The complete genome sequence of probiotic Lactobacillus acidophilus ATCC 9224 isolated from sour milk.

We present the complete genome sequence of the probiotic strain Lactobacillus acidophilus ATCC 9224. The genome sequence provides a valuable resource for investigating the phylogenetic evolution of this lineage and conducting comparative genomics with other Lactobacillus strains and species.

The Genome Sequence of an Algal Strain of Nannochloropsis QH25.

Species of Nannochloropsis are single-celled Stramenopiles commonly used in microalgae-based technologies for the manufacturing of bioproducts. Nannochloropsis oceanica QH25 was isolated from an algal cultivation pond located in Imperial, Texas (USA). We used PacBio continuous long read (CLR) sequencing to produce a highly contiguous 29.34 Mb genome.

High-Quality Genome Assembly of Nannochloris desiccata 2437 and Its Associated Bacterial Community.

High-quality genome sequences were generated for the nonaxenic marine microalga Nannochloris desiccata UTEX 2437 and eight of its associated environmental bacterial species. N. desiccata UTEX 2437 is diploid, and its 20.738-Mbp nuclear genome sequence is assembled in 29 contigs.

Phylogenetic analyses and reclassification of the oleaginous marine species Nannochloris sp. "desiccata" (Trebouxiophyceae, Chlorophyta), formerly Chlorella desiccata, supported by a high-quality genome assembly.

Microalgae are diverse, with many gaps remaining in phylogenetic and physiological understanding. Thus, studying new microalgae species increases our broader comprehension of biological diversity, and evaluation of new candidates as algal production platforms can lead to improved productivity under a variety of cultivation conditions. Chlorella is a genus of fast-growing species often isolated from freshwater habitats and cultivated as a source of nutritional supplements. However, the use of freshwater increases competition with other freshwater needs. We identified Chlorella desiccata to be worthy of further investigation as a potential algae production strain, due to its isolation from a marine environment and its promising growth and biochemical composition properties. Long-read genomic sequencing was conducted for C. desiccata UTEX 2526, resulting in a high-quality, near chromosome level, diploid genome with an assembly length of 21.55 Mbp in only 18 contigs. We also report complete circular mitochondrial and chloroplast genomes. Phylogenomic and phylogenetic analyses using nuclear, chloroplast, 18S rRNA, and actin sequences revealed that this species clades within strains currently identified as Nannochloris (Trebouxiophyceae, Chlorophyta), leading to its reclassification as Nannochloris sp. "desiccata" UTEX 2526. The mode of cell division for this species is autosporulation, differing from the type species N. bacillaris. As has occurred across multiple microalgae genera, there are repeated examples of Nannochloris species reclassification in the literature. This high-quality genome assembly and phylogenetic analysis of the potential algal production strain Nannochloris sp. "desiccata" UTEX 2526 provides an important reference and useful tool for further studying this region of the phylogenetic tree.

Inhibition of DNA Methylation in Picochlorum soloecismus Alters Algae Productivity.

Eukaryotic organisms regulate the organization, structure, and accessibility of their genomes through chromatin remodeling that can be inherited as epigenetic modifications. These DNA and histone protein modifications are ultimately responsible for an organism's molecular adaptation to the environment, resulting in distinctive phenotypes. Epigenetic manipulation of algae holds yet untapped potential for the optimization of biofuel production and bioproduct formation; however, epigenetic machinery and modes-of-action have not been well characterized in algae. We sought to determine the extent to which the biofuel platform species Picochlorum soloecismus utilizes DNA methylation to regulate its genome. We found candidate genes with domains for DNA methylation in the P. soloecismus genome. Whole-genome bisulfite sequencing revealed DNA methylation in all three cytosine contexts (CpG, CHH, and CHG). While global DNA methylation is low overall (∼1.15%), it occurs in appreciable quantities (12.1%) in CpG dinucleotides in a bimodal distribution in all genomic contexts, though terminators contain the greatest number of CpG sites per kilobase. The P. soloecismus genome becomes hypomethylated during the growth cycle in response to nitrogen starvation. Algae cultures were treated daily across the growth cycle with 20 µM 5-aza-2'-deoxycytidine (5AZA) to inhibit propagation of DNA methylation in daughter cells. 5AZA treatment significantly increased optical density and forward and side scatter of cells across the growth cycle (16 days). This increase in cell size and complexity correlated with a significant increase (∼66%) in lipid accumulation. Site specific CpG DNA methylation was significantly altered with 5AZA treatment over the time course, though nitrogen starvation itself induced significant hypomethylation in CpG contexts. Genes involved in several biological processes, including fatty acid synthesis, had altered methylation ratios in response to 5AZA; we hypothesize that these changes are potentially responsible for the phenotype of early induction of carbon storage as lipids. This is the first report to utilize epigenetic manipulation strategies to alter algal physiology and phenotype. Collectively, these data suggest these strategies can be utilized to fine-tune metabolic responses, alter growth, and enhance environmental adaption of microalgae for desired outcomes.

