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.     

Clustering of mutations blocking synthesis of xanthan gum by Xanthomonas campestris.

Mutations that block the synthesis of xanthan gum by Xanthomonas campestris B1459S-4L-II were isolated as nonmucoid colonies after treatment with ethyl methanesulfonate. Complete libraries of DNA fragments from wild-type X. campestris were cloned into Escherichia coli by using a broad-host-range cosmid vector and then transferred into each mutant strain by conjugal mating. Cloned fragments that restored xanthan gum synthesis (Xgs+; mucoidy) were compared according to restriction pattern, DNA sequence homology, and complementation of a subset of Xgs- mutations. Groups of clones that contained overlapping homologous DNA were found to complement specific Xgs- mutations. The results suggest clustering of the genetic loci involved in xanthan synthesis. The clustering occurred within three unlinked regions. Two forms of complementation were observed. In most instances, independently isolated cosmid clones that complemented a single mutation were found to be partially homologous. Less frequent was the second form of complementation, in which two cosmid clones that lacked any homologous sequences restored the mucoid phenotype to a single mutant. Finally, xanthan production was measured for wild-type X. campestris carrying multiple plasmid copies of the cloned xanthan genes.     

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.     

Genetic and physical analyses of a cluster of genes essential for xanthan gum biosynthesis in Xanthomonas campestris.

Xanthomonas campestris produces copious amounts of a complex exopolysaccharide, xanthan gum. Nonmucoid mutants, defective in synthesis of xanthan polysaccharide, were isolated after nitrosoguanidine mutagenesis. To isolate genes essential for xanthan polysaccharide synthesis (xps), a genomic library of X. campestris DNA, partially digested with SalI and ligated into the broad-host-range cloning vector pRK293, was constructed in Escherichia coli. The pooled clone bank was conjugated en masse from E. coli into three nonmucoid mutants by using pRK2013, which provides plasmid transfer functions. Kanamycin-resistant exconjugants were then screened for the ability to form mucoid colonies. Analysis of plasmids from several mucoid exconjugants indicated that overlapping segments of DNA had been cloned. These plasmids were tested for complementation of eight additional nonmucoid mutants. A 22-kilobase (kb) region of DNA was defined physically by restriction enzyme analysis and genetically by ability to restore mucoid phenotype to 10 of the 11 nonmucoid mutants tested. This region was further defined by subcloning and by transposon mutagenesis with mini-Mu(Tetr), with subsequent analysis of genetic complementation of nonmucoid mutants. A region of 13.5 kb of DNA was determined to contain at least five complementation groups. The effect of plasmids containing cloned xps genes on xanthan gum synthesis was evaluated. One plasmid, pCHC3, containing a 12.4-kb insert and at least four linked xanthan biosynthetic genes, increased the production of xanthan gum by 10% and increased the extent of pyruvylation of the xanthan side chains by about 45%. This indicates that a gene affecting pyruvylation of xanthan gum is linked to this cluster of xps genes.     

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.     

野油菜黄单胞菌NK-01编码生物合成多糖基因的克隆

采用甲基磺酸乙酯诱变野油菜黄单胞菌ＮＫ－０１得到多糖合成缺陷的不产粘突变株。经ＳａｌⅠ部分酶切得到的野生型ＮＫ－０１染色体ＤＮＡ片段,连接到广泛寄主载体ｐＲＫ２９３上,在Ｅ．ｃｏｌｉ中建立完整的ＮＫ－０１基因库。通过使一株不产多糖突变株在协助质粒ｐＲＫ２０１３存在下,分别与含基因库的Ｅ．ｃｏｌｉ菌落群体进行三亲结合转移筛选具有卡那霉素抗性的产粘接合后体,对接合后体进行的重组质粒的酶切分析表明,     

The Blight-Associated Epitope and DNA Fragment from Xanthomonas campestris pv. campestris are not Required for Blight

