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As opposed to typical bacteria exhibiting chemotaxis towards low-molecular-weight substances, such as amino acids and mono/oligosaccharides, gram-negative Sphingomonas sp. strain A1 shows chemotaxis towards alginate and pectin polysaccharides. To identify the mechanism of chemotaxis towards macromolecules, a genomic fragment was isolated from the wild-type strain A1 through complementation with the mutant strain A1-M5 lacking chemotaxis towards pectin. This fragment contained several genes including sph1118. Through whole-genome sequencing of strain A1-M5, sph1118 was found to harbour a mutation. In fact, sph1118 disruptant lost chemotaxis towards pectin, and this deficiency was recovered by complementation with wild-type sph1118. Interestingly, the gene disruptant also exhibited decreased pectin assimilation. Furthermore, the gene product SPH1118 was expressed in recombinant E. coli cells, purified and characterised. Differential scanning fluorimetry and UV absorption spectroscopy revealed that SPH1118 specifically binds to pectin with a dissociation constant of 8.5 μM. Using binding assay and primary structure analysis, SPH1118 was predicted to be a periplasmic pectin-binding protein associated with an ATP-binding cassette transporter. This is the first report on the identification and characterisation of a protein triggering chemotaxis towards the macromolecule pectin as well as its assimilation.

Gram-negative Sphingomonas sp. strain A1, originally identified as a non-motile and aflagellate bacterium, possesses two sets of genes required for flagellar formation. In this study, we characterized the flagellar genes and flagellum formation in strain A1. Flagellar gene cluster set I contained 35 flagellar genes, including one flagellin gene (p6), where the gene assembly structure resembled that required for the formation of lateral flagella in gammaproteobacteria. The set II flagellar genes were arranged in eight shorter clusters with 46 flagellar genes, including two flagellin genes (p5 and p5') and flhF, which is required for polar flagella. Our molecular phylogenetic analysis of the bacterial flagellins also demonstrated that, in contrast to p5 and p5', p6 was categorized as a lateral flagellin group. The motile phenotype appeared in strain A1 cells when they were subcultured on semisolid media. The motile strain A1 cells produced a single flagellum at the cell pole. DNA microarray analyses using non-motile and motile strain A1 cells indicated that flagellar formation was accompanied by increased transcription of both flagellar gene sets. The two flagellins p5 and p6 were major components of the flagellar filaments isolated from motile strain A1 cells, indicating that the polar flagellum is formed by lateral and non-lateral flagellins.

Sphingomonas sp. HXN-200 bacteria were immobilized onto gelatin and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) microspheres in an aqueous system for industrial scale biohydroxylation. Scanning electron microscopy and confocal laser scanning microscopy were used to confirm the bacterial immobilization. The gelatin microsphere-immobilized cells successfully transformed N-benzyloxycarbonyl-pyrrolidine and N-tert-butoxycarbonyl-pyrrolidine into R-N-benzyloxycarbonyl-3-hydroxypyrrolidine and R-N-tert-butoxycarbonyl-3-pyrrolidinol at various substrate concentrations showing an improved conversion yield and product efficiency with respect to the freely suspended cells system at each reaction interval time. Additionally, in the immobilized cells system, it showed a faster reaction rate as compared to the freely suspended cells system for reaching the same product concentration. The inhibition effect due to substrate and product concentrations was significantly lower after immobilizing the bacteria onto the microspheres, which is beneficial for continuous reaction. The efficient whole-cell biocatalytic process was feasibly conducted in a 500mL preparative scale bioreactor and the conversion yield of N-benzyloxycarbonyl-pyrrolidine did not decrease after consecutive repeated use, suggesting that the microsphere-cells immobilization system was stable and reusable., (Copyright © 2014 Elsevier B.V. All rights reserved.)

