Biomedical subjects

P Liras

Publications and source records attributed to P Liras.

At least 19 recordsLinked to original sources

PubMed2004

Two proteins with ornithine acetyltransferase activity show different functions in Streptomyces clavuligerus: Oat2 modulates clavulanic acid biosynthesis in response to arginine.

The oat2 gene, located in the clavulanic acid gene cluster in Streptomyces clavuligerus, is similar to argJ, which encodes N-acetylornithine:glutamic acid acetyltransferase activity. Purified proteins obtained by expression in Escherichia coli of the argJ and oat2 genes of S. clavuligerus posses N-acetyltransferase activity. The kinetics and substrate specificities of both proteins are very similar. Deletion of the oat2 gene did not affect the total N-acetylornithine transferase activity and slightly reduced the formation of clavulanic acid under standard culture conditions. However, the oat2 mutant produced more clavulanic acid than the parental strain in cultures supplemented with high levels (above 1 mM) of arginine. The purified S. clavuligerus ArgR protein bound the arginine box in the oat2 promoter, and the expression of oat2 was higher in mutants with a disruption in argR (arginine-deregulated), confirming that the Arg boxes of oat2 are functional in vivo. Our results suggest that the Oat2 protein or one of its reaction products has a regulatory role that modulates clavulanic acid biosynthesis in response to high arginine concentrations.

Acetyltransferases↗

PubMed2000

A cephalosporin C acetylhydrolase is present in the cultures of Nocardia lactamdurans.

Two protein bands with strong esterase activity are present in broths of Nocardia lactamidurans MA4213 cultures. One of them shows cephalosporin C acetylhydrolase (CAH) activity. This activity is maximal at 48 h of growth and shows a pattern of regulation slightly different from that of cephamycin production in medium supplemented with glucose (166 mM), glycerol (326 mM) or ammonium chloride (60 mM). The CAH activity was purified to homogeneity by DEAE-Sepharose ion-exchange, Sephadex G-75 gel filtration, and phenyl-Sepharose hydrophobic interaction chromatography. It showed a molecular mass of 72,100 Da. The N-terminus of the protein was determined and showed the amino acid sequence GGAAPGGPGAHPLWLPAGKD. The enzyme showed Km values of 7.0 mM and 8.3 mM for cephalosporin C and 7-aminocephalosporanic acid respectively but was not active on cephamycin C.

Amino Acid Sequence↗

PubMed2000

Clavulanic acid, a beta-lactamase inhibitor: biosynthesis and molecular genetics.

Clavulanic acid is a secondary metabolite produced by Streptomyces clavuligerus. It possesses a clavam structure and a characteristic 3R,5R stereochemistry essential for action as a beta-lactamase inhibitory molecule. It is produced from glyceraldehyde-3-phosphate and arginine in an eight step biosynthetic pathway. The pathway is carried out by unusual enzymes, such as (1) the enzyme condensing both precursors, N2-(2-carboxyethyl)-arginine (CEA) synthetase, (2) the beta-lactam synthetase cyclizing CEA and (3) the clavaminate synthetase, a well-characterized multifunctional enzyme. Genes for biosynthesis of clavulanic acid and other clavams have been cloned and characterized. They offer new possibilities for modification of the pathway and for obtaining new molecules with a clavam structure. The state of the regulatory proteins controlling clavulanic acid biosynthesis, as well as the relationship between the biosynthetic pathway of clavulanic acid and other clavams, is discussed.

Anti-Bacterial Agents↗

PubMed2000

Overexpression of the lat gene in Nocardia lactamdurans from strong heterologous promoters results in very high levels of lysine-6-aminotransferase and up to two-fold increase in cephamycin C production.

The level of lysine-6-aminotransferase (encoded by the lat gene), an enzyme that commits lysine to the cephamycin biosynthesis pathway, is very low in wild type Nocardia lactamdurans. Two lat overexpression systems (pAMEXlat and pSAFlat) were constructed to express the promoterless lat gene of N. lactamdurans from the strong promoters amyP (of the alpha-amylase gene) and safP (of the secretion activating factor gene) of Streptomyces griseus. Both constructions led to very high levels of lysine-6-aminotransferase (between 8- and 15-fold) in the cells. Expression of lat from the amy promoter was optimal in glycerol-containing medium and was negatively regulated by glucose. The high levels of lysine-6-aminotransferase resulted in a 50-200% increase in cephamycin C production in the standard fermentation conditions. Onset of cephamycin C biosynthesis occurred at the same time in control and in lat-overexpressing strains, but the cephamycin production rate was clearly higher in transformants overexpressing the lat gene. Furthermore, HPLC analysis of cephamycin C in the culture broths revealed an early depletion of biosynthetic intermediates and an accumulation of cephamycin C when the lat gene was overexpressed. These results indicate that lysine-6-aminotransferase activity is limiting for cephamycin C biosynthesis under some culture conditions.

