Comparative Genomics Approach Research Articles

Multidisciplinary Methods for Screening Toxic Proteins from Phages and Their Potential Molecular Targets.

This chapter presents a comprehensive methodology for the identification, characterization, and functional analyses of potentially toxic hypothetical proteins of unknown function (toxHPUFs) in phages. The methods begin with in vivo toxicity verification of toxHPUFs in bacterial hosts, utilizing conventional drop tests and following growth curves. Computational methods for structural and functional predictions of toxHPUFs are outlined, incorporating the use of tools such as Phyre2, HHpred, and AlphaFold2. To ascertain potential targets, a comparative genomic approach is described using bioinformatics toolkits for sequence alignment and functional annotation. Moreover, steps are provided to predict protein-protein interactions and visualizing these using PyMOL. The culmination of these methods equips researchers with an effective pipeline to identify and analyze toxHPUFs and their potential targets, laying the groundwork for future experimental confirmations.

Comparative Genomics of Clostridioides difficile.

Clostridioides difficile, a Gram-positive spore-forming anaerobic bacterium, has rapidly emerged as the leading cause of nosocomial diarrhoea in hospitals. The availability of large numbers of genome sequences, mainly due to the use of next-generation sequencing methods, has undoubtedly shown their immense advantages in the determination of C. difficile population structure. The implementation of fine-scale comparative genomic approaches has paved the way for global transmission and recurrence studies, as well as more targeted studies, such as the PaLoc or CRISPR/Cas systems. In this chapter, we provide an overview of recent and significant findings on C. difficile using comparative genomic studies with implications for epidemiology, infection control and understanding of the evolution of C. difficile.

Speeding genomic island discovery through systematic design of reference database composition.

Genomic islands (GIs) are mobile genetic elements that integrate site-specifically into bacterial chromosomes, bearing genes that affect phenotypes such as pathogenicity and metabolism. GIs typically occur sporadically among related bacterial strains, enabling comparative genomic approaches to GI identification. For a candidate GI in a query genome, the number of reference genomes with a precise deletion of the GI serves as a support value for the GI. Our comparative software for GI identification was slowed by our original use of large reference genome databases (DBs). Here we explore smaller species-focused DBs. With increasing DB size, recovery of our reliable prophage GI calls reached a plateau, while recovery of less reliable GI calls (FPs) increased rapidly as DB sizes exceeded ~500 genomes; i.e., overlarge DBs can increase FP rates. Paradoxically, relative to prophages, FPs were both more frequently supported only by genomes outside the species and more frequently supported only by genomes inside the species; this may be due to their generally lower support values. Setting a DB size limit for our SMAll Ranked Tailored (SMART) DB design speeded runtime ~65-fold. Strictly intra-species DBs would tend to lower yields of prophages for small species (with few genomes available); simulations with large species showed that this could be partially overcome by reaching outside the species to closely related taxa, without an FP burden. Employing such taxonomic outreach in DB design generated redundancy in the DB set; as few as 2984 DBs were needed to cover all 47894 prokaryotic species. Runtime decreased dramatically with SMART DB design, with only minor losses of prophages. We also describe potential utility in other comparative genomics projects.

Cuticular proteins in codling moth (Cydia pomonella) respond to insecticide and temperature stress

The insect cuticle consists of chitin and cuticular proteins (CPs), which stabilize the body shape and provide an effective physical barrier against the external environment. They are also potential target sites for developing environmentally friendly insect management through the utilization of physiology-based methods. The codling moth, Cydia pomonella, is a pest afflicting fruit orchards worldwide. This study used a comparative genomic approach, whole-genome resequencing, and transcriptome data to understand the role that CPs played in the environmental adaptation of the codling moth. A total of 182 putative CPs were identified in the codling moth genome, which were classified into 12 CP families. 119 CPR genes, including 54 RR-1, 60 RR-2, and 5 RR-3 genes were identified and accounted for 65.4% of the total CPs. Eight and seven gene clusters are formed in RR1 and RR2 subfamily and the ancestor-descendant relationship was explained. Five CPAP genes were highly expressed during the egg stage and exposed to high temperature, which indicated their potential role in aiding codling moth eggs in acclimating to varying external heat conditions. Moreover, six CPs belonging to the CPR and CPLCP families were identified in association with insecticide resistance by population resequencing. Their expression levels increased after exposure to insecticides, suggesting they might be involved in codling moth resistance to the insecticides azinphos-methyl or deltamethrin. Our results provide insight into the evolution of codling moth CPs and their association with high temperature adaptation and insecticide resistance, and provide an additional information required for further analysis of CPs in environmental adaptation.

