Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S₁₀₅, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD₆₀₀, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S₁₀₅, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.
Rhamnose/pharmacology* ; Plasmids/genetics* ; Pseudomonas aeruginosa ; Escherichia coli/metabolism*

α-L-rhamnosidase is a very important industrial enzyme that is widely distributed in a variety of organisms. α-L-rhamnosidase of different origins show functional diversity. For example, the optimal pH of α-L-rhamnosidase from bacteria is close to neutral or alkaline, while the optimal pH of α-L-rhamnosidase from fungi is in the acidic range. Furthermore, the enzymatic properties of α-L-rhamnosidases of different origins differ in terms of the optimal temperature, the thermal stability, and the substrate specificity, which determine the different applications of these enzymes. In this connection, it is crucial to elucidate the similarities and differences in the catalytic mechanism and substrate specificity of α-L-rhamnosidase of different origins through analyzing its enzymatic properties. Moreover, it is important to explore and understand the effects of aglycon and metal cations on enzyme activity and the competitive inhibition of L-rhamnose and glucose on enzymes. These knowledge can help discover α-L-rhamnosidase of industrial significance and promote its industrial application.
Glycoside Hydrolases/metabolism* ; Hydrogen-Ion Concentration ; Rhamnose ; Substrate Specificity ; Temperature

BACKGROUND: Pigment production and acidification of ribose are most frequently used biochemical tests for the differentiation of three enterococcal species carrying vanC genes such as Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens. However, pigment production may occasionally be negative in E. casseliflavus, and some of E. casseliflavus may be negative or delayed reaction with ribose fermentation test. So, we performed this study to find out biochemical tests capable of distinguishing the strains possessing vanC genotypes. METHOD: A total of 17 enterococci composed of 14 clinical isolates with motility or pigment positive strains and three ATCC strains(E. gallinarum ATCC 49573, E. casseliflavus ATCC 25788, and E. flavescens ATCC 49997) Were tested by multiplex PCR of the vanC genes(vanC-1, vanC-2 and vanC-3)and various biochemical tests. RESULTS: Among the 17 isolates including three ATCC control strains, four were genotyped as VanC-1, 11 were VanC-2, one were vanC-2/3, and any of vanC genes were not detected in one clinical isolate, respectively, Among the enterococci with vanC genotype, acid production from alphaD-cyclodextrin and hippurate hydrolysis were positive only in VanC-1 gneotype(E. gallinarum), acid production from glycerol and methyl-alpha-D-mannopyranoside were positive only in vanC-2 genotype(E. casseliflavus), and acid production from rhamnose and pigment production were negative only in VanC-1 genotype. Acid production from alphaD-cyclodextrin was negative only in vanC-2 genotype. The positive rate of ribose fermentation of VanC-1, VanC-2, and VanC-2/3(E. flavescens) genotype were 100%, 82%, and 0%, respectively. CONCLUSION: Acid production from rhamnose, alphaD-cyclodextrin, betaD-cyclodextrin, glycerol and methly-alphaD-mannopyranoside, pigment production, and hippurate hydrolysis test were useful biochemical tests for differentitating E. gallinarum form E. casseliflavus. The production of acid from alphaD-cyclodextrin, glycerol, methyl-alpha-D-mannopyranoside and were suitable biochemical tests for differentiating E. casseliflavus from E. flavescens.
Enterococcus ; Fermentation ; Genotype ; Glycerol ; Hydrolysis ; Multiplex Polymerase Chain Reaction ; Phenotype* ; Rhamnose ; Ribose

OBJECTIVETo study the chemical constituents from the active fractions against HIV in vitro, a crude ethanolic extract of Illicium simonsii.

METHODThe compounds were isolated with column chromatography methods. MS and NMR spectroscopic methods were used to determine the structures of the compounds.

RESULTSeven compounds were isolated from the active fractions against HIV in vitro of the 90% ethanol extract and their structures were elucidated as (+)-catechin (1), (-)-epicatechin (2), (+)-catechin 3-O-alpha-L-rhamnopyranoside (3), kaempferol 3-O-alpha-L-rhamnopyranoside (4), quercetin 3-O-alpha-L-rhamnopyranoside (5), erigeside C (6) and daucosterol (7).

