Cloning, expression and characterization of trehalose-6-phosphate phosphatase from a psychrotrophic bacterium, Arthrobacter strain A3.

A trehalose-6-phosphate phosphatase (TPP) gene, otsB, from a psychrotrophic bacterium, Arthrobacter strain A3, was identified. The product of this otsB gene is 266 amino acids in length with a calculated molecular weight of 27,873 Da. The protein was expressed in Escherichia coli and purified to apparent homogeneity. The purified recombinant TPP catalyzed the dephosphorylation of trehalose-6-phosphate to form trehalose and showed a broad optimum pH range from 5.0 to 7.5. This enzyme also showed an absolute requirement for Mg(2+) or Co(2+) for catalytic activity. The recombinant TPP had a maximum activity at 30 °C and maintained activity over a temperature range of 4-30 °C. TPP was generally heat-labile, losing 70 % of its activity when subjected to heat treatment at 50 °C for 6 min. Kinetic analysis of the Arthrobacter strain A3 TPP showed ~tenfold lower K (m) values when compared with values derived from other bacterial TPP enzymes. The highest k (cat)/K (m) value was 37.5 mM(-1) s(-1) (repeated three times), which is much higher than values published for mesophilic E. coli TPP, indicating that the Arthrobacter strain A3 TPP possessed excellent catalytic activity at low temperatures. Accordingly, these characteristics suggest that the TPP from the Arthrobacter strain A3 is a new cold-adapted enzyme. In addition, this is the first report characterizing the enzymatic properties of a TPP from a psychrotrophic organism.

Effect of inoculation with the endophyte Clavibacter sp. strain Enf12 on chilling tolerance in Chorispora bungeana.

Endophytic bacteria have been shown to increase resistance against biotic stress and tolerance to abiotic stress in many plants. The objective of this study was to evaluate the effect of an endophytic bacterium, Clavibacter sp. strain Enf12, in regenerated plantlets of Chorispora bungeana subjected to chilling stress (0°C). Aerial biomass and physiological markers for chilling stress, such as electrolyte leakage, lipid peroxidation, reactive oxygen species (ROS) accumulation, proline content and activities of superoxide dismutase (EC 1.15.1.1), catalase (EC 1.11.1.6), guaiacol peroxidase (EC 1.11.1.7) and ascorbate peroxidase (EC 1.11.1.11), were assessed. We demonstrated that Clavibacter sp. strain Enf12 was capable of colonizing internal tissues of regenerated plantlets of C. bungeana and maintained stable population densities under both normal (20°C) and chilling (0°C) conditions. Inoculation enhanced plantlet growth under both conditions and significantly attenuated the chilling-induced electrolyte leakage, lipid peroxidation and ROS accumulation. The endophyte significantly increased the activities of antioxidant enzymes and proline content in C. bungeana plantlets under chilling stress. These findings suggest that Clavibacter sp. strain Enf12 inoculation stimulates the growth of C. bungeana plantlets and improves their tolerance to chilling stress through enhancing the antioxidant defense system.

Analysis of the composition and characteristics of culturable endophytic bacteria within subnival plants of the Tianshan Mountains, northwestern China.

This study first described the composition and characteristics of culturable endophytic bacteria isolated from wild alpine-subnival plant species growing under extreme environmental conditions (i.e., on the border of a glacier with frequently fluctuating and freezing temperatures, strong wind, and high ultraviolet radiation). Using a cultivation-dependent approach and 16S rRNA gene amplification techniques, 93 bacterial isolates showing different phenotypic properties were obtained from 20 different subnival plant species, of which gram-positive bacteria (61.5%), psychrotolerant bacteria (67.3%), and pigmented isolates (70.9%) accounted for a large proportion. All these characteristics of endophytes were closely related to the survival environment of their host plants and were in good agreement with microbes occurring in other cold environments. Phylogenetic analysis indicated that the endophytic isolates consisted of five phylogenetic groups comprising α-proteobacteria, γ-proteobacteria, the high G+C content gram-positive bacteria, the low G+C content gram-positive bacteria, and Flavobacterium-Bacteroides-Cytophaga. The largest generic diversity was found in the HGC group, while Clavibacter, Agreia, Rhodococcus, Sphingomonas, and Pseudomonas were the most prevalent genera. Of all isolates, 46.4% showed a high sequence similarity (98-100%) to strains discovered from other cold environments such as glaciers, tundra, and polar seas. Furthermore, 36.4% of the isolates produced Indole-3-acetic acid and 76.3% were able to solubilize mineral phosphate, which revealed that endophytic bacteria with multiple physiological functions were abundant and widespread in subnival plants. These results are essential for understanding the ecological roles of endophytes and as a foundation for further studying the interactions with plants and environment.

