Biosynthesis of bile acids in a variety of marine bacterial taxa

Several marine bacterial strains, which were isolated from seawater off the island Dokdo, Korea, were screened to find new bioactive compounds such as antibiotics. Among them, Donghaeana dokdonensis strain DSW-6 was found to produce antibacterial agents, and the agents were then purified and analyzed by LC-MS/MS and 1D- and 2D-NMR spectrometries. The bioactive compounds were successfully identified as cholic acid and glycine-conjugated glycocholic acid, the 7alpha-dehydroxylated derivatives (deoxycholic acid and glycodeoxycholic acid) of which were also detected in relatively small amounts. Other masine isolates, taxonomically different from DSW-6, were also able to produce the compounds in a quite different production ratio from DSW-6. As far as we are aware of, these bile acids are produced by specific members of the genus Streptomyces and Myroides, and thought to be general secondary metabolites produced by a variety of bacterial taxa that are widely distributed in the sea.     

Cloning, sequencing, and characterization of the pradimicin biosynthetic gene cluster of Actinomadura hibisca P157-2

Pradimicins are potent antifungal antibiotics having an unusual dihydrobenzo[alpha]naphthacenequinone aglycone substituted with D-alanine and sugars. Pradimicins are polyketide antibiotics produced by Actinomadura hibisca P157-2. The gene cluster involved in the biosynthesis of pradimicins was cloned and sequenced. The pradimicin gene cluster was localized to a 39-kb DNA segment and its involvement in the biosynthesis of pradimicin was proven by gene inactivation of prmA and prmB (ketosynthases alpha and beta). The pradimicin gene cluster consists of 28 open reading frames (ORFs), encoding a type II polyketide synthase (PKS), the enzymes involved in sugar biosynthesis and tailoring enzymes as well as two resistance proteins. The deduced proteins showed strong similarities to the previously validated gene clusters of angucyclic polyketides such as rubromycin, griseorhodin, and fredericamycin. From the pradimicin gene cluster, prmP3 encoding a component of the acetyl-CoA carboxylase complex was disrupted. The production levels of pradimicins of the resulting mutants decreased to 62% of the level produced by the wild-type strain, which indicate that the acetyl-CoA carboxylase gene would have a significant role in the production of pradimicins through supplying the extender unit precursor, malonyl-CoA.     

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.     

Study on persistent infection of Japanese encephalitis virus Beijing-1 strain in serum-free Sf9 cell cultures

Sf9 cells have obvious advantages for the conventional production technology of vaccine. They are useful tools for high concentration and large-scale cultures. Sf9 cells were grown to maximal concentration, 8 x 10(6) cells/ml in a 500ml spinner flask, with a doubling time at the exponentially growing phase of 24.5 hours, using serum-free media. To explore the ability of Sf9 cells to be infected by the Japanese encephalitis (JE) virus Beijing-1 strain, Sf9 cells were infected with the virus. By 4-5 days post-infection, 10-15% of the Sf9 cells showed cytopathic effect (CPE), from granularity to the formation of syncytia and multinucleated giant cells continuously observed over a period of 35 days. Positive fluorescent reactions were detected in 30-40% of cells infected with the JE virus Beijing-1 strain, and the uninfected Sf9 cells were completely negative. Virus particles, propagated in Sf9 and Vero cells, were concentrated by sedimentation on 40% trehalose cushions by ultracentrifugation, and showed identical patterns of viral morphogenesis. Complete virus particles, 40 to 50 nm in diameter, were observed, and JE virus envelope (E) proteins, at 53 kDa, were found in the western blot analysis to the anti-JE virus E protein monoclonal antibody and reacted as a magenta band in the same position to the glycoprotein staining. To evaluate whether the infectious virus was produced in Sf9 cells inoculated with the JE virus Beijing-1 stain, Sf9 cells were inoculated with the virus, and sample harvested every 5 days. The titers of the JE virus Beijing-1 strain rose from 1.0 x 10(5) to 1.5 x 10(6) pfu/ml. The infected Sf9 cells could be sub-cultured in serum-free medium, with no change in the plaque sizes formed by the JE virus Beijing-1 strain in the plaque assay. It is suggested that the ability of the JE virus Beijing-1 strain to infect Sf9 cells in serum-free media will provide a useful insect cell system, where the JE virus replication, cytopathogenicity and vaccine immunogen can be studied.     

Denaturing gradient gel electrophoresis analysis of bacterial populations in 5-stage biological nutrient removal process with step feed system for wastewater treatment

Changes in the bacterial populations of a 5-stage biological nutrient removal (BNR) process, with a step feed system for wastewater treatment, were monitored by denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S ribosomal DNA fragments. DGGE analysis indicated seasonal community changes were observed, however, community profiles of the total bacteria of each reactor showed only minor differences in the samples obtained from the same season. The number of major bands was higher in the summer samples, and decreased during the winter period, indicating that the microbial community structure became simpler at low temperatures. Since the nitrogen and phosphate removal efficiencies were highly maintained throughout the winter operation period, the bacteria which still remaining in the winter sample can be considered important, playing a key role in the present 5-stage BNR sludge. The prominent DGGE bands were excised, and sequenced to gain insight into the identities of the predominant bacterial populations present, and most were found to not be closely related to previously characterized bacteria. These data suggest the importance of culture-independent methods for the quality control of wastewater treatment.     

Screening and characterization of a novel fibrinolytic metalloprotease from a metagenomic library

A metagenomic library was constructed using total genomic DNA extracted from the mud in the west coast of Korea and was used together with a fosmid vector, pCC1FOS in order to uncover novel gene sources. One clone from approximately 30,000 recombinant Escherichia coli clones was identified that showed proteolytic activity. The gene for the proteolytic enzyme was subcloned into pUC19 and sequenced, and a database search for homologies revealed it to be a zinc-dependent metalloprotease. The cloned gene included the intact coding gene for a novel metalloproteinase and its own promoter. It comprised an open reading frame of 1,080 base pairs, which encodes a protein of 39,490 Da consisting of 359 amino acid residues. A His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent metalloproteases, was found in the deduced amino acid sequence of the gene, suggesting that the enzyme is a zinc-dependent metalloprotease. The purified enzyme showed optimal activity at 50°C for 1 h and pH 7.0. The enzyme activity was inhibited by metal-chelating reagents, such as EDTA, EGTA and 1,10-phenanthroline. The enzyme hydrolyzed azocasein as well as fibrin. Thus, the enzyme could be useful as a therapeutic agent to treat thrombosis. The sequence reported in this paper has been deposited in the GenBank database (Accession number: EF100137).     

Fed-batch culture of Bacillus thuringiensis for thuringiensin production in a tower type bioreactor

Fed-batch culture of Bacillus thuringiensis in a modified airlift reactor has been developed by using adaptive control of glucose concentration in the reactor. The glucose concentration was estimated via a correlation equation between carbon dioxide production rate and glucose consumption rate. The estimated glucose concentration as the output variable was fed back to computer for calculation of substrate addition. The modified reactor was an airlift reactor with a net draft tube. The airlift reactor had high oxygen transfer rate and low shear stress which were important factors for production of thuringiensin. Fed-batch culture of Bacillus thuringiensis in the modified airlift reactor provided significant improvement of thuringiensin production. (c) 1995 John Wiley & Sons, Inc.