Enterobacter sp. YSU was grown in four M-9 minimal medium cultures. During early log phase after 1.5 hours of growth, selenite or an equal volume of water was added to give the following growth conditions: no L-cysteine and no selenite (NCNS), no L-cysteine and selenite (NCS), L-cysteine and no selenite (CNS), and L-cysteine and selenite (CS). Every 45 minutes, culture samples were diluted and plated to follow viable cell count versus time (Fig 1). Each experiment was performed four times, and each plotted point was the average of cell counts from at least three of the four experiments. Both conditions without selenite (NCNS and CNS) demonstrated normal growth curves. The bacteria in the culture lacking L-cysteine and containing selenite (NCS) were killed by the selenite, and 3.5 hours after adding selenite, the number of viable cells decreased on average by 98%. The culture containing both L-cysteine and selenite (CS), on the other hand, demonstrated a normal growth curve, and 3.5 hours after adding selenite, the number of viable cells increased on average by 890%. These results clearly showed that without L-cysteine, 40 mM selenite is toxic to Enterobacter sp. YSU.

Two stationary phase cultures of Enterobacter sp. YSU grown in M-9 minimal medium containing and lacking 40 μg/ml L-cysteine were diluted 1:20 into fresh medium containing and lacking L-cysteine. Growth was followed by measuring turbidity every 30 minutes. After 2.5 hours of growth, the NCNS and CNS samples were taken for protein analysis. The cultures were split into equal volumes, and selenite or water was added to give the four conditions. One hour after selenite was added, the NCS and CS samples were taken for protein analysis.

These growth curves also showed that L-cysteine played a role in selenite resistance (Fig 2). The two positive control cultures, NCNS and CNS, demonstrated a typical growth curve and increased on average to final cell densities of 160 ± 19 and 138 ± 38 Klett units, respectively. The cultures that lacked L-cysteine and contained selenite, NCS, increased in cell density on average by only12 Klett units to 46 ± 13 Klett units 3.5 hours after selenite was added. However, the cultures that contained L-cysteine and selenite (CS) were able to grow. This culture increased in cell density to 193 ± 81 Klett units 3.5 hours after selenite was added.

Protein samples from all 4 culture conditions were analyzed by 2DGE between the pI ranges of 4 and 7 (Fig 3). Differences in protein expression were detected by comparing the size and intensity of the same spot on each gel. Spots that appeared with equal intensities or higher intensities compared to other spots in the same location on other gels were excised, digested with trypsin and analyzed by mass spectrometry (Nano-LC/MS/MS analysis). Analysis of the peptides from each spot using the Mascot software package 33 identified significant matches to known proteins. For a match to be considered significant, it contained at least two different peptide fragments matching part of an entire known sequence.

Two landmark spots, which are identified by blue arrows (Fig 3), appeared at equal intensities in all four gels and were selected to demonstrate accuracy. Spot 1 from all four gels contained peptides that matched to an OmpF porin from Enterobacter cloacae with Mascot scores ranging from 496-520 and sequence coverages ranging from 22-23% (Table 1). Spot 2 from all four gels contained peptides that matched to a protein chain elongation factor EF-Ts from Salmonella typhimurium (S. typhimurium) with Mascot scores ranging from 654-1090 and sequence coverages ranging from 44-60%.

Spot 3, which is identified by a purple arrow (Fig 3), appeared at a higher intensity in the gels of samples prepared from cells grown in the absence of L-cysteine (NCNS and NCS). This spot contained peptides that matched to a conserved hypothetical lipobinding protein from Erwinia pyrifoliae with a Mascot score of 153 and a sequence coverage of 7% (Table 1). Spot 3 from the NCS gel also contained peptides that matched to two other proteins: a branched-chain amino-acid aminotransferase from Yersinia pestis with a Mascot score of 277 and a sequence coverage of 25% and an outer membrane protein II from Enterobacter aerogenes (E. aerogenes) with a Mascot score of 104 and a sequence coverage of 9%.