The catalytic efficiency of trehalose-6-phosphate synthase is effected by the N-loop at low temperatures.

The enzyme OtsA (trehalose-6-phosphate synthase) is ubiquitous in both prokaryotic and eukaryotic organisms, where it plays a critical role in stress resistance and glucose metabolism. Here, we cloned the otsA gene from Arthrobacter sp. Cjts, and expressed and then purified the recombinant proteins. Enzyme activity analysis indicated that the high catalytic efficiency of OtsA from Arthrobacter sp. Cjts resulted from the high affinity of the enzyme for uridine 5'-diphosphoglucose (UDP-Glc) at low temperatures. We also confirmed that the N-loop sequence of OtsA has a large effect on its affinity for UDP-Glc. Sequence analysis indicated that the flexibility of the N-loop may be directly related to the catalytic efficiency of OtsA at low temperatures.

BYPASS1-LIKE, A DUF793 Family Protein, Participates in Freezing Tolerance via the CBF Pathway in Arabidopsis.

The C-REPEAT BINDING FACTOR signaling pathway is strictly modulated by numerous factors and is essential in the cold response of plants. Here, we show that the DUF793 family gene BYPASS1-LIKE modulates freezing tolerance through the CBFs in Arabidopsis. The expression of B1L was rapidly induced under cold treatment. Comparing to wild type, B1L knockout mutants were more sensitive to freezing treatment, whereas B1L-overexpressing lines were more tolerant. The expression of CBFs and CBF target genes was significantly decreased in b1l mutant. Using yeast two-hybrid screening system, 14-3-3λ was identified as one of proteins interacting with B1L. The interaction was confirmed with bimolecular fluorescence complementation assay and co-immunoprecipitation assay. Biochemical assays revealed that b1l mutation promoted the degradation of CBF3 compared to wild type, whereas 14-3-3κλ mutant and b1l 14-3-3κλ mutant suppressed the degradation of CBF3. Consistently, 14-3-3κλ and b1l 14-3-3κλ mutants showed enhanced freezing tolerance compared to wild type. These results indicate that B1L enhances the freezing tolerance of plants, at least partly through stabilizing CBF. Our findings improve our understanding of the regulation of CBF in response to cold stress.

Phylogenetic analysis of lichen-forming fungi Rhizoplaca zopf from China based on ITS data and morphology.

A molecular phylogenetic analysis of Rhizoplaca melanophthalma, Rhizoplaca chrysoleuca, Rhizoplaca peltata and Rhizoplaca haydenii is presented based on the nuclear ribosomal internal transcribed spacer (ITS) regions and morphology. Rhizoplaca species were collected at 3400-3900 m in Tianshan Mountains, Xinjiang province, China. Rhizoplaca haydenii is reported for the first time in China. Maximum parsimony (MP) analysis of ITS sequences obtained from Tianshan Mountains samples and GenBank reveals that the evolution relationship of Rhizoplaca melanophthalma and Rhizoplaca chrysoleuca is closer to each other than to Rhizoplaca peltata, and Rhizoplaca haydenii showed closer relatedness to Rhizoplaca melanophthalma. When the four species groups from Tianshan Mountains were analyzed alone through the neighbour-joining (NJ) and minimum evolution method, we obtained the same result. The morphology analysis of Rhizoplaca Zopf which reveals the pruinose discs and apothecial discs of species did not show convincing evidences to prove phylogenetic relationship among Rhizoplaca species In our study, the result further proved that Rhizoplaca should be rejected as a genus separate from Lecanora.

