Effects of Farmhouse Hotel and Paper Mill Effluents on Bacterial Community Structures in Sediment and Surface Water of Nanxi River,China

The pyrosequencing technique was used to evaluate bacterial community structures in sediment and surface water samples taken from Nanxi River receiving effluents from a paper mill and a farmhouse hotel, respectively. For each sample, 4,610 effective bacterial sequences were selected and used to do the analysis of diversity and abundance, respectively. Bacterial phylotype richness in the sediment sample without effluent input was higher than the other samples, and the surface water sample with addition of effluent from the paper mill contained the least richness. Effluents from both the paper mill and farmhouse hotel have a potential to reduce the bacterial diversity and abundance in the sediment and surface water, especially it is more significant in the sediment. The effect of the paper mill effluent on the sediment and surface water bacterial communities was more serious than that of the farmhouse hotel effluent. Characterization of microbial community structures in the sediment and surface water from two tributaries of the downstream river indicated that various effluents from the paper mill and farmhouse hotel have the similar potential to decrease the natural variability in riverine microbial ecosystems.     

Extensive profiling of a complex microbial community by high-throughput sequencing

Complex microbial communities remain poorly characterized despite their ubiquity and importance to human and animal health, agriculture, and industry. Attempts to describe microbial communities by either traditional microbiological methods or molecular methods have been limited in both scale and precision. The availability of genomics technologies offers an unprecedented opportunity to conduct more comprehensive characterizations of microbial communities. Here we describe the application of an established molecular diagnostic method based on the chaperonin-60 sequence, in combination with high-throughput sequencing, to the profiling of a microbial community: the pig intestinal microbial community. Four libraries of cloned cpn60 sequences were generated by two genomic DNA extraction procedures in combination with two PCR protocols. A total of 1,125 cloned cpn60 sequences from the four libraries were sequenced. Among the 1,125 cloned cpn60 sequences, we identified 398 different nucleotide sequences encoding 280 unique peptide sequences. Pairwise comparisons of the 398 unique nucleotide sequences revealed a high degree of sequence diversity within the library. Identification of the likely taxonomic origins of cloned sequences ranged from imprecise, with clones assigned to a taxonomic subclass, to precise, for cloned sequences with 100% DNA sequence identity with a species in our reference database. The compositions of the four libraries were compared and differences related to library construction parameters were observed. Our results indicate that this method is an alternative to 16S rRNA sequence-based studies which can be scaled up for the purpose of performing a potentially comprehensive assessment of a given microbial community or for comparative studies.     

Draft genome sequence of Novosphingobium nitrogenifigens Y88(T)

Novosphingobium nitrogenifigens was originally isolated from pulp and paper mill wastewater, a low-nitrogen, high-carbon environment. N. nitrogenifigens is the first known nitrogen-fixing, polyhydroxyalkanoate-accumulating sphingomonad, and we report the annotated draft genome sequence of the type strain Y88(T) here.     

Influence of Long Term Irrigation with Pulp and Paper Mill Effluent on the Bacterial Community Structure and Catabolic Function in Soil

Microbial communities play a vital role in maintaining soil health. A multiphasic approach to assess the effect of pulp and paper mill effluent on both the structure and function of microbial soil communities is taken. Bacterial communities from agricultural soils irrigated with pulp and paper mill effluent were compared to communities form soils irrigated with well water. Samples were taken from fields in the state of Uttarakhand, India, where pulp and paper mill effluent has been used for irrigation for over 25 years. Comparisons of bacterial community structure were conducted using sequencing of the 16S rRNA gene from both isolates and clone libraries attained from the soil. Community-level physiological profiling was used to characterize the functional diversity and catabolic profile of the bacterial communities. The multiphasic approach using both physiological and molecular techniques proved to be a powerful tool in evaluating the soil bacterial community population and population differences therein. A significant and consistent difference in the population structure and function was found for the bacterial communities from soil irrigated with effluent in comparison to fields irrigated with well water. The diversity index parameters indicated that the microbial community in pulp and paper mill effluent irrigated fields were more diverse in both structure and function. This suggests that the pulp and paper mill effluent is not having a negative effect on the soil microbial community, but in fact may have a positive influence. In terms of soil health, this finding supports the continued use of pulp and paper mill effluent for irrigation. This is however only one aspect of soil health which was evaluated. Further studies on soil resistance and robustness could be undertaken to holistically evaluate soil health in this situation.     

