No abstract

Inherited disorders of vitamin B(12) (cobalamin) have provided important clues to how this vitamin, which is essential for hematological and neurological function, is transported and metabolized. We describe a new disease that results in failure to release vitamin B(12) from lysosomes, which mimics the cblF defect caused by LMBRD1 mutations. Using microcell-mediated chromosome transfer and exome sequencing, we identified causal mutations in ABCD4, a gene that codes for an ABC transporter, which was previously thought to have peroxisomal localization and function. Our results show that ABCD4 colocalizes with the lysosomal proteins LAMP1 and LMBD1, the latter of which is deficient in the cblF defect. Furthermore, we show that mutations altering the putative ATPase domain of ABCD4 affect its function, suggesting that the ATPase activity of ABCD4 may be involved in intracellular processing of vitamin B(12).

Work on soils suppressive to Thielaviopsis basicola-mediated tobacco black root rot has focused on antagonistic pseudomonads to date. The role of non-Pseudomonas rhizosphere populations has been neglected, and whether they differ in black root rot-suppressive versus -conducive soils is unknown. To assess this possibility, tobacco was grown in a suppressive and a conducive soil of similar physicochemical properties, and rhizobacterial community composition was compared using a 16S rRNA taxonomic microarray. The microarray contains 1033 probes and targets 19 bacterial phyla. Among them, 398 probes were designed for Proteobacteria, Firmicutes, Actinomycetes, Cyanobacteria and Bacteroidetes genera/species known to include strains relevant for plant protection or plant growth promotion. Hierarchical clustering as well as principal component analysis of microarray data discriminated clearly between black root rot-suppressive and -conducive soils. In contrast, T. basicola inoculation had no impact on rhizobacterial community composition. In addition to fluorescent Pseudomonas, the taxa Azospirillum, Gluconacetobacter, Burkholderia, Comamonas and Sphingomonadaceae, which are known to comprise strains with plant-beneficial properties, were more prevalent in the suppressive soil. Mycobacterium, Bradyrhizobium, Rhodobacteraceae, Rhodospirillum and others were more prevalent in the conducive soil. For selected taxa, microarray results were largely corroborated by quantitative PCR and cloning/sequencing. In conclusion, this work identified novel bacterial taxa that could serve as indicators of disease suppressiveness in soil-quality assessments, and it extends the range of bacterial taxa hypothesized to participate in black root rot suppression.

Hydrogen cyanide (HCN) is a broad-spectrum antimicrobial compound involved in biological control of root diseases by many plant-associated fluorescent pseudomonads. The HCN synthase is encoded by three biosynthetic genes (hcnA, hcnB, and hcnC), but little is known about the diversity of these genes in fluorescent Pseudomonas spp. and in other bacteria. Here, the partial hcnBC sequence was determined for a worldwide collection of biocontrol fluorescent Pseudomonas spp. Phylogenies based on hcnBC and deduced protein sequences revealed four main bacterial groups, but topological incongruences were found between hcnBC and rrs-based phylogenies, suggesting past lateral transfer of hcnBC among saprophytic root-colonizing pseudomonads. Three of the four groups included isolates from different countries and host plants. Yet, these groups corresponded to distinct, ecologically-adapted populations of HCN-producing biocontrol fluorescent pseudomonads, as indicated by high hcnBC distinctness ratio values and the differences in production levels of HCN in vitro found between groups. This is in accordance with previous results on catabolic properties and biocontrol abilities of these strains. HCN synthase gene diversity may thus reflect the adaptive radiation of HCN+ biocontrol fluorescent pseudomonads. Positive correlations were found between HCN production in vitro and plant protection in the cucumber/Pythium ultimum and tomato/Fusarium oxysporum f. sp. radicis-lycopersici pathosystems.

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%-42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide-responsiveness, and 24 abnormal kinetics of S-adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N-terminal catalytic domain, whereas missense mutations in the C-terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S-adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.