Limits...
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_3312 and MSMEG_6212 have redundant roles in erythromycin susceptibility; MSMEG_3312, but not MSMEG_6212, has a redundant role in H2O2 susceptibility.(A) Killing curve for mc2155, mc2155:Δ3312, mc2155:Δ6212 and mc2155:Δ3312-6212 in 7H9 medium with 31.25 mg/L erythromycin. (B) Relative expression levels of mtrA in mc2155 and mc2155:Δ3312-6212 with (+) or without (−) erythromycin treatment. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviations of three replicates (*p < 0.05). (C) Relative expression levels of msmeg_6212 in mc2155:Δ3312. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviation of three replicates (**p < 0.01). (D) Relative expression levels of msmeg_3312 in mc2155:Δ6212. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviation of three replicates. (E) H2O2 resistance phenotype of mc2155, mc2155:Δ3312, pMV261-3312/mc2155, pMV261/mc2155:Δ2415, pMV261-3312/mc2155:Δ2415. The pictures shown are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4660385&req=5

f5: MSMEG_3312 and MSMEG_6212 have redundant roles in erythromycin susceptibility; MSMEG_3312, but not MSMEG_6212, has a redundant role in H2O2 susceptibility.(A) Killing curve for mc2155, mc2155:Δ3312, mc2155:Δ6212 and mc2155:Δ3312-6212 in 7H9 medium with 31.25 mg/L erythromycin. (B) Relative expression levels of mtrA in mc2155 and mc2155:Δ3312-6212 with (+) or without (−) erythromycin treatment. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviations of three replicates (*p < 0.05). (C) Relative expression levels of msmeg_6212 in mc2155:Δ3312. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviation of three replicates (**p < 0.01). (D) Relative expression levels of msmeg_3312 in mc2155:Δ6212. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviation of three replicates. (E) H2O2 resistance phenotype of mc2155, mc2155:Δ3312, pMV261-3312/mc2155, pMV261/mc2155:Δ2415, pMV261-3312/mc2155:Δ2415. The pictures shown are representative of three independent experiments.

Mentions: In addition, we measured changes in the mRNA levels of MtrA regulon genes msmeg_1875 (encoding sensor histidine kinase MtrB) and msmeg_0637 (encoding iron-sulfur binding oxidoreductase)17 in response to erythromycin treatment in mc2155, mc2155:Δ6212, mc2155:Δ3312 and complemented strains pMV361-3312/mc2155:Δ3312 and pMV361-6212/mc2155:Δ6212. The level of msmeg_1875 and msmeg_0637 mRNA increased in mc2155 in response to erythromycin, but induction of msmeg_1875 and msmeg_0637 was abrogated in both mc2155:Δ6212 and mc2155:Δ3312 in response to erythromycin (Fig. 4B,C,E,F). Correspondingly, induction of msmeg_1875 and msmeg_0637 was restored in both the complemented strain pMV361-3312/ mc2155:Δ3312 and pMV361-6212/mc2155:Δ6212 in response to erythromycin (Fig. 4B,C,E,F). Taken together, those results indicate that both MSMEG_6212 and MSMEG_3312 are required for the MtrA-mediated erythromycin response. To characterize the relationship between MSMEG_3312 and MSMEG_6212, we constructed a double knockout mutant strain mc2155:Δ3312-6212 and assayed its resistance to erythromycin. As shown in Figs 5A and 6A, the resistance of the double-knockout mutant mc2155:Δ3312-6212 to erythromycin appeared to be comparable to mc2155:Δ3312. Moreover, a significant increase of mtrA mRNA in mc2155 was observed in response to erythromycin (Fig. 5B), while no significant changes of mRNA level in mc2155:Δ3312-6212 was observed in response to erythromycin (Fig. 5B)


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_3312 and MSMEG_6212 have redundant roles in erythromycin susceptibility; MSMEG_3312, but not MSMEG_6212, has a redundant role in H2O2 susceptibility.(A) Killing curve for mc2155, mc2155:Δ3312, mc2155:Δ6212 and mc2155:Δ3312-6212 in 7H9 medium with 31.25 mg/L erythromycin. (B) Relative expression levels of mtrA in mc2155 and mc2155:Δ3312-6212 with (+) or without (−) erythromycin treatment. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviations of three replicates (*p < 0.05). (C) Relative expression levels of msmeg_6212 in mc2155:Δ3312. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviation of three replicates (**p < 0.01). (D) Relative expression levels of msmeg_3312 in mc2155:Δ6212. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviation of three replicates. (E) H2O2 resistance phenotype of mc2155, mc2155:Δ3312, pMV261-3312/mc2155, pMV261/mc2155:Δ2415, pMV261-3312/mc2155:Δ2415. The pictures shown are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660385&req=5

f5: MSMEG_3312 and MSMEG_6212 have redundant roles in erythromycin susceptibility; MSMEG_3312, but not MSMEG_6212, has a redundant role in H2O2 susceptibility.(A) Killing curve for mc2155, mc2155:Δ3312, mc2155:Δ6212 and mc2155:Δ3312-6212 in 7H9 medium with 31.25 mg/L erythromycin. (B) Relative expression levels of mtrA in mc2155 and mc2155:Δ3312-6212 with (+) or without (−) erythromycin treatment. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviations of three replicates (*p < 0.05). (C) Relative expression levels of msmeg_6212 in mc2155:Δ3312. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviation of three replicates (**p < 0.01). (D) Relative expression levels of msmeg_3312 in mc2155:Δ6212. Levels of mRNA expression were determined by qRT-PCR. Results are shown as the mean ± standard deviation of three replicates. (E) H2O2 resistance phenotype of mc2155, mc2155:Δ3312, pMV261-3312/mc2155, pMV261/mc2155:Δ2415, pMV261-3312/mc2155:Δ2415. The pictures shown are representative of three independent experiments.
Mentions: In addition, we measured changes in the mRNA levels of MtrA regulon genes msmeg_1875 (encoding sensor histidine kinase MtrB) and msmeg_0637 (encoding iron-sulfur binding oxidoreductase)17 in response to erythromycin treatment in mc2155, mc2155:Δ6212, mc2155:Δ3312 and complemented strains pMV361-3312/mc2155:Δ3312 and pMV361-6212/mc2155:Δ6212. The level of msmeg_1875 and msmeg_0637 mRNA increased in mc2155 in response to erythromycin, but induction of msmeg_1875 and msmeg_0637 was abrogated in both mc2155:Δ6212 and mc2155:Δ3312 in response to erythromycin (Fig. 4B,C,E,F). Correspondingly, induction of msmeg_1875 and msmeg_0637 was restored in both the complemented strain pMV361-3312/ mc2155:Δ3312 and pMV361-6212/mc2155:Δ6212 in response to erythromycin (Fig. 4B,C,E,F). Taken together, those results indicate that both MSMEG_6212 and MSMEG_3312 are required for the MtrA-mediated erythromycin response. To characterize the relationship between MSMEG_3312 and MSMEG_6212, we constructed a double knockout mutant strain mc2155:Δ3312-6212 and assayed its resistance to erythromycin. As shown in Figs 5A and 6A, the resistance of the double-knockout mutant mc2155:Δ3312-6212 to erythromycin appeared to be comparable to mc2155:Δ3312. Moreover, a significant increase of mtrA mRNA in mc2155 was observed in response to erythromycin (Fig. 5B), while no significant changes of mRNA level in mc2155:Δ3312-6212 was observed in response to erythromycin (Fig. 5B)

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