Abstract: It has been reported that all tested naturally occurring strains of Xanthomonas campestris pv. campestris that are known to be capable of inducing blight symptoms in cabbage react with MAb A11 and hybridize with a 5.4-kb DNA fragment (in plasmid pJC41), cloned from the Xanthomonas campestris species type strain, Xcc528^T, whereas all tested naturally occurring strains that do not cause blight react with MAb X21 and do not hybridize to pJC41. The roles of the 5.4-kb DNA in pJC41 and the epitope recognized by MAb A11 in the pathogenicity of X. c. campestris strains that cause blight were examined by mutational analyses. A 4.0-kb deletion of the pJC41 region on the Xcc528^T chromosome was created by marker exchange, but the derivatives were evidently not affected in their ability to elicit blight symptoms. Nitrosoguanidine was used to mutagenize two blight strains, Xcc528^T and CAM19, and mutants were selected that were not reactive to MAb A11. The MAb A11-negative mutant of Xcc528^T was reactive to MAb X21, but was evidently not affected in the ability to elicit blight symptoms. MAb A11-negative mutants of CAM19, however, were not reactive to MAb X21, and showed reduction or loss of virulence, which suggested the requirement for at least one of the two antigens (to MAbs A11 or X21) for pathogenicity. A genomic library of CAM19 was made and screened for genes responsible for the production of the A11 antigen. Cosmid clones were identified that restored MAb A11 reactivity to the mutants. None of these cosmids restored the virulence of the mutant strains that had lost virulence. Therefore, neither the blight-associated 5.4-kb DNA fragment nor the MAb A11 antibody marker were required for blight symptom elicitation or pathogenicity.     

A Xanthomonas campestris pv. campestris protein similar to catabolite activation factor is involved in regulation of phytopathogenicity.

A DNA fragment from Xanthomonas campestris pv. campestris that partially restored the carbohydrate fermentation pattern of a cya crp Escherichia coli strain was cloned and expressed in E. coli. The nucleotide sequence of this fragment revealed the presence of a 700-base-pair open reading frame that coded for a protein highly similar to the catabolite activation factor (CAP) of E. coli (accordingly named CLP for CAP-like protein). An X. campestris pv. campestris clp mutant was constructed by reverse genetics. This strain was not affected in the utilization of various carbon sources but had strongly reduced pathogenicity. Production of xanthan gum, pigment, and extracellular enzymes was either increased or decreased, suggesting that CLP plays a role in the regulation of phytopathogenicity.     

Functional characterization of GumK, a membrane-associated beta-glucuronosyltransferase from Xanthomonas campestris required for xanthan polysaccharide synthesis

Xanthomonas campestris is a Gram-negative bacterium that produces an exopolysaccharide known as xanthan gum. Xanthan is involved in a variety of biological functions, including pathogenesis, and is widely used in the industry as thickener and viscosifier. Although the genetics and biosynthetic process of xanthan are well documented, the enzymatic components have not been examined and no data on glycosyltransferases have been reported. We describe the functional characterization of the gumK gene product, an essential protein for xanthan synthesis. Immunoblots and complementation studies showed that GumK is a 44-kDa protein associated to the membrane fraction. This value corresponds to the expected molecular mass for GumK encoded by an extended open reading frame than proposed from previous genetic data and in X. campestris published complete genome. The protein was expressed in Escherichia coli cells. The purified protein catalyzed the transfer of a glucuronic acid residue from UDP-glucuronic acid to mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl with formation of a glucuronic acid-beta-mannose linkage. We examined the acceptor substrate specificity. GumK was unable to use the trisaccharide acceptor freed from the pyrophosphate lipid moiety. Replacement of the natural lipid moiety by phytanyl showed that the catalytic function could proceed with glucuronic acid transfer. These results suggest the enzyme does not show specificity for the lipidic portion of the acceptor. GumK showed diminished activity when tested with 6-O-acetyl-mannose-alpha-1,3-glucose-beta-1,4-glucose-P-P-polyisoprenyl, a putative intermediate in the synthesis of xanthan. This could indicate that acetylation of the internal mannose takes place after the formation of the GumK product.     