Analysis of microbial diversity in tomato paste wastewater through PCR-DGGE

This study was conducted to evaluate the relationship between the flora and the changes in the microbial communities during tomato paste wastewater treatment. The bio-analytical techniques like Polymerase chain reaction denaturing gradient gel electrophoresis (PCR-DGGE), adenosine-triphosphate (ATP) analysis, and testing of mixed liquid and suspended solids (MLSS) were simultaneously conducted to analyze the dynamics of the microbial communities during tomato paste wastewater treatment process. The study suggests that the combined approaches of PCR-DGGE, ATP, and MLSS provided a simple and accurate method to evaluate the changes in microbial activity, microbial structure, and population size with the shift in contaminants in different treatment processes. The study also demonstrates that the structure and quantity of a microbial community are influenced by MLSS during the wastewater treatment process, which consequently determines the overall functionality of the system.     

Coliform bacteria and nitrogen fixation in pulp and paper mill effluent treatment systems

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N(2) fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N(2) fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N(2)-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N(2) fixation (0.87 to 4.90 mg of N liter(-1) day(-1)) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.     

The effect of Ni(II) on properties of bulking activated sludge and microbial analysis of sludge using 16S rDNA gene

This study investigated the effect of nickel on properties and microbial community of bulking activated sludge when 60-240 mg/L Ni(II) was dosed continuously in a sequencing batch reactor (SBR) over 350 days. Results showed that 120 mg/L nickel did not significantly inhibited removal of organic pollutant by activated sludge. However, the system was completely upset when 240 mg/L Ni(II) was dosed. Improvement of settling and dewatering ability was also observed with the addition of Ni(II). In addition, investigations by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) of 16S rDNA of bacteria strain demonstrated that Ni(II) significantly affected microbial community of bulking activated sludge, judging from the elimination of original species and emergence of possible new nickel-resistant bacteria. The effect of nickel on shift of microbial community was an important cause resulted in the improvement of sludge properties in this bulking activated sludge system.     

Resolution of Phenotypically Distinct Strains of Enterococcus spp. in a Complex Microbial Community Using cpn60 Universal Target Sequencing

Characterization of complex microbial communities is frequently based on the examination of polymerase chain reaction amplified sequences from a single phylogenetic marker, usually the 16S rRNA gene. However, this commonly used target often does not offer robust resolution of species or sub-species and is thus not a sufficiently informative target for understanding microbial population dynamics occurring at the strain level. We have used the cpn60 universal target sequence to characterize Enterococcus isolates from feces of growing pigs and have shown that sub-species groups, not detected using 16S rRNA sequences, can be resolved. Furthermore, groups resolved by cpn60-based phylogenetic analysis have distinct phenotypes. We report changes in the structure and function of Enterococcus communities in pig feces sampled from individual animals at three times, from suckling through to maturity. Enterococcus faecalis was largely replaced by Enterococcus hirae between suckling and 9 weeks of age, and a shift from one sub-species group of E. hirae to another was observed in all animals between 9 and 15 weeks. Conversely, E. faecalis strains remained consistent throughout the study period. Our results demonstrate that cpn60 sequences can be used to detect strain level changes in Enterococcus populations during succession in the fecal microbiota of growing pigs.     

