Proteomic profiling of L-cysteine induced selenite resistance in Enterobacter sp. YSU

Background Enterobacter sp. YSU is resistant to several different heavy metal salts, including selenite. A previous study using M-9 minimal medium showed that when the selenite concentration was 100,000 times higher than the sulfate concentration, selenite entered Escherichia coli cells using two pathways: a specific and a non-specific pathway. In the specific pathway, selenite entered the cells through a yet to be characterized channel dedicated for selenite. In the non-specific pathway, selenite entered the cells through a sulfate permease channel. Addition of L-cystine, an L-cysteine dimer, appeared to indirectly decrease selenite import into the cell through the non-specific pathway. However, it did not affect the level of selenite transport into the cell through the specific pathway. Results Growth curves using M-9 minimal medium containing 40 mM selenite and 1 mM sulfate showed that Enterobacter sp. YSU grew when L-cysteine was present but died when it was absent. Differential protein expression analysis by two dimensional gel electrophoresis showed that CysK was present in cultures containing selenite and lacking L-cysteine but absent in cultures containing both selenite and L-cysteine. Additional RT-PCR studies demonstrated that transcripts for the sulfate permease genes, cysA, cysT and cysW, were down-regulated in the presence of L-cysteine. Conclusion L-cysteine appeared to confer selenite resistance upon Enterobacter sp. YSU by decreasing the level of selenite transport into the cell through the non-specific pathway.


Introduction
Selenium is an important cofactor in some mammalian and bacterial enzymes [1][2][3][4][5]. It is found in mammalian glutathione peroxidase [6] and bacterial formate dehydrogenase [7] in the form of selenocysteine. A series of Escherichia coli (E. coli) proteins, SelA, SelB, SelC and SelD, incorporate selenium into selenocysteine which is then inserted into proteins. The mechanisms that transport selenite into the cell are not well understood. Once it enters the cell, it may be reduced to selenide by glutathione [8] or thioredoxin [9]. Then, SelD, a selenophosphate synthetase, catalyzes a reaction with selenide and ATP to make selenophosphate [10,11]. SelA, a selenocysteine synthetase, uses selenophosphate to convert ser-ine to selenocysteine [12], which is carried by SelC, a special tRNA [13]. Finally, SelB, a translation factor, inserts selenocysteine at a UGA stop codon. The mRNAs of proteins that encode selenocysteine also contain a special stem loop structure or selenocysteine insertion sequence (SECIS) which stalls the ribosome and allows SelB to insert selenocysteine at the UGA stop codon [14,15].
Although selenium is an important cofactor, too much can be toxic. Selenium inhibits the growth of E. coli when it is present at overabundant concentrations as selenite, but non-toxic when it is present as elemental selenium [16]. Selenite reacts with glutathione and other thiol containing proteins to produce highly reactive superoxides [16][17][18][19], which may kill the cells by damaging their DNA and lipids [18,20].
Selenite and selenate resistant bacteria appear to remove excess selenium by reducing it to elemental selenium or methylating it [5]. A strain of Stenotrophomonas maltophilia (S. maltophilia) isolated from a selenium-contaminated drainage pond reduces selenate and selenite to elemental selenium and deposits it near the cell surface and in the surrounding growth medium [21]. In addition, Cupriavidus metallidurans (C. metallidurans) CH34, a multi-metal resistant strain [22] from a zinc contaminated site, is resistant to selenate and selenite and reduces both oxyanions to elemental selenium and alkyl selenide under aerobic conditions [23,24]. A C. metallidurans CH34 protein, DedA, which was identified by transposon mutagenesis, may be involved in selenite uptake [25]. A set of E. coli proteins, YgfK, YgfM and YgfN, which were also identified by transposon mutagenesis, act as an enzyme that reduces selenate to elemental selenium [26]. Finally, some bacterial strains also methylate selenite and selenate by encoding a thiopurine methyltransferase, which produces volatile dimethyl selenide and dimethyl diselenide [27,28].
Understanding how selenite enters the cell is important to understanding the mechanisms of selenite resistance. An early study in E. coli shows that selenite and sulfate follow a Michaelis-Menten model when they are transported into the cell and compete competitively with each other [29]. Using a low sulfate, M-9 minimal medium containing 10 μM sulfate and 75 Se-labeled selenite mixed with 1 M unlabeled selenite, Muller et al define two pathways for selenium incorporation in E. coli: (1) specific and (2) nonspecific [30,31]. In the specific pathway, selenite enters the cell through a pathway dedicated for selenite and becomes incorporated into selenocysteine intentionally. In the non-specific pathway, selenite enters the cell through the sulfate transport system and becomes incorporated unintentionally to produce selenocysteine instead of L-cysteine. When 10 μg/ml of L-cystine, an Lcysteine dimer, is added to the medium, the wild type E. coli strain incorporates selenium randomly into cellular proteins, presumably via the non-specific pathway. However, when 60 μg/ml L-cystine is added to the medium, the amount of randomly incorporated selenium in wild type E. coli decreases dramatically. These results suggest that feedback inhibition blocks both the biosynthesis pathway for L-cysteine and the accidental incorporation of selenium into proteins. In support of this hypothesis, cysK mutants, which lack the ability to add sulfide to Oacetylserine to generate L-cysteine, fail to randomly incorporate radioactive selenite into protein through the nonspecific pathway [30,31]. Thus, under most conditions when selenite concentrations are lower than sulfate concentrations, selenite is excluded from the non-specific pathway. However, when selenite concentrations are higher than sulfate concentrations, selenite is transported into the cell through the non-specific pathway instead of sulfate.
Enterobacter sp. YSU is a multi-metal resistant strain that grows in the presence of mercury, cadmium, zinc, gold, silver, arsenic and selenium [32]. It does not grow in M-9 minimal medium containing 1 mM sulfate and 40 mM selenite. However, growth inhibition is relieved by the addition of L-cysteine. Here, we define L-cysteine-dependent selenite resistance in Enterobacter sp. YSU using growth curves, 2-D gel electrophoresis and reverse-transcriptasepolymerase chain reactions (RT-PCR).

