We report the results of taxonomic studies on members of the family Micrococcaceae that, according to the 16S rRNA, internal transcribed spacer 1 (ITS1), average nucleotide identity (ANI), and average amino acid identity (AAI) tests, are related to Kocuria rosea strain RCAM04488, a plant-growth-promoting rhizobacterium (PGPR) isolated from the rhizosphere of potato (Solanum tuberosum L.). In these studies, we used whole-genome phylogenetic tests and pangenomic analysis. According to the ANI > 95 % criterion, several known members of K. salina, K. polaris, and K. rosea (including K. rosea type strain ATCC 186T) that are related most closely to isolate RCAM04488 in the ITS1 test should be assigned to the same species with appropriate strain verification. However, these strains were isolated from strongly contrasting ecological and geographical habitats, which could not but affect their genotypes and phenotypes and which should be taken into account in evaluation of their systematic position. This contradiction was resolved by a pangenomic analysis, which showed that the strains differed strongly in the number of accessory and strain-specific genes determining their individuality and possibly their potential for adaptation to different ecological niches. Similar results were obtained in a full-scale AAI test against the UniProt database (about 250 million records), by using the AAI-profiler program and the proteome of K. rosea strain ATCC 186T as a query. According to the AAI > 65 % criterion, members of the genus Arthrobacter and several other genera belonging to the class Actinomycetes, with a very wide geographical and ecological range of sources of isolation, should be placed into the same genus as Kocuria. Within the paradigm with vertically inherited phylogenetic markers, this could be regarded as a signal for their following taxonomic reclassification. An important factor in this case may be the detailing of the gene composition of the strains and the taxonomic ratios resulting from analysis of the pangenomes of the corresponding clades.
Исследованы представители семейства Micrococcaceae, родственные, согласно тестам 16S рРНК, ITS1 (транскрибируемый межгенный спейсер), средней нуклеотидной идентичности (ANI) и средней аминокислотной идентичности (AAI), штамму RCAM04488 Kocuria rosea – стимулирующей рост растений ризобактерии (PGPR), изолированному из ризосферы картофеля (Solanum tuberosum L.) с использованием полногеномных филогенетических тестов и пангеномного анализа. Согласно критерию ANI > 95 %, ряд известных представителей видов K. salina, K. polaris и K. rosea (включая типовой штамм K. rosea ATCC 186T), наиболее близкородственных изоляту в тесте ITS1, должны быть приписаны к одному и тому же виду с соответствующей верификацией штаммов. Однако указанные штаммы были выделены из весьма контрастных по экологии и географии мест обитания, что не могло не сказаться на их генотипе и фенотипе и должно быть так или иначе учтено в оценках их систематического положения. Данное противоречие проясняют результаты пангеномного анализа, продемонстрировавшие существенные различия в этих штаммах количества акцессорных и штамм-специфичных генов, определяющих их индивидуальность и, возможно, потенциал для адаптации к различным экологическим нишам с соответствующими фенотипическими признаками. Аналогичные результаты получены в тесте AAI в полномасштабном варианте его применения против базы данных UniProt (около 250 млн записей) с использованием программы AAI-profiler и протеома штамма K. rosea ATCC 186T в качестве запроса. Согласно критерию AAI > 65 %, в один и тот же род с Kocuria должны быть объединены представители рода Arthrobacter и некоторых других родов, относящихся к классу актиномицетов, с весьма широким географическим и экологическим спектром источников их выделения. В рамках парадигмы о вертикально наследуемых филогенетических маркерах это можно трактовать как сигнал для их последующей таксономической переквалификации. Важным фактором при этом может быть детализация генного состава штаммов и таксономических соотношений, получаемых в результате анализа пангеномов соответствующих клад.

Microbial pangenome analysis identifies present or absent genes in prokaryotic genomes. However, current tools are limited when analyzing species with higher sequence diversity or higher taxonomic orders such as genera or families. The Roary ILP Bacterial core Annotation Pipeline (RIBAP) uses an integer linear programming approach to refine gene clusters predicted by Roary for identifying core genes. RIBAP successfully handles the complexity and diversity of Chlamydia, Klebsiella, Brucella, and Enterococcus genomes, outperforming other established and recent pangenome tools for identifying all-encompassing core genes at the genus level. RIBAP is a freely available Nextflow pipeline at github.com/hoelzer-lab/ribap and zenodo.org/doi/10.5281/zenodo.10890871.

