鞘氨醇单胞菌的研究进展

鞘氨醇单胞菌(Sphingomonas)不仅细胞膜含有比脂多糖更疏水的鞘糖脂,而且具有高效的代谢调控机制和基因调控能力,使其在威兰胶合成、环境修复和促进植物生长等方面具有巨大的应用潜力。目前国内在鞘氨醇单胞菌代谢机制方面的研究尚无新突破。本文主要综述了鞘氨醇单胞菌的系统分类、基因组学、基因调控机制及其应用等方面的研究,从基因层面分析鞘氨醇单胞菌产威兰胶的合成机制,为后续鞘氨醇单胞菌高密度发酵、工业化生产等研究提供理论基础,以便进一步发掘其在生物技术上的应用潜力。     

结冷胶生物合成机理研究进展

结冷胶是少动鞘氨醇单胞菌(Sphingomonas paucimobilis)产生的一种新型微生物多糖,其独特的流变特性使结冷胶具有广泛的工业用途。虽然在结冷胶的理化特性方面的研究比较详尽,但是对结冷胶的发酵生产及其生物合成机制还缺乏深入了解。主要关注最近在结冷胶生物合成途径分子生物学方面的研究,用于编码结冷胶生物合成所需蛋白质的基因主要有三类:与糖核苷酸合成有关的基因、与四碳重复单元合成有关的基因及与长链聚合和多糖分泌有关的基因。基因工程是结冷胶分子改造和产量增加最具前景的方法。     

少动鞘氨醇单胞菌引起小儿败血症合并脑膜炎1例

本文报道1例罕见的由少动鞘氨醇单胞菌引起的小儿败血症合并脑膜炎。该患儿突发高热起病,伴有皮肤散在瘀点。根据实验室微生物分离和鉴定,确诊为少动鞘氨醇单胞菌引起的败血症;同时根据脑脊液生化常规结果,诊断为合并细菌性脑膜炎。少动鞘氨醇单胞菌引起的小儿败血症合并脑膜炎属罕见病例,且该患儿有类似流行性脑脊髓膜炎的症状,易被误诊为春季小儿流行性脑脊髓膜炎,需引起临床及实验室工作人员的重视。     

鞘磷脂代谢与脑缺血

鞘磷脂特别是鞘脂是髓鞘的主要成分,高度集中在中枢神经系统。在生理和病理生理条件下,具有生物活性的鞘磷脂及其代谢产物以及信号传导过程的重要性正在逐步被人们所认识。鞘脂代谢产物鞘氨醇及其前体物质神经酰胺与细胞生长停滞和凋亡有关,而1-磷酸鞘氨醇与增强细胞增殖、分化和细胞生存以及调节细胞的生理和病理过程有关,具有细胞外第一信使和细胞内第二信使的双重功能。这三者之间的相互转换、鞘脂代谢物的相对水平以及细胞的命运,受到鞘氨醇激酶的活性的强烈影响。鞘氨醇激酶可催化磷酸鞘氨醇产生1-磷酸鞘氨醇。1-磷酸鞘氨醇在中枢神经系统中与G蛋白偶联受体家族结合对中枢神经系统发挥作用。本文对鞘磷脂代谢过程中的鞘氨醇激酶、1-磷酸鞘氨醇及其受体与脑缺血之间的关系进行概述。     

狗小肠鞘氨醇糖脂中长链碱组成的研究

 利用气相色谱、气相色谱-质谱联用等方法测定了狗小肠鞘氨醇糖脂中的长链碱组成。其主要的长链碱为鞘氨醇(Sphingosine)、异鞘氨醇(isosphingosine)、二氢鞘氨醇(Sphinganine)和植物鞘氨酸(Phyto-sphingosine)。一共分离出十三个鞘氨醇糖脂。在唯一的五糖基神经酰胺中异鞘氨醇是主要成份。在一个一糖基神经酰胺中植物鞘氨醇是主要成份。植植物鞘氨酸也是两个二糖基神经酰胺和一个三糖基神酰胺的主要长链碱。说明它不仅存于植物体内。     

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...     