Comparative genomics reveals evolutionary drivers of the dietary shift in Hemiptera.

Hemiptera insects exhibit a close relationship to plants and demonstrate a diverse range of dietary preferences, encompassing phytophagy as the predominant feeding habit while a minority engages in carnivorous or haematophagous behaviour. To counteract the challenges posed by phytophagous insects, plants have developed an array of toxic compounds, causing significant evolutionary selection pressure on these insects. In this study, we employed a comparative genomics approach to analyse the expansion and contraction of gene families specific to phytophagous insect lineages, along with their adaptive evolutionary traits, utilising representative species from the Hemiptera order. Our investigation revealed substantial expansions of gene families within the phytophagous lineages, especially in the Pentatomomorpha branch represented by Oncopeltus fasciatus and Riptortus pedestris. Notably, these expansions of gene families encoding enzymes are potentially involved in hemipteran-plant interactions. Moreover, the adaptive evolutionary analysis of these lineages revealed a higher prevalence of adaptively evolved genes in the Pentatomomorpha branch. The observed branch-specific gene expansions and adaptive evolution likely contribute significantly to the diversification of species within Hemiptera. These results help enhance our understanding of the genomic characteristics of the evolution of different feeding habits in hemipteran insects.

Repositioning of centromere-associated repeats during karyotype evolution in Oryzias fishes.

The karyotype, which is the number and shape of chromosomes, is a fundamental characteristic of all eukaryotes. Karyotypic changes play an important role in many aspects of evolutionary processes, including speciation. In organisms with monocentric chromosomes, it was previously thought that chromosome number changes were mainly caused by centric fusions and fissions, whereas chromosome shape changes, that is, changes in arm numbers, were mainly due to pericentric inversions. However, recent genomic and cytogenetic studies have revealed examples of alternative cases, such as tandem fusions and centromere repositioning, found in the karyotypic changes within and between species. Here, we employed comparative genomic approaches to investigate whether centromere repositioning occurred during karyotype evolution in medaka fishes. In the medaka family (Adrianichthyidae), the three phylogenetic groups differed substantially in their karyotypes. The Oryzias latipes species group has larger numbers of chromosome arms than the other groups, with most chromosomes being metacentric. The O. javanicus species group has similar numbers of chromosomes to the O. latipes species group, but smaller arm numbers, with most chromosomes being acrocentric. The O. celebensis species group has fewer chromosomes than the other two groups and several large metacentric chromosomes that were likely formed by chromosomal fusions. By comparing the genome assemblies of O. latipes, O. javanicus, and O. celebensis, we found that repositioning of centromere-associated repeats might be more common than simple pericentric inversion. Our results demonstrated that centromere repositioning may play a more important role in karyotype evolution than previously appreciated.

Genetics of human longevity: From variants to genes to pathways.

The current increase in lifespan without an equivalent increase in healthspan poses a grave challenge to the healthcare system and a severe burden on society. However, some individuals seem to be able to live a long and healthy life without the occurrence of major debilitating chronic diseases, and part of this trait seems to be hidden in their genome. In this review, we discuss the findings from studies on the genetic component of human longevity and the main challenges accompanying these studies. We subsequently focus on results from genetic studies in model organisms and comparative genomic approaches to highlight the most important conserved longevity-associated pathways. By combining the results from studies using these different approaches, we conclude that only five main pathways have been consistently linked to longevity, namely (1) insulin/insulin-like growth factor 1 signalling, (2) DNA-damage response and repair, (3) immune function, (4) cholesterol metabolism and (5) telomere maintenance. As our current approaches to study the relevance of these pathways in humans are limited, we suggest that future studies on the genetics of human longevity should focus on the identification and functional characterization of rare genetic variants in genes involved in these pathways.

Complete genome sequence of Paramuribaculum intestinale DSM 100749T, isolated from feces of C57BL/6 laboratory mice.

Here, the complete genome of Paramuribaculum intestinale type strain DSM 100749T(=JCM 33114T) is presented. P. intestinale is a recently described species of Muribaculaceae and was isolated from the gut of C57BL/6 laboratory mice. The genome can serve as an important resource for comparative genomics approaches.

