Juan Miranda

Presence of a membrane bound pyroglutamyl amino peptidase degrading thyrotropin releasing hormone in rat brain

In the present work we studied the pattern of degradation of [3H-Pro]-TRH by soluble and membrane fractions from rat brain. Demonstration of the membrane bound or soluble nature of the activities was obtained by comparing their distribution to that of lactate dehydrogenase and by looking at the effect of NaCl washes on the membrane fractions. We observed that the pyroglutamyl amino peptidase activity detected in brain homogenates is a result of two different enzymes. One of them is a soluble enzyme previously characterized, that needs DTT and EDTA for its expression, is inhibited by SH-blocking agents such as iodoacetamide and utilizes p-glu-beta-naphtylamide as a substrate. The other one, a membrane enzyme, is inhibited by chelating agents such as EDTA and DTT, is not affected by iodoacetamide and does not degrade p-glu-beta-naphtylamide. The later presents some specificity towards TRH as shown by competition experiments with TRH analogs. We were able to corroborate that the post proline cleaving enzyme acting on TRH is a soluble enzyme. In membranes we demonstrated also the presence of a post-proline dipeptidyl aminopeptidase. The membrane bound pyroglutamidase activity is a potential new source of L-his-L-pro-diketopiperazine in brain. The presence of a TRH degrading enzyme in membrane fractions is of particular importance in searching an inactivation mechanism of this peptide once it is released into the synaptic cleft.

Nodulating ability of Rhizobium tropici is conditioned by a plasmid‐encoded citrate synthase

Rhizobium species elicit the formation of nitrogen‐fixing root nodules through a complex interaction between bacteria and plants. Various bacterial genes involved in the nodulation and nitrogen‐fixation processes have been described and most have been localized on the symbiotic plasmids (pSym). We have found a gene encoding citrate synthase on the pSym plasmid of Rhizobium tropici, a species that forms nitrogen‐fixing nodules on the roots of beans (PhasBoius vuigaris) and trees (Leucaena spp.). Citrate synthase is a key metabolic enzyme that incorporates carbon into the tricarboxylic acid cycle by catalysing the condensation of acetyl‐CoA and oxalo‐acetic acid to form citrate. R. tropici pcsA (the plasmid citrate synthase gene) is closely related to the corresponding genes of Proteobacteria. pcsA inactivation by a Tn5‐mob insertion causes the bacteria to form fewer nodules (30–50% of the original strain) and to have a decreased citrate synthase activity in minimal medium with sucrose. A clone carrying the pcsA gene complemented ail the phenotypic alterations of the pcsA mutant, and conferred Rhizobium iegumino‐sarum bv. phaseoli (which naturally lacks a plasmid citrate synthase gene) a higher nodulation and growth capacity in correlation with a higher citrate synthase activity. We have also found that pcsA gene expression is sensitive to iron availability, suggesting a possible role of pcsA in iron uptake.

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli

Abstract A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb3, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb3 terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb3 in R. etli.

Rhizobium etli cytochrome mutants with derepressed expression of cytochrome terminal oxidases and enhanced symbiotic nitrogen accumulation

A method to isolate mutants with derepressed expression of cytochrome oxidases and better symbiotic performance is presented. A mutant of Rhizobium etli, CFN030, isolated by its azide-resistant phenotype, was obtained by transposon Tn5 -mob mutagenesis. This mutant has a derepressed expression of cytochrome aa3, higher respiratory activities when cultured microaerobically and an improved symbiotic nitrogen fixation capacity. This phenotype was similar to the previously described mutant CFN037, which was isolated by its increased capacity to oxidize N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) [Soberón M et al. (1990) J Bacteriol 172:1676–1680]. We show here that although both mutants have a similar symbiotic phenotype, they are affected in different genes. Strain CFN030 has the Tn5 inserted in the chromosome while in strain CFN037 the transposon was located in plasmid b. Cytochrome spectral analysis of both mutant strains in the post-exponential phase of growth, showed the expression of an additional terminal oxidase (cbb3) that is not expressed in the wild-type strain.

