In this automated single-run enzymatic procedure for specific determination of triglycerides in serum, free glycerol is removed from the reaction mixture by pre-incubation with glycerol phosphate oxidase and peroxidase. The subsequent addition of lipase and the chromogen, 4-aminoantipyrine, results in the formation of color proportional to the amount of triglycerides in serum. Standards containing triolein in aqueous detergent are used to calibrate the method. For serum pools from the Centers for Disease Control with target values of 0.74, 1.41, and 2.63 mmol/L, the method produced biases of +0.01, -0.05, and 0.00 mmol/L, respectively (mean: -0.01 mmol/L or -0.4%). The mean coefficient of variation was 1.4% within and 2.5% between days; the combined CV, 2.9%. Ninety 6-microL serum samples can be analyzed per hour. The method is more accurate and precise than one based on an NADH-coupled enzyme system with separate addition of lipase.

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Sinorhizobium meliloti 1021 is a Gram-negative alphaproteobacterium with a robust capacity for carbohydrate metabolism. The enzymes that facilitate these reactions assist in the survival of the bacterium across a range of environmental niches, and they may also be suitable for use in industrial processes. SmoS is a dehydrogenase that catalyzes the oxidation of the commonly occurring sugar alcohols sorbitol and galactitol to fructose and tagatose, respectively, using NAD+ as a cofactor. The main objective of this study was to evaluate SmoS using biochemical techniques. The nucleotide sequence was codon-optimized for heterologous expression in Escherichia coli BL21 (DE3) Gold cells and the protein was subsequently overexpressed and purified. Size-exclusion chromatography and X-ray diffraction experiments suggest that SmoS is a tetramer. SmoS was crystallized, and crystals obtained in the absence of substrate diffracted to 2.1 Å resolution and those of a complex with sorbitol diffracted to 2.0 Å resolution. SmoS was characterized kinetically and shown to have a preference for sorbitol despite having a higher affinity for galactitol. Computational ligand-docking experiments suggest that tagatose binds the protein in a more energetically favourable complex than fructose, which is retained in the active site over a longer time frame following oxidation and reduces the rate of the reaction. These results supplement the inventory of biomolecules with potential for industrial applications and enhance the understanding of metabolism in the model organism S. meliloti.

Mesorhizobia are soil bacteria that establish nitrogen-fixing symbioses with various legumes. Novel symbiotic mesorhizobia frequently evolve following horizontal transfer of symbiosis-gene-carrying integrative and conjugative elements (ICESyms) to indigenous mesorhizobia in soils. Evolved symbionts exhibit a wide range in symbiotic effectiveness, with some fixing nitrogen poorly or not at all. Little is known about the genetic diversity and symbiotic potential of indigenous soil mesorhizobia prior to ICESym acquisition. Here we sequenced genomes of 144 Mesorhizobium spp. strains cultured directly from cultivated and uncultivated Australian soils. Of these, 126 lacked symbiosis genes. The only isolated symbiotic strains were either exotic strains used previously as legume inoculants, or indigenous mesorhizobia that had acquired exotic ICESyms. No native symbiotic strains were identified. Indigenous nonsymbiotic strains formed 22 genospecies with phylogenomic diversity overlapping the diversity of internationally isolated symbiotic Mesorhizobium spp. The genomes of indigenous mesorhizobia exhibited no evidence of prior involvement in nitrogen-fixing symbiosis, yet their core genomes were similar to symbiotic strains and they generally lacked genes for synthesis of biotin, nicotinate and thiamine. Genomes of nonsymbiotic mesorhizobia harboured similar mobile elements to those of symbiotic mesorhizobia, including ICESym-like elements carrying aforementioned vitamin-synthesis genes but lacking symbiosis genes. Diverse indigenous isolates receiving ICESyms through horizontal gene transfer formed effective symbioses with Lotus and Biserrula legumes, indicating most nonsymbiotic mesorhizobia have an innate capacity for nitrogen-fixing symbiosis following ICESym acquisition. Non-fixing ICESym-harbouring strains were isolated sporadically within species alongside effective symbionts, indicating chromosomal lineage does not predict symbiotic potential. Our observations suggest previously observed genomic diversity amongst symbiotic Mesorhizobium spp. represents a fraction of the extant diversity of nonsymbiotic strains. The overlapping phylogeny of symbiotic and nonsymbiotic clades suggests major clades of Mesorhizobium diverged prior to introduction of symbiosis genes and therefore chromosomal genes involved in symbiosis have evolved largely independent of nitrogen-fixing symbiosis.

Bradyrhizobium japonicum strain FN1 was found to produce bacteriocin-like zones of clearing when tested against other strains of bradyrhizbia. The genome was sequenced, and several putative bacteriocin-producing genes, in addition to the expected genes involved in nodulation and nitrogen fixation, were identified.