Cephamycins↗

PubMed2000

Characterization and expression of the arginine biosynthesis gene cluster of Streptomyces clavuligerus.

A cluster of genes argCJBDRGH containing most of the arginine biosynthesis genes has been found in Streptomyces clavuligerus after sequencing a 8.3 kb DNA region containing overlapping sequences of two DNA fragments known to contain arginine biosynthesis genes. Subcloning, complementation of E. coli arginine auxotrophic strains and enzymatic assays confirmed the identity of each gene. S1 nuclease mapping studies and Northern hybridization analysis revealed the formation of two large transcripts corresponding to argCJBDR and argGH. The amount of each of these mRNAs is 10 to 44 times higher in a S. clavuligerus argR-disrupted mutant than in the wild type confirming the existence of an ArgR-mediated control of arginine biosynthesis gene expression. A low level constitutive monocistronic transcript of argR was observed in S. clavuligerus cells. Most of the argGH transcript initiating at an adenine 29 nt upstream of the argG initiation codon appears to stop at a termination stem and loop structure present downstream of the argG gene.

Acetyltransferases↗

PubMed1999

Deletion of the pyc gene blocks clavulanic acid biosynthesis except in glycerol-containing medium: evidence for two different genes in formation of the C3 unit.

The beta-lactamase inhibitor clavulanic acid is formed by condensation of a pyruvate-derived C3 unit with a molecule of arginine. A gene (pyc, for pyruvate converting) located upstream of the bls gene in the clavulanic acid gene cluster of Streptomyces clavuligerus encodes a 582-amino-acid protein with domains recognizing pyruvate and thiamine pyrophosphate that shows 29.9% identity to acetohydroxyacid synthases. Amplification of the pyc gene resulted in an earlier onset and higher production of clavulanic acid. Replacement of the pyc gene with the aph gene did not cause isoleucine-valine auxotrophy in the mutant. The pyc replacement mutant did not produce clavulanic acid in starch-asparagine (SA) or in Trypticase soy broth (TSB) complex medium, suggesting that the pyc gene product is involved in the conversion of pyruvate into the C3 unit of clavulanic acid. However, the beta-lactamase inhibitor was still formed at the same level as in the wild-type strain in defined medium containing D-glycerol, glutamic acid, and proline (GSPG medium) as confirmed by high-pressure liquid chromatography and paper chromatography. The production of clavulanic acid by the replacement mutant was dependent on addition of glycerol to the medium, and glycerol-free GSPG medium did not support clavulanic acid biosynthesis, suggesting that an alternative gene product catalyzes the incorporation of glycerol into clavulanic acid in the absence of the Pyc protein. The pyc replacement mutant overproduces cephamycin.

Acetolactate Synthase↗

PubMed1999

Inducing effect of diamines on transcription of the cephamycin C genes from the lat and pcbAB promoters in Nocardia lactamdurans.

The diamines putrescine, cadaverine, and diaminopropane stimulate cephamycin biosynthesis in Nocardia lactamdurans, in shake flasks and fermentors, without altering cell growth. Intracellular levels of the P7 protein (a component of the methoxylation system involved in cephamycin biosynthesis) were increased by diaminopropane, as shown by immunoblotting studies. Lysine-6-aminotransferase and piperideine-6-carboxylate dehydrogenase activities involved in biosynthesis of the alpha-aminoadipic acid precursor were also greatly stimulated. The diamine stimulatory effect is exerted at the transcriptional level, as shown by low-resolution S1 protection studies. The transcript corresponding to the pcbAB gene and to a lesser extent also the lat transcript were significantly increased in diaminopropane-supplemented cultures, whereas transcription from the cefD promoter was not affected. Coupling of the lat and pcbAB promoters to the reporter xylE gene showed that expression from the lat and pcbAB promoters was increased by addition of diaminopropane in Streptomyces lividans. Intracellular accumulation of diamines in Nocardia may be a signal to trigger antibiotic production.

Cadaverine↗

PubMed1998

The claR gene of Streptomyces clavuligerus, encoding a LysR-type regulatory protein controlling clavulanic acid biosynthesis, is linked to the clavulanate-9-aldehyde reductase (car) gene.

