Long-term field fertilization alters the diversity of autotrophic bacteria based on the ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO) large-subunit genes in paddy soil

Carbon dioxide (CO₂) assimilation by autotrophic bacteria is an important process in the soil carbon cycle with major environmental implications. The long-term impact of fertilizer on CO₂ assimilation in the bacterial community of paddy soils remains poorly understood. To narrow this knowledge gap, the composition and abundance of CO₂-assimilating bacteria were investigated using terminal restriction fragment length polymorphism and quantitative PCR of the cbbL gene [that encodes ribulose-1,5-biphosphate carboxylase/oxygenase (RubisCO)] in paddy soils. Soils from three stations in subtropical China were used. Each station is part of a long-term fertilization experiment with three treatments: no fertilizer (CK), chemical fertilizers (NPK), and NPK combined with rice straw (NPKM). At all of the stations, the cbbL-containing bacterial communities were dominated by facultative autotrophic bacteria such as Rhodopseudomonas palustris, Bradyrhizobium japonicum, and Ralstonia eutropha. The community composition in the fertilized soil (NPK and NPKM) was distinct from that in unfertilized soil (CK). The bacterial cbbL abundance (3–8?×?10⁸ copies g soil^?1) and RubisCO activity (0.40–1.76 nmol CO₂ g soil^?1 min^?1) in paddy soils were significantly positively correlated, and both increased with the addition of fertilizer. Among the measured soil parameters, soil organic carbon and pH were the most significant factors influencing the community composition, abundance, and activity of the cbbL-containing bacteria. These results suggest that long-term fertilization has a strong impact on the activity and community of cbbL-containing bacterial populations in paddy soils, especially when straw is combined with chemical fertilizers.     

Production of 3-O-xylosyl quercetin in Escherichia coli

Quercetin, a flavonol aglycone, is one of the most abundant flavonoids with high medicinal value. The bioavailability and pharmacokinetic properties of quercetin are influenced by the type of sugars attached to the molecule. To efficiently diversify the therapeutic uses of quercetin, Escherichia coli was harnessed as a production factory by the installation of various plant and bacterial UDP-xylose sugar biosynthetic genes. The genes encoding for the UDP-xylose pathway enzymes phosphoglucomutase (nfa44530), glucose-1-phosphate uridylyltransferase (galU), UDP-glucose dehydrogenase (calS8), and UDP-glucuronic acid decarboxylase (calS9) were overexpressed in E. coli BL21 (DE3) along with a glycosyltransferase (arGt-3) from Arabidopsis thaliana. Furthermore, E. coli BL21(DE3)/?pgi, E. coli BL21(DE3)/?zwf, E. coli BL21(DE3)/?pgi?zwf, and E. coli BL21(DE3)/?pgi?zwf?ushA mutants carrying the aforementioned UDP-xylose sugar biosynthetic genes and glycosyltransferase and the galU-integrated E. coli BL21(DE3)/?pgi host harboring only calS8, calS9, and arGt-3 were constructed to enhance whole-cell bioconversion of exogeneously supplied quercetin into 3-O-xylosyl quercetin. Here, we report the highest production of 3-O-xylosyl quercetin with E. coli BL21 (DE3)/?pgi?zwf?ushA carrying UDP-xylose sugar biosynthetic genes and glycosyltransferase. The maximum concentration of 3-O-xylosyl quercetin achieved was 23.78 mg/L (54.75 μM), representing 54.75 % bioconversion, which was an ~4.8-fold higher bioconversion than that shown by E. coli BL21 (DE3) with the same set of genes when the reaction was carried out in 5-mL culture tubes with 100 μM quercetin under optimized conditions. Bioconversion was further improved by 98 % when the reaction was scaled up in a 3-L fermentor at 36 h.     

