The next generation of biopanning: next gen sequencing improves analysis of bacterial display libraries.

BACKGROUND: Bacterial surface display libraries are a popular tool for novel ligand discovery due to their ease of manipulation and rapid growth rates. These libraries typically express a scaffold protein embedded within the outer membrane with a short, surface-exposed peptide that is either terminal or is incorporated into an outer loop, and can therefore interact with and bind to substrates of interest. RESULTS: In this study, we employed a novel bacterial peptide display library which incorporates short 15-mer peptides on the surface of E. coli, co-expressed with the inducible red fluorescent protein DsRed in the cytosol, to investigate population diversity over two rounds of biopanning. The naive library was used in panning trials to select for binding affinity against 3D printing plastic coupons made from polylactic acid (PLA). Resulting libraries were then deep-sequenced using next generation sequencing (NGS) to investigate selection and diversity. CONCLUSIONS: We demonstrated enrichment for PLA binding versus a sapphire control surface, analyzed population composition, and compared sorting rounds using a binding assay and fluorescence microscopy. The capability to produce and describe display libraries through NGS across rounds of selection allows a deeper understanding of population dynamics that can be better directed towards peptide discovery.

Toward Spider Glue: Long Read Scaffolding for Extreme Length and Repetitious Silk Family Genes AgSp1 and AgSp2 with Insights into Functional Adaptation.

An individual orb weaving spider can spin up to seven different types of silk, each with unique functions and material properties. The capture spiral silk of classic two-dimensional aerial orb webs is coated with an amorphous glue that functions to retain prey that get caught in a web. This unique modified silk is partially comprised of spidroins (spider fibroins) encoded by two members of the silk gene family. The glue differs from solid silk fibers as it is a viscoelastic, amorphic, wet material that is responsive to environmental conditions. Most spidroins are encoded by extremely large, highly repetitive genes that cannot be sequenced using short read technology alone, as the repetitive regions are longer than read length. We sequenced for the first time the complete genomic Aggregate Spidroin 1 (AgSp1) and Aggregate Spidroin 2 (AgSp2) glue genes of orb weaving spider Argiope trifasciata using error-prone long reads to scaffold for high accuracy short reads. The massive coding sequences are 42,270 bp (AgSp1) and 20,526 bp (AgSp2) in length, the largest silk genes currently described. The majority of the predicted amino acid sequence of AgSp1 consists of two similar but distinct motifs that are repeated ∼40 times each, while AgSp2 contains ∼48 repetitions of an AgSp1-similar motif, interspersed by regions high in glutamine. Comparisons of AgSp repetitive motifs from orb web and cobweb spiders show regions of strict conservation followed by striking diversification. Glues from these two spider families have evolved contrasting material properties in adhesion (stickiness), extensibility (stretchiness), and elasticity (the ability of the material to resume its native shape), which we link to mechanisms established for related silk genes in the same family. Full-length aggregate spidroin sequences from diverse species with differing material characteristics will provide insights for designing tunable bio-inspired adhesives for a variety of unique purposes.

Arabinose-Induced Catabolite Repression as a Mechanism for Pentose Hierarchy Control in Clostridium acetobutylicum ATCC 824.

