scholarly journals In Vivo Reversion to the Wild-Type β-Lactam Resistance Phenotype Mediated by a Plasmid Carrying ampR and qnrA1 in Enterobacter cloacae

2006 ◽  
Vol 50 (9) ◽  
pp. 3175-3178
Author(s):  
J. J. González-López ◽  
M. Sabaté ◽  
S. Lavilla ◽  
M. N. Larrosa ◽  
R. M. Bartolomé ◽  
...  
Keyword(s):  
Enterobacter Cloacae ◽  
The Other ◽  
Resistance Pattern ◽  
Wild Type ◽  
Resistance Phenotype ◽  
Amoxicillin Clavulanate

ABSTRACT Resistance to β-lactams and quinolones in two isogenic Enterobacter cloacae isolates was studied. One was susceptible to cefoxitin and amoxicillin-clavulanate. The other one showed its natural β-lactam resistance pattern. Both isolates had a nonfunctional AmpR regulator. However, within the second one, the presence of a plasmid carrying ampR and qnrA1 allowed reversion to the wild-type β-lactam resistance phenotype and decreased susceptibility to fluoroquinolones.

HNF-1α and endodermal transcription factors cooperatively activate Fabpl: MODY3 mutations abrogate cooperativity

2003 ◽  
Vol 285 (1) ◽  
pp. G62-G72 ◽  
Author(s):  
Joyce K. Divine ◽  
Sean P. McCaul ◽  
Theodore C. Simon
Keyword(s):  
Transcription Factors ◽  
Gene Activation ◽  
The Other ◽  
Wild Type ◽  
Critical Determinant ◽  
Fatty Acid Binding ◽  
Hepatic Expression ◽  
Synergistic Activation ◽  
Five Factors

Hepatocyte nuclear factor (HNF)-1α plays a central role in intestinal and hepatic gene regulation and is required for hepatic expression of the liver fatty acid binding protein gene ( Fabpl). An Fabpl transgene was directly activated through cognate sites by HNF-1α and HNF-1β, as well as five other endodermal factors: CDX-1, C/EBPβ, GATA-4, FoxA2, and HNF-4α. HNF-1α activated the Fabpl transgene by as much as 60-fold greater in the presence of the other five endodermal factors than in their absence, accounting for up to one-half the total transgene activation by the group of six factors. This degree of synergistic interaction suggests that multifactor cooperativity is a critical determinant of endodermal gene activation by HNF-1α. Mutations in HNF-1α that result in maturity onset diabetes of the young (MODY3) provide evidence for the in vivo significance of these synergistic interactions. An R131Q HNF-1α MODY3 mutant exhibits complete loss of synergistic activation in concert with the other endodermal transcription factors despite wild-type transactivation ability in their absence. Furthermore, whereas wild-type HNF-1α exhibited pairwise cooperative synergy with each of the other five factors, the R131Q mutant could synergize only with GATA-4 and C/EBPβ. Selective loss of synergy with other endodermal transcription factors accompanied by retention of native transactivation ability in an HNF-1α MODY mutant suggests in vivo significance for cooperative synergy.

scholarly journals Mutational Replacement of Leu-293 in the Class C Enterobacter cloacae P99 β-Lactamase Confers Increased MIC of Cefepime

2002 ◽  
Vol 46 (6) ◽  
pp. 1966-1970 ◽  
Author(s):  
Sergei B. Vakulenko ◽  
Dasantila Golemi ◽  
Bruce Geryk ◽  
Maxim Suvorov ◽  
James R. Knox ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Broad Spectrum ◽  
Random Mutagenesis ◽  
Enterobacter Cloacae ◽  
The Other ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Wide Range ◽  
Class C

ABSTRACT The class C β-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum β-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the E. cloacae P99 ampC gene, we obtained a Leu-293-Pro mutant of the P99 β-lactamase conferring a higher MIC of cefepime (MIC, 8 μg/ml, compared with 0.5 μg/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other β-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified E. cloacae P99 β-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (k cat/Km ) of the Leu-293-Pro mutant β-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 β-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant β-lactamase.