Effect of long-term different fertilization on bacterial community structures and diversity in citrus orchard soil of volcanic ash

This study was conducted to assess bacterial species richness, diversity and community distribution according to different fertilization regimes for 16 years in citrus orchard soil of volcanic ash. Soil samples were collected and analyzed from Compost (cattle manure, 2,000 kg/10a), 1/2 NPK+compost (14-20-14+2,000 kg/10a), NPK+compost (28-40-28+2,000 kg/10a), NPK (28-40-28 kg/10a), 3 NPK (84-120-84 kg/10a), and Control (no fertilization) plot which have been managed in the same manners with compost and different amount of chemical fertilization. The range of pyrosequencing reads and OTUs were 4,687–7,330 and 1,790–3,695, respectively. Species richness estimates such as Ace, Chao1, and Shannon index were higher in 1/2 NPK+compost than other treatments, which were 15,202, 9,112, 7.7, respectively. Dominant bacterial groups at level of phylum were Proteobacteria, Acidobacteria, and Actinobacteria. Those were occupied at 70.9% in 1/2 NPK+compost. Dominant bacterial groups at level of genus were Pseudolabrys, Bradyrhizobium, and Acidobacteria. Those were distributed at 14.4% of a total of bacteria in Compost. Soil pH displayed significantly closely related to bacterial species richness estimates such as Ace, Chao1 (p<0.05) and Shannon index (p<0.01). However, it showed the negative correlation with exchangeable aluminum contents (p<0.05). In conclusion, diversity of bacterial community in citrus orchard soil was affected by fertilization management, soil pH changes and characteristics of volcanic ash.     

长期有机无机肥配施对稻田杂草生长动态的影响

利用中国农业科学院红壤实验站红壤稻田长期定位试验,于2011年研究了在以无机肥(化肥NPK)与有机肥(M)氮磷钾养分等量条件下,长期有机无机肥配施水稻生育期间杂草种类和生物量变化。结果表明:30a后,早稻和晚稻施肥处理中:PK+M处理下杂草种类最多,NPK+M处理下杂草种类和优势杂草种类较少,且种类数量稳定,早稻和晚稻杂草种类数量前者比后者分别高出19.7%和9.8%;施肥处理中:两季杂草总生物量NPK+M处理最高,NP+M处理最低,且NPK+M、NK+M、PK+M和CK处理比NP+M处理分别高出31.3%、26.5%、8.3%和5.6%,早稻NPK+M处理杂草总生物量和浮生杂草总生物量最多,NK+M处理湿生杂草总生物量最多,晚稻NK+M处理杂草总生物量和湿生杂草总生物量最多,NPK+M处理浮生杂草总生物量最多;土壤碱解氮和有效磷与杂草总生物量、湿生杂草总生物量、浮生杂草总生物量显著正相关(相关系数依次分别为0.508*和0.578**、0.552*和0.453*、0.410*和0.802**),p H值与三者显著负相关(相关系数依次分别为-0.516*、-0.531*和-0.698*)。土壤p H受土壤有效磷和碱解氮及其他因子的共同作用对杂草总生物量产生影响。通过施肥措施调节土壤适宜p H及碱解氮和有效磷含量,能有效调控农田中湿生和浮生杂草生长,使杂草种类和生物量在农业生产中达到有益平衡。     

Differential response of radish plants to supplemental ultraviolet-B radiation under varying NPK levels: chlorophyll fluorescence, gas exchange and antioxidants

Current and projected increases in ultraviolet‐B (UV‐B; 280–315 nm) radiation may alter crop growth and yield by modifying the physiological and biochemical functions. This study was conducted to assess the possibility of alleviating the negative effects of supplemental UV‐B (sUV‐B; 7.2 kJ m^?2 day^?1; 280–315 nm) on radish (Raphanus sativus var Pusa Himani) by modifying soil nitrogen (N), phosphorus (P) and potassium (K) levels. The N, P and K treatments were recommended dose of N, P and K, 1.5 times recommended dose of N, P and K, 1.5 times recommended dose of N and 1.5 times recommended dose of K. Plants showed variations in their response to UV‐B radiation under varying soil NPK levels. The minimum damaging effects of sUV‐B on photosynthesis rate and stomatal conductance coupled with minimum reduction in chlorophyll content were recorded for plants grown at recommended dose of NPK. Flavonoids increased under sUV‐B except in plants grown at 1.5 times recommended dose of N. Lipid peroxidation (LPO) also increased in response to sUV‐B at all NPK levels with maximum at 1.5 times recommended dose of K and minimum at recommended dose of NPK. This study revealed that sUV‐B radiation negatively affected the radish plants by reducing the photosynthetic efficiency and increasing LPO. The plants grown at 1.5 times recommended dose of NPK/N/K could not enhance antioxidative potential to the extent as recorded at recommended dose of NPK and hence showed more sensitivity to sUV‐B.     

