Investigating the impact of insertion sequences and transposons in the genomes of the most significant phytopathogenic bacteria.

Genetic variability in phytopathogens is one of the main problems encountered for effective plant disease control. This fact may be related to the presence of transposable elements (TEs), but little is known about their role in host genomes. Here, we performed the most comprehensive analysis of insertion sequences (ISs) and transposons (Tns) in the genomes of the most important bacterial plant pathogens. A total of 35 692 ISs and 71 transposons were identified in 270 complete genomes. The level of pathogen-host specialization was found to be a significant determinant of the element distribution among the species. Some Tns were identified as carrying virulence factors, such as genes encoding effector proteins of the type III secretion system and resistance genes for the antimicrobial streptomycin. Evidence for IS-mediated ectopic recombination was identified in Xanthomonas genomes. Moreover, we found that IS elements tend to be inserted in regions near virulence and fitness genes, such ISs disrupting avirulence genes in X. oryzae genomes. In addition, transcriptome analysis under different stress conditions revealed differences in the expression of genes encoding transposases in the Ralstonia solanacearum, X. oryzae, and P. syringae species. Lastly, we also investigated the role of Tns in regulation via small noncoding regulatory RNAs and found these elements may target plant-cell transcriptional activators. Taken together, the results indicate that TEs may have a fundamental role in variability and virulence in plant pathogenic bacteria.

Uncovering the Secrets of Slow-Growing Bacteria in Tropical Savanna Soil Through Isolation and Genomic Analysis.

One gram of soil holds ten billion bacteria of thousands of different species, but most remain unknown, and one of the serious issues is intrinsic to slow-growing bacteria. In this study, we aimed to isolate and characterize slow-growing bacteria from Brazilian Cerrado soil. Over a period of 4 weeks, we conducted an incubation process and selected a total of 92 isolates. These isolates, consisting mostly of slow-growing bacteria, have the ability to thrive in low-water conditions and possess features that promote plant growth. To identify the isolated bacteria, we performed 16S rRNA sequencing analysis and found that the slow-growing strains were genetically similar to known bacterial species but also belonged to a novel group of species. The new strains identified were Caballeronia sp., Neobacillus sp., Bradyrhizobium sp., and high GC Gram-positive species. Furthermore, we conducted growth experiments using various culture media and temperature conditions. These experiments revealed an extended lag phase for five strains, indicating their slow growth characteristics. Genomic analysis of these five slow-growing bacteria showed their potential to participate in biogeochemical cycles, metabolize various carbohydrates, encode proteins with a role in promoting plant growth and have biosynthetic potential for secondary metabolites. Taken together, our findings reveal the untapped potential of slow-growing bacteria in tropical savanna soils.

Harnessing Novel Soil Bacteria for Beneficial Interactions with Soybean.

It is claimed that one g of soil holds ten billion bacteria representing thousands of distinct species. These bacteria play key roles in the regulation of terrestrial carbon dynamics, nutrient cycles, and plant productivity. Despite the overwhelming diversity of bacteria, most bacterial species remain largely unknown. Here, we used an oligotrophic medium to isolate novel soil bacteria for positive interaction with soybean. Strictly 22 species of bacteria from the soybean rhizosphere were selected. These isolates encompass ten genera (Kosakonia, Microbacterium, Mycobacterium, Methylobacterium, Monashia, Novosphingobium, Pandoraea, Anthrobacter, Stenotrophomonas, and Rhizobium) and have potential as novel species. Furthermore, the novel bacterial species exhibited plant growth-promoting traits in vitro and enhanced soybean growth under drought stress in a greenhouse experiment. We also reported the draft genome sequences of Kosakonia sp. strain SOY2 and Agrobacterium sp. strain SOY23. Along with our analysis of 169 publicly available genomes for the genera reported here, we demonstrated that these bacteria have a repertoire of genes encoding plant growth-promoting proteins and secondary metabolite biosynthetic gene clusters that directly affect plant growth. Taken together, our findings allow the identification novel soil bacteria, paving the way for their application in crop production.

