- Research article
- Open Access
The small heat shock proteins from Acidithiobacillus ferrooxidans: gene expression, phylogenetic analysis, and structural modeling
© Ribeiro et al; licensee BioMed Central Ltd. 2011
- Received: 21 July 2011
- Accepted: 7 December 2011
- Published: 7 December 2011
Acidithiobacillus ferrooxidans is an acidophilic, chemolithoautotrophic bacterium that has been successfully used in metal bioleaching. In this study, an analysis of the A. ferrooxidans ATCC 23270 genome revealed the presence of three sHSP genes, Afe_1009, Afe_1437 and Afe_2172, that encode proteins from the HSP20 family, a class of intracellular multimers that is especially important in extremophile microorganisms.
The expression of the sHSP genes was investigated in A. ferrooxidans cells submitted to a heat shock at 40°C for 15, 30 and 60 minutes. After 60 minutes, the gene on locus Afe_1437 was about 20-fold more highly expressed than the gene on locus Afe_2172. Bioinformatic and phylogenetic analyses showed that the sHSPs from A. ferrooxidans are possible non-paralogous proteins, and are regulated by the σ32 factor, a common transcription factor of heat shock proteins. Structural studies using homology molecular modeling indicated that the proteins encoded by Afe_1009 and Afe_1437 have a conserved α-crystallin domain and share similar structural features with the sHSP from Methanococcus jannaschii, suggesting that their biological assembly involves 24 molecules and resembles a hollow spherical shell.
We conclude that the sHSPs encoded by the Afe_1437 and Afe_1009 genes are more likely to act as molecular chaperones in the A. ferrooxidans heat shock response. In addition, the three sHSPs from A. ferrooxidans are not recent paralogs, and the Afe_1437 and Afe_1009 genes could be inherited horizontally by A. ferrooxidans.
- Chaperone Activity
- Acidithiobacillus Ferrooxidans
- sHSP Gene
- Craig Venter Institute
- Extremophile Microorganism
Acidithiobacillus ferrooxidans is an acidophilic, chemolithoautotrophic bacterium that derives energy from the oxidation of ferrous iron, elemental sulfur and reduced sulfur compounds . This bacterium has been successfully used in bioleaching to recover metals from low-grade sulfide ores. During the bioleaching process, A. ferrooxidans is subjected to extreme growth conditions, such as temperature increase, pH fluctuations, nutrient starvation, and the presence of heavy metals , all of which can affect the efficiency of metal recovery.
Temperature change is one of the most common environmental stresses that can influence essential bacterial processes such as energy transduction and growth. All organisms tend to respond to environmental stresses with a rapid transient increase in heat shock protein (HSP) synthesis. HSPs act either as molecular chaperones, mediating the correct folding and assembly of proteins, or as proteases, irreversibly degrading unfolded proteins . The HSPs are usually classified according to their molecular weights, and the small HSPs (denoted sHSPs) include the categories HSP100, HSP90, HSP70, HSP60, and HSP20.
The sHSPs are characterized by a molecular mass of between 12 and 43 kDa and the presence of 80 to 100 residues that constitute the α-crystallin domain, which is flanked by C- and N-terminals that present lower similarity. The N-terminus is critical to α-HSP activity in vivo, playing a role in α-HSP oligomerization and substrate binding [4, 5]. The α-crystallin domain is known to possess a molecular chaperone role , and the C-terminal extension maintains α-HSP solubility, stability, and chaperone activity .
The sHSPs have been extensively studied due to their importance in protecting cellular proteins and maintaining cellular viability under intensive stress conditions, which is particularly important for extremophile microorganisms. Interestingly, most extremophiles posses one or two sHSPs, and species harboring at least 3 sHSP genes are mostly from the Archea domain. However, three sHSP genes have been identified in the genome of A. ferrooxidans ATCC 23270 .
