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F Bolivar

Publications and source records attributed to F Bolivar.

At least 19 recordsLinked to original sources
PubMed2001

Construction of protein overproducer strains in Bacillus subtilis by an integrative approach.

We evaluated the effect of several genetic factors reported as having a role in the induction of the expression of significant levels of recombinant protein in Bacillus subtilis. We utilized the beta-galactosidase reporter protein from Escherichia coli as our model for measuring the overproduction of heterologous proteins in B. subtilis. The lacZ gene was expressed in B. subtilis using the regulatory region of the subtilisin gene aprE. In this study, we considered factors known to modulate the transcription and translation initiation rates and genetic and mRNA stability. We also consider the effects of different genetic backgrounds, such as degU32 and hpr2, that until now have been studied independently. By changing the native -35 promoter box to the consensus TTGACA sequence of the aprE promoter, a significant 100-fold increase in the beta-galactosidase activity was obtained. On the other hand, changes such as the GTG to ATG start codon, the construction of a consensus AAGGAGG ribosome binding site, and the addition of the cryIIIA transcription terminator at the 3' end of the lacZ gene, produced only marginal effects on the final beta-galactosidase activity.

Bacillus subtilis↗
PubMed2001

Characterization of sugar mixtures utilization by an Escherichia coli mutant devoid of the phosphotransferase system.

Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid and efficient manner. Therefore, the development of mutant strains that avoid this regulatory system is of special interest to fermentation processes. In the present study, the utilization of sugar mixtures by an Escherichia coli mutant strain devoid of the phosphotransferase system (PTS) was characterized. This mutant can transport glucose (PTS- Glucose+ phenotype) by a non-PTS mechanism as rapidly as its wild-type parental strain. In cultures grown in minimal medium supplemented with glucose-xylose or glucose-arabinose mixtures, glucose repressed arabinose- or xylose-utilization in the wild-type strain. However, under the same culture conditions with the PTS- Glucose+ mutant, glucose and arabinose were co-metabolized, but glucose still exerted a partial repressive effect on xylose consumption. In cultures growing with a triple mixture of glucose-arabinose-xylose, the wild-type strain sequentially utilized glucose, arabinose and finally, xylose. In contrast, the PTS- Glucose+ strain co-metabolized glucose and arabinose, whereas xylose was utilized after glucose-arabinose depletion. As a result of glucose-arabinose co-metabolism, the PTS- Glucose+ strain consumed the total amount of sugars contained in the culture medium 16% faster than the wild-type strain. [14C]-Xylose uptake experiments showed that in the PTS- Glucose+ strain, galactose permease increases xylose transport capacity and the observed partial repression of xylose utilization depends on the presence of intracellular glucose.

Carbohydrate Metabolism↗
PubMed2000

A family of removable cassettes designed to obtain antibiotic-resistance-free genomic modifications of Escherichia coli and other bacteria.

Modifications of microbial genomes often require the use of the antibiotic-resistance (Anb(R))-encoding genes and other easily selectable markers. We have developed a set of such selectable markers (Cm(R), Km(R) and Gm(R)), which could easily be inserted into the genome and subsequently removed by using the Cre/loxP site-specific recombination system of bacteriophage P1. In this manner the same marker could be used more than once in the same background, while the resulting strain could or would remain Anb(R) marker-free. Three plasmids were constructed, each containing a cassette consisting of the Cm(R), Km(R), or Gm(R) gene flanked by two parallel loxP sites and two polylinkers (MCS). To test insertion and excision, cassettes were inserted into the lacZ or galE genes carried on an origamma/pir-dependent suicide plasmid, which contained a dominant Sm(R) gene. The cassettes were crossed into the E. coli genome by homologous recombination (allelic exchange), in a manner analogous to that described by Pósfai et al. [Nucl. Acids Res. 22 (1994) 2392-2398], selecting for the Cm(R), Km(R), or Gm(R), for the LacZ(-) or GalE(-) and for the Sm(S) phenotypes (the latter to assure allelic exchange rather than insertion of the entire plasmid). When required, after selecting the strain with the desired modification, the Cm(R), Km(R), or Gm(R) marker was excised by supplying the Cre function. Cre was provided by the thermosensitive plasmid pJW168, which was transformed into the Anb(R) host at 30 degrees C, and was subsequently eliminated at 42 degrees C. Thus the Anb(R) marker was removed, whereas the lacZ or galE gene remained interrupted by the retained loxP site.

