Identification and characterization of the zosA gene involved in copper uptake in Bacillus subtilis 168

Figures & data

Table 1. Effects of dl-penicillamine on generation time in log phase B. subtilis 168.

dl-Penicillamine (%)
	0	0.3	0.5
Complete	35	46	71
w/o CuCl2	35	88	213
w/o ZnCl2	29	43	59
w/o CoCl2	34	45	63
w/o Na2MoO4	36	49	75
w/o MnSO4	40	54	56
w/o CaCl2	33	49	69

Notes: B. subtilis 168 was grown in the synthetic medium without one of the metals with the designated concentrations of dl-penicillamine. The generation time (min) was measured by monitoring OD₆₀₀, the optical density at 600 nm, during the log phase.

Fig. 1. Effects of metals and 0.1% dl-penicillamine on growth of B. subtilis 168.

Notes: The number of viable cells was counted on agar plates after serial dilutions. Cells were grown in the complete synthetic medium (◆, ◇) and in media containing 0.1 mM CuSO₄ (▲, △), 0.5 mM ZnSO₄ (●, ○), and 5 mM MnSO₄ (■, □). Symbols represent growth in the presence (closed) or absence (open) of 0.1% (6.7 mM) dl-penicillamine.

Table 2. B. subtilis 168 genes responding to 0.1% dl-penicillamine^a.

Gene	Ratio 1^b	Ratio 2^b	Regulon		Operon description
>10-fold up-regulation
yciA	18.2	12.2	Zur	yciAB	Putative Zn(II) uptake system
yciB	34.0	16.9	–	yciAB	Putative Zn(II) uptake system
yciC	178.7	239.6	Zur		Putative Zn(II) uptake system
ycdH	6.6	7.6	Zur	ycdHI	Putative ABC transporter for Zn(II)
ycdI	11.3	11.0	–	ycdHI	Putative ABC transporter for Zn(II)
yceA	15.0	11.1	Zur		ABC transporter subunit
zosA	12.3	11.9	PerR		Putative zinc transporting ATPase
mrgA	7.1	12.9	PerR		Dps-like DNA-binding protein
katA	9.3	15.2	PerR		Vegetable catalase
ahpC	7.8	15.0	PerR		Alkylhydroperoxide reductase
ahpF	6.6	15.4	PerR		Alkylhydroperoxide reductase
mntA	22.3	19.3	MntR	mntABCD	ABC transporter for Mn(II)
mntB	16.6	15.3	–	mntABCD	ABC transporter for Mn(II)
mntC	18.1	19.4	–	mntABCD	ABC transporter for Mn(II)
mntH	9.5	5.4	MntR		Proton-coupled metal ion transporter
yhzZ	24.2	114.1			Ribosomal protein S14
ydeG	219.9	343.5			Unknown
ydeH	21.0	46.7			Unknown
yrpE	160.4	59.7			Unknown
<10-fold up-regulation
opuAA	5.4	5.4	Glycine, betaine-binding ABC transporter
opuAB	3.0	6.2	Glycine, betaine-binding ABC transporter
opuAC	5.5	8.0	Glycine, betaine-binding protein
aroA	5.3	13.8	3-deoxy-D-arabino-heptulosonate7-phosphate
			Synthase
yvcE	5.8	4.1	Unknown, cell wall-binding protein

^a RNA was isolated from B. subtilis 168 grown to the exponential phase with or without 0.1% dl-penicillamine.

^b Ratios of signal intensities observed for cells grown in the dl-penicillamine-containing synthetic medium to those grown in the medium without the copper chelator.

Fig. 2. Characterization of the zosA mutant.

Notes: (A) The zosA mutant was grown in copper-limited (▲), zinc-limited (◇), and complete (●) synthetic media at 37 °C under aeration at 200 rpm. The inset is the growth of wild-type strain under the same conditions. (n = 3) (B) Complementation of the zosA mutation by IPTG induction. Growth curves for wild-type (circle symbols) and the zosA mutant (triangle symbols) in the presence (closed) and absence (open) of 0.3 mM IPTG. The concentration of dl-penicillamine was 0.1% (6.7 mM) for the time course study. (n = 4) (C) Growth in the Cu-omitted synthetic media for the zosA mutant and wild-type (wt) strains in the absence (open bar) and presence (closed bar) of 0.3 mM IPTG with varied concentrations of dl-penicillamine as designated on the x-axis. (n = 3).

Fig. 3. The wild-type strain (open symbols) and zosA mutant (closed symbols) were grown overnight on LB media containing either CuSO₄ (triangles) or ZnSO₄ (circles) at the concentrations designated on the x-axis.

Fig. 4. Effect of zosA mutation on the metal tolerance of B. subtilis 168.

Notes: A series of cell suspensions were diluted by 10²- to 10⁷-fold, and 5-μl aliquots were spotted on synthetic media containing (A) 0.1 mM CuSO₄, (B) 0.5 mM ZnSO₄, or (C) no excess metal. n.d.: colonies not observed.

Fig. 5. Effect of the csoR-copZA deletion on Cu-sensitivity of B. subtilis 168.

Notes: The wild-type strain (●), zosA mutant (○), ΔcsoR-copZA mutant (▲), and zosA/ΔcsoR-copZA double knockout (Δ) were spotted in a series of 10-fold dilutions. The number of viable cells that survived on the media containing designated concentrations of CuSO₄ was counted.

Fig. 6 Modeling of ZosA.

Notes: (A) Cartoon representation of overall structure with A-, N-, and P-domains in yellow, red, and blue, respectively. The transmembrane helices are displayed in pink (HA), cyan (HBa), blue (HBb), wheat (H1–3), and orange (H4). Key residues are shown in sphere form. Arrows indicate the suggested Cu-transport conduit, and lines indicate the approximate position of the membrane. (B) Putative entrance for copper on top of the helices. Surface representation for loops and helices are designated in green and wheat, respectively. Copper ions are led to the inner cavity through the gateway opened by the movement of Met88 and Met612. (C) The cavity for the Cu-departure port with putative amino acid residues that can trap copper, Met302, Met585, and Glu249. (D) Amphipathic HBb helix at the Cu-departure port is shown with hydrophilic residues in blue and hydrophobic residues in yellow.