Identification of a fourth gene involved in dTDP-rhamnose synthesis in Streptococcus mutans.

We had isolated three genes (rmlA, rmlB, and rmlC) involved in dTDP-rhamnose synthesis in Streptococcus mutans and found that three genes were insufficient for dTDP-rhamnose synthesis (Y. Tsukioka, Y. Yamashita, T. Oho, Y. Nakano, and T. Koga, J. Bacteriol. 179:1126-1134, 1997). The rmlD gene of S. mutans, encoding the enzyme which catalyzes the last step of dTDP-rhamnose synthesis, has been cloned and sequenced. The cell extract of Escherichia coli expressing the rmlD gene of S. mutans exhibited enzymatic activity corresponding to its counterpart in Shigella flexneri, a gram-negative bacterium. Rhamnose was not detected in the cell wall preparation purified from the mutant in which the cloned gene was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with autoclaved extracts from the mutant. The rmlD gene product of S. mutans compensated for the incompleteness of dTDP-rhamnose synthesis by the three previously isolated genes. These results indicate that the rmlD gene product is indispensable for the dTDP-rhamnose pathway and subsequently for the synthesis of serotype-specific antigen in S. mutans. Furthermore, conservation of the rmlD gene in Streptococcus species was demonstrated by Southern blot analysis.     

Disruption of dTDP-rhamnose biosynthesis modifies lipopolysaccharide core, exopolysaccharide production, and root colonization in Azospirillum brasilense

The interaction between Azospirillum brasilense and plants is not fully understood, although several bacterial surface components like exopolysaccharides (EPS), flagella, and capsular polysaccharides are required for attachment and colonization. While in other plant-bacteria associations (Rhizobium-legume, Pseudomonas-potato), lipopolysaccharides (LPS) play a key role in the establishment of an effective association, their role in the root colonization by Azospirillum had not been determined. In this study, we isolated a Tn5 mutant of A. brasilense Cd (EJ1) with an apparently modified LPS core structure, non-mucoid colony morphology, increased EPS production, and affected in maize root colonization. A 3790-bp region revealed the presence of three complete open reading frames designated rmlC, rmlB and rmlD. The beginning of a fourth open reading frame was found and designated rmlA. These genes are organized in a cluster which shows homology to the cluster involved in the synthesis of dTDP-rhamnose in other bacteria. Additionally, the analysis of the monosaccharide composition of LPSs showed a diminution of rhamnose compared to the wild-type strain.     

大肠杆菌O141 O-抗原基因簇的破译及进化分析

对大肠杆菌O141 O-抗原基因簇进行测序,序列全长15601bp,用生物信息学的方法进行序列分析,共发现12个基因：鼠李糖合成酶基因(rmlB,rmlD,rmlA,rmlC)、甘露糖合成酶基因(manB,manC),糖基转移酶基因(orf6,orf7,orf9,orf10)、O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy)。用PCR的方法筛选出了针对大肠杆菌O141的特异基因,可以用于基因芯片或PCR方法对大肠杆菌O141的快速检测。通过对大肠杆菌O141的O-抗原基因簇及甘露糖和鼠李糖合成酶基因的进化分析发现：大肠杆菌O141 O-抗原基因簇是低GC含量的片段,仅O-抗原特异的基因才出现在O-抗原基因簇;并且这些基因可能介导了O-抗原基因簇间的重组及以O141 O-抗原基因簇的形成。     

Biosynthesis of UDP-4-keto-6-deoxyglucose and UDP-rhamnose in pathogenic fungi Magnaporthe grisea and Botryotinia fuckeliana

