Exploration of chlamydial type III secretion system reconstitution in Escherichia coli

BACKGROUND: Type III secretion system is a virulent factor for many pathogens, and is thought to play multiple roles in the development cycle and pathogenesis of chlamydia, an important human pathogen. However, due to the obligate intracellular parasitical nature of chlamydiae and a lack of convenient genetic methodology for the organisms, very limited approaches are available to study the chlamydial type III secretion system. In this study, we explored the reconstitution of a chlamydial type III secretion in Escherichia coli. RESULTS: We successfully cloned all 6 genomic DNA clusters of the chlamydial type III secretion system into three bacterial plasmids. 5 of the 6 clusters were found to direct mRNA synthesis from their own promoters in Escherichia coli transformed with the three plasmids. Cluster 5 failed to express mRNA using its own promoters. However, fusion of cluster 5 to cluster 6 resulted in the expression of cluster 5 mRNA. Although only two of the type III secretion system proteins were detected transformed E. coli due to limited antibody availability, type III secretion system-like structures were detected in ultrathin sections in a small proportion of transformed E. coli. CONCLUSIONS: We have successfully generated E. coli expressing all genes of the chlamydial type III secretion system. This serves as a foundation for optimal expression and assembly of the recombinant chlamydial type III secretion system, which may be extremely useful for the characterization of the chlamydial type III secretion system and for studying its role in chlamydial pathogenicity

Climate change, water management and stakeholder analysis in the Dongjiang River basin in South China

This article proposes a systematic analysis of water management and allocation on the scale of a river basin, considering climate impacts and stakeholder networks in the Dongjiang River basin in South China. Specific approaches are integrated to explore various subtopics. Findings indicate a slight increase of precipitation in the basin and strong fluctuations in this century due to climate extremes, which may lead to seasonal or quality-related water shortages. It is highlighted that alternative options for holistic water management are needed in the basin, and participatory water allocation mechanisms and establishment of a basin-wide management framework could be helpful

Biodesulfurization using Pseudomonas delafieldii in magnetic polyvinyl alcohol beads

Aims: To immobilize Pseudomonas delafieldii R-8 cells in magnetic polyvinyl alcohol (PVA) beads for biodesulfurization

Vector information.

<p>Vectors used for experiments to determine cT3SS expression and T3S are shown in boldface.</p

Detection of CT557 and CT672 in <i>E. coli</i> transformed with pcT3SS-C1/C5/C6, pcT3SS-C2/C3 and pcT3SS-C4 by western blotting.

<p>Control bacteria were transformed with empty pACYC184 and pBAD18 plasmids.</p

Lack of secretion of cT3S effectors in cT3SS plasmids-transformed <i>E. coli.</i>

<p>(A) Secretion of Flag-tagged T3S effectors from <i>E. coli</i> could not be induced by using DMEM (medium) containing the calcium-depleting reagent EGTA and/or FBS for 6 h. An anti-Flag antibody was used to detect the C-terminally tagged cT3S effectors. (B) Secretion of non-tagged TARP from <i>E. coli</i> was not induced under the same conditions as in (A). (C) TARP secretion from EBs suspended in PBS and PBS containing Ca²⁺. (D) Secretion of non-tagged TARP from <i>E. coli</i> was not detected using PBS or PBS containing Ca²⁺.</p

cT3SS expression vectors.

<p>Plasmids in panels A–D contain different cT3SS clusters. The tetracycline-resistance gene (tet) and chloramphenicol-resistance gene (cml) are inactivated in A and B, respectively, as results of the insertion of cT3SS fragments. Thick black arrows show cT3SS genes, as numbered in the <i>C. trachomatis</i> serovar D genome. Genes of which mRNA was detected by RT-PCR are underlined. Lengths of genes are not in scale. Line arrows signify locations of promoters and direction of transcription in operons as previously established <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050833#pone.0050833-Hefty1" target="_blank">[39]</a>. Dotted line in C indicates no detectable transcription from the promoter shown. Transcription from two internal promoters in panels A and B is unlikely to occur in <i>E. coli</i> (shown in thinner line arrows). The activities of these two promoters were not specifically examined in this study. Endorestriction sites used for cloning are shown in italics. The orientations of the cT3SS clusters in the plasmids were not determined and are shown arbitrarily.</p

RT-PCR analysis of cT3SS gene transcription in <i>E. coli</i> transformed with pcT3SS-C1/C6-C5FU, pcT3SS-C2/C3 and pcT3SS-C4.

<p>Cluster (C) numbers and operon (O) numbers are shown. Note amplification occurred only in reaction for which cDNA but not RNA was used as template.</p

Improvement of Biodesulfurization Rate by Assembling Nanosorbents on the Surfaces of Microbial Cells

To improve biodesulfurization rate is a key to industrialize biodesulfurization technology. The biodesulfurization rate is partially affected by transfer rate of substrates from organic phase to microbial cell. In this study, γ-Al(2)O(3) nanosorbents, which had the ability to selectively adsorb dibenzothiophene (DBT) from organic phase, were assembled on the surfaces of Pseudomonas delafieldii R-8 cell, a desulfurization strain. γ-Al(2)O(3) nanosorbents have the ability to adsorb DBT from oil phase, and the rate of adsorption was far higher than that of biodesulfurization. Thus, DBT can be quickly transferred to the biocatalyst surface where nanosorbents were located, which quickened DBT transfer from organic phase to biocatalyst surface and resulted in the increase of biodesulfurization rate. The desulfurization rate of the cells assembled with nanosorbents was approximately twofold higher than that of original cells. The cells assembled with nanosorbents were observed by a transmission electron microscope