Random mutagenesis in Corynebacterium glutamicum ATCC 13032 using an IS6100-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

BACKGROUND: Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. RESULTS: A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. CONCLUSION: The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100

Revisiting Corynebacterium glyciniphilum (ex Kubota et al. 1972) sp. nov., nom. rev., isolated from putrefied banana

Al-Dilaimi A, Bednarz H, Lömker A, Niehaus K, Kalinowski J, Rückert C. Revisiting Corynebacterium glyciniphilum (ex Kubota et al. 1972) sp. nov., nom. rev., isolated from putrefied banana. International journal of systematic and evolutionary microbiology. 2015;65(Pt 1):177-182.: A corynebacterium, designated AJ 3170(T), was isolated in the eighties from putrefied bananas. Since then, no further updates concerning the strain description or phylogenetic classification of this species were made. However, phylogenetic analysis of this strain using 16S rRNA and in silico DNA-DNA hybridization confirms the membership to the genus Corynebacterium and clusters AJ 3170(T) with Corynebacterium variabile DSM 44702(T), Corynebacterium terpenotabidum IFO 14764(T) and Corynebacterium nuruki S6-4(T) in one subgroup. Furthermore, a combination of enzymatic, chemical, and morphological characterization technics was applied to describe the strain AJ 3170(T). The strain grows well at pH 6-10 and at 30-41 °C. The predominant major fatty acids were C16:0 (42.15 %), C18:1 ω9c (41.6 %) and C18:0 10-methyl (TBSA) (8.56 %). Cell wall and whole-cell sugar composition could be determined. On the basis of phenotypic, chemotaxonomic and phylogenetic characterization, it is proposed that the strain AJ 3170(T) represents a novel species, for which the name Corynebacterium glyciniphilum sp. nov. is proposed. The type strain is AJ 3170(T) (=DSM 45795(T) =ATCC 21341(T))

Random mutagenesis in <it>Corynebacterium glutamicum </it>ATCC 13032 using an IS<it>6100</it>-based transposon vector identified the last unknown gene in the histidine biosynthesis pathway

Abstract Background Corynebacterium glutamicum, a Gram-positive bacterium of the class Actinobacteria, is an industrially relevant producer of amino acids. Several methods for the targeted genetic manipulation of this organism and rational strain improvement have been developed. An efficient transposon mutagenesis system for the completely sequenced type strain ATCC 13032 would significantly advance functional genome analysis in this bacterium. Results A comprehensive transposon mutant library comprising 10,080 independent clones was constructed by electrotransformation of the restriction-deficient derivative of strain ATCC 13032, C. glutamicum RES167, with an IS6100-containing non-replicative plasmid. Transposon mutants had stable cointegrates between the transposon vector and the chromosome. Altogether 172 transposon integration sites have been determined by sequencing of the chromosomal inserts, revealing that each integration occurred at a different locus. Statistical target site analyses revealed an apparent absence of a target site preference. From the library, auxotrophic mutants were obtained with a frequency of 2.9%. By auxanography analyses nearly two thirds of the auxotrophs were further characterized, including mutants with single, double and alternative nutritional requirements. In most cases the nutritional requirement observed could be correlated to the annotation of the mutated gene involved in the biosynthesis of an amino acid, a nucleotide or a vitamin. One notable exception was a clone mutagenized by transposition into the gene cg0910, which exhibited an auxotrophy for histidine. The protein sequence deduced from cg0910 showed high sequence similarities to inositol-1(or 4)-monophosphatases (EC 3.1.3.25). Subsequent genetic deletion of cg0910 delivered the same histidine-auxotrophic phenotype. Genetic complementation of the mutants as well as supplementation by histidinol suggests that cg0910 encodes the hitherto unknown essential L-histidinol-phosphate phosphatase (EC 3.1.3.15) in C. glutamicum. The cg0910 gene, renamed hisN, and its encoded enzyme have putative orthologs in almost all Actinobacteria, including mycobacteria and streptomycetes. Conclusion The absence of regional and sequence preferences of IS6100-transposition demonstrate that the established system is suitable for efficient genome-scale random mutagenesis in the sequenced type strain C.glutamicum ATCC 13032. The identification of the hisN gene encoding histidinol-phosphate phosphatase in C. glutamicum closed the last gap in histidine synthesis in the Actinobacteria. The system might be a valuable genetic tool also in other bacteria due to the broad host-spectrum of IS6100.</p

