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The virus severe acute respiratory syndrome coronavirus 2, SARS-CoV-2, is the causative agent of the current COVID-19 pandemic. It possesses a large 30 kilobase (kb) genome that encodes structural, non-structural, and accessory proteins. Although not necessary to cause disease, these accessory proteins are known to influence viral replication and pathogenesis. Through the synthesis of novel infectious clones of SARS-CoV-2 that lack one or more of the accessory proteins of the virus, we have found that one of these accessory proteins, ORF8, is critical for the modulation of the host inflammatory response. Mice infected with a SARS-CoV-2 virus lacking ORF8 exhibit increased weight loss and exacerbated macrophage infiltration into the lungs. Additionally, infection of mice with recombinant SARS-CoV-2 viruses encoding ORF8 mutations found in variants of concern reveal that naturally occurring mutations in this protein influence disease severity. Our studies with a virus lacking this ORF8 protein and viruses possessing naturally occurring point mutations in this protein demonstrate that this protein impacts pathogenesis., Competing Interests: M.B.F. has collaborative research agreements with Novavax, AstraZeneca, Regeneron, and Irazu Bio. These do not have any effect on the planning or interpretations of the work presented in this manuscript., (Copyright: © 2024 McGrath et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Mycoplasmas are atypical bacteria with small genomes that necessitate colonization of their respective animal or plant hosts as obligate parasites, whether as pathogens, or commensals. Some can grow axenically in specialized complex media yet show only host-cell-dependent growth in cell culture, where they can survive chronically and often through interactions involving surface colonization or internalization. To develop a mycoplasma-based system to identify genes mediating such interactions, we exploited genetically tractable strains of the goat pathogen Mycoplasma mycoides ( Mmc ) with synthetic designer genomes representing the complete natural organism (minus virulence factors; JCVI-syn1.0) or its reduced counterpart (JCVI-syn3B) containing only those genes supporting axenic growth. By measuring growth of surviving organisms, physical association with cultured human cells (HEK-293T, HeLa), and induction of phagocytosis by human myeloid cells (dHL-60), we determined that JCVI-syn1.0 contained a set of eight genes ( MMSYN1-0179 to MMSYN1-0186 , dispensable for axenic growth) conferring survival, attachment, and phagocytosis phenotypes. JCVI-syn3B lacked these phenotypes, but insertion of these genes restored cell attachment and phagocytosis, although not survival. These results indicate that JCVI-syn3B may be a powerful living platform to analyze the role of specific gene sets, from any organism, on the interaction with diverse mammalian cells in culture.

The ongoing COVID-19 pandemic is a major public health crisis. Despite the development and deployment of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pandemic persists. The continued spread of the virus is largely driven by the emergence of viral variants, which can evade the current vaccines through mutations in the spike protein. Although these differences in spike are important in terms of transmission and vaccine responses, these variants possess mutations in the other parts of their genome that may also affect pathogenesis. Of particular interest to us are the mutations present in the accessory genes, which have been shown to contribute to pathogenesis in the host through interference with innate immune signaling, among other effects on host machinery. To examine the effects of accessory protein mutations and other nonspike mutations on SARS-CoV-2 pathogenesis, we synthesized both viruses possessing deletions in the accessory genes as well as viruses where the WA-1 spike is replaced by each variant spike gene in a SARS-CoV-2/WA-1 infectious clone. We then characterized the in vitro and in vivo replication of these viruses and compared them to both WA-1 and the full variant viruses. Our work has revealed that the accessory proteins contribute to SARS-CoV-2 pathogenesis and the nonspike mutations in variants can contribute to replication of SARS-CoV-2 and pathogenesis in the host. This work suggests that while spike mutations may enhance receptor binding and entry into cells, mutations in accessory proteins may alter clinical disease presentation.