Three spots, which are identified by dark red arrows (Fig 3), appeared at higher intensities on gels that contained samples prepared from cells grown in the absence of selenite (NCNS and CNS). Spot 4 from the NCNS and CNS gels was unique. This spot contained peptides that matched to an EF-Tu protein from S. typhimurium with Mascot scores of 1143 and 1467 and sequence coverages of 64% and 52%, respectively (Table 1). In addition, spots 5 and 6 were more intense in gels containing samples prepared from cells grown in the absence of selenite (NCNS and CNS). They contained peptides that matched to EF-Tu from S. typhimurium with Mascot scores of 611 and 666 and sequence coverages of 25% and 30%, respectively. Spot 6 also contained peptides that matched to 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase (MetE) from Erwinia carotovora with a Mascot score of 173 and a sequence coverage of 10%.

Five spots, which are identified by red arrows (Fig 3), appeared at higher intensities in gels of samples prepared from cells grown in the presence of selenite (NCS and CS). Spot 7 was the largest in the CS gels. In the no L-cysteine, selenite gel (NCS), it contained peptides that matched to 4 proteins (Table 1): an outer membrane protein II from E. aerogenes with a Mascot score of 921 and a sequence coverage of 86%, OmpA from E. sakazakii with a Mascot score of 700 and a sequence coverage of 40%, a putative membrane component hydrogenase from S. typhimurium with a Mascot score of 561 and a sequence coverage of 25% and a CysK protein from E. coli with a Mascot score of 245 and a sequence coverage of 16%. In the CS gels, it contained a similar set of peptides for 3 proteins: an outer membrane protein II from E. aerogenes with a Mascot score of 960 and a sequence coverage of 73%, OmpA from E. sakazakii with a Mascot score of 696 and a sequence coverage of 38% and a putative membrane component hydrogenase from S. typhimurium with a Mascot score of 549 and a sequence coverage of 25%. It lacked peptides that matched to CysK.

Under selenite sensitive condition (NCS), spots 8 and 9 were unique (Fig 3). They did not appear in gels of samples prepared from cells grown under the other three conditions. Spot 8 contained peptides that matched to 4 proteins (Table 1): a putative tellurium resistance protein C from E. coli 34 with a Mascot score of 516 and a sequence coverage of 52%, a protein similar to GroES from Klebsiella pneumoniae with a Mascot score of 184 and a sequence coverage of 46%, an outer membrane protein II from E. aerogenes with a Mascot score of 191 and a sequence coverage of 18% and OmpA from E. sakazakii with a Mascot score of 165 and a sequence coverage of 8%. Spot 9 contained peptides that matched to a small heat shock protein from S. typhimurium with a Mascot score of 299 and a sequence coverage of 32%.

Spots 10 and 11 were unique on the gels containing protein samples prepared from cells treated with selenite (NCS and CS). Spot 10 contained peptides that matched to 4 proteins: an outer membrane protein II from E. aerogenes with a Mascot score of 917 and a sequence coverage of 73%, OmpA from E. sakazakii with a Mascot score of 633 and a sequence coverage of 35%, a putative membrane component hydrogenase from S. typhimurium with a Mascot score of 501 and a sequence coverage of 22% and GroEL from Pantoea agglomerans with a Mascot score of 333 and a sequence coverage of 15%. Spot 11 contained peptides that matched to 3 proteins: outer membrane protein II from E. aerogenes with a Mascot score of 641 and a sequence coverage of 52%, OmpA from E. sakazakii with a Mascot score of 434 and a sequence coverage of 37% and a putative membrane component hydrogenase from S. typhimurium with a Mascot score of 362 and a sequence coverage of 20%. It lacked peptides for GroEL.