There is increasing evidence that in several fungi, rhamnose-containing glycans are involved in processes that affect host-pathogen interactions, including adhesion, recognition, virulence, and biofilm formation. Nevertheless, little is known about the pathways for the synthesis of these glycans. We show that rhamnose is present in glycans isolated from the rice pathogen Magnaporthe grisea and from the plant pathogen Botryotinia fuckeliana. We also provide evidence that these fungi produce UDP-rhamnose. This is in contrast to bacteria where dTDP-rhamnose is the activated form of this sugar. In bacteria, formation of dTDP-rhamnose requires three enzymes. Here, we demonstrate that in fungi only two genes are required for UDP-Rha synthesis. The first gene encodes a UDP-glucose-4,6-dehydratase that converts UDP-glucose to UDP-4-keto-6-deoxyglucose. The product was shown by time-resolved (1)H NMR spectroscopy to exist in solution predominantly as a hydrated form along with minor amounts of a keto form. The second gene encodes a bifunctional UDP-4-keto-6-deoxyglucose-3,5-epimerase/-4-reductase that converts UDP-4-keto-6-deoxyglucose to UDP-rhamnose. Sugar composition analysis and gene expression studies at different stages of growth indicate that the synthesis of rhamnose-containing glycans is under tissue-specific regulation. Together, our results provide new insight into the formation of rhamnose-containing glycans during the fungal life cycle. The role of these glycans in the interactions between fungal pathogens and their hosts is discussed. Knowledge of the metabolic pathways involved in the formation of rhamnose-containing glycans may facilitate the development of drugs to combat fungal diseases in humans, as to the best of our knowledge mammals do not make these types of glycans.     

Identification and functional characterization of the Lactococcus lactis rfb operon, required for dTDP-rhamnose Biosynthesis

dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis. We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli, the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis. Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.     

Arabinose content of extracellular polysaccharide plays a role in cell aggregation of Azospirillum brasilense

Extracellular polysaccharides play an important role in aggregation and surface colonization of plant-associated bacteria. In this work, we report the time course production and monomer composition of the exopolysaccharide (EPS) produced by wild type strain and several mutants of the plant growth promoting rhizobacterium (PGPR) Azospirillum brasilense. In a fructose synthetic medium, wild type strain Sp7 produced a glucose-rich EPS during exponential phase growth and an arabinose-rich EPS during stationary and death phase growth. D-glucose or L-arabinose did not support cell growth as sole carbon sources. However, glucose and arabinose-rich EPSs, when used as carbon source, supported bacterial growth. Cell aggregation of Sp7 correlated with the synthesis of arabinose-rich EPS. exoB (UDP-glucose 4'-epimerase), exoC (phosphomannomutase) and phbC (poly-beta-hydroxyburyrate synthase) mutant strains, under tested conditions, produced arabinose-rich EPS and exhibited highly cell aggregation capability. A mutant defective in LPS production (dTDP 4-rhamnose reductase; rmlD) produced glucose-rich EPS and did not aggregate. These results support that arabinose content of EPS plays an important role in cell aggregation. Cell aggregation appears to be a time course phenomenon that takes place during reduced metabolic cell activity. Thus, aggregation could constitute a protected model of growth that allows survival in a hostile environment. The occurrence of exoC and rmlD was detected in several species of Azospirillum.     

A gene (wbbL) from Serratia marcescens N28b (O4) complements the rfb-50 mutation of Escherichia coli K-12 derivatives.

A cosmid-based genomic library of Serratia marcescens N28b was introduced into Escherichia coli DH5alpha, and clones were screened for serum resistance. One clone was found resistant to serum, to bacteriocin 28b, and to bacteriophages TuIa and TuIb. This clone also showed O antigen in its lipopolysaccharide. Subcloning and sequencing experiments showed that a 2,124-bp DNA fragment containing the rmlD and wbbL genes was responsible for the observed phenotypes. On the basis of amino acid similarity, we suggest that the 288-residue RmlD protein is a dTDP-L-rhamnose synthase. Plasmid pJT102, containing only the wbbL gene, was able to induce O16-antigen production and serum resistance in E. coli DH5alpha. These results suggest that the 282-residue WbbL protein is a rhamnosyltransferase able to complement the rJb-50 mutation in E. coli K-12 derivatives, despite the low level of amino acid identity between WbbL and the E. coli rhamnosyltransferase (24.80%). S. marcescens N28b rmlD and wbbL mutants were constructed by mobilization of suicide plasmids containing a portion of rmlD or wbbL. These insertion mutants were unable to produce O antigen; since strain N28b produces O4 antigen, these results suggest that both genes are involved in O4-antigen biosynthesis.     