Two genes, claR and car, encoding proteins involved in clavulanic acid biosynthesis, have been found in a 2.8-kb BglII-EcoRI DNA fragment of Streptomyces clavuligerus adjacent to the region containing the cephamycin and clavulanic acid biosynthesis gene cluster. claR encoded a protein of 431 amino acids (deduced Mr 47080), that showed a significant degree of homology with several transcriptional activators of the LysR family. The ClaR protein contained two helix-turn-helix (HTH) motifs in the amino and carboxyl terminal regions. The second gene, car, encoded a protein of 247 amino acids (Mr 26629) that showed a strong similarity to oxydoreductases of the SDR family. Twelve amino acids of the amino-terminal region were identical to those previously obtained by Edman degradation of the purified clavulanic-9-aldehyde reductase of S. clavuligerus. Amplification of the claR gene in multicopy plasmids resulted in a threefold increase in clavulanic acid production and in a five- to sixfold increase of alanylclavam biosynthesis, whereas cephamycin production was significantly reduced both in defined and in complex media. By contrast, amplification of the car gene had no significant effect on clavulanic acid and alanylclavam or cephamycin production. Both claR and car are expressed as monocistronic transcripts; the level of transcript declined rapidly after 48h in complex media, but low sustained levels of both transcripts were observed in defined GSPG medium until 96h. claR and car were not significantly expressed in mutants disrupted in the ccaR gene, a regulatory gene that controls positively clavulanic acid and cephamycin biosynthesis. These results indicate that clavulanic acid and cephamycin biosynthesis in S. clavuligerus is controlled by a cascade of regulatory proteins that include CcaR and ClaR.

Alcohol Oxidoreductases↗

PubMed1998

The pcd gene encoding piperideine-6-carboxylate dehydrogenase involved in biosynthesis of alpha-aminoadipic acid is located in the cephamycin cluster of Streptomyces clavuligerus.

Three open reading frames (ORFs) have been located downstream of cefE in the cephamycin C gene cluster of Streptomyces clavuligerus. ORF13 (pcd) encodes a 496-amino-acid protein (molecular weight [MW], 52,488) with an N-terminal amino acid sequence identical to that of pure piperideine-6-carboxylate dehydrogenase. ORF14 (cmcT) encodes a 523-amino-acid protein (MW, 54,232) analogous to Streptomyces proteins for efflux and resistance to antibiotics. ORF15 (pbp74) encodes a high molecular weight penicillin-binding protein (MW, 74, 094).

2-Aminoadipic Acid↗

PubMed1998

The nine genes of the Nocardia lactamdurans cephamycin cluster are transcribed into large mRNAs from three promoters, two of them located in a bidirectional promoter region.

The nine biosynthesis genes of the Nocardia lactamdurans cephamycin cluster are expressed as three different mRNAs initiating at promoters latp, cefDp, and pcbABp, as shown by low-resolution S1 nuclease protection assays and Northern blotting analysis. Bidirectional expression occurred from divergent promoters (latp and cefDp) located in a 629-bp intergenic region that contains three heptameric direct repeats similar to those recognized by members of the SARP (Streptomyces antibiotic regulatory proteins) family. The lat gene is transcribed in a single monocistronic transcript initiating at latp. A second unusually long polycistronic mRNA (more than 16 kb) corresponding to six biosynthesis genes (pcbAB, pcbC, cmcI, cmcJ, cefF, and cmcH) started at pcbABp. A third polycistronic mRNA corresponding to the cefD and cefE genes started at cefDp.

Base Sequence↗

PubMed1998

Evolution of the clusters of genes for beta-lactam antibiotics: a model for evolutive combinatorial assembly of new beta-lactams.

beta-Lactam antibiotics are produced by prokaryotic and eukaryotic organisms. The genes for beta-lactam biosynthesis are organized in clusters but the location of the different genes is not identical. Biosynthesis genes are clustered with genes for resistance (bla, pbp) and for the efflux of the antibiotic (cmcT) in prokaryotes. Comparison of proteins reveals much larger differences for primary metabolism enzymes than for beta-lactam biosynthesis enzymes in producing organisms. This suggests a horizontal transfer of the beta-lactam antibiotic biosynthesis genes.

Actinomycetales↗

PubMed1997

Delta-1-piperideine-6-carboxylate dehydrogenase, a new enzyme that forms alpha-aminoadipate in Streptomyces clavuligerus and other cephamycin C-producing actinomycetes.