The absence of CysK in protein samples of cells grown in the presence of L-cysteine and selenite (CS) suggested that the addition of L-cysteine down-regulated the expression of the L-cysteine synthesis and the sulfate permease genes. To verify this hypothesis, Enterobacter sp. YSU was grown in M-9 minimal medium in the presence and absence of L-cysteine. The untreated samples (NCNS and CNS) were harvested immediately before selenite was added, and the selenite treated samples (NCS and CS) were harvested one hour after selenite was added. Equal amounts of purified RNA were converted to cDNA. Then, PCR reactions using equal amounts of cDNA and primers specific for cysK, cysE, cysT, cysA and cysW were analyzed on a 1% agarose gel (Fig 4). All PCR products were between 730 and 930 bp in length, and each experiment was repeated 3 times. As expected for E. coli 35, the cysE transcript, which served as an internal control, was expressed equally under all 4 conditions whether L-cysteine was present or absent (Lanes 1-4). The cysK transcript was sometimes but not always expressed at higher levels in the absence of L-cysteine (Lanes 1-2) than in the presence of L-cysteine (Lanes 3-4). The transcripts for cysA and cysT were consistently expressed at higher levels in the absence of L-cysteine (Lanes 1-2) than in its presence (Lanes 3-4). Finally, the transcripts for cysW were not always detectable but when they were visible, they were only observed in samples from cells grown in the absence of L-cysteine (Lanes 1-2) and not in samples from cells grown in the presence of L-cysteine (Lanes 3-4). Thus, it appeared that the addition of L-cysteine repressed the expression of the sulfate permease transcripts, cysA, cysT and cysW.

M-9 minimal medium 40 was described previously, and 5X M-9 Salts were obtained from Becton, Dickinson and Company (Sparks, MD). When required, M-9 medium was supplemented with 0.04 mg/ml L-cysteine (Fisher Scientific, Fair Lawn, NJ) and 40 mM sodium selenite (MP Biomedicals, Aurora, OH). Luria-Bertani (LB) medium 40 and agar were obtained from Fisher Scientific. Enterobacter sp. YSU was described previously 32.

Four Enterobacter sp. YSU cultures, two containing L-cysteine and two lacking L-cysteine, were grown in M-9 minimal overnight at 37°C and diluted 1:20 into corresponding fresh M-9 minimal medium containing or lacking L-cysteine. These four new cultures were grown at 37°C with shaking. After 1.5 hours of growth, selenite or an equal volume of water was added to give the following culture conditions: no L-cysteine and no selenite (NCNS), no L-cysteine and selenite (NCS), L-cysteine and no selenite (CNS), and L-cysteine and selenite (CS). Samples from each culture were removed every 45 minutes, serially diluted and plated on LB-agar medium in triplicate. Plates were incubated overnight at 37°C, and colony forming units (CFUs) were counted.

Two Enterobacter sp. YSU cultures, one containing L-cysteine and the other lacking L-cysteine, were grown in M-9 minimal overnight at 37°C and diluted 1:20 into corresponding fresh M-9 minimal medium containing or lacking L-cysteine. These new cultures were grown at 37°C with shaking, and turbidity was measured every 0.5 hour using a Klett Colorimeter with a KS-54 filter. After 2.5 hours of growth, the two cultures were divided into equal volumes. Sodium selenite or an equal volume of water was added to give the four NCNS, NCS, CNS and SC growth conditions. Immediately before and one hour after the addition of sodium selenite, samples were harvested by centrifugation at 5,000 × g and 4°C for 10 minutes, and cell pellets were stored at -80°C.

