Identification and Analysis of the DoxDA Manipulator of Thiobacillus Acidophilus

Abstract: Reverse transcriptase-PCR experiments suggest that the two clusters of genes potentially involved in the oxidation of reduced sulfur compounds are organized as operons in strain of the acidophilic, chemolithoautotrophic bacterium Acidithiobacillus ferrooxidans ATCC 23270, the two clusters of genes including such the ORF of Putative sulfate-thiosulfate-molybdate binding proteins, the ORF of putative thiosulfate: quinone oxidoreductase and the ORF of the rhodanese-like protein (P21). Bioinformatic analyses have predicted the possible promoters sequences and the possible +1 start site of transcription for the doxDA Operons.

In recent years, due to the metal sulfide ore bioleaching importance of resource utilization and a metal sulfide ore dumps environmental problems caused by acidic fluid seepage, leaching of biological oxidation and biological mechanisms related to metal sulfide ore attracted the attention of the relevant scientists. At present, research on aspects of energy metabolism important function of sulfide ore bioleaching bacteria Lactobacillus acidophilus oxidation of ferrous sulfur (A.ferrooxidans) as the representative of the genus Thiobacillus acidophilus is the most extensive and in-depth ^[1]. Thiobacillus ferrooxidans is a bacterium of the genus Thiobacillus, which belongs to Gram-negative bacteria, aerobic acidophilus, mainly grown in the environment of pH 1-3, and is more than 20 kinds of immersed so far. The most studied leaching bacteria in mineral bacteria. In the acidic environment of leaching, A. ferrooxidans relies on Fe ²⁺ , elemental sulfur produced by decomposition of metal sulfide ore and other various reducing sulfides to provide growth energy under aerobic conditions, and promote the growth of acidophilic sulfur oxidizing bacteria; At the same time, it maintains the high-iron ions and protons required for the continuous leaching of metal ions in the leaching environment ^[2,3] . Compared to the ferrous oxide system ^[4] , the sulfur oxidation system is more complicated, and there are still many unresolved issues in related research.

The oxidation of elemental sulfur by acidophilic sulfur oxidizing bacteria is an intricate process. In this process, the extracellular material of the bacteria mediates the adsorption of elemental sulfur by the bacteria. The outer membrane protein of the bacteria transports the adsorbed sulfur to the periplasmic space of the cell. The elemental sulfur is oxidized to sulfate through a series of biooxidation pathways. Ions are released into the extracellular medium. Annotated analysis of the functional genome function of A. ferrooxidans ATCC23270 revealed no detailed annotation of functional genes associated with the elemental sulfur oxidation system. Despite a deeper understanding of the sulfur reduction function gene and the sulfur reduction pathway, the understanding of the sulfur biooxidation pathway is still sporadic and incomplete.

Ramírez P et al. ^[5] studied the two-dimensional electrophoresis protein differential display of A. ferrooxidans bacteria grown in different energy matrices and found that a kind of rhodanese P21 is highly expressed in the bacterial cells grown in the elemental sulfur matrix, and the protein is There is almost no expression in the ferrous matrix. It is speculated that this type of rhodanese P21 is located in the periplasmic space of the cell. Analysis of the locus of the p21 gene revealed that there are some possible open reading frames (ORFs) related to sulfur oxidation before and after the p21 gene, encoding such as sulfate-thiosulfate-molybdate binding proteins (SBP-). 1 and SBP-2) of sbp-1 and sbp-2, doxDA-1 and doxDA-2 encoding membrane-bound thiosulfate-quinooxidoreductase (TQO-1 and TQO-2) Wait for the open reading frame. It is speculated that the encoded products of these genes are closely related to sulfur oxidation. In the high-throughput biochip study of ferrous oxidation and sulfur oxidation, it was also verified that some of the above ORFs in the locus of p21 are related to sulfur oxidation, and the expression level in the sulfur-oxidized matrix is ​​relative to the expression in the ferrous matrix. The levels are significantly increased ^[6] , and since they are sequentially arranged in the A. ferrooxidans genome, these genes are predicted to be co-transcribed at the time of transcription, belonging to the predicted doxDA-1 operon and the doxDA-2 operon, respectively. Based on the existing research results, the doxDA-1 operon of p21 gene and the doxDA-2 operon in the coding order of doxDA-1 operon were identified by RT-PCR. and to use the knowledge of bioinformatics for doxDA operon promoter sequence may be predicted and analyzed.

