Compared with the wild-type strain, growth assays showed that the deletion mutant had a decreased growth rate when cultured aerobically, but not affected under anaerobic conditions. Whole-genome expression (RNA and protein) profiles revealed numerous gene and protein expression changes relative to the wild-type control, including some involved in iron metabolism, oxidative damage protection and respiratory electron transfer, e. g. complex IV of the respiration chain. Although total intracellular iron levels remained unchanged, whole-cell electron paramagnetic resonance (EPR) demonstrated that the level of free iron in mutant cells was 3 times less than that of the wild-type strain. Siderophore excretion in the mutant also decreased in iron-depleted medium. The mutant was more sensitive to hydrogen peroxide and gave rise to 100 times more colonies resistant to gentamicin or kanamycin.

When grown under aerobic conditions in MR2A medium, the deletion mutant WG3 had a growth deficiency compared to the parental strain DSP-10 (Figure ​(Figure2).2). Under this condition, the average generation time of WG3 was 2.9 hours while that of DSP-10 was 1.7 hours. WG3 failed to reach the same final optical density as DSP-10. Similar results were obtained in LB under aerobic conditions (data not shown). However, under anaerobic conditions, no difference in growth rates was observed between WG3 and DSP-10 in the defined medium M1 supplemented with lactate (20 mM) as the electron donor and nitrate (10 mM) or fumarate (10 mM) as the electron acceptor (data not shown). As well, the ability to reduce soluble Fe(III) or Co(II) was not affected in WG3 in anaerobic M1 medium using lactate (20 mM) as the electron donor. This infers that the function of SO1377 is not involved in dissimilatory reduction of metals.

To investigate alterations in protein expression as a result of the SO1377 gene mutation, both mutant WG3 and its parental strain DSP-10 cells were subjected to proteomic profile analysis using two-dimensional (2-D) polyacrylamide gel electrophoresis (PAGE) followed by micro liquid chromatography-electrospray ionization tandem mass spectrometry (micro-LC-ESI-MS/MS). Representative 2-D gels of the two strains are presented in Figure ​Figure3.3. Four polypeptides (spot 235, 426, 741 and 748) increased significantly (P < 0.001) in the mutant cells relative to the control. In addition, five polypeptides (spot 202, 422, 482, 553 and 1160) displayed decreased expression and two polypeptides (spot 333 and 433) were not detected at all in WG3 cells (Table ​(Table4).4). Micro-LC-ESI-MS/MS was used to identify proteins showing significant differences in amount on 2-D gels (Table ​(Table4).4). Proteins (spot 333 and 433) completely absent in the deletion mutant were identified as SO1377 (conserved hypothetical protein) and SO2767 (AsnB-1, glutamine-hydrolyzing asparagine synthetase B), respectively. Therefore, similar to microarray analysis, the protein profile verified the deletion of the SO1377 gene. Among other identified proteins, those exhibiting lower gel abundance in the mutant, included SO1637 (bacterial surface antigen), SO1117 (putative cytosol aminopeptidase), SO4749 (AtpA, alpha subunit of ATP synthase F1), and SO3466 (RibH, beta subunit of riboflavin synthase). Note that consistent with microarray data, the lower gel abundance of SO4749, the alpha subunit of ATP synthase F1, suggested inhibition of aerobic respiration. The increased gel abundance of proteins included SO2407 (conserved hypothetical protein) and SO3505 (NagA). Again, the increased gel abundance of SO3505 agreed with the microarray data, in which the transcription of SO3505 gene displayed an increase at the magnitude of 2.72-fold in the mutant cells (Table ​(Table3).3). However, the remaining proteins shown in the proteomics data did not exhibit transcriptional changes. One possible explanation for this inconsistence between microarray and proteomics data is that the expression of these proteins is subjected to post-transcriptional regulation.

E. coli strains were grown at 37°C in LB medium overnight [29]. S. oneidensis strains were grown at 30°C in either LB or modified R2A (MR2A) medium [30]. Antibiotics were used at the following final concentrations: 15-μg/ml gentamicin (Gm), 50-μg/ml kanamycin (Kan) or 10-μg/ml rifamycin (Rif). For anaerobic growth of S. oneidensis MR-1 and its mutants, the defined medium M1 [per liter: 1.19-g (NH₄)₂SO₄, 0.56-g KH₂PO₄, 0.1 ml (0.115 M) Na₂SeO₄ and 10 ml 10X mineral solution) was used as described previously [30]. The electron donor in M1 medium was lactate (20 mM, pH7.0), and electron acceptors were ferric citrate (10 mM), KNO₃ (10 mM) or fumarate (10 mM).

