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.     

X-Ray fibre diffraction studies of members of the gellan family of polysaccharides

X-Ray fibre diffraction studies are reported for gellan gum and the family of related polysaccharides S-130, S-198, S-88 and S-194. Whereas the linear gellan molecules yield highly crystalline patterns, the branched polysaccharides yield well-aligned but poorly crystalline patterns. These patterns are consistent with the proposed 3-fold double helical structure of gellan with small changes in pitch dependent upon type and position of branches.     

合成生物聚合物的重要微生物资源-鞘氨醇单胞菌

摘要：鞘氨醇单胞菌属的许多菌株能够合成结冷胶、沃仑胶、迪特胶等多种结构相似,物理性能多样的生物聚合物,统称为鞘氨醇胶。目前,结冷胶已经大规模的生产和应用,由于鞘氨醇单胞菌属的提出仅有十几年的历史,其他种类鞘氨醇胶的研究和开发才刚刚起步。本文综述了鞘氨醇单胞菌属分类研究的最新进展,以及鞘氨醇胶的结构、特性、生物合成途径、分子遗传学和基因工程的研究现状,并对今后的研究重点和方向进行了展望。     

X-ray and computer modeling studies on gellan-related polymers: molecular structures of welan, S-657, and rhamsan

The primary structures of the four bacterial polysaccharides gellan, welan, S-657, and rhamsan are the same with respect to their backbones, but have different side-chains. This difference has a profound influence on their behavior in aqueous media. Solutions of gellan gum form stable aqueous gels under appropriate ionic conditions. By contrast, welan, S-657, and rhamsan do not gel but give very viscous solutions over a wide range of thermal, pH, and salt conditions. X-Ray fiber diffraction analysis and computer modeling of these branched polysaccharides demonstrate that they all have the same half-staggered, double-helical conformations as in the unbranched gellan, suggesting, therefore, that the side chains are responsible for diminishing gelling behavior. Depending on the size and location, the side chains shield the carboxylate groups to varying degrees; this shielding is substantial in welan and S-657, but less in rhamsan. In all cases, side-chain-main-chain interactions within the double helix prevent the carboxylate-mediated aggregation of double helices that is necessary for the gelation.     

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.     

Identification and organization of genes for diutan polysaccharide synthesis from <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. ATCC 53159

A cluster of genes for diutan polysaccharide synthesis was isolated from a library of Sphingomonas sp. ATCC 53159 genomic DNA by complementation of glucosyl-isoprenylphosphate transferase-deficient mutants of Sphingomonas elodea ATCC 31461 (producing gellan) and Xanthomonas campestris (producing xanthan). The synthesis of polysaccharide in these strains shares a common first step, transfer of glucose-1-phosphate from UDP-glucose to the isoprenylphosphate lipid. The cluster of 24 genes was compared to genes for biosynthesis of gellan, and S-88 sphingan from Sphingomonas sp. ATCC 31554. Diutan, gellan and S-88 sphingan have a common four-sugar backbone but different side chains, one rhamnose for S-88 sphingan, a two-rhamnose side chain for diutan and no side chain for gellan. The genes for biosynthesis of diutan, gellan and S-88 sphingan were similar in general organization but differed in location of some genes, in particular, dpsG (putative polymerase), dpsR (putative lyase) and dpsS (putative repeat unit transporter). An unidentified reading frame urf31, present in the gene clusters for diutan and S-88 sphingan but not gellan, had similarity to glycosyl transferase group 2 proteins, and was detrimental when cloned in Sphingomonas elodea producing gellan that lacks a side chain, but not in Sphingomonas ATCC 31554 producing S-88 sphingan with a rhamnose side chain. Gene urf31 could possibly encode a side-chain rhamnosyl transferase. Another gene urf31.4 was unique to the diutan gene cluster. A plasmid containing 20 of the 24 genes resulted in a slight increase in the amount of diutan produced, but a significant increase in the rheological properties of diutan.     