Requirement of L-cysteine for selenite resistance
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), Lcysteine 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 Lcysteine 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.

Growth curves used for proteomic analysis
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.

Two-dimensional gel electrophoresis (2DGE)
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.

Proteins in spots that appeared at equivalent intensities in gels of samples prepared from cells grown under all four conditions
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 Viable cell count growth curves Figure 1 Viable cell count growth curves. Overnight cultures were diluted 1:20 in fresh M-9 minimal medium and grown at 37°C. After 1.5 hours of growth, selenite or water was added to each culture. Samples were diluted and spread on plates containing LB medium every 45 minutes. Values at each time point are the average of at least 3 different experiments, and error was calculated using the student t test at a 95% confidence level. Figure 2 Turbidity growth curves. Overnight cultures were diluted 1:20 in fresh M-9 minimal medium and grown at 37°C. After 2.5 hours of growth, selenite or water was added to each culture. Turbidity was measured every 30 minutes using a Klett colorimeter. Values at each time point are the average of 6 different experiments, and error was calculated using the student t test at a 95% confidence level.

Turbidity growth curves
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%.

Proteins in a spot that appeared at a higher intensity in gels of samples prepared from cells grown in the absence of L-cysteine
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 pro-tein II from Enterobacter aerogenes (E. aerogenes) with a Mascot score of 104 and a sequence coverage of 9%.

Proteins in spots that appeared at higher intensities in gels of samples from cells grown in the absence of selenite
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%.

Proteins in spots that appeared at higher intensities in gels of samples prepared from cells grown in the presence of selenite
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 Negative images of Enterobacter sp. YSU total protein sepa-rated by 2DGE over a pI range of 4-7 and with L-Cysteine and Selenite (CS) were harvested after 3.5 hours of growth. Spots identified with blue arrows appeared with equal intensities under all 4 conditions. The spot identified by a purple arrow appeared at a higher intensity in gels containing samples from cells grown in the absence of L-cysteine. Spots identified by dark red arrows appeared at higher intensities in gels containing samples from cells grown in the absence of selenite. Spots identified by red arrows appeared at higher intensities in gels containing samples from cells grown in the presence of selenite. The spot numbers correspond to the spot numbers in Table 1.

Detection of cysK, cysE and sulfate permease transcripts in cells grown in the presence and absence of L-cysteine
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 Lcysteine (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 Lcysteine (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.