Optimizing cadmium and mercury specificity of CadR-based E. coli biosensors by redesign of CadR

The metalloprotein, CadR, was redesigned to optimize cadmium and mercury specificity of CadR-based E. coli biosensors. By truncating 10 and 21 amino acids from the C-terminal extension of CadR, CadR-TC10 and CadR-TC21 were obtained, respectively. The genes cadR, cadR-TC10 and cadR-TC21 were used as sensing elements to construct green fluorescent protein based E.coli biosensors. Induction at 30 °C for 4 h in supplemented M9 medium was the optimized condition for the biosensor. Compared with CadR-based biosensor, there was a clear decline in induction coefficient for CadR-TC21-based biosensor (decreased by 86 % in Zn(II), 44 % in Hg(II), and only 37 % in Cd(II)). While in CadR-TC10-based biosensor, the induction coefficient decreased by 95 % in Zn(II), 70 % in Hg(II), and 67 % in Cd(II). Improved performances of CadR mutants based E. coli biosensors indicated that truncating C-terminal extension of CadR could improve the specificity.     

Impact of ultraviolet-B radiation on photosynthetic capacity,antioxidative potential and metabolites in Solanum tuberosum L. under varying levels of soil NPK

The impact of supplemental ultraviolet-B (sUV-B; 280–315 nm; +7.2 kJ m^?2 d^?1) radiation was studied on various physiological parameters, antioxidative potential and metabolites of Solanum tuberosum L. cv. Kufri Badshah plants under varying levels of soil NPK. The N, P and K treatments were: the recommended dose of N, P and K; 1.5 times the recommended dose of N, P and K; 1.5 times the recommended dose of N and 1.5 times the recommended dose of K. The recommended NPK level provided maximum protection to photosynthetic assimilation under sUV-B radiation, while stomatal conductance was best at 1.5 times the recommended NPK. Carbon dioxide assimilation declined maximally at 1.5 times the recommended N/K under sUV-B radiation. Plants grown at the recommended NPK and 1.5 times the recommended NPK levels showed higher superoxide dismutase, peroxidase and ascorbate peroxidase activities under sUV-B radiation compared to 1.5 times the recommended N/K levels. sUV-B significantly increased total phenolics and flavonoids in plants at the recommended and 1.5 times the recommended NPK, while flavonoids declined at 1.5 times the recommended N. This study clearly showed that NPK amendment provided maximum protection to photosynthetic assimilation of potato plants under sUV-B radiation, activating the antioxidative defense system as well as flavonoids. NPK at 1.5 times the recommended dose, however, did not cause any additional benefit to photosynthetic carbon fixation; hence the recommended dose of NPK is found to be the best suited dose of fertilizer under ambient as well as sUV-B regime.     

Novel Cyanobacterial Biosensor for Detection of Herbicides

The aim of this work was to generate a cyanobacterial biosensor that could be used to detect herbicides and other environmental pollutants. A representative freshwater cyanobacterium, Synechocystis sp. strain PCC6803, was chromosomally marked with the luciferase gene luc (from the firefly Photinus pyralis) to create a novel bioluminescent cyanobacterial strain. Successful expression of the luc gene during growth of Synechocystis sp. strain PCC6803 cultures was characterized by measuring optical density and bioluminescence. Bioluminescence was optimized with regard to uptake of the luciferase substrate, luciferin, and the physiology of the cyanobacterium. Bioassays demonstrated that a novel luminescent cyanobacterial biosensor has been developed which responded to a range of compounds including different herbicide types and other toxins. This biosensor is expected to provide new opportunities for the rapid screening of environmental samples or for the investigation of potential environmental damage.     