A three-phase fluidized bed reactor (TPFBR) was designed to evaluate the potential of CoQ(10) production by gel-entrapped Sphingomonas sp. ZUTE03 via a conversion-extract coupled process. In the reactor, the CoQ(10) yield reached 46.99 mg/L after 8 h of conversion; a high-level yield of about 45 mg/L was maintained even after 15 repetitions (8 h/batch). To fully utilize the residual precursor (para-hydroxybenzoic acid, PHB) in the aqueous phase, the organic phase was replaced with new solution containing 70 mg/L solanesol for each 8 h batch. The CoQ(10) yield of each batch was maintained at a level of about 43 mg/L until the PHB ran out. When solid solanesol was fed to the organic phase for every 8 h batch, CoQ(10) could accumulate and reach a yield of 171.52 mg/L. When solid solanesol and PHB were fed to the conversion system after every 8 h batch, the CoQ(10) yield reached 441.65 mg/L in the organic phase after 20 repetitions, suggesting that the conversion-extract coupled process could enhance CoQ(10) production in the TPFBR., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Despite growing interest in the electro-bioremediation of contaminated soil it is still largely unknown to which degree weak electric fields influence the fate of contaminant-degrading microorganisms in the sub-surface. Here we evaluate the factors influencing the electrokinetic transport and deposition of fluorene-degrading Sphingomonas sp. LB126 in a laboratory model aquifer exposed to a direct current (DC) electric field (1 V cm(-1)) typically used in electro-bioremediation measures. The influence of cell size, cell membrane integrity, cell chromosome contents (all assessed by flow cytometry), cell surface charge and cell hydrophobicity on the spatial distribution of the suspended and matrix-bound cells after 15 h of DC-treatment was evaluated. In presence of DC the cells were predominantly mobilised by electroosmosis to the cathode with an apparent velocity of 0.6 cm h(-1), whereas a minor fraction only of the cells augmented was mobilised to the anode by electrophoresis. Different electrokinetic behaviour of individual cells could be solely attributed to intra-population heterogeneity of the cell surface charge. In the absence of DC by contrast, a Gaussian-type distribution of bacteria around the point of injection was found. DC had no influence on the deposition efficiency, as the glass beads in presence and absence of an electric field retained quasi-equal fractions of the cells. Propidium iodide staining and flow cytometry analysis of the cells indicated the absence of negative influences of DC on the cell wall integrity of electrokinetically mobilised cells and thus point at unchanged physiological fitness of electrokinetically mobilised bacteria.

Chlorinated organophosphate ester (OPE)-degrading enrichment cultures were obtained using tris(2-chloroethyl) phosphate (TCEP) or tris(1,3-dichloro-2-propyl) phosphate (TDCPP) as the sole phosphorus source. In cultures with 46 environmental samples, significant TCEP and TDCPP degradation was observed in 10 and 3 cultures, respectively, and successive subcultivation markedly increased their degradation rates. 67E and 45D stable enrichment cultures obtained with TCEP and TDCPP, respectively, completely degraded 20 muM of the respective compounds within 6 h and also the other, although the degradation rate of TCEP by 45D was relatively slow. We confirmed chloride ion generation on degradation in both cases and the generation of 2-chloroethanol (2-CE) and 1,3-dichloro-2-propanol (1,3-DCP) as metabolites of TCEP and TDCPP, respectively. 67E and 45D also showed dehalogenation ability toward 2-CE and 1,3-DCP, respectively. Addition of inorganic phosphate did not significantly influence their ability to degrade the chlorinated OPEs but markedly increased their dehalogenation ability, which was maximum at 0.2 mM of inorganic phosphate and decreased at a higher concentration. Denaturing gradient gel electrophoresis analysis showed that dominant bacteria in 67E are related to Acidovorax spp. and Sphingomonas spp. and those in 45D are Acidovorax spp., Aquabacterium spp., and Sphingomonas spp. This analysis indicated the relationship of the Sphingomonas- and Acidovorax-related bacteria with the cleavage of the phosphoester bond and dehalogenation, respectively, in both cultures. This is the first report on bacterial enrichment cultures capable of degrading both TCEP and TDCPP.

In spite of an increasing interest in fucoidans as biologically active compounds, no convenient commercial sources with fucoidanase activity are yet available. A marine bacterial strain that showed confluent growth on a minimal medium containing fucoidan, prepared from Korean Undaria pinnatifida sporophylls, as the sole carbon source was isolated and identified based on a 16S rDNA sequence analysis as a strain of Sphingomonas paucimobilis, and named Sphingomonas paucimobilis PF-1. The strain depolymerized fucoidan into more than 7 distinct lowmolecular- mass fucose-containing oligosaccharides, ranging from 305 to 3,749 Da. The enzyme activity was shown to be associated with the whole cell, suggesting the possibility of a surface display of the enzyme. However, a whole-cell enzyme preparation neither released the monomer Lfucose from the fucoidan nor hydrolyzed the chromogenic substrate p-nitrophenyl-alpha-L-fucoside, indicating that the enzyme may be an endo-acting fucoidanase rather than an alpha-L-fucosidase. Therefore, this would appear to be the first report on fucoidanolytic activity by a Sphingomonas species and also the first report on the enzymatic degradation of the Korean Undaria pinnatifida sporophyll fucoidan. Moreover, this enzyme activity may be very useful for structural analyses of fucose-containing polysaccharides and the production of bioactive fucooligosaccharides.

