Comparative whole plastome and low copy number phylogenetics of the core Saccharinae and Sorghinae.

Despite over 60 years' worth of taxonomic efforts, the relationships between sugarcane (Saccharum hybrid cultivars), Sorghum and their closest evolutionary relatives remain largely unresolved. Even relationships between generally accepted genera such as Miscanthus and Saccharum have not been examined in any large-scale molecular detail. Genera such as Erianthus, Miscanthidium and Narenga pose even greater taxonomic contention. Erianthus is not monophyletic and Erianthus sect. Ripidium (Valdés and Scholz 2006, Lloyd Evans et al. 2019a; Welker et al. 2019) represents a distinct and separate genus, Tripidium Scholz. Miscanthidium is placed within Miscanthus by many workers, whilst the New World Erianthus species and Narenga are currently placed within Saccharum. As these species represent a significant portion of the gene pool that sugarcane breeders use for introgression into sugarcane, their taxonomic placement and relationships to Saccharum are of significant economic import. Erianthus species from the Americas have not been significantly employed in sugarcane breeding and may represent an untapped genetic resource. In an attempt to resolve the taxonomic relationships of these genera, we have assembled three novel chloroplasts, from Miscanthidium capense, Miscanthidium junceum and Narenga porphyrocoma (this latter assembled from transcriptomic and long read data). In parallel, five low copy number loci have been assembled from species within Saccharum, Miscanthus, Sarga and Sorghum. Phylogenetic analyses were performed using both low copy number genes and whole chloroplasts. The phylogenetic results were compared with karyotype data to circumscribe the genera most closely related to sugarcane. We reveal that genera Miscanthus and Saccharum are monophyletic and have never undergone polyploidization outside their own genera. Genera Erianthus, Miscanthidium and Narenga are allopolyploids, which excludes them from being members of Saccharum and Miscanthus. Moreover, all three of these genera have divergent evolutionary histories. We therefore support the use of the genera Miscanthus, Miscanthidium, Erianthus (for the New World Species) and Narenga for those species and genera most closely allied to Saccharum. Our data demonstrate that all these genera should be excluded from Saccharum sensu lato.

Measuring the invisible - The sequences causal of genome size differences in eyebrights (Euphrasia) revealed by k-mers

Genome size variation within plant (and other) taxa may be due to presence/absence variation in low-copy sequences or copy number variation in genomic repeats of various frequency classes. However, identifying the sequences underpinning genome size variation has been challenging because genome assemblies commonly contain collapsed representations of repetitive sequences and because genome skimming studies miss low-copy number sequences. Here, we take a novel approach based on k-mers, short sub-sequences of equal length k, generated from whole genome sequencing data of diploid eyebrights (Euphrasia), a group of plants which have considerable genome size variation within a ploidy level. We compare k-mer inventories within and between closely related species, and quantify the contribution of different copy number classes to genome size differences. We further assign high-copy number k-mers to specific repeat types as retrieved from the RepeatExplorer2 pipeline. We find complex patterns of k-mer differences between samples. While all copy number classes contributed to genome size variation, the largest contribution came from repeats with 1000-10,000 genomic copies including the 45S rDNA satellite DNA and, unexpectedly, a repeat associated with an Angela transposable element. We also find size differences in the low-copy number class, likely indicating differences in gene space between our samples. In this study, we demonstrate that it is possible to pinpoint the sequences causing genome size variation within species without use of a reference genome. Such sequences can serve as targets for future cytogenetic studies. We also show that studies of genome size variation should go beyond repeats and consider the whole genome. To allow future work with other taxonomic groups, we share our analysis pipeline, which is straightforward to run, relying largely on standard GNU command line tools.