Increase of xanthan production by cloning xps genes into wild-type Xanthomonas campestris

Previously, genomic banks of Xanthomonas campestris were constructed in Escherichia coli, using mobilizable broad-host-range cosmids as the vectors. Following conjugal transfer, genes involved in the biosynthesis of xanthan polysaccharide (XPS) were cloned by the ability to restore the mucoid phenotype to the non-mucoid mutants. In this study, all clones were transferred into the wild-type strain Xc17 to evaluate the effects of the cloned genes on XPS production. Most clones showed no significant effect; however, two plasmids, pP2401 and pP2201, caused 10 and 15% yield increases, respectively, compared with that of controls. While it was not clear how pP2201 caused the yield increase, the effect of pP2401 seemed to result from elevated phosphomannose isomerase activity. Since XPS synthesis in X. campestris is a very efficient process, only relatively small increases are to be expected; an enhancement of productivity by 10-15% is important to the commercial production of xanthan.     

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。     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey.

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Construction of lactose-utilizing Xanthomonas campestris and production of xanthan gum from whey

Xanthomonas campestris pv. campestris possesses a low level of beta-galactosidase and therefore is not able to grow and produce significant amounts of xanthan gum in a medium containing lactose as the sole carbon source. In this study, a beta-galactosidase expression plasmid was constructed by ligating an X. campestris phage phi LO promoter with pKM005, a ColE1 replicon containing Escherichia coli lacZY genes and the lpp ribosome-binding site. It was then inserted into an IncP1 broad-host-range plasmid, pLT, and subsequently transferred by conjugation to X. campestris 17, where it was stably maintained. The lacZ gene under the control of the phage promoter was expressed at a high level, enabling the cells to grow in a medium containing lactose. Production of xanthan gum in lactose or diluted whey by the engineered strain was evaluated, and it was found to produce as much xanthan gum in these substrates as the cells did in a medium containing glucose.     

Isolation and characterization of a xanthan-degrading Microbacterium sp. strain XT11 from garden soil

AIMS: Isolation and characterization of the xanthan-degrading Microbacterium sp. XT11. METHODS AND RESULTS: The bacterial isolate XT11, capable of fragmenting xanthan, has been isolated from soil sample. Morphological and biochemical analyses, as well as 16S rRNA gene sequence comparisons, demonstrated that strain XT11 should be grouped in the genus Microbacterium, and represented a new member in this family. Xanthan could be degraded by the xanthan-degrading enzyme released from strain XT11. It has been shown that xantho-oligosaccharides fragmented from xanthan had both elicitor activity and antibacterial effect against Xanthomonas campestris pv. campestris. CONCLUSIONS: The xanthan-degrading enzyme produced by the newly isolated XT11 could fragment xanthan to form oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: Xanthan-degrading products would be useful for potential application in the control of black rot of cruciferous plants caused by X. campestris pv. campestris and, as an oligosaccharide elicitor, in making these plants resistant to disease.     

Location and cloning of the ketal pyruvate transferase gene of Xanthomonas campestris.

Genes required for xanthan polysaccharide synthesis (xps) are clustered in a DNA region of 13.5 kb in the chromosome of Xanthomonas campestris. Plasmid pCHC3 containing a 12.4-kb insert of xps genes has been suggested to include a gene involved in the pyruvylation of xanthan gum (N.E. Harding, J.M. Cleary, D.K. Caba?as, I. G. Rosen, and K. S. Kang, J. Bacteriol. 169:2854-2861, 1987). An essential step toward understanding the biosynthesis of xanthan gum and to enable genetic manipulation of xanthan structure is the determination of the biochemical function encoded by the xps genes. On the basis of biochemical characterization of an X. campestris mutant which produces pyruvate-free xanthan gum, complementation studies, and heterologous expression, we have identified the gene coding for the ketal pyruvate transferase (kpt) enzyme. This gene was located on a 1.4-kb BamHI fragment of pCHC3 and cloned in the broad-host-range cloning vector pRK404. An X. campestris kpt mutant was constructed by mini-Mu(Tetr) mutagenesis of the cloned gene and then by recombination of the mutation into the chromosome of the wild-type strain.     

Coexpression of colanic acid and serotype-specific capsular polysaccharides in Escherichia coli strains with group II K antigens.

