Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (Al-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the Al-2 synthase was cloned from TX1 and designated luxS(Et). LuxS(Et) was able to complement the Al-2 mutant phenotype of Escherichia coli strain DH5 alpha. Expression Of luxS(Et) correlated with Al-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS(Et) expression. Overexpression of luxS(Et) enhanced Al-2 activity in TX1, whereas disruption of luxS(Et) expression by antisense RNA interference (i) reduced the level of Al-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous Al-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the Al-2 activity in TX1 is controlled at least in part at the level of luxS(Et) expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS(Et) is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/Al-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

Two thymosin-repeated molecules with structural and functional diversity coexist in Chinese mitten crab Eriocheir sinensis

Recently, beta-thymosin-like proteins with multiple thymosin domains (defined as thymosin-repeated proteins) have been identified from invertebrate. In the present study, the cDNAs of two thymosin-repeated proteins (designated EsTRP1 and EsTRP2) were cloned from Chinese mitten crab by expressed sequence tags (EST) techniques. BLAST analysis presented three and two thymosin domains in EsTRP1 and EsTRP2, respectively, with the identities amongst the five domains varying from 47% to 100%. Both EsTRP1 and EsTRP2 shared high similarities with previously identified vertebrate beta-thynnosins and invertebrate thymosin-repeated proteins (TRPs) with the identities ranging from 43% to 78%, indicating that EsTRPs were new members of the beta-thymosin family. Real-time RT-PCR assay was adopted to determine the tissue distribution of EsTRPs and their temporal expression profile in hemocytes after pathogen stimulation and injury challenge. The expression of EsTRP1 transcript was predominantly detectable in the tissues of hemocytes, hepatopancreas and gonad with the highest expression in hemocytes, while the highest expression level of EsTRP2 was found in heart. EsTRP1 mRNA expression in hemocytes significantly increased at 3 and 48 h after Listonella anguillarum challenge, but there was no significant variation in EsTRP2 temporal expression profile. The injury challenge reduced the mRNA expression of EsTRPs, with the down-regulation of EsTRP2 expression occurred earlier than that of EsTRP1. The cDNA fragments encoding their mature peptides of EsTRP1 and EsTRP2 were recombined and expressed in Escherichia coli. The activities of recombinant proteins (rEsTRP1 and rEsTRP2) were examined by MTT (3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazoliumbromide) and lysoplate assay. rEsTRP2 could significantly accelerate the growth of human hepatocellular carcinoma cell line, but there was no significant effect of rEsTRP1 on the tumor cell proliferation. Both rEsTRP1 and rEsTRP2 did not possess the ability of killing Micrococcus luteus and L. anguillarum. The differences in the tissue distribution of mRNA transcripts, the response to pathogen stimulation and injury challenge, and the effect of recombinant proteins on human cell proliferation, indicated that there were functional diversity between the two structurally different molecules, EsTRP1 and EsTRP2. (C) 2009 Elsevier Ltd. All rights reserved.

Rapid detection of virulent protease secreted by Vibrio anguillarum by dot enzyme-linked immunosorbent assay

Dot enzyme-linked immunosorbent assay (dot-ELISA), indirect ELISA and Western blot were performed to detect the virulent protease secreted by Vibrio anguillarum which was isolated from the diseased left-eyed flounder, Paralichthys olivaceous. Sensitivity results showed that dot-ELISA is a more sensitive, rapid and simple technique for the protease detection. The minimal detectable amount of protease is about 7 pg in the dot-ELISA test, while 7.8 ng in the indirect ELISA and 6.25 ng in the Western blot respectively. Protease could be detected 2 h after incubation of V. anguillarum in the 2216E liquid medium but enzyme activity was very low at that period. From 6 to 12 h, the amount and enzyme activity of protease increased markedly and reached maximum at stationary phase. Analysis of serum samples periodically collected from the infected flounders showed that after 2 h of infection by V. anguillarum, the pathogenic bacteria could be detected in the blood of the infected flounders but no protease was found. It was 5 similar to 6 h after infection that the protease was detected in blood and then the amount increased as infection advanced. Quantitative detection of protease either incubation in the medium or from the blood of infected flounders could be accomplished in virtue of positive controls of quantificational protease standards ("marker") so that the alterations of protease secretion both in vitro and in vivo could be understood generally. In addition, the indirect ELISA and dot-ELISA were also performed to detect V. anguillarum cells. Results indicated that the sensitivity of indirect ELISA to bacteria cells is higher than that of the dot-ELISA, and that the minimal detectable amount is approximately 10(4) cell/mL in the indirect ELISA, while 10(5) cell/mL in the dot-ELISA.