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), resulting in up to 100% mortality in pigs. Although endemic in most sub-Saharan African countries, where all known ASFV genotypes have been reported, the disease has caused pandemics of significant economic impact in Eurasia, and no vaccines or therapeutics are available to date. In endeavors to develop live-attenuated vaccines against ASF, deletions of several of the ~170 ASFV genes have shown contrasting results depending on the genotype of the investigated ASFV. Here, we report the in vivo outcome of a single deletion of the A238L (5EL) gene and double deletions of A238L (5EL) and EP402R (CD2v) genes from the genome of a highly virulent genotype IX ASFV isolate. Domestic pigs were intramuscularly inoculated with (i) ASFV-Ke-ΔA238L to assess the safety of A238L deletion and (ii) ASFV-Ke-ΔEP402RΔA238L to investigate protection against challenge with the virulent wildtype ASFV-Ke virus. While A238L (5EL) gene deletion did not yield complete attenuation, co-deletion of A238L (5EL) and EP402R (CD2v) improved the safety profile of the single deletions, eliciting both humoral and cellular immune responses and conferred partial protection against challenge with the virulent wildtype ASFV-Ke virus.

Staphylococcus aureus is an opportunistic pathogen that causes a wide range of infections and food poisoning in humans with antibiotic resistance, specifically to methicillin, compounding the problem. Bacteriophages (phages) provide an alternative treatment strategy, but these only infect a limited number of circulating strains and may quickly become ineffective due to bacterial resistance. To overcome these obstacles, engineered phages have been proposed, but new methods are needed for the efficient transformation of large DNA molecules into S. aureus to "boot-up" (i.e., rescue) infectious phages. We presented a new, efficient, and reproducible DNA transformation method, NEST (non-electroporation Staphylococcus transformation), for S. aureus to boot-up purified phage genomic DNA (at least 150 kb in length) and whole yeast-assembled synthetic phage genomes. This method was a powerful new tool for the transformation of DNA in S. aureus and will enable the rapid development of engineered therapeutic phages and phage cocktails against Gram-positive pathogens. IMPORTANCE The continued emergence of antibiotic-resistant bacterial pathogens has heightened the urgency for alternative antibacterial strategies. Phages provide an alternative treatment strategy but are difficult to optimize. Synthetic biology approaches have been successfully used to construct and rescue genomes of model phages but only in a limited number of highly transformable host species. In this study, we used a new, reproducible, and efficient transformation method to reconstitute a functional nonmodel Siphophage from a constructed synthetic genome. This method will facilitate the engineering of Staphylococcus and Enterococcus phages for therapeutic applications and the engineering of Staphylococcus strains by enabling transformation of higher molecular weight DNA to introduce more complex modifications.

African swine fever virus (ASFV) is the etiological agent of a contagious and fatal disease of domestic pigs that has significant economic consequences for the global swine industry. Due to the lack of effective treatment and vaccines against African swine fever, there is an urgent need to leverage cutting-edge technologies and cost-effective approaches for generating and purifying recombinant virus to fast-track the development of live-attenuated ASFV vaccines. Here, we describe the use of the CRISPR/Cas9 gene editing and a cost-effective cloning system to produce recombinant ASFVs. Combining these approaches, we developed a recombinant virus lacking the non-essential gene A238L (5EL) in the highly virulent genotype IX ASFV (ASFV-Kenya-IX-1033) genome in less than 2 months as opposed to the standard homologous recombination with conventional purification techniques which takes up to 6 months on average. Our approach could therefore be a method of choice for less resourced laboratories in developing nations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Abkallo, Svitek, Oduor, Awino, Henson, Oyola, Mwalimu, Assad-Garcia, Fuchs, Vashee and Steinaa.)

Genomically minimal cells, such as JCVI-syn3.0, offer a platform to clarify genes underlying core physiological processes. Although this minimal cell includes genes essential for population growth, the physiology of its single cells remained uncharacterized. To investigate striking morphological variation in JCVI-syn3.0 cells, we present an approach to characterize cell propagation and determine genes affecting cell morphology. Microfluidic chemostats allowed observation of intrinsic cell dynamics that result in irregular morphologies. A genome with 19 genes not retained in JCVI-syn3.0 generated JCVI-syn3A, which presents morphology similar to that of JCVI-syn1.0. We further identified seven of these 19 genes, including two known cell division genes, ftsZ and sepF, a hydrolase of unknown substrate, and four genes that encode membrane-associated proteins of unknown function, which are required together to restore a phenotype similar to that of JCVI-syn1.0. This result emphasizes the polygenic nature of cell division and morphology in a genomically minimal cell., Competing Interests: Declaration of interests The authors declare no competing interests., (Published by Elsevier Inc.)

