Effect of 2-deoxy-d-glucose on gellan gum biosynthesis by Sphingomonas paucimobilis

Bioprocess and Biosystems Engineering - 2-Deoxy-d-glucose (2-DG) is a non-metabolizable glucose analogue and competitive inhibitor of glycolysis. Effect of 2-DG on gellan gum biosynthesis by...     

少动鞘脂单胞菌S1胞外多糖发酵工艺条件研究

研究了摇瓶培养条件下少动鞘脂单胞菌引的胞外多糖的发酵工艺。Ｓ１菌的发酵产胶可采用二步发酵法：第一阶段,培养基成分为：蔗糖１０ｇ,ＮＨ_４ＮＯ_３　０．５ｇ,ＫＨ_２ＯＰ_４　０．５ｇ,ＭｎＳＯ_４　０．３ｇ,ＭｇＳＯ_４　０．１ｇ,吐温８０ ０．０２ｇ溶于蒸馏水并定容至１０００ｍＬ,ｐＨ７．２±０．１,温度３３℃～３５℃,高溶氧。第二阶段,发酵 １０～１２ｈ后,补加蔗糖４０ｇ／Ｌ,温度     

Enhanced Degradation of Hexachlorocyclohexane Isomers by Sphingomonas paucimobilis

Hexachlorocyclohexane (HCH) has been banned for use in technologically advanced countries; however, it is still in use in tropical countries like India. Earlier we reported the degradation of HCH isomers by Sphingomonas paucimobilis within 12 days of incubation. Here we report the role of different factors that could enhance the degradation rate of HCH isomers. We found that an increase in the cell number from 10² to 10⁸ cells/ml resulted in an increased degradation rate of HCH isomers viz. α, β, γ, and δ-HCH. While α-HCH and γ-HCH disappeared completely from the medium within 3 days of incubation, a maximum of only 90% and 85% degradation was observed for β and δ-HCH, respectively. We have also observed that adapted cultures degraded HCH isomers more efficiently than did the normal cultures. Received: 16 February 2000 / Accepted: 23 May 2000     

Oxidation of Methyl-Substituted Naphthalenes: Pathways in a Versatile Sphingomonas paucimobilis Strain

Aromatic compounds with alkyl substituents are abundant in fossil fuels. These compounds become important environmental sources of soluble toxic products, developmental inhibitors, etc. principally through biological activities. To assess the effect of methyl substitution on the completeness of mineralization and accumulation of pathway products, an isolate from a phenanthrene enrichment culture, Sphingomonas paucimobilis 2322, was used. Washed cell suspensions containing cells grown on 2,6-dimethylnaphthalene in mineral medium were incubated with various mono-, di-, and trimethylnaphthalene isomers, and the products were identified and quantified by gas chromatography and mass spectrometry. The data revealed enzymes with relaxed substrate specificity that initiate metabolism either by methyl group monoxygenation or by ring dioxygenation. Congeners with a methyl group on each ring initially hydroxylate a methyl, and this is followed by conversion to a carboxyl; when there are two methyl groups on a single ring, the first reaction is aryl dioxygenation of the unsubstituted ring. Intermediates are channeled to primary ring fission via dihydrodiols to form methyl-substituted salicylates. Further evidence that there are multiple pathways comes from the fact that both phthalate and (methyl)salicylate are formed from 2-methylnaphthalene.     