Pyrosequencing of the Chaperonin-60 Universal Target as a Tool for Determining Microbial Community Composition

We compared dideoxy sequencing of cloned chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.Scientific interest in human microbial communities is growing, and basic concepts about the “human microbiome” are evolving rapidly (3, 34). Molecular phylogenetic analysis of 16S rRNA-encoding DNA sequences has revealed a vast diversity of uncultured microbial symbionts that influence animal physiology in ways only beginning to be understood. In particular, microbial species inhabiting the human vagina are thought to play an important role in host health (10). A shift in the composition of the vaginal microbiota from “normal” (Lactobacillus dominated) to a state defined as bacterial vaginosis (BV; increased abundance of gram-negative organisms) is associated with a range of negative outcomes, including pelvic inflammatory disease, preterm births, and the acquisition of sexually transmitted diseases (21, 22, 37). This observation has led to an increased interest in determining the composition of the vaginal microbiota by culture-independent methods (8, 11, 17, 25, 30, 35, 36). However, established cloning and sequencing techniques remain time- and labor-intensive, severely limiting the reach of phylogenetic or functional surveys of microbial communities across body sites, individuals, geographic areas, and scales of time.The advent of next-generation ultra-high-throughput sequencing technologies, in particular, the GS FLX (454 Life Sciences, Branford, CT), has removed an important quantitative barrier in molecular analysis by increasing the number of reads from a gene or genome by orders of magnitude in a single run (20). Unfortunately, the short average length of pyrosequencing reads (∼200 bp compared to ∼700 bp using dideoxy sequencing) presents a new set of problems. The results of recent application of this technology to analysis of 16S rRNA gene sequences from microbes in vaginal samples have demonstrated that short reads are more likely to generate matches to multiple sequences in the rRNA sequence database and that taxonomic and phylogenetic resolution was limited due to strong similarities between 16S rRNA sequences from closely related species (32).An alternative molecular target for microbial identification and phylogenetic analysis is cpn60, a gene that encodes the 60-kDa chaperonin or heat shock protein (HSP60/GroEL) (13). The cpn60 gene is universal in eubacteria and eukaryotes and an extensive, curated reference database is available (13) (http://cpndb.cbr.nrc.ca). The cpn60 universal target (UT) offers key advantages, including short target length (549 to 567 bp), sufficient resolving power to distinguish closely related species and subspecies, and a relatively uniform distribution of variability across the entire length of the target (9, 12). The use of the cpn60 UT has been well established for phylogenetic analysis of complex samples (4, 14) and has recently been applied to vaginal microbial communities (11). In the present study, we examined the feasibility of pyrosequencing for determining the composition of the vaginal microbiota using the cpn60 UT. We compared the microbial community structure generated by pyrosequencing of cpn60 amplicons using the GS FLX with dideoxy sequencing based on clone libraries generated from the same samples. In addition, we evaluated the microbial community profiles generated by pyrosequencing of cpn60 UT amplicons and 16S rRNA amplicons from the same vaginal samples.     

Community analysis of a full-scale anaerobic bioreactor treating paper mill wastewater

To get insight into the microbial community of an Upflow Anaerobic Sludge Blanket reactor treating paper mill wastewater, conventional microbiological methods were combined with 16S rRNA gene analyses. Particular attention was paid to microorganisms able to degrade propionate or butyrate in the presence or absence of sulphate. Serial enrichment dilutions allowed estimating the number of microorganisms per ml sludge that could use butyrate with or without sulphate (10(5)), propionate without sulphate (10(6)), or propionate and sulphate (10(8)). Quantitative RNA dot-blot hybridisation indicated that Archaea were two-times more abundant in the microbial community of anaerobic sludge than Bacteria. The microbial community composition was further characterised by 16S rRNA-gene-targeted Denaturing Gradient Gel Electrophoresis (DGGE) fingerprinting, and via cloning and sequencing of dominant amplicons from the bacterial and archaeal patterns. Most of the nearly full length (approximately 1.45 kb) bacterial 16S rRNA gene sequences showed less than 97% similarity to sequences present in public databases, in contrast to the archaeal clones (approximately. 1.3 kb) that were highly similar to known sequences. While Methanosaeta was found as the most abundant genus, also Crenarchaeote-relatives were identified. The microbial community was relatively stable over a period of 3 years (samples taken in July 1999, May 2001, March 2002 and June 2002) as indicated by the high similarity index calculated from DGGE profiles (81.9+/-2.7% for Bacteria and 75.1+/-3.1% for Archaea). 16S rRNA gene sequence analysis indicated the presence of unknown and yet uncultured microorganisms, but also showed that known sulphate-reducing bacteria and syntrophic fatty acid-oxidising microorganisms dominated the enrichments.     

