Actinobacteria of the acI lineage are the most abundant microbes in freshwater systems, but there are so far no pure living cultures of these organisms, possibly because of metabolic dependencies on other microbes. This, in turn, has hampered an in-depth assessment of the genomic basis for their success in the environment. Here we present genomes from 16 axenic cultures of acI Actinobacteria. The isolates were not only of minute cell size, but also among the most streamlined free-living microbes, with extremely small genome sizes (1.2-1.4 Mbp) and low genomic GC content. Genome reduction in these bacteria might have led to auxotrophy for various vitamins, amino acids and reduced sulphur sources, thus creating dependencies to co-occurring organisms (the 'Black Queen' hypothesis). Genome analyses, moreover, revealed a surprising degree of inter-and intraspecific diversity in metabolic pathways, especially of carbohydrate transport and metabolism, and mainly encoded in genomic islands. The striking genotype microdiversification of acI Actinobacteria might explain their global success in highly dynamic freshwater environments with complex seasonal patterns of allochthonous and autochthonous carbon sources. We propose a new order within Actinobacteria ('Candidatus Nanopelagicales') with two new genera ('Candidatus Nanopelagicus' and 'Candidatus Planktophila') and nine new species. DOI: https://doi.org/10. 1038/ismej.2017.156 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-144935 Journal Article Accepted Version Originally published at: Neuenschwander, Stefan M; Ghai, Rohit; Pernthaler, Jakob; Salcher, Michaela M (2018). Microdiversification in genome-streamlined ubiquitous freshwater Actinobacteria. The ISME journal,

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The Escherichia coli F18 receptor locus (ECF18R) has been genetically mapped to the halothane linkage group on porcine Chromosome (Chr) 6. In an attempt to obtain candidate genes for this locus, we isolated 5 cosmids containing the alpha (1,2)fucosyltransferase genes FUT1, FUT2, and the pseudogene FUT2P from a porcine genomic library. Mapping by fluorescence in situ hybridization placed all these clones in band q11 of porcine Chr 6 (SSC6q11). Sequence analysis of the cosmids resulted in the characterization of an open reading frame (ORF), 1098 bp in length, that is 82.3% identical to the human FUT1 sequence; a second ORF, 1023 bp in length, 85% identical to the human FUT2 sequence; and a third FUT-like sequence thought to be a pseudogene. The FUT1 and FUT2 loci therefore seem to be the porcine equivalents of the human blood group H and Secretor loci. Direct sequencing of the two ORFs in swine being either susceptible or resistant to adhesion and colonization by F18 fimbriated Escherichia coli (ECF18) revealed two polymorphisms at bp 307 (M307) and bp 857 (M857) of the FUT1 ORF. Analysis of these mutations in 34 Swiss Landrace families with 221 progeny showed close linkage with the locus controlling resistance and susceptibility to E. coli F18 adhesion and colonization in the small intestine (ECF18R), and with the locus of the blood group inhibitor S. A high linkage disequilibrium of M307-ECF18R in Large White pigs makes the M307 mutation a good marker for marker-assisted selection of E. coli F18 adhesion-resistant animals in this breed. Whether the FUT1 or possibly the FUT2 gene products are involved in the synthesis of carbohydrate structures responsible for bacterial adhesion remains to be determined.

Methylotrophic planktonic bacteria fulfill a particular role in the carbon cycle of lakes via the turnover of single-carbon compounds. We studied two planktonic freshwater lineages (LD28 and PRD01a001B) affiliated with Methylophilaceae (Betaproteobacteria) in Lake Zurich, Switzerland, by a combination of molecular and cultivation-based approaches. Their spatio-temporal distribution was monitored at high resolution (n = 992 samples) for 4 consecutive years. LD28 methylotrophs constituted up to 11 × 10 7 cells l − 1 with pronounced peaks in spring and autumn-winter, concomitant with blooms of primary producers. They were rare in the warm water layers during summer but abundant in the cold hypolimnion, hinting at psychrophilic growth. Members of the PRD01a001B lineage were generally less abundant but also had maxima in spring. More than 120 axenic strains from these so far uncultivated lineages were isolated from the pelagic zone by dilution to extinction. Phylogenetic analysis separated isolates into two distinct genotypes. Isolates grew slowly (μ max = 0.4 d − 1 ), were of conspicuously small size, and were indeed psychrophilic, with higher growth yield at low temperatures. Growth was enhanced upon addition of methanol and methylamine to sterile lake water. Genomic analyses of two strains confirmed a methylotrophic lifestyle with a reduced set of genes involved in C1 metabolism. The very small and streamlined genomes (1.36 and 1.75 Mb) shared several pathways with the marine OM43 lineage. As the closest described taxa (Methylotenera sp.) are only distantly related to either set of isolates, we propose a new genus with two species, that is, 'Candidatus Methylopumilus planktonicus' (LD28) and 'Candidatus Methylopumilus turicensis' (PRD01a001B).

