F Bastarrachea

Universidad Nacional Autónoma de México, Mexico City, The Federal District, Mexico

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Publications (16)19.41 Total impact

  • F Bastarrachea
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    ABSTRACT: Many antibiotic resistance mutations arise in pathogenic bacteria that harbor plasmids (R-plasmids). Resistance to third generation cephalosporins, for instance, largely occurs by one or more point mutations in plasmid bla genes that expand the resistance spectrum of beta-lactamases. Here I review relevant evidence underlying the worldwide emergence of extended spectrum beta-lactamases (ESBLs). The conclusion reached is that the origin of these resistance-conferring mutations cannot be explained by a series of single point mutation and selection events. Instead, highly advantageous stochastic processes might exist that generate alterations in the sequence or the conformation of particular regions in chromosomal or plasmid genomes such as bla, i.e., recombination or mutation. Several explanations for the origin of ESBLs are reviewed but direct experimental evidence to support or to invalidate them is still lacking. The cellular conditions under which ESBLs arise are unknown; however, involvement of nutritional stresses inside natural animal hosts and of plasmid conjugal functions appear likely.
    Journal of Theoretical Biology 02/1998; 190(4):379-87. · 2.35 Impact Factor
  • L Camarena, S Poggio, A Campos, F Bastarrachea, A Osorio
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    ABSTRACT: An insertion element (IS)4 insertion selected as suppressor of the rpoN73::Tn5 alelle was located inside the control region of the glnA gene in Escherichia coli. In the rpoN73::Tn5 background the IS4 insertion promotes glnA transcription at a low constitutive level sufficient to sustain glutamine-independent growth. The IS4 insertion mutation in either rpoN73::Tn5 or wild-type backgrounds promotes glnA transcription from a new start site located two bases downstream of the glnAp2 start site. Analysis of sequences flanking the insertion point showed a promoter sequence whose -35 region was located inside the IS4 sequence and the -10 region was inside the glnA control region. Site-directed mutagenesis of relevant nucleotide residues of the newly created promoter impaired transcription of a reporter gene. The results support our contention that IS4 carries a -35 promoter region that is able to create functional hybrid promoters. We propose that this mechanism could be one of the molecular reasons of the suppressor activity previously reported for IS4.
    Plasmid 02/1998; 39(1):41-7. · 1.76 Impact Factor
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    ABSTRACT: Escherichia coli cells carrying the gltX351 allele are unable to grow at 42 degrees C (Ts phenotype) due to an altered glutamyl-tRNA synthetase. We found that gltX351 cells display a new phenotype termed Gsd-, i.e. an inability to raise glutamine synthetase activity above low constitutive levels in minimal medium with 6.8 mM glutamine as sole nitrogen source. When 0.5 mM NH4+ or 12 mM glutamate replaced glutamine, the glutamine synthetase activities of gltX351 cells were raised to wild-type levels. Northern experiments showed that the Gsd- phenotype is the result of an impairment in transcription initiation from the Ntr-regulated promoter, glnAp2. Intragenic and extragenic secondary mutations appeared frequently in gltX351 cells, which suppressed their Gsd- but not their Ts phenotype. Moreover, in heterozygous gltX+/gltX351 partial diploids, gltX351 was dominant for the Gsd- phenotype and recessive for the Tr phenotype. A slight increase in the glutamine pool and in the intracellular glutamine: 2-oxoglutarate ratio was also observed but this could not account for the Gsd- phenotype of gltX351 cells. In cells carrying gltX351 and a suppressor of the Gsd- phenotype, sup-1, tightly linked to gltX351, the glutamine pool and glutamine: 2-oxoglutarate intracellular ratio were even higher than in the gltX351 single mutant. These results indicate that the gltX351 mutant polypeptide may be the direct cause of the Gsd- phenotype. The possibility that it interacts with one or more components that trigger the Ntr response is discussed.
    MGG - Molecular and General Genetics 07/1993; 239(3):400-8.