A comparison of genes involved in sphingan biosynthesis brought up to date

Microbial polysaccharides have a wide range of functional properties and show high relevance in industrial applications. The possibility to create tailor-made polysaccharides by genetic engineering will further enhance the product portfolio and may open new fields of application. Here, we have examined in detail the recently sequenced genome of the welan-producing strain Sphingomonas sp. ATCC 31555 to identify the complete welan cluster and further genes involved in EPS production. The corresponding genes were compared on the nucleotide and amino acid sequence level to the EPS clusters of the described gellan-producing Sphingomonas elodea ATCC 31461, diutan-producing Sphingomonas sp. ATCC 53159, and the S-88-producing Sphingomonas sp. ATCC 31554 strains. We also compared the previously mentioned strains to each other and included the genes upstream of the main cluster in gellan and welan cluster. The cluster organization of Sphingomonas strain S-7 was also compared based on previous hybridization experiments, without nucleotide sequences. We have found that the occurrence of genes in all biosynthesis clusters is connected to the structures of the various produced sphingans. Along these lines, homologous genes responsible for the assembly of the identical repeating unit generally show high sequence identity, whereas genes for putative side chain attachment urf31, urf31.4, and urf34 vary more in distinct areas. Moreover, gene clusters for biosynthesis of diutan, welan, gellan, and S-88 as well as S-7 are similar in general organization but differ in location and arrangement of some genes. Finally, we summarized genetic and mutational engineering approaches toward modified sphingan variants as described in literature.     

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.     

Comparative analysis of the behavior of gellan gum (S-60) and welan gum (S-130) in dilute aqueous solution

Optical rotation, circular dichroism, and microcalorimetric data clearly and consistently show that gellan gum, S-60 (Me₄N⁺ form), undergoes in water at 25° a rather sharp conformational transition upon increasing the concentration of added Me₄NCl. Similar data show that S-60 behaves anomalously upon addition of Ca²⁺ ions with, eventually, formation of aggregates and/or gels. The Me₄NCl-induced conformational change of S-60 is thermally reversible with no hysteresis. In contrast, with welan gum, S-130 (Me₄N⁺ form), no evidence could be found for a dependence of chain conformation of the main external variables considered. Comparison of the circular-dichroism spectra of the two polysaccharides suggests that S-130 in water might be present in a stiff conformation similar to that assumed by S-60 in aqueous Me₄NCl.     

Analysis of structure and function of gellans with different substitution patterns

Chemical mutagenesis or exposure to antibiotic stress of Sphingomonas paucimobilis ATCC 31461 and R40 have been used to isolate mutants producing modified gellan gum polysaccharides. N.m.r. and conventional carbohydrate analysis methods have been used to characterise these polysaccharides. The ¹H and ¹³C n.m.r. spectra of gellan gum have been fully assigned and the anomeric regions have been shown to be very sensitive to the type and location of non-carbohydrate substituents. Analysis of the gellan gum mutants suggests that they differ in the nature of acetate and glycerate substitution. Such gellan-related polysaccharides have been used to test the selective effect of acyl substituents on the gelation of gellan gum.     

Evidence for microbial polysaccharide preparations containing polyester substituents

CPMAS 13C-n.m.r. spectroscopy was employed to characterize the composition and solid phase morphology of gellan, welan, rhamsan and NW11. Spectra indicated that commercial preparations of these polysaccharides, which share a similar molecular backbone, contain a non-carbohydrate component exhibiting four inequivalent carbon atoms. Isolation of this component, followed by 13C-n.m.r. in CHCl3 and MS analysis, revealed its structure to be poly(beta-hydroxybutyrate). Evidence is presented which suggests that this polyester may be a covalent adduct to the above polysaccharides, although this cannot be unambiguously determined at this time. Further experimentation is in progress.     

Exopolysaccharide of the gellan family: prospects and potential

The use of microbial polysaccharides in the food, pharmaceutical and chemical industries has increased steadily during the past decade. The biopolymer gellan is a more recent addition to the family of microbial polysaccharides that is gaining much importance due to its novel property of forming thermo-reversible gels when heated and cooled. It is produced and marketed by some companies of Europe, USA, etc under trade names such as Gelrite, Phytagel and Kelcogel. It has applications in diverse fields in the food, pharmaceutical and many other industries. Further research and development in biopolymer technology is expected to expand its use. This article presents a critical review of the available published information on the gellan exopolysaccharide synthesized by Pseudomonas species. In particular information on its structure, physico-chemical properties and the rheology of its solutions etc. is critically assessed. Emphasis has also been paid to characterization of gellan. A brief historical background of the polymer and the biochemical and physiological characteristics of several different existing bacterial isolates which secrete gellan and related polysaccharides are discussed. An attempt has also been made to review the potential and future prospects, highlighting some novel techniques adopted to overcome the mass transfer problems associated with the fermentative production of gellan gum. The efficient downstream processes used for obtaining purified gellan are also highlighted. Attention has also been drawn to the problem associated with the fermentation processes due to the highly viscous nature of gellan gum and effect of different impeller systems on gellan fermentation kinetics and rheological properties.     

