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Using a new expression construct, rhamnose isomerase from Escherichia coli was purified and crystallized. The crystal structure was solved by multiple isomorphous replacement and refined to a crystallographic residual of 17.4 % at 1.6 A resolution. Rhamnose isomerase is a tight tetramer of four (beta/alpha)(8)-barrels. A comparison with other known structures reveals that rhamnose isomerase is most similar to xylose isomerase. Alignment of the sequences of the two enzymes based on their structures reveals a hitherto undetected sequence identity of 13 %, suggesting that the two enzymes evolved from a common precursor. The structure and arrangement of the (beta/alpha)(8)-barrels of rhamnose isomerase are very similar to xylose isomerase. Each enzyme does, however, have additional alpha-helical domains, which are involved in tetramer association, and largely differ in structure. The structures of complexes of rhamnose isomerase with the inhibitor l-rhamnitol and the natural substrate l-rhamnose were determined and suggest that an extended loop, which is disordered in the native enzyme, becomes ordered on substrate binding, and may exclude bulk solvent during catalysis. Unlike xylose isomerase, this loop does not extend across a subunit interface but contributes to the active site of its own subunit. It illustrates how an interconversion between inter and intra-subunit complementation can occur during evolution. In the crystal structure (although not necessarily in vivo) rhamnose isomerase appears to bind Zn(2+) at a "structural" site. In the presence of substrate the enzyme also binds Mn(2+) at a nearby "catalytic" site. An array of hydrophobic residues, not present in xylose isomerase, is likely to be responsible for the recognition of l-rhamnose as a substrate. The available structural data suggest that a metal-mediated hydride-shift mechanism, which is generally favored for xylose isomerase, is also feasible for rhamnose isomerase., (Copyright 2000 Academic Press.)

Pseudomonas aeruginosa produces a large variety of virulence factors and is characterized by its capacity to rapidly develop resistance when exposed to antibiotics. In order to evaluate a possible correlation between acquired resistance to antibiotics and virulence, we examined the virulence of four isogenic variants of P. aeruginosa O12 that differ in their resistance phenotypes to various beta-lactam antibiotics in a mouse model of acute pneumonia. Strains overproducing a chromosomal type 1 beta-lactamase were less virulent in both immunocompetent and immunosuppressed animals. Whereas the production of the exopolysaccharide alginate was similar between the four strains, extracellular virulence factors (elastase, rhamnolipid) that are controlled by the cell-to-cell signaling system circuit were detected in reduced amounts in the supernatant of the two isolates overproducing type 1 beta-lactamase. These results suggest that strains overexpressing the chromosomal type 1 beta-lactamase could be less virulent because of a reduction of cell-to-cell signaling dependent virulence factor production.

The Escherichia coli rhaBAD operon encodes the enzymes for catabolism of the sugar L-rhamnose. Full rhaBAD activation requires the AraC family activator RhaS (bound to a site that overlaps the -35 region of the promoter) and the cyclic AMP receptor protein (CRP; bound immediately upstream of RhaS at -92.5). We tested alanine substitutions in activating regions (AR) 1 and 2 of CRP for their effect on rhaBAD activation. Some, but not all, of the substitutions in both AR1 and AR2 resulted in approximately twofold defects in expression from rhaBAD promoter fusions. We also expressed a derivative of the alpha subunit of RNA polymerase deleted for the entire C-terminal domain (alpha-Delta235) and assayed expression from rhaBAD promoter fusions. The greatest defect (54-fold) occurred at a truncated promoter where RhaS was the only activator, while the defect at the full-length promoter (RhaS plus CRP) was smaller (13-fold). Analysis of a plasmid library expressing alanine substitutions at every residue in the carboxyl-terminal domain of the alpha subunit (alpha-CTD) identified 15 residues (mostly in the DNA-binding determinant) that were important at both the full-length and truncated promoters. Only one substitution was defective at the full-length but not the truncated promoter, and this residue was located in the DNA-binding determinant. Six substitutions were defective only at the promoter activated by RhaS alone, and these may define a protein-contacting determinant on alpha-CTD. Overall, our results suggest that CRP interaction with alpha-CTD may not be required for rhaBAD activation; however, alpha-CTD does contribute to full activation, probably through interactions with DNA and possibly RhaS.

Lipopolysaccharides (LPS) were isolated by hot phenol-water extraction from Danish Helicobacter pylori strains D1, D3, and D6, which were nontypeable using a variety of anti-Lewis and anti-blood-group monoclonal antibodies. An atypical O-chain polysaccharide (PS) was liberated from the LPS of the three strains by acid under mild conditions and found to contain D-rhamnose (D-Rha), L-rhamnose (L-Rha), and a branched sugar, 3-C-methyl-D-mannose (D-Man3CMe). The last sugar, which has not hitherto been found in Nature, was identified using GLC-MS of the derived alditol acetate and the partially methylated alditol acetate, and (1)H and (13)C NMR spectroscopy, including NOESY and (1)H,(13)C HMBC experiments. The following structure of the trisaccharide repeating unit of the PS was established: -->2)-alpha-D-Manp3CMe-(1-->3)-alpha-L-Rhap-(1-->3)-alpha-D- Rhap-(1-- >. In contrast to the pathogenic importance of the Lewis antigen mimicry exhibited by the PS of H. pylori strains previously investigated, the biological relevance of the atypical PS for H. pylori pathogenesis is unclear. The production of a differing surface PS may represent a form of antigenic variation by these particular H. pylori strains and/or may reflect the adaptation of these strains to a particular human population.

There is a clinical need for early detection of tumor response to therapy. This study aimed to determine whether metabolites of fluorodeoxyglucose (FDG) detected in solid mouse tumors in situ by I9F magnetic resonance spectroscopy (19F MRS) correlated with response to 5-fluorouracil chemotherapy. After injection of FDG (1.4 mmol/kg i.p.), uptake and metabolism was monitored for 2 h in RIF-1 tumors. FDG was detectable immediately, and after 10 min, a second broad peak was detected 5-6 ppm upfield. 19F MRS analysis of cell and tumor extracts in vitro showed that the upfield peak (> or =15% of the total detectable 19F signal) consisted of the epimer alpha-fluorodeoxymannose (FDM) and various conjugates. Mice treated with 5-fluorouracil (130 mg/kg) received, 48 h later, a repeat dose of FDG. The change in the rate of FDM formation, but not the FDG or total 19F signal, correlated significantly with the response to 5-fluorouracil (P = 0.032), suggesting that 19F MRS of FDM metabolism in vivo may be a novel means of predicting tumor response.