Evaluation of rheological properties of the exopolysaccharide of Sphingomonas paucimobilis GS-1 for application in oil exploration

Analysis of an exopolysaccharide of Sphingomonas paucimobilis GS-1 (EPS/GS-1) with respect to its rheological properties, cross-linking ability with chrome alum and performance test at 75 ± 5°C revealed its strong suspending ability, shear thinning property, and thixotrophic nature which are required to impart desirable rheology to drilling mud. The organism fulfilled all the specified requirements and its properties were superior to those of currently-used XC polymer (a xanthan product) for oil drilling applications. However, EPS/GS-1 was unstable in the presence of bentonite at 100 ± 5°C during performance tests, in contrast to XC polymer. Received 14 April 1999/ Accepted in revised form 26 July 1999     

The production of gellan exopolysaccharide with Sphingomonas paucimobilis E2 (DSM 6314)

Summary A new screening technique was used to isolate the bacterium Sphingomonas paucimobilis E2 (DSM 6314), which produces the exopolysaccharide gellan. The productivity was found to be about four times higher than that of the industrially used strain Auromonas elodea (ATCC 31461) it was isolated from. The polysaccharide formation was found to be predominantly growth-related. Correspondence to: W.-D. Deckwer     

少动鞘氨醇单胞菌致感染性心内膜炎1例

少动鞘氨醇单胞菌(Sphingomonas paucimobilis) 是一种少见的条件致病菌,可引起手术后感染、创伤后腿部溃疡、菌血症、脑膜炎、慢性蜂窝织炎、手术后眼内炎等,未查见累及心瓣膜的报道.本文报道1例由少动鞘氨醇单胞菌所致感染性心内膜炎的病例.该患者为中年男性,因"反复发热2月余"入院.以发热伴左上腹痛为首...     

Sphingomonas paucimobilis BPSI-3 mutant AN2 produces a red catabolite during biphenyl degradation

The biphenyl degradation pathway of Sphingomonas paucimobilis BPSI-3 was investigated using a degradation-deficient mutant generated by 1-methyl-3-nitro-1-nitrosoguanidine (NTG) mutagenesis. The mutant, designated AN2, was confirmed as originating from BPSI-3 through the use of ERIC (Enterobacterial Repetitive Intergenic Consensus) PCR and by detection of the diagnostic pigment, nostoxanthin, in cellular methanol extracts. Mutant AN2 produced a yellow followed by red extracellular substance when grown in the presence of biphenyl. In the presence of 2,3-dihydroxybiphenyl, yellow followed by red then yellow compounds were formed over time. This colour change was consistent with the characteristics of a quinone, 1-phenyl-2,3-benzoquinone, which could arise from the oxidation of 2,3-dihydroxybiphenyl. A quinone was synthesised from 2,3-dihydroxybiphenyl and compared to the red compound produced by mutant AN2. Gas chromatography-mass spectrophotometry (GC-MS) confirmed that a similar quinone (4,5-dimethoxy-3-phenyl-1,2-benzoquinone) compared to the structure of the proposed biogenic compound, had been formed. This compound was also found after GC-MS analysis of mutant AN2 culture extracts. Spectrophotometric analysis of the quinone synthesised and the red product produced revealed almost identical spectral profiles. A likely inference from this evidence is that the mutant AN2 is blocked, or its activity altered, in the first gene cluster, bphA to C, of the biphenyl degradation pathway. Received 19 April 1999/ Accepted in revised form 25 July 1999     

Construction of mutants of Sphingomonas paucimobilis defective in terminal mannose in the glycosphingolipid

Mutants of Sphingomonaspaucimobilis defective in a part of the carbohydrate moiety of the glycosphingolipid (GSL) were constructed by transposon (Tn5)-insertional mutagenesis. Defective mutants were selected by ELISA using the antibody recognizing the tetrasaccharide-type GSL (GSL-4A) of S. paucimobilis. Eight defective mutants were selected from about 8,000 kanamycin-resistant strains, and seven of them were found to lack the terminal mannose of GSL-4A. The chemical structure of the mutant GSL was investigated, and proved that the rest of the structure was not changed by the mutation.     