[Effect of Manual Acupuncture Stimulation of Single Acupoint or Acupoints Combination on Intestinal Movement in Slow Transit Constipation Mice].

OBJECTIVE: To observe the effect of acupuncture stimulation of different acupoints or acupoint groups on intestinal locomotor in slow transit constipation (STC) mice, so as to provide experimental evidence for demonstrating the synergic function of acupoint combination. METHODS: A total of 190 Kunming mice were randomized into 19 groups:normal control, model, uni-"Quchi" (LI 11), uni-"Shangjuxu" (ST 37), uni-"Tianshu"(ST 25), and uni-"Dachangshu"(BL 25) groups, for analyzing the effect of single acupoint; normal control, model, uni-ST 25+uni-BL 25, uni-LI 11+uni-ST 37, bi-ST 25, bi-BL 25, bi-LI 11, and bi-ST 37 groups, for comparing the effect between single acupoint and two acupoints combination; normal control, model, bi-ST 25+bi-BL 25, bi-LI 11+bi-ST 37, and uni-LI 11+uni-ST 37+uni-ST 25 +uni-BL 25 groups, for comparing the effect between four acupoints and two acupoints combinations (10 mice/group). The STC model was established by intragastric administration of compound diphenoxylate suspension (1 mg/100 g), once daily for 2 days. Manual acupuncture needle stimulation (200 twirling times/min for 1 min) was applied to the abovementioned unilateral or bilateral acupoints twice daily for 7 times. The first defecation time, the defecation grain number in 12 h and the intestinal propulsion rate were determined using activated carbon gavage. RESULTS: Compared with the normal group, the first defecation time was significantly increased in the STC model group (P<0.01), while the 12 h defecation grain number and the intestinal propulsion rate were considerably decreased in the model group (P<0.01). After manual acupuncture intervention, the first defecation time was notably shortened in the uni-LI 11, uni-ST 37, uni-ST 25, uni-BL 25, bi-ST 25, bi-BL 25, bi-LI 11, bi-ST 37, uni-ST 25+uni-BL 25, uni-LI 11+uni-ST 37, bi-ST 25+bi-BL 25, bi-LI 11+bi-ST 37 and the uni-ST 25+uni-BL 25+uni-LI 11+uni-ST 37 groups compared with the model group (P<0.05), while both the 12 h defecation grain number and intestinal propulsion rate were considerably increased in the abovementioned 13 acupuncture groups (P<0.05, P<0.01). The effects of uni-ST 25+uni-BL 25 and uni-LI 11+uni-ST 37 were significantly superior to those of bi-ST 25, bi-BL 25, bi-LI 11 and bi-ST 37 groups, and those of the uni-ST 25+uni-BL 25+uni-LI 11+uni-ST 37 group were markedly superior to those of bi-ST 25+bi-BL 25 and bi-LI 11+bi-ST 37 groups in shortening the first defecation time and in increasing the 12 h defecation grain number and intestinal propulsion rate (P<0.05). CONCLUSIONS: Acupuncture stimulation of LI 11, ST 37, ST 25 and BL 25 can improve constipation in STC mice, and the effect of two acupoints combination is significantly superior to that of the corresponding bilateral single acupoint, and that of the 4 unilateral single acupoint combination is obviously superior to that of two bilateral acupoints combination, suggesting a synergistic action of acupuncture stimulation.

[Exterior-interior correlation of "heart" and body surface based on researches of classic literature and modern animal experiments].

Exterior-interior relationship of meridians and zangfu organs is an important component of the theory of acupuncturology in Chinese medicine. According to the descriptions in classic works of Chinese medicine and findings of modern experimental researches, in the present paper, the authors analyzed the close association between the "Heart" and "Small Intestine", exterior-interior correlation between the Heart Meridian and Small Intestine Meridian, and between the "Heart" and body surface (acupoints), limbs, five sensory organs, etc. In addition, the authors also summarized the underlying mechanisms of the above-mentioned exterior-interior relationship of the "Heart" and put forward some proposals for the future researches.