Global transposon mutagenesis is a valuable tool for identifying genes required for cell viability. Here we present a global analysis of the orientation of viable Tn 5 -Puro r (Tn 5 -puromycin resistance) insertions into the near-minimal bacterial genome of JCVI-syn2.0. Sixteen of the 478 protein-coding genes show a noticeable asymmetry in the orientation of disrupting insertions of Tn 5 -Puro r Ten of these are located in operons, upstream of essential or quasi-essential genes. Inserts transcribed in the same direction as the downstream gene are favored, permitting read-through transcription of the essential or quasi-essential gene. Some of these genes were classified as quasi-essential solely because of polar effects on the expression of downstream genes. Three genes showing asymmetry in Tn 5 -Puro r insertion orientation prefer the orientation that avoids collisions between read-through transcription of Tn 5 -Puro r and transcription of an adjacent gene. One gene (JCVISYN2_0132 [abbreviated here as "_0132"]) shows a strong preference for Tn 5 -Puro r insertions transcribed upstream, away from the downstream nonessential gene _0133. This suggested that expression of _0133 due to read-through from Tn 5 -Puro r is lethal when _0132 function is disrupted by transposon insertion. This led to the identification of genes _0133 and _0132 as a toxin-antitoxin pair. The three remaining genes show read-through transcription of Tn 5 -Puro r directed downstream and away from sizable upstream intergenic regions (199 bp to 363 bp), for unknown reasons. In summary, polar effects of transposon insertion can, in a few cases, affect the classification of genes as essential, quasi-essential, or nonessential and sometimes can give clues to gene function. IMPORTANCE In studies of the minimal genetic requirements for life, we used global transposon mutagenesis to identify genes needed for a minimal bacterial genome. Transposon insertion can disrupt the function of a gene but can also have polar effects on the expression of adjacent genes. In the Tn 5 -Puro r construct used in our studies, read-through transcription from Tn 5 -Puro r can drive expression of downstream genes. This results in a preference for Tn 5 -Puro r insertions transcribed toward a downstream essential or quasi-essential gene within the same operon. Such polar effects can have an impact on the classification of genes as essential, quasi-essential, or nonessential, but this has been observed in only a few cases. Also, polar effects of Tn 5 -Puro r insertion can sometimes give clues to gene function., (Copyright © 2019 Hutchison et al.)

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects, and plants. Here, we demonstrate that highly virulent Mycoplasma mycoides subspecies capri ( Mmc ) can be fully attenuated via targeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately 10% of the original Mmc genome, were successively deleted using Saccharomyces cerevisiae as an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually non-essential in the JCVI-Syn3.0 minimal cell, were excised from the genome. In vitro characterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though 10% of the genome content were removed. A novel in vivo challenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reached endpoint criteria within 6 days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of a Mycoplasma strain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequent in vitro or in vivo trials employing the Mycoplasma chassis will allow us to dissect the role of individual candidate Mycoplasma virulence factors and lead the way for the development of an attenuated designer vaccine.

Bacteria are indispensable for the study of fundamental molecular biology processes due to their relatively simple gene and genome architecture. The ability to engineer bacterial chromosomes is quintessential for understanding gene functions. Here we demonstrate the engineering of the small-ribosomal subunit (16S) RNA of Mycoplasma mycoides, by combining the CRISPR/Cas9 system and the yeast recombination machinery. We cloned the entire genome of M. mycoides in yeast and used constitutively expressed Cas9 together with in vitro transcribed guide-RNAs to introduce engineered 16S rRNA genes. By testing the function of the engineered 16S rRNA genes through genome transplantation, we observed surprising resilience of this gene to addition of genetic elements or helix substitutions with phylogenetically-distant bacteria. While this system could be further used to study the function of the 16S rRNA, one could envision the "simple" M. mycoides genome being used in this setting to study other genetic structures and functions to answer fundamental questions of life.