Delta-1-Piperideine-6-carboxylate (P6C) dehydrogenase activity, which catalyses the conversion of P6C into alpha-aminoadipic acid, has been studied in the cephamycin C producer Streptomyces clavuligerus by both spectrophotometric and radiometric assays. The enzyme has been purified 124-fold to electrophoretic homogeneity with a 26% yield. The native protein is a monomer of 56.2 kDa that efficiently uses P6C (apparent Km 14 microM) and NAD+ (apparent Km 115 microM), but not NADP+ or other electron acceptors, as substrates. The enzyme activity was inhibited (by 66%) by its end product NADH at 0.1 mM concentration. It did not show activity towards pyrroline-5-carboxylate and was separated by Blue-Sepharose chromatography from pyrroline-5-carboxylate dehydrogenase, an enzyme involved in the catabolism of proline. P6C dehydrogenase reached maximal activity later than other early enzymes of the cephamycin pathway. The P6C dehydrogenase activity was decreased in ammonium (40 mM)-supplemented cultures, as was that of lysine 6 amino-transferase. P6C dehydrogenase activity was also found in other cephamycin C producers (Streptomyces cattleya and Nocardia lactamdurans) but no in actinomycetes that do no produce beta-lactams, suggesting that it is an enzyme specific for cephamycin biosynthesis, involved in the second stage of the two-step conversion of lysine to alpha-aminoadipic acid.

2-Aminoadipic Acid↗

PubMed1997

Partial purification, characterization and nitrogen regulation of the lysine epsilon-aminotransferase of Streptomyces clavuligerus.

The L-lysine epsilon-aminotransferase (LAT) of Streptomyces clavuligerus was partially purified and characterized. The 51.3-kDa enzyme exhibited optimal activity at pH 7.0-7.5 and 30 degrees C. It catalyzed transfer of the terminal amino group of L-lysine or L-ornithine to alpha-ketoglutarate. Oxalacetate and pyruvate were also used as acceptors of the amino group but with very low efficiency. Increasing ammonium concentrations added to chemically-defined medium MM enhanced the formation of LAT and decreased production of cephalosporins by S. clavuligerus. In cultures grown in the absence of lysine, greater enhancement of LAT formation by ammonium and less repression of cephalosporin biosynthesis were observed. In the chemically-defined GSPG medium, ammonium ions decreased cephalosporin production without showing an effect on LAT formation.

Ammonium Chloride↗

PubMed1997

Arginine boxes and the argR gene in Streptomyces clavuligerus: evidence for a clear regulation of the arginine pathway.

The argR gene of Streptomyces clavuligerus has been located in the upstream region of argG. It encodes a protein of 160 amino acids with a deduced M(r) of 17117 for the monomer. Transformants containing the amplified argR gene showed lower activity (50%) of the biosynthetic ornithine carbamoyltransferase (OTC) activity and higher levels (380%) of the catabolic ornithine aminotransferase (OAT) activity than control strains. Amplification of an arginine (ARG) box-containing sequence results in a 2- to 2.5-fold derepression of ornithine acetyltransferase and OTC, suggesting that the repressor is titrated out. Footprinting experiments using the pure homologous arginine repressor (AhrC) of B. subtilis showed a protected 38 nt region (ARG box) in the coding strand upstream of argC. The protected region contained two tandemly repeated imperfect palindromic 18-nt ARG boxes. The repressor-operator interaction was confirmed by bandshift experiments of the DNA fragment containing the protected region. By computer analysis of the Streptomyces sequences available in the databases, a consensus ARG box has been deduced for the genus Streptomyces. This is the first example of a clear regulation of an amino acid biosynthetic pathway in Streptomyces species, challenging the belief that actinomycetes do not have a well-developed regulatory system of these pathways.

Amino Acid Sequence↗

PubMed1997

Amy as a reporter gene for promoter activity in Nocardia lactamdurans: comparison of promoters of the cephamycin cluster.

Promoter probe vectors containing the pA origin of replication and the Streptomyces griseus promoterless amy gene (encoding alpha-amylase) as reporter have been constructed to study transcription initiation regions in Nocardia lactamdurans. In some of the promoter probe vectors the phage fd terminator has been introduced to avoid readthrough expression from upstream sequences. By using these vectors, four different transcription initiation regions of the cephamycin gene cluster have been studied in N. lactamdurans. The bla gene encoding a beta-lactamase has a relatively strong promoter. Two other separate promoters corresponding to the lat and cefD genes (encoding, respectively, lysine-6-aminotransferase and isopenicillin N-epimerase) showed weak transcription initiation ability. These two promoters are arranged in a bidirectional transcription initiation region located in the center of the cephamycin gene cluster. The cmcH gene (encoding 3-hydroxymethylcephem carbamoyltransferase) upstream region did not contain a functional promoter, suggesting that cmcH is transcribed as a part of a polycistronic mRNA. The native amy promoter is used very efficiently in N. lactamdurans, resulting in secretion of high levels of extracellular alpha-amylase.

