Bioreactor Expansion Affects Microbial Succession of Mixotrophic Acidophiles and Bioremediation of Cadmium-Contaminated Soils.

Microbial scale-up cultivation is the first step to bioremediating cadmium (Cd)-contaminated soils at the industrial scale. However, the changes in the microbial community as the bioreactor volume expands and their associations with soil Cd removal remain unclear. Herein, a six-stage scale-up cultivation process of mixotrophic acidophiles was conducted, scaling from 0.1 L to 10 m3, to remediate Cd-contaminated soils. The findings showed that bioreactor expansion led to a delay in sulfur and glucose oxidations, resulting in a reduced decline in solution pH and cell density. There were minimal differences observed in bacterial alpha-diversity and community structure as the bioreactor volume increased, except for the 10 m3 scale. However, bioreactor expansion decreased fungal alpha-diversity, changed the community structure, and simplified fungal community compositions. At the family level, Acidithiobacillaceae and Debaryomycetaceae dominated the bacterial and fungal communities throughout the scale-up process, respectively. Correlation analysis indicated that the indirect effect of mixotrophic acidophiles played a significant role in soil Cd removal. Bacterial community shifts, driven by changes in bioreactor volume, decreased the pH value through sulfur oxidation, thereby indirectly enhancing Cd removal efficiency. This study will contribute to the potential industrial application of mixotrophic acidophiles in bioremediating Cd-contaminated soils.

Bioremediation of paddy soil with amphitropic mixture markedly attenuates rice cadmium: Effect of soil cadmium removal and Fe/S-cycling bacteria in rhizosphere.

Cadmium (Cd) pollution in paddy soil can easily lead to excessive Cd in rice, thereby considerably threatening human health. Microbial leaching is an effective pathway for the mobilization and removal of Cd from soil. In this study, an amphitropic mixture (AM) composed of autotrophic and heterotrophic microbial strains was used to leach Cd-contaminated paddy fields. Chemical analysis showed that the AM effectively removed 52 % of the total Cd, 39 % of the available Cd, and 60 % of the exchangeable and carbonate-bound Cd from the paddy soil. After bioleaching, the Cd in the discarded AM solution was adsorbed using a metal adsorbent. Effects of remediation on the soil nutrients or secondary pollution were not significant. Microbial analysis showed that >96 % and 67 % of the indigenous bacteria and fungi, respectively, remained in the AM-remediated soil. Double-cropped rice was cultivated to evaluate the Cd removal efficiency of grains using AM remediation. The Cd in early and late brown rice decreased by 86 % and 56 %, respectively, which was higher than that found for a series of biochemical remediation materials reported in other studies. Furthermore, the AM remediation promoted the growth of iron (Fe)- and sulfur (S)-cycling bacteria in the rice rhizosphere, such as Sulfuricurvum, Desulfurivibrio and Geobacter etc., which reduced the Cd availability in the soil and rice uptake. This study shows that AM has potential applications in the remediation of Cd-contaminated paddy fields and provides a new pathway for safe rice production.

Streptomyces longhuiensis sp. nov, a novel species isolated from soil of Lilium brownii in Hunan Province, PR China.

