The Palaeognathae comprise the flightless ratites and the volant tinamous, and together with the Neognathae constitute the extant members of class Aves. It is commonly believed that Palaeognathae originated in Gondwana since most of the living species are found in the Southern Hemisphere [1-3]. However, this hypothesis has been questioned because the fossil paleognaths are mostly from the Northern Hemisphere in their earliest time (Paleocene) and possessed many putative ancestral characters [4]. Uncertainties regarding the origin and evolution of Palaeognathae stem from the difficulty in estimating their divergence times [1, 2] and their remarkable morphological convergence. Here, we recovered nuclear genome fragments from extinct elephant birds, which enabled us to reconstruct a reliable phylogenomic time tree for the Palaeognathae. Based on the tree, we identified homoplasies in morphological traits of paleognaths and reconstructed their morphology-based phylogeny including fossil species without molecular data. In contrast to the prevailing theories, the fossil paleognaths from the Northern Hemisphere were placed as the basal lineages. Combined with our stable divergence time estimates that enabled a valid argument regarding the correlation with geological events, we propose a new evolutionary scenario that contradicts the traditional view. The ancestral Palaeognathae were volant, as estimated from their molecular evolutionary rates, and originated during the Late Cretaceous in the Northern Hemisphere. They migrated to the Southern Hemisphere and speciated explosively around the Cretaceous-Paleogene boundary. They then extended their distribution to the Gondwana-derived landmasses, such as New Zealand and Madagascar, by overseas dispersal. Gigantism subsequently occurred independently on each landmass.

We identified the nfsA gene, encoding the major oxygen-insensitive nitroreductase in Escherichia coli, and determined its position on the E. coli map to be 19 min. We also purified its gene product, NfsA, to homogeneity. It was suggested that NfsA is a nonglobular protein with a molecular weight of 26,799 and is associated tightly with a flavin mononucleotide. Its amino acid sequence is highly similar to that of Frp, a flavin oxidoreductase from Vibrio harveyi (B. Lei, M. Liu, S. Huang, and S.-C. Tu, J. Bacteriol. 176:3552-3558, 1994), an observation supporting the notion that E. coli nitroreductase and luminescent-bacterium flavin reductase families are intimately related in evolution. Although no appreciable sequence similarity was detected between two E. coli nitroreductases, NfsA and NfsB, NfsA exhibited a low level of the flavin reductase activity and a broad electron acceptor specificity similar to those of NfsB. NfsA reduced nitrofurazone by a ping-pong Bi-Bi mechanism possibly to generate a two-electron transfer product.The oxygen-insensitive nitroreductase activity in Escherichia coli consists of one major and two minor components (5). The major component is an NADPH-linked enzyme encoded by nfsA, while minor components, encoded by nfsB and an unidentified gene (5, 19), can use both NADH and NADPH as electron donors. We have cloned and mapped nfsB and analyzed biochemical properties of the purified gene product (NfsB) (29). Our analysis suggested that NfsB is similar in sequence and many biochemical properties to FRase I, the major flavin reductase in Vibrio fischeri (31). NfsB was also found to have a low level of flavin reductase activity (29). Furthermore, a single amino acid substitution of Phe-124 of NfsB changed NfsB into an FRase I-like flavin reductase whose activity was three times higher than that of the authentic FRase I (28). Thus, it is reasonable to assume that genes coding for V. fischeri FRase I and E. coli NfsB nitroreductase are derivatives of a common progenitor gene. Since luminescent bacteria contain several species of flavin reductase (9, 30, 31) and nitroreductase in E. coli forms a family consisting of functionally redundant members (5), as with the NfsB-FRase I pair (29, 31), other nitroreductase members in E. coli might have their counterparts in the flavin reductase family of luminescent bacteria.To clarify the evolutionary and biochemical relationships between flavin reductases of luminescent bacteria and E. coli nitroreductases, gene cloning and biochemical characterization of E. coli nitroreductases other than NfsB may be necessary. Here, we identified the nfsA gene, encoding the major nitroreductase in E. coli, and characterized its gene product, NfsA.Our results showed NfsA nitroreductase to be a flavoprotein associated tightly with flavin mononucleotide (FMN) and to be the ortholog in E. coli of Frp, a flavin oxidoreductase from Vibrio harveyi (11,12,18), an observation supporting a close evolutionary relation between E. coli nitroreductase and luminescent-bacterium flav...

Ustiloxin B is a secondary metabolite known to be produced by Ustilaginoidea virens. In our previous paper, we observed the production of this compound by Aspergillus flavus, and identified two A. flavus genes responsible for ustiloxin B biosynthesis (Umemura et al., 2013). The compound is a cyclic tetrapeptide of Tyr-Ala-Ile-Gly, whose tyrosine is modified with a non-protein coding amino acid, norvaline. Although its chemical structure strongly suggested that ustiloxin B is biosynthesized by a non-ribosomal peptide synthetase, in the present study, we observed its synthesis through a ribosomal peptide synthetic (RiPS) pathway by precise sequence analyses after experimental validation of the cluster. The cluster possessed a gene (AFLA_094980), termed ustA, whose translated product, UstA, contains a 16-fold repeated peptide embedding a tetrapeptide, Tyr-Ala-Ile-Gly, that is converted into the cyclic moiety of ustiloxin B. This result strongly suggests that ustiloxin B is biosynthesized through a RiPS pathway and that UstA provides the precursor peptide of the compound. The present work is the first characterization of RiPS in Ascomycetes and the entire RiPS gene cluster in fungi. Based on the sequence analyses, we also proposed a biosynthetic mechanism involving the entire gene cluster. Our finding indicates the possibility that a number of unidentified RiPSs exist in Ascomycetes as the biosynthetic genes of secondary metabolites, and that the feature of a highly repeated peptide sequence in UstA will greatly contribute to the discovery of additional RiPS.

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