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. Sulfur is an essential element for any microorganism. It is required for the synthesis of the sulfur-containing amino acids (L-cysteine and L-methionine) together with that of the sulfur-containing coenzymes or prosthetic groups and other compounds. In the context of applied microbiology, the assimilation of sulfur is crucial in at least two settings. One is related to pathogenic microorganisms in which there is an indication that such organisms face sulfur limitation in their natural environment. This is probably the case for the facultative intracellular pathogen Mycobacterium tuberculosis in its preferred host, the macrophage, since under conditions of sulfur limitation, the mycobacterial cysDNC operon enabling sulfur assimilation is upregulated (1). In addition to L-cysteine and L-methionine synthesis, the sulfate assimilation pathway of M. tuberculosis produces a number of sulfur-containing metabolites with important contributions to pathogenesis and survival (2). In different clinical M. tuberculosis isolates, significant differences in the abundance of sulfur (and iron) assimilatory proteins highlight the relevance of sulfur availability for the bacilli (3). In the opportunistic pathogen Pseudomonas aeruginosa E601, which causes infections of cystic fibrosis in patients, 43 proteins, among them a sulfatase with mucin as the substrate which makes the sulfur of the glycopeptide available, are upregulated more than 10-fold under conditions of sulfur limitation (4).Sulfur availability is also of relevance for the large-scale synthesis of L-cysteine and L-methionine. The bacterium Corynebacterium glutamicum is a prime candidate for amino acid production, because proce...

Development of a new platform for secretory production of recombinant proteins in Corynebacterium glutamicum

“…Previous studies have reported secretome analyses of C. glutamicum ATCC 13032 and C. glutamicum R, but those studies were performed with the samples of flask cultivations (Hansmeier et al, 2006;Suzuki et al, 2009). Expression of the cg1514 gene has been shown to be up-regulated by CysR which is activated by O-acetyl-L-serine (OAS) or O-acetyl-L-homoserine (OAH) under sulfur limiting condition (Hoffmann et al, 2013;R€ uckert et al, 2008). A similar number of protein spots (approximately 90 spots) were found, with most of the proteins having an acidic pI in the 3.5-4.5 range, however a few proteins had acidic pIs in the 7-8 range (Fig.…”

“…Previous studies indicated that Cg1514 is related to sulfur metabolism in C. glutamicum however the exact function of the protein is still unknown (R€ uckert et al, 2008). Expression of the cg1514 gene has been shown to be up-regulated by CysR which is activated by O-acetyl-L-serine (OAS) or O-acetyl-L-homoserine (OAH) under sulfur limiting condition (Hoffmann et al, 2013;R€ uckert et al, 2008). In our work, however, there was no expectation for elevated cytosolic OAS or OAH concentrations during fed-batch cultivations because the medium used for cultivation, contained 10 g/L of ammonium sulfate and this concentration was high enough not to be limited during the cultivation.…”

Expression regulation of multiple key genes to improve l-threonine in Escherichia coli

“…To sense and regulate intracellular or extracellular chemicals, bacteria evolve a variety of transcription factors. Based on this principle, various biosensors for amino acids and their precursors, including l-methionine, l-leucine, l-isoleucine, l-valine [13,14], l-lysine, l-arginine, l-serine, O-acetyl-l-serine [15], O-acetyl homoserine [16], and oxygen [17] have been constructed, and some of these sensors have been successfully applied to the high-throughput screening [12,18,19] and systems metabolic engineering [20]. l-Threonine production in E. coli can be enhanced by regulating IclR [8,9], a transcription factor.…”

Screening of an Escherichia coli promoter library for a phenylalanine biosensor

“…In this context, biosensor-based strategies were shown to enable the identification of novel and non-intuitive targets for engineering and improving of production strains. Furthermore, the efficient screening of feedback-resistant enzymes (Schendzielorz et al 2014), dynamic pathway regulation (Dahl et al 2013;Liu et al 2015b;Xu et al 2014;Zhang et al 2012), and the analysis of population heterogeneity at the single-cell level (Hoffmann et al 2013;Mustafi et al 2014) demonstrate the broad applicability of biosensors (Liu et al 2015a;Mahr and Frunzke 2016).…”