Manure and mineral fertilization change enzyme activity and bacterial community in millet rhizosphere soils

Fertilization is a key agricultural practice for increasing millet yields and influencing soil properties, enzyme activities and rhizosphere bacterial communities. High throughput Illumina sequencing of the 16S rDNA was applied to compare the bacterial community structures and diversities among six different soil samples. The experiments involved the following: no fertilizer (CK), phosphate (P) and potassium (K) plus organic manure (M) (PKM), nitrogen (N) and K plus M (NKM), NPM, NPK and NPKM fertilization. The results showed that the NPKM fertilization of the millet field had a maximal yield of 3617 kg ha^?1 among the six different treatments. The abundances of the Actinobacteria and Bacteroidetes phyla, especially the Devosia, Mycobacterium, Opitutus and Chitinophaga genera, were higher in NPKM than those in the other samples. Redundancy analysis showed that the soil organic matter (SOM), available phosphorus (AP), and urease (UR) activity were significantly correlated with bacterial communities, while SOM and AP were strongly correlated with soil enzyme activities. Pearson’s correlation showed that the available nitrogen was strongly correlated with Devosia and Mycobacterium, and SOM was strongly correlated with Opitutus and Chitinophaga. Besides, catalase was significantly related to Iamia, the UR was significantly related to Devosia, phosphatase was significantly related to Luteimonas and Chitinophaga. Based on the soil quality and millet yield, NPKM treatment was a better choice for the millet field fertilization practices. These findings provide a better understanding of the importance of fertilization in influencing millet yield, soil fertility and microbial diversity, and they lead to a choice of scientific fertilization practices for sustainable development of the agroecosystem.     

An improved embryo-rescue protocol for hybrid progeny from seedless Vitis vinifera grapes × wild Chinese Vitis species

A highly efficient technique of embryo rescue is critical when using stenospermocarpic Vitis vinifera cultivars (female parents) to breed novel, disease-resistant, seedless grape cultivars by hybridizing with wild Chinese Vitis species (male parents) having many disease-resistance alleles. The effects of various factors on the improvement of embryo formation, germination, and plantlet development for seven hybrid combinations were studied. The results indicated that Beichun and Shuangyou were the best male parents. The best sampling time for ovule inoculation differed among the female parents. When hybrid ovules were cultured on a double-phase medium with five different solid medium types, percent embryo formation was highest (11.3–28.3%) on a modified MM3 medium. Percentages of embryo germination (15.4–55.4%) and plantlet development (11.15–44.6%) were all highest when embryos were cultured on Woody Plant Medium?+?5.7 μM indole-3-acetic acid?+?4.4 μM 6-benzylaminopurine?+?1.4 μM gibberellic acid?+?2% sucrose?+?0.05% casein hydrolysate?+?0.3% activated charcoal?+?0.7% agar. In the absence of other amino acids, the addition of proline significantly increased embryo formation (36.1%), embryo germination (64.6%), and plantlet development (90.5%). A highly efficient protocol has been developed for hybrid embryo rescue from seedless V. vinifera grapes?×?wild Chinese Vitis species that results in a significant improvement in breeding efficiency for new disease-resistant seedless grapes.     

Interaction between the Microbial Community and Invading Escherichia coli O157:H7 in Soils from Vegetable Fields

