Remediation of toxic metals by bacteria offers a relatively inexpensive and efficient way for the decontamination of soil and associated environments. The present study was carried out to investigate the surface characteristics, adsorption, and remobilization of Cd and Cu on bacteria and their composites with soil colloidal components, which are the most active constituents in soils. The bacterial strain NTG-01 (Enterobacter aerogenes), which was both Cd-and Cu-resistant, was isolated from a heavily Cu-contaminated soil of the mining area in Daye suburb of Hubei Province, China. Batch laboratory experiments with NTG-01 and soil colloids were performed to quantify adsorption of Cu and Cd. The surface area of kaolinite and the soil colloids from an Alfisol and Ultisol increased by 3.0-8.8% after the introduction of the bacteria. In the presence of bacterial cells, the negative charges of soil colloid systems increased and the positive charges decreased, shifting pH from 4.0 to 6.5. Our results demonstrate that bacteria promote the adsorption of Cd and Cu by kaolinite and soil colloid systems. However, the heavy metals bound by the bacterial composites could also be easily released by NH4NO3 and EDTA. Caution should be taken when using such bacterial strains in bioremediation of heavy metalcontaminated soils.

Soybean rhizobia and their biodiversities were studied based on a systematicinvestigation of microbial flora at different altitudes of Shennongjia, a forest reserve incentral China. The objectives of this work were to investigate the distribution and thegenetic variation of the indigenous soybean rhizobia in an unexploited virgin forest withno soybean-planting history, and to employ this biodiversity to improve innoculantstrains that are applied to increase biological nitrogen fixation of soybean. Samples werecollected respectively from the soils at 500, 1,060, 1,500, 1,950, 2,400 and 3,100 metersabove sea level in Shennongjia Forest Reserve. Their corresponding pHs were 5.50, 4.91, 5.64, 5.28, 5.49 and 4.60. Results showed that the number of microorganisms in all thesoil samples studied followed the order: bacteria>actinomyces>fungi. Soybean rhizobiawere isolated only from the soils above elevations of 1,500 meter. A total of 25 strainswere isolated using the plant trap method with four different trapping plants. They wereall Sinorhizobium fredii. Their genetic biodiversities were characterized by 16S-23SrDNA intergenic region PCR-RFLP and RAPD analysis. After having been washed twotimes, bacterial suspensions were used as a template for the PCR amplification. Onepair of opposite highly conserved 16S and 23S ribosomal primerspHr(rev) andp23SR01 were used to amplify the intergenic region between 16S and 23S rDNA byPCR. All the tested strains produced a 2.1Kb 16S-23S rDNA fragment. After digestionwith three restriction enzymes (HaeIII, MspI and CfoI) respectively, several differentrestriction patterns were observed. Great variations in 16S-23S RFLP-patterns wereobserved. The tested S. fredii strains could be differentiated in eleven differentgenotypes. Then twelve primers were applied to RAPD analysis and a dendrogram wasobtained. These isolates showed a great diversity. Since Shennongjia is an unexploitedforest region in central China and the gene center of soybean is located in China, thesymbiotic genes harbored by these strains may be of great importance. Further studiesto characterize these isolates are being carried out.

The adsorption and binding of plasmid p34S DNA on four different colloidal fractions from a Brown soil and clay minerals in the presence of various Ca2+ concentrations, the ability of bound DNA to transform competent cells of CaCl2-treated Escherichia coli, and the resistance of bound DNA to degradation by DNase I were studied. DNA adsorption on soil colloids and clay minerals was promoted in the presence of Ca2+. Kaolinite exhibited the highest adsorption affinity for DNA among the examined soil colloids and clay minerals. In comparison with organo-mineral complexes (organic clays) and fine clays (0.02mm), DNA was tightly adsorbed by H2O2-treated clays (inorganic clays) and coarse clays (0.2-2mm). The transformation efficiency of bound DNA increased with increasing concentrations of Ca2+at which soil colloid or clay mineral-DNA complexes were formed. DNA bound by kaolinite showed the lowest transformation efficiency, and especially no transformants were observed with kaolinite-DNA complex prepared at 5-100mM Ca2+. Compared to organic clays and fine clays, DNA bound on inorganic clays and coarse clays showed a lower capacity to transform E. coli at different Ca2+ concentrations. The presence of soil colloids and minerals provided protection to DNA against degradation by DNase I. Montmorillonite, organic clays and fine clays showed stronger protective effects for DNA than inorganic clays and coarse clays. The protection mechanisms as well as the differences in transforming efficiency of plasmid DNA molecules bound on various soil colloidal particles are discussed. The information obtained in this study is of fundamental significance for the understanding of the horizontal dissemination of recombinant DNA and the fate of extracellular DNA in soil environments.

