Engineering Bacillus subtilis for the formation of a durable living biocomposite material.

Engineered living materials (ELMs) are a fast-growing area of research that combine approaches in synthetic biology and material science. Here, we engineer B. subtilis to become a living component of a silica material composed of self-assembling protein scaffolds for functionalization and cross-linking of cells. B. subtilis is engineered to display SpyTags on polar flagella for cell attachment to SpyCatcher modified secreted scaffolds. We engineer endospore limited B. subtilis cells to become a structural component of the material with spores for long-term storage of genetic programming. Silica biomineralization peptides are screened and scaffolds designed for silica polymerization to fabricate biocomposite materials with enhanced mechanical properties. We show that the resulting ELM can be regenerated from a piece of cell containing silica material and that new functions can be incorporated by co-cultivation of engineered B. subtilis strains. We believe that this work will serve as a framework for the future design of resilient ELMs.

Characterization of Tomato Necrotic Spot Virus, a Subgroup 1 Ilarvirus Causing Necrotic Foliar, Stem, and Fruit Symptoms in Tomatoes in the United States.

The genomic, biological, and serological characterization of tomato necrotic spot virus (ToNSV), a virus first described infecting tomato in California, was completed. The complete genomic sequence identified ToNSV as a new subgroup 1 ilarvirus distinct from the previously described tomato-infecting ilarviruses. We identified ToNSV in Indiana in 2017 and 2018 and in Ohio in 2018. The coat protein coding region of the isolates from California, Indiana, and Ohio have 94 to 98% identity, while the same isolates had 99% amino acid identity. ToNSV is serologically related to TSV, a subgroup 1 ilarvirus, and shows no serological relationship to ilarviruses in the other subgroups. In tomato, ToNSV caused symptoms of necrotic spots and flecks on leaves, necrotic streaking on stems, and necrotic spots and circular patterns on fruit resulting in a yield loss of 1 to 13%. These results indicate that ToNSV is a proposed new subgroup 1 ilarvirus causing a necrotic spotting disease of tomato observed in California, Indiana, and Ohio.

Characterization of a New Nepovirus Causing a Leaf Mottling Disease in Petunia hybrida.

Icosahedral virus-like particles were isolated from Petunia hybrida cuttings with interveinal chlorotic mottling. The virus was transmitted by mechanical inoculation from infected to healthy P. hybrida, and was found to contain a bipartite RNA genome of 7.6 and 3.8 kilobases. Full genomic sequence was obtained by high-throughput sequencing combined with RACE amplification of the 5'-termini of RNAs 1 and 2, and reverse-transcription PCR amplification of the 3'-termini with oligo-dT and sequence specific primers. Based on particle morphology, genome organization, and phylogenetic analyses, it was concluded that the new virus is a member of the genus Nepovirus, subgroup A. This new virus causing a leaf mottling disease of petunia was provisionally named Petunia chlorotic mottle virus (PCMoV).

The two suborders of chiropterans have the canonical heavy-chain immunoglobulin (Ig) gene repertoire of eutherian mammals.

Bats comprise 20% of all mammals, yet little is known about their immune system and virtually nothing about their immunoglobulin genes. We show that four different bat species transcribe genes encoding IgM, IgE, IgA and IgG subclasses, the latter which have diversified after speciation; the canonical pattern for eutherian mammals. IgD transcripts were only recovered from insectivorous bats and were comprised of CH1, CH3 and two hinge exons; the second hinge exon was fused to CH3. IgA in all species resembles human IgA2 with the putative cysteine forming the bridge to the light chain found at position 77. Sequence comparisons yielded no evidence for a diphyletic origin of the suborders. Bats show no close similarity to another mammalian order; the strongest association was with carnivores. Data reveal that CH diversity and VDJ and CDR3 organization are similar to other eutherian mammals, although the expressed VH3 family repertoire was unusually diverse.

The little brown bat, M. lucifugus, displays a highly diverse V H, D H and J H repertoire but little evidence of somatic hypermutation.

Myotis lucifugus populations in Northeastern US are being decimated by a fungal disease. Since almost nothing is known about the immune system of bats, we are characterizing the immunoglobulin genes of bats. We show that M. lucifugus has a diverse V(H) gene repertoire comprised of five of the seven human V(H) gene families and an estimated 236V(H)3 genes. 95% of these germline VH3 genes differ in FR3. A comparison of 67 expressed V(H)3 genes with 75 germline V(H)3 genes revealed a mutation frequency similar to fetal piglets never exposed to environmental antigens. Analysis of CDR3 regions identified at least 13 putative J(H) segments and a large D(H) repertoire. The low mutation frequency, highly diverse V(H), D(H), and J(H) germline repertoire suggests that this species may rely more on combinatorial and junctional diversity than on somatic hypermutation raising questions about the ability of M. lucifugus to respond rapidly to emerging pathogens.