Two distinct non-ribosomal peptide synthetase-independent siderophore synthetase gene clusters identified in Armillaria and other species in the Physalacriaceae.

Siderophores are important for ferric iron solubilization, sequestration, transportation, and storage, especially under iron-limiting conditions such as aerobic conditions at high pH. Siderophores are mainly produced by non-ribosomal peptide synthetase-dependent siderophore pathway, non-ribosomal peptide synthetase-independent siderophore synthetase pathway, or the hybrid non-ribosomal peptide synthetases/non-ribosomal peptide synthetases-independent siderophore pathway. Outcompeting or inhibition of plant pathogens, alteration of host defense mechanisms, and alteration of plant-fungal interactions have been associated with fungal siderophores. To understand these mechanisms in fungi, studies have been conducted on siderophore biosynthesis by ascomycetes with limited focus on the basidiomycetes. Armillaria includes several species that are pathogens of woody plants and trees important to agriculture, horticulture, and forestry. The aim of this study was to investigate the presence of non-ribosomal peptide synthetases-independent siderophore synthetase gene cluster(s) in genomes of Armillaria species using a comparative genomics approach. Iron-dependent growth and siderophore biosynthesis in strains of selected Armillaria spp. were also evaluated in vitro. Two distinct non-ribosomal peptide synthetases-independent siderophore synthetase gene clusters were identified in all the genomes. All non-ribosomal peptide synthetases-independent siderophore synthetase genes identified putatively encode Type A' non-ribosomal peptide synthetases-independent siderophore synthetases, most of which have IucA_IucC and FhuF-like transporter domains at their N- and C-terminals, respectively. The effect of iron on culture growth varied among the strains studied. Bioassays using the CAS assay on selected Armillaria spp. revealed in vitro siderophore biosynthesis by all strains irrespective of added FeCl3 concentration. This study highlights some of the tools that Armillaria species allocate to iron homeostasis. The information generated from this study may in future aid in developing molecular based methods to control these phytopathogens.

Newly discovered genomic mutation patterns in radiation-induced small intestinal tumors of ApcMin/+ mice.

Among the small intestinal tumors that occur in irradiated mice of the established mouse model B6/B6-Chr18MSM-F1 ApcMin/+, loss of heterozygosity analysis can be utilized to estimate whether a deletion in the wild-type allele containing the Adenomatous polyposis coli (Apc) region (hereafter referred to as Deletion), a duplication in the mutant allele with a nonsense mutation at codon 850 of Apc (Duplication), or no aberration (Unidentified) has occurred. Previous research has revealed that the number of Unidentified tumors tends to increase with the radiation dose. In the present study, we investigated the molecular mechanisms underlying the development of an Unidentified tumor type in response to radiation exposure. The mRNA expression levels of Apc were significantly lower in Unidentified tumors than in normal tissues. We focused on epigenetic suppression as the mechanism underlying this decreased expression; however, hypermethylation of the Apc promoter region was not observed. To investigate whether deletions occur that cannot be captured by loss of heterozygosity analysis, we analyzed chromosome 18 using a customized array comparative genomic hybridization approach designed to detect copy-number changes in chromosome 18. However, the copy number of the Apc region was not altered in Unidentified tumors. Finally, gene mutation analysis of the Apc region using next-generation sequencing suggested the existence of a small deletion (approximately 3.5 kbp) in an Unidentified tumor from a mouse in the irradiated group. Furthermore, nonsense and frameshift mutations in Apc were found in approximately 30% of the Unidentified tumors analyzed. These results suggest that radiation-induced Unidentified tumors arise mainly due to decreased Apc expression of an unknown regulatory mechanism that does not depend on promoter hypermethylation, and that some tumors may result from nonsense mutations which are as-yet undefined point mutations.

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Evolution End Classification of tfd Gene Clusters Mediating Bacterial Degradation of 2,4-Dichlorophenoxyacetic Acid (2,4-D).