Non-Genetic Determinants of Clonotypic T Cell Expansion Following Allogeneic Stem Cell Transplant

Background: The immunologic basis of acute GVHD fundamentally involves alloreactive donor T cells that recognize foreign major histocompatibility complex (MHC)-peptide structures derived from both major and minor antigen mismatches with the host. Within this paradigm, the relationship between the donor and recipient genetics represents a closed system that dictates the potential ability of any given T cell receptor (TCR) to expand, raising the question of whether there are predictable aspects of TCR reconstitution at a clonal level. Take a hypothetical example - if genetically identical twins were to receive allogeneic grafts from the same donor and both recipients develop GVHD, would one expect similar TCRs to be clonally expanded? It has been challenging to rigorously explore this phenomenon, however, because of the vast combinatorial diversity of αβ TCRs, the high prevalence of low copy number TCRs, and sampling constraints - all of which render tracking and comparing TCR expansion between the donor and host difficult. Methods: We address these challenges in order to better understand the predictability TCR clonal dynamics through an analysis platform utilizing 1) a series of matched and mismatched murine transplant experiments where genetically identical littermates receive T cells from the same polyclonal donor pool, thus creating multiple transplant replicates simulating the twin transplant system describe above (Fig 1), and 2) probabilistic modeling of individual TCR frequencies to account for partitioning stochasticity (variation in how low copy number TCRs are distributed from donor to recipient). We conduct high-throughput DNA-based TCR amplicon sequencing for both donor and post-transplant recipient samples to generate over 20 million TCRs and model the expansion rates of all identifiable TCRs in each transplant system using a Bayesian approach. Results: While overall V and J gene usage were similar amongst identical recipients (Fig 2), we find that a small fraction of TCR clonotypes appears to have widely disparate clone counts amongst identical recipients receiving the same donor T cell pool. For example, we saw 9,739, 129 and 0 copies of a particular TCR in 3 different recipients in our B6->B6D2F1 system (Fig 3). In order to distinguish whether TCR count discrepancies seen across identical recipients is simply a reflection of donor partitioning stochasticity or true differential expansion (Fig 4), we apply a Bayesian algorithm to identify differential expanders, which represent TCRs that are asymmetrically expanded between recipients of a genetically identical pair (Fig 5). These TCRs can be generated from both memory and naïve T cell compartments. The presence of these differentially expanded clones amongst identical recipients suggests that non-genetic dependent mechanisms may influence which TCRs expand post-transplant. We next show that broad gut decontamination of microbiota with peri-transplant vancomycin, gentamicin, cefoxitin and metronidazole dramatically reduced the fraction of differential expanders (p<0.0001). However, the change in inflammation from microbiome depletion did not appear to drive this difference, as 1) MyD88/TRIF double knockout recipients (deficient TLR signaling) did not show a reduction in differential expanders, and 2) altering conditioning intensity (900cGy to 1300 cGy TBI) also did not change the fraction of differential expanders. Rather, the difference is likely antigenically driven, as differential expanders are enriched in antigen specificity compared to other TCR sequences (p<0.0001) based on published algorithm that identify TCRs with similar amino acid sequence overlap. Conclusions: These results refine our current understanding of clonal T cell selection and expansion after allogeneic BMT and suggests that for a given transplant system, individual TCR selection is not solely dictated by genetic donor and recipient major and/or minor histocompatibility disparities. Rather, microbiota-derived molecules appear to behave as minor antigens to direct systemic clonal TCR selection. These data suggest a novel mechanism by which the microbiome may modulate transplant outcome, challenging current paradigms suggesting the microbiota primarily drive inflammation via their PAMP activities. Figure 1 Figure 1. Disclosures Hill: Applied Molecular Transport: Research Funding; Syndax Pharmaceuticals: Research Funding; Compass Therapeutics: Research Funding; NapaJun Pharma: Consultancy; Generon corporation: Consultancy; iTeos Therapeutics: Consultancy, Research Funding; Neoleukin Therapeutics: Consultancy; Roche: Research Funding.

