Regulation of intracellular free calcium concentration during heterocyst differentiation by HetR and NtcA in Anabaena sp. PCC 7120

Abstract

Calcium ions are important to some prokaryotic cellular processes, such as heterocyst differentiation of cyanobacteria. Intracellular free Ca(2+)concentration, [Ca(2+)](i), increases several fold in heterocysts and is regulated by CcbP, a Ca(2+)-binding protein found in heterocyst-forming cyanobacteria. We demonstrate here that CcbP is degraded by HetR, a serine-type protease that controls heterocyst differentiation. The degradation depends on Ca(2+) and appears to be specific because HetR did not digest other tested proteins. CcbP was found to bind two Ca(2+) per molecule with K(D) values of 200 nM and 12.8 microM. Degradation of CcbP releases bound Ca(2+) that contributes significantly to the increase of [Ca(2+)](i) during the process of heterocyst differentiation in Anabaena sp. strain PCC 7120. We suggest that degradation of CcbP is a mechanism of positive autoregulation of HetR. The down-regulation of ccbP in differentiating cells and mature heterocysts, which also is critical to the regulation of [Ca(2+)](i), depends on NtcA. Coexpression of ntcA and a ccbP promoter-controlled gfp in Escherichia coli diminished production of GFP, and the decrease is enhanced by alpha-ketoglutarate. It was also found that NtcA could bind a fragment of the ccbP promoter containing an NtcA-binding sequence in a alpha-ketoglutarate-dependent fashion. Therefore, [Ca(2+)](i) is regulated by a collaboration of HetR and NtcA in heterocyst differentiation in Anabaena sp. strain PCC 7120.

Fig. 1.

Measurement of Ca²⁺ content, cellular free Ca²⁺ concentration, and expression of hetR during heterocyst differentiation of Anabaena 7120. (A) Measurement of cellular Ca²⁺ content. Total cellular Ca²⁺ of Anabaena 7120 (■) was determined with ⁴⁵Ca²⁺, and [Ca²⁺]_i was determined with Ca²⁺-dependent fluorescence emission at 460 nm in Anabaena 7120 expressing the obelin gene (●). The values of fluorescence emission were normalized to the initial value at time 0, i.e., when combined nitrogen was removed. (B) Relative amount of the hetR mRNA during heterocyst differentiation as determined by quantitative PCR. Each point represents an average of six individual measurements, and all values were normalized to the value at time 0 of nitrogen step-down.

Fig. 2.

SDS/PAGE analysis of CcbP degradation by HetR. (A) Degradation of CcbP by wild-type HetR under the conditions indicated above the gel. The duration of incubation at 37°C was 2 h. The initial concentrations of HetR, CcbP, NtcA, and BSA were 1, 2, 1, and 2 mg·ml⁻¹, respectively. The concentrations of EGTA and PMSF were 5 and 0.2 mM, respectively. Lanes: 1 and 2, HetR before and after incubation at 37°C, respectively; 3 and 4, CcbP after incubation without or with HetR at 37°C, respectively; 5 and 6, CcbP after incubation with HetR in the presence of 5 mM EGTA or 0.2 mM PMSF, respectively; 7 and 8, NtcA after incubation without or with HetR at 37°C, respectively; 9 and 10, BSA after incubation without or with HetR at 37°C, respectively. The thin bands below the major BSA bands in lanes 9 and 10 were from BSA stock and were present before treatment. (B) No digestion of CcbP by two mutant HetR proteins that lack autodegradation activity. The concentrations of CcbP, HetR_S152A, and HetR_S179N and the digestion conditions were the same as for CcbP and HetR in A. Lanes: 1 and 2, HetR_S152A before and after incubation at 37°C, respectively; 3 and 4, HetR_S179N before and after incubation at 37°C, respectively; 5 and 6, incubation of CcbP with HetR_S152A and HetR_S179N at 37°C, respectively. The upper arrows in A and B indicate the position of the HetR dimer, the middle arrows indicate the position of HetR monomer, and the lower arrows indicate the position of CcbP.