An actinobacterium strain, designated BH-MK-02T, was isolated from the soil of Lilium brownii. The taxonomic position was determined using a polyphasic approach. Strain BH-MK-02T grew well on International Streptomyces Project series media and formed well-developed, branched substrate hyphae and aerial mycelium that differentiated into straight spore chains with a wrinkled surface. The diagnostic diamino acid was ll-diaminopimelic acid. The major menaquinones were MK-9(H4), MK-9(H6) and MK-9(H8). The polar lipids were diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol mannosides, phosphatidylglycerol and unidentified lipid spots. The predominant fatty acids were anteiso-C15 : 0, iso-C16 : 0, C16 : 0 and C16 : 1 ω7c/C16 : 1 ω6c. The phenotypic characteristics of strain BH-MK-02T indicated that it belonged to the genus Streptomyces. Phylogenetic analysis based on the 16S rRNA gene sequence indicated that strain BH-MK-02T was most closely related to Streptomyces aureus CGMCC 4.1833T (99.7 %). However, the average nucleotide identity and digital DNA-DNA hybridization values between the whole-genome sequences of strain BH-MK-02T and S. aureus CGMCC 4.1833T were 78.1 and 23.2 %, respectively, below the 96.7 and 70 % cut-off points respectively recommended for delineating Streptomyces species. Furthermore, the novel isolate could be distinguished from S. aureus CGMCC 4.1833T by morphological, physiological and biochemical characteristics. Based on all these data, strain BH-MK-02T (=MCCC 1K06237T=JCM 34789T) clearly represents a novel species within the genus Streptomyces, for which the name Streptomyces longhuiensis sp. nov. is proposed.

Purification and water resource circulation utilization of Cd-containing wastewater during microbial remediation of Cd-polluted soil.

The purification and water resource circulation utilization of cadmium-containing leachate is a key link in the field application of microbial remediation in Cd-polluted soil. In this study, through a simulation experiment of microbial remediation of Cd-polluted paddy soil, the feasibility of the purification and recycling process of wastewater derived from microbial remediation of Cd-polluted soil was explored. The results of the microbial mobilization and removal experiment showed that the concentrations of Cd, N, P, and K in the leachate were 88.51 µg/L, 38.06, 0.53, and 98.87 mg/L, respectively. The leachate also contained a large number of microbial resources, indicating that it had high recovery values. To recycle this wastewater, activated carbon (C), humic acid (H), and self-assembled monolayers on mesoporous supports (SAMMS; S) were used as adsorbents. The results showed that the co-existing cations in the leachate had a major influence on the adsorption of Cd. In the ternary system of Fe, Al, and Cd, the removal efficiency of Cd increased to 91.2% when the S dosage was increased to 5‰, and the sorption of Cd occurred after Fe and Al. However, C and H exhibited poor adsorption performances. The isotherm models further showed that the maximum adsorption capacities of S, H, and C were 13.96, 6.41 and 2.94 mg/g, respectively. The adsorption kinetics of S showed that adsorption was a rapid process, and the C-H and O-Si-O of S were the key functional groups. The pH of the leachate significantly affected the adsorption efficiency of Cd. Finally, the purified leachate was successfully applied to microbial cultivation and soil remediation. Overall, the reclamation of Cd-containing wastewater can not only dampen the impacts of water shortages, but also achieve the purposes of Cd removal and resource recovery to lower costs by approximately 1166-3499 yuan per mu.

A review on the bioleaching of toxic metal(loid)s from contaminated soil: Insight into the mechanism of action and the role of influencing factors.

The anthropogenic activities in agriculture, industrialization, mining, and metallurgy combined with the natural weathering of rocks, have led to severe contamination of soils by toxic metal(loid)s. In an attempt to remediate these polluted sites, a plethora of conventional approaches such as Solidification/Stabilization (S/S), soil washing, electrokinetic remediation, and chemical oxidation/reduction have been used for the immobilization and removal of toxic metal(loid)s in the soil. However, these conventional methods are associated with certain limitations. These limitations include high operational costs, high energy demands, post-waste disposal difficulties, and secondary pollution. Bioleaching has proven to be a promising alternative to these conventional approaches in removing toxic metal(loid)s from contaminated soil as it is cost-effective, environmentally friendly, and esthetically pleasing. The bioleaching process is influenced by factors including pH, temperature, oxygen, and carbon dioxide supply, as well as nutrients in the medium. It is crucial to monitor these parameters before and throughout the reaction since a change in any, for instance, pH during the reaction, can alter the microbial activity and, therefore, the rate of metal leaching. However, research on these influencing factors and recent innovations has brought significant progress in bioleaching over the years. This critical review, therefore, presents the current approaches to bioleaching and the mechanisms involved in removing toxic metal(loid)s from contaminated soil. We further examined and discussed the fundamental principles of various influencing factors that necessitate optimization in the bioleaching process. Additionally, the future perspectives on adding omics for bioleaching as an emerging technology are discussed.

