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     

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...     

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.     

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     

Differences in dynamics between indigenous and inoculated Sphingomonas paucimobilis strain SS86 in soils

Abstract Survival of γ-HCH-degrading Sphingomonas paucimobilis strain SS86 indigenous or inoculated to soil was examined under laboratory conditions. Strain SS86 inoculated to soil declined to undectectable levels though amendment of soil with γ-HCH or starvation-treatment of the cells enhanced its survivability. About 10³–10⁴ cells/g soil of strain SS86 indigenous to soil survived stably for long periods and was more tolerant to soil treatment by desiccation, percolation, and chloroform-fumigation than inoculated SS86. The haritable micro-pores in soil and/or physiological properties peculiar to the indigenous SS86 were supposed to ensure its good ability to survive by protecting it against protozoan grazing.     

Degradation of 3-O-methylgallate in Sphingomonas paucimobilis SYK-6 by pathways involving protocatechuate 4,5-dioxygenase

Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate and 3-O-methylgallate (3MGA), respectively. 3MGA is metabolized via multiple pathways involving 3MGA 3,4-dioxygenase, protocatechuate 4,5-dioxygenase (LigAB), and gallate dioxygenase whereas protocatechuate is degraded via the protocatechuate 4,5-cleavage pathway. Here the secondary role of LigAB in syringate metabolism is investigated. The reaction product of 3MGA catalyzed by His-tagged LigAB was identified as 4-carboxy-2-hydroxy-6-methoxy-6-oxohexa-2,4-dienoate (CHMOD) and 2-pyrone-4,6-dicarboxylate (PDC), indicating that 3MGA is transformed to CHMOD and PDC by both reactions catalyzed by DesZ and LigAB. Mutant analysis revealed that the 3MGA catabolic pathways involving LigAB are functional in SYK-6.     

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     

Identification of soil micro-habitats for growth, death and survival of a bacterium, γ-1,2,3,4,5,6-hexachlorocyclohexane-assimilating Sphingomonas paucimobilis, by fractionation of soil

Abstract A soil bacterium, Sphingomonas paucimobilis , is known to be the only bacterium which can aerobically assimilate γ-1,2,3,4,5,6-hexachlorocyclohexane (γ-HCH). Indigenous γ-HCH-assimilating S. paucimobilis survives in the soil where γ-HCH has been annually applied since 1973. In contrast, γ-HCH-assimilating S. paucimobilis strain SS86 cannot survive when inoculated into the control soil, although it can multiply in the presence of γ-HCH. Micro-habitats of γ-HCH-assimilating S. paucimobilis indigenous or inoculated into the soils were identified by fractionation of the soils. When γ-HCH was added to the soil, indigenous γ-HCH-assimilating S. paucimobilis grew. Most of the growing indigenous bacteria were found in fractions smaller than 0.025 mm which corresponded to soil inter-aggregate pores, and died afterwards. However, the indegenous bacteria which survived for a long period were found mainly in fractions larger than 0.025 mm which contained soil aggregates. When γ-HCH-assimilating strain SS86 was inoculated, the bacteria were located in inter-aggregate pores and died quickly. Consequently, association of the bacteria with soil aggregates was suggested to be related to the long-term survival of γ-HCH-assimilating S. paucimobilis .     

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 in the attachment of sphingomonas paucimobilis

The importance of exopolymers in the adhesion of Sphingomonas paucimobilis was established by studying the attachment to glass of three mutants with defective gellan production. The attachment assays were performed in either phosphate buffered saline (controls) or in the exopolymeric solutions produced by the mutants. The exopolymer was found to have surface active properties, changing the glass surface from hydrophilic to hydrophobic, making adhesion thermodynamically favourable. Only the cells that had a substantial polymeric layer surrounding their walls were able to significantly colonise glass coated with the exopolymer. It is hypothesised that the exopolymer bound to the glass and the exopolymer present at the surface of the bacteria bound together, overcoming the energy barrier created by the negative charge of both surfaces. It is concluded that the exopolymer from S. paucimobilis has a dual role in the process of adhesion by both coating the surface thereby strengthening adhesion and by enhancing adhesion through the establishment of polymeric bridges.     

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     

A Common Structure of Substrate Shared by Lignostilbenedioxygenase Isozymes from Sphingomonas paucimobilis TMY1009

A common structure of substrates of lignostilbenedioxygenases was investigated using synthesized stilbenes. Cell-free extracts of Sphingomonas paucimobilis TMY1009 degraded only trans-4-hydroxystilbene and trans-4-hydroxy-3-methoxystilbene. Other stilbenes that had no 4-hydroxyl group and had a cis structure were not substrates for lignostilbenedioxygenases. These results indicate that a 4-hydroxyl group and trans-structure is necessary for the common structure for substrates of lignostilbenedioxygenases.     

