Regulation of the physiology and virulence of Ralstonia solanacearum by the second messenger 2',3'-cyclic guanosine monophosphate.

Previous studies have demonstrated that bis-(3',5')-cyclic diguanosine monophosphate (bis-3',5'-c-di-GMP) is a ubiquitous second messenger employed by bacteria. Here, we report that 2',3'-cyclic guanosine monophosphate (2',3'-cGMP) controls the important biological functions, quorum sensing (QS) signaling systems and virulence in Ralstonia solanacearum through the transcriptional regulator RSp0980. This signal specifically binds to RSp0980 with high affinity and thus abolishes the interaction between RSp0980 and the promoters of target genes. In-frame deletion of RSp0334, which contains an evolved GGDEF domain with a LLARLGGDQF motif required to catalyze 2',3'-cGMP to (2',5')(3',5')-cyclic diguanosine monophosphate (2',3'-c-di-GMP), altered the abovementioned important phenotypes through increasing the intracellular 2',3'-cGMP levels. Furthermore, we found that 2',3'-cGMP, its receptor and the evolved GGDEF domain with a LLARLGGDEF motif also exist in the human pathogen Salmonella typhimurium. Together, our work provides insights into the unusual function of the GGDEF domain of RSp0334 and the special regulatory mechanism of 2',3'-cGMP signal in bacteria.

Molecular linkages between chemodiversity and MCPA complexation behavior of dissolved organic matter in paddy soil: Effects of land conversion.

Complexation of dissolved organic matter (DOM) plays a crucial role in regulating the fate and risk of agrochemicals. Here, taking a toxic herbicide MCPA (4-chloro-2- methylphenoxyacetic acid) as the target, the effect of land conversion on complexation behavior of DOM to agrochemicals was investigated in paddy soil. Furthermore, the mechanisms were explored in a new perspective of DOM chemodiversity. Soil DOMs were selected from four long-term cropping systems, including paddy field (PF), vegetable field (VF), rice-vegetable rotation (RV) and abandoned land (AL). The results showed that the DOMs in PF and AL were rich in hydrophilic substances (e.g., carbohydrates or protein-like molecules) with low aromaticity. However, after converting PF to VF and RV, abundant aromatic macromolecules and aliphatic alkanes were observed in DOM. Due to those changes in DOM chemodiversity, the binding site and capability of DOM were highest in VF and RV, and were positively correlated with DOM aromaticity, MW, humus and polar groups (e.g., amino). This was because the complexation of "DOM-MCPA" was static binding via ligand exchange and H-bonding among polar groups and hydrophobic interaction among aromatic skeletons. The EEM-PARAFAC confirmed that microbial humic-like substances dominated the complexation of DOM rather than terrestrial humic-like and tryptophan-like matters. The 2D-COS analysis further revealed that the complexation of DOM preferentially occurred in amino, polysaccharide C-O and aliphatic C-H for PF and AL, but in aromatic C=C, amide C=N for RV and VF. In summary, these findings provide molecular insight into the effect of land conversion on DOM complexation activity, which highlight the importance of DOM chemodiversity. These results will contribute to the risk assessments of agrochemicals in paddy soil.

Ralstonia solanacearum promotes pathogenicity by utilizing l-glutamic acid from host plants.

Ralstonia solanacearum is an important bacterial pathogen that can infect a broad range of plants worldwide. A previous study showed that R. solanacearum could respond to exogenous organic acids or amino acids to modulate cell motility. However, it was unclear whether R. solanacearum uses these compounds to control infection. In this study, we found that R. solanacearum GMI1000 uses host plant metabolites to enhance the biosynthesis of virulence factors. We demonstrated that l-glutamic acid from host plants is the key active component associated with increased extracellular polysaccharide production, cellulase activity, swimming motility, and biofilm formation in R. solanacearum GMI1000. In addition, l-glutamic acid also promoted colonization of R. solanacearum cells in the roots and stems of tomato plants and accelerated disease incidence. Furthermore, genetic screening and biochemical analysis suggested that RS01577, a hybrid sensor histidine kinase/response regulator, is involved in l-glutamic acid signalling in R. solanacearum. Mutations in RS01577 and exogenous addition of l-glutamic acid to the GMI1000 wild-type strain had overlapping effects on both the transcriptome and biological functions of R. solanacearum, including on motility, biofilm formation, and virulence. Thus, our results have established a new interaction mechanism between R. solanacearum and host plants that highlights the complexity of the virulence regulation mechanism and may provide new insight into disease control.

Anthranilic acid from Ralstonia solanacearum plays dual roles in intraspecies signalling and inter-kingdom communication.

Quorum sensing (QS) signals are widely utilized by bacteria to regulate biological functions in response to cell population density. Previous studies have demonstrated that Ralstonia solanacearum employs two different types of QS systems. We report here that anthranilic acid controls important biological functions and the production of QS signals in R. solanacearum. It was demonstrated that the biosynthesis of anthranilic acid is mainly performed by TrpEG. The accumulation of anthranilic acid and the transcription of trpEG occur in a cell density-dependent manner in R. solanacearum. Both the anthranilic acid and TrpEG homologues are conserved in various bacterial species. Moreover, we show that Sporisorium scitamineum sexual mating and hypha formation are strongly inhibited by the addition of exogenous anthranilic acid. Our results suggest that anthranilic acid is important for the physiology of bacteria in addition to its role in inter-kingdom communication.