The survival of Escherichia coli O157:H7 in soils can contaminate vegetables, fruits, drinking water, etc. However, data on the impact of E. coli O157:H7 on soil microbial communities are limited. In this study, we monitored the changes in the indigenous microbial community by using the phospholipid fatty acid (PLFA) method to investigate the interaction of the soil microbial community with E. coli O157:H7 in soils. Simple correlation analysis showed that the survival of E. coli O157:H7 in the test soils was negatively correlated with the ratio of Gram-negative (G⁻) to Gram-positive (G⁺) bacterial PLFAs (G⁻/G⁺ ratio). In particular, levels of 14 PLFAs were negatively correlated with the survival time of E. coli O157:H7. The contents of actinomycetous and fungal PLFAs in the test soils declined significantly (P, <0.05) after 25 days of incubation with E. coli O157:H7. The G⁻/G⁺ ratio declined slightly, while the ratio of bacterial to fungal PLFAs (B/F ratio) and the ratio of normal saturated PLFAs to monounsaturated PLFAs (S/M ratio) increased, after E. coli O157:H7 inoculation. Principal component analysis results further indicated that invasion by E. coli O157:H7 had some effects on the soil microbial community. Our data revealed that the toxicity of E. coli O157:H7 presents not only in its pathogenicity but also in its effect on soil microecology. Hence, close attention should be paid to the survival of E. coli O157:H7 and its potential for contaminating soils.     

长期不同施肥处理对苹果产量、品质及土壤肥力的影响

通过连续7年(2003-2010年)的田间定位试验,研究了不同施肥处理\[不施肥对照,CK;不施N肥只施PK肥,PK;不施P肥只施NK肥,NK;不施K肥只施NP肥,NP;单施NPK化肥,NPK;单施有机肥(猪粪),M;化肥有机肥配施(化肥有机肥氮各占一半),NPKM\]对渭北旱塬富士苹果产量、品质及果园土壤养分含量变化的影响.结果表明: 施肥可以提高苹果产量,连续7年不同施肥处理苹果平均产量较对照提高14.4%～63.8%,各处理苹果年平均产量顺序为:NPKM＞NPK≥M＞NP≥NK＞PK＞CK.NPKM、M、NPK处理随着试验时间的推移,果实可溶性糖、维生素C、可溶性固形物含量呈上升趋势,NPKM、M处理不同年际间波动相对较小;NPKM处理糖酸比5年较对照提高了30.9%,维生素C含量提高了17.5%.长期合理施肥有利于提高土壤有机质,NPKM、M处理0～20 cm土层有机质含量提高幅度最大,分别提高了28.8%和29.3%. NPK、NPKM、M处理土壤各层速效氮、有效磷、速效钾含量较试验前均有显著提高,NPK处理0～20 cm、20～40 cm和40～60 cm土层速效氮含量分别提高了22.7%、37.3%和53.4%.与NPK处理相比,NPKM处理的土壤速效磷含量提高了18.7%,且不同处理土壤速效磷含量上层显著大于下层.
     

Potassium release characteristics, potassium balance, and fingermillet (Eleusine coracana G.) yield sustainability in a 27- year long experiment on an Alfisol in the semi-arid tropical India

Aims

In Alfisols, potassium (K) deficiency limits productivity, as these soils are poor in K-bearing minerals such as mica. As nutrient management practices greatly influence K nutrition of crops especially in the longer term, we evaluated the effects of 27 (1978–2004) years of cropping fingermillet (Eleusine coracana G.) under different manure and mineral fertilizer treatments on K release, balance and yield sustainability on K deficient Alfisols in the semi-arid tropical region of southern India.

Methods

Fingermillet (variety: PR-202) was grown each year under rainfed conditions with 5 different nutrient management treatments: control (no amendment), 10 Mg ha^?1 farm yard manure (FYM), 10 Mg ha^?1 FYM +50 % NPK, 10 Mg ha^?1 FYM +100 % NPK and 100 % NPK. Potassium release characteristics in the soil profile were determined using 1 N boiling HNO₃ (strong extracting solution), 0.01 M HCl (medium extracting solution) and 0.01 M CaCl₂ (mild extracting solution).

Results

Continuous cropping of Alfisols for 27 years resulted in a decrease in K supplying capacity due to soil K depletion through crop K uptake. In soils without K addition, inherent soil supply could not meet the K requirement of fingermillet; thus, a negative K balance following 27 years of cropping affected K nutrition of the crop in all the treatments. As a result, the highest sustainable yield index (SYI) was observed using an integrated nutrient supply (combined application of nutrients from organic and inorganic sources), and the lowest index was obtained without K additions.