Screening experiments identified several bacteria which were able to use residual oil from biotechnological rhamnose production as a carbon source for growth. Ralstonia eutropha H16 and Pseudomonas oleovorans were able to use this waste material as the sole carbon source for growth and for the accumulation of polyhydroxyalkanoic acids (PHA). R. eutropha and P. oleovorans accumulated PHA amounting to 41.3% and 38.9%, respectively, of the cell dry mass, when these strains were cultivated in mineral salt medium with the oil from the rhamnose production as the sole carbon source. The accumulated PHA isolated from R. eutropha consisted of only 3-hydroxybutyric acid, whereas the PHA isolated from P. oleovorans consisted of 3-hydroxyhexanoic acid, 3-hydroxyoctanoic acid, 3-hydroxydecanoic acid, and 3-hydroxydodecanoic acid. The composition was confirmed by gas chromatography of the isolated polyesters. Batch and fed-batch cultivations in stirred-tank reactors were done.

Noninvasive assessment of intestinal permeability in vivo is based on the measurement of urinary excretion of orally administered sugar probes. It is expressed as a ratio, usually lactulose/rhamnose or 3-O-methyl-D-glucose (3-OMG)/rhamnose. In both endotoxemic and control rats that were receiving fluid, we observed an increase in the recovery of lactulose and 3-OMG but not rhamnose in both groups, suggesting an enhancement of intestinal permeability. In the measurement of intestinal permeability, all pre- and postmucosal factors are considered equal for all sugars. We hypothesized that postmucosal factors and not changes in intestinal permeability caused the increased urinary lactulose and 3-OMG recoveries observed during fluid loading. Therefore, the effects of fluid loading on urinary excretion of the sugar probes were studied in healthy rats receiving the sugars intravenously. After intravenous injection, fluid loading increased urinary lactulose recovery threefold but not that of 3-OMG and rhamnose. In conclusion, fluid loading increases the lactulose/rhamnose ratio independent of changes in intestinal permeability. The 3-OMG/rhamnose ratio is not influenced by fluid loading.

Background: Sugar absorption tests are an effective, noninvasive way to assess intestinal permeability. The role of intestinal barrier integrity in complications and outcome of short-bowel syndrome is not known. The purpose of the study was to evaluate whether such tests provide information on the status of intestinal mucosa of these patients., Methods: Six children with short-bowel syndrome--median age, 12 months, and median small bowel length at birth, 30 cm--had a sugar test with 3-o-methyl-D-glucose, D-xylose, D-rhamnose, and melibiose approximately 2 months after operation. The melibiose/L-rhamnose ratio was used as an index of permeability, and percentages of 3-o-methyl-D-glucose and D-xylose absorbed were used as indices of absorption. Parenteral nutrition requirement, bowel length, liver disease, recent sepsis, and bacterial overgrowth were recorded., Results: Three patients had increased permeability, and all of them had had a recent episode of sepsis and severe liver disease. All subjects had malabsorption of 3-o-methyl-D-glucose, and five of six had malabsorption of D-xylose and L-rhamnose. The absorption of 3-o-methyl-D-glucose correlated with bowel length (r2 = 0.78; P = 0.04), whereas the absorption of D-xylose correlated with parenteral requirement (r2 = 0.66; P = 0.04) at that time., Conclusions: Increased permeability was observed in three of six patients with short-bowel syndrome associated with a recent episode of sepsis and severe liver disease. Other indices of malabsorption correlated significantly with different clinical features of the disease. A prospective larger scale study in a homogeneous population is indicated to assess at multiple points during the disease course whether the test can be helpful in the management of these patients.

Pseudomonas aeruginosa produces exoproducts correlated with its pathogenicity. One of these virulence-associated traits is the surfactant rhamnolipid. The production of alginate and lipopolysaccharide (LPS) are also of importance for P. aeruginosa virulence. The product of the algC gene (which is involved in alginate production through its phosphomannomutase activity and in LPS synthesis through its phosphoglucomutase activity) participates in rhamnolipid production, presumably catalyzing the first step in the deoxy-thymidine-diphospho-L-rhamnose (dTDP-L-rhamnose) pathway, the conversion of glucose-6-phosphate to glucose-1-phosphate. Other structural alg genes, encoded in the alg operon, are not involved in rhamnolipid nor LPS production. These results show that the AlgC protein plays a central role in the production of the three P. aeruginosa virulence-associated saccharides: alginate, LPS and rhamnolipid.

The pH-stat fed-batch culture of Pseudomonas aeruginosa YPJ-80 was done to produce a rhamnolipid biosurfactant. With glucose as the sole carbon source, the final concentrations of cells and rhamnolipid biosurfactant obtained in 25 h were 25 g cell dry weight/l and 4.4 g/l, respectively.

dTDP-rhamnose is made from glucose-1-phosphate and dTTP by four enzymes encoded by rmIA-D. An Escherichia coli rmIC mutant was constructed and a crude enzyme extract prepared from it did not produce dTDP-4-keto-rhamnose, in contrast to a crude enzyme extract prepared from a wild-type E. coli strain where small amounts of this intermediate were found after incubation with dTDP-glucose in the absence of NADPH. These results showed that dTDP-4-keto-rhamnose, the product of RmIC, exists as a free intermediate. Further, the Mycobacterium tuberculosis rmIC gene was expressed and incubation of the resulting purified M. tuberculosis RmIC enzyme with dTDP-4-keto-6-deoxyglucose resulted in the conversion of approximately 7% of dTDP-4-keto-6-deoxyglucose to dTDP-4-keto-rhamnose. The enzyme also allowed for the incorporation of two deuterium atoms from deuterium oxide solvent into dTDP-4-keto-glucose. Thus the rmIC gene encodes dTDP-4-keto-6-deoxyglucose epimerase capable of epimerizing at both C-3' and C-5'; this enzyme produces free dTDP-4-keto-rhamnose but the equilibrium of the 4-keto sugar nucleotides lies strongly on the side of the gluco configuration.

Background: The lactulose/rhamnose (L/R) intestinal permeability test is widely used. However, different quantities and proportions of lactulose and rhamnose are used. The aim of this study was to determine whether a low dosage of lactulose is able to discriminate between normal and increased permeability., Materials and Methods: Two groups of 10 healthy subjects were studied. In group 1, three different iso-osmolar test solutions were administered on 3 days. The solutions consisted of 10 g of L with 1 g of R, 5 g of L with 0.5 g of R and 1 g of L with 0.1 g of R in 65 mL of water. Group 2 ingested these solutions 1 h after ingestion of 750 mg of chenodeoxycholeic acid (CDCA), which is known to increase permeability. The urinary L/R ratio was determined using high-performance liquid chromatography. Data are presented as medians (range)., Results: In group 1, no differences were observed between the three solutions. In Group 2, there was a significant difference (P = 0.045) between the three solutions. The L/R ratios were 0.0079 (0.0024-0.0152) (1L to 0.1R), 0.0138 (0.0066-0.0192) (5L to 0.5R) and 0.0144 (0.0074-0.0374) (10L to 1R). The L/R ratio differed significantly between Groups 1 and 2 (P < 0.001) using the 5L to 0.5R and 10L to 1R solutions respectively., Conclusion: If the permeability is increased, the urinary L/R ratio depends on the quantity of lactulose and rhamnose administered in equal proportion. 5L to 0.5R is sufficient to discriminate between a normal and a moderately increased permeability.

