Fatty acid DSF binds and allosterically activates histidine kinase RpfC of phytopathogenic bacterium Xanthomonas campestris pv. campestris to regulate quorum-sensing and virulence.

As well as their importance to nutrition, fatty acids (FA) represent a unique group of quorum sensing chemicals that modulate the behavior of bacterial population in virulence. However, the way in which full-length, membrane-bound receptors biochemically detect FA remains unclear. Here, we provide genetic, enzymological and biophysical evidences to demonstrate that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, a medium-chain FA diffusible signal factor (DSF) binds directly to the N-terminal, 22 amino acid-length sensor region of a receptor histidine kinase (HK), RpfC. The binding event remarkably activates RpfC autokinase activity by causing an allosteric change associated with the dimerization and histidine phosphotransfer (DHp) and catalytic ATP-binding (CA) domains. Six residues were found essential for sensing DSF, especially those located in the region adjoining to the inner membrane of cells. Disrupting direct DSF-RpfC interaction caused deficiency in bacterial virulence and biofilm development. In addition, two amino acids within the juxtamembrane domain of RpfC, Leu172 and Ala178, are involved in the autoinhibition of the RpfC kinase activity. Replacements of them caused constitutive activation of RpfC-mediated signaling regardless of DSF stimulation. Therefore, our results revealed a biochemical mechanism whereby FA activates bacterial HK in an allosteric manner, which will assist in future studies on the specificity of FA-HK recognition during bacterial virulence regulation and cell-cell communication.

Two-Component Signaling System VgrRS Directly Senses Extracytoplasmic and Intracellular Iron to Control Bacterial Adaptation under Iron Depleted Stress.

Both iron starvation and excess are detrimental to cellular life, especially for animal and plant pathogens since they always live in iron-limited environments produced by host immune responses. However, how organisms sense and respond to iron is incompletely understood. Herein, we reveal that in the phytopathogenic bacterium Xanthomonas campestris pv. campestris, VgrS (also named ColS) is a membrane-bound receptor histidine kinase that senses extracytoplasmic iron limitation in the periplasm, while its cognate response regulator, VgrR (ColR), detects intracellular iron excess. Under iron-depleted conditions, dissociation of Fe3+ from the periplasmic sensor region of VgrS activates the VgrS autophosphorylation and subsequent phosphotransfer to VgrR, an OmpR-family transcription factor that regulates bacterial responses to take up iron. VgrR-VgrS regulon and the consensus DNA binding motif of the transcription factor VgrR were dissected by comparative proteomic and ChIP-seq analyses, which revealed that in reacting to iron-depleted environments, VgrR directly or indirectly controls the expressions of hundreds of genes that are involved in various physiological cascades, especially those associated with iron-uptake. Among them, we demonstrated that the phosphorylated VgrR tightly represses the transcription of a special TonB-dependent receptor gene, tdvA. This regulation is a critical prerequisite for efficient iron uptake and bacterial virulence since activation of tdvA transcription is detrimental to these processes. When the intracellular iron accumulates, the VgrR-Fe2+ interaction dissociates not only the binding between VgrR and the tdvA promoter, but also the interaction between VgrR and VgrS. This relieves the repression in tdvA transcription to impede continuous iron uptake and avoids possible toxic effects of excessive iron accumulation. Our results revealed a signaling system that directly senses both extracytoplasmic and intracellular iron to modulate bacterial iron homeostasis.

Optimization of Maximum Light Use Efficiency in Inner Mongolian Steppe.

For the case that the value of the maximum light use efficiency (MLUE) is not optimized for different steppes, we simulated the MLUE for meadow steppe, typical steppe and desert steppe in Inner Mongolia based on the field observed NPP and CASA ecosystem model, and analyzed the spatial and temporal pattern of the LUE and net primary productivity (NPP) in Inner Mongolia. The result indicate that the MLUE is optimized to be 0.654，0.553 and 0.511 gC·MJ-1 for meadow steppe, typical steppe and desert steppe in Inner Mongolia, respectively, with an average of 0.573 gC·MJ-1. Compared to the result that used same value of 0.541 gC·MJ-1 for MLUE, the correlation coefficient and relative mean square error was improved 0.024 and 2.62 gC·(m2·month-1)-1, respectively after optimization. Affected by the hydrothermal condition and distribution of grassland types, the LUE and NPP in Inner Mongolia decreased from northeast to southwest, and showed one crest shape. However, the maximum value of LUE and NPP was appeared in August and July. This difference could be attributed to the difference in the maximum value between absorbed photosynthetically active radiation and LUE. The LUE and NPP decreased by meadow steppe, typical steppe and desert steppe.

[Chemical Constituents from the Roots of Lindera glauca and Their Antitumor Activity on Four Different Cancer Cell Lines].

Objective: To study the chemical constitutes from the roots of Lindera glauca and the alkaloids influence on proliferation of HT-29,SGC-7901,SMMC-7721 and A549 cell lines. Methods: The constituents were isolated by column chromatography such as RP-18,Sephadex LH-20 and silica gel,and their structures were elucidated by spectroscopic data analysis and compared with literature data. The antitumor activity was determined by MTT assay. Results: Ten compounds had been isolated and identified as(-)-magnocurarine( 1),N-methyl-laurotetanine( 2),laurotetanine( 3),( +)-boldine( 4),(-)-norisoboldine( 5),( +)-norisocorydine( 6),pmethane-3,8-trans-diol( 7),p-methane-3,8-cis-diol( 8),eudesm-4( 15)-ene-7,11-diol( 9) and 4ß,6ß-dihydroxy-1α,5ß( H)-guai-9-ene( 10). Compounds 2 ~ 4 showed significant inhibitory activities against HT-29,SGC-7901,SMMC-7721 and A549 cells. Conclusion: Compound 1,9 and 10 are isolated from this plant for the first time. The IC50 value of compound 2 against HT-29 and SGC-7901 cell lines is even lower than VP-16.