Xanthomonas campestris Promotes Diffusible Signal Factor Biosynthesis and Pathogenicity by Utilizing Glucose and Sucrose from Host Plants.

The plant pathogen Xanthomonas campestris pv. campestris produces diffusible signal factor (DSF) quorum sensing (QS) signals to regulate its biological functions and virulence. Our previous study showed that X. campestris pv. campestris utilizes host plant metabolites to enhance the biosynthesis of DSF family signals. However, it is unclear how X. campestris pv. campestris benefits from the metabolic products of the host plant. In this study, we observed that the host plant metabolites not only boosted the production of the DSF family signals but also modulated the expression levels of DSF-regulated genes in X. campestris pv. campestris. Infection with X. campestris pv. campestris induced changes in the expression of many sugar transporter genes in Arabidopsis thaliana. Exogenous addition of sucrose or glucose, which are the major products of photosynthesis in plants, enhanced DSF signal production and X. campestris pv. campestris pathogenicity in the Arabidopsis model. In addition, several sucrose hydrolase-encoding genes in X. campestris pv. campestris and sucrose invertase-encoding genes in the host plant were notably upregulated during the infection process. These enzymes hydrolyzed sucrose to glucose and fructose, and in trans expression of one of these enzymes, CINV1 of A. thaliana or XC_0805 of X. campestris pv. campestris, enhanced DSF signal biosynthesis in X. campestris pv. campestris in the presence of sucrose. Taken together, our findings demonstrate that X. campestris pv. campestris applies multiple strategies to utilize host plant sugars to enhance QS and pathogenicity.

Modulation of Inter-kingdom Communication by PhcBSR Quorum Sensing System in Ralstonia solanacearum Phylotype I Strain GMI1000.

Ralstonia solanacearum is a ubiquitous soil-borne plant pathogenic bacterium, which frequently encounters and interacts with other soil cohabitants in competition for environmental niches. Ralsolamycin, which is encoded by the rmy genes, has been characterized as a novel inter-kingdom interaction signal that induces chlamydospore development in fungi. In this study, we provide the first genetic evidence that the rmy gene expression is controlled by the PhcBSR quorum sensing (QS) system in strain GMI1000. Mutation of phcB could lead to significant reduction of the expression levels of the genes involved in ralsolamycin biosynthesis. In addition, both the phcB and rmy mutants were attenuated in induction of chlamydospore formation in Fusarium oxysporum f. cubense and diminished in the ability to compete with the sugarcane pathogen Sporisorium scitamineum. Agreeable with the pattern of QS regulation, transcriptional expression analysis showed that the transcripts of the rmy genes were increased along with the increment of the bacterial population density. Taken together, the above findings provide new insights into the regulatory mechanisms that the QS system involves in governing the ralsolamycin inter-kingdom signaling system.

Diffusible signal factor family signals provide a fitness advantage to Xanthomonas campestris pv. campestris in interspecies competition.

Diffusible signal factor (DSF) represents a new class of widely conserved quorum sensing signals, which regulates various biological functions through intra- or interspecies signaling. The previous studies identified that there is an antagonistic interaction between Xanthomonas and Bacillus species bacteria in natural ecosystem, but the detailed molecular mechanism of interspecies competition is not clear. This study showed that Xanthomonas campestris pv. campestris (Xcc) interfered with morphological transition and sporulation of Bacillus thuringiensis in mixed cultures, whereas abrogation of the DSF synthase RpfF reduced the interference. DSF inhibited B. thuringiensis cell division and sporulation through modulation of ftsZ, which encodes an important cell division protein in bacterial cells. In addition, RpfF is essential for production of six DSF-family signals in Xcc, which employ the same signaling pathways to regulate biological functions in Xcc and play similar effects on reduction of cell division, sporulation and antibiotic resistance of B. thuringiensis. Furthermore, abrogation of RpfF decreased the competitive capability of Xcc against B. thuringiensis on the surface of Chinese cabbage leaves. Our findings provide new insights into the role of DSF-family signals in interspecies competition and depict molecular mechanisms with which Xcc competes with B. thuringiensis.

[Analysis of the risk factors for hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation for beta-thalassemia in children].

OBJECTIVE: To analyze the risk factors of hemorrhagic cystitis after allogeneic hematopoietic stem cell transplantation for beta-thalassemia in children. METHODS: The clinical records of 30 children with beta-thalassemia undergoing allogeneic hematopoietic stem cell transplantation between December, 2008 and November, 2009 were analyzed. RESULTS: Hemorrhagic cystitis occurred in 8 of the 33 patients with an incidence of 24.24%, including 1 with grade I, 6 with grade II and 1 with grade III hemorrhagic cystitis. The median time of hemorrhagic cystitis onset was 22.9 days (range 6-35 days) and the median duration was 11.9 days(range 3-27 days). Univariate analysis indicated that the different types of transplantation and acute graft-versus-host disease affect the occurrence of hemorrhagic cystitis. The children with Allo-PBSCT had higher incidence than those receiving Allo-PBSCT+Allo-UBT and Allo-BMT (P<0.05). The children at an age >or=6 years had obviously higher incidence of hemorrhagic cystitis than those at younger ages. CONCLUSION: Age is the major factor that affects the occurrence of hemorrhagic cystitis in children undergoing allogeneic hematopoietic stem cell transplantation for beta-thalassemia.