Biosurfactants containing rhamnose and beta-hydroxydecanoic acid and called rhamnolipids are reviewed with respect to microbial producers, their physiological role, biosynthesis and genetics, and especially their microbial overproduction, physicochemical properties and potential applications. With Pseudomonas species, more than 100 g l-1 rhamnolipids were produced from 160 g l-1 soybean oil at a volumetric productivity of 0.4 g l-1 h-1. The individual rhamnolipids are able to lower the surface tension of water from 72 mN m-1 to 25-30 mN m-1 at concentrations of 10-200 mg l-1. After initial testing, rhamnolipids seem to have potential applications in combating marine oil pollution, removing oil from sand and in combating zoosporic phytopathogens. Rhamnolipids are also a source of L-rhamnose, which is already used for the industrial production of high-quality flavor components.

The serotype c antigen of Actinobacillus actinomycetemcomitans consists of 6-deoxy-l-talose. A gene cluster involved in the synthesis of serotype-specific polysaccharide antigen was cloned from the chromosomal DNA of A. actinomycetemcomitans NCTC 9710 (serotype c). This cluster consisted of 17 open reading frames. Escherichia coli produced the polysaccharide that reacts with the serotype c-specific antibody when transformed with a plasmid containing the cluster. Comparing the structure of the gene cluster with a similar cluster from A. actinomycetemcomitans Y4 (serotype b), which produces a polysaccharide consisting of l-rhamnose and d-fucose, revealed that a 5.7 kb region containing seven genes in the cluster from strain Y4 was replaced by a 3.8 kb region containing three genes in strain NCTC 9710. The results suggest that these region, as well as dTDP-6-deoxy-l-talose-forming dTDP-4-keto-l-rhamnose reductase, is essential to the production of extracellular polysaccharide specific to serotype c.

L-fucose and L-rhamnose are two 6-deoxyhexoses naturally occurring in several complex carbohydrates. In prokaryotes both of them are found in polysaccharides of the cell wall, while in animals only L-fucose has been described, which mainly participates to the structure of glycoconjugates, either in the cell membrane or secreted in biological fluids, such as ABH blood groups and Lewis system antigens. L-fucose and L-rhamnose are synthesized by two de novo biosynthetic pathways starting from GDP-D-mannose and dTDP-D-glucose, respectively, which share several common features. The first step for both pathways is a dehydration reaction catalyzed by specific nucleotide-sugar dehydratases. This leads to the formation of unstable 4-keto-6-deoxy intermediates, which undergo a subsequent epimerization reaction responsible for the change from D- to L-conformation, and then a NADPH-dependent reduction of the 4-keto group, with the consequent formation of either GDP-L-fucose or dTDP-L-rhamnose. These compounds are then the substrates of specific glycosyltransferases which are responsible for insertion of either L-fucose or L-rhamnose in the corresponding glycoconjugates. The enzyme involved in the first step of GDP-L-fucose biosynthesis in E. coli, i.e., GDP-D-mannose 4,6 dehydratase, has been recently expressed as recombinant protein and characterized in our laboratory. We have also cloned and fully characterized a human protein, formerly named FX, and an E. coli protein, WcaG, which display both the epimerase and the reductase activities, thus indicating that only two enzymes are required for GDP-L-fucose production. Fucosylated complex glycoconjugates at the cell surface can then be recognized by specific counter-receptors in interacting cells, these mechanisms initiating important processes including inflammation and metastasis. The second pathway starting from dTDP-D-glucose leads to the synthesis of antibiotic glycosides or, alternatively, to the production of dTDP-L-rhamnose. While several sets of data are available on the first enzyme of the pathway, i.e., dTDP-D-glucose dehydratase, the enzymes involved in the following steps still need to be identified and characterized.

A yeast strain isolated from the air in Japan was found to represent a new species, and was named Trichosporon japonicum. This species produced arthroconidia and appressoria. T. japonicum formed a cluster with the appressorium-forming species Trichosporon inkin and Trichosporon ovoides in a phylogenetic tree constructed using small-subunit rDNA sequences. However, they had low relatedness to each other in DNA-DNA hybridization experiments. T. japonicum is distinguished from both T. inkin and T. ovoides by its ability to assimilate inulin, and its inability to assimilate L-rhamnose. JCM 8357T is the type strain of the species.

Two L-rhamnose-binding lectins named STL1 and STL2 were isolated from eggs of steelhead trout (Oncorhynchus mykiss) by affinity chromatography and ion exchange chromatography. The apparent molecular masses of purified STL1 and STL2 were estimated to be 84 and 68 kDa, respectively, by gel filtration chromatography. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and matrix-assisted laser desorption ionization time of flight mass spectrometry of these lectins revealed that STL1 was composed of noncovalently linked trimer of 31.4-kDa subunits, and STL2 was noncovalently linked trimer of 21.5-kDa subunits. The minimum concentrations of STL1, a major component, and STL2, a minor component, needed to agglutinate rabbit erythrocytes were 9 and 0.2 microg/ml, respectively. The most effective saccharide in the hemagglutination inhibition assay for both STL1 and STL2 was L-rhamnose. Saccharides possessing the same configuration of hydroxyl groups at C2 and C4 as that in L-rhamnose, such as L-arabinose and D-galactose, also inhibited. The amino acid sequence of STL2 was determined by analysis of peptides generated by digestion of the S-carboxamidomethylated protein with Achromobacter protease I or Staphylococcus aureus V8 protease. The STL2 subunit of 195 amino acid residues proved to have a unique polypeptide architecture; that is, it was composed of two tandemly repeated homologous domains (STL2-N and STL2-C) with 52% internal homology. These two domains showed a sequence homology to the subunit (105 amino acid residues) of D-galactoside-specific sea urchin (Anthocidaris crassispina) egg lectin (37% for STL2-N and 46% for STL2-C, respectively). The N terminus of the STL1 subunit was blocked with an acetyl group. However, a partial amino acid sequence of the subunit showed a sequence similarity to STL2. Moreover, STL2 also showed a sequence homology to the ligand binding domain of the vitellogenin receptor. We have also employed surface plasmon resonance biosensor methodology to investigate the interactions between STL2 and major egg yolk proteins from steelhead trout, lipovitellin, and beta'-component, which are known as vitellogenin digests. Interestingly, STL2 showed distinct interactions with both egg yolk proteins. The estimated values for the affinity constant (Ka) of STL2 to lipovitellin and beta' component were 3.44 x 10(6) and 4.99 x 10(6), respectively. These results suggest that the fish egg lectins belong to a new family of animal lectin structurally related to the low density lipoprotein receptor super- family.

