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Differential roles of the hemerythrin-like proteins of Mycobacterium smegmatis in hydrogen peroxide and erythromycin susceptibility.

Li X, Li J, Hu X, Huang L, Xiao J, Chan J, Mi K - Sci Rep (2015)

Bottom Line: Hemerythrin-like proteins are oxygen-carrying non-heme di-iron binding proteins and their functions have effect on oxidation-reduction regulation and antibiotic resistance.In this study, we have systematically analyzed all three hemerythrin-like proteins in M. smegmatis and our results identified and characterized two functional classes: MSMEG_2415 plays an important role in H2O2 susceptibility, and MSMEG_3312 and MSMEG_6212 are associated with erythromycin susceptibility.Here, combined with biological and phylogenetic analyses, our results provide new insights into the evolutionary divergence of the hemerythrin-like proteins in M. smegmatis.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, CAS, Beijing 100101, China.

ABSTRACT
Hemerythrin-like proteins are oxygen-carrying non-heme di-iron binding proteins and their functions have effect on oxidation-reduction regulation and antibiotic resistance. Recent studies using bioinformatic analyses suggest that multiple hemerythrin-like protein coding sequences might have been acquired by lateral gene transfer and the number of hemerythrin-like proteins varies amongst different species. Mycobacterium smegmatis contains three hemerythrin-like proteins, MSMEG_3312, MSMEG_2415 and MSMEG_6212. In this study, we have systematically analyzed all three hemerythrin-like proteins in M. smegmatis and our results identified and characterized two functional classes: MSMEG_2415 plays an important role in H2O2 susceptibility, and MSMEG_3312 and MSMEG_6212 are associated with erythromycin susceptibility. Phylogenetic analysis indicated that these three proteins have different evolutionary origins, possibly explaining their different physiological functions. Here, combined with biological and phylogenetic analyses, our results provide new insights into the evolutionary divergence of the hemerythrin-like proteins in M. smegmatis.

No MeSH data available.


Related in: MedlinePlus

MSMEG_6212 is associated with erythromycin susceptibility.(A) Growth curve of mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium. (B) Growth curve of mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium with 1.56 mg/L erythromycin (*p < 0.05, **p < 0.01). (C) Killing curve for mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium with 31.25 mg/L erythromycin (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Killing curve for pMV261/mc2155 and pMV261-6212/mc2155 in 7H9 medium with 15.6 mg/L erythromycin (*p < 0.05, **p < 0.01, ***p < 0.001).
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f3: MSMEG_6212 is associated with erythromycin susceptibility.(A) Growth curve of mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium. (B) Growth curve of mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium with 1.56 mg/L erythromycin (*p < 0.05, **p < 0.01). (C) Killing curve for mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium with 31.25 mg/L erythromycin (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Killing curve for pMV261/mc2155 and pMV261-6212/mc2155 in 7H9 medium with 15.6 mg/L erythromycin (*p < 0.05, **p < 0.01, ***p < 0.001).