Genetic and Biochemical Characterizations of Enzymes Involved in Streptococcus pneumoniae Serotype 2 Capsule Synthesis Demonstrate that Cps2T (WchF) Catalyzes the Committed Step by Addition of β1-4 Rhamnose,the Second Sugar Residue in the Repeat Unit

Five genes (cps2E, cps2T, cps2F, cps2G, and cps2I) are predicted to encode the glycosyltransferases responsible for synthesis of the Streptococcus pneumoniae serotype 2 capsule repeat unit, which is polymerized to yield a branched surface structure containing glucose-glucuronic acid linked to a glucose-rhamnose-rhamnose-rhamnose backbone. Cps2E is the initiating glycosyltransferase, but experimental evidence supporting the functions of the remaining glycosyltransferases is lacking. To biochemically characterize the glycosyltransferases, the donor substrate dTDP-rhamnose was first synthesized using recombinant S. pneumoniae enzymes Cps2L, Cps2M, Cps2N, and Cps2O. In in vitro assays with each of the glycosyltransferases, only reaction mixtures containing recombinant Cps2T, dTDP-rhamnose, and the Cps2E product (undecaprenyl pyrophosphate glucose) generated a new product, which was consistent with lipid-linked glucose-rhamnose. cps2T, cps2F, and cps2I deletion mutants produced no detectable capsule, but trace amounts of capsule were detectable in Δcps2G mutants, suggesting that Cps2G adds a nonbackbone sugar. All Δcps2F, Δcps2G, and Δcps2I mutants contained different secondary suppressor mutations in cps2E, indicating that the initial mutations were lethal in the absence of reduced repeat unit synthesis. Δcps2T mutants did not contain secondary mutations affecting capsule synthesis. The requirement for secondary mutations in mutants lacking Cps2F, Cps2G, and Cps2I indicates that these activities occur downstream of the committed step in capsule synthesis and reveal that Cps2T catalyzes this step. Therefore, Cps2T is the β1-4 rhamnosyltransferase that adds the second sugar to the repeat unit and, as the committed step in type 2 repeat unit synthesis, is predicted to be an important point of capsule regulation.     

Mutational analysis of exopolysaccharide biosynthesis by Lactobacillus sakei 0-1

Lactobacillus sakei strain 0-1 produces an exopolysaccharide (EPS) consisting of glucose and rhamnose in a ratio of 3:2. As part of a biochemical and molecular analysis of the EPS biosynthetic pathway in L. sakei strain 0-1, we have isolated a random set of EPS-negative mutants. Following treatment of cells with the mutagen ethylmethane sulfonic acid, a total of 10 mutants were identified that lacked the clear ropy appearance of wild-type colonies on agar plates. Their characterization revealed that eight mutants had completely lost the ability to synthesize the normal EPS. Six of these mutants lacked activities of enzymes involved in the biosynthesis of dTDP-rhamnose, required for EPS production. Only mutant strains 12 and 20 were directly affected in EPS synthesis. Strain 12 synthesized EPS with a different sugar composition, however. Interestingly, strain 12 showed temperature-dependent EPS synthesis, with the highest amounts synthesized at 12°C, and low amounts at the optimal temperature for growth (30°C). Two mutants were in fact EPS-positive, producing the normal EPS, but displayed a different cell morphology (elongated cells), indicating a modification in cell wall synthesis.     