A rhamnolipid-producing strain of Pseudomonas aeruginosa GL1 was isolated from a bacterial community growing on a mixture of polycyclic aromatic hydrocarbons (PAH) as sole carbon source. Strain GL1 did not grow on PAH but grew on known degradation metabolites of phenanthrene (O-phthalic acid) and of naphthalene (salicylic acid). In co-culture with a phenanthrene-degrading strain, Ps. aeruginosa GL1 accelerated the degradation of phenanthrene. Strain GL1 was resistant to toxic amphiphilic compounds such as cationic and anionic detergents. Rhamnolipid production took place in a late stage growth in cultures of strain GL1 on glycerol or n-hexadecane. It coincided with a substantial decrease in cell hydrophobicity and with morphological changes of the outer membrane as observed by transmission electronic microscopy. The rhamnolipids produced inhibited the growth of bacteria such as Rhodococcus erythropolis, Bacillus cereus and Ps. fluorescens. The overall results suggested an outer membrane origin for the rhamnolipids. They also indicate that the utilization of PAH metabolites by strain GL1 is important for the stability of the PAH-degrading community.

Ristocetin A is used clinically as a tool for the measurement of von Willebrand protein due to its ability to induce the aggregation of platelets in plasma being proportional to the concentration of von Willebrand protein. The enzymatically induced cleavage of alpha-L-rhamnose from the tetrasaccharide of ristocetin A removes this ability to induce platelet aggregation in plasma, providing a possible insight into the structural basis for this effect.

Bacterial translocation from the intestinal lumen has been demonstrated in humans. Three mechanisms have been suggested to explain the phenomenon: altered intestinal barrier function, bacterial overgrowth, and impaired host defense. The aim of this study was to determine whether changes in intestinal barrier function assessed by measurement of intestinal permeability and morphology were associated with alteration in bacterial translocation. Intestinal permeability was assessed in 43 patients by the lactulose/L-rhamnose test with a 5-h urine collection. Mucosal atrophy was assessed from the villus height-to-mucosal thickness ratio in small-bowel biopsies. Bacterial translocation was determined by microbiologic analysis of harvested mesenteric lymph nodes. No significant differences were apparent in the incidence of bacterial translocation in patients with normal permeability (5 [23%] of 22 patients translocated) compared with patients with increased permeability (4 [19%] of 21 patients translocated). Similarly, no correlation was apparent between the incidence of bacterial translocation and the index of villus atrophy. The degree of villus atrophy failed to correlate with gastrointestinal permeability. These data suggest that the incidence of bacterial translocation is not related to increased intestinal permeability or mucosal atrophy.

Escherichia coli genes regulated by environmental inorganic phosphate (Pi) levels form the phosphate (Pho) regulon. This regulation requires seven proteins, whose synthesis is under autogenous control, including response regulator PhoB, its partner, histidine sensor kinase PhoR, all four components of the Pi-specific transport (Pst) system (PstA, PstB, PstC, and PstS), and a protein of unknown function called PhoU. Here we examined the effects of uncoupling PhoB synthesis and PhoR synthesis from their normal controls by placing each under the tight control of the arabinose-regulated P(araB) promoter or the rhamnose-regulated P(rhaB) promoter. To do this, we made allele replacement plasmids that may be generally useful for construction of P(araB) or P(rhaB) fusions and for recombination of them onto the E. coli chromosome at the araCBAD or rhaRSBAD locus, respectively. Using strains carrying such single-copy fusions, we showed that a P(rhaB) fusion is more tightly regulated than a P(araB) fusion in that a P(rhaB)-phoR+ fusion but not a P(araB)-phoR+ fusion shows a null phenotype in the absence of its specific inducer. Yet in the absence of induction, both P(araB)-phoB+ and P(rhaB)-phoB+ fusions exhibit a null phenotype. These data indicate that less PhoR than PhoB is required for transcriptional activation of the Pho regulon, which is consistent with their respective modes of action. We also used these fusions to study PhoU. Previously, we had constructed strains with precise delta phoU mutations. However, we unexpectedly found that such delta phoU mutants have a severe growth defect (P. M. Steed and B. L. Wanner, J. Bacteriol. 175:6797-6809, 1993). They also readily give rise to compensatory mutants with lesions in phoB, phoR, or a pst gene, making their study particularly difficult. Here we found that, by using P(araB)-phoB+, P(rhaB)-phoB+, or P(rhaB)-phoR+ fusions, we were able to overcome the extremely deleterious growth defect of a Pst+ delta phoU mutant. The growth defect is apparently a consequence of high-level Pst synthesis resulting from autogenous control of PhoB and PhoR synthesis in the absence of PhoU.

The stereochemical course of hydrolysis catalysed by four Aspergillus aculeatus enzymes acting on alpha-L-rhamnosyl and alpha-D-galacturonosyl linkages in the hairy regions of pectins has been determined using 1H-NMR. Exogalacturonase acts with inversion of anomeric configuration (e-->a), shown by the initial release of beta-D-GalpA from the non-reducing end of polygalacturonic acid. Similarly, rhamnogalacturonan (RG) hydrolase also acts with inversion of anomeric configuration (e-->a) during hydrolysis of alpha-D-GalpA-(1-->2)-alpha-L-Rhap linkages in RG, initially releasing oligosaccharides with beta-D-GalpA at the reducing end. This result is consistent with the recently solved crystal structure of this enzyme, as well as its classification based on amino acid sequence similarity into glycosyl hydrolase family 28. alpha-L-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration (a-->e), initially releasing beta-L-Rhap from p-nitrophenyl alpha-L-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.

An enzyme with alpha-L-rhamnosidase activity was purified by anion exchange chromatography from an Aspergillus niger commercial preparation. The alpha-L-rhamnosidase was shown to be N-glycosylated, and had a molecular mass of 85 kD on sodium dodecylsulfate-polyacrylamide gel electrophoresis of which approximately 12% was contributed by carbohydrate. The enzyme was optimally active at pH 4.5 and 65 degrees C. When tested towards p-nitrophenyl-alpha-L-rhamnopyranoside it showed Km and Vmax values of 2.9 mM and 20.6 U mg-1, respectively whereas it was inhibited competitively by L-rhamnose (Ki 3.5 mM). Substrate specificity studies showed alpha-L-rhamnosidase to be active both on alpha-1,2 and alpha-1,6 linkages to beta-D-glucose. Moreover, the enzyme was able to release L-rhamnose from geranyl-beta-D-rutinoside and 2-phenylethyl-beta-D-rutinoside.

