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Carotenoid biosynthesis in Corynebacteriumglutamicum is controlled by the MarR-type regulator CrtR, which represses transcription of the promoter of the crt operon (PcrtE) and of its own gene (PcrtR). Geranylgeranyl pyrophosphate (GGPP), and to a lesser extent other isoprenoid pyrophosphates, interfere with the binding of CrtR to its target DNA in vitro, suggesting they act as inducers of carotenoid biosynthesis. CrtR homologs are encoded in the genomes of many other actinobacteria. In order to determine if and to what extent the function of CrtR, as a metabolite-dependent transcriptional repressor of carotenoid biosynthesis genes responding to GGPP, is conserved among actinobacteria, five CrtR orthologs were characterized in more detail. EMSA assays showed that the CrtR orthologs from Corynebacteriumcallunae, Acidipropionibacteriumjensenii, Paenarthrobacternicotinovorans, Micrococcusluteus and Pseudarthrobacterchlorophenolicus bound to the intergenic region between their own gene and the divergently oriented gene, and that GGPP inhibited these interactions. In turn, the CrtR protein from C. glutamicum bound to DNA regions upstream of the orthologous crtR genes that contained a 15 bp DNA sequence motif conserved between the tested bacteria. Moreover, the CrtR orthologs functioned in C. glutamicum in vivo at least partially, as they complemented the defects in the pigmentation and expression of a PcrtE_gfpuv transcriptional fusion that were observed in a crtR deletion mutant to varying degrees. Subsequently, the utility of the PcrtE_gfpuv transcriptional fusion and chromosomally encoded CrtR from C. glutamicum as genetically encoded biosensor for GGPP was studied. Combined FACS and LC-MS analysis demonstrated a correlation between the sensor fluorescent signal and the intracellular GGPP concentration, and allowed us to monitor intracellular GGPP concentrations during growth and differentiate between strains engineered to accumulate GGPP at different concentrations.

Additional file 1 Supplementary Figures. Includes all supplementary figures.

Additional file 1: Method S1. 5′-library preparation, sequencing and data processing. Material S1. Adapters and primers used for 5′-library preparation. Material S2. Control primer for Colony PCR and Sanger sequencing. Material S3. Gibson Assembly primers for the amplification of inserts for pKC1139 expression system. Material S4. Gibson Assembly primer for the amplification of inserts with native promoters for pSET152. Material S5. Gibson Assembly primer for the gusA reporter system. Material S6. Gibson Assembly primer for acbC expression by strong promoters in pSET152. Material S7. Primers used in RT-qPCR. Data S1. PCR unveils vector-integration of pKC1139-constructs by homologous recombination. Data S2. Reduced transcription of acb genes downstream of the locus of vector integration shown by RT-qPCR for the mutant Actinoplanes sp. SE50/110 [pKC1139::PermE*::acbL]. Data S3. Results of promoter screening by GUS-assay. Data S4. Determination of transcription start sites of heterologous promoters in Actinoplanes sp. SE50/110 by 5‘-end specific transcriptome sequencing. Data S5. Growth and acarbose formation of pSET152-based acbC-overexpression mutants. Data S6. Smart formula analysis of the isotopic pattern of mass = 255.03 [M−H+].

Corynebacterium glutamicum is a commonly applied host for the industrial production of amino acids. While valued for its robustness, it is somewhat inferior to competing strains such as Escherichia coli because of the relatively low growth rate of 0.40 h(-1) in synthetic, industrial media. Accordingly, adaptive laboratory evolution (ALE) experiments were performed in continuous cultivation mode to select for a growth-improved host. To ensure industrial attractiveness, this ALE study aimed at a reduction of dependency on costly growth-boosting additives such as protocatechuate (RCA) or complex media supplements. Consequently, double selection pressures were installed consisting of a steady increase in growth rate demands and a parallel reduction of complex medium fractions. Selection yielded C. glutamicum EVO5 achieving 0.54 h(-1) and 1.03g(Glc) g(CDW)(-1) h(-1) in minimal medium without above mentioned supplements. Sequencing revealed 10 prominent mutations, three of them in key regulator genes.