Effects of substrate composition on the structure of microbial communities in wastewater using fluorescence in situ hybridisation

The microbial composition in a pulp and paper wastewater aerated lagoon system was analysed using fluorescence in situ hybridisation (FISH) to gain further understanding of the effect of substrate composition on microbial diversity for improved management of wastewater treatment systems. Few experiments have been conducted to tease apart the factors influencing the composition and abundance of certain groups within these wastewaters. Specific probes were used to investigate and enumerate the different bacterial groups present at particular stages through the treatment system over an extended period. Community composition and abundance of specific groups differed through the system however temporal stability was retained despite significant variability in the wastewater. Middle stream wastewater samples were enriched to explore the impact of different carbon/nitrogen/phosphorus (C:N:P) ratios on community composition and provide functionality to groups of micro-organisms within the microbial consortia. Nitrogen and phosphorus conditions did not impact community composition of methanol-fed cultures, which exhibited a dominance of Betaproteobacteria (>75%), namely Methylotrophic bacteria. This was confirmed through 16S rRNA gene sequencing and specific FISH probing, reflecting population observations at the beginning of the treatment system. We conclude that the nutrient and carbon combinations used in the enrichments created an interactive effect, altering the community composition and mimicking the main substrate load in the different stages of the treatment system. Finally, pulp and paper wastewater microbial composition was highly variable across the treatment system but was stable within the time sampled, with the enrichments emulating the substrate loads in the full scale system.     

Metal biosorption in lignocellulosic biofuel biorefinery effluent: an initial step towards sustainability of water resources

Biosorption of metals by microorganisms is a promising technology to remove accumulated non-process elements in highly recycled biorefinery process water. Removal of these elements would enable greater water reuse and reduce the environmental impact of effluent discharge. A model lignocellulosic ethanol biorefinery wastewater was created based on pulp mill effluent. This generated a wastewater with an environmentally realistic high loading of dissolved natural organic matter (900?mg/l), a potentially important factor influencing metal biosorption. Analysis of feedstock and pulp mill effluent indicated that Mn and Zn are likely to be problematic in highly recycled lignocellulosic ethanol biorefinery process water. Therefore, the growth of several bacteria and fungi from existing collections, and some isolated from pulp mill effluent were tested in the model wastewater spiked with Mn and Zn (0.2?mM). Wastewater isolates grew the best in the wastewater. Metal uptake varied by species and was much greater for Zn than Mn. A bacterium, Novosphingobium nitrogenifigens Y88(T), removed the most metal per unit biomass, 35 and 17?mg?Mn/g. No other organism tested decreased the Mn concentration. A yeast, Candida tropicalis, produced the most biomass and removed the most total metal (38?% of Zn), while uptake per unit biomass was 24?mg?Zn/g. These results indicate that microorganisms can remove significant amounts of metals in wastewater with high concentrations of dissolved natural organic matter. Metal sorption by autochthonous microorganisms in an anaerobic bioreactor may be able to extend water reuse and therefore lower the water consumption of future biorefineries.     

Coliform Bacteria and Nitrogen Fixation in Pulp and Paper Mill Effluent Treatment Systems

