Characterization of the response to zinc deficiency in the cyanobacterium Anabaena sp. strain PCC 7120

Abstract

Zur regulators control zinc homeostasis by repressing target genes under zinc-sufficient conditions in a wide variety of bacteria. This paper describes how part of a survey of duplicated genes led to the identification of the open reading frame all2473 as the gene encoding the Zur regulator of the cyanobacterium Anabaena sp. strain PCC 7120. All2473 binds to DNA in a zinc-dependent manner, and its DNA-binding sequence was characterized, which allowed us to determine the relative contribution of particular nucleotides to Zur binding. A zur mutant was found to be impaired in the regulation of zinc homeostasis, showing sensitivity to elevated concentrations of zinc but not other metals. In an effort to characterize the Zur regulon in Anabaena, 23 genes containing upstream putative Zur-binding sequences were identified and found to be regulated by Zur. These genes are organized in six single transcriptional units and six operons, some of them containing multiple Zur-regulated promoters. The identities of genes of the Zur regulon indicate that Anabaena adapts to conditions of zinc deficiency by replacing zinc metalloproteins with paralogues that fulfill the same function but presumably with a lower zinc demand, and with inducing putative metallochaperones and membrane transport systems likely being involved in the scavenging of extracellular zinc, including plasma membrane ABC transport systems and outer membrane TonB-dependent receptors. Among the Zur-regulated genes, the ones showing the highest induction level encode proteins of the outer membrane, suggesting a primary role for components of this cell compartment in the capture of zinc cations from the extracellular medium.

Fig 1

Structure of a divalent metal-responsive gene cluster of Anabaena. A diagram of the gene cluster is at the top. +, presence of a putative metal cofactor (specified in parentheses in cases where it is predictable). Anabaena genes homologous to those in the cluster are indicated with their locus tags or with an asterisk when many paralogues are present in the genome.

Fig 2

Transcriptional response of the genes in the all4729-all4721 cluster to the chelation of divalent metals. Anabaena cells growing in BG11 medium were split into two cultures; one of them was supplemented with TPEN at a final concentration of 20 μM, and both were further cultured for 24 h. Aliquots were extracted at the times indicated at the top of the panels; RNA was extracted and subjected to Northern hybridization with probes for the genes indicated at the left. m1, m2, m3, and m4 refer to the membranes used for the hybridization. Numbers on the right indicate the positions of RNA molecular weight markers. Hybridization of the four membranes (m1, m2, m3, and m4) with the rnpB probe, used as a control for RNA loading, is shown at the bottom.

Fig 3

Determination of the limits of the operon by RT-PCR and promoter mapping. (A) Products of RT-PCRs. + and −, reactions in which retrotranscriptase was added or omitted, respectively. C, control PCRs using genomic DNA as the template. The letters A to L identify the fragments on the electrophoresis and correspond to the fragments in panel B. (B) Fragments that were amplified by RT-PCR are depicted with solid lines, and those that failed to be amplified are depicted with gray lines. The primers used for retrotranscription are indicated by arrowheads. Bent arrows indicate the position of transcription start points, mapped by 5′-RACE. (C) Promoter mapping by 5′-RACE. RNA samples from Anabaena cells cultured for 24 h in the presence or absence of TPEN were treated with TAP or left untreated and subjected to 5′-RACE as described in Materials and Methods. The reverse oligonucleotides used in the PCR step annealed with the 5′ region of the ORF indicated to the left of each panel. Numbers indicate the positions of DNA size markers. Arrowheads indicate the positions of major differential bands.

Fig 4

Binding of Fur proteins to the promoter regions of genes in the all4725-all4721 operon. (A) EMSA of FurA, FurB, and FurC with a DNA fragment from the promoter region of all4725. C, control assay in which no protein was added to the reaction mixture. The amounts (pmol) of dimer protein in 15-μl assay mixtures are indicated at the top. (B) Binding to 0.25 fmol of a labeled fragment of the promoter region of all4725 generated with primers all4725_2F and all4725_2R was assayed with no protein in the reaction mixture (C−) or with 0.1 pmol FurB, in the absence (C+) or presence (C+) of a 10-, 50-, 100-, or 200-fold excess of cold all4725 promoter fragment (left) or cold unrelated competitor DNA (right) generated by PCR with primers all4726_2F and all4726_1R. (C) EMSA of FurB with DNA fragments of the promoter regions of all4723, all4722, and all4721. The amount (pmol) of FurB dimer in each reaction is indicated on top of each lane.

Fig 5

Phenotype of the zur mutant. (A) Northern hybridizations of RNA from cells of the WT or the zur mutant treated with TPEN for 24 h or left untreated. Probes used for hybridization are shown above the panels. Hybridizations of the membranes with the rnpB gene used as a RNA loading control are shown at the bottom of each panel. (B) Growth curves of the WT (squares) and the zur mutant (circles) in the presence of 25 μM ZnSO₄, 25 μM CuSO₄, 25 μM CoCl₂, or 25 μM NiSO₄. The data are representative of three independent repeats. (C) EMSA reaction mixtures (15 μl) containing 0.1 pmol of FurB dimer were incubated in the presence (+) or absence (−) of 500 μM TPEN for 1 h at 25°C and subsequently supplemented with increasing concentrations (100, 250, or 500 μM) of ZnSO₄, incubated 30 min at 25°C, and resolved in a native acrylamide gel.

Fig 6

Mapping and characterization of Zur-binding sequences. (A) The sequences of the promoter regions of all4725, all4723, and all4721 are depicted. The bent arrow indicates the position of the transcription start point, bold indicates putative −35 and −10 sequences, and putative Zur-binding sequences are boxed. (B) The binding of FurB to different DNA sequences was assayed by EMSA. The plot shows the curves resulting from the adjustment of the percentage of retarded band to a simplified version of the Hill equation. The colors correspond to the sequences at the bottom. The top sequence is that of the all4725 promoter. Mutations introduced to generate the other DNA fragments are indicated by colors corresponding to the graph. The K_d of Zur for each fragment is indicated. The curve for the sequence at the very bottom is not shown.