In eukaryotic genomes, rDNA generally resides as a highly repetitive and dynamic structure, making it difficult to study. Here, a synthetic rDNA array on chromosome III in budding yeast was constructed to serve as the sole source of rRNA. Utilizing the loxPsym site within each rDNA repeat and the Cre recombinase, we were able to reduce the copy number to as few as eight copies. Additionally, we constructed strains with two or three rDNA arrays and found that the presence of multiple arrays did not affect the formation of a single nucleolus. Although alteration of the position and number of rDNA arrays did impact the three-dimensional genome structure, the additional rDNA arrays had no deleterious influence on cell growth or transcriptomes. Overall, this study sheds light on the high plasticity of rDNA organization and opens up opportunities for future rDNA engineering.

A key step for comparative genomics is to group open reading frames into functionally and evolutionarily meaningful gene clusters. Gene clustering is complicated by intraspecific duplications and horizontal gene transfers that are frequent in prokaryotes. In consequence, gene clustering methods must deal with a trade-off between identifying vertically transmitted representatives of multicopy gene families, which are recognizable by synteny conservation, and retrieving complete sets of species-level orthologs. We studied the implications of adopting homology, orthology, or synteny conservation as formal criteria for gene clustering by performing comparative analyses of 125 prokaryotic pangenomes.
Clustering criteria affect pangenome functional characterization, core genome inference, and reconstruction of ancestral gene content to different extents. Species-wise estimates of pangenome and core genome sizes change by the same factor when using different clustering criteria, allowing robust cross-species comparisons regardless of the clustering criterion. However, cross-species comparisons of genome plasticity and functional profiles are substantially affected by inconsistencies among clustering criteria. Such inconsistencies are driven not only by mobile genetic elements, but also by genes involved in defense, secondary metabolism, and other accessory functions. In some pangenome features, the variability attributed to methodological inconsistencies can even exceed the effect sizes of ecological and phylogenetic variables.
Choosing an appropriate criterion for gene clustering is critical to conduct unbiased pangenome analyses. We provide practical guidelines to choose the right method depending on the research goals and the quality of genome assemblies, and a benchmarking dataset to assess the robustness and reproducibility of future comparative studies.

Stenotrophomonas maltophilia is a species with immensely broad phenotypic and genotypic diversity that could widely distribute in natural and clinical environments. However, little attention has been paid to reveal their genome plasticity to diverse environments. In the present study, a comparative genomic analysis of S. maltophilia isolated from clinical and natural sources was systematically explored its genetic diversity of 42 sequenced genomes. The results showed that S. maltophilia owned an open pan-genome and had strong adaptability to different environments. A total of 1612 core genes were existed with an average of 39.43% of each genome, and the shared core genes might be necessary to maintain the basic characteristics of those S. maltophilia strains. Based on the results of the phylogenetic tree, the ANI value, and the distribution of accessory genes, genes associated with the fundamental process of those strains from the same habitat were found to be mostly conserved in evolution. Isolates from the same habitat had a high degree of similarity in COG category, and the most significant KEGG pathways were mainly involved in carbohydrate and amino acid metabolism, indicating that genes related to essential processes were mostly conserved in evolution for the clinical and environmental settings. Meanwhile, the number of resistance and efflux pump gene was significantly higher in the clinical setting than that of in the environmental setting. Collectively, this study highlights the evolutionary relationships of S. maltophilia isolated from clinical and environmental sources, shedding new light on its genomic diversity.