鞘脂与细胞凋亡

随着生物技术的不断发展,近年来对鞘脂类物质的研究不断深入。鞘脂质除了在细胞骨架的迁移、血管发生、胚胎发育和信号转导等方面起重要作用外,最近的研究发现鞘脂及其代谢物(神经酰胺、鞘氨醇、鞘氨醇-1-磷酸)能诱导多种肿瘤和恶性增殖细胞(如腺癌、结肠癌、肝肿瘤、肺癌、鼻咽癌等)的凋亡。本文着重对鞘脂与细胞凋亡相关的最新研究进展进行综述。     

四乙酰基植物鞘氨醇生物合成的研究进展

四乙酰基植物鞘氨醇(tetraacetyl phytosphingosine, TAPS)是一种性能卓越的天然护肤品原料,经去乙酰化后生成的植物鞘氨醇可作为前体合成保湿护肤品神经酰胺,因此广泛应用于护肤化妆品行业。非常规酵母威克汉姆西弗酵母(Wickerhamomyces ciferrii)是已知的唯一可天然分泌四乙酰基植物鞘氨醇的微生物,目前已成为四乙酰基植物鞘氨醇工业生产的宿主。本文介绍了四乙酰基植物鞘氨醇的发现、功能及其生物合成途径,综述了近年来利用单倍体筛选、诱变育种和代谢工程改造威克汉姆西弗酵母高产四乙酰基植物鞘氨醇的研究进展,并展望了实现四乙酰基植物鞘氨醇工业生产的未来发展方向。     

水环境中微囊藻毒素的生物降解

微囊藻毒素在水环境中的生物降解是决定其环境归趋和影响其毒性的重要因素。本文综述了水细菌、鱼类、水生植物、水生无脊椎动物、浮游动物等水生生物对微囊藻毒素生物降解方面的研究进展。目前报道的微囊藻毒素降解菌有鞘氨醇单胞菌、铜绿假单胞菌和青枯菌。鞘氨醇单胞菌和铜绿假单胞菌分别以微囊藻毒素酶和碱性蛋白酶降解毒素,青枯菌降解机理未明;而鱼类、水生植物、水生无脊椎动物、浮游动物等水生生物主要通过谷胱甘肽S-转移酶催化形成低毒性的微囊藻毒素-谷胱甘肽结合物进行转化。本文还对水环境微囊藻毒素的生物修复方式进行了初步的探讨。     

鞘氨醇杆菌的研究进展

鞘氨醇杆菌是一类革兰氏阴性非发酵杆状细菌,很少引起人类感染,它的主要特点是含有大量的细胞膜鞘磷脂。由于其广泛的生态分布与石油降解能力,已引起了环境微生物学者的重视。本综述总结分析了鞘氨醇杆菌的分类学地位及其主要成员的进化亲缘关系,重点阐述了它们的生理生化特征方面的研究进展,最后总结了8个鞘氨醇杆菌的基因组特征,以期为深入研究鞘氨醇杆菌的功能及其泛基因组提供理论指导,并进一步对鞘氨醇杆菌的深入研究进行了展望。     

Occurrence, production, and applications of gellan: current state and perspectives

Bacterial exopolysaccharides (EPS) are products of biotechnology that are of high interest due to their rheological properties. This is the case of sphingans, a group of structurally related EPS secreted by members of the genus Sphingomonas. Among these, gellan is a multifunctional gelling agent produced in high yields by the non-pathogenic strain Sphingomonas elodea ATCC 31461. In its native form, gellan is a linear anionic EPS based on a tetrasaccharide repeat unit composed of two molecules of D: -glucose, one of L: -rhamnose and one of D: -glucuronic acid. The native gellan is partially esterified with acyl substituents (1 mol of glycerate and 0.5 mol of acetate) per repeat unit. Gellan has unique characteristics and has many applications, particularly in the food, pharmaceutical, and biomedical fields. This review summarizes current knowledge on the structure and properties of gellan and provides details about the biosynthesis of this exopolysaccharide. In addition, a highlight of the importance of gellan in industrial and medicinal applications is given.     

Linkage of genes essential for synthesis of a polysaccharide capsule in Sphingomonas strain S88.

Several structurally related capsular polysaccharides that are secreted by members of the genus Sphingomonas are being developed as aqueous rheological control agents for diverse industrial and food applications. They include gellan (S-60), welan (S-130), rhamsan (S-194), S-657, S-88, S-198, S-7, and NW-11. We refer to these polysaccharides as sphingans, after the genus name. This paper characterizes the first gene cluster isolated from a Sphingomonas species (S88) that is required for capsule synthesis. Overlapping DNA segments which spanned about 50 kbp of S88 DNA restored the synthesis of sphingan S-88 in capsule-negative mutants. The mutations were mapped into functional complementation groups, and the contiguous nucleotide sequence for the 29-kbp cluster was determined. The genetic complementation map and the DNA sequences were interpreted as an extended multicistronic locus containing genes essential for the assembly and secretion of polysaccharide S-88. Many of the deduced amino acid sequences were similar to gene products from other polysaccharide-secreting bacteria such as Rhizobium meliloti (succinoglycan), Xanthomonas campestris (xanthan gum), and Salmonella enterica (O antigen). The S88 locus contained a four-gene operon for the biosynthesis of dTDP-L-rhamnose, an essential precursor for the sphingans. Unexpectedly, there were also two genes for secretion of a lytic or toxin-like protein nested within the polysaccharide cluster. The conservation and linkage of genes that code for a defensive capsule and genes for secretion of an offensive lysin or toxin suggest a heretofore unknown pathogenic life history for Sphingomonas strain S88.     