Acetyltransferases↗
PubMed2000

Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis.

The aprE gene of Bacillus subtilis encodes the major serine alkaline protease known as subtilisin. It is expressed during the transition state and transcribed by the sigma(A) form of the RNA polymerase (RNAP). In this work, we characterized the regulatory region of the aprE gene (rraprE) from B. subtilis. By computer analysis and site-directed mutagenesis, we localized the aprE promoter sequence 7 bp upstream from its transcription initiation site (TIS). We also characterized the static curvature properties of the rraprE DNA and found two different areas of DNA bending, within the first 400 bp upstream of its TIS. We postulate that these particular curved DNA regions could play a role in the interaction with some regulatory proteins and discuss possible implications related to aprE transcription regulation.

Bacillus subtilis↗
PubMed2000

Relationship between whole proteome aminoacid composition and static DNA curvature.

To study possible relationships between an organism's genomic DNA curvature and the aminoacid composition of its proteome, every peptidic sequence from fully determined genomes was retrotranslated using the E. coli codon preferences, and the curvature profiles of the resulting DNA sequences were calculated and compared. A clear interdependence between these two variables was observed, as each retrotranslated proteome presented a distinctive, statistically significant DNA curvature profile biased toward its natural DNA curvature profile. In addition, by comparing the profiles arising from real and randomly permuted proteomes, we also found a position-dependent contribution of the peptidic sequence to DNA curvature. The implications of these results support the idea of a possible selection toward a specific global curvature of genomes.

Amino Acids↗
PubMed1998

pBRINT-Ts: a plasmid family with a temperature-sensitive replicon, designed for chromosomal integration into the lacZ gene of Escherichia coli.

A pBRINT-Ts family of integrative vectors for Escherichia coli was constructed by using a temperature-sensitive replicon derived from pSC101, a region of homology to the lacZ gene, and various antibiotic resistance markers (kanamycin, chloramphenicol and gentamycin) for selection of the integrants. The gene or group of genes to be integrated can be inserted into a multiple cloning site, flanked by an antibiotic resistance marker and lacZ sequences. A simple and rapid procedure was developed for the selection of cells where allelic exchange had occurred. With this procedure, the efficiency of integration of around 10-3 was observed, using several E. coli strains. From colonies with an integrated pBRINT-Ts plasmid, we detected an average allelic exchange event frequency of 7.5%. As a test for this system, we integrated the amy gene that codes for the alpha-amylase from B. stearothermophilus, into the lacZ gene of E. coli W3110. Production of alpha-amylase was found to be proportional to copy number; at up to 10 copies per chromosome.

Chromosomes, Bacterial↗
PubMed1998

Relationship between codon usage and sequence-dependent curvature of genomes.

Static DNA curvature distributions of full-sequenced genomes and large DNA contigs from different organisms were calculated. Very distinctive differences among histogram profiles coming from archaebacteria, eubacteria, and eukaryotes were observed. Eubacterial profiles were, on average, more curved than were archaeal and eukaryotic profiles. A comparative analysis between real and randomized DNA sequences revealed that eubacterial genomes presented, overall, higher curvature values than random sequences. An opposite portrait was exhibited by archaeal and eukaryotic genomes. They displayed a lower frequency of curved regions than their corresponding randomized sequences. The contributions of coding and intergenic regions to the curvature profile were also analyzed. Intergenic regions, on average, were found to be more curved than the overall genomic sequences, especially in prokaryotic organisms. Nevertheless, because of their small size with respect to coding regions, the contribution of intergenic sequences to the overall curvature profile tended to be minor. A clear relationship between codon usage and DNA curvature was demonstrated, and a proposal of the possible coevolution of both systems is discussed. Finally, we present a procedure to quantify the deviation of a curvature profile from randomness through a formal statistical analysis.