Adsorption of Pseudomonas putida on minerals including montmorillonite, kaolinite and goethite was studied. The adsorption isotherms of P. putida on the examined minerals conformed to the Langmuir equation. The amount of P. putida adsorbed followed the order: goethite>kaolinite>montmorillonite. A greater extent of P. putida adsorption on minerals was observed in the range of temperature from 15 to 35℃. The adsorption of P. putida on minerals decreased with the increase of pH from 3.0 to 10.0. Magnesium ion was more efficient than sodium ion in promoting P. putida adsorption on minerals. The results suggest that electrostatic interactions play a vital role in P. putida adsorption by soil colloidal factions. The information obtained in this study is of fundamental significance for the understanding of the survival and transport of bacteria in soil systems.

3’-Phosphoadenosine-5’-phosphatase (PAPase) is required for the removal of toxic 3’-phosphoadenosine- 5’-phosphate (PAP) produced during sulfur assimilation in various eukaryotic organisms. This enzyme is a well-known target of lithium and sodium toxicity and has been used for the production of salt-resistant transgenic plants. In addition, PAPase has also been proposed as a target in the treatment of manic-depressive patients. One gene, halA, which could encode a protein closely related to the PAPases of yeasts and plants, was identified from the cyanobacterium Arthrospira (Spirulina) platensis. Phylogenic analysis indicated that proteins related to PAPases from several cyanobacteria were found in different clades, suggesting multiple origins of PAPases in cyanobacteria. The HalA polypeptide from A. platensis was overproduced in Escherichia coli and used for the characterization of its biochemical properties. HalA was dependent on Mg2+for its activity and could use PAP or 3’-phosphoadenosine-5’-phosphosulfate as a substrate. HalA is sensitive to Li+ (50% inhibitory concentration [IC50=3.6mM) but only slightly sensitive to Na (IC50=600mM). The salt sensitivity of HalA was thus different from that of most of its eukaryotic counterparts, which are much more sensitive to both Li+ and Na+, but was comparable to the PAPase AtAHL (Hal2p-like protein) from Arabidopsis thaliana. The properties of HalA could help us to understand the structure-function relationship underlying the salt sensitivity of PAPases. The expression of halA improved the Li+ tolerance of E. coli, suggesting that the sulfur-assimilating pathway is a likely target of salt toxicity in bacteria as well.

Protein intein is widespread in a variety of organisms. Several intein elements are also present in cyanobacteria, and some of them have been studied biochemically in vitro. However, no evidence is available for intein removal in vivo in cyanobacteria. In the filamentous cyanobacterium Anabaena sp. strain PCC 7120, the DNA replication factor DnaE is encoded by two split open reading frames (ORFs) far apart from each other on the chromosome, and each of them could contain a split intein element. This organism can undergo a developmental process leading to the formation of nitrogen-fixing cells, or heterocysts. Heterocysts are terminally differentiated cells with arrest of cell cycle. Since DnaE is an important cell cycle element involved in DNA replication, we would like to provide in vivo evidence for DnaE intein removal in cyanobacteria and determine whether mature DnaE protein is still present in heterocysts. In this study, we showed that the products of these two ORFs were joined together to form a complete DnaE protein through the process of protein trans-splicing. More interestingly, protein trans-splicing could be detected in vivo for the first time in cyanobacteria, which allowed us to compare the formation of mature DnaE protein in heterocysts and vegetative cells, and show that mature DnaE protein could be formed in both cell types. Transcriptional fusion between the promoter regions of the two split ORFs and gfp reporter also demonstrate that both ORFs are transcribed in vegetative cells and heterocysts, without strong variation during the process of heterocyst differentiation. Although heterocysts are terminally differentiated and may not replicate its chromosome, the expression and maturation of DnaE in these cells may underlie the need for DNA replication machinery in processes such as DNA recombination and repair.

sp. PCC 7120 is a cyanobacterium capable of performing several important biological functions: photosynthesis, nitrogen fixation, cell differentiation, cell^cell communication, etc. These activities require an extensive signaling capability in order to respond to the changing environment. Based on the genomic data, we have retrieved several gene families encoding signaling components. It is estimated that 211 genes encode two-component signaling elements, and 66 genes encode Ser/Thr kinases and phosphatases. These genes together represent 4.2% of the coding capacity of the whole genome, making Anabaena PCC 7120 a leading member among prokaryotes in terms of its signaling potential. It is known that two-component systems are composed of a few basic modules that can arrange into different structures best adapted for each signaling system. Many proteins in Anabaena PCC 7120 have incorporated both modules of two-component systems and catalytic domains of either Ser/Thr kinases or phosphatases. A family of 13 genes encode proteins with both a Ser/Thr kinase domain and a His kinase domain, and another four genes were also found whose products have both a response regulator domain and a Ser/Thr phosphatase domain. Of all the signaling proteins in Anabaena PCC 7120, about one third (35%) are conserved in the genome of the unicellular cyanobacterium strain Synechocystis sp. PCC 6803. Interestingly, one subfamily of His kinases and two subfamilies of response regulators are found in Anabaena PCC 7120 but are absent in Synechocystis PCC 6803. This study constitutes a basis for analyses of signal transduction in Anabaena PCC 7120 using functional genomic approaches.

