Comparative genomic analysis of Citrobacter sp. XT1-2-2 reveals insights into the molecular mechanism of microbial immobilization of heavy metals.

BACKGROUND: In our previous study, Citrobacter sp. XT1-2-2 was isolated from high cadmium-contaminated soils, and demonstrated an excellent ability to decrease the bioavailability of cadmium in the soil and inhibit cadmium uptake in rice. In addition, the strain XT1-2-2 could significantly promote rice growth and increase rice biomass. Therefore, the strain XT1-2-2 shows great potential for remediation of cadmium -contaminated soils. However, the genome sequence of this organism has not been reported so far.  RESULTS: Here the basic characteristics and genetic diversity of the strain XT1-2-2 were described, together with the draft genome and comparative genomic results. The strain XT1-2-2 is 5040459 bp long with an average G + C content of 52.09%, and contains a total of 4801 genes. Putative genomic islands were predicted in the genome of Citrobacter sp. XT1-2-2. All genes of a complete set of sulfate reduction pathway and various putative heavy metal resistance genes in the genome were identified and analyzed. CONCLUSIONS: These analytical results provide insights into the genomic basis of microbial immobilization of heavy metals.

Impacts of a Compound Amendment on Cd Immobilization, Enzyme Activities and Crop Uptake in Acidic Cd-Contaminated Paddy Soils.

This study assessed the effectiveness of limestone-montmorillonite-rapeseed residue-Si fertilizer compound amendment on the bioavailability and crop uptake of cadmium (Cd) and enzyme activities in acidic paddy soils. Applying the compound amendment at ratios of 1%-3% increased soil pH by 0.1-1.9 units, decreased leaching ratios of soil Cd 4.0%-22%, and decreased exchangeable and carbonated Cd 42%-55% and 27%-49%, respectively. Organic matter-bound Cd increased 47%-62% (p < 0.05). Cadmium concentrations decreased in the roots, culms, leaves, and grains of rice grown in the Cd-contaminated soils by 37%-81%, 18%-73%, 29%-64% and 27%-63%, respectively, (p < 0.05). Catalase and urease activities increased 2.5%-63% and 3.9%-36%, (p < 0.05), respectively. Applying this compound amendment may significantly mitigate soil acidification and decrease the bioavailability and crop uptake of Cd in acidic Cd-contaminated paddy soils.

Draft Genome Sequence of Bacillus thuringiensis subsp. aizawai HD133.

We report here the 6,512,057-bp draft genome sequence of Bacillus thuringiensis subsp. aizawai HD133. This strain contains at least 6 cry genes and 13 candidate biosynthetic gene clusters.

Enhancement of Bacillus thuringiensis insecticidal activity by combining Cry1Ac and bi-functional toxin HWTX-XI from spider.

In order to assess the potency of bi-functional HWTX-XI toxin from spider Ornithoctonus huwena in improving the insecticidal activity of Bacillus thuringiensis, a fusion gene of cry1Ac and hwtx-XI was constructed and expressed in an acrystalliferous B. thuringiensis strain Cry(-)B. Western blot analysis and microscopic observation revealed that the recombinant strain could express 140-kDa Cry1Ac-HWTX-XI fusion protein and produce parasporal inclusions during sporulation. Bioassay using the larvae of Helicoverpa armigera and Spodoptera exigua showed that the Cry1Ac-HWTX-XI fusion was more toxic than the control Cry1Ac protoxin, as revealed by 95% lethal concentration. Our study indicated that the HWTX-XI from spider might be a candidate for enhancing the toxicity of B. thuringiensis products.

The distribution pattern of DNA and protoxin in Bacillus thuringiensis as revealed by laser confocal microscopy analysis.