A bifunctional 3,5-epimerase/4-keto reductase for nucleotide-rhamnose synthesis in Arabidopsis

l-Rhamnose is a component of plant cell wall pectic polysaccharides, diverse secondary metabolites, and some glycoproteins. The biosynthesis of the activated nucleotide-sugar form(s) of rhamnose utilized by the various rhamnosyltransferases is still elusive, and no plant enzymes involved in their synthesis have been purified. In contrast, two genes (rmlC and rmlD) have been identified in bacteria and shown to encode a 3,5-epimerase and a 4-keto reductase that together convert dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose. We have identified an Arabidopsis cDNA that contains domains that share similarity to both reductase and epimerase. The Arabidopsis gene encodes a protein with a predicated molecular mass of approximately 33.5 kD that is transcribed in all tissue examined. The Arabidopsis protein expressed in, and purified from, Escherichia coli converts dTDP-4-keto-6-deoxy-Glc to dTDP-beta-l-rhamnose in the presence of NADPH. These results suggest that a single plant enzyme has both the 3,5-epimerase and 4-keto reductase activities. The enzyme has maximum activity between pH 5.5 and 7.5 at 30 degrees C. The apparent K(m) for NADPH is 90 microm and 16.9 microm for dTDP-4-keto-6-deoxy-Glc. The Arabidopsis enzyme can also form UDP-beta-l-rhamnose. To our knowledge, this is the first example of a bifunctional plant enzyme involved in sugar nucleotide synthesis where a single polypeptide exhibits the same activities as two separate prokaryotic enzymes.     

Metabolic engineering of the heterologous production of clorobiocin derivatives and elloramycin in Streptomyces coelicolor M512

The aminocoumarin antibiotic clorobiocin is a potent inhibitor of bacterial gyrase. Two new analogs of clorobiocin could be obtained by deletion of a methyltransferase gene, involved in deoxysugar biosynthesis, from the biosynthetic gene cluster of clorobiocin, followed by expression of the modified cluster in the heterologous host Streptomyces coelicolor M512. However, only low amounts of the desired glycosides were formed, and aminocoumarins accumulated predominantly in form of aglyca. In the present study, we clarified the limiting steps for aminocoumarin glycoside formation, and devised strategies to improve glycosylation efficiency. Heterologous expression of a partial elloramycin biosynthetic gene cluster indicated that the rate of dTDP-l-rhamnose synthesis, rather than the rate of glycosyl transfer, was limiting for glycoside formation in this strain. Introduction of plasmid pRHAM which contains four genes from the oleandomycin biosynthetic gene cluster, directing the synthesis of dTDP-rhamnose, led to a 26-fold increase of the production of glycosylated aminocoumarins. Expression of the 4-ketoreductase gene oleU alone resulted in an 8-fold increase. Structural investigation of the resulting deoxysugars confirmed that both the endogeneous and the heterologous pathway involve a 3,5-epimerization of the deoxysugar, a hypothesis which had recently been questioned.     

Characterization of the dTDP-rhamnose biosynthetic genes encoded in the rfb iocus of Shigella flexneri

The nucleotide sequence of the proximal half of the rfb region of Shigella flexneri has been determined, and the genes encoding enzymes involved in the biosynthesis of dTDP-rhamnose have been identified. These genes show strong homology to the rfb genes encoding dTDP-rhamnose biosynthesis in Salmonella enterica serovar typhimurium (strain LT2) and S. enterica serovar anatum (strain M32) (Jiang et al., 1991; Wang et al., 1992). An open reading frame upstream of rfbB was also identified which encoded a protein having strong similarity with GalU, and has been designated galF. GalF has 92% amino acid sequence identity with an S. enterica LT2 gene, orf2X8, which is similarly situated upstream of rfbB (Jiang et al., 1991). The T7 expression system was utilized to identify proteins corresponding to those predicted from DNA sequence analysis. The similarity of the predicted proteins with proteins that are functionally identical or related, and with others of unknown function from the Yersinia enterocolitica O3 rfb region, and in the Escherichia coli K-12 rff region are also described. We have re-addressed the assignment of each gene of the dTDP-rhamnose pathway with the known enzymes of the pathway, in particular rfbC and rfbD. A reporter plasmid to detect genes encoding enzymes of the dTDP-rhamnose pathway is described. An analysis of the intergenic region between galF and rfbB has been made, and comparison with the same region from S. enterica LT2 discussed.     