Actinoplanes sp. SE50/110 is the wild type of industrial production strains of the fine-chemical acarbose (acarviosyl-maltose), which is used as α-glucosidase inhibitor in the treatment of type II diabetes. Although maltose is an important building block of acarbose, the maltose/maltodextrin metabolism has not been studied in Actinoplanes sp. SE50/110 yet. Bioinformatic analysis located a putative maltase gene amlE (ACSP50_2474, previously named malL; Wendler et al., 2015a), in an operon with an upstream PurR/LacI-type transcriptional regulator gene, named amlR (ACSP50_2475), and a gene downstream (ACSP50_2473) encoding a GGDEF-EAL-domain-containing protein putatively involved in c-di-GMP signaling. Targeted gene deletion mutants of amlE and amlR were constructed by use of the CRISPR/Cas9 technology. By growth experiments and functional assays of ΔamlE, we could show that AmlE is essential for the maltose utilization in Actinoplanes sp. SE50/110. Neither a gene encoding a maltose phosphorylase (MalP) nor MalP enzyme activity were detected in the wild type. By this, the maltose/maltodextrin system appears to be fundamentally different from other described prokaryotic systems. By sequence similarity analysis and functional assays from the species Streptomyces lividans TK23, S. coelicolor A3(2) and S. glaucescens GLA.O, first hints for a widespread lack of MalP and presence of AmlE in the class Actinobacteria were given. Transcription of the aml operon is significantly repressed in the wild type when growing on glucose and repression is absent in an ΔamlR deletion mutant. Although AmlR apparently is a local transcriptional regulator of the aml operon, the ΔamlR strain shows severe growth inhibitions on glucose and – concomitantly – differential transcription of several genes of various functional classes. We ascribe these effects to ACSP50_2473, which is localized downstream of amlE and presumably involved in the metabolism of the second messenger c-di-GMP. It can be assumed, that maltose does not only represent the most important carbon source of Actinoplanes sp. SE50/110, but that its metabolism is coupled to the nucleotide messenger system of c-di-GMP.

Background The eighth step of l-histidine biosynthesis is carried out by an enzyme called histidinol-phosphate phosphatase (HolPase). Three unrelated HolPase families are known so far. Two of them are well studied: HAD-type HolPases known from Gammaproteobacteria like Escherichia coli or Salmonella enterica and PHP-type HolPases known from yeast and Firmicutes like Bacillus subtilis. However, the third family of HolPases, the inositol monophosphatase (IMPase)-like HolPases, present in Actinobacteria like Corynebacterium glutamicum (HisN) and plants, are poorly characterized. Moreover, there exist several IMPase-like proteins in bacteria (e.g. CysQ, ImpA, and SuhB) which are very similar to HisN but most likely do not participate in l-histidine biosynthesis. Results Deletion of hisN, the gene encoding the IMPase-like HolPase in C. glutamicum, does not result in complete l-histidine auxotrophy. Out of four hisN homologs present in the genome of C. glutamicum (impA, suhB, cysQ, and cg0911), only cg0911 encodes an enzyme with HolPase activity. The enzymatic properties of HisN and Cg0911 were determined, delivering the first available kinetic data for IMPase-like HolPases. Additionally, we analyzed the amino acid sequences of potential HisN, ImpA, SuhB, CysQ and Cg0911 orthologs from bacteria and identified six conserved sequence motifs for each group of orthologs. Mutational studies confirmed the importance of a highly conserved aspartate residue accompanied by several aromatic amino acid residues present in motif 5 for HolPase activity. Several bacterial proteins containing all identified HolPase motifs, but showing only moderate sequence similarity to HisN from C. glutamicum, were experimentally confirmed as IMPase-like HolPases, demonstrating the value of the identified motifs. Based on the confirmed IMPase-like HolPases two profile Hidden Markov Models (HMMs) were build using an iterative approach. These HMMs allow the fast, reliable detection and differentiation of the two paralog groups from each other and other IMPases. Conclusion The kinetic data obtained for HisN from C. glutamicum, as an example for an IMPase-like HolPases, shows remarkable differences in enzyme properties as compared to HAD- or PHP-type HolPases. The six sequence motifs and the HMMs presented in this study can be used to reliably differentiate between IMPase-like HolPases and IMPase-like proteins with no such activity, with the potential to enhance current and future genome annotations. A phylogenetic analysis reveals that IMPase-like HolPases are not only present in Actinobacteria and plant but can be found in further bacterial phyla, including, among others, Proteobacteria, Chlorobi and Planctomycetes. Electronic supplementary material The online version of this article (doi:10.1186/s12866-017-1069-4) contains supplementary material, which is available to authorized users.

Data collection for motif analysis. This data collection contains all sequence data of IMPase-like proteins belonging to the group Cg0911, CysQ, HisN, ImpA, and SuhB used for motif analysis. Furthermore, HisN orthologues not used for the motif analysis were listed. (DOCX 120 kb)

Supplementary Figures and Tables. The supplementary figures comprise the SDS-PAGE of purified HisN Cg and Cg0911, several alignments of IMPase-like proteins in C. glutamicum and other species, and the complementation assay for various hisN Cg gene mutants. Furthermore, supplementary tables carry the used strains, plasmids and primers. (DOCX 200 kb)