[Prokaryote diversity in water environment of land-ocean ecotone of Zhuhai City].

By constructing 16S rDNA clone library with PCR-RFLP, the prokaryote diversity in the seawater and groundwater of land-ocean ecotone of Zhuhai City was investigated, and the similarity and cluster analyses were implemented with the database of the sequences in Genbank. In the seawater, Proteobacteria was dominant, followed by Archaeon, Gemmatimonadetes, Candidate division OP3 and OP8, and Planctomycetes, etc.; while in the groundwater, Archaeon was dominant, followed by Proteobacteria, Sphingobacteria, Candidate division OP3, Actinobacterium, and Pseudomonas. The dominant taxa in the groundwater had high similarity to the unculturable groups of marine microorganisms. Large amount of bacteria capable of degrading organic matter and purifying water body existed in the groundwater, suggesting that after long-term evolution, the land-ocean ecotone of Zhuhai City had the characteristics of both land and ocean.

The ClpP protease homologue is required for the transmission traits and cell division of the pathogen Legionella pneumophila.

BACKGROUND: Legionella pneumophila, the intracellular bacterial pathogen that causes Legionnaires' disease, exhibit characteristic transmission traits such as elevated stress tolerance, shortened length and virulence during the transition from the replication phase to the transmission phase. ClpP, the catalytic core of the Clp proteolytic complex, is widely involved in many cellular processes via the regulation of intracellular protein quality. RESULTS: In this study, we showed that ClpP was required for optimal growth of L. pneumophila at high temperatures and under several other stress conditions. We also observed that cells devoid of clpP exhibited cell elongation, incomplete cell division and compromised colony formation. Furthermore, we found that the clpP-deleted mutant was more resistant to sodium stress and failed to proliferate in the amoebae host Acanthamoeba castellanii. CONCLUSIONS: The data present in this study illustrate that the ClpP protease homologue plays an important role in the expression of transmission traits and cell division of L. pneumophila, and further suggest a putative role of ClpP in virulence regulation.

[Spatial characteristics of microbial groups associated with the groundwater flow in a small watershed].

Few reports of microbial groups associated with the groundwater flow system are available in China. 16S rRNA gene library was constructed by the cultured-independent approach to investigate gene sequences of microorganism in groundwater samples from the recharge (R), intermediate (M) and discharge (D) zones of an experimental watershed at Zhuhai campus of Sun Yat-sen University. Proteobacterium, Candidate division OPx, uncultured archaeon (uncultured Crenarchaeote and Euryarchaeote) and Actinobacterium are found predominant in all these three zones with the Proteobacterium accounting for 23.21%, 36.21%, and 28.84% in R, M, D zone respectively. The other predominant microbial groups were identified for varied zones, e. g. Eubacterium and Nitrospira in the R wells, Eubacterium and Acidobacterium in the M wells, and Bacteroidetes bacterium in the D wells. Linkages and potential evolution of microbial groups among three zones were examined by using the genetic neighbor-joining tree. Environmental adaptation along the groundwater flow contributes to the similarity and discrepancy of microorganism in term of the genetic tree, and the ecological functions of the microbial groups need further assessment.

The Legionella pneumophila Dps homolog is regulated by iron and involved in multiple stress tolerance.

Iron homeostasis is essential to almost all organisms. In this study, we identified the putative homolog of the iron-storage protein-encoding gene, dpsL, in the intracellular pathogen Legionella pneumophila and demonstrated its expression under iron-limited conditions and its responses to multiple stresses. Quantitative real-time PCR analysis indicated that the expression of dpsL was enhanced under iron limitation regardless of the growth phase. Compared with the wild-type cells, the cells devoid of dpsL were heat and H(2)O(2)-sensitive. In contrast to the dps mutants of other bacteria, the growth of the dpsL mutant in an iron-deprived medium was delayed but finally reached the same cell density as wild-type cells during the stationary phase of growth. The finding that the dpsL mutant is salt resistant suggested the involvement of DpsL in virulence.

Construction of a Monascus purpureus mutant showing lower citrinin and higher pigment production by replacement of ctnA with pks1 without using vector and resistance gene.

Monascus products are used as both natural colorants and food additives all over the world. However, its safety is really an issue because of the presence of citrinin, which is considered to be hepatotoxic and nephrotoxic. Therefore, the objective of the present study was to develop an approach for using a fragment of pigment-related gene pks1 to replace citrinin-activator gene ctnA. The character of citrinin antibacterial activity to Bacillus subtilis was used for primary screening of transformants based on the foundation that the inhibition zone formed around the mutant colonies with low citrinin products will become smaller. The selected mutants were then further confirmed by polymerase chain reaction and high-performance liquid chromatography methods. During all of the processes, antibiotic markers and vectors were avoided. The results showed that the citrinin products of mutants were reduced to 42%, while the pigment products were improved to 33.9%, respectively, over those of the wild-type strains.

A new hTopo I isomerase inhibitor produced by a mangrove endophytic fungus no. 2240.