Genomic analysis reveals the role of integrative and conjugative elements in plant pathogenic bacteria.

BACKGROUND: ICEs are mobile genetic elements found integrated into bacterial chromosomes that can excise and be transferred to a new cell. They play an important role in horizontal gene transmission and carry accessory genes that may provide interesting phenotypes for the bacteria. Here, we seek to research the presence and the role of ICEs in 300 genomes of phytopathogenic bacteria with the greatest scientific and economic impact. RESULTS: Seventy-eight ICEs (45 distinct elements) were identified and characterized in chromosomes of Agrobacterium tumefaciens, Dickeya dadantii, and D. solani, Pectobacterium carotovorum and P. atrosepticum, Pseudomonas syringae, Ralstonia solanacearum Species Complex, and Xanthomonas campestris. Intriguingly, the co-occurrence of four ICEs was observed in some P. syringae strains. Moreover, we identified 31 novel elements, carrying 396 accessory genes with potential influence on virulence and fitness, such as genes coding for functions related to T3SS, cell wall degradation and resistance to heavy metals. We also present the analysis of previously reported data on the expression of cargo genes related to the virulence of P. atrosepticum ICEs, which evidences the role of these genes in the infection process of tobacco plants. CONCLUSIONS: Altogether, this paper has highlighted the potential of ICEs to affect the pathogenicity and lifestyle of these phytopathogens and direct the spread of significant putative virulence genes in phytopathogenic bacteria.

The coexistence of monopartite integrative and conjugative elements in the genomes of Acidobacteria.

Soil bacteria can rapidly adapt to environmental perturbations through horizontal gene transfer. Acidobacteria is one of the most persistent dominant phyla in the soil. However, the role of these organisms in terrestrial ecosystems remains elusive. Here we identified and describe the integrative and conjugative elements (ICEs) in the published complete genomes of Acidobacteria. In total, ten novel ICEs were identified, in which nine were found integrated as three separated monopartite ICEs in the single chromosome sequences of three Acidobacteria. These ICEs carry a repertoire of genes with potential environmental roles, including heavy metal resistance, iron uptake, secondary metabolism, and antibiotic resistance. To our knowledge, these are the first evidence of three monopartite ICEs identified in the single chromosome, and this might be due to the absence of recognizable entry exclusion systems. We hypothesis that the coexistence of multiples ICEs in the chromosome of Acidobacteria might reflect a major advantage for the survival, resistance, and persistence of phylum in the environment.

Widespread distribution of prophages signaling the potential for adaptability and pathogenicity evolution of Ralstonia solanacearum species complex.

Integrated bacteriophages (prophages) can impact host cells, affecting their lifestyle, genomic diversity, and fitness. However, many basic aspects of how these organisms affect the host cell remain poorly understood. Ralstonia solanacearum is a gram-negative plant pathogenic bacterium that encompasses a great diversity of ecotypes regarded as a species complex (R. solanacearum Species Complex - RSSC). RSSC genomes have a mosaic structure containing numerous elements, signaling the potential for its evolution through horizontal gene transfer. Here, we analyzed 120 Ralstonia spp. genomes from the public database to identify prophage sequences. In total, 379 prophage-like elements were found in the chromosome and megaplasmid of Ralstonia spp. These elements encode genes related to host fitness, virulence factors, antibiotic resistance, and niche adaptation, which might contribute to RSSC adaptability. Prophage-like elements are widespread into the complex in different species and geographic origins, suggesting that the RSSC phages are ancestrally acquired. Complete prophages belonging to the families Inoviridae, Myoviridae, and Siphoviridae were found, being the members of Inoviridae the most abundant. Analysis of CRISPR-Cas spacer sequences demonstrated the presence of prophages sequences that indicate successive infection events during bacterial evolution. Besides complete prophages, we also demonstrated 14 novel putative prophages integrated into Ralstonia spp. genomes. Altogether, our results provide insights into the diversity of prophages in RSSC genomes and suggest that these elements may deeply affect the virulence and host adaptation and shaping the genomes among the strains of this important pathogen.

