Iron uptake in proteobacteria by TonB-dependent outer membrane transporters represents a well-explored subject. In contrast, the same process has been scarcely investigated in cyanobacteria. The heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120 is known to secrete the siderophore schizokinen, but its transport system has remained unidentified. Inspection of the genome of strain PCC 7120 shows that only one gene encoding a putative TonB-dependent iron transporter, namely alr0397, is positioned close to genes encoding enzymes involved in the biosynthesis of a hydroxamate siderophore. The expression of alr0397, which encodes an outer membrane protein, was elevated under iron-limited conditions. Inactivation of this gene caused a moderate phenotype of iron starvation in the mutant cells. The characterization of the mutant strain showed that Alr0397 is a TonB-dependent schizokinen transporter (SchT) of the outer membrane and that alr0397 expression and schizokinen production are regulated by the iron homeostasis of the cell.

The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Days 11 and 12 of gestation. Concomitant with trophoblast elongation, production of conceptus estrogen, the porcine embryonic pregnancy recognition signal, increases. Conceptus attachment to the uterine surface epithelium starts after Day 13, initiating epitheliochorial placentation. To analyze the transcriptome changes in the endometrium in the course of maternal recognition of pregnancy, deep sequencing of endometrial RNA samples of Day 12 pregnant animals (n = 4) and corresponding nonpregnant controls (n = 4) was performed using RNA sequencing (RNA-Seq). Between 30 000 000 and 35 000 000 sequence reads per sample were produced and mapped to the porcine genome (Sscrofa10.2). Analysis of read counts revealed 2593 differentially expressed genes (DEGs). Expression of selected genes was validated by the use of quantitative real-time RT-PCR. Bioinformatics analysis identified several functional terms specifically overrepresented for up-regulated or down-regulated genes. Comparison of the RNA-Seq data from Days 12 and 14 of pregnancy was performed at the level of all expressed genes, the level of the DEG, and the level of functional categories. This revealed specific gene expression patterns reflecting the different functions of the endometrium during these stages (i.e., recognition of pregnancy and preparation for conceptus attachment). Genes related to mitosis, immune response, epithelial cell differentiation and development, proteolysis, and prostaglandin signaling and metabolism are discussed in detail. This study identified comprehensive transcriptome changes in porcine endometrium associated with establishment of pregnancy and could be a resource for targeted studies of genes and pathways potentially involved in regulation of this process.

In pigs, conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of initial conceptus attachment, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding cyclic controls (n = 4) was performed using Illumina RNA-Seq. The obtained sequence reads were mapped to the porcine genome, and relative expression values were calculated for the analysis of differential gene expression. Statistical analysis revealed 1933 differentially expressed genes (false discovery rate 1%), 1229 with higher and 704 with lower mRNA concentration, in the samples from pregnant animals. Expression of selected genes was validated by the use of quantitative real-time RT-PCR. The RNA-Seq data were compared to results of a microarray study of bovine endometrium on Day 18 of pregnancy and additional related data sets. Bioinformatics analysis revealed for the genes with higher mRNA concentration in pregnant samples strong overrepresentation, particularly for immune-related functional terms but also for apoptosis and cell adhesion. Overrepresented terms for the genes with lower mRNA concentration in pregnant samples were related to extracellular region, ion transport, cell adhesion, and lipid and steroid metabolic process. In conclusion, RNA-Seq analysis revealed comprehensive transcriptome differences in porcine endometrium between Day 14 of pregnancy and corresponding cyclic endometrium and highlighted new processes and pathways probably involved in regulation of noninvasive implantation in the pig.

Iron uptake is essential for Gram-negative bacteria including cyanobacteria. In cyanobacteria, however, the iron demand is higher than in proteobacteria due to the function of iron as a cofactor in photosynthesis and nitrogen fixation, but our understanding of iron uptake by cyanobacteria stands behind the knowledge in proteobacteria. Here, two genes involved in this process in the heterocyst-forming cyanobacterium Anabaena sp. PCC 7120 were identified. ORF all4025 encodes SchE, a putative cytoplasmic membrane-localized transporter involved in TolC-dependent siderophore secretion. Inactivation of schE resulted in an enhanced sensitivity to high metal concentrations and decreased secretion of hydroxamate-type siderophores. ORF all4026 encodes a predicted outer membrane-localized TonB-dependent iron transporter, IacT. Inactivation of iacT resulted in decreased sensitivity to elevated iron and copper levels. Expression of iacT from the artificial trc promoter (P(trc)) resulted in sensitization against tested metals. Further analysis showed that iron and copper effects are synergistic because a decreased supply of iron induced a significant decrease of copper levels in the iacT insertion mutant but an increase of those levels in the strain carrying P(trc)-iacT. Our results unravel a link between iron and copper homeostasis in Anabaena sp. PCC 7120.