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    L Velázquez, L Camarena, J L Reyes, F Bastarrachea
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    ABSTRACT: Individual mutations which affected each of the two Shine-Dalgarno sequences at the 5' untranslated region of the gltB gene of Escherichia coli were characterized. They were isolated in plasmids carrying a gltB'-'lacZ protein fusion preceded by the regulatory region of the gltBDF operon. Subcloning and nucleotide sequencing of approximately 1,206 bp of DNA encompassing the gltBDF regulatory region showed that the mutations affected the first base at each of the two identical Shine-Dalgarno sequences, SD1 and SD2, located 40 and 8 bases, respectively, upstream from the putative gltB open reading frame. Only mutation gltB2r227, an adenine in place of a guanine, affecting the first base of SD2, lowered beta-galactosidase expression significantly, i.e., about fivefold. The results suggest that SD2 is the preferred functional site at which ribosomes initiate gltB mRNA translation.
    Journal of Bacteriology 06/1991; 173(10):3261-4. · 3.19 Impact Factor
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    I Castaño, F Bastarrachea, A A Covarrubias
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    ABSTRACT: A 2.0-kilobase DNA fragment carrying antibiotic resistance markers was inserted into the gltB gene of Escherichia coli previously cloned in a multicopy plasmid. Replacement of the chromosomal gltB+ gene by the gltB225::omega mutation led to cells unable to synthesize glutamate synthase, utilize growth rate-limiting nitrogen sources, or derepress their glutamine synthetase. The existence of a gltBDF operon encoding the large (gltB) and small (gltD) subunits of glutamate synthase and a regulatory peptide (gltF) at 69 min of the E. coli linkage map was deduced from complementation analysis. A plasmid carrying the entire gltB+D+F+ operon complemented cells for all three of the mutant phenotypes associated with the polar gltB225::omega mutation in the chromosome. By contrast, plasmids carrying gltB+ only complemented cells for glutamate synthase activity. A major tricistronic mRNA molecule was detected from Northern (RNA blot) DNA-RNA hybridization experiments with DNA probes containing single genes of the operon. A 30,200-dalton polypeptide was identified as the gltF product, the lack of which was responsible for the inability of cells to use nitrogen-limiting sources associated with gltB225::omega.
    Journal of Bacteriology 03/1988; 170(2):821-7. · 3.19 Impact Factor
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    ABSTRACT: Cells of Escherichia coli K12 were sensitive to 100 mM-methylammonium when cultured under nitrogen limitation, and resistant when grown with an excess of either NH4Cl or glutamine. Glutamine synthetase activity was required for expression of the methylammonium-sensitive phenotype. Mutants were isolated which were resistant to 100 mM-methylammonium, even when grown under nitrogen limitation. P1 bacteriophage transduction and F' complementation analysis revealed that the resistance-conferring mutations mapped either inside the glnA structural gene and/or elsewhere in the E. coli chromosome. Glutamine synthetase was purified from the wild-type and from some of the mutant strains. Strains carrying glnA-linked mutations that were solely responsible for the methylammonium-resistant phenotype yielded an altered enzyme, which was less active biosynthetically with either ammonium or methylammonium as substrate. Sensitivity to methylammonium appeared to be due to synthesis of gamma-glutamylmethylamide by glutamine synthetase, which was synthesized poorly, if at all, by mutants carrying an altered glutamine synthetase enzyme.
    Journal of general microbiology 07/1987; 133(6):1631-9.
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    ABSTRACT: The spontaneous gln-76 mutation of Escherichia coli (Osorio et al., Mol. Gen. Genet. 194:114-123, 1984) was previously shown to be responsible for the cis-dominant constitutive expression of the glnA gene in the absence of a glnG-glnF activator system. Nucleotide sequence analysis has now revealed that gln-76 is a single transversion T.A to A.T, an up-promoter mutation affecting the -10 region of glnAp1, the upstream promoter of the glnALG control region. Both, wild-type and gln-76 DNA control regions were cloned into the promoter-probe plasmid pKO1. Galactokinase activity determinations of cells carrying the fused plasmids showed 10-fold more effective expression mediated by gln-76 than by the glnA wild-type control region. Primer extension experiments with RNA from strains carrying the gln-76 control region indicated that the transcription initiation sites were the same in both the gln-76 mutant and the wild type.