Application of mixotrophic acidophiles for the bioremediation of cadmium-contaminated soils elevates cadmium removal, soil nutrient availability, and rice growth.

A major challenge in radically alleviating the threats posed by Cd-contaminated paddy fields to human health is to reduce the Cd levels in both soils and rice grains. In this study, the microbial extraction (ME) treatment using a mixotrophic acidophilic consortium was used for the bioremediation of Cd-contaminated soils. The results showed that the ME treatment enhanced the total Cd (40%) and diethylenetriamine pentaacetic acid-soluble Cd (DTPA-Cd, 64%) removal efficiencies in contaminated soils. In addition, ME treatment decreased the levels of Cd acid-soluble and reducible fractions and thereby reduced Cd uptake in rice tissues. Microbial community analysis indicated that the indigenous soil microbial diversity and composition were not changed after the ME treatment, but the relative abundance of functional microbes associated with Cd removal was improved. Notably, soil available nutrient levels were elevated upon inoculation with mixotrophic acidophiles, resulting in an increase in rice growth and grain weight. This study provides a scientific basis for the potential application and evaluation of ME treatment in the field for remediating Cd-contaminated paddy soils.

Biogeographic pattern and relevant environmental factors for rhizobial communities in the rhizosphere and root nodules of kudzu (Pueraria lobata).

Kudzu (Pueraria lobata) is an important medicinal plant, which can associate with rhizobia for nitrogen fixation. The mutualistic symbiosis between rhizobium and kudzu is not well understood, but it is necessary to fully utilize kudzu. Nodules and rhizosphere soils collected from 16 sampling sites were characterized based on phylogenetic analyses of the rpoB gene; 16S rRNA gene; the housekeeping genes SMc00019, truA, and thrA; and the symbiotic genes nodA and nifH. The relationships between biogeographic pattern, nitrogenase activity, and environmental factors were studied. Results indicated that a clear biogeographic pattern of rhizobial communities in the kudzu rhizosphere existed in southern China; latitude and soil pH were found to be the most important factors affecting the biogeographic pattern. Bradyrhizobium diazoefficiens and Bradyrhizobium erythrophlei were the dominant species in kudzu rhizosphere. The symbiotic rhizobia in kudzu nodules mainly belonged to B. lablabi, B. elkanii, B. pachyrhizi, and B. japonicum. Nitrogenase activities in the nodules of kudzu in the Jiangxi sampling region were significantly higher than those in the Guangxi and Hunan sampling regions, and they were significantly negatively correlated to pH and exchangeable Ca. These results constitute the first report of the existence of symbiotic genes in kudzu bradyrhizobia, which are similar to those in B. elkanii and B. pachyrhizi. Our findings could improve the understanding of kudzu-rhizobium symbiosis and could advance the application of rhizobial inoculation in medicinal legumes in terms of increasing the content of active ingredients.

Cultivar-dependent rhizobacteria community and cadmium accumulation in rice: Effects on cadmium availability in soils and iron-plaque formation.

