l-Histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. For decades l-histidine biosynthesis has been studied mainly in Escherichia coli and Salmonella typhimurium, revealing fundamental regulatory processes in bacteria. Furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid-producing bacterium Corynebacterium glutamicum, revealing similarities to E. coli and S. typhimurium, as well as differences. This review summarizes the current knowledge of l-histidine biosynthesis in C. glutamicum. The genes involved and corresponding enzymes are described, in particular focusing on the imidazoleglycerol-phosphate synthase (HisFH) and the histidinol-phosphate phosphatase (HisN). The transcriptional organization of his genes in C. glutamicum is also reported, including the four histidine operons and their promoters. Knowledge of transcriptional regulation during stringent response and by histidine itself is summarized and a translational regulation mechanism is discussed, as well as clues about a histidine transport system. Finally, we discuss the potential of using this knowledge to create or improve C. glutamicum strains for the industrial l-histidine production.

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The Hologic Panther Fusion® Open Access™ functionality allows implementation of laboratory-developed tests (LDTs), with fully automated sample extraction, real-time PCR, and result interpretation. We report the development and validation of a multiplex LDT for norovirus G1, norovirus G2, and rotavirus from stool samples on this system. The LDT was optimized for primer and probe sequences, salt concentration, and PCR annealing temperature. Reproducibility of the PCR and extraction process was assessed. Performance of the multiplex LDT assay was evaluated with external quality assessment (EQA) samples and compared to a commercial multiplex assay (Allplex™ GI-Virus Assay, Seegene) in clinical samples. Salt concentrations and annealing/extension temperature were optimized to 4 mM MgCl2, 70 mM KCl, 20 mM Tris, and 60 °C, respectively. The user-prepared part of the LDT PCR mix (containing salts, probes, and primers) was stable for ≥ 11 days onboard the instrument. We observed reproducible results of PCR and the extraction process. The LDT had a sensitivity comparable to or greater than the commercial Allplex™ assay and showed excellent linearity. Forty-five EQA samples yielded the expected result with the LDT. There was 100% concordance between LDT and Allplex™ results in 160 clinical samples. Results from the suspension and direct swab stool sample preparation methods were highly concordant in the LDT. We report the successful development and validation of a multiplex PCR LDT for detection of norovirus G1, norovirus G2, and rotavirus from stool samples on the Panther Fusion® system.

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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.