Xiao et al.  showed that there could be significant differences in the expression levels of A. ferrooxidans ATCC 23270 sHSP genes in response to heat shock. These findings suggest that A. ferrooxidans sHSP genes may be controlled by different regulatory mechanisms, which could be related to specialized functions of the genes. In this study, the expression levels of three sHSP genes (Afe_1009, Afe_1437, and Afe_2172) were investigated in the A. ferrooxidans LR strain subjected to heat shock. Phylogenetic analysis and comparative molecular modeling were used to provide new insights concerning the structure and function of the sHSPs from A. ferrooxidans.
Bacterial strain and growth conditions
The Brazilian strain A. ferrooxidans LR  was grown at 30°C and 250 rpm in modified T&K liquid medium  containing 0.4 g/L K2HPO4.3H2O, 0.4 g/L MgSO4.7H2O, 0.4 g/L (NH4)2SO4, and 33.4 g/L FeSO4.7H2O. The pH was adjusted to 1.8 with sulfuric acid. For the heat shock experiments, A. ferrooxidans LR cells were grown in T&K liquid medium until 50% oxidation of Fe2+ was reached. The cells were then collected, inoculated into 100 ml of T&K liquid medium, and incubated at 40°C and 250 rpm for 15, 30 and 60 minutes.
The total RNA was isolated from three independent A. ferrooxidans cultures, according to the procedure described by Paulino et al. . The cells were suspended in a solution containing 1 mM EDTA, 100 mM LiCl, and 100 mM Tris-HCl, at pH 7.5. The RNA fraction was extracted with phenol/chloroform/isoamyl alcohol (25:24:1, v/v/v) containing 10% (w/v) SDS, precipitated at -20°C with 2% (w/v) potassium acetate at pH 5.5 and 100% (v/v) ethanol, and resuspended in DEPC-treated water. The RNA was treated with DNase (Invitrogen) for 1 h at 37°C, and stored at -70°C.
Quantitative real-time PCR (qRT-PCR)
Primers used in the real-time PCR experiments.
Forward primer (5' → 3')
Reverse primer (5 '→ 3')
Amplicon length (bp)
The qRT-PCR experiments were performed in triplicate using a 7500 Real Time PCR System (Applied Biosystems), and threshold cycle (Ct) numbers were determined using Real Time System RQ Study Software v. 1.3.1 (Applied Biosystems). The qRT-PCR reactions were performed in triplicate using Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). After thermal cycling, a dissociation (melting) curve analysis was performed to ensure the specificity of the amplifications and the absence of primer-dimer and unspecific amplifications. The relative gene expression was calculated according to the comparative critical threshold method (ΔΔTC) described by Livak and Schmittgen . The statistical significance of the qRT-PCR data was determined using the Student's t-test (p-value ≤ 0.05).
The A. ferrooxidans ATCC 23270 genome (J. Craig Venter Institute - http://cmr.jcvi.org/cgi-bin/CMR/Genome) was used to search for genes encoding sHSPs. CLUSTAL W was employed to align the sHSP sequences from A. ferrooxidans with sequences found in other bacteria. The alignment was edited with the GeneDoc program .
Prediction of the transcription start site was performed with BPROM software (Softberry, Inc.). A widely accepted theoretical informational approach was adopted to identify potential σ32 sites [16, 17]. Since the σ32 binding site comprises two conserved blocks (-35 and -10), separated by a gap of variable length, two positional weight matrices (PWM) were generated, each one based on complementary information from the -35 and -10 binding sites. The frequency matrix was based on a set of eighteen V. cholerae σ32 promoters , including the extended σ32 promoter, with 6 positions in the -35 element and 8 positions in the -10 element, separated by a spacer of variable length. Using the PWMs as a scoring function, putative -35 and -10 regions of σ32 were searched on 200 bases upstream from the ATG start codon of the A. ferrooxidans sHSP genes. Each site was scored for its degree of matching to the σ32 -35 and -10 PWMs.