Acidiferrobacter spp. are facultatively anaerobic acidophiles that belong to a distinctive Acidiferrobacteraceae family, which are similar to Ectothiorhodospiraceae phylogenetically, and are closely related to Acidithiobacillia class/subdivision physiologically. The limited genome information has kept them from being studied on molecular taxonomy and environmental adaptation in depth. Herein, Af. thiooxydans ZJ was isolated from acid mine drainage (AMD), and the complete genome sequence was reported to scan its genetic constitution for taxonomic and adaptative feature exploration. The genome has a single chromosome of 3,302,271 base pairs (bp), with a GC content of 63.61%. The phylogenetic tree based on OrthoANI highlighted the unique position of Af. thiooxydans ZJ, which harbored more unique genes among the strains from Ectothiorhodospiraceae and Acidithiobacillaceae by pan-genome analysis. The diverse mobile genetic elements (MGEs), such as insertion sequence (IS), clustered regularly interspaced short palindromic repeat (CRISPR), prophage, and genomic island (GI), have been identified and characterized in Af. thiooxydans ZJ. The results showed that Af. thiooxydans ZJ may effectively resist the infection of foreign viruses and gain functional gene fragments or clusters to shape its own genome advantageously. This study will offer more evidence of the genomic plasticity and improve our understanding of evolutionary adaptation mechanisms to extreme AMD environment, which could expand the potential utilization of Af. thiooxydans ZJ as an iron and sulfur oxidizer in industrial bioleaching.

Genome plasticity is a key determinant that Acinetobacter johnsonii could widely distribute in natural and clinical environments. However, little attention has been paid to figure out the changes in the genome during A. johnsonii\'s evolution. Here, a comparative genomic analysis of A. johnsonii isolated from clinical and environmental sources was conducted. In this study, we found A. johnsonii has an open pan-genome and has great adaptability to different environments. Based on the results of the phylogenetic tree, ANI value and the distribution of accessory genes, we found that strains from the same habitat had a high degree of similarity. Though genes associated with the fundamental process were mostly conserved in evolution, clinical-derived isolates accumulate more genes associated with translational modification, β-lactamase and defense mechanisms, whereas environmental-derived isolates enriched more genes related to substances degradation. In addition, clinical-derived strains harbored some \"strong\" virulence islands and resistance islands. This study highlights the evolutionary relationship of A. johnsonii isolates from clinical and environmental sources.

Salmonella enterica remains one of the leading causes of foodborne bacterial disease. Retail meat is a major source of human salmonellosis. However, comparative genomic analyses of S. enterica isolates from retail meat from different sources in China are lacking. A total of 341 S. enterica strains were isolated from retail meat in sixteen districts of Beijing, China, at three different time points (January 1st, May 1st, and October 1st) in 2017. Comparative genomics was performed to investigate the genetic diversity, virulence and antimicrobial resistance gene (ARG) profiles of these isolates. The most common serotype was S. Enteritidis (203/341, 59.5%), which dominated among isolates from three different time points during the year. Laboratory retesting confirmed the accuracy of the serotyping results predicted by the Salmonella In Silico Typing Resource (SISTR) (96.5%). The pangenome of the 341 S. enterica isolates contained 13,931 genes, and the core genome contained 3,635 genes. Higher Salmonella phage 118970 sal3 (219/341, 64.2%) and Gifsy-2 (206/341, 60.4%) prevalence contributed to the diversity of the accessory genes, especially those with unknown functions. IncFII(S), IncX1, and IncFIB(S) plasmid replicons were more common in these isolates and were major sources of horizontally acquired foreign genes. The virulence gene profile showed fewer virulence genes associated with type III secretion systems in certain isolates from chicken. A total of 88 different ARGs were found in the 341 isolates. Three beta-lactamases, namely, bla CTX - M - 55 (n = 15), bla CTX - M - 14 (n = 11), and bla CTX - M - 65 (n = 11), were more prevalent in retail meats. The emergence of qnrE1 and bla CTX - M - 123 indicated a potential increase in the prevalence of retail meats. After the prohibition of colistin in China, three and four isolates were positive for the colistin resistance genes mcr-1.1 and mcr-9, respectively. Thus, we explored the evolution and genomic features of S. enterica isolates from retail meats in Beijing, China. The diverse ARGs of these isolates compromise food security and are a clinical threat.

