Gene order of the TOL catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xylA product.

TOL plasmid pWW0 specifies enzymes for the oxidative catabolism of toluene and xylenes. The upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. The synthesis of these enzymes is positively regulated by the product of xylR. Determination of upper pathway enzyme levels in bacteria carrying Tn5 insertion mutant derivatives of plasmid pWW0-161 has shown that the genes for upper pathway enzymes are organized in an operon with the following order: promoter-xylC (benzaldehyde dehydrogenase gene[s])-xylA (xylene oxygenase gene[s])-xylB (benzyl alcohol dehydrogenase gene). Subcloning of the upper pathway genes in a lambda pL promoter-containing vector and analysis of their expression in Escherichia coli K-12 confirmed this order. Two distinct enzymes were found to attack benzyl alcohol, namely, xylene oxygenase and benzyl alcohol dehydrogenase; and their catalytic activities were additive in the conversion of benzyl alcohol to benzaldehyde. The fact that benzyl alcohol is both a product and a substrate of xylene oxygenase indicates that this enzyme has a relaxed substrate specificity.     

A simple procedure for transferring genes cloned in Escherichia coli vectors into other gram-negative bacteria: phenotypic analysis and mapping of TOL plasmid gene xylK

A simple method to transfer non-conjugative Escherichia coli plasmids to other Gram-negative bacteria and their maintenance is described. This method involves generation of inverse transposition-mediated cointegrates of the non-conjugative E. coli plasmid with a conjugative IncW broad-host-range plasmid, R388, carrying Tn10. Isolation of such cointegrates was readily effected by conjugal transfer from an E. coli donor containing the two plasmids to an E. coli recipient, with selection for transconjugants expressing a marker of the E. coli plasmid. This method is particularly useful when large series of E. coli vector-based clones need to be expressed in other Gram-negative bacteria to be functionally analysed, either by complementation or recombination. Utility of the method is shown by a functional analysis in Pseudomonas putida of pBR322 hybrid plasmids containing catabolic genes of TOL plasmid pWW0.     

Comparison of the nucleotide sequences of the meta-cleavage pathway genes of TOL plasmid pWW0 from Pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution

TOL plasmid pWW0 from Pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. The structural genes for these catabolic enzymes are clustered into two operons, the xylCMABN operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylXYZLTEGFJQKIH operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to Krebs cycle intermediates. The latter operon can be divided physically and functionally into two parts, the xylXYZL cluster, which is involved in the transformation of benzoate/toluates to (methyl)catechols, and the xylTEGFJQKIH cluster, which is involved in the transformation of (methyl)catechols to Krebs cycle intermediates. Genes isofunctional to xylXYZL are present in Acinetobacter calcoaceticus, and constitute a benzoate-degradative pathway, while xylTEGFJQKIH homologous encoding enzymes of a methylphenol-degradative pathway and a naphthalene-degradative pathway are present on plasmid pVI150 from P. putida CF600, and on plasmid NAH7 from P. putida PpG7, respectively. Comparison of the nucleotide sequences of the xylXYZLTEGFJQKIH genes with other isofunctional genes suggested that the xylTEGFJQKIH genes on the TOL plasmid diverged from these homologues 20 to 50 million years ago, while the xylXYZL genes diverged from the A. calcoaceticus homologues 100 to 200 million years ago. In codons where amino acids are not conserved, the substitution rate in the third base was higher than that in synonymous codons. This result was interpreted as indicating that both single and multiple nucleotide substitutions contributed to the amino acid-substituting mutations, and hence to enzyme evolution. This observation seems to be general because mammalian globin genes exhibit the same tendency.     

Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseudomonas putida.

