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 to 10 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-β-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.

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.

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.

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.

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.