Fig. 3.

Release of bound Ca²⁺ from CcbP during its digestion by HetR. Solutions (50 mM Tris·HCl, pH 7.4/100 mM KCl) containing both HetR at 0.1 mg·ml⁻¹ and CcbP at 0.43 mg·ml⁻¹ (3 μM) (●), HetR only (■), or CcbP only (▴) were incubated at 37°C, and the concentrations of free Ca²⁺ were measured with a Ca²⁺ electrode. The initial concentration of free Ca²⁺ was adjusted to 1.0 μM.

Fig. 4.

Measurement of the number of bound Ca²⁺ per molecule of CcbP. (A) Determination of the stoichiometry of CcbP and bound Ca²⁺. CaCl₂ from a stock solution of 1 M was added incrementally to a 10-ml 0.83 mg·ml⁻¹ CcbP solution in 10 mM Tris·HCl buffer (pH 7.5) containing 100 mM KCl, and free Ca²⁺ in solution was measured with a Ca²⁺ electrode. An average of 1.7 Ca²⁺ bound per CcbP was determined. (B) Scatchard plot for the determination of Ca²⁺ dissociation constants (K_d) of CcbP. Y represents the percentage of CcbP with bound Ca²⁺ and [L] represents the free Ca²⁺ concentration in micromolar. Curves I and II were obtained by curve fitting. Curve I has a slope of −4.99, corresponding to a K_d of 200 nM; curve II has a slope of −0.078, corresponding to a K_d of 12.8 μM.

Fig. 5.

Down-regulation of the ccbP gene of Anabaena 7120 by NtcA. (A) GFP fluorescence spectra of E. coli cells expressing a gfp gene under control of the ccbP promoter and the ntcA gene from Anabaena 7120 inducible by IPTG. Curves: 1, fluorescence spectrum obtained from the E. coli cells containing pPccbP-gfp; 2, emission spectrum obtained when the E. coli cells containing pPccbP-gfp and pET-psaE were in the presence of 0.1 mM IPTG and 0.5 mM 2-OG; 3 and 4, emission spectra obtained when the E. coli cells containing both pPccbP-gfp and pET-ntcA were in the presence of 0.1 mM IPTG without or with 0.5 mM 2-OG, respectively; 5, a spectrum obtained from E. coli cells containing pRL25C (from which pPccbP-gfp is derived) and pET-ntcA. No GFP fluorescence emission peak was obtained. The optical densities at 600 nm of all cultures were adjusted to 1.0 before the measurement of the fluorescence spectra. (B) Quantitative PCR analysis of ccbP expression in the wild type (■) and ntcA⁻ (●) after nitrogen step-down. Total RNA was isolated at the times indicated for quantitative PCR. All values were normalized to that at time 0. (C) NtcA-induced gel mobility shift of a 100-bp DNA fragment in the ccbP promoter region of Anabaena 7120. DNA (400 ng) was incubated with 3 nM NtcA in the binding buffer with 2-OG at the concentrations described below for 10 min before analysis with polyacrylamide gel (6%) electrophoresis. Lanes: 1–5, incubation of the DNA fragment with NtcA in the presence of 2-OG at concentrations of 0, 0.05, 0.1, 0.2, and 0.5 mM, respectively; 6, the DNA fragment incubated with BSA alone. (D) The NtcA-binding sequence was required for the NtcA-induced gel mobility shift. The conditions for EMSA were the same as in C, except that synthetic DNA fragments of the ccbP promoter region (from nucleotides −179 to −130 upstream of the start codon) were used. The sequence GTN₁₁ACA was retained in one of the fragments (lane 2), and it was changed to CCN₁₁CCC in the other fragment (lane 3). The conditions for lane 1 were the same as those for lane 2, except that no NtcA was included in the binding buffer. (C and D) The upper arrows indicate the positions of the shifted DNA bands and the lower arrows indicate the free DNA fragments.