Degradation of substituted naphthalenesulfonic acids by Sphingomonas xenophaga BN6

Sphingomonas xenophaga BN6 was isolated from the river Elbe as a member of a multispecies bacterial culture which mineralized 6-aminonaphthalene-2-sulfonate. Pure cultures of strain BN6 converted a wide range of amino- and hydroxynaphthalene-2-sulfonates via a catabolic pathway similar to that described for the metabolism of naphthalene to salicylate by Pseudomonas putida NAH7 or Pseudomonas sp NCIB 9816. In contrast to the naphthalene-degrading pseudomonads, S. xenophaga BN6 only partially degraded the naphthalenesulfonates and excreted the resulting amino- and hydroxysalicylates in almost stoichiometric amounts. Enzymes that take part in the degradative pathway of the naphthalenesulfonates by strain BN6 were purified, characterized and compared with the isofunctional enzymes from the naphthalene-degrading pseudomonads. According to the enzyme structures and the catalytic constants, no fundamental differences were found between the 1,2-dihydroxynaphthalene dioxygenase or the 2′-hydroxybenzalpyruvate aldolase from strain BN6 and the isofunctional enzymes from the naphthalene-degrading pseudomonads. The limited available sequence information about the enzymes from strain BN6 suggests that they show about 40–60% sequence identity to the isofunctional enzymes from the pseudomonads. In addition to the gene for the 1,2-dihydroxynaphthalene dioxygenase, the genes for two other extradiol dioxygenases were cloned and sequenced from strain BN6 and the corresponding gene products were studied. S. xenophaga BN6 has also been used as a model organism to study the mechanism of the non-specific reduction of azo dyes under anaerobic conditions and to establish combined anaerobic/aerobic treatment systems for the degradation of sulfonated azo dyes. Furthermore, the degradation of substituted naphthalenesulfonates by mixed cultures containing strain BN6 was studied in continuous cultures and was described by mathematical models. Received 02 April 1999/ Accepted in revised form 09 July 1999     

一株多环芳烃降解菌的鉴定及GST基因克隆和序列分析

由石油污染土壤中分离到一株能以多环芳烃（菲、芴、萘）为唯一碳源的细菌,经形态观察、生理生化（BiologGN）和 G+C mol%分析,鉴定该菌为少动鞘氨醇单胞菌(Sphingomonas paucimobilis)。与16S rDNA序列同源性的比较进一步确证了鉴定结果。经菲诱导后的细菌谷胱甘肽S转移酶（Glutathione Stransferase, GST）酶活明显高于未诱导前,表明谷胱甘肽S转移酶可能与多环芳烃的降解有关。根据该酶基因的同源性序列设计引物,PCR扩增出编码谷胱甘肽S转移酶基因片段,进一步证实在该菌中有GST的存在。测序后基于编码GST的基因所进行的系统发育分析表明,该多环芳烃降解菌与其它多环芳烃降解菌在进化上亲缘关系较近。     

少动鞘脂单胞菌产结冷胶发酵培养基的响应面法优化

通过单因素试验分析不同碳源、氮源、无机盐对（Sphingomonas paucimobilisFJAT-5627）产胶量的影响,确定最适碳源、氮源、无机盐,并在单因素筛选试验的基础上,利用Box-Benhnken设计和响应面分析法对碳源、氮源和无机盐进行优化,得到少动鞘脂单胞菌产生结冷肢发酵培养基最佳优化组合.实验结果表明,少动鞘脂单胞菌产胶量发酵最适碳源、氮源和无机盐分别为淀粉、豆饼粉和KH₂PO₄.响应面法得到产胶量（Y）与碳源淀粉（x₁）、氮源豆饼粉（x₂）和无机盐KH₂PO₄（x₃）的回归方程为:Y=13.87+0.54x₁+0.22x₂-0.42x₃-3.26x₁²-1.85x₂²-1.51x₃²+0.053x₁x₂+0.067x₁x₃+0.4x₂x₃.优化培养基组合为:淀粉浓度为30g/L,豆饼粉浓度为5 g/L,KH₂PO₄的浓度为0.7g/L,且此组合下少动鞘脂假单胞发酵得到结冷胶可达23.87g/L.     

Convergent and divergent points in catabolic pathways involved in utilization of fluoranthene, naphthalene, anthracene, and phenanthrene by Sphingomonas paucimobilis var. EPA505