Discussion
A previous proteomic study on selenite tolerant E. coli cells used M-9 minimal medium that lacked L-cysteine [17]. When two derivative K12 strains, MC4100 and GC4468, growing at logarithmic phase were pretreated with 0.25 mM selenite for 60 minutes and then exposed to 25 mM selenite, they were able to tolerate the increased selenite concentrations and were even able to grow for one hour. After this point, the cells entered into a stationary phase and began to die. This increased tolerance was attributed to higher expression levels of superoxide dismutase in response to selenite induced oxidative stress. The current work carried these E. coli studies one step further by demonstrating that Enterobacter sp. YSU was also sensitive to selenite in M-9 minimal medium, but the selenite inhibition was relieved by supplementing the medium with L-cysteine (Figs 1 and 2).
The role of L-cysteine in the observed selenite resistance/ sensitivity phenotypes was investigated using proteomics. Interestingly, spot 7 from cells grown under the selenite sensitive conditions (NCS) contained peptides for CysK (Fig 3 and Table 1), whereas the same spot from cells grown under selenite resistant conditions (CS) did not. This result supported the hypothesis that L-cysteine conferred selenite resistance to Enterobacter sp. YSU by preventing selenite uptake through the non-specific pathway or sulfate permease system. The presence of L-cysteine probably caused feedback inhibition of CysE, which converted serine to O-acetylserine [30,35]. Then, lower levels of the O-acetylserine, the derivative of N-acetylserine, which acted as an inducer for the L-cysteine synthesis genes, resulted in decreased expression of cysK and the sulfate transport genes. Although the RT-PCR experiments did not conclusively show that the addition of L-cysteine to the growth medium reduced the level of cysK transcripts, they did suggest that L-cysteine indirectly repressed the expression of the sulfate transport genes. By decreasing the level of uptake through the sulfate permease system, L-cysteine allowed the cells to grow even when the selenite concentrations were 40 times higher than the sulfate concentrations.
Under the selenite sensitive conditions (NCS), Enterobacter sp. YSU appeared to respond to selenite by expressing a putative tellurium resistance protein C [5,34] in spot 8. Since tellurium is located directly under selenium in the periodic table of elements and is chemically similar to selenium [36], this protein may have been expressed nonspecifically in response to selenite but was not successful in conferring resistance. The association of a GroES-like protein with the tellurium resistance protein and the appearance of the small heat shock protein in unique spot 9 suggested that these cells were probably experiencing selenite-induced oxidative stress [17].
The function of other identified proteins in spots 7, 10 and 11, which were present at higher intensities in cultures grown in the presence of selenite (NCS and CS) than in cultures grown in the absence of selenite (NCNS and CNS), was not clear. They all contained peptides for 3 proteins: outer membrane protein II (OmpA) from E. aerogenes [37], OmpA from E. sakazakii and a putative component membrane hydrogenase of OmpA. Basic Local Alignment Search Tool (BLAST) analysis and the references associated with the BLAST results did not provide any additional information about these polypeptides. However, an anaerobic strain of Clostridium pasteurianum reduced selenite using a hydrogenase during anaerobic respiration [38], and other Gram negative bacteria such as Stenotrophomonas maltophilia and Enterobacter sp. SLD1a-1 reduced selenite and selenate and deposited elemental selenium just inside or outside the cell surface [21,39].
Since the three proteins in spots 7, 10 and 11 shared many of the same peptides (Table 1), they may form a single OmpA-like protein that reduced selenite to elemental selenium using the hydrogenase component. Further studies are needed to understand the role that they played in selenite resistance.

Conclusion
Viable cell count and turbidometric growth curves in M-9 minimal medium showed that Enterobacter sp. YSU required L-cysteine to be resistant to 40 mM selenite. Selenite can enter E. coli through a specific, undefined pathway and a non-specific sulfate permease pathway [30]. Proteomic and RT-PCR analysis of Enterobacter sp. YSU cultures grown in the absence of L-cysteine and presence of selenite (NCS) and in the presence of L-cysteine and selenite (CS) suggested that L-cysteine conferred selenite resistance by feedback inhibition of the synthesis of N-acetylserine. This intermediate in L-cysteine synthesis acted as an inducer for cysK and the sulfate permease genes, cysA, cysT, and cysW. The lower levels of inducer decreased the expression of sulfate permease and may have limited selenite transport into the cells through the non-specific pathway, allowing the bacteria survive. This work linked studies on selenite tolerance in M-9 medium lacking L-cysteine [17] with research on selenite transport in E. coli [30].

Viable cell count growth curves
Four Enterobacter sp. YSU cultures, two containing Lcysteine 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.

Proteomic analysis growth curves
Two Enterobacter sp. YSU cultures, one containing Lcysteine 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.

Protein extraction
Cells were thawed and resuspended in lysing buffer con-  [41,42] was used to determine protein concentrations before 2DGE analysis.

Two-dimensional gel electrophoresis (2DGE)
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.