Most in silico evolutionary studies commonly assumed that core genes are essential for cellular function, while accessory genes are dispensable, particularly in nutrient-rich environments. However, this assumption is seldom tested genetically within the pangenome context. In this study, we conducted a robust pangenomic Tn-seq analysis of fitness genes in a nutrient-rich medium for Sinorhizobium strains with a canonical open pangenome. To evaluate the robustness of fitness category assignment, Tn-seq data for three independent mutant libraries per strain were analyzed by three methods, which indicates that the Hidden Markov Model (HMM)-based method is most robust to variations between mutant libraries and not sensitive to data size, outperforming the Bayesian and Monte Carlo simulation-based methods. Consequently, the HMM method was used to classify the fitness category. Fitness genes, categorized as essential (ES), advantage (GA), and disadvantage (GD) genes for growth, are enriched in core genes, while nonessential genes (NE) are over-represented in accessory genes. Accessory ES/GA genes showed a lower fitness effect than core ES/GA genes. Connectivity degrees in the cofitness network decrease in the order of ES, GD, and GA/NE. In addition to accessory genes, 1599 out of 3284 core genes display differential essentiality across test strains. Within the pangenome core, both shared quasi-essential (ES and GA) and strain-dependent fitness genes are enriched in similar functional categories. Our analysis demonstrates a considerable fuzzy essential zone determined by cofitness connectivity degrees in Sinorhizobium pangenome and highlights the power of the cofitness network in understanding the genetic basis of ever-increasing prokaryotic pangenome data.

BACKGROUND: Transposable Elements (TEs) are segments of DNA, typically a few hundred base pairs up to several tens of thousands bases long, that have the ability to generate new copies of themselves in the genome. Most existing methods used to identify TEs in a newly sequenced genome are based on their repetitive character, together with detection based on homology and structural features. As new high quality assemblies become more common, including the availability of multiple independent assemblies from the same species, an alternative strategy for identification of TE families becomes possible in which we focus on the polymorphism at insertion sites caused by TE mobility.
RESULTS: We develop the idea of using the structural polymorphisms found in pangenomes to create a library of the TE families recently active in a species, or in a closely related group of species. We present a tool, pantera, that achieves this task, and illustrate its use both on species with well-curated libraries, and on new assemblies.
CONCLUSIONS: Our results show that pantera is sensitive and accurate, tending to correctly identify complete elements with precise boundaries, and is particularly well suited to detect larger, low copy number TEs that are often undetected with existing de novo methods.

To assess the genomic diversity of Fusarium oxysporum f. sp. lini strains and compile a comprehensive gene repertoire, we constructed a pangenome using 13 isolates from four different clonal lineages, each exhibiting distinct levels of virulence. Syntenic analyses of two selected genomes revealed significant chromosomal rearrangements unique to each genome. A comprehensive examination of both core and accessory pangenome content and diversity points at an open genome state. Additionally, Gene Ontology (GO) enrichment analysis indicated that non-core pangenome genes are associated with pathogen recognition and immune signaling. Furthermore, the Folini pansecterome, encompassing secreted proteins critical for fungal pathogenicity, primarily consists of three functional classes: effector proteins, CAZYmes, and proteases. These three classes account for approximately 3.5% of the pangenome. Each functional class within the pansecterome was meticulously annotated and characterized with respect to pangenome category distribution, PFAM domain frequency, and strain virulence assessment. This analysis revealed that highly virulent isolates have specific types of PFAM domains that are exclusive to them. Upon examining the repertoire of SIX genes known for virulence in other formae speciales, it was found that all isolates had a similar gene content except for two, which lacked SIX genes entirely.

Microbial strains of variable functional capacities coexist in microbiomes. Current bioinformatics methods of strain analysis cannot provide the direct linkage between strain composition and their gene contents from metagenomic data. Here we present Strain-level Pangenome Decomposition Analysis (StrainPanDA), a novel method that uses the pangenome coverage profile of multiple metagenomic samples to simultaneously reconstruct the composition and gene content variation of coexisting strains in microbial communities. We systematically validate the accuracy and robustness of StrainPanDA using synthetic data sets. To demonstrate the power of gene-centric strain profiling, we then apply StrainPanDA to analyze the gut microbiome samples of infants, as well as patients treated with fecal microbiota transplantation. We show that the linked reconstruction of strain composition and gene content profiles is critical for understanding the relationship between microbial adaptation and strain-specific functions (e.g., nutrient utilization and pathogenicity). Finally, StrainPanDA has minimal requirements for computing resources and can be scaled to process multiple species in a community in parallel. In short, StrainPanDA can be applied to metagenomic data sets to detect the association between molecular functions and microbial/host phenotypes to formulate testable hypotheses and gain novel biological insights at the strain or subspecies level.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen responsible for complicated UTIs and exhibits high antibiotic resistance, leading to increased mortality rates, especially in cases of multidrug-resistant strains. This study aimed to investigate the antibiotic susceptibility patterns and genomic characterization of XDR strains identified in end-stage liver disease patients who underwent liver transplants.
METHODS: In this study, a number of 30 individuals who underwent liver transplants were registered. Ninety urine and 60 wound site swab samples were collected and processed for culturing, identification, and antimicrobial sensitivity. Extensively drug-resistant strain EMARA01 was confirmed through Sanger sequencing and was then processed for whole genome sequencing to characterize the genomic pattern. Sequencing data were processed for de novo assembly using various tools and databases, including genome annotation, serotype identification, virulence factor genes, and antimicrobial resistance gene. Pangenome analysis of randomly selected 147 reference strains and EMAR01 sequenced strain was performed using the Bacterial Pan Genome Analysis (BPGA) software.
RESULTS: Of these total examined samples, nosocomial infection due to P. aeruginosa was detected in twelve patients\' samples. AST analysis showed that P. aeruginosa strains exhibit resistance to tobramycin, erythromycin, and gentamicin, followed by piperacillin and ofloxacin, and no strains exhibit resistance to meropenem and imipenem. The CARD database identified 59 AMR genes similar to the EMAR01 strain genome and mostly belong to the family involved in the resistance-nodulation-cell division (RND) antibiotic efflux pump. Five genes; nalC, nalD, MexR, MexA, and MexB, exhibit resistance to 14 classes of antibiotics, while two AMR; CpxR, and OprM, exhibit resistance to 15 classes of drugs. Pangenome analysis revealed that the pan-genome remained open, suggesting the potential for acquiring accessory and unique genes. Notably, the genes predominantly involved in amino acid transport metabolism were identified using the KEGG database.
CONCLUSIONS: This study provides valuable insights into the antimicrobial resistance profile, genetic features, and genomic evolution of P. aeruginosa strains causing UTIs in liver transplant patients. The findings emphasize the significance of comprehending AMR mechanisms and genetic diversity in P. aeruginosa for developing effective treatment strategies and infection control measures.