Pseudorapidity densities of charged particles with transverse momentum thresholds in pp collisions at √ s = 5.02 and 13 TeV

The pseudorapidity density of charged particles with minimum transverse momentum (pT) thresholds of 0.15, 0.5, 1, and 2 GeV/c is measured in pp collisions at the center of mass energies of √s=5.02 and 13 TeV with the ALICE detector. The study is carried out for inelastic collisions with at least one primary charged particle having a pseudorapidity (η) within 0.8pT larger than the corresponding threshold. In addition, measurements without pT-thresholds are performed for inelastic and nonsingle-diffractive events as well as for inelastic events with at least one charged particle having |η|2GeV/c), highlighting the importance of such measurements for tuning event generators. The new measurements agree within uncertainties with results from the ATLAS and CMS experiments obtained at √s=13TeV.

Measurement of the radius dependence of charged-particle jet suppression in Pb–Pb collisions at sNN=5.02\sqrt{s_{\rm NN}} = 5.02 TeV

The ALICE Collaboration reports a new differential measurement of inclusive jet suppression using pp and Pb–Pb collision data at center-of-mass energy per nucleon–nucleon collision $\sqrt{s_{\rm NN}} = 5.02$ TeV. Charged-particle jets are reconstructed using the anti-$k_{\rm T}$ algorithm with resolution parameters $R$ = 0.2, 0.3, 0.4, 0.5, and 0.6 in pp collisions and $R$ = 0.2, 0.4, 0.6 in central (0–10\%), semi-central (30–50\%), and peripheral (60–80\%) Pb–Pb collisions. The analysis uses a novel approach based on machine learning to mitigate the influence of jet background in central heavy-ion collisions, which enables measurements of inclusive jet suppression for jet $p_{\rm T} \ge 40$ GeV/$c$ in central collisions at a resolution parameter of $R$ = 0.6. This is the lowest value of jet $p_{\rm T}$ achieved for inclusive jet measurements at $R$ = 0.6 at the LHC, and is an important step for discriminating different models of jet quenching in the quark-gluon plasma. The transverse momentum spectra, nuclear modification factors, and derived cross section and nuclear modification factor ratios for different jet resolution parameters of charged-particle jets are presented and compared to model predictions. A mild dependence of the nuclear modification factor ratios on collision centrality and resolution parameter is observed. The results are compared to a variety of jet quenching models with varying levels of agreement, demonstrating the effectiveness of this observable to discriminate between models.The ALICE Collaboration reports a new differential measurement of inclusive jet suppression using pp and Pb$-$Pb collision data at center-of-mass energy per nucleon-nucleon collision $\sqrt{s_{\rm NN}} = 5.02$ TeV. Charged-particle jets are reconstructed using the anti-$k_{\rm T}$ algorithm with resolution parameters $R =$ 0.2, 0.3, 0.4, 0.5, and 0.6 in pp collisions and $R =$ 0.2, 0.4, 0.6 in central (0$-$10%), semi-central (30$-$50%), and peripheral (60$-$80%) Pb$-$Pb collisions. The analysis uses a novel approach based on machine learning to mitigate the influence of jet background in central heavy-ion collisions, which enables measurements of inclusive jet suppression for jet $p_{\rm T} \geq 40$ GeV/$c$ in central collisions at a resolution parameter of $R = 0.6$. This is the lowest value of jet $p_{\rm T}$ achieved for inclusive jet measurements at $R=0.6$ at the LHC, and is an important step for discriminating different models of jet quenching in the quark-gluon plasma. The transverse momentum spectra, nuclear modification factors, and derived cross section and nuclear modification factor ratios for different jet resolution parameters of charged-particle jets are presented and compared to model predictions. A mild dependence of the nuclear modification factor ratios on collision centrality and resolution parameter is observed. The results are compared to a variety of jet quenching models with varying levels of agreement, demonstrating the effectiveness of this observable to discriminate between models