Rhizobium etli cycHJKL gene locus involved in c-type cytochrome biogenesis: sequence analysis and characterization of two cycH mutants

The cycHJKL gene locus was cloned from Rhizobium etli by the rescue of a Tn5mob insertion of a mutant (IFC01) which was affected in the production of c-type cytochromes. The cycH, cycJ, cycK and cycL genes are proposed to code for different subunits of a haem lyase complex involved in the attachment of haem to cytochrome c apoproteins. CycH of 365 aa shared 27, 36, 47 and 63% identity with CycH from Paracoccus denitrificans, Bradyrhizobium japonicum, R. meliloti, and R. leguminosarum, respectively. CycJ of 153 aa shared 52, 71, and 85% identity to the cycJ gene product of B. japonicum, R. meliloti, R. leguminosarum, respectively. CycK of 666 aa shared 62, 73, and 90% homology with CycK from B. japonicum, R. meliloti, and R. leguminosarum, respectively, while CycL of 151 aa shared 57, 67 and 86% hómology with CycL from the abovementioned species. The Tn5mob insertion present in the IFC01 strain was located in the cycH gene. This strain was able to infect bean plants, but unable to fix nitrogen during symbiosis. A previously described R. etli cytochrome c-deficient MuD1lac-induced mutant (CFN4202) that induced empty nodules on Phaseolus vulgaris, also have lesions in cycH. Complementation analysis suggested that the MuD1lac insertion of the CFN4202 strain was polar on expression of genes downstream of cycH in contrast with the Tn5mob insertion present in IFC01, which showed no polarity on cycJKL. Our data suggest that CycH may not be essential for the infection process, but is necessary for nitrogen fixation.

A Purine-Related Metabolite Negatively Regulates fixNOQP Expression in Sinorhizobium meliloti by Modulation of fixK Expression

5-aminoimidazole-4-carboxamide nucleotide (AICAR) is a negative effector of cytochrome terminal oxidase cbb3 production in Rhizobium etli. In this work, the effect of AICAriboside (AICAr), the precursor of AICAR on the expression of the Sinorhizobium meliloti fixNOQP operon encoding the symbiotic terminal oxidase cbb3, was analyzed. AICAr reduced the microaerobic induction levels of fixN-lacZ and fixT-lacZ gene fusions 18- and seven-fold respectively, and both genes were activated by the transcriptional activator FixK. A fixK-lacZ fusion presented 14-fold-reduced induction levels in microaerobic cell cultures in the presence of AICAr. AICAr also reduced three-fold the microaerobic expression levels of the nifA-lacZ fusion, whose expression as well as that of fixK is controlled by the two-component system FixL-FixJ. In contrast, AICAr had no effect on the expression levels of a hemA-lacZ fusion. These data suggest that AICAr prevents fixNOQP induction by the inhibition of fixK transcription.

Expression pattern of Rhizobium etli ccmIEFH genes involved in c-type cytochrome maturation.

In different bacterial species, ccmIEFH genes have been suggested to code for subunits of a bacterial haem-lyase catalyzing the covalent attachment of haem to c-type apoproteins. In Rhizobium etli CE3 there are two copies of ccmIEFH: one in the chromosome and the other located in plasmid pf. However, the null phenotype of chromosomal ccmF mutant indicates that the gene locus of plasmid pf is not functional. Two ccmI chromosomal mutants, previously isolated, produced detectable levels of c-type cytochromes under certain culture conditions in contrast with the ccmF mutant, suggesting that ccmF could be transcribed independently. The transcriptional organization of ccmIEFH operon was established. Two promoters from the chromosomal locus were mapped by primer extension, one located upstream of ccmI and the second located upstream of ccmF. The regulation of the expression of both promoters was studied using appropriate lacZ gene fusions (ccmI-lacZ and ccmEF-lacZ). The ccmI-lacZ gene fusion was expressed in complex medium, during exponential growth, under microaerobic conditions and in a R. etli mutant that accumulates reducing power, conditions where a higher respiration rate could be limited by c-type cytochrome content. The ccmEF-lacZ fusion was also primarily expressed in complex medium and under microaerophilic conditions. The finding of two independent promoters in this gene locus could suggest that the step catalyzed by CcmFH could be a rate-limiting step for c-type cytochrome assembly under certain culture conditions.