In-frame deletion of the SO1377 gene was generated by the method of Link et al [31] and its schematic presentation is shown in Figure ​Figure1.1. The deletion process was carried out as follows. In the first step (Fig. ​(Fig.1A),1A), PCR primers were used to amplify 5'- and 3'- end fragments of SO1377 gene, respectively. Primers SO1377-No (5'-CGCGAGCTCGCCTTAGCCCTCCTCATCGG-3') and Ni (5'-tgtttaaacttagtggatgggTCCTTTCGTTTACCTACACA-3') were for the 5'-end fragment, and SO1377-Ci (5'-cccatccactaagtttaaacaTTGAGCCAAAGATAAGTAAT-3') and Co (5'-CGCGAGCTCGCTCCATGGCAAATAGCCGC-3') for the 3'-end fragment. The outside primers (No and Co) harbor a SacI restriction site. The inside primers (Ni and Ci) contain complementary 21-nt tags at their respective 5' termini as shown in lower case letters. The 21-nt complementary tags used in this study also contain a PmeI restriction site. Amplification was performed in a 20-μl volume containing 0.2-μl Taq polymerase (Sigma), 2-μl 10X PCR buffer (Promega), 1-μl each primer (20 pmol/μl), 1.2-μl 25 mM MgCl₂, 1-μl genomic DNA of S. oneidensis MR-1 as template (50 ng/μl), 0.5-μl 10 mM dNTP Mix (Clontech) and 13.1-μl ddH₂O. The PCR mixture was denatured at 94°C for 2 minutes, followed by 30 cycles of 94°C for 30 seconds, 58°C for 1 minute and 72°C for 1 minute, followed by 7 minutes at 72°C. The PCR products were then purified from agarose gel using QIAquick Gel Purification Kit (QIAGEN Inc.). In the second step (Fig. ​(Fig.1A),1A), the 5'- and 3'- end fragments were annealed at their overlapping region and amplified by PCR as a single fragment, using the outer primers (SO1377-No and SO1377-Co). The PCR amplification conditions were as described above except that the genomic DNA template was replaced by adding 0.5-μl each of 5'- and 3'- end fragments of SO1377 amplified in the first round PCR. The fusion product was purified from agarose gel using QIAquick Gel Purification Kit (QIAGEN Inc.), resuspended in 50-μl of 1X SacI restriction buffer containing 5 U of SacI restriction enzyme, and digested for two hours at 37°C. The digested fragment was gel purified, ligated into SacI-digested and phosphatase-treated suicide vector pDS3.0, electroporated into E. coli S17-1/λ_pir, spread on LB gentamicin agar plates and incubated at 37°C overnight. The transformants were screened for inserts by PCR with SO1377-No and Co as primers. In the third step, the suicide plasmid construct was integrated into S. oneidensis DSP-10 chromosome by homologous recombination (Fig. ​(Fig.1B).1B). This process was facilitated by conjugal transfer between E. coli S17-1/λ_pir cells harboring the suicide plasmid construct (donor) with strain DSP-10, a spontaneous rifampicin-resistant (Rif^r) derivative of S. oneidensis MR-1 (recipient). E. coli transformants and DSP-10 cells were grown separately in LB medium with proper antibiotics overnight, washed in fresh medium, and mixed in a 1:3 ratio (donor: recipient) by spotting onto 0.2 μm Millipore membrane disks. Following an eight-hour incubation at 30°C, the cells were removed from the filter disks, resuspended in medium, and plated onto LB agar supplemented with Gm (15-μg/ml) and Rif (10-μg/ml). Correct integration of the suicide vector was verified by PCR amplification. PCR confirmation of the inserts was accomplished by comparing the sizes of the products amplified from the wild-type and mutant DNA using primers flanking the insertion sites (Fo and Ro) (Fig ​(Fig1B).1B). The Fo primer sequence is: 5'-TGCAGCCAAAAGCACAGCAC-3'. The Ro primer sequence is: 5'-CGAACTGTTATGCCATAAAT-3'. In the fourth step, to obtain the deletion mutation of SO1377 gene, the integrated suicide plasmid had to be resolved from chromosome through recombination (Fig. ​(Fig.1C).1C). This was accomplished by growing the strain carrying the integrated suicide vector in NaCl-less LB liquid overnight, followed by 10, 100 and 1000 dilutions of the culture, and plating 100 μl of each dilution onto LB agar containing 5% sucrose. The plates were then incubated at 30°C for two days. Screen for deletion was accomplished by PCR amplification using the outside Fo and Ro primers. Finally, the deletion was verified through DNA sequencing. The sequencing reaction was performed with ABI Prism Dye Terminator Sequencing Kit (Applied Biosystems, Foster City, CA) and analyzed by ABI 3700 sequencer.

Microarray fabrication, hybridization, probe labeling, image acquisition and processing were carried out as described previously [11,32,33]. Gene expression analysis was performed using 3 independent microarray experiments, with each slide containing 2 replicate arrays of S. oneidemis MR-1 genome [34]. Total cellular RNA, from the SO1377 gene deletion mutant WG3 and its parental strain DSP-10 were grown to mid-log-phase in the MR2A medium, then isolated and purified using the TRIzol Reagent (Gibco BRL) according to the manufacturer's instruction. The ratios of mutant samples to the wild-type control were normalized by a trimmed geometric mean [11,32,33]. Standard t-test was carried out to determine the significance of gene expression.