Sphinganase, a new endoglycanase that cleaves specific members of the gellan family of polysaccharides.

A sporeforming gram-positive aerobic bacterium was isolated from soil and shown to secrete an endoglycanase that cleaves the tetrasaccharide backbone structure of specific members within the gellan family of related bacterial exopolysaccharides. We refer to these polysaccharides as sphingans. The structures of the sphingans differ by the type and position of side groups that are attached to the backbone. The new enzyme named sphinganase degrades welan, gellan, deacylated gellan, and polysaccharides S-88, S-7, and S-198. However, the enzyme does not attack rhamsan or polysaccharide NW11. Methods for growing the bacteria, isolating the enzyme, and assaying sphinganase activity are presented, and uses for the enzyme are proposed.     

Genome sequence of Sphingomonas elodea ATCC 31461, a highly productive industrial strain of gellan gum

The commercial gelling agent gellan gum is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. However, the genes involved in the biosynthesis, regulation, and modification of gellan gum have not been fully characterized. Here we describe the draft genome sequence of stain ATCC 31461 and major findings from its annotation.     

Mechanism of bacitracin resistance in gram-negative bacteria that synthesize exopolysaccharides.

Four representative species from three genera of gram-negative bacteria that secrete exopolysaccharides acquired resistance to the antibiotic bacitracin by stopping synthesis of the exopolysaccharide. Xanthomonas campestris, Sphingomonas strains S-88 and NW11, and Escherichia coli K-12 secrete xanthan gum, sphingans S-88 and NW11, and colanic acid, respectively. The gumD gene in X. campestris is required to attach glucose-P to C55-isoprenyl phosphate, the first step in the assembly of xanthan. A recombinant plasmid carrying the gumD gene of X. campestris restored polysaccharide synthesis to bacitracin-resistant exopolysaccharide-negative mutants of X. campestris and Sphingomonas strains. Similarly, a newly cloned gene (spsB) from strain S-88 restored xanthan synthesis to the same X. campestris mutants. However, the intergeneric complementation did not extend to mutants of E. coli that were both resistant to bacitracin and nonproducers of colanic acid. The genetic results also suggest mechanisms for assembling the sphingans which have commercial potential as gelling and viscosifying agents.     

Gellan gum

For decades microbial exopolysaccharides have been invaluable ingredients in the food industry, as well as having many attractive pharmaceutical and chemical applications. Gellan gum is a comparatively new gum elaborated by the Gram-negative bacterium Sphingomonas paucimobilis. Although its physico-chemical properties have been well characterized, the ecology and physiology of Sphingomonas, and the factors influencing the fermentation process for production of this gum have received much less attention. This review focuses on the metabolism and the enzymic activity of this bacterium, as well as the factors that influence gellan production, including process temperature, pH, stirring rate, oxygen transfer, and composition of the production medium. Potential strategies for improving the production process are discussed in the context of processes for the production of other microbial biopolymers, particularly exopolysaccharides. In addition, the importance and potential utility of gellan lyases in modification of gellan and in other applications is critically evaluated.     

Production and rheological characteristics of the microbial polysaccharide gellan

B. MANNA, A. GAMBHIR AND P. GHOSH. 1996. Sphingomonas paucimobilis was used for the synthesis of the microbial polysaccharide gellan. In a 60 h fermentation, polysaccharide yield and productivity obtained were 0.45 g gellan per g glucose consumed and 0.21 g I⁻¹ h⁻¹ respectively. The broth showed pseudoplastic behaviour with yield stress. The requirement of the solvent propanol to precipitate gellan from the broth depended on the volume of the broth rather than on gellan concentration. The addition of salt to the broth reduced the propanol requirement. Attempts to separate cells from the highly viscous broth by microfiltration were not successful. Ultrafiltration reduced the propanol requirement but appreciable membrane fouling and loss of gellan was observed.     