Background-Living autologous vascular grafts with the capacity for regeneration and growth may overcome the limitations of contemporary artificial prostheses. Particularly in congenital cardiovascular surgery, there is an unmet medical need for growing replacement materials. Here we investigate growth capacity of tissue-engineered living pulmonary arteries in a growing lamb model. Methods and Results-Vascular grafts fabricated from biodegradable scaffolds (ID 18Ϯl mm) were sequentially seeded with vascular cells. The seeded constructs were grown in vitro for 21days using biomimetic conditions. Thereafter, these tissue-engineered vascular grafts (TEVGs) were surgically implanted as main pulmonary artery replacements in 14 lambs using cardiopulmonary bypass and followed up for Յ100 weeks. The animals more than doubled their body weight during the 2-year period. The TEVG showed good functional performance demonstrated by regular echocardiography at 20, 50, 80, and 100 weeks and computed tomography-angiography. In particular, there was no evidence of thrombus, calcification, stenosis, suture dehiscence, or aneurysm. There was a significant increase in diameter by 30% and length by 45%. Histology showed tissue formation reminiscent of native artery. Biochemical analysis revealed cellularity and proteoglycans and increased collagen contents in all of the groups, analogous to those of native vessels. The mechanical profiles of the TEVG showed stronger but less elastic tissue properties than native pulmonary arteries. Conclusions-This

Development of the mammary gland during puberty, pregnancy, and lactation is controlled by steroid and peptide hormones and growth factors. To determine the role of the insulin-like growth factors (IGFs) in this process we developed a transgenic model using the whey acidic protein (WAP) gene to direct expression of rat IGF-I and human IGF binding protein-3 (IGFBP-3) to mammary tissue during late pregnancy and throughout lactation.High levels of expression of transgenic IGF-I and IGFBP-3 were seen in lobular-alveolar cells by in situ hybridization. There was no obvious effect on mammary development during pregnancy and lactation; indeed, mothers were capable of nursing their pups normally and the only structural difference seen in the mammary glands at peak lactation was an overall smaller size of the alveoli. We also evaluated the role of IGF-I and IGFBP-3 in the remodeling of mammary tissue during involution. Compared with control animals, the process of involution was modified in both transgenic lines. The degree of apoptotic cells was lower in the WAP-IGF-I and WAP-BP-3 expressing mice. In addition, there was a more quiescent pattern of involution with residual lobular secretory ability and a muted host inflammatory reaction with fewer lumenal microcalcifications.These results demonstrate that IGF-I and IGFBP-3 may modulate the involutionary process of the lactating mammary gland. ( J. Clin. Invest. 1996. 97:2225-2232).

The alpha(1,2)fucosyltransferases (FUT1 and FUT2) contribute to the formation of blood group antigen structures, which are present on cell membranes and in secretions. In the present study we demonstrate that both FUT1 and FUT2 are expressed in the pig small intestine. FUT1 polymorphisms influence adhesion of F18 fimbriated Escherichia coli (ECF18) to intestinal mucosa, and FUT2 is associated with expression of erythrocyte antigen 0. The FUT1 polymorphisms result in amino acid substitutions at positions 103 (Ala-->Thr) and 286 (Arg-->Glu). Tightly controlled expression of the FUT2 gene results in either an abundance or an absence of mRNA in small intestinal mucosa. ECF18-resistant animals were shown to be homozygous for threonine at amino acid 103 of the FUT1 enzyme. Susceptibility to ECF18 adhesion appeared to be solely dependent on the activity of FUT1 in intestinal epithelia. In intestinal mucosae of ECF18-resistant pigs which expressed FUT1 but not FUT2 RNA, the levels of alpha(1,2)fucosyltransferase activity were significantly lower (28- to 45-fold, P<0.001) than in susceptible pigs. Moreover, lysates of CHO cells transfected with FUT1 constructs encoding threonine at amino acid position 103 also showed significantly reduced enzyme activity compared with constructs encoding alanine at this position. Our genetic and enzymatic studies support the hypothesis that the FUT1 enzyme, and particularly the amino acid at position 103, is likely important in the synthesis of a structure that enables adhesion of ECF18 bacteria to small intestinal mucosa.