CONCLUSIONSeven compounds were isolated from this plant for the first time, but none of them exhibited active against HIV in vitro. Compounds 3 and 6 were isolated from this genus for the first time.

Catechin ; chemistry ; Drugs, Chinese Herbal ; chemistry ; Ethanol ; chemistry ; Glycosides ; chemistry ; Illicium ; chemistry ; Magnetic Resonance Spectroscopy ; Mass Spectrometry ; Rhamnose ; analogs & derivatives ; chemistry ; Sitosterols ; chemistry

OBJECTIVETo study the chemical features of CPB-4, a heteropolysaccharide obtained from Cynanchum paniculatum.

METHODSugar composition analysis, methylation analysis, partial hydrolysis and carbon-13 nuclear magnetic resonance were used to determine the sugar composition, linkages, main chain, branch chains and branching points.

RESULTCPB-4 is composed of L-arabinose, L-xylose, L-rhamnose and D-galactose in closely molar ratios of 0.8:0.2:0.2:1.0. Its main chain is comprised of 1, 5 linked galactose and side chains are comprised of terminal xylose, terminal arabinose, oligosaccharide of arabinose and oligosaccharide of arabinose, rhamnose and galactose. The branching points are located at C-6 and C-2 of galactose.

CONCLUSIONCPB-4 is a new heteropolysaccharide from C. paniculatum.

Arabinose ; isolation & purification ; Cynanchum ; chemistry ; Methylation ; Plant Roots ; chemistry ; Plants, Medicinal ; chemistry ; Polysaccharides ; chemistry ; isolation & purification ; Rhamnose ; isolation & purification ; Xylose ; isolation & purification

AIMTo study the chemical structure of SC3, an acidic polysaccharide from Salvia chinesis.

METHODSBased on chemical (including sugar composition analysis, methylation analysis, uronic acid reduction and partial acid hydrolysis) and spectral analysis (IR, NMR, ESI-MS), the structural characterization of SC3 was investigated.

RESULTSSC3 was composed Rha, Ara, Gal and GalA, with its mean molecular weight of 7.7 x 10(4). By means of methylation analysis, partial acid hydrolysis, NMR and ESI-MS spectrum, the linkages and sequence information of SC3 were obtained.

CONCLUSIONSC3 is an complicated acidic polysaccharide, obtained for the first time from the plant.

Arabinose ; chemistry ; isolation & purification ; Galactose ; chemistry ; isolation & purification ; Molecular Structure ; Molecular Weight ; Plants, Medicinal ; chemistry ; Polysaccharides ; chemistry ; isolation & purification ; Rhamnose ; chemistry ; isolation & purification ; Salvia ; chemistry

AIMTo study the bioactive constituents of the flower of Castanea mollissima Blume.

METHODSCompounds were isolated and purified by column chromatography of silica gel and TLC. Structures were determined by various spectroscopic data, including IR, 1HNMR and 13CNMR, EIMS, FABMS and HMBC as well as comparison of the data with those reported in literatures.

RESULTSFive compounds were isolated and elucidated as myricetin (I), quercetin (II), gallic acid (III), 4-quinolinone-2-caboxylic acid (IV), (+) -isolariciresinol-9'-O-alpha-L-rhamnoside (V).

CONCLUSIONThese compounds were separated from the flower for the first time and compound V is a new compounds, named chestnutlignansoide.

