Open access peer-reviewed chapter

Two-Component Systems in the Regulation of Sulfur and Ferrous Iron Oxidation in Acidophilic Bacteria

By Lifeng Li and Zhaobao Wang

Submitted: February 2nd 2021Reviewed: February 10th 2021Published: May 19th 2021

DOI: 10.5772/intechopen.96553

Downloaded: 98


The two-component system (TCS) is a regulatory system composed of a sensor histidine kinase (HK) and a cytoplasmic response regulator (RR), which participates in the bacterial adaptation to external stimuli. Sulfur oxidation and ferrous iron oxidation are basic energy metabolism systems for chemoautotrophic acidophilic bacteria in acid mine environments. Understanding how these bacteria perceive and respond to complex environmental stimuli offers insights into oxidization mechanisms and the potential for improved applications. In this chapter, we summarized the TCSs involved in the regulation of sulfur and ferrous iron metabolic pathways in these acidophilic bacteria. In particular, we examined the role and molecular mechanism of these TCSs in the regulation of iron and sulfur oxidation in Acidithiobacillus spp.. Moreover, research perspectives on TCSs in acidophilic bacteria are discussed in this section.


  • Acidithiobacillus
  • two-component system
  • ferrous iron oxidation
  • sulfur oxidation
  • transcriptional regulation

1. Introduction

Acidithiobacillusgenus is composed of high acid-tolerance chemolithotrophic bacteria that can oxidize various reduced inorganic sulfur compounds (RISCs) and ferrous iron to obtain electrons for carbon dioxide fixation and energy production [1]. The composition and comparison of the members in this genus has been reviewed [2]. As reported, the bacteria can be classified into two groups based on their energy resources: the sulfur-oxidizing-only species and the sulfur- and ferrous- oxidizing species [3]. Sulfur-oxidizing-only bacteria include Acidithiobacillus caldus, Acidithiobacillus thiooxidansand Acidithiobacillus albertensis, whereas sulfur- and ferrous oxidizing bacteria include Acidithiobacillus ferrooxidans, Acidithiobacillus ferrivorans, Acidithiobacillus ferriphilus, and Acidithiobacillus ferridurans. These bacteria are widespread in the bioleaching heap and acid mine drainage water environments and play critical roles in bioleaching and wastewater treatment [3, 4, 5, 6]. Sulfur and iron oxidation capacities are critical physiological features of these bacteria, which are also the basis for their applications. The oxidation of reduced inorganic sulfur compounds can dissolve ore and produce sulfuric acid, whereas the oxidation of ferrous iron (Fe II) produces ferric iron (Fe III), in which sulfuric acid and ferric iron products can attack minerals, releasing metal ions [7]. Sulfur metabolism and iron oxidation are complicated and various metabolic genes are involved. Thus, the regulation and mechanism of the sulfur and iron oxidation in Acidithiobacillusspp. have drawn increasing attention.

Sensing and responding to environmental stimuli is necessary for bacteria to adjust the expression of related genes and adapt to changing habitats. The two-component systems (TCSs) are the most widespread regulation system in bacteria [8]. The TCS is mainly composed of two proteins, histidine kinase (HK) and their cognate response regulator (RR) (Figure 1). Histidine kinase is a membrane protein that can sense extracellular signals and autophosphorylate its histidine. The phosphorylated HK can transfer the phosphoryl group to its cognate RR protein leading to the phosphorylation of the RR protein at the aspartate residue (Asp) and the activation of RR protein. The activated RR protein is able to change its conformation by dimerization or multimerization and regulates the expression of its target genes. In general, the RR protein can regulate gene transcription by binding to specific sequences in the promoter region of related genes located upstream of the RNA polymerase binding region.

Figure 1.

Two-component system regulation mechanism.

Although not completely understood, the study of molecular regulation mechanisms in acidophilic bacteria has recently been progressing. In this chapter, we discuss the occurrence of the TCS in these bacteria, the regulation mechanism of sulfur and iron oxidation, and the future prospects in the TCS regulation research.