The sno gene cluster in Streptomyces nogalater ATCC 27451 contains the nogalamycin biosynthesis genes. A set of plasmid constructions carrying fragments of the sno cluster that lie downstream of snoD were used to complement the S. galilaeus mutant H039, which is blocked in rhodosamine and 2-deoxyfucose biosynthesis in the aclacinomycin pathway. Sequence analysis of this cluster revealed three contiguous open reading frames (ORFs) that were designated snoF, snoG, and snoH. Only those plasmid constructs that expressed SnoG were able to complement H039. SnoG shows similarity to GalE, a UDP-glucose-4-epimerase catalyzing the epimerization of UDP-glucose to UDP-galactose. The putative SnoF protein is similar to 3,5-epimerases involved in rhamnose biosynthesis. The deduced product of snoH is a 489-amino acid polypeptide. It is similar to the product of dau ORF3 found in the daunomycin cluster. However its function is still unclear. Based on the complementation experiments and sequence analysis, this part of the sno cluster is suggested to be involved in the biosynthesis of the sugar portion of nogalamycin. Interestingly, SnoA, a transcriptional activator for the sno minimal polyketide synthase, is also needed to express this cluster.

Background: The purpose of this study was to investigate whether the increase intestinal permeability (IP) seen in patients with cystic fibrosis (CF) is correlated with the basic defect, as revealed by the patient's genotype, and/or whether the intestinal disturbance reflects secondary abnormalities such as essential fatty acid deficiency., Methods: Nineteen CF patients were compared with nine age- and sex-matched healthy controls. IP was evaluated by studying urinary excretion for 5 hours after a test meal containing lactulose, L-rhamnose and xylose in water. Urine was analyzed for carbohydrates, and blood samples were taken for determination of the fatty acid pattern of serum phospholipids. The CF patients were grouped according to genotype: homozygous for delta F508, heterozygous for alpha F508, or unidentified., Results: Patients who were homozygous (n = 9) or heterzygous (n = 6) for delta F508 had significantly higher lactulose/L-rhamnose excretion ratios (mean(range) values of 0.08(0.05-0.13) and 0.09(0.03-0.13), respectively) than patients (n = 4) with unidentified genotypes [0.03(0.02-0.05); p = 0.005] or healthy controls [0.02(0.003-0.06); p = 0.002]. CF patients with EFAD (n = 6) did not differ from those with a normal pattern of serum phospholipid fatty acids, the lactulose/L-rhamnose excretion ratio being 0.08(0.02-0.13) and 0.07(0.03-0.12), respectively., Conclusions: These data show that the IP in CF was related to patient genotype; those homozygozous or heterozygous for delta F508 having a significantly increased IP compared with patients with unidentified genotypes, who had IP values within the normal range.

Pseudomonas aeruginosa produces rhamnolipids which are tenso-active compounds with potential industrial and environmental applications. There are two main types of rhamnolipids produced in liquid cultures, rhamnosyl-beta-hydroxydecanoyl-beta-hydroxydecanoate (mono-rhamnolipid) and rhamnosyl-rhamnosyl-beta-hydroxydecanoyl-beta-hydroxyd ecanoate (di-rhamnolipid). In this work we report the selective isolation of a rhamnolipid deficient mutant (IBT8), which does not accumulate mono-rhamnolipid while still producing di-rhamnolipid. IBT8 was selected after random mutagenesis with Tn501; yet, its mono-rhamnolipid deficiency was found associated neither with its Tn501 insertion nor with a possible alteration in the rhlABRI genes for rhamnosyl-transferase 1 synthesis. Different possibilities to explain IBT8 phenotype are discussed.

The preparation of 17beta-substituted 14beta-hydroxysteroid C-3 alpha-L-rhamnopyranosides is described. These derivatives have a 14beta,20-ether, 14beta,20-lactone, or 17beta-CH2CH2OH, -CH2CH2NH2, -CH=CHNO2(E), -CH=CHCOOH(E), -CH(OH)CH2NO2(R), -CH(OMe)CH2NO2(R), -CH2-CH2COOH, or -CH(OH)CH2NH2(R) group. Derivatives were assayed in a radioligand binding assay for [3H]ouabain in membranes from canine heart muscle. The digitalis "receptor" comprises isoenzymes of the ion-pumping enzyme, Na+,K+-ATPase. The 17beta-CH=CHNO2(E), 17beta-CH=CHCOOH(E), and 17beta-CH(OMe)CH2NO2(R) derivatives were the most potent and equivalent to ouabain with low-nanomolar IC50 values. The very potent binding affinity of the disubstituted compound 17beta-CH(OMe)CH2NO2(R) further demonstrates that 17beta-unsaturated substitution is not required for potent binding affinity. This observation may be of value in the separation of cardiotonic and cardiotoxic effects. Tosylation of the 17beta-CH2OH, prepared from the 17beta-CHO by lithium aluminum hydride reduction, yielded the 14beta,17beta-ether. Synthesis of the 17beta-CH2COOH gave the epimeric 14alpha,17alpha- and 14beta,17beta-lactones. Structures have been established by NMR analysis.

Unlabelled: Intestinal permeability can be assessed non-invasively using the lactulose-rhamnose (L-R) test, which is a reliable measure of small intestinal integrity., Aims: To determine risk factors for abnormal intestinal permeability in kwashiorkor, and to measure changes in L-R ratios with inpatient rehabilitation., Design: A case-control study of 149 kwashiorkor cases and 45 hospital controls. The L-R test was adapted to study kwashiorkor in Malawi, with testing at weekly intervals during nutritional rehabilitation. Urine sugars were measured by thin layer chromatography in London., Results: The initial geometric mean L-R ratios (x100) (with 95% confidence interval) in kwashiorkor were 17.3 (15.0 to 19.8) compared with 7.0 (5.6 to 8.7) for controls. Normal ratios are < 5, so the high ratios in controls indicate tropical enteropathy syndrome. Abnormal permeability in kwashiorkor was associated with death, oliguria, sepsis, diarrhoea, wasting and young age. Diarrhoea and death were associated with both decreased L-rhamnose absorption (diminished absorptive surface area) and increased lactulose permeation (impaired barrier function) whereas nutritional wasting affected only L-rhamnose absorption. Despite, clinical recovery, mean L-R ratios improved little on treatment, with mean weekly ratios of 16.3 (14.0 to 19.0), 13.3 (11.1 to 15.9) and 14.4 (11.0 to 18.8)., Conclusion: Abnormal intestinal permeability in kwashiorkor correlates with disease severity, and improves only slowly with nutritional rehabilitation.

The mycobacterial cell wall core consists of an outer lipid layer of mycolic acids connected, via arabinogalactan polysaccharide, to an inner peptidoglycan layer. An alpha-L-rhamnopyranosyl residue has been shown to be a key component linking the mycolated arabinogalactan to the peptidoglycan and, therefore, the biosynthesis of L-rhamnose (Rha) in mycobacteria was investigated as the first step of developing inhibitors of its biosynthesis. Biochemical assays were used to show that dTDP-Rha was synthesized in Mycobacterium smegmatis from alpha-D-glucose 1-phosphate (alpha-D-Glc-1-P) and dTTP by the same four enzymic steps used by Escherichia coli and other bacteria. PCR primers based on consensus regions of known sequences of the first enzyme in this series, alpha-D-Glc-1-P thymidylytransferase (RfbA) were used to amplify rfbA DNA from M. tuberculosis. The entire rfbA gene was then cloned and sequenced. The deduced amino acid sequence revealed a 31362 Da putative protein product which showed similarity to RfbA proteins of other bacteria (59% identity to that found in E. coli). Sequencing of DNA flanking the rfbA gene did not reveal any of the other rfb genes required for dTDP-Rha biosynthesis. Therefore, the four Rha biosynthetic genes are not clustered in M. tuberculosis. The enzymic activity of the sequenced gene product was confirmed by transformation of E. coli with pBluescript KS(-) containing the rfbA gene from M. tuberculosis. Analysis of enzyme extracts prepared from this transformant revealed an 11-fold increase in alpha-D-Glc-1-P thymidylyltransferase activity.