Archaea↗
PubMed1996

A pBRINT family of plasmids for integration of cloned DNA into the Escherichia coli chromosome.

Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gene of Escherichia coli [Balbás et al., Gene 136(1993) 211-213]. A family of related plasmids containing different antibiotic-resistance markers (CmR or GmR or KmR) and a larger multiple cloning site (MCS) has been constructed. This set of plasmids, whose integration efficiencies are as good as those obtained with the prototype plasmid pBRINT, constitutes a collection of tools that allow rapid and easy integration of cloned DNA, at the chromosomal level. Their functionality as integration vectors has been ascertained by integrating the Vitreoscilla sp. hemoglobin-encoding gene and the Photobacterium leiognathi lux genes. To evaluate the level of expression obtained after chromosomal integration, we constructed strains carrying one or two copies of the cat gene integrated in the chromosome, and compared their enzymatic activities with those obtained from a strain carrying cat on a multicopy plasmid.

Base Sequence↗
PubMed1996

Pathway engineering for the production of aromatic compounds in Escherichia coli.

Glucose is the preferred substrate for certain fermentation processes. During its internalization and concomitant formation of glucose-6-phosphate through the glucose phosphotransferase system (PTS), one molecule of phosphoenolpyruvate (PEP) is consumed. Together with erythrose 4-phosphate (E4P), PEP is condensed to form 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP), the first intermediate of the common segment of the aromatic pathway. From this metabolic route, several commercially important aromatic compounds can be obtained. We have selected Escherichia coli mutants that can transport glucose efficiently by a non-PTS uptake system. In theory, this process should increase the availability of PEP for other biosynthetic reactions. Using these mutants, in a background where the DAHP synthase (the enzyme that catalyzes the condensation of PEP and E4P into DAHP) was amplified, we were able to show that at least some of the PEP saved during glucose transport, can be redirected into the aromatic pathway. This increased carbon commitment to the aromatic pathway was enhanced still further upon amplification of the E. coli tktA gene that encodes for a transketolase involved in the biosynthesis of E4P.

3-Deoxy-7-Phosphoheptulonate Synthase↗
PubMed1994

Production in Escherichia coli of a rat chimeric proinsulin polypeptide carrying human A and B chains and its preparative chromatography.

A pseudohuman proinsulin coding DNA sequence (MMRPI) carrying human A and B chains, was constructed via directed mutagenesis of a previously modified rat proinsulin cDNA (MRPI) and expressed as a tryptophan (Trp)LE-proinsulin fusion protein in Escherichia coli W3110. Expression of the hybrid gene was achieved by depletion of tryptophan from the medium. The heterologous fusion protein, accumulated as insoluble inclusion bodies within the cell, was obtained by differential centrifugation and then solubilized using formic acid. At the junction of the two peptides, a methionine residue allowed proinsulin to be released from the carrier protein by cyanogen bromide treatment. The sulfonated form of this proinsulin polypeptide was easily purified, at a preparative level, using ion exchange chromatography.

Amino Acid Sequence↗
PubMed1993

The genomic region encoding toxin gamma from the scorpion Tityus serrulatus contains an intron.

The gene encoding toxin gamma from the scorpion, Tityus serrulatus, was amplified by PCR from genomic DNA employing synthetic oligonucleotides designed from the reported cDNA sequence. The nucleotide sequence of this gene reveals the presence of an intron of 475 base pairs (bp) which interrupts the region that encodes the signal peptide of the precursor toxin. A comparison of the intron boundary sequences of the gamma toxin gene with ones from other arachnid genes is also presented.

Animals↗
PubMed1993

Cloning and characterization of cDNAs that code for Na(+)-channel-blocking toxins of the scorpion Centruroides noxius Hoffmann.