    Journal of Bacteriology 01/1986; 164(3):1032-8. · 3.19 Impact Factor
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    L Servín-González, F Bastarrachea
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    ABSTRACT: Uptake of 14CH3NH+3 (methylammonium) was measured as a probe of NH+4 transport in intact Escherichia coli cells and derivatives impaired in the Ntr regulatory system. The results suggest that expression of the high affinity 14CH3NH+3 transport system (a) requires de novo polypeptide synthesis, (b) is activated by the glnG and glnF regulatory products under nitrogen limitation, and (c) is repressed under nitrogen excess by the glnL product. Cells deficient in glutamate synthase activity by virtue of their harbouring the gltB31 mutation were unable to activate synthesis of 14CH3NH+3 transport. This could explain the inability of cells carrying gltB mutations to grow on low concentrations of NH+4.
    Journal of general microbiology 01/1985; 130(12):3071-7.
  • I Castaño, F Bastarrachea
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    ABSTRACT: The regulatory gene, glnF, of Escherichia coli was fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mudl (Ap lac). Analysis of two of these fusions showed that the glnF gene is expressed constitutively, i.e., independent of either the nitrogen source in the growth medium or the availability of the glnA, glnL, glnG or glnF functional gene products. The orientation of the Mud1 (Ap lac) insertions was determined by chromosome mobilization in F-merogenotes carrying either of the two glnF::Mud1 chromosomal insertions isolated, and either one of a pair of F'lacZ::Mucts62 episomes; the two episomes differing in that their Mucts62 insertions are located in opposite orientations with regard to lacZ. The direction of chromosome mobilization by the Hfrs that were probably formed via Mu homology demonstrated that orientation of the glnF gene is clockwise relative to that of the chromosome.
    MGG - Molecular and General Genetics 02/1984; 195(1-2):228-33.
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    ABSTRACT: Mutants resistant to 80 microM L-methionine-DL-sulfoximine (MS) were isolated on glucose-minimal 15 mM NH4+ medium plates from Escherichia coli cells which were hypersensitive to this concentration of the analogue by virtue of their harboring glnG mutations. MS-resistant mutants derived from strain MX902 carried, in addition to its glnG74 ::Tn5 allele, mutations tightly linked to glnA, as shown by P1-mediated transduction experiments. One particular allele, gln-76, which suppressed the MS-sensitivity conferred by glnG74 ::Tn5 but not its Ntr- phenotype (inability to transport and utilize compounds such as arginine or proline as the only nitrogen sources), was shown to allow constitutive expression of glutamine synthetase in the absence not only of a functional glnG product but also of a functional glnF product. This behavior was found to be cis-dominant in complementation experiments with F'14 merogenotes . In an otherwise wild-type genetic background as in MX929 (gln-76 glnA+ glnL+ glnG+ glnF +), however, normal activation, mediated by the glnG and glnF products was preferred over that mediated by gln-76.
    MGG - Molecular and General Genetics 02/1984; 194(1-2):114-23.
  • Alejandra A. Covarrubias, Fernando Bastarrachea
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    ABSTRACT: The RNA polymerase binding sites present along a DNA segment encompasing the glnA, glnL, and glnG genes have been identified in a hybrid plasmid carrying this chromosomal region of Escherichia coli. The DNA sequence was determined of an 817 base pair segment that contains the region coding for the first 42 amino acids of the NH2-terminal and of the glnA structural gene, as well as its regulatory region. Analysis of this nucleotide sequence revealed three probable RNA polymerase recognition sites, imperfect palindromes, inverted repeats, and direct repeated sequences.
    MGG - Molecular and General Genetics 03/1983; 190(1):171-175.
  • A A Covarrubias, M Rocha, F Bolivar, F Bastarrachea
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    ABSTRACT: We have isolated part of the glnA region of Escherichia coli K-12 as a 6.4 Md DNA fragment on the ColE1 hybrid plasmid pACR1. DNA fragments from pACR1 obtained by cleavage with certain restriction endonucleases were subcloned into the pBR322 cloning vehicle. Recognition sites for the endonucleases BamHI, SmaI, BglII, and EcoRI were localized inside the glnA gene sequence. De novo synthesized polypeptides, employing minicells that carried some of these plasmids, allowed us to determine the direction of transcription of the glnA gene relative to an adjoining gene that codes for a 65 000 dalton protein.