Detection of Abrin-Like and Prepropulchellin-Like Toxin Genes and Transcripts Using Whole Genome Sequencing and Full-Length Transcript Sequencing of Abrus precatorius.

The sequenced genome and the leaf transcriptome of a near relative of Abrus pulchellus and Abrus precatorius was analyzed to characterize the genetic basis of toxin gene expression. From the high-quality genome assembly, a total of 26 potential coding regions were identified that contain genes with abrin-like, pulchellin-like, and agglutinin-like homology, with full-length transcripts detected in leaf tissue for 9 of the 26 coding regions. All of the toxin-like genes were identified within only five isolated regions of the genome, with each region containing 1 to 16 gene variants within each genomic region (<1 Mbp). The Abrusprecatorius cultivar sequenced here contains genes which encode for proteins that are homologous to certain abrin and prepropulchellin genes previously identified, and we observed substantial diversity of genes and predicted gene products in Abrus precatorius and previously characterized toxins. This suggests diverse toxin repertoires within Abrus, potentially the results of rapid toxin evolution.

Complete Genome Sequences of Historic Clostridioides difficile Food-Dwelling Ribotype 078 Strains in Canada Identical to That of the Historic Human Clinical Strain M120 in the United Kingdom.

Clostridioides (Clostridium) difficile is a spore-forming anaerobic bacterium that causes severe intestinal diseases in humans. Here, we report the complete genome sequence of the first C. difficile foodborne type strain (PCR ribotype 078) isolated from food animals in Canada in 2004, which has 100% similarity to the genome sequence of the historic human clinical strain M120.

Actin restructuring during Salmonella typhimurium infection investigated by confocal and super-resolution microscopy.

We have used super-resolution optical microscopy and confocal microscopy to visualize the cytoskeletal restructuring of HeLa cells that accompanies and enables Salmonella typhimurium internalization. Herein, we report the use of confocal microscopy to verify and explore infection conditions that would be compatible with super-resolution optical microscopy, using Alexa-488 labeled phalloidin to stain the actin cytoskeletal network. While it is well known that actin restructuring and cytoskeletal rearrangements often accompany and assist in bacterial infection, most studies have employed conventional diffraction-limited fluorescence microscopy to explore these changes. Here we show that the superior spatial resolution provided by single-molecule localization methods (such as direct stochastic optical reconstruction microscopy) enables more precise visualization of the nanoscale changes in the actin cytoskeleton that accompany bacterial infection. In particular, we found that a thin (100-nm) ring of actin often surrounds an invading bacteria 10 to 20 min postinfection, with this ring being transitory in nature. We estimate that a few hundred monofilaments of actin surround the S. typhimurium in this heretofore unreported bacterial internalization intermediate.

Rapid prototyping of robust and versatile microfluidic components using adhesive transfer tapes.