Bacterial fermentation of carbohydrates from sustainable lignocellulosic biomass into commodity chemicals by the anaerobic bacterium Clostridium acetobutylicum is a promising alternative source to fossil fuel-derived chemicals. Recently, it was demonstrated that xylose is not appreciably fermented in the presence of arabinose, revealing a hierarchy of pentose utilization in this organism (L. Aristilde, I. A. Lewis, J. O. Park, and J. D. Rabinowitz, Appl Environ Microbiol 81:1452-1462, 2015, https://doi.org/10.1128/AEM.03199-14). The goal of the current study is to characterize the transcriptional regulation that occurs and perhaps drives this pentose hierarchy. Carbohydrate consumption rates showed that arabinose, like glucose, actively represses xylose utilization in cultures fermenting xylose. Further, arabinose addition to xylose cultures led to increased acetate-to-butyrate ratios, which indicated a transition of pentose catabolism from the pentose phosphate pathway to the phosphoketolase pathway. Transcriptome sequencing (RNA-Seq) confirmed that arabinose addition to cells actively growing on xylose resulted in increased phosphoketolase (CA_C1343) mRNA levels, providing additional evidence that arabinose induces this metabolic switch. A significant overlap in differentially regulated genes after addition of arabinose or glucose suggested a common regulation mechanism. A putative open reading frame (ORF) encoding a potential catabolite repression phosphocarrier histidine protein (Crh) was identified that likely participates in the observed transcriptional regulation. These results substantiate the claim that arabinose is utilized preferentially over xylose in C. acetobutylicum and suggest that arabinose can activate carbon catabolite repression via Crh. Furthermore, they provide valuable insights into potential mechanisms for altering pentose utilization to modulate fermentation products for chemical production. IMPORTANCE Clostridium acetobutylicum can ferment a wide variety of carbohydrates to the commodity chemicals acetone, butanol, and ethanol. Recent advances in genetic engineering have expanded the chemical production repertoire of C. acetobutylicum using synthetic biology. Due to its natural properties and genetic engineering potential, this organism is a promising candidate for converting biomass-derived feedstocks containing carbohydrate mixtures to commodity chemicals via natural or engineered pathways. Understanding how this organism regulates its metabolism during growth on carbohydrate mixtures is imperative to enable control of synthetic gene circuits in order to optimize chemical production. The work presented here unveils a novel mechanism via transcriptional regulation by a predicted Crh that controls the hierarchy of carbohydrate utilization and is essential for guiding robust genetic engineering strategies for chemical production.

Engineered integrative and conjugative elements for efficient and inducible DNA transfer to undomesticated bacteria.

Engineering microorganisms to promote human or plant health will require manipulation of robust bacteria that are capable of surviving in harsh, competitive environments. Genetic engineering of undomesticated bacteria can be limited by an inability to transfer DNA into the cell. Here we developed an approach based on the integrative and conjugative element from Bacillus subtilis (ICEBs1) to overcome this problem. A donor strain (XPORT) was built to transfer miniaturized integrative and conjugative elements (mini-ICEBs1) to undomesticated bacteria. The strain was engineered to enable inducible control over conjugation, to integrate delivered DNA into the chromosome of the recipient, to restrict spread of heterologous DNA through separation of the type IV secretion system from the transferred DNA, and to enable simple isolation of engineered bacteria through a D-alanine auxotrophy. Efficient DNA transfer (10-1 to 10-7 conjugation events per donor) is demonstrated using 35 Gram-positive strains isolated from humans (skin and gut) and soil. Mini-ICEBs1 was used to rapidly characterize the performance of an isopropyl-ß-D-thiogalactoside (IPTG)-inducible reporter across dozens of strains and to transfer nitrogen fixation to four Bacillus species. Finally, XPORT was introduced to soil to demonstrate DNA transfer under non-ideal conditions.

The Heme Transport Capacity of LHR1 Determines the Extent of Virulence in Leishmania amazonensis.

Leishmania spp. are trypanosomatid parasites that replicate intracellularly in macrophages, causing serious human morbidity and mortality throughout the world. Trypanosomatid protozoa cannot synthesize heme, so must acquire this essential cofactor from their environment. Earlier studies identified LHR1 as a Leishmania amazonensis transmembrane protein that mediates heme uptake. Null mutants of LHR1 are not viable and single knockout strains have reduced virulence, but very little is known about the properties of LHR1 directly associated with heme transport. Here, we use functional assays in Saccharomyces cerevisiae to show that specific tyrosine residues within the first three predicted transmembrane domains of LHR1 are required for efficient heme uptake. These tyrosines are unique to LHR1, consistent with the low similarity between LHR1 and its corresponding homologs in C. elegans and human. Substitution of these tyrosines in LHR1 resulted in varying degrees of heme transport inhibition, phenotypes that closely mirrored the impaired ability of L. amazonensis to replicate as intracellular amastigotes in macrophages and generate cutaneous lesions in mice. Taken together, our results imply that the mechanism for heme transport by LHR1 is distinctive and may have adapted to secure heme, a limiting cofactor, inside the host. Since LHR1 is significantly divergent from the human heme transporter HRG1, our findings lay the groundwork for selective targeting of LHR1 by small molecule antagonists.