The tfd (tfdI and tfdII) are gene clusters originally discovered in plasmid pJP4 which are involved in the bacterial degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) via the ortho-cleavage pathway of chlorinated catechols. They share this activity, with respect to substituted catechols, with clusters tcb and clc. Although great effort has been devoted over nearly forty years to exploring the structural diversity of these clusters, their evolution has been poorly resolved to date, and their classification is clearly obsolete. Employing comparative genomic and phylogenetic approaches has revealed that all tfd clusters can be classified as one of four different types. The following four-type classification and new nomenclature are proposed: tfdI, tfdII, tfdIII and tfdIV(A,B,C). Horizontal gene transfer between Burkholderiales and Sphingomonadales provides phenomenal linkage between tfdI, tfdII, tfdIII and tfdIV type clusters and their mosaic nature. It is hypothesized that the evolution of tfd gene clusters proceeded within first (tcb, clc and tfdI), second (tfdII and tfdIII) and third (tfdIV(A,B,C)) evolutionary lineages, in each of which, the genes were clustered in specific combinations. Their clustering is discussed through the prism of hot spots and driving forces of various models, theories, and hypotheses of cluster and operon formation. Two hypotheses about series of gene deletions and displacements are also proposed to explain the structural variations across members of clusters tfdII and tfdIII, respectively. Taking everything into account, these findings reconstruct the phylogeny of tfd clusters, have delineated their evolutionary trajectories, and allow the contribution of various evolutionary processes to be assessed.

Convergent reductive evolution and host adaptation in Mycoavidus bacterial endosymbionts of Mortierellaceae fungi

Intimate associations between fungi and intracellular bacterial endosymbionts are becoming increasingly well understood. Phylogenetic analyses demonstrate that bacterial endosymbionts of Mucoromycota fungi are related either to free-living Burkholderia or Mollicutes species. The so-called Burkholderia-related endosymbionts or BRE comprise Mycoavidus, Mycetohabitans and Candidatus Glomeribacter gigasporarum. These endosymbionts are marked by genome contraction thought to be associated with intracellular selection. However, the conclusions drawn thus far are based on a very small subset of endosymbiont genomes, and the mechanisms leading to genome streamlining are not well understood. The purpose of this study was to better understand how intracellular existence shapes Mycoavidus and BRE functionally at the genome level. To this end we generated and analyzed 14 novel draft genomes for Mycoavidus living within the hyphae of Mortierellomycotina fungi. We found that our novel Mycoavidus genomes were significantly reduced compared to free-living Burkholderiales relatives. Using a genome-scale phylogenetic approach including the novel and available existing genomes of Mycoavidus, we show that the genus is an assemblage composed of two independently derived lineages including three well supported clades of Mycoavidus. Using a comparative genomic approach, we shed light on the functional implications of genome reduction, documenting shared and unique gene loss patterns between the three Mycoavidus clades. We found that many endosymbiont isolates demonstrate patterns of vertical transmission and host-specificity, but others are present in phylogenetically disparate hosts. We discuss how reductive evolution and host specificity reflect convergent adaptation to the intrahyphal selective landscape, and commonalities of eukaryotic endosymbiont genome evolution.

Specific and sensitive detection tools for Xanthomonas arboricola pv. corylina, the causal agent of bacterial blight of hazelnut, developed with comparative genomics.

Xanthomonas arboricola pv. corylina (Xac; formerly Xanthomonas campestris pv. corylina) is the causal agent of the bacterial blight of hazelnuts, a devastating disease of trees in plant nurseries and young orchards. Currently, there are no PCR assays to distinguish Xac from all other pathovars of X. arboricola. A comparative genomics approach with publicly available genomes of Xac was used to identify unique sequences, conserved across the genomes of the pathogen. We identified a 2,440 bp genomic region that was unique to Xac and designed identification and detection systems for conventional PCR, qPCR (SYBR® Green and TaqMan™), and loop-mediated isothermal amplification (LAMP). All PCR assays performed on genomic DNA isolated from eight X. arboricola pathovars and closely related bacterial species confirmed the specificity of designed primers. These new multi-platform molecular diagnostic tools may be used by plant clinics and researchers to detect and identify Xac in pure cultures and hazelnut tissues rapidly and accurately.