scholarly journals Involvement of the AcrAB-TolC Efflux Pump in the Resistance, Fitness, and Virulence of Enterobacter cloacae

2012 ◽  
Vol 56 (4) ◽  
pp. 2084-2090 ◽  
Author(s):  
Astrid Pérez ◽  
Margarita Poza ◽  
Ana Fernández ◽  
Maria del Carmen Fernández ◽  
Susana Mallo ◽  
...  
Keyword(s):  
Antimicrobial Resistance ◽  
Pathogenic Bacteria ◽  
Efflux Pump ◽  
Enterobacter Cloacae ◽  
Efflux Pumps ◽  
Clinical Isolates ◽  
Wild Type ◽  
Content Type

ABSTRACTMultidrug efflux pumps have emerged as important mechanisms of antimicrobial resistance in bacterial pathogens. In order to cause infection, pathogenic bacteria require mechanisms to avoid the effects of host-produced compounds, and express efflux pumps may accomplish this task. In this study, we evaluated the effect of the inactivation of AcrAB-TolC on antimicrobial resistance, fitness, and virulence inEnterobacter cloacae, an opportunistic pathogen usually involved in nosocomial infections. Two different clinical isolates ofE. cloacaewere used, EcDC64 (multidrug resistance overexpressing the AcrAB-TolC efflux pump) and Jc194 (basal AcrAB-TolC expression). TheacrAandtolCgenes were deleted in strains EcDC64 and Jc194 to produce, respectively, EcΔacrAand EcΔtolCand JcΔacrAand JcΔtolCknockout (KO) derivatives. Antibiotic susceptibility testing was performed with all isolates, and we discovered that these mechanisms are involved in the resistance ofE. cloacaeto several antibiotics. Competition experiments were also performed with wild-type and isogenic KO strains. The competition index (CI), defined as the mutant/wild-type ratio, revealed that theacrAandtolCgenes both affect the fitness ofE. cloacae, as fitness was clearly reduced in theacrAandtolCKO strains. The median CI values obtainedin vitroandin vivowere, respectively, 0.42 and 0.3 for EcDC64/EcΔacrA, 0.24 and 0.38 for EcDC64/EcΔtolC, 0.15 and 0.11 for Jc194/JcΔacrA, and 0.38 and 0.39 for Jc194/JcΔtolC. Use of an intraperitoneal mouse model of systemic infection revealed reduced virulence in bothE. cloacaeclinical strains when either theacrAortolCgene was inactivated. In conclusion, the structural components of the AcrAB-TolC efflux pump appear to play a role in antibiotic resistance as well as environmental adaptation and host virulence in clinical isolates ofE. cloacae.

scholarly journals BACTERIOPHAGE T4 ENDONUCLEASES II AND IV, OPPOSITELY AFFECTED BY dCMP HYDROXYMETHYLASE ACTIVITY, HAVE DIFFERENT ROLES IN THE DEGRADATION AND IN THE RNA POLYMERASE-DEPENDENT REPLICATION OF T4 CYTOSINE-CONTAINING DNA

Genetics ◽  
1986 ◽  
Vol 114 (3) ◽  
pp. 669-685
Author(s):  
Karin Carlson ◽  
Aud Ȗvervatin
Keyword(s):  
Rna Polymerase ◽  
Dna Synthesis ◽  
Bacteriophage T4 ◽  
The Other ◽  
Wild Type ◽  
E Coli ◽  
Phage Dna ◽  
Dna Template ◽  
Do So

