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PUL0485 |
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19717629, Appl Environ Microbiol. 2009 Nov;75(21):6864-75. doi: 10.1128/AEM.01495-09. Epub 2009 Aug 28. |
| | Novel features of the polysaccharide-digesting gliding bacterium Flavobacterium johnsoniae as revealed by genome sequence analysis. |
| | McBride MJ, Xie G, Martens EC, Lapidus A, Henrissat B, Rhodes RG, Goltsman E, Wang W, Xu J, Hunnicutt DW, Staroscik AM, Hoover TR, Cheng YQ, Stein JL |
| | The 6.10-Mb genome sequence of the aerobic chitin-digesting gliding bacterium Flavobacterium johnsoniae (phylum Bacteroidetes) is presented. F. johnsoniae is a model organism for studies of bacteroidete gliding motility, gene regulation, and biochemistry. The mechanism of F. johnsoniae gliding is novel, and genome analysis confirms that it does not involve well-studied motility organelles, such as flagella or type IV pili. The motility machinery is composed of Gld proteins in the cell envelope that are thought to comprise the "motor" and SprB, which is thought to function as a cell surface adhesin that is propelled by the motor. Analysis of the genome identified genes related to sprB that may encode alternative adhesins used for movement over different surfaces. Comparative genome analysis revealed that some of the gld and spr genes are found in nongliding bacteroidetes and may encode components of a novel protein secretion system. F. johnsoniae digests proteins, and 125 predicted peptidases were identified. F. johnsoniae also digests numerous polysaccharides, and 138 glycoside hydrolases, 9 polysaccharide lyases, and 17 carbohydrate esterases were predicted. The unexpected ability of F. johnsoniae to digest hemicelluloses, such as xylans, mannans, and xyloglucans, was predicted based on the genome analysis and confirmed experimentally. Numerous predicted cell surface proteins related to Bacteroides thetaiotaomicron SusC and SusD, which are likely involved in binding of oligosaccharides and transport across the outer membrane, were also identified. Genes required for synthesis of the novel outer membrane flexirubin pigments were identified by a combination of genome analysis and genetic experiments. Genes predicted to encode components of a multienzyme nonribosomal peptide synthetase were identified, as were novel aspects of gene regulation. The availability of techniques for genetic manipulation allows rapid exploration of the features identified for the polysaccharide-digesting gliding bacteroidete F. johnsoniae. |
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37269952, J Biol Chem. 2023 Jul;299(7):104885. doi: 10.1016/j.jbc.2023.104885. Epub 2023 Jun 2. |
| | Bacteroidota polysaccharide utilization system for branched dextran exopolysaccharides from lactic acid bacteria. |
| | Nakamura S, Kurata R, Tonozuka T, Funane K, Park EY, Miyazaki T |
| | Dextran is an alpha-(1-->6)-glucan that is synthesized by some lactic acid bacteria, and branched dextran with alpha-(1-->2)-, alpha-(1-->3)-, and alpha-(1-->4)-linkages are often produced. Although many dextranases are known to act on the alpha-(1-->6)-linkage of dextran, few studies have functionally analyzed the proteins involved in degrading branched dextran. The mechanism by which bacteria utilize branched dextran is unknown. Earlier, we identified dextranase (FjDex31A) and kojibiose hydrolase (FjGH65A) in the dextran utilization locus (FjDexUL) of a soil Bacteroidota Flavobacterium johnsoniae and hypothesized that FjDexUL is involved in the degradation of alpha-(1-->2)-branched dextran. In this study, we demonstrate that FjDexUL proteins recognize and degrade alpha-(1-->2)- and alpha-(1-->3)-branched dextrans produced by Leuconostoc citreum S-32 (S-32 alpha-glucan). The FjDexUL genes were significantly upregulated when S-32 alpha-glucan was the carbon source compared with alpha-glucooligosaccharides and alpha-glucans, such as linear dextran and branched alpha-glucan from L. citreum S-64. FjDexUL glycoside hydrolases synergistically degraded S-32 alpha-glucan. The crystal structure of FjGH66 shows that some sugar-binding subsites can accommodate alpha-(1-->2)- and alpha-(1-->3)-branches. The structure of FjGH65A in complex with isomaltose supports that FjGH65A acts on alpha-(1-->2)-glucosyl isomaltooligosaccharides. Furthermore, two cell surface sugar-binding proteins (FjDusD and FjDusE) were characterized, and FjDusD showed an affinity for isomaltooligosaccharides and FjDusE for dextran, including linear and branched dextrans. Collectively, FjDexUL proteins are suggested to be involved in the degradation of alpha-(1-->2)- and alpha-(1-->3)-branched dextrans. Our results will be helpful in understanding the bacterial nutrient requirements and symbiotic relationships between bacteria at the molecular level. |
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38661728, FEBS J. 2024 Jul;291(14):3267-3282. doi: 10.1111/febs.17139. Epub 2024 Apr 25. |
| | Structural insights into alpha-(1-->6)-linkage preference of GH97 glucodextranase from Flavobacterium johnsoniae. |
| | Nakamura S, Kurata R, Miyazaki T |
| | Glycoside hydrolase family 97 (GH97) comprises enzymes like anomer-inverting alpha-glucoside hydrolases (i.e., glucoamylase) and anomer-retaining alpha-galactosidases. In a soil bacterium, Flavobacterium johnsoniae, we previously identified a GH97 enzyme (FjGH97A) within the branched dextran utilization locus. It functions as an alpha-glucoside hydrolase, targeting alpha-(1-->6)-glucosidic linkages in dextran and isomaltooligosaccharides (i.e., glucodextranase). FjGH97A exhibits a preference for alpha-(1-->6)-glucoside linkages over alpha-(1-->4)-linkages, while Bacteroides thetaiotaomicron glucoamylase SusB (with 69% sequence identity), which is involved in the starch utilization system, exhibits the highest specificity for alpha-(1-->4)-glucosidic linkages. Here, we examined the crystal structures of FjGH97A in complexes with glucose, panose, or isomaltotriose, and analyzed the substrate preferences of its mutants to identify the amino acid residues that determine the substrate specificity for alpha-(1-->4)- and alpha-(1-->6)-glucosidic linkages. The overall structure of FjGH97A resembles other GH97 enzymes, with conserved catalytic residues similar to anomer-inverting GH97 enzymes. A comparison of active sites between FjGH97A and SusB revealed differences in amino acid residues at subsites +1 and +2 (specifically Ala195 and Ile378 in FjGH97A). Among the three mutants (A195S, I378F, and A195S-I378F), A195S and A195S-I378F exhibited increased activity toward alpha-(1-->4)-glucoside bonds compared to alpha-(1-->6)-glucoside bonds. This suggests that Ala195, located on the Gly184-Thr203 loop (named loop-N) conserved within the GH97 subgroup, including FjGH97A and SusB, holds significance in determining linkage specificity. The conservation of alanine in the active site of the GH97 enzymes, within the same gene cluster as the putative dextranase, indicates its crucial role in determining the specificity for alpha-(1-->6)-glucoside linkage. |