1230. Evaluation of the Interplay Between β-lactamases and Porin Production on β-Lactam MICs in Klebsiella pneumoniae

Background K. pneumoniae can emerge resistant to β-lactam antibiotics through the production of β-lactamase enzymes and/or loss of the outer membrane porins, OmpK35, OmpK36, and/or PhoE. While both mechanisms are hypothesized to work synergistically, β-lactamases have been the focus of previous studies. As a result, the contribution of outer membrane porin loss to the β-lactam minimum inhibitory concentration (MIC) is unknown. The objective of this study was to evaluate the contribution of specific β-lactamases and porin production to β-lactam susceptibility. We hypothesize that production of a β-lactamase in a clinical isolate deficient in 3 major porins will result in higher β-lactam MICs but not always a resistant phenotype. Methods The structural gene and promoter of CTX-M-14, CTX-M-15, and CMY-2 were cloned into a low copy number vector and transformed into Kp 23, a wild-type clinical isolate, and KPM 20, a clinical isolate deficient in OmpK35/36 and PhoE. MICs to ceftolozane/tazobactam, cefotaxime, ceftazidime, cefepime, and meropenem were determined by E-test. Kp 23 and KPM 20 were characterized by Western blot and whole genome sequencing. Results Production of CMY-2 alone led to a resistant phenotype for ceftolozane/tazobactam, cefotaxime, and ceftazidime regardless of porin production (Figure 1). CMY-2 production in KPM 20 resulted in non-susceptibility to meropenem. Both clones were susceptible to cefepime. Production of CTX-M-14 and CTX-M-15 in Kp 23 resulted in only cefotaxime resistance. Production of CTX-M-14 and CTX-M-15 in KPM 20 resulted in isolates non-susceptible to all antibiotics tested. Figure 1. MICs of K. pneumoniae clones against panel of β-lactam antibiotics. Conclusion When evaluating clinical isolates, it is impossible to determine the contribution of individual resistance mechanisms in the susceptibility pattern. This study demonstrated that resistance is not solely dependent on the β-lactamase produced and that the impact of porin deficiency varies with the antibiotic being evaluated. These data suggest that antibiotic selection may be more nuanced and that a broader range of therapeutics may be available given the appropriate diagnostic tools. Understanding the contributions of all resistance mechanisms is necessary to inform selection of the most appropriate antibiotic therapy. Disclosures Nancy D. Hanson, PhD, Merck (Grant/Research Support)

Comparison of swab types for collection and analysis of microorganisms

The human microbiome has begun to emerge as a potential forensic tool, with varied applications ranging from unique identification to investigative leads that link individuals and/or locations. The relative abundance of the combined DNA of the microbiome, compared to human nuclear DNA, may expand potential sources of biological evidence, especially in cases with transfer or low-copy number DNA samples. This work sought to determine the optimal swab type for the collection and analysis of the microbiome. A bacterium (Proteus mirabilis) was deposited by pipette onto four swab types (cotton, flocked, dental applicators, and dissolvable), and extraction and real-time PCR quantitation of the bacterial DNA were performed, which allowed for absolute microbial DNA recovery and comparison of yields across the four sampling substrates. Flocked swabs had the highest yield (~1240 ng) compared to the cotton swabs (~184 ng), dental applicators (~533 ng), and dissolvable swabs (~430 ng). The collection efficiency was further evaluated for cotton and flocked swabs using dried microbial samples spotted onto non-porous surfaces (treated wood, glass, plastic, and tile). Flocked swabs performed consistently better across wood, glass, and tile, but showed decreased recovery from plastic. The cotton swabs failed in the recovery of P. mirabilis DNA across all surfaces. Knowing the appropriate sampling substrate will be useful as others continue to investigate the use of the microbiome as a forensics tool.

