Fernando Bastarrachea

Universidad Autónoma de Yucatán, Mérida, Yucatan, Mexico

Are you Fernando Bastarrachea?

Claim your profile

Publications (15)16.73 Total impact

  • Marcela Zamudio · Aracely González · Fernando Bastarrachea
    [Show abstract] [Hide abstract]
    ABSTRACT: Phytases catalyze the release of phosphate from phytate (myo-inositol hexakisphosphate) to inositol polyphosphates. Raoultella terrigena comb.nov. phytase activity is known to increase markedly after cells reach the stationary phase. In this study, phytase activity measurements made on single batch cultures indicated that specific enzyme activity was subject to catabolite repression. Cyclic AMP (cAMP) showed a positive effect in expression during exponential growth and a negative effect during stationary phase. RpoS exhibited the opposite effect during both growth phases; the induction to stationary phase decreased twofold in the rpoS::Tn10 mutant, but the effect of RpoS was not clearly determined. Two phy::MudI1734 mutants, MW49 and MW52, were isolated. These formed small colonies in comparison with the MW25 parent strain when plated on Luria-Bertani (LB) or LB supplemented with glucose. They did not grow in minimal media or under anaerobiosis, but did grow aerobically on LB and LB glucose at a lower rate than did MW25. The beta-galactosidase activity level in these mutants increased three to four fold during stationary growth in LB glucose and during anaerobiosis. Addition of cAMP during the exponential growth of MW52 on LB glucose provoked a decrease in beta-galactosidase activity during the stationary phase, confirming its negative effect on phytase expression during stationary growth.
    No preview · Article · Feb 2002 · Canadian Journal of Microbiology
  • L Camarena · S Poggio · A Campos · F Bastarrachea · A Osorio
    [Show abstract] [Hide abstract]
    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.
    No preview · Article · Feb 1998 · Plasmid
  • Marcela Zamudio · Fernando Bastarrachea
    [Show abstract] [Hide abstract]
    ABSTRACT: A binding assay for Azospirillum cells adsorbed to wheat roots was developed. It consisted of incubation exposing germinating seedlings to bacterial cells in liquid Fahreaus solution at 30°C for 2 h. The seedlings were removed and washed four times in Fahreaus solution followed by centrifugation for 15 min at 300 rev min−1 after every washing. The number of Azospirilla that remained attached to the roots was estimated by plating serial dilutions on agar media of root homogenates to enable colony counts. Optimal assay conditions for inoculum size, pH, age of the cultures and exposure time were determined. Binding kinetic data best fitted a Langmuir adsorption isotherm thus indicating a saturable, specific number of available sites on the roots for the adsorption of Azospirillum brasilense Sp245. Competition assays with different Rhizobium species were carried out showing a preferential adsorption for Azospirillum.
    No preview · Article · Jun 1994 · Soil Biology and Biochemistry
  • [Show abstract] [Hide abstract]
    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.
    No preview · Article · Jul 1993 · MGG - Molecular and General Genetics
  • Source
    L Velázquez · L Camarena · J L Reyes · F Bastarrachea
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jun 1991 · Journal of Bacteriology
  • Source
    I Castaño · F Bastarrachea · A A Covarrubias
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Mar 1988 · Journal of Bacteriology
  • Source
    L Servín-González · M Ortiz · A González · F Bastarrachea
    [Show abstract] [Hide abstract]
    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.
    Preview · Article · Jul 1987 · Journal of general microbiology
  • Source
    P León · D Romero · A Garciarrubio · F Bastarrachea · A A Covarrubias
    [Show abstract] [Hide abstract]
    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.
    Full-text · Article · Jan 1986 · Journal of Bacteriology
  • Source
    L Servín-González · F Bastarrachea
    [Show abstract] [Hide abstract]
    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.
    Preview · Article · Jan 1985 · Journal of general microbiology
  • Irene Castaño · Fernando Bastarrachea
    [Show abstract] [Hide abstract]
    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.
    No preview · Article · Feb 1984 · MGG - Molecular and General Genetics
  • [Show abstract] [Hide abstract]
    ABSTRACT: Mutants resistant to 80 μM L-methionine-DL-sulfoximine (MS) were isolated on glucose-minimal 15 mM NH +4medium 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.
    No preview · Article · Feb 1984 · MGG - Molecular and General Genetics
  • M Rocha · F Bastarrachea · A A Covarrubias

    No preview · Article · Jul 1983 · Boletín de estudios médicos y biológicos
  • Alejandra A. Covarrubias · Fernando Bastarrachea
    [Show abstract] [Hide abstract]
    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.
    No preview · Article · Mar 1983 · MGG - Molecular and General Genetics
  • [Show abstract] [Hide abstract]
    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.
    No preview · Article · Apr 1980 · Plasmid
  • Source
    [Show abstract] [Hide abstract]
    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
    Preview · Article ·

Publication Stats

252 Citations
16.73 Total Impact Points


  • 2002
    • Universidad Autónoma de Yucatán
      • Facultad de Ingeniería Química
      Mérida, Yucatan, Mexico
  • 1980-1994
    • Universidad Nacional Autónoma de México
      • Departament of Molecular Biology and Biotechnology
      Ciudad de México, Mexico City, Mexico
  • 1983-1993
    • Instituto de Investigaciones Biomedicas de Barcelona
      Barcino, Catalonia, Spain