Microbial polysaccharides with actual potential industrial applications

The microbial polysaccharides reviewed include xanthan gum, scleroglucan, PS-10, PS-21 and PS-53 gums, polysaccharides from Alcaligenes sp., PS-7 gum, gellan gum, curdlan, bacterial alginate, dextran, pullulan, Baker's Yeast Glycan, 6-deoxy-hexose-containing polysaccharides and bacterial cellulose. Factors limiting the commercial potential of certain microbial polysaccharides such as availability, rheological properties, and polyvalency are outlined. The polysaccharides are classified according to their uses as viscosity-increasing agents and as gelling agents. A third category includes polysaccharides with specific applications such as tailor-made dextran and pullulan and polysaccharides used as substrates for the preparation of rare sugars. The difficulties encountered in development of a polysaccharide at the industrial level are pointed out.     

Agarose and gellan as morphology-directing agents for the preparation of selenium nanowires in water

Commercially available polysaccharides, agarose and gellan, were used as morphology-directing agents for the synthesis of t-Se nanowires in water at room temperature in the presence of ascorbic acid as reducing agent. The nanostructures were characterized using XRD, SEM, and TEM. The diameter of the nanowires varied from 100 to 208 nm for nanowires obtained in the presence of agarose and from 51 to 145 nm for nanowires from gellan, as evidenced by SEM and TEM. Agarose and gellan have then a potential as environmentally acceptable morphology-directing agents to generate Se nanostructures in water.     

Deproteinization of gellan gum produced by Sphingomonas paucimobilis ATCC 31461

Deproteinization is a technical bottleneck in the purification of viscous water-soluble polysaccharides. The aim of this work is to provide an appropriate approach to deproteinize crude gellan gum. Several methods of deproteinization were investigated, including Sevag method, alkaline protease, papain and neutral protease. The results revealed that Sevag method had high deproteinization efficiency (87.9%), but it showed dissatisfactory recovery efficiency of gellan gum (28.6%), which made it less advisable in industrial applications. The deproteinization by alkaline protease was demonstrated in this work for the first time, indicating alkaline protease was preferred in the deproteinization of crude gellan gum with high polysaccharide recovery (89.3%) and high deproteinization efficiency (86.4%).     

Organization of genes required for gellan polysaccharide biosynthesis in<Emphasis Type="Italic"> Sphingomonas elodea</Emphasis> ATCC 31461

Sphingomonas elodea ATCC 31461 produces gellan, a capsular polysaccharide that is useful as a gelling agent for food and microbiological media. Complementation of nonmucoid S. elodea mutants with a gene library resulted in identification of genes essential for gellan biosynthesis. A cluster of 18 genes spanning 21 kb was isolated. These 18 genes are homologous to genes for synthesis of sphingan polysaccharide S-88 from Sphingomonas sp. ATCC 31554, with predicted amino acid identities varying from 61% to 98%. Both polysaccharides have the same tetrasaccharide repeat unit, comprised of [4)--l-rhamnose-(13)--d-glucose-(14)--d-glucuronic acid-(14)--d-glucose-(1]. Polysaccharide S-88, however, has mannose or rhamnose in the fourth position and has a rhamnosyl side chain, while gellan has no sugar side chain but is modified by glyceryl and acetyl substituents. Genes for synthesis of the precursor dTDP-l-rhamnose were highly conserved. The least conserved genes in this cluster encode putative glycosyl transferases III and IV and a gene of unknown function, gelF. Three genes (gelI, gelM, and gelN) affected the amount and rheology of gellan produced. Four additional genes present in the S-88 sphingan biosynthetic gene cluster did not have homologs in the gene cluster for gellan biosynthesis. Three of these gene homologs, gelR, gelS, and gelG, were found in an operon unlinked to the main gellan biosynthetic gene cluster. In a third region, a gene possibly involved in positive regulation of gellan biosynthesis was identified.     

