High-salinity-induced iron limitation in Bacillus subtilis

Abstract

Proteome analysis of Bacillus subtilis cells grown at low and high salinity revealed the induction of 16 protein spots and the repression of 2 protein spots, respectively. Most of these protein spots were identified by mass spectrometry. Four of the 16 high-salinity-induced proteins corresponded to DhbA, DhbB, DhbC, and DhbE, enzymes that are involved in the synthesis of 2,3-dihydroxybenzoate (DHB) and its modification and esterification to the iron siderophore bacillibactin. These proteins are encoded by the dhbACEBF operon, which is negatively controlled by the central iron regulatory protein Fur and is derepressed upon iron limitation. We found that iron limitation and high salinity derepressed dhb expression to a similar extent and that both led to the accumulation of comparable amounts of DHB in the culture supernatant. DHB production increased linearly with the degree of salinity of the growth medium but could still be reduced by an excess of iron. Such an excess of iron also partially reversed the growth defect exhibited by salt-stressed B. subtilis cultures. Taken together, these findings strongly suggest that B. subtilis cells grown at high salinity experience iron limitation. In support of this notion, we found that the expression of several genes and operons encoding putative iron uptake systems was increased upon salt stress. The unexpected finding that high-salinity stress has an iron limitation component might be of special ecophysiological importance for the growth of B. subtilis in natural settings, in which bioavailable iron is usually scarce.

FIG. 1.

Influence of salinity on the protein profile of the B. subtilis strain JH642. Bacteria were grown in SMM or SMM supplemented with either 0.7 M NaCl, 1.2 M NaCl, or 0.7 M NaCl and 1 mM glycine betaine (GB). Crude protein extracts were prepared and separated by 2D gel electrophoresis. After being stained with silver nitrate, the gels were scanned with an imaging system and analyzed with the Melanie 3.0 software package. Protein spots induced or repressed by high salinity are marked with arrowheads or boxes, respectively. Proteins that were identified by peptide mass fingerprinting are labeled with their gene names. The image in the left section of the figure displays a gel obtained with an extract from cells grown in the presence of 1.2 M NaCl. The right part of the figure displays selected regions of gels prepared with extracts from cells grown under the conditions indicated above the columns. sr, salt-repressed protein; si, salt-induced protein; co, control.

FIG. 2.

Induction of the dhb operon by salt. (A) Structure of the dhbACEBF operon. Genes whose products were identified by peptide mass fingerprinting are marked in black. The dhbC (labeled in gray) gene product was identified by comparison with a 2D reference map of B. subtilis (10); the dhbF gene product was not detected on our 2D gels. Segments of the dhbA and dhbF genes used as probes in hybridization experiments are indicated. (B) Transcription of the dhbA and dhbF genes in response to increased salinity in the absence or presence of the osmoprotectant glycine betaine (GB).

FIG. 3.

Synthesis pathway of the iron siderophore bacillibactin. Production of bacillibactin starts with chorismate and proceeds through the enzymatic actions of the DhbC, DhbB, and DhbA proteins to DHB. This intermediate has a weak iron siderophore activity (48) and is activated by DhbE-mediated adenylation. A modular peptide synthetase then modifies the resulting 2,3-dihydroxybenzoyladenylate through the addition of glycine and threonine residues and finally esterifies three of these intermediates to form bacillibactin (39).

FIG. 4.

Salt-induced synthesis of DHB. Supernatants of overnight cultures of strain JH642 were analyzed for DHB. The cells were grown either in iron-free medium or in iron-free medium supplemented with 5 μM FeCl₃. The cells were grown in either the absence or presence of iso-osmotic concentrations of NaCl (0.7 M), KCl, sucrose (sucr.), maltose (malt.), or NaCl with 1 mM glycine betaine (NaCl/GB). The data presented are the averages for two independent experiments.

FIG. 5.

Modulation of DHB production by salt and iron. (A) Cells of strain JH642 were grown overnight in the modified MM of Chen et al. (11) containing either 5 μM FeCl₃ (open circles) or 250 μM FeCl₃ (closed circles), and DHB was assayed in the culture supernatant. (B) Cells of strain JH642 were grown in modified MM containing the indicated concentrations of FeCl₃ in the absence (closed circles) or the presence (open circles) of 0.7 M NaCl, and culture supernatants were subsequently assayed for DHB.

FIG. 6.

Growth of salt-stressed B. subtilis cells in the presence or absence of excess iron. Cultures of strain JH642 were grown in modified MM containing the following salt, glycine betaine, and iron concentrations: 5 μM FeCl₃ (○); 5 μM FeCl₃, 1 mM glycine betaine (•); 5 μM FeCl₃ and 0.7 M NaCl (▵); 5 μM FeCl₃, 1 mM glycine betaine, and 0.7 M NaCl (▴); 250 μM FeCl₃ and 0.7 M NaCl (□); and 250 μM FeCl₃, 1 mM glycine betaine, and 0.7 M NaCl (▪).

FIG. 7.

Consensus-directed search for Fur boxes. The B. subtilis genome sequence was searched for the occurrence of Fur boxes (ATAAT) with the program Motif-Finder from Decodon GmbH. Several copies of this pentamer are required for effective recognition by Fur (–18, 24). The search revealed multiple Fur-box-like sequences in front of the translation initiation codons of dhbA, fhuB, fhuD, feuA, yfiY, and yfmC that are indicated in bold uppercase letters in the sequences. The −10 and −35 regions of potential SigA-type promoters are indicated with lines and shading. The genes (dhbACBEF; fhuD) marked by an asterisk have been shown experimentally to be under Fur control (8, 48).

FIG. 8.

Salt-induced induction of iron-controlled B. subtilis genes. Total RNA was isolated from cells of strain JH642 grown in modified MM in the absence (lanes 1) or presence of either 5 μM FeCl₃ (lanes 2 and 3) or 50 μM FeCl₃ (lanes 4). In addition, 0.7 M NaCl was added to the cultures whose RNA was used for the experiments displayed in lanes 3 and 4. The RNA was dot blotted onto a nylon membrane and hybridized to gene-specific antisense RNA probes labeled with digoxigenin. The signal intensity was quantified using a Storm860 fluorimager and the software ImageQuant. For each gene investigated, the highest level of gene expression was set to 100%.

FIG. 9.

DHB production in a B. subtilis fur mutant and a bacillibactin-producing strain. DHB production was assayed in culture supernatants of strain JH642 (fur⁺ sfp⁰), TMB1 (fur::kan sfp⁰), and KE10 (fur⁺ sfp⁺) grown in modified MM in the absence (lanes 1) or presence of either 5 μM FeCl₃ (lanes 2 and 3) or 50 μM FeCl₃ (lanes 4). In addition, 0.7 M NaCl was added to the culture used for the DHB assays displayed in lanes 3 and 4. The bacillibactin producer KE10 has an undefined auxotrophy and fails to grow in defined MM. Therefore, we included 0.02% Casamino Acids in the precultures of all strains.