Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gen... more 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.
Glutamate dehydrogenase (GDH) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammo... more Glutamate dehydrogenase (GDH) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammonia. The complete nucleotide sequence of the Escherichia coli gdhA gene, as well as its 5' and 3' flanking regions have been previously reported [Valle et al., Gene 23 (1983) 199-209; 27 (1984) 193-199]. In this paper we present data on the GDH specific activities using both excess and limiting concentrations of ammonia as nitrogen sources. Evidence is presented on the regulation of the mRNA levels for this enzyme by the ammonia concentration in the growth medium. We have identified a single and apparently invariant transcript for several metabolic growth conditions. We also report the identification of a functional promoter and the corresponding transcription start point under several growth conditions. Finally, possible regulatory sequences located at the 5' flanking region of the gdhA gene are discussed.
Quantitative analysis of specific pac mRNA and a lacZ fusion to the 5’-terminal region of the pac... more 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.
Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid a... more 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.
We have determined the complete nucleotide sequence of a 6.3-kb chromosomal HpaI-EcoRI fragment, ... more We have determined the complete nucleotide sequence of a 6.3-kb chromosomal HpaI-EcoRI fragment, that contains the structural genes for both the large and small subunits of the Escherichia coli K-12 glutamate synthase (GOGAT) enzyme, as well as the 5'- and 3'-flanking and intercistronic DNA regions. The Mrs of the two subunits, as deduced from the nucleotide (nt) sequence, were estimated as 166,208 and 52,246. Partial amino acid sequence of the GOGAT enzyme revealed that the large subunit starts with a cysteine residue that is probably generated by a proteolytic cleavage. Northern blotting experiments revealed a transcript of approximately 7300 nt, that at least contains the cistrons for both subunits. A transcriptional start point and a functional promoter were identified in the 5' DNA flanking region of the large subunit gene. The messenger RNA nontranslated leader region has 120 nt and shares identity with the leader regions of E. coli ribosomal operons, in particular around the so-called boxA sequence implicated in antitermination. Other possible regulatory sequences are described.
Penicillin amidase (PA) is the enzyme used commercially for the production of semisynthetic penic... more Penicillin amidase (PA) is the enzyme used commercially for the production of semisynthetic penicillins. During the past decade, a detailed picture of the structure and regulation of the gene encoding this enzyme has emerged, revealing a variety of interesting features that are unique among microorganisms. Clues to the biological role of this enzyme have been provided, as well as new strategies for the commercial production and utilization of PA.
We report here the construction and analysis of insertional mutations in each of the three genes ... more 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 26350Da, 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.
Deletions of the 3' flanking DNA region of the glutamate dehydrog... more Deletions of the 3' flanking DNA region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, have been produced on a plasmid that carries the complete gdhA gene. Those deletions include part of the repetitive extragenic palindromic (REP) sequences proposed by Stern et al. [Cell 37 (1984) 1015-1026], as a novel and major feature of the bacterial genome. The effect of these deletions on the final GDH level in the cell, has been determined. A broader compilation, analysis and alternative functions of the REP sequences, is also presented.
Escherichia coli strains devoid of one or both of the two pyruvate kinase isoenzymes (PKA and PKF... more Escherichia coli strains devoid of one or both of the two pyruvate kinase isoenzymes (PKA and PKF), were grown on minimal media in batch fermentations. The strain lacking both PKs showed a 28% decrease on its specific growth rate when compared to the wild type. However, protein and CO2 yields did not change. Using radioactive 1-C14 glucose and collecting the CO2 produced by the cultures, it was found that the mutant lacking both pyruvate kinases, metabolized glucose mainly through the pentose pathway (PP). The increased participation of the PP in glucose metabolism in this strain, was also reflected on the levels of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases.
The aprE gene of Bacillus subtilis codes for the serine alkaline protease known as subtilisin. It... more The aprE gene of Bacillus subtilis codes for the serine alkaline protease known as subtilisin. Its expression is regulated by a complex network of activators and repressors that includes the products of hpr, degU and sinR. In order to understand the effect of these gene products on subtilisin expression, strains carrying combinations of the degU32(Hy), hpr2 and sinR null mutations, were constructed. We found that in all the genetic backgrounds tested, the sinR null mutation decreased aprE expression. Also, by measuring alkaline phosphatase synthesis and the formation of heat-resistant spores, as indicators of sporulation, we found that some of the mutant strains showed alterations in the sporulation process. These results suggest that these alterations are partially responsible for some of the observed changes in aprE expression.
Plasmid pBRINT is an efficient vector for chromosomal integration of cloned DNA into the lacZ gen... more 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.
