High-level chromate resistance in Arthrobacter sp. strain FB24 requires previously uncharacterized accessory genes

<p>Abstract</p> <p>Background</p> <p>The genome of <it>Arthrobacter </it>sp. strain FB24 contains a chromate resistance determinant (CRD), consisting of a cluster of 8 genes located on a 10.6 kb fragment of a 96 kb plasmid. The CRD includes <it>chrA</it>, which encodes a putative chromate efflux protein, and three genes with amino acid similarities to the amino and carboxy termini of ChrB, a putative regulatory protein. There are also three novel genes that have not been previously associated with chromate resistance in other bacteria; they encode an oxidoreductase (most similar to malate:quinone oxidoreductase), a functionally unknown protein with a WD40 repeat domain and a lipoprotein. To delineate the contribution of the CRD genes to the FB24 chromate [Cr(VI)] response, we evaluated the growth of mutant strains bearing regions of the CRD and transcript expression levels in response to Cr(VI) challenge.</p> <p>Results</p> <p>A chromate-sensitive mutant (strain D11) was generated by curing FB24 of its 96-kb plasmid. Elemental analysis indicated that chromate-exposed cells of strain D11 accumulated three times more chromium than strain FB24. Introduction of the CRD into strain D11 conferred chromate resistance comparable to wild-type levels, whereas deletion of specific regions of the CRD led to decreased resistance. Using real-time reverse transcriptase PCR, we show that expression of each gene within the CRD is specifically induced in response to chromate but not by lead, hydrogen peroxide or arsenate. Higher levels of <it>chrA </it>expression were achieved when the <it>chrB </it>orthologs and the WD40 repeat domain genes were present, suggesting their possible regulatory roles.</p> <p>Conclusion</p> <p>Our findings indicate that chromate resistance in <it>Arthrobacter </it>sp. strain FB24 is due to chromate efflux through the ChrA transport protein. More importantly, new genes have been identified as having significant roles in chromate resistance. Collectively, the functional predictions of these additional genes suggest the involvement of a signal transduction system in the regulation of chromate efflux and warrants further study.</p

SO2426 is a positive regulator of siderophore expression in \u3cem\u3eShewanella oneidensis\u3c/em\u3e MR-1

Background The Shewanella oneidensis MR-1 genome encodes a predicted orphan DNA-binding response regulator, SO2426. Previous studies with a SO2426-deficient MR-1 strain suggested a putative functional role for SO2426 in the regulation of iron acquisition genes, in particular, the siderophore (hydroxamate) biosynthesis operon so3030-3031-3032. To further investigate the functional role of SO2426 in iron homeostasis, we employed computational strategies to identify putative gene targets of SO2426 regulation and biochemical approaches to validate the participation of SO2426 in the control of siderophore biosynthesis in S. oneidensis MR-1. Results In silico prediction analyses revealed a single 14-bp consensus motif consisting of two tandem conserved pentamers (5\u27-CAAAA-3\u27) in the upstream regulatory regions of 46 genes, which were shown previously to be significantly down-regulated in a so2426 deletion mutant. These genes included so3030 and so3032, members of an annotated siderophore biosynthetic operon in MR-1. Electrophoretic mobility shift assays demonstrated that the SO2426 protein binds to its motif in the operator region of so3030. A short form of SO2426, beginning with a methionine at position 11 (M11) of the originally annotated coding sequence for SO2426, was also functional in binding to its consensus motif, confirming previous 5\u27 RACE results that suggested that amino acid M11 is the actual translation start codon for SO2426. Alignment of SO2426 orthologs from all sequenced Shewanella spp. showed a high degree of sequence conservation beginning at M11, in addition to conservation of a putative aspartyl phosphorylation residue and the helix-turn-helix (HTH) DNA-binding domain. Finally, the so2426 deletion mutant was unable to synthesize siderophores at wild-type rates upon exposure to the iron chelator 2,2\u27-dipyridyl. Conclusions Collectively, these data support the functional characterization of SO2426 as a positive regulator of siderophore-mediated iron acquisition and provide the first insight into a coordinate program of multiple regulatory schemes controlling iron homeostasis in S. oneidensis MR-1

Effect of Cr-AuNPs on the growth of <i>Shewanella oneidensis</i> MR-1.

<p>Either 3.5 nm (closed symbols) or 13 nm (open symbols) Cr-AuNPs were added to wells at volumes of 0, 5, 10, and 50 µl. Error bars represent the standard error from three independent cultures.</p

Thin-section TEM Images of <i>S. oneidensis</i> MR-1.

<p>A. without particles, B. plain 13 nm gold Nanoparticles, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g004" target="_blank">Fig. 4C–4D</a>. Chromate coated gold nanoparticles, Cr-AuNp:13 nm, (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g004" target="_blank">Fig. 4E–4F</a>) 3.5 nm Cr-AuNp. Red arrows indicate extracellularly bound Cr-AuNp and green arrows/circle indicate internalized particles. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g004" target="_blank">Fig. 4G–4H</a>) show 3.5 nm and 13 nm probes used in Cr-AuNp preparation.</p

Confocal Raman Mapping.

<p>Raman Intensity Maps averaged over a wide wavenumber region (162–1953 cm⁻¹) covering most of bio-molecular components in cells to obtain a Raman chemical image of the cell (A), Phonon Plasmon peak (207–297 cm⁻¹) originating from gold depicting the presence of Cr-AuNps (B), Cr(VI) - hexavalent chromium (C, 837–873 cm⁻¹), reduced non-toxic trivalent Cr(III) (D, 531–567 cm⁻¹). Raman images in grid format, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g006" target="_blank">Fig. 6E and 6F</a> are representations of 6B and 6C respectively. 6-E* and F* represent magnified pixel plots to demonstrate the overlap in signal of Au and Cr(VI) peaks within cells.</p

Inductively Coupled Mass-Spectrometry.

<p>A. ICP-MS Calibration curve for Cr quantification. B. Intracellularly trapped Cr(VI) and Cr(III) at time t = 0 and t = 12 h after Cr-AuNp treatment.</p

Effect of Cr-AuNPs on chromate reduction by <i>S. oneidensis</i> MR-1.

<p>Abiotic controls were included to rule out the reduction of chromate by media components and the Cr-AuNPs (open symbols). There was no adverse effect on chromate reduction ability induced by the nanoparticles (closed symbols). In addition, the nanoparticles did not directly reduce the chromate in the medium in the absence of cells (open symbols). Error bars represent the standard error from three independent cultures.</p

Confocal Fluorescence Lifetime Imaging.

<p><i>S. oneidensis</i> MR-1 incubated with 3.5 nm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g007" target="_blank">Fig. 7A & 7C</a>) and 13 nm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016634#pone-0016634-g007" target="_blank">Fig. 7B & 7D</a>) Cr-AuNp probes show scattered low-lifetime (blue) distribution indicating the presence of gold nanoparticles (both internalized and externally bound) compared to the control incubated with plain gold nanoparticles (inset - 7A).</p