Pathways of iron acquisition and utilization in Leishmania.

Iron is essential for many metabolic pathways, but is toxic in excess. Recent identification of the ferric iron reductase LFR1, the ferrous iron transporter LIT1, and the heme transporter LHR1 greatly advanced our understanding of how Leishmania parasites acquire iron and regulate its uptake. LFR1 and LIT1 have close orthologs in plants, and are required for Leishmania virulence. Consistent with the lack of heme biosynthesis in trypanosomatids, LHR1 and LABCG5, a protein involved in heme salvage from hemoglobin, seem essential for Leishmania survival. LFR1, LIT1 and LHR1 are upregulated under low iron availability, in agreement with the need to prevent excessive iron uptake. Future studies should clarify how Leishmania interacts with the iron homeostasis machinery of its host cell, the macrophage.

Heme uptake by Leishmania amazonensis is mediated by the transmembrane protein LHR1.

Trypanosomatid protozoan parasites lack a functional heme biosynthetic pathway, so must acquire heme from the environment to survive. However, the molecular pathway responsible for heme acquisition by these organisms is unknown. Here we show that L. amazonensis LHR1, a homolog of the C. elegans plasma membrane heme transporter HRG-4, functions in heme transport. Tagged LHR1 localized to the plasma membrane and to endocytic compartments, in both L. amazonensis and mammalian cells. Heme deprivation in L. amazonensis increased LHR1 transcript levels, promoted uptake of the fluorescent heme analog ZnMP, and increased the total intracellular heme content of promastigotes. Conversely, deletion of one LHR1 allele reduced ZnMP uptake and the intracellular heme pool by approximately 50%, indicating that LHR1 is a major heme importer in L. amazonensis. Viable parasites with correct replacement of both LHR1 alleles could not be obtained despite extensive attempts, suggesting that this gene is essential for the survival of promastigotes. Notably, LHR1 expression allowed Saccharomyces cerevisiae to import heme from the environment, and rescued growth of a strain deficient in heme biosynthesis. Syntenic genes with high sequence identity to LHR1 are present in the genomes of several species of Leishmania and also Trypanosoma cruzi and Trypanosoma brucei, indicating that therapeutic agents targeting this transporter could be effective against a broad group of trypanosomatid parasites that cause serious human disease.

Cell death in bovine parvovirus-infected embryonic bovine tracheal cells is mediated by necrosis rather than apoptosis.

The helper-independent bovine parvovirus (BPV) was studied to determine its effect on host embryonic bovine tracheal (EBTr) cells: whether the ultimate outcome of infection results in apoptotic cell death or cell death by necrosis. Infected cells were observed for changes marking apoptosis. Observations of alterations in nuclear morphology, membrane changes, apoptotic body formation, membrane phosphatidylserine inversions, caspase activation and cell DNA laddering in infected cells were not indicative of apoptosis. On the other hand, at the end of the virus replication cycle, infected cells released viral haemagglutinin and infectious virus particles, as would be expected from cell membrane failure. Moreover, the infected cells released lactate dehydrogenase (LDH), release of which is a marker of necrosis. LDH release into the cell medium correlated directly with viral m.o.i. and time post-infection. Furthermore, assessment of mitochondrial dehydrogenase activity was consistent with cell death by necrosis. Taken together, these findings indicate that cell death in BPV-infected EBTr cells is due to necrosis, as defined by infected-cell membrane failure and release of the cell contents into the extracellular environment.