I. Materials and methods

(1) Strains, culture media and culture conditions

The strain A. ferrooxidans ATCC 23270 was derived from the American model strain collection center. The experiment used 9K medium for liquid culture, and the strain was activated and passaged. In the 9K medium containing 5.0 g/L elemental sulfur, the components were: (NH ₄ ) ₂ SO ₄ , 3.0 g / L; MgSO ₄ · 7H ₂ O, 0.5g / L; KCl, 0.1g / L; K ₂ HPO ₄ , 0.5g / L; Ca (NO ₃ ) ₂ , 0.01g / L and FeSO ₄ · 7H ₂ O, 44.5g / L . The initial pH of the culture solution was adjusted to 2.5 with 5 mol/L of H ₂ SO ₄ . The bacteria were incubated with a 250 mL Erlenmeyer flask at 30 ° C, 160 r / min shaker at constant temperature.

(B) bacterial DNA and RNA extraction and cDNA synthesis

The bacterial liquid grown to the middle and the middle of the logarithm was cultured, and the elemental sulfur particles were filtered off. The filtrate was centrifuged to collect the cell pellet, and the cells were washed twice with 5 mmol/L H ₂ SO _{4 and} then cryopreserved at low temperature as a sample for extracting bacterial DNA and RNA.

Bacterial DNA extraction: The cell pellet was resuspended 400μLTE, added 80μL20% SDS, after 3μL20g / L proteinase K mixing, placing 1h 55 ℃; were added 100μL5mol / LNaCl and 80μL cetyl trimethyl ammonium bromide ( CTAB) -NaCl solution (0.7mol / L NaCl containing 10% CTAB), thoroughly mixed, placed 10min 65 ℃; adding an equal volume of chloroform / isopropanol - ethanol, vortex mixed; 12000r / min centrifugal 15min; transfer after the supernatant was added to a fresh tube 2 to 3 volumes of isopropanol precipitated DNA; 12000r / min precipitated DNA was collected by centrifugation 10min; 70% ethanol, the DNA pellet was washed 2 times; after drying in vacuo was added 100μL of sterilized distilled water were dissolved spare.

Bacterial RNA Extraction: Total RNA was extracted using Trizol kit (GIBCO, LifeTechnologies), carried out according to the method of Trizol kit instructions. Finally, RNase-free water to dissolve RNA, were stored at -70 ℃.

Synthesis of cDNA: Total RNA was treated with DNase for 30 min at room temperature for cDNA synthesis. The total RNA (1 μg to 3 μg) was used as a template, and cDNA was obtained by reverse transcription of a random primer hexamer in a cDNA synthesis kit (MBI). The reverse transcription method was referred to the specification.

(III) Primer design and PCR reaction

Primers were designed based on the A. ferrooxidans ATCC23270 genome-wide open reading frame sequence. The possible open reading frame gene sequences in the doxDA-1 and doxDA-2 operon sequences in the A.ferrooxidans genome are: AFE_2973, AFE_2974, AFE_2975, AFE_2976, AFE_2977, AFE_2978, AFE_2979, AFE_2980, AFE_2981, AFE_2982 and AFE_2983. The primers designed according to these sequences are shown in Table 1. The synthesis of oligonucleotide primers was completed by Shanghai Shenggong Bioengineering Co., Ltd.

PCR amplification reaction system (50 μL): 1.0 μL (10 mmol/L) primer, 2.0 U (2.0 μL) Taq DNA polymerase (Fermentas), 5 μL 10× PCR buffer, 1 μL (10 mmol/L) dNTPs, 4 μL (25 mmol/L) ) MgCl ₂ , 0.5 μL template (DNA template and cDNA template), supplemented with water to 50 μL. DNA amplification process: 93 ° C 3 min; 93 ° C 30 s, 56 ° C 30 s, 72 ° C 30 s, 32 cycles; 72 ° C 10 min; 4 ° C preservation after the end of the program. The PCR product was detected on a 2.0% agarose gel loaded with ethidium bromide, and the results were observed under a UV detector.

(four) sequence analysis

The gene sequence in the genome-wide database of Thiobacillus ferrooxidans ATCC23270 is from the TIGR database. For the proteins encoded by the open reading frame sequences to be analyzed, the Protparam module (http://) was used to analyze the molecular weight and isoelectric point of the protein, TMPRED (http:// The ) and TMHMM (http://) modules predict protein transmembrane helix sequences, as well as SignalP (http:// The dtu.dk/services/SignalP) module analyzes the signal peptide sequence of the protein. The predictive analysis software module for prokaryotic promoters is BDGP (http://).