Dual Regulatory Role Exerted by Cyclic Dimeric GMP To Control FsnR-Mediated Bacterial Swimming.

Bacterial motility has great medical and ecological significance because of its essential role in bacterial survival and pathogenesis. Cyclic dimeric GMP (c-di-GMP), a second messenger in bacteria, is the predominant regulator of flagellar synthesis and motility and possesses turnover mechanisms that have been thoroughly investigated. Therefore, much attention has been focused on identifying the upstream stimulatory signals and downstream modules that respond to altered c-di-GMP levels. Here, we systematically analyzed c-di-GMP cyclases and phosphodiesterases in Stenotrophomonas maltophilia to screen for motility regulators. Of these enzymes, we identified and characterized a new phosphodiesterase named SisP, which was found to facilitate bacterial swimming upon stimulation with ferrous iron. SisP-mediated degradation of c-di-GMP leads to FsnR-dependent transcription of flagellar genes. Remarkably, c-di-GMP controls FsnR via two independent mechanisms: by direct binding and indirectly by modulating its phosphorylation state. In this study, we deciphered a novel "one stone, two birds" regulatory strategy of c-di-GMP and uncovered the signal that stimulates c-di-GMP hydrolysis. Facilitation of bacterial swimming motility by ferrous iron might contribute to the higher risk of bacterial infection in acutely ill patients. IMPORTANCE Stenotrophomonas maltophilia has become a great threat to human health because of the high mortality of infected patients. Swimming motility plays a crucial role in regulating bacterial virulence and adaptation. However, limited progress has been made in cyclic dimeric GMP (c-di-GMP) controlling swimming motility of S. maltophilia. Here, we characterized c-di-GMP turnover enzymes encoded by S. maltophilia and dissected the regulatory details of a phosphodiesterase named SisP. We demonstrated that SisP degrades c-di-GMP to fully activate FsnR through directly releasing FsnR from the FsnR-c-di-GMP complex and indirectly increasing its phosphorylation level. This finding uncovered a quantitative, rather than an on-off, regulatory manner employed by c-di-GMP to regulate activities of its effectors. Identification of the specific activation of SisP by ferrous iron proposes SisP as a putative drug-target for controlling bacterial infection and ferrous iron at the wounds or cuts as a putative factor contributing to the higher risk of bacterial infection.

Comparison and quantitative analysis of microstructure parameters between original loess and remoulded loess under different wetting-drying cycles.

The microstructural evolution of loess had a significant impact on the collapsibility of loess during wetting-drying cycles. Based on the analysis of scanning electron microscope (SEM) images by using Image-Pro Plus, the present study quantitatively compared the microstructural parameters of original loess and remoulded loess with different moisture content before and after wetting-drying cycles in size, shape, and arrangement. In size, the average diameter of both original loess particles and remoulded loess particles increased with the increasing of initial moisture content. However, the average diameter of original loess particles was slightly larger than that of remoulded loess particles before wetting-drying cycles. In contrast, the average diameter of both original loess particles and remoulded loess particles were very close to each other after three wetting-drying cycles. In shape, before wetting-drying cycles, the average shape factor of original loess particles was higher than that of remoulded loess particles. After three wetting-drying cycles, the difference in the average shape factor of both two loess samples with 5% initial moisture content is similar to that before wetting-drying cycles. Nevertheless, the average shape factor of both original loess particles and remouled loess particles with 15% initial moisture content were very close to that with 25% initial moisture content. In the arrangement, directional frequency indicated remoulded loess appeared to be more vertically aligned than original before and after three wetting-drying cycles. Furthermore, the directed anisotropy rate of remoulded loess was higher than that of the original loess before and after three wetting-drying cycles. In summary, the size, shape, and arrangement of both original loess particles and remoulded loess particles varied in different degrees before and after three wetting-drying cycles. Combined with the water retention curve of the loess, we analyzed the microstructural evolution mechanism of two loess particles during wetting-drying cycles. It is an excellent significance to study the engineering properties of original loess and remoulded loess.

[Investigation and analysis of the bacteria community in silkworm intestine].

Majority of the environmental microorganisms are still unculturable. To shed a light on the bacteria community in the silkworm intestine more completely, culture-dependent and culture-independent methods were used to perform the investigation. As the latter, 16s rRNA gene was amplified and a library was constructed by using the meta-genomic DNA extracted from the bacteria in the silkworm intestine as template. Restriction fragment length polymorphism (RFLP) method was used to screen recombinants originated from different bacteria, and the nucleotide sequences were determined. A BLAST searching in the GenBank and an analysis on phylogenesis were performed. The taxonomy of these bacteria and their physiological function to silkworm were speculated. Results showed that the bacteria in silkworm intestine are mainly belong to the genera Arthrobacter, Lactobacillus, Pseudomonas, Escherichia, Micrococcus, Bacillus, and Staphylococcus. They may play important roles to the host silkworm in the utilization of their food mulberry and disease prevention. Both the culture-dependent and culture-independent methods have their advantages and disadvantages, and they are complemented with each other.