Molecular cloning, characterization and expression of a serine protease with clip-domain homologue from scallop Chlamys farreri

Serine proteases play critical roles in a variety of invertebrate immune defense responses, including hemolymph coagulation, antimicrobial peptide synthesis, and melanization. The first mollusk serine protease with clip-domain (designated CFSP1) cDNA was obtained from the scallop Chlamys farreri challenged with Vibrio anguillarum by randomly sequencing a whole tissue cDNA library and rapid amplification of cDNA ends (RACE). The full-length cDNA of the C. farreri serine protease was 1211 bp, consisting of a 5-terminal untranslated region (UTR) of 72 bp, a 3'-terminal UTR of 77 bp with a canonical polyadenylation signal sequence AATAAA and a poly (A) tail, and an open reading frame of 1062 bp. The CFSP1 cDNA encoded a polypeptide of 354 amino acids with a putative signal peptide of 19 amino acids and a mature protein of 335 amino acids. The deduced amino acid sequence of CFSP1 contained an amino-terminal clip domain, a low complexity region, and a carboxyl-terminal serine protease domain. CFSP1 mRNA was mainly expressed constitutively in the hemocytes and was up-regulated and increased 2.9- and 1.9-fold at 16 h after injury and injection of bacteria. (c) 2006 Elsevier Ltd. All rights reserved.

Microbial modulation in the biomass and toxin production of a red-tide causing alga

The effect of S-10, a strain of marine bacteria isolated from sediment in the Western Xiamen Sea, on the growth and paralytic shellfish poison (PSP) production in the alga Alexandrium tamarense (A. tamarense) was studied under controlled experimental conditions. The results of these experiments have shown that the growth of A. tamarense is obviously inhibited by S-10 at high concentrations, however no evident effect on its growth was observed at low concentrations. Its PSP production was also inhibited by S 10 at different concentrations, especially at low concentrations. The toxicity of this strain of A. tamarense is about (0.9512.14) x 10(-6) MU/cell, a peak toxicity value of 12.14 x 10(-6) MU/cell appeared on the 14th day, after which levels decreased gradually. The alga grew well in conditions of pH 6-8 and salinities of 20-34 parts per thousand. The toxicity of the alga varied markedly at different pH and salinity levels. Toxicity decreased as pH increased, while it increased with salinity and reached a peak value at a salinity of 30 parts per thousand, after which it declined gradually. S-10 at a concentration of 1.02 x 10(9) cells/ml inhibited growth and the PSP production of A. tamarense at different pH and salinity levels. S-10 had the strongest inhibitory function on the growth of A. tamarense under conditions of pH 7 and a salinity of 34 parts per thousand. The best inhibitory effect on PSP production by A. tamarense was at pH 7, this inhibitory effect on PSP production did not relate to salinity. Interactions between marine bacteria and A. tamarense were also investigated using the flow cytometer technique (FCM) as well as direct microscope counting. S-10 was identitied as being a member of the genus Bacillus, the difference in 16S rDNA between S-10 and Bacillus halmapalus was only 2%. The mechanism involved in the inhibition of growth and PSP production of A. tamarense by this strain of marine bacteria, and the prospect of using it and other marine bacteria in the biocontrol of red-tides was discussed. (c) 2005 Elsevier Ltd. All rights reserved.

Seasonal variations in growth and clearance rate of the Zhikong scallop Chlamys farreri suspended in the deep water of Haizhou Bay, China

The growth and survival of the Zhikong scallop Chlamys farreri suspended in deep water of Haizhou Bay were studied from July 2007 to June 2008, and the biodeposition method was used to estimate the clearance rate of C. farreri under field conditions. Results showed that the scallop grew fast during all the culture time, with the exception of summer. The condition index of the scallop increased with time and reached the highest value in spring of the second year. The survival of scallops was 60.8 +/- A 3.9% at the end of this study, mortality occurring mainly during the summer and autumn of the first year. The clearance rate fluctuated obviously with season,with the highest value in September 2007, and the lowest value in March 2008. Factors accounting for variations in growth and clearance rate of scallops are also discussed.