Unlabelled: The dual sugar test of intestinal permeability is a reliable non-invasive way of assessing the response of the small intestinal mucosa to nutritional rehabilitation., Aim: To compare a local mix of maize-soya-egg to the standard milk diet in the treatment of kwashiorkor., Design: The diets were alternated three monthly in the sequence milk-maize-milk. There were a total of 533 kwashiorkor admissions of at least five days during the study who received either milk or maize. Intestinal permeability was assessed at weekly intervals by the lactulose-rhamnose test in 100 kwashiorkor cases, including 55 on milk and 45 on the maize diet., Results: Permeability ratios (95% confidence interval) on the milk diet improved by a mean of 6.4 (1.7 to 11.1) compared with -6.8 (-16.8 to 5.0) in the maize group. The improved permeability on milk occurred despite more diarrhoea, which constituted 34.8% of hospital days (29.8 to 39.8) compared with 24.3% (17.8 to 30.8) in the maize group. Case fatality rates for all 533 kwashiorkor admissions were 13.6% v 20.9%, respectively, giving a relative risk of death in the maize group of 1.54 (1.04 to 2.28). The maize group also had more clinical sepsis (60% v 31%) and less weight gain (2.9 v 4.4 g/kg/day) than the milk group., Implications: Milk is superior to a local maize based diet in the treatment of kwashiorkor in terms of mortality, weight gain, clinical sepsis, and improvement in intestinal permeability.

Bacillus circulans, a soil bacterium, produced an exocellular polysaccharide of high viscosity. On the basis of the results of methylation analysis, mild acid hydrolysis, and 1D and 2D 1H-NMR spectroscopy, it was concluded that the polysaccharide has a basic repeating unit composed of beta-L-rhamnopyranose, alpha-D-mannopyranose, alpha-D-galactopyranose, and alpha-D-glucopyranuronic acid with the following structure. [symbol: see text]

Enhanced intestinal permeability has been associated with gastrointestinal disorders in long-distance runners. The primary purpose of this study was to evaluate the effect of running intensity on small intestinal permeability by using the lactulose and rhamnose differential urinary excretion test. Secondary purposes included assessing the relationship between small intestinal permeability and gastrointestinal symptoms and evaluating gastric damage by using sucrose as a probe. Six healthy volunteers [5 men, 1 woman; age = 30 +/- 2 yr; peak O2 uptake (VO2peak) = 57.7 +/- 2.1 ml.kg-1.min-1] rested or performed treadmill exercise at 40, 60, or 80% VO2peak for 60 min in a moderate environment (22 degrees C, 50% relative humidity). At 30 min into rest or exercise, the permeability test solution (5 g sucrose, 5 g lactulose, 2 g rhamnose in 50 ml water, approximately 800 mosM) was ingested. Urinary excretion rates (6 h) of the lactulose-to-rhamnose ratio were used to assess small intestinal permeability, and concentrations of each probe were determined by using high-performance liquid chromatography. Running at 80% VO2peak increased (P < 0.05) small intestinal permeability compared with rest, 40, and 60% VO2peak with mean values expressed as percent recovery of ingested dose of 0.107 +/- 0.021 (SE), 0.048 +/- 0.009, 0.056 +/- 0.005, and 0.064 +/- 0.010%, respectively. Increases in small intestinal permeability did not result in a higher prevalence of gastrointestinal symptoms, and urinary recovery of sucrose did not reflect increased gastric permeability. The significance and mechanisms involved in increased small intestinal permeability after high-intensity running merit further investigation.

A field study was carried out with the objective of investigating the prevalence of enteropathogenic Escherichia coli (EPEC) with the eae gene in diarrhoeic rabbits. EPEC eae+ were isolated from 60 (74%) of 81 diarrhoeic rabbits sampled in 30 industrial fattening farms localized in the four provinces of Galicia (northwestern Spain). Attaching and effacing lesions were found in 44 of 50 animals processed for histology. The 111 E. coli strains identified belonged to 19 different O serogroups and 13 biotypes. However, 53 (48%) of the strains belonged to serogroup O103 and 36 (32%) showed the serobiotype O103:B14. The eae gene was significantly more frequent (100%; 47 of 47) among the highly pathogenic rhamnose-negative strains of serobiotypes O103:B6 and O103:B14 than among the E. coli strains belonging to other serobiotypes (36%; 23 of 64) (P < 0.001). In this first report about the prevalence of EPEC with the eae gene in rabbits, we conclude that the class of E. coli strains observed is a common cause of diarrhoea in Galician rabbit farms, and that highly pathogenic rhamnose-negative strains of serotype O103:K-:H2 and biotype B14 are specially predominant.

Pseudomonas aeruginosa GS3 produced rhamnolipid biosurfactants during growth on carbohydrates, higher chain length n-alkanes and l-alkenes, petroleum crude oil and vegetable oils. With glucose as the substrate, maximum surfactant production (0.44 g/l) was observed during the stationary phase of growth. Partially purified rhamnolipids showed excellent surface-active properties in terms of reduction in the interfacial tension between them and a variety of hydrocarbons, hydrocarbon mixtures and vegetable oils and formation of stable emulsion.

A well-known radiopharmaceutical 2-deoxy-2-fluoro-D-glucose widely used for positron emission tomography diagnosis in terms of glucose utilization, was re-evaluated here as a nuclear magnetic resonance pharmaceutical for cancer detection. The uptake and metabolism of FDG in the experimental tumor, MH134, transplanted to the peritoneum of C3H mice as an ascitic tumor was studied extensively by ex vivo 19F NMR. Prolonged retention of FDG and its metabolites over 2 days was confirmed in the tumor cells as well as in the heart. In these tissues, the 6-phosphate of the injected compound was converted reversibly to its epimer 2-deoxy-2-fluoro-D-mannose and further to their NDP bound forms. The metabolites were almost cleared within a day from the other healthy organs where the formation of NDP-2-deoxy-2-fluoro-D-mannose was low. Thus, the 19F NMR signal of NDP-FDM detected 1 day after the FDG injection could be used as a target signal for tumor detection. Through the use of in vivo 19F NMR spectra and 19F chemical shift images, the feasibility of this proposal was demonstrated. It was concluded that FDG-NMR has a potential for tumor diagnosis in animals.