A search was performed against all complete bacterial genomes (1295 genomes on 08/03/2010), using NCBI's microbial genome BLAST tool http://0-www.ncbi.nlm.nih.gov.brum.beds.ac.uk/sutils/genom_table.cgi?organism=microb and the protein sequences from Afe_1009, Afe_1437 and Afe_2172 as queries. The 20 best hits for each A. ferrooxidans sHSP were selected to build an alignment using MAFFT v6.717b http://align.bmr.kyushu-u.ac.jp/mafft/software/. The alignment containing 76 aligned residues was used to produce a maximum likelihood (ML) tree using PhyML 3.0 software http://atgc.lirmm.fr/phyml/. The PAM matrix procedure  was used to calculate genetic distances, and statistical support for the nodes employed aLRT statistics .
PSI-BLAST search against the Protein Data Bank (PDB) using the three A. ferrooxidans sHSPs (Afe_1009, Afe_1437, and Afe_2172) resulted only in templates with low sequence identity (< 28%). However, fold assignment searches using the pGenTHREADER algorithm implemented in the PSIPRED server  returned two structures that had significant scores, both of which displayed well-conserved α-crystallin domains. The crystal structures of HSP16.9 from wheat (wHSP16.9, PDB entry code: 1GME)  and HSP16.5 from Methanococcus jannaschii (MjHSP16.5, PDB entry code: 1SHS) were used as three-dimensional templates for molecular modeling of the α-crystallin domain. The N-terminal region was modeled using only the wHSP16.9 structure as template. Template and target sequences were aligned using the mGenThreader server , and were carefully examined to confirm the alignment accuracy. Comparative protein modeling by satisfaction of spatial restraints was carried out using the program MODELLER 9v7 . Fifty models were built for each sHSP from A. ferrooxidans, and all models were evaluated with the DOPE potential. Models of each protein with the lower global score were selected for explicit solvent molecular dynamics (MD) simulation, using GROMACS  to check for stability and consistency. The overall and local quality of the final model was assessed by VERIFY3D , PROSA  and VADAR . Three-dimensional structures were displayed, analyzed, and compared using the programs COOT  and PyMoL .
The sHSPs from A. ferrooxidans
Search of the A. ferrooxidans ATCC 23270 genome (J. Craig Venter Institute) revealed the presence of three sHSP genes (Afe_1009, Afe_1437, and Afe_2172) belonging to the HSP20 family. According to Han and co-workers , about 71% of the microbial organisms with completed annotated genomes possess one or two sHSP genes, and 10% of the Archaea species have more than three sHSP-related genes. Notably, the genome of Bradyrhizobium japonicum (a rhizobial species) possesses 13 sHSP-related genes .
Physical and chemical parameters of the three sHSPs from A. ferrooxidans.
Molecular weight (Da)
Identity/similarity to Afe_1009
Identity/similarity to Afe_1437
Identity/similarity to Afe_2172
Afe_1009, Afe_1437, and Afe_2172 are not organized in an operon in the A. ferrooxidans genome. Indeed, most of the known sHSP genes are not arranged in operons [33, 34], with some exceptions such as the Escherichia coli ibpAB operon, which contains two sHSP genes (ibpA and ibpB) [35, 36], and Bradyrhizobium japonicum, which has sHSP genes found as independent units and others grouped in the same operon .
sHSP genes expression in A. ferrooxidans LR cells subjected to heat shock
In A. ferrooxidans, the -35 motif at the σ32 binding site appears to be more conserved than the -10 motif. The same occurs for the V. cholerae and the E. coli σ32 consensus sequences . In spite of the different expression levels observed for the A. ferrooxidans sHSP genes, the bioinformatics analyses did not reveal any other type of regulation mechanism (data not shown). However, within the σ32-regulated genes, alternative mechanisms of regulation are possible. Münchbach and co-workers  used subtractive two-dimensional gel electrophoresis to identify a set of 10 sHSPs in B. japonicum subjected to a temperature shift from 28°C to 43°C. These authors observed that the amounts of the sHSPs were quite dissimilar, suggesting the existence of a diverse regulatory repertoire.