Mentions: To investigate whether the three M. smegmatis hemerythrin-like proteins, MSMEG_3312, MSMEG_2415 and MSMEG_6212, have distinct or overlapping functions, we used a series of strains overexpression individual genes and knockout mutants. The specialized transduction strategy for the sequential deletion of the three genes encoding hemerythrin-like proteins in M. smegmatis, and the overexpression of individual genes encoding hemerythrin-like proteins is shown in Fig. 1. We have previously shown that MSMEG_2415 is involved in the SigF-mediated H2O2 pathway and that MSMEG_3312 is associated with erythromycin susceptibility1213. Here, to characterize the function of MSMEG_6212, we constructed a msmeg_6212 knockout strain (mc2155:Δ6212). Knockout mutants were confirmed by PCR analysis (Fig. 2B). A msmeg_6212 gene fragment was not amplified and no msmeg_6212 mRNA was detected in an assay of its mRNA expression (data not shown). The msmeg_6212 mutant, mc2155:Δ6212, was complemented with a single integrated copy using pMV361-6212. The constructed M. smegmatis mutant strain mc2155:Δ6212 was initially tested for growth in rich 7H9 medium and defined Sauton medium. Growth of mc2155:Δ6212 appeared to have no discernable phenotypic difference from the wild type strain mc2155, in either rich (Fig. 3A) or defined media (data not shown). These results indicate that msmeg_6212, like the previous investigated msmeg_3312 and msmeg_2415, is not an essential gene for M. smegmatis growth in either 7H9 rich medium or Sauton defined medium. In order to characterize the potential roles of MSMEG_6212, we compared the minimum inhibitory concentrations (MICs) of eleven antibiotic drugs, and H2O2 in the msmeg_6212 knockout strain mc2155:Δ6212 and wild type strain mc2155 (Table S1). Surprisingly, a difference in MIC values was detected only for the macrolides erythromycin and azithromycin (AZM) (Table S1). To clarify the effect of MSMEG_6212 on erythromycin susceptibility, we performed drug exposure experiments to compare the growth rates of wild-type strain mc2155, msmeg_6212 knockout strain mc2155:Δ6212, and the complemented strain pMV361-6212/mc2155:Δ6212 in the presence of 1.56mg/L erythromycin (Fig. 3B). The strain mc2155:Δ6212 showed a growth advantage compared with wild type mc2155, which was partially reversed in the complemented strain pMV361-6212/ mc2155:Δ6212 in the presence of erythromycin (Fig. 3B). Furthermore, we compared the survival of various M. smegmatis strains every few hours under treatment with 31.2 mg/L (10x MIC) erythromycin. As shown in Fig. 3C and Fig. S1, the percentage survival of mc2155:Δ6212 was greater than that of wild type mc2155, whereas the complemented strain pMV361-6212/mc2155:Δ6212 did not grow well and its survival was partially reversed to that of the wild-type. As overexpression of MSMEG_6212 increased susceptibility to erythromycin, we used 15.6 mg/L (5 × MIC) to perform the killing experiment: overexpression of msmeg_6212 caused greater susceptibility to erythromycin and lower survival than in wild type mc2155 under the same treatment (Fig. 3D and Fig. S1). Taken together, these results show that, like MSMEG_3312, MSMEG_6212 negatively impacts erythromycin resistance.


Differential roles of the hemerythrin-like proteins of Mycobacterium smegmatis in hydrogen peroxide and erythromycin susceptibility.

Li X, Li J, Hu X, Huang L, Xiao J, Chan J, Mi K - Sci Rep (2015)

