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

Abstract

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.

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.

Figure 3

Negative images of Enterobacter sp. YSU total protein separated by 2DGE over a pI range of 4-7. Cultures were grown as in Fig 2. Cultures grown with No L-Cysteine and No Selenite (NCNS) and with L-Cysteine and No Selenite (CNS) were harvested after 2.5 hours of growth. The cultures grown with No L-Cysteine and Selenite (NCS) 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.

Figure 4

Detection of the cysK, cysA, cysT, cysW and cysE transcripts using RT-PCR. Cells were grown and harvested as in Fig 3. Equal volumes of cDNA synthesized from 0.5 μg of total RNA were used in PCR reactions containing primers specific for each gene, and 10 μl of each PCR reaction were analyzed by agarose gel electrophoresis. Lanes: (1) No L-Cysteine, No Selenite (NCNS); (2) No L-Cysteine, Selenite (NCS); (3) L-Cysteine, No Selenite (CNS); (4) L-Cysteine, Selenite (CS). The gene, cysE, was used as an internal control.