A new hTopo I isomerase inhibitor, (+)-3,3',7,7',8,8'-hexahydroxy-5,5'-dimethylbianthraquinone (2240A), was isolated from the mangrove endophytic fungus no. 2240 collected from an estuarine mangrove at the South China Sea coast. Its structure was elucidated by spectral analyses including two-dimensional NMR, HR-EI-MS, IR, and UV. The hTopo I isomerase inhibition experiment showed that 2240A (1) possessed strong inhibiting activity. When its inhibition concentration was 4.65 micromol/l, its percent inhibition rate was 59.1%, while the lowest inhibition concentration of the positive control camptothecin was 1.00 x 10(3) micromol/l.

Five nitro-phenyl compounds from the South China Sea mangrove fungus.

A novel nitro-phenyl glucoside (1) was isolated from mangrove endophytic fungus (fungus B60), collected from the Shenzhen mangrove Acanthus ilicifolius linn. Four related nitro-phenyl compounds (2-5) were also obtained, which were isolated for the first time as natural products. Their structures were established on the basis of NMR spectroscopic, mass spectrometric data and some chemical transformations. In the preliminary bioassay, compound 1 had a slight inhibitory effect on alpha-glucosidase with an IC(50) of 160.3 microM.

[The metabolites of cyclic peptides from three endophytic mangrove fungi].

Nine secondary metaboites of cyclic peptide were isolated from three mangrove endophytic fungi Paecilomyces sp. (treel-7), 4557,ZZF65. They were viscumamide(1),cyclo(Pro-Iso)(2),cyclo(Phe-Gly)(3),cyclo(Phe-Ana)(4),cyclo(Gly-Pro) (5),cyclo(Gly-Leu)(6), cyclo(Trp-Ana)(7),neoechinulin A(8),cyclo(Pro-Thr)(9). The compounds 1,7,8,9 were firstly isolated from marine fungus.

Lactones from a brown alga endophytic fungus (No. ZZF36) from the South China Sea and their antimicrobial activities.

Two new metabolites named 6-oxo-de-O-methyllasiodiplodin (1) and (E)-9-etheno-lasiodiplodin (2), with three known compounds lasiodiplodin (3), de-O-methyllasiodiplodin (4), and 5-hydroxy-de-O-methyllasiodiplodin (5), were isolated from the mycelium extracts of a brown alga endophytic fungus (No. ZZF36) obtained from the South China Sea. Their structures were elucidated using spectroscopic methods, mainly 1D and 2D NMR. Additionally, the structure of compound 1 was confirmed by single crystal X-ray diffraction analysis. The antimicrobial activities of lasiodiplodins, and the 13-acetyl and 12,14-dibromo derivatives of lasiodiplodin were tested for the first time and the results were compared to each other.

[Study on the sterols from a brown alga endophytic fungus (NO. ZZF36) from the South China Sea].

Four sterols were isolated from a brown alga endophytic fungus NO. ZZF36 from the South China Sea. Their structures were identified as brassicaterol(2), ergosterol(2), ergosterol peroxide(3),7,22(E)-ergostadiene-beta,5alpha,6beta-triol(4) by spectroscopic methods.

[Optimization of the expression of human DNA topoisomerase I in Pichia pastoris].

Human DNA Topoisomerase I (hTopo I) has been identified to be an efficient target of many effective antitumor drugs. Natural hTopo I is not convenient to be used in screening because of its low concentration in cells. In order to fast screen new anticancer drugs targeting at hTopo I from natural compounds in vitro, hTopo I gene open reading frame (ORF) has been successfully cloned and overexpressed in Pichia pastoris. Total RNA extracted from Hela cells was reversely transcripted to synthesize cDNA with the hTopo I specific antisense primer and the hTopo I ORF was synthesized by PCR. After digestion with EcoR I and Kpn I, the synthesized fragment was inserted into pPICZaA, gave rise to pPICZalpha-hTopoI. After digestion with Sac I, the lined pPICZalpha-hTopoI was transformed into Pichia pastoris strains (KM71, X33 and SMD1168) by electroporation and integrated into their genome. After screened on YPDS plates (containing 1000 ug/mL zeocin), the high-copy recombinant strains (KM-hTopoI, X33-hTopoI and SMD-hTopol) could overexpress recombinant hTopo I, which was fused to the alpha-factor secretion signal and could be secreted into the supernatant in the culture. alpha-factor could be cleaved from the expressed protein during secretion. A higher activity amount of the enzyme was secreted by the particular strain SMD-hTopoI because of its absence of proteimase A than by other strains which possess proteinase A activity. After optimizing the fermentation conditions, a relatively higher enzyme activity in the culture supernatant could be obtained when SMD-hTopoI was induced in BMMY (pH7.25) at 20 degrees C , with addition of 0.5% (V/V) methanol and 3% (V/V) nutrient liquid every 24h. The enzyme activity reached 43 000 u/mL, the yield reached 11 mg/L, achieving approximate 10% of total protein in the culture supernatant. SDS-PAGE and Western blot analyses showed that the mass of the recombinant hTopo I was 91 kD with no glycosylation.