The association between the rhizospheric microbial community and Cd accumulation in rice is poorly understood. A field trial was conducted to investigate the different rhizobacterial communities of two rice cultivars with high Cd accumulation (HA) and low Cd accumulation (LA) at four growth stages. Results showed that the Cd content in the roots of the HA cultivar was 1.23 - 27.53 higher than that of the LA cultivar (0.08 - 10.5 µg/plant) at four stages. The LA cultivar had a significantly lower Cd availability in rhizosphere and a higher quantity of iron plaque (IP) on the root surface than the HA cultivar at four stages. This resulted in the reduction of Cd concentration in IPs and Cd translocation from IP-to-root. Microbial analysis indicated that the LA cultivar formed a distinct rhizobacterial community from the HA cultivar and had less α-diversity. The rhizosphere of the LA cultivar was enriched in specific bacterial taxa (e.g., Massilia and Bacillus) involved in Cd immobilization by phosphate precipitation and IP formation by iron oxidization. However, the rhizosphere in the HA cultivar assembled abundant sulfur-oxidizing bacteria (e.g., Sulfuricurvum) and iron reduction bacteria (Geobacter). They promoted Cd mobilization and reduced IP formation via the metal redox process. This study reveals a potential approach in which specific rhizobacteria decrease or increase Cd accumulation in rice on contaminated soil and provides a new perspective for secure rice production.

Ecological responses of bacterial assembly and functions to steep Cd gradient in a typical Cd-contaminated farmland ecosystem.

The response of soil bacterial communities from farmland ecosystems to cadmium (Cd) pollution, in which a steep concentration gradient of more than 100 mg/kg has naturally formed, has not previously been fully reported. In this study, a field investigation was conducted in a typical severe Cd-polluted farmland ecosystem, and the bacterial community response to the steep Cd gradient was analyzed. The results showed that Cd concentration sharply decreased from 159.2 mg/kg to 4.18 mg/kg among four sampling sites alongside an irrigation canal over a distance of 150 m. Bacterial diversity and richness were significantly lower in highly polluted sites, and random forest analysis indicated that Cd gradient played a decisive role in reducing alpha diversity. Redundancy analysis (RDA) and co-occurrence network indicated that the synergistic effects of pH, Cd, and phosphorus were the main drivers shaping community structure. The functional results predicted by BugBase suggested that the bacterial community may adapt to the harsh environment by recruiting Cd-resistant microbes and improving oxidative stress tolerance of the whole community. Cd-resistant microorganisms such as Burkholderia, Bradyrhizobium, and Sulfurifustis, which directly or indirectly participate in diminishing oxidative damage of Cd, may play essential roles in maintaining community stability and might be potential bacterial resources for the bioremediation of Cd pollution.

Cadmium Speciation Distribution Responses to Soil Properties and Soil Microbes of Plow Layer and Plow Pan Soils in Cadmium-Contaminated Paddy Fields.

Cadmium (Cd) speciation ratio in arable land determines the Cd exposure risk and Cd uptake in crops. However, the driving mechanisms of Cd speciation change on the vertical scale of paddy fields remain poorly understood. In this study, the effects of plow layer and plow pan on Cd speciation distribution were investigated in a long-term Cd-contaminated rice ecosystem. The Cd accumulative effect within rice grain was enhanced with high levels of activated Cd speciation ratios in soils. Activated Cd speciation ratios were higher in plow layer soils, while stabilized Cd speciation ratios were elevated in plow pan soils. Soil physicochemical properties and soil microbes synergistically affected the Cd speciation changes in different ways between the two soil layers. Soil pH and organic elements in plow layer environment directly hindered the transformation of stabilized Cd speciation, while in plow pan environment, soil pH and organic elements indirectly decreased activated Cd speciation ratios and resulted in the accumulation of stabilized Cd speciation via regulating the predominant bacterial taxa. This study will improve our understanding of how soil environments regulate Cd speciation distributions in rice ecosystems and help to seek effective remediation methods of Cd-contaminated paddy fields to reduce the Cd accumulation in rice.

Reduction mechanism of Cd accumulation in rice grain by Chinese milk vetch residue: Insight into microbial community.

