural phage proteins, including some tail and collar components, are deficient or missing, and host-cell lysis is delayed and slow. Almost all phage that can overcome the host block carry mutantions that map in functionally undefined 'nonessential' regions of the T4 genome, most near gene 39. The mutant host strains are temperature sensitive for growth and show simultaneous reversion of the ts phenotype and the inability to propagate T4+. The host mutations are cotransduced with ilv (83 min) and may lie in the gene for transcription termination factor rho. Images PMID:6999171

  13. Transcriptomic analysis of triclosan-susceptible and -tolerant Escherichia coli O157:H19 in response to triclosan exposure.

    PubMed

    Lenahan, Mary; Sheridan, Áine; Morris, Dermot; Duffy, Geraldine; Fanning, Séamus; Burgess, Catherine M

    2014-04-01

    Triclosan is an active agent that is commonly found in biocide formulations which are used by the food industry to control microbial contamination. The aim of this study was to use microarray analysis to compare gene expression between a triclosan-susceptible Escherichia coli O157:H19 isolate (minimum inhibitory concentration [MIC] 6.25 μg/ml) and its in vitro generated triclosan-tolerant mutant (MIC >8,000 μg/ml). Gene expression profiling was performed on the wild-type and mutant isogenic pairs after 30 min exposure to the parent MIC for triclosan and an untreated control. Microarray analysis was carried out using the Affymetrix GeneChip E. coli Genome 2.0 Array, and differential expression of genes was analyzed using the pumaDE method in Bioconductor R software. Wild-type gene expression was found to be significantly different from the triclosan-tolerant mutant for a large number of genes, even in the absence of triclosan exposure. Significant differences were observed in the expression of a number of pathway genes involved in metabolism, transport, and chemotaxis. In particular, gene expression in the triclosan-tolerant mutant was highly up-regulated for 33 of 38 genes belonging to the flagellar assembly pathway. The presence of extended flagella in the mutant isolate was confirmed visually by transmission electron microscopy, although no significant difference was observed in the motility of the parent and mutant at low levels of triclosan. Data from this study show that at a transcriptomic level, a triclosan-tolerant E. coli O157:H19 mutant is significantly different from the wild-type strain in a number of different pathways, providing an increased understanding of triclosan tolerance.

  14. Nonphotic phase shifting in hamster clock mutants.

    PubMed

    Mrosovsky, N; Salmon, P A; Menaker, M; Ralph, M R

    1992-01-01

    Golden hamsters with the tau mutation were kept in the dark and induced to become active through confinement to a novel running wheel for 3 hr. The response of the mutants to this nonphotic phase-shifting stimulus differed from that of wild-type hamsters. The mutants showed larger phase shifts, and their phase response curves differed in shape, with an advance portion at about circadian time 24, a phase at which wild types show delays. The results establish that the tau mutation, in addition to its already known effects, alters the response of the circadian system to nonphotic events.

  15. Fluoroquinolone-resistant mutants of Burkholderia cepacia.

    PubMed

    Pope, C F; Gillespie, S H; Pratten, J R; McHugh, T D

    2008-03-01

    Fluoroquinolone-resistant Burkholderia cepacia mutants were selected on ciprofloxacin. The rate of mutation in gyrA was estimated to be 9.6 x 10(-11) mutations per division. Mutations in gyrA conferred 12- to 64-fold increases in MIC, and an additional parC mutation conferred a large increase in MIC (>256-fold). Growth rate, biofilm formation, and survival in water and during drying were not impaired in strains containing single gyrA mutations. Double mutants were impaired only in growth rate (0.85, relative to the susceptible parent).

  16. Three New Escherichia coli Phages from the Human Gut Show Promising Potential for Phage Therapy

    PubMed Central

    Dalmasso, Marion; Strain, Ronan; Neve, Horst; Franz, Charles M. A. P.; Cousin, Fabien J.; Ross, R. Paul; Hill, Colin

    2016-01-01

    With the emergence of multi-drug resistant bacteria the use of bacteriophages (phages) is gaining renewed interest as promising anti-microbial agents. The aim of this study was to isolate and characterize phages from human fecal samples. Three new coliphages, ɸAPCEc01, ɸAPCEc02 and ɸAPCEc03, were isolated. Their phenotypic and genomic characteristics, and lytic activity against biofilm, and in combination with ciprofloxacin, were i