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

Ashley Jasenec,Nabeel Shaik,Venkataramana Konda,Samatha Kulkarni,Swarnalatha Moparthi,Nathaniel Barasa,Jonathan Caguiat

doi:10.1186/1477-5956-7-30

Abstract

BackgroundEnterobacter 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.ResultsGrowth 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.ConclusionL-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.

Highlights

Selenium is an important cofactor in some mammalian and bacterial enzymes [1,2,3,4,5]
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
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

Summary

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. 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]. 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. It did not affect the level of selenite transport into the cell through the specific pathway

Methods

Results

Discussion

Conclusion