Phylogenetic analysis and comparative sequence analysis
The N-terminal region showed no significant sequence similarity to other sHSPs with well-defined chaperone activity (groups C and D), but secondary structure prediction tools indicated that all of the sequences analyzed had the propensity to form the α-helical structures that are considered key elements for substrate binding and stabilization of the oligomeric structure. Furthermore, the N-terminal region alone was capable of interacting with denatured proteins , and its truncation reduces the chaperone activity of sHSPs . These findings emphasize that this region contains the substrate binding site, and is therefore important for the chaperone activity.
Structural modeling of the sHSPs from A. ferrooxidans
In silico three-dimensional models of the proteins encoded by Afe_1009, Afe_1437, and Afe_2172 displayed excellent global and local stereochemical properties, with a Z-score (PROSA server) of around -3.5 and all residues lying within the allowed regions of the Ramachandran plot. A good Z-score means that it is within the range of scores typically found for native proteins of similar size. RMSD analysis of the template crystal structures and the developed models resulted in values below 0.5 Å for the main-chain backbone of the α-crystallin domain, suggesting that the models were suitable for structural and comparative analyses.
In order to gain insights into the oligomeric state of the proteins encoded by Afe_1437 and Afe_1009, which possess the extended C-terminus, analysis was performed of the structural determinants required for assembling into either a dodecameric double disk (wHSP16.9) or a spherical shell composed of 24 monomers (MjHSP16.5). In both the wHSP16.9 and the MjHSP16.5 structures, the intermolecular interactions made by the C-terminal extension are virtually identical, despite the fact that the C-terminus of wHSP16.9 requires two different orientations to form the oligomer. This ability of the C-terminus to adopt two conformations resides in the amino acid segment between the strands β 9 and β 10, which permits a hinge movement. Analysis of the C-terminus contacts in the MjHSP16.5 structure showed that the segment between the strands β 9 and β 10 adopts a conformation stabilized by hydrogen bonds between the OεGlu137 and NεGln52 atoms, and the carbonyl oxygen of the Glu137 and NζLys142 atoms. Surprisingly, these contacts are not found in the wHSP16.9 structure, due to the presence of a second Pro residue at position 142 that enables the segment to fold into a stable motif, generating a 6-residue segment (KAEVKK) with high flexibility, which allows the hinge movement. In both Afe_1437 and Afe_1009 protein sequences, this segment does not contain a proline residue at the same relative position, and the residues populating this segment have all the requirements to form a stable motif in the same way as the MjHSP16.5 structure. Thus, based on our structural findings, we suggest that both Afe_1437 and Afe_1009 proteins behave like the prokaryotic sHSP from M. jannaschii, adopting a 24-molecule hollow spherical shell. However, additional experimental data obtained using techniques that can provide insights into hydrodynamic behavior, such as dynamic light scattering, ultra-centrifugation, size-exclusion chromatography and small angle X-ray scattering, are required to confirm our in silico predictions.
In this study, we have demonstrated that the expression level of the A. ferrooxidans Afe_1437 gene is considerable higher than that of the Afe_2172 gene, and that the three sHSP genes harbor possible σ32-dependent promoters. The three sHSPs from A. ferrooxidans are not recent paralogs, while the genes Afe_1437 and Afe_1009 can be inherited horizontally by A. ferrooxidans. This suggests that the sHSPs encoded by Afe_1437 and Afe_1009 are more likely to act as molecular chaperones in the A. ferrooxidans heat shock response. These findings were corroborated by molecular modeling showing that both Afe_1437 and Afe_1009 proteins behave like the prokaryotic sHSP from M. jannaschii, a well characterized sHSP with chaperone activity.
This work was supported by grant 02/07642-3 from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP). DAR had a fellowship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). LMMO received a research fellowship from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).
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