Mobile Genetic Elements Drive Antimicrobial Resistance Gene Spread in Pasteurellaceae Species.

Mobile genetic elements (MGEs) and antimicrobial resistance (AMR) drive important ecological relationships in microbial communities and pathogen-host interaction. In this study, we investigated the resistome-associated mobilome in 345 publicly available Pasteurellaceae genomes, a large family of Gram-negative bacteria including major human and animal pathogens. We generated a comprehensive dataset of the mobilome integrated into genomes, including 10,820 insertion sequences, 2,939 prophages, and 43 integrative and conjugative elements. Also, we assessed plasmid sequences of Pasteurellaceae. Our findings greatly expand the diversity of MGEs for the family, including a description of novel elements. We discovered that MGEs are comparable and dispersed across species and that they also co-occur in genomes, contributing to the family's ecology via gene transfer. In addition, we investigated the impact of these elements in the dissemination and shaping of AMR genes. A total of 55 different AMR genes were mapped to 721 locations in the dataset. MGEs are linked with 77.6% of AMR genes discovered, indicating their important involvement in the acquisition and transmission of such genes. This study provides an uncharted view of the Pasteurellaceae by demonstrating the global distribution of resistance genes linked with MGEs.

Summer school: a warm journey through teaching microbiology to undergraduate students.

The Núcleo de Estudos em Microbiologia Agrícola (NEMA) is an academic-scientific group created by graduate students in the Post Graduate in Agricultural Microbiology in the Department of Microbiology at Universidade Federal de Viçosa, Brazil. NEMA's purposes include promoting and sharing research and knowledge on microbiology in different fields of application. Here, we will comment on our experience in organizing the Summer School on Microbiology and teaching microbiology to undergraduate students during the program. NEMA offers this annual event to disseminate and stimulate knowledge about microbiology for undergraduate students in a participatory, collaborative and interactive way.

Potential evolutionary impact of integrative and conjugative elements (ICEs) and genomic islands in the Ralstonia solanacearum species complex.

Ralstonia solanacearum, a soil-borne plant pathogen, encompasses a large number of strains known as R. solanacearum species complex (RSSC). Although it has been suggested that mobile genetic elements (MGEs) may play an important role in the RSSC genome, the evolutionary impact of these elements remains unknown. Here, we identified and analysed Integrative and Conjugative Elements (ICEs) and Genomic Islands (GIs) in the 121 genomes published for Ralstonia spp., including RSSC strains and three other non-plant pathogenic Ralstonia spp. Our results provided a dataset of 12 ICEs and 31 GIs distributed throughout Ralstonia spp. Four novel ICEs in RSSC were found. Some of these elements cover 5% of the host genome and carry accessory genes with a potential impact on the fitness and pathogenicity of RSSC. In addition, phylogenetic analysis revealed that these MGEs clustered to the same species, but there is evidence of strains from different countries that host the same element. Our results provide novel insight into the RSSC adaptation, opening new paths to a better understanding of how these elements affect this soil-borne plant pathogen.

Transposable elements contribute to the genome plasticity of Ralstonia solanacearum species complex.

The extensive genetic diversity of Ralstonia solanacearum, a serious soil-borne phytopathogen, has led to the concept that R. solanacearum encompasses a species complex [R. solanacearum species complex (RSSC)]. Insertion sequences (ISs) are suggested to play an important role in the genome evolution of this pathogen. Here, we identified and analysed transposable elements (TEs), ISs and transposons, in 106 RSSC genomes and 15 Ralstonia spp. We mapped 10 259 IS elements in the complete genome of 62 representative RSSC strains and closely related Ralstonia spp. A unique set of 20 IS families was widespread across the strains, IS5 and IS3 being the most abundant. Our results showed six novel transposon sequences belonging to the Tn3 family carrying passenger genes encoding antibiotic resistance and avirulence proteins. In addition, internal rearrangement events associated with ISs were demonstrated in Ralstonia pseudosolanacearum strains. We also mapped IS elements interrupting avirulence genes, which provided evidence that ISs plays an important role in virulence evolution of RSSC. Additionally, the activity of ISs was demonstrated by transcriptome analysis and DNA hybridization in R. solanacearum isolates. Altogether, we have provided collective data of TEs in RSSC genomes, opening a new path for understanding their evolutionary impact on the genome evolution and diversity of this important plant pathogen.

