Vibrio natriegens has recently emerged as an alternative to Escherichia coli for molecular biology and biotechnology, but low-efficiency genetic tools hamper its development. Here, we uncover how to induce natural competence in V. natriegens and describe methods for multiplex genome editing by natural transformation (MuGENT). MuGENT promotes integration of multiple genome edits at high-efficiency on unprecedented time scales. Also, this method allows for generating highly complex mutant populations, which can be exploited for metabolic engineering efforts. As a proof-of-concept, we attempted to enhance production of the value added chemical poly-β-hydroxybutyrate (PHB) in V. natriegens by targeting the expression of nine genes involved in PHB biosynthesis via MuGENT. Within 1 week, we isolated edited strains that produced ∼100 times more PHB than the parent isolate and ∼3.3 times more than a rationally designed strain. Thus, the methods described here should extend the utility of this species for diverse academic and industrial applications.

Development of antibody protection during SARS-CoV-2 infection is a pressing question for public health and for vaccine development. We developed highly sensitive SARS-CoV-2-specific antibody and neutralization assays. SARS-CoV-2 Spike protein or Nucleocapsid protein specific IgG antibodies at titers more than 1:100,000 were detectable in all PCR+ subjects (n = 115) and were absent in the negative controls. Other isotype antibodies (IgA, IgG1-4) were also detected. SARS-CoV-2 neutralization was determined in COVID-19 and convalescent plasma at up to 10,000-fold dilution, using Spike protein pseudotyped lentiviruses, which were also blocked by neutralizing antibodies (NAbs). Hospitalized patients had up to 3000-fold higher antibody and neutralization titers compared to outpatients or convalescent plasma donors. Interestingly, some COVID-19 patients also possessed NAbs against SARS-CoV Spike protein pseudovirus. Together these results demonstrate the high specificity and sensitivity of our assays, which may impact understanding the quality or duration of the antibody response during COVID-19 and in determining the effectiveness of potential vaccines.

The genus Alphavirus consists of a group of enveloped, single-stranded RNA viruses, many of which are transmitted by arthropods to a wide range of vertebrate host species. Here we report that Sindbis virus (SINV) produced from a representative mammalian cell line consists of at least two unique particle subpopulations, separable on the basis of virion density. In contrast, mosquito-derived SINV consists of a homogeneous population of particles. Our findings indicate that the denser particle subpopulation, SINV Heavy , is more infectious on a per-particle basis than SINV Light . SINV produced in mosquito cell lines (SINV C6/36 ) exhibited particle-to-PFU ratios similar to those observed for SINV Heavy . In mammalian cells, viral RNA was synthesized and accumulated more rapidly following infection with SINV Heavy or SINV C6/36 than following infection with SINV Light , due partly to enhanced translation of viral genomic RNA early in infection. Analysis of the individual particle subpopulations indicated that SINV Heavy and SINV C6/36 contain host-derived factors whose presence correlates with the enhanced translation, RNA synthesis, and infectivity observed for these particles. Members of the genus Alphavirus, of the family Togaviridae, are a group of enveloped positive-sense RNA viruses with a wide host range. For the mosquito-borne species, the virus is maintained in the enzootic cycle through transmission between a sylvatic reservoir and the mosquito host (1). The maintenance of this cycle directly affects the genetic fitness of the mosquito-borne alphaviruses. Prolonged disruption of this cycle leads to deleterious effects on viral transmission as the virus becomes adapted to a single host (2-5). Spillover from the enzootic cycle often results in the tangential infection of both humans and equines, which can result in significant outbreaks of disease. The outcome of alphaviral infection is dependent on the host system (6-12). Infection of mosquito cells does not result in the shutoff of host macromolecular synthesis and often culminates in persistent infection for the majority of mosquito cell lines (12-15). Nevertheless, cell death as a result of infection has been reported for several members of the genus in whole mosquitoes (16)(17)(18)(19)(20)(21). In contrast, infection of mammalian cells induces the shutoff of host macromolecular synthesis, resulting in a predominantly cytolytic infection. In vertebrates, the immune response to infection generally results in virus clearance. This is initiated by the recognition of viral doublestranded RNA and a rapid type I interferon (IFN-␣/␤) response (22)(23)(24)(25).Previously, we reported that the infectivity of Sindbis virus (SINV), as measured by the ratio of particles to infectious units, depends on the host cell line from which it is derived (26). SINV derived from mammalian cell lines exhibited a higher particle-to-PFU ratio, on average, than SINV generated from mosquito cell lines. This was due largely to differences in the quantity of total virus particles pr...

SUMMARY Vibrio cholerae is a natural resident of the aquatic environment, where a common nutrient is the chitinous exoskeletons of microscopic crustaceans. Chitin utilization requires chitinases, which degrade this insoluble polymer into soluble chitin oligosaccharides. These oligosaccharides also serve as an inducing cue for natural transformation in Vibrio species. There are 7 predicted endochitinase-like genes in the V. cholerae genome. Here, we systematically dissect the contribution of each gene to growth on chitin as well as induction of natural transformation. Specifically, we created a strain that lacks all 7 putative chitinases and from this strain, generated a panel of strains where each expresses a single chitinase. We also generated expression plasmids to ectopically express all 7 chitinases in our chitinase deficient strain. Through this analysis, we found that low levels of chitinase activity are sufficient for natural transformation, while growth on insoluble chitin as a sole carbon source requires more robust and concerted chitinase activity. We also assessed the role that the three uptake systems for the chitin degradation products GlcNAc, (GlcNAc)2, and (GlcN)2, play in chitin utilization and competence induction. Cumulatively, this study provides mechanistic details for how this pathogen utilizes chitin to thrive and evolve in its environmental reservoir.

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