With the purpose of studying the organization and characteristics of the genes that code for toxins present in the venom of the Mexican scorpion, Centruroides noxius Hoffmann (CnH), we prepared a lambda gt11 cDNA library from the venom glands. Using specific oligodeoxyribonucleotides (oligos) designed according to known amino acid (aa) sequences of CnH toxins (STox), we detected several positive clones, determined their nucleotide (nt) sequences and deduced their aa sequences. A comparative analysis of these sequences with previously reported STox revealed that CnH cDNAs code for a family of very similar STox. The cDNA coding for a known STox, II-10, was cloned. Additionally, three other complete (new) nt sequences were obtained for cDNAs encoding peptides similar to STox 1 from CnH or variants 2 and 3 from Centruroides sculpturatus Ewing. Southern blot genomic DNA analysis showed a minimum size of approximately 600 bp as EcoRI fragments for elements of this family. PCR amplifications of CnH genomic DNA and hybridization of PCR products with specific probes indicated that the genomic structural regions that code for these genes do not contain introns, or at least not large introns.

Amino Acid Sequence↗
PubMed1993

Recombinant protein production in cultures of an Escherichia coli trp- strain.

Fermentation conditions were developed in order to achieve simultaneously a high biomass concentration and high-level expression of a hybrid cI-human insulin B peptide gene. In our system, this hybrid gene is under control of the Escherichia coli trp promoter, in a trp- derivative strain of E. coli W3110. The dual role of tryptophan concentration on cellular growth and hybrid gene regulation was studied in 10-1 batch fermentations. In the best batch conditions, a biomass concentration of 12 g dry weight/l can be obtained, and 0.53 g/l of cI-insulin B hybrid protein is produced. Tryptophan in the culture medium is consumed by the growing culture, until a level is reached that causes induction of the hybrid gene. Plasmid loss was detected, as only 62% of the cells retained the recombinant plasmid. In order to increase the hybrid protein production level, a fed-batch culture strategy was developed whereby the specific growth rate of the cells was restrained. Using the same amount of nutrients as in the batch fermentations, it was possible to increase the final biomass concentration to 20 g/l, plasmid-bearing cells in the population to 90% and recombinant hybrid protein to 1.21 g/l.

Biotechnology↗
PubMed1992

Carbon regulation and the role in nature of the Escherichia coli penicillin acylase (pac) gene.

Quantitative analysis of specific pac mRNA and a lacZ fusion to the 5'-terminal region of the pac gene demonstrated that both phenylacetic acid induction and catabolite repression by glucose are involved, at the transcriptional level, in the regulation of the pac gene. The studies presented here suggest that this regulation is also present in Escherichia coli transformed strains in which the pac gene was not originally present. Analysis of the nucleotide sequence of the 5'-terminal region of this gene, with a statistical algorithm, confirms that the putative promoter previously proposed by our group is the most feasible within this region. We demonstrate that penicillin acylase activity can confer on E. coli the ability to use penicillin G as a metabolic substrate, by detaching the phenylacetic group which can be used as a carbon source. Based on these data, the regulation properties of the pac gene studied in this work, and the specificity profile of the penicillin acylase enzyme we suggest a role for it in E. coli as a scavenger enzyme for phenylacetylated compounds.

Base Sequence↗
PubMed1992

gltF, a member of the gltBDF operon of Escherichia coli, is involved in nitrogen-regulated gene expression.

We report here the construction and analysis of insertional mutations in each of the three genes of the gltBDF operon and the nucleotide sequence of the region downstream from gltD. Two open reading frames were identified, the first of which corresponds to gltF. The gltB and gltD genes code for the large and small subunits, respectively, of the enzyme glutamate synthase (GOGAT). gltF codes for a protein, with a molecular mass of 26,350 Da, which is required for Ntr induction. Histidase synthesis was determined as a measure of Ntr function. First, insertions in gltB, gltD or gltF all prevent Ntr induction. Second, complementation analysis indicates that high-level expression of both the gltD and gltF genes is required for the induction of the Ntr enzymes under nitrogen-limiting conditions, indicating that the phenotype of the gltB insertion probably results from polarity on gltD and gltF. Third, glutamate-dependent repression of the glt operon appears to be mediated by the product of the gltF gene. Thus, the gltBDF operon of Escherichia coli is involved in induction of the so-called Ntr enzymes in response to nitrogen deprivation, as well as in glutamate biosynthesis.

Amino Acid Sequence↗