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The genera Microbispora and Sphaerimonospora were examined for GyrB and RecA amino-acid signatures to determine whether molecular-signature analyses support the recent establishment of the genus Sphaerimonospora . The creation of Sphaerimonospora was based mainly upon morphological differences between Microbispora and Sphaerimonospora and the clustering of the type strains of the two genera in phylogenetic trees based on a multilocus sequence analysis. The molecular-signature analyses showed that all members of Sphaerimonospora can be distinguished from all members of Microbispora at 14 amino acid positions in the GyrB protein and at four positions in the shorter RecA protein. These amino acid differences can be used as signatures to differentiate the members of these genera from each other and thus provide support for the establishment of the genus Sphaerimonospora . This is the first demonstration of the use of molecular signatures to support the establishment of a new genus in the family Streptosporangiaceae . Following the transfer of Microbispora mesophila and Microbispora thailandensis from Microbispora to Sphaerimonospora , all species in the genus Microbispora are characterised by the insertion of a small, hydrophobic amino acid after position 208 in the GyrB protein. This insertion is absent from the GyrB protein of members of the genus Sphaerimonospora . [ABSTRACT FROM AUTHOR]

The family Streptosporangiaceae (suborder Streptosporangineae ) comprises 13 genera and 100 species with validly published names. In a recent study, gyrB gene sequences were obtained for members of the family Streptosporangiaceae and the GyrB amino acid sequences were analysed for molecular signatures. In this study, recA gene sequences (895 nt) were determined for the type strains of members of the family Streptosporangiaceae . The sequences used represent 81% of the full-length recA gene of Streptosporangium roseum DSM 43021 T . The recA gene sequences were used for phylogenetic analyses and the trees were compared to the corresponding 16S-rRNA and gyrB gene trees. RecA amino acid alignments (298 amino acids) were generated and inspected for unique amino acid signatures to distinguish the genera in the family from each other. As was observed for the gyrB gene trees, the recA gene trees generally supported the division of the members of the family Streptosporangiaceae into 13 genera. The genus Nonomuraea was not monophyletic in any of the recA gene trees, while the genera Planomonospora and Streptosporangium were not monophyletic in the maximum likelihood and maximum parsimony trees. The gyrB - recA concatenated-gene tree was more robust than the recA gene tree, with 63 nodes in the gyrB - recA tree having bootstrap values ≥95%. The only insertions in the recA gene sequences were inteins identified in the type strains of Acrocarpospora phusangensis , Acrocarpospora pleiomorpha and Microbispora mesophila . Examination of the RecA sequence alignments for genus-specific amino acid sequences showed that the genera Herbidospora , Planobispora , Planomonospora and Streptosporangium contain unique amino acid sequences that distinguish these genera from all other genera in the family Streptosporangiaceae . The results of this investigation extend the results of the GyrB study and will be useful in future taxonomic studies in the family Streptosporangiaceae by providing additional genus-specific molecular signatures. [ABSTRACT FROM AUTHOR]

In this study, six E. coli strains, which harboring enhanced green fluorescence protein (EGFP), an intein (INT) and ice nucleation protein (INP), were used to express the EGFP protein. The effect of E. coli strains on the surfaced displayed EGFP via INP-INT system was first investigated. Among these strains, E. coli JM109(DE3) was found the best strain for EGFP production (67.9 mg/L), two-fold as compared to that produced by E. coli DH1(DE3). It was concluded that lon mutation could stabilize recombinant proteins. Thus, the deletion of the gene coding for proteolysis might be a critical factor when using host for surface-expression production. The optimal conditions for cell cultivation, gene induction and intein cleavage for EGFP production were systematically studied and discussed. The objective function (O) value of 26.5 can be reached at a 12-h induction and 20-h cleavage process, where the EGFP yield is 58.6 mg/L with a purity of 45.3%. Furthermore, results indicated that the higher intein activity was obtained in the shorter cleavage duration; however, the shorter cleavage time sacrificed the production yield as expected. To compromise between the production yield and purity, the optimal conditions for the EGFP production were: 12-h induction and 20-h cleavage process is for the production of higher purity EGFP. [ABSTRACT FROM AUTHOR]

Abstract: Subtype-selective thyromimetics have potential as new pharmaceuticals for the prevention or treatment of heart disease, high LDL cholesterol and obesity, but there are only a few methods that can detect agonistic behavior of TR-active compounds. Among these are the rat pituitary GH3 cell assay and transcriptional activation assays in engineered yeast and mammalian cells. We report the construction and validation of a newly designed TRα-1 bacterial biosensor, which indicates the presence of thyroid active compounds through their impacts on the growth of an engineered Escherichia coli strain in a simple defined medium. This biosensor couples the configuration of a hormone receptor ligand-binding domain to the activity of a thymidylate synthase reporter enzyme through an engineered allosteric fusion protein. The result is a hormone-dependent growth phenotype in the expressing E. coli cells. This sensor can be combined with our previously published TRβ-1 biosensor to detect potentially therapeutic subtype-selective compounds such as GC-1 and KB-141. To demonstrate this capability, we determined the half-maximal effective concentration (EC50) for the compounds T3, Triac, GC-1 and KB-141 using our biosensors, and determined their relative potency in each biosensor strain. Our results are similar to those reported by mammalian cell reporter gene assays, confirming the utility of our assay in identifying TR subtype-selective therapeutics. This biosensor thus provides a high-throughput, receptor-specific, and economical method (less than US$ 0.10 per well at laboratory scale) for identifying important therapeutics against these targets. [Copyright &y& Elsevier]

Subtype‐selective thyromimetics have potential as new pharmaceuticals for the prevention or treatment of heart disease, high LDL cholesterol and obesity, but there are only a few methods that can detect agonistic behavior of TR‐active compounds. Among these are the rat pituitary GH3 cell assay and transcriptional activation assays in engineered yeast and mammalian cells. We report the construction and validation of a newly designed TRα‐1 bacterial biosensor, which indicates the presence of thyroid active compounds through their impacts on the growth of an engineered Escherichia coli strain in a simple defined medium. This biosensor couples the configuration of a hormone receptor ligand‐binding domain to the activity of a thymidylate synthase reporter enzyme through an engineered allosteric fusion protein. The result is a hormone‐dependent growth phenotype in the expressing E. coli cells. This sensor can be combined with our previously published TRβ‐1 biosensor to detect potentially therapeutic subtype‐selective compounds such as GC‐1 and KB‐141. To demonstrate this capability, we determined the half‐maximal effective concentration (EC50) for the compounds T3, Triac, GC‐1 and KB‐141 using our biosensors, and determined their relative potency in each biosensor strain. Our results are similar to those reported by mammalian cell reporter gene assays, confirming the utility of our assay in identifying TR subtype‐selective therapeutics. This biosensor thus provides a high‐throughput, receptor‐specific, and economical method (less than US$ 0.10 per well at laboratory scale) for identifying important therapeutics against these targets. [ABSTRACT FROM AUTHOR]