Glutamate dehydrogenase (GDH) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammo... more Glutamate dehydrogenase (GDH) catalyzes the synthesis of L-glutamate from 2-oxoglutarate and ammonia. The complete nucleotide sequence of the Escherichia coli gdhA gene, as well as its 5' and 3' flanking regions have been previously reported [Valle et al., Gene 23 (1983) 199-209; 27 (1984) 193-199]. In this paper we present data on the GDH specific activities using both excess and limiting concentrations of ammonia as nitrogen sources. Evidence is presented on the regulation of the mRNA levels for this enzyme by the ammonia concentration in the growth medium. We have identified a single and apparently invariant transcript for several metabolic growth conditions. We also report the identification of a functional promoter and the corresponding transcription start point under several growth conditions. Finally, possible regulatory sequences located at the 5' flanking region of the gdhA gene are discussed.
Quantitative analysis of specific pac mRNA and a lacZ fusion to the 5’-terminal region of the pac... more 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.
Due to catabolite repression in microorganisms, sugar mixtures cannot be metabolized in a rapid a... more 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.
We have determined the complete nucleotide sequence of a 6.3-kb chromosomal HpaI-EcoRI fragment, ... more We have determined the complete nucleotide sequence of a 6.3-kb chromosomal HpaI-EcoRI fragment, that contains the structural genes for both the large and small subunits of the Escherichia coli K-12 glutamate synthase (GOGAT) enzyme, as well as the 5'- and 3'-flanking and intercistronic DNA regions. The Mrs of the two subunits, as deduced from the nucleotide (nt) sequence, were estimated as 166,208 and 52,246. Partial amino acid sequence of the GOGAT enzyme revealed that the large subunit starts with a cysteine residue that is probably generated by a proteolytic cleavage. Northern blotting experiments revealed a transcript of approximately 7300 nt, that at least contains the cistrons for both subunits. A transcriptional start point and a functional promoter were identified in the 5' DNA flanking region of the large subunit gene. The messenger RNA nontranslated leader region has 120 nt and shares identity with the leader regions of E. coli ribosomal operons, in particular around the so-called boxA sequence implicated in antitermination. Other possible regulatory sequences are described.
Penicillin amidase (PA) is the enzyme used commercially for the production of semisynthetic penic... more Penicillin amidase (PA) is the enzyme used commercially for the production of semisynthetic penicillins. During the past decade, a detailed picture of the structure and regulation of the gene encoding this enzyme has emerged, revealing a variety of interesting features that are unique among microorganisms. Clues to the biological role of this enzyme have been provided, as well as new strategies for the commercial production and utilization of PA.
We report here the construction and analysis of insertional mutations in each of the three genes ... more 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 26350Da, 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.
Deletions of the 3' flanking DNA region of the glutamate dehydrog... more Deletions of the 3' flanking DNA region of the glutamate dehydrogenase (GDH) structural gene from Escherichia coli K-12, have been produced on a plasmid that carries the complete gdhA gene. Those deletions include part of the repetitive extragenic palindromic (REP) sequences proposed by Stern et al. [Cell 37 (1984) 1015-1026], as a novel and major feature of the bacterial genome. The effect of these deletions on the final GDH level in the cell, has been determined. A broader compilation, analysis and alternative functions of the REP sequences, is also presented.
Escherichia coli strains devoid of one or both of the two pyruvate kinase isoenzymes (PKA and PKF... more Escherichia coli strains devoid of one or both of the two pyruvate kinase isoenzymes (PKA and PKF), were grown on minimal media in batch fermentations. The strain lacking both PKs showed a 28% decrease on its specific growth rate when compared to the wild type. However, protein and CO2 yields did not change. Using radioactive 1-C14 glucose and collecting the CO2 produced by the cultures, it was found that the mutant lacking both pyruvate kinases, metabolized glucose mainly through the pentose pathway (PP). The increased participation of the PP in glucose metabolism in this strain, was also reflected on the levels of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases.
The aprE gene of Bacillus subtilis codes for the serine alkaline protease known as subtilisin. It... more The aprE gene of Bacillus subtilis codes for the serine alkaline protease known as subtilisin. Its expression is regulated by a complex network of activators and repressors that includes the products of hpr, degU and sinR. In order to understand the effect of these gene products on subtilisin expression, strains carrying combinations of the degU32(Hy), hpr2 and sinR null mutations, were constructed. We found that in all the genetic backgrounds tested, the sinR null mutation decreased aprE expression. Also, by measuring alkaline phosphatase synthesis and the formation of heat-resistant spores, as indicators of sporulation, we found that some of the mutant strains showed alterations in the sporulation process. These results suggest that these alterations are partially responsible for some of the observed changes in aprE expression.
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Papers by Fernando Valle