The objective of this study was to evaluate the progression of the uterine microbiota from calving until establishment of metritis. Uterine swabs (n ‫؍‬ 72) collected at 0, 2, and 6 ؎ 2 days postpartum (dpp) from 12 metritic and 12 healthy cows were used for metagenomic sequencing of the 16S rRNA gene on the Illumina MiSeq platform. A heat map showed that uterine microbiota was established at calving. The microbiota changed rapidly from 0 to 6 ؎ 2 dpp, with a decrease in the abundance of Proteobacteria and an increase in the abundance of Bacteroidetes and Fusobacteria, which were dominant in metritic cows. Uterine microbiota composition was shared; however, metritic and healthy cows could be discriminated using relative abundance of bacterial genera at 0, 2, and 6 ؎ 2 dpp. Bacteroides was the main genus associated with metritis because it was the only genus that showed significantly greater abundance in cows with metritis. As the abundance of Bacteroides organisms increased, the uterine discharge score, a measure of uterine health, worsened. Fusobacterium was also an important genus associated with metritis because Fusobacterium abundance increased as Bacteroides abundance increased and the uterine discharge score worsened as the abundance increased. The correlation with uterine discharge score and the correlation with Bacteroides or Fusobacterium showed that other bacteria, such as Helcoccocus, Filifactor, and Porphyromonas, were also associated with metritis. There were also bacteria associated with uterine health, such as "Candidatus Blochmannia," Escherichia, Sneathia, and Pedobacter. Metritis is a huge concern for the dairy industry worldwide because it is highly prevalent (25 to 40%) and negatively affects the productivity, survival, and welfare of dairy cows (1). Diverse bacteria, including anaerobes and facultative anaerobes, were observed in the uteri of dairy cows within the first 2 weeks postpartum, but they were naturally cleared out within 60 days postpartum (dpp) (1). Culture-based studies observed that Escherichia coli, Trueperella pyogenes, Fusobacterium necrophorum, and Bacteroides spp. (e.g., Prevotella melaninogenica, formerly Bacteroides melaninogenicus) were commonly associated with endometritis or pyometra (1-3).Although culture-based studies have laid out the foundation of our understanding of the uterine microbiota, previous studies might have underestimated the microbial complexity of the intrauterine environment of cows postpartum, given that less than 1% of the microorganisms in many environments are readily cultured under standard laboratory conditions (4). In recent years, cultureindependent techniques such as clone library sequencing (5, 6) and pyrosequencing (7, 8) have been used to characterize the uterine microbiota of cows with metritis (5-7) and endometritis (7,8). Sequencing using the Illumina platform allows for deeper sequencing than has previously been feasible even with pyrosequencing (9). Indeed, evaluating the rarefaction curves from previous 16S rRNA sequencing studies ...

Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/ Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila. This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ. While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ.

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Legionella pneumophila survives and replicates inside host cells by secreting ~300 effectors through the Dot/Icm type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography (ECT) and immunofluorescence microscopy (IF) to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. ECT mapped the location of the core and accessory components of the Legionella core-transmembrane subcomplex revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. IF deciphered an early-stage assembly process that begins with targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that interestingly are homologs of the T6SS membrane complex components TssL and TssM, suggesting the Dot/Icm T4BSS is a hybrid system. Together these results revealed that the Dot/Icm complex assembles in an “axial-to-peripheral” pattern.