Prediction of the secretomes of endophytic and nonendophytic fungi reveals similarities in host plant infection and colonization strategies.

Endophytic fungi are microorganisms that inhabit internal plant tissues without causing apparent damage. During the infection process, both endophytic and phytopathogenic fungi secrete proteins to resist or supplant the plant's defense mechanisms. This study analyzed the predicted secretomes of six species of endophytic fungi and compared them with predicted secretomes of eight fungal species with different lifestyles: saprophytic, necrotrophic, hemibiotrophic, and biotrophic. The sizes of the predicted secretomes varied from 260 to 1640 proteins, and the predicted secretomes have a wide diversity of CAZymes, proteases, and conserved domains. Regarding the CAZymes in the secretomes of the analyzed fungi, the most abundant CAZyme families were glycosyl hydrolase and serine proteases. Several predicted proteins have characteristics similar to those found in small, secreted proteins with effector characteristics (SSPEC). The most abundant conserved domains, besides those found in the SSPEC, have oxidation activities, indicating that these proteins can protect the fungus against oxidative stress, against domains with protease activity, which may be involved in the mechanisms of nutrition, or against lytic enzymes secreted by the host plant. This study demonstrates that secretomes of endophytic and nonendophytic fungi share an arsenal of proteins important in the process of infection and colonization of host plants.

Comparative Genomics of Actinobacillus pleuropneumoniae Serotype 8 Reveals the Importance of Prophages in the Genetic Variability of the Species.

Actinobacillus pleuropneumoniae is the etiologic agent of porcine pleuropneumonia. Currently, there are 18 different serotypes; the serotype 8 is the most widely distributed in the United States, Canada, United Kingdom, and southeastern Brazil. In this study, genomes of seven A. pleuropneumoniae serotype 8 clinical isolates were compared to the other genomes of twelve serotypes. The analyses of serotype 8 genomes resulted in a set of 2352 protein-coding sequences. Of these sequences, 76.6% are present in all serotypes, 18.5% are shared with some serotypes, and 4.9% were differential. This differential portion was characterized as a series of hypothetical and regulatory protein sequences: mobile element sequence. Synteny analysis demonstrated possible events of gene recombination and acquisition by horizontal gene transfer (HGT) in this species. A total of 30 sequences related to prophages were identified in the genomes. These sequences represented 0.3 to 3.5% of the genome of the strains analyzed, and 16 of them contained complete prophages. Similarity analysis between complete prophage sequences evidenced a possible HGT with species belonging to the family Pasteurellaceae. Thus, mobile genetic elements, such as prophages, are important components of the differential portion of the A. pleuropneumoniae genome and demonstrate a central role in the evolution of the species. This study represents the first study done to understand the genome of A. pleuropneumoniae serotype 8.

Colletotrichum: species complexes, lifestyle, and peculiarities of some sources of genetic variability.

The genus Colletotrichum comprises species with different lifestyles but is mainly known for phytopathogenic species that infect crops of agronomic relevance causing considerable losses. The fungi of the genus Colletotrichum are distributed in species complexes and within each complex some species have particularities regarding their lifestyle. The most commonly found and described lifestyles in Colletotrichum are endophytic and hemibiotrophic phytopathogenic. Several of these phytopathogenic species show wide genetic variability, which makes long-term maintenance of resistance in plants difficult. Different mechanisms may play an important role in the emergence of genetic variants but are not yet fully understood in this genus. These mechanisms include heterokaryosis, a parasexual cycle, sexual cycle, transposable element activity, and repeat-induced point mutations. This review provides an overview of the genus Colletotrichum, the species complexes described so far and the most common lifestyles in the genus, with a special emphasis on the mechanisms that may be responsible, at least in part, for the emergence of new genotypes under field conditions.