Characterization of Sphingomonas paucimobilis SYK-6 genes involved in degradation of lignin-related compounds

Sphingomonas paucimobilis SYK-6 is able to grow on a wide variety of dimeric lignin compounds. These compounds are degraded via vanillate and syringate by a unique enzymatic system, composed of etherases, O demethylases, ring cleavage oxygenases and side chain cleaving enzymes. These unique and specific lignin modification enzymes are thought to be powerful tools for utilization of the most abundant aromatic biomass, lignin. Here, we focus on the genes and enzymes involved in β-aryl ether cleavage and biphenyl degradation. Two unique etherases are involved in the reductive cleavage of β-aryl ether. These two etherases have amino acid sequence similarity with the glutathione S-transferases, and use glutathione as a hydrogen donor. It was found that 5,5′-dehydrodivanillate, which is a typical lignin-related biphenyl structure, was transformed into 5-carboxyvanillate by the reaction sequence of O-demethylation, meta-ring cleavage, and hydrolysis, and the genes involved in the latter two reactions have been characterized. Vanillate and syringate are the most common intermediate metabolites in lignin catabolism. These compounds are initially O-demethylated and the resulting diol compounds, protocatechuate (PCA) and 3-O-methylgallate, respectively, are subjected to ring cleavage catalyzed by PCA 4,5-dioxygenase. The ring cleavage products generated are further degraded through the PCA 4,5-cleavage pathway. We have isolated and characterized genes for enzymes involved in this pathway. Disruption of a gene for 2-pyrone-4,6-dicarboxylate hydrolase (ligI) in this pathway suggested that an alternative route for 3-O-methylgallate degradation, in which ligI is not involved, would play a role in syringate catabolism. In this article, we describe the genetic and biochemical features of the S. paucimobilis SYK-6 genes involved in degradation of lignin-related compounds. A possible application of the SYK-6 lignin degradation system to produce a valuable chemical material is also described. Received 01 May 1999/ Accepted in revised form 29 July 1999     

The Role of Exopolymers Produced by Sphingomonas paucimobilis in Biofilm Formation and Composition

Exopolymers have been associated with the initial adhesion of bacteria, which is the primary step for biofilm formation. Moreover, the polymeric matrix of biofilms has a considerable influence on some of the most important physical and physiological properties of biofilms. The role of extracellular polymers in biofilm formation was studied using three mutants of Sphingomonas paucimobilis with increasing capabilities for exopolymer production. The physical, biochemical and physiological properties of three different layers of each biofilm were determined. The layers were detached by submitting the biofilm to increasing shear stress. The results revealed that the presence of exopolymers in the growth medium was essential for biofilm formation. The mutant producing the highest amount of exopolymer formed very thick biofilms, while the biofilms formed by the medium exopolymer producer were on average 8 times thinner. The lowest exopolymer producer did not form biofilm. In both types of biofilms, exopolymer density increased with depth, although this tendency was more significant in thinner biofilms. Cell distribution was also more heterogeneous in thinner biofilms, exhibiting a greater accumulation of cells in the inner layers. The thicker biofilms had very low activity in the inner layer. This was related to a high accumulation of proteins and DNA in this layer due to cell lysis and hydrolytic activity. Activity in the thin biofilm was constant throughout its depth, suggesting that there was no nutrient limitation. The production of exopolymers by each cell was constant throughout the depth of the biofilms, although it was greater in the case of the higher producer.     

Synergistic mineralization of biphenyl by Alcaligenes faecalis type II BPSI-2 and Sphingomonas paucimobilis BPSI-3

Polychlorinated biphenyls (PCBs) are important environmental pollutants and have been found to have adverse effects on a number of different organisms. Aerobic biodegradation of PCBs occurs through direct oxidation of the biphenyl nucleus. Biphenyl degraders are instrumental in the mineralization of PCBs to CO₂ and water. Here two bacteria, Alcaligenes faecalis type II strain BPSI-2 and Sphingomonas paucimobilis strain BPSI-3, are described that exhibit synergistic mineralization of biphenyl (using ¹⁴C-UL-biphenyl) when grown as a co-culture. Mineralization rates (23·7 and 9·1 nmol nmol⁻¹ h⁻¹, respectively) and extent of mineralization (38·1% and 24·4%, respectively) were significantly different between the strains as well as when compared to the co-culture (35·2 nmol nmol⁻¹ h⁻¹ and 45·2%). Both strains were originally isolated from an enrichment culture, BSEN-2. The co-culture of BPSI-2 and 3 showed a threefold increase in mineralization rate compared with the parent culture and a decrease in the time taken for ¹⁴CO₂ evolution to occur. There was no significant difference in the extent of mineralization between the co-culture and BSEN-2. Examination of enrichment cultures at the community level may play a role in optimizing bioremediation programmes.     