Red soil and cinnamon soil were collected from Chenzhou of Hunan and Gongyi of Henan, respectively. Soils were treated with Cu (NO3)2, Zn (NO3)2 or Cd (NO3)2, respectively, for two weeks. Rhizobium fredii strain HN01 was inoculated into the two soils polluted with three heavy metals. Sequential extraction method was employed to investigate the forms of copper (Cu), zinc (Zn), and cadmium (Ca) in the examined soils with the absence and presence of rhizobia. Results showed that the total amount of solid-bound Zn decreased 10% after the inoculation. The decrease for the amount of Zn associated with carbonate, manganese (Mn) oxides, and organic matter fraction was from 9 to 26%. No significant change was observed for the total amount of Zn combined with solid phase of red soil in the presence of rhizobia. However, the amount of specifically adsorbed and Mn oxides bound Zn decreased, while the amount of exchangeable Zn increased. Inoculation of rhizobia depressed the release of Cu to the soil solution and increased the total amount of Cu associated with solid phase in cinnamon soil. The increase for the amount of exchangeable Cu and the Cu in fractions of carbonate, Mn oxides, and organic matter ranged from 20 to 54%. There was no significant change for the level of Cd in the solution in both soils after rhizobia inoculation. The amount of Cd in the fractions of exchangeable and organic increased 22 and 11%, while that in the fractions of specific and Mn oxides decreased 14 and 29%, respectively. The different influence of rhizobia on the distribution of three heavy metals in two soils was mainly ascribed to the growth status and pH changes exerted by the metabolites of rhizobia. These data are helpful for the understanding of the chemical behavior and biogeochemical cycle of heavy metals affected by microorganisms in soil environment, which is fundamental for heavy metal bioremediation.

Adsorption of acid phosphatase on goethite, kaolinite and two colloids from the soils in central and south China in the presence of organic acids and phosphate was studied. With the increase of anion concentration, the ability in decreasing enzyme adsorption followed the sequence: phosphate > tartrate> oxalate >acetate. Acetate showed promotive effect on enzyme adsorption at lower anion concentrations whereas oxalate, tartrate and phosphate compete effectively with enzyme in a broad range of anion concentration. The adsorption isotherms of enzyme in most of the anionic systems studied conformed to the Langmuir equation. Phosphate reduced the affinity of enzyme on goethite more significantly than the other anions. However, tartrate decreased the affinity of enzyme on soil colloids and kaolinite to a greater extent than phosphate, oxalate and acetate. This observation suggested that the impact of anions on enzyme adsorption varies with anionic type and the surface characteristics of soil components. The influence of the addition order of ligand on enzyme adsorption was found greater in tartrate and phosphate systems. In general, simultaneous introduction of ligand and enzyme into the system had the lowest enzyme adsorption, showing more competition between ligand and enzyme molecules in this system. Data from this work indicated that the status and activity of enzyme in certain soil microenvironments especially the rhizosphere where various organic and inorganic ligands are active can be altered and may be completely different from the bulk soil.

The aim of this work was to study the influence of phosphate and citrate, which are common inorganic and organic anions in soils, on the adsorption of acid phosphatase by kaolin, goethite and the colloids separated from yellow-brown soil (YBS) and latosol (LS) in centrM-south China. The YBS colloid has the major clay mineral composition of 1.4 nm mineral, illite and kaolinite while the LS colloid mainly contains kaolinite and oxides. The adsorption isotherm of acid phosphatase on the examined soil colloids and minerals fitted to the Langmuir model. The amount of enzyme adsorbed in the absence of ligands was in the order of YBS colloid>LS colloid>kaolin goethite. In the presence of phosphate or citrate, the amounts of the enzyme adsorbed followed the sequence YBS colloid>kaolin>LS colloid>goethite. The presence of ligands also decreased the binding energy" between the enzyme and soil colloids or minerals. With the increase of ligand concentration from 10 mmol L-1 to 400 m mol L-l, different behaviors for the adsorption of enzyme were found in the colloid and mineral systems studied. A sharp decrease in enzyme adsorption was observed on goethite while gradual decreases of enzyme adsorption were recorded in the two soil colloid systems. However, no any decrease was found for the amount of enzyme adsorbed on kaolin at higher ligand concentrations. When phosphate or citrate was introduced to the system before the addition of enzyme, the ligands usually enhanced the adsorption of enzyme. The results obtained in this study suggested the important role of kaolinite mineral in the adsorption of enzyme molecules in acidic soils in the presence of various ligands.