2. Discovery of two-component system in acidophilic bacteria

The occurrence of the TCSs in the acidophilic bacteria was compared among different species on basis of the reported TspS-TspR, RsrS-RsrR, and RegB-RegA two-component systems [7, 9, 10] (Figure 2). The sulfur oxidization (Sox) system is a critical sulfur oxidization pathway of chemotrophic sulfur-oxidizing bacteria, and the regulation of the Sox system in A. caldusby the TspS-TspR two-component system has been reported [10]. Meanwhile, genome sequences were used to compare the occurrence of TCS similar to that of TspS-TspR in the Acidithiobacillusspp.. tspS-tspR-soxgene clusters were found in all the sulfur-oxidizing bacteria (Acidithiobacillus caldus, Acidithiobacillus thiooxidansand Acidithiobacillus albertensis), and in one type of the sulfur- and ferrous- oxidizing bacteria, Acidithiobacillus ferrivorans. The amino acid similarity of the TspS/TspR ranged from 63% to 81%.

Figure 2.

Distribution of two-component system in acidophilic bacteria. The identities of corresponding protein were indicated by the percentage values with the first line of each part set as 100%. Accession numbers (GenBank) for proteins in Sox pathway are as follows,A. caldusMTH-04, sox (A5904_11270–11305); A. thiooxidans ATCC19377, sox (ATHIO_RS0101665-RS0101630);A. albertensisDSM 14366, sox (BLW97_RS11430-RS11465);A. ferrivoransSS3, sox (Acife_2487–2494). Accession numbers (GenBank) for proteins in S4I pathway are as follows,A. caldusMTH-04, RsrR (ANJ65973.1), RsrS (ANJ65974.1), TetH (OAN03451.1), DoxDA (OAN03452.1) (GenBank: MK165448);A. caldusATCC 51756, RsrR (ABP38227.1), RsrS (ABP38226.1), TetH (ABP38225.1), DoxDA (ABP38224.1);A. thiooxidansATCC 19377, TetH (WP_029316048.1), DoxDA (WP_010638552.1);Acidithiobacillus ferrooxidansATCC 23270, TcsS (ACK79489.1), TcsR (ACK79259.1), TetH (ACK80599.1), DoxDA_2 (ACK79881.1), DoxDA_1 (ACK78481.1);A. ferrooxidansATCC 53993, TcsS (ACH82290.1), TscR (ACH82291.1), TetH (ACH82292.1), DoxDA_2(ACH82311.1), DoxDA_1(ACH82307.1);Acidithiobacillus ferriduransJCM 18981, TcsS (BBF65177.1), TcsR (BBF65176.1), TetH (BBF65175.1), DoxDA_2 (BBF65156.1), DoxDA_1 (BBF65160.1). Accession numbers (GenBank) for proteins in iron oxidation pathway are as follows,Acidithiobacillus ferrooxidansATCC 23270, AFE_RS14375-AFE_RS16285;A. ferrooxidansATCC 53993, LFERR_RS13505-LFERR_RS15550;A. ferrivoransSS3, ACIFE_RS08920-ACIFE_RS09010.

The S4I pathway is also an important thiosulfate oxidization pathway composed of tetrathionate hydrolase (TetH) and thiosulfate: quinone oxidoreductase (DoxDA), and its regulation by the RsrS-RsrR system was reported [9, 11]. However, a similar distribution of this gene cluster was only found in A. caldus. No regulatory system was found in A. thiooxidans. In contrast, a different kind of TCS was found before the tetHgene in Acidithiobacillus ferrooxidansand Acidithiobacillus ferriduranswith two doxDAgenes separated in a different gene cluster.

The RegB-RegA is a well-studied global redox responding regulatory system in A. ferrooxidans, and plays roles in the iron and sulfur oxidization regulation [12, 13]. RegB-RegA located in the ctaand rusoperon, which was composed of genes in the biogenesis of aa3 type oxidase and iron oxidation pathway. Similarly, the regB-regA-cta-ruscluster was only found in the sulfur- and ferrous- oxidizing bacteria A. ferrooxidansand A. ferrivoranswith high identity.

Hence, the TCSs are widespread in the sulfur and iron oxidization bacteria, while different distributions are revealed by bioinformatics analysis and different regulation mechanism maybe adapted, which deserves further studies.


3. Roles of two-component system in sulfur oxidation

Gene transcription is a fundamental process in bacteria, which is carried out by multi-subunit RNA polymerase (RNAP). σ factors determine transcription specificity by recognizing specific promoter sequences. Bacterial σ factors can be divided into two distinct classes: σ70 and σ54 [14]. σ70 recognizes the consensus −10 and − 35 regions and recruits RNAP to a specific promoter region to initiate gene transcription [15]. σ70 controls transcription of most housekeeping genes, whereas σ54 regulates the genes involved in nitrogen assimilation [16], phage shock response [17], infection [18], and other cellular stresses [19, 20]. σ54 recognizes distinct sequences in the −12 (GC) and − 24 (GG) regions of the promoter. The requirement of the bacterial enhancer binding proteins (bEBPs) is a remarkable feature of σ54-dependent transcription initiation [20]. Accordingly, two kinds of transcription regulation were reported in acidophilic bacteria (Figure 3).

