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Visualization of imbalances in sulfur assimilation and synthesis of sulfur-containing amino acids at the single-cell level.

ABSTRACT: We describe genetically encoded sensors which transmit elevated cytosolic concentrations of O-acetyl serine (OAS) and O-acetyl homoserine (OAH)-intermediates of l-cysteine and l-methionine synthesis-into an optical output. The sensor pSenOAS3 elicits 7.5-fold-increased fluorescence in cultures of a Corynebacterium glutamicum strain that excrete l-cysteine. Determination of the cytosolic OAS concentration revealed an increase to 0.13 mM, whereas the concentration in the reference strain was below the detection limit, indicating that incorporation of assimilatory sulfur is limited in the strain studied. In another strain, overexpression of metX encoding homoserine acetyltransferase resulted in an 8-fold increase in culture fluorescence at a cytosolic OAH concentration of 0.76 mM. We also assayed for consequences of extracellular sulfur supply and observed a graded fluorescence increase at decreasing sulfur concentrations below 400 ?M. Overall, this demonstrates the usefulness of the sensors for monitoring intracellular sulfur availability. The sensors also enable monitoring at the single-cell level, and since related and close homologs of the transcription factor used in the constructed sensors are widespread among bacteria, this technology offers a new possibility of assaying in vivo for sulfur limitation and of doing this at the single-cell level.

SUBMITTER: Hoffmann K

PROVIDER: S-EPMC3811485 | biostudies-literature | 2013 Nov

REPOSITORIES: biostudies-literature

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Visualization of imbalances in sulfur assimilation and synthesis of sulfur-containing amino acids at the single-cell level.

Hoffmann Kristina K Grünberger Alexander A Lausberg Frank F Bott Michael M Eggeling Lothar L

Applied and environmental microbiology 20130830 21

We describe genetically encoded sensors which transmit elevated cytosolic concentrations of O-acetyl serine (OAS) and O-acetyl homoserine (OAH)-intermediates of l-cysteine and l-methionine synthesis-into an optical output. The sensor pSenOAS3 elicits 7.5-fold-increased fluorescence in cultures of a Corynebacterium glutamicum strain that excrete l-cysteine. Determination of the cytosolic OAS concentration revealed an increase to 0.13 mM, whereas the concentration in the reference strain was below ...[more]

PMID: 23995919

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Project description:The sulfur-containing amino acids methionine and cysteine play an important role in food industry. These amino acids are used to confer a sulfur smell or meat-related aroma to food products. Besides their use as food additives, methionine and cysteine participate in flavor formation in dairy fermentations. For instance, the characteristic aroma of Cheddar cheeses is derived from methionine. Therefore, bacterial strains with the ability to overproduce and secrete these amino acids are relevant for the food industry. In addition, the quantification of these compounds in food matrices is a laborious task that involves sample preparation and specific analytical methods such as high-performance liquid chromatography. The ability of bacteria to naturally sense metabolites has successfully been exploited to develop biosensors. The presence of a specific metabolite is sensed by the biosensors, and it is subsequently translated into the expression of one or more reporter genes. In this study we aim to develop biosensors to detect methionine and cysteine, which are produced and secreted by wild-type Lactococcus lactis strains. We employed two strategies to create L. lactis biosensors, the first one is based on the methionine auxotrophy of this bacterium and the second strategy is based on a cysteine-responsive promoter. The characterization of the biosensors showed their specific response to the presence of these amino acids. Subsequently, we applied the methionine biosensor to quantify the presence of methionine in bacterial supernatants of wild-type L. lactis that naturally secretes methionine to benchmark the performance of our biosensors. The methionine biosensor responded linearly to the amounts of methionine present in the bacterial supernatants, i.e., the increases in the biosensor cell densities were proportional to the amounts of methionine present in the supernatants. The biosensors developed in this study tackle the limitations of amino acid quantification and the selection of strains with secretion of amino acids. These biosensors may eventually be used for screening of engineered strains to increase methionine and cysteine production, and may facilitate the detection of these amino acids in complex food matrices.

Dataset Information

Visualization of imbalances in sulfur assimilation and synthesis of sulfur-containing amino acids at the single-cell level.

Publications

Visualization of imbalances in sulfur assimilation and synthesis of sulfur-containing amino acids at the single-cell level.

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