A rapid prototyping technique of microfluidic devices is presented using adhesive transfer tapes. Lab on a chip systems can integrate multiple microfluidic functions in a single platform. Therefore, any rapid prototyping technique should be flexible and robust to accommodate different aspects of microfluidic integrations. In this work, the versatility of using adhesive transfer tapes for microfluidic applications is demonstrated by fabricating a wide range of platform. Prototypes demonstrating microfluidic mixing, dielectrophoretic trapping, complex microchannel networks and biologically relevant high temperature reactions were fabricated in less than 30 min. A novel ready to use world-to-chip interface was also developed using the same fabrication platform. All components (e.g. tapes, electrodes, acoustic sources or heaters) were obtained as finished products alleviating any chemical or clean-room specific processing. Only a 2D CAD software, a CO2 laser cutter and a seam roller was utilized to fabricate the devices. Adhesive transfer tapes provide additional flexibility compared to common double sided tapes as they do not contain any carrier material layer. Demonstrated ability to sustain in a wide range of dynamic physical processes (mechanical, electrical, or thermal) validates the robustness and the versatility of adhesive transfer tapes as an option for developing integrated lab on a chip systems.

A rapid multiplex assay for nucleic acid-based diagnostics.

We have developed a rapid (under 4 hours), multiplex, nucleic acid assay, adapted to a microsphere array detection platform. We call this assay multiplex oligonucleotide ligation-PCR (MOL-PCR). Unlike other ligation-based assays that require multiple steps, our protocol consists of a single tube reaction, followed by hybridization to a Luminex microsphere array for detection. We demonstrate the ability of this assay to simultaneously detect diverse nucleic acid signatures (e.g., unique sequences, single nucleotide polymorphisms) in a single multiplex reaction. Detection probes consist of modular components that enable target detection, probe amplification, and subsequent capture onto microsphere arrays. To demonstrate the utility of our assay, we applied it to the detection of three biothreat agents, B. anthracis, Y. pestis, and F. tularensis. Combined with the ease and robustness of this assay, the results presented here show a strong potential of our assay for use in diagnostics and surveillance.

Soluble MD2 increases TLR4 levels on the epithelial cell surface.

The accessory protein MD2 has been implicated in LPS-mediated activation of the innate immune system by functioning as a co-receptor with TLR4 for LPS binding at the cell surface. Epithelial cells that play a role in primary immune response, such as in the lung or gut, often express TLR4, but are dependent on circulating soluble MD2 (sMD2) to bind TLR4 to assemble the functional receptor. In this study, we show that sMD2 incubation with HEK293 epithelial cells transfected with TLR4 increases the cell surface levels of TLR4 in the absence of LPS. Dose response studies reveal that a threshold sMD2 concentration (approximately 450 nM) stimulates maximal TLR4 levels on the cell surface, whereas higher concentrations of sMD2 (approximately 1800 nM) reduce these enhanced TLR4 levels. We show evidence that MD2 multimer formation is increased at these higher concentrations of sMD2 and that addition of LPS to sMD2-stimulated cells masks the enhanced TLR4 cell surface levels, most likely due to the LPS-induced downregulation of TLR4 by endocytosis following receptor stimulation. All together, these results support a model in which sMD2 binds to TLR4 and increases TLR4 levels at the cell surface by preventing TLR4 turnover through the endocytic pathway. Thus, sMD2 may prime epithelial cells for enhanced immunoresponsive function prior to LPS exposure.

Upregulation of I-CAM1 in response to beryllium exposure in small airway epithelial cells.

Chronic Beryllium Disease (CBD) is a delayed-type hypersensitivity immune reaction that leads to granuloma formation in the lungs and potentially severe loss of pulmonary function. Although the molecular mechanisms that mediate beryllium (Be)-stimulated granuloma formation are not well understood, cell adhesion molecules are likely to play a key role in the migration of immune cells to sites of inflammation. In this study, we examined the role of the cell adhesion molecule I-CAM1 in Be-stimulated small airway epithelial cells (SAECs). These epithelial cells line the airway and represent the first point of contact for inhaled foreign substances. We find that Be exposure specifically induced I-CAM1 expression on the cell surface of SAEC and release of soluble I-CAM1 into the extracellular medium. Furthermore, anti-I-CAM1 antibodies inhibited Be-stimulated adhesion of SAEC to the macrophage cell-line THP1, indicating that the Be-induced adhesive properties of SAEC are at least partly due to I-CAM1 expression. These studies support a model in which I-CAM1 cell adhesion functions may play a role in directing immune cells to the lung and activating a Be-specific immune response in Be hypersensitivity disease.