Characterization of antibiotic resistance genes in the species of the rumen microbiota.

Infections caused by multidrug resistant bacteria represent a therapeutic challenge both in clinical settings and in livestock production, but the prevalence of antibiotic resistance genes among the species of bacteria that colonize the gastrointestinal tract of ruminants is not well characterized. Here, we investigate the resistome of 435 ruminal microbial genomes in silico and confirm representative phenotypes in vitro. We find a high abundance of genes encoding tetracycline resistance and evidence that the tet(W) gene is under positive selective pressure. Our findings reveal that tet(W) is located in a novel integrative and conjugative element in several ruminal bacterial genomes. Analyses of rumen microbial metatranscriptomes confirm the expression of the most abundant antibiotic resistance genes. Our data provide insight into antibiotic resistange gene profiles of the main species of ruminal bacteria and reveal the potential role of mobile genetic elements in shaping the resistome of the rumen microbiome, with implications for human and animal health.

Genomic analysis and lactose transporter expression in Kluyveromyces marxianus CCT 7735.

Kluyveromyces marxianus CCT 7735 has been used to produce ethanol, aromatic compounds, enzymes and heterologous proteins besides assimilates lactose as carbon source. Its genome has 10.7 Mb and encodes 4787 genes distributed in 8 nuclear chromosomes and one mitochondrial. Contrary to Kluyveromyces lactis, which has a unique LAC12 gene (encodes lactose permease), K. marxianus possesses four. The presence of degenerated copies and Solo-LTRs related to retrotransposon TKM close to the LAC12 genes in K. marxianus indicates ectopic recombinations. The Lac12 permeases of K. marxianus and K. lactis are conserved, however the conservation is higher between the copy of the left side of the chromosome three and the unique copy of K. lactis, indicating that this copy is the ancestor. The expression of the four LAC12 genes occurred in aerobiosis and hypoxia. Notably, the high lactose consumption in hypoxia seems to be related to the high expression of the LAC12 genes.

Desulfovibrio alaskensis prophages and their possible involvement in the horizontal transfer of genes by outer membrane vesicles.

Desulfovibrio alaskensis is a Gram-negative bacterial species that belongs to the group of Sulphate Reducing Bacteria (SRB) and presents prophages in genomes, a common characteristic of the genus Desulfovibrio. Genetic material can be transported by outer membrane vesicles, however, no data regarding the production of these vesicles has been reported for D. alaskensis. To verify the expression of D. alaskensis prophages and their involvement with outer membrane vesicles, the DSM16109 strain was used. The DSM16109 strain had three prophages and presented reduced growth after mitomycin C addition when compared to the control culture. This reduction was accompanied by the presence of virus-like particles (VLPs), indicating mitomycin C dependent prophage induction. The increase in the number of cap gene copies and transcriptions of the three prophages was verified in the control sample, however, without the formation of VLPs. Prophage genes were identified in outer membrane vesicles from cultures treated and not treated with mitomycin C. DSM16109 prophages are expressed spontaneously but only in the presence of mitomycin C was it possible to observe VLP formation. Due to the genetic material detection from the prophages within outer membrane vesicles, this property may be related to the horizontal transfer of viral genes.

The repertoire of effector candidates in Colletotrichum lindemuthianum reveals important information about Colletotrichum genus lifestyle.

