Genome context influences evolutionary flexibility of nearly identical type III effectors in two phytopathogenic Pseudomonads

Integrative Conjugative Elements (ICEs) are replicons that can insert and excise from chromosomal locations in a site specific manner, can conjugate across strains, and which often carry a variety of genes useful for bacterial growth and survival under specific conditions. Although ICEs have been identified and vetted within certain clades of the agricultural pathogen Pseudomonas syringae, the impact of ICE carriage and transfer across the entire P. syringae species complex remains underexplored. Here we identify and vet an ICE (PmaICE-DQ) from P. syringae pv. maculicola ES4326, a strain commonly used for laboratory virulence experiments, demonstrate that this element can excise and conjugate across strains, and contains loci encoding multiple type III effector proteins. Moreover, genome context suggests that another ICE (PmaICE-AOAB) is highly similar in comparison with and found immediately adjacent to PmaICE-DQ within the chromosome of strain ES4326, and also contains multiple type III effectors. Lastly, we present passage data from in planta experiments that suggests that genomic plasticity associated with ICEs may enable strains to more rapidly lose type III effectors that trigger R-gene mediated resistance in comparison to strains where nearly isogenic effectors are not present in ICEs. Taken together, our study sheds light on a set of ICE elements from P. syringae pv. maculicola ES4326 and highlights how genomic context may lead to different evolutionary dynamics for shared virulence genes between strains.

Stability of DNA methylation and chromatin accessibility in structurally diverse maize genomes

Accessible chromatin and unmethylated DNA are associated with many genes and cis-regulatory elements. Attempts to understand natural variation for accessible chromatin regions (ACRs) and unmethylated regions (UMRs) often rely upon alignments to a single reference genome. This limits the ability to assess regions that are absent in the reference genome assembly and monitor how nearby structural variants influence variation in chromatin state. In this study, de novo genome assemblies for four maize inbreds (B73, Mo17, Oh43 and W22) are utilized to assess chromatin accessibility and DNA methylation patterns in a pan-genome context. A more complete set of UMRs and ACRs can be identified when chromatin data is aligned to the matched genome rather than a single reference genome. While there are UMRs and ACRs present within genomic regions that are not shared between genotypes, these features are 6-12 fold enriched within regions between genomes. Characterization of UMRs present within shared genomic regions reveals that most UMRs maintain the unmethylated state in other genotypes with only ∼5% being polymorphic between genotypes. However, the majority (71%) of UMRs that are shared between genotypes only exhibit partial overlaps suggesting that the boundaries between methylated and unmethylated DNA are dynamic. This instability is not solely due to sequence variation as these partially overlapping UMRs are frequently found within genomic regions that lack sequence variation. The ability to compare chromatin properties among individuals with structural variation enables pan-epigenome analyses to study the sources of variation for accessible chromatin and unmethylated DNA.

Florigen revisited: proteins of the FT/CETS/PEBP/PKIP/YbhB family may be the enzymes of small molecule metabolism

Flowering signals are sensed in plant leaves and transmitted to the shoot apical meristems, where the formation of flowers is initiated. Searches for a diffusible hormone-like signaling entity ("florigen") went on for many decades, until in the 1990s a product of plant gene FT was identified as the key component of florigen, based on genetic evidence and protein localization studies. Sequence homologs of FT protein are found throughout prokaryotes and eukaryotes; some eukaryotic family members appear to bind phospholipids or interact with the components of the signal transduction cascades. We studied molecular features of the FT homologs in prokaryotes and analyzed their genome context, to find tentative evidence connecting the bacterial family members with small molecule metabolism, often involving sugar- or ribonucleoside-containing substrates. Most FT homologs share a constellation of five charged residues, three of which, i.e., two histidines and an aspartic acid, circumfere the rim of a well-defined cavity on the protein surface. We argue that this conserved feature is more likely to be an enzymatic active center than a catalytically inactive ligand-binding site. We propose that most of FT-related proteins are enzymes operating on small diffusible molecules, which may constitute an overlooked essential ingredient of the florigen signal.

DeepTrio: Variant Calling in Families Using Deep Learning

Every human inherits one copy of the genome from their mother and another from their father. Parental inheritance helps us understand the transmission of traits and genetic diseases, which often involve de novo variants and rare recessive alleles. Here we present DeepTrio, which learns to analyze child-mother-father trios from the joint sequence information, without explicit encoding of inheritance priors. DeepTrio learns how to weigh sequencing error, mapping error, and de novo rates and genome context directly from the sequence data. DeepTrio has higher accuracy on both Illumina and PacBio HiFi data when compared to DeepVariant. Improvements are especially pronounced at lower coverages (with 20x DeepTrio roughly equivalent to 30x DeepVariant). As DeepTrio learns directly from data, we also demonstrate extensions to exome calling solely by changing the training data. DeepTrio includes pre-trained models for Illumina WGS, Illumina exome, and PacBio HiFi.

