Transmissible plasmids spread genes encoding antibiotic resistance and other traits to new bacterial species. Here we report that laboratory populations of Escherichia coli with a newly acquired IncQ plasmid often evolve ‘satellite plasmids’ with deletions of accessory genes and genes required for plasmid replication. Satellite plasmids are molecular parasites: their presence reduces the copy number of the full-length plasmid on which they rely for their continued replication. Cells with satellite plasmids gain an immediate fitness advantage from reducing burdensome expression of accessory genes. Yet, they maintain copies of these genes and the complete plasmid, which potentially enables them to benefit from and transmit the traits they encode in the future. Evolution of satellite plasmids is transient. Cells that entirely lose accessory gene function or plasmid mobility dominate in the long run. Satellite plasmids also evolve in Snodgrassella alvi colonizing the honey bee gut, suggesting that this mechanism may broadly contribute to the importance of IncQ plasmids as agents of bacterial gene transfer in nature.

Plant pathogenic Ralstonia spp. colonize plant xylem and cause wilt diseases on a broad range of host plants. To identify genes that promote growth of diverse Ralstonia strains in xylem sap from tomato plants, we performed genome-scale genetic screens (random barcoded transposon mutant sequencing screens; RB-TnSeq) in Ralstonia pseudosolanacearum GMI1000 and R. syzygii PSI07. Contrasting mutant fitness phenotypes in culture media versus in xylem sap suggest that Ralstonia strains are adapted to sap and that culture media impose foreign selective pressures. Although wild-type Ralstonia grew in sap and in rich medium with similar doubling times and to a similar carrying capacity, more genes were essential for growth in sap than in rich medium. Multiple mutants lacking amino acid biosynthesis and central metabolism functions had fitness defects in xylem sap and minimal medium. Our screen identified > 26 genes in each strain that contributed to growth in xylem sap but were dispensable for growth in culture media. Many sap-specific fitness factors are associated with bacterial stress responses: envelope remodeling and repair processes such as peptidoglycan peptide formation (murI and RSc1177), LPS O-antigen biosynthesis (RSc0684), periplasmic protein folding (dsbA), drug efflux (tolA and tolR), and stress responses (cspD3). Our genome-scale genetic screen identified Ralstonia fitness factors that promote growth in xylem sap, an ecologically relevant condition.ImportanceTraditional transposon mutagenesis genetic screens pioneered molecular plant pathology and identified core virulence traits like the type III secretion system. TnSeq approaches that leverage next-generation sequencing to rapidly quantify transposon mutant phenotypes are ushering in a new wave of biological discovery. Here we have adapted a genome-scale approach, random barcoded transposon mutant sequencing (RB-TnSeq), to discover fitness factors that promote growth of two related bacterial strains in a common niche, tomato xylem sap. Fitness of wild-type and mutants show that Ralstonia spp. are adapted to grow well in xylem sap from their natural host plant, tomato. Our screen identified multiple sap-specific fitness factors with roles in maintaining the bacterial envelope. These factors are putative adaptations to resist plant defenses, including antimicrobial proteins and specialized metabolites that damage bacterial membranes.

Traditional transposon mutagenesis genetic screens pioneered molecular plant pathology and identified core virulence traits like the type III secretion system. TnSeq approaches that leverage next-generation sequencing to rapidly quantify transposon mutant phenotypes are ushering in a new wave of biological discovery.