ABSTRACT Bacteriophage T4 mutants defective in gene 56 (dCTPase) synthesize DNA where cytosine (Cyt) partially or completely replaces hydroxymethylcytosine (HmCyt). This Cyt-DNA is degraded in vivo by T4 endonucleases II and IV, and by the exonuclease coded or controlled by genes 46 and 47.—Our results demonstrate that T4 endonuclease II is the principal enzyme initiating degradation of T4 Cyt-DNA. The activity of endonuclease IV, but not that of endonuclease II, was stimulated in the presence of a wild-type dCMP hydroxymethylase, also when no HmCyt was incorporated into phage DNA, suggesting the possibility of direct endonuclease IV-dCMP hydroxymethylase interactions. Endonuclease II activity, on the other hand, was almost completely inhibited in the presence of very small amounts of HmCyt (3-9% of total Cyt + HmCyt) in the DNA. Possible mechanisms for this inhibition are discussed.—The E. coli RNA polymerase modified by the products of T4 genes 33 and 55 was capable of initiating DNA synthesis on a Cyt-DNA template, although it probably cannot do so on an HmCyt template. In the presence of an active endonuclease IV, Cyt-DNA synthesis was arrested 10-30 min after infection, probably due to damage to the template. Cyt-DNA synthesis dependent on the unmodified (33  -  55  -) RNA polymerase was less sensitive to endonuclease IV action.

scholarly journals Independent Regulation of Two Genes in Escherichia coli by Tetracyclines and Tet Repressor Variants

2004 ◽  
Vol 186 (13) ◽  
pp. 4399-4401 ◽  
Author(s):  
Annette Kamionka ◽  
Miriam Sehnal ◽  
Oliver Scholz ◽  
Wolfgang Hillen
Keyword(s):  
Escherichia Coli ◽  
Cross Talk ◽  
Reporter Genes ◽  
The Other ◽  
Regulation System ◽  
The Novel ◽  
Wild Type ◽  
Tet Repressor

ABSTRACT We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities. One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline. The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline. We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk.

SDQR migrations in Caenorhabditis elegans are controlled by multiple guidance cues and changing responses to netrin UNC-6

Development ◽  
1999 ◽  
Vol 126 (17) ◽  
pp. 3881-3890 ◽  
Author(s):  
S. Kim ◽  
X.C. Ren ◽  
E. Fox ◽  
W.G. Wadsworth
Keyword(s):  
Caenorhabditis Elegans ◽  
Cell Body ◽  
The Other ◽  
Wild Type ◽  
Multiple Signals ◽  
C Elegans ◽  
Guidance Cues ◽  
Guidance Cue ◽  
Responsive Cell

The netrin guidance cue, UNC-6, and the netrin receptors, UNC-5 and UNC-40, guide SDQR cell and axon migrations in C. elegans. In wild-type larvae, SDQR migrations are away from ventral UNC-6-expressing cells, suggesting that UNC-6 repels SDQR. In unc-6 null larvae, SDQR migrations are towards the ventral midline, indicating a response to other guidance cues that directs the migrations ventrally. Although ectopic UNC-6 expression dorsal to the SDQR cell body would be predicted to cause ventral SDQR migrations in unc-6 null larvae, in fact, more migrations are directed dorsally, suggesting that SDQR is not always repelled from the dorsal source of UNC-6. UNC-5 is required for dorsal SDQR migrations, but not for the ventral migrations in unc-6 null larvae. UNC-40 appears to moderate both the response to UNC-6 and to the other cues. Our results show that SDQR responds to multiple guidance cues and they suggest that, besides UNC-6, other factors influence whether an UNC-6 responsive cell migrates toward or away from an UNC-6 source in vivo. We propose that multiple signals elicited by the guidance cues are integrated and interpreted by SDQR and that the response to UNC-6 can change depending on the combination of cues encountered during migration. These responses determine the final dorsoventral position of the SDQR cell and axon.