Conclusion

For balanced nutrient management in cereal production systems, K nutrition needs urgent attention in the K deficient Alfisol region of southern India. Addition of any amount of organic manures available at field level offers an alternative strategy for maintaining soil K fertility to improve and sustain crop productivity.     

Additive effect of heparin on the photoinactivation of Escherichia coli using tricationic P-porphyrins

Polycationic porphyrins have received substantial attention in developing singlet oxygen-sensitizers for biological use such as in the photoinactivation of bacteria and photodynamic therapy (PDT) of tumor cells because they have strong binding affinities for DNA and proteins. However, these strong cellular interactions can retard elimination of the drug after PDT. Therefore, the studies on the interactions of porphyrins with other molecules present much interest, in order to modulate the sensitizers’ activity or even remove them from the human body after PDT. Here, we studied the additive effect of heparin on the photoinactivation by polycationic porphyrins using Escherichia coli as a model cell. Tricationic P-porphyrin sensitizers substituted with an N-alkylpyridinium group (alkyl?=?pentyl (1a), hexyl (1b), and heptyl (1c)) or N-hexylammonium (1d) as the axial ligand were used. Additionally, dicationic Sb-porphyrin substituted with an N-hexylpyridinium group (1e) was prepared. We studied the additive effect of heparin on the photoinactivation of E. coli by 1a–1e. The bactericidal activities were evaluated using the half-life (T_1/2 in min) of E. coli and the minimum effective concentrations ([P]) of the porphyrin sensitizers. In the absence of heparin, the [P] values were determined to be 0.4–0.5?μM for 1a?1c and 2.0?μM for 1d?1e. The bactericidal activity of 1a?1c was completely retarded by the addition of heparin (1.0?μM). However, the addition of heparin (1.0?μM) could not completely retard the bactericidal activity of 1d?1e whose [P] values were relatively large. It is suggested that tricationic 1a?1c adsorbed onto the anionic heparin through electrostatic interactions. The adsorption of 1 on heparin disturbs the uptake of 1 into E. coli cells. Thus, the addition of heparin was found to be a useful method for retarding photoinactivation.     

Rapid label-free detection of E. coli using a novel SPR biosensor containing a fragment of tail protein from phage lambda

Abstract

In efforts to speed up the assessment of microorganisms, researchers have sought to use bacteriophages as a biosensing tool, due to their host-specificity, wide abundance, and safety. However, the lytic cycle of the phage has limited its efficacy as a biosensor. Here, we cloned a fragment of tail protein J from phage lambda and characterized its binding with the host, E. coli K-12, and other microorganism. The N-terminus of J was fused with a His-tag (6HN-J), overexpressed, purified, and characterized using anti-His monoclonal antibodies. The purified protein demonstrated a size of ～38?kDa upon SDS-PAGE and bound with the anti-His monoclonal antibodies. ELISA, dot blot, and TEM data revealed that it specifically bound to E. coli K-12, but not to Pseudomonas aeruginosa. The observed protein binding occurred over a concentration range of 0.01–5?μg/ml and was found to inhibit the in vivo adsorption of phage to host cells. This specific binding was exploited by surface plasmon resonance (SPR) to generate a novel 6HN-J-functionalized SPR biosensor. This biosensor showed rapid label-free detection of E. coli K-12 in the range of 2?×?10⁴ ?2?×?10⁹ CFU/ml, and exhibited a lower detection limit of 2?×?10⁴ CFU/ml.     

