Identification and Characterization of Mycobacterium smegmatis and Mycobacterium avium subsp. paratuberculosis Zinc Transporters

Abstract

Zinc uptake in bacteria is essential to maintain cellular homeostasis and survival. ZnuABC is an important zinc importer of numerous bacterial genera, which is expressed to restore zinc homeostasis when the cytosolic concentration decreases beyond a critical threshold. Upon zinc limitation the fast-growing nonpathogenic organism Mycobacterium smegmatis (MSMEG) as well as the ruminant pathogen M. avium subsp. paratuberculosis (MAP) increases expression of genes encoding ZnuABC homologues, but also of genes encoding other transporters. This suggests an involvement of these transporters in zinc homeostasis. Here we characterized the putative zinc transporters of MSMEG (ZnuABC and ZnuABC2) and MAP (ZnuABC, MptABC, and MAP3774-76). Deletion of either ZnuABC or ZnuABC2 in MSMEG did not lead to growth defects, but to an increased expression of zinc marker genes in MSMEGΔznuABC, indicating cytosolic zinc limitation. However, chromatin immunoprecipitation proved direct binding of the global zinc regulator Zur to promoter regions of both znuABC and znuABC2. Simultaneous deletion of both transporters caused severe growth defects, which could be restored either by homologous complementation with single ZnuABC transporters or supplementation of growth media with zinc but not iron, manganese, cobalt, or magnesium. Heterologous complementation of the double mutant with MAP transporters also resulted in reconstitution of growth. Nonradioactive FluoZin^TM-3AM zinc uptake assays directly revealed the competence of all transporters to import zinc. Finally, structural and phylogenetic analyses provided evidence of a novel class of ZnuABC transporters represented by the ZnuABC2 of MSMEG, which is present only in actinobacteria, mainly in the genera Nocardia, Streptomyces and fast growing Mycobacteria IMPORTANCEZinc is necessary for bacterial growth but simultaneously toxic when in excess. Hence, bacterial cells have developed systems to alter intracellular concentration. Regulation of these systems is primarily executed at transcriptional level by regulator proteins which sense femtomolar changes in the zinc level. In environmental and pathogenic mycobacteria zinc starvation induces expression of common zinc import systems such as the ZnuABC transporter, but also of other additional not yet characterized transport systems. In this study, we characterized the role of such systems in zinc transport. We showed that transport systems of both species whose transcription is induced upon zinc starvation can exchangeably restore cellular zinc homeostasis in transporter deficient mutants by transporting zinc into the cell.

FIG 1

Growth of MSMEG znuABC single mutants in different media. MSwt, mutant strains MSΔ1 and MSΔ2, and the complemented strains MSΔ1::A and MSΔ2::B were grown in MB to an OD₆₀₀ of approximately 2.0, washed twice with PBS, and inoculated at an OD₆₀₀ of 0.05 in MB (A) or 0.1 in SM (B). Growth was monitored daily by measurement of OD₆₀₀ for 4 (A) or 8 (B) days. Shown are the results of three independent replicates in duplicate. (C and D) Substitution of znuABC transporters at the transcriptional level. Expression of MSMEG_RS29170 (znuA2, primers 27 and 28) or MSMEG_RS29150 (znuC, primers 23 and 24) and MSMEG_RS29260 (rpmG, primers 37 and 38) in MSΔ1 (C) and MSΔ2 (D) and the complemented strains MSΔ1::A or MSΔ2::B, respectively, was analyzed by qRT-PCR. Shown are the results of at least three independent replicates in duplicate. Statistical analysis was performed using Student's t test. *, P < 0.05; ****, P < 0.001.

FIG 2

Impact of Zur on MSMEG znuABC and znuABC2 expression. MSΔ6 complemented with HA-tagged Zur (MSΔ6::L) was grown in standard MB (control) or treated with 10 μM TPEN or 500 μM ZnSO₄. ChIP from cross-linked lysates was performed using anti-HA and anti-IgG1κ (isotype control). DNA from ChIP and input control was subjected to qRT-PCR with primers targeting the promoter of znuABC, znuABC2, or an intragenic region (znuint). Shown are the results of at least three independent experiments in duplicate; statistical analysis of relative fold enrichment from promoter regions compared to the intragenic control was performed using Student's t test (unpaired). ***, P < 0.0005.