Figure 3.

Different TCS regulation mechanisms between the sox system and the S4I pathway. Sox system and the S4I pathway are important sulfur oxidation system inA. caldus, and the regulation mechanism of which has been revealed. The regulation mechanism was summarized in this diagram, the left part represented the regulation mechanism of TspS-TspR on the Sox system inA. caldussummarized according to literature [10]. The right part was the model of RsrS-RsrR regulating the S4I pathway inA. caldus[9]. The activation signals, the interaction between the regulators and key binding motifs of the promoters were showed.

The rsrS-rsrR-tetH-doxDAgene cluster in A. calduswas reported in 2007 [11]. The genes in this cluster were proven to be cotranscribed using RT-PCR (Reverse transcription PCR), and the results of quantitative PCR and Western blot indicated that the gene cluster was tetrathionate induced. The promoter before tetHgene was mapped by primer extension. The verification of the regulation role and mechanism of the S4I pathway by the RsrS-RsrR two component system was reported in 2016 [9]. ΔrsrRand ΔrsrSstrains were constructed using a marker-less gene knockout method in A. caldus. The transcription levels of rsrS, rsrR, tetH, and doxDAwere analyzed by RT-qPCR under the stimulation of K2S4O6 in the wild type and two gene knockout strains, and the results indicated that the RsrS-RsrR regulated the transcription of tetHand doxDAin a K2S4O6–dependent manner. The regulatory protein RsrR was expressed and purified to verify protein and promoter DNA binding using electrophoretic mobility shift assays (EMSAs). A 19 bp (AACACCTGTTACACCTGTT) inverted repeat sequence (IRS) was identified to be the binding motif of RsrR through EMSA and promoter probe plasmid analysis in vitroand in vivo, respectively. Hence, as summarized in Figure 3, the RsrS can sense the extracellular tetrathionate signal and autophosphorylated, then RsrR is activated by receiving the phosphate from RsrS, the phosphorylated RsrR dimerizes and binds on the IRS region of tetHoperon and initiates the transcription of the genes together with the RNA polymerase.

The RR protein of TCS can function as the activator of σ54-dependent transcription initiation, which converts the closed RNAP-σ54 holoenzyme complex to open state to initiate transcription. σ54 -dependent RR proteins have been reported in several bacteria [21, 22, 23]. It was reported that the two-component system TspS-TspR could regulate the sulfur oxidization (Sox) system in Acidithiobacillus caldusand some chemolithotrophic bacteria in a σ54-dependent manner [10]. RT-PCR was used to analyze the composition of the soxoperon. Results indicated that the genes in the soxoperon were cotranscribed whereas the transcription of tspRwas independent. The activation of σ54 on the transcription of the soxgenes was verified by the higher transcripts of the operon genes in the constructed rpoN-overexpression strain. Following, the transcription initiation site (TSS) of the first gene (sox-X) in the operon was verified using 5’RACE (Rapid amplification of cDNA ends). Upstream of the TSS (G, +1), the potential −12 (GC) and − 24 (GG) sites were also identified, which was a typical feature of the σ54-dependent promoter. Promoter-probe plasmids were constructed to analyze the promoter activity in A. caldusby comparing the wild type (P1) and the mutated promoter (GG/GC mutated to AA, P12M, P24M, and P12/24M) containing strains. Hence, the σ54-dependent promoter was verified by 5’RACE and promoter-probe plasmid activity analysis. As reported, the σ54-dependent transcription requires binding of the enhancer binding protein (EBP) to upstream activator sequences (UASs) to activate transcription initiation. TspR protein was then expressed and purified to analyze the binding of P1 promoter by EMSA. The binding of TspR protein with different length promoters with two different predicted UASs was analyzed, and only the promoter containing UAS1 (TGTCCCAAATGGGACA) showed a shift lane on the native PAGE. To verify the critical sites in UAS1, UASM1 and UASM2 mutants were constructed, which converted the bases TGT/ACA to GAG/GTG and changed the variable bases CCC to TTG, respectively. TGT/ACA was identified as the critical sites of UAS1 by analyzing the activity of the wild type, UASM1 and UASM2 mutants. Thus, the experiments confirmed that the Sox system was regulated by the σ54-dependent two-component system TspS/TspR. A signal transduction and transcriptional regulation model for the Sox system in A. caldusis depicted in Figure 3. TspS can sense the signal stimuli such as thiosulfate and other sulfur substrates, and phosphorylate at a proposed conserved His residue. TspS is activated TspR by the transferring phosphoryl group from its His residue to the conserved Asp residue of TspR. Subsequently, the activated TspR is dimerized and binds to the UAS sequence of promoter P1, meanwhile changing the conformation of the holoenzyme (σ54-RNA polymerase), which binds to the −12/−24 region to activate the transcription of soxgenes. Interestingly, potential −12/−24 region and UAS sequences were predicted in other bacteria with similar tspS-tspR-soxgene composition, which may indicate the importance of TCS in the regulation the Sox oxidation system.


