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Regulation of arsenite oxidation by the phosphate two-component system PhoBR in Halomonas sp. HAL1.

Chen F, Cao Y, Wei S, Li Y, Li X, Wang Q, Wang G - Front Microbiol (2015)

Bottom Line: HAL1.Using 15 consensus Pho box sequences, a putative Pho box was found in the aioBA regulation region.The new regulation model further implies the close metabolic connection between As and Pi.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University Wuhan, China.

ABSTRACT
Previously, the expression of arsenite [As(III)] oxidase genes aioBA was reported to be regulated by a three-component regulatory system, AioXSR, in a number of As(III)-oxidizing bacterial strains. However, the regulation mechanism is still unknown when aioXSR genes are absent in some As(III)-oxidizing bacterial genomes, such as in Halomonas sp. HAL1. In this study, transposon mutagenesis and gene knock-out mutation were performed, and two mutants, HAL1-phoR 931 and HAL1-▵phoB, were obtained in strain HAL1. The phoR and phoB constitute a two-component system which is responsible for phosphate (Pi) acquisition and assimilation. Both of the mutants showed negative As(III)-oxidation phenotypes in low Pi condition (0.1 mM) but not under normal Pi condition (1 mM). The phoBR complementation strain HAL1-▵phoB-C reversed the mutants' phenotypes back to wild type status. Meanwhile, lacZ reporter fusions using pCM-lacZ showed that the expression of phoBR and aioBA were both induced by As(III) but were not induced in HAL1-phoR 931 and HAL1-▵phoB. Using 15 consensus Pho box sequences, a putative Pho box was found in the aioBA regulation region. PhoB was able to bind to the putative Pho box in vivo (bacterial one-hybrid detection) and in vitro (electrophoretic mobility gel shift assay), and an 18-bp binding sequence containing nine conserved bases were determined. This study provided the evidence that PhoBR regulates the expression of aioBA in Halomonas sp. HAL1 under low Pi condition. The new regulation model further implies the close metabolic connection between As and Pi.

No MeSH data available.


Related in: MedlinePlus

Quantitative lacZ reporter gene analysis of aioBA::lacZ expression in strains HAL1, HAL1-phoR931, HAL1-ΔphoB, and HAL1-ΔphoB-C. β-galactosidase activity is presented as Miller units. Data are shown as the mean of three replicates, with the error bars represent ± 1 SD. (A) Bacteria were cultured in MMNH4 medium containing 0.1 mM Pi and 0.8 M NaCl, with or without the addition of 1 mM As(III). With the addition of As(III), the mean values of strain HAL1 and HAL1-▵phoB-C were significantly different from the ones with the absence of As(III) (*p < 0.05). (B) Bacteria were cultured in MMNH4 medium containing 1 mM Pi and 0.8 M NaCl, with or without the addition of 1 mM As(III). With the addition of As(III), the mean values of all the four strains were significantly different from the ones with the absence of As(III) (*p < 0.05).
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Figure 5: Quantitative lacZ reporter gene analysis of aioBA::lacZ expression in strains HAL1, HAL1-phoR931, HAL1-ΔphoB, and HAL1-ΔphoB-C. β-galactosidase activity is presented as Miller units. Data are shown as the mean of three replicates, with the error bars represent ± 1 SD. (A) Bacteria were cultured in MMNH4 medium containing 0.1 mM Pi and 0.8 M NaCl, with or without the addition of 1 mM As(III). With the addition of As(III), the mean values of strain HAL1 and HAL1-▵phoB-C were significantly different from the ones with the absence of As(III) (*p < 0.05). (B) Bacteria were cultured in MMNH4 medium containing 1 mM Pi and 0.8 M NaCl, with or without the addition of 1 mM As(III). With the addition of As(III), the mean values of all the four strains were significantly different from the ones with the absence of As(III) (*p < 0.05).

Mentions: To understand how phoBR and aioBA respond to As(III) in Halomonas sp. HAL1, quantitative lacZ reporter gene analyses were performed with the lacZ reporter vector pCM-lacZ constructed in this study (Figure S2). The expressions of phoBR::lacZ and aioBA::lacZ were not induced (Figure 4A) in 5− or 6-h cultivations in the low Pi condition without As(III). However, in the low Pi condition with the addition of 1 mM As(III), the expressions of phoBR::lacZ and aioBA::lacZ increased with increasing induction time, and the phoBR::lacZ expression appeared to be higher than that of aioBA::lacZ (Figure 4A). Meanwhile, in the low Pi condition, the expression of aioBA::lacZ was significantly induced by As(III) after 6 h of cultivation in strains HAL1 and HAL1-ΔphoB-C, but no statistically significant inductions in the mutants HAL1-phoR931 and HAL1-ΔphoB were observed (Figure 5A). However, in the normal Pi condition, the expression levels of phoBR::lacZ were similar in the presence or absence of As(III), and the expression of aioBA::lacZ was again significantly induced by As(III) (Figure 4B). In addition, the disruption of phoB and phoR had no effect on the expression of aioBA::lacZ in the normal Pi condition (Figure 5B). The expression of aioBA in low Pi and normal Pi conditions were consistent with the As(III) oxidation phenotypes (Figures 2, 3). The above results indicated that PhoBR regulates the expression of aioBA in low Pi condition but not in normal Pi condition.