The natural occurring Y129F polymorphism in Rhizopus oryzae (R. arrhizus) Cyp51Ap accounts for its intrinsic voriconazole resistance

2021 ◽  
Author(s):  
Daiana Macedo ◽  
Florencia Leonardelli ◽  
Matias S Cabeza ◽  
Soledad Gamarra ◽  
Guillermo Garcia-Effron
Keyword(s):  
Experimental Data ◽  
Amino Acid ◽  
Rhizopus Oryzae ◽  
The Other ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Fluconazole Resistance ◽  
Resistance Phenotype ◽  
Naturally Occurring

Abstract Rhizopus oryzae (heterotypic synonym: R. arrhizus) intrinsic voriconazole and fluconazole resistance has been linked to its CYP51A gene. However, the amino acid residues involved in this phenotype have not yet been established. A comparison between R. oryzae and Aspergillus fumigatus Cyp51Ap sequences showed differences in several amino acid residues. Some of them were already linked with voriconazole resistance in A. fumigatus. The objective of this work was to analyze the role of two natural polymorphisms in the intrinsic voriconazole resistance phenotype of R. oryzae (Y129F and T290A, equivalent to Y121F and T289A seen in triazole-resistant A. fumigatus). We have generated A. fumigatus chimeric strains harboring different R. oryzae CYP51A genes (wild-type and mutants). These mutant R. oryzae CYP51A genes were designed to carry nucleotide changes that produce mutations at Cyp51Ap residues 129 and 290 (emulating the Cyp51Ap protein of azole susceptible A. fumigatus). Antifungal susceptibilities were evaluated for all the obtained mutants. The polymorphism T290A (alone or in combination with Y129F) had no impact on triazole MIC. On the other hand, a > 8-fold decrease in voriconazole MICs was observed in A. fumigatus chimeric strains harboring the RoCYP51Ap-F129Y. This phenotype supports the assumption that the naturally occurring polymorphism Y129F at R. oryzae Cyp51Ap is responsible for its voriconazole resistance phenotype. In addition, these chimeric mutants were posaconazole hypersusceptible. Thus, our experimental data demonstrate that the RoCYP51Ap-F129 residue strongly impacts VRC susceptibility and that it would be related with posaconazole-RoCYP51Ap interaction. Lay summary Rhizopus oryzae is intrinsically resistant to voriconazole, a commonly used antifungal agent. In this work, we analyze the role of two natural polymorphisms present in the target of azole drugs. We established that F129 residue is responsible of the intrinsic voriconazole resistance in this species.

Abstract 1114: Deregulation of MicroRNAs in the Heart of c-kit-Defective Mice

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Toru Hosoda ◽  
Saori Yasuzawa-Amano ◽  
Katsuya Amano ◽  
Alessandro Boni ◽  
Konrad Urbanek ◽  
...  
Keyword(s):  
Cell Cycle Progression ◽  
Transgenic Animals ◽  
Ventricular Myocardium ◽  
The Other ◽  
Transcriptional Level ◽  
Wild Type ◽  
Protein Levels ◽  
Novel Mirna ◽  
Kit Receptor

MicroRNAs (miRNAs) are a family of non-coding RNAs that regulate gene expression by repressing mRNA function at the post-transcriptional level. Recently, the involvement of miRNAs in the modulation of cardiac function has been described in vivo in the developing and adult heart. Whether miRNAs play an important role in the control of the growth and differentiation of c-kit-positive cardiac progenitor cells (CPCs) is currently unknown. Therefore, two transgenic mouse models characterized by defects in the c-kit receptor or its ligand, stem cell factor (SCF), were studied in combination with wild-type littermates. The first one has a spontaneous point mutation of the c-kit receptor in one allele (W V ) coupled with an amino acid deletion in the other allele (W); and the second has a complete deletion of the SCF gene locus in one allele (Sl) and deletion of the membrane-bound ligand in the other (Sl d ). The ventricular myocardium of transgenic and non-transgenic mice was analyzed with respect to miRNA expression by microarray. We have identified three miRNAs that showed an extremely high level of expression in the heart of transgenic and non-transgenic animals: miR-150, miR-451 and miR-805. These miRNAs have not been described previously in the myocardium and were consistently upregulated in the heart of wild-type mice; a 1.4 –1.8-fold difference was found with respect to transgenic animals. The major difference was detected for miR-150. So far, this miRNA was considered to be restricted to hematopoietic progenitors. Surveying the predicted targets of miR-150, we identified a cluster of genes involved in cell proliferation that were expected to be upregulated in W/W V and Sl/Sl d mice: Akt3, ELK1, FOXP1, Pim-1, IGF-1, Myb and PA2G4. Consistently, W/W V mice showed a two-fold or larger mRNA and protein levels for genes implicated in cell cycle progression. A similar analysis was not possible for miR-451 and miR-805 since the predicted targets of these miRNAs remain to be recognized. In conclusion, a novel miRNA critical for hematopoiesis has been identified in the heart and this miRNA is downregulated when defects of c-kit signaling are present.