Second, the results

(1) Bioinformatics analysis of polypeptides encoded by open reading frames in doxDA-1 and doxDA-2 operons

Some basic biological information of the open reading frame of the doxDA-1 and doxDA-2 operons and the sequences of their adjacent open reading frames are shown in Table 2. Their sequence of gene loci in the whole genome of A. ferrooxidans ATCC23270 is shown in Figure 1. Shown.

Figure 1 shows the open reading frame in the doxDA-1 and doxDA-2 operons and its adjacent open reading frame sequences.

Arrangement order in the A.ferrooxidans genome

(2) Co-transcriptional analysis of open reading frames in doxDA-1 and doxDA-2 operons

Using the primers in Table 1, PCR was carried out by using genomic DNA and cDNA obtained by reverse transcription of RNA as a template to amplify the desired product. The result is shown in Figure 2.

(III) Sequence analysis of promoters in doxDA-1 and doxDA-2 operons

The possible genes represented by the open reading frames AFE_2978, AFE_2979, AFE_2980, AFE_2981, AFE_2982 and AFE_2983 contained in the doxDA-1 operon are unknown, P21, doxDA-1, sbp-1, tat-1 and cdt genes, respectively. The primers designed according to their sequences were subjected to PCR reaction with genomic DNA and cDNA template obtained by reverse transcription of RNA, respectively, and the products were obtained in the same size as expected, as shown in Fig. 2, and cdt and its adjacent open reading frame AFE_2984 The reaction product can be obtained by PCR reaction using genomic DNA as a template, and there is no amplification product when cDNA is used as a template. This indicates that the gene transcription of the six open reading frames of unknown, P21, doxDA-1, sbp-1, tat-1 and cdt should belong to the co-transcription sharing a single promoter sequence. When the promoter sequence information was analyzed upstream of the cdt open reading frame, a nucleotide sequence very similar to that of the prokaryotic promoter was found, with a typical -10 sequence and a -35 sequence conserved region, as shown in FIG. .

Figure 2: Open reading frame of doxDA-1 and doxDA-2 operons and its electrophoresis effect of PCR and RT-PCR products in its adjacent open reading frame

a: a product obtained by performing a PCR reaction using genomic DNA as a template;

b: a product obtained by PCR reaction using cDNA obtained by reverse transcription of total RNA as a template

The genes represented by the open reading frames AFE_2974, AFE_2975, AFE_2976, and AFE_2977 contained in the doxDA-2 operon represent tat-2, sbp-2, doxDA-2, and unknown, respectively. Primers designed according to their sequences were ligated with genomic DNA and cDNA obtained by reverse transcription of RNA as a template to obtain a product with the same size as expected, as shown in Figure 2, and tat-2 and its adjacent open reading. PCR amplification can be performed between the frame AFE_2973 using genomic DNA as a template, and there is no amplification product when cDNA is used as a template. This suggests that the four open reading frames of tat-2, sbp-2, doxDA-2 and unknown should belong to co-transcription when the gene is transcribed.

It should be particularly noted that when PCR amplification was carried out using cDNA as a template under the same conditions of template concentration, enzyme amount, and number of cycles, it was found that the PCR product concentration of the sequence in the operon doxDA-2 was significantly lower than that of manipulation. The sequence PCR product concentration in the sub-doxDA-1 is shown in Figure 2. It is indicated that the transcription units of the tat-2, sbp-2, doxDA-2 and unknown genes in the doxDA-2 operon are significantly different in the amount of transcription of the elemental sulfur growth substrate relative to the transcription of the doxDA-1 operon transcription unit.

Nucleotide sequence between the tat-2 open reading frame and the adjacent open reading frame AFE_2974 in the doxDA-1 operon, the translation initiation codon ATG of the tat-2 open reading frame and the open reading frame AFE_2974 An overlap is formed between the translation start codons ATG, as shown in FIG. When the promoter sequence information was analyzed for the open reading frame AFE_2974, two possible promoter sequences were predicted, as shown in FIG. However, none of the two possible promoter sequences have an oligonucleotide fragment that clearly conforms to the prokaryotic promoter characteristics of the -35 sequence and the -10 sequence.