Nodule development induced by Mesorhizobium loti mutant strains affected in polysaccharide synthesis

The role of Mesorhizobium loti surface polysaccharides on the nodulation process is not yet fully understood. In this article, we describe the nodulation phenotype of mutants affected in the synthesis of lipopolysaccharide (LPS) and beta(1,2) cyclic glucan. M. loti lpsbeta2 mutant produces LPS with reduced amount of O-antigen, whereas M. loti lpsbeta1 mutant produces LPS totally devoid of O-antigen. Both genes are clustered in the chromosome. Based on amino acid sequence homology, LPS sugar composition, and enzymatic activity, we concluded that lpsbeta2 codes for an enzyme involved in the transformation of dTDP-glucose into dTDP-rhamnose, the sugar donor of rhamnose for the synthesis of O-antigen. On the other hand, lpsbeta1 codes for a glucosyltransferase involved in the biosynthesis of the O-antigen. Although LPS mutants elicited normal nodules, both show reduced competitiveness compared with the wild type. M. loti beta(1-2) cyclic glucan synthase (cgs) mutant induces white, empty, ineffective pseudonodules in Lotus tenuis. Cgs mutant induces normal root hair curling but is unable to induce the formation of infection threads. M. loti cgs mutant was more sensitive to deoxycholate and displayed motility impairment compared with the wild-type strain. This pleiotropic effect depends on calcium concentration and temperature.     

Biological function of the dTDP-rhamnose synthesis pathway in Streptococcus mutans.

We have cloned a new gene locus that comprises three genes concerned with the biosynthesis of the serotype c-specific polysaccharide antigen in Streptococcus mutans. The genes encode proteins exhibiting significant homology to the rfbA, rfbB, and rfbD gene products that are involved in the anabolism of dTDP-L-rhamnose from D-glucose-1-phosphate. This anabolism pathway pertains to biosynthesis of the O antigen of lipopolysaccharide in gram-negative bacteria. The cell extract of Escherichia coli expressing each of the cloned genes of S. mutans exhibited enzymatic activity corresponding to the homologous counterpart of the rfb gene products. Rhamnose was not detected in the cell wall preparation purified from the mutant in which each of the three cloned genes was insertionally inactivated. Rabbit antiserum against S. mutans serotype c-specific antigen did not react with the autoclaved extracts from these mutants. These results indicate that the gene products identified in the present study are involved in the dTDP-L-rhamnose synthesis pathway and that the pathway relates to the biosynthesis of the serotype-specific polysaccharide antigen of S. mutans. Southern hybridization analysis revealed that genes homologous to the cloned genes involved in the dTDP-L-rhamnose synthesis pathway were widely distributed in a variety of streptococci. This is the first report of the biological function of the dTDP-rhamnose pathway in streptococci.     

大肠杆菌O150 O-抗原基因簇的破译和dTDP-鼠李糖合成酶基因的鉴定

利用鸟枪法对大肠杆菌O150 O-抗原基因簇进行测序,序列全长13551bp,用生物信息学的方法进行序列分析,共发现11个基因,分别为鼠李糖合成酶基因(rmlB、rmlD、rmlA、rmlC)糖基转移酶基因(3个)、O-抗原转运酶基因(wzx)和O-抗原聚合酶基因(wzy),另外还有两个基因功能未知。用PCR的方法筛选出了针对大肠杆菌O150的特异基因,可以用于基因芯片或PCR方法对大肠杆菌O150的快速检测。另外,通过进化分析发现大肠杆菌O150的O-抗原基因簇中携带有典型的大肠杆菌鼠李糖合成酶基因,并且这些基因参与了O-抗原基因簇间的重组以形成新的基因簇的过程。     

Genetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster: identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene.