The bacterial wilt pathogens in the Ralstonia solanacearum species complex (RSSC) have broad but finite host ranges. Population genetic surveys of RSSC pathogens show that many sequevars (subspecies groups) are predominantly recovered from wilting solanaceous plants. In contrast, strains in the IIB-4 sequevar have been isolated from plants in over a dozen families. Certain IIB-4 lineages have been classified as banana-virulent or “not pathogenic to banana (NPB)”. Prior analysis suggested that the NPB lineage has diverged from the banana-virulent IIB-4 strains. To test this model, we analyzed the phenotypes and phylogeny of a diverse collection of 19 IIB-4 isolates. We used Illumina sequencing to assemble draft genomes of 12 new strains. Based on whole genome phylogenetic analysis, these IIB-4 strains clustered into five subclades. We quantified virulence of each strain on tomato, banana, melon, and impatiens plants. Overall, the virulence patterns correlated with phylogeny. Banana virulence was restricted to the 4/4 IIB-4D subclade (N=4/4 strains) and IIB-4E subclade (N=1/2 strains). Subclades IIB-4D and IIB-4E are sister sublcades and their closest relative, the IIB-4A-C subclade, lacked virulence on banana. Our data support a revised model in which banana virulence is an innovation within the IIB4D/E subclades.

12Plasmids play a principal role in the spread of antibiotic resistance and other traits by horizontal 13 gene transfer in bacteria. However, newly acquired plasmids generally impose a fitness burden 14 on a cell, and they are lost from a population rapidly if there is not selection to maintain a unique 15 function encoded on the plasmid. Mutations that ameliorate this fitness cost can sometimes 16 eventually stabilize a plasmid in a new host, but they typically do so by inactivating some of its 17 novel accessory genes. In this study, we identified an additional evolutionary pathway that can 18 prolong the maintenance of newly acquired genes encoded on a plasmid. We discovered that 19 propagation of an RSF1010-based IncQ plasmid in Escherichia coli often generated 'satellite 20 plasmids' with spontaneous deletions of accessory genes and genes required for plasmid 21 replication. These smaller plasmid variants are nonautonomous genetic parasites. Their presence 22 Significance Statement 30Plasmids are multicopy DNA elements found in bacteria that replicate independently of a cell's 31 chromosome. The spread of plasmids carrying antibiotic-resistance genes to new bacterial 32 pathogens is a challenge for treating life-threatening infections. Often plasmids or their accessory 33 genes encoding unique functions are lost soon after transfer into a new cell because they impose 34 a fitness burden. We report that a family of transmissible plasmids can rapidly evolve 'satellite 35 plasmids' that replicate as genetic parasites of the original plasmid. Satellite plasmid formation 36 reduces the burden from the newly acquired genes, which may enable them to survive intact for 37 longer after transfer into a new cell and thereby contribute to the spread of antibiotic resistance 38 and other traits within bacterial populations. 39Recently acquired plasmids generally impose a fitness burden on a new host cell, which may 53 be due to the cost of expressing antibiotic resistance genes (6) or due to plasmid-encoded genes 54 interfering with host processes (7-9). Therefore, purifying selection can rapidly lead to the loss 55 of genes encoded on a plasmid from a new host unless there is positive selection for traits they 56 confer (10). Continuous conjugative transfer to spread a plasmid to new cells within a population 57 can counteract the purifying selection process to some extent, but there is also a fitness cost of 58 increasing the conjugation rate on donor cells (11). Compensatory mutations on the chromosome 59 and/or plasmid can sometimes ameliorate the cost of a plasmid and favor its persistence (12-17). 60However, it takes a long time, typically several hundred cell generations, with constant selection 61 5 for novel plasmid function to evolve and fix these types of compensatory mutations in laboratory 62 experiments (12, 18, 19). These conditions may be unrealistic in most natural settings. 63Most experimental work examining BHR plasmid stability has used IncP plasmids as a 64 model (13, 14, 17). Fewer studies have focu...