Posttranslational processing of polysaccharide lyase: maturation route for gellan lyase in Bacillus sp. GL1

Cells of Bacillus sp. GL1 extracellularly secrete a gellan lyase with a molecular mass of 130 kDa responsible for the depolymerization of a heteropolysaccharide (gellan), although the gene is capable of encoding a huge protein with a molecular mass of 263 kDa. A maturation route for gellan lyase in the bacterium was determined using anti-gellan lyase antibodies. The fluid of the bacterial exponentially growing cultures on gellan contained two proteins with molecular masses of 260 and 130 kDa, both of which reacted with the antibodies. The 260 kDa protein was purified from the cultured fluid and characterized. The protein exhibited gellan lyase activity and showed similar enzyme properties, such as optimal pH and temperature, thermal stability, and substrate specificity, to those of the 130 kDa gellan lyase. The N-terminal amino acid sequences of the 260 and 130 kDa enzymes were found to be identical. Determination of the C-terminal amino acid of the 130 kDa enzyme indicated that the 260 kDa enzyme is cleaved between the 1205Gly and 1206Leu residues to yield the mature form (130 kDa) of the gellan lyase. Therefore, the mature enzyme consists of 1170 amino acids (36Ala-1205Gly) with a molecular weight of 125,345, which is in good agreement with that calculated from SDS-PAGE analysis. Judging from these results, gellan lyase is first synthesized as a preproform (263 kDa) and then secreted as a precursor (260 kDa) into the medium through cleavage of the signal peptide. Finally, the precursor is post-translationally processed into the N-terminal half domain of 130 kDa as the mature form, the function of C-terminal half domain being unclear.     

Increasing the yield and viscosity of exopolysaccharides secreted by Sphingomonas by augmentation of chromosomal genes with multiple copies of cloned biosynthetic genes

Certain bacteria of the Sphingomonas genus secrete structurally related capsular polysaccharides. Due to their unique properties, three (gellan, welan and rhamsan) are produced commercially by submerged fermentation and are used as modifiers of aqueous rheology and as gelling agents. However, conversion of glucose into these polysaccharides is relatively inefficient. To identify general methods for increasing the productivity of Sphingomonas, we augmented the normal chromosomal copy of the phosphoglucomutase gene (pgm) and the cluster of genes (sps) required for assembly of the carbohydrate repeat unit for strain S7 with multiple copies of plasmids carrying these genes. Although a sixfold increase in Pgm activity only lead to a small percentage increase in conversion of glucose to the S-7 polysaccharide, multiple sps genes caused a nearly 20% increase in the yield from glucose and an even larger increase in culture viscosity. The increased viscosity was accompanied by a change in the sugar composition of the secreted polymer. Journal of Industrial Microbiology & Biotechnology (2000) 25, 49–57. Received 18 February 2000/ Accepted in revised form 29 April 2000     

Characterization of alpha-L-rhamnosidase of Bacillus sp. GL1 responsible for the complete depolymerization of gellan.

A bacterium, Bacillus sp. GL1, depolymerizes a heteropolysaccharide (gellan) to a tetrasaccharide (unsaturated glucuronyl-glucosyl-rhamnosyl-glucose) by extracellular gellan lyase. The resultant tetrasaccharide was degraded to the constituent monosaccharides by subsequent reactions of unsaturated glucuronyl hydrolase, beta-d-glucosidase, and alpha-l-rhamnosidase. alpha-l-Rhamnosidase was substantially induced in the bacterial cells when grown in a medium containing gellan as a carbon source. The purified enzyme from the cells was a monomer with a molecular mass of about 100 kDa and was most active at pH 7.0 and 50 degrees C. The enzyme acted on the gellan-degrading product (rhamnosyl-glucose) formed after successive reactions catalyzed by gellan lyase, unsaturated-glucuronyl hydrolase and beta-d-glucosidase, and released rhamnose from the disaccharide. Therefore, the alpha-l-rhamnosidase is found to be responsible as the final enzyme for the complete depolymerization of gellan.     

Structural studies of the capsular polysaccharide from Streptococcus pneumoniae types 15B and 15C

The structures of the capsular polysaccharides (S-15B and S-15C) from Streptococcus pneumoniae types 15B and 15C have been investigated by using n.m.r. spectroscopy, methylation analysis, and various specific degradations. It is concluded that the polysaccharides are composed of pentasaccharide repeating-units having the following structure: (Formula: see text). In this structure, R is H (80%) or CH2CH2N+Me3 (20%). S-15B further contains O-acetyl groups, approximately 0.7 per repeating unit, which have not been located. The capsular polysaccharides S-15F and S-15A, which have been studied previously, are also composed of pentasaccharide repeating-units, containing the same sequence of sugars, but in a linear arrangement.