Maria Luisa Tabche

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.

RNA-Seq analysis of the multipartite genome of Rhizobium etli CE3 shows different replicon contributions under heat and saline shock.

BackgroundRegulation of transcription is essential for any organism and Rhizobium etli (a multi-replicon, nitrogen-fixing symbiotic bacterium) is no exception. This bacterium is commonly found in the rhizosphere (free-living) or inside of root-nodules of the common bean (Phaseolus vulgaris) in a symbiotic relationship. Abiotic stresses, such as high soil temperatures and salinity, compromise the genetic stability of R. etli and therefore its symbiotic interaction with P. vulgaris. However, it is still unclear which genes are up- or down-regulated to cope with these stress conditions. The aim of this study was to identify the genes and non-coding RNAs (ncRNAs) that are differentially expressed under heat and saline shock, as well as the promoter regions of the up-regulated loci.ResultsAnalysing the heat and saline shock responses of R. etli CE3 through RNA-Seq, we identified 756 and 392 differentially expressed genes, respectively, and 106 were up-regulated under both conditions. Notably, the set of genes over-expressed under either condition was preferentially encoded on plasmids, although this observation was more significant for the heat shock response. In contrast, during either saline shock or heat shock, the down-regulated genes were principally chromosomally encoded. Our functional analysis shows that genes encoding chaperone proteins were up-regulated during the heat shock response, whereas genes involved in the metabolism of compatible solutes were up-regulated following saline shock. Furthermore, we identified thirteen and nine ncRNAs that were differentially expressed under heat and saline shock, respectively, as well as eleven ncRNAs that had not been previously identified. Finally, using an in silico analysis, we studied the promoter motifs in all of the non-coding regions associated with the genes and ncRNAs up-regulated under both conditions.ConclusionsOur data suggest that the replicon contribution is different for different stress responses and that the heat shock response is more complex than the saline shock response. In general, this work exemplifies how strategies that not only consider differentially regulated genes but also regulatory elements of the stress response provide a more comprehensive view of bacterial gene regulation.

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.

Molecular and structural considerations of TF-DNA binding for the generation of biologically meaningful and accurate phylogenetic footprinting analysis: the LysR-type transcriptional regulator family as a study model

BackgroundThe goal of most programs developed to find transcription factor binding sites (TFBSs) is the identification of discrete sequence motifs that are significantly over-represented in a given set of sequences where a transcription factor (TF) is expected to bind. These programs assume that the nucleotide conservation of a specific motif is indicative of a selective pressure required for the recognition of a TF for its corresponding TFBS. Despite their extensive use, the accuracies reached with these programs remain low. In many cases, true TFBSs are excluded from the identification process, especially when they correspond to low-affinity but important binding sites of regulatory systems.ResultsWe developed a computational protocol based on molecular and structural criteria to perform biologically meaningful and accurate phylogenetic footprinting analyses. Our protocol considers fundamental aspects of the TF-DNA binding process, such as: i) the active homodimeric conformations of TFs that impose symmetric structures on the TFBSs, ii) the cooperative binding of TFs, iii) the effects of the presence or absence of co-inducers, iv) the proximity between two TFBSs or one TFBS and a promoter that leads to very long spurious motifs, v) the presence of AT-rich sequences not recognized by the TF but that are required for DNA flexibility, and vi) the dynamic order in which the different binding events take place to determine a regulatory response (i.e., activation or repression). In our protocol, the abovementioned criteria were used to analyze a profile of consensus motifs generated from canonical Phylogenetic Footprinting Analyses using a set of analysis windows of incremental sizes. To evaluate the performance of our protocol, we analyzed six members of the LysR-type TF family in Gammaproteobacteria.ConclusionsThe identification of TFBSs based exclusively on the significance of the over-representation of motifs in a set of sequences might lead to inaccurate results. The consideration of different molecular and structural properties of the regulatory systems benefits the identification of TFBSs and enables the development of elaborate, biologically meaningful and precise regulatory models that offer a more integrated view of the dynamics of the regulatory process of 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.

Analysis of Spo0M function in Bacillus subtilis

Spo0M has been previously reported as a regulator of sporulation in Bacillus subtilis; however, little is known about the mechanisms through which it participates in sporulation, and there is no information to date that relates this protein to other processes in the bacterium. In this work we present evidence from proteomic, protein-protein interaction, morphological, subcellular localization microscopy and bioinformatics studies which indicate that Spo0M function is not necessarily restricted to sporulation, and point towards its involvement in other stages of the vegetative life cycle. In the current study, we provide evidence that Spo0M interacts with cytoskeletal proteins involved in cell division, which suggest a function additional to that previously described in sporulation. Spo0M expression is not restricted to the transition phase or sporulation; rather, its expression begins during the early stages of growth and Spo0M localization in B. subtilis depends on the bacterial life cycle and could be related to an additional proposed function. This is supported by our discovery of homologs in a broad distribution of bacterial genera, even in non-sporulating species. Our work paves the way for re-evaluation of the role of Spo0M in bacterial cell.

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.