Molecular Cloning of Novel Cellulase Genes <Emphasis Type="Italic">cel</Emphasis>9A and <Emphasis Type="Italic">cel</Emphasis>12A from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> GXN151 and Synergism of Their Encoded Polypeptides

A thermophilic bacterial strain GXN151 which could degrade Avicel efficiently was isolated and identified as Bacillus licheniformis. A genomic library of GXN151 was constructed and two novel endoglucanase genes designated cel9A and cel12A were isolated by screening the library on carboxylmethyl cellulase indicator plates. The analysis of amino acid sequences deduced from the genes indicated that Cel9A consisted of a catalytic domain belonging to glycosyl hydrolase family 9, a linker domain, and a carbohydrate binding module family 3 from N-terminal to C-terminal; Cel12A had only one catalytic domain belonging to glycosyl hydrolase family 12. The combinations of Cel9A and Cel12A produced by the recombinant E. coli exhibited synergistic action against substrates of carboxylmethyl cellulose as well as Avicel.     

Sediment quality criteria in use around the world

 There have been numerous sediment quality guidelines (SQGs) developed during the past 20 years to assist regulators in dealing with contaminated sediments. Unfortunately, most of these have been developed in North America. Traditionally, sediment contamination was determined by assessing the bulk chemical concentrations of individual compounds and often comparing them with background or reference values. Since the 1980s, SQGs have attempted to incorporate biological effects in their derivation approach. These approaches can be categorized as empirical, frequency-based approaches to establish the relationship between sediment contamination and toxic response, and theoretically based approaches that attempt to account for differences in bioavailability through equilibrium partitioning (EqP) (i.e., using organic carbon or acid volatile sulfides). Some of these guidelines have been adopted by various regulatory agencies in several countries and are being used as cleanup goals in remediation activities and to identify priority polluted sites. The original SQGs, which compared bulk chemical concentrations to a reference or to background, provided little insight into the ecosystem impact of sediment contaminants. Therefore, SQGs for individual chemicals were developed that relied on field sediment chemistry paired with field or laboratory-based biological effects data. Although some SQGs have been found to be relatively good predictors of significant site contamination, they also have several limitations. False positive and false negative predictions are frequently in the 20% to 30% range for many chemicals and higher for others. The guidelines are chemical specific and do not establish causality where chemical mixtures occur. Equilibrium-based guidelines do not consider sediment ingestion as an exposure route. The guidelines do not consider spatial and temporal variability, and they may not apply in dynamic or larger-grained sediments. Finally, sediment chemistry and bioavailability are easily altered by sampling and subsequent manipulation processes, and therefore, measured SQGs may not reflect in situ conditions. All the assessment tools provide useful information, but some (such as SQGs, laboratory toxicity and bioaccumulation, and benthic indices) are prone to misinterpretation without the availability of specific in situ exposure and effects data. SQGs should be used only in a “screening” manner or in a “weight-of-evidence” approach. Aquatic ecosystems (including sediments) must be assessed in a “holistic” manner in which multiple components are assessed (e.g., habitat, hydrodynamics, resident biota, toxicity, and physicochemistry, including SQGs) by using integrated approaches. Received: December 26, 2000 / Accepted: December 28, 2001     

<Emphasis Type="Italic">Phellinus</Emphasis> extracts inhibit migration and matrix metalloproteinase secretion in porcine coronary artery endothelial cells

Phellinus linteus is a fungus which is found primarily in tropical regions of the Americas, Africa, and Asia.P. linteus has been used in traditional medical practice for the treatment of arthritis, liver damage and cancer. Angiogenesis is a process that involves migration, proliferation and cell differentiation, as well as the formation of new capillary structures. The anti-angiogenic activities evidenced by natural compounds may actually be a critical effect for the inhibition of angiogenesis-dependent disease by these agents via the blockage of vascular development. This study assessed the effects of water extracts fromP. linteus (Phellinus extracts) on primary cultured porcine coronary artery endothelial cells (PCAECs).Phellinus extracts induced no changes in DNA synthesis or cell numbers, but inhibited the migration of PCAECs.Phellinus extracts also induced a reduction in the secretion of matrix metalloproteinase-2 (MMP-2) and MMP-9. Our results show that, in endothelial cells,Phellinus extracts may inhibit angiogenesis by reducing levels of MMP-2 and MMP-9 secretion.     

Eutrophication assessment and bioremediation strategy in a marine fish cage culture area in Nansha Bay,China

On the basis of field data measured during four cruises from January to November 2007, variations in the characteristics of dissolved inorganic nitrogen and phosphate were analyzed in Nansha marine fish cage culture area, Ningbo City, China. Dissolved inorganic nitrogen (DIN) was selected as the parameter to balance seaweed absorption and fish DIN production. The contents of DIN and phosphate varied with different seasons, and eutrophication index (E) value ranged from 2.41 to 15.99, indicating serious eutrophication conditions; the annual average value of N/P of 32.95 indicates a nitrogen surplus in this system. The eutrophication condition in Nansha Bay was mainly caused by the fish cage culture activities. Based on their biological characteristics, Laminaria and Gracilaria were selected as the bioremediation species in winter and spring and summer and autumn, respectively. The optimal co-cultivation proportion of fish cage to Laminaria and Gracilaria in this bay was 1 cage, 450 m² and one cage, 690 m², respectively.     