An alpha-L-rhamnosidase (EC 3.2.1.40) was purified 1500-fold from Bacteroides JY-6, an intestinal anaerobic bacterium of human. The specific activity of purified enzyme was 89.9 mumol/min/mg protein. The enzyme (M.W. 240000) is composed of two subunits of M.W. 120000 with pI and optimal pH values of 4.2 and 7.0, respectively. The apparent Kms for naringin, rutin and p-nitrophenyl-alpha-L-rhamnopyranoside were determined to be 0.89, 1.44 and 0.29 mM, respectively. The enzyme was strongly inhibited by L-rhamnose, L-fucose, saccharic acid 1,4-lactone, p-chlormercuriphenylsulfonic acid and Pb2+.

Rhamnogalacturonan hydrolase expression in A. aculeatus can be induced by pectin, but also by a combination of two constituent monosaccharides of pectin, rhamnose and galacturonic acid. The rhgA promoter was fused to the A. niger glucose oxidase coding sequence and a single copy of the hybrid gene was integrated at the rhgA locus in the genome of A. aculeatus. The gene product was subsequently used as reporter in a screening assay for the selection of rhamnogalacturonan hydrolase-overproducing mutant strains. At least four of the mutations were recessive and could be assigned to different loci. One mutation (rgr25) showed linkage with the rhgA locus. Inducible rhamnogalacturonan hydrolase expression levels of about 5-10 times that in the wild-type were found in the mutants rgr48, rgr25 and rgr34 after growth on a combination of rhamnose and galacturonic acid with or without fructose as a carbon source. In mutant rgr48 elevated levels of rhgA transcription were found.

Objective: To assess gastrointestinal permeability and functional absorptive capacity in patients with sepsis., Design: Case control study to analyze gastrointestinal permeability and functional absorptive capacity of septic patients by differential saccharide absorption (from an oral test solution) and excretion., Setting: The intensive Therapy Unit of St. Thomas' Hospital, London, UK., Patients: Twenty patients with a mean Acute Physiology and Chronic Health Evaluation (APACHE) II score of 18.4 who were admitted to the intensive care unit with a diagnosis of sepsis. All patients were on enteral feeding. Patients with abdominal pathology were excluded., Interventions: An oral test solution containing 5 g of lactulose, 1 g of L-rhamnose, 0.5 g of D-xylose, and 0.2 g of 3-O-methyl-D-glucose dissolved in water to a final volume of 100 mL was administered to patients and controls. Urine was collected for 5 hrs starting immediately after administration of the test solution and the saccharide content of the urine was estimated and expressed as a percentage recovery of the oral test solution., Measurements and Main Results: Septic patients had increased lactulose/L-rhamnose urine excretion ratios (0.23 +/- 0.19) compared with control subjects (0.03 +/- 0.01, p < .001), consistent with increased gastrointestinal permeability in sepsis. Septic patients had decreased L-rhamnose/3-O-methyl-D-glucose urine excretion ratios (0.14 +/- 0.07) compared with normal controls (0.28 +/- 0.08, p < .001), consistent with decreased gastrointestinal functional absorptive capacity in sepsis., Conclusions: Patients with acute sepsis exhibit increased gastrointestinal permeability and decreased gastrointestinal functional absorptive capacity in comparison with healthy control subjects. These abnormalities may contribute to the pathophysiology of sepsis.

Transcriptional regulation of the rhaT gene, one of the operons forming the rhamnose regulon in Escherichia coli, was studied by fusing its complete or deleted promoter to the reporter gene lacZ. Analysis of beta-galactosidase activities induced in these constructions grown under different conditions predicted the presence of two putative control elements: one for the RhaS regulatory protein and activating the gene not only by L-rhamnose but also by L-lyxose or L-mannose, the other for cAMP-catabolite repression protein and activating this gene in the absence of glucose. Anaerobiosis increased the promoter function two- to threefold with respect to the aerobic condition. Experiments involving complementation of strains containing the rhaT-promoter fusion and carrying a deletion in the rhaS and/or rhaR genes with plasmids bearing the rhamnose regulatory genes showed that rhaT is controlled by a regulatory cascade, in which RhaR induces rhaSR and the accumulated RhaS directly activates rhaT.

Different approaches were used to examine the function of teichoic acids (TA) as phage receptors among selected Listeria strains, and to identify and characterize specific receptor structures of host cells belonging to different serovars. This included successive removal of cell wall constituents, preparation and purification of TA, and GLC analysis of TA components. Adsorption of Listeria monocytogenes bacteriophages could be inhibited by polyvalent antisera, specific lectins and addition of purified TA. The results confirmed the necessity of TA in general and of rhamnose and glucosamine in particular for adsorption of Listeria phage A118, which is a temperate Siphovirus (morphotype B1), attacking predominantly serovars 1/2. Host binding of siphoviral phage A500 (predominantly lysing serovars 4b), was also dependent on cell wall TA. A phage-resistant L. monocytogenes strain was shown to lack glucosamine in its TA. These results support the view that TA substituents may play an important role not only in antigenicity of Listeria cells, but also in specificity of host recognition by two temperate Listeria phages. In contrast, the broad-host-range virulent phage A511 (Myovirus, morphotype A1) uses the listerial peptidoglycan as primary receptor. This corresponds well with the observation that A511 is capable of lysing the majority of L. monocytogenes strains.

Objectives: To determine whether intestinal permeability increases during cardiac operations, and whether the degree of endotoxemia is related to this increase. Furthermore, to determine whether intestinal permeability is related to the hemodynamic state during operation and to postoperative systemic responses., Design: Prospective study., Setting: University hospital., Participants: Twenty-three male patients undergoing elective coronary artery bypass surgery., Interventions: Before surgery and during the fifth postoperative day, 100 mL of a solution containing L-rhamnose and cellobiose were administered orally., Measurements and Main Results: Intestinal permeability was assessed by measuring the urinary excretion of L-rhamnose and cellobiose. Endotoxin concentrations in blood and prime fluid, hemodynamics, oxygen consumption, gas exchange, fluid balance, and the dose of vasoactive drugs were measured. Systemic responses were assessed by measuring hypermetabolism, circulatory support, and gas exchange. Intestinal permeation of cellobiose, reflecting paracellular transport, significantly increased during operation (p < 0.01), and correlated with the amount of circulating endotoxin (r2 = 0.46; p < 0.01). A high dose of ephedrine administered during operation, low baseline central venous pressure, and a less positive fluid balance during operation were associated with high intestinal permeability (r2 = 0.7; p < 0.01). Intestinal permeability was related to postoperative systemic responses (r2 = 0.49; p < 0.01)., Conclusions: This study shows that during elective coronary artery bypass operations intestinal permeability between cells may increase. The degree of endotoxemia is related to this increase. Increased intestinal permeability is related to the use of ephedrine, especially during hypovolemia, and to postoperative systemic responses. Although a causative relation is not shown, these results might indicate that hypovolemia and vasoconstriction should be avoided during the operation.