SYMBIOTIC TERMINAL OXIDASE CBB3 PRODUCTION IS NEGATIVELY MODULATED BY A PURINE RELATED METABOLITE IN SINORHIZOBIUM MELILOTI

The genes which code for a bacteroid terminal oxidase, denoted as the cytochrome terminal oxidase cbb 3 have been identified as the fixNOQP operon. In Sinorhizobium meliloti, the expression of the fixNOQP genes is mainly regulated in response to O2 availability, through the oxygen sensing cascade fixL, fixJ, and fixK gene products. fixL codes for a membrane bound haem-containing protein which in response to low O2 phosphorylates itself and FixJ, activating the capacity of FixJ to promote the transcription of fixK. In turn FixK activates the transcription of the fixNOQP operon and negatively regulates its own promoter (Batut et al., 1989). fixK codes for a transcriptional activator homologous to Fnr and Crp (Batut et al., 1989). The nature of the possible effector which interacts with FixK is unknown. In R. leguminosarum biovar viciae, an homologous but different transcriptional activator, FnrN, activates the two fixNOQP copies (Collona-Romano et al., 1990; Patschowski et al., 1996; Gutierrez et al., 1997). In contrast with FixK, FnrN conserves the N-terminal domain involved in O2 sensing present in Fnr from E. colt suggesting that in R. leguminosarum FnrN transcriptional activity is negatively modulated by O2 (Collona-Romano et al., 1990). Analysis of the transcription of the fixNOQP operon in different R. etli mutants of the purine-thiamin biosynthetic pathway, suggested that 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) is a negative effector of the expression of these genes (Soberon et al., 1997; Miranda-Rios et al., 1997).

Characterization of R. etli Mutants in the Purine-Thiamin Metabolism Suggest That 5-Aminoimidazole-4-Carboxamide Ribonucleotide (AICAR) is a Negative Effector of Symbiotic Cytochrome Terminal Oxidase cbb3 Production

Two Rhizobium etli Tn5mob -induced mutants (CFN035 and CFN037) exhibited enhanced capacity to oxidize N,N,N′,N′,tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase. Sequence of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl-transferase enzyme (purF), catalyzing the first step of the purine biosynthetic pathway (1). In CFN037 the Tn5mob insertion was located in the promoter region of thethiCOGE gene cluster and promotes a constitutive expression of thiC (thiC C mutant). 4-methyl-5-(s-hydroxyethyl)thiazole monophosphate (THZ-P) and 4-amino-5-hydroxymethylpyrimidine pyrophosphate (HMP-P), are coupled to form thiamin monophosphate, which is phosphorylated to make thiamin pyrophosphate. ThiC from R. etli shared significant homology with thiC from E. coli which is involved in the synthesis of HMP from the purine intermediate 5-Aminoimidazole-ribonucleotide. The second ORF of 327 residues is the product of a novel gene which is denoted as thiO. Analysis of the protein sequence suggests that ThiO catalyzes the oxidative deamination of some intermediate of thiamin biosynthesis. ThiG and ThiE from R. etli shared significant homology with ThiG and thiE from E. coli which are involved in the synthesis of THZ and in the condensation of HMP-P with THZ-P respectively. CFN035 and CFN037 produced the cbb 3 terminal oxidase as did the wild-type R. etli strain expressing the B. japonicum fixNOQP genes, which code for the symbiotic cbb 3 terminal oxidase. A blockade in the first step of the purine biosynthetic pathway and the constitutive expression of thiC would lower the concentration of several metabolites of the purine biosynthetic pathway. In order to identify the possible metabolic effector involved in cbb 3, production, the expression of a R. etli fixN-lacZ gene fusion was measured in several mutants affected in different steps of the purine biosynthetic pathway.