No abstract

Urochordates are the only animals that produce cellulose, a polysaccharide existing primarily in the extracellular matrices of plant, algal, and bacterial cells. Here we report a Ciona intestinalis homolog of cellulose synthase, which is the core catalytic subunit of multi-enzyme complexes where cellulose biosynthesis occurs. The Ciona cellulose synthase gene, Ci-CesA, is a fusion of a cellulose synthase domain and a cellulase (cellulose-hydrolyzing enzyme) domain. Both the domains have no animal homologs in public databases. Exploiting this fusion of atypical genes, we provided evidence of a likely lateral transfer of a bacterial cellulose synthase gene into the urochordate lineage. According to fossil records, this likely lateral acquisition of the cellulose synthase gene may have occurred in the last common ancestor of extant urochordates more than 530 million years ago. Whole-mount in situ hybridization analysis revealed the expression of Ci-CesA in C. intestinalis embryos, and the expression pattern of Ci-CesA was spatiotemporally consistent with observed cellulose synthesis in vivo. We propose here that urochordates may use a laterally acquired "homologous" gene for an analogous process of cellulose synthesis.

Tunicates are the only animals that perform cellulose biosynthesis. The tunicate gene for cellulose synthase, Ci-CesA, was likely acquired by horizontal transfer from bacteria and was a key innovation in the evolution of tunicates. Transposon-based mutagenesis in an ascidian, Ciona intestinalis, has generated a mutant, swimming juvenile (sj). Ci-CesA is the gene responsible for the sj mutant, in which a drastic reduction in cellulose was observed in the tunic. Furthermore, during metamorphosis, which in ascidians convert the vertebrate-like larva into a sessile filter feeder, sj showed abnormalities in the order of metamorphic events. In normal larvae, the metamorphic events in the trunk region are initiated after tail resorption. In contrast, sj mutant larvae initiated the metamorphic events in the trunk without tail resorption. Thus, sj larvae show a ''swimming juvenile'' phenotype, the juvenile-like trunk structure with a complete tail and the ability to swim. It is likely that ascidian cellulose synthase is required for the coordination of the metamorphic events in the trunk and tail in addition to cellulose biosynthesis.Minos ͉ swimming juvenile ͉ Ci-CesA ͉ metamorphosis T unicates are the only animals able to perform cellulose biosynthesis (1-3). Cellulose synthase is an enzyme that has a central role in cellulose biosynthesis; the gene (Ci-CesA) for cellulose synthase is encoded in the Ciona intestinalis genome and expressed in the larval ectoderm (4). Recent molecular phylogenetic analyses of Ci-CesA and Cs-CesA of Ciona savignyi suggest that the gene has been acquired by horizontal transfer from bacteria (5, 6). Ascidian cellulose synthase may be involved in the formation of the tunic, the cellulose-containing structure that surrounds the surface of the body to protect against predators. However, functional analysis showing that ascidian cellulose synthase contributes to the tunic formation in vivo has not been reported.Mutant analysis is a powerful way to understand gene function. In an ascidian, C. savignyi, mutagenesis with a chemical mutagen N-ethyl-N-nitrosourea, successfully revealed the function of prickle (7) and tyrosinase (8). Recently, germ-line transgenesis has been achieved in two ascidians, C. intestinalis and C. savignyi, with a Tc1͞mariner superfamily transposon Minos (9-14). The insertion of Minos into the Ciona genome indicates that Minos can be used as a mutagen, because Minos disrupts the gene function if it is inserted into a genomic region that encodes a gene.Here, we have isolated a mutant, swimming juvenile (sj), which was caused by an insertion of Minos into the C. intestinalis genome. sj shows defects in the tunic structure of swimming larvae. In addition to the tunic phenotype, they showed an abnormal order of metamorphic events, suggesting that the regulation of the timing of these events is affected in sj mutants. Ci-CesA is the gene responsible for the sj mutant, and the drastic loss of cellulose was observed in the tunic of sj mutants. These results indicate that ascidian ...

Bacteriorhodopsin (BR) and halorhodopsin (HR) are light-driven outward proton and inward chloride pumps, respectively. They have similar protein architecture, being composed of seven-transmembrane helices that bind an all-trans-retinal. BR can be converted into a chloride pump by a single amino acid replacement at position 85, suggesting that BR and HR share a common transport mechanism, and the ionic specificity is determined by the amino acid at that position. However, HR cannot be converted into a proton pump by the corresponding reverse mutation. Here we mutated 6 and 10 amino acids of HR into BR-like, whereas such multiple HR mutants never pump protons. Light-induced Fourier transform infrared spectroscopy revealed that hydrogen bonds of the retinal Schiff base and water are both strong for BR and both weak for HR. Multiple HR mutants exhibit strong hydrogen bonds of the Schiff base, but the hydrogen bond of water is still weak. We concluded that the cause of nonfunctional conversion of HR is the lack of strongly hydrogen-bonded water, the functional determinant of the proton pump.

Mammalian gut microbiota are integral to host health. However, how this association began remains unclear. We show that in basal chordates the gut space is radially compartmentalized into a luminal part where food microbes pass and an almost axenic peripheral part, defined by membranous delamination of the gut epithelium. While this membrane, framed with chitin nanofibers, structurally resembles invertebrate peritrophic membranes, proteome supports its affinity to mammalian mucus layers, where gut microbiota colonize. In ray-finned fish, intestines harbor indigenous microbes, but chitinous membranes segregate these luminal microbes from the surrounding mucus layer. These data suggest that chitin-based barrier immunity is an ancient system, the loss of which, at least in mammals, provided mucus layers as a novel niche for microbial colonization. These findings provide a missing link for intestinal immune systems in animals, revealing disparate mucosal environment in model organisms and highlighting the loss of a proven system as innovation.