We used whole-genome design and complete chemical synthesis to minimize the 1079-kilobase pair synthetic genome of Mycoplasma mycoides JCVI-syn1.0. An initial design, based on collective knowledge of molecular biology combined with limited transposon mutagenesis data, failed to produce a viable cell. Improved transposon mutagenesis methods revealed a class of quasi-essential genes that are needed for robust growth, explaining the failure of our initial design. Three cycles of design, synthesis, and testing, with retention of quasi-essential genes, produced JCVI-syn3.0 (531 kilobase pairs, 473 genes), which has a genome smaller than that of any autonomously replicating cell found in nature. JCVI-syn3.0 retains almost all genes involved in the synthesis and processing of macromolecules. Unexpectedly, it also contains 149 genes with unknown biological functions. JCVI-syn3.0 is a versatile platform for investigating the core functions of life and for exploring whole-genome design., (Copyright © 2016, American Association for the Advancement of Science.)

The availability of genetically tractable organisms with simple genomes is critical for the rapid, systems-level understanding of basic biological processes. Mycoplasma bacteria, with the smallest known genomes among free-living cellular organisms, are ideal models for this purpose, but the natural versions of these cells have genome complexities still too great to offer a comprehensive view of a fundamental life form. Here we describe an efficient method for reducing genomes from these organisms by identifying individually deletable regions using transposon mutagenesis and progressively clustering deleted genomic segments using meiotic recombination between the bacterial genomes harbored in yeast. Mycoplasmal genomes subjected to this process and transplanted into recipient cells yielded two mycoplasma strains. The first simultaneously lacked eight singly deletable regions of the genome, representing a total of 91 genes and ∼ 10% of the original genome. The second strain lacked seven of the eight regions, representing 84 genes. Growth assay data revealed an absence of genetic interactions among the 91 genes under tested conditions. Despite predicted effects of the deletions on sugar metabolism and the proteome, growth rates were unaffected by the gene deletions in the seven-deletion strain. These results support the feasibility of using single-gene disruption data to design and construct viable genomes lacking multiple genes, paving the way toward genome minimization. The progressive clustering method is expected to be effective for the reorganization of any mega-sized DNA molecules cloned in yeast, facilitating the construction of designer genomes in microbes as well as genomic fragments for genetic engineering of higher eukaryotes., (© 2015 Suzuki et al.; Published by Cold Spring Harbor Laboratory Press.)

We report the discovery of a broadly reactive antibody-binding protein (Protein M) from human mycoplasma. The crystal structure of the ectodomain of transmembrane Protein M differs from other known protein structures, as does its mechanism of antibody binding. Protein M binds with high affinity to all types of human and nonhuman immunoglobulin G, predominantly through attachment to the conserved portions of the variable region of the κ and λ light chains. Protein M blocks antibody-antigen union, likely because of its large C-terminal domain extending over the antibody-combining site, blocking entry to large antigens. Similar to the other immunoglobulin-binding proteins such as Protein A, Protein M as well as its orthologs in other Mycoplasma species could become invaluable reagents in the antibody field.

Understanding how complex phenotypes arise from individual molecules and their interactions is a primary challenge in biology that computational approaches are poised to tackle. We report a whole-cell computational model of the life cycle of the human pathogen Mycoplasma genitalium that includes all of its molecular components and their interactions. An integrative approach to modeling that combines diverse mathematics enabled the simultaneous inclusion of fundamentally different cellular processes and experimental measurements. Our whole-cell model accounts for all annotated gene functions and was validated against a broad range of data. The model provides insights into many previously unobserved cellular behaviors, including in vivo rates of protein-DNA association and an inverse relationship between the durations of DNA replication initiation and replication. In addition, experimental analysis directed by model predictions identified previously undetected kinetic parameters and biological functions. We conclude that comprehensive whole-cell models can be used to facilitate biological discovery., (Copyright © 2012 Elsevier Inc. All rights reserved.)

A suicide plasmid, pExp1-ctpA::tetM-recAec, employing recA from Escherichia coli and tetM as a selection marker, was used to generate ctpA knockout mutants in Mycoplasma mycoides subsp. capri through targeted gene disruption. Inclusion of E. coli recA greatly enhanced both the consistency and the recovery of mutants generated by homologous recombination.