FIG 3

Growth experiments with MSΔΔ4 and complemented strains. (A) Strains MSwt and MSΔΔ4 as well as homologously complemented strain MSΔΔ4::A::B (znuABC and znuABC2) were grown in MB. Growth was monitored daily by OD₆₀₀ determination for 4 days. Shown are the results of three independent replicates in duplicate. Statistical analysis was performed using Student's t test. *, P < 0.05. (B) MSwt, MSΔΔ4, and homologously complemented strains MSΔΔ4::A (znuABC), MSΔΔ4::B (znuABC2), and MSΔΔ4::A::B were grown in MB, diluted in PBS to an OD₆₀₀ of 0.1, diluted 10-fold to 10⁻⁴, and plated on MB standard agar (control) and MB supplemented with 1 or 5 μM TPEN or 25 or 100 μM ZnSO₄, MnSO₄, CoCl₂, MgSO₄, or FeNH₃-citrate. Agar plates were incubated for 4 days at 37°C. The control was incubated for up to 10 days. Assays were repeated three times in duplicate. Shown are the results of the 10⁻¹ dilution of a representative replicate.

FIG 4

Growth of MSΔΔ4 with different putative MAP zinc transporters. (A) MSwt, MSΔΔ4, and heterologously complemented strains MSΔΔ4::C (MptABC), MSΔΔ4::D (MAP3774-76), and MSΔΔ4::E (ZnuABC^MAP) were grown in MB, diluted in PBS to an OD₆₀₀ of 0.1, diluted 10-fold up to 10⁻⁴, and plated on MB standard agar (control) and MB supplemented with 1 or 5 μM TPEN or 25 or 100 μM ZnSO₄, MnSO₄, CoCl₂, MgSO₄, or FeNH₃-citrate. Agar plates were incubated for 4 days at 37°C. The control was incubated for up to 10 days. Assays were repeated three times in duplicate. Shown are the results of the 10⁻¹ dilution of a representative replicate. (B to E) Growth of MSΔΔ4 in Sauton’s medium (SM). Strains were grown in MB, washed and inoculated to an initial OD₆₀₀ of 0.1 into fresh SM. SM standard (B) was supplemented with (C) 10 μM TPEN or (D) 1 μM or (E) 10 μM ZnSO₄. Growth was monitored daily for 8 days by OD₆₀₀ measurement. Shown are the results of at least three independent replicates in duplicate.

FIG 5

FluoZin-3AM zinc uptake assay. MSMEG strains were grown in SM for 3 days, washed with PBS, adjusted to an OD₆₀₀ of 2.0, and treated with 1 μM FluoZin-3AM or left untreated (MSwt w/o FluoZin [control]) for 30 min. Samples of each strain were adjusted to an OD₆₀₀ of 0.1 (approximately 1 × 10⁷ bacteria) and subjected to a zinc uptake assay. ZnSO₄ (25 μM) was added after 1 h of incubation (blue arrow) for 3 h; subsequently, 1 μM TPEN was added for 1 h. Fluorescence (relative light units [RLU]) was measured at extinction/excitation wavelengths of 495/516 nm every 10 min. RLU was normalized to OD₆₀₀ (input). Shown are the results of three experiments in triplicate (with standard errors of the means [SEM]). Statistical analysis was performed using one-way analysis of variance (ANOVA). *, P < 0.05.

FIG 6

Structural analysis of ZnuABC transporters. (A) Protein prediction of transmembrane (gray peaks), cytoplasmic (green lines), and noncytoplasmic domains (blue lines) as well as signal sequences (red lines) by Phobius analysis of ZnuABC transporter proteins from MSMEG, SCO, MVAN, MAP, MTB (abbreviated Rv), S. Typhimurium (EIL) and E. coli (b). (B) Organization of znuABC transporter genes of MVAN (top), MSMEG (middle), and MAP (bottom). Genes of an operon are colored the same. Bars indicate predicted Zur binding sites; the colors indicate affiliations with operons. Green arrowheads indicate locations of gene-overlapping primers used in Fig. S2. (C) Clustal Omega alignment of ZnuA/ZnuA2 proteins of MSMEG, SCO, MVAN, MAP, MTB, S. Typhimurium, and E. coli. Highly conserved histidine (H) residues for zinc binding are highlighted in green; H/E/D loops are highlighted in yellow.

FIG 7

Phylogenetic tree of ZnuB2 homologous proteins. Shown are the results of NCBI blastp analysis with MSMEG_RS29175 and subsequent processing of data by the neighbor-joining method (maximum sequence difference, 0.75), illustrated in iTOL (version 5.7). Mycobacteria are in red, Nocardia species are in blue, and Streptomyces species are in green.