The Two-Component Signal Transduction System CopRS of Corynebacterium glutamicum Is Required for Adaptation to Copper-Excess Stress

Copper is an essential cofactor for many enzymes but at high concentrations it is toxic for the cell. Copper ion concentrations ≥50 µM inhibited growth of Corynebacterium glutamicum. The transcriptional response to 20 µM Cu2+ was studied using DNA microarrays and revealed 20 genes that showed a ≥ 3-fold increased mRNA level, including cg3281-cg3289. Several genes in this genomic region code for proteins presumably involved in the adaption to copper-induced stress, e. g. a multicopper oxidase (CopO) and a copper-transport ATPase (CopB). In addition, this region includes the copRS genes (previously named cgtRS9) which encode a two-component signal transduction system composed of the histidine kinase CopS and the response regulator CopR. Deletion of the copRS genes increased the sensitivity of C. glutamicum towards copper ions, but not to other heavy metal ions. Using comparative transcriptome analysis of the ΔcopRS mutant and the wild type in combination with electrophoretic mobility shift assays and reporter gene studies the CopR regulon and the DNA-binding motif of CopR were identified. Evidence was obtained that CopR binds only to the intergenic region between cg3285 (copR) and cg3286 in the genome of C. glutamicum and activates expression of the divergently oriented gene clusters cg3285-cg3281 and cg3286-cg3289. Altogether, our data suggest that CopRS is the key regulatory system in C. glutamicum for the extracytoplasmic sensing of elevated copper ion concentrations and for induction of a set of genes capable of diminishing copper stress

Two-component signal transduction in Corynebacterium glutamicum and other corynebacteria: on the way towards stimuli and targets

In bacteria, adaptation to changing environmental conditions is often mediated by two-component signal transduction systems. In the prototypical case, a specific stimulus is sensed by a membrane-bound histidine kinase and triggers autophosphorylation of a histidine residue. Subsequently, the phosphoryl group is transferred to an aspartate residue of the cognate response regulator, which then becomes active and mediates a specific response, usually by activating and/or repressing a set of target genes. In this review, we summarize the current knowledge on two-component signal transduction in Corynebacterium glutamicum. This Gram-positive soil bacterium is used for the large-scale biotechnological production of amino acids and can also be applied for the synthesis of a wide variety of other products, such as organic acids, biofuels, or proteins. Therefore, C. glutamicum has become an important model organism in industrial biotechnology and in systems biology. The type strain ATCC 13032 possesses 13 two-component systems and the role of five has been elucidated in recent years. They are involved in citrate utilization (CitAB), osmoregulation and cell wall homeostasis (MtrAB), adaptation to phosphate starvation (PhoSR), adaptation to copper stress (CopSR), and heme homeostasis (HrrSA). As C. glutamicum does not only face changing conditions in its natural environment, but also during cultivation in industrial bioreactors of up to 500 m3 volume, adaptability can also be crucial for good performance in biotechnological production processes. Detailed knowledge on two-component signal transduction and regulatory networks therefore will contribute to both the application and the systemic understanding of C. glutamicum and related species

Proteomic study uncovers molecular principles of single-cell-level phenotypic heterogeneity in lipid storage of Nannochloropsis oceanica

Abstract Background Nannochloropsis oceanica belongs to a large group of photoautotrophic eukaryotic organisms that play important roles in fixation and cycling of atmospheric CO2. Its capability of storing solar energy and carbon dioxide in the form of triacylglycerol (TAG) of up to 60% of total weight under nitrogen deprivation stress sparked interest in its use for biofuel production. Phenotypes varying in lipid accumulation among an N. oceanica population can be disclosed by single-cell analysis/sorting using fluorescence-activated cell sorting (FACS); yet the phenomenon of single cell heterogeneity in an algae population remains to be fully understood at the molecular level. In this study, combination of FACS and proteomics was used for identification, quantification and differentiation of these heterogeneities on the molecular level. Results For N. oceanica cultivated under nitrogen deplete (−N) and replete (+N) conditions, two groups differing in lipid content were distinguished. These differentiations could be recognized on the population as well as the single-cell levels; proteomics uncovered alterations in carbon fixation and flux, photosynthetic machinery, lipid storage and turnover in the populations. Although heterogeneity patterns have been affected by nitrogen supply and cultivation conditions of the N. oceanica populations, differentiation itself seems to be very robust against these factors: cultivation under +N, −N, in shaker bottles, and in a photo-bioreactor all split into two subpopulations. Intriguingly, population heterogeneity resumed after subpopulations were separately recultivated for a second round, refuting the possible development of genetic heterogeneity in the course of sorting and cultivation. Conclusions This work illustrates for the first time the feasibility of combining FACS and (prote)-omics for mechanistic understanding of phenotypic heterogeneity in lipid-producing microalgae. Such combinatorial method can facilitate molecular breeding and design of bioprocesses