The gellan gum biosynthetic genes gelC and gelE encode two separate polypeptides homologous to the activator and the kinase domains of tyrosine autokinases

The high-molecular-weight exopolysaccharide gellan is an important commercial gelling agent produced in high yield by the Gram-negative bacterium Sphingomonas elodea ATCC 31461. The cluster of genes required for gellan biosynthesis contains the genes gelC and gelE. These encode for two polypeptides homologous to the activator domain and the kinase domain, respectively, of bacterial autophosphorylating tyrosine kinases involved in polysaccharide chain length determination. The GelC/GelE pair is an exception to the biochemically characterized Gram-negative tyrosine autokinases since it consists of two polypeptides instead of a single one. The deletion of gelC or gelE resulted in the abolishment of gellan in the culture medium confirming their role in gellan biosynthesis. In addition, ATP-binding assays confirmed the predicted ATP-binding ability of GelE. Interestingly, GelE contains an unusual Walker A sequence (ASTGVGCS), where the invariant lysine is replaced by a cysteine. This residue was replaced by alanine or lysine and although both mutant proteins were able to restore gellan production by complementation of the gelE deletion mutant to the production level observed with native GelE, only the mutated GelE where the cysteine was replaced by alanine was demonstrated to bind ATP in vitro. The importance of specific tyrosine residues present in the C-terminal domain of GelE in gellan assembly was also determined. The tyrosine residue at position 198 appears to be essential for the synthesis of high-molecular-weight gellan, although other tyrosine residues may additionally contribute to GelE biological function.     

The complex of Sphingomonas elodea ATCC 31461 glucose-1-phosphate uridylyltransferase with glucose-1-phosphate reveals a novel quaternary structure, unique among nucleoside diphosphate-sugar pyrophosphorylase members

Gellan gum is a widely used commercial material, available in many different forms. Its economic importance has led to studies into the biosynthesis of exopolysaccharide gellan gum, which is industrially prepared in high yields using Sphingomonas elodea ATCC 31461. Glucose-1-phosphate uridylyltransferase mediates the reversible conversion of glucose-1-phosphate and UTP into UDP-glucose and pyrophosphate, which is a key step in the biosynthetic pathway of gellan gums. Here we present the X-ray crystal structure of the glucose-1-phosphate uridylyltransferase from S. elodea. The S. elodea enzyme shares strong monomeric similarity with glucose-1-phosphate thymidylyltransferase, several structures of which are known, although the quaternary structures of the active enzymes are rather different. A detailed comparison between S. elodea glucose-1-phosphate uridylyltransferase and available thymidylyltransferases is described and shows remarkable structural similarities, despite the low sequence identities between the two divergent groups of proteins.     

Proteins encoded by Sphingomonas elodea ATCC 31461 rmlA and ugpG genes, involved in gellan gum biosynthesis, exhibit both dTDP- and UDP-glucose pyrophosphorylase activities

The commercial gelling agent gellan is a heteropolysaccharide produced by Sphingomonas elodea ATCC 31461. In this work, we carried out the biochemical characterization of the enzyme encoded by the first gene (rmlA) of the rml 4-gene cluster present in the 18-gene cluster required for gellan biosynthesis (gel cluster). Based on sequence homology, the putative rml operon is presumably involved in the biosynthesis of dTDP-rhamnose, the sugar necessary for the incorporation of rhamnose in the gellan repeating unit. Heterologous RmlA was purified as a fused His6-RmlA protein from extracts prepared from Escherichia coli IPTG (isopropyl-beta-D-thiogalactopyranoside)-induced cells, and the protein was proven to exhibit dTDP-glucose pyrophosphorylase (Km of 12.0 microM for dTDP-glucose) and UDP-glucose pyrophosphorylase (Km of 229.0 microM for UDP-glucose) activities in vitro. The N-terminal region of RmlA exhibits the motif G-X-G-T-R-X2-P-X-T, which is highly conserved among bacterial XDP-sugar pyrophosphorylases. The motif E-E-K-P, with the conserved lysine residue (K163) predicted to be essential for glucose-1-phosphate binding, was observed. The S. elodea ATCC 31461 UgpG protein, encoded by the ugpG gene which maps outside the gel cluster, was previously identified as the UDP-glucose pyrophosphorylase involved in the formation of UDP-glucose, also required for gellan synthesis. In this study, we demonstrate that UgpG also exhibits dTDP-glucose pyrophosphorylase activity in vitro and compare the kinetic parameters of the two proteins for both substrates. DNA sequencing of ugpG gene-adjacent regions and sequence similarity studies suggest that this gene maps with others involved in the formation of sugar nucleotides presumably required for the biosynthesis of another cell polysaccharide(s).