The cell envelope structure of the lipopolysaccharide-lacking gram-negative bacterium Sphingomonas paucimobilis.

From the cell envelope preparation of Sphingomonas paucimobilis two membrane fractions with different densities were separated by sucrose density gradient ultracentrifugation. The high-density fraction contained several major proteins, phospholipids, and glycosphingolipids, which are the only glycolipids of this lipopolysaccharide-lacking gram-negative bacterium. The low-density fraction showed many minor bands of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and NADH oxidase activity was localized in this fraction. Combined with morphological data of vesicles formed by these membrane fractions, the high-density and low-density fractions were proposed to be an outer membrane and a cytoplasmic membrane, respectively. The localization of the glycosphingolipid was investigated also by means of immunoelectron microscopic analysis using a glycosphingolipid-specific antibody. The glycosphingolipid was shown to localize at the cell envelope, and the antigenic sugar portion was exposed to the bacterial cell surface. From these results the glycosphingolipid was assumed to have a function similar to that of the lipopolysaccharide of other gram-negative bacteria.     

Degradation of alpha, beta, gamma and delta-hexachlorocyclohexanes by Sphingomonas paucimobilis

Sphingomonas paucimobilis degrades aerobically , , and -hexachlorocyclohexane. With -HCH, complete degradation occurred after 3 days but with and , and with -HCH, 98 and 56 % degradation occurred after 12 and 8 days of incubation, respectively. Pentachlorocyclohexene was formed as the primary metabolite during the degradation of all the HCH isomers. © Rapid Science Ltd. 1998     

Enhanced gellan gum production by hydrogen peroxide (H2O2) induced oxidative stresses in Sphingomonas paucimobilis

In this study, the effect of H₂O₂-induced oxidative stress on gellan gum production and cell growth were investigated. Gellan gum production was improved and cell growth was inhibited by H₂O₂. A multiple H₂O₂ stresses with different concentrations were developed to optimize gellan gum production. A maximal gellan gum yield (22.52 g/L), which was 35.58 % higher than the control, was observed with 2, 2, 3, 4 mmol/L H₂O₂ added at 6, 12, 18, 24 h, respectively. Moreover, UDP-glucose pyrophosphorylase activity and glucosyltransferase activity were increased with H₂O₂ stresses. This new strategy of multiple H₂O₂-induced oxidative stresses would be further applied to gellan gum production in future study.     

Catalytic mechanism of the maloalkane dehalogenase LinB from Sphingomonas paucimobilis UT26

Haloalkane dehalogenases are bacterial enzymes capable of carbon-halogen bond cleavage in halogenated compounds. To obtain insights into the mechanism of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB), we studied the steady-state and presteady-state kinetics of the conversion of the substrates 1-chlorohexane, chlorocyclohexane, and bromocyclohexane. The results lead to a proposal of a minimal kinetic mechanism consisting of three main steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond with simultaneous formation of an alkyl-enzyme intermediate, and (iii) hydrolysis of the alkyl-enzyme intermediate. Release of both products, halide and alcohol, is a fast process that was not included in the reaction mechanism as a distinct step. Comparison of the kinetic mechanism of LinB with that of haloalkane dehalogenase DhlA from Xantobacter autotrophicus GJ10 and the haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 shows that the overall mechanisms are similar. The main difference is in the rate-limiting step, which is hydrolysis of the alkylenzyme intermediate in LinB, halide release in DhlA, and liberation of an alcohol in DhaA. The occurrence of different rate-limiting steps for three enzymes that belong to the same protein family indicates that extrapolation of this important catalytic property from one enzyme to another can be misleading even for evolutionary closely related proteins. The differences in the rate-limiting step were related to: (i) number and size of the entrance tunnels, (ii) protein flexibility, and (iii) composition of the halide-stabilizing active site residues based on comparison of protein structures.     