Regulation of arsenite oxidation by the phosphate two-component system PhoBR in Halomonas sp. HAL1.

Chen F, Cao Y, Wei S, Li Y, Li X, Wang Q, Wang G - Front Microbiol (2015)

Quantitative lacZ reporter gene analysis of aioBA::lacZ expression in strains HAL1, HAL1-phoR931, HAL1-ΔphoB, and HAL1-ΔphoB-C. β-galactosidase activity is presented as Miller units. Data are shown as the mean of three replicates, with the error bars represent ± 1 SD. (A) Bacteria were cultured in MMNH4 medium containing 0.1 mM Pi and 0.8 M NaCl, with or without the addition of 1 mM As(III). With the addition of As(III), the mean values of strain HAL1 and HAL1-▵phoB-C were significantly different from the ones with the absence of As(III) (*p < 0.05). (B) Bacteria were cultured in MMNH4 medium containing 1 mM Pi and 0.8 M NaCl, with or without the addition of 1 mM As(III). With the addition of As(III), the mean values of all the four strains were significantly different from the ones with the absence of As(III) (*p < 0.05).
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Figure 5: Quantitative lacZ reporter gene analysis of aioBA::lacZ expression in strains HAL1, HAL1-phoR931, HAL1-ΔphoB, and HAL1-ΔphoB-C. β-galactosidase activity is presented as Miller units. Data are shown as the mean of three replicates, with the error bars represent ± 1 SD. (A) Bacteria were cultured in MMNH4 medium containing 0.1 mM Pi and 0.8 M NaCl, with or without the addition of 1 mM As(III). With the addition of As(III), the mean values of strain HAL1 and HAL1-▵phoB-C were significantly different from the ones with the absence of As(III) (*p < 0.05). (B) Bacteria were cultured in MMNH4 medium containing 1 mM Pi and 0.8 M NaCl, with or without the addition of 1 mM As(III). With the addition of As(III), the mean values of all the four strains were significantly different from the ones with the absence of As(III) (*p < 0.05).
Mentions: To understand how phoBR and aioBA respond to As(III) in Halomonas sp. HAL1, quantitative lacZ reporter gene analyses were performed with the lacZ reporter vector pCM-lacZ constructed in this study (Figure S2). The expressions of phoBR::lacZ and aioBA::lacZ were not induced (Figure 4A) in 5− or 6-h cultivations in the low Pi condition without As(III). However, in the low Pi condition with the addition of 1 mM As(III), the expressions of phoBR::lacZ and aioBA::lacZ increased with increasing induction time, and the phoBR::lacZ expression appeared to be higher than that of aioBA::lacZ (Figure 4A). Meanwhile, in the low Pi condition, the expression of aioBA::lacZ was significantly induced by As(III) after 6 h of cultivation in strains HAL1 and HAL1-ΔphoB-C, but no statistically significant inductions in the mutants HAL1-phoR931 and HAL1-ΔphoB were observed (Figure 5A). However, in the normal Pi condition, the expression levels of phoBR::lacZ were similar in the presence or absence of As(III), and the expression of aioBA::lacZ was again significantly induced by As(III) (Figure 4B). In addition, the disruption of phoB and phoR had no effect on the expression of aioBA::lacZ in the normal Pi condition (Figure 5B). The expression of aioBA in low Pi and normal Pi conditions were consistent with the As(III) oxidation phenotypes (Figures 2, 3). The above results indicated that PhoBR regulates the expression of aioBA in low Pi condition but not in normal Pi condition.

Bottom Line: HAL1.Using 15 consensus Pho box sequences, a putative Pho box was found in the aioBA regulation region.The new regulation model further implies the close metabolic connection between As and Pi.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University Wuhan, China.

ABSTRACT
Previously, the expression of arsenite [As(III)] oxidase genes aioBA was reported to be regulated by a three-component regulatory system, AioXSR, in a number of As(III)-oxidizing bacterial strains. However, the regulation mechanism is still unknown when aioXSR genes are absent in some As(III)-oxidizing bacterial genomes, such as in Halomonas sp. HAL1. In this study, transposon mutagenesis and gene knock-out mutation were performed, and two mutants, HAL1-phoR 931 and HAL1-▵phoB, were obtained in strain HAL1. The phoR and phoB constitute a two-component system which is responsible for phosphate (Pi) acquisition and assimilation. Both of the mutants showed negative As(III)-oxidation phenotypes in low Pi condition (0.1 mM) but not under normal Pi condition (1 mM). The phoBR complementation strain HAL1-▵phoB-C reversed the mutants' phenotypes back to wild type status. Meanwhile, lacZ reporter fusions using pCM-lacZ showed that the expression of phoBR and aioBA were both induced by As(III) but were not induced in HAL1-phoR 931 and HAL1-▵phoB. Using 15 consensus Pho box sequences, a putative Pho box was found in the aioBA regulation region. PhoB was able to bind to the putative Pho box in vivo (bacterial one-hybrid detection) and in vitro (electrophoretic mobility gel shift assay), and an 18-bp binding sequence containing nine conserved bases were determined. This study provided the evidence that PhoBR regulates the expression of aioBA in Halomonas sp. HAL1 under low Pi condition. The new regulation model further implies the close metabolic connection between As and Pi.

No MeSH data available.


Related in: MedlinePlus