Cells were thawed and resuspended in lysing buffer containing 8 M urea, 2 M thiourea, 2% (w/v) 3-[(3-cholamidopropyl)dimethy-ammonio]-1-propanesulfonate (CHAPS), 2% (w/v) SB 3-10, 40 mM tris(hydroxymethyl)aminomethane (Tris) (Amresco, Solon, OH), 0.2% (v/v) Bio-Lyte^® 3/10 ampholyte (Bio Rad), 1% (v/v) tributylphosphine (TBP) (Bio Rad) and 1% (v/v) Halt Protease Inhibitor Cocktail (Pierce, Rockford, IL). Cells were lysed using a MiniBeadbeater-8 (BioSpec, Bartlesville, OK) and mixed to final concentrations of 0.2 μg/ml RNase (Amresco) and 200 U/ml DNase I (Pierce). After centrifuging the lysate for 10 minutes at 16,000 × g, the supernatant was treated with Bio-Rad's 2D Clean Up Kit™ (Bio-Rad) and the final pellet was resuspended in rehydration buffer containing 8 M urea, 1% (w/v) CHAPS, 15 mM dithiothreitol (DTT), trace Bromophenol Blue (Amresco), and 0.2% (w/v) Bio-Lyte^® 3/10 ampholyte (Amresco). A modified Bradford assay 4142 was used to determine protein concentrations before 2DGE analysis.

Isoelectric focusing 43 was carried out using a Bio-Rad Protean IEF Cell (Bio-Rad). A total of 150 μg of protein was separated using an 11 cm IPG Ready Strip (Bio-Rad) with a fixed pH range of 4-7. After active rehydration at 50 V and 20°C for 12 hours, the sample was focused, starting at 0 V and ending at 8,000 V for a total of 40,000 volt hours.

After focusing, the IPG Ready Strip was washed with Equilibration Buffer I containing 6 M urea, 2% (w/v) SDS, 0.375 M Tris-HCl pH 8.8, 20% (v/v) glycerol and 130 mM DTT for 10 minutes and with Equilibration Buffer II containing 6 M urea, 2% (w/v) SDS, 0.375 M Tris-HCl pH 8.8, 20% (v/v) glycerol, and 135 mM iodoacetamide for an additional 10 minutes. The IPG strip was then submerged in 1× tris glycine SDS (TGS) buffer containing 0.025 M tris base, 0.192 M glycine and 0.1% (w/v) sodium dodecyl sulfate (SDS) (Amresco) before being placed in a 10.5%-14% Criterion precast gel (Bio-Rad) for protein size separation at 100 V.

After size separation, gels were stained with SYPRO^® Ruby Protein Stain (Bio-Rad), imaged using the Bio-Rad Gel Chemidoc™ XRS Gel Documentation System and analyzed for matchsets using the Bio-Rad PD Quest 2-D Image Analysis Software 42. The matchsets were used to select and excise protein spots which were sent to The Ohio State University Proteomics Facility to be digested with trypsin and analyzed by capillary-liquid chromatography-nanospray tandem mass spectrometry (Nano-LC/MS/MS). The sequences of the peptide fragments were analyzed by the Mascot software package (Matrix Science Inc., Boston, MA) to determine the potential identity of the proteins in each excised spot 32.

Cells were grown and harvested as in Fig 2. RNA was extracted using Bio-Rad's Aurum™ Total RNA Mini Kit, and cDNA from 0.5 μg of purified RNA was synthesized using Bio-Rad's iScript™ Select cDNA Synthesis Kit. PCR reactions containing 1.5 μl of cDNA, GoTaq^® Green Master Mix (Promega, Madison, WI) and primers for cysK (5'-CTCGCTGACTATCGGTCA-3' and 5'-GATACCCGCAAGAATACC-3'), cysA (5'-ATGAGCATTGAGATTGCC-3' and 5'-ACGACTAATTGGGTGTAG-3'), cysT (5'-GCTGTTTGTGTGCCTGAT-3' and 5'-CGACTTTGCAGAGTGTTA-3'), cysW (5'-CGTGCAGGCGTTCAGCAA-3' and 5'-CCTGTTGCGCGCGTTTTT-3') or cysE (5'-ATGCCGTGTGAAGAACTG-3' and 5'-CTCGAAGGTATGGTGAAT-3') were performed for 30 cycles of 95°C for 1 minute, 50°C for 1 minute and 72°C for 1 minute. Equal volumes of the resulting PCR reactions were analyzed using a 1% agarose (Amresco) gel.