MSMEG_6212 is associated with erythromycin susceptibility.(A) Growth curve of mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium. (B) Growth curve of mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium with 1.56 mg/L erythromycin (*p < 0.05, **p < 0.01). (C) Killing curve for mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium with 31.25 mg/L erythromycin (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Killing curve for pMV261/mc2155 and pMV261-6212/mc2155 in 7H9 medium with 15.6 mg/L erythromycin (*p < 0.05, **p < 0.01, ***p < 0.001).
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f3: MSMEG_6212 is associated with erythromycin susceptibility.(A) Growth curve of mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium. (B) Growth curve of mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium with 1.56 mg/L erythromycin (*p < 0.05, **p < 0.01). (C) Killing curve for mc2155, mc2155:Δ6212 and the complemented strain pMV361-6212/mc2155:Δ6212 in 7H9 medium with 31.25 mg/L erythromycin (*p < 0.05, **p < 0.01, ***p < 0.001). (D) Killing curve for pMV261/mc2155 and pMV261-6212/mc2155 in 7H9 medium with 15.6 mg/L erythromycin (*p < 0.05, **p < 0.01, ***p < 0.001).
Mentions: To investigate whether the three M. smegmatis hemerythrin-like proteins, MSMEG_3312, MSMEG_2415 and MSMEG_6212, have distinct or overlapping functions, we used a series of strains overexpression individual genes and knockout mutants. The specialized transduction strategy for the sequential deletion of the three genes encoding hemerythrin-like proteins in M. smegmatis, and the overexpression of individual genes encoding hemerythrin-like proteins is shown in Fig. 1. We have previously shown that MSMEG_2415 is involved in the SigF-mediated H2O2 pathway and that MSMEG_3312 is associated with erythromycin susceptibility1213. Here, to characterize the function of MSMEG_6212, we constructed a msmeg_6212 knockout strain (mc2155:Δ6212). Knockout mutants were confirmed by PCR analysis (Fig. 2B). A msmeg_6212 gene fragment was not amplified and no msmeg_6212 mRNA was detected in an assay of its mRNA expression (data not shown). The msmeg_6212 mutant, mc2155:Δ6212, was complemented with a single integrated copy using pMV361-6212. The constructed M. smegmatis mutant strain mc2155:Δ6212 was initially tested for growth in rich 7H9 medium and defined Sauton medium. Growth of mc2155:Δ6212 appeared to have no discernable phenotypic difference from the wild type strain mc2155, in either rich (Fig. 3A) or defined media (data not shown). These results indicate that msmeg_6212, like the previous investigated msmeg_3312 and msmeg_2415, is not an essential gene for M. smegmatis growth in either 7H9 rich medium or Sauton defined medium. In order to characterize the potential roles of MSMEG_6212, we compared the minimum inhibitory concentrations (MICs) of eleven antibiotic drugs, and H2O2 in the msmeg_6212 knockout strain mc2155:Δ6212 and wild type strain mc2155 (Table S1). Surprisingly, a difference in MIC values was detected only for the macrolides erythromycin and azithromycin (AZM) (Table S1). To clarify the effect of MSMEG_6212 on erythromycin susceptibility, we performed drug exposure experiments to compare the growth rates of wild-type strain mc2155, msmeg_6212 knockout strain mc2155:Δ6212, and the complemented strain pMV361-6212/mc2155:Δ6212 in the presence of 1.56mg/L erythromycin (Fig. 3B). The strain mc2155:Δ6212 showed a growth advantage compared with wild type mc2155, which was partially reversed in the complemented strain pMV361-6212/ mc2155:Δ6212 in the presence of erythromycin (Fig. 3B). Furthermore, we compared the survival of various M. smegmatis strains every few hours under treatment with 31.2 mg/L (10x MIC) erythromycin. As shown in Fig. 3C and Fig. S1, the percentage survival of mc2155:Δ6212 was greater than that of wild type mc2155, whereas the complemented strain pMV361-6212/mc2155:Δ6212 did not grow well and its survival was partially reversed to that of the wild-type. As overexpression of MSMEG_6212 increased susceptibility to erythromycin, we used 15.6 mg/L (5 × MIC) to perform the killing experiment: overexpression of msmeg_6212 caused greater susceptibility to erythromycin and lower survival than in wild type mc2155 under the same treatment (Fig. 3D and Fig. S1). Taken together, these results show that, like MSMEG_3312, MSMEG_6212 negatively impacts erythromycin resistance.

Bottom Line: Hemerythrin-like proteins are oxygen-carrying non-heme di-iron binding proteins and their functions have effect on oxidation-reduction regulation and antibiotic resistance.In this study, we have systematically analyzed all three hemerythrin-like proteins in M. smegmatis and our results identified and characterized two functional classes: MSMEG_2415 plays an important role in H2O2 susceptibility, and MSMEG_3312 and MSMEG_6212 are associated with erythromycin susceptibility.Here, combined with biological and phylogenetic analyses, our results provide new insights into the evolutionary divergence of the hemerythrin-like proteins in M. smegmatis.

View Article: PubMed Central - PubMed

Affiliation: CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, CAS, Beijing 100101, China.

ABSTRACT
Hemerythrin-like proteins are oxygen-carrying non-heme di-iron binding proteins and their functions have effect on oxidation-reduction regulation and antibiotic resistance. Recent studies using bioinformatic analyses suggest that multiple hemerythrin-like protein coding sequences might have been acquired by lateral gene transfer and the number of hemerythrin-like proteins varies amongst different species. Mycobacterium smegmatis contains three hemerythrin-like proteins, MSMEG_3312, MSMEG_2415 and MSMEG_6212. In this study, we have systematically analyzed all three hemerythrin-like proteins in M. smegmatis and our results identified and characterized two functional classes: MSMEG_2415 plays an important role in H2O2 susceptibility, and MSMEG_3312 and MSMEG_6212 are associated with erythromycin susceptibility. Phylogenetic analysis indicated that these three proteins have different evolutionary origins, possibly explaining their different physiological functions. Here, combined with biological and phylogenetic analyses, our results provide new insights into the evolutionary divergence of the hemerythrin-like proteins in M. smegmatis.

No MeSH data available.


Related in: MedlinePlus