Catabolic pathways for utilization of naphthalene (NAP), anthracene (ANT), phenanthrene (PHE), and fluoranthene (FLA) by Sphingomonas paucimobilis EPA505 were identified. Accumulation of catabolic intermediates was investigated with three classes of Tn5 mutants with the following polycyclic aromatic hydrocarbon (PAH)-negative phenotypes; (class I NAP(-) PHE(-) FLA(-), class II NAP(-) PHE(-), and class III FLA(-)). Class I mutant 200pbhA had a Tn5 insertion within a meta ring fission dioxygenase (pbhA), and a ferredoxin subunit gene (pbhB) resided directly downstream. Mutant 200pbhA and other class I mutants lost the ability to catalyze the initial dihydroxylation step and did not transform NAP, ANT, PHE, or FLA. Class I mutant 401 accumulated salicylic acid, 2-hydroxy-3-naphthoic acid, 1-hydroxy-2-naphthoic acid, and hydroxyacenaphthoic acid during incubation with NAP, ANT, PHE, or FLA, respectively. Class II mutant 132pbhC contained the Tn5 insertion in an aldolase hydratase (pbhC) and accumulated what appeared to be meta ring fission products: trans-o-hydroxybenzylidene pyruvate, trans-o-hydroxynaphylidene pyruvate, and trans-o-hydroxynaphthyl-oxobutenoic acid when incubated with NAP, ANT, and PHE, respectively. When mutant 132pbhC was incubated with 1-hydroxy-2-naphthoic acid, it accumulated trans-o-hydroxybenzylidene pyruvate. Class III mutant 104ppdk had a Tn5 insertion in a pyruvate phosphate dikinase gene that affected expression of a FLA-specific gene and accumulated a proposed meta ring fission product; trans-o-hydroxyacenaphyl-oxobutenoic acid during incubation with FLA. Trans-o-hydroxyacenaphyl-oxobutenoic acid was degraded to acenaphthenone that accumulated with class III mutant 611. Acenaphthenone was oxidized via incorporation of one molecule of dioxygen by another oxygenase. 2,3-Dihydroxybenzoic acid was the final FLA-derived catabolic intermediate detected. Analysis of PAH utilization mutants revealed that there are convergent and divergent points involved in NAP, ANT, PHE, and FLA utilization by S. paucimobilis EPA505.     

Degradation of beta-Hexachlorocyclohexane by Haloalkane Dehalogenase LinB from Sphingomonas paucimobilis UT26

Beta-Hexachlorocyclohexane (beta-HCH) is the most recalcitrant among the alpha-, beta-, gamma-, and delta-isomers of HCH and causes serious environmental pollution problems. We demonstrate here that the haloalkane dehalogenase LinB, reported earlier to mediate the second step in the degradation of gamma-HCH in Sphingomonas paucimobilis UT26, metabolizes beta-HCH to produce 2,3,4,5,6-pentachlorocyclohexanol.     

Degradation of azo dyes containing aminonaphthol by Sphingomonas sp strain 1CX

Sphingomonas sp strain 1CX was isolated from a wastewater treatment plant and is capable of aerobically degrading a suite of azo dyes, using them as a sole source of carbon and nitrogen. All azo dyes known to be decolorized by strain 1CX (Orange II, Acid Orange 8, Acid Orange 10, Acid Red 4, and Acid Red 88) have in their structure either 1-amino-2-naphthol or 2-amino-1-naphthol. In addition, an analysis of the structures of the dyes degraded suggests that there are certain positions and types of substituents on the azo dye which determine if degradation will occur. Growth and dye decolorization occurs only aerobically and does not occur under fermentative or denitrification conditions. The mechanism by which 1CX decolorizes azo dyes appears to be through reductive cleavage of the azo bond. In the case of Orange II, the initial degradation products were sulfanilic acid and 1-amino-2-naphthol. Sulfanilic acid, however, was not used by 1CX as a growth substrate. The addition of glucose or inorganic nitrogen inhibited growth and decoloration of azo dyes by 1CX. Attempts to grow the organism on chemically defined media containing several different amino acids and sugars as sources of nitrogen and carbon were not successful. Phylogenetic analysis of Sphingomonas sp strain 1CX shows it to be related to, but distinct from, other azo dye-decolorizing Sphingomonas spp strains isolated previously from the same wastewater treatment facility. Received 19 May 1999/ Accepted in revised form 11 August 1999     

Simultaneous and rapid monitoring of biomass and biopolymer production by Sphingomonas paucimobilis using Fourier transform-near infrared spectroscopy

The application of Fourier Transform near infrared spectroscopy (FT-NIRS) to near real-time monitoring of polysaccharide and biomass concentration was investigated using a gellan-producing strain of Sphingomonas paucimobilis grown in a stirred tank reactor. Successful models for both biomass and gellan were constructed despite the physichochemical complexity of the viscous process fluid. Modelling of biomass proved more challenging than for gellan, partly because of the low range of biomass concentration but a model with a good correlation coefficient (0.94) was formulated based on second derivative spectra. The gellan model was highly satisfactory, with an excellent correlation coefficient (0.98), again based on second derivative spectra. No sample pre-treatment was required and all spectral scanning was carried out on whole broth. Additionally, both models should be robust in practice since both were formulated using low numbers of factors. Thus, the near real time simultaneous monitoring of gellan and biomass in this highly complex matrix using FT-NIRS potentially opens the way to greatly improved process control strategies.