Figure 3 Promoter sequence information upstream of the cdt. open reading frame in the doxDA-1 operon

Figure 4 Promoter sequences that may be present in the nucleotide sequence upstream of the tat-2 open reading frame in the doxDA-2 operon

Third, discussion

Under different environmental conditions, the energy metabolism of Thiobacillus ferrooxidans is mainly caused by the ferrous oxide system and sulfur oxidation system in the body. QuatriniR et al. ^[6] used genomic microarray technology to study the transcriptional differential spectroscopy of the NAD(P) reduction pathway-related enzyme system and some open reading frames on the electron transport chain during the oxidation of ferrous and reducing sulfur compounds. In the reducing sulfur compound matrix, the expression levels of the open reading frames doxDA-1 and P21 in the doxDA-1 operon were significantly higher, and also higher than the expression level of the open reading frame doxDA-2 in the doxDA-2 operon. The double-copy genes doxDA-1 and doxDA-2 genes similar to the archaeal A.ambivalens-encoded thiosulfate-coenzyme Q oxidoreductase gene were found in the genome ^[7] , which are distributed in the coding sequence relative to the operon doxDA. -1 and doxDA-2. An interesting phenomenon is the simultaneous presence of two sulfate-thiosulfate-binding proteins encoding the double-copy genes sbp-1 and sbp-2 upstream of the doxDA-1 and doxDA-2 genes, in addition to SBP-1 and SBP. when the simulation analysis of the structure-2, was found SBP-1 and SBP-2 protein structure ModA in E.coli, thiosulfates molybdenum atoms and key binding sites are very similar three-dimensional structure ^{[ 8]} . The presence of thiosulfate-coenzyme Q oxidoreductase and sulphate-thiosulfate-binding proteins may be closely related to the transport and oxidation utilization of sulfates, thiosulfates. In the doxDA-1 operon, there is a thiocyanate hydrolase-encoding gene p21 downstream of doxDA-1. P21 is highly expressed in sulfide, sulfur and thiosulfate matrix, indicating that P21 has important correlation with sulfur metabolism. Sexually, but thiocyanate hydrolase activity could not be detected by P21 expressed in large amounts in vitro ^[5] . There has been no research on tat-1 and cdt upstream of the doxDA-1 operon, which together with sbp-1, doxDA-1 and p21 constitute doxDA-1 operons, and there may be interactions between the proteins encoded by them, or Other proteins in the body form a complex system that may play a role in the acquisition, transport and absorption of sulfur compounds in the sulfur oxidation system of A. ferrooxidans.

In the doxDA-2 operon, an overlap occurs between the translation initiation codon ATG of the tat-2 open reading frame and the translation initiation codon ATG of the open reading frame AFE_2974, causing them to compete in the transcriptional sequence, resulting in The difference in the amount of transcription. In fact, the difference in this amount of transcription is mainly controlled by transcriptional regulation. During cell growth and development, gene expression can occur in a certain time sequence, and changes with the environmental conditions inside and outside the cell, forming timing regulation and adaptive regulation. The transcription and translation of prokaryotes occur almost simultaneously, and the regulation of transcription levels is even more important. QuatriniR et al ^[6] and AcostaM et al ^[9] used doxDA-1 and p21 as reference targets in the analysis of sulfur oxidation-related gene expression, but not doxDA-2 in doxDA-2 operon. This may be due to the fact that the gene expression level in the doxDA-2 operon is too low, which is consistent with the low overall transcription level of each gene in the doxDA-2 operator described above. This phenomenon may also be a promoter sequence and operon doxDA-1, respectively, have related transcription factor σ prokaryotes need to recognize and properly guided stable RNA polymerase binds to the DNA promoter, the promoter sequence results in the difference The sigma factor-directed RNA polymerase correctly recognizes and stably binds to the DNA promoter to form a difference, resulting in a difference in transcription levels. The -35 sequence and the -10 sequence with sigma-specific recognition in the nucleotide sequence upstream of the cdt open reading frame in the doxDA-1 operon, but the possible promoter sequence of the doxDA-2 operon does not have the -35 sequence and -10 nucleotide fragments characterized by a sequence. Why do double-copying doxDA genes, double-copy sbp genes exhibit different transcripts, and whether the functions of the products encoded by double-copy genes are identical, etc., remains to be further studied. The solution of these problems, as well as the separation of some key enzymes in the sulfur oxidation system and the identification of genes, the description of the enzyme catalytic mechanism of various sulfur compounds under the action of the enzyme system will provide the perfection of the sulfur oxidation system of acidophilic sulfur oxide bacteria. Help.

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