Survival, growth and immune activity of scallop Chlamys farreri cultured at different depths in Haizhou Bay (Yellow Sea, China) during hot season

Survival, growth and immune response of the scallop, Chlamys farreri, cultured in lantern nets at five different depths (2, 5, 10, 15, and 20 m below the sea surface) were studied in Haizhou Bay during the hot season (summer and autumn) of 2007. Survival and growth rates were quantified bimonthly. Immune activities in hemolymph (superoxide dismutase (SOD) and acid phosphatase (ACP)) were measured to evaluate the health of scallops at the end of the study. Environmental parameters at the five depths were also monitored during the experiment. Mortalities mainly occurred during summer. Survival of scallops suspended at 15 m (78.0%) and 20 m (86.7%) was significantly higher than at 2 m (62.9%), 5 m (60.8%) or 10 m (66.8%) at the end of the study. Mean shell height grew significantly faster at 10 m (205.0 mu m/d) and 20 m (236.9 mu m/d) than at 2, 5 or 15 m in summer (July 9 to September 1); however, shell growth rate at 20 m was significantly lower than at the other four depths in autumn (September 2 to November 6). In contrast to summer, scallops at 5 m grew faster (262.9 mu m/d) during autumn. The growth of soft tissue at different depths showed a similar trend to the shell. Growth rates of shell height and soft tissue were faster in autumn than in summer, with the exception of shell height at 20 m. SOD activity of scallops increased with depth, and ACP activity was significantly higher at 15 and 20 m than at other depths, which suggests that scallops were healthier near the bottom. Factors explaining the depth-related mortality and growth of scallops are also discussed. We conclude that the mass mortality of scallop, C. farreri, during summer can be prevented by moving the culture area to deeper water and yield can be maximized by suspending the scallops in deep water during summer and then transferring them to shallow water in autumn.

Fucoidan protects against dopaminergic neuron death in vivo and in vitro

Parkinson's disease is a neurodegenerative disorder of uncertain pathogenesis characterized by a loss of dopaminergic neurons in substantia nigra pars compacta, and can be modeled by the neurotoxin 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP). Oxidative stress may contribute to MPTP- and Parkinson's disease-related neurodegeneration. Fucoidan is a sulfated polysaccharide extracted from brown seaweeds which possesses a wide variety of biological activities including potent antioxidative effects. Here we investigated the effect of fucoidan treatment on locomoter activities of animals, striatal dopamine and its metabolites and survival of nigral dopaminergic neurons in MPTP-induced animal model of Parkinsonism in C57/BL mice in vivo and on the neuronal damage induced by 1-methyl-4-phenylpyridinium (MPP+) in vitro, and to study the possible mechanisms. When administered prior to MPTP, fucoidan reduced behavioral deficits, increased striatal dopamine and its metabolites levels, reduced cell death, and led to a marked increase in tyrosine hydroxylase expression relative to mice treated with MPTP alone. Furthermore, we found that fucoidan inhibited MPTP-induced lipid peroxidation and reduction of antioxidant enzyme activity. In addition, pre-treatment with fucoidan significantly protected against MPP+-induced damage in MN9D cells. Taken together, these findings suggest that fucoidan has protective effect in MPTP-induced neurotoxicity in this model of Parkinson's disease via its antioxidative activity. (C) 2009 Elsevier B.V. All rights reserved.