Chinese milk vetch is an efficient approach to reduce Cd accumulation in rice, nevertheless, its reduction mechanism is not well understood. In this study, we investigated the rice grain Cd, soil properties and microbial community in a Cd-polluted paddy field amended with milk vetch residue (MV) or without (CK) during rice growth period. We found that milk vetch residue averagely decreased the Cd content in rice grain by 45%. Decrease of Cd in rice mainly attributed to the inhibition of Cd activation by milk vetch residue at heading stage probably by the formation of HA-Cd (Humic Acid) and CdS. Increased pH and organic matter (OM) promoted the reduction of available Cd. In addition, nonmetric multidimensional scaling (NMDS) analysis revealed that microbial community structure was significantly different between MV and CK treatment (r = 0.187, p = 0.002), and the core functions of differentially abundant genera were mainly associated with N-cycling, organic matter degradation and sulfate-reducing. The application of milk vetch residue increased the abundance of sulfate-reducing bacteria (SRB) by 8-112% during the rice growth period, which may involve in promoting the transformation of Cd to a more stably residual Cd (CdS). Canonical correspondence analysis (CCA) and mantel test analysis indicated that available K (p = 0.004) and available N (p = 0.005) were the key environmental factors of shaping the SRB. Altogether, changes in soil properties affected microbial structure and functional characteristics, especially the response of SRB in MV treatment would provide valuable insights into reducing the bioavailability of Cd in soil.

Bioremediation of cadmium-contaminated paddy soil using an autotrophic and heterotrophic mixture.

Cadmium (Cd) pollution poses a serious risk to human health and ecological security. Bioremediation can be a promising and effective remediation technology for treating Cd contaminated soils. In this study, seven heterotrophic strains were isolated from Cd contaminated soil and 7 autotrophic strains were isolated from acid mine drainage. Cd removal efficiencies were compared after leaching with autotrophic bacteria (Att-sys), heterotrophic isolates (Htt-sys) and cooperative leaching systems (Co-sys) in laboratory agitating reactors. The results indicated that Cd removal efficiency of Co-sys (32.09%) was significantly higher than that of Att-sys (23.24%) and Htt-sys (0.74%). By analyzing the soil microbial community in different bioleaching systems, we found that the addition of heterotrophic isolates significantly promoted the growth of some heavy metal resistant inhabitants (Massilia, Alicyclobacillus, Micromonospora, etc.), and Co-sys had a minor effect on the growth of soil indigenous microbes. In Co-sys, the content of the four Cd fractions all decreased compared with other leaching systems. The analysis of soil physicochemical parameters during the leaching process showed that pH and ORP (oxidation reduction potential) were not the only determinants for Cd removal efficiency in Co-sys, synergistic metabolic activities of autotrophic and heterotrophic strains may be other determinants. This study demonstrated that cooperative bioremediation may prove to be a safe and efficient technique for field application in heavy metal soil pollution.

Spatial distribution and risk assessment of heavy metals in contaminated paddy fields - A case study in Xiangtan City, southern China.

An extensive investigation on spatial distribution and environmental risk assessment based on total content and fractions of heavy metals, as well as the cancer risk of Cd from seven adjacent contaminated paddy fields at Xiangtan City, southern China, was conducted in this study. A total of 63 soil samples were analyzed for soil physical properties and concentrations of eight heavy metals (Cd, Cr, Co, Cu, Mn, Ni, Pb, Zn). The results showed that concentrations of metals except for Cr, Mn and Ni exceeded the background values to varying degrees, and particularly, content of Cd was as 57.4-612 times higher than background values. Principal components analysis and correlation analysis revealed three groups: industry activities for Cd and Zn; natural sources mainly for Cu, Pb, Ni and Cr, with some slight anthropogenic activities for Cu and Pb accumulation; and manganese ore associated with cobalt for Co and Mn. Combined with different indices, Cd and Zn were the major contributors to the ecological risk, and cancer risk of Cd indicated an unacceptable degree in this area. Altogether, results from this study will facilitate a better understanding of metals distribution characteristics and provide a scientific basis for further comprehensive management for these paddy fields. Combination of functional microbial agent and plants promises to be a feasible and effective remediation method for cadmium pollution in the study area.