Autoregulation of switching behavior by cellular compartment size

Many kinds of cellular compartments comprise decision making mechanisms that control growth and shrinkage of the compartment in response to external signals. Key examples include synaptic plasticity mechanisms that regulate the size and strength of synapses in the nervous system. However, when synaptic compartments and postsynaptic densities are small such mechanisms operate in a regime where chemical reactions are discrete and stochastic due to low copy numbers of the species involved. In this regime, fluctuations are large relative to mean concentrations, and inherent discreteness leads to breakdown of mass action kinetics. Understanding how synapses and other small compartments achieve reliable switching in the low copy number limit thus remains a key open problem. We propose a novel self regulating signaling motif that exploits the breakdown of mass action kinetics to generate a reliable size-regulated switch. We demonstrate this in simple two and three-species chemical reaction systems and uncover a key role for inhibitory loops among species in generating switching behavior. This provides an elementary motif that could allow size dependent regulation in more complex reaction pathways and may explain discrepant experimental results on well-studied biochemical pathways.

Dangerous Liaisons: Long-Term Replication with an Extrachromosomal HPV Genome

Papillomaviruses cause persistent, and usually self-limiting, infections in the mucosal and cutaneous surfaces of the host epithelium. However, in some cases, infection with an oncogenic HPV can lead to cancer. The viral genome is a small, double-stranded circular DNA molecule that is assembled into nucleosomes at all stages of infection. The viral minichromosome replicates at a low copy number in the nucleus of persistently infected cells using the cellular replication machinery. When the infected cells differentiate, the virus hijacks the host DNA damage and repair pathways to replicate viral DNA to a high copy number to generate progeny virions. This strategy is highly effective and requires a close association between viral and host chromatin, as well as cellular processes associated with DNA replication, repair, and transcription. However, this association can lead to accidental integration of the viral genome into host DNA, and under certain circumstances integration can promote oncogenesis. Here we describe the fate of viral DNA at each stage of the viral life cycle and how this might facilitate accidental integration and subsequent carcinogenesis.

Suggested Absence of Horizontal Transfer of Retrotransposons between Humans and Domestic Mammal Species

Transposable element sequences are usually vertically inherited but have also spread across taxa via horizontal transfer. Previous investigations of ancient horizontal transfer of transposons have compared consensus sequences, but this method resists detection of recent single or low copy number transfer events. The relationship between humans and domesticated animals represents an opportunity for potential horizontal transfer due to the consistent shared proximity and exposure to parasitic insects, which have been identified as plausible transfer vectors. The relatively short period of extended human–animal contact (tens of thousands of years or less) makes horizontal transfer of transposons between them unlikely. However, the availability of high-quality reference genomes allows individual element comparisons to detect low copy number events. Using pairwise all-versus-all megablast searches of the complete suite of retrotransposons of thirteen domestic animals against human, we searched a total of 27,949,823 individual TEs. Based on manual comparisons of stringently filtered BLAST search results for evidence of vertical inheritance, no plausible instances of HTT were identified. These results indicate that significant recent HTT between humans and domesticated animals has not occurred despite the close proximity, either due to the short timescale, inhospitable recipient genomes, a failure of vector activity, or other factors.

CTP and parS coordinate ParB partition complex dynamics and ParA-ATPase activation for ParABS-mediated DNA partitioning

ParABS partition systems, comprising the centromere-like DNA sequence parS, the parS-binding ParB-CTPase and the nucleoid-binding ParA-ATPase, ensure faithful segregation of bacterial chromosomes and low-copy-number plasmids. F-plasmid partition complexes containing ParBF and parSF move by generating and following a local concentration gradient of nucleoid-bound ParAF. However, the process through which ParBF activates ParAF-ATPase has not been defined. We studied CTP- and parSF-modulated ParAF-ParBF complex assembly, in which DNA-bound ParAF-ATP dimers are activated for ATP hydrolysis by interacting with two ParBF N-terminal domains. CTP or parSF enhances the ATPase rate without significantly accelerating ParAF-ParBF complex assembly. Together, parSF and CTP accelerate ParAF-ParBF assembly without further significant increase in ATPase rate. Magnetic-tweezers experiments showed that CTP promotes multiple ParBF loading onto parSF-containing DNA, generating condensed partition complex-like assemblies. We propose that ParBF in the partition complex adopts a conformation that enhances ParBF-ParBF and ParAF-ParBF interactions promoting efficient partitioning.