Stability of DNA methylation and chromatin accessibility in structurally diverse maize genomes

Accessible chromatin and unmethylated DNA are associated with many genes and cis-regulatory elements. Attempts to understand natural variation for accessible chromatin regions (ACRs) and unmethylated regions (UMRs) often rely upon alignments to a single reference genome. This limits the ability to assess regions that are absent in the reference genome assembly and monitor how nearby structural variants influence variation in chromatin state. In this study, de novo genome assemblies for four maize inbreds (B73, Mo17, Oh43 and W22) are utilized to assess chromatin accessibility and DNA methylation patterns in a pan-genome context. The number of UMRs and ACRs that can be identified is more accurate when chromatin data is aligned to the matched genome rather than a single reference genome. While there are UMRs and ACRs present within genomic regions that are not shared between genotypes, these features are substantially enriched within shared regions, as determined by chromosomal alignments. Characterization of UMRs present within shared genomic regions reveals that most UMRs maintain the unmethylated state in other genotypes with only a small number being polymorphic between genotypes. However, the majority of UMRs between genotypes only exhibit partial overlaps suggesting that the boundaries between methylated and unmethylated DNA are dynamic. This instability is not solely due to sequence variation as these partially overlapping UMRs are frequently found within genomic regions that lack sequence variation. The ability to compare chromatin properties among individuals with structural variation enables pan-epigenome analyses to study the sources of variation for accessible chromatin and unmethylated DNA.Article summaryRegions of the genome that have accessible chromatin or unmethylated DNA are often associated with cis-regulatory elements. We assessed chromatin accessibility and DNA methylation in four structurally diverse maize genomes. There are accessible or unmethylated regions within the non-shared portions of the genomes but these features are depleted within these regions. Evaluating the dynamics of methylation and accessibility between genotypes reveals conservation of features, albeit with variable boundaries suggesting some instability of the precise edges of unmethylated regions.

Diagnostic Sequences That Distinguish M. avium Subspecies Strains

Over a decade ago Mycobacterium avium subspecies paratuberculosis (Map) specific genes were initially identified in a whole genome context by comparing draft genome sequences of Map strain K-10 with Mycobacterium avium subspecies hominissuis (Mah) strain 104. This resulted in identification of 32 Map specific genes, not including repetitive elements, based on the two-genome comparison. The goal of this study was to define a more complete catalog of M. avium subspecies-specific genes. This is important for obtaining additional diagnostic targets for Johne's disease detection and for understanding the unique biology, evolution and niche adaptation of these organisms. There are now over 28 complete genome sequences representing three M. avium subspecies, including avium (Maa), Mah, and Map. We have conducted a comprehensive comparison of these genomes using two independent pan genomic comparison tools, PanOCT and Roary. This has led to the identification of more than 250 subspecies defining genes common to both analyses. The majority of these genes are arranged in clusters called genomic islands. We further reduced the number of diagnostic targets by excluding sequences having high BLAST similarity to other mycobacterial species recently added to the National Center for Biotechnology Information database. Genes identified as diagnostic following these bioinformatic approaches were further tested by DNA amplification PCR on an additional 20 M. avium subspecies strains. This combined approach confirmed 86 genes as Map-specific, seven as Maa-specific and three as Mah-specific. A single-tube PCR reaction was conducted as a proof of concept method to quickly distinguish M. avium subspecies strains. With these novel data, researchers can classify isolates in their freezers, quickly characterize clinical samples, and functionally analyze these unique genes.

Iterative Variable Selection for High-Dimensional Data: Prediction of Pathological Response in Triple-Negative Breast Cancer

Over the last decade, regularized regression methods have offered alternatives for performing multi-marker analysis and feature selection in a whole genome context. The process of defining a list of genes that will characterize an expression profile remains unclear. It currently relies upon advanced statistics and can use an agnostic point of view or include some a priori knowledge, but overfitting remains a problem. This paper introduces a methodology to deal with the variable selection and model estimation problems in the high-dimensional set-up, which can be particularly useful in the whole genome context. Results are validated using simulated data and a real dataset from a triple-negative breast cancer study.

The Salmonella enterica serovar Typhimurium virulence factor STM3169 is a hexuronic acid binding protein component of a TRAP transporter

The intracellular pathogen S. Typhimurium is a leading cause of foodborne illness across the world and is known to rely on a range of virulence factors to colonize the human host and cause disease. The gene coding for one such factor, stm3169, was determined to be upregulated upon macrophage entry and its disruption reduces survival in the macrophage. In this study we characterize the STM3169 protein, which forms the substrate binding protein (SBP) of an uncharacterized tripartite ATP-independent periplasmic (TRAP) transporter. Genome context analysis of the genes encoding this system in related bacteria suggests a function in sugar acid transport. We demonstrate that purified STM3169 binds d-glucuronic acid with high affinity and specificity. S. Typhimurium LT2 can use this sugar acid as a sole carbon source and the genes for a probable catabolic pathway are present in the genome. As this gene was previously implicated in macrophage survival, it suggests a role for d-glucuronate as an important carbon source for S. Typhimurium in this environment.

TonB-Dependent Transporters in Sphingomonads: Unraveling Their Distribution and Function in Environmental Adaptation

TonB-dependent transport system plays a critical role in the transport of nutrients across the energy-deprived outer membrane of Gram-negative bacteria. It contains a specialized outer membrane TonB-dependent transporter (TBDT) and energy generating (ExbB/ExbD) and transducing (TonB) inner membrane multi-protein complex, called TonB complex. Very few TonB complex protein-coding sequences exist in the genomes of Gram-negative bacteria. Interestingly, the TBDT coding alleles are phenomenally high, especially in the genomes of bacteria surviving in complex and stressful environments. Sphingomonads are known to survive in highly polluted environments using rare, recalcitrant, and toxic substances as their sole source of carbon. Naturally, they also contain a huge number of TBDTs in the outer membrane. Out of them, only a few align with the well-characterized TBDTs. The functions of the remaining TBDTs are not known. Predictions made based on genome context and expression pattern suggest their involvement in the transport of xenobiotic compounds across the outer membrane.