Mixotrophic acidophiles increase cadmium soluble fraction and phytoextraction efficiency from cadmium contaminated soils.

A profound concern in developing microbially-assisted phytoextraction is that introduced microbes not only remove heavy metals from contaminated soils but also enhance metal uptake into plant tissues from the treated soils. Cadmium (Cd) removal efficiencies were compared after leaching with deionized water (CK), acidified basal salts medium (acid control), cell-free spent medium (spent bioleaching) and mixotrophic acidophiles (two-step bioleaching). Two-step bioleaching using the mixotrophic acidophiles removed 34% of total Cd and 87% of available Cd, significantly more than CK (3% and 4%), acid control (12% and 51%) and spent bioleaching (26% and 75%). Pot experiments of water spinach growing in four treated soils were conducted to evaluate the Cd uptake performance in plants. Notably, the mixotrophic acidophiles increased Cd concentration in plant tissues by 78% compared to the CK group. More interestingly, the mixotrophic acidophiles were not colonized in soils but caused the compositional increase of indigenous microbes such as the genera of Alicyclobacillus, Clostridium sensu strict and Streptacidiphilus. Meanwhile, two-step bioleaching had little effects on soil structure and physicochemical properties determined by the spectroscopy characteristics analysis. These results implied that the mixotrophic acidophiles facilitated the development of microbially-assisted phytoextraction technology.

Combined use of Bacillus subtilis strain B-001 and bactericide for the control of tomato bacterial wilt.

BACKGROUND: Tomato bacterial wilt caused by Ralstonia solanacearum poses a serious threat to tomato production. However, no effective control measures are available. In this study, the bactericide Saisentong was combined with an effective biological control agent, Bacillus subtilis B-001, to control tomato bacterial wilt under greenhouse and field conditions. RESULTS: Growth of B-001 in vitro was unaffected by Saisentong. In greenhouse experiments, the combined application of B-001 and Saisentong via root irrigation or spray resulted in better disease control compared with either agent alone. In two field trials, at a Saisentong concentration of 400 or 500 mg kg-1 , the combined treatment was more effective than expected and showed a synergistic effect. A lower concentration of Saisentong (200 or 300 mg kg-1 ) in combination with B-001 resulted in an antagonistic effect. However, disease control was significantly greater compared with either treatment alone. CONCLUSION: The combination of Saisentong and B-001 effectively controls tomato bacterial wilt. The integrated strategy represents a promising new tool to control this disease. © 2016 Society of Chemical Industry.

Effects of Arsenite Resistance on the Growth and Functional Gene Expression of Leptospirillum ferriphilum and Acidithiobacillus thiooxidans in Pure Culture and Coculture.

The response of iron-oxidizing Leptospirillum ferriphilum YSK and sulfur-oxidizing Acidithiobacillus thiooxidans A01 to arsenite under pure culture and coculture was investigated based on biochemical characterization (concentration of iron ion and pH value) and related gene expression. L. ferriphilum YSK and At. thiooxidans A01 in pure culture could adapt up to 400 mM and 800 mM As(III) after domestication, respectively, although arsenite showed a negative effect on both strains. The coculture showed a stronger sulfur and ferrous ion oxidation activity when exposed to arsenite. In coculture, the pH value showed no significant difference when under 500 mM arsenite stress, and the cell number of At. thiooxidans was higher than that in pure culture benefiting from the interaction with L. ferriphilum. The expression profile showed that the arsenic efflux system in the coculture was more active than that in pure culture, indicating that there is a synergetic interaction between At. thiooxidans A01 and L. ferriphilum YSK. In addition, a model was proposed to illustrate the interaction between arsenite and the ars operon in L. ferriphilum YSK and At. thiooxidans A01. This study will facilitate the effective application of coculture in the bioleaching process by taking advantage of strain-strain communication and coordination.

Comparative study of nickel resistance of pure culture and co-culture of Acidithiobacillus thiooxidans and Leptospirillum ferriphilum.