Construction of a ColD cda Promoter-Based SOS-Green Fluorescent Protein Whole-Cell Biosensor with Higher Sensitivity toward Genotoxic Compounds than Constructs Based on recA, umuDC, or sulA Promoters

Four different green fluorescent protein (GFP)-based whole-cell biosensors were created based on the DNA damage inducible SOS response of Escherichia coli in order to evaluate the sensitivity of individual SOS promoters toward genotoxic substances. Treatment with the known carcinogen N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) revealed that the promoter for the ColD plasmid-borne cda gene had responses 12, 5, and 3 times greater than the recA, sulA, and umuDC promoters, respectively, and also considerably higher sensitivity. Furthermore, we showed that when the SOS-GFP construct was introduced into an E. coli host deficient in the tolC gene, the minimal detection limits toward mitomycin C, MNNG, nalidixic acid, and formaldehyde were lowered to 9.1 nM, 0.16 μM, 1.1 μM, and 141 μM, respectively, which were two to six times lower than those in the wild-type strain. This study thus presents a new SOS-GFP whole-cell biosensor which is not only able to detect minute levels of genotoxins but, due to its use of the green fluorescent protein, also a reporter system which should be applicable in high-throughput screening assays as well as a wide variety of in situ detection studies.     

Killing of Escherichia coli by Myxococcus xanthus in Aqueous Environments Requires Exopolysaccharide-Dependent Physical Contact

Nutrient or niche-based competition among bacteria is a widespread phenomenon in the natural environment. Such interspecies interactions are often mediated by secreted soluble factors and/or direct cell–cell contact. As ubiquitous soil bacteria, Myxococcus species are able to produce a variety of bioactive secondary metabolites to inhibit the growth of other competing bacterial species. Meanwhile, Myxococcus spp. also exhibit sophisticated predatory behavior, an extreme form of competition that is often stimulated by close contact with prey cells and largely depends on the availability of solid surfaces. Myxococcus spp. can also be isolated from aquatic environments. However, studies focusing on the interaction between Myxococcus and other bacteria in such environments are still limited. In this study, using the well-studied Myxococcus xanthus DK1622 and Escherichia coli as model interspecies interaction pair, we demonstrated that in an aqueous environment, M. xanthus was able to kill E. coli in a cell contact-dependent manner and that the observed contact-dependent killing required the formation of co-aggregates between M. xanthus and E. coli cells. Further analysis revealed that exopolysaccharide (EPS), type IV pilus, and lipopolysaccharide mutants of M. xanthus displayed various degrees of attenuation in E. coli killing, and it correlated well with the mutants' reduction in EPS production. In addition, M. xanthus showed differential binding ability to different bacteria, and bacterial strains unable to co-aggregate with M. xanthus can escape the killing, suggesting the specific nature of co-aggregation and the targeted killing of interacting bacteria. In conclusion, our results demonstrated EPS-mediated, contact-dependent killing of E. coli by M. xanthus, a strategy that might facilitate the survival of this ubiquitous bacterium in aquatic environments.     

Human β-Defensin-2 Induction in Human Foreskin Keratinocyte by Synergetic Stimulation with Foods and Escherichia Coli

We established a real-time quantitative RT-PCR assay that permits rapid and sensitive screening of foods that increase the human β-defensin-2 (hBD-2) mRNA level in human foreskin keratinocyte (HFK) cells. The range of hBD-2 mRNA concentrations suitable for the assay was between 8 × 10^?11 M (39-cycle amplification) and 8 × 10^?18 M (13-cycle amplification) as calibrated with standard hBD-2 cDNA. With this assay system, it was found that the stimulation of HFK cells by the addition of yeast powder at 5 g l^?1 to the culture medium resulted in about 40 times increase in hBD-2 mRNA level, though stimulation with Escherichia coli attained the same level of induction. The active component of yeast was insoluble in water. Simultaneous co-stimulation of HFK cells with E. coli and grains, such as amaranth, millet, soybean and sesame, boosted hBD-2 mRNA induction significantly (6.1, 2.5, 3.3, and 3.3 times, respectively) above the level attained with E. coli alone. The results of successive fractionations of amaranth grain powder by ether extraction and amylase digestion showed that the boosting activity of amaranth grain resided in its insoluble fraction. Significant boosting of hBD-2 mRNA induction in epithelial cells with foods opens a new possibility of developing functional foods that can protect the human body against microbial infection at the oral cavity, skin, and respiratory tract among others.