Two aldehyde dehydrogenases involved in the degradation of toluene and xylenes, namely, benzaldehyde dehydrogenase and 2-hydroxymuconic semialdehyde dehydrogenase, are encoded by the xylC and xylG genes, respectively, on TOL plasmid pWW0 of Pseudomonas putida. The nucleotide sequence of xylC was determined in this study. A protein exhibiting benzaldehyde dehydrogenase activity had been purified from cells of P. putida (pWW0) (J. P. Shaw and S. Harayama, Eur. J. Biochem. 191:705-714, 1990); however, the amino-terminal sequence of this protein does not correspond to that predicted from the xylC sequence but does correspond to that predicted from the xylG sequence. The protein purified in the earlier work was therefore 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product). This conclusion was confirmed by the fact that this protein oxidized 2-hydroxymuconic semialdehyde (kcat/Km = 1.6 x 10(6) s-1 M-1) more efficiently than benzaldehyde (kcat/Km = 3.2 x 10(4) s-1 M-1). The xylC product, the genuine benzaldehyde dehydrogenase, was purified from extracts of P. putida (pWW0-161 delta rylG) which does not synthesize 2-hydroxymuconic semialdehyde dehydrogenase. The amino-terminal sequence of the purified protein corresponds to the amino-terminal sequence deduced from the xylC sequence. This enzyme efficiently oxidized benzaldehyde (kcat/Km = 1.7 x 10(7) s-1 M-1) and its analogs but did not oxidize 2-hydroxymuconic semialdehyde or its analogs.     

Divergent evolution of chloroplast-type ferredoxins

The TOL plasmid pWW0 of Pseudomonas putida encodes a set of enzymes required for the oxidation of toluene to Krebs cycle intermediates. The structural genes for these enzymes are encoded in two operons which comprise the xylCMABN and xylXYZLTEGFJQKIH genes, respectively. The function of the xylT gene has not yet been identified. The nucleotide sequence of xylT was determined in this study and putative gene product was shown to contain a sequence characteristic for chloroplast-type ferredoxins. The nahT gene, the homologue of xylT, present on NAH plasmid NAH7 encoding naphthalene-degrading enzymes, was also sequenced. The sequence conservation between xylT and nahT strongly suggests that both gene products have some physiological function. Chloroplast-type ferredoxins have been discovered in photosynthetic organisms (plants, algae, cyanobacteria and Rhodobacter) and Halobacterium species. Furthermore, chloroplast-type ferredoxin-like sequences have been found in the electron-transfer components of some oxygenases. The sequences of XylT and NahT were compared with those of the previously identified chloroplast-type ferredoxins, in order to examine their evolutionary relationships.     

Opinion Formation Models on a Gradient

Statistical physicists have become interested in models of collective social behavior such as opinion formation, where individuals change their inherently preferred opinion if their friends disagree. Real preferences often depend on regional cultural differences, which we model here as a spatial gradient g in the initial opinion. The gradient does not only add reality to the model. It can also reveal that opinion clusters in two dimensions are typically in the standard (i.e., independent) percolation universality class, thus settling a recent controversy about a non-consensus model. However, using analytical and numerical tools, we also present a model where the width of the transition between opinions scales , not as in independent percolation, and the cluster size distribution is consistent with first-order percolation.     

The K-Segments of the Wheat Dehydrin DHN-5 are Essential for the Protection of Lactate Dehydrogenase and β-Glucosidase Activities In Vitro

The wheat dehydrin DHN-5 has been previously shown to exhibit heat protecting effect on enzymatic activities. In order to understand the molecular mechanism by which DHN-5 exerts its protective function, we performed an approach to dissect the functional domains of DHN-5 responsible for this feature. In two distinct enzymatic assays, we found that the truncated forms of DHN-5 containing only one K- or two K-segments are able to protect albeit to less extent than the wild type protein, lactate dehydrogenase and β-glucosidase against damage induced by various stresses in vitro. However, the YS- and Φ-segments alone have no protective effects on these enzymes. Therefore, our study provides the evidence that the protective function of DHN-5 seems to be directly linked to its K-segments which through their amphipatic α-helical structure, may act to prevent protein aggregation.     