HETEROKARYON ANALYSIS OF THE GENETIC CONTROL OF PIGMENT SYNTHESIS IN CHICK EMBRYO MELANOCYTES

Genetics ◽  
1982 ◽  
Vol 102 (3) ◽  
pp. 557-569
Author(s):  
L M Wilkins ◽  
J A Brumbaugh ◽  
J W Moore
Keyword(s):  
Chick Embryo ◽  
Genetic Control ◽  
Regulatory Elements ◽  
The Other ◽  
Wild Type ◽  
Mutant Type ◽  
Pigment Synthesis ◽  
Dominant Mutant

ABSTRACT The genetic control of pigmentation was analyzed using five unlinked mutants, namely, c, pk, Bl, ey and I. Each mutant blocks or reduces pigmentation. Chick melanocyte cultures of each mutant type were fused to produce all ten possible pair combinations of nondividing heterokaryons. Heterokaryons were identified autoradiographically. (One partner in each pair was labeled with 3H-thymidine.) Crosses produced comparable pairs of double heterozygotes that were analyzed in vivo and in vitro. Heterokaryon pairs were compared to their corresponding double heterozygotes.—Some combinations showed complementation and produced wild-type pigment. Others showed noncomplementation having little or no pigment. Double heterozygotes complemented each other except in the cases involving the dominant mutant, I. Four heterokaryon pairs gave different results from their corresponding double heterozygotes. The pk-Bl and pk-ey combinations failed to complement as heterokaryons but did complement as double heterozygotes. On the other hand, the I-c and I-Bl combinations complemented as heterokaryons but not as double heterozygotes. Based on these differences it is hypothesized that the pk and I loci are nuclearly restricted regulatory elements. Examples in the literature from other systems are cited to support such a hypothesis.

scholarly journals The Heat Shock Protein Ssa2p Is Required for Import of Fructose-1,6-Bisphosphatase into Vid Vesicles

2000 ◽  
Vol 150 (1) ◽  
pp. 65-76 ◽  
Author(s):  
C. Randell Brown ◽  
Jameson A. McCann ◽  
Hui-Ling Chiang
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Heat Shock Protein ◽  
Binding Proteins ◽  
The Other ◽  
Atp Binding ◽  
Wild Type ◽  
Fructose 1,6 Bisphosphatase

Fructose-1,6-bisphosphatase (FBPase) is targeted to the vacuole for degradation when Saccharomyces cerevisiae are shifted from low to high glucose. Before vacuolar import, however, FBPase is sequestered inside a novel type of vesicle, the vacuole import and degradation (Vid) vesicles. Here, we reconstitute import of FBPase into isolated Vid vesicles. FBPase sequestration into Vid vesicles required ATP and cytosol, but was inhibited if ATP binding proteins were depleted from the cytosol. The heat shock protein Ssa2p was identified as one of the ATP binding proteins involved in FBPase import. A Δssa2 strain exhibited a significant decrease in the rate of FBPase degradation in vivo as compared with Δssa1, Δssa3, or Δssa4 strains. Likewise, in vitro import was impaired for the Δssa2 strain, but not for the other Δssa strains. The cytosol was identified as the site of the Δssa2 defect; Δssa2 cytosol did not stimulate FBPase import into import competent Vid vesicles, but wild-type cytosol supported FBPase import into competent Δssa2 vesicles. The addition of purified recombinant Ssa2p stimulated FBPase import into Δssa2 Vid vesicles, providing Δssa2 cytosol was present. Thus, Ssa2p, as well as other undefined cytosolic proteins are required for the import of FBPase into vesicles.

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