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Fluorescence in situ hybridization (FISH) has been employed to identify microorganisms at the single cell level under a microscope. Extensive efforts have been made to improve and extend the FISH technique; however, the development of a widely applicable protocol is a continuing challenge. The present study evaluated the effects of divalent cations in the hybridization solution on the FISH-based detection of various species of bacteria and archaea with rRNA-targeted probes. A flow cytometric analysis after FISH with a standard hybridization buffer detected positive signals from less than 30% of Escherichia coli IAM 1264 cells. However, the number of cells with positive signals increased to more than 90% after the addition of calcium chloride to the hybridization buffer. Mn2+ also had positive effects, whereas Mg2+ did not. The positive effects of Ca2+ were similarly observed for bacteria belonging to Enterobacteriaceae, including Enterobacter sakazakii IAM 12660T, E. aerogenes IAM 12348, Klebsiella planticola IAM 14202, and Salmonella enterica subsp. enterica serovar Typhimurium strain LT2. These results indicate that the supplementation of Ca2+ to the hybridization buffer for FISH contributes to the efficient detection of Enterobacteriaceae cells.

Additional file 12: Figure S7. Substrate utilization profiles of yeast strains expressing crfax1 and crfax2.

H-NS family proteins encoded on the chromosome of Pseudomonas putida KT2440 (TurA and TurB) and the IncP-7 plasmid pCAR1 (Pmr) commonly have an N-terminal dimerization/oligomerization domain constituted by a central and a terminal dimerization sites. To clarify the dimerization manner at the central dimerization sites of the three homologs, we performed chemical cross-linking analyses with protein variants inactivated at the terminal dimerization site. Comparison of the hetero-dimer ratios among them suggested stronger affinities between the central dimerization sites of TurA and TurB monomers than between TurA and Pmr or TurB and Pmr. Furthermore, analyses of the interaction between truncated TurB containing only a functional terminal dimerization site and full-length proteins suggested that TurB exhibited higher affinities for oligomer complex formation with TurB itself and TurA but not Pmr. Altogether, we revealed stronger interaction between the N-terminal domains of TurA and TurB than between either of them and Pmr. Binding affinities between the chromosomally-encoded H-NS family proteins TurA and TurB were stronger than between either of them and the plasmid-encoded Pmr.

Representative western blotting results of the three H-NS family proteins. The amounts of TurA, TurB, and His-tagged Pmr were monitored at various points in the growth curve of KT2440 (A), KT2440(pCAR1) (B), and KT2440(pCAR1pmrHis) (C). Results of western blotting analyses using anti-TurA (left panels in A and B), anti-TurB (right panels in A and B), and anti-His antibodies (C) are shown in the black boxes. The loading amounts of whole cell protein were 80 μg for KT2440 and KT2440(pCAR1) and 40 μg for KT2440(pCAR1pmrHis) per lane. Specific amounts of purified His-tagged proteins (shown in red) were used to quantify the number of protein molecules. For each strain, 5 μg of whole cell protein were loaded and detected by anti-RNA polymerase α subunit (RNAP) antibody (green boxes) as a loading control. (PDF 83 kb)

Sweet sorghum is a C4 plant with high efficiency of photosynthesis and accumulation of photosynthetic products during growth stage. Until now, little is known about photosynthetic characteristics of sweet sorghum subjected to heavy metal contaminated soil. In this study, photosynthetic modification of sweet sorghum was measured in response to cadmium (Cd) under greenhouse conditions. The experiment was concentrated specifically on Keller (KE) and E-Tian (ET), two cultivars of sweet sorghum. The plants subjected to 100 mg/kg Cd in soil were compared with the control plants without Cd treatment. The results indicated that both cultivars of sweet sorghum display similar photosynthetic responses to Cd exposure. The high Cd concentration resulted in significant decline of photosynthesis net rate (Pn). Pn of ET declines more than Cd-treated KE plants. Moreover, Cd-treated plants were detected to have lower stomata conductance (gs) and intercellular CO2 concentrations (Ci) than control plants. Chlorophyll content also displayed significant differences between control and Cd-treated plants. In ET, the reduction of both chlorophyll-a and chlorophyll-b was observed under Cd stress, compared with the controls. In contrast, chlorophyll content was significantly enhanced by Cd stress in KE. In conclusion, through the comparison between two sweet sorghum cultivars, it is demonstrated the photosynthetic activity of ET more sensitive to Cd stress than that of KE. Therefore, KE has a potential for biofuel production and phytoremediation of heavy metal in soil.

Ralstonia solanacearum is the second most destructive bacterial plant pathogens worldwide and HrpG is the master regulator of its pathogenicity. PrhG is a close paralogue of HrpG and both belong to OmpR/PhoB family of two-component response regulators. Despite a high similarity (72% global identity and 96% similarity in helix-loop-helix domain), they display distinct roles in pathogenicity. HrpG is necessary for the bacterial growth in planta and pathogenicity, while PrhG is dispensable for bacterial growth in planta and contributes little to pathogenicity. The main difference between HrpG and PrhG is the 50-amino-acid-long C-terminal extension in PrhG (amino-acid residues 230-283), which is absent in HrpG. When this extension is deleted, truncated PrhGs (under the control of its native promoter) allowed complete recovery of bacterial growth in planta and wild-type virulence of hrpG mutant. This novel finding demonstrates that the extension region in PrhG is responsible for the functional difference between HrpG and PrhG, which may block the binding of PrhG to target promoters and result in impaired activation of hrp expression by PrhG and reduced virulence of R. solanacearum.

Sweet sorghum has become a candidate species for combining phytoremediation with biofuel production due to heavy metal resistance, great biomass and sugar enrichment in stem. However, the influence of heavy metals on sugar accumulation in sweet sorghum is still unknown. In this study, Cd induced modification of sugar storage and translocation in stem was monitored using two sweet sorghum cultivars Keller (KE) and E-tian (ET). Brix degree, which represents sugar content in stem juice, displays significant reduction in response to exogenous Cd treatment in both lines. The sugar enrichment is more obviously repressed in the lower internodes while it is hardly affected in the top internodes. Excessive Cd results in drastically enhanced Cd accumulation, preferentially in the lower internodes. More interestingly, negative correlation between Cd and sugar content is detected along internodes, indicating antagonistic translocation between Cd and sugar along stem of sweet sorghum. The preferential compartmentation of Cd and sugar in different regions of stem provides novel insights into understanding and application of sweet sorghum for combining biofuel production with phytoremediation of heavy metal in soil.