Analysis of a ferric uptake regulator (Fur) mutant of Desulfovibrio vulgaris Hildenborough

Abstract

Previous experiments examining the transcriptional profile of the anaerobe Desulfovibrio vulgaris demonstrated up-regulation of the Fur regulon in response to various environmental stressors. To test the involvement of Fur in the growth response and transcriptional regulation of D. vulgaris, a targeted mutagenesis procedure was used for deleting the fur gene. Growth of the resulting Deltafur mutant (JW707) was not affected by iron availability, but the mutant did exhibit increased sensitivity to nitrite and osmotic stresses compared to the wild type. Transcriptional profiling of JW707 indicated that iron-bound Fur acts as a traditional repressor for ferrous iron uptake genes (feoAB) and other genes containing a predicted Fur binding site within their promoter. Despite the apparent lack of siderophore biosynthesis genes within the D. vulgaris genome, a large 12-gene operon encoding orthologs to TonB and TolQR also appeared to be repressed by iron-bound Fur. While other genes predicted to be involved in iron homeostasis were unaffected by the presence or absence of Fur, alternative expression patterns that could be interpreted as repression or activation by iron-free Fur were observed. Both the physiological and transcriptional data implicate a global regulatory role for Fur in the sulfate-reducing bacterium D. vulgaris.

FIG. 1.

Map of pMO707 containing the fur deletion construct. A 2,706-bp PCR product containing the pSC27 Km^r determinant flanked by DNA sequences upstream (768 bp) and downstream (831 bp) of the D. vulgaris fur gene was inserted into the EcoRV site of the pBluescript SK(+) multicloning site. Both pBluescript and pMO707 are unstable in D. vulgaris. Numbers indicate positions of sequences upstream and downstream of the fur gene; BC indicates molecular barcodes allowing mutant tracking in a mixed population.

FIG. 2.

(A) PCR analysis of Km^r D. vulgaris transformants using primers targeting regions outside of the fur knockout cassette (P7/P8). Lane 1, wild type; lane 2, transformant A; lane 3, transformant B; lane 4, pMO707; lane 5, no DNA; lane M, 1-kb marker. An increase in product size from 2,168 bp to 2,839 bp indicates exchange of the fur gene for the Km^r determinant, creating JW707. (B) Northern analyses of total RNA (10 μg) from JW707. Lane 1, wild type; lane 2, JW707; lane M, RNA marker. Probes used for hybridization are indicated above the blot. nt, nucleotides.

FIG. 3.

Growth curves of the D. vulgaris wild type (open symbols) and mutant strain JW707 (filled symbols) under various conditions. (A) Growth under iron-replete conditions (30 μM FeCl₂). (B) Response to osmolarity stress, with sodium as 300 mM NaCl (circles) or 300 mM NaCl plus 2 mM glycine betaine (squares). (C) Response to osmolarity stress, with potassium as 300 mM KCl (circles) or 300 mM KCl plus 2 mM glycine betaine (squares). (D) Response to nitrite at 2 mM NaNO₂ (circles) and 5 mM NaNO₂ (squares). Curves are representative of three trials at 37°C.

FIG. 4.

Growth response of the D. vulgaris wild type (open symbols) and mutant strain JW707 (filled symbols) to MnCl₂ treatment. Curves are representative of three trials at 37°C.

FIG. 5.

(A) Genes differentially expressed threefold in the Δfur mutant, JW707, compared to wild-type cells, under iron-replete (+Fe; 60 μM) and iron-limited (−Fe; 5 μM) conditions. (B) Overlap of genes differentially expressed threefold in JW707 iron-limited (−Fe; 5 μM) conditions compared with JW707 iron-replete conditions versus those differentially expressed in iron-limited wild-type cells compared to iron-replete wild-type cells.