Impact of astaxanthin-enriched algal powder of Haematococcus pluvialis on memory improvement in BALB/c mice

The impact of astaxanthin-enriched algal powder on auxiliary memory improvement was assessed in BALB/c mice pre-supplemented with different dosages of cracked green algal (Haematococcus pluvialis) powder daily for 30 days. The supplemented mice were first tested over 8 days to find a hidden platform by swimming in a Morris water maze. Then, for 5 days, the mice were used to search for a visible platform in a Morris water maze. After that, the mice practised finding a safe place-an insulated platform in a chamber-for 2 days. During these animal experimental periods, similar algal meals containing astaxanthin at 0, 0.26, 1.3 and 6.4 mg/kg body weight were continuously fed to each group of tested mice. Profiles of latency, distance, speed and the direction angle to the platforms as well as the diving frequency in each group were measured and analyzed. The process of mice jumping up onto the insulated platform and diving down to the copper-shuttered bottom with a 36 V electrical charge were also monitored by automatic video recording. The results of the Morris maze experiment showed that middle dosage of H. pluvialis meals (1.3 mg astaxanthin/kg body weight) significantly shortened the latency and distance required for mice to find a hidden platform. However, there was no obvious change in swim velocity in any of the supplemented groups. In contrast, the visible platform test showed a significant increase in latency and swim distance, and a significant decrease in swim speed for all groups of mice orally supplemented with H. pluvialis powder compared to the placebo group (P < 0.05 or P < 0.01). Mice supplemented with the algal meal hesitantly turned around the original hidden platform, in contract to mice supplemented with placebo, who easily forgot the original location and accepted the visible platform as a new safe place. These results illustrate that astaxanthin-enriched H. pluvialis powder has the auxiliary property of memory improvement. The results from the platform diving test showed that the low and middle dosage of H. pluvialis powder, rather that the high dosage, increased the latency and reduced the frequency of diving from the safe insulated platform to the electrically stimulated copper shutter, especially in the low treatment group (P < 0.05). These results indicate that H. pluvialis powder is associated with dose-dependent memory improvement and that a low dosage of algal powder (<= middle treatment group) is really good for improving the memory.

Effects of different seed pretreatments on germination of ten Calligonum species

In order to determine the optimum seed pre??sowing treatments and patterns of germination,a greenhouse experiment was conducted to study the effects of abrasion,sulphuric acid,boiling water,cold stratification and seed exudate treatments on the germination of ten Calligonum species.The results showed that the response of seed germination to the different pretreatments was similar for all ten Calligonum species.The abrasion,sulphuric acid and cold stratification treatments significantly promoted overall germinability.Compared with the control,the exudate treatment significantly decreased the percent germination,hampered the rate of germination and bolstered dormancy for almost all species.The cold stratification treatment can break the dormancy of viable Calligonum seeds and increased the germination,but it has a little lethal effect on viable seeds probably as well as the boiling water treatments.The germinaton patterns performed significant difference between the pre-sowing treatments for all ten Calligonum species,and the speed and percent germination of the Calligonum species can be greatly increased by mechanical scarification or sulphuric acid treatments.The results showed that seeds of C.junceum have strong ability of germinating.

Effect of plateau zokors on vegetation characteristics and productivity of alpine meadow

This research was conducted on alpine meadow site at Menyuan county, Qinghai Province, People's Republic of China to determine the effects of native, subterranean rodent of Qinghai-Tibet grasslands, the plateau zokors (Myospalax baileyi), on seasonal above-and below-ground plant biomass, plant species diversity and productivity. Both total peaks of above-and below-ground biomass were the greatest (413.600 g/m~2 and 2297.502 g/m~2) in the patch no any plateau zokors colonized by plateau zokors over 10 years in August and October, respectively. Both above-and below-ground biomass were significantly increased in the patches where plateau zokors were removed or the burrow systems were abandoned for five years compared to the patches plateau zokors colonized over 10 years. However, both above-and below-ground biomass in abandoned patches were significantly lower than that in uncolonized patches. Monocotyledonous biomass was reduced greatly, but the non-palatable dicots were significantly increased in colonized patches. The palatable biomass of monocots and dicots were increased in abandoned patches. Total plant species diversity was the greatest in uncolonized patchesand least in abandoned patch. The total net primary production in colonized patches was reduced by 68.98% compared with uncolonized patches. Although the patches were without any plateau zokors disturbance for fives years, the total net primary production just reached 58.69% of the uncolonized patches. The above-ground net primary production in abandoned patches increased 28.74% and the below-ground increased 54.91% compared with the colonized patches. We suggest that plateau zokor-induced changes in plant above- and below-ground biomass and species diversity may lead to further alterations of nutrient cycling and trophic dynamics in this alpine meadow ecosystem.