Upon the addition of beta-lactam antibiotics at concentrations that caused a 50% reduction in the dry weight, beta-haemolytic streptococci produced increased amount of rhamnose, though the hexosamine content remained unchanged. These sugars are components of C-carbohydrate. Sialic acid content also increased in group B streptococcal surfaces and penicillin treatment generated new accessible surface sialic acid residues.

Background: The purpose of this study was to assess the structure and function of the small intestine before and after enteral feeding given via a percutaneous feeding gastrostomy (PEG). It is not known whether this method of feeding provides a good luminal drive to the small intestine., Methods: Studies were performed of patients at the time of PEG placement, in a cross-sectional group after a period of feeding and in a smaller longitudinal subgroup. Enteral feeds were adjusted in volume and caloric content for each patient. Duodenal biopsies were taken during endoscopy for quantitative morphometry, and lactulose-rhamnose permeability tests were performed during the next day. Duodenal fluid was cultured quantitatively in the first study, and disaccharidases determined in the second study., Results: The first study of 15 patients, who had enteral feeding for a median (range) period of 13 (8 to 104) weeks, showed partial villous atrophy with normal crypt length, no increase in duodenal bacteriology, and abnormal lactulose-rhamnose sugar permeability due to rhamnose malabsorption. These changes were also present in 38 similar patients before enteral feeding. A second study before enteral feeding showed lowered maltase activity (24 patients), and similar intestinal permeability findings (22 patients). Twelve of these patients were followed longitudinally for 3 months of enteral feeding that maintained but did not improve nutrition, as assessed by body mass index and mid-arm muscle circumference, and there was no change in duodenal morphometry (11 patients), rhamnose malabsorption (4 patients), or disaccharidases (11 patients)., Conclusions: These studies suggest villous atrophy was not due to an inflammatory enteropathy but resulted from a poor luminal "drive" associated with the enteral feeding. Enteral feeding maintained but did not improve nutrition status.

Fecal Escherichia (E.) coli strains from calves were tested for simply detectable phenotypical features associated with Shiga-like toxin (SLT) genes. DNA hybridization with SLT-specific oligonucleotide gene probes (detection of genes for SLT-I and SLT-II) was the "gold standard" for the evaluation of Vero cell cytotoxicity, fermentation of several saccharides, beta-D-glucuronidase activity and production of alpha-hemolysin (alpha-Hly) or enterohemolysin (E-Hly). While SLTEC and non-SLTEC did not significantly differ in production of alpha-Hly, beta-D-glucuronidase activity and fermentation of D-sorbitol, production of E-Hly and non-fermentation of L-rhamnose (Rha) and D-sucrose (Suc) were associated with SLT genes. Sensitivity and specificity of the E-Hly+ phenotype were 53% and 88% for identification of calf SLTEC. When three markers were combined to form the parameter ["E-Hly+ or (Rha- and Suc-)"], sensitivity was higher (65%) and specificity was almost the same (85%). Production of enterohemolysin and inability to ferment rhamnose and sucrose were more often associated with the SLT-I gene than with SLT-II genes. Approximately 71% SLT-I+ E. coli were positive in the enterohemolysin assay. The test combination "E-Hly+ or (Rha- and Suc-)" and Suc-)" was most valuable for the presumptive identification of SLT-I+ E. coli (sensitivity 85%, specificity 83%). These data suggest that the phenotype "E-Hly+ or (Rha- and Suc-)" may be a helpful marker for the detection of SLT-I+ E. coli in SLTEC associated diarrhoea of calves.

S. typhimurium isolates obtained during a large outbreak of human salmonellosis associated with smoked mackerels in the Czech Republic as well as strains of S. typhimurium isolated from black headed gull (Larus ridibundus) were examined following biotyping, phage typing, plasmid profiling and restriction endonuclease analysis (Eco RI, Hind III and Bam HI) of plasmid DNA. The epidemic strain of S. typhimurium and two isolates from environment of nesting colony black-headed gull were meso-inositol and L-rhamnose negative, phage type 141. The isolates from human and environment of nesting colony were found to share the same plasmid profile and REA.

A bacterial strain FP2001 isolated from the exudate of land reclaimed for municipal waste was identified as Pseudomonas paucimobilis. Cells of strain FP2001 were mobile by means of polar monotrichous flagellum, only when rhamnose was added as a carbon source in the liquid medium. The replacement of rhamnose by arabinose, galactose, glucose or xylose did not lead to the formation of flagella.

A silent gene encoding a kinase that specifically phosphorylates L-xylulose was activated and rendered constitutive in mutant cells of Escherichia coli. L-Xylulose kinase was purified to homogeneity and found to be a dimer of two subunits of 55 kDa, highly specific for L-xylulose with a Km of 0.8 mM, a Vmax of 33 mumol/min/mg, and an optimum pH of 8.4. Physical (thin layer chromatography) and spectroscopic (nuclear magnetic resonance and optical rotation) characterization of the product of L-xylulose kinase indicated that the enzyme phosphorylated the sugar at position 5. The gene encoding L-xylulose kinase was mapped in the 80.2 min region of the chromosome by conjugation and transduction. Cloning and comparison of the restriction map with the Kohara map (Kohara, Y., Akiyame, K., and Isono, K. (1987) Cell 50, 495-501) located the gene between positions 3963 and 3965 kilobases. The molecular and functional features of L-xylulose kinase together with the location of the corresponding gene indicate that this enzyme did not derive from mutation of any other known kinase. The new kinase opens a route for the utilization of L-lyxose through the action of rhamnose permease, rhamnose isomerase, and the phosphorylation of the L-xylulose formed to L-xylulose 5-phosphate, which is then introduced into the pentose phosphate pathway for subsequent metabolism.

Previous work has indicated that the RhaS protein directly activates the L-rhamnose catabolic operon, rhaBAD, and that the likely RhaS binding site lies downstream of position -84 relative to the rhaBAD transcription start point. Biochemical analysis of RhaS binding to this DNA site had not been possible due to the extreme insolubility of overproduced RhaS protein. Here we have been able to analyze directly the DNA binding properties of RhaS by developing a method to refold insoluble RhaS protein into a form with specific DNA binding activity. We found that active RhaS protein could be recovered only if the renaturation reaction was performed in the presence of DNA. We also found that the recovery of DNA-binding activity from the related AraC protein, after denaturation in urea, was dependent upon added DNA. To test the specificity of the recovered RhaS DNA-binding activity, and to define the binding site for comparison with other AraC family binding sites, we then investigated the details of the RhaS binding site. Using refolded RhaS protein in a DNase footprinting assay, we found that RhaS protects a region of the rhaBAD promoter from position -83 to -28. Analysis of the effects of single base mutations in the rhaBAD promoter region indicates that RhaS binds to an inverted repeat of two 17 bp half-sites separated by 16 bp, located between -81 and -32 relative to the rhaBAD transcription start site.