Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.     

Escherichia coli mutants with defects in the biosynthesis of 5-methylaminomethyl-2-thio-uridine or 1-methylguanosine in their tRNA.

Two tRNA methyltransferase mutants, isolated as described in the accompanying paper (G.R. Bj?rk and K. Kjellin-Str?by, J. Bacteriol. 133:499-207, 1978), are biochemicaaly and genetically characterized. tRNA from mutant IB13 lacks 5-methylaminomethyl-2-thio-uridine in vivo due to a permanently nonfunctional methyltransferase. Thus tRNA from this mutant is a specific substrate for the corresponding tRNA methyltransferase in vitro. In spite of this defect in tRNA, such a mutant is viable. Mutant IB11 is conditionally defective in the biosynthesis of 1-methylguanosine in tRNA due to a temperature-sensitive tRNA (1-methyl-guanosine) methyltransferase. In mutant cells grown at a high temperature, the level of 1-methylguanosine in bulk tRNA is 20% of that of the wild type, demonstrating that in this mutant an 80% deficiency of 1-methylguanosine in tRNA is not lethal. Genetically these two distinct lesions, trmC2, causing 5=methylaminomethyl-2-thio-uridine deficiency, and trmD1, giving a temperature-sensitive tRNA (1-methylguanosine)methyltransferase, are both located between 50 and 61 min on the Escherichia coli chromosome.     

Metagenomic insights into microbial metabolism affecting arsenic dispersion in Mediterranean marine sediments

Microorganisms dwelling in sediments have a crucial role in biogeochemical cycles and are expected to have a strong influence on the cycle of arsenic, a metalloid responsible for severe water pollution and presenting major health risks for human populations. We present here a metagenomic study of the sediment from two harbours on the Mediterranean French coast, l'Estaque and St Mandrier. The first site is highly polluted with arsenic and heavy metals, while the arsenic concentration in the second site is below toxicity levels. The goal of this study was to elucidate the potential impact of the microbial community on the chemical parameters observed in complementary geochemical studies performed on the same sites. The metagenomic sequences, along with those from four publicly available metagenomes used as control data sets, were analysed with the RAMMCAP workflow. The resulting functional profiles were compared to determine the over‐represented Gene Ontology categories in the metagenomes of interest. Categories related to arsenic resistance and dissimilatory sulphate reduction were over‐represented in l'Estaque. More importantly, despite very similar profiles, the identification of specific sequence markers for sulphate‐reducing bacteria and sulphur‐oxidizing bacteria showed that sulphate reduction was significantly more associated with l'Estaque than with St Mandrier. We propose that biotic sulphate reduction, arsenate reduction and fermentation may together explain the higher mobility of arsenic observed in l'Estaque in previous physico‐chemical studies of this site. This study also demonstrates that it is possible to draw sound conclusions from comparing complex and similar unassembled metagenomes at the functional level, even with very low sequence coverage.     

Caenorhabditis elegans ZC376.5 encodes a tRNA (m2/2G(26))dimethyltransferance in which (246)arginine is important for the enzyme activity

It has been estimated that eukaryotes carry more than 50 genes for tRNA modifying enzymes. Of the few so far identified most come from yeast, a lower eukaryote. In Saccharomyces cerevisiae, the TRM1 gene is a nuclear gene encoding the tRNA(m2/ 2G(26))dimethyltransferase, which catalyses the formation of the N2, N2-dimethylguanosine at position 26 in tRNA. We have isolated and characterized the corresponding gene ZC376.5 in Caenorhabditis elegans. Via RTPCR the cDNA sequence of the full length ZC376.5 has now been cloned, expressed in Escherichia coli and demonstrated to encode a tRNA(m2/2G(26))dimethyltransferase that produces dimethyl-G26 in vivo and in vitro with tRNA from yeast and bacteria as substrates. This is the first example of a complete gene sequence coding for a tRNA modifying enzyme from a multicellular organism. A point mutation in exon IV in the C. elegans genome sequence coding for the tRNA(m2/2G(26))methyltransferase that substituted arginine246 for glycine eliminated the modification activity. Exchanging the corresponding lysine residue in the yeast Trm1p for alanine caused a severe loss of activity, indicating that the identity of the amino acid at this position is important for enzyme activity.     

The human tRNA(m(2)(2)G(26))dimethyltransferase: functional expression and characterization of a cloned hTRM1 gene

This paper presents the first example of a complete gene sequence coding for and expressing a biologically functional human tRNA methyltransferase: the hTRM1 gene product tRNA(m²₂G)dimethyltransferase. We isolated a human cDNA (1980 bp) made from placental mRNA coding for the full-length (659 amino acids) human TRM1 polypeptide. The sequence was fairly similar to Saccharomyces cerevisiae Trm1p, to Caenorhabditis elegans TRM1p and to open reading frames (ORFs) found in mouse and a plant (Arabidopsis thaliana) DNA. The human TRM1 gene was expressed at low temperature in Escherichia coli as a functional recombinant protein, able to catalyze the formation of dimethylguanosine in E.coli tRNA in vivo. It targeted solely position G₂₆ in T7 transcribed spliced and unspliced human tRNA^Tyr in vitro and in yeast trm1 mutant tRNA. Thus, the human TRM1 protein is a tRNA(m²₂G₂₆)dimethyltransferase. Compared with yeast Trm1p, hTRM1p has a C-terminal protrusion of ~90 amino acids which shows similarities to a mouse protein related to RNA splicing. A deletion of these 90 C-terminal amino acids left the modification activity in vitro intact. Among point mutations in the hTRM1 gene, only those located in conserved regions of hTRM1p completely eliminated modification activity.     

The Boar-Operated-System: a Novel Method to Deliver Baits to Wild Pigs

ABSTRACT Bait-delivered pharmaceuticals, increasingly used to manage populations of wild boar (Sus scrofa) and feral pigs, may be ingested by nontarget species. Species-specificity could be achieved through a delivery system. We designed the BOS^TM (Boar-Operated-System) as a device to deliver baits to wild pigs. The BOS^TM consists of a metal pole onto which a round perforated base is attached. A metal cone with a wide rim slides up and down the pole and fully encloses the base onto which the baits are placed. We conducted a pilot, captive trial and found that captive wild boar fed from the BOS^TM either directly, by lifting the cone, or indirectly, by feeding once another animal had lifted the cone. Thus, we tested whether free-living wild boar fed from the BOS^TM and whether the BOS^TM could prevent bait uptake by nontarget species. We observed that free-living wild boar fed regularly from the BOS^TM and that the device successfully prevented bait uptake by nontarget species. The BOS^TM should be trialed more extensively to confirm its effectiveness and species-specificity to distribute pharmaceuticals to wild suids. If successful, the BOS^TM could be used to deliver vaccines in disease control programs as well as contraceptives to manage overabundant populations of wild suids.