This study investigated the feasibility of a slow-release inoculation approach as a bioaugmentation strategy for the degradation of lindane (gamma-hexachlorocyclohexane [gamma-HCH]). Slow-release inoculation of Sphingomonas sp. gamma 1-7 was established in both liquid and soil slurry microcosms using open-ended silicone tubes in which the bacteria are encapsulated in a protective nutrient-rich matrix. The capacity of the encapsulated cells to degrade lindane under aerobic conditions was evaluated in comparison with inoculation of free-living cells. Encapsulation of cells in tubes caused the removal of lindane by adsorption to the silicone tubes but also ensured prolonged biodegradation activity. Lindane degradation persisted 2.2 and 1.4 times longer for liquid and soil slurry microcosms, respectively, than that for inoculation with free cells. While inoculation of free-living cells led to a loss in lindane-degrading activity in limited time intervals, encapsulation in tubes allowed for a more stable actively degrading community. The loss in degrading activity was linked to the loss of the linA gene, encoding gamma-HCH dehydrochlorinase (LinA), which is involved in the initial steps of the lindane degradation pathway. This work shows that a slow-release inoculation approach using a catabolic strain encapsulated in open-ended tubes is a promising bioaugmentation tool for contaminated sites, as it can enhance pollutant removal and can prolong the degrading activity in comparison with traditional inoculation strategies.

Hydroxylation of N-benzylpyrrolidine 8 with resting cells of Sphingomonas sp. HXN-200 gave N-benzyl-3-hydroxypyrrolidine 15 in 53% ee (S) with an activity of 5.8 U/g CDW. By changing the "docking/protecting group" in pyrrolidines, hydroxylation activity and enantioselectivity were further improved and the enantiocomplementary formation of 3-hydroxypyrrolidines was achieved: hydroxylation of N-benzoyl-, N-benzyloxycarbonyl-, N-phenoxycarbonyl-, and N-tert-butoxycarbonyl-pyrrolidines 9-12 gave the corresponding 3-hydroxypyrrolidines 16-19 in ee of 52% (R), 75% (R), 39% (S), and 23% (R), respectively, with an activity of 2.2, 16, 14, and 24 U/g CDW, respectively. Simple crystallizations increased the ee of 16-18 to 95% (R), 98% (R), and 96% (S), respectively. Hydroxylation of pyrrolidines 8-12 with soluble cell-free extracts of Sphingomonas sp. HXN-200 and equimolar NADH gave 3-hydroxypyrrolidines 15-19 in nearly the same ee as the products generated by whole cell transformation, suggesting that this strain possesses a novel soluble alkane monooxygenase. Cells of Sphingomonas sp. HXN-200 were produced in large amounts and could be stored at -80 degrees C for 2 years without significant loss of activity. The frozen cells can be thawed and resuspended for biohydroxylation, providing a highly active and easy to handle biocatalyst for the regio- and stereoselective hydroxylation of nonactivated carbon atoms. These cells were used to prepare 1.0-3.2 g (66.4-93.5% yield) of 3-hydroxypyrrolidines 16-19 by hydroxylation of pyrrolidines 9-12 on 0.9-2 L scale. Preparative hydroxylation was also achieved with growing cells as biocatalysts; hydroxylation of pyrrolidine 11 on 1 L scale gave 1.970 g (79.7% yield) of 3-hydroxypyrrolidine 18.

In a soil isolate, Sphingomonas sp. A1, the transport of a macromolecule (alginate: 27 kDa) is mediated by a pit-dependent ATP-binding cassette (ABC) transporter. The transporter is different from other ABC transporters so far analyzed in that its function is dependent on the pit, a mouth-like organ formed on the cell surface only when the cells are compelled to assimilate macromolecules, and in that it allows direct import of macromolecules into cells. The ABC transporter coupled with the pit, which functions as a funnel and/or concentrator of macromolecules to be imported, was designated as the "Super-channel", and in this review, we discuss the three-dimensional structure and specific function of the "Super-channel" for macromolecule import found for the first time in a bacterium.