Mid-log-phase cells of WG3 and DSP-10, grown in aerobic MR2A medium, were collected and washed three times with 50 nM Tris-HCL (pH = 7.4) and the cell pellets were frozen until used. Frozen cell pellets were mixed with five volumes of a solution containing 9 M urea, 2% (v/v) 2-mercaptoethanol, 2% (v/v) pH 8–10 ampholytes (Biorad), and 4% (v/v) Nonidet P40. The lysed cells were centrifuged for 10 min at 400,000 g in a Beckman TL100 ultracentrifuge to sediment particulates. The supernatants were analyzed by 2-D PAGE. Isoelectric focusing (IEF) gels were cast as described by Anderson and Anderson [35] using a 2:1 mixture of pH 5–7 and pH 3–10 ampholytes (Biorad). Aliquots of samples containing 40 μg of protein were loaded, in triplicate, onto each gel. After IEF, the gels were equilibrated in a buffer containing sodium dodecyl sulfate (SDS) as described by O'Farrell (1975) [36]. The second-dimension slab gels were cast using a linear gradient of 9–17% PAGE. The equilibrated tube gels were secured to the slab gels using agarose and SDS-PAGE as described previously [35]. The proteins were fixed in the gels by soaking in a solution of 20% ethanol (v/v) with 1% formaldehyde (v/v) and detected by silver stain [37]. The 2-DE images were digitized using an Eikonixl412 scanner interfaced with a VAX 4000-90 workstation, and resulting image files were converted to tif format. Data analysis was done using Progenesis software for 2-DE pattern analysis (Nonlinear USA). Statistical analysis of the relative abundance of each matched protein spot across the data set was done using a two-tailed Student t-test as previously described [37]. Proteins showing statistically significant differences in abundance (i.e., P < 0.05) were then identified on the basis of their tryptic peptide masses and amino acid sequences. Proteins to be identified were cut from one to five replicate gels (number of spots required varied with the abundance of individual proteins), reduced at room temperature with tris (2-carboxyethyl) phosphine (Pierce, Rockport, IL), alkylated with iodoacetamide (Sigma), and digested in situ with modified trypsin (Promega Corp., Madison, WI) [38]. The resulting peptides were eluted from the gel with 25 mM ammonium bicarbonate and 5% formic acid in 50% acetonitrile and analyzed by micro liquid chromatography-electrospray ionization tandem mass spectrometry (μ-LC-ESI-MS/MS). For μ-LC-ESI-MS/MS, samples were loaded onto a 365 × 100 μm fused silica capillary (FSC) column packed with 5 μm Zorbax XDB-C18 packing material (Agilent Technologies, Palo Alto, CA) at a length of 7–8 cm. The tryptic peptides were separated with a 30-min linear gradient of 0–60% solvent B (80% acetonitrile/0.02% heptafluorobutyric acid), and then entered a LCQ ion-trap mass spectrometer (Thermo Finnigan, San Jose, CA). Tandem mass spectra were automatically collected under computer control during 30-min LC-MS runs. MS/MS spectra were then directly subjected to SEQUEST [39,40] database searches to identify proteins by correlating experimental MS/MS spectra to protein sequences predicted by the S. oneidensis MR-1 ORE database available in Genbank.

Assay of hydrogen peroxide challenge was carried out as described previously [23]. Cells grown in LB medium to mid-log-phase stage were distributed into 50 ml Erlenmeyer flasks (5 ml each), and hydrogen peroxide was added at concentrations from 0.1 mM to 10 mM. After 20 min of incubation with shaking at 30°C, treatment was stopped by addition of catalase (Roche Molecular Biochemicals) at 400 U/ml and chilling. Samples were diluted in cold 10^-2 MgSO₄ containing 400 U/ml catalase, followed by plating on LB plates for viable count. Colonies were counted after two days of incubation at 30°C.

Intracellular iron concentration was determined as described previously with some modifications [42]. Inoculated cultures were grown in 20 ml of liquid LB, either with no iron supplement or with an iron supplement of 0.05, 0.1, 0.5, 1, 5 and 10 mM ferric citrate, at 30°C for 12 hours with shaking (120 rpm). The cells were washed four times with fresh cold LB medium (twice with 10 ml and twice with 1 ml), and their OD₆₀₀ was determined. The cell pellet was then dispensed in 10 ml 1 N HNO₃ and incubated in a waterbath at 95°C for 30 min. After spinning down the cellular debris, at 4000 rpm for 10 min, the supernatants were recovered for determination of iron concentration using a Perkin Elmer Optima 3100XL instrument. Iron concentrations were expressed as ppm/10⁹ cells.

CAS (chrome azurol S) blue agar with iron depletion was used to determine the siderophore secretion of the wild-type and deletion mutant strains [43]. Bacterial cultures grown in LB medium, to mid-log-phase, were collected and washed twice with 0.7% NaCl solution and resuspended in 0.7% NaCl solution. A 5 μl suspension of each strain was spotted on the CAS agar, followed by incubation overnight at 30°C. The excretion of siderophore was indicated by the formation of a yellowish halo area surrounding the colony.