It was reported that the parasporal crystal from Bacillus thuringiensis contained DNA fragments. To investigate the distribution of protoxin and DNA in B. thuringiensis cells at different growth stages, a cry1Ac-gfp fusion gene was constructed and expressed in an acrystalliferous B. thuringiensis strain, in which the localization of DNA and protoxin were indicated by DNA-specific dye and green fluorescent protein, respectively. When the recombinant cells were at the vegetative growth stage, the Cry1Ac-GFP fusion protein was not expressed and the DNA fluorescent signal was evenly distributed throughout the cell. At the initial stage of sporulation, the Cry1Ac-GFP fusion protein was expressed and accumulated as inclusion body, while two condensed DNA signals existed at each pole of the cell. With the extension of culture time, it seemed that the DNA fluorescence from the region of spore development gradually became faint or vanishing, while the DNA signal was still present in the other pole or the remaining area of the mother cell. Interestingly and unexpectedly, there was no DNA fluorescence signal in the region of the growing and mature inclusion body of Cry1Ac-GFP in B. thuringiensis cell, which might indicate that the DNA embodied in the inclusion body was not accessible to the DNA-specific dye. This was the first investigation devoted exclusively to the in vivo distribution of protoxin and DNA in B. thuringiensis at different growth stages. These data shed light on deeply understanding the process of sporulation and parasporal crystal formation as well as further exploring the interaction of DNA and protoxin in B. thuringiensis.

Evaluating the insecticidal genes and their expressed products in Bacillus thuringiensis strains by combining PCR with mass spectrometry.

By a combination of PCR and mass spectrometry, a total of five cry genes (cry1Aa, cry1Ac, cry2Aa, cry2Ab, and cry1Ia) were detected in genomic DNA from the wild-type Bacillus thuringiensis strain 4.0718, and three protoxins (Cry1Aa, Cry1Ac, and Cry2Aa) were identified in the strain's parasporal crystals. These results indicated that this complementary method may be useful in evaluating B. thuringiensis strains at both the gene and protein levels.

Assessment of protoxin composition of Bacillus thuringiensis strains by use of polyacrylamide gel block and mass spectrometry.

Assessment of protoxin composition in Bacillus thuringiensis parasporal crystals is principally hampered by the fact that protoxins in a single strain usually possess high sequence homology. Therefore, new strategies towards the identification of protoxins have been developed. Here, we established a powerful method through embedding solubilized protoxins in a polyacrylamide gel block coupled to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of in-gel-generated peptides for protoxin identification. Our model study revealed that four protoxins (Cry1Aa, Cry1Ab, Cry1Ac and Cry2Aa) and six protoxins (Cry4Aa, Cry4Ba, Cry10Aa, Cry11Aa, Cyt1Aa, and Cyt2Ba) could be rapidly identified from B. thuringiensis subsp. kurstaki HD1 and subsp. israelensis 4Q2-72, respectively. The experimental results indicated that our method is a straightforward tool for analyzing protoxin expression profile in B. thuringiensis strains. Given its technical simplicity and sensitivity, our method might facilitate the present screening program for B. thuringiensis strains with new insecticidal properties.

Identification of cry-type genes on 20-kb DNA associated with Cry1 crystal proteins from Bacillus thuringiensis.

Heterologous DNA fragments (20-kb) associated with Cry1 crystal proteins (protoxins) from a soil-isolated Bacillus thuringiensis strain 4.0718 were isolated and analyzed. RFLP patterns of the PCR products showed that the 20-kb DNA fragments harbored cry1Aa, cry1Ac, cry2Aa, and cry2Ab genes. Furthermore, a 4.2-kb DNA fragment, which contained the promoter, the coding region, and the terminator of cry1Ac gene, was cloned from the 20-kb DNAs by PCR, and then the cry1Ac gene was expressed in an acrystalliferous B. thuringiensis strain 4Q7 by using E. coli-B. thuringiensis shuttle vector pHT3101. SDS-PAGE and microscopy studies revealed that the recombinant could express 130-kDa Cry1Ac protoxin and produce bipyramidal crystals during sporulation. Bioassay results proved that crystal-spore mixture from the recombinant was toxic to Plutella xylostella. This was the first report of cry-type genes present on 20-kb DNA associated with Cry1 protoxins of B. thuringiensis.