Fagaceae ; chemistry ; Flavonoids ; chemistry ; isolation & purification ; Flowers ; chemistry ; Molecular Structure ; Naphthols ; chemistry ; isolation & purification ; Plants, Medicinal ; chemistry ; Quercetin ; chemistry ; isolation & purification ; Rhamnose ; analogs & derivatives ; chemistry ; isolation & purification

Development of a new and effecient tuberculosis vaccine is very important since the efficacy of the only available vaccine against tuberculosis, BCG, is variable among races and different ages. Attempts to develop attenuated vaccines by disrupting virulence gene(s) specifically in Mycobacterium tuberculosis are now actively being tried after the release of whole genome sequence of M. tuberculosis in 1998. However, disruption of specific genes in M. tuberculosis is still very difficult due to the lack of effective gene knock-out system(s) in mycobacteria. In this study, we developed a novel method to delete specific genes in both Escherichia coli and mycobacteria. This knock-out system is operated by a sequence-specific recombinase FLP and its recognition sequence FRT (FLP/FRT system). Two shuttle vectors (an FLP expressing vector and a gene targeting vector) between Escherichia coli and Mycobacteria were developed. The gene targeting vector contains a kanamycin resistance gene (KmR) flanked by two neighboring genes and two FRTs (FLPrecognition targets). We applied this system to knock-out the rhamnose biosynthetic gene rmlD of Escherichia coli. The upstream and downstream genes of rmlD, rmlB and rmlA, were cloned into the gene targeting vector. After and allelic exchange of E. coli chromosomal rmlB, rmlD, rmlA with vectoral rmlB, FRT-KmR-FRT, rmlA by homologous recombination, FLP-expressing plasmid was introduced to induce the excision of KmR cassette remaining one FRT sequence between rmlB and rmlA. We also demonstrated our shuttle vector could disrupt a target gene (kanamycin resistance gene) in M. smegmatis. These results suggest that our gene knock-out system can be used for the development of an attenuated tuberculosis vaccines and for the functional genomic study of mycobacteria.
Clone Cells ; Continental Population Groups ; Escherichia coli ; Gene Targeting ; Genes, vif ; Genetic Vectors ; Genome ; Homologous Recombination ; Humans ; Kanamycin Resistance ; Mycobacterium bovis ; Mycobacterium tuberculosis ; Plasmids ; Recombinases* ; Rhamnose* ; Tuberculosis ; Tuberculosis Vaccines ; Vaccines, Attenuated ; Virulence

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.
Bacterial Proteins ; genetics ; Batch Cell Culture Techniques ; Decanoates ; Escherichia coli ; metabolism ; Fermentation ; Glycolipids ; biosynthesis ; Hexosyltransferases ; genetics ; Industrial Microbiology ; methods ; Multigene Family ; Promoter Regions, Genetic ; Pseudomonas aeruginosa ; Rhamnose ; analogs & derivatives ; biosynthesis ; Surface-Active Agents ; metabolism

BACKGROUND: The MK1 strain, a novel bacterial isolate from soft-rotten tissue of the Neungee mushroom, produces copious amounts of exopolysaccharide (EPS) in a dextrose minimal medium. This study examined the molecular characteristics and immunomodulatory activity of MK1 EPS. METHODS: The EPS in the culture supernatant was purified by cold ethanol precipitation, and characterized by SDS-PAGE/silver staining and Bio-HPLC. The immunomodulatory activities of the EPS were examined using the mouse monocytic cell line, RAW 264.7 cells. RESULTS: The molecular weights of the purified EPS were rather heterogeneous, ranging from 10.6 to 55 kDa. The EPS was composed of glucose, rhamnose, mannose, galactose, and glucosamine at an approximate molar ratio of 1.00:0.8:0.71:0.29:0.21. EPS activated the RAW cells to produce cytokines, such as TNF-alpha and IL-1beta, and nitric oxide (NO). EPS also induced the expression of co-stimulatory molecules, such as B7-1, B7-2 and ICAM-1, and increased the phagocytic activity. The macrophage-activating activity of EPS was not due to endotoxin contamination because the treatment of EPS with polymyin B did not reduce the macrophage-activating activity. CONCLUSION: The EPS produced from the MK1 strain exerts macrophage-activating activity.
Agaricales ; Animals ; Cell Line ; Cold Temperature ; Cytokines ; Ethanol ; Galactose ; Glucosamine ; Glucose ; Intercellular Adhesion Molecule-1 ; Macrophages ; Mannose ; Mice ; Molar ; Molecular Weight ; Nitric Oxide ; Rhamnose ; Sprains and Strains ; Tumor Necrosis Factor-alpha