Most gene knockouts in mycoplasmas are achieved through labor-intensive transposon mutagenesis. Here, we describe a method for making targeted deletions in Mycoplasma pneumoniae by use of homologous recombination. In this method, M. pneumoniae is transformed with a plasmid carrying an antibiotic resistance marker flanked by 1-kb regions surrounding the target gene. Following selection for the antibiotic resistance, colonies are screened for double crossovers which indicate complete deletion of the target open reading frame.

We report the design, synthesis, and assembly of the 1.08-mega-base pair Mycoplasma mycoides JCVI-syn1.0 genome starting from digitized genome sequence information and its transplantation into a M. capricolum recipient cell to create new M. mycoides cells that are controlled only by the synthetic chromosome. The only DNA in the cells is the designed synthetic DNA sequence, including "watermark" sequences and other designed gene deletions and polymorphisms, and mutations acquired during the building process. The new cells have expected phenotypic properties and are capable of continuous self-replication.

Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation.

Over the past several years, significant advances have been made in the molecular genetics of the Mollicutes (the simplest cells that can be grown in axenic culture). Nevertheless, a number of basic molecular tools are still required before genetic manipulations become routine. Here we describe the development of a new dominant selectable marker based on the enzyme puromycin-N-acetyltransferase from Streptomyces alboniger. Puromycin is an antibiotic that mimics the 3'-terminal end of aminoacylated tRNAs and attaches to the carboxyl terminus of growing protein chains. This stops protein synthesis. Because puromycin conscripts rRNA recognition elements that are used by all of the various tRNAs in a cell, it is unlikely that spontaneous antibiotic resistance can be acquired via a simple point mutation--an annoying issue with existing mycoplasma markers. Our codon-optimized cassette confers pronounced puromycin resistance on all five of the mycoplasma species we have tested so far. The resistance cassette was also designed to function in Escherichia coli, which simplifies the construction of shuttle vectors and makes it trivial to produce the large quantities of DNA generally necessary for mycoplasma transformation. Due to these and other features, we expect the puromycin marker to be a widely applicable tool for studying these simple cells and pathogens.

We recently reported the chemical synthesis, assembly, and cloning of a bacterial genome in yeast. To produce a synthetic cell, the genome must be transferred from yeast to a receptive cytoplasm. Here we describe methods to accomplish this. We cloned a Mycoplasma mycoides genome as a yeast centromeric plasmid and then transplanted it into Mycoplasma capricolum to produce a viable M. mycoides cell. While in yeast, the genome was altered by using yeast genetic systems and then transplanted to produce a new strain of M. mycoides. These methods allow the construction of strains that could not be produced with genetic tools available for this bacterium.

Mycoplasma genitalium has the smallest genome of any organism that can be grown in pure culture. It has a minimal metabolism and little genomic redundancy. Consequently, its genome is expected to be a close approximation to the minimal set of genes needed to sustain bacterial life. Using global transposon mutagenesis, we isolated and characterized gene disruption mutants for 100 different nonessential protein-coding genes. None of the 43 RNA-coding genes were disrupted. Herein, we identify 382 of the 482 M. genitalium protein-coding genes as essential, plus five sets of disrupted genes that encode proteins with potentially redundant essential functions, such as phosphate transport. Genes encoding proteins of unknown function constitute 28% of the essential protein-coding genes set. Disruption of some genes accelerated M. genitalium growth.

The microprojectile bombardment method was used to transfer DNA into embryogenic callus of asparagus (Asparagus officcinalis L.) and to produce stably transformed asparagus plants. Embryogenic callus, derived from UC 157 and UC72 asparagus cultivars, was bombarded with tungsten particles coated with plasmid DNA that contained genes encoding hygromycin phosphotransferase, phosphinothricin acetyl transferase and β-glucuronidase. Putatively transformed calli were identified from the bombarded tissue after 4 months selection on 25 mg/L hygromycin B plus 4 mg/L phosphinothricin (PPT). By selecting embryogenic callus on hygromycin plus PPT the overall transformation and selection efficiencies were substantially improved over selection with hygromycin or PPT alone, where no transgenic clones were recovered. The transgenic nature of the selected material was demonstrated by GUS histochemical assays and Southern blot hybridization analysis. Transgenic asparagus plants were found to withstand the prescribed levels of the PPT-based herbicide BASTATM for weed control.