Competition between Stipa grandis and Cleistogenes squarrosa

Competitive strategy for resources between Cleistogenes squarrosa (Trin.) Keng which is a dominant species of grasslands degraded by moderate-heavy grazing, and Stipa grandis P. Smirnov, which is a dominant species of ungrazed communities, was studied using a replacement series method in a greenhouse. The knowledge would be helpful in managing grasslands and restoring the degraded C. squarrosa grassland. Although there was neither inter- nor intra-specific competition between the two species when no nutrients were added, intra-specific competition of C. squarrosa was observed and increased with increased nutrient availability and more sulfur (S) was allocated to the aboveground partition of the plant. Relative competitive ability of C. squarrosa was greater than that of S. grandis when nutrients were supplied regardless of S. There was no significant difference between shoot and root competition based on dry matter yields. However, root competition was significantly greater than that of shoot based on S uptake under all treatments. A significant interaction was not observed between shoot and root competition. Therefore, nutrients addition benefits the restoration of degraded grassland of C. squarrosa, which may not exclude S. grandis. Also productivity and forage quality of the community will be increased. (C) 2007 Elsevier Ltd. All rights reserved.

Seasonal and interannual variation in water vapor and energy exchange over a typical steppe in Inner Mongolia, China

In this study, we conducted eddy covariance (EC) measurements of water vapor exchange over a typical steppe in a semi-arid area of the Inner Mongolia Plateau, China. Measurement sites were located within a 25-year-old enclosure with a relatively low leaf area index (similar to 1. 5 m(2) m(-2)) and dominated by Leymus chinensis. Energy balance closure was (H + LE) = 17.09 + 0.69 x (Rn - G) (W/m(2); r(2) = 0.95, n = 6596). Precipitation during the two growing seasons of the study period was similar to the long-term average. The peak evapotranspiration in 2004 was 4 mm d(-1), and 3.5 mm d(-1) in 2003. The maximum latent heat flux was higher than the sensible heat flux, and the sensible heat flux dominated the energy budget at midday during the entire growing season in 2003; latent heat flux was the main consumption component for net radiation during the 2004 growing season. During periods of frozen soil in 2003 and 2004, the sensible heat flux was the primary consumption component for net radiation. The soil heat flux component was similar in 2003 and 2004. The decoupling coefficient (between 0.5 and 0.1) indicates that evapotranspiration was strongly controlled by saturation water vapor pressure deficit (VPD) in this grassland. The results of this research suggest that energy exchange and evapotranspiration were controlled by the phenology of the vegetation and soil water content. In addition, the amount and frequency of rainfall significantly affect energy exchange and evapotranspiration upon the Inner Mongolia plateau. (c) 2007 Published by Elsevier B.V.

Reproduction and behavior of plateau pikas (Ochotona curzoniae Hodgson) under predation risk: A field experiment

The mixture of the feces and urine of the red fox (Vulpes vulpes Linnaeus) was used to increase the perception of predation risk of plateau pikas (Ochotona curzoniae Hodgson) in the field. The influence of the predation risk on the reproduction and behavior of plateau pikas was examined through comparing reproductive characteristics and five different kinds of behavior between treatment and control plots. The results showed that 1) the body weight of the pikas was not significantly different between treatment and control plots. 2) The reproductive period of the pikas extended from March to later August in both treatment and control plots. The pregnant ratio, developed testes ratio, reproductive success and sex ratio of the pikas were not significantly different between the treatment and control plots. 3) The pikas increased their observing and calling frequencies and decreased their moving and feeding frequencies when exposed to red fox's feces and urine. 4) The increased red fox's feces and urine had no influence on the behavior of the pikas when the number of their natural enemies increased; the pikas obviously increased the observing frequencies and sharply decreased the calling frequency so as to decrease the direct predation risk. 5) There were no significantly behavioral differences between males and females as well as between adults and young. 6) The results reject the hypothesis 1 that the red fox's feces and urine as indirect predation risk suppresses the reproduction of the pikas and support the hypothesis 2 that the pikas can make decision by changing behavior to avoid the predation risk they encountered whenever.