Rapid detection of the rice false smut fungus Ustilaginoidea virens by lateral flow strip-based recombinase polymerase amplification assay1

Rice false smut, caused by Ustilaginoidea virens, is a devastating disease that greatly reduces rice yield and quality. However, controlling rice false smut disease is challenging due to the unique infection mode of U. virens. Therefore, there is a need for early diagnosis and monitoring techniques to prevent the spread of this disease. Lateral flow strip-based recombinase polymerase amplification (LF-RPA) overcomes the limitations of current U. virens detection technologies, which are time-consuming, require delicate equipment, and have a high false-positive rate. In this study, we used a comparative genomics approach to identify Uv_3611, a specific gene of U. virens, as the target for the LF-RPA assay. The designed primers and probe efffectively detected the genomic DNA (gDNA) of U. virens and demonstrated no cross-reactivity with related pathogens. Under optimal conditions, the LF-RPA assay demonstrated a sensitivity of 10 pg of U. virens gDNA. Additionally, by incorporating a simplified PEG-NaOH method for plant DNA extraction, the LF-RPA assay enabled the detection of U. virens in rice spikelets within 30 minutes, without the need for specialized equipment. Furthermore, the LF-RPA assay successfully detected U. virens in naturally infected rice and seed samples in the field. Therefore, the LF-RPA assay is sensitive, efficient, and convenient, and could be developed as a kit for monitoring rice false smut disease in the field.

Comparative genomics of bacteria from amphibian skin associated with inhibition of an amphibian fungal pathogen, Batrachochytrium dendrobatidis.

Chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd), is a skin disease associated with worldwide amphibian declines. Symbiotic microbes living on amphibian skin interact with Bd and may alter infection outcomes. We completed whole genome sequencing of 40 bacterial isolates cultured from the skin of four amphibian species in the Eastern US. Each isolate was tested in vitro for the ability to inhibit Bd growth. The aim of this study was to identify genomic differences among the isolates and generate hypotheses about the genomic underpinnings of Bd growth inhibition. We identified sixty-five gene families that were present in all 40 isolates. Screening for common biosynthetic gene clusters revealed that this set of isolates contained a wide variety of clusters; the two most abundant clusters with potential antifungal activity were siderophores (N=17 isolates) and Type III polyketide synthases (N=22 isolates). We then examined various subsets of the 22 isolates in the phylum Proteobacteria for genes encoding specific compounds that may inhibit fungal growth, including chitinase and violacein. We identified differences in Agrobacterium and Sphingomonas isolates in the chitinase genes that showed some association with anti-Bd activity, as well as variation in the violacein genes in the Janthinobacterium isolates. Using a comparative genomics approach, we generated several testable hypotheses about differences among bacterial isolates from amphibian skin communities that could contribute to variation in the ability to inhibit Bd growth. Further work is necessary to explore and uncover the various mechanisms utilized by amphibian skin bacterial isolates to inhibit Bd.

Genomic tools for comparative conservation genetics among three recently diverged stag beetles (Lucanus, Lucanidae)

Abstract We are witnessing a rapid decline in global biodiversity. International protocols and local conservation laws have been installed to counter such an unprecedent rate of decline. However, quantitatively evaluating how much biodiversity has been lost due to climatic and anthropogenic effects and how much biodiversity has been restored due to conservation efforts remains challenging. We applied a comparative conservation genomic approach to statistically and quantitatively address these questions using three geographical taxa from a stag beetle species complex. We found that the three sky‐island taxa formed three independently evolving units without detectable post‐divergence gene flow; furthermore, the three taxa, which have been divergent from each other since the mid‐Pleistocene, have experienced episodes of demographic decline in the past. More importantly, even though idiosyncratic anthropogenic exploitations have been hypothesised to impact the recent demographic history (<100 years) differently, we found a shared pattern of continuous decline in effective population size among the three geographical taxa. We argue that future empirical studies should include more taxa, in addition to the focal species, that may or may not be affected by the focal historical events to avoid making biased conservation plans.

High-quality haploid genomes corroborate 29 chromosomes and highly conserved synteny of genes in Hyles hawkmoths (Lepidoptera: Sphingidae)