Gellan gum biosynthesis in Sphingomonas paucimobilis ATCC 31461: genes,enzymes and exopolysaccharide production engineering

The commercial gelling agent, gellan, is an extracellular polysaccharide (EPS) produced by Sphingomonas paucimobilis ATCC 31461. In recent years, significant progress in understanding the relationship between gellan structure and properties and elucidation of the biosynthesis and engineering of this recent product of biotechnology has been made. This review focuses on recent advances in this field. Emphasis is given to identification and characterization of genes and enzymes involved, or predicted to be involved, in the gellan biosynthetic pathway, at the level of synthesis of sugar-activated precursors, of the repeat unit assembly and of gellan polymerization and export. Identification of several genes, biochemical characterization of the encoded enzymes and elucidation of crucial steps of the gellan pathway indicate that possibilities now exist for exerting control over gellan production at any of the three levels of its biosynthesis. However, a better knowledge of the poorly understood steps and of the bottlenecks and regulation of the pathway, the characterization of the composition, structure and functional properties of gellan-like polymers produced either by the industrial strain under different culture conditions or by mutants are still required for eventual success of the metabolic engineering of gellan production. Journal of Industrial Microbiology & Biotechnology (2002) 29, 170–176 doi:10.1038/sj.jim.7000266 Received 11 February 2002/ Accepted in revised form 09 April 2002     

Identification of the catalytic triad in the haloalkane dehalogenase from Sphingomonas paucimobilis UT26

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the gamma-hexachlorocyclohexane degradation. This enzyme hydrolyses a broad range of halogenated aliphatic compounds via an alkyl-enzyme intermediate. LinB is believed to belong to the family of alpha/beta-hydrolases which employ a catalytic triad, i.e. nucleophile-histidine-acid, during the catalytic reaction. The position of the catalytic triad within the sequence of LinB was probed by a site-directed mutagenesis. The catalytic triad residues of the haloalkane dehalogenase LinB are proposed to be D108, H272 and E132. The topological location of the catalytic acid (E132) is after the beta-strand six which corresponds to the location of catalytic acid in the pancreatic lipase, but not in the haloalkane dehalogenase of Xanthobacter autotrophicus GJ10 which contains the catalytic acid after the beta-strand seven.     

Characterization of ligV essential for catabolism of vanillin by Sphingomonas paucimobilis SYK-6

The vanillin dehydrogenase gene (ligV), which conferred the ability to transform vanillin into vanillate on Escherichia coli, was isolated from Sphingomonas paucimobilis SYK-6. The ligV gene consists of a 1,440-bp open reading frame encoding a polypeptide with a molecular mass of 50,301 Da. The deduced amino acid sequence of ligV showed about 50% identity with the known vanillin dehydrogenases of Pseudomonas vanillin degraders. The gene product of ligV (LigV) produced in E. coli preferred NAD+ to NADP+ and exhibited a broad substrate preference, including vanillin, benzaldehyde, protocatechualdehyde, m-anisaldehyde, and p-hydroxybenzaldehyde, but the activity toward syringaldehyde was less than 5% of that toward vanillin. Insertional inactivation of ligV in SYK-6 indicated that ligV is essential for normal growth on vanillin. On the other hand, growth on syringaldehyde was only slightly affected by ligV disruption, indicating the presence of a syringaldehyde dehydrogenase gene or genes in SYK-6.