HIV-1 drug resistance genotypic tests have primarily been performed by Sanger sequencing of gene segments encoding different drug target proteins. Since the number of targets has increased with the addition of a new class of antiretroviral drugs, a simple high-throughput system for assessing nucleotide sequences throughout the HIV-1 genome is required. Here, we developed a new solution using nanopore sequencing of viral pangenomes amplified by PCR. Benchmark tests using HIV-1 molecular clones demonstrated an accuracy of up to 99.9%. In addition, validation tests of our protocol in 106 clinical samples demonstrated high concordance of drug resistance and tropism genotypes (92.5% and 98.1%, respectively) between the nanopore sequencing-based results and archived clinical determinations made based on Sanger sequencing data. These results suggest that our new approach will be a powerful solution for the comprehensive survey of HIV-1 drug resistance mutations in clinical settings.

The strain Gordonia polyisoprenivorans 135 is able to utilize a wide range of aromatic compounds. The aim of this work was to study the features of genetic organization and biotechnological potential of the strain G. polyisoprenivorans 135 as a degrader of aromatic compounds. The study of the genome of the strain 135 and the pangenome of the G. polyisoprenivorans species revealed that some genes, presumably involved in PAH catabolism, are atypical for Gordonia and belong to the pangenome of Actinobacteria. Analyzing the intergenic regions of strain 135 alongside the \"panIGRome\" of G. polyisoprenivorans showed that some intergenic regions in strain 135 also differ from those located between the same pairs of genes in related strains. The strain G. polyisoprenivorans 135 in our work utilized naphthalene (degradation degree 39.43%) and grew actively on salicylate. At present, this is the only known strain of G. polyisoprenivorans with experimentally confirmed ability to utilize these compounds.

A newly isolated bacterium Acinetobacter pittii S-30 was recovered from waste-contaminated soil in Ranchi, India. The isolated bacterium belongs to the ESKAPE organisms which represent the major nosocomial pathogens that exhibit high antibiotic resistance. Furthermore, average nucleotide identity (ANI) analysis also showed its closest match (>95%) to other A. pittii genomes. The isolate showed metal-resistant behavior and was able to survive up to 5 mM of ZnSO4. Whole genome sequencing and annotations revealed the occurrence of various genes involved in stress protection, motility, and metabolism of aromatic compounds. Moreover, genome annotation identified the gene clusters involved in secondary metabolite production (biosynthetic gene clusters) such as arylpolyene, acinetobactin like NRP-metallophore, betalactone, and hserlactone-NRPS cluster. The metabolic potential of A. pittii S-30 based on cluster of orthologous, and Kyoto Encyclopedia of Genes and Genomes indicated a high number of genes related to stress protection, metal resistance, and multiple drug-efflux systems etc., which is relatively rare in A. pittii strains. Additionally, the presence of various carbohydrate-active enzymes such as glycoside hydrolases (GHs), glycosyltransferases (GTs), and other genes associated with lignocellulose breakdown suggests that strain S-30 has strong biomass degradation potential. Furthermore, an analysis of genetic diversity and recombination in A. pittii strains was performed to understand the population expansion hypothesis of A. pittii strains. To our knowledge, this is the first report demonstrating the detailed genomic characterization of a heavy metal-resistant bacterium belonging to A. pittii. Therefore, the A. pittii S-30 could be a good candidate for the promotion of plant growth and other biotechnological applications.