BackgroundMorphological and traditional genetic studies of the young Pliocene genus Hyles have led to the understanding that despite its importance for taxonomy, phenotypic similarity of wing patterns does not correlate with phylogenetic relationship. To gain insights into various aspects of speciation in the Spurge Hawkmoth (Hyles euphorbiae), we assembled a chromosome-level genome and investigated some of its characteristics.ResultsThe genome of a male H. euphorbiae was sequenced using PacBio and Hi-C data, yielding a 504 Mb assembly (scaffold N50 of 18.2 Mb) with 99.9% of data represented by the 29 largest scaffolds forming the haploid chromosome set. Consistent with this, FISH analysis of the karyotype revealed n = 29 chromosomes and a WZ/ZZ (female/male) sex chromosome system. Estimates of chromosome length based on the karyotype image provided an additional quality metric of assembled chromosome size. Rescaffolding the published male H. vespertilio genome resulted in a high-quality assembly (651 Mb, scaffold N50 of 22 Mb) with 98% of sequence data in the 29 chromosomes. The larger genome size of H. vespertilio (average 1C DNA value of 562 Mb) was accompanied by a proportional increase in repeats from 45% in H. euphorbiae (measured as 472 Mb) to almost 55% in H. vespertilio. Several wing pattern genes were found on the same chromosomes in the two species, with varying amounts and positions of repetitive elements and inversions possibly corrupting their function.ConclusionsOur two-fold comparative genomics approach revealed high gene synteny of the Hyles genomes to other Sphingidae and high correspondence to intact Merian elements, the ancestral linkage groups of Lepidoptera, with the exception of three simple fusion events. We propose a standardized approach for genome taxonomy using nucleotide homology via scaffold chaining as the primary tool combined with Oxford plots based on Merian elements to infer and visualize directionality of chromosomal rearrangements. The identification of wing pattern genes promises future understanding of the evolution of forewing patterns in the genus Hyles, although further sequencing data from more individuals are needed. The genomic data obtained provide additional reliable references for further comparative studies in hawkmoths (Sphingidae).

Pathovar-Specific PCR Method for Detection and Identification of Xanthomonas translucens pv. undulosa.

Bacterial leaf streak disease caused by Xanthomonas translucens pv. undulosa is an economically important disease threatening wheat and barley crops around the globe. Thus far, specific PCR-based detection and identification tests for X. translucens pathovars are not available. In this study, we used comparative genomics approach to design a pathovar-specific primer pair for detection of X. translucens pv. undulosa in naturally infected seeds and its differentiation from other pathovars of the species. For this aim, complete genome sequences of strains of different X. translucens pathovars were compared and the specific PCR primer pair XtuF/XtuR was designed. These primers were strictly specific to X. translucens pv. undulosa because the expected 229-bp DNA fragment was not amplified in the closely related pathovars or in other xanthomonads, wheat-pathogenic bacteria, and other plant-pathogenic bacteria. High sensitivity of the primer pair XtuF/XtuR allowed detection of pure DNA of the pathogen in a concentration as low as 4.5 pg/μl. The pathogen was also detected in water suspension at a concentration of 8.6 × 102 CFU/ml. The PCR test was capable of detecting the pathogen in extracts of naturally infected wheat seeds at a concentration of 3.5 × 104 CFU/g while a culture-plate method was able to detect the pathogen at a concentration of 50 × 105 CFU/g of the same seeds. The PCR test developed in this study is a step forward for precise detection and identification of X. translucens pv. undulosa to prevent outbreaks of the bacterial leaf streak disease.

Feature architecture aware phylogenetic profiling indicates a functional diversification of type IVa pili in the nosocomial pathogen Acinetobacter baumannii.

The Gram-negative bacterial pathogen Acinetobacter baumannii is a major cause of hospital-acquired opportunistic infections. The increasing spread of pan-drug resistant strains makes A. baumannii top-ranking among the ESKAPE pathogens for which novel routes of treatment are urgently needed. Comparative genomics approaches have successfully identified genetic changes coinciding with the emergence of pathogenicity in Acinetobacter. Genes that are prevalent both in pathogenic and a-pathogenic Acinetobacter species were not considered ignoring that virulence factors may emerge by the modification of evolutionarily old and widespread proteins. Here, we increased the resolution of comparative genomics analyses to also include lineage-specific changes in protein feature architectures. Using type IVa pili (T4aP) as an example, we show that three pilus components, among them the pilus tip adhesin ComC, vary in their Pfam domain annotation within the genus Acinetobacter. In most pathogenic Acinetobacter isolates, ComC displays a von Willebrand Factor type A domain harboring a finger-like protrusion, and we provide experimental evidence that this finger conveys virulence-related functions in A. baumannii. All three genes are part of an evolutionary cassette, which has been replaced at least twice during A. baumannii diversification. The resulting strain-specific differences in T4aP layout suggests differences in the way how individual strains interact with their host. Our study underpins the hypothesis that A. baumannii uses T4aP for host infection as it was shown previously for other pathogens. It also indicates that many more functional complexes may exist whose precise functions have been adjusted by modifying individual components on the domain level.