4. Roles of two-component system in ferrous iron oxidation

A. ferrooxidansis an important iron and sulfur oxidizing bacterium in the Acidithiobacillusgenus, which can oxidize Fe (II) and reduced sulfur compounds to obtain energy for growth. Compared with Fe (II), sulfur seems to be a better energy source because it can provide more ATP at the same molar level [24]. Understanding the function and regulation mechanism of the two energy production systems is critical in coordinating sulfur and iron oxidation process to avoid the S0 deposition and improve the efficiency of bioleaching.

When A. ferrooxidanswas cultivated in the presence of both Fe (II) and S0 as electron donors, the Fe (II) concentration, bacterial concentration, and pH were measured along with the growth process [7]. The results indicated that ferrous iron was oxidized before S0. The redox potential increased in the Fe (II) oxidization process, while kept stable during sulfur oxidation. Additionally, RT-qPCR analysis showed that the Fe (II) oxidization genes (rus, cup, petC1and cta) transcribed before the sulfur oxidation genes (cyoB, hdrA, hdrC, hdrB, sqrand tetH). The sensor/regulator two-component signal transducing system RegBA consisting of a redox-sensing RegB and a DNA binding RegA, located near the rusoperon, was also studied. The recombinant RegA was produced and purified and was used in the EMSA experiments to analyze its binding with related gene promoters. The retarded bands could be detected with the regulatory regions of the Fe (II) oxidization genes (rus, petI, cta) and sulfur oxidation genes (cyo, hdr, hdrB, tetHand sqr), which indicated the regulation roles of RegA on these genes. As a result, an initial model of the RegBA regulation on A. ferrooxidanssulfur and iron oxidation was proposed. Both the full-length RegA and the DNA binding domain of RegA could bind the rusand hdroperon regulatory regions in the phosphorylated and unphosphorylated state [13]. However, the recombinant RegA tagged with six histidines had signs of aggregation and precipitation. Moinier et al. attempted to purify the RegA protein using the SUMO tag and compared its binding affinity to the target genes in four different states [12]. Similarly, different forms of RegA (DNA binding domain, wild-type, unphosphorylated and phosphorylated-like forms) are able to specifically bind to the regulatory region of the rus, cta, petI, reg, tet, cyo, hdr,and sqrgenes/operons, and the binding of the target genes leads to the formation of multimeric complexes as shown by EMSA results. Further, dynamic light scattering (DLS) analysis also confirmed that 6His-SUMO-RegA protein was polydispersed according to the increased size of hydrodynamic diameters. The protein in the solution (94.6%) had a mean diameter of 7.58 nm, indicating that it was a stable dimer without target binding, whereas it multimerized in the presence of its target DNA, which was consistent with the EMSA analysis. Acetyl phosphate and amino acid mutation was used to change the phosphorylation state of RegA and all the treatments showed that the phosphorylation state of RegA had no effect on the binding affinity for the targets. TSS was determined for the iron oxidation genes (rus, cta, petI,and regoperons) and RISC oxidation genes (tetH, cyo, hdr, hdrB, cyd, sqrand doxIIoperons) using 5′ RACE experiments. The main promoters were σ70-dependent whereas the tetHgene and the cydoperons had predicted σ54-dependent promoters and the cydoperon also had a σ70-dependent promoter. The sequences and the downstream sequences of the RNA polymerase binding site of each gene were amplified and used in the analysis. Results indicated that RegA mainly binded upstream of the −10 (−12) /−35 (−24) region, except for the PI promoter of the rusoperon and the tetHoperon promoter. However, no RegA binding motifs could be found in the binding gene promoter region using several bioinformatics analysis methods. Hence, the RegBA is a global regulatory system regulating the expression of genes involved in the energy production.