What governs the reaction center excitation wavelength of photosystems I and II?

The sun’s spectrum harvested through photosynthesis is the primary source of energy for life on earth. Plants, green algae, and cyanobacteria—the major primary producers on earth—utilize reaction centers that operate at wavelengths of 680 and 700 nm. Why were these wavelengths “chosen” in evolution? This study analyzes the efficiency of light conversion into chemical energy as a function of hypothetical reaction center absorption wavelengths given the sun’s spectrum and the overpotential cost associated with charge separation. Surprisingly, it is found here that when taking into account the empirical charge separation cost the range 680–720 nm maximizes the conversion efficiency. This suggests the possibility that the wavelengths of photosystem I and II were optimized at some point in their evolution for the maximal utilization of the sun’s spectrum.     

Caspofungin reduces the incidence of fungal contamination in cell culture

Fungal contamination is a major problem in cell culture, and the antifungal compounds currently in use can affect cultured cells. Echinocandins are antifungal drugs that inhibit fungal cell wall synthesis by targeting an enzyme that has no counterpart in mammalian cells. We evaluated whether the echinocandin caspofungin affected the growth or morphology of six murine cell lines (a macrophage-like cell line (J774.16) and five hybridoma lines), or primary human endothelial cells. The antifungal did not influence cellular characteristics at concentrations less than 512 μg/ml, while effectively reducing the incidence of fungal contamination. Also, caspofungin did not affect the production of antibody by hybridoma cells, or alter the cytokine production of J774.16 cells, although modest increases in IL-4 and IFN-γ occurred upon LPS stimulation. Hence, echinocandins appear to be relatively non-toxic, and protect against fungal contamination in cell culture.     

Characterization and development of EST-SSR markers to study the genetic diversity and populations analysis of Jerusalem artichoke (<Emphasis Type="Italic">Helianthus tuberosus</Emphasis> L.)

In recent years, Jerusalem artichoke has received widespread attention as a novel source of sugar, biofuel, and animal feed. Currently, only few gDNA-SSRs derived from sunflower were verified in the Jerusalem artichoke; therefore, it is particularly important to develop SSR primer markers that belonged to Jerusalem artichoke resources. Using EST data to develop EST-SSR markers is simple and effective. In order to understand the general characteristics of SSR markers in Jerusalem artichoke EST sequences and accelerate the use of SSR markers in Jerusalem artichoke research. This study used 40,370 sequenced unigene fragments and MISA software to identify SSR loci. The 48 pairs of EST-SSR primers assessed for the identification of 45 varieties of Jerusalem artichoke. Cluster, genetic diversity parameters and AMOVA analysis was conducted using the genetic similarity coefficient, revealing genetic differences between 48 genetic material. A total of 1204 SSR loci were identified with 13 different types of repeats, distributed among 1020 EST sequences, of which trinucleotide repeats were the most common, accounting for 38.21% of the total SSR loci. Among the 44 repeat motifs, AG/CT, AAG/CTT, and ATC/ATG motifs had the highest frequencies, accounting for 22.45, 14.71, and 7.84% of all motifs, respectively. From these sequences, 48 pairs of EST-SSR primers were designed, and 22 primer pairs for loci with high polymorphism were selected to analyze the genetic diversity of 45 Jerusalem artichoke germplasm sources. The results indicated that the variation range of the effective number of alleles for 22 primers ranged between 1.7502 and 4.5660. The Shannon’s information index ranged between 0.6200 and 1.6423. The variation range of PIC ranged between 0.3121 and 0.6662 with an average of 0.5184. Cluster analysis was conducted using the genetic similarity coefficient, revealing significant genetic differences between Asian and European genetic material. Cluster analysis revealed a relationship between the genotypes and geographic origins of the Jerusalem artichoke. The results of AMOVA as well as the genetic identity and genetic distance in the Jerusalem artichoke population showed that there presented certain genetic heterogeneity in Jerusalem artichoke genetic structure of 45 samples from seven different geographic populations. The Jerusalem artichoke EST-SSR marker system established in this study provides an effective molecular marker system for future research focused on Jerusalem artichoke genetic diversity and the breeding of new varieties.     