The O-repeating unit of the Escherichia coli O7-specific lipopolysaccharide is made of galactose, mannose, rhamnose, 4-acetamido-4,6-dideoxyglucose, and N-acetyglucosamine. We have recently characterized the genes involved in the biosynthesis of the sugar precursor GDP-mannose occurring in the E. coli O7:K1 strain VW187 (C. L. Marolda and M. A. Valvano, J. Bacteriol. 175:148-158, 1993). In the present study, we identified and sequenced the rfbBDAC genes encoding the enzymes for the biosynthesis of another precursor, dTDP-rhamnose. These genes are localized on the upstream end of the rfbEcO7 region, and they are strongly conserved compared with similar genes found in various enteric and nonenteric bacteria. Upstream of rfbB we identified a DNA segment containing the rfb promoter and a highly conserved untranslated leader sequence also present in the promoter regions of other surface polysaccharide gene clusters. Also, we have determined that rfbB and rfbA have homologs, rffG (o355) and rffH (o292), respectively, located on the rff cluster, which is involved in the synthesis of enterobacterial common antigen. We provide biochemical evidence that rffG and rffH encode dTDP-glucose dehydratase and glucose-1-phosphate thymidylyltransferase activities, respectively, and we also show that rffG complemented the rfbB defect in the O7+ cosmid pJHCV32. We also demonstrate that rffG is distinct from rffE and map the rffE gene to the second gene of the rff cluster.     

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).     

The identification of cryptic rhamnose biosynthesis genes in Neisseria gonorrhoeae and their relationship to lipopolysaccharide biosynthesis.

Neisseria gonorrhoeae synthesizes a rough lipopolysaccharide that does not contain any of the repetitive units characteristic of the smooth lipopolysaccharide of members of the family Enterobacteriaceae. Three gonococcal homologs of Salmonella serovar typhimurium genes involved in the synthesis of the rhamnose component of the repetitive subunits have been isolated. Gonococcal homologs for rfbB, rfbA, and rfbD were found downstream of the galE gene in a region of the chromosome which shows overall homology with the meningococcal capsule gene complex region D. Sequence alignment demonstrated that the gonococcal gene products have 69, 65, and 54% amino acid identity with the Salmonella proteins RfbB, RfbA, and RfbD. The gonococcal RfbB and RfbA amino acid sequences share even more identical residues (73 and 65%, respectively) with the amino acid sequences derived from Escherichia coli genes o355 and o292, respectively. These genes are clustered with the genes involved in the biosynthesis of enterobacterial common antigen, and o355 is listed in the GenBank and Swiss Protein data banks as rffE (encoding UDP-GlcNAc-2-epimerase). However, complementation studies demonstrated that o355 does not encode the enzyme UDP-GlcNAc-2-epimerase. Gonococcal strains constructed with null mutations in the rfbBAD genes were unchanged in lipopolysaccharide phenotype and in the synthesis of gonococcal carbohydrate-containing C antigen. We were unable to detect any changes in gonococcal phenotype with respect to lipopolysaccharide sialylation, monoclonal-antibody binding, serum sensitivity, or interaction with eukaryotic cells in vitro. We conclude that the absence of a homolog for rfbC precludes the existence of a functional dTDP-rhamnose biosynthesis pathway in the gonococcal strains examined and that these genes are only maintained in N. gonorrhoeae either because of the presence of the galE gene or because of another as yet unrecognized function.     

大肠杆菌O138 O-抗原基因簇的破译及Gne的生物信息学鉴定

利用鸟枪法对大肠杆菌E .coliO138O 抗原基因簇进行测序 ,序列全长 14 139bp ,用生物信息学的方法进行序列分析 ,共发现 11个基因 ,分别为鼠李糖合成酶基因 (rmlB ,rmlD ,rmlA ,rmlC)、UDP GalNAcA合成酶基因 (gne ,gna)、糖基转移酶基因 (3个 )、O 抗原转运酶基因 (wzx)和O 抗原聚合酶基因 (wzy)。发现一种稀有单糖UDP Gal NAcA的合成途径 ,对合成该糖的第一种酶Gne进行了生物信息学鉴定 ,另外用PCR方法筛选出了针对大肠杆菌O138的特异基因