Moreover, other regulatory proteins may be involved in the regulation of these genes. The transcription factor Fur was proven to control the transcription of petIoperon by binding to the promoter region in EMSA experiments [25]. Fur may inhibit the binding of RNA polymerase and repress the transcription of petIin the presence of high intracellular levels of Fe (II). RegA may impede Fur binding on the regulatory region of the petIoperon when Fe (II) is present and activate its transcription. However, the interaction between RegA and Fur requires further studies. Interestingly, the identification of σ54-dependent promoters in tetHoperon was parallel to the occurrence of a σ54-dependent transcriptional response regulator and cognate histidine kinase at the upstream of tetHoperon whereas the role of the TCS has not been verified. A σ54-dependent transcriptional regulator was also predicted at the upstream of the cydoperon consistent with the existence of this type promoter whereas no histidine kinase was found near the gene [26].

Based on the reported results, the regulation model for RegBA two-component system is portrayed in Figure 4. When Fe (II) is used as the electro donor, RegB is able to sense the low potential state and activate through autophosphorylation. It then activates the RegA protein by transferring the phosphoryl group to the conserved Asp residue of RegA. Phosphorylated RegA protein multimerizes and binds to the promoter region of the target genes, which may activate iron oxidation genes by repressing the binding of other repressor proteins such as Fur for petIas well as repress sulfur oxidation by interaction with other activator proteins such as σ54-dependent transcriptional regulator for tetH. In the absence of Fe (II), RegB is not activated and other proteins may act together with RegA, leading to the activation of sulfur oxidation genes and repression of the iron oxidation genes. The interaction between RegBA and other regulatory proteins should be studied further to fully understand the regulation mechanism of the iron and sulfur oxidation pathways.

Figure 4.

Regulation of sulfur and ferrous iron oxidation by the TCS system. The regulation inA. ferrooxidansis complicated because it can oxidize Fe (II) and reduced sulfur compounds to obtain energy for growth. Hence, the regulation in this bacterium can be divided according to the energy resources used. The left part represents the regulation mechanism when Fe is the electro donor, and the right part represents the case when sulfur is the electro donor. Several genes are involved in the regulation summarized according to the studies reported [7,12,13,25,27,28,29].


5. Conclusions

Two component systems possess critical roles in the regulation of sulfur and iron oxidation in acidophilic bacteria. In the sulfur oxidizing species A. caldus, two typical regulatory modes were identified, the σ54-dependent TCS regulation in the Sox system and the σ70-dependent TCS regulation in the S4I pathway. Meanwhile, research on the global regulatory RegBA system indicates that it could control the transcription of several important genes relevant to iron and sulfur oxidation pathways in A. ferrooxidans. Although it has been verified that three different two-component systems can participate the regulation of energy production processes in Acidithiobacillusspp., further studies are required in the following aspects: (i) the distribution of similar regulatory systems such as TspS-R, RsrS-R, and RegB-A were identified, but the verification of their regulatory roles in relative genes awaits further research; (ii) the detailed regulation mechanism of the different two-component systems in the iron oxidation and sulfur oxidation bacteria merits investigation, for example, the σ54-dependent TCS in the tetHoperon in A. ferrooxidans; (iii) studies should examine the interactions between the TCS systems and other regulatory proteins to understand the concrete mechanism of energy regulation in Acidithiobacillusspp.; (iv) studies should identify the signaling molecules and reveal the interaction between the signals and the response proteins; (v) the structural studies of the TCSs in Acidithiobacillusspp. await further research. Therefore, studies on important TCSs in acidophilic bacteria will benefit the understanding of the mechanisms of their environmental adaption and growth as well as facilitate applications that take advantage their special properties.



This work was supported by grants from the National Natural Science Foundation of China (31900116), the Scientific and Technological Projects of Henan Province (202102310395), the Natural Science Foundation of Shandong Province (6622320549) and the Medical Science and Technology Projects of Henan Province (LHGJ20190955).


Conflict of interests

The authors declare no conflict of interests.

© 2021 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution 3.0 License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Lifeng Li and Zhaobao Wang (May 19th 2021). Two-Component Systems in the Regulation of Sulfur and Ferrous Iron Oxidation in Acidophilic Bacteria, Acidophiles - Fundamentals and Applications, Jianqiang Lin, Linxu Chen and Jianqun Lin, IntechOpen, DOI: 10.5772/intechopen.96553. Available from:

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