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The maltooligosaccharide (MOS) utilization locus in Lactobacillus acidophilus NCFM, a model for human small-intestine lactobacilli, encodes three glycoside hydrolases (GHs) a putative maltogenic α-amylase of family 13 subfamily 20 (LaGH13_20), a maltose phosphorylase of GH65 (LaGH65) and a family 13 subfamily 31 member (LaGH13_31B), annotated as a 1,6-α-glucosidase. Here, we reveal that LaGH13_31B is a 1,4-α-glucosyltransferase that disproportionates MOS of degree of polymerization (DP) ≥2, with preference for maltotriose. Kinetic analyses of the three GHs encoded by the MOS locus, revealed that the substrate preference of LaGH13_31B towards maltotriose, complements the about 40-fold lower k cat of LaGH13_20 towards this substrate, thereby enhancing the conversion of odd-numbered MOS to maltose. The concerted action of LaGH13_20 and LaGH13_31B confers the efficient conversion of MOS to maltose that is phosphorolysed by LaGH65. Structural analyses revealed the presence of a flexible elongated loop, which is unand a phosphorylase. The intriguing involvement of a glucosyltransferase is likely to allow fine-tuning the regulation of MOS catabolism for optimal harnessing of this key metabolic resource in the human small intestine. The study extends the suite of specificities, which have been identified in GH13_31 and highlights amino acid signatures underpinning the evolution of 1,4-α-glucosyl transferases that have been recruited in the MOS catabolism pathway in lactobacilli.Anaerobic degradation of polycyclic aromatic hydrocarbons has been mostly investigated with naphthalene as a model compound. Naphthalene degradation by sulphate-reducing bacteria proceeds via carboxylation to 2-naphthoic acid, formation of a coenzyme A thioester and subsequent reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA), which is further reduced to hexahydro-2-naphthoyl-CoA (HHNCoA) by tetrahydronaphthoyl-CoA reductase (THNCoA reductase), an enzyme similar to class I benzoyl-CoA reductases. When analysing THNCoA reductase assays with crude cell extracts and NADH as electron donor via LC-MS, scanning for putative metabolites, we could show that small amounts of the product of an HHNCoA hydratase are formed in the assays, but the downstream conversion by an NAD+-dependent β-hydroxyacyl-CoA dehydrogenase was prevented by the excess of NADH present in those assays. Experiments with alternative electron donors indicated that 2-oxoglutarate can serve as an indirect electron donor for the THNCoA-reducinextracts of anaerobic naphthalene degraders. The identified metabolites provide evidence that ring reduction terminates at the stage of hexahydro-2-naphthoyl-CoA and a sequence of β-oxidation-like degradation reactions starts with a hydratase acting on this intermediate. The final product of this reaction sequence was identified as cis-2-carboxycyclohexylacetyl-CoA, a compound for which a further downstream degradation pathway has recently been published (see reference 33). The current manuscript reveals the first ring-cleaving reaction in the anaerobic naphthalene degradation pathway. It closes the gap between the reduction of the first ring of 2-naphthoyl-CoA by 2-napthoyl-CoA reductase and the lower degradation pathway starting from cis-2-carboxycyclohexylacetyl-CoA, where the second ring cleavage takes place.Rhizobia are nitrogen fixing bacteria that engage in symbiotic relationships with plant hosts but can also persist as free-living bacteria with the soil and rhizosphere. Here we show that free living Rhizobium leguminosarum SRDI565 can grow on the sulfosugar sulfoquinovose (SQ), or the related glycoside SQ-glycerol, using a sulfoglycolytic Entner-Doudoroff (sulfo-ED) pathway resulting in production of sulfolactate (SL) as the major metabolic end-product. Comparative proteomics supports the involvement of a sulfo-ED operon encoding an ABC transporter cassette, sulfo-ED enzymes and an SL exporter. Consistent with an oligotrophic lifestyle, proteomics data revealed little change in expression of the sulfo-ED proteins during growth on SQ versus mannitol, a result confirmed through biochemical assay of sulfoquinovosidase activity in cell lysates. Metabolomics analysis showed that growth on SQ involves gluconeogenesis to satisfy metabolic requirements for glucose-6-phosphate and fructose-6-phosphate. Metabolomics aff-cycle sulfoglycolytic species were also detected pointing to the complexity of metabolic processes within cells under conditions of sulfoglycolysis. Thus rhizobial metabolism of the abundant sulfosugar SQ may contribute to persistence of the bacteria in the soil and to mobilization of sulfur in the pedosphere.Insects are frequently infected by bacterial symbionts that greatly affect their physiology and ecology. Most of these endosymbionts are however barely tractable outside of their native host, rendering functional genetics studies difficult or impossible. Spiroplasma poulsonii is a facultative bacterial endosymbiont of Drosophila melanogaster that manipulates its host reproduction by killing its male progeny at the embryonic stage. S. CBR-470-1 poulsonii, although being a very fastidious bacteria, is closely related to pathogenic Spiroplasma species that are cultivable and genetically modifiable. In this work, we present the transformation of S. poulsonii with a plasmid bearing a fluorescence cassette, leveraging techniques adapted from those used to modify the pathogenic species S. citri. We demonstrate the feasibility of S. poulsonii transformation and discuss approaches for mutant selection and fly colonization, which are persisting hurdles that will need to be overcome to allow functional bacterial genetics studies of this endosymbiont in vivo. Importance Dozens of bacterial endosymbiont species are described and estimated to infect about the half of all insect species. Yet only a handful of them are tractable in vitro, which hampers the understanding of the bacterial determinants of the host-symbiont interaction. Developing a transformation method for S. poulsonii is a major step towards genomic engineering of this symbiont, which will foster basic research on endosymbiosis. This could also open the way to practical uses of endosymbiont engineering through paratransgenesis of vector or pest insects.