Amino Acid Isomerases↗

PubMed1997

The bla gene of the cephamycin cluster of Streptomyces clavuligerus encodes a class A beta-lactamase of low enzymatic activity.

A gene (bla) encoding a beta-lactamase is present in the cephamycin gene cluster of Streptomyces clavuligerus, the strain producing clavulanic acid and a beta-lactamase inhibitory protein. The bla gene is located 5.1 kb downstream from and in the opposite orientation to cefE, encoding the deacetoxycephalosporin C synthase. The bla gene encodes a 332-residue protein (Mr, 35,218), similar to other class A beta-lactamases produced by actinomycetes. Modification (to SDG) of the SDN conserved motif of class A beta-lactamases as well as of amino acids in otherwise conserved regions in the molecule may explain the low penicillinase and cephalosporinase activities of the protein. The beta-lactamase has been purified to homogeneity and found to bind [3H]benzylpenicillin, a result reflecting a rate-limiting deacylation step. Nucleotide sequences homologous to bla were found in all tested cephamycin producers, but several other Streptomyces species which produce a beta-lactamase do not contain genes for beta-lactam antibiotic biosynthesis.

Amino Acid Sequence↗

PubMed1997

A regulatory gene (ccaR) required for cephamycin and clavulanic acid production in Streptomyces clavuligerus: amplification results in overproduction of both beta-lactam compounds.

A regulatory gene (ccaR), located within the cephamycin gene cluster of Streptomyces clavuligerus, is linked to a gene (blp) encoding a protein similar to a beta-lactamase-inhibitory protein. Expression of ccaR is required for cephamycin and clavulanic acid biosynthesis in S. clavuligerus. The ccaR-encoded protein resembles the ActII-ORF4, RedD, AfsR, and DnrI regulatory proteins of other Streptomyces species, all of which share several motifs. Disruption of ccaR by targeted double recombination resulted in the loss of the ability to synthesize cephamycin and clavulanic acid. Complementation of the disrupted mutant with ccaR restored production of both secondary metabolites. ccaR was expressed as a monocistronic transcript at 24 and 48 h in S. clavuligerus cultures (preceding the phase of antibiotic accumulation), but no transcript hybridization signals were observed at 72 or 96 h. This expression pattern is consistent with those of regulatory proteins required for antibiotic biosynthesis. Amplification of ccaR in S. clavuligerus resulted in a two- to threefold increase in the production of cephamycin and clavulanic acid.

Amino Acid Sequence↗

PubMed1996

Interaction of the two proteins of the methoxylation system involved in cephamycin C biosynthesis. Immunoaffinity, protein cross-linking, and fluorescence spectroscopy studies.

Cephamycin C-producing microorganisms contain a two-protein enzyme system that converts cephalosporins to 7-methoxycephalosporins. Interaction between the two component proteins P7 (Mr 27,000) and P8 (Mr 32,000) has been studied by immunoaffinity chromatography using anti-P7 and anti-P8 antibodies, cross-linking with glutaraldehyde, and fluorescence spectroscopy analysis. Co-renaturation of the P7 and P8 polypeptides resulted in the formation of a protein complex with a molecular mass of 59 kDa, which corresponds to a heterodimer of P7 and P8. Glutaraldehyde cross-linking of the polypeptides after assembly of the protein complex showed the presence of a single heterodimer form that reacted with antibodies against P7 and P8. Each separate protein did not associate with itself into multimers. The P7.P8 complex co-purified by immunoaffinity chromatography from extracts of Nocardia lactamdurans and Streptomyces clavuligerus, suggesting that both proteins are present as an aggregate in vivo. Fluorescence spectroscopy studies of 5-methylaminonaphthalene-1-sulfonyl-P7 in response to increasing concentrations of P8 showed a blue shift in the fluorophore emission, indicating a conformational change of P7 in response to the interaction of P8 with an apparent dissociation constant of 47 microM. NADH showed affinity for the P7 component. The P7.P8 complex interacted strongly with the substrates S-adenosylmethionine and cephalosporin C, differently from that occurring with the separate P7 or P8 components, resulting in a strong blue shift in the fluorescence emission spectra of the complex.

Cephamycins↗