Influence of Nutrient Availability and Quorum Sensing on the Formation of Metabolically Inactive Microcolonies Within Structurally Heterogeneous Bacterial Biofilms: An Individual-Based 3D Cellular Automata Model

The resistance of bacterial biofilms to antibiotic treatment has been attributed to the emergence of structurally heterogeneous microenvironments containing metabolically inactive cell populations. In this study, we use a three-dimensional individual-based cellular automata model to investigate the influence of nutrient availability and quorum sensing on microbial heterogeneity in growing biofilms. Mature biofilms exhibited at least three structurally distinct strata: a high-volume, homogeneous region sandwiched between two compact sections of high heterogeneity. Cell death occurred preferentially in layers in close proximity to the substratum, resulting in increased heterogeneity in this section of the biofilm; the thickness and heterogeneity of this lowermost layer increased with time, ultimately leading to sloughing. The model predicted the formation of metabolically dormant cellular microniches embedded within faster-growing cell clusters. Biofilms utilizing quorum sensing were more heterogeneous compared to their non-quorum sensing counterparts, and resisted sloughing, featuring a cell-devoid layer of EPS atop the substratum upon which the remainder of the biofilm developed. Overall, our study provides a computational framework to analyze metabolic diversity and heterogeneity of biofilm-associated microorganisms and may pave the way toward gaining further insights into the biophysical mechanisms of antibiotic resistance.     

<Emphasis Type="Italic">Nibribacter flagellatus</Emphasis> sp. nov., isolated from rhizosphere of <Emphasis Type="Italic">Hibiscus syriacus</Emphasis> and emended description of the genus <Emphasis Type="Italic">Nibribacter</Emphasis>

A Gram-stain negative, aerobic, motile by flagella, rod-shaped strain (THG-T16^T) was isolated from rhizosphere of Hibiscus syriacus. Growth occurred at 10–40 °C (optimum 28–30 °C), at pH 6.0–8.0 (optimum 7.0) and at 0–1.0% NaCl (optimum 0%). Based on 16S rRNA gene sequence analysis, the near phylogenetic neighbours of strain THG-T16^T were identified as Nibribacter koreensis KACC 16450^T (98.6%), Rufibacter roseus KCTC 42217^T (94.7%), Rufibacter immobilis CCTCC AB 2013351^T (94.5%) and Rufibacter tibetensis CCTCC AB 208084^T (94.4%). The DNA G+C content of strain THG-T16^T was determined to be 46.7 mol%. DNA–DNA hybridization values between strain THG-T16^T and N. koreensis KACC 16450^T, R. roseus KCTC 42217^T, R. immobilis CCTCC AB 2013351^T, R.tibetensis CCTCC AB 208084^T were 33.5?±?0.5% (31.7?±?0.7% reciprocal analysis), 28.1?±?0.2% (25.2?±?0.2%), 17.1?±?0.9% (10.2?±?0.6%) and 8.1?±?0.3% (5.2?±?0.1%). The polar lipids were identified as phosphatidylethanolamine, two unidentified aminophospholipids, an unidentified aminolipid and three unidentified lipids. The quinone was identified as MK-7 and the polyamine as sym-homospermidine. The major fatty acids were identified as C_16:1 ω5c, C_17:1 ω6c, iso-C_15:0, summed feature 3 (C_16:1 ω7c and/or C_16:1 ω6c) and summed feature 4 (iso-C_17:1 I and/or anteiso-C_17:1 B). On the basis of the phylogenetic analysis, chemotaxonomic data, physiological characteristics, and DNA–DNA hybridization data, strain THG-T16^T represents a novel species of the genus Nibribacter, for which the name Nibribacter flagellatus sp. nov. is proposed. The type strain is THG-T16^T(=?KACC 19188^T?=?CCTCC AB 2016246^T).     

Very high gravity ethanol and fatty acid production of <Emphasis Type="Italic">Zymomonas mobilis</Emphasis> without amino acid and vitamin

Very high gravity (VHG) fermentation is the mainstream technology in ethanol industry, which requires the strains be resistant to multiple stresses such as high glucose concentration, high ethanol concentration, high temperature and harsh acidic conditions. To our knowledge, it was not reported previously that any ethanol-producing microbe showed a high performance in VHG fermentations without amino acid and vitamin. Here we demonstrate the engineering of a xylose utilizing recombinant Zymomonas mobilis for VHG ethanol fermentations. The recombinant strain can produce ethanol up to 136 g/L without amino acid and vitamin with a theoretical yield of 90 %, which is significantly superior to that produced by all the reported ethanol-producing strains. The intracellular fatty acids of the bacterial were about 16 % of the bacterial dry biomass, with the ratio of ethanol:fatty acids was about 273:1 (g/g). The recombinant strain was achieved by a multivariate-modular strategy tackles with the multiple stresses which are closely linked to the ethanol productivity of Z. mobilis. The over-expression of metB/yfdZ operon enabled the growth of the recombinant Z. mobilis in a chemically defined medium without amino acid and vitamin; and the fatty acids overproduction significantly increased ethanol tolerance and ethanol production. The coupled production of ethanol with fatty acids of the Z. mobilis without amino acid and vitamin under VHG fermentation conditions may permit a significant reduction of the production cost of ethanol and microbial fatty acids.