The majority of pulp and paper mills now biotreat their combined effluents using activated sludge. On the assumption that their wood-based effluents have negligible fixed N, and that activated-sludge microorganisms will not fix significant N, these mills routinely spend large amounts adding ammonia or urea to their aeration tanks (bioreactors) to permit normal biomass growth. N₂ fixation in seven Eastern Canadian pulp and paper mill effluent treatment systems was analyzed using acetylene reduction assays, quantitative nitrogenase (nifH) gene probing, and bacterial isolations. In situ N₂ fixation was undetectable in all seven bioreactors but was present in six associated primary clarifiers. One primary clarifier was studied in greater detail. Approximately 50% of all culturable cells in the clarifier contained nifH, of which >90% were Klebsiella strains. All primary-clarifier coliform bacteria growing on MacConkey agar were identified as klebsiellas, and all those probed contained nifH. In contrast, analysis of 48 random coliform isolates from other mill water system locations showed that only 24 (50%) possessed the nifH gene, and only 13 (27%) showed inducible N₂-fixing activity. Thus, all the pulp and paper mill primary clarifiers tested appeared to be sites of active N₂ fixation (0.87 to 4.90 mg of N liter⁻¹ day⁻¹) and a microbial community strongly biased toward this activity. This may also explain why coliform bacteria, especially klebsiellas, are indigenous in pulp and paper mill water systems.     

污水生物处理中的噬菌体及其功能研究进展

污水生物处理是一种利用微生物分解污水中的污染物、实现污水净化的方法。噬菌体是侵染细菌的病毒,在污水生物处理系统中广泛存在,它们能够特异性地控制微生物菌群,影响污水处理效果和调控污泥性状。因此,研究污水生物处理中噬菌体的分布及其功能具有重要意义。本文介绍了不同污水生物处理中噬菌体的分布,简要分析了噬菌体分离、培养与鉴定方法及其优缺点,详细总结了噬菌体在污水生物处理中的功能,包括：（1）调节微生物群落结构,影响污水处理效果;（2）作为环境监测的指示生物;（3）控制病原菌、污泥膨胀、污泥发泡和膜污染;（4）减少污泥产量,重点分析了影响噬菌体功能的因素,探讨了污水生物处理中噬菌体功能应用存在的问题及其解决方法,最后对噬菌体未来应用的发展方向进行了展望,以期为污水生物处理技术和工艺的开发与应用提供参考,促进污水处理健康发展。     

Effect of PCR amplicon size on assessments of clone library microbial diversity and community structure

PCR-based surveys of microbial communities commonly use regions of the small-subunit ribosomal RNA (SSU rRNA) gene to determine taxonomic membership and estimate total diversity. Here we show that the length of the target amplicon has a significant effect on assessments of microbial richness and community membership. Using operational taxonomic unit (OTU)- and taxonomy-based tools, we compared the V6 hypervariable region of the bacterial SSU rRNA gene of three amplicon libraries of c. 100, 400 and 1000 base pairs (bp) from each of two hydrothermal vent fluid samples. We found that the smallest amplicon libraries contained more unique sequences, higher diversity estimates and a different community structure than the other two libraries from each sample. We hypothesize that a combination of polymerase dissociation, cloning bias and mispriming due to secondary structure accounts for the differences. While this relationship is not linear, it is clear that the smallest amplicon libraries contained more different types of sequences, and accordingly, more diverse members of the community. Because divergent and lower abundant taxa can be more readily detected with smaller amplicons, they may provide better assessments of total community diversity and taxonomic membership than longer amplicons in molecular studies of microbial communities.     

Improved template representation in cpn60 polymerase chain reaction (PCR) product libraries generated from complex templates by application of a specific mixture of PCR primers

Some classes of high G+C content organisms such as the Actinobacteria, which are known through culture-based studies to be present in large numbers in particular microbial communities, are under-represented or even absent from 16S rRNA or cpn60 polymerase chain reaction (PCR) product libraries derived from these templates. Using reference cpn60 sequence data from organisms with high G+C content genomes, a pair of PCR primers were designed which, when used in combination with the previously developed degenerate, universal cpn60 primers, improve the representation of templates with high G+C content. The primers were validated using a combination of traditional and quantitative real-time PCR on both manufactured template mixtures and biological samples. The development and optimization of this specific primer mixture represents an improvement of established methods and a significant advance in the ability to generate cpn60 PCR product libraries that more closely represent the sequence diversity in complex templates.     