Differentiation of Fanconi anemia and aplastic anemia using mitomycin C test in Tunisia

Fanconi anemia (FA) is a recessive chromosomal instability syndrome that is clinically characterized by multiple symptoms. Chromosome breakage hypersensitivity to alkylating agents is the gold standard test for FA diagnosis. In this study, we provide a detailed laboratory protocol for accurate assessment of FA diagnosis based on mitomycin C (MMC) test. Induced chromosomal breakage study was successful in 171 out of 205 aplastic anemia (AA) patients. According to the sensitivity of MMC at 50 ng/ml, 38 patients (22.22%) were diagnosed as affected and 132 patients (77.17%) as unaffected. Somatic mosaicism was suspected in an 11-year-old patient with a FA phenotype. Twenty-six siblings of FA patients were also evaluated and five of them (19.23%) were diagnosed as FA. From this study, a standard protocol for diagnosis of FA was developed. It is routinely used as a diagnostic test of FA in Tunisia.     

Fanconi anemia: contribution of molecular analyses to the identification of bone marrow graft donors and the study of chimerism in grafted patients

We report on the effectiveness of molecular studies regarding Fanconi anemia (FA) for a better selection of bone marrow graft donors and for post-transplant follow up. Ten unrelated FA patients and their families were analyzed by microsatellite markers. In 9 cases, the cytogenetic investigation of potential human leukocyte antigen (HLA)-identical related donors was normal, and the molecular analyses confirmed that they were also either normal or heterozygous carriers. For 1 patient, cytogenetic analysis of an HLA-identical sibling donor yielded ambiguous results with a relatively high number of chromosomal breakages using cross-linking agents. However, genotyping of this potential donor demonstrated his heterozygous state. Nine patients have received allogeneic bone marrow transplantation from HLA-matched related donors. Microsatellite analysis showed complete chimerism (CC) in all cases. The median follow up was 54 months (range 8-144 months). One patient out of 9 with CC rejected her graft without prior detection of a transitional mixed chimerism. Among these patients, 1 died 25 months after the transplantation of a chronic graft-versus-host-disease (GVHD). We conclude that, when the cytogenetic studies are not conclusive, molecular analyses are crucial to distinguish heterozygous carriers from asymptomatic FA Tunisian patients. Molecular analyses also allowed the evaluation of hematopoietic chimerism after allogeneic bone marrow transplantation and might be of value to identify patients with a high risk for graft rejection.     

Sequence Polymorphism of Cytochrome b Gene in Theileria annulata Tunisian Isolates and Its Association with Buparvaquone Treatment Failure

Background

Buparvaquone (BW 720C) is the major hydroxynaphtoquinone active against tropical theileriosis (Theileria annulata infection). Previous studies showed that buparvaquone, similarly to others hydroxynaphtoquinone, probably acts by binding to cytochrome b (cyt b) inhibiting the electron transport chain in the parasite. Several observations suggested that T. annulata is becoming resistant to buparvaquone in many endemic regions (Tunisia, Turkey and Iran), which may hinder the development of bovine livestock in these areas.

Methodology/Principal Findings

In the present study we sought to determine whether point mutations in T. annulata cytochrome b gene could be associated to buparvaquone resistance. A total of 28 clones were studied in this work, 19 of which were obtained from 3 resistant isolates (ST2/12, ST2/13 and ST2/19) collected at different time after treatment, from a field treatment failure and nine clones isolated from 4 sensitive stocks of T. annulata (Beja, Battan, Jed4 and Sousse). The cytochrome b gene was amplified and sequenced. We identified five point mutations at the protein sequences (114, 129, 253, 262 and 347) specific for the clones isolated from resistant stocks. Two of them affecting 68% (13/19) of resistant clones, are present in the drug-binding site Q₀₂ region at the position 253 in three resistant clones and at the position 262 in 11 out of 19 resistant clones. These two mutations substitute a neutral and hydrophobic amino acids by polar and hydrophilic ones which could interfere with the drug binding capabilities. When we compared our sequences to the Iranian ones, the phylogenetic tree analyses show the presence of a geographical sub-structuring in the population of T. annulata.

Conclusions/Significance

Taken together, our results suggest that the cytochrome b gene may be used as a tool to discriminate between different T. annulata genotypes and also as a genetic marker to characterize resistant isolates of T. annulata.