The bacterial wilt pathogens in the Ralstonia solanacearum species complex (RSSC) have broad but finite host ranges. Population genetic surveys of RSSC pathogens show that many sequevars (subspecies groups) are predominantly recovered from wilting solanaceous plants. In contrast, strains in the IIB-4 sequevar have been isolated from plants in over a dozen families. We characterized the natural variation in host range of nineteen IIB-4 strains to explore the molecular determinants of host range within the RSSC. We used Illumina sequencing to assemble draft genomes of 12 strains. Based on whole genome phylogenetic analysis, these IIB-4 strains cluster into five subclades. We quantified virulence of each strain on tomato cv. Moneymaker (Solanaceae), banana cv. Dwarf Cavendish (Musaceae), melon cv. Sweet Granite (Cucurbitaceae), and impatiens cv. Beacon Orange (Balsaminaceae). To enable future meta-analyses that identify genetic factors that drive host-range, the raw virulence data is included as a supplemental table. Overall, the virulence patterns correlated with phylogeny. Strains from Martinique, Dominican Republic, Brazil, as well as multiple strains imported into Florida were highly virulent on tomato, melon, and impatiens. Several strains from Peru were highly virulent on tomato and had moderate-to-low virulence on banana, melon, and impatiens. One strain from Colombia was highly virulent on all hosts. Our findings reinforce the phenotypic plasticity within the Ralstonia solanacearum species complex.

Introduction: Multiple myeloma (MM) is the second most common blood cancer in the United States. Recent breakthroughs in immunotherapy have the potential to transform MM treatment. An immunotherapy target that shows considerable promise in myeloma is the B-cell maturation antigen (BCMA). BCMA is specifically expressed in myeloma cells and plasma cells, making it an ideal target in myeloma. Results from two early-phase clinical trials using anti-BCMA therapy, showed remarkable response in most patients. Although immunotherapy has been promising, recent findings suggest that patients can develop resistance to such therapies by lowering the levels of the target. Here we employ our innovative CRISPR-interference/activation (CRISPRi/a)-based functional genomics platform to identify mechanisms that regulate BCMA expression, which would enable us to design strategies to improve the efficacy of available BCMA immunotherapy agents. Methods: The CRISPRi/a platform utilizes a catalytically dead Cas9 (dCas9) fused to a transcriptional repressor domain to silence genes (CRISPRi) or to activator domains to activate transcription (CRISPRa). The CRISPR machinery is targeted to specific genes using single guide-RNAs (sgRNA). We have engineered a panel of myeloma cell lines to express components of the CRISPRi system. Here we transduced CRISPRi-AMO1 cell line with sgRNAs targeting the human genome. The sgRNA expressing cells were stained with a fluorescent-tagged BCMA antibody and FACS sorted into cells expressing high and low levels of BCMA. The cells were processed for next generation sequencing to determine the frequency of sgRNA in each of these populations. To develop BCMA chimeric antigen receptor-T (CAR-T) cells, CD8+ T cells were transduced with BCMA CAR construct specifically recognizing BCMA. To examine the anti-myeloma activity of the BCMA CAR-T cells, CAR-T cells were co-cultured with MM cell lines at a ratio of 1:2 (Effector:Target) for 24hrs. The cells were analyzed by flow cytometry for expression of CD69, an activation marker on T cells and for apoptosis of cancer cells using propidium iodide. Results: Our FACS-based genome-wide CRISPRi screen identified several genes and pathways regulating BCMA cell surface expression. In addition to previously reported gamma-secretase complex and transcription factor POU2AF1 we identified genes involved in peroxisome biogenesis, subunits of the proteasome, transcription factors and a few druggable targets regulating cell surface expression of BCMA. We are currently validating the novel genes identified from our primary genome-wide screen in a panel of MM cell lines and developing MM cell lines expressing CRISPRa machinery to perform gain-of-function screens that will complement our CRISPRi screen. Furthermore, we have developed active CAR-T cells targeting BCMA and demonstrated its efficacy in MM cell lines expressing different levels of BCMA. We are currently testing the novel genes identified from our CRISPRi screen in combination with BCMA CAR-T cells to identify genes that alter sensitivity to BCMA immunotherapy. Conclusions: Our studies have identified several novel genes and pathways regulating BCMA expression including some druggable targets. Through these studies, we expect to uncover mechanisms regulating expression of BCMA and its impact on sensitivity to BCMA immunotherapy, and pinpoint potential combination therapy targets that pre-empt resistance to BCMA immunotherapy. Disclosures Wiita: Sutro Biopharma: Research Funding; TeneoBio: Research Funding.