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In mammals, an efficient exchange of molecular signals between the embryo and the maternal environment plays a crucial role for the implantation and development of early embryos as well as for recognition and maintenance of pregnancy. So far, only a few molecular signals involved in this process have been identified. To address the underlying biochemical processes in pigs at the protein level, we performed a quantitative proteome study with endometrial tissue samples from non-pregnant and pregnant (Day 14) sows. Endometrium samples (lamina epithelialis, lamina propria and tela submucosa; n = 4 per group) were taken from sites of embryonic attachment and from comparable locations in nonpregnant animals. Proteome data were generated by iTRAQ labelling and nano-LC-MS/MS measurements of tryptic endometrium peptides on a high-resolution Orbitrap XL mass spectrometer. To further address and visualize protein isoforms, LC-MS/MS experiments were complemented by 2D gel-based analyses. To enhance the accuracy of protein quantification, the 2D fluorescence difference gel electrophoresis (2D-DIGE) technique was used, including internal pooled standards for inter-gel matching and normalization. The statistical and bioinformatics analysis of 2D-DIGE and iTRAQ data revealed 14 proteins being significantly altered in abundance (fold-change values >1.5, maximum fold-change 13; P < 0.05) between the endometrium proteomes of pregnant and non-pregnant animals. Several of the affected proteins are already known to play an important role in embryo-maternal communication in other species; for example, signal transducer and activator of transcription 1 (STAT1), a protein mediating the cellular response of cells to interferons (IFN) or aldose reductase (AKR1B1), for which a key role in the synthesis of endometrial prostaglandin F is supposed. Several other proteins showing alterations in abundance between pregnant and nonpregnant endometrial tissues were not described previously and represent new and interesting targets for further functional studies addressing their role during early pregnancy.

Establishment of pregnancy in mammals requires prolongation of luteal life span for sustained progesterone (P4) production. P4 stimulates secretory functions of the endometrium required for conceptus growth and development and is essential for endometrial receptivity. The porcine conceptus undergoes rapid differentiation and expansion of its trophoblastic membranes between Day 11 and Day 12 of gestation. With trophoblast elongation production of estrogen, the porcine embryonic pregnancy recognition signal, by the conceptus increases. A complex interplay of estrogen signaling and prostaglandin (PG) metabolism in the endometrium finally results in prevention of luteolysis. Conceptus attachment to the uterine surface epithelium starts around Day 14 of pregnancy, preceded by a pronounced vascularization at the implantation zones, initiating the epitheliochorial placentation. To characterize the complex transcriptome changes in the endometrium in the course of maternal recognition of pregnancy and initial placentation, deep sequencing of endometrial RNA samples of pregnant animals (n = 4) and corresponding non-pregnant controls (n = 4) was performed on Day 12 and Day 14 after ovulation using Illumina RNA-Seq (Illumina Inc., San Diego, CA, USA). Between 21 and 36 million 76-bp single-end reads were produced per sample. Reads were mapped with TopHat to the porcine genome assembly Sscrofa10.2 and relative expression values were calculated based on the National Center for Biotechnology Information’s gene annotation for the analysis of differential gene expression. Statistical analysis revealed more than 3500 differentially expressed genes (DEG) for Day 12 and almost 2500 DEG for Day 14 of pregnancy (DESeq, FDR 1%). Differential expression of selected genes was validated by the use of quantitative real-time PCR (RT-PCR). Comparison of the RNA-Seq data from Day 12 and Day 14 of pregnancy based on a comparison of the DEGs and of overrepresented functional categories revealed distinct differences in gene regulation, reflecting the different functions of the endometrium during these stages; that is, recognition of pregnancy on Day 12 and preparation for conceptus implantation on Day 14. The data from Day 14 was also compared to the published microarray data set from Day 14 of pregnancy (Østrup et al. 2010 Biol. Reprod.) and other sets of DEG by the use of gene set enrichment analysis (GSEA). Overall, a very good agreement with the results of the previous microarray study was found. Furthermore, a significant overlap with genes upregulated in bovine endometrium on Day 18 of pregnancy was found for the genes upregulated in porcine endometrium on Day 14. More than half of these genes were known interferon-regulated genes. In conclusion, RNA-Seq revealed distinct transcriptome changes on Day 12 and Day 14 of pregnancy in porcine endometrium associated with MRP and initiation of implantation.