The effect of Ni²âº on the growth and functional gene expression of the pure culture and co-culture of Acidithiobacillus thiooxidans and Leptospirillum ferriphilum has been studied. Compared with the pure culture, the co-culture showed a stronger sulfur and ferrous ion oxidation activity. At 100 mM, A. thiooxidans in co-culture grew faster and had 48 h shorter lag phases. The cell number of A. thiooxidans in co-culture was about 5 times higher than that in pure culture. The existence of A. thiooxidans in co-culture activated the expression of some metal resistance genes in L. ferriphilum at least 16 h in advance. A. thiooxidans in co-culture tends to chose more efficient pathways to transport nickel ion, ensuring the export of heavy metal was faster and more effective than that in pure culture. All the data indicated that there were synergetic interactions between iron- and sulfur-oxidizing bacteria under the stress of Ni²âº.

The co-culture of Acidithiobacillus ferrooxidans and Acidiphilium acidophilum enhances the growth, iron oxidation, and CO2 fixation.

Although the synergetic interactions between chemolithoautotroph Acidithiobacillus ferrooxidans and heterotroph Acidiphilium acidophilum have drawn a share of attention, the influence of Aph. acidophilum on growth and metabolic functions of At. ferrooxidans is still unknown on transcriptional level. To assess this influence, a co-culture composed by At. ferrooxidans and Aph. acidophilum was successfully acclimated in this study. Depending on the growth dynamics, At. ferrooxidans in co-culture had 2 days longer exponential phase and 5 times more cell number than that in pure culture. The ferrous iron concentration in culture medium and the expression of iron oxidation-related genes revealed that the energy acquisition of At. ferrooxidans in co-culture was more efficient than that in pure culture. Besides, the analysis of CO2 fixation-related genes in At. ferrooxidans indicated that the second copy of RuBisCO-encoding genes cbbLS-2 and the positive regulator-encoding gene cbbR were up-regulated in co-culture system. All of these results verified that Aph. acidophilum could heterotrophically grow with At. ferrooxidans and promote the growth of it. By means of activating iron oxidation-related genes and the second set of cbbLS genes in At. ferrooxidans, the Aph. acidophilum facilitated the iron oxidation and CO2 fixation by At. ferrooxidans.

Asp97 is a crucial residue involved in the ligation of the [Fe4S4] cluster of IscA from Acidithiobacillus ferrooxidans.

IscA was proposed to be involved in the iron-sulfur cluster assembly encoded by the iscSUA operon, but the role of IscA in the iron-sulfur cluster assembly still remains controversial. In our previous study, the IscA from A. ferrooxidans was successfully expressed in Escherichia coli, and purified to be a [Fe4S4]-cluster-containing protein. Cys35, Cys99, and Cys101 were important residues in ligating with the [Fe4S4] cluster. In this study, Asp97 was found to be another ligand for the iron-sulfur cluster binding according to site-directed mutagenesis results. Molecular modeling for the IscA also showed that Asp97 was a strong ligand with the [Fe4S4] cluster, which was in good agreement with the experimental results. Thus, the [Fe4S4] cluster in IscA from A. ferrooxidans was ligated by three cysteine residues and one aspartic acid.

In vitro assembly of [Fe4S4] cluster in high potential iron-sulfur protein from Acidithiobacillus ferrooxidans.

High-potential iron-sulfur protein (HiPIP) has been proposed to be involved in the iron respiratory electron transport chain in Acidithiobacillus ferrooxidans, which contains an [Fe(4)S(4)] cluster. We report here the assembly of an [Fe(4)S(4)] cluster in HiPIP from A. ferrooxidans ATCC 23270 in vitro in the presence of Fe(2+) and sulfide. The spectra and matrix-assisted laser desorption ionization-time of flight mass spectrometry results of holoHiPIP confirmed that the iron-sulfur cluster was correctly assembled into the protein.