The fungus Colletotrichum lindemuthianum is the causal agent of anthracnose in the common bean (Phaseolus vulgaris), and anthracnose is one of the most devastating diseases of this plant species. However, little is known about the proteins that are essential for the fungus-plant interactions. Knowledge of the fungus' arsenal of effector proteins is of great importance for understanding this pathosystem. In this work, we analyzed for the first time the arsenal of Colletotrichum lindemuthianum effector candidates (ClECs) and compared them with effector proteins from other species of the genus Colletotrichum, providing a valuable resource for studying the infection mechanisms of these pathogens in their hosts. Isolates of two physiological races (83.501 and 89 A2 2-3) of C. lindemuthianum were used to predict 353 and 349 ClECs, respectively. Of these ClECs, 63% were found to be rich in cysteine, have repetitive sequences of amino acids, and/or possess nuclear localization sequences. Several conserved domains were found between the ClECs. We also applied the effector prediction to nine species in the genus Colletotrichum, and the results ranged from 247 predicted effectors in Colletotrichum graminicola to 446 in Colletotrichum orbiculare. Twelve conserved domains were predicted in the effector candidates of all analyzed species of Colletotrichum. An expression analysis of the eight genes encoding the effector candidates in C. lindemuthianum revealed their induction during the biotrophic phase of the fungus on the bean.

Screening and characterization of prophages in Desulfovibrio genomes.

Bacteria of the genus Desulfovibrio belong to the group of Sulphate Reducing Bacteria (SRB). SRB generate significant liabilities in the petroleum industry, mainly due to their ability to microbiologically induce corrosion, biofilm formation and H2S production. Bacteriophages are an alternative control method for SRB, whose information for this group of bacteria however, is scarce. The present study developed a workflow for the identification of complete prophages in Desulfovibrio. Poly-lysogenesis was shown to be common in Desulfovibrio. In the 47 genomes analyzed 53 complete prophages were identified. These were classified within the order Caudovirales, with 69.82% belonging to the Myoviridade family. More than half the prophages identified have genes coding for lysozyme or holin. Four of the analyzed bacterial genomes present prophages with identity above 50% in the same strain, whose comparative analysis demonstrated the existence of colinearity between the sequences. Of the 17 closed bacterial genomes analyzed, 6 have the CRISPR-Cas system classified as inactive. The identification of bacterial poly-lysogeny, the proximity between the complete prophages and the possible inactivity of the CRISPR-Cas in closed bacterial genomes analyzed allowed the choice of poly-lysogenic strains with prophages belonging to the Myoviridae family for the isolation of prophages and testing of related strains for subsequent studies.

Comparative analysis of the mitochondrial genome of the fungus Colletotrichum lindemuthianum, the causal agent of anthracnose in common beans.

Fungi of the genus Colletotrichum are economically important and are used as models in plant-pathogen interaction studies. In this study, the complete mitochondrial genomes of two Colletotrichum lindemuthianum isolates were sequenced and compared with the mitochondrial genomes of seven species of Colletotrichum. The mitochondrial genome of C. lindemuthianum is a typical circular molecule 37,446 bp (isolate 89 A2 2-3) and 37,440 bp (isolate 83.501) in length. The difference of six nucleotides between the two genomes is the result of a deletion in the ribosomal protein S3 (rps3) gene in the 83.501 isolate. In addition, substitution of adenine for guanine within the rps3 gene in the mitochondrial genome of the 83.501 isolate was observed. Compared to the previously sequenced C. lindemuthianum mitochondrial genome, an exon no annotated in the cytochrome c oxidase I (cox1) gene and a non-conserved open reading frame (ncORF) were observed. The size of the mitochondrial genomes of the seven species of Colletotrichum was highly variable, being attributed mainly to the ncORF, ranging from one to 10 and also from introns ranging from one to 11 and which encode a total of up to nine homing endonucleases. This paper reports for the first time by means of transcriptome that then ncORFs are transcribed in Colletotrichum spp. Phylogeny data revealed that core mitochondrial genes could be used as an alternative in phylogenetic relationship studies in Colletotrichum spp. This work contributes to the genetic and biological knowledge of Colletotrichum spp., which is of great economic and scientific importance.