Genomic islands (GIs) play an important role in the chromosome diversity of Enterococcus. In the current study, we aimed to investigate the spread of GIs between Enterococcus strains and their correlation with antibiotic resistance genes (ARGs). Bitsliced Genomic Signature Indexes (BIGSI) were used to screen the NCBI Sequence Read Archive (SRA) for multiple resistant Enterococcus. A total of 37 pairs of raw reads were screened from 457,000 whole-genome sequences (WGS) in the SRA database, which come from 37 Enterococci distributed in eight countries. These raw reads were assembled for the prediction and analysis of GIs, ARGs, plasmids and prophages.
The results showed that GIs were universal in Enterococcus, with an average of 3.2 GIs in each strain. Network analysis showed that frequent genetic information exchanges mediated by GIs occurred between Enterococcus strains. Seven antibiotic-resistant genomic islands (ARGIs) were found to carry one to three ARGs, mdtG, tetM, dfrG, lnuG, and fexA, in six strains. These ARGIs were involved in the spread of antibiotic resistance in 45.9% of the 37 strains, although there was no significant positive correlation between the frequency of GI exchanges and the number of ARGs each strain harboured (r = 0. 287, p = 0.085). After comprehensively analysing the genome data, we found that partial GIs were associated with multiple mobile genetic elements (transposons, integrons, prophages and plasmids) and had potential natural transformation characteristics.
All of these results based on genomic sequencing suggest that GIs might mediate the acquisition of some ARGs and might be involved in the high genome plasticity of Enterococcus through transformation, transduction and conjugation, thus providing a fitness advantage for Enterococcus hosts under complex environmental factors.

Magnaporthe oryzae causes blast disease on more than 50 species of monocot plants, including important crops such as rice, millet, and most recently wheat. Additionally, it is an important model system for studying host-pathogen interaction. Here, we report a high-quality genome assembly and annotation of a laboratory strain 2539 of M. oryzae, which is a widely used progeny of a rice-infecting isolate and a grass-infecting isolate. The genome sequence of strain 2539 will be useful for studying the evolution, host adaption, and pathogenicity of M. oryzae, which will be beneficial for a better understanding of the mechanisms of host-pathogen interaction.

Electronic waste (e-waste) has caused a severe worldwide pollution problem. Despite increasing isolation of degradative microorganisms from e-waste contaminated environments, the mechanisms underlying their adaptive evolution in such habitats remain unclear. Sphingomonads generally have xenobiotic-degrading ability and may play important roles in bioremediation. Sphingobium hydrophobicum C1T, characterized with superior cell surface hydrophobicity, was recently isolated from e-waste contaminated river sediment. To dissect the mechanisms driving its adaptive evolution, we evaluated its stress resistance, sequenced its genome and performed comparative genomic analysis with 19 other Sphingobium strains. Strain C1T can feed on several kinds of e-waste-derived xenobiotics, exhibits a great resistance to heavy metals and possesses a high colonization ability. It harbors abundant genes involved in environmental adaptation, some of which are intrinsic prior to experiencing e-waste contamination. The extensive genomic variations between strain C1T and other Sphingobium strains, numerous C1T-unique genes, massive mobile elements and frequent genome rearrangements reflect a high genome plasticity. Positive selection, gene duplication, and especially horizontal gene transfer drive the adaptive evolution of strain C1T. Moreover, presence of type IV secretion systems may allow strain C1T to be a source of beneficial genes for surrounding microorganisms. This study provides new insights into the adaptive evolution of sphingomonads, and potentially guides bioremediation strategies.

Plant diseases cause extensive yield loss of crops worldwide, and secretory \'warfare\' occurs between plants and pathogenic organisms all the time. Filamentous plant pathogens have evolved the ability to manipulate host processes and facilitate colonization through secreting effectors inside plant cells. The stresses from hosts and environment can drive the genome dynamics of plant pathogens. Remarkable advances in plant pathology have been made owing to these adaptable genome regions of several lineages of filamentous phytopathogens. Characterization new effectors and interaction analyses between pathogens and plants have provided molecular insights into the plant pathways perturbed during the infection process. In this mini-review, we highlight promising approaches of identifying novel effectors based on the genome plasticity. We also discuss the interaction mechanisms between plants and their filamentous pathogens and outline the possibilities of effector gene expression under epigenetic control that will be future directions for research.