    Gene 12/1980; 11(3-4):239-51. · 2.20 Impact Factor
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    ABSTRACT: The Clarke-Carbon bank of Escherichia coli strains carrying ColE1 hybrid plasmids was screened for complementation of gdh, gltB, and glnA mutations affecting nitrogen metabolism in E. coli. Plasmids which complemented each one of these mutations were isolated. In every case, the plasmids conferred to otherwise mutant cells the capacity to synthesize the corresponding wild-type enzymes: glutamate dehydrogenase, glutamate synthase, and glutamine synthetase (GS), respectively. For three representative plasmids, endonuclease restriction maps were constructed. One of the plasmids, pACR1, which complemented glnA mutations, including the glnA21::Tn5 insertion, was deemed to carry the glnA+ allele. GS synthesis by pACR1 heterozygous merodiploids was subjected to repression by growth on 15 mm NH4+ and had a twofold high derepressed level than wild-type (glnA+) haploid cells when grown on 0.5 mm NH4+ or on glutamate as only nitrogen sources. The presence of glutamine as sole nitrogen source promoted repressed GS synthesis in the merodiploids. By contrast, glutamine allowed almost fully derepressed synthesis of GS in glnA+ haploid cells.
    Plasmid 04/1980; 3(2):150-64. · 1.76 Impact Factor
  • Plasmid 01/1980; 3(2):150-164. · 1.76 Impact Factor
  • M Rocha, F Bastarrachea, A A Covarrubias
    Boletín de estudios médicos y biológicos 32(7-8 Suppl 1):299-307.
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    LAURA VELAZQUEZ, LAURA CAMARENA, FERNANDO BASTARRACHEA
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    ABSTRACT: Individual mutations whichaffected eachofthetwoShine-Dalgarno sequencesatthe5'untranslated region ofthegl&B geneofEscherichia coli were characterized. Theywere isolated inplasmids carrying agltB'-'lacZ protein fusion preceded bytheregulatory region ofthegltBDF operon.Subcloning andnucleotide sequencing of-1,206 bpofDNAencompassing thegltBDFregulatory region showedthatthemutations affected thefirst baseateachofthetwoidentical Shine-Dalgarno sequences,SD1andSD2,located 40and8bases, respectively, upstream fromtheputative gWtB open reading frame. Onlymutation gltB2r227, an adenine inplace ofa guanine, affecting thefirst baseofSD2,lowered I-galactosidase expression significantly, i.e., aboutfivefold. Theresults suggest that SD2isthepreferred functional site atwhichribosomes initiate gltBmRNA translation. Glutamate synthase (EC2.6.1.53) ofEscherichia coli catalyzes theformation oftwoL-glutamate molecules from L-glutamine and2-ketoglutarate and,together withglu- tamine synthetase (EC6.3.1.2), isresponsible forassimila- tionoflowammoniaconcentrations (reviewed inreference 20). Glutamate synthase iscomposed offourdimers, eachof themformed bynonidentical subunits withMrsof135,000 and53,000 (14, 17). We haveestablished theexistence ofa gltBDF operoninE.coli (4)whichiscomposed ofgenes encoding large (gltB) andsmall (gltD) subunits ofglutamate synthase, aswell as a third downstream gene,gltF, whose product appears tobeinvolved inpositive regulation of glutamine synthetase, i.e., thegInALGoperon,aswell asin negative regulation ofits own gltBDF operon (2). PartofthegltBDF operon,including 430bpofits non- translated leader region, hasbeensequenced (19); sequence consensuswitha &70-RNA polymerase holoenzyme-recog- nizable promoter, aboxA,andtworibosome-binding (Shine- Dalgarno) sites (25), each onefollowed byaninitiator codon, were identified through a computer-based databankpro- gram. Inthiswork,we isolated andcharacterized mutations whichaffected either one ofthetwoShine-Dalgarno se- quencesofthenontranslated gltBDF operonleader region. Thepresent study addsknowledge regarding thefunctional- ityofsuchsequences. Toisolate mutations affecting expression ofthegltBDF operon,we constructed plasmid pLLT1(Fig. 1).pLLT1 allowed us toestimate gltBDF operonexpression bymoni- toring P-galactosidase madefromthegltB'-'lacZ protein fusion. CSH3cells (16) harboring pLLT1were mutagenized with ethyl methanesulfonate (5)andspread on plates ofNN- minimal agarmedium(6)with0.2%lactose, 11mM L-gluta- mate,20,ugofchloramphenicol ml-', and40,ugof5-bromo- 4-cloro-3-indolyl-3-D-galactopyranoside (SigmaChemical