Increase in bacterial community diversity in subsurface aquifers receiving livestock wastewater input

Despite intensive studies of microbial-community diversity, the questions of which kinds of microbial populations are associated with changes in community diversity have not yet been fully solved by molecular approaches. In this study, to investigate the impact of livestock wastewater on changes in the bacterial communities in groundwater, bacterial communities in subsurface aquifers were analyzed by characterizing their 16S rDNA sequences. The similarity coefficients of restriction fragment length polymorphism (RFLP) patterns of the cloned 16S ribosomal DNAs showed that the bacterial communities in livestock wastewater samples were more closely related to those in contaminated aquifer samples. In addition, calculations of community diversity clearly showed that bacterial communities in the livestock wastewater and the contaminated aquifer were much more diverse than those in the uncontaminated aquifer. Thus, the increase in bacterial-community diversity in the contaminated aquifer was assumed to be due to the infiltration of livestock wastewater, containing high concentrations of diverse microbial flora, into the aquifer. Phylogenetic analysis of the sequences from a subset of the RFLP patterns showed that the Cytophaga-Flexibacter-Bacteroides and low-G+C gram-positive groups originating from livestock wastewater were responsible for the change in the bacterial community in groundwater. This was evidenced by the occurrence of rumen-related sequences not only in the livestock wastewater samples but also in the contaminated-groundwater samples. Rumen-related sequences, therefore, can be used as indicator sequences for fecal contamination of groundwater, particularly from livestock.     

Expression of plant chaperonin-60 genes in Escherichia coli.

We have examined the expression in Escherichia coli of genes encoding a plant chloroplast molecular chaperone, chaperonin-60. Purified plant chaperonin-60 is distinct in that it contains two polypeptides, p60cpn-60 alpha and p60cpn-60 beta, which have divergent amino acid sequences (Hemmingsen, S. M., and Ellis, R. J. (1986) Plant Physiol. 80, 269-276; Martel, R., Cloney, L. P., Pelcher, L. E., and Hemmingsen, S. M. (1990) Gene (Amst.) 94, 181-187). The precise polypeptide composition(s) of the active tetradecameric specie(s) (cpn60(14)) has not been determined. Genes encoding the mature forms of the Brassica napus chaperonin polypeptides have been expressed separately and in combination in E. coli to produce three novel strains: alpha, beta, and alpha beta. The plant cpn60 polypeptides accumulated in soluble forms and to similar high levels in each. There was no conclusive evidence that p60cpn-60 alpha assembled into cpn60(14) species in alpha cells. In beta and alpha beta cells, the plant gene products assembled efficiently into cpn60(14) species. Thus, the assembly of p60cpn-60 alpha required the presence of p60cpn-60 beta, whereas the assembly of p60cpn-60 beta could occur in the absence of p60cpn-60 alpha. Significant proportions of the endogenous groEL polypeptides were not assembled into tetradecameric groEL14 in beta and alpha beta cells. Analysis of the tetradecameric species that did form indicated the presence of novel hybrid cpn6014 species that contained both plant and bacterial cpn60 polypeptides.     

Affinity purification of fusion chaperonin Cpn60-(His)(6) from thermophilic bacterium Bacillus strain MS and its use in facilitating protein refolding and preventing heat denaturation

The cpn60 gene from Bacillus strain MS, which is highly homologous to Bacillus stearothermophilus, was cloned. Cpn60 with a hexahistidine affinity tag (His)(6) fused to its C-terminus (cpn60-(His)(6)) was overproduced in Escherichia coli. Cpn60-(His)(6) was expressed in a soluble form in E. coli. and purified to homogeneity in a single step by nickel chelate affinity chromatography. Cpn60-(His)(6) formed a tetradecamer and had ATPase activity. Cpn60-(His)(6) mediated refolding of guanidine hydrochloride unfolded pig heart malic dehydrogenase (MDH) and Thermus flavus MDH at 25 and 70 degrees C, respectively, in an ATP-dependent manner. In addition, cpn60-(His)(6) prevented heat denaturation of pig heart MDH and T. flavus MDH at 30 and 80 degrees C, respectively, in an ATP-dependent manner. Therefore, cpn60-(His)(6) facilitates protein refolding and prevents heat denaturation of proteins across a wide temperature range.