In Escherichia coli K-12, the rcsA and rcsB gene products are positive regulators in expression of the slime polysaccharide colanic acid. We have previously demonstrated the presence of rcsA sequences in E. coli K1 and K5, strains with group II capsular K antigens, and shown that introduction of multicopy rcsA into these strains results in the expression of colanic acid. We report here the presence of rcsB sequences in E. coli K1 and K5 and demonstrate that RcsB also plays a role in the biosynthesis of colanic acid in strains with group II K antigens. In E. coli K1 and K5 grown at 37 degrees C, multicopy rcsB and the resulting induction of colanic acid synthesis had no significant effect on synthesis of the group II K antigens. K-antigen-specific sugar transferase activities were not significantly different in the presence or absence of multicopy rcsB, and introduction of a cps mutation to eliminate colanic acid biosynthesis in a K1-derivative strain did not influence the activity of the polysialyltransferase enzyme responsible for synthesis of the K1 polymer. Furthermore, immunoelectron microscopy showed no detectable difference in the size or distribution of the group II K-antigen capsular layer in cells which produced colanic acid. Colanic acid expression therefore does not appear to significantly affect synthesis of the group II K-antigen capsule and, unlike for group I K antigens, expression of group II K antigens is not positively regulated by the rcs system.     

Constitutive expression of Pasteurella multocida toxin

Abstract The introduction of a plasmid containing skc (streptokinase-coding gene) fused with ompA signal sequence into Escherichia coli K-12 strains, rendered the bacteria mucoid. Measurement of the synthesis of β-galactosidase from a cps-lacZ fusion ( lacZ fusion to a gene necessary for capsule synthesis) showed that the mucoid phenotype was due to induction of the capsular polysaccharide colanic acid synthesis. The introduction of a plasmid carrying skc fused with malE (gene encoding maltose-binding protein) also induced cps-lacZ expression, but intracellular expression of streptokinase in E. coli did not. The cps expression by secretion of streptokinase was diminished to the basal level in a cps-lacZ strain carrying a rcsC mutation. These results show that the secretion of streptokinase in E. coli induces colanic acid synthesis through the RcsC-dependent pathway.     

The ypdI gene codes for a putative lipoprotein involved in the synthesis of colanic acid in Escherichia coli

In Escherichia coli, the synthesis of colanic acid, an extracellular polysaccharide imminent in biofilm development, is a complicated process involving numerous genes and not yet wholly elucidated. Using a plasmid-borne E. coli K-12 gene library, we have identified a clone whose presence conferred mucoid colony phenotype onto E. coli CM2555 strain. Our results indicate that overexpression of a gene previously catalogued as ypdI, which encodes a putative lipoprotein, is responsible for this phenotype. We show that the mucoidy of ypdI -overexpressing bacteria is due to increased production of colanic acid. This phenotype depends on the function of the rcsA gene, but not on that of rcsF. These results suggest that the ypdI gene product might be an additional factor playing a role in colanic acid synthesis, indicating that this process can be even more complicated than supposed to date. However, no obvious phenotype was observed in the DeltaypdI::kan mutant cultivated under standard laboratory conditions.     

Xanthomonas campestris pv. campestris secretes the endoglucanases ENGXCA and ENGXCB: construction of an endoglucanase-deficient mutant for industrial xanthan production

Xanthomonas campestris pv. campestris secretes at least two cellulose-degrading endoglucanases. One of these endoglucanases is encoded by the engXCA gene of X. c. pv. campestris 8400 that was previously characterized by Gough et al. [Gene (1990) 89: 53-59]. An additional endoglucanase encoded by the engXCB gene was identified in X. c. pv. campestris 8400 and FC2. The engXCB gene product that was grouped into the endoglucanase family E contains a putative N-terminal signal peptide, suggesting a secretion by the type II secretion system. The ENGXCB protein contributed approximately 8% to the cellulase activity in xanthan preparations. Deletion of engXCA and engXCB resulted in a fivefold reduction of the cellulose-degrading activity in xanthan preparations. The cellulase activity determined in xanthan preparations of the engXCA-engXCB mutant was only slightly higher than the activity found in preparations that were subjected to heat treatment. Mutations in engXCA and engXCB did not affect the growth rate and xanthan production of X. c. pv. campestris FC2 under several cultivation conditions. The engXCA-engXCB deletion mutant is markerless, which makes this mutant a valuable strain for xanthan production and approaches aimed at inactivating further genes encoding extracellular enzymes.